WO2004037251A1 - Inhibitors of 11-beta-hydroxy steroid dehydrogenase type 1 and type 2 - Google Patents

Inhibitors of 11-beta-hydroxy steroid dehydrogenase type 1 and type 2 Download PDF

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Publication number
WO2004037251A1
WO2004037251A1 PCT/GB2003/004590 GB0304590W WO2004037251A1 WO 2004037251 A1 WO2004037251 A1 WO 2004037251A1 GB 0304590 W GB0304590 W GB 0304590W WO 2004037251 A1 WO2004037251 A1 WO 2004037251A1
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Prior art keywords
arh
compound according
formula
hsd
group
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PCT/GB2003/004590
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French (fr)
Inventor
Nigel Vicker
Xiangdong Su
Dharshini Ganeshapillai
Atul Purohit
Michael John Reed
Barry Victor Lloyd Potter
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Sterix Limited
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Priority claimed from GBGB0224830.0A external-priority patent/GB0224830D0/en
Application filed by Sterix Limited filed Critical Sterix Limited
Priority to MXPA05004434A priority Critical patent/MXPA05004434A/en
Priority to CA002501228A priority patent/CA2501228A1/en
Priority to JP2004546183A priority patent/JP2006514614A/en
Priority to EP03758357A priority patent/EP1556040A1/en
Priority to AU2003274373A priority patent/AU2003274373A1/en
Priority to BR0315605-2A priority patent/BR0315605A/en
Publication of WO2004037251A1 publication Critical patent/WO2004037251A1/en
Priority to NO20052469A priority patent/NO20052469L/en

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    • C07D263/52—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings condensed with carbocyclic rings or ring systems
    • C07D263/54—Benzoxazoles; Hydrogenated benzoxazoles
    • C07D263/56—Benzoxazoles; Hydrogenated benzoxazoles with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 2
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    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
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    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
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    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/38—Drugs for disorders of the endocrine system of the suprarenal hormones
    • A61P5/42—Drugs for disorders of the endocrine system of the suprarenal hormones for decreasing, blocking or antagonising the activity of mineralocorticosteroids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/08—Indoles; Hydrogenated indoles with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to carbon atoms of the hetero ring
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    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/30—Indoles; Hydrogenated indoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to carbon atoms of the hetero ring
    • C07D209/42—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
    • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
    • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
    • C07D235/06—Benzimidazoles; Hydrogenated benzimidazoles with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 2
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
    • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
    • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
    • C07D235/06—Benzimidazoles; Hydrogenated benzimidazoles with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 2
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    • C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
    • C07D277/62—Benzothiazoles
    • C07D277/68—Benzothiazoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 2
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    • C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
    • C07D277/62—Benzothiazoles
    • C07D277/68—Benzothiazoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 2
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    • C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
    • C07D277/62—Benzothiazoles
    • C07D277/68—Benzothiazoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 2
    • C07D277/82—Nitrogen atoms

Definitions

  • the present invention relates to a compound.
  • the present invention provides compounds capable of inhibiting 11 ⁇ -hydroxysteroid dehydrogenase (11 ⁇ - HSD).
  • Glucocorticoids are synthesised in the adrenal cortex from cholesterol.
  • the principle glucocorticoid in the human body is cortisol, this hormone is synthesised and secreted in response to the adrenocortictrophic hormone (ACTH) from the pituitary gland in a circadian, episodic manner, but the secretion of this hormone can also be stimulated by stress, exercise and infection.
  • Cortisol circulates mainly bound to transcortin (cortisol binding protein) or albumin and only a small fraction is free (5-10%) for biological processes [1].
  • Cortisol has a wide range of physiological effects, including regulation of carbohydrate, protein and lipid metabolism, regulation of normal growth and development, influence on cognitive function, resistance to stress and mineralocorticoid activity. Cortisol works in the opposite direction compared to insulin meaning a stimulation of hepatic gluconeogenesis, inhibition of peripheral glucose uptake and increased blood glucose concentration. Glucocorticoids are also essential in the regulation of the immune response. When circulating at higher concentrations glucocorticoids are immunosuppressive and are used pharmacologically as anti-inflammatory agents.
  • Glucocorticoids like other steroid hormones are lipophilic and penetrate the cell membrane freely. Cortisol binds, primarily, to the intracellular glucocorticoid receptor (GR) that then acts as a transcription factor to induce the expression of glucocorticoid responsive genes, and as a result of that protein synthesis.
  • GR glucocorticoid receptor
  • 11 ⁇ -HSD Localisation of the 11 ⁇ -HSD showed that the enzyme and its activity is highly present in the MR dependent tissues, kidney and parotid. However in tissues where the MR is not mineralocorticoid specific and is normally occupied by glucocorticoids, 11 ⁇ -HSD is not present in these tissues, for example in the heart and hippocampus [5]. This research also showed that inhibition of 11 ⁇ -HSD caused a loss of the aldosterone specificity of the MR in these mineralocorticoid dependent tissues.
  • 11 ⁇ -HSD type 2 acts as a dehydrogenase to convert the secondary alcohol group at the C-11 position of cortisol to a secondary ketone, so producing the less active metabolite cortisone.
  • 11 ⁇ -HSD type 1 is thought to act mainly in vivo as a reductase, that is in the opposite direction to type 2 [6] [see below].
  • 11 ⁇ - HSD type 1 and type 2 have only a 30% amino acid homology.
  • cortisol The intracellular activity of cortisol is dependent on the concentration of glucocorticoids and can be modified and independently controlled without involving the overall secretion and synthesis of the hormone.
  • 11 ⁇ -HSD type 1 The direction of 11 ⁇ -HSD type 1 reaction in vivo is generally accepted to be opposite to the dehydrogenation of type 2. In vivo homozygous mice with a disrupted type 1 gene are unable to convert cortisone to cortisol, giving further evidence for the reductive activity of the enzyme [7]. 11 ⁇ -HSD type 1 is expressed in many key glucocorticoid regulated tissues like the liver, pituitary, gonad, brain, adipose and adrenals .however, the function of the enzyme in many of these tissues is poorly understood [8].
  • cortisone in the body is higher than that of cortisol , cortisone also binds poorly to binding globulins, making cortisone many times more biologically available.
  • cortisol is secreted by the adrenal cortex, there is a growing amount of evidence that the intracellular conversion of E to F may be an important mechanism in regulating the action of glucocorticoids [9].
  • 11 ⁇ -HSD type 1 allows certain tissues to convert cortisone to cortisol to increase local glucocorticoid activity and potentiate adaptive response and counteracting the type 2 activity that could result in a fall in active glucocorticoids [10]. Potentiation of the stress response would be especially important in the brain and high levels of 11 ⁇ - HSD type 1 are found around the hippocampus, further proving the role of the enzyme. 11 ⁇ -HSD type 1 also seems to play an important role in hepatocyte maturation [8].
  • the 11 ⁇ -HSD type 1 enzyme is in the detoxification process of many non-steroidal carbonyl compounds, reduction of the carbonyl group of many toxic compounds is a common way to increase solubility and therefore increase their excretion.
  • the 11 ⁇ -HSD typel enzyme has recently been shown to be active in lung tissue [11]. Type 1 activity is not seen until after birth, therefore mothers who smoke during pregnancy expose their children to the harmful effects of tobacco before the child is able to metabolically detoxify this compound.
  • the 11 ⁇ -HSD type 2 converts cortisol to cortisone, thus protecting the MR in many key regulatory tissues of the body.
  • the importance of protecting the MR from occupation by glucocorticoids is seen in patients with AME or liquorice intoxification.
  • Defects or inactivity of the type 2 enzyme results in hypertensive syndromes and research has shown that patients with an hypertensive syndrome have an increased urinary excretion ratio of cortisol : cortisone. This along with a reported increase in the half life of radiolabelled cortisol suggests a reduction of 11 ⁇ -HSD type 2 activity [12].
  • cortisol opposes the action of insulin meaning a stimulation of hepatic gluconeogenesis, inhibition of peripheral glucose uptake and increased blood glucose concentration.
  • the effects of cortisol appear to be enhanced in patients suffering from glucose intolerance or diabetes mellitus.
  • Inhibition of the enzyme 11 ⁇ -HSD type 1 would increase glucose uptake and inhibit hepatic gluconeogenesis, giving a reduction in circulatory glucose levels.
  • the development of a potent 11 ⁇ -HSD type 1 inhibitor could therefore have considerable therapeutic potential for conditions associated with elevated blood glucose levels.
  • a specific 11 ⁇ -HSD type 1 inhibitor might be of some importance by reducing neuronal dysfunctions and the loss of cognitive functions associated with ageing, by blocking the conversion of cortisone to cortisol.
  • Glucocorticoids also have an important role in regulating part of the immune response [13]. Glucocorticoids can suppress the production of cytokines and regulate the receptor levels. They are also involved in determining whether T-helper (Th) lymphocytes progress into either Th1 or Th2 phenotype. These two different types of Th cells secrete a different profile of cytokines, Th2 is predominant in a glucocorticoid environment. By inhibiting 11 ⁇ -HSD type 1 , Th1 cytokine response would be favoured. It is also possible to inhibit 11 ⁇ -HSD type 2 , thus by inhibiting the inactivation of cortisol, it may be possible to potentiate the anti-inflammatory effects of glucocorticoids.
  • the present invention provides a compound having Formula Formula I
  • R 4 is selected from H and hydrocarbyl
  • R 5 is a hydrocarbyl group and L is an optional linker group, or R-, and R 2 together form a ring substituted with the group
  • R 3 is H or a substituent, and wherein X is selected from S, O, NR 6 and C(R 7 )(R 8 ). wherein R 6 is selected from H and hydrocarbyl groups, wherein each of R 7 and R 8 are independently selected from H and hydrocarbyl groups.
  • the present invention provides a pharmaceutical composition comprising
  • R 4 is selected from H and hydrocarbyl
  • R 5 is a hydrocarbyl group and L is an optional linker group, or R.
  • R 2 together form a ring substituted with the group
  • R 3 is H or a substituent, and wherein X is selected from S, O, NR 6 and C(R 7 )(R 8 ), wherein R 6 is selected from H and hydrocarbyl groups, wherein each of R 7 and R 8 are independently selected from H and hydrocarbyl groups.
  • the present invention provides a compound having Formula I Formula
  • R., and R 2 are a group of the formula wherein R 4 is selected from H and hydrocarbyl, R 5 is a hydrocarbyl group and L is an optional linker group, or R., and R 2 together form a ring substituted with the group
  • R 3 is H or a substituent, and wherein X is selected from S, O, NR 6 and C(R 7 )(R 8 ), wherein R 6 is selected from H and hydrocarbyl groups, wherein each of R 7 and R 8 are independently selected from H and hydrocarbyl groups, for use in medicine.
  • the present invention provides a use of a compound in the manufacture of a medicament for use in the therapy of a condition or disease associated with 11 ⁇ -HSD, wherein the compound has Formula I Formula
  • R 4 is selected from H and hydrocarbyl
  • R 5 is a hydrocarbyl group and L is an optional linker group, or R.
  • R 2 together form a ring substituted with the group wherein R 3 is H or a substituent
  • X is selected from S, O, NR 6 and C(R 7 )(R 8 ), wherein R 6 is selected from H and hydrocarbyl groups, wherein each of R 7 and R 8 are independently selected from H and hydrocarbyl groups.
  • the compounds of the present invention can act as 11 ⁇ -HSD inhibitors.
  • the compounds may inhibit the interconversion of inactive 11-keto steroids with their active hydroxy equivalents.
  • present invention provides methods by which the conversion of the inactive to the active form may be controlled, and to useful therapeutic effects which may be obtained as a result of such control. More specifically, but not exclusively, the invention is concerned with interconversion between cortisone and cortisol in humans.
  • Another advantage of the compounds of the present invention is that they may be potent 11 ⁇ -HSD inhibitors in vivo.
  • Some of the compounds of the present invention are also advantageous in that they may be orally active.
  • the present invention may provide for a medicament for one or more of (i) regulation of carbohydrate metabolism, (ii) regulation of protein metabolism, (iii) regulation of lipid metabolism, (iv) regulation of normal growth and/or development, (v) influence on cognitive function, (vi) resistance to stress and mineralocorticoid activity.
  • Some of the compounds of the present invention may also be useful for inhibiting hepatic gluconeogenesis.
  • the present invention may also provide a medicament to relieve the effects of endogenous glucocorticoids in diabetes mellitus, obesity (including centripetal obesity), neuronal loss and/or the cognitive impairment of old age.
  • the invention provides the use of an inhibitor of 11 ⁇ -HSD in the manufacture of a medicament for producing one or more therapeutic effects in a patient to whom the medicament is administered, said therapeutic effects selected from inhibition of hepatic gluconeogenesis, an increase in insulin sensitivity in adipose tissue and muscle, and the prevention of or reduction in neuronal loss/cognitive impairment due to glucocorticoid- potentiated neurotoxicity or neural dysfunction or damage.
  • the invention provides a method of treatment of a human or animal patient suffering from a condition selected from the group consisting of: hepatic insulin resistance, adipose tissue insulin resistance, muscle insulin resistance, neuronal loss or dysfunction due to glucocorticoid potentiated neurotoxicity, and any combination of the aforementioned conditions, the method comprising the step of administering to said patient a medicament comprising a pharmaceutically active amount of a compound in accordance with the present invention.
  • Some of the compounds of the present invention may be useful for the treatment of cancer, such as breast cancer, as well as (or in the alternative) non-malignant conditions, such as the prevention of auto-immune diseases, particularly when pharmaceuticals may need to be administered from an early age.
  • cancer such as breast cancer
  • non-malignant conditions such as the prevention of auto-immune diseases, particularly when pharmaceuticals may need to be administered from an early age.
  • the present invention provides a compound having Formula Formula I
  • R 4 is selected from H and hydrocarbyl
  • R 5 is a hydrocarbyl group and L is an optional linker group, or R.
  • R 2 together form a ring substituted with the group wherein R 3 is H or a substituent
  • X is selected from S, O, NR 6 and C(R 7 )(R 8 ), wherein R 6 is selected from H and hydrocarbyl groups, wherein each of R 7 and R 8 are independently selected from H and hydrocarbyl groups.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising
  • the present invention provides a compound having Formula I defined above, for use in medicine.
  • the present invention provides a use of a compound having Formula I defined above in the manufacture of a medicament for use in the therapy of a condition or disease associated with 11 ⁇ -HSD.
  • the present invention provides a use of a compound having Formula I defined above in the manufacture of a medicament for use in the therapy of a condition or disease associated with adverse 11 ⁇ -HSD levels.
  • the present invention provides a use of a compound having Formula I defined above in the manufacture of a pharmaceutical for inhibiting 11 ⁇ -HSD activity.
  • the present invention provides a use of a compound having Formula I defined above in the manufacture of a pharmaceutical for inhibiting 11 ⁇ -HSD activity.
  • the present invention provides a method comprising (a) performing a 11 ⁇ - HSD assay with one or more candidate compounds having Formula I defined above; (b) determining whether one or more of said candidate compounds is/are capable of modulating 11 ⁇ -HSD activity; and (c) selecting one or more of said candidate compounds that is/are capable of modulating 11 ⁇ -HSD activity.
  • the present invention provides a method comprising (a) performing a 11 ⁇ - HSD assay with one or more candidate compounds having Formula I defined above; (b) determining whether one or more of said candidate compounds is/are capable of inhibiting 11 ⁇ -HSD activity; and (c) selecting one or more of said candidate compounds that is/are capable of inhibiting 11 ⁇ -HSD activity.
  • the present invention provides
  • composition comprising the said compound, optionally admixed with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant,
  • the compound of the present invention has Formula II Formula II
  • R 4 is selected from H and hydrocarbyl, and R 5 is a hydrocarbyl group; or R and R 2 together form a ring substituted with the group
  • R- is H or a substituent
  • R 1 and R 2 together form a ring substituted with the group
  • R-, and R 2 together form a carbocyclic ring.
  • R-, and R 2 together form a six membered ring.
  • R-i and R 2 together form a six membered carbocyclic ring. In one preferred aspect the compound of the present invention wherein R., and R 2 together form an aryl ring.
  • Preferred compounds of the present invention are those having one of the following formulae.
  • R 3 is selected from H, hydrocarbyl, -S- hydrocarbyl, -S-H, halogen and N(R 9 )(R 10 ), wherein each of R 9 and R 10 are independently selected from H and hydrocarbyl groups.
  • R 3 is selected from H, hydroxy, alkyl especially C,-C 10 alkyl groups, C,-C 6 alkyl, e.g. C.,-C 3 alkyl group, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl and other pentyl isomers, and n-hexyl and other hexyl isomers, alkoxy especially C r C 10 alkoxy groups, C r C 6 alkoxy, e.g. C 1 -C 3 alkoxy group, methoxy, ethoxy, propoxy etc., alkinyl, e.g. ethinyl, or halogen, e.g. fluoro substituents.
  • R3 is -S-hydrocarbyl, preferably R3 is selected from -S-alkyl, -S-carboxylic acid, - S-ether, and -S-amide, preferably selected from -S-C ⁇ alkyl, -S- CnoCarboxcylic acid, -S- C-noether, and -S- C ⁇ amide.
  • R 3 is -CH 3 .
  • R 3 is selected from O, hydrocarbyl, and N(R 9 ) wherein R 9 is selected from H and hydrocarbyl groups. More preferably R 3 is selected from O, 0,-0, 0 alkenyl groups, such as C,-C 6 alkenyl group, and C,-C 3 alkenyl group, NH and N-C-,-C 10 alkyl groups, such as N-C,-C 6 alkyl group, and N-C,-C 3 alkyl groups.
  • R 4 is selected from H and 0,-0, 0 alkyl groups, such as C,-C 6 alkyl group, and C,-C 3 alkyl group.
  • R 4 is H.
  • R 4 is a group of the formula.
  • each R 5 is independently selected from hydrocarbyl groups.
  • Each R 5 may be the same of different to the other R 5 .
  • the two R 5 groups are the same.
  • R 5 is a cyclic hydrocarbyl group.
  • R 5 is a cyclic hydrocarbyl group comprising a hydrocarbon ring.
  • R 5 may be a substituted ring or an unsubstituted ring. In some preferred aspects of the invention R 5 is substituted ring. Preferably R 5 is a carbocyclic ring.
  • R 5 is a six membered ring.
  • R 5 is a six membered carbocyclic ring. More preferably R 5 is a substituted six membered carbocyclic ring.
  • R 5 is an aryl ring.
  • R 5 is a substituted aryl ring.
  • R 5 is a group having the formula
  • R ⁇ , R 12 , R 13 , R 14 and R 15 are independently selected from H, halogen, and hydrocarbyl groups.
  • each of R ⁇ , R 12 , R 13 , R 14 and R 15 are independently selected from H, halogen, alkyl, such as C 1-6 alkyl, phenyl, O-alkyl, O-phenyl, nitrile, haloalkyl, such as CF 3 , CCI 3 and CBr 3 , carboxyalkyl, -CO 2 H, CO 2 alkyl, and NH-acetyl groups.
  • R 11; R 12 , R 13 , R-, 4 and R 15 may join to form a ring.
  • R 1 4 and Ri5 ma Y °r a y not be adjacent.
  • the ring may be carbocyclic or heterocyclic ring.
  • the ring may be optionally substituted by any of the R ⁇ , R 12 , R 13 , R 14 and R 15 substituents listed above.
  • quinolyl may provide a naphthyl, quinolyl, tetrahydroquinolyl, or benzothtrahydropyranyl, each of which may be substituted or unsubstituted.
  • the compound of the present invention may have substituents other than those of the ring systems show herein.
  • the ring systems herein are given as general formulae and should be interpreted as such.
  • the absence of any specifically shown substituents on a given ring member indicates that the ring member may substituted with any moiety of which H is only one example.
  • the ring system may contain one or more degrees of unsaturation, for example is some aspects one or more rings of the ring system is aromatic.
  • the ring system may be carbocyclic or may contain one or more hetero atoms.
  • the compound of the invention in particular the ring system compound of the invention of the present invention may contain substituents other than those show herein.
  • substituents may be one or more of: one or more halo groups, one or more O groups, one or more hydroxy groups, one or more amino groups, one or more sulphur containing group(s), one or more hydrocarbyl group(s) - such as an oxyhydrocarbyl group.
  • the ring system of the present compounds may contain a variety of non- interfering substituents.
  • the ring system may contain one or more hydroxy, alkyl especially lower (C,-C 6 ) alkyl, e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, sec- butyl, tert-butyl, n-pentyl and other pentyl isomers, and n-hexyl and other hexyl isomers, alkoxy especially lower (C r C 6 ) alkoxy, e.g. methoxy, ethoxy, propoxy etc., alkinyl, e.g.
  • hydrocarbylsulphanyl means a group that comprises at least hydrocarbyl group (as herein defined) and sulphur, preferably -S- hydrocarbyl, more preferably -S-hydrocarbon. That sulphur group may be optionally oxidised.
  • the hydrocarbylsulphanyl group is -S-C 1-10 alkyl, more preferably -S-C 1-5 alkyl, more preferably -S-C 1-3 alkyl, more preferably -S-CH 2 CH 2 CH 3 , -S-CH 2 CH 3 or -SCH 3
  • the compounds have a reversible action.
  • the compounds have an irreversible action.
  • the compounds of the present invention are useful for the treatment of breast cancer.
  • the compounds of the present invention may be in the form of a salt.
  • the present invention also covers novel intermediates that are useful to prepare the compounds of the present invention.
  • the present invention covers novel alcohol precursors for the compounds.
  • the present invention covers bis protected precursors for the compounds. Examples of each of these precursors are presented herein.
  • the present invention also encompasses a process comprising each or both of those precursors for the synthesis of the compounds of the present invention.
  • 11 ⁇ Steroid dehydrogenase may be referred to as “11 ⁇ -HSD” or “HD” for short
  • 11 ⁇ -HSD is preferably 11 ⁇ -HSD Type 1.
  • 11 ⁇ -HSD is preferably 11 ⁇ -HSD Type 2.
  • the term “inhibit” includes reduce and/or eliminate and/or mask and/or prevent the detrimental action of HD.
  • the compound of the present invention is capable of acting as an HD inhibitor.
  • the term "inhibitor” as used herein with respect to the compound of the present invention means a compound that can inhibit HD activity - such as reduce and/or eliminate and/or mask and/or prevent the detrimental action of HD.
  • the HD inhibitor may act as an antagonist.
  • the compound of the present invention may have other beneficial properties in addition to or in the alternative to its ability to inhibit HD activity.
  • hydrocarbyl group means a group comprising at least C and H and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo, alkoxy, nitro, an alkyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen and oxygen. A non- limiting example of a hydrocarbyl group is an acyl group.
  • a typical hydrocarbyl group is a hydrocarbon group.
  • hydrocarbon means any one of an alkyl group, an alkenyl group, an alkynyl group, which groups may be linear, branched or cyclic, or an aryl group.
  • the term hydrocarbon also includes those groups but wherein they have been optionally substituted. If the hydrocarbon is a branched structure having substituent(s) thereon, then the substitution may be on either the hydrocarbon backbone or on the branch; alternatively the substitutions may be on the hydrocarbon backbone and on the branch.
  • one or more hydrocarbyl groups is independently selected from optionally substituted alkyl group, optionally substituted haloalkyl group, aryl group, alkylaryl group, alkylarylakyl group, and an alkene group.
  • one or more hydrocarbyl groups is independently selected from C r C 10 alkyl group, such as C,-C 6 alkyl group, and C,-C 3 alkyl group.
  • Typical alkyl groups include C, alkyl, C 2 alkyl, C 3 alkyl, C 4 alkyl, C 5 alkyl, C 7 alkyl, and C 8 alkyl.
  • one or more hydrocarbyl groups is independently selected from 0,-0, 0 haloalkyl group, C C 6 haloalkyl group, C r C 3 haloalkyl group, C,-C 10 bromoalkyl group, C,-C 6 bromoalkyl group, and C,-C 3 bromoalkyl group.
  • Typical haloalkyl groups include C, haloalkyl, C 2 haloalkyl, C 3 haloalkyl, C 4 haloalkyl, C 5 haloalkyl, C 7 haloalkyl, C 8 haloalkyl, C, bromoalkyl, C 2 bromoalkyl, C 3 bromoalkyl, C 4 bromoalkyl, C 5 bromoalkyl, C 7 bromoalkyl, and C 8 bromoalkyl.
  • one or more hydrocarbyl groups is independently selected from aryl groups, alkylaryl groups, alkylarylakyl groups, -(CH 2 ).,. 10 -aryl, -(CH 2 ) 1-10 -Ph, (CH 2 ) 1-10 -Ph-C 1-10 alkyl, -(CH 2 ) 1-5 -Ph, (CH 2 ) 1 . 5 -Ph-C 1-5 alkyl, -(CH 2 ) 1-3 - Ph, (CH 2 ) 1-3 -Ph-C 1-3 alkyl, -CH 2 -Ph, and -CH 2 -Ph-C(CH 3 ) 3 .
  • the aryl groups may contain a hetero atom.
  • the aryl group or one or more of the aryl groups may be carbocyclic or more may heterocyclic. Typical hetero atoms include O, N and S, in particular N.
  • one or more hydrocarbyl groups is independently selected from -(CH 2 ) 1-10 -cycloalkyl, -(CH 2 ) 1-10 -C 3-1 ocycloalkyl, -(CH 2 ) 1 . 7 -C 3- 7 cycloalkyl, -(CH 2 ) 1 . 5 -C 3 . 5 cycloalkyl, -(CH 2 ) 1 . 3 -C 3-5 cycloalkyl, and -CH 2 - C 3 cycloalkyl.
  • one or more hydrocarbyl groups is independently selected from alkene groups.
  • Typical alkene groups include 0,-0, 0 alkene group, C,-C 6 alkene group, C,-C 3 alkene group, such as C,, C 2 , C 3 , C 4 , C 5 , C 6 , or C 7 alkene group.
  • one or more hydrocarbyl groups is independently selected from oxyhydrocarbyl groups.
  • oxyhydrocarbyl group as used herein means a group comprising at least C, H and O and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the oxyhydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the oxyhydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur and nitrogen.
  • the oxyhydrocarbyl group is a oxyhydrocarbon group.
  • oxyhydrocarbon means any one of an alkoxy group, an oxyalkenyl group, an oxyalkynyl group, which groups may be linear, branched or cyclic, or an oxyaryl group.
  • the term oxyhydrocarbon also includes those groups but wherein they have been optionally substituted. If the oxyhydrocarbon is a branched structure having substituent(s) thereon, then the substitution may be on either the hydrocarbon backbone or on the branch; alternatively the substitutions may be on the hydrocarbon backbone and on the branch.
  • the oxyhydrocarbyl group is of the formula C ⁇ O (such as a C 1-3 O).
  • the compounds of the present invention may be studied using an animal model, in particular in ovariectomised rats.
  • an animal model in particular in ovariectomised rats.
  • compounds which are oestrogenic stimulate uterine growth.
  • the compound (10 mg/Kg/day for five days) was administered orally to rats with another group of animals receiving vehicle only (propylene glycol).
  • a further group received the estrogenic compound EMATE subcutaneously in an amount of 10 ⁇ g/day for five days.
  • uteri were obtained and weighed with the results being expressed as uterine weight/whole body weight x 100.
  • reporter gene may encode an enzyme which catalyses a reaction which alters light absorption properties.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescent activated cell sorting
  • reporter molecules include but are not limited to ( ⁇ -galactosidase, invertase, green fluorescent protein, luciferase, chloramphenicol, acetyltransferase, (- glucuronidase, exo-glucanase and glucoamylase.
  • radiolabelled or fluorescent tag-labelled nucleotides can be incorporated into nascent transcripts which are then identified when bound to oligonucleotide probes.
  • reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like. Patents teaching the use of such labels include US-A-3817837; US-A-3850752; US-A-3939350; US-A-3996345; US-A-4277437; US-A-4275149 and US-A-4366241.
  • host cell in relation to the present invention includes any cell that could comprise the target for the agent of the present invention.
  • a further embodiment of the present invention provides host cells transformed or transfected with a polynucleotide that is or expresses the target of the present invention.
  • said polynucleotide is carried in a vector for the replication and expression of polynucleotides that are to be the target or are to express the target.
  • the cells will be chosen to be compatible with the said vector and may for example be prokaryotic (for example bacterial), fungal, yeast or plant cells.
  • the gram negative bacterium E. coli is widely used as a host for heterologous gene expression.
  • large amounts of heterologous protein tend to accumulate inside the cell.
  • Subsequent purification of the desired protein from the bulk of E.coli intracellular proteins can sometimes be difficult.
  • bacteria from the genus Bacillus are very suitable as heterologous hosts because of their capability to secrete proteins into the culture medium.
  • Other bacteria suitable as hosts are those from the genera Streptomyces and Pseudomonas.
  • eukaryotic hosts such as yeasts or other fungi may be preferred.
  • yeast cells are preferred over fungal cells because they are easier to manipulate.
  • some proteins are either poorly secreted from the yeast cell, or in some cases are not processed properly (e.g. hyperglycosylation in yeast). In these instances, a different fungal host organism should be selected.
  • suitable expression hosts within the scope of the present invention are fungi such as Aspergillus species (such as those described in EP-A-0184438 and EP-A- 0284603) and Trichoderma species; bacteria such as Bacillus species (such as those described in EP-A-0134048 and EP-A-0253455), Streptomyces species and Pseudomonas species; and yeasts such as Kluyveromyces species (such as those described in EP-A-0096430 and EP-A-0301670) and Saccharomyces species.
  • typical expression hosts may be selected from Aspergillus niger, Aspergillus niger var. tubigenis, Aspergillus niger var.
  • suitable host cells - such as yeast, fungal and plant host cells - may provide for post-translational modifications (e.g. myristoylation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the present invention.
  • post-translational modifications e.g. myristoylation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation
  • organism in relation to the present invention includes any organism that could comprise the target according to the present invention and/or products obtained therefrom. Examples of organisms may include a fungus, yeast or a plant.
  • transgenic organism in relation to the present invention includes any organism that comprises the target according to the present invention and/or products obtained.
  • the host organism can be a prokaryotic or a eukaryotic organism.
  • suitable prokaryotic hosts include E. coli and Bacillus subtilis. Teachings on the transformation of prokaryotic hosts is well documented in the art, for example see Sambrook et al (Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press) and Ausubel et al., Current Protocols in Molecular Biology (1995), John Wiley & Sons, Inc.
  • nucleotide sequence may need to be suitably modified before transformation - such as by removal of introns.
  • the transgenic organism can be a yeast.
  • yeast have also been widely used as a vehicle for heterologous gene expression.
  • the species Saccharomyces cerevisiae has a long history of industrial use, including its use for heterologous gene expression.
  • Expression of heterologous genes in Saccharomyces cerevisiae has been reviewed by Goodey et al (1987, Yeast Biotechnology, D R Berry et al, eds, pp 401-429, Allen and Unwin, London) and by King et al (1989, Molecular and Cell Biology of Yeasts, E F Walton and G T Yarronton, eds, pp 107-133, Blackie, Glasgow).
  • Saccharomyces cerevisiae is well suited for heterologous gene expression. First, it is non-pathogenic to humans and it is incapable of producing certain endotoxins. Second, it has a long history of safe use following centuries of commercial exploitation for various purposes. This has led to wide public acceptability. Third, the extensive commercial use and research devoted to the organism has resulted in a wealth of knowledge about the genetics and physiology as well as large-scale fermentation characteristics of Saccharomyces cerevisiae.
  • yeast vectors include integrative vectors, which require recombination with the host genome for their maintenance, and autonomously replicating plasmid vectors.
  • expression constructs are prepared by inserting the nucleotide sequence into a construct designed for expression in yeast.
  • constructs used for heterologous expression have been developed.
  • the constructs contain a promoter active in yeast fused to the nucleotide sequence, usually a promoter of yeast origin, such as the GAL1 promoter, is used.
  • a promoter of yeast origin such as the GAL1 promoter
  • a signal sequence of yeast origin such as the sequence encoding the SUC2 signal peptide, is used.
  • a terminator active in yeast ends the expression system.
  • transgenic Saccharomyces can be prepared by following the teachings of Hinnen et al (1978, Proceedings of the National Academy of Sciences of the USA 75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito, H et al (1983, J Bacteriology 153, 163-168).
  • the transformed yeast cells are selected using various selective markers.
  • markers used for transformation are a number of auxotrophic markers such as LEU2, HIS4 and TRP1 , and dominant antibiotic resistance markers such as aminoglycoside antibiotic markers, e.g. G418.
  • Another host organism is a plant.
  • the basic principle in the construction of genetically modified plants is to insert genetic information in the plant genome so as to obtain a stable maintenance of the inserted genetic material.
  • Several techniques exist for inserting the genetic information the two main principles being direct introduction of the genetic information and introduction of the genetic information by use of a vector system.
  • a review of the general techniques may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27). Further teachings on plant transformation may be found in EP- A-0449375.
  • the present invention also provides a method of transforming a host cell with a nucleotide sequence that is to be the target or is to express the target.
  • Host cells transformed with the nucleotide sequence may be cultured under conditions suitable for the expression of the encoded protein.
  • the protein produced by a recombinant cell may be displayed on the surface of the cell.
  • expression vectors containing coding sequences can be designed with signal sequences which direct secretion of the coding sequences through a particular prokaryotic or eukaryotic cell membrane.
  • the present invention also encompasses the use of variants, homologue and derivatives thereof.
  • the term “homology” can be equated with “identity”.
  • an homologous sequence is taken to include an amino acid sequence which may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical.
  • homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
  • Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.
  • % homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
  • a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
  • An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs.
  • GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
  • % homology preferably % sequence identity.
  • the software typically does this as part of the sequence comparison and generates a numerical result.
  • the sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
  • the nucleotide sequence for use as the target or for expressing the target can be incorporated into a recombinant replicable vector.
  • the vector may be used to replicate and express the nucleotide sequence in and/or from a compatible host cell. Expression may be controlled using control sequences which include promoters/enhancers and other expression regulation signals. Prokaryotic promoters and promoters functional in eukaryotic cells may be used. Tissue specific or stimuli specific promoters may be used. Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above.
  • the protein produced by a host recombinant cell by expression of the nucleotide sequence may be secreted or may be contained intracellularly depending on the sequence and/or the vector used.
  • the coding sequences can be designed with signal sequences which direct secretion of the substance coding sequences through a particular prokaryotic or eukaryotic cell membrane.
  • the target amino acid sequence may be produced as a fusion protein, for example to aid in extraction and purification.
  • fusion protein partners include glutathione-S- transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and (-galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. Preferably the fusion protein will not hinder the activity of the target.
  • the fusion protein may comprise an antigen or an antigenic determinant fused to the substance of the present invention.
  • the fusion protein may be a non-naturally occurring fusion protein comprising a substance which may act as an adjuvant in the sense of providing a generalised stimulation of the immune system.
  • the antigen or antigenic determinant may be attached to either the amino or carboxy terminus of the substance.
  • the amino acid sequence may be ligated to a heterologous sequence to encode a fusion protein.
  • a heterologous sequence for example, for screening of peptide libraries for agents capable of affecting the substance activity, it may be useful to encode a c imeric substance expressing a heterologous epitope that is recognised by a commercially available antibody.
  • the compounds of the present invention may be used as therapeutic agents - i.e. in therapy applications.
  • the term "therapy” includes curative effects, alleviation effects, and prophylactic effects.
  • the therapy may be on humans or animals, preferably female animals.
  • the present invention provides a pharmaceutical composition, which comprises a compound according to the present invention and optionally a pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof).
  • the pharmaceutical compositions may be for human or animal usage in human and veterinary medicine and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier, or excipient.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
  • the choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
  • Preservatives may be provided in the pharmaceutical composition.
  • preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
  • Antioxidants and suspending agents may be also used.
  • the pharmaceutical composition of the present invention may be formulated to be delivered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestable solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route.
  • the formulation may be designed to be delivered by both routes.
  • the agent is to be delivered mucosally through the gastrointestinal mucosa, it should be able to remain stable during transit though the gastrointestinal tract; for example, it should be resistant to proteolytic degradation, stable at acid pH and resistant to the detergent effects of bile.
  • compositions can be administered by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intravenously, intramuscularly or subcutaneously.
  • compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood.
  • compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.
  • the compound of the present invention may be used in combination with one or more other active agents, such as one or more other pharmaceutically active agents.
  • the compounds of the present invention may be used in combination with other 11 ⁇ -HSD inhibitors and/or other inhibitors such as an aromatase inhibitor (such as for example, 4hydroxyandrostenedione (4-OHA)), and/or a steroid sulphatase inhibitors such as EMATE and/or steroids - such as the naturally occurring stemeursteroids dehydroepiandrosterone sulfate (DHEAS) and pregnenolone sulfate (PS) and/or other structurally similar organic compounds.
  • an aromatase inhibitor such as for example, 4hydroxyandrostenedione (4-OHA)
  • a steroid sulphatase inhibitors such as EMATE and/or steroids - such as the naturally occurring stemeurosteroids dehydroepiandrosterone sulfate (DHEAS) and pregnenolone sulfate (PS) and/or other structurally similar organic compounds.
  • DHEAS dehydroepian
  • the compound of the present invention may be used in combination with a biological response modifier.
  • biological response modifier includes cytokines, immune modulators, growth factors, haematopoiesis regulating factors, colony stimulating factors, chemotactic, haemolytic and thrombolytic factors, cell surface receptors, ligands, leukocyte adhesion molecules, monoclonal antibodies, preventative and therapeutic vaccines, hormones, extracellular matrix components, fibronectin, etc.
  • the biological response modifier is a cytokine.
  • cytokines examples include: interleukins (IL) - such as IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL- 9, IL-10, IL-11 , IL-12, IL-19; Tumour Necrosis Factor (TNF) - such as TNF- ⁇ ; Interferon alpha, beta and gamma; TGF- ⁇ .
  • TNF Tumour Necrosis Factor
  • the cytokine is tumour necrosis factor (TNF).
  • the TNF may be any type of TNF - such as TNF- ⁇ , TNF- ⁇ , including derivatives or mixtures thereof. More preferably the cytokine is TNF- ⁇ . Teachings on TNF may be found in the art - such as WO-A-98/08870 and WO-A-98/13348.
  • a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular patient.
  • the dosages below are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited.
  • compositions of the present invention may be administered by direct injection.
  • the composition may be formulated for parenteral, mucosal, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration.
  • the agent may be administered at a dose of from 0.01 to 30 mg/kg body weight, such as from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.
  • the agents of the present invention may be administered in accordance with a regimen of 1 to 4 times per day, preferably once or twice per day.
  • the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.
  • administered also includes delivery by techniques such as lipid mediated transfection, liposomes, immunoliposomes, lipofectin, cationic facial amphiphiles (CFAs) and combinations thereof.
  • routes for such delivery mechanisms include but are not limited to mucosal, nasal, oral, parenteral, gastrointestinal, topical, or sublingual routes.
  • administered includes but is not limited to delivery by a mucosal route, for example, as a nasal spray or aerosol for inhalation or as an ingestable solution; a parenteral route where delivery is by an injectable form, such as, for example, an intravenous, intramuscular or subcutaneous route.
  • the compounds of the present invention can be formulated in any suitable manner utilising conventional pharmaceutical formulating techniques and pharmaceutical carriers, adjuvants, excipients, diluents etc. and usually for parenteral administration.
  • Approximate effective dose rates may be in the range from 1 to 1000 mg/day, such as from 10 to 900 mg/day or even from 100 to 800 mg/day depending on the individual activities of the compounds in question and for a patient of average (70Kg) bodyweight. More usual dosage rates for the preferred and more active compounds will be in the range 200 to 800 mg/day, more preferably, 200 to 500 mg/day, most preferably from 200 to 250 mg/day.
  • the compounds may be given in single dose regimes, split dose regimes and/or in multiple dose regimes lasting over several days.
  • oral administration they may be formulated in tablets, capsules, solution or suspension containing from 100 to 500 mg of compound per unit dose.
  • the compounds will be formulated for parenteral administration in a suitable parenterally administrable carrier and providing single daily dosage rates in the range 200 to 800 mg, preferably 200 to 500, more preferably 200 to 250 mg.
  • Such effective daily doses will, however, vary depending on inherent activity of the active ingredient and on the bodyweight of the patient, such variations being within the skill and judgement of the physician.
  • the compounds of the present invention may be useful in the method of treatment of a cell cycling disorder.
  • Yeast cells can divide every 120 min., and the first divisions of fertilised eggs in the embryonic cells of sea urchins and insects take only 1530 min. because one large pre-existing cell is subdivided. However, most growing plant and animal cells take 10-20 hours to double in number, and some duplicate at a much slower rate. Many cells in adults, such as nerve cells and striated muscle cells, do not divide at all; others, like the fibroblasts that assist in healing wounds, grow on demand but are otherwise quiescent. Still, every eukaryotic cell that divides must be ready to donate equal genetic material to two daughter cells. DNA synthesis in eukaryotes does not occur throughout the cell division cycle but is restricted to a part of it before cell division.
  • FACS fluorescence-activated cell sorter
  • the stages of mitosis and cytokinesis in an animal cell are as follows
  • Each contains a centromere that is linked by a spindle fibre to one pole, to which it moves. Thus one copy of each chromosome is donated to each daughter cell.
  • Cytokinesis begins as the cleavage furrow starts to form.
  • cell cycling is an extremely important cell process. Deviations from normal cell cycling can result in a number of medical disorders. Increased and/or unrestricted cell cycling may result in cancer. Reduced cell cycling may result in degenerative conditions. Use of the compound of the present invention may provide a means to treat such disorders and conditions. Thus, the compound of the present invention may be suitable for use in the treatment of cell cycling disorders such as cancers, including hormone dependent and hormone independent cancers.
  • the compound of the present invention may be suitable for the treatment of cancers such as breast cancer, ovarian cancer, endometrial cancer, sarcomas, melanomas, prostate cancer, pancreatic cancer etc. and other solid tumours.
  • cancers such as breast cancer, ovarian cancer, endometrial cancer, sarcomas, melanomas, prostate cancer, pancreatic cancer etc. and other solid tumours.
  • cell cycling is inhibited and/or prevented and/or arrested, preferably wherein cell cycling is prevented and/or arrested.
  • cell cycling may be inhibited and/or prevented and/or arrested in the G 2 /M phase.
  • cell cycling may be irreversibly prevented and/or inhibited and/or arrested, preferably wherein cell cycling is irreversibly prevented and/or arrested.
  • irreversibly prevented and/or inhibited and/or arrested it is meant after application of a compound of the present invention, on removal of the compound the effects of the compound, namely prevention and/or inhibition and/or arrest of cell cycling, are still observable. More particularly by the term “irreversibly prevented and/or inhibited and/or arrested” it is meant that when assayed in accordance with the cell cycling assay protocol presented herein, cells treated with a compound of interest show less growth after Stage 2 of the protocol 1 than control cells. Details on this protocol are presented below.
  • the present invention provides compounds which: cause inhibition of growth of oestrogen receptor positive (ER+) and ER negative (ER-) breast cancer cells in vitro by preventing and/or inhibiting and/or arresting cell cycling; and/or cause regression of nitroso-methyl urea (NMU)-induced mammary tumours in intact animals (i.e. not ovariectomised), and/or prevent and/or inhibit and/or arrest cell cycling in cancer cells; and/or act in vivo by preventing and/or inhibiting and/or arresting cell cycling and/or act as a cell cycling agonist.
  • NMU nitroso-methyl urea
  • CELL CYCLING ASSAY PROTOCOL 2 Procedure Stage 1 MCF-7 breast cancer cells are seeded into multi-well culture plates at a density of 105 cells/well. Cells were allowed to attach and grown until about 30% confluent when they are treated as follows:
  • Cells are grown for 6 days in growth medium containing the COI with changes of medium/COI every 3 days. At the end of this period cell numbers were counted using a Coulter cell counter.
  • the compounds of the present invention may be useful in the treatment of a cell cycling disorder.
  • a particular cell cycling disorder is cancer.
  • Cancer remains a major cause of mortality in most Western countries. Cancer therapies developed so far have included blocking the action or synthesis of hormones to inhibit the growth of hormone-dependent tumours. However, more aggressive chemotherapy is currently employed for the treatment of hormone-independent tumours.
  • the compound of the present invention provides a means for the treatment of cancers and, especially, breast cancer.
  • the compound of the present invention may be useful in the blocking the growth of cancers including leukaemias and solid tumours such as breast, endometrium, prostate, ovary and pancreatic tumours.
  • the compound or composition of the present invention may be useful in the treatment of the disorders listed in WO-A-99/52890 - viz:
  • the compound or composition of the present invention may be useful in the treatment of the disorders listed in WO-A-98/05635.
  • diabetes including Type II diabetes, obesity, cancer, inflammation or inflammatory disease, dermatological disorders, fever, cardiovascular effects, haemorrhage, coagulation and acute phase response, cachexia, anorexia, acute infection, HIV infection, shock states, graft-versus-host reactions, autoimmune disease, reperfusion injury, meningitis, migraine and aspirin-dependent anti-thrombosis; tumour growth, invasion and spread, angiogenesis, metastases, malignant, ascites and malignant pleural effusion; cerebral ischaemia, ischaemic heart disease, osteoarthritis, rheumatoid arthritis, osteoporosis, asthma, multiple sclerosis, neurodegeneration, Alzheimer's disease, atherosclerosis, stroke, vasculitis, Crohn's disease and ulcerative colitis; periodontitis, gingivitis
  • the compound or composition of the present invention may be useful in the treatment of disorders listed in WO-A-98/07859.
  • cytokine and cell proliferation/differentiation activity e.g. for treating immune deficiency, including infection with human immune deficiency virus; regulation of lymphocyte growth; treating cancer and many autoimmune diseases, and to prevent transplant rejection or induce tumour immunity
  • regulation of haematopoiesis e.g. treatment of myeloid or lymphoid diseases
  • promoting growth of bone, cartilage, tendon, ligament and nerve tissue e.g.
  • follicle-stimulating hormone for healing wounds, treatment of burns, ulcers and periodontal disease and neurodegeneration; inhibition or activation of follicle-stimulating hormone (modulation of fertility); chemotactic/chemokinetic activity (e.g. for mobilising specific cell types to sites of injury or infection); haemostatic and thrombolytic activity (e.g. for treating haemophilia and stroke); antiinflammatory activity (for treating e.g. septic shock or Crohn's disease); as antimicrobials; modulators of e.g. metabolism or behaviour; as analgesics; treating specific deficiency disorders; in treatment of e.g. psoriasis, in human or veterinary medicine.
  • composition of the present invention may be useful in the treatment of disorders listed in WO-A-98/09985.
  • macrophage inhibitory and/or T cell inhibitory activity and thus, anti-inflammatory activity i.e.
  • inhibitory effects against a cellular and/or humoral immune response including a response not associated with inflammation; inhibit the ability of macrophages and T cells to adhere to extracellular matrix components and fibronectin, as well as up-regulated fas receptor expression in T cells; inhibit unwanted immune reaction and inflammation including arthritis, including rheumatoid arthritis, inflammation associated with hypersensitivity, allergic reactions, asthma, systemic lupus erythematosus, collagen diseases and other autoimmune diseases, inflammation associated with atherosclerosis, arteriosclerosis, atherosclerotic heart disease, reperfusion injury, cardiac arrest, myocardial infarction, vascular inflammatory disorders, respiratory distress syndrome or other cardiopulmonary diseases, inflammation associated with peptic ulcer, ulcerative colitis and other diseases of the gastrointestinal tract, hepatic fibrosis, liver cirrhosis or other hepatic diseases, thyroiditis or other glandular diseases, glomerulonephritis or other renal and urologic diseases, otitis or other oto-rhino-
  • retinitis or cystoid macular oedema retinitis or cystoid macular oedema, sympathetic ophthalmia, scleritis, retinitis pigmentosa, immune and inflammatory components of degenerative fondus disease, inflammatory components of ocular trauma, ocular inflammation caused by infection, proliferative vitreo-retinopathies, acute ischaemic optic neuropathy, excessive scarring, e.g.
  • monocyte or leukocyte proliferative diseases e.g. leukaemia
  • monocytes or lymphocytes for the prevention and/or treatment of graft rejection in cases of transplantation of natural or artificial cells, tissue and organs such as cornea, bone marrow, organs, lenses, pacemakers, natural or artificial skin tissue.
  • the present invention provides use of a compound as described herein in the manufacture of a medicament for use in the therapy of a condition or disease associated with 11 ⁇ -HSD.
  • condition or disease is selected from the list consisting of:
  • glaucoma inflammatory disorders such as arthritis or asthma immune disorders bone disorders, such as osteoporosis cancer intra-uterine growth retardation apparent mineralocorticoid excess syndrome (AME) polycystic ovary syndrome (PCOS) hirsutism acne oligo- or amenorrhea adrenal cortical adenoma and carcinoma
  • AME osteoporosis cancer intra-uterine growth retardation apparent mineralocorticoid excess syndrome
  • PCOS polycystic ovary syndrome
  • Cushing's syndrome pituitary tumours invasive carcinomas breast cancer; and endometrial cancer.
  • the present invention provides compounds for use as steroid dehydrogenase inhibitors, and pharmaceutical compositions for the same.
  • Figure 1 is of graph 1 which shows the amount of protein per ⁇ L of rat liver and rat kidney.
  • Figure 2 is of graph 2 which shows the enzyme concentration and time-dependency course, E to F, in rat liver, 11 ⁇ -HSD type 1 activity.
  • Figure 3 is of graph 3 which shows the enzyme concentration and time-dependency course, F to E, in rat kidney, 11 ⁇ -HSD type 2 activity.
  • Figure 4 is a graph showing extraction efficiencies obtained with four extraction methods.
  • Figure 5 is a graph showing a comparison of 11 ⁇ -HSD1 activity in rat and human hepatic microsomes.
  • Figure 6 is a series of graphs showing the effect of incubation time on human microsomal 11 ⁇ -HSD1 activity.
  • Figure 7 is a series of graphs showing the effect of microsomal protein concentration on human microsomal 11 ⁇ -HSD1 activity.
  • Figure 8 is a graph showing the substrate (cortisone) saturation curve for human hepatic microsomal 11 ⁇ HSD1.
  • Figure 9 is a Lineweaver-Burke plot of substrate saturation data for human hepatic microsomal 11 ⁇ HSD1.
  • Figure 10 is a graph showing the IC 50 determination for glycyrrhetinic acid.
  • Figure 11 is a graph showing the IC 50 determination for carbenoxolone.
  • Figures 12(A), 12(B) and 12(C) are graphs showing the 11 ⁇ -HSD1 activity measured by Immunoassay.
  • Figure 12(A) shows the effect of protein
  • Figure 12(B) shows the effect of cortisone
  • Figure 12(C) shows the effect of Tween-80.
  • Figure 13 is a graph showing the evaluation of the Assay Designs Cortisol
  • Figure 14 is a graph showing the effect of increasing microsomal protein on measurement of 11 ⁇ HSD1 activity detected by Assay Designs Immunoassay.
  • Figure 15 is a graph showing the detection of 11 ⁇ HSD1 activity by RIA using the
  • Figure 16 is a graph showing the effect of lowering the Immunotech antibody concentration on the signal to noise (microsome group compared to GA blank group).
  • Figure 17 is a graph showing the Immunotech antibody saturation curve for detection of 11 ⁇ HSD1 activity by RIA.
  • Figure 18 is a graph showing the linearity of human hepatic microsomal 11 ⁇ HSD1 activity detected by RIA.
  • Figure 19 is a graph showing the effect of Tween 80 on detection of human hepatic microsomal 11 ⁇ HSD1 activity by RIA.
  • Figure 20 is a graph showing the effect of buffer systems on detection of human hepatic microsomal 11 ⁇ HDS1 activity by RIA.
  • Figure 21 is a graph showing the linearity of human hepatic microsomal 11 ⁇ HSD1 activity with incubation time detected by RIA.
  • Figure 22 is a graph showing the substrate saturation curve for human hepatic microsomal 11 ⁇ HDS1 activity detected by RIA.
  • Figure 23 is a Lineweaver-Burke plot of substrate saturation data for human hepatic microsomal 11 ⁇ HDS1 activity detected by RIA.
  • Figure 24 is a graph showing the DMSO tolerance of human hepatic microsomal 11 ⁇ HSD1 activity.
  • Figure 25 is an IC 50 curve for inhibition of human hepatic microsomal 11 ⁇ HSD1 activity by glycyrrhetinic acid.
  • TLC aluminium sheets 20 x 20 cm silica gel 60 F 254 , Merck, Germany.
  • Scintillation vials 20 ml polypropylene vials with caps, SARSTEDT, Germany.
  • Scintillation counter Beckman LS 6000 SC, Beckman Instruments Inc., USA.
  • Assay medium PBS-sucrose buffer, Dulbecco's Phosphate Buffered Saline, 1 tablet/100 ml with 0,25 M sucrose, pH 7,4 BDH Laboratory supplies, UK.
  • Extraction fluid Di-ethylether, Fischer Chemicals, UK.
  • Inhibitors compounds were synthesised in accordance with the synthetic routes below .
  • Labelled cortisol (F) ( 3 H-F and C-F) was oxidised at the C-11 position with CrO 3 in order to synthesize to the corresponding labelled cortisone ( 3 H-E and 1 C-E).
  • the experiment was done according to the Bradford method [14]. The following method was used: first a BSA (protein) solution was prepared (1 mg/ml). Protein solutions containing 10 to 100 ⁇ g protein were pipetted into tubes and volumes adjusted with distilled water. Then 5 ml of protein reagent was added to the tubes and vortex mixed. The absorbance was measured at 595 nm after 15 minutes and before 1 hour in 3 ml cuvettes against a reagent blank. The weight of the protein was plotted against the corresponding absorbance resulting in a standard curve used to determine the protein concentration in rat liver and rat kidney cytosols.
  • 11 ⁇ -HSD type 1 is the enzyme responsible for the conversion E to F and this type of enzyme is present in rat liver.
  • the substrate solution used in this assay contained 70,000 cpm/ml 3 H-E in PBS-sucrose and 0.5 ⁇ M of unlabelled E and co-factor NADPH (9 mg/10 ml of substrate solution). 1 ml of the substrate solution and the different amounts of rat liver homogenate was added to all tubes.
  • the amount of rat liver homogenate needed for an assay was determined by incubating the substrate solution with 25, 50, 100 and 150 ⁇ l for 30, 60, 90 and 120 minutes at 37°C in a water bath with the tubes being mechanically shaken. After the incubation 50 ⁇ L of recovery solution was added, containing about 8,000 cpm/ 50 ⁇ L of 14 C-F and 50 ⁇ g/50 ⁇ L of unlabelled F for visualising the spot on the TLC-plate, to correct for the losses made in the next two steps. F was then extracted from the aqueous phase with 4 ml of ether (2 x 30 sec cycle, vortex mix). The aqueous phase was then frozen using dry-ice and the organic layer was decanted and poured into smaller tubes and evaporated.
  • TLC-plate aluminium thin layer chromatography plate
  • the TLC-plate was developed in a TLC tank under saturated conditions.
  • the solvent system used was chloroform : methanol 9:1 (v/v).
  • the spots from the TLC- plate were then put into scintillation vials and 0.5 ml of methanol was added to all vials to elute the radioactivity from the TLC-plate for 5 minutes.
  • the substrate solution contained about 50,000 cpm/ml 3 H-F in PBS-sucrose and 0.5 ⁇ M F. 1ml of the substrate solution was added to each tube, the inhibitors were also added, at a 10 ⁇ M concentration, to each tube except to the "control" and "blank” tubes. 150 ⁇ L was added to all tubes except to the blanks, this was done to correct for the amount of 3 H-F spontaneously formed. The tubes were incubated for 60 minutes in a mechanically shaken water bath at 37°C. The amount of kidney liver homogenate and incubation time used resulted from the enzyme- and time-dependency assay.
  • the TLC-plate was developed in chloroform : methanol (9:1 v/v) solvent system, the TLC-plate ran for about 90 minutes until the solvent front had moved about 18 cm.
  • the position of the product E was visualised under UV-light and cut out from the TLC-plate and put into scintillation vials. Radioactivity was eluted over 5 minutes with 0.5 ml methanol.
  • 0.5 ml of PBS- sucrose and 10 ml of Ecoscint were then added and vortex mixed before counting in the scintillation counter. Before counting the samples, two total activity vials were prepared.
  • Buffer 1 from Barf [15]: 30 mM Tris-HCL, pH 7.2, containing 1 mM EDTA Buffer 2, from the Sterix protocol: PBS (pH 7.4) containing 0.25M sucrose Buffer 3, from the Sigma RIA protocol: 50 mM Tris-HCL, pH 8, containing 0.1 M NaCL and 0.1 % gelatin Stop solution, from Barf [15]: 1 mM glycyrrhetinic acid in 100 % DMSO
  • Enzyme assays were carried out in the presence of 181 ⁇ M NADPH, 1 mM Glucose-6- Phosphate and cortisone concentrations indicated for each experiment.
  • Enzyme assay buffer 30 mM Tris-HCL, pH 7.2 containing 1 mM EDTA
  • Antibody binding buffer 50 mM Tris-HCL, pH 8, containing 0.1 M NaCl and 0.1 % gelatin
  • Compound preparation Prepare 10 mM stock solutions in 100% DMSO at 100 times the required assay concentration. Dilute into assay buffer 1 in 25. Also dilute neat DMSO 1 in 25 into assay buffer for controls.
  • Substrate preparation Prepare a solution of cortisone in ethanol 600 times the required assay concentration (175 nM). Dilute this 1 in 50 into assay buffer. Prepare NADPH as a 1.8 mg/ml solution in assay buffer. Prepare G-6-P as a 3.65 mg/ml solution in assay buffer.
  • Microsome preparation Dilute stock 20 mg/ml solution 1 in 100 with assay buffer.
  • Antibody preparation Dilute stock antibody solution to 17 ⁇ g/ml in antibody binding buffer.
  • Dextran coated charcoal preparation Make a 20 mg/ml solution in antibody binding buffer and chill on ice.
  • Enzyme assay To a u-bottom polypropylene 96 well plate add:
  • the 11 ⁇ HSD1 enzyme assay was carried out following the standard operating procedure described above in u-bottom polypropylene 96 well plates or 1.5 ml Eppendorf tubes as indicated for each experiment. Subsequent to stopping the enzyme reaction, 100 ⁇ l antibody prepared in buffer 3 unless otherwise indicated was added to test samples and 100 ⁇ l buffer 3 was added to the NSB samples. The samples were incubated for 1 hour at 37°C and the chilled on ice for 15 mins. Dextran coated charcoal (50 ⁇ l / sample) prepared to the indicated concentration in buffer 3 was added and the samples were mixed (vortex for tubes and aspiration 5 times with an 8-channel pipette for 96 well plates) and chilled for a further 10 min.
  • [ 1 C]-labelled cortisol was obtained from NEN.
  • a stock was prepared in phosphate buffered saline (PBS) containing 4000 DPM in 50 ⁇ l with cold cortisol (1 ⁇ g) added as a carrier.
  • the final ethanol concentration was 0.4%.
  • Aliquots of this solution were added to glass tubes (100 ⁇ l) and the following extractions were carried out: 1. 1 ml CH 2 CI 2t vortex and pass through phase separating filter paper (Whatman, IPS) 2. 1ml ethyl acetate, vortex and pass through phase separating filter paper 3.
  • the 11 ⁇ -HSD1 activities in rat and human microsomes were similar, 0.7pmol/mg/min and 0.5pmol/mg/min for rat and human microsomes respectively.
  • the activity in human microsomes is apparently not related to microsomal protein concentration, which may suggest that that the protein concentration range examined is too high.
  • the standard compounds glycyrrhetinic acid and carbenoxolone were examined in this assay system, as part of the validation process.
  • the assay was performed using 175nM cortisone substrate, with 10 ⁇ g microsomal protein and a 30 minute incubation at 37°C, as described by Barf [15]. Although the data in figures 8 and 9 above suggest that this substrate concentration is not saturating under these assay conditions.
  • Glycyrrhetinic acid and carbenoxolone were tested at concentrations from 0.012 ⁇ M to 3 ⁇ M , the DMSO concentration was 1% in all samples. The results are shown in figures 10 and 11.
  • Glycyrrhetinic acid and carbenoxolone give IC 50 values of 40nM and 119nM respectively.
  • the IC 50 reported for carbenoxolone by Barf et al. using the SPA format and recombinant 11 ⁇ -HSD is 330nM [15], approximately three-fold less potent.
  • the difference in potency in the two assay systems is probably due to the different assay conditions, SPA compared to tic end point, and also the enzyme source, native hepatic enzyme compared to recombinant enzyme.
  • the assay conditions described above support good enzyme activity however, which should be transferable to a 96 well plate format.
  • An Assay Designs enzyme immunoassay system was evaluated as a potential assay format.
  • the basis of the assay is competition for antibody binding between sample cortisol, generated by 11 ⁇ -HSD1 , and labelled cortisol binding.
  • the anti-cortisol detection antibody provided in the kit is a mouse monoclonal, reported to cross react less than 0.1% with cortisone.
  • the kit is designed for the analysis of cortisol levels in saliva, urine, serum and plasma and also in tissue culture media, rather than for determining enzyme activity however.
  • Figure 12(A) shows the effect of protein. Data taken from the 700 ⁇ M cortisone group tested in the presence of Tween-80.
  • Figure 12(B) shows the effect of cortisone. Data taken from the 25 ⁇ g microsomal protein group tested in the presence of Tween-80.
  • Figure 12(C) shows the effect of Tween-80.
  • the assay detected cortisol in the standard curve (313 pg/ml to 10,000 pg/ml) as expected but the signal obtained from the enzyme assay samples decreased with increasing microsomal protein concentration, suggesting that the microsomal protein may interfere with the immunoassay, figure 12(A).
  • Addition of exogenous cortisone had no effect on levels of cortisol detected in the enzyme assay samples, suggesting the antibody does not cross react with cortisone, figure 12(B).
  • Inclusion of detergent in the enzyme assay buffer had little effect, figure 12(C).
  • the assay conditions were varied to determine if it was feasible to use the immunoassay system to detect 11 ⁇ -HSD1 activity; 24 ⁇ g microsomal protein per sample and 2 ⁇ M cortisone substrate in Buffer 2. Enzyme activity was also measured in samples following the addition of steroid displacement reagent; a kit component which releases cortisol from cortisol binding protein, if present in the sample. The assay detected the cortisol in the standard curve (313pg/ml to 10,000pg/ml). Figure 13 shows the absorbance at 405m obtained for the different groups:
  • Glycyrrhetinic acid (GA) in the presence of the top concentration of cortisol standard has no effect on the ability of the kit to measure cortisol concentrations.
  • the 11 ⁇ HSD1 assay was carried out using 10 ⁇ g/well human hepatic microsomal protein.
  • the Immunotech antibody was used in the RIA at concentrations from 6.25 ⁇ g/well to 25 ⁇ g/well, the results are shown in figure 15.
  • the Immunotech antibody worked well in the assay and gave good signal to background at all the concentrations tested.
  • the signal to noise with 12.5 and 6.1 ⁇ g antibody per well was similar suggesting it may be possible to reduce the antibody concentration.
  • the antibody titre at concentrations from 0.67 ⁇ g/well to 6.7 ⁇ g/well, was examined.
  • the 11 ⁇ HSD1 assay was carried out using human microsomal protein at 20 ⁇ g/well, to generate the optimum signal to background.
  • Each antibody concentration was tested against a "no enzyme" blank (buffer substituted for microsomes), a "GA blank” (10 ⁇ l stop solution added prior to microsomes) and a control group. The results are shown in figures 16 and 17.
  • the saturation curve indicates that there is no difference in the detection of enzyme activity above 1.68 ⁇ g/well.
  • the signal to background ratio with this antibody concentration is good, (6 fold).Consequently the antibody will be used at 1.7 ⁇ g/well in future assays.
  • the 11 ⁇ HSD1 assay was carried with microsomal protein concentrations varying from 1 ⁇ g/well to 40 ⁇ g/well. 11 ⁇ HSD1 activity was linear with protein up to concentrations of 20 ⁇ g/well, figure 18, confirming the results obtained with the classical enzyme assay (figure 7).
  • the optimal concentration of human microsomal protein to use in the assay appears to be 10 ⁇ g/well.
  • Tween 80 in the enzyme assay buffer was also investigated. This assay was carried out in parallel with the assay above and under the same conditions except that the enzyme assay buffer (Buffer 2) contained 0.05 % Tween 80. Microsomal protein was tested at four concentrations. Tween 80 was found to increase the blank CPM, reducing the signal to noise of the assay. Representative data, from the group tested 10 ⁇ g/well microsomal protein, are shown in figure 19. Similar results were obtained with all the microsome protein concentrations examined, consequently Tween will not be used in future studies..
  • both phases were carried out in either enzyme assay buffer (Buffer 2) or Buffer 3 (RIA buffer).
  • Buffer 2 enzyme assay buffer
  • RIA buffer Buffer 3
  • the microsomal protein concentration used was 10 ⁇ g/well and the cortisone concentration was 175 nM. Performing both enzyme assay and RIA in Buffer 3 appears to improve the data slightly, figure 20.
  • IC 50 value was generated for the standard inhibitor glycyrrhetinic acid, the compound was tested at concentrations between 0.012 ⁇ M and 3 ⁇ M, with a final DMSO concentration of 1 %, figure 25.
  • An IC 50 value of 30nM has been reported for glycyrrhetinic acid inhibition of 11 ⁇ HSD1 in human hepatic microsomes, using dehydro-dexamethasone as the substrate [19]. However, these values are lower than the value reported by Barf et al. [15].
  • DGS03062B (STX469) To a stirred solution of DGS03022A (50 mg, 0.14 mmol, 1 eq.) in anhy. DMF (5 ml) and NaH (7 mg, 0.16 mmol, 1.1 eq.) was added Mel (3 ml, 0.21 mmol, 1.5 eq.) and the mixture was stirred for 1h. The resulting mixture was poured into water and the organic layer was extracted with ethyl acetate, dried (MgSO 4 ), filtered and concentrated under reduced pressure to give a yellow suspension.
  • 2,4-Dichloro benzoic acid (10 g, 0.0523 mol, 1 eq.) was heated to 115 °C with excess chlorosulphonic acid (10.5 mL, 0.1571 mol, 3 eq.) under N 2 for 18 h. The resulting mixture was cooled and consciously poured into ice-water. The resulted white precipitate was filtered out, washed with plenty of water and dried under vacuum over night.
  • DGS03116A (STX580) Synthesised by method B. Pale yellow crystals of DGS03116A (151 mg; 44%). mp 153 °C; TLC R f : 0.55 CH 2 CI 2 /EtOAc (4:1 ); 1 H NMR (DMSO-d 6 ) ⁇ 10.96 (s, 1 H, NH, Ex.
  • DGS03122B (STX583) Yellow crystals of DGS03122B (47 mg; 16%). mp 204-206 °C; TLC R f : 0.48 CH 2 CI 2 /EtOAc (4:1 ); 1 H NMR (DMSO-d 6 ) ⁇ 10.95 (s, 1H, NH, Ex.
  • DGS03130B (STX701) Pale yellow crystals of DGS03130A (45 mg; 14%). mp 169 °C; TLC R f : 0.42 CH 2 CI 2 /EtOAc (4:1 ); 1 H NMR (DMSO-d 6 ) ⁇ 10.63 (s, 1H, NH, Ex.
  • R -CH 2 COOC 2 H 5
  • R -CH 2 COOC 2 H 5
  • R -H
  • R -CH 3
  • Ar 3-CI-2-CH 3 -phenyl
  • R -H
  • R -CH 2 COOC 2 H 5 a) RX, NaH, THF r.t. b) ArS0 3 CI, DC .Pyridine or ArS0 3 CI, DCM.Pyridine/DMAP c) Diethylamine, DCM, AICI 3 d) RX, K 2 C0 3 , Acetone, reflux
  • R -NHCH 3
  • R -NHCH 3
  • R -N(C 2 H 5 ) 2
  • R -N(C 2 H 5 ) 2
  • Ar 3-CI-2-CH 3 -phenyl
  • Ar 2,5-dichlorophenyl a) HNO3, H 2 S0 4 -5 - 0°C b) H 2 , 5% Pd/C, C 2 H 5 OH, c) ArS0 3 CI, DCM.Pyridine or ArS0 3 CI, DCM,Pyridine/DMAP d) amine, THF, reflux
  • STX987 5-(3-Chloro-2-methyl-benzenesulfonylamino)-1-ethyl-2,3-dihydro-1H-indolium chloride (STX987, XDS02031)
  • STX987 The free base of STX987 was synthesized as above.
  • 1 -Acetyl-5-aminoindoline The solution of 1 -acetyl-5-nitroindoline (1.0 g, 4.85 mmol) in ethanol-THF (100 mL : 30 mL) was hydrogenated over 5% Pd/C (600 mg) at atmosphere pressure for 2h, filtered through Celite and concentrated in vacuo to give a white solid which was recrystalllized from ethanol. White crystalline solid (580 mg, 68%) was obtained.
  • 5-amino-1H-indole-3-carboxylic acid methyl ester (KRB01131): To a solution of 5- nitro-1W-indole-3-carboxylic acid methyl ester (206 mg, 0.940 mmol) in methanol (40 mL) was added 5% palladium on carbon (40 mg) and the mixture was stirred under 1 atm H 2 for 5h. The mixture was filtered through celite and the filtrate evaporated to yield a brown solid that was used without further purification (173 mg, 97%), single spot at R f 0.64 (ethyl acetate).
  • Ar 2-Me-3-CI-phenyl
  • R -CH 2 C 6 H 5
  • R -CH 2 C00C 2 H 5
  • Ar 4-n-propylphenyl
  • R -CH 3
  • R -Cri ⁇ CgHs
  • N 1 -Phenyl-benzene-1 ,2,4-triamine 800 mg, 4 mmol was dissolved in acetic acid (10 mL), acetic anhydride (1.0 mL) was added to the solution. The mixture was stirred at
  • the compound was prepared with general method of benzenesulphonamide formation.

Abstract

There is provided a compound having Formula (I): wherein one of R1 and R2 is a group of the Formula (a), wherein R4 is selected from H and hydrocarbyl, R5 is a hydrocarbyl group and L is an optional linker group, or R1 and R2 together form a ring substituted with the group (Formula (a)) wherein R3 is H or a substituent, and wherein X is selected from S, O, NR6 and C(R7)(R8), wherein R6 is selected from H and hydrocarbyl groups, wherein each of R7 and R8 are independently selected from H and hydrocarbyl groups.

Description

INHIBITORS OF 11-BETA-HYDROXY STEROID DEHYDROGENASE TYPE 1 AND TYPE 2
FIELD OF INVENTION
The present invention relates to a compound. In particular the present invention provides compounds capable of inhibiting 11 β-hydroxysteroid dehydrogenase (11 β- HSD).
Introduction
The role of glucocorticoids
Glucocorticoids are synthesised in the adrenal cortex from cholesterol. The principle glucocorticoid in the human body is cortisol, this hormone is synthesised and secreted in response to the adrenocortictrophic hormone (ACTH) from the pituitary gland in a circadian, episodic manner, but the secretion of this hormone can also be stimulated by stress, exercise and infection. Cortisol circulates mainly bound to transcortin (cortisol binding protein) or albumin and only a small fraction is free (5-10%) for biological processes [1].
Cortisol has a wide range of physiological effects, including regulation of carbohydrate, protein and lipid metabolism, regulation of normal growth and development, influence on cognitive function, resistance to stress and mineralocorticoid activity. Cortisol works in the opposite direction compared to insulin meaning a stimulation of hepatic gluconeogenesis, inhibition of peripheral glucose uptake and increased blood glucose concentration. Glucocorticoids are also essential in the regulation of the immune response. When circulating at higher concentrations glucocorticoids are immunosuppressive and are used pharmacologically as anti-inflammatory agents.
Glucocorticoids like other steroid hormones are lipophilic and penetrate the cell membrane freely. Cortisol binds, primarily, to the intracellular glucocorticoid receptor (GR) that then acts as a transcription factor to induce the expression of glucocorticoid responsive genes, and as a result of that protein synthesis. The role of the 11β-HSD enzyme
The conversion of cortisol (F) to its inactive metabolite cortisone (E) by 11 β-HSD was first described in the 1950's, however it was not until later that the biological importance for this conversion was suggested [2]. In 1983 Krozowski et al. showed that the mineralocorticoid receptor (MR) has equal binding affinities for glucocorticoids and mineralocorticoids [3]. Because the circulating concentration of cortisol is a 100 times higher than that of aldosterone and during times of stress or high activity even more, it was not clear how the MR remained mineralocorticoid specific and was not constantly occupied by glucocorticoids. Earlier Ulick et al. [4] had described the hypertensive condition known as, "apparent mineralocorticoid excess" (AME), and observed that whilst secretion of aldosterone from the adrenals was in fact low the peripheral metabolism of cortisol was disrupted. These discoveries lead to the suggestion of a protective role for the enzymes. By converting cortisol to cortisone in mineralocorticoid dependent tissues 11 β-HSD enzymes protects the MR from occupation by glucocorticoids and allows it to be mineralcorticoid specific. Aldosterone itself is protected from the enzyme by the presence of an aldehyde group at the C-18 position.
Congenital defects in the 11 β-HSD enzyme results in over occupation of the MR by cortisol and hypertensive and hypokalemic symptoms seen in AME.
Localisation of the 11 β-HSD showed that the enzyme and its activity is highly present in the MR dependent tissues, kidney and parotid. However in tissues where the MR is not mineralocorticoid specific and is normally occupied by glucocorticoids, 11 β-HSD is not present in these tissues, for example in the heart and hippocampus [5]. This research also showed that inhibition of 11 β-HSD caused a loss of the aldosterone specificity of the MR in these mineralocorticoid dependent tissues.
It has been shown that two iso-enzymes of 11 β-HSD exist. Both are members of the short chain alcohol dehydrogenase (SCAD) superfamily which have been widely conserved throughout evolution. 11 β-HSD type 2 acts as a dehydrogenase to convert the secondary alcohol group at the C-11 position of cortisol to a secondary ketone, so producing the less active metabolite cortisone. 11 β-HSD type 1 is thought to act mainly in vivo as a reductase, that is in the opposite direction to type 2 [6] [see below]. 11 β- HSD type 1 and type 2 have only a 30% amino acid homology.
Figure imgf000004_0001
11 β-HSD enzyme activity
The intracellular activity of cortisol is dependent on the concentration of glucocorticoids and can be modified and independently controlled without involving the overall secretion and synthesis of the hormone.
The role of 11 β-HSD Type 1
The direction of 11 β-HSD type 1 reaction in vivo is generally accepted to be opposite to the dehydrogenation of type 2. In vivo homozygous mice with a disrupted type 1 gene are unable to convert cortisone to cortisol, giving further evidence for the reductive activity of the enzyme [7]. 11 β-HSD type 1 is expressed in many key glucocorticoid regulated tissues like the liver, pituitary, gonad, brain, adipose and adrenals .however, the function of the enzyme in many of these tissues is poorly understood [8].
The concentration of cortisone in the body is higher than that of cortisol , cortisone also binds poorly to binding globulins, making cortisone many times more biologically available. Although cortisol is secreted by the adrenal cortex, there is a growing amount of evidence that the intracellular conversion of E to F may be an important mechanism in regulating the action of glucocorticoids [9].
It may be that 11 β-HSD type 1 allows certain tissues to convert cortisone to cortisol to increase local glucocorticoid activity and potentiate adaptive response and counteracting the type 2 activity that could result in a fall in active glucocorticoids [10]. Potentiation of the stress response would be especially important in the brain and high levels of 11 β- HSD type 1 are found around the hippocampus, further proving the role of the enzyme. 11 β-HSD type 1 also seems to play an important role in hepatocyte maturation [8]. Another emerging role of the 11 β-HSD type 1 enzyme is in the detoxification process of many non-steroidal carbonyl compounds, reduction of the carbonyl group of many toxic compounds is a common way to increase solubility and therefore increase their excretion. The 11 β-HSD typel enzyme has recently been shown to be active in lung tissue [11]. Type 1 activity is not seen until after birth, therefore mothers who smoke during pregnancy expose their children to the harmful effects of tobacco before the child is able to metabolically detoxify this compound.
The role of 11 β-HSD Type 2
As already stated earlier the 11 β-HSD type 2 converts cortisol to cortisone, thus protecting the MR in many key regulatory tissues of the body. The importance of protecting the MR from occupation by glucocorticoids is seen in patients with AME or liquorice intoxification. Defects or inactivity of the type 2 enzyme results in hypertensive syndromes and research has shown that patients with an hypertensive syndrome have an increased urinary excretion ratio of cortisol : cortisone. This along with a reported increase in the half life of radiolabelled cortisol suggests a reduction of 11 β-HSD type 2 activity [12].
Rationale for the development of 11 β-HSD inhibitors
As said earlier cortisol opposes the action of insulin meaning a stimulation of hepatic gluconeogenesis, inhibition of peripheral glucose uptake and increased blood glucose concentration. The effects of cortisol appear to be enhanced in patients suffering from glucose intolerance or diabetes mellitus. Inhibition of the enzyme 11 β-HSD type 1 would increase glucose uptake and inhibit hepatic gluconeogenesis, giving a reduction in circulatory glucose levels. The development of a potent 11 β-HSD type 1 inhibitor could therefore have considerable therapeutic potential for conditions associated with elevated blood glucose levels.
An excess in glucocorticoids can result in neuronal dysfunctions and also impair cognitive functions. A specific 11 β-HSD type 1 inhibitor might be of some importance by reducing neuronal dysfunctions and the loss of cognitive functions associated with ageing, by blocking the conversion of cortisone to cortisol.
Glucocorticoids also have an important role in regulating part of the immune response [13]. Glucocorticoids can suppress the production of cytokines and regulate the receptor levels. They are also involved in determining whether T-helper (Th) lymphocytes progress into either Th1 or Th2 phenotype. These two different types of Th cells secrete a different profile of cytokines, Th2 is predominant in a glucocorticoid environment. By inhibiting 11 β-HSD type 1 , Th1 cytokine response would be favoured. It is also possible to inhibit 11 β-HSD type 2 , thus by inhibiting the inactivation of cortisol, it may be possible to potentiate the anti-inflammatory effects of glucocorticoids.
Aspects of the invention are defined in the appended claims.
SUMMARY ASPECTS OF THE PRESENT INVENTION
In one aspect the present invention provides a compound having Formula Formula I
Figure imgf000006_0001
wherein one of R-, and R2 is a group of the formula
Figure imgf000006_0002
wherein R4 is selected from H and hydrocarbyl, R5 is a hydrocarbyl group and L is an optional linker group, or R-, and R2 together form a ring substituted with the group
Figure imgf000006_0003
wherein R3 is H or a substituent, and wherein X is selected from S, O, NR6 and C(R7)(R8). wherein R6 is selected from H and hydrocarbyl groups, wherein each of R7 and R8 are independently selected from H and hydrocarbyl groups. In one aspect the present invention provides a pharmaceutical composition comprising
(i) a compound having Formula I Formula
Figure imgf000007_0001
wherein one of R., and R2 is a group of the formula
Figure imgf000007_0002
wherein R4 is selected from H and hydrocarbyl, R5 is a hydrocarbyl group and L is an optional linker group, or R., and R2 together form a ring substituted with the group
Figure imgf000007_0003
wherein R3 is H or a substituent, and wherein X is selected from S, O, NR6 and C(R7)(R8), wherein R6 is selected from H and hydrocarbyl groups, wherein each of R7 and R8 are independently selected from H and hydrocarbyl groups.
(ii) optionally admixed with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
In one aspect the present invention provides a compound having Formula I Formula
Figure imgf000007_0004
wherein one of R., and R2 is a group of the formula
Figure imgf000008_0001
wherein R4 is selected from H and hydrocarbyl, R5 is a hydrocarbyl group and L is an optional linker group, or R., and R2 together form a ring substituted with the group
Figure imgf000008_0002
wherein R3 is H or a substituent, and wherein X is selected from S, O, NR6 and C(R7)(R8), wherein R6 is selected from H and hydrocarbyl groups, wherein each of R7 and R8 are independently selected from H and hydrocarbyl groups, for use in medicine.
In one aspect the present invention provides a use of a compound in the manufacture of a medicament for use in the therapy of a condition or disease associated with 11 β-HSD, wherein the compound has Formula I Formula
Figure imgf000008_0003
wherein one of R., and R2 is a group of the formula
Figure imgf000008_0004
wherein R4 is selected from H and hydrocarbyl, R5 is a hydrocarbyl group and L is an optional linker group, or R., and R2 together form a ring substituted with the group
Figure imgf000009_0001
wherein R3 is H or a substituent, and wherein X is selected from S, O, NR6 and C(R7)(R8), wherein R6 is selected from H and hydrocarbyl groups, wherein each of R7 and R8 are independently selected from H and hydrocarbyl groups.
SOME ADVANTAGES
One key advantage of the present invention is that the compounds of the present invention can act as 11 β-HSD inhibitors. The compounds may inhibit the interconversion of inactive 11-keto steroids with their active hydroxy equivalents. Thus present invention provides methods by which the conversion of the inactive to the active form may be controlled, and to useful therapeutic effects which may be obtained as a result of such control. More specifically, but not exclusively, the invention is concerned with interconversion between cortisone and cortisol in humans.
Another advantage of the compounds of the present invention is that they may be potent 11 β-HSD inhibitors in vivo.
Some of the compounds of the present invention are also advantageous in that they may be orally active.
The present invention may provide for a medicament for one or more of (i) regulation of carbohydrate metabolism, (ii) regulation of protein metabolism, (iii) regulation of lipid metabolism, (iv) regulation of normal growth and/or development, (v) influence on cognitive function, (vi) resistance to stress and mineralocorticoid activity.
Some of the compounds of the present invention may also be useful for inhibiting hepatic gluconeogenesis. The present invention may also provide a medicament to relieve the effects of endogenous glucocorticoids in diabetes mellitus, obesity (including centripetal obesity), neuronal loss and/or the cognitive impairment of old age. Thus, in a further aspect, the invention provides the use of an inhibitor of 11 β-HSD in the manufacture of a medicament for producing one or more therapeutic effects in a patient to whom the medicament is administered, said therapeutic effects selected from inhibition of hepatic gluconeogenesis, an increase in insulin sensitivity in adipose tissue and muscle, and the prevention of or reduction in neuronal loss/cognitive impairment due to glucocorticoid- potentiated neurotoxicity or neural dysfunction or damage.
From an alternative point of view, the invention provides a method of treatment of a human or animal patient suffering from a condition selected from the group consisting of: hepatic insulin resistance, adipose tissue insulin resistance, muscle insulin resistance, neuronal loss or dysfunction due to glucocorticoid potentiated neurotoxicity, and any combination of the aforementioned conditions, the method comprising the step of administering to said patient a medicament comprising a pharmaceutically active amount of a compound in accordance with the present invention.
Some of the compounds of the present invention may be useful for the treatment of cancer, such as breast cancer, as well as (or in the alternative) non-malignant conditions, such as the prevention of auto-immune diseases, particularly when pharmaceuticals may need to be administered from an early age.
DETAILED ASPECTS OF THE PRESENT INVENTION
In one aspect the present invention provides a compound having Formula Formula I
Figure imgf000010_0001
wherein one of R-i and R2 is a group of the formula
Figure imgf000010_0002
wherein R4 is selected from H and hydrocarbyl, R5 is a hydrocarbyl group and L is an optional linker group, or R., and R2 together form a ring substituted with the group
Figure imgf000011_0001
wherein R3 is H or a substituent, and wherein X is selected from S, O, NR6 and C(R7)(R8), wherein R6 is selected from H and hydrocarbyl groups, wherein each of R7 and R8 are independently selected from H and hydrocarbyl groups.
In one aspect the present invention provides a pharmaceutical composition comprising
(i) a compound having Formula I defined above
(ii) optionally admixed with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
In one aspect the present invention provides a compound having Formula I defined above, for use in medicine.
In one aspect the present invention provides a use of a compound having Formula I defined above in the manufacture of a medicament for use in the therapy of a condition or disease associated with 11 β-HSD.
In one aspect the present invention provides a use of a compound having Formula I defined above in the manufacture of a medicament for use in the therapy of a condition or disease associated with adverse 11 β-HSD levels.
In one aspect the present invention provides a use of a compound having Formula I defined above in the manufacture of a pharmaceutical for inhibiting 11 β-HSD activity.
In one aspect the present invention provides a use of a compound having Formula I defined above in the manufacture of a pharmaceutical for inhibiting 11 β-HSD activity.
In one aspect the present invention provides a method comprising (a) performing a 11β- HSD assay with one or more candidate compounds having Formula I defined above; (b) determining whether one or more of said candidate compounds is/are capable of modulating 11 β-HSD activity; and (c) selecting one or more of said candidate compounds that is/are capable of modulating 11 β-HSD activity. In one aspect the present invention provides a method comprising (a) performing a 11β- HSD assay with one or more candidate compounds having Formula I defined above; (b) determining whether one or more of said candidate compounds is/are capable of inhibiting 11 β-HSD activity; and (c) selecting one or more of said candidate compounds that is/are capable of inhibiting 11 β-HSD activity.
In one aspect the present invention provides
• a compound identified by the above method, • the use of the said compound in medicine,
• a pharmaceutical composition comprising the said compound, optionally admixed with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant,
• use of the said compound in the manufacture of a medicament for use in the therapy of a condition or disease associated with 11 β-HSD, and • use of the said compound in the manufacture of a medicament for use in the therapy of a condition or disease associated with adverse 11 β-HSD levels.
For ease of reference, these and further aspects of the present invention are now discussed under appropriate section headings. However, the teachings under each section are not necessarily limited to each particular section.
PREFERABLE ASPECTS
In one preferred aspect the compound of the present invention has Formula II Formula II
Figure imgf000012_0001
In one preferred aspect L is not present. In this aspect the present invention provides a compound having Formula I Formula
Figure imgf000013_0001
wherein one of R., and R2 is a group of the formula
Figure imgf000013_0002
wherein R4 is selected from H and hydrocarbyl, and R5 is a hydrocarbyl group; or R and R2 together form a ring substituted with the group
Figure imgf000013_0003
wherein R-, is H or a substituent
In one preferred aspect the compound of the present invention R1 and R2 together form a ring substituted with the group
Figure imgf000013_0004
In one preferred aspect the compound of the present invention R-, and R2 together form a carbocyclic ring.
In one preferred aspect the compound of the present invention R-, and R2 together form a six membered ring.
In one preferred aspect the compound of the present invention R-i and R2 together form a six membered carbocyclic ring. In one preferred aspect the compound of the present invention wherein R., and R2 together form an aryl ring.
Preferred compounds of the present invention are those having one of the following formulae.
Formula III
Formula IV
Formula V
Formula VI
Formula Via
Figure imgf000014_0001
Formula VII
Figure imgf000015_0001
Figure imgf000015_0002
In preferred aspects of the present invention R3 is selected from H, hydrocarbyl, -S- hydrocarbyl, -S-H, halogen and N(R9)(R10), wherein each of R9 and R10 are independently selected from H and hydrocarbyl groups.
In preferred aspects of the present invention R3 is selected from H, hydroxy, alkyl especially C,-C10 alkyl groups, C,-C6 alkyl, e.g. C.,-C3 alkyl group, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl and other pentyl isomers, and n-hexyl and other hexyl isomers, alkoxy especially CrC10 alkoxy groups, CrC6 alkoxy, e.g. C1-C3 alkoxy group, methoxy, ethoxy, propoxy etc., alkinyl, e.g. ethinyl, or halogen, e.g. fluoro substituents.
When R3 is -S-hydrocarbyl, preferably R3 is selected from -S-alkyl, -S-carboxylic acid, - S-ether, and -S-amide, preferably selected from -S-C^^alkyl, -S- CnoCarboxcylic acid, -S- C-noether, and -S- C^^amide.
In preferred aspects of the present invention R3 is -CH3.
Further preferred compounds of the present invention are those having one of the following formulae. Formula la
Figure imgf000015_0003
Formula VIII
Formula IX
Formula X
Formula Xa
Formula XI
Formula Xla
Figure imgf000016_0001
In further preferred aspects of the present invention, such as when the compound has Formula la, Formula VIII, Formula IX, Formula X, Formula Xa, Formula XI, or Formula Xla, R3 is selected from O, hydrocarbyl, and N(R9) wherein R9 is selected from H and hydrocarbyl groups. More preferably R3 is selected from O, 0,-0,0 alkenyl groups, such as C,-C6 alkenyl group, and C,-C3 alkenyl group, NH and N-C-,-C10 alkyl groups, such as N-C,-C6 alkyl group, and N-C,-C3 alkyl groups.
In further preferred aspects of the present invention R4 is selected from H and 0,-0,0 alkyl groups, such as C,-C6 alkyl group, and C,-C3 alkyl group. Preferably R4 is H.
In further preferred aspects of the present invention R4 is a group of the formula.
O:
5
In these aspects the group shown above as
Figure imgf000017_0001
may be of the formula
Figure imgf000017_0002
wherein each R5 is independently selected from hydrocarbyl groups. Each R5 may be the same of different to the other R5. In one aspect the two R5 groups are the same.
In some preferred aspects of the invention R5 is a cyclic hydrocarbyl group. Preferably R5 is a cyclic hydrocarbyl group comprising a hydrocarbon ring.
R5 may be a substituted ring or an unsubstituted ring. In some preferred aspects of the invention R5 is substituted ring. Preferably R5 is a carbocyclic ring.
Preferably R5 is a six membered ring.
Preferably R5 is a six membered carbocyclic ring. More preferably R5 is a substituted six membered carbocyclic ring.
In some preferred aspects of the invention R5 is an aryl ring. Preferably R5 is a substituted aryl ring.
In one highly preferred aspect R5 is a group having the formula
Figure imgf000018_0001
wherein each of R^, R12, R13, R14 and R15 are independently selected from H, halogen, and hydrocarbyl groups.
Preferably each of R^, R12, R13, R14 and R15 are independently selected from H, halogen, alkyl, such as C1-6 alkyl, phenyl, O-alkyl, O-phenyl, nitrile, haloalkyl, such as CF3, CCI3 and CBr3, carboxyalkyl, -CO2H, CO2alkyl, and NH-acetyl groups..
Two or more of R11; R12, R13, R-,4 and R15 may join to form a ring. The two or more of R-,-,, R12. R 13. R14 and Ri5 maY °r ay not be adjacent. The ring may be carbocyclic or heterocyclic ring. The ring may be optionally substituted by any of the R^, R12, R13, R14 and R15 substituents listed above. When two or more of R„, R12, R13, R14 and R15 may join to form a ring the group
Figure imgf000019_0001
may provide a naphthyl, quinolyl, tetrahydroquinolyl, or benzothtrahydropyranyl, each of which may be substituted or unsubstituted.
SUBSTITUENTS
The compound of the present invention may have substituents other than those of the ring systems show herein. Furthermore the ring systems herein are given as general formulae and should be interpreted as such. The absence of any specifically shown substituents on a given ring member indicates that the ring member may substituted with any moiety of which H is only one example. The ring system may contain one or more degrees of unsaturation, for example is some aspects one or more rings of the ring system is aromatic. The ring system may be carbocyclic or may contain one or more hetero atoms.
The compound of the invention, in particular the ring system compound of the invention of the present invention may contain substituents other than those show herein. By way of example, these other substituents may be one or more of: one or more halo groups, one or more O groups, one or more hydroxy groups, one or more amino groups, one or more sulphur containing group(s), one or more hydrocarbyl group(s) - such as an oxyhydrocarbyl group.
In general terms the ring system of the present compounds may contain a variety of non- interfering substituents. In particular, the ring system may contain one or more hydroxy, alkyl especially lower (C,-C6) alkyl, e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, sec- butyl, tert-butyl, n-pentyl and other pentyl isomers, and n-hexyl and other hexyl isomers, alkoxy especially lower (CrC6) alkoxy, e.g. methoxy, ethoxy, propoxy etc., alkinyl, e.g. ethinyl, or halogen, e.g. fluoro substituents. For some compounds of the present invention, the compound may be substituted with a hydrocarbylsulphanyl group. The term "hydrocarbylsulphanyl" means a group that comprises at least hydrocarbyl group (as herein defined) and sulphur, preferably -S- hydrocarbyl, more preferably -S-hydrocarbon. That sulphur group may be optionally oxidised.
Preferably the hydrocarbylsulphanyl group is -S-C1-10 alkyl, more preferably -S-C1-5 alkyl, more preferably -S-C1-3 alkyl, more preferably -S-CH2CH2CH3, -S-CH2CH3 or -SCH3
FURTHER ASPECTS
For some applications, preferably the compounds have a reversible action.
For some applications, preferably the compounds have an irreversible action.
In one embodiment, the compounds of the present invention are useful for the treatment of breast cancer.
The compounds of the present invention may be in the form of a salt.
The present invention also covers novel intermediates that are useful to prepare the compounds of the present invention. For example, the present invention covers novel alcohol precursors for the compounds. By way of further example, the present invention covers bis protected precursors for the compounds. Examples of each of these precursors are presented herein. The present invention also encompasses a process comprising each or both of those precursors for the synthesis of the compounds of the present invention.
STEROID DEHYDROGENASE
11 β Steroid dehydrogenase may be referred to as "11 β-HSD" or "HD" for short
In some aspects of the invention 11 β-HSD is preferably 11 β-HSD Type 1.
In some aspects of the invention 11 β-HSD is preferably 11 β-HSD Type 2. STEROID DEHYDROGENASE INHIBITION
It is believed that some disease conditions associated with HD activity are due to conversion of a inactive, cortisone to an active, cortisol. In disease conditions associated with HD activity, it would be desirable to inhibit HD activity.
Here, the term "inhibit" includes reduce and/or eliminate and/or mask and/or prevent the detrimental action of HD.
HD INHIBITOR
In accordance with the present invention, the compound of the present invention is capable of acting as an HD inhibitor.
Here, the term "inhibitor" as used herein with respect to the compound of the present invention means a compound that can inhibit HD activity - such as reduce and/or eliminate and/or mask and/or prevent the detrimental action of HD. The HD inhibitor may act as an antagonist.
The ability of compounds to inhibit steroid dehydrogenase activity can be assessed using the suitable Assay Protocol presented in the Examples section.
It is to be noted that the compound of the present invention may have other beneficial properties in addition to or in the alternative to its ability to inhibit HD activity.
HYDROCARBYL
The term "hydrocarbyl group" as used herein means a group comprising at least C and H and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo, alkoxy, nitro, an alkyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen and oxygen. A non- limiting example of a hydrocarbyl group is an acyl group.
A typical hydrocarbyl group is a hydrocarbon group. Here the term "hydrocarbon" means any one of an alkyl group, an alkenyl group, an alkynyl group, which groups may be linear, branched or cyclic, or an aryl group. The term hydrocarbon also includes those groups but wherein they have been optionally substituted. If the hydrocarbon is a branched structure having substituent(s) thereon, then the substitution may be on either the hydrocarbon backbone or on the branch; alternatively the substitutions may be on the hydrocarbon backbone and on the branch.
In some aspects of the present invention, one or more hydrocarbyl groups is independently selected from optionally substituted alkyl group, optionally substituted haloalkyl group, aryl group, alkylaryl group, alkylarylakyl group, and an alkene group.
In some aspects of the present invention, one or more hydrocarbyl groups is independently selected from CrC10 alkyl group, such as C,-C6 alkyl group, and C,-C3 alkyl group. Typical alkyl groups include C, alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C7 alkyl, and C8 alkyl.
In some aspects of the present invention, one or more hydrocarbyl groups is independently selected from 0,-0,0 haloalkyl group, C C6 haloalkyl group, CrC3 haloalkyl group, C,-C10 bromoalkyl group, C,-C6 bromoalkyl group, and C,-C3 bromoalkyl group. Typical haloalkyl groups include C, haloalkyl, C2 haloalkyl, C3 haloalkyl, C4 haloalkyl, C5 haloalkyl, C7 haloalkyl, C8 haloalkyl, C, bromoalkyl, C2 bromoalkyl, C3 bromoalkyl, C4 bromoalkyl, C5 bromoalkyl, C7 bromoalkyl, and C8 bromoalkyl.
In some aspects of the present invention, one or more hydrocarbyl groups is independently selected from aryl groups, alkylaryl groups, alkylarylakyl groups, -(CH2).,. 10-aryl, -(CH2)1-10-Ph, (CH2)1-10-Ph-C1-10 alkyl, -(CH2)1-5-Ph, (CH2)1.5-Ph-C1-5 alkyl, -(CH2)1-3- Ph, (CH2)1-3-Ph-C1-3 alkyl, -CH2-Ph, and -CH2-Ph-C(CH3)3. The aryl groups may contain a hetero atom. Thus the aryl group or one or more of the aryl groups may be carbocyclic or more may heterocyclic. Typical hetero atoms include O, N and S, in particular N.
In some aspects of the present invention, one or more hydrocarbyl groups is independently selected from -(CH2)1-10-cycloalkyl, -(CH2)1-10-C3-1ocycloalkyl, -(CH2)1.7-C3- 7cycloalkyl, -(CH2)1.5-C3.5cycloalkyl, -(CH2)1.3-C3-5cycloalkyl, and -CH2- C3cycloalkyl. In some aspects of the present invention, one or more hydrocarbyl groups is independently selected from alkene groups. Typical alkene groups include 0,-0,0 alkene group, C,-C6 alkene group, C,-C3 alkene group, such as C,, C2, C3, C4, C5, C6, or C7 alkene group. In a preferred aspect the alkene group contains 1 , 2 or 3 C=C bonds. In a preferred aspect the alkene group contains 1 C=C bond. In some preferred aspect at least one C=C bond or the only C=C bond is to the terminal C of the alkene chain, that is the bond is at the distal end of the chain to the ring system.
In some aspects of the present invention, one or more hydrocarbyl groups is independently selected from oxyhydrocarbyl groups.
OXYHYDROCARBYL
The term "oxyhydrocarbyl" group as used herein means a group comprising at least C, H and O and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the oxyhydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the oxyhydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur and nitrogen.
In one embodiment of the present invention, the oxyhydrocarbyl group is a oxyhydrocarbon group.
Here the term "oxyhydrocarbon" means any one of an alkoxy group, an oxyalkenyl group, an oxyalkynyl group, which groups may be linear, branched or cyclic, or an oxyaryl group. The term oxyhydrocarbon also includes those groups but wherein they have been optionally substituted. If the oxyhydrocarbon is a branched structure having substituent(s) thereon, then the substitution may be on either the hydrocarbon backbone or on the branch; alternatively the substitutions may be on the hydrocarbon backbone and on the branch.
Typically, the oxyhydrocarbyl group is of the formula C^O (such as a C1-3O). ANIMAL ASSAY MODEL FOR DETERMINING OESTROGENIC ACTIVITY
(PROTOCOL S
Lack of in vivo oestrogenicity
The compounds of the present invention may be studied using an animal model, in particular in ovariectomised rats. In this model, compounds which are oestrogenic stimulate uterine growth.
The compound (10 mg/Kg/day for five days) was administered orally to rats with another group of animals receiving vehicle only (propylene glycol). A further group received the estrogenic compound EMATE subcutaneously in an amount of 10μg/day for five days. At the end of the study uteri were obtained and weighed with the results being expressed as uterine weight/whole body weight x 100.
Compounds having no significant effect on uterine growth are not oestrogenic.
REPORTERS
A wide variety of reporters may be used in the assay methods (as well as screens) of the present invention with preferred reporters providing conveniently detectable signals (e.g. by spectroscopy). By way of example, a reporter gene may encode an enzyme which catalyses a reaction which alters light absorption properties.
Other protocols include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilising monoclonal antibodies reactive to two non- interfering epitopes may even be used. These and other assays are described, among other places, in Hampton R et al (1990, Serological Methods, A Laboratory Manual, APS Press, St Paul MN) and Maddox DE et al (1983, J Exp Med 15 8:121 1).
Examples of reporter molecules include but are not limited to (β-galactosidase, invertase, green fluorescent protein, luciferase, chloramphenicol, acetyltransferase, (- glucuronidase, exo-glucanase and glucoamylase. Alternatively, radiolabelled or fluorescent tag-labelled nucleotides can be incorporated into nascent transcripts which are then identified when bound to oligonucleotide probes.
By way of further examples, a number of companies such as Pharmacia Biotech (Piscataway, NJ), Promega (Madison, Wl), and US Biochemical Corp (Cleveland, OH) supply commercial kits and protocols for assay procedures. Suitable reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like. Patents teaching the use of such labels include US-A-3817837; US-A-3850752; US-A-3939350; US-A-3996345; US-A-4277437; US-A-4275149 and US-A-4366241.
HOST CELLS
The term "host cell" - in relation to the present invention includes any cell that could comprise the target for the agent of the present invention.
Thus, a further embodiment of the present invention provides host cells transformed or transfected with a polynucleotide that is or expresses the target of the present invention.
Preferably said polynucleotide is carried in a vector for the replication and expression of polynucleotides that are to be the target or are to express the target. The cells will be chosen to be compatible with the said vector and may for example be prokaryotic (for example bacterial), fungal, yeast or plant cells.
The gram negative bacterium E. coli is widely used as a host for heterologous gene expression. However, large amounts of heterologous protein tend to accumulate inside the cell. Subsequent purification of the desired protein from the bulk of E.coli intracellular proteins can sometimes be difficult.
In contrast to E.coli, bacteria from the genus Bacillus are very suitable as heterologous hosts because of their capability to secrete proteins into the culture medium. Other bacteria suitable as hosts are those from the genera Streptomyces and Pseudomonas.
Depending on the nature of the polynucleotide encoding the polypeptide of the present invention, and/or the desirability for further processing of the expressed protein, eukaryotic hosts such as yeasts or other fungi may be preferred. In general, yeast cells are preferred over fungal cells because they are easier to manipulate. However, some proteins are either poorly secreted from the yeast cell, or in some cases are not processed properly (e.g. hyperglycosylation in yeast). In these instances, a different fungal host organism should be selected.
Examples of suitable expression hosts within the scope of the present invention are fungi such as Aspergillus species (such as those described in EP-A-0184438 and EP-A- 0284603) and Trichoderma species; bacteria such as Bacillus species (such as those described in EP-A-0134048 and EP-A-0253455), Streptomyces species and Pseudomonas species; and yeasts such as Kluyveromyces species (such as those described in EP-A-0096430 and EP-A-0301670) and Saccharomyces species. By way of example, typical expression hosts may be selected from Aspergillus niger, Aspergillus niger var. tubigenis, Aspergillus niger var. awamori, Aspergillus aculeatis, Aspergillus nidulans, Aspergillus orvzae, Trichoderma reesei, Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Kluyveromyces lactis and Saccharomyces cerevisiae.
The use of suitable host cells - such as yeast, fungal and plant host cells - may provide for post-translational modifications (e.g. myristoylation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the present invention.
ORGANISM
The term "organism" in relation to the present invention includes any organism that could comprise the target according to the present invention and/or products obtained therefrom. Examples of organisms may include a fungus, yeast or a plant.
The term "transgenic organism" in relation to the present invention includes any organism that comprises the target according to the present invention and/or products obtained.
TRANSFORMATION OF HOST CELLS/HOST ORGANISMS
As indicated earlier, the host organism can be a prokaryotic or a eukaryotic organism. Examples of suitable prokaryotic hosts include E. coli and Bacillus subtilis. Teachings on the transformation of prokaryotic hosts is well documented in the art, for example see Sambrook et al (Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press) and Ausubel et al., Current Protocols in Molecular Biology (1995), John Wiley & Sons, Inc.
If a prokaryotic host is used then the nucleotide sequence may need to be suitably modified before transformation - such as by removal of introns.
In another embodiment the transgenic organism can be a yeast. In this regard, yeast have also been widely used as a vehicle for heterologous gene expression. The species Saccharomyces cerevisiae has a long history of industrial use, including its use for heterologous gene expression. Expression of heterologous genes in Saccharomyces cerevisiae has been reviewed by Goodey et al (1987, Yeast Biotechnology, D R Berry et al, eds, pp 401-429, Allen and Unwin, London) and by King et al (1989, Molecular and Cell Biology of Yeasts, E F Walton and G T Yarronton, eds, pp 107-133, Blackie, Glasgow).
For several reasons Saccharomyces cerevisiae is well suited for heterologous gene expression. First, it is non-pathogenic to humans and it is incapable of producing certain endotoxins. Second, it has a long history of safe use following centuries of commercial exploitation for various purposes. This has led to wide public acceptability. Third, the extensive commercial use and research devoted to the organism has resulted in a wealth of knowledge about the genetics and physiology as well as large-scale fermentation characteristics of Saccharomyces cerevisiae.
A review of the principles of heterologous gene expression in Saccharomyces cerevisiae and secretion of gene products is given by E Hinchcliffe E Kenny (1993, "Yeast as a vehicle for the expression of heterologous genes", Yeasts, Vol 5, Anthony H Rose and J Stuart Harrison, eds, 2nd edition, Academic Press Ltd.).
Several types of yeast vectors are available, including integrative vectors, which require recombination with the host genome for their maintenance, and autonomously replicating plasmid vectors.
In order to prepare the transgenic Saccharomyces, expression constructs are prepared by inserting the nucleotide sequence into a construct designed for expression in yeast. Several types of constructs used for heterologous expression have been developed. The constructs contain a promoter active in yeast fused to the nucleotide sequence, usually a promoter of yeast origin, such as the GAL1 promoter, is used. Usually a signal sequence of yeast origin, such as the sequence encoding the SUC2 signal peptide, is used. A terminator active in yeast ends the expression system.
For the transformation of yeast several transformation protocols have been developed. For example, a transgenic Saccharomyces according to the present invention can be prepared by following the teachings of Hinnen et al (1978, Proceedings of the National Academy of Sciences of the USA 75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito, H et al (1983, J Bacteriology 153, 163-168).
The transformed yeast cells are selected using various selective markers. Among the markers used for transformation are a number of auxotrophic markers such as LEU2, HIS4 and TRP1 , and dominant antibiotic resistance markers such as aminoglycoside antibiotic markers, e.g. G418.
Another host organism is a plant. The basic principle in the construction of genetically modified plants is to insert genetic information in the plant genome so as to obtain a stable maintenance of the inserted genetic material. Several techniques exist for inserting the genetic information, the two main principles being direct introduction of the genetic information and introduction of the genetic information by use of a vector system. A review of the general techniques may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27). Further teachings on plant transformation may be found in EP- A-0449375.
Thus, the present invention also provides a method of transforming a host cell with a nucleotide sequence that is to be the target or is to express the target. Host cells transformed with the nucleotide sequence may be cultured under conditions suitable for the expression of the encoded protein. The protein produced by a recombinant cell may be displayed on the surface of the cell. If desired, and as will be understood by those of skill in the art, expression vectors containing coding sequences can be designed with signal sequences which direct secretion of the coding sequences through a particular prokaryotic or eukaryotic cell membrane. Other recombinant constructions may join the coding sequence to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins (Kroll DJ et al (1993) DNA Cell Biol 12:441-53). VARIANTS/HOMOLOGUES/DERIVATIVES
In addition to the specific amino acid sequences and nucleotide sequences mentioned herein, the present invention also encompasses the use of variants, homologue and derivatives thereof. Here, the term "homology" can be equated with "identity".
In the present context, an homologous sequence is taken to include an amino acid sequence which may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.
% homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local homology.
However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - will achieve a higher score than one with many gaps. "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package (see below) the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.
Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid - Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). However it is preferred to use the GCG Bestfit program.
A further useful reference is that found in FEMS Microbiol Lett 1999 May 15;174(2):247- 50 (and a published erratum appears in FEMS Microbiol Lett 1999 Aug 1 ;177(1 ): 187-8).
Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result. The sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:
Table 1
Figure imgf000031_0001
EXPRESSION VECTORS
The nucleotide sequence for use as the target or for expressing the target can be incorporated into a recombinant replicable vector. The vector may be used to replicate and express the nucleotide sequence in and/or from a compatible host cell. Expression may be controlled using control sequences which include promoters/enhancers and other expression regulation signals. Prokaryotic promoters and promoters functional in eukaryotic cells may be used. Tissue specific or stimuli specific promoters may be used. Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above.
The protein produced by a host recombinant cell by expression of the nucleotide sequence may be secreted or may be contained intracellularly depending on the sequence and/or the vector used. The coding sequences can be designed with signal sequences which direct secretion of the substance coding sequences through a particular prokaryotic or eukaryotic cell membrane.
FUSION PROTEINS
The target amino acid sequence may be produced as a fusion protein, for example to aid in extraction and purification. Examples of fusion protein partners include glutathione-S- transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and (-galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. Preferably the fusion protein will not hinder the activity of the target.
The fusion protein may comprise an antigen or an antigenic determinant fused to the substance of the present invention. In this embodiment, the fusion protein may be a non-naturally occurring fusion protein comprising a substance which may act as an adjuvant in the sense of providing a generalised stimulation of the immune system. The antigen or antigenic determinant may be attached to either the amino or carboxy terminus of the substance.
In another embodiment of the invention, the amino acid sequence may be ligated to a heterologous sequence to encode a fusion protein. For example, for screening of peptide libraries for agents capable of affecting the substance activity, it may be useful to encode a c imeric substance expressing a heterologous epitope that is recognised by a commercially available antibody.
THERAPY
The compounds of the present invention may be used as therapeutic agents - i.e. in therapy applications.
The term "therapy" includes curative effects, alleviation effects, and prophylactic effects.
The therapy may be on humans or animals, preferably female animals. PHARMACEUTICAL COMPOSITIONS
In one aspect, the present invention provides a pharmaceutical composition, which comprises a compound according to the present invention and optionally a pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof).
The pharmaceutical compositions may be for human or animal usage in human and veterinary medicine and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier, or excipient. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
Preservatives, stabilisers, dyes and even flavouring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.
There may be different composition/formulation requirements dependent on the different delivery systems. By way of example, the pharmaceutical composition of the present invention may be formulated to be delivered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestable solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route. Alternatively, the formulation may be designed to be delivered by both routes.
Where the agent is to be delivered mucosally through the gastrointestinal mucosa, it should be able to remain stable during transit though the gastrointestinal tract; for example, it should be resistant to proteolytic degradation, stable at acid pH and resistant to the detergent effects of bile. Where appropriate, the pharmaceutical compositions can be administered by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intravenously, intramuscularly or subcutaneously. For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.
COMBINATION PHARMACEUTICAL
The compound of the present invention may be used in combination with one or more other active agents, such as one or more other pharmaceutically active agents.
By way of example, the compounds of the present invention may be used in combination with other 11 β-HSD inhibitors and/or other inhibitors such as an aromatase inhibitor (such as for example, 4hydroxyandrostenedione (4-OHA)), and/or a steroid sulphatase inhibitors such as EMATE and/or steroids - such as the naturally occurring stemeurosteroids dehydroepiandrosterone sulfate (DHEAS) and pregnenolone sulfate (PS) and/or other structurally similar organic compounds.
In addition, or in the alternative, the compound of the present invention may be used in combination with a biological response modifier.
The term biological response modifier ("BRM") includes cytokines, immune modulators, growth factors, haematopoiesis regulating factors, colony stimulating factors, chemotactic, haemolytic and thrombolytic factors, cell surface receptors, ligands, leukocyte adhesion molecules, monoclonal antibodies, preventative and therapeutic vaccines, hormones, extracellular matrix components, fibronectin, etc. For some applications, preferably, the biological response modifier is a cytokine. Examples of cytokines include: interleukins (IL) - such as IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL- 9, IL-10, IL-11 , IL-12, IL-19; Tumour Necrosis Factor (TNF) - such as TNF-α; Interferon alpha, beta and gamma; TGF-β. For some applications, preferably the cytokine is tumour necrosis factor (TNF). For some applications, the TNF may be any type of TNF - such as TNF-α, TNF-β, including derivatives or mixtures thereof. More preferably the cytokine is TNF-α. Teachings on TNF may be found in the art - such as WO-A-98/08870 and WO-A-98/13348.
ADMINISTRATION
Typically, a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular patient. The dosages below are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited.
The compositions of the present invention may be administered by direct injection. The composition may be formulated for parenteral, mucosal, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration. Depending upon the need, the agent may be administered at a dose of from 0.01 to 30 mg/kg body weight, such as from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.
By way of further example, the agents of the present invention may be administered in accordance with a regimen of 1 to 4 times per day, preferably once or twice per day. The specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.
Aside from the typical modes of delivery - indicated above - the term "administered" also includes delivery by techniques such as lipid mediated transfection, liposomes, immunoliposomes, lipofectin, cationic facial amphiphiles (CFAs) and combinations thereof. The routes for such delivery mechanisms include but are not limited to mucosal, nasal, oral, parenteral, gastrointestinal, topical, or sublingual routes.
The term "administered" includes but is not limited to delivery by a mucosal route, for example, as a nasal spray or aerosol for inhalation or as an ingestable solution; a parenteral route where delivery is by an injectable form, such as, for example, an intravenous, intramuscular or subcutaneous route.
Thus, for pharmaceutical administration, the compounds of the present invention can be formulated in any suitable manner utilising conventional pharmaceutical formulating techniques and pharmaceutical carriers, adjuvants, excipients, diluents etc. and usually for parenteral administration. Approximate effective dose rates may be in the range from 1 to 1000 mg/day, such as from 10 to 900 mg/day or even from 100 to 800 mg/day depending on the individual activities of the compounds in question and for a patient of average (70Kg) bodyweight. More usual dosage rates for the preferred and more active compounds will be in the range 200 to 800 mg/day, more preferably, 200 to 500 mg/day, most preferably from 200 to 250 mg/day. They may be given in single dose regimes, split dose regimes and/or in multiple dose regimes lasting over several days. For oral administration they may be formulated in tablets, capsules, solution or suspension containing from 100 to 500 mg of compound per unit dose. Alternatively and preferably the compounds will be formulated for parenteral administration in a suitable parenterally administrable carrier and providing single daily dosage rates in the range 200 to 800 mg, preferably 200 to 500, more preferably 200 to 250 mg. Such effective daily doses will, however, vary depending on inherent activity of the active ingredient and on the bodyweight of the patient, such variations being within the skill and judgement of the physician.
CELL CYCLING
The compounds of the present invention may be useful in the method of treatment of a cell cycling disorder.
As discussed in "Molecular Cell Biology" 3rd Ed. Lodish et al. pages 177-181 different eukaryotic cells can grow and divide at quite different rates. Yeast cells, for example, can divide every 120 min., and the first divisions of fertilised eggs in the embryonic cells of sea urchins and insects take only 1530 min. because one large pre-existing cell is subdivided. However, most growing plant and animal cells take 10-20 hours to double in number, and some duplicate at a much slower rate. Many cells in adults, such as nerve cells and striated muscle cells, do not divide at all; others, like the fibroblasts that assist in healing wounds, grow on demand but are otherwise quiescent. Still, every eukaryotic cell that divides must be ready to donate equal genetic material to two daughter cells. DNA synthesis in eukaryotes does not occur throughout the cell division cycle but is restricted to a part of it before cell division.
The relationship between eukaryotic DNA synthesis and cell division has been thoroughly analysed in cultures of mammalian cells that were all capable of growth and division. In contrast to bacteria, it was found, eukaryotic cells spend only a part of their time in DNA synthesis, and it is completed hours before cell division (mitosis). Thus a gap of time occurs after DNA synthesis and before cell division; another gap was found to occur after division and before the next round of DNA synthesis. This analysis led to the conclusion that the eukaryotic cell cycle consists of an M (mitotic) phase, a G-, phase (the first gap), the S (DNA synthesis) phase, a G2 phase (the second gap), and back to M. The phases between mitoses (G-,, S, and G2) are known collectively as the interphase.
Many nondividing cells in tissues (for example, all quiescent fibroblasts) suspend the cycle after mitosis and just prior to DNA synthesis; such "resting" cells are said to have exited from the cell cycle and to be in the G0 state.
It is possible to identify cells when they are in one of the three interphase stages of the cell cycle, by using a fluorescence-activated cell sorter (FACS) to measure their relative DNA content: a cell that is in G-, (before DNA synthesis) has a defined amount x of DNA; during S (DNA replication), it has between x and 2x; and when in G2 (or M), it has 2x of DNA.
The stages of mitosis and cytokinesis in an animal cell are as follows
(a) Interphase. The G2 stage of interphase immediately precedes the beginning of mitosis. Chromosomal DNA has been replicated and bound to protein during the S phase, but chromosomes are not yet seen as distinct structures. The nucleolus is the only nuclear substructure that is visible under light microscope. In a diploid cell before DNA replication there are two morphologic chromosomes of each type, and the cell is said to be 2n. In G2, after DNA replication, the cell is 4n. There are four copies of each chromosomal DNA. Since the sister chromosomes have not yet separated from each other, they are called sister chromatids. b) Early prophase. Centrioles, each with a newly formed daughter centriole, begin moving toward opposite poles of the cell; the chromosomes can be seen as long threads. The nuclear membrane begins to disaggregate into small vesicles.
(c) Middle and late prophase. Chromosome condensation is completed; each visible chromosome structure is composed of two chromatids held together at their centromeres. Each chromatid contains one of the two newly replicated daughter DNA molecules. The microtubular spindle begins to radiate from the regions just adjacent to the centrioles, which are moving closer to their poles. Some spindle fibres reach from pole to pole; most go to chromatids and attach at kinetochores.
(d) Metaphase. The chromosomes move toward the equator of the cell, where they become aligned in the equatorial plane. The sister chromatids have not yet separated.
(e) Anaphase. The two sister chromatids separate into independent chromosomes.
Each contains a centromere that is linked by a spindle fibre to one pole, to which it moves. Thus one copy of each chromosome is donated to each daughter cell.
Simultaneously, the cell elongates, as do the pole-to-pole spindles. Cytokinesis begins as the cleavage furrow starts to form.
(f) Telophase. New membranes form around the daughter nuclei; the chromosomes uncoil and become less distinct, the nucleolus becomes visible again, and the nuclear membrane forms around each daughter nucleus. Cytokinesis is nearly complete, and the spindle disappears as the microtubules and other fibres depolymerise. Throughout mitosis the "daughter" centriole at each pole grows until it is full-length. At telophase the duplication of each of the original centrioles is completed, and new daughter centrioles will be generated during the next interphase.
(g) Interphase. Upon the completion of cytokinesis, the cell enters the G, phase of the cell cycle and proceeds again around the cycle.
It will be appreciated that cell cycling is an extremely important cell process. Deviations from normal cell cycling can result in a number of medical disorders. Increased and/or unrestricted cell cycling may result in cancer. Reduced cell cycling may result in degenerative conditions. Use of the compound of the present invention may provide a means to treat such disorders and conditions. Thus, the compound of the present invention may be suitable for use in the treatment of cell cycling disorders such as cancers, including hormone dependent and hormone independent cancers.
In addition, the compound of the present invention may be suitable for the treatment of cancers such as breast cancer, ovarian cancer, endometrial cancer, sarcomas, melanomas, prostate cancer, pancreatic cancer etc. and other solid tumours.
For some applications, cell cycling is inhibited and/or prevented and/or arrested, preferably wherein cell cycling is prevented and/or arrested. In one aspect cell cycling may be inhibited and/or prevented and/or arrested in the G2/M phase. In one aspect cell cycling may be irreversibly prevented and/or inhibited and/or arrested, preferably wherein cell cycling is irreversibly prevented and/or arrested.
By the term "irreversibly prevented and/or inhibited and/or arrested" it is meant after application of a compound of the present invention, on removal of the compound the effects of the compound, namely prevention and/or inhibition and/or arrest of cell cycling, are still observable. More particularly by the term "irreversibly prevented and/or inhibited and/or arrested" it is meant that when assayed in accordance with the cell cycling assay protocol presented herein, cells treated with a compound of interest show less growth after Stage 2 of the protocol 1 than control cells. Details on this protocol are presented below.
Thus, the present invention provides compounds which: cause inhibition of growth of oestrogen receptor positive (ER+) and ER negative (ER-) breast cancer cells in vitro by preventing and/or inhibiting and/or arresting cell cycling; and/or cause regression of nitroso-methyl urea (NMU)-induced mammary tumours in intact animals (i.e. not ovariectomised), and/or prevent and/or inhibit and/or arrest cell cycling in cancer cells; and/or act in vivo by preventing and/or inhibiting and/or arresting cell cycling and/or act as a cell cycling agonist.
CELL CYCLING ASSAY (PROTOCOL 2) Procedure Stage 1 MCF-7 breast cancer cells are seeded into multi-well culture plates at a density of 105 cells/well. Cells were allowed to attach and grown until about 30% confluent when they are treated as follows:
Control - no treatment
Compound of Interest (COI) 20μM
Cells are grown for 6 days in growth medium containing the COI with changes of medium/COI every 3 days. At the end of this period cell numbers were counted using a Coulter cell counter.
Stage 2
After treatment of cells for a 6-day period with the COI cells are re-seeded at a density of 104 cells/well. No further treatments are added. Cells are allowed to continue to grow for a further 6 days in the presence of growth medium. At the end of this period cell numbers are again counted.
CANCER
As indicated, the compounds of the present invention may be useful in the treatment of a cell cycling disorder. A particular cell cycling disorder is cancer.
Cancer remains a major cause of mortality in most Western countries. Cancer therapies developed so far have included blocking the action or synthesis of hormones to inhibit the growth of hormone-dependent tumours. However, more aggressive chemotherapy is currently employed for the treatment of hormone-independent tumours.
Hence, the development of a pharmaceutical for anti-cancer treatment of hormone dependent and/or hormone independent tumours, yet lacking some or all of the side- effects associated with chemotherapy, would represent a major therapeutic advance.
We believe that the compound of the present invention provides a means for the treatment of cancers and, especially, breast cancer.
In addition or in the alternative the compound of the present invention may be useful in the blocking the growth of cancers including leukaemias and solid tumours such as breast, endometrium, prostate, ovary and pancreatic tumours.
OTHER THERAPIES
It is also to be understood that the compound/composition of the present invention may have other important medical implications.
For example, the compound or composition of the present invention may be useful in the treatment of the disorders listed in WO-A-99/52890 - viz:
In addition, or in the alternative, the compound or composition of the present invention may be useful in the treatment of the disorders listed in WO-A-98/05635. For ease of reference, part of that list is now provided: diabetes including Type II diabetes, obesity, cancer, inflammation or inflammatory disease, dermatological disorders, fever, cardiovascular effects, haemorrhage, coagulation and acute phase response, cachexia, anorexia, acute infection, HIV infection, shock states, graft-versus-host reactions, autoimmune disease, reperfusion injury, meningitis, migraine and aspirin-dependent anti-thrombosis; tumour growth, invasion and spread, angiogenesis, metastases, malignant, ascites and malignant pleural effusion; cerebral ischaemia, ischaemic heart disease, osteoarthritis, rheumatoid arthritis, osteoporosis, asthma, multiple sclerosis, neurodegeneration, Alzheimer's disease, atherosclerosis, stroke, vasculitis, Crohn's disease and ulcerative colitis; periodontitis, gingivitis; psoriasis, atopic dermatitis, chronic ulcers, epidermolysis bullosa; corneal ulceration, retinopathy and surgical wound healing; rhinitis, allergic conjunctivitis, eczema, anaphylaxis; restenosis, congestive heart failure, endometriosis, atherosclerosis or endosclerosis.
In addition, or in the alternative, the compound or composition of the present invention may be useful in the treatment of disorders listed in WO-A-98/07859. For ease of reference, part of that list is now provided: cytokine and cell proliferation/differentiation activity; immunosuppressant or immunostimulant activity (e.g. for treating immune deficiency, including infection with human immune deficiency virus; regulation of lymphocyte growth; treating cancer and many autoimmune diseases, and to prevent transplant rejection or induce tumour immunity); regulation of haematopoiesis, e.g. treatment of myeloid or lymphoid diseases; promoting growth of bone, cartilage, tendon, ligament and nerve tissue, e.g. for healing wounds, treatment of burns, ulcers and periodontal disease and neurodegeneration; inhibition or activation of follicle-stimulating hormone (modulation of fertility); chemotactic/chemokinetic activity (e.g. for mobilising specific cell types to sites of injury or infection); haemostatic and thrombolytic activity (e.g. for treating haemophilia and stroke); antiinflammatory activity (for treating e.g. septic shock or Crohn's disease); as antimicrobials; modulators of e.g. metabolism or behaviour; as analgesics; treating specific deficiency disorders; in treatment of e.g. psoriasis, in human or veterinary medicine.
In addition, or in the alternative, the composition of the present invention may be useful in the treatment of disorders listed in WO-A-98/09985. For ease of reference, part of that list is now provided: macrophage inhibitory and/or T cell inhibitory activity and thus, anti-inflammatory activity; anti-immune activity, i.e. inhibitory effects against a cellular and/or humoral immune response, including a response not associated with inflammation; inhibit the ability of macrophages and T cells to adhere to extracellular matrix components and fibronectin, as well as up-regulated fas receptor expression in T cells; inhibit unwanted immune reaction and inflammation including arthritis, including rheumatoid arthritis, inflammation associated with hypersensitivity, allergic reactions, asthma, systemic lupus erythematosus, collagen diseases and other autoimmune diseases, inflammation associated with atherosclerosis, arteriosclerosis, atherosclerotic heart disease, reperfusion injury, cardiac arrest, myocardial infarction, vascular inflammatory disorders, respiratory distress syndrome or other cardiopulmonary diseases, inflammation associated with peptic ulcer, ulcerative colitis and other diseases of the gastrointestinal tract, hepatic fibrosis, liver cirrhosis or other hepatic diseases, thyroiditis or other glandular diseases, glomerulonephritis or other renal and urologic diseases, otitis or other oto-rhino-laryngological diseases, dermatitis or other dermal diseases, periodontal diseases or other dental diseases, orchitis or epididimo-orchitis, infertility, orchidal trauma or other immune-related testicular diseases, placental dysfunction, placental insufficiency, habitual abortion, eclampsia, pre-eclampsia and other immune and/or inflammatory-related gynaecological diseases, posterior uveitis, intermediate uveitis, anterior uveitis, conjunctivitis, chorioretinitis, uveoretinitis, optic neuritis, intraocular inflammation, e.g. retinitis or cystoid macular oedema, sympathetic ophthalmia, scleritis, retinitis pigmentosa, immune and inflammatory components of degenerative fondus disease, inflammatory components of ocular trauma, ocular inflammation caused by infection, proliferative vitreo-retinopathies, acute ischaemic optic neuropathy, excessive scarring, e.g. following glaucoma filtration operation, immune and/or inflammation reaction against ocular implants and other immune and inflammatory-related ophthalmic diseases, inflammation associated with autoimmune diseases or conditions or disorders where, both in the central nervous system (CNS) or in any other organ, immune and/or inflammation suppression would be beneficial, Parkinson's disease, complication and/or side effects from treatment of Parkinson's disease, AIDS-related dementia complex HIV-related encephalopathy, Devic's disease, Sydenham chorea, Alzheimer's disease and other degenerative diseases, conditions or disorders of the CNS, inflammatory components of stokes, post-polio syndrome, immune and inflammatory components of psychiatric disorders, myelitis, encephalitis, subacute sclerosing pan-encephalitis, encephalomyelitis, acute neuropathy, subacute neuropathy, chronic neuropathy, Guillaim-Barre syndrome, Sydenham chora, myasthenia gravis, pseudo-tumour cerebri, Down's Syndrome, Huntington's disease, amyotrophic lateral sclerosis, inflammatory components of CNS compression or CNS trauma or infections of the CNS, inflammatory components of muscular atrophies and dystrophies, and immune and inflammatory related diseases, conditions or disorders of the central and peripheral nervous systems, post-traumatic inflammation, septic shock, infectious diseases, inflammatory complications or side effects of surgery, bone marrow transplantation or other transplantation complications and/or side effects, inflammatory and/or immune complications and side effects of gene therapy, e.g. due to infection with a viral carrier, or inflammation associated with AIDS, to suppress or inhibit a humoral and/or cellular immune response, to treat or ameliorate monocyte or leukocyte proliferative diseases, e.g. leukaemia, by reducing the amount of monocytes or lymphocytes, for the prevention and/or treatment of graft rejection in cases of transplantation of natural or artificial cells, tissue and organs such as cornea, bone marrow, organs, lenses, pacemakers, natural or artificial skin tissue.
As previously mentioned, in one aspect the present invention provides use of a compound as described herein in the manufacture of a medicament for use in the therapy of a condition or disease associated with 11 β-HSD.
Conditions and diseases associated with 11 β-HSD have been reviewed in Walker, E. A,; Stewart, P. M.; Trends in Endocrinology and Metabolism, 2003, 14 (7), 334-339.
In a preferred aspect, the condition or disease is selected from the list consisting of:
• metabolic disorders, such as diabetes and obesity • cardiovascular disorders, such as hypertension
• glaucoma inflammatory disorders, such as arthritis or asthma immune disorders bone disorders, such as osteoporosis cancer intra-uterine growth retardation apparent mineralocorticoid excess syndrome (AME) polycystic ovary syndrome (PCOS) hirsutism acne oligo- or amenorrhea adrenal cortical adenoma and carcinoma
Cushing's syndrome pituitary tumours invasive carcinomas breast cancer; and endometrial cancer.
SUMMARY
In summation, the present invention provides compounds for use as steroid dehydrogenase inhibitors, and pharmaceutical compositions for the same.
BRIEF DESCRIPTION OF THE FIGURES
The present invention will be described in further detail by way of example only with reference to the accompanying figures in which:-
Figure 1 is of graph 1 which shows the amount of protein per μL of rat liver and rat kidney. Figure 2 is of graph 2 which shows the enzyme concentration and time-dependency course, E to F, in rat liver, 11 β-HSD type 1 activity.
Figure 3 is of graph 3 which shows the enzyme concentration and time-dependency course, F to E, in rat kidney, 11 β-HSD type 2 activity.
Figure 4 is a graph showing extraction efficiencies obtained with four extraction methods. Figure 5 is a graph showing a comparison of 11 β-HSD1 activity in rat and human hepatic microsomes.
Figure 6 is a series of graphs showing the effect of incubation time on human microsomal 11β-HSD1 activity. Figure 7 is a series of graphs showing the effect of microsomal protein concentration on human microsomal 11 β-HSD1 activity.
Figure 8 is a graph showing the substrate (cortisone) saturation curve for human hepatic microsomal 11β HSD1.
Figure 9 is a Lineweaver-Burke plot of substrate saturation data for human hepatic microsomal 11 β HSD1.
Figure 10 is a graph showing the IC50 determination for glycyrrhetinic acid.
Figure 11 is a graph showing the IC50 determination for carbenoxolone.
Figures 12(A), 12(B) and 12(C) are graphs showing the 11 β-HSD1 activity measured by Immunoassay. Figure 12(A) shows the effect of protein; Figure 12(B) shows the effect of cortisone; and Figure 12(C) shows the effect of Tween-80.
Figure 13 is a graph showing the evaluation of the Assay Designs Cortisol
Immunoassay.
Figure 14 is a graph showing the effect of increasing microsomal protein on measurement of 11 β HSD1 activity detected by Assay Designs Immunoassay. Figure 15 is a graph showing the detection of 11 β HSD1 activity by RIA using the
Immunotech anti-cortisol antibody.
Figure 16 is a graph showing the effect of lowering the Immunotech antibody concentration on the signal to noise (microsome group compared to GA blank group).
Figure 17 is a graph showing the Immunotech antibody saturation curve for detection of 11 β HSD1 activity by RIA.
Figure 18 is a graph showing the linearity of human hepatic microsomal 11 β HSD1 activity detected by RIA.
Figure 19 is a graph showing the effect of Tween 80 on detection of human hepatic microsomal 11 β HSD1 activity by RIA. Figure 20 is a graph showing the effect of buffer systems on detection of human hepatic microsomal 11 β HDS1 activity by RIA.
Figure 21 is a graph showing the linearity of human hepatic microsomal 11 β HSD1 activity with incubation time detected by RIA.
Figure 22 is a graph showing the substrate saturation curve for human hepatic microsomal 11 β HDS1 activity detected by RIA. Figure 23 is a Lineweaver-Burke plot of substrate saturation data for human hepatic microsomal 11β HDS1 activity detected by RIA.
Figure 24 is a graph showing the DMSO tolerance of human hepatic microsomal 11 β HSD1 activity. Figure 25 is an IC50 curve for inhibition of human hepatic microsomal 11 β HSD1 activity by glycyrrhetinic acid.
EXAMPLES
The present invention will now be described only by way of example.
MATERIALS AND METHODS
Materials
Enzymes - Rat livers and rat kidneys were obtained from normal Wistar rats (Harlan
Olac, Bicester, Oxon,UK). Both the kidneys and livers were homogenised on ice in PBS- sucrose buffer (1g/ 10 ml) using an Ultra-Turrax. After the livers and kidneys were homogenised the homogenate was centrifuged for five minutes at 4000 rpm. The supernatant obtained was removed and stored in glass vials at -20°C. The amount of protein per μl of rat liver and kidney cytosol was determined using the Bradford method
[14].
Apparatus
Incubator: mechanically shaken water bath, SW 20, Germany. Evaporator, Techne Driblock DB 3A, UK
TLC aluminium sheets 20 x 20 cm silica gel 60 F254, Merck, Germany. Scintillation vials: 20 ml polypropylene vials with caps, SARSTEDT, Germany. Scintillation counter: Beckman LS 6000 SC, Beckman Instruments Inc., USA.
Solutions
• Assay medium: PBS-sucrose buffer, Dulbecco's Phosphate Buffered Saline, 1 tablet/100 ml with 0,25 M sucrose, pH 7,4 BDH Laboratory supplies, UK.
• Scintillation fluid: Ecoscint A (National Diagnostics, USA). • Radioactive compound solutions: [1 ,2,6,7-3H]-cortisol (Sp. Ac. 84 Ci/mmol) NEN Germany, [4-1 C]-cortisol (Sp. Ac. 53 mCi/mmol) NEN Germany. • CrO3 and Acetic acid (Sigma Chemical Co., UK).
• Extraction fluid: Di-ethylether, Fischer Chemicals, UK.
• Bradford Reagent solution: Coomassie Brilliant Blue G-250, 100 mg in 95% ethanol with 100 ml of phosphoric acid (85% w/v) diluted to 1 litre.
Compounds
• Inhibitors: compounds were synthesised in accordance with the synthetic routes below .
• Cofactor: NADPH and NADP, Sigma Chemical Co., UK.
Methods
Synthesis of radio labelled cortisone
Labelled cortisol (F) (3H-F and C-F) was oxidised at the C-11 position with CrO3 in order to synthesize to the corresponding labelled cortisone (3H-E and 1 C-E).
For this reaction F was oxidised in a 0,25% CrO3 (w/v) dissolved in a 50% acetic- acid/distilled water (v/v) solution. The labelled F was then added to 1 ml of the CrO3 solution, vortex mixed and put in an incubator for 20 minutes at 37°C. The aqueous reaction mixture was extracted twice with 4 ml of di-ethylether, the di-ethylether was then evaporated and the residue transferred to a TLC-plate, which was developed in the following system, chloroform : methanol 9:1 (v/v). Unlabelled cortisone (E) was also run on the TLC-plate to locate the position of the labelled steroids. After locating the spot of the labelled steroids this area is cut out from the TLC-plate and eluted with 0,5 ml of methanol.
The amount of protein per μL of rat liver and rat kidney
The amount of protein in rat liver and rat kidney needed to be determined. The experiment was done according to the Bradford method [14]. The following method was used: first a BSA (protein) solution was prepared (1 mg/ml). Protein solutions containing 10 to 100 μg protein were pipetted into tubes and volumes adjusted with distilled water. Then 5 ml of protein reagent was added to the tubes and vortex mixed. The absorbance was measured at 595 nm after 15 minutes and before 1 hour in 3 ml cuvettes against a reagent blank. The weight of the protein was plotted against the corresponding absorbance resulting in a standard curve used to determine the protein concentration in rat liver and rat kidney cytosols.
Assay validation - Enzyme concentration and time-dependency of 11 β-HSD activity
Before carrying out 11 β-HSD assays to examine the conversion E to F and F to E and the influence that different inhibitors have on these conversions the amount of rat liver homogenate and rat kidney homogenate and their incubation time need to be determined.
11 β-HSD type 1 is the enzyme responsible for the conversion E to F and this type of enzyme is present in rat liver. The substrate solution used in this assay contained 70,000 cpm/ml 3H-E in PBS-sucrose and 0.5 μM of unlabelled E and co-factor NADPH (9 mg/10 ml of substrate solution). 1 ml of the substrate solution and the different amounts of rat liver homogenate was added to all tubes.
The amount of rat liver homogenate needed for an assay was determined by incubating the substrate solution with 25, 50, 100 and 150 μl for 30, 60, 90 and 120 minutes at 37°C in a water bath with the tubes being mechanically shaken. After the incubation 50 μL of recovery solution was added, containing about 8,000 cpm/ 50 μL of 14C-F and 50 μg/50 μL of unlabelled F for visualising the spot on the TLC-plate, to correct for the losses made in the next two steps. F was then extracted from the aqueous phase with 4 ml of ether (2 x 30 sec cycle, vortex mix). The aqueous phase was then frozen using dry-ice and the organic layer was decanted and poured into smaller tubes and evaporated. 6 drops of ether were then added to the small tubes to re-dissolve the residue which was transferred to an aluminium thin layer chromatography plate (TLC-plate). The TLC-plate was developed in a TLC tank under saturated conditions. The solvent system used was chloroform : methanol 9:1 (v/v). The F spots on the TLC-plate were visualised under UV- light and cut out from the TLC-plate (Rf=0.45). The spots from the TLC- plate were then put into scintillation vials and 0.5 ml of methanol was added to all vials to elute the radioactivity from the TLC-plate for 5 minutes. 10 ml of Ecoscint was added to the scintillation vials and they were put into the scintillation counter to count amount of product formed. The same procedure was used for the 11 β-HSD type 2 assay, the conversion F to E, to determine the amount of rat kidney to be used and the incubation time. Except this time the substrate solution contained 3H-F and unlabelled F and the recovery contained 14C-E and unlabelled E and cortisone has a Rf value of 0.65 on the TLC-plate.
Assay procedure - The 11 β-HSD inhibitors
In these assays the influence of different inhibitors on the 11 β-HSD activity both in reductive (type 1 ) and oxidative (type 2) directions were assessed. In the reductive direction E is the substrate and F the product and visa versa in the case of oxidation. The method described here is for the oxidative direction.
The substrate solution contained about 50,000 cpm/ml 3H-F in PBS-sucrose and 0.5 μM F. 1ml of the substrate solution was added to each tube, the inhibitors were also added, at a 10 μM concentration, to each tube except to the "control" and "blank" tubes. 150 μL was added to all tubes except to the blanks, this was done to correct for the amount of 3H-F spontaneously formed. The tubes were incubated for 60 minutes in a mechanically shaken water bath at 37°C. The amount of kidney liver homogenate and incubation time used resulted from the enzyme- and time-dependency assay. After incubation 50 μL of recovery was added to correct for the losses made in the next steps, containing 5000 cpm/50 μL of 14C-E and 50μg/50 μL of unlabelled E (to visualise the spot on the TLC- plate). The aqueous mixture was then extracted with 4 ml of ether (2 x 30 sec cycle, vortex mix). After freezing the aqueous phase, the ether (upper) layer was decanted into smaller tubes and evaporated at 45°C until completely dry. The residue was then re- dissolved in 6 drops of ether and transferred to a TLC-plate. The TLC-plate was developed in chloroform : methanol (9:1 v/v) solvent system, the TLC-plate ran for about 90 minutes until the solvent front had moved about 18 cm. The position of the product E was visualised under UV-light and cut out from the TLC-plate and put into scintillation vials. Radioactivity was eluted over 5 minutes with 0.5 ml methanol. 0.5 ml of PBS- sucrose and 10 ml of Ecoscint were then added and vortex mixed before counting in the scintillation counter. Before counting the samples, two total activity vials were prepared. These contained 0.5 ml of the substrate solution, 50 μL of the recovery, 0.5 ml of methanol and 10 ml of Ecoscint. These two total activity vials were needed to determine the amount of 14C-E and 3H-F added in the beginning to make the calculations. In case of the reductive direction, E to F ,the same method was used. Only the substrate solution containing 3H-E and unlabelled E and the recovery containing 14C-F and unlabelled F are different to the method used in the oxidative direction.
After testing all the inhibitors at 10 μM a dose-response experiment was done for the most potent 11 β-HSD type 1 and type 2 inhibitors. To look at the percentage of inhibition four different concentrations, 1, 5, 10 and 20 μM, were used. The method for both the rat liver, type 1 the reductive, and rat kidney, type 2 the oxidative, stay the same throughout the entire experiment.
RESULTS
The amount of protein per μL of rat liver and rat kidney
An initial experiment was carried out to determine the amount of protein in rat liver cytosol and rat kidney cytosol, to be added to each tube. Graph 1 in figure 1 shows the standard curve from which the amount of protein used in both experiments was calculated. The amount of protein added to each tube in the rat liver experiment was 75.5 μg (per 25 μL). In the rat kidney experiment the amount of protein added to each tube was 135.6 μg (per 150μL).
Enzyme concentration and time-dependency of 11 β-HSD activity
In this experiment the amount of rat liver homogenate and rat kidney homogenate added to each tube and the incubation time was determined. Graph 2 in figure 2 shows the enzyme concentration and time-dependency course of the rat liver experiment E to F, 11 β-HSD type 1 activity. Graph 3 in figure 3 shows the enzyme concentration and time- dependency course F to E, 11 β-HSD type 2 activity. After drawing the graphs the optimal amount of rat liver cytosol and rat kidney cytosol and both their incubation times were selected. One important rule when selecting both variables, to select an amount of rat liver and rat kidney and incubation time on a linear part of the graph. This is done to avoid fluctuations in enzyme activity. The amount of rat liver cytosol selected was 25 μL and 90 minutes of incubation time, the amount of rat kidney cytosol selected was 150 μL and 60 minutes of incubation time. The 11 β-HSD inhibitors
In this experiment the influence of different inhibitors on the conversion E to F and F to E was determined. The reason why inhibition in both directions was examined was to make a comparison between the inhibitors and which type of 11 β-HSD they inhibit more. Compounds were screened for their ability to inhibit 11 β-HSD type 1 (E to F) and type 2 (F to E). All the inhibitors were initially tested at a 10 μM concentration. The percent of inhibition was calculated as the percentage of decrease in radio labelled 3H-E and 3H-F of product formed, compared with the control activity (the tubes without an inhibitor in it). All the results calculated are means, n=2.
Table 2: Inhibitory Effect
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
BIOLOGICAL ASSAY DEVELOPMENT USING HUMAN 11 β-HYDROXYSTEROID DEHYDROGENASE TYPE 1.
Standard Operating Procedure for the 11 β-Hydroxysteroid Dehydrogenase Type 1 cortisol Radioimmunoassay.
11β HSD1 cortisol RIA
Reagents: Cortisone, Cortisol (Hydrocortisone), NADPH, Glucose-6-phosphate, Glycyrrhetinic acid (GA), Dextran coated charcoal (C6197) and DMSO were obtained from Sigma Aldrich, Carbenoxolone was obtained from ICN Biomedicals, Product 215493001 , 3H-cortisone was obtained from American Radiolabelled Componds Inc, Product ART-743, 3H-cortisol was obtained from NEN, Product NET 396, 14C-cortisol was obtained from NEN, Product NEC 163, human hepatic microsomes were obtained from XenoTech, product H0610 / Lot 0210078, rat hepatic microsomes were obtained from XenoTech, SPA beads were obtained from Amersham, Product RPNQ0017, the Immunoassay kit was obtained from Assay Designs, Product 900-071 , the Immunologicals Direct anti-cortisol antibody was Product OBT 0646, the Sigma anti- cortisol antibody was Product C8409 and the Immunotech antibody was supplied by Beckman, Product IMBULK3 6D6.
Buffer Solutions
Buffer 1 , from Barf [15]: 30 mM Tris-HCL, pH 7.2, containing 1 mM EDTA Buffer 2, from the Sterix protocol: PBS (pH 7.4) containing 0.25M sucrose Buffer 3, from the Sigma RIA protocol: 50 mM Tris-HCL, pH 8, containing 0.1 M NaCL and 0.1 % gelatin Stop solution, from Barf [15]: 1 mM glycyrrhetinic acid in 100 % DMSO
Enzyme assays were carried out in the presence of 181 μM NADPH, 1 mM Glucose-6- Phosphate and cortisone concentrations indicated for each experiment. Enzyme assay buffer: 30 mM Tris-HCL, pH 7.2 containing 1 mM EDTA Antibody binding buffer: 50 mM Tris-HCL, pH 8, containing 0.1 M NaCl and 0.1 % gelatin
Compound preparation: Prepare 10 mM stock solutions in 100% DMSO at 100 times the required assay concentration. Dilute into assay buffer 1 in 25. Also dilute neat DMSO 1 in 25 into assay buffer for controls.
Substrate preparation: Prepare a solution of cortisone in ethanol 600 times the required assay concentration (175 nM). Dilute this 1 in 50 into assay buffer. Prepare NADPH as a 1.8 mg/ml solution in assay buffer. Prepare G-6-P as a 3.65 mg/ml solution in assay buffer.
Mix these 3 solutions 1 :1 :1 to make a solution of sufficient volume for 25 μl additions to each sample. Add 0.5 μCi tritiated cortisone per 25 μl and mix the solution well.
Microsome preparation: Dilute stock 20 mg/ml solution 1 in 100 with assay buffer. Antibody preparation: Dilute stock antibody solution to 17 μg/ml in antibody binding buffer.
Dextran coated charcoal preparation: Make a 20 mg/ml solution in antibody binding buffer and chill on ice.
Enzyme assay: To a u-bottom polypropylene 96 well plate add:
25 μl compound dilution or diluted DMSO to controls, NSB's and blanks
10 μl 1 mM GA in DMSO (enzyme stop solution) to blanks 25 μl substrate mixture to all samples
50 μl diluted microsomes to all samples
Incubate plate for 30 min at 37°C shaking
Add 10 μl enzyme stop solution to all wells except the blanks
Add 100 μl antibody solution to all wells except the NSB's, add antibody binding buffer to these wells
Incubate at 37°C for 1 h
Chill plate on ice for 15 min
Add 50 μl / well charcoal solution and mix with an 8-channel pipette (4 - 5 aspirations)
Chill the plate on ice Centrifuge at 4°C, 2000 x g for 15 min
Transfer 100 μl supernatant into an Optiplate, also add 25 μl substrate mixture to 2 empty wells to indicate counting efficiency
Add 200 μl Microscint-40 to all wells and count on a Topcount
Radioimmunoassay
The 11 β HSD1 enzyme assay was carried out following the standard operating procedure described above in u-bottom polypropylene 96 well plates or 1.5 ml Eppendorf tubes as indicated for each experiment. Subsequent to stopping the enzyme reaction, 100 μl antibody prepared in buffer 3 unless otherwise indicated was added to test samples and 100 μl buffer 3 was added to the NSB samples. The samples were incubated for 1 hour at 37°C and the chilled on ice for 15 mins. Dextran coated charcoal (50 μl / sample) prepared to the indicated concentration in buffer 3 was added and the samples were mixed (vortex for tubes and aspiration 5 times with an 8-channel pipette for 96 well plates) and chilled for a further 10 min. The samples were centrifuged at 2000 x g for 15 min at 4°C to pellet the charcoal. Aliquots of the supernatant (100 μl) were transferred to an Optiplate and counted on the Topcount in 150-200 μl Microscint 40. In some experiments, aliquots of supernatant were transferred to scintillation vials and counted on the Tricarb LSC in 5 ml Ultima Gold scintillant.
11 β HSD1 ASSAY DEVELOPMENT
11β HSD1 TLC format assay
Separation of Cortisone and Cortisol
Prior to performing an enzyme assay, solvent systems reported in the literature for separation of cortisone from cortisol were investigated [16, 17]. Solutions of cortisone and cortisol at 10 mg/ml were prepared in methanol, and aliquots spotted onto a silica gel TLC plate. The plate was run in CH2CI2: IMS 92 : 8 7V (2). The plate was then air dried and sprayed with 0.1% Rhodamine B in methanol to visualise the spots. The table below describes the separation obtained.
Table 3: Separation of cortisone from cortisol by TLC
Figure imgf000059_0001
This separation was considered adequate for use in an enzyme assay.
The literature details several methods of extracting cortisol from aqueous solution [16, 17]. In order to select a method for use, [1 C]-labelled cortisol was obtained from NEN. A stock was prepared in phosphate buffered saline (PBS) containing 4000 DPM in 50μl with cold cortisol (1μg) added as a carrier. The final ethanol concentration was 0.4%. Aliquots of this solution were added to glass tubes (100μl) and the following extractions were carried out: 1. 1 ml CH2CI2t vortex and pass through phase separating filter paper (Whatman, IPS) 2. 1ml ethyl acetate, vortex and pass through phase separating filter paper 3. 1 ml CH2CI2 and 200μl 0.05% CaCI2, vortex, centrifuge (500g for 5 min) and remove upper aqueous phase 4. 1 ml ethyl acetate and 200μl 0.05 % CaCI2, vortex, centrifuge (500g for 5 min) and collect upper organic phase. The organic phases were dried and the residues were taken up in 100μl IMS. An aliquot of this was spotted onto a TLC plate and the plate run as before. Following visualisation with Rhodamine B, the spots were scraped into scintillation vials and counted on a liquid scintillation counter (Packard TriCarb) in 5ml Ultima gold scintillant. Extraction efficiencies were calculated and are given in Figure 4.
From these results it appears that 90 % of the cortisol is lost by phase separating filtration. Ethyl acetate appears to extract cortisol more efficiently than CH2CI2, possibly because the organic phase is easier to collect. Ethyl acetate appears to be a suitable method of extraction.
Human and Rat Hepatic Microsomal 11β-HSD1 Activity.
11 β-HSD1 activity in rat and human hepatic microsomes was evaluated, to determine the minimum microsomal protein concentrations required for measurement of enzyme activity. The experiment was done according to the Bradford Method [14]. The assay was performed in Buffer 2 and the cortisone concentration used was 2μM containing O.δμCi [3H]-cortisone per incubation. Microsomes were tested at concentrations ranging from 50μg to 400μg protein per incubation in a final incubation volume of 100μl in glass tubes. Samples were incubated for 1 h in a shaking water bath at 37°C and the assay was stopped by addition of 1ml ethyl acetate. To correct for recovery, 50μl [1 C]-cortisol was added to the samples followed by 200μl 0.05 % CaCI2. The samples were vortex mixed and centrifuged as described above. The upper organic phase was removed and dried down, and the residue dissolved in 100μl methanol and 50μl aliquots were spotted onto TLC plates, which were run as described above. Samples were counted on a TriCarb liquid scintillation counter using a dual label programme. Recovery efficiency was determined from the DPM obtained in 50μl [14C]-cortisol solution, which was counted with the samples. Results are shown in Figure 5.
The 11 β-HSD1 activities in rat and human microsomes were similar, 0.7pmol/mg/min and 0.5pmol/mg/min for rat and human microsomes respectively. The activity in human microsomes is apparently not related to microsomal protein concentration, which may suggest that that the protein concentration range examined is too high.
Lower human microsome protein concentrations were evaluated; 3.7μg to 100 μg per sample. The time course of activity was also determined, from 0 to 60 minutes at 37°C. The extraction conditions were as described above. The results from these experiments are shown in figures 6 and 7.
The results shown in figures 6 and 7 demonstrate that enzyme activity is linear at incubation times up to 30 min at all the microsomal protein concentrations tested, and that enzyme activity is linear at microsomal protein concentrations below 30μg per sample.
The influence of substrate concentration on activity was examined. The [3H]-cortisone concentration was kept constant at O.δμCi/sample, and unlabelled cortisone varied from 44nM to 2μM. The assay was carried out with 10μg microsomal protein per sample with an incubation time of 30 minutes at 37°C. The results are shown in figure 8. A double reciprocal plot (Lineweaver-Burke) of these data gives an apparent Km for cortisone of 660nM, figure 9.
The standard compounds glycyrrhetinic acid and carbenoxolone were examined in this assay system, as part of the validation process. The assay was performed using 175nM cortisone substrate, with 10μg microsomal protein and a 30 minute incubation at 37°C, as described by Barf [15]. Although the data in figures 8 and 9 above suggest that this substrate concentration is not saturating under these assay conditions. Glycyrrhetinic acid and carbenoxolone were tested at concentrations from 0.012μM to 3μM , the DMSO concentration was 1% in all samples. The results are shown in figures 10 and 11.
Glycyrrhetinic acid and carbenoxolone give IC50 values of 40nM and 119nM respectively. The IC50 reported for carbenoxolone by Barf et al. using the SPA format and recombinant 11 β-HSD is 330nM [15], approximately three-fold less potent. The difference in potency in the two assay systems is probably due to the different assay conditions, SPA compared to tic end point, and also the enzyme source, native hepatic enzyme compared to recombinant enzyme. The assay conditions described above support good enzyme activity however, which should be transferable to a 96 well plate format.
Development of High Throughput 11β HSD1 Assays
Supply of the antibody used by Barf [15] in the Scintillation Proximity assay (SPA) proved problematic. A sample batch of the antibody (from Immunotech) was tested for suitability and a second order was placed for a larger quantity. A robust 96 well plate assay using Radioimmunoassay (RIA) format was developed using the Immunotech antibody available, this is described below.
Immunoassay Format
An Assay Designs enzyme immunoassay system was evaluated as a potential assay format. The basis of the assay is competition for antibody binding between sample cortisol, generated by 11 β-HSD1 , and labelled cortisol binding. The anti-cortisol detection antibody provided in the kit is a mouse monoclonal, reported to cross react less than 0.1% with cortisone. The kit is designed for the analysis of cortisol levels in saliva, urine, serum and plasma and also in tissue culture media, rather than for determining enzyme activity however.
The 11 β-HSD1 enzyme assay conditions described by Barf et al [15] were used; human hepatic microsomes in Buffer 1 at protein concentrations from 25μg to 200μg, cortisone at concentrations from 44nM to 700nM incubated for 60 minutes at 37°C. The effect of 0.9% Tween 80 was also investigated, as this detergent is reported to improve the activity of enzymes involved in steroid metabolism. Results are shown in Figure 12.
Figure 12(A) shows the effect of protein. Data taken from the 700μM cortisone group tested in the presence of Tween-80.
Figure 12(B) shows the effect of cortisone. Data taken from the 25μg microsomal protein group tested in the presence of Tween-80.
Figure 12(C) shows the effect of Tween-80. Data taken from the 25μg microsomal protein group tested in the presence of 700 μM cortisone. The assay detected cortisol in the standard curve (313 pg/ml to 10,000 pg/ml) as expected but the signal obtained from the enzyme assay samples decreased with increasing microsomal protein concentration, suggesting that the microsomal protein may interfere with the immunoassay, figure 12(A). Addition of exogenous cortisone had no effect on levels of cortisol detected in the enzyme assay samples, suggesting the antibody does not cross react with cortisone, figure 12(B). Inclusion of detergent in the enzyme assay buffer had little effect, figure 12(C).
The assay conditions were varied to determine if it was feasible to use the immunoassay system to detect 11 β-HSD1 activity; 24μg microsomal protein per sample and 2μM cortisone substrate in Buffer 2. Enzyme activity was also measured in samples following the addition of steroid displacement reagent; a kit component which releases cortisol from cortisol binding protein, if present in the sample. The assay detected the cortisol in the standard curve (313pg/ml to 10,000pg/ml). Figure 13 shows the absorbance at 405m obtained for the different groups:
The lowest and highest concentrations of the cortisol standard have been included in Figure 13 as 313 pg/ml and 1000 pg/ml together with the NSB absorbance to show the dynamic range obtained in the assay.
Absorbance obtained in the presence of reaction mixture taken from samples incubated with microsomal protein ("Enzyme") are lower than those in the presence of reaction mixture not containing microsomal protein ("No enzyme") indicating increases in levels of cortisol.
In the presence of the kit steroid displacement reagent ("DR") these two reaction mixtures show the same pattern but the signal is depressed.
Glycyrrhetinic acid (GA) in the presence of the top concentration of cortisol standard has no effect on the ability of the kit to measure cortisol concentrations.
Although the signal to background ratio of 2.5 for the assay is rather poor, these data demonstrate that the antibody can bind the cortiso AP conjugate and that this can be displaced by cortisol. An experiment was carried out to examine the effect of increasing microsomal protein concentration, in an attempt to improve the signal to noise obtained. Microsomal protein was tested from 100μg/incubation down to 5μg/incubation using 2μM cortisone in Buffer 2. All other conditions were identical to those detailed above. The results are shown in figure 14.
Decreasing microsomal protein from 10μg/incubation to 5μg/incubation results in a corresponding decrease in enzyme activity. Increasing microsomal protein above 10μg/incubation results in a quenching of signal which may be due to the colour of the microsomes. Therefore the dynamic range of this assay cannot be improved by increasing the microsomal protein concentration.
RIA development using Immunotech Antibody
The 11 β HSD1 assay was carried out using 10μg/well human hepatic microsomal protein. The Immunotech antibody was used in the RIA at concentrations from 6.25μg/well to 25μg/well, the results are shown in figure 15.
The Immunotech antibody worked well in the assay and gave good signal to background at all the concentrations tested. The signal to noise with 12.5 and 6.1 μg antibody per well was similar suggesting it may be possible to reduce the antibody concentration.
The antibody titre, at concentrations from 0.67μg/well to 6.7μg/well, was examined. The 11β HSD1 assay was carried out using human microsomal protein at 20μg/well, to generate the optimum signal to background. Each antibody concentration was tested against a "no enzyme" blank (buffer substituted for microsomes), a "GA blank" (10 μl stop solution added prior to microsomes) and a control group. The results are shown in figures 16 and 17.
The saturation curve indicates that there is no difference in the detection of enzyme activity above 1.68 μg/well. The signal to background ratio with this antibody concentration is good, (6 fold).Consequently the antibody will be used at 1.7 μg/well in future assays.
Linearity of enzyme activity with human hepatic microsomal protein concentration using
RIA detection was examined. The 11 β HSD1 assay was carried with microsomal protein concentrations varying from 1 μg/well to 40 μg/well. 11 β HSD1 activity was linear with protein up to concentrations of 20 μg/well, figure 18, confirming the results obtained with the classical enzyme assay (figure 7).
The optimal concentration of human microsomal protein to use in the assay appears to be 10μg/well.
The effect of including Tween 80 in the enzyme assay buffer was also investigated. This assay was carried out in parallel with the assay above and under the same conditions except that the enzyme assay buffer (Buffer 2) contained 0.05 % Tween 80. Microsomal protein was tested at four concentrations. Tween 80 was found to increase the blank CPM, reducing the signal to noise of the assay. Representative data, from the group tested 10μg/well microsomal protein, are shown in figure 19. Similar results were obtained with all the microsome protein concentrations examined, consequently Tween will not be used in future studies..
To simplify the protocol such that both enzyme assay and RIA stages are carried out in the same buffer, both phases were carried out in either enzyme assay buffer (Buffer 2) or Buffer 3 (RIA buffer). The microsomal protein concentration used was 10 μg/well and the cortisone concentration was 175 nM. Performing both enzyme assay and RIA in Buffer 3 appears to improve the data slightly, figure 20.
Linearity of enzyme activity with incubation time was investigated. The enzyme assay was carried out with 10μg/well microsomal protein and with 175nM cortisone, and stopped at varying time points, the results are shown in figure 21.
With microsome protein concentrations of 10μg/well and 175nM substrate, the reaction is linear at time points up to 30 minutes. These results indicate that a substrate concentration of 175nM is too low. The apparent Km observed in the classical 11 β HSD1 assay was 660 nM (figures 8 and 9), although these assays are end-point measurement, hence it is not certain that initial rates were measured in the low substrate groups with a 30 minute incubation time. However, published Km values for cortisone in human hepatic microsomal 11β HSD1 assays are in the micromolar range [18, 19]. Although 175nM substrate is well below the apparent Km, it may not be possible to increase the concentration significantly for two reasons: (i) If the compounds are competitive with cortisone, the measured inhibition will fall if the substrate is increased above the concentration used in Reference 1.
(ii) Increasing the substrate concentration will reduce the specific activity of the label, reducing the sensitivity of the assay. This could be overcome by adding higher concentrations of [3H]-cortisone, but the protocol uses 0.5μCi/well and there is a cost implication if higher levels of radioactivity are used.
Substrate saturation was examined. The enzyme assay was carried out exactly described in the methods section, in Buffer 3 with 10μg/well microsomal protein and with [cold cortisone] as indicated. [3H]-cortisone was O.δμCi/sample throughout. The reaction was stopped after 30 min by the addition of 10μl stop solution. The RIA was carried out exactly as indicated in the methods section. The results are shown in figures 22 and 23.
The apparent Km (700 nM), determined from the Lineweaver-Burke plot of these data shown in figure 23 is very similar to that determined in the tic format 11 β HSD1 assay (Figure 9, apparent Km ~660 nM). The data suggests that at 10μg microsomal protein, the enzyme is not saturated at 175nM cortisone, over an incubation period of 30 minutes.
Lowering the microsomal protein concentration or the incubation time to bring the reaction within the linear range would partly overcome the problem. However either of these adjustment adjustments would decrease the assay sensitivity, and decrease the apparent potency of inhibitors. Consequently the initial experiments were performed with 175nM cortisone.
11B-HSP1 Assay Validation
Prior to compound testing, the tolerance of the enzyme assay to DMSO was determined. Inclusion of DMSO at 1 % in the enzyme assay does not affect total or blank values, but slightly increases enzyme activity and the signal to noise ratio (Table 4). The experiment was repeated over a range of DMSO concentrations from 0.3 to 10%, figure 24. Table 4:Control and blank CPM obtained in the Glycyrrhetinic acid IC50 assay showing effect of 1% DMSO and signal to noise ratio obtained.
Figure imgf000067_0002
There is a slight increase in microsomal enzyme activity in the presence of 0.3% and 1 % DMSO. At DMSO concentrations above 1%, there is a linear reduction in enzyme activity. It is reported that DMSO can both increase and reduce microsomal enzyme activity, depending on the concentration, presumably due to effects on the microsomal membranes. On the basis of these data, it is intended that compounds be screened in the presence of 1 % DMSO.
An IC50 value was generated for the standard inhibitor glycyrrhetinic acid, the compound was tested at concentrations between 0.012μM and 3μM, with a final DMSO concentration of 1 %, figure 25.
Glycyrrhetinic acid gives a concentration-related inhibition of the enzyme with an IC50 of 41 nM, with good curve fit values (r2 = 0.962) and Hillslope. This is similar to the value of 40nM generated using the tic format assay, (see figure 10). An IC50 value of 30nM has been reported for glycyrrhetinic acid inhibition of 11 β HSD1 in human hepatic microsomes, using dehydro-dexamethasone as the substrate [19]. However, these values are lower than the value reported by Barf et al. [15].
Table 5: Inhibition Data
Figure imgf000067_0001
Figure imgf000068_0001
STX No. Structure % inhibition of Human 11β HSD1 @ 10 μM typical sd ± 5% N=2
469 66
Figure imgf000069_0001
1018 66
Figure imgf000069_0002
986 65
Figure imgf000069_0003
1020 64
Figure imgf000069_0004
996 63
Figure imgf000069_0005
STX No. Structure % inhibition of Human 11β HSD1 @ 10 μM typical sd ± 5% N=2
987 62
Figure imgf000070_0001
1030 61
Figure imgf000070_0002
523 61
Figure imgf000070_0003
992 61
Figure imgf000070_0004
985 61
Figure imgf000070_0005
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000078_0001
Figure imgf000079_0001
SULPHONAMIDE SYNTHESIS
Method A
To the amine (1 eq.) dissolved in pyridine (3 eq.) was added the corresponding sulphonyl chloride (1.2 eq.) and the reaction mixture was stirred at RT under N2 overnight. The resulting mixture was poured into aq. HCI and the organic layer was extracted with ethyl acetate, dried (MgSO4), filtered and concentrated under reduced pressure to give the desired sulphonamide as crystalline solid or as a thick syrup. The crude compound was then purified by flash chromatography using EtOAc/hexane (3:2) or CH2CI2/EtOAc (4:1) as eluent to give crystalline solid.
Method B
To the amine (1 eq.) dissolved in Et3N (5 eq.) was added the corresponding sulphonyl chloride (1.2 eq.) and the reaction mixture was stirred at RT under N2 overnight. The resulting mixture was poured into water and the organic layer was extracted with ethyl acetate, dried (MgSO4), filtered and concentrated under reduced pressure to give the desired sulphonamide as crystalline solid or as a thick syrup. The crude compound was then purified by flash chromatography using EtOAc/hexane (3:2) or CH2CI2/EtOAc (4:1) as eluent to give crystalline solid. Note : Insoluble amines and sulphonyl chlorides were dissolved in minimum amount of CH2CI2, THF or DMF.
Method C To a solution Arylsulphonyl chloride (1.1 eq.) in DCM were added Pyridine (2.2 eq.) and catalytic amount of DMAP. The solution was stirred at room temperature under nitrogen for 10 minutes. Then the amine (1 eq.) was added and the reaction mixture was stirred at room temperature under nitrogen for 4-16 hrs. The resulting mixture was partitioned between DCM and 5% sodium bicarbonate. The organic layer was washed with brine, dried over MgSO4, and concentrated to give a solid or a thick syrup. The crude compound was then purified by flash chromatography to give desired arylsulphonamide as crystalline solid.
DGS03020A (STX412) Synthesised by method A. Off-white crystals of DGS03020A (186 mg; 55%). mp 189- 190 °C; TLC Rf : 0.68 EtOAc/Hexane (3:2); 1H NMR (CDCI3) δ 2.80 (s, 3H, CH3), 7.13 (s, 1 H, N-H, exchanged with D2O), 7.212 (dd,1 H, Ar-H, J = 2.34 Hz and 8.59 Hz), 7.27 (dd, 1H, Ar-H, J = 1.95 Hz and 8.59 Hz), 7.51 (d, 1H, Ar-H, J = 1.95 Hz), 7.65 (d, 1H, Ar-H, J = 1.95 Hz), 7.69 (d, 1 H, Ar-H, J = 8.59 Hz), 7.91 (d, 1H, Ar-H, J = 8.59 Hz); MS (FAB+) 372.9 [100, (M+H)+]; HRMS m/z (FAB+) 372.9627, C14H10 35CI2N2O2S2 requires 372.9639, 376.9574, C14H10 37CI2N2O2S2 requires 376.9580; HPLC tr 3.65 min (92 : 08 = MeOH : H2O).
DGS03022A (STX413) Synthesised by method A. Off-white crystals of DGS03022A (233 mg; 72%). mp 178 °C; TLC Rf : 0.71 EtOAc/Hexane (3:2); 1H NMR (CDCI3) δ 2.75 (s, 3H, CH3), 2.80 (s, 3H, CH3), 6.75 (s, 1 H, N-H, exchanged with D2O), 7.11 (dd, 1 H, Ar-H, J = 1.95 Hz and 8.59 Hz), 7.17 - 7.21 (m, 1H, Ar-H), 7.53 (d, 1H, Ar-H, J = 1.17 Hz), 7.55 (d, 1H, Ar-H, J = 1.95 Hz), 7.68 (d, 1 H, Ar-H, J = 8.20 Hz), 7.92 (dd, 1H, Ar-H, J = 1.17 Hz and 7.81 Hz); MS (FAB+) 164.1 [35, (5-Amino-2-methyl benzothiazole)+], 353.0 [100, (M+H)+]; HRMS m/z (FAB+) 353.0176, C15H14 35CIN2O2S2 requires 353.0185, 355.0155, C15H14 37CIN2O2S2 requires 355.0156; HPLC tr 3.78 min (92 : 08 = MeOH : H2O).
DGS03024A (STX421) Synthesised by method A. White crystals of DGS03024A (240 mg; 76%). mp 133-134 °C; TLC Rf : 0.7 EtOAc/Hexane (3:2); Η NMR (CDCI3) δ 0.90 (t, 3H, CH3CH2CH2, J = 7.42 Hz), 1.56 - 1.66 (m, 2H, CH3CH2CH2), 2.59 (t, 2H, CH3CH2CH2, J = 7.42 Hz), 2.80 (s, 3H, CH3), 6.71 (s, 1H, N-H, exchanged with D2O), 7.17 (d 1H, Ar-H, J = 2.34 Hz and 8.59 Hz), 7.201 - 7.214 (m, 1H, Ar-H), 7.218- 7.223 (m, 1H, Ar-H), 7.57 (d, 1H, Ar-H, J - 2.34 Hz), 7.76-7.69 (m, 3H, Ar-H); MS (FAB+) 347.1 [100, (M+H)+]; HRMS m/z (FAB+) 347.0881, C17H19N2O2S2 requires 347.0887; HPLC tr 3.69 min (92 : 08 = MeOH : H2O).
DGS03034A (STX424)
Synthesised by method A. White crystals of DGS03034A (262 mg; 86%). mp 152 °C;
TLC Rf : 0.48 EtOAc/Hexane (3:2); 1H NMR (CDCI3) δ 10.31 (s, 1H, NH, Ex. With D2O), 7.85 (d, 1 H, Ar-H, J = 8.59 Hz), 7.66 - 7.69 (m, 2H, Ar-H), 7.57 (d, 1H, Ar-H, J = 1.95
Hz), 7.11 (dd, 1H, Ar-H, J = 2.34 Hz and 8.59 Hz), 7.02 - 7.05 (m, 2H, Ar-H), 3.76 (s,
3H, OCH3), 2.73 (s, 3H, CH3); MS (FAB+) 164.0[25 (Amine SM+)], 335.0 [100, (M+H)+];
HRMS m/z (FAB+) 335.0519, C15H15N2O3S2 requires 335.0524; HPLC tr 1.94 min (80 :
20 = MeOH : H2O).
DGS03036A (STX425)
Synthesised by method A. White crystals of DGS03036A (136 mg; 42%). mp 295-296
°C; TLC Rf : 0.56 EtOAc/Hexane (3:2); 1H NMR (DMSO-d6) δ 10.66 (s, 1H, NH, Ex. With
D2O), 7.87 (d, 1H, Ar-H, J = 8.59 Hz), 7.52 (d, 1H, Ar-H, J = 1.95 Hz), 7.32 - 7.44 (m, 3H, Ar-H), 7.12 (dd, 1H, Ar-H, J = 2.3 Hz and 8.59 Hz), 2.73 (s, 3H, CH3), 2.64 (s, 3H,
CH3); MS (FAB+) 164.0 [40, (Starting amine)+], 353.0 [100, (M+H)+]; HRMS m/z (FAB+)
353.0187, C15H14 35CIN2O2S2 requires 353.0185, 355.0165, C15H14 37CIN2O2S2 requires
355.0155; HPLC tr 1.94 min (80 : 20 = MeOH : H2O).
DGS03058A (STX519)
Synthesised by method A. White crystals of DGS03058A (199 mg; 57%). mp 172 °C; TLC Rf : 0.56 EtOAc/Hexane (3:2); 1H NMR (DMSO-d6) δ 10.53 (s, 1H, NH, Ex. With D2O), 7.88 (d, 1 H, Ar-H, J = 8.59 Hz), 7.74 - 7.77 (m, 2H, Ar-H), 7.64 - 7.68 (m, 2H, Ar- H), 7.58 (d, 1H, Ar-H, J = 1.95 Hz), 7.11 (dd, 1 H, Ar-H, J = 1.95 Hz and 8.59 Hz), 2.74 (s, 3H, CH3); MS (FAB+) 384.9 [100, (M+H)+]; HRMS m/z (FAB+) 384.9494, C14H12 8 BrN2O2S2 requires 384.9503, 382.9501 , C14H12 79BrN2O2S2 requires 382.9523; HPLC tr 2.64 min (90 : 10 = MeOH : H2O).
DGS03062B (STX469) To a stirred solution of DGS03022A (50 mg, 0.14 mmol, 1 eq.) in anhy. DMF (5 ml) and NaH (7 mg, 0.16 mmol, 1.1 eq.) was added Mel (3 ml, 0.21 mmol, 1.5 eq.) and the mixture was stirred for 1h. The resulting mixture was poured into water and the organic layer was extracted with ethyl acetate, dried (MgSO4), filtered and concentrated under reduced pressure to give a yellow suspension. The crude compound (70 mg) was purified by flash chromatography using EtOAc/hexane (3:2) as eluent to give white crystals of DGS03062A (36 mg; 69%). mp 97-98 °C; TLC Rf : 0.61 EtOAc/Hexane (3:2); 1H NMR (CDCI3) δ 7.73 (dd, 1H, Ar-H, J = 1.17 Hz and 7.81 Hz), 7.37 (d, 1H, Ar-H, J = 8.59 Hz), 7.59 (d, 1 H, Ar-H, J = 1.95 Hz), 7.49 (dd, 1 H, Ar-H, J = 1.17 Hz and 8.2 Hz), 7.24 (dd, 1H, Ar-H, J = 2.34 Hz and 8.59 Hz), 7.11 - 7.15 (m, 1H, Ar-H), 3.24 (s, 3H, CH3), 2.76 (s, 3H, CH3), 2.35 (s, 3H, CH3); MS (FAB+) 366.9 [100, (M+H)+]; HRMS m/z (FAB+) 366.0262, C16H15 35CIN2O2S2 requires 366.0262, 368.0300, C16H15 37CIN2O2S2 requires 368.0234; HPLC tr 1.93 min (96 : 04 = MeOH : H2O).
DGS03072A (STX470)
To a stirred solution of DGS03022A (50 mg, 0.14 mmol, 1 eq.) in anhy. DMF (5 ml) and NaH (10 mg, 0.16 mmol, 1.1 eq.) was added Etl (23 mg, 0.21 mmol, 1.5 eq.) and the mixture was stirred for 1h. The resulting mixture was poured into water and the organic layer was extracted with ethyl acetate, dried (MgSO4), filtered and concentrated under reduced pressure to give a yellow suspension. The crude compound (75 mg) was purified by flash chromatography using EtOAc/hexane (3:2) as eluent to give a pale yellow thick syrup of DGS03072A (16 mg; 30%). TLC Rf : 0.71 EtOAc/Hexane (3:2); 1H
NMR (CDCI3) δ 7.76 - 7.78 (m, 2H, Ar-H), 7.66 (m, 1 H, Ar-H), 7.53 - 7.55 (m, 1 H, Ar-H),
7.27 - 7.28 (m, 1 H, Ar-H), 7.14 - 7.18 (m, 1 H, Ar-H), 7.11 - 7.18 (m, 1H, Ar-H), 5.30 (s,
1H, NH, Ex. with D2O), 3.74 (q, 2H, Ar-H, J = 7.42 Hz and 7.03 Hz), 2.83 (s, 3H, CH3),
2.53 (s, 3H, CH3), 1.12 (t, 3H, CH3> J = 7.03 Hz), MS (FAB+) 381.1 [100, (M+H)+]; HRMS m/z (FAB+) 381.1062, C17H17 35CIN2O2S2 requires 381.1058, 385.0952, C17H17 37CIN2O2S2 requires 385.0949.
DGS03082A (STX521)
Synthesised by method A. White crystals of DGS03082A (230 mg; 67%). mp 85-86 °C; TLC Rf : 0.64 EtOAc/Hexane (3:2); 1H NMR (CDCI3) δ 10.54 (s, 1 H, NH, Ex. With D2O), 7.87 (d, 1H, Ar-H, J = 8.59 Hz), 7.84 (broad s, 4H, Ar-H), 7.67 - 7.69 (m, 2H, Ar-H), 7.62 (d, 1 H, Ar-H, J = 1.95 Hz), 7.39 - 7.49 (m, 3H, Ar-H), 7.17 (dd, 1H, Ar-H, J = 1.95 Hz and 8.59 Hz), 2.73 (s, 3H, CH3); MS (FAB+) 381.2 [100, (M+H)+]; HRMS m/z (FAB+) 381.0730, C20H17N2O2S2 requires 381.0731; HPLC tr 1.36 min (96 : 04 = MeOH : H2O). DGS03084A (STX522)
Synthesised by method A. Yellow crystals of DGS03084A (46 mg; 10%). mp 253-254 °C; TLC Rf : 0.74 EtOAc/Hexane (3:2); 1H NMR (DMSO-d6) δ 11.09 (s, 1 H, NH, Ex. with D2O), 7.91 (d, 1H, Ar-H, J = 8.59 Hz), 7.86 (s, 2H, Ar-H), 7.59 (d, 1H, Ar-H, J = 2.34 Hz), 7.15 (dd, 1H, Ar-H, J = 1.95 Hz and 8.59 Hz), 2.74 (s, 3H, CH3); MS (FAB+) 409.1 [100, (M+H)+]; MS (FAB-) 407.0 [100, (M-H)+]; HRMS m/z (FAB+) 406.9176, C14H19 35CI3N2O2S2 requires 406.9167, 408.9136, C14H19 37CI3N2O2S2 requires 408.9140.
DGS03086A (STX523)
Synthesised by method A. Pale yellow crystals of DGS03086A (101 mg; 57%). mp 219 °C; TLC Rf : 0.71 EtOAc/Hexane (3:2); 1H NMR (DMSO-d6) δ 10.68 (s, 1H, NH, Ex. With D2O), 7.87 (d, 1 H, Ar-H, J = 8.59 Hz), 7.79 (d, 1 H, Ar-H, J = 8.59 Hz), 7.64 (d, 1H, Ar-H, J = 1.95 Hz), 7.54 - 7.57 (m, 2H, Ar-H), 7.11 (dd, 1 H, Ar-H, J = 2.34 Hz and 8.59 Hz), 2.73 (s, 3H, CH3), 2.59 (s, 3H, CH3); MS (FAB+) 399.0 [100, (M+H)+], 164.1 [50, (Starting amine)+]; HRMS m/z (FAB+) 398.9663, C15H13 81BrN2O2S2 requires 398.9569, 396.9684, C15H13 79BrN2O2S2 requires 396.9680; HPLC t 1.39 min (96 : 04 = MeOH : H2O).
DGS03064
2,4-Dichloro benzoic acid (10 g, 0.0523 mol, 1 eq.) was heated to 115 °C with excess chlorosulphonic acid (10.5 mL, 0.1571 mol, 3 eq.) under N2 for 18 h. The resulting mixture was cooled and consciously poured into ice-water. The resulted white precipitate was filtered out, washed with plenty of water and dried under vacuum over night. The crude DGS03064 (11.5 g, 76 %) was used for the subsequent reaction without further purification, mp 173-174 °C; TLC Rf ; 0.48 (4:1 , CH2CI2/EtOAc); 1H NMR (CDCI3) δ 8.28 (1 H, s, Ar-H), 7.65 (1 H, s, Ar-H); MS m/z (FAB+) 286.9 [100, (M+H)+]; HRMS m/z (FAB+) 287.8798, C7H3 35CI3O4S requires 287.8818, 291.8755, C7H3 37CI3O4S requires 291.8759.
DGS03088A (STX524)
Synthesised by method B. Two compounds were isolated - DGS03088A and DGS03088A. White crystals of DGS03088A (48 mg; 13%). mp 153-155 °C; TLC Rf : 0.79 EtOAc/Hexane (3:2); 1H NMR (CDCI3) δ 8.31 (s, 1 H, NH, Ex. With D2O), 8.07 (s, 1H, NH, Ex. With D2O), 8.07 (s, 1 H, Ar-H), 7.71 - 7.79 (m, 4H, Ar-H), 7.67 (d, 1H, Ar-H, J = 1.95 Hz), 7.58 (s, 1 H, Ar-H), 7.27 (dd, 1 H, Ar-H, J = 2.72 Hz and 8.59 Hz), 2.83 (s, 3H, CH3), 2.79 (s, 3H, CH3); MS (FAB+) 562.9 [100, (M+H)+]; HRMS m/z (FAB+) 562.9825, C23H17 35CI2N4O3S3 requires 562.9839, 566.9778, C23H17 37CI2N4O3S3 requires 566.9781; HPLC tr 1.33 min (96 : 04 = MeOH : H2O).
DGS03088-1 (STX575)
White crystals of DGS03088-1 (31 mg; 12%). mp 147-148 °C; TLC Rf : 0.45 EtOAc/Hexane (3:2); 1H NMR (CDCI3) δ 8.45 (s, 1H, NH, Ex. With D2O), 8.17 (d, 1H, Ar- H, J = 8.09 Hz), 8.04 (s, 1H, Ar-H), 7.77 (s, 1H, Ar-H), 7.50 (d, 1 H, Ar-H, J = 1.83 Hz), 7.35 (dd, 1H, Ar-H, J = 1.83 Hz and 8.05 Hz), 2.85 (s, 3H, CH3); LC-MS 418.1 [100, (M+)]; HPLC tr 1.97 min (96 : 04 = MeOH : H2O).
DGS03100A (STX552)
Synthesised by method B. White crystals of DGS03100A (224 mg; 69%). mp 222-223 °C; TLC Rf : 0.56 CH2CI2/EtOAc (4:1 ); 1H NMR (DMSO-d6) δ 10.27 (s, 1 H, NH, Ex. With D2O), 9.16 - 9.17 (m, 1 H, Ar-H), 8.48 - 8.51 (m, 2H, Ar-H), 8.36 - 8.38 (m, 2H, Ar-H), 8.23 - 8.25 (m, 1H, Ar-H), 7.67 - 7.34 (m, 3H, Ar-H), 7.51 - 7.12 (m, 1 H, Ar-H), 7.09 - 7.12 (m, 1H, Ar-H), 2.67 (s, 3H, CH3); LC-MS 355.7 [(M)+]; MS (FAB+) 356.0 [100, (M+H)+]; HRMS m/z (FAB+) 356.0531 , C17H14N3O2S2 requires 356.0527; HPLC tr 1.86 min (96 : 04 = MeOH : H2O).
DGS03102A (STX553)
Synthesised by method B. Pale yellow crystals of DGS03102A (170 mg; 52%). mp 89-90 °C; TLC Rf : 0.55 CH2CI2/EtOAc (4:1); 1H NMR (DMSO-d6) δ 10.87 (s, 1 H, NH, Ex. With D2O), 8.28 - 8.24 (m, 1H, Ar-H), 8.06 - 8.22 (m, 2H, Ar-H), 8.05 (d, 1H, Ar-H, J = 8.20 Hz), 7.60 - 7.77 (m, 2H, Ar-H), 7.47 (d, 1 H, Ar-H, J = 1.95 Hz), 7.04 (dd, 1 H, Ar-H, J = 1.95 Hz and 8.59 Hz), 2.69 (s, 3H, CH3); MS (FAB+) 355.0 [100, (M+H)+]; HRMS m/z (FAB+) 355.0576, C18H15N2O2S2 requires 355.0575; HPLC tr 1.93 min (96 : 04 = MeOH : H2O).
DGS03104A (STX554)
Synthesised by method B. Yellow crystals of DGS03104A (230 mg; 63%). mp 85-86 °C; TLC Rf : 0.65 CH2CI2/EtOAc (4:1); 1H NMR (DMSO-d6) δ 10.84 (s, 1H, NH, Ex. With D2O), 8.40 - 8.42 (m, 2H, Ar-H), 8.22 - 8.23 (m, 1H, Ar-H), 7.76 - 7.78 (m, 1H, Ar-H), 7.58 - 7.65 (m, 2H, Ar-H), 7.51 - 7.56 (m, 1H, Ar-H), 7.23 - 7.25 (m, 1H, Ar-H), 7.05 - 7.07 (m, 1H, Ar-H), 2.79 (s, 6H, 2χCH3), 2.69 (s, 3H, CH3); MS (FAB+) 398.1 [100, (M+H)+]; HRMS m/z (FAB+) 398.0978, C20H20N3O2S2 requires 398.0997; HPLC tr 2.01 min (96 : 04 = MeOH : H2O).
DGS03116A (STX580) Synthesised by method B. Pale yellow crystals of DGS03116A (151 mg; 44%). mp 153 °C; TLC Rf : 0.55 CH2CI2/EtOAc (4:1 ); 1H NMR (DMSO-d6) δ 10.96 (s, 1 H, NH, Ex. With D2O), 8.00 (d, 1H, Ar-H, J = 2.34 Hz), 7.90 (d, 1 H, Ar-H, J = 8.59 Hz), 7.66 - 7.73 (m, 2H, Ar-H), 7.58 (d, 1H, Ar-H, J = 2.34 Hz), 7.17 (dd, 1H, Ar-H, J = 2.34 Hz and 8.59 Hz), 2.74 (s, 3H, CH3); MS (FAB+) 372.8 [100, (M+H)+]; HRMS m/z (FAB+) 375.9599, CMHH^C^N SJ, requires 375.9502, 372.9606, C^H^CI^^S;, requires 372.9639; HPLC tr 2.98 min (90 : 10 = MeOH : H2O).
DGS03118A (STX581)
Synthesised by method B. White crystals of DGS03118A (416 mg; 42%). mp 88-89 °C; TLC Rf : 0.49 CH2CI2/EtOAc (4:1 ); 1H NMR (DMSO-d6) δ 10.47 (s, 1 H, NH, Ex. With D2O), 7.74 (d, 1 H, Ar-H, J = 8.59 Hz), 7.58 - 7.61 (m, 2H, Ar-H), 7.35 - 7.38 (m, 1 H, Ar- H), 7.13 - 7.17 (m, 1H, Ar-H), 4.76 - 4.78 (m, 2H, CH2), 3.75 - 3.79 (m, 2H, CH2), 2.90 - 2.93 (m, 2H, CH2), 2.73 (s, 3H, CH3); MS (FAB+) 456.0 [100, (M+H)+]; HRMS m/z (FAB+) 456.0663, C19H17F3N3O3S2 requires 456.0663; HPLC tr 1.63 min (96 : 04 = MeOH : H2O).
DGS03120A (STX582)
Synthesised by method B. Pale yellow crystals of DGS03120A (185 mg; 55%). mp 91-92 °C; TLC Rf : 0.51 CH2CI2/EtOAc (4:1 ); 1H NMR (DMSO-d6) δ 10.35 (s, 1H, NH, Ex. With D2O), 7.85 (d, 1H, Ar-H, J = 8.98 Hz), 7.69 (d, 1H, Ar-H, J = 2.34 Hz), 7.61 (dd, 1H, Ar- H, J = 2.73 Hz and 8.98 Hz), 7.55 (d, 1 H, Ar-H, J = 2.73 Hz), 7.20 (d, 1 H, Ar-H, J = 8.98 Hz), 7.15 (dd, 1 H, Ar-H, J = 2.3 Hz and 8.59 Hz), 3.89 (s, 3H, OCH3), 2.73 (s, 3H, CH3); MS (FAB+) 369.0 [100, (M+H)+]; HRMS m/z (FAB+) 371.0114, C15H14 37CIN2O3S2 requires 371.0105, 369.0135, C15H14 35CIN2O3S2 requires 369.0134; HPLC tr 1.68 min (96 : 04 = MeOH : H2O).
DGS03122A (STX731)
Synthesised by method B. Two compounds were isolated - DGS03122A and
DGS03122B. Yellow crystals of DGS03122A (67 mg; 22%). mp 272-273 °C; TLC Rf : 0.59 CH2CI2/EtOAc (4:1); 1H NMR (DMSO-d6) δ 8.15 (m, 5H, , Ar-H), 8.02 - 8.09 (m, 4H, Ar-H), 7.74 (d, 1 H, Ar-H, J = 2.3 Hz), 7.14 (dd, 1H, Ar-H, J - 1.95 Hz and 8.59 Hz), 2.84 (s, 3H, CH3); MS (FAB+) 495.0 [100, (M+H)+]; HPLC tr 1.79 min (90 : 10 = MeOH : H2O).
DGS03122B (STX583) Yellow crystals of DGS03122B (47 mg; 16%). mp 204-206 °C; TLC Rf : 0.48 CH2CI2/EtOAc (4:1 ); 1H NMR (DMSO-d6) δ 10.95 (s, 1H, NH, Ex. With D2O), 8.03 - 8.07 (m, 2H, Ar-H), 7.86 - 7.91 (m, 2H, Ar-H), 7.77 - 7.81 (m, 1 H, Ar-H), 7.55 (d, 1H, Ar-H, J = 1.95 Hz), 7.12 (dd, 1H, Ar-H, J = 2.34 Hz and 8.59 Hz), 2.74 (s, 3H, CH3); MS (FAB+) 330.0 [100, (M+H)+]; HRMS m/z (FAB+) 330.0370, C15H12N3O2S2 requires 330.0371 ; HPLC tr 1.84 min (90 : 10 = MeOH : H2O).
DGS03124A (STX584)
Synthesised by method B. Pale yellow crystals of DGS03124A (125 mg; 55%). mp 188- 189 °C; TLC Rf : 0.37 CH2CI2/EtOAc (4:1); H NMR (DMSO-d6) δ 10.09 (s, 1 H, NH, Ex. With D2O), 7.81 (d, 1H, Ar-H, J = 8.59 Hz), 7.63 (d, 1H, Ar-H, J = 8.20 Hz), 7.56 (d, 1H, Ar-H, J = 1.95 Hz), 7.14 (dd, 1 H, Ar-H, J = 1.95 Hz and 8.59 Hz), 6.96 (s, 1 H, Ar-H), 6.81 (d, 1H, Ar-H, J = 8.59 Hz), 3.87 (s, 3H, OCH3), 2.72 (s, 3H, CH3), 2.28 (s, 3H, CH3); MS (FAB+) 219.1 [20, (sulphonyl chloride-H)+], 349.0 [100, (M+H)+]; HRMS m/z (FAB+) 349.0678, C16H17N2O3S2 requires 349.0681 ; HPLC tr 1.80 min (96 : 04 = MeOH : H2O).
DGS03126A (STX585)
Synthesised by method B. Pale yellow crystals of DGS03126A (145 mg; 40%). mp 84-86 °C; TLC Rf : 0.71 CH2CI2/EtOAc (4:1); 1H NMR (DMSO-d6) δ 10.42 (s, 1 H, NH, Ex. With D2O), 7.88 (d, 1H, Ar-H, J = 8.59 Hz), 7.73 - 7.77 (m, 2H, Ar-H), 7.59 (d, 1H, Ar-H, J = 1.95 Hz), 7.41 - 7.46 (m, 2H, Ar-H), 7.22 - 7.26 (m, 2H, Ar-H), 7.13 (dd, 1 H, Ar-H, J = 8.59 Hz and 2.34 Hz), 7.02 - 7.10 (m, 4H, Ar-H), 2.75 (s, 3H, CH3); MS (FAB+) 397.0 [100, (M+H)+]; HRMS m/z (FAB+) 397.0671 , C20H17N2O3S2 requires 397.0681 ; HPLC t 1.93 min (96 : 04 = MeOH : H2O).
DGS03130A (STX730)
Synthesised by method B. Two compounds were isolated - DGS03130A and DGS03130B. Synthesised by method B. Pale yellow crystals of DGS03130A (105 mg; 33%). mp 125-126 °C; TLC Rf : 0.55 CH2CI2/EtOAc (4:1 ); 1H NMR (DMSO-d6) δ 8.21 - 8.24 (m, 4H, Ar-H), 8.13 (d, 1H, Ar-H, J = 8.59 Hz), 7.99 - 8.03 (m, 4H, Ar-H), 7.57 (d, 1H, Ar-H, J = 1.95 Hz), 7.03 (dd, 1 H, Ar-H, J = 8.59 Hz and 1.95 Hz), 2.82 (s, 3H, CH3); 2.69 (s, 6H, 2xCH3); MS (FAB+) 529.0 [100, (M+H)+]; MS (FAB-) 527.1 [70, (M-H)+], 345.0 [100, (M-2-Acetyl sulphonyl chloride)+]; HPLC tr 1.81 min (96 : 04 = MeOH : H2O).
DGS03130B (STX701) Pale yellow crystals of DGS03130A (45 mg; 14%). mp 169 °C; TLC Rf : 0.42 CH2CI2/EtOAc (4:1 ); 1H NMR (DMSO-d6) δ 10.63 (s, 1H, NH, Ex. With D2O), 8.04 - 8.07 (m, 2H, Ar-H), 7.86 - 7.89 (m, 3H, Ar-H), 7.59 (d, 1 H, Ar-H, J = 1.95 Hz), 7.13 (dd, 1 H, Ar-H, J = 8.9 Hz and 2.3 Hz), 3.73 (s, 3H, CH3); 2.56 (s, 3H, CH3); MS (FAB+) 347.0 [100, (M+H)+], 219.1 [10, (sulphonyl chloride+H)+]; HRMS m/z (FAB+) 347.0522, C16H15N2O3S2 requires 347.0524; HPLC tr 1.77 min (96 : 04 = MeOH : H2O).
DGS03134A (STX703)
Synthesised by method B. Pale yellow crystals of DGS03134A (91 mg; 23%). mp 206- 207 °C; TLC Rf : 0.81 CH2CI2/EtOAc (4:1 ); 1H NMR (DMSO-d6) δ 10.37 (s, 1H, NH, Ex. With D2O), 7.87 (d, 1 H, Ar-H, J = 8.59 Hz), 7.46 (d, 1 H, Ar-H, J = 1.95 Hz), 7.19 (s, 2H, Ar-H), 7.07 (dd, 1H, Ar-H, J = 8.59 Hz and 1.95 Hz), 4.13 - 4.20 (m, 2H, 2χ(CH3)2H), 2.83 - 2.89 (m, 1 H, (CH3)2H), 2.72 (s, 3H, CH3), 1.15 (d, 12H, 4x(CH3)2, J = 7.03 Hz), 1.11 (d, 9H, 2χ(CH3)2, J = 6.64 Hz); LC-MS 429.72 (M)+; HPLC t 2.84 min (90 : 10 = MeOH : H2O).
DGS03136A (STX704)
Synthesised by method B. Pale yellow crystals of DGS03136A (225 mg; 71%). mp 54-55 °C; TLC Rf : 0.50 CH2CI2/EtOAc (4:1); 1H NMR (CDCI3) δ 7.65 (m, 3H, Ar-H), 7.58 (d, 1 H, Ar-H, J = 2.34 Hz), 7.18 (dd, 1 H, Ar-H, J = 8.6 Hz and 1.95 Hz), 6.84 - 6.85 (m, 2H, Ar- H), 6.82 (s, 1 H, NH, Ex. With D2O), 4.51 - 4.60 (m, 1 H, (CH3)2H), 2.80 (s, 3H, CH3), 1.31 (s, 6H, (CH3)2); LC-MS 347.6 (M)+; HRMS m/z (FAB+) 347.0847, C17H19N2O2S2 requires 347.0837; HPLC tr 2.39 min (90 : 10 = MeOH : H2O).
DGS03138B (STX705) Synthesised by method B. Pale yellow crystals of DGS03138B (24 mg; 7%). mp 248 °C; TLC Rf : 0.52 CH2CI2/EtOAc (4:1); 1H NMR (CDCI3) δ 8.18 (d, 1 H, Ar-H, J = 8.59 Hz), 8.15 (d, 1H, Ar-H, J = 1.95 Hz), 7.89 - 8.04 (m, 4H, Ar-H), 7.51 (dd, 1H, Ar-H, J = 8.20 Hz and 1.95 Hz), 7.27 (s, 1H, NH, Ex. With D2O), 2.89 (s, 3H, CH3), 1.59 (s, 3H, CH3); LC-MS 372.90 (M+CH3CN)+; HRMS m/z (FAB+) 371.2281 , C16H15N2O4S2 requires 371.2278; HPLC tr 2.22 min (90 : 10 = MeOH : H2O). DGS03140A (STX711)
Synthesised by method B. Brown crystals of DGS03140A (85 mg; 26%). mp 73-75 °C; TLC Rf : 0.59 CH2CI2/EtOAc (4:1 ); 1H NMR (CDCI3) δ 7.85 (d, 1H, Ar-H, J = 8.59 Hz), 7.80 (d, 1H, Ar-H, J = 8.59 Hz), 7.68 (s, 1H, NH, Ex. With D2O), 7.57 (d, 1H, Ar-H, J = 1.95 Hz), 7.54 (s, 1 H, NH, Ex. With D2O), 7.24 (d, 1 H, Ar-H, J = 2.34 Hz), 7.18 (dd, 1H, Ar-H, J = 8.20 Hz and 1.95 Hz), 7.03 (dd, 1H, Ar-H, J = 8.59 Hz and 2.34 Hz), 6.77 (dd, 1H, Ar-H, J = 8.59 Hz and 2.34 Hz), 2.78 (s, 3H, CH3), 2.24 (s, 3H, CH3); LC-MS 362.32 (M)+; HRMS AT?/Z (FAB+) 361.0587, C16H16N3O3S2 requires 361.0636; HPLC tr 2.09 min (90 : 10 = MeOH : H2O).
DGS03142A (STX706)
Synthesised by method B. Pale yellow crystals of DGS03142A (79 mg; 24%). mp 89-91 °C; TLC Rf : 0.65 CH2CI2/EtOAc (4:1); 1H NMR (CDCI3) δ 7.78 (d, 1 H, Ar-H, J = 8.20 Hz), 7.61 (d, 1H, Ar-H, J = 1.56 Hz), 6.98 (dd, 1 H, Ar-H, J = 1.95 Hz and 8.20 Hz), 6.93 (s, 1 H, Ar-H), 6.92 (s, 1 H, NH, Ex. With D2O), 3.99 (s, 6H, 2xCH3), 3.93 (s, 6H, 2xCH3), 2.85 (s, 3H, CH3); LC-MS 361.48 (M)+; HRMS m/z (FAB+) 361.1605, C18H21N2O2S2 requires 361.1606; HPLC tr 2.26 min (90 : 10 = MeOH : H2O).
DGS03144A (STX707) Synthesised by method B. Pale yellow crystals of DGS03144A (79 mg; 24%). mp 89-91 °C; TLC Rf : 0.69 CH2CI2/EtOAc (4:1); 1H NMR (CDCI3) δ 7.70 (d, 1 H, Ar-H, J = 8.59 Hz),
7.58 (d, 1H, Ar-H, J = 2.34 Hz), 7.39 (dd, 1H, Ar-H, J = 2.34 Hz and 8.59 Hz), 7.19 (d, 1 H, Ar-H, J = 1.95 Hz), 7.17 (t, 1H, Ar-H, J = 1.95 Hz), 6.83 (d, 1H, Ar-H, J = 8.59 Hz),
6.59 (s, 1 H, NH, Ex. With D2O), 3.89 (s, 3H, OCH3), 3.76 (s, 3H, OCH3), 2.81 (s, 3H, CH3); LC-MS 363.02 (M)+; HRMS m/z (FAB+) 365.0642, C16H17N2O4S2 requires
365.0585; HPLC tr 2.15 min (90 : 10 = MeOH : H2O).
DGS03146A (STX708)
Synthesised by method B. Pale yellow crystals of DGS03146A (181 mg; 51%). mp 175 °C; TLC Rf : 0.57 CH2CI2/EtOAc (4:1); 1H NMR (CDCI3) δ 7.71 (dd, 1H, Ar-H, J = 2.3 Hz and 8.98 Hz), 7.59 (d, 1 H, Ar-H, J = 1.95 Hz), 7.43 (d, 1 H, Ar-H, J = 8.98 Hz), 7.21 (dd, 1H, Ar-H, J = 1.95 Hz and 8.59 Hz), 6.67 (s, 1H, NH, Ex. With D2O), 2.81 (s, 3H, OCH3), 1.59 (s, 6H, 2xCH3), 1.29 (s, 6H, 2χCH3); LC-MS 377.01 (M)+; HRMS m/z (FAB+) 377.0988, C18H21N2O3S2 requires 377.0994; HPLC tr 2.53 min (90 : 10 = MeOH : H2O). DGS03148A (STX709)
Synthesised by method B. Off-white crystals of DGS03148A (102 mg; 31%). mp 214- 215 °C; TLC Rf : 0.62 CH2CI2/EtOAc (4:1); 1H NMR (CDCI3) δ 7.71 - 7.73 (m, 2H, Ar-H), 7.60 - 7.61 (m, 1H, Ar-H), 7.44 - 7.46 (m, 2H, Ar-H), 7.21 - 7.24 (m, 2H, Ar-H), 6.61 (s, 1 H, NH, Ex. With D2O), 2.83 (s, 3H, CH3), 1.31 (s, 9H, (CH3)3); LC-MS 360.12 (M)+; HRMS m/z (FAB+) 361.1057, C18H21N2O3S2 requires 361.1044; HPLC tr 2.67 min (90 : 10 = MeOH : H2O).
DGS03150A (STX710)
Synthesised by method B. Pale yellow crystals of DGS03150A (101 mg; 30%). mp 200- 201 °C; TLC Rf : 0.50 CH2CI2/EtOAc (4:1); 1H NMR (CDCI3) δ 7.65 (d, 1H, Ar-H, J = 8.59 Hz), 7.44 (d, 1H, Ar-H, J = 1.95 Hz), 7.09 (dd, 1 H, Ar-H, J = 1.95 Hz and 8.59 Hz), 6.75 (s, 1 H, NH, Ex. With D2O), 2.79 (s, 3H, CH3), 2.57 (s, 6H, 2xCH3), 2.24 (s, 3H, CH3), 2.19 (s, 6H, 2χCH3); LC-MS 374.10 (M)+; HRMS m/z (FAB+) 375.1195, C19H23N2O2S2 requires 375.1201 ; HPLC tr 3.15 min (80 : 20 = MeOH : H2O).
DGS03152A (STX712)
Synthesised by method B. Pale yellow crystals of DGS03152A (120 mg; 33%). mp 181- 182 °C; TLC Rf : 0.65 CH2CI2/EtOAc (4:1 ); 1H NMR (CDCI3) δ 7.63 (d, 1 H, Ar-H, J = 8.59 Hz), 7.59 (d, 1 H, Ar-H, J = 2.3 Hz), 7.22 (dd, 1H, Ar-H, J = 2.3 Hz and 8.59 Hz), 4.91 (s, 1 H, NH, Ex. With D2O), 3.82 (s, 3H, CH3); LC-MS 392.96 (M)+; HRMS m/z (FAB+) 394.9941 , C14H8F5N2O2S2 requires 394.9947; HPLC tr 2.49 min (90 : 10 = MeOH : H2O).
DGS03158A (STX713)
Synthesised by method B. Yellow crystals of DGS03158A (158 mg; 40%). mp 334-335 °C; TLC Rf : 0.47 CH2CI2/EtOAc (4:1); 1H NMR (CDCI3) δ 7.65 (d, 1H, Ar-H, J = 8.59 Hz), 7.47 (d, 1H, Ar-H, J = 2.3 Hz), 7.10 (dd, 1H, Ar-H, J = 2.3 Hz and 8.59 Hz), 6.69 (s, 1 H, NH, Ex. With D2O), 2.79 (s, 3H, CH3), 2.61 (t, 2H, CH2, J = 6.64 Hz), 2.55 (s, 3H, CH3), 2.51 (s, 3H, CH3), 2.08 (s, 3H, CH3), 1.79 (t, 2H, CH2, J = 7.03 Hz), 1.29 (s, 6H, 2χCH3); LC-MS 431.11 (M)+; HPLC tr 3.24 min (90 : 10 = MeOH : H2O). Synthesis of Benzothiazole Arylsulphonamide Derivatives
Figure imgf000090_0001
R = -C2H5 R = -C2H5
R — -C2H OCH3 R = -C2H4OCH3
R = -CH2COOC2H5 R = -CH2COOC2H5
Figure imgf000090_0002
R = -H R = -CH3 Ar = 3-CI-2-CH3-phenyl, R = -H
Ar = 3-CI-2-CH3-phenyl, R = -CH3,
Ar = 4-n-propylρhenyl, R = -CH ,
Ar = 2,5-dichlorophenyl, R = -CH3,
Figure imgf000090_0003
R = -CH3
R = -CH2COOC2H5 a) RX, NaH, THF r.t. b) ArS03CI, DC .Pyridine or ArS03CI, DCM.Pyridine/DMAP c) Diethylamine, DCM, AICI3 d) RX, K2C03, Acetone, reflux
Figure imgf000091_0001
J
H,N
-Cl
Figure imgf000091_0003
Figure imgf000091_0002
R = -NHCH3 R = -NHCH3 R = -N(C2H5)2 R = -N(C2H5)2
Figure imgf000091_0004
Ar = 3-CI-2-CH3-phenyl Ar = 2,5-dichlorophenyl a) HNO3, H2S04 -5 - 0°C b) H2, 5% Pd/C, C2H5OH, c) ArS03CI, DCM.Pyridine or ArS03CI, DCM,Pyridine/DMAP d) amine, THF, reflux
Figure imgf000092_0001
Ar = 3-CI-2-CH3-phenyl Ar = 2,5-dichlorophenyl Ar = 4-n-propylphenyl
Figure imgf000092_0002
a) N-chlorosuccinimide, IPA b) ArS03CI, DCM.Pyridine or ArS03Cl, DCM,Pyridine/DMAP c) N-bromosucinimide, CCI4, benzoyl peroxide d) 3-chloro-2methylbenzenesulphomamide, K2C03, CH3CN
Figure imgf000093_0001
STX751.XDS01141 STX752, XDS01142 STX754.XDS01144
Figure imgf000093_0002
STX755, XDS01145A STX763, XDS01145B
Figure imgf000093_0003
Figure imgf000093_0004
STX768.XDS01151B STX834.XDS01168
Figure imgf000093_0005
STX767, XDS01151A
Figure imgf000094_0001
STX833, XDS01167 STX835, XDS01176 STX836, XDS01177
Figure imgf000094_0002
Figure imgf000094_0003
Figure imgf000094_0004
General Method for the Preparation of N-Benzothiazole Benzenesulphonamide derivatives:
To a solution arylsulphonyl chloride (1.1 eq.) in DCM (5-10 mL) were added pyridine (2.2 eq.) and catalytic amount of DMAP. The solution was stirred at room temperature under nitrogen for 10 minutes. Then the amine (1 eq.) was added and the reaction mixture was stirred at room temperature under nitrogen for 4-16 hrs. The resulting mixture was partitioned between DCM and 5% sodium bicarbonate. The organic layer was washed with brine, dried over MgSO4, and concentrated to give a yellow residue. The crude compound was then purified by flash chromatography to give desired benzenesulphonamide as crystalline solid. (Yield 40-90%).
Synthesis of 2-Alkylsulfanyl-benzothiazol-6-yl-amine
To a solution of 6-amino-2-merceptobenzothiazole (273mg, 1.5 mmol) in anhydrous THF (10 mL) was added NaH (60% dispersion, 1.5 mmol), followed by alkyl halide (1.5 mmol). The mixture was stirred at rt for 24h, partitioned between ethyl acetate and 5% sodium bicarbonate. The organic phase was washed with brine, dried over sodium sulphate and concentrated in vacuo to a yellow solid, which was purified with recrystallization or flash chromatography. (Yield 60-90%).
The following amines were synthesized with the method described above:
2-Ethylsulfanylbenzothiazol-6-ylamine
Yellow crystalline solid, mp 77-78°C (lit.77°C). TLC single spot at Rf 0.78 (8% methanol/DCM); 1H NMR (270 MHz, DMSO): δ 7.50 (1 H, d, J = 8.5 Hz, 4-H), 6.98 (1 H, d, J = 2.2 Hz, 7-H), 6.69 (1 H, dd, J = 8.5, 2.2 Hz, 5-H), 5.33 (2H, broad, NH2), 3.23 (2H, q, J = 7.3 Hz, SCH2), 1.35 (3H, t, J = 7.3 Hz, CH3). (Francolor, S.A.; US 2500093; 1945)
2-(2-Methoxyethylsulfanyl)-benzothiazol-6-ylamine
Yellow thick syrup. TLC single spot at Rf 0.65 (30% ethyl acetate/DCM); H NMR (270 MHz, DMSO): δ 7.49 (1H, d, J = 8.8 Hz, 4-H), 6.97 (1 H, d, J = 2.2 Hz, 7-H), 6.69 (1 H, dd, J = 8.8, 2.2 Hz, 5-H), 5.34 (2H, broad, NH2), 3.63 (2H, t, J = 6.3 Hz, CH2), 3.43 (2H, t, J = 6.3 Hz, CH2), 3.26 (3H, s, CH3). (6-aminobenzothiazol-2-ylmercapto)-acetic acid ethyl ester
Off white solid, mp 87-89°C (lit.92°C, [20]); TLC single spot at R, 0.72 (8% methanol/DCM); 1H NMR (270 MHz, DMSO): δ 7.48 (1H, d, J = 8.7 Hz, 4-H), 7.01 (1H, d, J = 1.8 Hz, 7-H), 6.71 (1H, dd, J = 8.7, 1.8 Hz, 5-H), 5.58 (2H, broad, NH2), 4.17 (2H, s, SCH2), 4.12 (2H, t, J = 7.3 Hz, CH2), 1.19 (3H, t, J = 7.3 Hz, CH3).
The following compounds were synthesized with the general method for N- benzothiazole benzenesulphonamide:
3-Chloro-N-(2-ethylsulfanylbenzothiazol-6-yl)-2-methylbenzenesulphonamide (STX751, XDS01141)
Off-white solid (220 mg; 55%). TLC single spot at Rf : 0.83 (17% EtOAc/DCM); HPLC purity 96% (tR 1.9 min in methanol); 1HNMR (400MHz, DMSO-d6) δ 10.8 (1H, s, NH), 7.89 (1 H, dd, J = 8.0, 1.0 Hz, 6'-H of benzene), 7.71 (1 H, d, J = 8 Hz, 4-H of benzothiazole), 7.70 (1H, d, J = 2 Hz, 7-H of benzothiazole), 7.70 (1H, dd, J = 8.0, 1.0 Hz, 4'-H of benzene), 7.36 (1H, t, J = 8 Hz, 5'-H of benzene), 7.15 (1 H, dd, J = 8.0, 2.0 Hz, 5-H of benzothiazole), 3.30 (2H, q, J = 7.0 Hz, SCH2), 2.66 (3H, s, CH3), 1.38 (3H, t, J = 7.0 Hz, CH3); APCI-MS 397.99 (M)+; FAB-HRMS calcd for C16H16CIN2O2S3 (MH+) 399.0062, found 399.0048.
[6-(3-Chloro-2-methylbenzenesulphonylamino)-benzothiazol-2-ylsulfanyl]-acetic acid ethyl ester (STX752, XDS01142)
White crystalline solid (210 mg; 46%). TLC single spot at Rf : 0.69 (17% EtOAc/DCM); HPLC purity 99% (tR 2.9 min in 10% water-methanol); 1HNMR (400MHz, DMSO-d6) δ 10.8 (1 H, s, SO2NH), 7.88 (1 H, dd, J = 8.0, 1.0 Hz, 6'-H of benzene), 7.72 (1 H, d, J = 2.0 Hz, 7-H of benzothiazole), 7.69 (1H, dd, J = 8.0, 1.0 Hz, 4'-H of benzene), 7.68 (1H, d, J = 8.0 Hz, 4-H of benzothiazole), 7.36 (1 H, t, J = 8.0 Hz, 5'-H of benzene), 7.15(1 H, dd, J = 8.0, 2.0 Hz, 5-H of benzothiazole), 4.25 (2H, s, 2-SCH2-), 4.13 (2H, q, J = 7.1 Hz, COOCH2), 2.64 (3H, s, CH3), 1.17 (3H, t, J = 7.1 Hz, 2-COOCH2CH3); APCI-MS 456.0 (M)+; FAB-HRMS calcd for C18H18CIN2O4S3 (MH+) 457.0117, found 457.0109.
3-Chloro-N-[2-(2-methoxyethylsulfanyl)-benzothiazol-6-yl]-2- methylbenzenesulphonamide (STX754, XDS01144) Off-White solid (150 mg; 77%). TLC single spot at Rf 0.60 (17% EtOAc/DCM); HPLC purity 94% (tR 3.1 min in 10% water-methanol); 1HNMR (400MHz, DMSO-d6) δ 10.8 (1 H, s, SO2NH), 7.88 (1 H, dd, J = 8, 1 Hz, 6'-H of benzene), 7.71 (1H, d, J = 8 Hz , 4-H of benzothiazole), 7.70 (1H, dd, J = 8, 1 Hz, 4'-H of benzene), 7.69 (1H, d, J = 2 Hz, 7-H of benzothiazole), 7.36 (1H, t, J = 8 Hz, 5'-H of benzene), 7.15(1H, dd, J = 8, 2 Hz, 5-H of benzothiazole), 3.64 (2H, t, J = 6 Hz, CH2), 3.50 (2H, t, J = 6 Hz, SCH2), 3.27 (3H, s, CH3), 2.65 (3H, s, CH3); APCI-MS 428.0 (M)+; FAB-HRMS calcd for C17H18CIN2O3S3 (MH+) 429.0168, found 429.0159.
2-[6-(3-Chloro-2-methylbenzenesulphonylamino)-benzothiazol-2-ylsulfanyl]-N,N- diethylacetamide (STX755, XDS01145) and 2-[6-(3-chloro-2-methyl- benzenesulphonylamino)-benzothiazol-2-yl]-N,N-diethylacetamide (STX763,
XDS01145B)
To a suspension of AICI3 (50 mg) in DCM (5 ml) was added diethylamine ( 0.4 ml). The solution was stirred under nitrogen at room temperature for 10 minutes. [6-(3-Chloro-2- methyl-benzenesulphonylamino)-benzothiazol-2-ylsulfanyl]-acetic acid ethyl ester (STX752, 100 mg) was added and the mixture was kept stirring at room temperature for 30 minutes. The reaction was quenched with water, partitioned between DCM and 5% NaHCO3. The organic phase was washed with water, dried over MgSO4 and evaporated in vacuo to give a yellow residue, which was purified with flash column chromatography using 20-30% ethyl acetate-DCM as eluting solvent. STX755 (50 mg, 47%) was obtained as white solid. TLC single spot at Rf 0.60 (25% EtOAc/DCM); HPLC purity 89% (tR 2.7 min in 10% water-methanol); 1HNMR (270MHz, DMSO-d6) δ 10.7 (1H, s, SO2NH), 7.86 (1H, d, J = 8 Hz, 6'-H of benzene), 7.64-7.68 (3H, m, 4'-H of benzene and 4,7-H of benzothiazole), 7.34 (1 H, t, J = 8 Hz, 5'-H of benzene), 7.12 (1H, dd, J = 8, 2 Hz, 5-H of benzothiazole), 4.42 (2H, s, 2-SCH2-), 3.26-3.38 (4H, m, -N(CH2)2-), 2.50 (3H, s, 1 '-CH3), 1.17 (3H, t, J = 7 Hz, -NCH,CH,). 1.00 (3H, t, J = 7 Hz, -NCH2CH3): APCI-MS 484.0 (M)+; FAB-HRMS calcd for C20H23CIN3O3S3 (MH+) 484.0590, found 484.0584. STX763 (25 mg, 25%) was obtained as white solid. TLC single spot at Rf 0.39 (25% EtOAc/DCM); LCMS purity 98% (tR 6.9 min in 10% water-CH3CN); HNMR (400 MHz, DMSO-d6) δ 10.8 (1H, s, SO2NH), 7.88 (1H, dd, J = 8.1, 1.2 Hz, 6'-H of benzene), 7.79 (1 H, d, J = 8.6 Hz, 4-H), 7.72 (1 H, d, J = 2.0 Hz, 7-H), 7.68 (1 H, dd, J = 8.1 , 1.2 Hz, 4'-H of benzene), 7.35 (1 H, t, J = 8.1 Hz, 5'-H of benzene), 7.17 (1H, dd, J = 8.6, 2 Hz, 5-H of benzothiazole), 4.2 (2H, s, 2-SCH2-), 3.26-3.38 (4H, m, -N(CH2)2-), 2.65 (3H, s, CH3), 1.10 (3H, t, J = 7 Hz, -NCH2CH3), 1.02 (3H, t, J = 7 Hz, -NCH2CH3); APCI-MS 451.0 (M)+; FAB-HRMS calcd for C20H23CIN3O3S2 (MH+) 452.0869, found 452.0870.
3-Chloro-N-benzothiazol-6-yl-2-methylbenzenesulphonamide (STX750, XDS01139) Light pink needles (260 mg; 77%). TLC single spot at Rf 0.46 (17% EtOAc/DCM); HPLC purity 99% (tR 2.5 min in 10% water-methanol); 1HNMR (400MHz, DMSO-d6) δ 10.9 (1 H, s, SO2NH), 9.25 (1 H, s, 2-H of benzothiazole), 7.95 (1H, d, J = 9 Hz, 4-H of benzothiazole), 7.92 (1 H, dd, J = 8.0, 1.0 Hz, 6'-H of benzene), 7.84 (1H, d, J = 2 Hz, 7- H of benzothiazole), 7.70 (1H, dd, J = 8.0, 1.0 Hz, 4'-H of benzene), 7.37(1 H, t, J = 8 Hz, 5'-H of benzene), 7.25 (1 H, dd, J = 9.0, 2.0 Hz, 5-H of benzothiazole), 2.66 (3H, s, CH3); APCI-MS 337.9 (M)+; FAB-HRMS calcd for C20H23CIN3O3S2 (MH+) 452.0869, found 452.0870.
3-Chloro-N-(2-methy lbenzothiazol-6-yl)-2-methylbenzenesulphonamide (STX886, XDS01187B)
Off-white solid. TLC single spot at Rf 0.65 (10% methanol/DCM); HPLC purity >99% (tR 2.4 min in 10% water-methanol); 1HNMR (270MHz, DMSO-d6) δ 10.7 (1 H, s, NH), 7.87 (1 H, dd, J = 7.8, 1.9 Hz, ArH), 7.75 (1H, d, J = 8.8 Hz, ArH), 7.69 (1H, d, J = 2.2 Hz, ArH), 7.68 (1 H, dd, J = 7.8, 1.9 Hz, ArH), 7.34 (1 H, t, J = 7.8 Hz, ArH), 7.15 (1H, dd, J = 8.8, 2.2 Hz, ArH), 2.71 (3H, s, CH3), 2.63 (3H, s, CH3); APCI-MS 351 (M-H)+; FAB- HRMS calcd for C15H14CIN2O2S2 (MH+) 353.0185, found 353.0197.
N-(2-methylbenzothiazol-6-yl)-N-(3-chloro-2-methylphenylsulphonyl)-3-chloro-2- methylbenzenesulphonamide (STX887, XDS01187A)
Off-white powder. TLC single spot at Rf 0.89 (10% methanol/DCM); HPLC purity 91% (tR 3.1 min in 10% water-methanol); 1HNMR (270MHz, DMSO-d6) δ 8.09 (1 H, d, J = 2.2 Hz, ArH), 7.92 (1 H, d, J = 8.6 Hz, ArH), 7.85-7.90 (4H, m, ArH), 7.46 (2H, t, J = 8.0 Hz, ArH), 7.35 (1 H, dd, J = 8.8, 2.2 Hz, ArH), 2.81 (3H, s, CH3), 2.33 (6H, s, 2 x CH3); APCI- MS 539 (M-H)+; FAB-HRMS calcd for C22H19CI2N2O4S3 (MH+) 540.9884, found 540.9897.
2,5-Dichloro-N-(2-methylbenzothiazol-6-yl)-benzenesulphonamide (STX888,
XDS01188B) White crystalline solid. TLC single spot at Rf 0.68 (10% methanol/DCM); HPLC purity >99% (tR 2.3 min in 10% water-methanol); 1HNMR (270MHz, DMSO-d6) δ 10.9 (1H, s, NH), 7.98 (1H, d, J = 2.3 Hz, ArH), 7.76 (1 H, d, J = 8.9 Hz, ArH), 7.74 (1H, d, J = 2.3 Hz, ArH), 7.69 (1H, dd, J = 8.6, 2.3 Hz, ArH), 7.66 (1 H, ά, J = 8.6 Hz, ArH), 7.18 (1 H, dd, J = 8.9, 2.3 Hz, ArH), 2.71 (3H, s, CH3); APCI-MS 371 (M-H)+; FAB-HRMS calcd for C14H11CI2N2O2S2 (MH+) 372.9639, found 372.9651. N-(2-Methylbenzothiazol-6-yl)-N-(2,5-dichlorophenylsulphonyl)-2,5-dichloro- benzenesulphonamide (STX889, XDS01188A)
Yellow solid. TLC single spot at Rf 0.72 (10% methanol/DCM); HPLC purity 94% (tR 2.9 min in 10% water-methanol); 1HNMR (270MHz, DMSO-d6) δ 8.13 (1 H, d, J = 2.2 Hz, ArH),, 7.99 (2H, d, J = 2.4 Hz, ArH), 7.90-7.94 (3H, m, ArH), 7.77 (2H, d, J = 8.4 Hz, ArH), 7.29 (1 H, dd, J = 8.6, 2.2 Hz, ArH), 2.86 (3H, s, CH3); APCI-MS 581 (M)+; FAB- HRMS calcd for C20H13CI4N2O4S3 (MH+) 580.8792, found 580.8777.
N-(2-Methylbenzothiazol-6-yl)-4-propylbenzenesulphonamide (STX890, XDS01189)
Off-white solid. TLC single spot at Rf 0.72 (10% methanol/DCM); HPLC purity 99% (tR 2.3 min in 10% water-methanol); 1HNMR (270MHz, DMSO-d6) δ 10.3 (1 H, s, NH), 7.67 (1 H, d, J = 8.7 Hz, ArH) 7.64 (1 H, d, J = 1.5 Hz, ArH), 7.59 (2H, d, J = 7.7 Hz, ArH), 7.27 (2H, d, J = 7.7 Hz, ArH), 7.09 (1 H, dd, J = 8.7, 1.5 Hz, ArH), 2.65 (3H, s, CH3), 2.49 (2H, t, J = 7.9 Hz, CH2), 1.47 (2H, sextet, J = 7.9 Hz, CH2), 0.58 (3H, t, J = 7.9 Hz, CH3); APCI-MS 345 (M-H)+; FAB-HRMS calcd for C17H19N2O2S2 (MH+) 347.0888, found 347.0904.
3-Chloro-2-methyl-N-(2-oxo-2,3-dihydro-benzothiazol-6-yl)-benzenesulphonamide (STX753, XDS01143)
White crystalline solid (160 mg; 45%). TLC single spot at Rf 0.42 (17% EtOAc/DCM); HPLC purity 98% (tR 2.3 min in 10% water-methanol); 1HNMR (400MHz, DMSO-d6) δ 11.8 (1 H, s, 3-NH), 10.5 (1 H, s, SO2NH), 7.81 (1 H, dd, J = 8, 1 Hz, 6'-H of benzene), 7.71 (1 H, dd, J = 8, 1 Hz, 4'-H of benzene), 7.36 (1 H, t, J = 8 Hz, 5'-H of benzene), 7.28 (1 H, d, J = 2 Hz, 7-H of benzothiazole), 6.93-6.98 (2H, m, 4,5-H of benzothiazole), 2.63 (3H, s, CH3); APCI-MS 353.7 (M)+; FAB-HRMS calcd for C14H12CIN2O3S2 (MH+) 354.9978, found 354.9980.
3-Chloro-N-methyl-N-(3-methyl-2-oxo-2,3-dihydro-benzothiazol-6-yl)-2- methylbenzenesulphonamide (STX831 , XDS01163)
To a solution of STX753 (66 mg, 0.19 mmol) in acetone (3 mL) was added potassium carbonate (66 mg), followed by methyl iodide (66 mg). The mixture was stirred at rt for 2 h, extracted into DCM and washed with brine. After drying over sodium sulphate, the solvent was removed in vacuo to give an oily residue that was purified with flash chromatography. Off white solid (59 mg, 80%) was obtained. TLC single spot at Rf 0.37 (100% DCM); HPLC purity 99% (tR 2.0 min in 10% water-methanol); 1HNMR (400MHz, DMSO-d6) δ 7.73-7.80 (2H, m, ArH), 7.61 (1H, d, J = 2.1 Hz, ArH), 7.42 (1H, t, J = 8.0 Hz, ArH), 7.29 (1H, d, J = 8.8 Hz, ArH), 7.21 (1H, dd, J = 8.1, 2.1 Hz, ArH), 3.38 (3H, s, CH3), 3.32 (3H, s, CH3), 2.33 (3H, s, CH3); APCI-MS 383 (MH)+; FAB-HRMS calcd for C-I6H16CIN2O3S2 (MH+) 383.0291 , found 383.0273.
[(3-Chloro-2-methylbenzenesulphonyl)-(3-ethoxycarbonylmethyl-2-oxo-2,3- dihydro-benzothiazol-6-yl)-amino]-acetic acid ethyl ester (STX764, XDS01149) To a solution of STX753 (20 mg, 0.056 mmol) in acetone (3 mL) was added potassium carbonate (20 mg), followed by methyl 2-bromoethyl acetate (50 μl). The mixture was stirred at rt for 4 h, extracted into EtOAc and washed with brine. After drying over sodium sulphate, the solvent was removed in vacuo to give an oily residue that was purified with flash chromatography. White crystalline solid (20 mg, 68%) was obtained. TLC single spot at Rf 0.51 (30% ethyl acetate/hexane); HPLC purity 98% (tR 2.6 min in 10% water-methanol); HNMR (400MHz, DMSO-d6) δ 7.75-7.78 (2H, m, ArH), 7.70 (1 H, d, J = 2.3 Hz, ArH), 7.37 (1 H, t, J = 8.2 Hz, ArH), 7.30 (1 H, d, J = 8.6 Hz, ArH), 7.24 (1 H, dd, J = 8.6, 2.3 Hz, ArH), 4.81 (2H, s, CH2), 4.56 (2H, s, CH2), 4.14 (2H, q, J = 7.0 Hz, CH2), 4.06 (2H, q, J = 7.0 Hz, CH2), 2.44 (3H, s, CH3), 1.19 (3H, t, J = 7.0 Hz, CH3), 1.13 (3H, t, J = 7.0 Hz, CH3); FAB-MS 527 (MH)+; FAB-HRMS calcd for C22H24CI 2O7S2 (MH+) 527.0713, found 527.0694.
Synthesis of 2-chlorobenzothiazol-6-yl-amine and 2-chloro-benzothiazol-5-yl- amine
To a solution of 2-chlorobenzothiazole (12.0 g, 70.7 mmol) in concentrated H2SO4 (60 mL) was added HNO3 (69% solution, 6 mL) dropwise at 0°C for 20 min. The mixture was stirred at 5°C for 3h, poured into ice-water (150 mL). The precipitate was collected and washed with 5% sodium bicarbonate and water, dried in vacuo. 1H NMR analysis showed the mixture contained 78% 6-nitro-2-chlorobenzothiazole and 8% 5-nitro-2- chlorobenzothiazole. Recrystallization from ethanol gave 6-nitro-2-chlorobenzothiazole as white crystalline solid (11 g, 72%). 3.5 g of the solid was dissolved in refluxing ethanol-acetic acid (150 : 15 mL), Iron powder was added in one portion.. The mixture was refluxed for 1.5h, filtered. The filtrate was concentrated in vacuo to half volume and neutralized with 10% NaOH to pH 7.5, extracted with ethyl acetate. The organic phase was washed with brine, dried over magnesium sulphate and evaporated to give a residue, which was recrystallized from ethanol. Light purple crystals (2.5 g, 83%) were obtained. Mp 160-164°C; TLC single spot at Rf 0.27 (30% EtOAc/hexane); ΗNMR (270 MHz, DMSO-d6) δ 7.58 (1 H, d, J = 9.0 Hz, 4-H), 7.03 (1 H, d, J = 2.0 Hz, 7-H), 6.77 (1 H, dd, J = 9.0, 2.0 Hz, 5-H), 5.55 (2H, s, NH2).
The mother liquor from the recrystallization of nitration product was evaporated and subjected to iron powder reduction as described above. The crude product was purified with flash chromatography (ethyl acetate-DCM gradient elution) to give 2-chloro- benzothiazol-5-yl-amine as yellow solid. Mp 146-149°C; TLC single spot at Rf 0.52 (10% EtOAc/DCM); 1HNMR (270 MHz, DMSO-d6) δ 7.63 (1 H, d, J = 8.6 Hz, 7-H), 7.05 (1 H, d, J = 2.3 Hz, 4-H), 6.78 (1 H, dd, J = 8.6, 2.3 Hz, 6-H), 5.40 (2H, s, NH2).
The following compounds were synthesized with the general method for N- benzothiazole benzenesulphonamide:
N-(2-chlorobenzothiazol-6-yl)-N-(3-chloro-2-methylphenylsulphonyl)-3-chloro-2- methylbenzenesulphonamide (STX767, XDS01151A) White crystalline solid. TLC single spot at Rf 0.78 (33% EtOAc/DCM); HPLC purity 95% (tR 6.4 min in 10% water-methanol); 1HNMR (270MHz, DMSO-d6) δ 8.21 (1H, d, J = 1.3 Hz, ArH), 8.00 (1 H, d, J = 8.8 Hz, ArH), 7.83-7.90 (4H, m, ArH), 7.46 (2H, t, J = 8.0 Hz, ArH), 7.37 (1 H, dd, J = 8.8, 1.8 Hz, ArH), 2.33 (6H, s, 2 x CH3); APCI-MS 560 (M)+; FAB-HRMS calcd for C21 H16CI3N2O4S3 (MH+) 560.9338, found 560.9344.
3-Chloro-N-(2-chlorobenzothiazol-6-yl)-2-methylbenzenesulphonamide (STX768,
XDS01151B)
Off-white crystalline solid. TLC single spot at Rf 0.68 (33% EtOAc/DCM); HPLC purity
99% (tR 1.7 min in methanol); 1HNMR (270MHz, DMSO-d6) δ 10.9 (1 H, s, NH), 7.91 (1H, d, J = 8.1 Hz, ArH), 7.82 (1H, d, J = 8.8 Hz, ArH), 7.80 (1H, d, J = 3.1 Hz, ArH), 7.70 (1 H, d, J = 8.1 Hz, ArH), 7.36 (1 H, t, J = 8.1 Hz, ArH), 7.23 (1H, dd, J = 8.8, 3.0 Hz, ArH), 2.63 (3H, s, CH3); APCI-MS 372 (M)+; FAB-HRMS calcd for C14H 1CI2N2O2S2 (MH+) 372.9639, found 372.9651.
3-Chloro-N-(2-chlorobenzothiazol-5-yl)-2-methylbenzenesulphonamide (STX834, XDS01168)
White crystalline solid. TLC single spot at Rf 0.52 (30% EtOAc/hexane); HPLC purity 99% (tR 1.7 min in methanol); 1HNMR (270MHz, DMSO-d6) δ 10.9 (1 H, s, NH), 7.91- 7.96 (2H, m, ArH), 7.71 (1H, d, J = 8.1 Hz, ArH), 7.58 (1H, d, J = 2.2 Hz, ArH), 7.39 (1 H, d, J = 8.1 Hz, ArH), 7.22 (1 H, dd, J = 8.1 , 2.2 Hz, ArH), 2.64 (3H, s, CH3); APCI-MS 371 (M-H)+; FAB-HRMS calcd for C14H11 CI2N2O2S2 (MH+) 372.9639, found 372.9656. 3-Chloro-2-methyl-N-(2-methylaminobenzothiazol-6-yl)-benzenesulphonamide (STX833, XDS01167)
The solution of 3-chloro-N-(2-chlorobenzothiazol-6-yl)-2-methylbenzenesulphonamide (STX768, 150 mg, 0.40 mmol) in CH3NH-THF (2M, 3 mL) was stirred at 82°C in a sealed tube for 24h, extracted with ethyl acetate. The organic phase was washed brine, dried over sodium sulphate and concentrated in vacuo to give a residue that was purified with flash chromatography (ethyl acetate/DCM gradient elution). White crystals (100 mg, 68%) were obtained. TLC single spot at Rf 0.27 (30% EtOAc/DCM); HPLC purity 99% (tR 1.8 min in 4% water-methanol); HNMR (270MHz, DMSO-d6) δ 10.2 (1 H, s, NH), 7.82 (1 H, q, J = 4.8 Hz, NH), 7.72 (1 H, d, J = 7.7 Hz, ArH), 7.61 (1 H, d, J = 7.7 Hz, ArH), 7.29 (1 H, d, J = 2.2 Hz, ArH), 7.26 (1 H, t, J = 8.0 Hz, ArH), 7.15 (1 H, d, J = 8.7 Hz, ArH), 6.79 (1H, dd, J = 8.7, 2.2 Hz, ArH), 2.80 (3H, d, J = 4.8 Hz, NCH3), 2.54 (3H, s, CH3); APCI-MS 366 (M-H)+; FAB-HRMS calcd for C15H15CIN3O2S2 (MH+) 368.0294, found 368.0292.
3-Chloro-2-methyl-N-(2-methylaminobenzothiazol-5-yl)-benzenesulphonamide (STX835, XDS01176)
The compound was prepared as described for STX833 using 3-chloro-N-(2- chlorobenzothiazol-5-yl)-2-methylbenzenesulphonamide (STX834, 80 mg, 0.21 mmol) as starting material. White crystals (60 mg, 78%) were obtained. TLC single spot at Rf 0.25 (30% EtOAc/DCM); HPLC purity 99% (tR 2.3 min in 10% water-methanol); 1HNMR (270MHz, DMSO-d6) δ 10.5 (1 H, s, NH), 7.96 (1H, q, J = 4.7 Hz, NH), 7.86 (1 H, d, J = 8.1 Hz, ArH), 7.69 (1 H, d, J = 8.1 Hz, ArH), 7.47 (1 H, d, J = 8.0 Hz, ArH), 7.37 (1H, t, J = 8.1 Hz, ArH), 7.05 (1 H, d, J = 1.9 Hz, ArH), 6.73 (1 H, dd, J = 8.0, 1.9 Hz, ArH), 2.88 (3H, d, J = 4.7 Hz, NCH3), 2.64 (3H, s, CH3); APCI-MS 368 (MH)+; FAB-HRMS calcd for C15H-15CIN3O2S2 (MH+) 368.0294, found 368.0292.
3-Chloro-N-(2-diethylaminobenzothiazol-5-yl)-2-methylbenzenesulphonamide (STX836. XDS01177)
The compound was prepared as described for STX833 using 3-chloro-N-(2- chlorobenzothiazol-5-yl)-2-methylbenzenesulphonamide (STX834, 70 mg, 0.18 mmol) and diethylamine-THF (3 mL) as starting material. White crystals (50 mg, 68%) were obtained. TLC single spot at Rf 0.60 (30% EtOAc/DCM); HPLC purity 97% (tR 3.0 min in 10% water-methanol)} 1HNMR (270MHz, DMSO-d6) δ 10.5 (1 H, s, NH), 7.85 (1 H, d, J = 7.9 Hz, ArH), 7.68 (1 H, d, J = 8.0 Hz, ArH), 7.52 (1 H, d, J = 8.4 Hz, ArH), 7.36 (1 H, t, J = 8.0 Hz, ArH), 7.06 (1H, d, J = 2.2 Hz, ArH), 6.75 (1H, dd, J = 8.3, 2.2 Hz, ArH), 3.45 (4H, q, J = 7.0 Hz, N(CH2)2), 2.63 (3H, s, CH3), 1.15 (6H, t, J = 7.0 Hz, 2 x CH3); APCI- MS 410 (MH)+; FAB-HRMS calcd for C18H21CIN3O2S2 (MH+) 410.0764, found 410.0753.
3-Chloro-N-(2-diethylaminobenzothiazol-6-yl)-2-methylbenzenesulphonamide (STX878, XDS01164)
The compound was prepared as described for STX833 using 3-chloro-N-(2- chlorobenzothiazol-6-yl)-2-methylbenzenesulphonamide (STX768, 240 mg, 0.64 mmol) and diethylamine-IPA (3 mL) as starting material. Off-white crystalline solid (128 mg, 49%) were obtained. TLC single spot at Rf 0.33 (30% EtOAc/hexane); HPLC purity 96% (tR 2.2 min in 4% water-methanol); 1HNMR (270MHz, DMSO-d6) δ 7.56-7.62 (3H, m, ArH), 7.18-7.27 (2H, m, ArH), 7.00 (1 H, dd, J = 8.5, 1.7 Hz, ArH), 5.52 (1 H, s, NH), 3.54 (4H, q, J = 7.0 Hz, N(CH2)2), 2.25 (3H, s, CH3), 1.22 (6H, t, J = 7.0 Hz, 2 x CH3); APCI- MS 409 (M)+; FAB-HRMS calcd for C18H21CIN3O2S2 (MH+) 410.0764, found 410.0698.
Synthesis of 2,6-Dimethylbenzothiazol-7-ylamine
To a solution of 2,6-dimethylbenzothiazol (350 mg, 2.15 mmol) in Cone. H2SO4 (4 mL) was added HNO3 (69%, 0.3 mmol) at 0°C. After stirred at 0°C for 0.5h, the mixture was poured over ice-water. The precipitate was collected and washed with 5% sodium bicarbonate and water, recrystallized from ethanol to give 7-nitro-2,6- dimethylbenzothiazol as yellow solid (160 mg). The product (150 mg) was hydrogenated over 5% Pd/C in ethanol-THF (10 : 2 mL) at atmosphere pressure to give 2,6-dimethyl- benzothiazol-7-ylamine as yellow solid (120 mg). TLC single spot at Rf 0.55 (10% EtOAc/DCM); 1H NMR (270 MHz, DMSO): δ 7.07 (2H, s, ArH), 5.23 (2H, s, NH2), 2.73 (3H, s, CH3), 2.20 (3H, s, CH3).
Synthesis of 6-Methoxy-2-methylbenzothiazol-7-ylamine
The compound was prepared as described above starting from 6-methoxy-2- methylbenzothiazol. Yellow solid was obtained, mp 117-119°C (lit.121-122°C); TLC single spot at Rf 0.55 (40% EtOAc/DCM); 1H NMR (270 MHz, DMSO): δ 7.14 (1 H, d, J =
8.7 Hz, ArH), 7.05 (1 H, d, J = 8.7 Hz, ArH), 5.10 (2H, broad, NH2), 3.83 (3H, s, OCH3),
2.71 (3H, s, CH3).
(Friedman, S.G. J Gen Chem USSR 31 , 1961 , 3162-3167) Synthesis of 2,5-Dimethylbenzothiazol-4-ylamine and 2,5-Dimethylbenzothiazol-6- ylamine
To a solution of 2,5-dimethylbenzothiazol (1.63 g, 10 mmol) in Cone. H2SO4 (12 mL) was added HNO3 (69%, 1 mmol) at -5°C. After stirred at -5 - 0°C for 2h, the mixture was poured over ice-water (150 mL). The precipitate was collected and washed with 5% sodium bicarbonate, water and 70% ethanol. The product (1.98 g) was a mixture of 4- nitro-2,5-dimethylbenzothiazol and 6-nitro-2,5-dimethylbenzothiazol in 1 : 1 ratio judged by NMR. The product (998 mg) was hydrogenated over 5% Pd/C (600 mg) in ethanol- THF (50 : 20 mL) at atmosphere pressure to give a yellow solid (880 mg). Separation with flash chromatography (EtOAc/DCM gradient elution) yielded 2,5- dimethylbenzothiazol-4-ylamine as yellow crystals (400 mg). TLC single spot at Rf 0.60 (15% EtOAc/DCM); 1H NMR (270 MHz, DMSO): δ 7.05 (1 H, d, J = 8.0 Hz, ArH), 6.99 (1H, d, J = 8.0 Hz, ArH), 5.26 (2H, s, NH2), 2.74 (3H, s, CH3), 2.18 (3H, s, CH3); APCI- MS 177 (M-H)+. 2,5-Dimethylbenzothiazol-6-ylamine was obtained as yellow solid (320 mg). TLC single spot at Rf 0.55 (15% EtOAc/DCM); 1H NMR (270 MHz, DMSO): δ 7.47 (1H, s, ArH), 7.05 (1 H, s, ArH), 5.05 (2H, s, NH2), 2.65 (3H, s, CH3), 2.16 (3H, s, CH3); APCI-MS 177 (M- H)+.
Synthesis of 4-chloro-2-methylbenzothiazol-5-ylamine and 4,6-dichloro-2- methylbenzothiazol-5-ylamine
To a solution of 5-amino-2-methylbenzothiazole (818 mg, 4.99 mmol) in isopropanol (12 mL) was added N-chlorosuccinimide (732 mg, 5.48 mmol). The mixture was stirred at 60°C for 15 min., partitioned between DCM and 5% sodium bicarbonate. The organic phase was washed with brine, dried over sodium sulphate and concentrated in vacuo to give a residue that was purified with flash chromatography (EtOAc/DCM gradient elution). 4-Chloro-2-methylbenzothiazol-5-ylamine was obtained as off-white crystalline solid (510 mg, 51 %). mp 121-122°C (lit.124°C); TLC single spot at Rf 0.51 (20% EtOAc/DCM); 1H NMR (270 MHz, DMSO): δ 7.61 (1 H, d, J = 8.6 Hz, ArH), 6.91 (1 H, d, J = 8.6 Hz, ArH), 5.49 (2H, s, NH2), 2.75 (3H, s, CH3); APCI-MS 199 (MH)+.
4,6-Dichloro-2-methylbenzothiazol-5-ylamine was obtained as yellow solid (60 mg, 5%). TLC single spot at Rf 0.57 (20% EtOAc/DCM); 1H NMR (270 MHz, DMSO): δ 7.89 (1 H, s, ArH), 5.26 (2H, s, NH2), 2.77 (3H, s, CH3); APCI-MS 233 (MH)+. The following compounds were synthesized with the general method for N- benzothiazole benzenesulphonamide.
3-Chloro-N-(6-methoxy-2-methylbenzothiazol-7-yl)-2-methylbenzenesulphonamide (STX989, XDS02038)
White crystalline solid. TLC single spot at Rf 0.71 (30% EtOAc/DCM); HPLC purity 99% (tR 2.3 min in 10% water-methanol); 1H NMR (270 MHz, DMSO-d6) δ 10.1 (1 H, s, NH), 7.76 (1 H, d, J = 8.9 Hz, ArH), 7.72 (1 H, d, J = 8.2 Hz, ArH), 7.53 (1 H, d, J = 8.2 Hz, ArH), 7.23 (1 H, t, J = 8.2 Hz, ArH), 7.05 (1H, d, J = 8.9 Hz, ArH), 3.29 (3H, s, OCH3), 2.74 (3H, s, CH3), 2.70 (3H, s, CH3); APCI-MS 381 (M-H)+; FAB-HRMS calcd for C16H16CIN2O3S2 (MH+) 383.0291 , found 383.0284.
3-Chloro-N-(2,6-dimethyl-benzothiazol-7-yl)-2-methyl-benzenesulphonamide (STX1021, XDS02069)
Off-white crystalline solid. TLC single spot at Rf 0.49 (10% EtOAc/DCM); HPLC purity 98% (tR 2.0 min in 20% water-methanol); 1H NMR (270 MHz, DMSO-d6) δ 10.3 (1 H, s, NH), 7.78 (1 H, d, J = 7.9 Hz, ArH), 7.74 (1 H, d, J = 8.4 Hz, ArH), 7.64 (1 H, d, J = 7.9 Hz, ArH), 7.34 (1 H, t, J = 7.9 Hz, ArH), 7.30 (1 H, d, J = 7.9 Hz, ArH), 2.68 (3H, s, CH3), 2.61 (3H, s, CH3), 2.03 (3H, s, CH3); FAB-MS 367 (MH)+; FAB-HRMS calcd for C16H16CIN2O2S2 (MH+) 367.0342, found 367.0347.
3-Chloro-N-(2,5-dimethyl-benzothiazol-4-yl)-2-methyl-benzenesulphonamide (STX996, XDS02047) White crystalline solid. TLC single spot at Rf 0.76 (10% EtOAc/DCM); HPLC purity >99% (tR 2.9 min in 10% water-methanol); Η NMR (270 MHz, DMSO-d6) δ 9.98 (1H, s, NH), 7.81 (1 H, d, J = 8.3 Hz, ArH), 7.64 (1 H, d, J = 7.9 Hz, ArH), 7.45 (1 H, d, J = 7.9 Hz, ArH), 7.31 (1 H, d, J = 8.3 Hz, ArH), 7.12 (1 H, t, J = 7.9 Hz, ArH), 2.73 (3H, s, CH3), 2.47 (3H, s, CH3), 2.44 (3H, s, CH3); APCI-MS 367 (MH)+; FAB-HRMS calcd for C16H16CIN2O2S2 (MH+) 367.0342, found 367.0342.
N-(2,5-dimethylbenzothiazol-6-yl)-N-(3-chloro-2-methylphenylsulphonyl)-3-chloro- 2-methylbenzenesulphonamide (STX997, XDS02048A)
Off-white syrup. TLC single spot at Rf 0.78 (10% EtOAc/DCM); HPLC purity 85% (tR 4.2 min in 10% water-methanol); 1H NMR (270 MHz, DMSO-d6) δ 8.06 (1 H, s, ArH), 7.86-
7.95 (5H, m, ArH), 7.68 (1 H, s, ArH), 7.52 (2H, t, J = 8.2 Hz, ArH), 2.83 (3H, s, CH3), 2.29 (6H, s, 2 x CH3), 2.05 (3H, s, CH3); APCI-MS 555 (MH)+; FAB-HRMS calcd for C23H21CI2N2O4S3 (MH+) 555.0040, found 555.0041.
3-Chloro-N-(2,5-dimethylbenzothiazol-6-yl)-2-methylbenzenesulphonamide (STX998, XDS02048B)
White crystalline solid. TLC single spot at Rf 0.39 (10% EtOAc/DCM); HPLC purity 96% (tR 2.1 min in 10% water-methanol); 1H NMR (270 MHz, DMSO-d6) δ 10.0 (1 H, s, NH), 7.74 (1 H, d, J = 7.9 Hz, ArH), 7.70 (1 H, s, ArH), 7.67 (1 H, d, J = 7.9 Hz, ArH), 7.63 (1 H, s, ArH), 7.33 (1 H, t, J = 7.9 Hz, ArH), 2.75 (3H, s, CH3), 2.60 (3H, s, CH3), 2.13 (3H, s, CH3); APCI-MS 367 (MH)+; FAB-HRMS calcd for C16H16CIN2O2S2 (MH+) 367.0342, found 367.0350.
2,5-Dichloro-N-(2,5-dimethylbenzothiazol-6-yl)-benzenesulphonamide (STX999, XDS02049) White crystalline solid. TLC single spot at Rf 0.43 (10% EtOAc/DCM); HPLC purity 98% (tR 2.0 min in 10% water-methanol); H NMR (270 MHz, DMSO-d6) δ 10.3 (1 H, s, NH), 7.76 (3H, s, ArH), 7.72 (1 H, s, ArH), 7.67 (1 H, s, ArH), 2.75 (3H, s, CH3), 2.23 (3H, s, CH3); APCI-MS 387 (MH)+; FAB-HRMS calcd for C15H13CI2N2O2S2 (MH+) 386.9795, found 386.9806.
N-(4-Chloro-2-methyl-benzothiazol-5-yl)-N-(3-chloro-2-methylphenylsuIphonyl)-3- chloro-2-methyl-benzenesulphonamide (STX991, XDS02042A) White powder. TLC single spot at Rf 0.75 (8% EtOAc/DCM); HPLC purity >99% (tR 4.4 min in 10% water-methanol); 1H NMR (270 MHz, DMSO-d6) δ 8.19 (1 H, d, J = 8.7 Hz, ArH), 7.93 (4H, d, J = 8.2 Hz„ ArH), 7.59 (1 H, d, J = 8.7 Hz, ArH), 7.50 (2H, d, J = 8.2 Hz, ArH), 2.85 (3H, s, CH3), 2.41 (6H, s, 2 x CH3); APCI-MS 575 (MH)+; FAB-HRMS calcd for C22HI8CI3N2O4S3 (MH+) 574.9494, found 574.9492.
3-Chloro-N-(4-chloro-2-methylbenzothiazol-5-yl)-2-methylbenzenesulphonamide (STX992, XDS02042B)
White crystalline solid. TLC single spot at Rf 0.69 (8% EtOAc/DCM); HPLC purity 99% (tR 2.5 min in 10% water-methanol); 1H NMR (270 MHz, DMSO-d6) δ 10.5 (1 H, s, NH), 7.96 (1 H, d, J = 8.7 Hz, ArH), 7.73 (1 H, d, J = 7.9 Hz„ ArH), 7.64 (1 H, d, J = 7.9 Hz, ArH), 7.30 (1 H, d, J = 8.7 Hz, ArH), 7.29 (1 H, t, J = 7.9 Hz„ ArH), 2.79 (3H, s, CH3), 2.70 (3H, s, CH3); APCI-MS 385 (M-H)+; FAB-HRMS calcd for C15H13CI2N2O2S2 (MH+) 386.9795, found 386.9790.
2,5-Dichloro-N-(4-chloro-2-methylbenzothiazol-5-yl)-benzenesulphonamide (STX993, XDS02043B)
White crystalline solid. TLC single spot at Rf 0.71 (8% EtOAc/DCM); HPLC purity 99% (tR 5.0 min in 10% water-methanol); 1H NMR (270 MHz, DMSO-d6) δ 10.7 (1 H, s, NH), 7.97 (1 H, d, J = 8.6 Hz, ArH), 7.71-7.78 (3H, m„ ArH), 7.28 (1H, d, J = 8.6 Hz, ArH), 2.81 (3H, s, CH3); APCI-MS 407 (MH)+; FAB-HRMS calcd for C-14H10CI3N2O2S2 (MH+) 406.9249, found 406.9234.
N-(4-Chloro-2-methylbenzothiazol-5-yl)-4-propylbenzenesulphonamide (STX994, XDS02044B)
White crystalline solid. TLC single spot at Rf 0.70 (8% EtOAc/DCM); HPLC purity 99% (tR 2.7 min in 10% water-methanol); 1H NMR (270 MHz, DMSO-d6) δ 10.1 (1 H, s, NH), 7.93 (1 H, d, J = 8.6 Hz, ArH), 7.60 (2H, d, J = 8.2 Hz, ArH), 7.35 (2H, d, J = 8.2 Hz, ArH), 7.28 (1H, d, J = 8.4 Hz, ArH), 2.79 (3H, s, CH3), 2.69 (2H, t, J = 7.2 Hz, CH2), 1.59 (2H, m, CH2), 0.86 (3H, t, J = 7.2 Hz, CH3); APCI-MS 381 (MH)+; FAB-HRMS calcd for C17H-I8CIN2O2S2 (MH+) 381.0498, found 381.0484.
N-(4-Chloro-2-methylbenzothiazol-5-yl)-N-(4-propylphenylsulphonyl)-4- propylbenzenesulphonamide (STX995, XDS02044A)
White powder. TLC single spot at Rf 0.70 (8% EtOAc/DCM); HPLC purity 99% (tR 3.8 min in 10% water-methanol); 1H NMR (270 MHz, DMSO-d6) δ 8.10 (1 H, d, J = 8.4 Hz, ArH), 7.74 (4H, d, J = 8.1 Hz, ArH), 7.50 (4H, d, J = 8.1 Hz, ArH), 7.08 (1H, d, J = 8.4 Hz, ArH), 2.91 (3H, s, CH3), 2.71 (4H, t, J = 7.1 Hz, 2 x CH2), 1.59 (4H, m, CH2), 0.86 (6H, t, J = 7.1 Hz, 2 x CH3); APCI-MS 561 (M-H)+; FAB-HRMS calcd for C26H28CIN2O4S3 (MH+) 563.0900, found 563.0886.
Synthesis of 3-Chloro-2-methyl-N-(2-methyl-benzothiazol-5-ylmethyl)- benzenesulphonamide (STX1029, XDS02070A) and 3-Chloro-2-methyl-N,N-bis-(2- methyl-benzothiazol-5-ylmethyl)-benzenesulphonamide (STX1030, XDS02070B)
To a solution of 3-chloro-2-methylbenzenesulphonamide (103 mg, 0.5 mmol) in CH3CN was added potassium carbonate (100 mg), followed 5-bromomethyl-2- methylbenzothiazole (121mg, 0.5 mmol). The mixture was refluxed under N2 for 6h, partitioned between ethyl acetate and water. The organic phase was washed brine, dried over sodium sulphate and concentrated in vacuo to give a yellow residue, which was separated with flash chromatography (ethyl acetate/DCM, gradient elution). STX1029 was obtained as white solid. TLC single spot at Rf 0.55 (10% EtOAc/DCM); HPLC purity >99% (tR 2.0 min in 10% water-methanol); 1HNMR (270 MHz, CDCI3) δ 7.89 (1H, d, J = 7.9 Hz, ArH), 7.66 (1 H, d, J = 7.9 Hz, ArH), 7.65 (1H, d, J - 1.3 Hz, ArH), 7.49 (1 H, d, J = 7.9 Hz, ArH), 7.17 (1H, t, J = 7.9 Hz, ArH), 7.13 (1 H, dd, J = 7.9, 1.5 Hz, ArH), 5.35 (1 H, t, J = 5.9 Hz, NH), 4.24 (2H, d, J = 5.9 Hz, CH2), 2.79 (3H, s, CH3), 2.62 (3H, s, CH3); APCI-MS 367 (MH)+; FAB-HRMS calcd for C16H16CIN2O2S2 (MH+) 367.0342, found 367.0330.
STX1030 was obtained as white solid. TLC single spot at Rf 0.50 (10% EtOAc/DCM); HPLC purity 99% (tR 6.1 min in 20% water-methanol); Η NMR (270 MHz, DMSO-d6) δ 7.87 (3H, d, J = 8.1 Hz, ArH), 7.75 (1H, d, J = 8.0 Hz, ArH), 7.59 (2H, broad w1/2 = 1.1 Hz, ArH), 7.38 (1 H, t, J = 8.0 Hz, ArH), 7.11 (2H, dd, J = 8.1 , 1.1 Hz, ArH), 4.56 (4H, s, 2 x NCH2), 2.78 (6H, s, 2 x CH3), 2.58 (3H, s, CH3); APCI-MS 528 (MH)+; FAB-HRMS calcd for C25H23CIN3O2S3 (MH+) 528.0641 , found 528.0630.
Synthesis of N-lndole or N-lndoline Arysulfonamide Derivatives
Figure imgf000108_0001
STX832, XDS01165 STX981 , XDS02019 STX982, XDS02020
Figure imgf000108_0002
. XDS02063 STX984, XDS02025 STX987, XDS02031 General method for synthesis N-indole or N-indoline arylsulphonamide derivatives (STX832, STX981-982, STX984, STX986-987, STX1018-1020):
To a solution arylsulphonyl chloride (1.1 eq.) in DCM were added pyridine (2.2 eq.) and catalytic amount of DMAP, followed by the corresponding amine (1 eq.). The reaction mixture was stirred at rt under nitrogen for 4-6 h, then partitioned between ethyl acetate and 5% sodium bicarbonate after TLC showed completion of the reaction. The organic layer was washed with brine, dried over sodium sulphate, and concentrated in vacuo to give crude product as solid or thick syrup. The compound was then purified by flash chromatography (methanol-DCM gradient elution) to give desired arylsulphonamide as crystalline solid. Yield ranges from 50-85%.
3-Chloro-2-methyl-N-(2-methyl-1H-indol-5-yl)-benzenesulphonamide (STX832,
XDS01165)
White crystalline solid. TLC single spot at Rf 0.68 (30% ethyl acetate/hexane); HPLC purity > 99 % (tR 1.8 min in 4 % water-methanol); 1H NMR (270 MHz, DMSO): δ 10.9 (1 H, s, NH), 9.98 (1 H, s, NH), 7.72 (1H, d, J = 8 Hz, ArH), 7.63 (1 H, d, J = 8 Hz, ArH), 7.27 (1 H, t, J = 8 Hz, ArH), 7.07 (1 H, d, J = 8 Hz, ArH), 7.05 (1 H, d, J = 2 Hz, ArH), 6.67 (1 H, dd, J=8, 2 Hz, ArH), 5.99 (1H, s, 3-H), 2.60 (3H, s, CH3), 2.29 (3H, s, CH3); APCI- MS 334 (M+); FAB-HRMS calcd for C16H16CIN2O2S (MH+) 335.0621 , found 335.0609
3-Chloro-2-methyl-N-(1H-indol-5-yl)-benzenesulphonamide (STX981, XDS02019) White crystalline solid. TLC single spot at Rf 0.72 (6% methanol/DCM); HPLC purity 98 % (tR 2.1 min in 10 % water-methanol); 1H NMR (270 MHz, DMSO): δ 11.1 (1 H, s, NH), 10.1 (1 H, s, NH), 7.76 (1 H, d, J = 7.9 Hz, ArH), 7.66 (1 H, d, J = 7.9 Hz, ArH), 7.22-7.32 (4H, m, ArH), 6.81 (1 H, dd, J=7.9, 1.2 Hz, ArH), 6.33 (1H, broad, 3-H), 2.64 (3H, s, CH3); APCI-MS 319 (M-H+); FAB-HRMS calcd for C15H14CIN2O2S (MH+) 321.0465, found 321.0453.
3-Chloro-2-methyl-N-(1 H-indol-6-yl)-benzenesulphonamide (STX982, XDS02020) White crystalline solid. TLC single spot at Rf 0.88 (10% methanol/DCM); HPLC purity 98 % (tR 2.5 min in 20 % water-methanol); 1H NMR (270 MHz, DMSO): δ 11.0 (1 H, s, NH), 10.3 (1 H, s, NH), 7.80 (1 H, d, J = 7.9 Hz, ArH), 7.67 (1 H, d, J = 7.9 Hz, ArH), 7.31-7.38 (2H, m, ArH), 7.26 (1 H, m, ArH), 7.80 (1H, d, J = 1.2 Hz, ArH), 6.75 (1H, dd, J=7.9, 1.2 Hz, ArH), 6.31 (1 H, broad, 3-H), 2.65 (3H, s, CH3); APCI-MS 319 (M-H+); FAB-HRMS calcd for C15H14CIN2O2S (MH+) 321.0465, found 321.0446. 5-(3-Chloro-2-methylbenzenesulfonylamino)-1 H-indole-2-carboxylic acid ethyl ester (STX986, XDS02030)
White crystalline solid. TLC single spot at Rf 0.82 (8% methanol/DCM); HPLC purity > 99 % (tR 2.3 min in 10% water-methanol); H NMR (270 MHz, DMSO): δ 11.9 (1 H, s, NH), 10.3 (1 H, s, NH), 7.78 (1 H, d, J = 7.9 Hz, ArH), 7.67 (1 H, d, J = 7.9 Hz, ArH), 7.28- 7.33 (3H, m, ArH), 7.06 (1H, d, J = 2.2 Hz, ArH), 7.00 (1 H, dd, J= 8.2, 2.2 Hz, ArH), 4.32 (2H, q, J = 6.9 Hz, OCH2), 2.63 (3H, s, CH3), 1.31 (3H, t, J = 6.9 Hz, CH3); APCI-MS 391 (M-H+); FAB-HRMS calcd for C18H18CIN2O4S (MH+) 393.0676, found 393.0659
3-Chloro-2-methyl-N-(2,3-dimethyl-1H-indol-5-yl)-benzenesulphonamide (STX1018, XDS02061)
White crystalline solid. TLC single spot at Rf 0.83 (30% ethyl acetate/hexane); HPLC purity 97 % (tR 2.9 min in 20% water-methanol); 1H NMR (270 MHz, DMSO): δ 10.6 (1H, s, NH), 10.0 (1 H, s, NH), 7.74 (1H, d, J = 7.5 Hz, ArH), 7.65 (1 H, d, J = 7.5 Hz, ArH), 7.29 (1H, t, J = 8.0 Hz, ArH), 7.05 (1H, d, J = 8.6 Hz, ArH), 6.99 (1H, d, J = 1.7 Hz, ArH), 6.66 (1 H, dd, J= 8.6, 1.7 Hz, ArH), 2.62 (3H, s, CH3), 2.25 (3H, s, CH3), 2.03 (3H, s, CH3); APCI-MS 349 (MH+); FAB-HRMS calcd for C17H-I8CIN2O2S (MH+) 349.0778, found 349.0737.
2,5-Dichloro-N-(2,3-dimethyl-1H-indol-5-yl)-benzenesulphonamide (STX1019,
XDS02062)
White amorphous powder. TLC single spot at Rf 0.82 (10% ethyl acetate/hexane); HPLC purity 98 % (tR 3.0 min in 20% water-methanol); 1H NMR (270 MHz, DMSO): δ 10.7 (1 H, s, NH), 10.2 (1 H, s, NH), 7.80 (1H, m, ArH), 7.67-7.70 (2H, m, ArH), 7.07 (1H, d, J = 8.5 Hz, ArH), 7.05 (1H, d, J = 1.7 Hz, ArH), 6.73 (1H, dd, J= 8.5, 1.7 Hz, ArH), 2.25 (3H, s, CH3), 2.04 (3H, s, CH3); APCI-MS 367 (M-H+); FAB-HRMS calcd for CI6H14CI2N2O2S (M+) 368.0153, found 368.0146
4-n-Propyl-N-(2,3-dimethyl-1 H-indol-5-yl)-benzenesulphonamide (STX1020, XDS02063)
Off-white crystalline solid. TLC single spot at Rf 0.82 (10% ethyl acetate/hexane); HPLC purity 97 % (tR 2.9 min in 20% water-methanol); 1H NMR (270 MHz, DMSO): δ 10.6 (1H, s, NH), 9.6 (1 H, s, NH), 7.56 (2H, d, J = 8.3 Hz, ArH), 7.30 (2H, d, J = 8.3 Hz, ArH), 7.03 (1H, d, J = 8.3 Hz, ArH), 6.95 (1 H, d, J = 1.7 Hz, ArH), 6.68 (1H, dd, J= 8.3, 1.7 Hz, ArH), 2.56 (2H, t, J = 7.3 Hz, CH2), 2.24 (3H, s, CH3), 2.01 (3H, s, CH3), 1.55 (2H, sextet, J = 7.3 Hz, CH2), 0.85 (3H, t, J = 7.3 Hz, CH3),; APCI-MS 343 (MH+); FAB-HRMS calcd for C19H22N2O2S (M+) 342.1402, found 342.1403
3-Chloro-2-methyl-N-(1-acetyl-2,3-dihydro-1H-indol-5-yl)-benzenesulphonamide (STX984, XDS02025)
White crystalline solid. TLC single spot at Rf 0.58 (5% methanol/DCM); HPLC purity 95 % (tR 2.2 min in 20% water-methanol); 1H NMR (270 MHz, DMSO): δ 10.4 (1H, s, NH), 7.80-7.86 (2H, m, ArH), 7.71 (1 H, d, J = 7.8 Hz, ArH), 7.36 (1 H, t, J = 8.0 Hz, ArH), 6.93 (1H, d, J = 1.8 Hz, ArH), 6.83 (1H, dd, J = 8.2, 1.8 Hz, ArH), 4.01 (2H, t, J = 8.3 Hz, CH2), 3.03 (2H, t, J = 8.4 Hz, CH2), 2.62 (3H, s, CH3), 2.09 (3H, s, CH3); APCI-MS 363 (M-H+); FAB-HRMS calcd for C17H-I8CIN2O3S (MH+) 365.0727, found 365.0796.
5-(3-Chloro-2-methyl-benzenesulfonylamino)-1-ethyl-2,3-dihydro-1H-indolium chloride (STX987, XDS02031) The free base of STX987 was synthesized as above. A purple amorphous powder was obtained; TLC single spot at Rf 0.79 (8% methanol/DCM); H NMR (270 MHz, CDCI3): δ 7.79 (1H, d, J = 7.9 Hz, ArH), 7.53 (1H, d, = 7.9 Hz, ArH), 7.15 (1 H, t, J = 8.0 Hz, ArH), 6.75 (1H, d, J = 1.8 Hz, ArH), 6.58 (1H, dd, J = 8.1 , 1.8 Hz, ArH), 6.35 (1H, s, NH), 6.22 (1H, d, J = 8.1 Hz, ArH), 3.30 (2H, t, J = 8.4 Hz, CH2), 3.05 (2H, q, J = 7.2 Hz, CH2), 2.84 (2H, t, J = 8.3 Hz, CH2), 2.67 (3H, s, CH3), 1.11 (3H, t, J = 7.2 Hz, CH3). The free base was treated with HCI-ether solution to give STX987 as light pink crystalline solid. HPLC purity 93 % (tR 3.6 min in 20 % water-methanol); 1H NMR (270 MHz, DMSO): δ 10.3 (1H, s, NH), 7.82 (1H, d, = 8.1 Hz, ArH), 7.72 (1 H, d, J = 8.1 Hz, ArH), 7.38 (1H, t, J = 8.1 Hz, ArH), 6.79-6.89 (3H, , broad, ArH), 3.42 (2H, t, J = 8.4 Hz, CH2), 3.16 (2H, q, J = 7.0 Hz, CH2), 2.91 (2H, t, J = 8.4 Hz, CH2), 2.62 (3H, s, CH3), 1.10 (3H, t, J = 7.0 Hz, CH3); APCI-MS 349 (M-HCI-H+); FAB-HRMS calcd for C17H20CIN2O2S (M-HCI+H+) 351.0934, found 351.0941.
1 -Acetyl-5-aminoindoline The solution of 1 -acetyl-5-nitroindoline (1.0 g, 4.85 mmol) in ethanol-THF (100 mL : 30 mL) was hydrogenated over 5% Pd/C (600 mg) at atmosphere pressure for 2h, filtered through Celite and concentrated in vacuo to give a white solid which was recrystalllized from ethanol. White crystalline solid (580 mg, 68%) was obtained. Mp 185-186.5°C (lit 184-185°C, [21]); 1H NMR (270 MHz, DMSO): δ 7.73 (1 H, d, J = 8.6 Hz, ArH), 6.45 (1H, s broad, w1/2 = 1.8 Hz, ArH), 6.33 (1 H, dd, J = 8.6, 1.8 Hz, ArH), 4.82 (2H, s, NH2), 3.97 ill
(2H, t, J = 8.4 Hz, CH2), 2.99 (2H, t, J = 8.4 Hz, CH2), 2.07 (3H, s, CH3); APCI-MS 175 (M-H+).
1-Ethyl-5-aminoindoline To a suspension of 1-acetyl-5-aminoindoline (130 mg, 0.74 mmol) in anhydrous THF (10 mL) was added LiAIH4 (42 mg, 1.11 mmol). The mixture was stirred at rt for 6h, quenched with saturated NH4CI and extracted with ethyl acetate. The organic phase was washed with brine, dried over sodium sulphate and concentrated in vacuo to give a purple residue (80 mg, 67%) that was used without further purification. 1H NMR (270 MHz, DMSO): δ 6.56 (1 H, s, ArH), 6.47 (1 H, d broad, J = 8.1 Hz, ArH), 6.37 (1 H, d, J = 8.0 Hz, ArH), 3.29 (2H, s, NH2), 3.20 (2H, t, J = 7.6 Hz, CH2), 3.02 (2H, q, J = 6.9 Hz, CH2), 2.86 (2H, t, J = 7.6 Hz, CH2), 1.17 (3H, t, J = 6.9 Hz, CH3).
Synthesis of 5-(3-chloro-2-methyl-benzenesulf onamino)-1 H-indole-3-carboxylic acid methyl ester, STX 1050 (KRB01132):
Figure imgf000112_0001
5-amino-1H-indole-3-carboxylic acid methyl ester (KRB01131): To a solution of 5- nitro-1W-indole-3-carboxylic acid methyl ester (206 mg, 0.940 mmol) in methanol (40 mL) was added 5% palladium on carbon (40 mg) and the mixture was stirred under 1 atm H2 for 5h. The mixture was filtered through celite and the filtrate evaporated to yield a brown solid that was used without further purification (173 mg, 97%), single spot at Rf 0.64 (ethyl acetate). H NMR (d6-DMSO): δ 11.50 (1H, s, N-H), 7.83 (1 H, d, =3.2 Hz), 7.17 (1H, d, J=2.0 Hz), 7.14 (1 H, d, J=8.4 Hz), 6.56 (1H, dd, J=8.6, 2.2 Hz), 4.77 (2H, s, N-V2), 3.76 (3H, s).
To a solution of 3-chloro-2-methylbenzenesulphonyl chloride (124 mg, 0.552 mmol) in dichloromethane (4 mL) was added pyridine (100 μL, 1.3 mmol) and the mixture was stirred under N2 for 5 min, after which time 5-amino-1H-indole-carboxylic acid methyl ester (100 mg, 0.526 mmol) was added. The resulting mixture was stirred for 1.5 h at room temperature, then saturated NaHCO3 solution (15 mL) was added and the mixture was extracted into ethyl acetate (20 mL). The organic phase was washed with brine, dried (Na2SO4), filtered and evaporated to give a residue that was purified using flash chromatography to afford a white solid (129 mg, 65%), single spot at Rf 0.84 (ethyl acetate), mp 216.8-219.3°C, [22] , HPLC purity 99+% (tR 2.07 min in 10% water- acetonitrile). 1H NMR (d6-DMSO): δ 11.91 (1H, s), 10.32 (1H, s), 8.03 (1H, d, =3.0 Hz), 7.82 (1H, d, J=7.9 Hz), 7.70-7.67 (2H, m), 7.37-7.31 (2H, m), 6.95 (1 H, dd, J=8.6, 2.0 Hz), 3.77 (3H, s), 2.65 (3H, s). LCMS: 377.09. FAB-MS (MH+, C17H15CIN2O4S): calcd 378.0441 , found 378.0439.
Synthesis of Benzimidazole Arysulphonamide Derivatives
Figure imgf000114_0001
Ar = 2-Me-3-CI-phenyl R = -CH2C6H5 R = -CH2C00C2H5
Ar = 4-n-propylphenyl R = -CH3 R = -Cri^CgHs
Ar = 2,5-dichlorophen yi R = -CH 3
Ar = 2,4-dichlorophenyl R = -CH3 Ar = 2-Me-4-Br-phenyl R = -CH3
Ar = 4-biphenyl R = -CH3
Figure imgf000114_0002
a) RX, K2C03, Acetone ,rt or reflux b)H2/ 5%Pd-C, Ethanol-THF r.t. or Fe, AcOH-ethanol c) ArS03CI, DCM.Pyridine or ArS03CI, DCM,Pyridine/DMAP d)HOBt, THF, r.t. e)N-chlorosuccimide, IPA
Figure imgf000115_0001
STX975, XDS02001 STX976, XDS02003 STX1121. XDS02102
Figure imgf000115_0002
STX1112, XDS02088 STX1113, XDS02089 STX1114, XDS02090
Figure imgf000115_0003
STX1115, XDS02091 STX1116. XDS02092 STX1110, XDS02084
Figure imgf000115_0004
STX1111, XDS02085 STX1119. XDS02100
Figure imgf000115_0005
STX1117, XDS02098
Figure imgf000115_0007
Figure imgf000115_0006
STX1118, XDS02099 STX879. XDS01173 STX985, XDS02026
Preparation of 1-AlkyI-5-amino-2-methylbenzimidazole and 1-alkyl-6-amino-2- methylbenzimidazole
To a solution of 5-nitrobenzimidazole (1.0 g, 5.6 mmol) in acetone (50 mL) was added potassium carbonate (1.0 g), followed by alkyl halide (1.2 - 1.5 equivalents). The mixture was stirred under nitrogen at rt, then partitioned between ethyl acetate and water after TLC showed completion of the reaction. The organic phase was washed with brine, dried over sodium sulphate and concentrated in vacuo to give a mixture of 1-alkyl- 5-nitro-2-methylbenzimidazole and 1-alkyl-6-nitro-2-methylbenzimidazole, which were dissolved in ethanol-THF (100 mL, 2 : 1) and hydrogenated over 5% Pd-C under atmosphere pressure for 8h. After filtration through celite®, the filtrate was evaporated to give a yellow solid that was separated with flash chromatography (Methanol-DCM gradient elution). 1-Alkyl-5-aminobenzimidazole and 1-alkyl-6-aminobenzimidazole were obtained as yellow solid or thick syrup.
5-Amino-1,2-dimethylbenzimidazole (XDS01191B, XDS02082B):
Yellow solid, mp 126-127°C (lit.128°C, [23]). TLC single spot at Rf 0.30 (5%methanol/DCM); 1H NMR (270 MHz, DMSO): δ 7.08 (1H, d, J = 8.7 Hz, 7-H), 7.65 (1H, d, J = 1.5 Hz, 4-H), 6.50 (1H, dd, J = 8.7, 1.5 Hz, 6-H), 4.63 (2H, broad, NH2), 3.58(3H, s, NCH3), 2.39 (3H, s, CH3).
6-Amino-1,2-dimethylbenzimidazole (XDS01191A, XDS02082A):
Yellow solid. TLC single spot at Rf 0.33 (5%methanol/DCM); 1H NMR (270 MHz, DMSO): δ 7.13 (1 H, d, J = 8.4 Hz, 4-H), 6.48 (1H, d, J = 2.0 Hz, 7-H), 6.43 (1H, dd, J = 8.4, 2.0 Hz, 5-H), 4.83 (2H, broad, NH2), 3.53(3H, s, NCH3), 2.39 (3H, s, CH3).
5-Amino-1-ethyl-2-methylbenzimidazole (XDS02079B):
Yellow syrup. TLC single spot at Rf 0.27 (5%methanol/DCM); 1H NMR (270 MHz, DMSO): δ 7.12 (1 H, d, J = 8.3 Hz, 7-H), 6.68 (1H, d, J = 2.0 Hz, 4-H), 6.51 (1H, dd, J = 8.3, 2.0 Hz, 6-H), 4.68 (2H, broad, NH2), 4.08 (2H, q, J = 7.2 Hz, NCH2), 2.43 (3H, s, CH3), 1.24(3H, t, J = 7.2 Hz, CH3); APCI-MS 175 (M+).
6-Amino-1-ethyl-2-methylbenzimidazole (XDS02079A):
Yellow solid. TLC single spot at Rf 0.30 (5%methanol/DCM); 1H NMR (270 MHz, DMSO): δ 7.16 (1 H, d, J = 8.4 Hz, 4-H), 6.68 (1H, d, J = 1.7 Hz, 7-H), 6.46 (1H, dd, J = 8.4, 1.7 Hz, 5-H), 4.85 (2H, broad, NH2), 4.02 (2H, q, J = 7.9 Hz, NCH2), 2.42 (3H, s, CH3), 1.24(3H, t, J = 7.9 Hz, CH3); APCI-MS 175 (M+).
5-Amino-1-i-butyl-2-methylbenzimidazole (XDS02093B):
Yellow syrup. TLC single spot at Rf 0.42 (10%methanol/DCM); 1H NMR (400 MHz, DMSO): δ 7.08 (1 H, d, J = 8.5 Hz, 7-H), 6.65 (1 H, d, J = 1.9 Hz, 4-H), 6.48 (1 H, dd, J = 8.5, 1.9 Hz, 6-H), 4.63 (2H, broad, NH2), 3.82 (2H, d, J = 7.4 Hz, NCH2), 2.41 (3H, s, CH3), 2.07 (1 H, m, CH), 0.84 (6H, d, J = 7.0 Hz, 2 x CH3); APCI-MS 204 (MH+)
6-Amino-1-i-butyl-2-methylbenzimidazole (XDS02093A): Yellow solid. TLC single spot at Rf 0.45 (10%methanol/DCM); 1H NMR (270 MHz, DMSO): δ 7.15 (1 H, d, J = 8.2 Hz, 4-H), 6.62 (1 H, d, J = 1.6 Hz, 7-H), 6.44 (1 H, dd, J = 8.2, 1.8 Hz, 5-H), 4.83 (2H, broad, NH2), 3.79 (2H, d, J = 7.7 Hz, NCH2), 2.41 (3H, s, CH3), 2.10 (1H, m, CH), 0.87 (6H, d, J = 6.6 Hz, 2 x CH3); APCI-MS 204 (MH+)
(5-Aminobenzoimidazol-1-yl)-acetic acid ethyl ester (XDS02012B)
Yellow solid. TLC single spot at Rf 0.36 (5%methanol/DCM); 1H NMR (270 MHz, DMSO): δ 7.08 (1 H, d, J = 8.4 Hz, 7-H), 6.69 (1 H, d, J = 2.2 Hz, 4-H), 6.49 (1 H, dd, J = 8.4, 2.2 Hz, 6-H), 5.02 (2H, s, NCH2), 4.68 (2H, s, NH2), 4.16 (2H, q, J = 7.2 Hz, CH2), 2.36 (3H, s, CH3), 1.21 (3H, t, J = 7.2 Hz, CH3); APCI-MS 234 (MH+).
(6-Aminobenzoimidazol-1-yl)-acetic acid ethyl ester (XDS02012A) Yellow solid. TLC single spot at Rf 0.40 (5%methanol/DCM); 1H NMR (270 MHz, DMSO): δ 7.17 (1 H, d, J = 9.0 Hz, 4-H), 6.45-6.48 (2H, m, 5 and 7-H), 4.95 (2H, s, NCH2), 4.87 (2H, s, NH2), 4.17 (2H, q, J = 7.1 Hz, CH2), 2.36 (3H, s, CH3), 1.22 (3H, t, J = 7.1 Hz, CH3); APCI-MS 234 (MH+).
5-Amino-1-benzyl-2-methylbenzimidazole (XDS02086B):
Yellow syrup. TLC single spot at Rf 0.27 (5%methanol/DCM); 1H NMR (400 MHz, DMSO): δ 7.26-7.32 (2H, m, ArH), 7.23 (1 H, tt, J = 7.5, 2.3 Hz, ArH), 7.05-7.09 (3H, m, ArH), 6.69 (1 H, d, J = 2.3 Hz, 4-H), 6.46 (1 H, dd, J = 8.2, 2.3 Hz, 6-H), 5.30 (2H, s, CH2), 4.68 (2H, broad, NH2), 2.40 (3H, s, CH3); APCI-MS 238 (MH+).
6-Amino-1-benzyl-2-methylbenzimidazole (XDS02086A):
Yellow solid. TLC single spot at Rf 0.30 (5%methanol/DCM); 1H NMR (400 MHz, DMSO): δ 7.28-7.32 (2H, m, ArH), 7.23 (1 H, tt, J = 7.5, 2.3 Hz, ArH), 7.17 (1 H, d, J = 8.2, Hz, ArH), 7.06 (2H, m ArH), 6.43-6.46 (2H, m, ArH), 5.26 (2H, s, CH2), 4.63 (2H, s, NH2), 2.40 (3H, s, CH3); APCI-MS 238 (MH+).
Preparation of 5-amino-4-chloro-1,2-dimethylbenzimidazole (XDS02096A) To a solution of 5-amino-1 ,2-dimethylbenzimidazole (600 mg, 3.73 mmol) in IPA (15 mL) was added N-chlorosuccinimide (548 mg, 4.10 mmol). The mixture was stirred at rt for 20 min, diluted with DCM (80 mL) and washed with 5% sodium bicarbonate and brine. The dark brown solution was dried over sodium sulphate and concentrated in vacuo to give a brown residue, which was subjected to flash chromatography (methanol-DCM gradient elution). Yellow solid (220 mg, 33%) was obtained. TLC single spot at Rf 0.69 (10%methanol/DCM); 1H NMR (270 MHz, DMSO): δ 7.16 (1H, d, J = 7.9 Hz, ArH), 6.72 (1 H, d, J - 7.9, Hz, ArH), 4.90 (2H, s, NH2), 3.64 (3H, s, NCH3), 2.40 (3H, s, CH3); APCI- MS 196 (MH+)
General method for synthesis of N-benzimidazole arylsulphonamide derivatives: To a solution arylsulphonyl chloride (1.1 eq.) in DCM were added pyridine (2.2 eq.) and catalytic amount of DMAP, followed by the corresponding amine (1 eq.). The reaction mixture was stirred at rt under nitrogen for 4-16 h, then partitioned between ethyl acetate and 5% sodium bicarbonate after TLC showed completion of the reaction. The organic layer was washed with brine, dried over sodium sulphate, and concentrated in vacuo to give crude product as solid or thick syrup. The compound was then purified by flash chromatography (Methanol-DCM gradient elution) to give desired arylsulphonamide as crystalline solid. Yield ranges from 50-80%.
3-Chloro-N-(1,2-dimethyl-1H-benzoimidazol-6-yl)-2-methylbenzenesulphonamide (STX975, XDS02001)
White crystalline solid. Mp 265-266°C; TLC single spot at Rf 0.43 (5% methanol/DCM); HPLC purity > 99% (tR 2. min in 10% water-methanol); 1H NMR (270 MHz, DMSO): δ 10.4 (1 H, s, NH), 7.84 (1H, d, J = 7.9 Hz, ArH), 7.67 (1 H, d, J = 7.9 Hz, ArH), 7.34 (1 H, d, J = 8.2 Hz, ArH), 7.32 (1H, t, J = 7.9 Hz, ArH), 7.14(1H, d, J = 2 Hz, ArH ), 6.80 (1H, dd, J= 8.2, 2.0 Hz, ArH), 3.61 (3H, s, NCH3), 2.64 (3H, s, CH3), 2.46 (3H, s, CH3); APCI- MS 348 (M-H+); FAB-HRMS calcd for C16H17CIN3O2S (MH+) 350.0730, found 350.0749.
3-Chloro-N-(1,2-dimethyl-1H-benzoimidazol-5-yl)-2-methylbenzenesulphonamide (STX976, XDS02003)
White crystalline solid. Mp 283-283.5°C; TLC single spot at Rf 0.38 (5% methanol/DCM); HPLC purity >99% (tR 2.0 min in 10% water-methanol); Η NMR (270 MHz, DMSO): δ 10.3 (1H, s, NH), 7.77 (1 H, d, J = 7.6 Hz, ArH), 7.66 (1 H, d, J = 7.6 Hz, ArH), 7.32 (1H, d, J = 8.4 Hz, ArH), 7.30 (1H, t, J = 7.6 Hz, ArH), 7.16(1 H, d, J = 2 Hz, ArH ), 6.90 (1H, dd, J= 8.4, 2.0 Hz, ArH), 3.64 (3H, s, NCH3), 2.64 (3H, s, CH3), 2.44 (3H, s, CH3); APCI-MS 348 (M-H+); FAB-HRMS calcd for C16H17CIN3O2S (MH+) 350.0730, found 350.0747.
3-Chloro-N-(4-chloro-1 ,2-dimethyl-1 H-benzoimidazol-5-yl)-2- methylbenzenesulphonamide (STX1121, XDS02102B)
Off-white crystalline solid. TLC single spot at Rf 0.50 (10% methanol/DCM); HPLC purity 95% (tR 2.1 min in 20% water-methanol); 1H NMR (270 MHz, DMSO): δ 10.1 (1 H, s, NH), 7.70 (1H, dd, J = 7.7, 1.7 Hz, ArH), 7.56 (1H, dd, J = 7.8, 1.7 Hz, ArH), 7.39 (1H, d, J = 8.2 Hz, ArH), 7.25 (1H, t, J = 7.7 Hz, ArH), 7.04 (1H, d, = 8.2 Hz, ArH), 3.69 (3H, s, NCH3), 2.67 (3H, s, CH3), 2.51 (3H, s, CH3); APCI-MS 384 (MH+).
N-(1 ,2-Dimethyl-1 H-benzoimidazol-5-yl)-4-propylbenzenesulphonamide (STX1112, XDS02088)
White crystalline solid. TLC single spot at Rf 0.38 (5% methanol/DCM); HPLC purity >99% (tR 2.1 min in 20% water-methanol); 1H NMR (270 MHz, DMSO): δ 9.90 (1H, s,
NH), 7.58 (2H, d, J = 8.3 Hz, ArH), 7.29-7.32 (3H, m, ArH), 7.17 (1 H, d, J = 1.5 Hz, ArH),
6.91 (1 H, dd, J = 8.6, 2.0 Hz, ArH), 3.64 (3H, s, NCH3), 2.55 (2H, m, CH2), 2.50 (3H, s,
CH3), 1.55 (2H, sextet, J = 7.6 Hz, CH2), 0.84 (3H, t, J = 7.6 Hz, CH3); APCI-MS 344
(MH+).
2,5-Dichloro-N-(1,2-dimethyl-1H-benzoimidazol-5-yl)-benzenesulphonamide
(STX1113, XDS02089)
White crystalline solid. TLC single spot at Rf 0.67 (10% methanol/DCM); HPLC purity
99% (tR 2.0 min in 20% water-methanol); 1H NMR (270 MHz, DMSO): δ 10.5 (1 H, s, NH), 7.84 (1 H, t, J = 1.4 Hz, ArH), 7.68 (2H, d, J = 2.0 Hz, ArH), 7.35 (1 H, d, J = 8.5 Hz,
ArH), 7.21 (1 H, d, J = 2.0 Hz, ArH), 6.97 (1 H, dd, J = 8.2, 2.0 Hz, ArH ), 3.64 (3H, s,
NCH3), 2.45 (3H, s, CH3); APCI-MS 370 (MH+).
2,4-Dichloro-N-(1,2-dimethyl-1H-benzoimidazol-5-yl)-benzenesulphonamide (STX1114, XDS02090)
Off-white crystalline solid. TLC single spot at Rf 0.59 (10% methanol/DCM); HPLC purity >99% (tR 2.0 min in 20% water-methanol); 1H NMR (270 MHz, DMSO): δ 10.4 (1H, s, NH), 7.88 (1 H, d, J = 8.7 Hz, ArH), 7.84 (2H, d, J = 1.9 Hz, ArH), 7.52 (1 H, dd, J - 8.7, 1.9 Hz, ArH), 7.33 (1H, d, J = 8.5 Hz, ArH), 7.20 (1 H, d, J = 1.7 Hz, ArH ), 6.95 (1H, dd, J = 8.7, 1.7 Hz, ArH ), 3.63 (3H, s, NCH3), 2.45 (3H, s, CH3); APCI-MS 370 (MH+). 4-Bromo-N-(1,2-dimethyl-1H-benzoimidazol-5-yl)-2-methylbenzenesulphonamide (STX1115, XDS02091)
White crystalline solid. TLC single spot at Rf 0.67 (10% methanol/DCM); HPLC purity >99% (tR 2.1 min in 20% water-methanol); 1H NMR (270 MHz, DMSO): δ 10.1 (1H, s, NH), 7.62-7.69 (2H, m, ArH), 7.50 (1 H, dd, J = 8.5, 2.2 Hz, ArH), 7.32 (1 H, d, J = 8.5 Hz, ArH), 7.14 (1 H, d, J = 1.9 Hz, ArH), 6.89 (1H, dd, J = 8.5, 1.9 Hz, ArH), 3.63 (3H, s, NCH3), 2.55 (3H, s, CH3), 2.45 (3H, s, CH3); APCI-MS 394 (MH+).
N-(1 ,2-Dimethyl-1 H-benzoimidazol-5-yl)-4-phenylbenzenesulphonamide (STX1116, XDS02092)
White crystalline solid. TLC single spot at Rf 0.72 (5% methanol/DCM); HPLC purity >99% (tR 2.1 min in 20% water-methanol); 1H NMR (270 MHz, DMSO): δ 10.0 (1 H, s, NH), 7.67-7.82 (6H, m, ArH), 7.41-7.50 (3H, m, ArH), 7.33 (1 H, d, J = 8.5 Hz, ArH), 7.22 (1H, d, J = 1.9 Hz, ArH), 6.95 (1H, dd, J = 8.5, 1.9 Hz, ArH), 3.64 (3H, s, NCH3), 2.44 (3H, s, CH3); APCI-MS 378 (MH+).
3-Chloro-N-(1 -ethyl-2-methyl-1 H-benzoimidazol-6-y l)-2- methylbenzenesulphonamide (STX1110, XDS02084)
Off-white solid. TLC single spot at Rf 0.45 (8% methanol/DCM); HPLC purity >99% (tR 2.2 min in 20% water-methanol); 1H NMR (270 MHz, DMSO): δ 10.4 (1H, s, NH), 7.84 (1H, d, J = 7.9 Hz, ArH), 7.67 (1H, d, J = 7.8 Hz, ArH), 7.35 (1 H, d, J = 8.5 Hz, ArH), 7.32 (1H, t, J = 7.9 Hz, ArH), 7.10 (1H, d, J = 2.2 Hz, ArH ), 6.82 (1H, dd, J= 8.5, 2.1 Hz, ArH), 4.09 (2H, q, J =7.1 Hz, CH2), 2.61 (3H, s, CH3), 2.46 (3H, s, CH3), 1.18 (3H, t, J =
7.1 Hz, CH3); APCI-MS 364 (MH+).
3-Chloro-N-(1-ethyl-2-methyl-1H-benzoimidazol-5-yl)-2- methylbenzenesulphonamide (STX1111, XDS02085)
Off-white solid. TLC single spot at Rf 0.42 (8% methanol/DCM); HPLC purity >99% (tR
2.2 min in 20% water-methanol); 1H NMR (270 MHz, DMSO): δ 10.3 (1H, s, NH), 7.78 (1 H, d, J = 7.9 Hz, ArH), 7.66 (1H, d, J = 7.9 Hz, ArH), 7.36 (1 H, d, J = 8.2 Hz, ArH),
7.32 (1H, t, J = 7.9 Hz, ArH), 7.16 (1 H, d, J = 1.9 Hz, ArH ), 6.90 (1H, dd, J= 7.9, 2.0 Hz, ArH), 4.12 (2H, q, J =7.1 Hz, CH2), 2.64 (3H, s, CH3), 2.46 (3H, s, CH3), 1.23 (3H, t, J = 7.1 Hz, CH3); APCI-MS 364 (MH+). 3-Chloro-N-(1-isobutyl-2-methyl-1H-benzoimidazol-6-yl)-2- methylbenzenesulphonamide (STX1119, XDS02100)
Off-white solid. TLC single spot at Rf 0.57 (8% methanol/DCM); HPLC purity 99% (tR 2.2 min in 20% water-methanol); 1H NMR (270 MHz, DMSO): δ 10.4 (1 H, s, NH), 7.80 (1 H, d, J = 8.9 Hz, ArH), 7.66 (1 H, d, J = 8.8 Hz, ArH), 7.36 (1H, d, J = 8.5 Hz, ArH), 7.29 (1 H, t, J = 7.9 Hz, ArH), 7.03 (1 H, d, J = 1.9 Hz, ArH ), 6.84 (1 H, dd, J= 8.5, 1.8 Hz, ArH), 3.85 (2H, d, J =7.2 Hz, NCH2), 2.61 (3H, s, CH3), 2.45 (3H, s, CH3), 1.91 (1H, m, CH), 0.81 (6H, d, J = 7.0 Hz, 2 x CH3); APCI-MS 392 (MH+).
3-Chloro-N-(1-isobutyl-2-methyl-1H-benzoimidazol-5-yl)-2- methylbenzenesulphonamide (STX1120, XDS02101)
Off-white solid. TLC single spot at Rf 0.52 (8% methanol/DCM); HPLC purity 99% (tR 2.3 min in 20% water-methanol); 1H NMR (270 MHz, DMSO): δ 10.3 (1H, s, NH), 7.80 (1 H, d, J = 7.9 Hz, ArH), 7.68 (1H, d, J = 7.9 Hz, ArH), 7.38 (1H, d, J - 8.8 Hz, ArH), 7.33 (1 H, t, J = 7.9 Hz, ArH), 7.16 (1 H, d, J = 1.9 Hz, ArH ), 6.90 (1 H, dd, J= 8.7, 1.9 Hz, ArH), 3.91 (2H, d, J =7.3 Hz, NCH2), 2.62 (3H, s, CH3), 2.47 (3H, s, CH3), 2.05 (1 H, m, CH), 0.83 (6H, d, J = 7.0 Hz, 2 x CH3); APCI-MS 392 (MH+).
[6-(3-Chloro-2-methylbenzenesulphonylamino)-2-methylbenzoimidazol-1-yl]-acetic acid ethyl ester (STX977, XDS02015)
Off-white solid. TLC single spot at Rf 0.46 (6% methanol/DCM); HPLC purity >99% (tR
2.0 min in 10% water-methanol); 1H NMR (270 MHz, DMSO): δ 10.4 (1H, s, NH), 7.82 (1 H, d, J = 8.0 Hz, ArH), 7.67 (1 H, d, J = 8.0 Hz, ArH), 7.37 (1 H, d, J = 8.5 Hz, ArH), 7.30 (1H, t, J = 8.0 Hz, ArH), 7.11 (1H, d, J = 2.0 Hz, ArH ), 6.83 (1 H, dd, J= 8.5, 2.0 Hz, ArH), 5.09 (2H, s, NCH2), 4.16 (2H, q, J = 7.1 Hz, CH2), 2.62 (3H, s, CH3), 2.45 (3H, s, CH3), 1.21 (3H, t, J = 7.1 Hz, CH3); APCI-MS 420 (M-H+); FAB-HRMS calcd for C19H21CIN3O4S (MH+) 422.0941 , found 422.0942.
[5-(3-Chloro-2-methylbenzenesulphonylamino)-2-methylbenzoimidazol-1-yl]-acetic acid ethyl ester (STX978, XDS02017)
Off-white solid. TLC single spot at Rf 0.40 (6% methanol/DCM); HPLC purity 99% (tR 2.0 min in 10% water-methanol); 1H NMR (270 MHz, DMSO): δ 10.3 (1 H, s, NH), 7.80 (1H, d, J = 7.9 Hz, ArH), 7.67 (1H, d, J = 7.9 Hz, ArH), 7.29-7.34 (2H, m, ArH), 7.18 (1 H, d, J = 1.7 Hz, ArH ), 6.90 (1 H, dd, J= 8.6, 1.7 Hz, ArH), 5.11 (2H, s, NCH2), 4.15 (2H, q, J =
7.1 Hz, CH2), 2.64 (3H, s, CH3), 2.40 (3H, s, CH3), 1.19 (3H, t, J = 7.1 Hz, CH3); APCI- MS 420 (M-H+); FAB-HRMS calcd for C19H21CIN3O4S (MH+) 422.0941 , found 422.0944.
3-Chloro-N-(1-benzyl-2-methyl-1H-benzoimidazol-6-yl)-2- methylbenzenesulphonamide (STX1117, XDS02098)
Off-white solid. TLC single spot at Rf 0.70 (10% methanol/DCM); HPLC purity 99% (tR 2.2 min in 20% water-methanol); 1H NMR (270 MHz, DMSO): δ 10.4 (1 H, s, NH), 7.66 (1H, d, J = 7.9 Hz, ArH), 7.64 (1 H, d, J = 7.9 Hz, ArH), 7.30-7.39 (4H, m, ArH), 7.21 (1H, t, J = 7.9 Hz, ArH), 7.11 (1 H, d, J = 2.0 Hz, ArH), 7.02-7.06 (2H, m, ArH ), 6.83 (1H, dd, J= 7.9, 2.0 Hz, ArH), 5.34 (2H, s, NCH2), 2.58 (3H, s, CH3), 2.45 (3H, s, CH3); APCI-MS 426 (MH+).
3-Chloro-N-(1 -benzyl-2-methyl-1 H-benzoimidazol-5-yl)-2- methylbenzenesulphonamide (STX1118, XDS02099) Off-white solid. TLC single spot at Rf 0.65 (10% methanol/DCM); HPLC purity 99% (tR 2.2 min in 10% water-methanol); 1H NMR (270 MHz, DMSO): δ 10.3 (1 H, s, NH), 7.80 (1 H, d, J = 7.9 Hz, ArH), 7.67 (1 H, d, = 7.9 Hz, ArH), 7.25-7.35 (5H, m, ArH), 7.19 (1H, d, J = 1.9 Hz, ArH), 7.06-7.09 (2H, m, ArH ), 6.87 (1 H, dd, J= 8.5, 1.9 Hz, ArH), 5.38 (2H, s, NCH2), 2.62 (3H, s, CH3), 2.45 (3H, s, CH3); APCI-MS 426 (MH+).
3-Chloro-N-(2-trifluromethyl-1H-benzoimidazol-5-yl)-2- methylbenzenesulphonamide (STX879, XDS01173)
White crystalline solid. TLC single spot at Rf 0.58 (20% ethyl acetate/DCM); HPLC purity 99% (tR 2.4 min in 20% water-methanol); 1H NMR (270 MHz, DMSO): δ 13.9 (1 H, s, NH), 10.7 (1H, s, NH), 7.85 (1 H, d, J = 8.0 Hz, ArH), 7.68 (1 H, d, J = 8.0 Hz, ArH), 7.60 (1 H, d broad, J = 8.1 Hz, ArH), 7.34 (2H, m, ArH), 7.10(1 H, d, J = 8.3 Hz, ArH ), 2.64 (3H, s, CH3); APCI-MS 388 (M-H+); FAB-HRMS calcd for C15H12CIF3N3O2S (MH+) 390.0291 , found 390.0291.
Preparation of 3-chloro-N-(2-methyl-1H-benzoimidazol-5-yl)-2- methylbenzenesulphonamide (STX985, XDS02026)
The coupling reaction of 3-chloro-2-methyibenzenesulphonyl chloride (2 eq.) with 2- methylbenzimidazoel (1 eq.) under the condition described above yielded a mixture of 3- chloro-N-[1-(3-chloro-2-methylbenzenesulphonyl)-2-methyl-1 H-benzoimidazol-5-yl]-2- methyl-benzenesulphonamide and 3-chloro-N-[1-(3-chloro-2-methylbenzenesulphonyl)- 2-methyl-1H-benzoimidazol-6-yl]-2-methyl-benzenesulphonamide in 1 : 1 ratio as judged by HNMR. 1H NMR (270 MHz, DMSO): δ 10.7 (2H, s, 2 x NH), 7.86-7.96 (3H, m, ArH), 7.65-7.78 (5H, m, ArH), 7.52-7.58 (5H, m, ArH), 7.25-7.40 (3H, m, ArH), 7.07 (2H, t, J = 8.2 Hz, ArH), 2.61 (3H, s, CH3), 2.58 (3H, s, CH3), 2.54 (6H, s, 2 x CH3), 2.39 (3H, s, CH3), 2.33 (3H, s, CH3). The mixture (200 mg) was dissolved in THF (15 mL), N- hydroxybenzotriazole (200 mg) was added. After stirred at rt for 48h, the mixture was partitioned between ethyl acetate and 5% sodium bicarbonate. The organic phase was washed with brine, dried over sodium sulphate and concentrate in vacuo to give a yellow residue, which was purified with flash chromatography (methanol/DCM gradient elution). Off white amorphous powder was obtained. TLC single spot at Rf 0.38 (10% methanol/DCM); HPLC purity 99% (tR 2.0 min in 10% water-methanol); Η NMR (270 MHz, DMSO): δ 12.1 (1H, s, NH), 10.3 (1H, s, NH), 7.78 (1 H, d, J = 7.9 Hz, ArH), 7.68 (1H, d, J = 7.9 Hz, ArH), 7.29-7.35 (2H, m, ArH), 7.12(1 H, s, ArH ), 6.83 (1H, dd, J= 8.4, 1.8 Hz, ArH), 2.63 (3H, s, CH3), 2.41 (3H, s, CH3); APCI-MS 334 (M-H+); FAB-HRMS calcd for C15H15CIN3O2S (MH+) 336.0573, found 336.0583.
Synthesis of N-Benzimidazole Arylsulphonamide Derivatives
Figure imgf000123_0001
STX977. XDS02015 STX1141, XDS02116 STX978, XDS02017 STX1142, XDS02116
a) Raney-Ni, NH2NH2Η20, ethanol, rt b) Ac20, AcOH, 80°C c) 6N HCI, 75°C d) 3-CI-2-Me-benzenesulphonyl chloride, DCM.Pyridine e) LIAIH4, THF, 0°C N1-Phenyl-benzene-1 ,2,4-triamine:
To a solution of 2,4-dinitrophenylamine (1.5 g, 5.8 mmol) in ethanol-THF (150 : 50 mL) were added hydrazine hydrate (2 mL, 65 mmol) and Raney Nickel (2.0 g). The reaction mixture was stirred at rt for 20 min, filtered through Celite. Evaporation of the solvent gave a black residue, which was purified by flash chromatography (methanol-DCM gradient elution). A black crystalline solid (1.0 g, 87%) was obtained. Mp 128-129°C; TLC single spot at Rf 0.46 (8% methanol/DCM); 1H NMR (270 MHz, DMSO): δ 7.25 (2H, t, J = 7.5 Hz, ArH), 6.73 (1 H, s, NH), 6.61 (1 H, d, J = 8.3 Hz, ArH), 6.49-6.55 (3H, m, ArH), 5.99 (1H, d, J = 2.5 Hz, ArH), 5.83 (1 H, dd, J = 8.2, 2.5 Hz, ArH), 4.66 (2H, s, NH2), 4.44 (2H, s, NH2); APCI-MS 198 (M-H+).
N-(2-Methy 1-1 -phenyl-1 H-benzimidazol-5-yl)-acetamide:
N1-Phenyl-benzene-1 ,2,4-triamine (800 mg, 4 mmol) was dissolved in acetic acid (10 mL), acetic anhydride (1.0 mL) was added to the solution. The mixture was stirred at
80°C for 6h, cooled to rt and neutralized with 5% sodium carbonate, then extracted with ethyl acetate. The organic phase was washed with brine, dried over magnesium sulphate and concentrated to give a residue, which was crystallized from ethanol. A brown crystalline solid (0.85 g, 80%) was obtained. Mp 231-232°C; TLC single spot at Rf
0.39 (10% methanol/DCM); 1H NMR (270 MHz, DMSO): δ 9.92 (1 H, s, NH), 7.97 (1 H, d,
J = 1.6 Hz, ArH), 7.51-7.67 (5H, m, ArH), 7.31 (1 H, dd, J = 8.3, 1.9 Hz, ArH), 7.04 (1 H, d, J = 8.3 Hz, ArH),, 2.41 (3H, s, CH3), 2.05 (3H, s, CH3); APCI-MS 264 (M-H+).
2-Methyl-1-phenyl-1H-benzoimidazol-5-ylamine:
The solution of N-(2-methyl-1 -phenyl-1 H-benzimidazol-5-yl)-acetamide (800 mg, 3 mmol) in 6N HCI (5 mL) was stirred at 75°C for 3h, cooled to rt and neutralized with sodium carbonate to pH 7, then extracted with ethyl acetate. The organic phase was washed with brine, dried over magnesium sulphate and concentrated to give a dark brown solid (600 mg, 90%). Mp 145-146°C; TLC single spot at Rf 0.47 (10% methanol/DCM); 1H NMR (270 MHz, DMSO): δ 7.58-7.64 (2H, m, ArH), 7.46-7.53 (3H, m, ArH), 6.82 (1 H, d, J = 8.5 Hz, ArH), 6.76 (1 H, d, J = 1.9 Hz, ArH), 6.51 (1 H, dd, J =
8.5, 1.9 Hz, ArH), 4.78 (2H, s, NH2), 2.36 (3H, s, CH3); APCI-MS 223 (M+).
3-Chloro-2-methyl-N-(2-methyl-1-phenyl-1H-benzoimidazol-5-yl)- benzenesulphonamide (STX1140, XDS02110)
The compound was prepared with general method of benzenesulphonamide formation.
Light pink crystalline solid was obtained. Mp 254-256°C; TLC single spot at Rf 0.62 (8% methanol/DCM); HPLC purity > 99% (tR 2.6 min in 20% water-methanol); 1H NMR (270
MHz, DMSO): δ 10.4 (1 H, s, NH), 7.83 (1 H, dd, J = 8.6, 1.8 Hz, ArH), 7.69 (1 H, dd, J =
8.6, 1.7 Hz, ArH), 7.58-7.63 (5H, m, ArH), 7.34 (1 H, t, J = 8.0 Hz, ArH), 7.28 (1 H, d, J = 1.9 Hz, ArH), 6.90-7.00 (2H, m, ArH), 2.66 (3H, s, CH3), 2.36 (3H, s, CH3); APCI-MS 412 (MH+). 3-Chloro-N-[1-(2-hydroxyethyl)-2-methyl-1H-benzoimidazol-6-yl]-2-methyl- benzenesulfonamide (STX1141, XDS02115)
To a solution of [6-(3-Chloro-2-methyl-benzenesulfonylamino)-2-methyl-benzoimidazol- 1-yl]-acetic acid ethyl ester (100 mg, 0.237 mmol) in anhydrous THF (10 mL) was added LiAIH4 (54 mg, 1.42 mmol) at 0°C. The mixture was stirred at 0°C for 0.5h, quenched with saturated ammonium chloride solution, neutralized with 6N HCI and extracted with ethyl acetate. The organic phase was washed with brine, dried over sodium sulphate and concentrated in vacuo to give a light pink crystalline solid (82 mg, 91%). Mp 213- 214.5°C; TLC single spot at Rf 0.39 (12% methanol/DCM); HPLC purity > 99% (tR 2.0 min in 20% water-methanol); 1H NMR (270 MHz, DMSO): δ 10.4 (1H, s, NH), 7.84 (1 H, d, J = 7.7 Hz, ArH), 7.67 (1H, d, J = 7.9 Hz, ArH), 7.28-7.35 (2H, m, ArH), 7.15 (1 H, d, J = 1.9 Hz, ArH), 6.80 (1H, dd, J = 8.5, 1.9 Hz, ArH), 4.94 (1H, t, J = 5.2 Hz, OH), 4.10 (2H, t, J = 5.0 Hz, NCH2), 3.59 (2H, q, J = 5.2 Hz, CH2), 2.51 (3H, s, CH3), 2.47 (3H, s, CH3); APCI-MS 380 (MH+).
3-Chloro-N-[1-(2-hydroxy-ethyl)-2-methyl-1H-benzoimidazol-5-yl]-2-methyl- benzenesulfonamide (STX1142, XDS02116)
The compound was prepared as above from [5-(3-Chloro-2-methyl- benzenesulfonylamino)-2-methyl-benzoimidazol-1-yl]-acetic acid ethyl ester (35 mg, 0.083 mmol). White crystalline solid (22 mg, 89%) was obtained. Mp 245-247°C; TLC single spot at Rf 0.38 (12% methanol/DCM); HPLC purity > 99% (tR 2.0 min in 20% water-methanol); 1H NMR (270 MHz, DMSO): δ 10.3 (1H, s, NH), 7.79 (1 H, d, J = 8.0 Hz, ArH), 7.67 (1H, d, J = 8.0 Hz, ArH), 7.29-7.35 (2H, m, ArH), 7.16 (1H, s, ArH), 6.89 (1 H, d, J = 8.5, ArH), 4.90 (1H, t, J = 5.0 Hz, OH), 4.14 (2H, t, J = 5.0 Hz, NCH2), 3.63 (2H, q, J = 5.2 Hz, CH2), 2.65 (3H, s, CH3), 2.48 (3H, s, CH3); APCI-MS 380 (MH+).
Synthesis of Benzoxazole Derivatives
Figure imgf000125_0001
STX 839: R.,=R2=H, R3=CI, R4=Me STX 842: R =R2=H, R3=CI, R4=Me STX 840: R-ι=R3=R4=H, R2=n-propyl STX 843. Rι=R3=R4=H, R2=n-propyl STX 841: R1=R4=CI, R2=R3=H STX 846: R1=R4=CI, R2=R3=H Synthesis of 3-chloro-2-methyl-W-(2-methyl-benzooxazol-6-yl)- benzenesulfonamide, STX 839 (KRB01009):
To a solution of 3-chloro-2-methylbenzenesulphonyl chloride (163 mg, 0.723 mmol) in dichloromethane (3 mL) was added pyridine (140 μL, 1.72 mmol) and the mixture was stirred under N2 for 5 min, after which time 6-amino-2-methylbenzoxazole (102 mg, 0.688 mmol) was added. The resulting mixture was stirred for 1 h at room temperature, then saturated NaHCO3 solution was added (8 mL) and the mixture was extracted into ethyl acetate (15 mL). The organic phase was washed with brine, dried (Na2SO4), filtered and evaporated to give a residue that was purified using flash chromatography to afford a white solid (151 mg, 65%), single spot at Rf 0.50 (60:40 hexane:ethyl acetate), mp 127.1-127.5°C, HPLC purity 97% (tR 2.05 min in 10% water-acetonitrile). 1H NMR (CDCI3): δ 7.85 (1H, dd, J=8.1 , 1.1 Hz), 7.53 (1H, dd, .7=8.1 , 1.3 Hz), 7.45 (1 H, d, J=8.4 Hz), 7.27 (1H, d, J=2.2 Hz), 7.17 (1H, t, =7.9 Hz), 6.93 (1H, s, N-H), 6.86 (1 H, dd, J=8.4, 2.2 Hz), 2.71 (3H, s), 2.58 (3H, s). LCMS: 335.14 (M-). FAB-MS (MH+, C15H13CIN2O3S): calcd 337.0413, found 337.0406.
Synthesis of Λ/-(2-methyl-benzooxazol-6-yl)-4-propyl-benzenesulfonamide, STX 840 (KRB01010):
Figure imgf000126_0002
To a solution of 4n-propylbenzenesulphonyl chloride (163 mg, 0.744 mmol) in dichloromethane (3 mL) was added pyridine (140 μL, 1.72 mmol) and the mixture was stirred under N2 for 5 min, after which time 6-amino-2-methylbenzoxazole (105 mg, 0.709 mmol) was added. The resulting mixture was stirred for 1 h at room temperature, then saturated NaHCO3 solution was added (8 mL) and the mixture was extracted into ethyl acetate (15 mL). The organic phase was washed with brine, dried (Na2SO4), filtered and evaporated to give a residue that was purified using flash chromatography to afford a pale pink solid (164 mg, 70%), single spot at Rf 0.49 (60:40 hexane:ethyl acetate), mp 101.7-102.3°C, HPLC purity 99% (tR 2.02 min in 10% water-acetonitrile). 1H NMR (CDCI3): δ 7.61 (2H, m), 7.43 (1H, d, J=8.4 Hz), 7.37 (1 H, d, J=1.8 Hz), 7.19 (2H, m), 6.83 (2H, m), 2.57 (5H, m), 1.58 (2H, sextet, J=7.3 Hz), 0.88 (3H, t, J=7.3 Hz). LCMS: 329.21 (M-). FAB-MS (MH+, C17H18N2O3S): calcd 331.1116, found 331.1107.
Synthesis of 2,5-dichloro-(2-methyl-benzooxazol-6-yl)-benzenesulfonamide, STX 841 (KRB01011):
Figure imgf000127_0001
To a solution of 2,5-dichlorobenzenesulphonyl chloride (174 mg, 0.709 mmol) in dichloromethane (3 mL) was added pyridine (140 μL, 1.72 mmol) and the mixture was stirred under N2 for 5 min, after which time 6-amino-2-methylbenzoxazole (100 mg, 0.675 mmol) was added. The resulting mixture was stirred for 1 h at room temperature, then saturated NaHCO3 solution was added (8 mL) and the mixture was extracted into ethyl acetate (15 mL). The organic phase was washed with brine, dried (Na2SO4), filtered and evaporated to give a residue that was purified using flash chromatography to afford a white solid (154 mg, 64%), single spot at Rf 0.50 (60:40 hexane:ethyl acetate), mp 167.0-167.3°C, HPLC purity 97% (tR 1.97 min in 10% water-acetonitrile). 1H NMR (CDCI3): δ 7.93 (1 H, d, J=2.3 Hz), 7.47 (1 H, d, J=8.6 Hz), 7.46-7.40 (4H, m), 7.00 (1 H, dd, J=8.6, 2.0 Hz), 2.61 (3H, s). LCMS: 355.07 (M-). FAB-MS (MH+, C14H10CI2N2O3S): calcd 356.9867, found 356.9875.
Synthesis of 3-chloro-2-methyl-/V-(2-methyl-benzooxazol-5-yl)- benzenesulfonamide, STX 842 (KRB01014):
Figure imgf000127_0002
To a solution of 3-chloro-2-methylbenzenesulphonyl chloride (96 mg, 0.43 mmol) in dichloromethane (2 mL) was added pyridine (80 μL, 1.0 mmol) and the mixture was stirred under N2 for 5 min, after which time 5-amino-2-methylbenzoxazole (60 mg, 0.40 mmol) was added. The resulting mixture was stirred for 1 h at room temperature, then saturated NaHCO3 solution was added (8 mL) and the mixture was extracted into ethyl acetate (15 mL). The organic phase was washed with brine, dried (Na2SO4), filtered and evaporated to give a residue that was purified using flash chromatography to afford a white solid (89 mg, 64%), single spot at Rf 0.52 (1 :1 hexane:ethyl acetate), mp 180.2- 180.5°C, HPLC purity 99% (tR 2.32 min in 10% water-acetonitrile). H NMR (CDCI3): δ 7.82 (1H, dd, J=8.1 , 1.1 Hz), 7.52 (1H, dd, J=7.7, 1.1 Hz), 7.32 (1H, d, J=8.4 Hz), 7.25 (1H, d, J-2.9 Hz (overlap with CHCI3)), 7.14 (1H, t, J=8.1 Hz), 6.99 (1H, dd, J=8.4, 2.2 Hz), 6.67 (1H, s, N-H), 2.71 (3Η, s), 2.58 (3H, s). LCMS: 335.01 (M-). FAB-MS (MH+, C15H13CIN2O3S): calcd 337.0413, found 337.0420.
Synthesis of /V-(2-methyl-benzooxazol-5-yl)-4-propyl-benzenesulfonamide, STX 843 (KRB01015):
Figure imgf000128_0001
To a solution of 4n-propylbenzenesulphonyl chloride (93 mg, 0.43 mmol) in dichloromethane (2 mL) was added pyridine (80 μL, 1.0 mmol) and the mixture was stirred under N2 for 5 min, after which time 5-amino-2-methylbenzoxazole (60 mg, 0.40 mmol) was added. The resulting mixture was stirred for 1 h at room temperature, then saturated NaHCO3 solution was added (8 mL) and the mixture was extracted into ethyl • acetate (15 mL). The organic phase was washed with brine, dried (Na2SO4), filtered and evaporated to give a residue that was purified using flash chromatography to afford a pale pink oil (112 mg, 85%), single spot at Rf 0.53 (1:1 hexane:ethyl acetate). HPLC purity 99+% (tR 2.38 min in 10% water-acetonitrile) 1H NMR (CDCI3): δ 7.64 (2H, dt, =8.1 , 1.8 Hz), 7.31 (2H, m), 7.18 (2H, d, J=8.4 Hz), 7.06 (1 H, dd, J=8.6, 2.4 Hz), 2.59 (5H, m), 1.58 (2H, sextet, J=7.3 Hz), 0.89 (3H, t, J=7.3 Hz). LCMS: 329.15 (M-). FAB- MS (MH+, C17H18N2O3S): calcd 331.1116, found 331.1118.
Synthesis of 2,5«dichloro-W-(2-methyl-benzooxazol-5-yl)-benzenesulfonamide, STX 846 (KRB01016):
Figure imgf000129_0001
To a solution of 2,5-dichlorobenzenesulphonyl chloride (52 mg, 0.21 mmol) in dichloromethane (1.5 mL) was added pyridine (40 μL, 0.5 mmol) and the mixture was stirred under N2 for 5 min, after which time 5-amino-2-methylbenzoxazole (30 mg, 0.20 mmol) was added. The resulting mixture was stirred for 1 h at room temperature, then saturated NaHCO3 solution was added (8 mL) and the mixture was extracted into ethyl acetate (15 mL). The organic phase was washed with brine, dried (Na2SO4), filtered and evaporated to give a residue that was purified using flash chromatography to afford a pale pink solid (45 mg, 63%), single spot at Rf 0.53 (1 :1 hexane:ethyl acetate), mp 193.5-193.9°C, HPLC purity 98% (tR 2.27 min in 10% water-acetonitrile). 1H NMR (CDCI3): δ 7.86 (1H, d, =2.2 Hz), 7.39 (4H, m), 7.12 (1H, dd, =8.4, 1.8 Hz), 2.58 (3H, s). LCMS: 355.07 (M-). FAB-MS (MH+, C14H10CI2N2O3S): calcd 356.9867, found 356.9878.
All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry or related fields are intended to be within the scope of the following claims.
REFERENCES
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Claims

1. A compound having Formula
Formula
Figure imgf000132_0001
wherein one of R-, and R2 is a group of the formula
Figure imgf000132_0002
wherein R4 is selected from H and hydrocarbyl, R5 is a hydrocarbyl group and L is an optional linker group, or R and R2 together form a ring substituted with the group
wherein R3 is H or a substituent and wherein X is selected from S, O, NR6 and C(R7)(R8), wherein R6 is selected from H and hydrocarbyl groups, wherein each of R7 and R8 are independently selected from H and hydrocarbyl groups.
2. A compound according to claim 1 having Formula II
Formula II
Figure imgf000132_0004
A compound according to claim 1 or 2 wherein L is not present.
A compound according to claim 1 , 2 or 3 wherein R1 and R2 together form a ring substituted with the group
Figure imgf000133_0001
5. A compound according to any one of the preceding claims wherein R., and R2 together form a carbocyclic ring.
6. A compound according to any one of the preceding claims wherein R, and R2 together form a six membered ring.
7. A compound according to any one of the preceding claims wherein R., and R2 together form an aryl ring.
A compound according to any one of the preceding claims having Formula Formula III
Figure imgf000133_0002
9. A compound according to any one of the preceding claims having Formula IV. Formula IV
Figure imgf000133_0003
10. A compound according to any one of the preceding claims having Formula V. Formula V
Figure imgf000134_0001
11. A compound according to any one of the preceding claims having Formula VI Formula VI
Figure imgf000134_0002
12. A compound according to any one of claims 1 to 10 having Formula VII Formula VII
Figure imgf000134_0003
13. A compound according to any one of the preceding claims wherein R3 is selected from H, hydrocarbyl, -S-hydrocarbyl, -S-H, halogen and N(R9)(R10), wherein each of R9 and R10 are independently selected from H and hydrocarbyl groups.
14. A compound according to any one of the preceding claims wherein R3 is selected from H and 0,-0,0 alkyl groups, such as C,-C6 alkyl group, and C,-C3 alkyl group.
15. A compound according to any one of the preceding claims wherein R3 is -CH3.
16. A compound according to any one of claims 1 to 7 having Formula VIII. Formula VIII
Figure imgf000135_0001
17. A compound according to according to any one of claims 1 to 7 having Formula IX.
Formula IX
Figure imgf000135_0002
18. A compound according to according to any one of claims 1 to 7 having Formula X Formula X
Figure imgf000135_0003
19. A compound according to any one of claims 1 to 7 having Formula XI Formula XI
Figure imgf000135_0004
20. A compound according to any one of claims 16 to 19 wherein R3 is selected from O, hydrocarbyl, and N(R9) wherein R9 is selected from H and hydrocarbyl groups.
21. A compound according to any one of the preceding claims wherein R3 is selected from O, C,-C10 alkenyl groups, such as C,-C6 alkenyl group, and C,-C3 alkenyl group, NH and N-C,-C10 alkyl groups, such as N-C,-C6 alkyl group, and N-C,-C3 alkyl groups.
22. A compound according to any one of the preceding claims wherein R4 is selected from H and C,-C10 alkyl groups, such as C C6 alkyl group, and C,-C3 alkyl group.
23. A compound according to any one of the preceding claims wherein R4 is H.
24. A compound according to any one of claims 1 to 21 wherein R4 is a group of the formula.
Figure imgf000136_0001
25. A compound according to any one of the preceding claims wherein R5 is a substituted ring.
26. A compound according to any one of the preceding claims wherein R5 is a carbocyclic ring.
27. A compound according to any one of the preceding claims wherein R5 is a six membered ring.
28 A compound according to any one of the preceding claims wherein R5 is an aryl ring.
29 A compound according to any one of the preceding claims wherein R5 is a group having the formula
Figure imgf000136_0002
wherein each of R-n, R12l R13, R14 and R15 are independently selected from H, halogen, and hydrocarbyl groups.
30 A compound according to claim 29 wherein each of R^, R12, R13, R1 and R15 are independently selected from H, halogen, alkyl, phenyl, O-alkyl, O-phenyl, nitrile, haloalkyl, carboxyalkyl, -CO2H, CO2alkyl, and NH-acetyl groups..
31. A pharmaceutical composition comprising a compound according to any one of claims 1 to 30 optionally admixed with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
32. A compound according to any one of claims 1 to 30 for use in medicine.
33. Use of a compound according to any one of claims 1 to 30 in the manufacture of a medicament for use in the therapy of a condition or disease associated with 11 β-HSD.
34. Use according to claim 33 wherein the condition or disease is selected from the group consisting of metabolic disorders such as diabetes and obesity; cardiovascular disorders such as hypertension; glaucoma; inflammatory disorders such as arthritis or asthma; immune disorders; bone disorders such as osteoporosis; cancer; intra-uterine growth retardation; apparent mineralocorticoid excess syndrome (AME); polycystic ovary syndrome (PCOS); hirsutism; acne; oligo- or amenorrhea; adrenal cortical adenoma and carcinoma; Cushing's syndrome; pituitary tumours; invasive carcinomas; breast cancer; and endometrial cancer.
35. Use of a compound according to any one of claims 1 to 30 in the manufacture of a medicament for use in the therapy of a condition or disease associated with adverse 11 β-HSD levels.
36. Use of a compound according to any one of claims 1 to 30 in the manufacture of a pharmaceutical for modulating 11 β-HSD activity.
37. Use of a compound according to any one of claims 1 to 30 in the manufacture of a pharmaceutical for inhibiting 11 β-HSD activity.
38. A method comprising (a) performing a 11 β-HSD assay with one or more candidate compounds having the formula as defined in any one claims 1 to 30; (b) determining whether one or more of said candidate compounds is/are capable of modulating 11 β-HSD activity; and (c) selecting one or more of said candidate compounds that is/are capable of modulating 11 β-HSD activity.
39. A method comprising (a) performing a 11 β-HSD assay with one or more candidate compounds having the formula as defined in any one of claims 1 to 30; (b) determining whether one or more of said candidate compounds is/are capable of inhibiting 11 β-HSD activity; and (c) selecting one or more of said candidate compounds that is/are capable of inhibiting 11 β-HSD activity.
40. A compound identified by the method according to claim 38 or claim 39.
41. A compound according to claim 40 for use in medicine.
42. A pharmaceutical composition comprising the compound according to claim 40 optionally admixed with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
43. Use of a compound according to claim 40 in the manufacture of a medicament for use in the therapy of a condition or disease associated with 11 β-HSD.
44. Use according to claim 43 wherein the condition or disease is selected from the group consisting of metabolic disorders such as diabetes and obesity; cardiovascular disorders such as hypertension; glaucoma; inflammatory disorders such as arthritis or asthma; immune disorders; bone disorders such as osteoporosis; cancer; intra-uterine growth retardation; apparent mineralocorticoid excess syndrome (AME); polycystic ovary syndrome (PCOS); hirsutism; acne; oligo- or amenorrhea; adrenal cortical adenoma and carcinoma; Cushing's syndrome; pituitary tumours; invasive carcinomas; breast cancer; and endometrial cancer.
45. Use of a compound according to claim 40 in the manufacture of a medicament for use in the therapy of a condition or disease associated with adverse 11 β-HSD levels.
46. The invention of any one of claims 33 to 45 wherein 11 β-HSD is 11 β-HSD Type 1.
47. The invention of any one of claims 33 to 45 wherein 11 β-HSD is 11 β-HSD Type 2.
48. A compound as substantially hereinbefore described with reference to any one of the Examples.
49. A composition as substantially hereinbefore described with reference to any one of the Examples.
50. A method as substantially hereinbefore described with reference to any one of the Examples.
51. A use as substantially hereinbefore described with reference to any one of the Examples.
PCT/GB2003/004590 2002-10-24 2003-10-23 Inhibitors of 11-beta-hydroxy steroid dehydrogenase type 1 and type 2 WO2004037251A1 (en)

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