EP1675844A1 - Phenyl carboxamide and sulfonamide derivatives for use as 11-beta-hydroxysteroid dehydrogenase - Google Patents

Phenyl carboxamide and sulfonamide derivatives for use as 11-beta-hydroxysteroid dehydrogenase

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Publication number
EP1675844A1
EP1675844A1 EP04791576A EP04791576A EP1675844A1 EP 1675844 A1 EP1675844 A1 EP 1675844A1 EP 04791576 A EP04791576 A EP 04791576A EP 04791576 A EP04791576 A EP 04791576A EP 1675844 A1 EP1675844 A1 EP 1675844A1
Authority
EP
European Patent Office
Prior art keywords
arh
compound according
mmol
ethyl acetate
hsd
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04791576A
Other languages
German (de)
French (fr)
Inventor
Nigel Vicker
Su Xiangdong
Dharshini Ganeshapillai
Atul Purohit
Michael John Reed
Barry Victor Lloyd Potter
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sterix Ltd
Original Assignee
Sterix Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB0324792.1A external-priority patent/GB0324792D0/en
Application filed by Sterix Ltd filed Critical Sterix Ltd
Publication of EP1675844A1 publication Critical patent/EP1675844A1/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/08—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms
    • C07D211/18—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D211/26—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms with hydrocarbon radicals, substituted by nitrogen atoms
    • C07D211/28—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms with hydrocarbon radicals, substituted by nitrogen atoms to which a second hetero atom is attached
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/64—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings
    • C07C233/76—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by doubly-bound oxygen atoms
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    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/42—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/44—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton with carbon atoms of carboxamide groups and singly-bound oxygen atoms bound to carbon atoms of the same non-condensed six-membered aromatic ring
    • C07C235/56—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton with carbon atoms of carboxamide groups and singly-bound oxygen atoms bound to carbon atoms of the same non-condensed six-membered aromatic ring having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a six-membered aromatic ring
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    • C07C311/15—Sulfonamides having sulfur atoms of sulfonamide groups bound to carbon atoms of six-membered aromatic rings
    • C07C311/21—Sulfonamides having sulfur atoms of sulfonamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring
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    • C07C311/30—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/37—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring
    • C07C311/38—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring having sulfur atoms of sulfonamide groups and amino groups bound to carbon atoms of six-membered rings of the same carbon skeleton
    • C07C311/39—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring having sulfur atoms of sulfonamide groups and amino groups bound to carbon atoms of six-membered rings of the same carbon skeleton having the nitrogen atom of at least one of the sulfonamide groups bound to hydrogen atoms or to an acyclic carbon atom
    • C07C311/40—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring having sulfur atoms of sulfonamide groups and amino groups bound to carbon atoms of six-membered rings of the same carbon skeleton having the nitrogen atom of at least one of the sulfonamide groups bound to hydrogen atoms or to an acyclic carbon atom to an acyclic carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
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    • C07D209/44—Iso-indoles; Hydrogenated iso-indoles
    • C07D209/48—Iso-indoles; Hydrogenated iso-indoles with oxygen atoms in positions 1 and 3, e.g. phthalimide
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    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom 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 to ring carbon atoms
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    • C07D261/00—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings
    • C07D261/02—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings
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    • C07D261/10—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings having two or more double bonds between ring members or between ring members and non-ring members 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 to ring carbon atoms
    • C07D261/14—Nitrogen atoms
    • C07D261/16—Benzene-sulfonamido isoxazoles
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    • C07D295/12—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms
    • C07D295/135—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms separated by carbocyclic rings or by carbon chains interrupted by carbocyclic rings
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    • C07D307/78—Benzo [b] furans; Hydrogenated benzo [b] furans
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    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/06—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2
    • C07D311/08—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring
    • C07D311/18—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring substituted otherwise than in position 3 or 7
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    • C07D319/00—Heterocyclic compounds containing six-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D319/10—1,4-Dioxanes; Hydrogenated 1,4-dioxanes
    • C07D319/14—1,4-Dioxanes; Hydrogenated 1,4-dioxanes condensed with carbocyclic rings or ring systems
    • C07D319/16—1,4-Dioxanes; Hydrogenated 1,4-dioxanes condensed with carbocyclic rings or ring systems condensed with one six-membered ring
    • C07D319/18—Ethylenedioxybenzenes, not substituted on the hetero ring
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    • C07D333/50—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom condensed with carbocyclic rings or ring systems
    • C07D333/52—Benzo[b]thiophenes; Hydrogenated benzo[b]thiophenes
    • C07D333/54—Benzo[b]thiophenes; Hydrogenated benzo[b]thiophenes with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to carbon atoms of the hetero ring
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    • C07D333/52—Benzo[b]thiophenes; Hydrogenated benzo[b]thiophenes
    • C07D333/62—Benzo[b]thiophenes; Hydrogenated benzo[b]thiophenes 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 to carbon atoms of the hetero ring
    • C07D333/68—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
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    • C07C2603/18—Fluorenes; Hydrogenated fluorenes

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 15 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 25 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.
  • 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.
  • the present invention provides a pharmaceutical composition
  • 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 I defined above.
  • 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 modulating 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
  • 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 ⁇ -HSD, and
  • Q is an atom selected from the group consisting of S, O, N and C, include the following structural moieties
  • the structural moiety may be saturated or may include unsaturation, such as one or more double bonds.
  • the structural moiety may be substituted.
  • the structural moiety may be part of a polycyclic system such as , for example
  • R ⁇ is an optionally substituted phenyl ring and R 2 is or comprises an optionally substituted aromatic ring.
  • R-j and R 2 are referred to collectively as the ring systems.
  • Ri is an optionally substituted phenyl ring.
  • Ri may be substituted or unsubstituted.
  • Ri is substituted.
  • Ri may be substituted with one or more hydrocarbyl substituents.
  • the substituents are selected from hydrocarbon groups, oxyhydrocarbon groups, halogens, amines and amides. More preferably the substituents are selected from aromatic hydrocarbon groups, alkyl groups, oxyalkyl groups, halogens, amines and amides, such as from aromatic hydrocarbon groups, alkyl groups, oxyalkyl groups and halogens.
  • the substituents are selected from phenyl groups, C 1-5 alkyl groups, oxy-C 1-5 -alkyl groups, chloro, bromo and iodo. More preferably the substituents are selected from phenyl, methyl, ethyl, propyl, O-methyl, O-ethyl, O-propyl and chloro.
  • Ri is selected from the following:
  • Ri is or comprises a group selected from the following wherein indicates the point of attachment to Z.
  • Ri is selected from the following:
  • R 2 is or comprises an optionally substituted aromatic ring.
  • the optionally substituted aromatic ring is a five or six membered ring.
  • R 2 is an optionally substituted five membered aromatic ring.
  • R 2 is an optionally substituted six membered aromatic ring.
  • the optionally substituted aromatic ring is a heterocyclic ring.
  • R 2 is an optionally substituted five or six membered aromatic heterocyclic ring.
  • the optionally substituted aromatic ring is a heterocyclic ring comprising a carbon and a hetero atom selected from O, S and N. More preferably the optionally substituted aromatic ring is a heterocyclic ring comprising a carbon and a hetero atom selected from O and N.
  • R 2 is or comprises:
  • R 2 is
  • R 2 is or comprises a group selected from the following wherein indicates the point of attachment to Z.
  • R 2 is an optionally substituted five membered heterocyclic aromatic ring. In this aspect preferably R 2 is or comprises:
  • R 2 is or comprises:
  • the optionally substituted aromatic ring is a carbocyclic ring.
  • R 2 may be substituted or unsubstituted.
  • R 2 is substituted, more preferably R 2 is a substituted aromatic carbocyclic ring.
  • the substituents are selected from hydrocarbon groups, oxyhydrocarbon groups, halogens, amines and amides. More preferably the substituents are selected from aromatic hydrocarbon groups, alkyl groups, oxyalkyl groups, halogens, amines and amides, such as from aromatic hydrocarbon groups, alkyl groups, oxyalkyl groups and halogens.
  • R 2 is substituted with two or more substituents.
  • R 2 is substituted with two or more substituents and the two or more substituents together form a ring which is fused to the carbocyclic ring of R 2 .
  • the carbocyclic ring is a five or six membered aromatic carbocyclic ring. More preferably the carbocyclic ring is a phenyl ring.
  • R 2 is a substituted phenyl ring.
  • R 2 is substituted with two or more substituents and the two or more substituents together form a ring which is fused to the phenyl ring of R 2 .
  • R 2 comprises the following structure:
  • R 2 comprises the following structure:
  • R 2 is or comprises a group selected from the following:
  • R 2 is or comprises a group selected from the following:
  • R 2 comprises the following structure:
  • R 2 is or comprises a group selected from the following:
  • R 2 is or comprises a group selected from the following:
  • R 2 is or comprises a group selected from the following:
  • R 2 is or comprises a group selected from the following:
  • R 2 is or comprises a group selected from the following wherein — indicates the point of attachment to Z.
  • 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.
  • Each ring system may contain one or more degrees of unsaturation, for example is some aspects one or more rings of a ring system is aromatic.
  • Each ring system may be carbocyclic or may contain one or more hetero atoms.
  • the compound of the invention in particular the ring systems of the compound of the 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 oxy hydrocarbyl group.
  • the ring systems of the present compounds may contain a variety of non- interfering substituents.
  • the ring systems 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 C 6 ) alkoxy, e.g. methoxy, ethoxy, propoxy etc., alkinyl, e.g. ethinyl, or halogen, e.g. fluoro substituents.
  • alkyl especially lower (C C 6 ) alkyl, e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl
  • X is -S-.
  • Z is selected from -S- C(R 4 )(R 5 )-, -S-C(R 4 )(R 5 )-L-, -C(R 4 )(R 5 )-S- and -C(R 4 )(R 5 )-S-L-.
  • Y is -NR 3 -.
  • Y is -C(R 4 )(R 5 )-.
  • X is -S- and Y is -C(R )(R 5 ) ⁇ .
  • Z is - SC(R 4 )(R 5 )- or -C(R 4 )(R 5 )S-, such as -SCH 2 - or -CH 2 S-
  • L is an optional linker.
  • L may be any suitable group, for example L may a hydrocarbyl group or a hetero atom, in particular L may be a hydrocarbon group such a C ⁇ o alkyl group.
  • 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 1 -C 1 0 alkyl group, such as C ⁇ -C 6 alkyl group, and C 1 -C 3 alkyl group.
  • Typical alkyl groups include Ci 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 C C 10 haloalkyl group, C C 6 haloalkyl group, C 1 -C 3 haloalkyl group, CrC bromoalkyl group, C ⁇ -C 6 bromoalkyl group, and C C 3 bromoalkyl group.
  • Typical haloalkyl groups include Ci haloalkyl, C 2 haloalkyl, C 3 haloalkyl, C haloalkyl, C 5 haloalkyl, C 7 haloalkyl, C 8 haloalkyl, Ci 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 ) ⁇ -5 -Ph-C ⁇ -5 alkyl, -(CHs 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 ) ⁇ - ⁇ o-C 3-1 ocycloalkyl, -(CH 2 ) 1-7 -C 3- 7 cycloalkyl, -(CH 2 ) 1-5 -C 3 . 5 cycloalkyl, -(CH 2 ) ⁇ - 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 C C 10 alkene group, C C 6 alkene group, C 1 -C 3 alkene group, such as C-i, 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 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 1-6 0 (such as a C-i -3 0).
  • R 3 is selected from H and hydrocarbon groups.
  • R 3 is selected from H and alkyl groups, preferably from H and C ⁇ oalkyl groups, preferably from H and C ⁇ _ 5 alkyl groups.
  • R 3 is H.
  • R 3 may be equivalent to -L-R 2 wherein L and R 2 are independently selected from the possibilities defined herein.
  • the present compound contains two groups of the formula -L-R 2 wherein each L and R 2 are selected independently of each other.
  • each of L and R 2 may be the same as the other of L and R 2 present in the compound. Examples of compounds meeting such requirements are shown below.
  • R 4 and R 5 are independently selected from H and hydrocarbon groups.
  • R and R 5 are independently selected from H and alkyl groups, preferably independently selected from H and C -10 alkyl groups, preferably independently selected from H and C 1-5 alkyl groups.
  • at least one of R and R 5 is H.
  • R and R 5 are both H.
  • 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 also encompasses a process comprising 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. 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.
  • 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 sterneursteroids 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 sterneurosteroids dehydroepiandrosterone sulfate (DHEAS) and pregnenolone sulfate (PS) and/or other structurally similar organic compounds.
  • DHEAS dehydro
  • biological response modifier includes cytokines, immune modulators, growth factors, haematopoiesis regulating factors, colony stimulating factors, chernotactic, 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, lL-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.
  • 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.
  • 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.
  • 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.
  • 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 I 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
  • 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 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 group consisting of:
  • 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.
  • AME apparent mineralocorticoid excess syndrome
  • PCOS polycystic ovary syndrome
  • 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 compounds for use as steroid dehydrogenase inhibitors, and pharmaceutical compositions for the same.
  • the present invention provides a pharmaceutical composition
  • Ri is an aromatic ring.
  • Ri is a five or six membered aromatic ring, more preferably a six membered aromatic ring.
  • Ri is a carbocyclic ring.
  • Ri is an optionally substituted phenyl ring.
  • R 2 is or comprises a ring, preferably an aromatic ring, more preferably an optionally substituted aromatic ring.
  • R 2 comprises the following structure:
  • A represents a heterocyclic ring, preferably a five or six membered heterocyclic ring.
  • Figure 1 is a graph showing extraction efficiencies obtained with four extraction methods.
  • Figure 2 is a graph showing a comparison of 11 ⁇ -HSD1 activity in rat and human hepatic microsomes.
  • Figure 3 is a series of graphs showing the effect of incubation time on human microsomal 11 ⁇ -HSD1 activity
  • Figure 4 is a series of graphs showing the effect of microsomal protein concentration on human microsomal 11 ⁇ -HSD1 activity.
  • Figure 5 is a graph showing the substrate (cortisone) saturation curve for human hepatic microsomal 11 ⁇ HSD1.
  • Figure 6 is a Lineweaver-Burke plot.
  • Figure 7 is a graph showing the IC 50 determination for Glycyrrhetinic acid.
  • Figure 8 is a graph showing the IC 50 determination for Carbenoxolone.
  • Figures 9(A), 9(B) and 9(C) are graphs showing the 11 ⁇ -HSD1 activity measured by
  • Figure 9(A) shows the effect of protein
  • Figure 9(B) shows the effect of cortisone
  • Figure 9(C) shows the effect of Tween-80.
  • Figure 10 is a graph showing the performance of the cortisol immunoassay: various experimental designs.
  • Figure 11 is a graph showing the effect of increasing microsomal protein on measurement of 11 ⁇ HSD1 activity detected by Assay Designs Immunoassay.
  • Figure 12 is a graph showing the detection of 11 ⁇ HSD1 activity by RIA using the Immunotech anti-cortisol antibody.
  • Figure 13 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 14 is a graph showing the Immunotech antibody saturation curve for detection of
  • Figure 15 is a graph showing the linearity of human hepatic microsomal 11 ⁇ HSD1 activity detected by RIA.
  • Figure 16 is a graph showing the effect of Tween 80 on detection of human hepatic microsomal 11 ⁇ HSD1 activity by RIA.
  • Figure 17 is a graph showing the effect of buffer systems on detection of human hepatic microsomal 11 ⁇ HDS1 activity by RIA.
  • Figure 18 is a graph showing the linearity of human hepatic microsomal 11 ⁇ HSD1 activity with incubation time detected by RIA.
  • Figure 19 is a graph showing the substrate saturation curve for human hepatic microsomal 11 ⁇ HDS1 activity detected by RIA.
  • Figure 20 is a Lineweaver-Burke plot
  • Figure 21 is an IC 50 curve for inhibition of human hepatic microsomal 11 ⁇ HSD1 activity by Glycyrrhetinic acid.
  • Figure 22 is an IC 5 o curve for inhibition of human hepatic microsomal 11 ⁇ HSD1 activity by Glycyrrhetinic acid in the presence of 350 nM cortisone.
  • Figure 23 is an IC 50 curve for inhibition of human hepatic microsomal 11 ⁇ HSD1 activity by Carbenoxolone in the presence of 350 nM cortisone.
  • 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 , 3 H-cortisone was obtained from American Radiolabelled Compounds Inc, Product ART-743, 3 H- cortisol was obtained from NEN, Product NET 396, 14 C-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
  • Buffer 1 from Barf et al., (2002) [14]: 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 et al., (2002) [14]: 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.
  • 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.
  • Thin layer chromatography was performed on precoated plates (Merck TLC aluminium sheets silica gel 60 F 254 , Art. No. 5554). Compounds were visualised by either viewing under UV light or treating with an ethanolic solution of phosphomolybdic acid (PMA) followed by heating. Flash chromatography was carried out using Sorbsil C60 silica gel or Isolute ® pre-packed Flash Si columns from Argonaut Technologies. Parallel synthesis was performed on either Radleys Carousel reaction stations or Radleys GreenHouse parallel synthesisers. Solvent removal from parallel syntheses was performed on a GeneVac DD4 evaporation system.
  • PMA ethanolic solution of phosphomolybdic acid
  • NMR spectra were recorded with a JEOL GX-270 or Varian-Mercury-400 spectrometer, and chemical shifts are reported in parts per million (ppm, ⁇ relative to tetramethylsilane (TMS) as an internal standard. Mass spectra were recorded at the Mass Spectrometry Service Centre, University of Bath. FAB-MS were carried out using m-nitrobenzyl alcohol (NBA) as the matrix. High performance liquid chromatography (HPLC) analysis was performed with a Waters Delta 600 liquid chromatograph with a Waters 996 photodiode Array Detector using a Waters Radialpack C18, 8x100 mm column. Melting points (Mp) were measured with a Reichert-Jung ThermoGalen Kofler block or a Sanyo Gallenkamp melting point apparatus and are uncorrected.
  • HPLC high performance liquid chromatography
  • 5-amino-benzo[b]thiophene-2-carboxylic acid methyl ester KRB01094: To a solution of 5-nitro-benzo[b]thiophene-2-carboxylic acid methyl ester [15] (130 mg, 0.548 mmol) in methanol (30 mL) was added 5% palladium on carbon (20 mg) and the mixture was stirred under 1 atm H 2 for 2 h. The mixture was filtered through celite and the filtrate evaporated.
  • 5-amino-benzofuran-2-carboxylic acid methyl ester KRB01099: To a solution of 5- nitrobenzofuran-2-carboxylic acid methyl ester (100 mg, 0.452 mmol) in methanol (20 mL) was added 5% palladium on carbon (20 mg) and the mixture was stirred under 1 atm H 2 for 4h. The mixture was filtered through celite and the filtrate evaporated.
  • 6-amino-2-methylquinoxaline KRB01078: To a solution of 2-methyl-6-nitroquinoxaline [23] (500 mg, 2.64 mmol) in methanol (22 mL) was added 10% palladium on carbon (50 mg) and the mixture was stirred under 1 atm H 2 for 4 h. The mixture was filtered through celite and the filtrate evaporated. The residue was passed through a silica plug and evaporated to afford 6-amino-2-methylquinoxaline as a yellow solid (376 mg, 89%), single spot at R f 0.33 (ethyl acetate), mp 163-164°C ([23] 164-165°C).
  • 6-aminoquinoxaline KRB01083: To a solution of 6-nitroquinoxaline [24] (500 mg, 2.86 mmol) in methanol (20 mL) was added 10% palladium on carbon (50 mg) and the mixture was stirred under 1 atm H 2 for 4 h. The mixture was filtered through celite and the filtrate evaporated. The residue was passed through a silica plug and evaporated to afford 6-aminoquinoxaline as a yellow solid (342 mg, 82%), single spot at R f 0.32 (ethyl acetate).
  • Method B A solution of the acid (3n mmol) in thionyl chloride is refluxed 3 hours. Thionyl chloride is then removed under reduced pressure. The crude product is diluted in dry THF and added to a solution of the amine (n mmol) and triethylamine in THF. After completion, ethyl acetate and water are added . The aqueous layer is extracted by EtOAc. The combined organic layers are then washed with brine, dried (MgSO4), filtered and evaporated under reduce pressure. The crude product is purified to give the amide. Biphenyl-4-carboxylic acid (2-thiophen-2-yl-ethyl -amide (STX1377.
  • Method A to a stirred solution of the amine (n mmol) in THF are added triethylamine (1.2n mmol) and the acyl chloride (1.2n mrr ⁇ ol) at room temperature. After completion, ethyl acetate and water are added. The aqueous layer is extracted by ethyl acetate. The combined organic layers are washed with brine, dried (MgSO4), filtered and evaporated under reduce pressure. The crude product is then purified to give the amide.
  • Method B A solution of the acid (3n mmol) in thionyl chloride is refluxed 3 hours. Thionyl chloride is then removed under reduced pressure.
  • the crude product is diluted in dry THF and added to a solution of the amine (n mmol) and triethylamine in THF. After completion, ethyl acetate and water are added . The aqueous layer is extracted by EtOAc. The combined organic layers are then washed with brine, dried (MgSO4), filtered and evaporated under reduce pressure. The crude product is purified to give the amide.
  • Method A to a stirred solution of the amine (n mmol) in THF are added triethylamine (1.2n mmol) and the acyl chloride (1.2n mmol) at room temperature. After completion, ethyl acetate and water are added. The aqueous layer is extracted by ethyl acetate. The combined organic layers are washed with brine, dried (MgSO4), filtered and evaporated under reduce pressure. The crude product is then purified to give the amide.
  • Method B A solution of the acid (3n mmol) in thionyl chloride is refluxed 3 hours. Thionyl chloride is then removed under reduced pressure.
  • the crude product is diluted in dry THF and added to a solution of the amine (n mmol) and triethylamine in THF. After completion, ethyl acetate and water are added . The aqueous layer is extracted by EtOAc. The combined organic layers are then washed with brine, dried (MgSO4), filtered and evaporated under reduce pressure. The crude product is purified to give the amide.
  • N-(4-Benzoyl-phenyl -3.5-dichloro-benzamide STX1431.
  • CCM0103n Reaction of 4-aminobenzophenone (100 mg, 0.51 mmol) in THF (6 mL) with 3,5- dicchlorobenzoyl chloride (136 mg, 0.65 mmol) in presence of triethylamine (90 ⁇ L, 0.65 mmol) according to method A gave N-(4-benzoyl-phenyl)-3,5-dichloro-benzamide (160 mg, 0.33 mmol, 63% yield) as a white powder after purification by crystallisation in
  • N-(4-Benzoyl-phenyl -3-methoxy-benzamide (STX1432. CCM01032 ⁇ ) Reaction of 4-aminobenzophenone (100 mg, 0.51 mmol) in THF (6 mL) with m-anisoyl chloride (136 mg, 0.65 mmol) in presence of triethylamine (90 ⁇ L, 0.65 mmol) according to method A gave N-(4-benzoyl-phenyl)-3-methoxy-benzamide (160 mg, 0.48 mmol, 94% yield) as a white powder after purification by cliromatography on silica gel (eluent EtOAc/DCM 0/100 to 5/95.
  • N-(4-Benzoyl-phenyl -2.5-dichloro-benzamide (STX1433.
  • CCM01034 Reaction of 2,5-dichlorobenzoic acid (300 mg, 1.50 mmol) in thionyl chloride (3.5 mL) then with 4-aminobenzophenone (100 mg, 0.51 mmol) in presence of triethylamine (90 ⁇ L) in THF (6 mL) according to method B gave N-(4-benzoyl-phenyl)-2,5-dichloro- benzamide (160 mg, 0.43 mmol, 84% yield) as a off-white powder after purification by cliromatography on silica gel (eluent : EtOAc/exane 1/9 to 3/7).
  • N-f4-Benzoyl-phenylV4-propyl-benzamide (STX1434.
  • CCM01036 Reaction of 4-aminobenzophenone (99 mg, 0.51 mmol) in THF (6 mL) with 4- propylbenzoyl chloride (100 ⁇ L, 0.65 mmol) in presence of triethylamine (90 ⁇ L, 0.65 mmol) according to method A gave N-(4-benzoyl-phenyl)-4-propyl-benzamide (136 mg, 0.40 mmol, 78% yield) as a white powder after purification by chromatography on silica gel (eluent EtOAc/DCM 0/100 to 5/95).
  • N-(4-Benzoyl-phenyl -4-methoxy-benzamide STX1435.
  • CCM01037 Reaction of 4-aminobenzophenone (98 mg, 0.51 mmol) in THF (6 mL) with -anisoyl chloride (90 ⁇ L, 0.65 mmol) in presence of triethylamine (90 ⁇ L, 0.65 mmol) according to method A gave N-(4-benzoyl-phenyl)-4-methoxy-benzamide (165 mg, 0.50 mmol, 98% yield) as a white powder after purification by washing the crude product with ethyl acetate and hexane.
  • Method A to a stirred solution of the amine (n mmol) in THF are added triethylamine (1.2n mmol) and the acyl chloride (1.2n mmol) at room temperature. After completion, ethyl acetate and water are added. The aqueous layer is extracted by ethyl acetate. The combined organic layers are washed with brine, dried (MgSO4), filtered and evaporated under reduce pressure. The crude product is then purified to give the amide.
  • Method B A solution of the acid (3n mmol) in thionyl chloride is refluxed 3 hours. Thionyl chloride is then removed under reduced pressure.
  • the crude product is diluted in dry THF and added to a solution of the amine (n mmol) and triethylamine in THF. After completion, ethyl acetate and water are added . The aqueous layer is extracted by EtOAc. The combined organic layers are then washed with brine, dried (MgSO4), filtered and evaporated under reduce pressure. The crude product is purified to give the amide.
  • Biphenyl-4-carboxylic acid (4-chloro-2-methyl-benzothiazol-5-yl)-amide (STX1364, CCM01009) Reaction of 4-biphenylacetic acid (160 mg, 0.76 mmol) in thionyl chloride (1 mL) then with 4-chloro-2-methyl-benzothiazol-5-ylamine (50 mg, 0.25 mmol) in presence of triethylamine (0.5 mL) in THF (2 mL) according to method B gave biphenyl-4-carboxylic acid (4-chloro-2-methyl-benzothiazol-5-yl)-amide (60 mg, 0.15 mmol, 60% yield) as a pink powder after purification by chromatography on silica gel (eluent : DCM).
  • Method A to a stirred solution of the amine (n mmol) in THF are added triethylamine (1.2n mmol) and the acyl chloride (1.2n mmol) at room temperature. After completion, ethyl acetate and water are added. The aqueous layer is extracted by ethyl acetate. The combined organic layers are washed with brine, dried (MgSO4), filtered and evaporated under reduce pressure. The crude product is then purified to give the amide.
  • Method B A solution of the acid (3n mmol) in thionyl chloride is refluxed 3 hours. Thionyl chloride is then removed under reduced pressure.
  • the crude product is diluted in dry THF and added to a solution of the amine (n mmol) and triethylamine in THF. After completion, ethyl acetate and water are added . The aqueous layer is extracted by EtOAc. The combined organic layers are then washed with brine, dried (MgSO4), filtered and evaporated under reduce pressure. The crude product is purified to give the amide.
  • 2,5-Dichloro-W-(5-oxo-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide (CCM01044, STX1465) Reaction of 2,5-dichlorobenzoic acid (300 mg, 1.50 mmol) in thionyl chloride (3.5 mL) then with 6-Amino-3,4-dihydro-2H-naphthalen-l-one (80 mg, 0.50 mmol) in presence of triethylamine (90 ⁇ L, 0.65 mmol) in T ⁇ F (6 mL) according to method B gave 2,5- dichloro-N-(5-oxo-5,6,7,8-tetraliydro-naphthalen-2-yl)-benzamide (150 mg, 0.48 mmol, 76% yield) as a grey powder after purification by crystallisation in DCM.
  • Method A to a stirred solution of the amine (n mmol) in THF are added triethylamine (1.2n mmol) and the acyl chloride (1.2n mmol) at room temperature. After completion, ethyl acetate and water are added. The aqueous layer is extracted by ethyl acetate. The combined organic layers are washed with brine, dried (MgSO4), filtered and evaporated under reduce pressure. The crude product is then purified to give the amide.
  • Method B A solution of the acid (3n mmol) in thionyl chloride is refluxed 3 hours. Thionyl chloride is then removed under reduced pressure.
  • the crude product is diluted in dry THF and added to a solution of the amine (n mmol) and triethylamine in THF. After completion, ethyl acetate and water are added . The aqueous layer is extracted by EtOAc. The combined organic layers are then washed with brine, dried (MgS04), filtered and evaporated under reduce pressure. The crude product is purified to give the amide.
  • This experiment was carried out to compare the enzyme activity in hepatic microsomes from human and rat and to assess minimum microsomal protein concentrations necessary for reasonable measurement of enzyme activity.
  • the assay was carried out in Buffer 2 and the cortisone concentration used was 2 ⁇ M containing 0.5 ⁇ Ci per incubation 3 H-cortisone.
  • Rat and human hepatic microsomes were tested at concentrations ranging from 400 ⁇ g to 50 ⁇ g microsomal protein per incubation in a final incubation volume of 100 ⁇ l in glass tubes. Buffer was substituted for microsomal protein for blanks.
  • Enzyme activity is linear with microsomal protein concentrations below 30 ⁇ g per sample
  • An enzyme immunoassay kit was obtained from Assay Designs, Inc.
  • the antibody provided in the kit is a mouse monoclonal reported to cross react 100% with cortisol (the enzyme product) and ⁇ 0.1 % with cortisone (the enzyme substrate).
  • the kit is designed for the analysis of cortisol levels in saliva, urine, serum and plasma and also in tissue culture media, not for microsomal enzyme activity.
  • Figure 9 (11 B-HSD1 activity measured by Immunoassay)
  • Figure 9(A) shows the effect of protein. Data taken from 700 ⁇ M cortisone group tested in the presence of Tween-80
  • Figure 9(B) shows the effect of cortisone. Data taken from the 25 ⁇ g microsomal protein group tested in the presence of Tween-80
  • Figure 9(C) shows the effect of Tween-80. Data taken from the 25 ⁇ g microsomal protein group tested in the presence of 700 ⁇ M cortisone
  • the enzyme assay was carried out in Buffer 2.
  • the substrate (cortisone) concentration of 175 nM was chosen from the SPA method described by Barf et al. [14] with 0.5 ⁇ Ci / well 3 H- cortisone.
  • the enzyme assay was carried out in a polypropylene plate in a final incubation volume of 100 ⁇ l containing 10 ⁇ g / well human hepatic microsomal protein. Blanks had either buffer substituted for microsomal protein or had 10 ⁇ l stop solution added prior to the microsomes.
  • the assay was incubated at 37°C for 30 mins and the reaction was terminated by the addition of the stop solution to all remaining wells.
  • the Immunotech antibody was diluted in Buffer 3 to give 25 ⁇ g / 100 ⁇ l down to 6.25 ⁇ g / 100 ⁇ l.
  • the antibody (100 ⁇ l) was added to test wells, 100 ⁇ l Buffer 3 was added to the antibody blank wells. The remainder of the procedures followed the 96 well plate RIA protocol exactly. Results demonstrating 11 ⁇ HSD1 activity using the Immunotech are shown in Figure 12.
  • the antibody titre was examined in the next test, investigating concentrations per well from 6.7 ⁇ g down to 0.67 ⁇ g.
  • the usual 11 ⁇ HSD1 assay was carried out except that the microsomal protein concentration was doubled to 20 ⁇ g / well in order to get the best signal to noise.
  • the cortisone concentration was 175 nM and the enzyme assay buffer was Buffer 2.
  • Each antibody concentration was tested against a "no enzyme" blank (buffer substituted for microsomes) and a "GA blank” (10 ⁇ l stop solution added prior to microsomes) and a control group.
  • the RIA was carried out exactly as indicated in the methods for assay in 96 wells. These results are shown in Figure 13 and Figure 14.
  • the antibody will be used at 1.7 ⁇ 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. The data in Figure 16 are taken from the group tested with 10 ⁇ g / well microsomal protein, but the same effect was seen with all protein concentrations examined.
  • both enzyme assay and RIA stages were 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 ⁇ g / well and the cortisone concentration was 175 nM.
  • Performing both enzyme assay and RIA in enzyme assay buffer gave similar data to the two buffers system but performing both enzyme assay and RIA in Buffer 3 appeared to improve the data slightly.
  • Figure 18 Linearity of human hepatic microsomal 11 ⁇ HSD1 activity with incubation time detected by RIA
  • the substrate saturation effects were examined in the next assay.
  • the enzyme assay was carried out exactly as indicated in the methods section in buffer 3 with 10 ⁇ g / well microsomal protein and with [cold cortisone] as indicated. 3H-cortisone was 0.5 ⁇ Ci / sample throughout. The reaction was stopped after 30 min by the addition of 10 ⁇ l stop solution. The RIA was carried out exactly as indicated in the methods section. Results are shown in Figure 19.
  • an IC 50 for Glycyrrhetinic acid was determined in the next test.
  • a 10 mM stock solution of Glycyrrhetinic acid was prepared in 100 % DMSO and was further diluted in 100 %
  • GA blanks (addition of 10 ⁇ l stop solution prior to the addition of microsomes) were included with and without the addition of 1 % DMSO.
  • Human hepatic microsomal protein was tested at 10 ⁇ g / well and the substrate concentration (cortisone) was 175 nM, 0.5 ⁇ Ci / well. All other procedures were as indicated in the methods section.
  • GA inhibition data are shown in Figure 21.
  • Figure 22 (ICm curve for inhibition of human hepatic microsomal 11 ⁇ HSD1 activity by Glycyrrhetinic acid in the presence of 350 nM cortisone) and Figure 23 (ICgn curve for inhibition of human hepatic microsomal 11 ⁇ HSD1 activity by Carbenoxolone in the presence of 350 nM cortisone)

Abstract

There is provided a compound having Formula (I) R1-Z-R2 Formula (I) wherein R1 is an optionally substituted phenyl ring; R2 is or comprises an optionally substituted aromatic ring; and Z is -X-Y-L- or -Y-X-L- wherein either X is selected from -S(=O)(=O)- and -C(=O)-, and Y is -NR3-; or X is selected from -S(=O)(=O)- and -S-, and Y is -C(R4)(R5)-; L is an optional linker; and R3, R4 and R5 are each independently selected from H and hydrocarbyl; and wherein when R2 comprises the following structural moiety, Formula (II) wherein Q is an atom selected from the group consisting of S, O, N and C; the compound is selected from compounds of the formulae R1-C(=O)-NR3-L-R2; R1-S(=O)(=O)-C(R4)(R5)-L-R2; R1-S-C(R4)(R5)-L-R2; R1-NR3-S(=O)(=O)-L-R2; R1-NR3-C(=O)-L-R2; R1-C(R4)(R5)-S(=O)(=O)-L-R2; and R1-C(R4)(R5)-S-L-R2. These compounds are useful as 11β-hydroxysteriod dehydrogenase inhibitors in the treatment of i.a. diabetes.

Description

PHENYL CARBOXAMIDE AND SULFONAMIDE DERIVATIVES FOR USE AS 11-BETA-HYDROXYSTEROID DEHYDROGENASE
FIELD OF INVENTION
5 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 10 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 15 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]. 20 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 25 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.
30 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 11 β-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.
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 I R Z-R2 Formula I wherein R^ is an optionally substituted phenyl ring; R2 is or comprises an optionally substituted aromatic ring; and Z is -X-Y-L- or -Y-X-L- wherein either X is selected from - S(=O)(=O)- and -C(=O)-, and Y is -NR3-; or X is selected from -S(=O)(=0)- and -S-, and Y is -C(R4)(R5)-; L is an optional linker; and R3, R4 and R5 are each independently selected from H and hydrocarbyl; and wherein when R2 comprises the following structural moiety
wherein Q is an atom selected from the group consisting of S, O, N and C; the compound is selected from compounds of the formulae R C(=0)-NR3-L-R2; R S(=O)(=O)-C(R4)(R5)-L-R2; R S-C(R4)(R5)-L-R2; R NR3-S(=O)(=O)-L-R2; R NR3- C(=O)-L-R2; R C(R4)(R5)-S(=O)(=0)-L-R2; and R C(R4)(R5)-S-L-R2.
In one aspect the present invention provides a pharmaceutical composition comprising (i) a compound having Formula I R Z-R2 Formula I wherein R^ is an optionally substituted phenyl ring; R2 is or comprises an optionally substituted aromatic ring; and Z is -X-Y-L- or -Y-X-L- wherein either X is selected from - S(=O)(=O)- and -C(=0)-, and Y is -NR3-; or X is selected from -S(=0)(=O)- and -S-, and Y is -C(R4)(R5)~; L is an optional linker; and R3, R4 and R5 are each independently selected from H and hydrocarbyl; and wherein when R2 comprises the following structural moiety
wherein Q is an atom selected from the group consisting of S, O, N and C; the compound is selected from compounds of the formulae R C(=0)-NR3-L-R2; R
S(=O)(=O)-C(R4)(R5)-L-R2; R S-C(R4)(R5)-L-R2; R NR3-S(=O)(=0)-L-R2; R NR3-
C(=O)-L-R2; R C(R4)(R5)-S(=O)(=O)-L-R2; and R1-C(R4)(R5)-S-L-R2. (ii) optionally admixed with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
In one aspect the present invention provides a compound for use in medicine wherein the compound has Formula I Rι-Z-R2 Formula I wherein R-i is an optionally substituted phenyl ring; R2 is or comprises an optionally substituted aromatic ring; and Z is -X-Y-L- or -Y-X-L- wherein either X is selected from - S(=O)(=O)- and -C(=O)-, and Y is -NR3-; or X is selected from -S(=0)(=O)- and -S-, and Y is -C(R4)(R5)~; L is an optional linker; and R3, R4 and R5 are each independently selected from H and hydrocarbyl; and wherein when R2 comprises the following structural moiety
wherein Q is an atom selected from the group consisting of S, O, N and C; the compound is selected from compounds of the formulae Rι-C(=0)-NR3-L-R2; R S(=O)(=O)-C(R4)(R5)-L-R2; R S-C(R4)(R5)-L-R2; R1-NR3-S(=O)(=0)-L-R2; R NR3- C(=O)-L-R2; R C(R4)(R5)-S(=O)(=0)-L-R2; and R1-C(R4)(R5)-S-L-R2.
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 R Z-R2 Formula I wherein Ri is an optionally substituted phenyl ring; R2 is or comprises an optionally substituted aromatic ring; and Z is -X-Y-L- or -Y-X-L- wherein either X is selected from - S(=O)(=O)- and -C(=O)-, and Y is -NR3-; or X is selected from -S(=O)(=O)- and -S-, and Y is -C(R )(R5)-; L is an optional linker; and R3, R and R5 are each independently selected from H and hydrocarbyl; and wherein when R2 comprises the following structural moiety
wherein Q is an atom selected from the group consisting of S, O, N and C; the compound is selected from compounds of the formulae RrC(=O)-NR3-L-R2; R S(=O)(=O)-C(R4)(R5)-L-R2; R S-C(R4)(R5)-L-R2; RrNR3-S(=0)(=O)-L-R2; R NR3- C(=O)-L-R2; RrC(R4)(R5)-S(=0)(=O)-L-R2; and R C(R4)(R5)-S-L-R2.
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
As previously mentioned, in one aspect the present invention provides a compound having Formula I defined above.
As previously mentioned, 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. As previously mentioned, in one aspect the present invention provides a compound having Formula I defined above, for use in medicine.
As previously mentioned, 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 modulating 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
Compound
As previously mentioned, in one aspect the present invention provides a compound having Formula I R Z-R2 Formula I wherein R-i is an optionally substituted phenyl ring; R2 is or comprises an optionally substituted aromatic ring; and Z is -X-Y-L- or -Y-X-L- wherein either X is selected from - S(=O)(=O)- and -C(=0)-, and Y is -NR3-; or X is selected from -S(=O)(=O)- and -S-, and Y is -C(R )(R5)-; L is an optional linker; and R3, R4 and R5 are each independently selected from H and hydrocarbyl; and wherein when R2 comprises the following structural moiety
wherein Q is an atom selected from the group consisting of S, O, N and C; the compound is selected from compounds of the formulae RrC(=0)-NR3-L-R2; R-r S(=O)(=O)-C(R4)(R5)-L-R2; R S-C(R4)(R5)-L-R2; R NR3-S(=O)(=0)-L-R2; RrNR3- C(=O)-L-R2; R C(R4)(R5)-S(=O)(=O)-L-R2; and RrC(R4)(R5)-S-L-R2.
It will be readily appreciated that references to the structural moiety
wherein Q is an atom selected from the group consisting of S, O, N and C, include the following structural moieties
The structural moiety may be saturated or may include unsaturation, such as one or more double bonds.
Thus, references to the structural moiety
include structural moieties such as
The structural moiety may be substituted. The structural moiety may be part of a polycyclic system such as , for example
R^ is an optionally substituted phenyl ring and R2 is or comprises an optionally substituted aromatic ring. R-j and R2 are referred to collectively as the ring systems.
Ri is an optionally substituted phenyl ring.
Ri may be substituted or unsubstituted. Preferably Ri is substituted. Ri may be substituted with one or more hydrocarbyl substituents. Preferably the substituents are selected from hydrocarbon groups, oxyhydrocarbon groups, halogens, amines and amides. More preferably the substituents are selected from aromatic hydrocarbon groups, alkyl groups, oxyalkyl groups, halogens, amines and amides, such as from aromatic hydrocarbon groups, alkyl groups, oxyalkyl groups and halogens.
In a highly preferred aspect the substituents are selected from phenyl groups, C1-5alkyl groups, oxy-C1-5-alkyl groups, chloro, bromo and iodo. More preferably the substituents are selected from phenyl, methyl, ethyl, propyl, O-methyl, O-ethyl, O-propyl and chloro.
Preferably Ri is selected from the following:
Preferably Ri is or comprises a group selected from the following wherein indicates the point of attachment to Z.
More preferably Ri is selected from the following:
R2 is or comprises an optionally substituted aromatic ring. Preferably the optionally substituted aromatic ring is a five or six membered ring. In one aspect preferably R2 is an optionally substituted five membered aromatic ring. In another aspect preferably R2 is an optionally substituted six membered aromatic ring.
In one preferred aspect, the optionally substituted aromatic ring is a heterocyclic ring. Preferably R2 is an optionally substituted five or six membered aromatic heterocyclic ring.
Preferably the optionally substituted aromatic ring is a heterocyclic ring comprising a carbon and a hetero atom selected from O, S and N. More preferably the optionally substituted aromatic ring is a heterocyclic ring comprising a carbon and a hetero atom selected from O and N. Preferably R2 is or comprises:
Preferably R2 is
Preferably R2 is or comprises a group selected from the following wherein indicates the point of attachment to Z.
In one preferred aspect, R2 is an optionally substituted five membered heterocyclic aromatic ring. In this aspect preferably R2 is or comprises:
In this aspect more preferably R2 is or comprises:
In one preferred aspect the optionally substituted aromatic ring is a carbocyclic ring.
R2 may be substituted or unsubstituted. Preferably R2 is substituted, more preferably R2 is a substituted aromatic carbocyclic ring.
In one aspect the substituents are selected from hydrocarbon groups, oxyhydrocarbon groups, halogens, amines and amides. More preferably the substituents are selected from aromatic hydrocarbon groups, alkyl groups, oxyalkyl groups, halogens, amines and amides, such as from aromatic hydrocarbon groups, alkyl groups, oxyalkyl groups and halogens.
In one aspect R2 is substituted with two or more substituents. In a preferred aspect R2 is substituted with two or more substituents and the two or more substituents together form a ring which is fused to the carbocyclic ring of R2.
Preferably the carbocyclic ring is a five or six membered aromatic carbocyclic ring. More preferably the carbocyclic ring is a phenyl ring.
In a preferred aspect R2 is a substituted phenyl ring. Preferably R2 is substituted with two or more substituents and the two or more substituents together form a ring which is fused to the phenyl ring of R2.
In this aspect preferably R2 comprises the following structure:
wherein A represents a heterocyclic ring, preferably a five or six membered heterocyclic ring. Thus, in one aspect preferably R2 comprises the following structure:
wherein A r 3epresents a five membered heterocyclic ring. In this aspect preferably R2 is or comprises a group selected from the following:
More preferably R2 is or comprises a group selected from the following:
In another preferred aspect R2 comprises the following structure:
wherein A r 3epresents a six membered heterocyclic ring. In this aspect preferably R2 is or comprises a group selected from the following:
More preferably R2 is or comprises a group selected from the following:
In one preferred aspect R2 is or comprises a group selected from the following:
In another preferred aspect R2 is or comprises a group selected from the following:
Preferably R2 is or comprises a group selected from the following wherein — indicates the point of attachment to Z.
Preferably in this aspect, Ri is a substituted phenyl and Z is -S(=0)(=O)NH-or NHS(=O)(=O) -.
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. Each ring system may contain one or more degrees of unsaturation, for example is some aspects one or more rings of a ring system is aromatic. Each ring system may be carbocyclic or may contain one or more hetero atoms.
The compound of the invention, in particular the ring systems of the compound of the 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 oxy hydrocarbyl group.
In general terms the ring systems of the present compounds may contain a variety of non- interfering substituents. In particular, the ring systems 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 (C C6) alkoxy, e.g. methoxy, ethoxy, propoxy etc., alkinyl, e.g. ethinyl, or halogen, e.g. fluoro substituents.
X. Y and L
As previously mentioned, Z in Formula I is -X-Y-L- or -Y-X-L-; wherein either X is selected from -S(=O)(=O)- and -C(=O)-, and Y is -NR3-; or X is selected from - S(=O)(=O)- and -S-, and Y is -C(R4)(R5)-; L is an optional linker; and R3, R4 and R5 are each independently selected from H and hydrocarbyl.
In one preferred aspect, X is -C(=O)-. In this aspect preferably Z is selected from - C(=O)-NR3-, -C(=O)-NR3-L-, -NR3-C(=O)-, and -NR3-C(=0)-L-.
In one preferred aspect, X is -S(=O)(=O)-. In this aspect preferably Z is selected from - S(=O)(=O)-NR3-, -S(=O)(=O)-NR3-L-, -NR3-S(=O)(=0)-, -NR3-S(=O)(=O)-L-, - S(=O)(=O)-C(R4)(R5)-, -S(=O)(=O)-C(R4)(R5)-L-, -C(R4)(R5)-S(=O)(=0)- and -C(R4)(R5)- S(=O)(=O)-L-. In one preferred aspect, X is -S-. In this aspect preferably Z is selected from -S- C(R4)(R5)-, -S-C(R4)(R5)-L-, -C(R4)(R5)-S- and -C(R4)(R5)-S-L-.
In one preferred aspect, Y is -NR3-. In this aspect preferably Z is selected from -C(=O)- NR3-, -C(=O)-NR3-L-, -NR3-C(=O)-, -NR3-C(=O)-L-, -S(=O)(=O)-NR3-, -S(=O)(=O)-NR3- L-, -NR3-S(=O)(=O)- and -NR3-S(=0)(=O)-L-.
In another preferred aspect, Y is -C(R4)(R5)-. In this aspect preferably Z is selected from -S(=O)(=O)-C(R4)(R5)-, -S(=O)(=O)-C(R4)(R5)-L-, -C(R4)(R5)-S(=0)(=O)- and -C(R4)(R5)- S(=O)(=O)-L-, -S-C(R4)(R5)-, -S-C(R4)(R5)-L-, -C(R4)(R5)-S- and -C(R4)(R5)-S-L-.
In one preferred aspect, X is -S(=0)(=O)- and Y is -NR3-. In this aspect preferably Z is - S(=O)(=O)NR3-or - NR3S(=O)(=O) -, such as -S(=O)(=O)NH-or - NHS(=O)(=O) -
In one preferred aspect, X is -S(=0)(=O)- and Y is -C(R4)(R5)~. In this aspect preferably Z is -S(=O)(=O)C(R4)(R5)- or -C(R4)(R5)S(=O)(=O)-, such as -S(=O)(=O)CH2- or - CH2S(=O)(=O)-
In one preferred aspect, X is -S- and Y is -C(R )(R5)~. In this aspect preferably Z is - SC(R4)(R5)- or -C(R4)(R5)S-, such as -SCH2- or -CH2S-
L is an optional linker. In one aspect L is present. L may be any suitable group, for example L may a hydrocarbyl group or a hetero atom, in particular L may be a hydrocarbon group such a C^o alkyl group. In one aspect, L is selected from -C(=O)-, - S(=O)(=O)-, -S-, -NR3-, -[C(R4)(R5)]n-, -C6H4- and combinations thereof, wherein R3, R4 and R5 are each independently selected from H and hydrocarbyl and wherein n is an integer from 1 to 10, preferably from 1 to 5, more preferably 1 or 2.. In one preferred aspect, L is selected from -C(=0)-, -S(=O)(=O)-, -S-, -NR3-, -C(R4)(R5)-, -C6H4- and combinations thereof, wherein R3, R4 and R5 are each independently selected from H and hydrocarbyl. In these aspects X-Y is preferably S(=O)(=O)NR3- or - NR3S(=0)(=O) -, such as -S(=0)(=O)NH-or - NHS(=O)(=0) -.
In a preferred aspect, L is selected from -NR3-C(=O)-C(R4)(R5)-S- and -S(=O)(=O)-NR3- NR3-C(=O)-. In these aspects X-Y is preferably S(=O)(=O)NR3- or - NR3S(=O)(=O)-, such as -S(=O)(=O)NH-or - NHS(=0)(=O) -. In a highly preferred aspect, L is selected from -NH-C(=O)-CH2-S- and -S(=O)(=O)-NH- NH-C(=O)-. In these aspects X-Y is preferably S(=O)(=O)NR3- or - NR3S(=O)(=O)-, such as -S(=O)(=O)NH-or -NHS(=O)(=O)-.
As previously mentioned, Z in Formula I is -X-Y-L- or -Y-X-L-; wherein either X is selected from -S(=0)(=O)- and -C(=O)-, and Y is -NR3-; or X is selected from - S(=O)(=O)- and -S-, and Y is -C(R4)(R5)-; L is an optional linker; and R3, R4 and R5 are each independently selected from H and 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 C1-C10 alkyl group, such as Cι-C6 alkyl group, and C1-C3 alkyl group. Typical alkyl groups include Ci 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 C C10 haloalkyl group, C C6 haloalkyl group, C1-C3 haloalkyl group, CrC bromoalkyl group, Cι-C6 bromoalkyl group, and C C3 bromoalkyl group. Typical haloalkyl groups include Ci haloalkyl, C2 haloalkyl, C3 haloalkyl, C haloalkyl, C5 haloalkyl, C7 haloalkyl, C8 haloalkyl, Ci 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)ι- 10-aryl, -(CH2)1-10-Ph, (CH2)1-10-Ph-C1-10 alkyl, -(CH2)1-5-Ph, (CH2)ι-5-Ph-Cι-5 alkyl, -(CHs 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)ι-ιo-C3-1ocycloalkyl, -(CH2)1-7-C3- 7cycloalkyl, -(CH2)1-5-C3.5cycloalkyl, -(CH2)ι-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 C C10 alkene group, C C6 alkene group, C1-C3 alkene group, such as C-i, 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. 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 C1-60 (such as a C-i-30).
In a preferred aspect, R3 is selected from H and hydrocarbon groups. Preferably, R3 is selected from H and alkyl groups, preferably from H and C^oalkyl groups, preferably from H and Cι_5alkyl groups. In a highly preferred aspect, R3 is H.
In a preferred aspect, R3 may be equivalent to -L-R2 wherein L and R2 are independently selected from the possibilities defined herein. In this aspect the present compound contains two groups of the formula -L-R2 wherein each L and R2 are selected independently of each other. However in one aspect, each of L and R2 may be the same as the other of L and R2 present in the compound. Examples of compounds meeting such requirements are shown below.
In a preferred aspect, R4 and R5 are independently selected from H and hydrocarbon groups. Preferably R and R5 are independently selected from H and alkyl groups, preferably independently selected from H and C -10alkyl groups, preferably independently selected from H and C1-5alkyl groups. In a preferred aspect at least one of R and R5 is H. In a highly preferred aspect, R and R5are both H.
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. The present invention also encompasses a process comprising precursors for the synthesis of the compounds of the present invention.
Steroid Dehydrogenase
11β 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 biological assay 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. 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 sterneurosteroids 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, chernotactic, 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, lL-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-i phase (the first gap), the S (DNA synthesis) phase, a G2 phase (the second gap), and back to M. The phases between mitoses (Gi, 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 (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 Gi 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 I 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
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 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.
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.
Summary
In summation, the present invention provides compounds for use as steroid dehydrogenase inhibitors, and pharmaceutical compositions for the same.
BROAD ASPECTS
In one broad aspect the present invention provides a compound having Formula I R Z-R2 Formula I wherein Z is -X-Y-L- or -Y-X-L-; Ri is an aromatic ring; R2 is or comprises a ring; X is selected from -C(=O)-, -S(=O)(=O)- and -S-; Y is selected from -NR3- and -C(R4)(R5)-; L is an optional linker; R3, R and R5 are each independently selected from H and hydrocarbyl.
In one broad aspect the present invention provides a pharmaceutical composition comprising (i) a compound having Formula I R Z-R2 Formula I wherein Z is -X-Y-L- or -Y-X-L-; R^ is an aromatic ring; R2 is or comprises a ring; X is selected from -C(=0)-, -S(=O)(=O)- and -S-; Y is selected from -NR3- and -C(R4)(R5)-; L is an optional linker; R3, R4 and R5 are each independently selected from H and hydrocarbyl; (ii) optionally admixed with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
In one broad aspect the present invention provides a compound for use in medicine wherein the compound has Formula I R Z-R2 Formula I wherein Z is -X-Y-L- or -Y-X-L-; Ri is an aromatic ring; R2 is or comprises a ring; X is selected from -C(=O)-, -S(=O)(=O)- and -S-; Y is selected from -NR3- and -C(R4)(R5)-; L is an optional linker; R3, R4 and R5 are each independently selected from H and hydrocarbyl.
In one broad 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 R Z-R2 Formula I wherein Z is -X-Y-L- or -Y-X-L-; Ri is an aromatic ring; R2 is or comprises a ring; X is selected from -C(=O)-, -S(=O)(=0)- and -S-; Y is selected from -NR3- and -C(R4)(R5)-; L is an optional linker; R , R and R5 are each independently selected from H and hydrocarbyl.
In these broad aspects, Ri is an aromatic ring. Preferably Ri is a five or six membered aromatic ring, more preferably a six membered aromatic ring. Preferably, Ri is a carbocyclic ring. In a highly preferred aspect, Ri is an optionally substituted phenyl ring.
In these broad aspects, R2 is or comprises a ring, preferably an aromatic ring, more preferably an optionally substituted aromatic ring.
In one broad aspect R2 comprises the following structure:
wherein A represents a heterocyclic ring, preferably a five or six membered heterocyclic ring.
In these broad aspects X is selected from -C(=O)-, -S(=O)(=O)- and -S-; and Y is selected from -NR3- and -C(R4)(R5)-. In a preferred aspect either X is selected from - S(=O)(=O)- and -C(=O)-, and Y is -NR3-; or X is selected from -S(=O)(=O)- and -S-, and Y is -C(R4)(R5)-.
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 a graph showing extraction efficiencies obtained with four extraction methods.
Figure 2 is a graph showing a comparison of 11 β-HSD1 activity in rat and human hepatic microsomes.
Figure 3 is a series of graphs showing the effect of incubation time on human microsomal 11 β-HSD1 activity Figure 4 is a series of graphs showing the effect of microsomal protein concentration on human microsomal 11β-HSD1 activity.
Figure 5 is a graph showing the substrate (cortisone) saturation curve for human hepatic microsomal 11 β HSD1.
Figure 6 is a Lineweaver-Burke plot. Figure 7 is a graph showing the IC50 determination for Glycyrrhetinic acid.
Figure 8 is a graph showing the IC50 determination for Carbenoxolone.
Figures 9(A), 9(B) and 9(C) are graphs showing the 11 β-HSD1 activity measured by
Immunoassay. Figure 9(A) shows the effect of protein; Figure 9(B) shows the effect of cortisone; and Figure 9(C) shows the effect of Tween-80. Figure 10 is a graph showing the performance of the cortisol immunoassay: various experimental designs.
Figure 11 is a graph showing the effect of increasing microsomal protein on measurement of 11 β HSD1 activity detected by Assay Designs Immunoassay.
Figure 12 is a graph showing the detection of 11 β HSD1 activity by RIA using the Immunotech anti-cortisol antibody.
Figure 13 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 14 is a graph showing the Immunotech antibody saturation curve for detection of
11 β HSD1 activity by RIA. Figure 15 is a graph showing the linearity of human hepatic microsomal 11 β HSD1 activity detected by RIA.
Figure 16 is a graph showing the effect of Tween 80 on detection of human hepatic microsomal 11 β HSD1 activity by RIA. Figure 17 is a graph showing the effect of buffer systems on detection of human hepatic microsomal 11 β HDS1 activity by RIA.
Figure 18 is a graph showing the linearity of human hepatic microsomal 11 β HSD1 activity with incubation time detected by RIA. Figure 19 is a graph showing the substrate saturation curve for human hepatic microsomal 11β HDS1 activity detected by RIA. Figure 20 is a Lineweaver-Burke plot
Figure 21 is an IC50 curve for inhibition of human hepatic microsomal 11 β HSD1 activity by Glycyrrhetinic acid. Figure 22 is an IC5o curve for inhibition of human hepatic microsomal 11 β HSD1 activity by Glycyrrhetinic acid in the presence of 350 nM cortisone.
Figure 23 is an IC50 curve for inhibition of human hepatic microsomal 11 β HSD1 activity by Carbenoxolone in the presence of 350 nM cortisone.
The present invention will now be described in further detail in the following examples.
EXAMPLES
The present invention will now be described only by way of example.
Biological Assays
Standard Operating Procedure for the 11 β-Hydroxysteroid Dehydrogenase Type 1 cortisol Radioimmunoassay (11 β 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 Compounds 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 et al., (2002) [14]: 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 et al., (2002) [14]: 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 jul substrate mixture to 2 empty wells to indicate counting efficiency Add 200 μl Microscint-40 to all wells and count on a Topcount
Radioimmunoassav
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.
Inhibition Data
Chemistry Experimental Section and Compound Examples
General. All chemicals were either purchased from the Aldrich Chemical Co. (Gillingham, UK), Lancaster Synthesis (Morecambe, UK) or ACROS Organics (Loughborough, UK). All organic solvents of A.R. grade were supplied by Fisher Scientific (Loughborough, UK).
Thin layer chromatography (TLC) was performed on precoated plates (Merck TLC aluminium sheets silica gel 60 F254, Art. No. 5554). Compounds were visualised by either viewing under UV light or treating with an ethanolic solution of phosphomolybdic acid (PMA) followed by heating. Flash chromatography was carried out using Sorbsil C60 silica gel or Isolute® pre-packed Flash Si columns from Argonaut Technologies. Parallel synthesis was performed on either Radleys Carousel reaction stations or Radleys GreenHouse parallel synthesisers. Solvent removal from parallel syntheses was performed on a GeneVac DD4 evaporation system. NMR spectra were recorded with a JEOL GX-270 or Varian-Mercury-400 spectrometer, and chemical shifts are reported in parts per million (ppm, δrelative to tetramethylsilane (TMS) as an internal standard. Mass spectra were recorded at the Mass Spectrometry Service Centre, University of Bath. FAB-MS were carried out using m-nitrobenzyl alcohol (NBA) as the matrix. High performance liquid chromatography (HPLC) analysis was performed with a Waters Delta 600 liquid chromatograph with a Waters 996 photodiode Array Detector using a Waters Radialpack C18, 8x100 mm column. Melting points (Mp) were measured with a Reichert-Jung ThermoGalen Kofler block or a Sanyo Gallenkamp melting point apparatus and are uncorrected.
Synthesis of Benzofuran and Benzo[ϋ]thiophene Derivatives
STX 971 STX 972
STX 973 STX 1015
STX 1038 STX 1049
Synthesis of 5-(3-chloro-2-methyl-benzenesulfonylamino) -benzorblthiophene-2-carboxylic acid methyl ester. STX 971 (KRB01096):
5-amino-benzo[b]thiophene-2-carboxylic acid methyl ester (KRB01094): To a solution of 5-nitro-benzo[b]thiophene-2-carboxylic acid methyl ester [15] (130 mg, 0.548 mmol) in methanol (30 mL) was added 5% palladium on carbon (20 mg) and the mixture was stirred under 1 atm H2 for 2 h. The mixture was filtered through celite and the filtrate evaporated. The residue was passed through a silica column to afford 5-amino- benzo[b]thiophene-2-carboxylic acid methyl ester as a pale yellow solid (94 mg, 83%), single spot at Rf 0.75 (95:5 dichloromethane:methanol). 1H NMR (CDCI3): δ 7.86 (1 H, s), 7.61 (1 H, d, J=8.7 Hz), 7.11 (1 H, d, J=2.2 Hz), 6.88 (1 H, dd, J=8.7, 2.5 Hz), 3.91 (3H, s), 3.76 (2H, s, N-H2) [16].
To a solution of 3-chloro-2-methylbenzenesulphonyl chloride (46 mg, 0.20 mmol) in dichloromethane (2 mL) was added pyridine (40 μL, 0.48 mmol) and the mixture was stirred under N2 for 5 min, after which time 5-amino-benzo[b]thiophene-2-carboxylic acid methyl ester (40 mg, 0.19 mmol) was added. The resulting mixture was stirred for 2 h at room temperature, then saturated NaHCO3 solution (6 mL) was added and the mixture was extracted into ethyl acetate (12 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 yellow solid (65 mg, 85%), single spot at Rf 0.52 (2:1 hexane:ethyl acetate), mp 173.6-174.2°C, HPLC purity 99% (tR 2.13 min in 10% water- acetonitrile). 1H NMR (CDCI3): δ 7.91 (1 H, s), 7.88 (1 H, d, J=7.9 Hz), 7.70 (1 H, d, J=8.7 Hz), 7.55-7.52 (2H, m), 7.18 (1 H, t, =8.0 Hz), 7.12 (1 H, dd, J=8.7, 2.2 Hz), 6.98 (1 H, s, N-H), 3.92 (3H, s), 2.73 (3H, s). LCMS: 394.12 (M-). FAB-MS (MH+, C17H14CINO4S2): calcd 395.0053, found 395.0045.
Synthesis of 3-chloro-2-methyl-Λ/-(3-methyl-benzofuran-5-yl) -benzenesulfonamide, STX 972 (KRB01097):
5-amino-3-methyl-benzofuran (KRB01095): To a solution of 3-methyl-5-nitro-benzofuran [17] (135 mg, 0.762 mmol) in methanol (30 mL) was added 5% palladium on carbon (30 mg) and the mixture was stirred under 1 atm H2 for 8 h. The mixture was filtered through celite and the filtrate evaporated. The residue was passed through a silica plug to afford 5-amino-3-methyl-benzofuran as a pale pink oil (85 mg, 76%), single spot at Rf 0.28 (2:1 hexane:ethyl acetate). 1H NMR (CDCI3): δ 7.31 (1 H, s), 7.22 (1H, d, J=8.7 Hz), 6.78 (1H, d, J=2.5 Hz), 6.66 (1H, dd, J=8.7, 2.5 Hz), 3.60 (2H, s, N-H2), 2.16 (3H, s).
To a solution of 3-chloro-2-methylbenzenesulphonyl chloride (128 mg, 0.571 mmol) in dichloromethane (3 mL) was added pyridine (110 μL, 1.36 mmol) and the mixture was stirred under N2 for 5 min, after which time 5-amino-3-methyl-benzofuran (80 mg, 0.54 mmol) was added. The resulting mixture was stirred for 2 h at room temperature, then saturated NaHCO3 solution (8 mL) was added and the mixture was extracted into ethyl acetate (15 mL). The organic phase was washed with brine, dried (Na2SO ), filtered and evaporated to give a residue that was purified using flash chromatography to afford a yellow solid (159 mg, 87%), single spot at R 0.65 (2:1 hexane:ethyl acetate), mp 119.5- 120.1°C, HPLC purity 99% (tR 2.22 min in 10% water-acetonitrile). 1H NMR (CDCI3): δ 7.82 (1 H, d, J=7.9 Hz), 7.54 (1 H, d, J=7.9 Hz), 7.38 (1 H, s), 7.28-7.22 (2H, m), 7.15 (1 H, t, J=8.0 Hz), 6.83 (1H, dd, J=8.7, 2.2 Hz), 6.56 (1 H, s, N-H), 2.72 (3H, s), 2.15 (3H, s). LCMS: 334.07 (M-). FAB-MS (MH+, C16H14CINO3S): calcd 335.0383, found 335.0383.
Synthesis of 5-(3-chloro-2-methyl-benzenesulfonylamino)-benzofuran-2-carboxylic acid methyl ester. STX 973 (KRB01100):
5-nitro-benzofuran-2-carboxylic acid methyl ester (KRB01098): To a suspension of NaH (258 mg, 60% dispersion in mineral oil, 6.46 mmol) in anhydrous DMF (10 mL) was added methyl glycolate (420 μL, 5.39 mmol) dropwise. After 10 min, a solution of 2- chloro-5-nitrobenzaldehyde (1.00 g, 5.39 mmol) in anhydrous DMF (3 mL) was added and the resulting solution was stirred at room temperature for 1 h followed by 100°C for 5 h. After cooling, the mixture was poured into 1N HCl (50 mL) and the resulting precipitate filtered and washed with water. The solid was purified by flash chromatography (4:1 hexane:ethyl acetate) and then recrystallized from hexane/ethyl acetate to yield 5-nitro-benzofuran-2-carboxylic acid methyl ester as yellow needles (450 mg, 37%). mp 168.0-168.4°C. 1H NMR (CDCI3): δ 8.65 (1 H, d, J=2.2 Hz), 8.37 (1 H, dd, =9.0, 2.2 Hz), 7.70 (1 H, d, J=9.1 Hz), 7.64 (1 H, s), 4.01 (3H, s). 5-amino-benzofuran-2-carboxylic acid methyl ester (KRB01099): To a solution of 5- nitrobenzofuran-2-carboxylic acid methyl ester (100 mg, 0.452 mmol) in methanol (20 mL) was added 5% palladium on carbon (20 mg) and the mixture was stirred under 1 atm H2 for 4h. The mixture was filtered through celite and the filtrate evaporated. The residue was passed through a silica plug to afford 5-amino-benzofuran-2-carboxylic acid methyl ester as an orange solid (87 mg, ~100%), single spot at Rf 0.34 (1 :1 hexane:ethyl acetate), mp 112.4-113.0°C. 1H NMR (CDCI3): δ 7.36 (1 H, s), 7.36 (1H, d, =8.2 Hz), 6.89 (1H, d, J=2.2 Hz), 6.83 (1H, dd, J=8.2, 2.2 Hz), 3.95 (3H, s), 3.66 (2H, s, N-tf2).
To a solution of 3-chloro-2-methylbenzenesulphonyl chloride (99 mg, 0.44 mmol) in dichloromethane (3 mL) was added pyridine (85 μL, 1.05 mmol) and the mixture was stirred under N2 for 5 min, after which time 5-amino-benzofuran-2-carboxylic acid methyl ester (80 mg, 0.42 mmol) was added. The resulting mixture was stirred for 2 h at room temperature, then saturated NaHCO3 solution (10 mL) was added 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 an off-white solid (122 mg, 77%), single spot at Rf 0.47 (2:1 hexane:ethyl acetate), mp 139.4-140.1°C , HPLC purity 99+% (tR 1.90 min in 10% water-acetonitrile). 1H NMR (CDCI3): δ 7.83 (1 H, d, =7.9 Hz), 7.55 (1 H, d, J=7.9 Hz), 7.44 (1H, d, J=9.0 Hz), 7.42 (1H, s), 7.39 (1H, d, =2.2 Hz), 7.17 (1H, t, J=8.0 Hz), 7.05 (1 H, dd, J=8.7, 2.2 Hz), 6.65 (1 H, s, N-H), 3.95 (3H, s), 2.72 (3H, s). LCMS: 378.16. FAB-MS (MH+, C17H14CIN05S): calcd 379.0281 , found 379.0281.
Synthesis of 3-chloro-2-methyl-Λ/-(3-methyl-benzorDlthiophen-5-yl)-benzenesulfonamide, STX 1015 (KRB01108):
3-methyl-benzo[b[thiophen-5-ylamine (KRB01106): To a solution of 3-methyl-5-nitro- benzo[ό]thiophene [18] (95 mg, 0.492 mmol) in methanol (20 mL) was added 5% palladium on carbon (20 mg) and the mixture was stirred under 1 atm H2 for 8.5h. The mixture was filtered through celite and the filtrate evaporated. The residue was passed through a silica plug to afford 3-methyl-benzo[b]thiophen-5-ylamine as a dark orange solid (31 mg, 39%), single spot at Rf 0.31 (4:1 hexane:ethyl acetate). 1H NMR (CDCI3): δ 7.60 (1H, d, J=8.4 Hz), 7.02 (1 H, s), 7.00 (1 H, d, J=2.2 Hz), 6.79 (1H, dd, J=8.4, 2.2 Hz), 2.37 (3H, s).
To a solution of 3-chloro-2-methylbenzenesulphonyl chloride (43 mg, 0.19 mmol) in dichloromethane (2 mL) was added pyridine (40 μL, 0.46 mmol) and the mixture was stirred under N2 for 5 min, after which time 3-methyl benzo[b]thiophen-5-ylamine (30 mg, 0.18 mmol) was added. The resulting mixture was stirred for 2 h at room temperature, then saturated NaHCO3 solution (10 mL) was added 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 brown solid (30 mg, 46%), single spot at Rf 0.39 (3:1 hexane:ethyl acetate), mp 152.6-153.0°C , HPLC purity 99% (tR 2.42 min in 10% water-acetonitrile). 1H NMR (CDCI3): δ 7.90 (1 H, d, J=7.9 Hz), 7.65 (1 H, d, J=8.7 Hz), 7.53 (1 H, d, J=7.9 Hz), 7.37 (1 H, d, J=2.0 Hz), 7.16 (1 H, t, J=7.9 Hz), 7.07 (1 H, s), 7.02 (1H, dd, =8.7, 2.0 Hz), 2.74 (3H, s), 2.32 (3H,. s). LCMS: 350.09. FAB-MS (MH+, C16H14CINO2S2): calcd 351.0155, found 351.0155
Synthesis of 3-chloro-2-methyl-Λ/-(2-methyl-benzofuran-5-yl)-benzenesulfonamide, STX 1038 (KRB01114):
5-amino-2-methylbenzofuran (KRB01112): To a solution of 2-methyl-5-nitro-benzofuran [19] (125 mg, 0.706 mmol) in methanol (20 mL) was added 5% palladium on carbon (20 mg) and the mixture was stirred under 1 atm H2 for 4h. The mixture was filtered through celite and the filtrate evaporated. The residue was passed through a silica plug to afford 5-amino-2-methyl benzofuran as a red-brown oil (78 mg, 75%), single spot at Rf 0.45 (1 :1 hexane:ethyl acetate). 1H NMR (CDCI3): δ 7.17 (1 H, d, J=8.7 Hz), 6.75 (1H, d, =2.5 Hz), 6.57 (1H, dd, J=8.7, 2.5 Hz), 6.20 (1 H, s), 3.53 (2H, s, NH2), 2.39 (3H, s). To a solution of 3-chloro-2-methylbenzenesulphonyl chloride (112 mg, 0.499 mmol) in dichloromethane (3 mL) was added pyridine (95 μL, 1.2 mmol) and the mixture was stirred under N2 for 5 min, after which time 5-amino-2-methyl benzofuran (70 mg, 0.48 mmol) was added. The resulting mixture was stirred for 2 h at room temperature, then saturated NaHCO3 solution (10 mL) was added and the mixture was extracted into ethyl acetate (15 mL). The organic phase was washed with brine, dried (Na2SO ), filtered and evaporated to give a residue that was purified using flash chromatography to afford a pale brown solid (120 mg, 75%), single spot at Rf 0.51 (3:1 hexane:ethyl acetate), mp 123.2-123.6°C , HPLC purity 98% (tR 3.14 min in 10% water-acetonitrile). 1H NMR (CDCI3): δ 7.81 (1 H, d, J=7.7 Hz), 7.52 (1 H, d, J=7.7 Hz), 7.21 (1 H, d, J=8.7 Hz), 7.16- 7.11 (2H, m), 6.78 (1 H, dd, J=8.7, 2.2 Hz), 6.48 (1 H, s, N-H), 6.27 (1 H, s), 2.70 (3H, s), 2.40 (3H, s). LCMS: 334.13. FAB-MS (MH+, C16H14CINO3S): calcd 335.0383, found 335.0381
Synthesis of Λ/-benzoffrlthiophen-5-yl-3-chloro-2-methyl- benzenesulfonamide, STX 1049 (KRB01121):
To a solution of 3-chloro-2-methylbenzenesulphonyl chloride (47 mg, 0.21 mmol) in dichloromethane (2 mL) was added pyridine (40 μL, 0.50 mmol) and the mixture was stirred under N2 for 5 min, after which time 5-amino-benzo[Jb]thiophene [20] (30 mg, 0.20 mmol) was added. The resulting mixture was stirred for 2 h at room temperature, then saturated NaHCO3 solution (10 mL) was added 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 dark brown solid (50 mg, 74%), single spot at Rf 0.58 (3:1 hexane:ethyl acetate), mp 108.5-109.0°C , HPLC purity 99% (tR 2.20 min in 10% water-acetonitrile). 1H NMR (CDCI3): δ 7.87 (1 H, d, J=7.9 Hz), 7.70 (1 H, d, J=8.7 Hz), 7.54-7.51 (2H, m), 7.44 (1 H, d, =5.4 Hz), 7.21 (1 H, d, J=5.4 Hz), 7.16 (1 H, t, =7.9 Hz), 6.98 (1 H, dd, J=8.7, 2.2 Hz), 6.73 (1 H, s, N-rτ), 2.74 (3H, s). LCMS: 336.07. FAB-MS (MH+, C15H12CINO2S2): calcd 336.9998, found 337.0004. Synthesis of Quinoline, Quinazoline, and Quinoxaline Derivatives
STX 932: R^R^H, R3=CI, R4=Me STX 935: R1=R2=H, R3=CI, R4=Me STX 933: R1=R3=R4=H, R2=n-propyl STX 936: R-,=R3=R4=H, R2=n-propyl STX 934: R1=R4=CI, R2=R3=H STX 937: R.,=R4=CI, R2=R3=H
STX 943: R1=R2=H, R3=CI, R4=Me STX 953: R1=R2=H, R3=CI, R4=Me STX 944: R1=R3=R4=H, R2=n-propy! STX 954: R-,=R3=R4=H, R2=n-propyl
STX 955: R.,=R2=H, R3=CI, R4=Me STX 957: R^R^H, R3=CI, R4=Me STX 956: R1=R3=R4=H, R2=n-propyl STX 958: R1=R3=R4=H, R2=n-propyl
Synthesis of 3-chloro-2-methyl-A/-guinolin-6-yl-benzenesulfonamide. STX 932 (KRB01058):
To a solution of 3-chloro-2-methylbenzenesulphonyl chloride (164 mg, 0.728 mmol) in dichloromethane (4 mL) was added pyridine (140 μL, 1.74 mmol) and the mixture was stirred under N2 for 5 min, after which time 6-aminoquinoline (100 mg, 0.694 mmol) was added. The resulting mixture was stirred for 2 h at room temperature, then saturated NaHCO3 solution (10 mL) was added and the mixture was extracted into ethyl acetate (20 mL). The organic phase was washed with brine, dried (Na2SO ), filtered and evaporated to give a residue that was purified using flash chromatography to afford a pale pink solid (48 mg, 21%), single spot at Rf 0.71 (ethyl acetate), mp 228.0-228.5°C, HPLC purity 99+% (tR 2.43 min in 10% water-acetonitrile). 1H NMR (CDCI3): δ 8.83 (1H, dd, =4.2, 1.7 Hz), 7.98 (3H, m), 7.54 (1H, d, J=7.9 Hz), 7.48 (1H, d, J=2.7 Hz), 7.39- 7.32 (2H, m), 7.20 (1 H, t, J=7.9 Hz), 7.05 (1 H, s, N-H), 2.75 (3H, s). LCMS: 331.16 (M-). FAB-MS (MH+, C16H13CIN2O2S): calcd 333.0464, found 333.0461.
Synthesis of 4-propyl-Λ/-quinolin-6-yl-benzenesulfonamide. STX 933 (KRB01059):
To a solution of 4n-propylbenzenesulphonyl chloride (159 mg, 0.728 mmol) in dichloromethane (4 mL) was added pyridine (140 μL, 1.74 mmol) and the mixture was stirred under N2 for 5 min, after which time 6-aminoquinoline (100 mg, 0.694 mmol) was added. The resulting mixture was stirred for 2 h at room temperature, then saturated NaHCO3 solution (10 mL) was added and the mixture was extracted into ethyl acetate (20 mL). The organic phase was washed with brine, dried (Na2S04), filtered and evaporated to give a residue that was purified using flash chromatography to afford a white solid (210 mg, 93%), single spot at Rf 0.71 (ethyl acetate), mp 180.1-180.7°C, HPLC purity 99+% (tR 2.37 min in 10% water-acetonitrile). 1H NMR (CDCI3): δ 8.83 (1 H, dd, J=4.2, 1.7 Hz), 8.05 (1 H, d, =8.4 Hz), 7.97 (1 H, d, J=9.2 Hz), 7.70 (2H, d, J=8.2 Hz), 7.57 (1 H, d, J=2.5 Hz), 7.40-7.34 (2H, m), 7.22 (2H, m), 6.85 (1 H, s, N-H), 2.57 (2H, t, J=7.2 Hz), 1.57 (2H, sextet, =7.2 Hz), 0.87 (3H, t, J=7.3 Hz). LCMS: 325.23 (M- ). FAB-MS (MH+, C18H18N2O2S): calcd 327.1167, found 327.1167.
Synthesis of 2,5-dichloro-Λ/-quinolin-6-yl-benzenesulfonamide, STX 934 (KRB01060):
To a solution of 2,5-dichlorobenzenesulphonyl chloride (179 mg, 0.728 mmol) in dichloromethane (4 mL) was added pyridine (140 μL, 1.74 mmol) and the mixture was stirred under N2 for 5 min, after which time 6-aminoquinoline (100 mg, 0.694 mmol) was added. The resulting mixture was stirred for 2 h at room temperature, then saturated NaHCO3 solution (10 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 (168 mg, 69%), single spot at Rf 0.74 (ethyl acetate), mp 213.4-213.8°C, HPLC purity 99+% (tR 2.28 min in 10% water-acetonitrile). 1H NMR (CDCI3): δ 8.85 (1 H, dd, J=4.2, 1.5 Hz), 8.09 (1 H, d, J=8.4 Hz), 8.03-8.00 (2H, m), 7.62 (1 H, d, J=2.5 Hz), 7.56 (1H, s, N-H), 7.47-7.38 (4H, m). LCMS: 351.10 (M-). FAB-MS (MH+, C15H10CI2N2O2S): calcd 352.9918, found 352.9922.
Synthesis of 3-chloro-2-methyl-Λ/-(2-methyl-quinolin-6-yl) -benzenesulfonamide. STX 935 (KRB01061):
To a solution of 3-chloro-2-methylbenzenesulphonyl chloride (149 mg, 0.664 mmol) in dichloromethane (4 mL) was added pyridine (130 μL, 1.58 mmol) and the mixture was stirred under N2 for 5 min, after which time 6-amino-2-methylquinoline (100 mg, 0.632 mmol) was added. The resulting mixture was stirred for 90 min at room temperature, then saturated NaHCO3 solution (10 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 an off-white solid (148 mg, 68%), single spot at Rf 0.64 (ethyl acetate), mp 178.1-
178.4°C, HPLC purity 99+% (tR 2.27 min in 10% water-acetonitrile). 1H NMR (CDCI3): δ
7.93-7.89 (3H, m), 7.53 (1 H, d, J=8.2 Hz), 7.44 (1 H, d, =2.5 Hz), 7.31-7.24 (2H, m),
7.18 (1 H, t, J=8.1 Hz), 2.73 (3H, s), 2.69 (3H, s). LCMS: 345.17 (M-). FAB-MS (MH+, C17H15CIN2O2S): calcd 347.0621 , found 347.0622. Synthesis of A/-(2-methyl-quinolin-6-yl)-4-propyl-benzenesulfonamide, STX 936 (KRB01062):
To a solution of 4n-propylbenzenesulphonyl chloride (145 mg, 0.664 mmol) in dichloromethane (4 mL) was added pyridine (130 μL, 1.58 mmol) and the mixture was stirred under N2 for 5 min, after which time 6-amino-2-methylquinoline (100 mg, 0.632 mmol) was added. The resulting mixture was stirred for 1 h at room temperature, then saturated NaHCO3 solution (10 mL) was added and the mixture was extracted into ethyl acetate (20 mL). The organic phase was washed with brine, dried (Na2S04), filtered and evaporated to give a residue that was purified using flash chromatography to afford an off-white solid (186 mg, 86%), single spot at Rf 0.70 (ethyl acetate), mp 173.7-174.0°C, HPLC purity 99+% (tR 2.32 min in 10% water-acetonitrile). 1H NMR (CDCI3): δ 7.94 (1 H, d, J=8.4 Hz), 7.89 (1 H, d, J=9.2 Hz), 7.67 (2H, d, J=8.4 Hz), 7.54 (1 H, d, J=2.5 Hz), 7.31 (1 H, dd, J=8.9, 2.5 Hz), 7.25 (1H, d, J=8.4 Hz), 7.19 (2H, d, J=8.4 Hz), 7.07 (1H, s, N- H), 2.70 (3H, s), 2.56 (2H, t, =7.5 Hz), 1.58 (2H, sextet, J=7.4 Hz), 0.87 (3H, t, J=7.4 Hz). LCMS: 339.24 (M-). FAB-MS (MH+, C19H20N2O2S): calcd 341.1323, found 341.1324.
Synthesis of 2,5-dichloro-Λ/-(2-methyl-quinolin-6-yl)- benzenesulfonamide. STX 937 (KRB01063):
To a solution of 2,5-dichlorobenzenesulphonyl chloride (163 mg, 0.664 mmol) in dichloromethane (4 mL) was added pyridine (130 μL, 1.58 mmol) and the mixture was stirred under N2 for 5 min, after which time 6-amino-2-methylquinoline (100 mg, 0.632 mmol) was added. The resulting mixture was stirred for 2 h at room temperature, then saturated NaHCO3 solution (10 mL) was added and the mixture was extracted into ethyl acetate (20 mL). The organic phase was washed with brine, dried (Na2S0 ), filtered and evaporated to give a residue that was purified using flash chromatography to afford an off-white solid (120 mg, 52%), single spot at Rf 0.68 (ethyl acetate), mp 124.6-125.0°C, HPLC purity 99+% (tR 2.22 min in 10% water-acetonitrile). 1H NMR (CDCI3): δ 7.98 (1 H, d, J=2.2 Hz), 7.94 (1 H, d, =8.4 Hz), 7.88 (1 H, d, J=8.9 Hz), 7.57 (1 H, d, =2.2 Hz), 7.45-7.35 (3H, m), 7.27-7.24 (2H, m), 2.68 (3H, s). LCMS: 365.10 (M-). FAB-MS (MH+, C16H12CI2N2O2S): calcd 367.0075, found 367.0074.
Synthesis of 3-chloro-2-methyl- -quinazolin-6-yl-benzenesulfonamide, STX 943 (KRB01068):
To a solution of 3-chloro-2-methylbenzenesulphonyl chloride (163 mg, 0.723 mmol) in dichloromethane (4 mL) was added pyridine (140 μL, 1.72 mmol) and the mixture was stirred under N2 for 5 min, after which time 6-aminoquinazoline [21] (100 mg, 0.689 mmol) was added. The resulting mixture was stirred for 4 h at room temperature, then saturated NaHCO3 solution (10 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 pale yellow solid (55 mg, 24%), single spot at R 0.56 (ethyl acetate), mp >270°C (appeared to decompose at about 150°C), HPLC purity 98% (tR 2.06 min in 10% water- acetonitrile). 1H NMR (CDCI3): δ 9.28 (1 H, s), 9.25 (1 H, s), 7.98 (1 H, d, J=4.4 Hz), 7.95 (1 H, d, J=5.2 Hz), 7.59-7.53 (3H, m), 7.25-7.20 (1 H, m), 2.76 (3H, s). LCMS: 332.11 (M- ). FAB-MS (MH+, C15H12CIN3O2S): calcd 334.0417, found 334.0420.
Synthesis of 4-propyl-Λ/-guinazolin-6-yl-benzenesulfonamide, STX 944 (KRB01069):
To a solution of 4n-propylbenzenesulphonyl chloride (151 mg, 0.723 mmol) in dichloromethane (4 mL) was added pyridine (140 μL, 1.72 mmol) and the mixture was stirred under N2 for 5 min, after which time 6-aminoquinazoline (100 mg, 0.689 mmol) was added. The resulting mixture was stirred for 4 h at room temperature, then saturated NaHCO3 solution (10 mL) was added and the mixture was extracted into ethyl acetate (20 mL). The organic phase was washed with brine, dried (Na2SO ), filtered and evaporated to give a residue that was purified using flash chromatography to afford a pale yellow solid (47 mg, 21%), single spot at Rf 0.57 (ethyl acetate), mp 202.1 -202.6°C, HPLC purity 96% (tR 2.14 min in 10% water-acetonitrile). 1H NMR (CDCI3): δ 9.30 (1 H, s), 9.24 (1 H, s), 7.99-7.92 (2H, m), 7.77 (2H, d, J=8.4 Hz), 7.68-7.63 (2H, m), 7.23 (1 H, d, =7.7 Hz), 2.57 (2H, t, J=7.6 Hz), 1.57 (2H, sextet, J=7.6 Hz), 0.87 (3H, t, J=7.4 Hz). LCMS: 326.24 (M-). FAB-MS (MH+, C17H17N3O2S): calcd 328.1119, found 328.1118.
Synthesis of 3-chloro-Λ/-(2,3-dimethyl-quinoxalin-6-yl)-2-methyl- benzenesulfonamide. STX 953 (KRB01074):
To a solution of 3-chloro-2-methylbenzenesulphonyl chloride (109 mg, 0.485 mmol) in dichloromethane (3 mL) was added pyridine (90 μL, 1.2 mmol) and the mixture was stirred under N2 for 5 min, after which time 6-amino-2,3-dimethylquinoxaline [22] (80 mg, 0.46 mmol) was added. The resulting mixture was stirred for 6 h at room temperature, then saturated NaHCO3 solution (8 mL) was added and the mixture was extracted into ethyl acetate (15 L). The organic phase was washed with brine, dried (Na2SO ), filtered and evaporated to give a residue that was purified using flash chromatography to afford an off-white solid (152 mg, 91 %), single spot at Rf 0.70 (ethyl acetate), mp 225.3- 225.7°C, HPLC purity 99+% (tR 2.29 min in 10% water-acetonitrile). 1H NMR (CDCI3): δ 7.99 (1 H, d, =7.9 Hz), 7.85 (1 H, d, J=9.2 Hz), 7.54-7.52 (2H, m), 7.40 (1H, dd, =9.1 , 2.6 Hz), 7.21 (1H, t, J=7.9 Hz), 6.95 (1 H, s, N-H), 2.75 (3H, s), 2.67 (6H, s). LCMS: 360.24 (M-). FAB-MS (MH+, C17H16CIN3O2S): calcd 362.0730, found 362.0732. Synthesis of Λ/-(2.3-dimethyl-quinoxalin-6-yl)-4-propyl- benzenesulfonamide. STX 954 (KRB01075):
To a solution of 4n-propylbenzenesulphonyl chloride (106 mg, 0.485 mmol) in dichloromethane (3 L) was added pyridine (90 μL, 1.2 mmol) and the mixture was stirred under N2 for 5 min, after which time 6-amino-2,3-dimethylquinoxaline (80 mg, 0.46 mmol) was added. The resulting mixture was stirred for 6 h at room temperature, then saturated NaHCO3 solution (8 mL) was added 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 an off-white solid (147 mg, 90%), single spot at Rf 0.69 (ethyl acetate), mp 198.4- 198.8°C, HPLC purity 99+% (tR 2.39 min in 10% water-acetonitrile). 1H NMR (CDCI3): δ 7.86 (1 H, d, J=8.9 Hz), 7.72 (2H, d, J=8.4 Hz), 7.56 (1 H, d, =2.5 Hz), 7.48 (1 H, dd, J=9.2, 2.5 Hz), 7.20 (2H, d, =7.9 Hz), 6.82 (1 H, s, N-H), 2.67 (6H, s), 2.56 (2H, t, J=7.5 Hz), 1.57 (2H, sextet, J=7.4 Hz), 0.87 (3H, t, J=7.4 Hz). LCMS: 354.31 (M-). FAB-MS (MH+, C19H21N3O2S): calcd 356.1432, found 356.1433.
Synthesis of 3-chloro-Λ/-(2-methyl-quinoxalin-6-yl)-2-methyl- benzenesulfonamide, STX 955 (KRB01079):
6-amino-2-methylquinoxaline (KRB01078): To a solution of 2-methyl-6-nitroquinoxaline [23] (500 mg, 2.64 mmol) in methanol (22 mL) was added 10% palladium on carbon (50 mg) and the mixture was stirred under 1 atm H2 for 4 h. The mixture was filtered through celite and the filtrate evaporated. The residue was passed through a silica plug and evaporated to afford 6-amino-2-methylquinoxaline as a yellow solid (376 mg, 89%), single spot at Rf 0.33 (ethyl acetate), mp 163-164°C ([23] 164-165°C). 1H NMR (CDCI3): δ 8.57 (1 H, s), 7.78 (1 H, d, J=8.4 Hz), 7.17-7.12 (2H, m), 4.11 (2H, s -NH2), 2.67 (3H, s). To a solution of 3-chloro-2-methylbenzenesulphonyl chloride (119 mg, 0.528 mmol) in dichloromethane (3 mL) was added pyridine (100 μL, 1.26 mmol) and the mixture was stirred under N2 for 5 min, after which time 6-amino-2-methylquinoxaline (80 mg, 0.50 mmol) was added. The resulting mixture was stirred for 5 h at room temperature, then saturated NaHCO3 solution (8 mL) was added and the mixture was extracted into ethyl acetate (15 mL). The organic phase was washed with brine, dried (Na2SO ), filtered and evaporated to give a residue that was purified using flash chromatography to afford a pale pink solid (161 mg, 92%), single spot at Rf 0.67 (ethyl acetate), mp 158.7-159.3°C, HPLC purity 99+% (tR 2.23 min in 10% water-acetonitrile). 1H NMR (CDCI3): δ 8.66 (1 H, s), 8.02 (1 H, d, J=7.7 Hz), 7.89 (1 H, d, J=8.9 Hz), 7.61 (1H, d, J=2.5 Hz), 7.55 (1H, d, J=8.2 Hz), 7.46 (1 H, dd, J=8.9, 2.5 Hz), 7.23 (1 H, t, J=7.9 Hz), 7.08 (1H, s, N-H), 2.76 (3H, s), 2.71 (3H, s). LCMS: 346.11 (M-). FAB-MS (MH+, C16H14CIN3O2S): calcd 348.0573, found 348.0589.
Synthesis of Λ/-(2-methyl-quinoxalin-6-yl)-4-propyl-benzenesulfonamide. STX 956 (KRB01080):
To a solution of 4n-propylbenzenesulphonyl chloride (115 mg, 0.528 mmol) in dichloromethane (3 mL) was added pyridine (100 μL, 1.26 mmol) and the mixture was stirred under N2 for 5 min, after which time 6-amino-2-methylquinoxaline (80 mg, 0.50 mmol) was added. The resulting mixture was stirred for 2 h at room temperature, then saturated NaHCO3 solution (8 mL) was added 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 (168 mg, 95%), single spot at Rf 0.72 (ethyl acetate), mp 135.6-136.2°C, HPLC purity 99+% (tR 2.23 min in 10% water-acetonitrile). 1H NMR (CDCI3): δ 8.66 (1 H, s), 7.89 (1H, d, J=9.2 Hz), 7.77 (2H, d, J=8.4 Hz), 7.67 (1H, d, J=2.5 Hz), 7.54 (1 H, dd, =8.9, 2.5 Hz), 7.22 (2H, d, =8.4 Hz), 2.71 (3H, s), 2.56 (2H, t, J=7.7 Hz), 1.58 (2H, sextet, =7.7 Hz), 0.87 (3H, t, J=7.4 Hz). LCMS: 340.25 (M-). FAB-MS (MH+, C18H19N3O2S): calcd 342.1276, found 342.1290. Synthesis of 3-chloro-2-methyl-Λ/-quinoxalin-6-yl-benzenesulfonamide, STX 957:
6-aminoquinoxaline (KRB01083): To a solution of 6-nitroquinoxaline [24] (500 mg, 2.86 mmol) in methanol (20 mL) was added 10% palladium on carbon (50 mg) and the mixture was stirred under 1 atm H2 for 4 h. The mixture was filtered through celite and the filtrate evaporated. The residue was passed through a silica plug and evaporated to afford 6-aminoquinoxaline as a yellow solid (342 mg, 82%), single spot at Rf 0.32 (ethyl acetate). 1H NMR (CDCI3): δ 8.65 (1 H, d, J= 1.7 Hz), 8.55 (1 H, d, J=1.7 Hz), 7.87 (1 H, d, J=8.9 Hz), 7.18 (1 H, dd, J=8.9, 2.5 Hz), 7.13 (1 H, d, =2.5 Hz), 4.20 (2H, br.s, -NH2) [25].
To a solution of 3-chloro-2-methylbenzenesulphonyl chloride (147 mg, 0.651 mmol) in dichloromethane (4 mL) was added pyridine (125 μL, 1.55 mmol) and the mixture was stirred under N2 for 5 min, after which time 6-aminoquinoxaline (90 mg, 0.62 mmol) was added. The resulting mixture was stirred for 5 h at room temperature, then saturated NaHCO3 solution (10 mL) was added and the mixture was extracted into ethyl acetate (20 mL). The organic phase was washed with brine, dried (Na2S0 ), filtered and evaporated to give a residue that was purified using flash chromatography to afford a pale yellow solid (180 mg, 87%), single spot at Rf 0.60 (ethyl acetate), mp 166.5- 166.9°C, HPLC purity 99+% (tR 2.15 min in 10% water-acetonitrile). 1H NMR (CDCI3): δ 8.77 (1 H, d, =1.7 Hz), 8.74 (1 H, d, J=1.7 Hz), 8.06 (1 H, d, J=8.2 Hz), 8.00 (1 H, d, J=9.2 Hz), 7.66 (1 H, d, =2.5 Hz), 7.56 (1 H, d, J=7.9 Hz), 7.50 (1 H, dd, J=9.1 , 2.6 Hz), 7.28- 7.22 (1 H, obscured under CHCI3), 2.76 (3H, s). LCMS: 332.24 (M-). FAB-MS (MH+, Cι5H12CIN3O2S): calcd 334.0417, found 334.0433.
Synthesis of 4-propyl-N-quinoxalin-6-yl-benzenesulfonamide. STX 958 (KRB01085):
To a solution of 4n-propylbenzenesulphonyl chloride (142 mg, 0.651 mmol) in dichloromethane (4 mL) was added pyridine (125 μL, 1.55 mmol) and the mixture was stirred under N2 for 5 min, after which time 6-aminoquinoxaline (90 mg, 0.62 mmol) was added. The resulting mixture was stirred for 4 h at room temperature, then saturated NaHCO3 solution (10 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 an off-white solid (190 mg, 94%), single spot at Rf 0.66 (ethyl acetate), mp 198.4-198.9°C, HPLC purity 99+% (tR 2.22 min in 10% water-acetonitrile). 1H NMR (CDCI3): δ 8.77 (1 H, d, J=1.7 Hz), 8.73 (1 H, d, J=1.7 Hz), 8.00 (1 H, d, J=8.9 Hz), 7.80 (2H, d, =8.2 Hz), 7.71 (1 H, d, =2.5 Hz), 7.59 (1 H, dd, J=8.9, 2.5 Hz), 7.23 (2H, obscured under CHCI3), 2.57 (2H, t, J=7.6 Hz), 1.58 (2H, sextet, J=7.5 Hz), 0.87 (3H, t, =7.4 Hz). LCMS: 326.24 (M- ). FAB-MS (MH+, C17H17N3O2S): calcd 328.1119, found 328.1136.
Synthesis of benzisothiazole derivatives
STX 929: R-,=R2=H, R3=CI, R4=Me STX 930: R^R^R^H, R2=n-propyl STX 931: R1=R4=CI, R2=R3=H
Synthesis of Λ/-acetyl-Λ/-(5-amino-benzo[c lisothiazol-3-yl)-acetamide (KRB01050):
/V-acetyl-Λ/-(5-nitro-benzo[o]isothiazol-3-yl)-acetamide (KRB01049): To a solution of 3- amino-5-nitrobenzisothiazole (1.00g, 5.12 mmol) in DMF (20 mL) was added acetic anhydride (1 mL) and triethylamine (1 mL) and the resulting solution was stirred for 4 h. The yellow precipitate was filtered, washed quickly with cold ethyl acetate, and dried in vacuo (1.139g, 80%). 1H NMR (CDCI3): δ 9.20 (1 H, d, J=2.5 Hz), 8.87 (1 H, d, J=9.4 Hz), 8.55 (1 H, dd, J=9.4, 2.5 Hz), 2.69 (3H, s), 2.63 (3H, s).
/V-acetyI-Λ/-(5-amino-benzo[α]isothiazol-3-yl)-acetamide: To a suspension of Λ/-acetyl-Λ/- (5-nitro-benzo[d]isothiazol-3-yl)-acetamide (1.133g, 4.06 mmol) in a 1 :1 acetic acid:ethyl acetate solution (70 mL) at 50°C was added iron powder (680 mg, 12.2 mmol). The resulting mixture was stirred at 50°C for 5 h, then cooled, poured into saturated sodium bicarbonate and extracted into ethyl acetate. The organic layers were combined, washed with water and then brine, dried (MgSO4), filtered, and evaporated. The residue was purified using flash chromatography on silica to afford the desired amine as an orange solid (383 mg, 38%), single spot at Rf 0.30 (1 :1 dichloromethane:ethyl acetate), mp 213.0-214.2°C. 1H NMR (CDCI3): δ 8.56 (1 H, d, J=9.2 Hz), 7.48 (d, J=2.7 Hz), 7.14 (1 H, dd, J=9.2, 2.7 Hz), 2.62 (3H, s), 2.56 (3H, s).
Synthesis of Λ/-r5-(3-chloro-2-methyl-benzenesulfonylamino)- benzorαlisothiazol-3-yll-acetamide, STX 929 (KRB01055):
To a solution of 3-chloro-2-methylbenzenesulphonyl chloride (95 mg, 0.42 mmol) in dichloromethane (3 mL) was added pyridine (80 μL, 1.0 mmol) and the mixture was stirred under N2 for 5 minutes, at which time Λ/-acetyl-Λ/-(5-amino-benzo[cf]isothiazol-3- yl)-acetamide (100 mg, 0.401 mmol) was added. The resulting mixture was stirred for 4 h at room temperature. 5% NaHCO3 solution (8 mL) was added 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 yellow solid (82 mg, 47%), single spot at Rf 0.71 (1 :1 hexane:ethyl acetate). This solid was stirred in a solution of 1:1 THF:6N HCl (6 mL) for 16 h. The solution was then made slightly basic by the addition of Na2CO3, and extracted into ethyl acetate (15 mL). The organic layer was dried (MgSO4), filtered, and evaporated. The residue was purified using flash chromatography to afford a dark brown solid (32 mg, 44%), single spot at Rf 0.84 (ethyl acetate), mp 142-146 °C, HPLC purity 96% (tR 2.34 min in 10% water-acetonitrile). 1H NMR (dβ DMSO): δ 12.32 (1 H, s), 10.61 (1 H, s), 7.93-7.88 (2H, m), 7.68 (1 H, d, J=8.2 Hz), 7.47 (1 H, d, J=9.4 Hz), 7.34 (1H, t, J=8.1 Hz), 7.13 (1 H, dd, J=9.4, 2.2 Hz), 2.68 (3H, s), 2.34 (3H, s). LCMS: 394.18 (M-). FAB-MS (MH+, C16H14CIN3O3S2): calcd 396.0243, found 396.0238.
Synthesis of A/-r5-(4-propyl-benzenesulphonylamino)-benzo[Qlisothiazol-3-yll-acetamide, STX 930 (KRB01056):
To a solution of 4n-propylbenzenesulphonyl chloride (92 mg, 0.42 mmol) in dichloromethane (3 mL) was added pyridine (80 μL, 1.0 mmol) and the mixture was stirred under N2 for 5 minutes, at which time Λ/-acetyl-Λ/-(5-amino-benzo[ t]isothiazol-3- yl)-acetamide (100 mg, 0.401 mmol) was added. The resulting mixture was stirred for 3 h at room temperature. 5% NaHCO3 solution (8 mL) was added and the mixture was extracted into ethyl acetate (15 mL). The organic phase was washed with brine, dried (Na2S04), filtered and evaporated to give a residue that was purified using flash chromatography to afford a yellow solid (115 mg, 66%), single spot at Rf 0.66 (1:1 hexane:ethyl acetate). This solid was stirred in a solution of 1 :1 THF:6N HCl (6 mL) for 6 h. The solution was then made slightly basic by the addition of Na2CO3, and extracted into ethyl acetate (15 mL). The organic layer was dried (MgSO ), filtered, and evaporated. The residue was purified using flash chromatography to afford a pale brown solid (88 mg, 91 %), single spot at Rf 0.71 (ethyl acetate), mp 254.3-255.0°C, HPLC purity 99+% (tR 2.32 min in 10% water-acetonitrile). 1H NMR (dβ DMSO): δ 12.35 (1 H, s), 10.23 (1 H, s), 7.94 (1 H, d, J=2.0 Hz), 7.70 (2H, d, J=8.2 Hz), 7.43 (1 H, d, J=9.4 Hz), 7.33 (2H, d, =8.2 Hz), 7.08 (1 H, dd, J=9.4, 2.0 Hz), 2.50 (2H, t, J=7.4 Hz), 2.35 (3H, s), 1.53 (2H, sextet, J=7.4 Hz), 0.82 (3H, t, J=7.3 Hz). LCMS: 388.25 (M-). FAB- MS (MH+, C18H19N3O3S2): calcd 390.0946, found 390.0941. Synthesis of Λ/-r5-(2,5-dichloro-benzenesulphonylamino)- benzo[o1isothiazol-3-vn-acetamide, STX 931 (KRB01057):
To a solution of 2,5-dichlorobenzenesulphonyl chloride (103 mg, 0.421 mmol) in dichloromethane (3 mL) was added pyridine (80 μL, 1.0 mmol) and the mixture was stirred under N2 for 5 minutes, at which time Λ/-acetyl-Λ/-(5-amino-benzo[ ]isothiazol-3- yl)-acetamide (100 mg, 0.401 mmol) was added. The resulting mixture was stirred for 6 h at room temperature. 5% NaHCO3 solution (8 mL) was added and the mixture was extracted into ethyl acetate (15 mL). The organic phase was washed with brine, dried (Na2SO ), filtered and evaporated to give a residue that was purified using flash chromatography to afford a yellow solid (114 mg, 62%), single spot at Rf 0.74 (1 :1 hexane:ethyl acetate). This solid was stirred in a solution of 1 :1 THF:6N HCl (6 mL) for 5 h. The solution was then made slightly basic by the addition of Na2CO3, and extracted into ethyl acetate (15 mL). The organic layer was dried (MgSO4), filtered, and evaporated. The residue was purified using flash chromatography to afford a pale brown solid (28 mg, 29%), single spot at Rf 0.64 (ethyl acetate), mp 169-172°C, HPLC purity 99+% (tR 2.85 min in 10% water-acetonitrile). 1H NMR (d6 DMSO): δ 12.34 (1 H, s), 10.84 (1 H, s), 8.12 (1 H, d, J=1.7 Hz), 7.89 (1 H, d, J=1.7 Hz), 7.68 (2H, m), 7.49 (1 H, d, J=9.4 Hz), 7.18 (1 H, dd, J=9.4, 2.0 Hz), 2.35 (3H, s). LCMS: 414.11 (M-). FAB-MS (MH+, dsHnClaNsOaSz): calcd 415.9697, found 415.9697.
Synthesis of Benzisoxazole Derivatives
STX 874: R!=R2=H, R3=CI, R4=Me STX 918: R1=R2=H, R3=CI, R4=Me STX 875: Rι=R3=R4=H, R2=n-propyl STX 919: Rι=R3=R =H, R2=n-propyl STX 876: R.,=R4=CI, R2=R3=H STX 920: R1=R4=CI, R2=R3=H
Synthesis of 3-chloro-2-methyl-/V-(3-methyl-benzo['oflisoxazol-5-yl)- benzenesulfonamide, STX 874 (KRB01027):
To a solution of 3-chloro-2-methylbenzenesulphonyl chloride (128 mg, 0.567 mmol) in dichloromethane (3 mL) was added pyridine (110 μL, 1.35 mmol) and the mixture was stirred under N2 for 5 min, after which time 5-amino-3-methyl-1 ,2-benzisoxazole [26] (80 mg, 0.54 mmol) was added. The resulting mixture was stirred for 4 h at room temperature, then saturated NaHCO3 solution (8 mL) was added 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 (88 mg, 48%), single spot at Rf 0.58 (1 :1 hexane:ethyl acetate), mp 162.8-163.2°C, HPLC purity 99+% (tR 2.26 min in 10% water- acetonitrile). 1H NMR (CDCI3): δ 7.80 (1 H, dd, J=7.9, 1.3 Hz), 7.55 (1 H, dd, J=8.1 , 1.1 Hz), 7.38 (2H, m), 7.17 (1 H, t, J=8.1 Hz), 7.10 (1 H, dd, J=8.8, 2.2 Hz), 6.72 (1 H, s, N-H), 2.72 (3H, s), 2.50 (3H, s). LCMS: 320.00 (M-CH3). FAB-MS (MH+, C15H13CIN2O3S): calcd 337.0413, found 337.0422. Synthesis of Λ/-(3-methyl-benzorα1isozazol-5-yl)-4-propyl- benzenesulfonamide. STX 875 (KRB01028):
To a solution of 4n-propylbenzenesulphonyl chloride (124 mg, 0.567 mmol) in dichloromethane (3 mL) was added pyridine (110 μL, 1.35 mmol) and the mixture was stirred under N2 for 5 min, after which time 5-amino-3-methyl-1 ,2-benzisoxazole (80 mg, 0.54 mmol) was added. The resulting mixture was stirred for 3 h at room temperature, then saturated NaHC03 solution (8 mL) was added 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 (150 mg, 84%), single spot at Rf 0.62 (1 :1 hexane:ethyl acetate), mp 119.5-120.0°C, HPLC purity 99+% (tR 2.29 min in 10% water-acetonitrile). 1H NMR (CDCI3): δ 7.57 (2H, d, J=8.4 Hz), 7.40 (1 H, d, J=2.2 Hz), 7.37 (1H, d, J=8.8 Hz), 7.20 (2H, d, J=8.4 Hz), 7.10 (1 H, dd, J=8.8, 2.2 Hz), 6.71 (1 H, s, N-H), 2.59 (2H, t, J=7.5 Hz), 2.51 (3H, s), 1.59 (2H, sextet, J= 7.5 Hz), 0.88 (3H, t, J=7.5 Hz). LCMS: 314.07 (M- CH3). FAB-MS (MH+, C17H18N2O3S): calcd 331.1116, found 331.1117.
Synthesis of 2,5-dichloro-A/-(3-methyl-benzofo1isozazol-5-yl)- benzenesulfonamide, STX 876 (KRB01030):
To a solution of 2,5-dichlorobenzenesulphonyl chloride (105 mg, 0.428 mmol) in dichloromethane (3 mL) was added pyridine (100 μL, 1.02 mmol) and the mixture was stirred under N2 for 5 min, after which time 5-amino-3-methyl-1 ,2-benzisoxazole (60 mg, 0.41 mmol) was added. The resulting mixture was stirred for 2 h at room temperature, then saturated NaHC03 solution (8 mL) was added 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 yellow solid (100 mg, 68%), single spot at Rf 0.62 (1 :1 hexane:ethyl acetate), mp 229.4-230.0°C, HPLC purity 94% (tR 2.18 min in 10% water-acetonitrile). 1H NMR (CDCI3): δ 7.89 (1 H, d, J=2.2 Hz), 7.43 (4H, m), 7.22 (1 H, m), 7.09 (1 H, s, N-H), 2.54 (3H, s). LCMS: 340.06 (M-CH3). FAB-MS (MH+, C14H10CI2N2O3S): calcd 356.9867, found 356.9860.
Synthesis of Λ/-benzo[o1isoxazol-5-yl-3-chloro-2-methyl- benzenesulfonamide, STX 918 (KRB01046):
To a solution of 3-chIoro-2-methylbenzenesulphonyl chloride (176 mg, 0.783 mmol) in dichloromethane (4 mL) was added pyridine (150 μL, 1.86 mmol) and the mixture was stirred under N2 for 5 min, after which time 5-amino-1 ,2-benzisoxazole [27] (100 mg, 0.746 mmol) was added. The resulting mixture was stirred for 2 h at room temperature, then saturated NaHCO3 solution (10 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 (178 mg, 74%), single spot at Rf 0.69 (1 :1 hexane:ethyl acetate), mp 111.9-112.4°C, HPLC purity 97% (tR 2.44 min in 10% water-acetonitrile). 1H NMR (CDCI3): δ 8.62 (1 H, d, J=1.0 Hz), 7.81 (1 H, dd, J=7.9, 1.2 Hz), 7.55 (1H, dd, J=7.9, 1.0 Hz), 7.45 (2H, m), 7.17 (2H, m), 6.77 (1 H, s, N-H), 2.72 (3H, s). LCMS: 321.01 (M-). FAB-MS (MH+, C14HnCIN2O3S): calcd 323.0257, found 323.0271.
Synthesis of Λ/-benzo[c lisoxazol-5-yl-4-propyl-benzenesulfonamide, STX 919 (KRB01047):
To a solution of 4n-propylbenzenesulphonyl chloride (171 mg, 0.783 mmol) in dichloromethane (4 mL) was added pyridine (150 μL, 1.86 mmol) and the mixture was stirred under N2 for 5 min, after which time 5-amino-1 ,2-benzisoxazole (100 mg, 0.746 mmol) was added. The resulting mixture was stirred for 2 h at room temperature, then saturated NaHCO3 solution (10 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 (170 mg, 72%), single spot at Rf 0.68 (1:1 hexane:ethyl acetate), mp 130.0- 130.6°C, HPLC purity 99+% (tR 2.44 min in 10% water-acetonitrile). 1H NMR (CDCI3): δ 8.62 (1 H, d, J=1.0 Hz), 7.60 (2H, d, J=8.4 Hz), 7.48 (2H, m), 7.19 (3H, m), 6.86 (1 H, s, N-H), 2.58 (2H, t, J=7.5 Hz), 1.58 (2H, sextet, J=7.4 Hz), 0.88 (3H, t, J=7.4 Hz). LCMS: 315.14 (M-). FAB-MS (MH+, C16H16N203S): calcd 317.0960, found 317.0962.
Synthesis of A/-benzo[o1isoxazol-5-yl-2.5-dichloro-benzenesulfonamide, STX 920 (KRB01048):
To a solution of 2,5-dichlorobenzenesulphonyl chloride (192 mg, 0.783 mmol) in dichloromethane (4 mL) was added pyridine (150 μL, 1.86 mmol) and the mixture was stirred under N2 for 5 min, after which time 5-amino-1 ,2-benzisoxazole (100 mg, 0.746 mmol) was added. The resulting mixture was stirred for 2 h at room temperature, then saturated NaHCO3 solution (10 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 (174 mg, 68%), single spot at Rf 0.68 (1 :1 hexane:ethyl acetate), mp 172.9-
173.6°C, HPLC purity 99+% (tR 2.41 min in 10% water-acetonitrile). 1H NMR (CDCI3): δ
8.64 (1 H, s), 7.89 (1H, d, =2.2 Hz), 7.56-7.28 (5H, m). LCMS: 341.07 (M-). FAB-MS
(MH+, C13H8CI2N2O3S): calcd 342.971 1 , found 342.9710. Synthesis of Arysulfonamide, Arylthiol Ether and Arylsulfone Derivatives
STX979 STX980
General method for arylsulphonamide formation:
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 the 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 40-90%. 3-Chloro-2-methyl-/V-(4-morpholin-4-yl-phenyl)-benzenesulfonamide (STX828.
XDS01161)
Off-white crystalline solid. TLC single spot at Rf 0.70 (70% ethyl acetate/hexane); HPLC purity > 99 % (tR 1.8 min in 4 % water-methanol); 1H NMR (400 MHz, DMSO): δ 10.1 (1 H, s, NH), 7.77 (1H, dd, J = 8.2, 1.2 Hz, ArH), 7.70 (1 H, dd, J=8.2, 1.2 Hz, ArH), 7.34 (1 H, t, J = 8.2 Hz, ArH), 6.78-6.92 (4H, m, ArH), 3.67 (4H, t, J = 5.1 Hz, N(CH2)2), 2.99 (4H, t, J = 5.1 Hz, O(CH2)2), 2.60 (3H, s, CH3); APCI-MS 365 (M-H+); FAB-HRMS calcd for C17H20CIN2O3S (MH+) 367.0883, found 367.O854
3-Chloro-2-methyl-Λ/-(3-oxo-1.3-dihydro-isobenzofuran-5-yl)-benzenesulfonamide (STX885, XDS01179)
White crystalline solid. TLC single spot at Rf 0.60 (20% ethyl acetate/hexane); HPLC purity > 99 % (tR 2.2 min in 10 % water-methanol); 1H NMR (270 MHz, DMSO): δ 11.0 (1 H, s, NH), 7.84 (1 H, d, J = 7.9 Hz, ArH), 7.66 (1 H, d, J = 7.9 Hz, ArH), 7.49 (1 H, d, J = 8.5 Hz, ArH), 7.31-7.46 (3H, m, ArH), 5.22 (2H, s, CH2), 2.57 (3H, s, CH3); APCI-MS 338 (M+H+); FAB-HRMS calcd for C15Hι3CIN04S (MH+) 338.0254, found 338.0270
3-Chloro-2-methyl-N-(2-methyl-1.3-dioxo-2.3-dihvdro-1 H-isoindol-5-yl)- benzenesulfonamide (STX979, XDS01180)
Yellow crystalline solid. TLC single spot at Rf 0.82 (20% ethyl acetate/hexane); HPLC purity > 99 % (tR 2.1 min in 10 % water-methanol); 1H NMR (270 MHz, DMSO): δ 11.5 (1 H, s, NH), 7.98 (1 H, d, J = 7.9 Hz, ArH), 7.76 (1 H, d, J = 8.3 Hz, ArH), 7.73 (1 H, d, J = 8.3 Hz, ArH), 7.45 (1 H, t, J = 8.3 Hz, ArH ), 7.41 (1 H, s, ArH), 7.39 (1 H, d, J = 7.9 Hz, ArH ),2.94 (3H, s, NCH3), 2.64 (3H, s, CH3); APCI-MS 363 (M-H+); FAB-HRMS calcd for C16H14CIN2O4S (MH+) 365.0363, found 365.0375
3-Chloro-2-methyl-N-(4-methyl-2-oxo-2H-chrornen-7-yl)-benzenesulfonamide (STX980, XDS02018)
White crystalline solid. TLC single spot at Rf 0.78 (6% methanol/DCM); HPLC purity >
99 % (tR 2.1 min in 10 % water-methanol); 1H NMR (270 MHz, DMSO): δ 11.3 (1 H, s, NH), 8.03 (1 H, d, J = 7.9 Hz, ArH), 7.76 (1 H, d, J = 7.9 Hz, ArH), 7.65 (1 H, d, J = 8.6 Hz, ArH), 7.46 (1 H, t, J = 7.9 Hz, ArH), 7.07 (1 H, dd, =8.6, 1.8 Hz, ArH), 6.98 (1 H, d, J = 1.5 Hz, ArH), 6.24 (1 H, s, 3-H), 2.69 (3H, s, CH3), 2.33 (3H, s, CH3); APCI-MS 362 (M- H+); FAB-HRMS calcd for C17H15CINO4S (MH+) 364.0410, found 364.0414
3-Chloro-N-(2,3-dihvdro-benzori .41dioxin-6-yl)-2-methyl-benzenesulfonamide (STX990. XDS02039)
White crystalline solid. TLC single spot at Rf 0.78 (30% ethyl acetate/DCM); HPLC purity 94 % (tR 2.3 min in 10 % water-methanol); 1H NMR (270 MHz, DMSO): δ 10.3 (1 H, s, NH), 7.82 (1 H, d, J = 7.9 Hz, ArH), 7.72 (1 H, dd, J = 7.9, 1.0 Hz, ArH), 7.39 (1 H, t, J = 7.9 Hz, ArH ), 6.72 (1 H, d, J = 8.4 Hz, ArH ), 6.49-6.55 (2H, m, ArH), 4.15 (4H, s, (CH2)2), 2.62 (3H, s, CH3); APCI-MS 338 (M-H+); FAB-HRMS calcd for C15H15CINO4S (MH+) 340.0410, found 340.0387
3-Chloro-2-methyl-N-(9-oxo-9H-fluoren-3-yl)-benzenesulfonamide (STX1033,
XDS02074)
Yellow solid. TLC single spot at Rf 0.55 (8% ethyl acetate/DCM); HPLC purity > 99 % (tR 2.5 min in 20 % water-methanol); 1H NMR (270 MHz, DMSO): δ 10.3 (1 H, s, NH), 7.82 (1 H, d, J = 7.9 Hz, ArH), 7.72 (1 H, dd, J = 7.9, 1.0 Hz, ArH), 7.39 (1 H, t, J = 7.9 Hz, ArH ), 6.72 (1 H, d, J = 8.4 Hz, ArH ), 6.49-6.55 (2H, m, ArH), 4.15 (4H, s, (CH2)2), 2.62 (3H, s, CH3); APCI-MS 382 (M-H+); FAB-HRMS calcd for C20H15CINO3S (MH+) 384.0461 , found 384.0457.
5-(2.5-Dichloro-phenylsulfanylmethyl)-2-methylbenzothiazole (STX1031. XDS02072)
To a solution of 2,5-dichlorobenzothiol (179 mg, 1.0 mmol) in absolute ethanol (3 mL) were added 5-(bromomethyl)-2-methylbenzothiazole (182 mg, 0.75 mmol) and triethylamine (0.15 mL). After stirred at rt for 3h, the mixture was partitioned between ethyl acetate and 1% KOH solution. The organic phase was washed with brine, dried over sodium sulphate and concentrated in vacuo to give a residue that was subjected to flash chromatography. Colorless needles (220 mg, 86%) were obtained. TLC single spot at Rf 0.55 (5% ethyl acetate/DCM); HPLC purity > 99% (tR 6.8 min in 20% water- methanol); 1H NMR (270 MHz, DMSO): δ 8.00 (1 H, d, J = 8.1 Hz, ArH), 7.97 (1 H, s, ArH), 7.53 (1 H, d, J = 2.5 Hz, ArH ), 7.46-7.49 (2H, m, ArH ), 7.24 (1 H, dd, J = 8.1 , 2.5 Hz, ArH), 4.54 (2H, s, CH2), 2.79 (3H, s, CH3); APCI-MS 338 (M+); FAB-HRMS calcd for C15H12CI2NS2 (MH+) 339.9788, found 339.9779.
5-(2,5-Dichloro-benzenesulfonylmethyl)-2-methylbenzothiazole (STX1032. XDS02075)
To a solution of 5-(2,5-Dichloro-phenylsulfanylmethyl)-2-methylbenzothiazole (STX1031 , 125 mg, 0.367 mmol) in DCM (5 mL) was added 3-cholroperoxybenzoic acid (370 mg, 2.14 mmol). The mixture was stirred at rt for 2h, then partitioned between DCM and saturated sodium carbonate solution. The organic phase was washed with brine, dried over sodium sulphate and concentrated to give a residue that was purified with flash chromatography. White crystals (55 mg, 40%) were obtained after recrystallization from DCM. TLC single spot at Rf 0.20 (5% ethyl acetate/DCM); HPLC purity 97% (tR 3.0 min in 20% water-methanol); 1H NMR (270 MHz, DMSO): δ 7.99 (1 H, d, J = 8.2 Hz, ArH), 7.82-7.83 (2H, m, ArH), 7.77 (1H, s, ArH), 7.69 (1H, d, J = 2.5 Hz, ArH ), 7.23 (1 H, dd, J = 8.2, 1.5 Hz, ArH), 5.07 (2H, s, CH2), 2.79 (3H, s, CH3); APCI-MS 370 (M+); FAB-HRMS calcd for C15H12CI2NO2S2 (MH+) 371.9686, found 371.9691.
Synthesis of N-2-Hydroxyethyl Arysulfonamides and Arysulfonic Acid Esters
STX648 XDS01111
General Method for coupling 4-amino-N-(2-hvdroxyethyl)benzenesulfonamide with arylsulphonyl chloride (STX646-STX653)
To a solution arylsulphonyl chloride (1.1 eq.) in DCM were added pyridine (10 eq.) and 4-amino-N-(2-hydroxy-ethyl)-benzenesulfonamide (1 eq.). The reaction mixture was stirred at rt under nitrogen for 20 h, then partitioned between DCM and 1% HCl solution. The organic layer was washed with brine, dried over sodium sulphate, and concentrated in vacuo to give crude oily product, which was seperated by flash chromatography (ethyl acetate-DCM gradient elution) to give arylsulphonamide (STX646, 648, 649, 651 , 653) and arylsulphonamide arylsulphonic acid ester (STX647, 650, 652) in 10 : 1 ratio.
N-f4-(2-Hydroxyethylsulfamoyl)phenyll-4'-methoxybenzenesulfonamide (STX646,
XDS01110)
White foam . TLC single spot at Rf 0.21 (50% DCM-ethyl acetate); HPLC purity > 99% (tR 2.0 min in 10% water-methanol); 1H NMR (400 MHz, DMSO): δ 10.8 (1 H, s, NH), 7.76 (2H, m, ArH), 7.64 (2H, d, J = 9 Hz, ArH), 7.46 (1 H, t, J = 5.9 Hz, NH), 7.24 (2H, d, J = 9 Hz, ArH), 7.08 (2H, m, ArH), 4.66 (1 H, t, J = 5.5 Hz, OH), 3.80 (3H, s, OCH3), 3.30 (2H, m, OCH2), 2.72 (2H, m, NCH2); FAB-MS 387 (MH+); FAB-HRMS calcd for C15H-19N2O6S2 (MH+) 387.0685, found 387.0685
4-Methoxy-benzenesulfonic acid 2-r4-(4-methoxybenzenesulfonylamino)- benzenesulfonylaminol ethyl ester (STX647. XDS0 1 10B)
White foam TLC single spot at Rf 0.62 (50% DCM-ethyl acetate); HPLC purity > 99% (tR 1.6 min methanol); 1H NMR (400 MHz, DMSO): δ 10.8 (1 H, s, NH), 7.82 (1 H, t, J = 5.9 Hz, NH), 7.75-7.81 (4H, m, ArH), 7.59 (2H, d, J = 9 Hz, ArH), 7.22-7.25 (2H, 2d, J = 9 Hz, ArH), 7.17 (2H, m, ArH), 7.08 (2H, m, ArH), 3.88 (2H, m, OCH2), 3.87 (3H, s, OCH3), 3.79 (3H, s, OCH3), 2.90 (2H, q, J = 5.9 Hz, NCH2); FAB-MS 557 (MH+); FAB-HRMS calcd for C22H25N2θgS3 (MH+) 557.0722, found 557.0717.
N-f4-(2-Hydroxyethylsulfamoyl)phenvn-2'-methyl-3'-chlorobenzenesulfonamide (STX648. XDS01111)
White foam. TLC single spot at Rf 0.28 (50% DCM-ethyl acetate); HPLC purity > 99% (tR 1.7 min in methanol); 1H NMR (400 MHz, DMSO): δ 11.2 (1 H, s, NH), 7.96 (1 H, dd, J = 8.2, 1.1 Hz, ArH), 7.75 (1H, dd, J = 8.2, 1.1 Hz, ArH), 7.65 (2H, m, ArH), 7.46 (1 H, t, J = 8.2 Hz, ArH ), 7.44 (1H, t, J = 6.4 Hz, NH), 7.22 (2H, m, ArH ), 4.66 (1 H, t, J = 5.8 Hz, OH), 3.32 (2H, m, OCH2), 2.71 (2H, m, NCH2), 2.65 (3H, s, CH3); APCI-MS 405 (MH+); FAB-HRMS calcd for C15H18CIN2O5S2 (MH+) 405.0345, found 405.0334
N-r4-(2-Hvdroxyethylsulfamoyl)phenyll-3'.4'-dimethoxy Benzene Sulfonamide (STX649. STX01112)
White foam. TLC single spot at Rf 0.17 (50% DCM-ethyl acetate); HPLC purity > 99 % (tR 1.6 min in methanol); 1H NMR (400 MHz, DMSO): δ 10.7 (1 H, s, NH), 7.64 (2H, d, J - 8.6 Hz, ArH), 7.46 (1H, t, J = 6.4 Hz, NH), 7.39 (1 H, dd, J = 8.5, 2.3 Hz, ArH), 7.28 (1H, d, J = 2.3 Hz, ArH), 7.26 (2H, d, J = 8.6 Hz, ArH), 7.08 (1H, d, J = 8.5 Hz, ArH ), 4.66 (1 H, t, J = 5.4 Hz, OH), 3.79 (3H, s, OCH3), 3.77 (3H, s, OCH3), 3.33 (2H, m, OCH2), 2.71 (2H, m, NCH2); FAB-MS 417 (MH+); FAB-HRMS calcd for C16H21 N2O7S2 (MH+) 417.0790, found 417.0783. 3,4-Dimethoxybenzenesulfonic acid 2-r4-(34-dimethoxybenzenesulfonylamino)- benzenesulfonylaminolethyl ester (STX650. XDS01112B)
White foam TLC single spot at Rf 0.50 (50% DCM-ethyl acetate); HPLC purity > 99 % (tR 1.6 min in methanol); H NMR (400 MHz, DMSO): δ 10.7 (1 H, s, NH), 7.80(1 H, t, J = 5.9 Hz, NH), ,7.60 (2H, d, J = 8.9 Hz, ArH), 7.38-7.45 (2H, m ArH), 7.23-7.29 (4H, m ArH), 7.17 (2H, d, J = 8.6 Hz, ArH), 7.07 (2H, d, J = 8.6 Hz, ArH), 3.92 (2H, t, J = 5.1 Hz, OCH2), 3.87 (3H, s, OCH3), 3.84 (3H, s, OCH3), 3.79 (3H, s, OCH3), 3.76 (3H, s, OCH3), 2.90 (2H, q, J = 5.9 Hz, NCH2); FAB-MS 617 (M-H+); FAB-HRMS calcd for C24H29N2O11S3 (MH+) 617.0933, found 617.0926
N-[4-(2-Hvdroxyethylsulfamoyl)phenyll-2'.3',6'-trimethyl-4'-methoxy-benzenesulfonamide (STX651. XDS01117)
White solid. TLC single spot at Rf 0.28 (60% DCM-ethyl acetate) ; HPLC purity > 99 % (tR 1.6 min in methanol); 1H NMR (400 MHz, DMSO): δ 10.8 (1H, s, NH), 7.60 (2H, d, J = 8.9 Hz, ArH), 7.41 (1 H, t, J = 5.8 Hz, NH), 7.07 (2H, d, J = 8.9 Hz, ArH), 6.83 (1 H, s, ArH), 4.65 (1 H, broad, OH), 3.81 (3H, s, OCH3), 3.33 (2H, m, OCH2), 2.70 (2H, m, NCH2), 2.65 (3H, s CH3), 2.53 (3H, s CH3), 2.05 (3H, s CH3); FAB-MS 429 (MH+); FAB- HRMS calcd for C18H25N2O6S2 (MH+) 429.1154, found 429.1143.
4-Methoxy-2,3,6-trimethylbenzenesulfonic acid 2-r4-(4-methoxy-2,3,6-trimethyl- benzenesulfonylamino)benzenesulfonylaminol ethyl ester (STX652, XDS01117B)
White foam TLC single spot at R 0.80 (60% DCM-ethyl acetate); HPLC purity > 99 % (tR 1.7 min in methanol); 1H NMR (400 MHz, DMSO): δ 10.8 (1H, s, NH), 7.72(1 H, t, J = 5.9 Hz, NH), 7.56 (2H, d, J = 9 Hz, ArH), 7.03 (2H, d, J = 9 Hz, ArH), 6.89 (1 H, s, ArH), 6.84 (1 H, s, ArH), 3.86 (3H, s, OCH3), 3.80 (5H, m, OCH3, and OCH2), 2.89 (2H, m, NCH2), 2.65 (3H, s CH3), 2.54 (3H, s CH3), 2.52 (3H, s CH3), 2.42 (3H, s CH3), 2.08 (3H, s CH3), 2.03 (3H, s CH3); FAB-MS 641 (MH+); FAB-HRMS calcd for C28H37N2O9S3 (MH+) 641.1661 , found 641.1642 N-f4-(2-Hvdroxy-ethylsulfamoyl)-phenvπ-2'.4'-dichlorobenzenesulfonamide (STX653. XDS01118)
White crystalline solid. TLC single spot at Rf 0.36 (60% DCM-ethyl acetate); HPLC purity > 99 % (tR 1.9 min in 4 % water-methanol); 1H NMR (400 MHz, DMSO): δ 11.3 (1H, s, NH), 8.11 (1 H, d, J = 8.2 Hz, ArH), 7.88 (1 H, d, J = 2.3 Hz, ArH), 7.62-7.68 (3H, m, ArH), 7.47 (1 H, t, J = 5.8 Hz, NH), 7.23 (2H, d, J = 8.6 Hz, ArH), 4.66 (1 H, t, J = 5.9 Hz, OH), 3.33 (2H, m, OCH2), 2.71 (2H, m, NCH2); FAB-MS 424.9 (MH+); FAB-HRMS calcd for C14H15CI2N2O5S2 (MH+) 424.9799, found 424.9800
Synthesis of N-(5- ethylisoxazol-3-yl)-arysulphonamides
STX 824 XDS0156B STX825 XDS01157
STX823 XDS01155B
STX606 XDS01096 STX608 XDS01099
STX643 XDS01107
General Method for coupling 3-amino-5-methylisooxazole with arylsulphonyl chloride (STX606, STX645. STX823-825)
To a solution arylsulphonyl chloride (1.1 eq.) in DCM were added pyridine (1 .2 eq.) and 3-amino-5-methylisooxazole (1 eq.). The reaction mixture was stirred at rt under nitrogen for 24 h, then partitioned between ethyl acetate and 5% sodium bicarbonate solution. The organic layer was washed with 1% HCl solution and brine, dried over sodium sulphate, and concentrated in vacuo to give crude product that was purified by flash chromatography (ethyl acetate-DCM gradient elution) to give arylsulphonamide as white or off-white crystalline solid (Yield 50-80%o).
2,5-Dichloro-N-(5-methylisoxazol-3-yl)benzenesulphonamide (STX823 XDS01 155B)
White crystalline solid. TLC single spot at Rf 0.45 (10% ethyl acetate-DCM); HPLC purity as rotational isomers > 99% (tR 2.2 min in 15% water-methanol); 1H NMR (270 MHz, DMSO-d6): δ 12.0 (1 H, s, NH), 8.01 (1H, d, J = 2 Hz, ArH), 7.78 (1 H, dd, J = 8.0, 2.0 Hz, ArH), 7.71 (1 H, d, J = 8.0 Hz, ArH), 6.04 (1 H, s, ArH), 2.27 (3H, s, CH3); APCI-MS 306 (M+); FAB-HRMS calcd for CιoHgCl2N2θ3S (MH+) 306.9711 , found 306.9718
N-(5-Methylisoxazo!-3-yl)-4-propylbenzenesulphonamide (STX824 XDS01156B)
White crystalline solid. TLC single spot at Rf 0.65 (10% ethyl acetate-DCM); HPLC purity > 99% (tR 2.3 min in 10% water-methanol); 1H NMR (270 MHz, DMSO-d6): δ 11.3 (1 H, s, NH), 7.72 (2H, d, J = 8.1 Hz, ArH), 7.38 (2H, d, J = 8.1 Hz, ArH), 6.10 (1 H, s, ArH), 2.58 (2H, t, J = 8.0 Hz, CH2), 2.25 (3H, s, CH3), 1.53 (2H, t, J = 7.9 Hz, CH2), 0.83 (3H, t, J = 7.9 Hz, CH3); APCI-MS 281 (MH+); FAB-HRMS calcd for C13H17N2O3S (MH+) 281.0960, found 281.0970 3-Chloro-2-methyl-N-(5-methylisoxazol-3-yl)benzenesulphonamide (STX825
XDS01157)
White crystalline solid. TLC single spot at Rf 0.50 (10% ethyl acetate-DCM); H PLC purity as rotational isomers > 99% (tR 2.4 min in 15% water-methanol); 1H NMR (270 MHz, DMSO-d6): δ 11.8 (1H, s, NH), 7.91 (1H, d, J = 8.1 Hz, ArH), 7.76 (1H, d, J = 8.1 Hz, ArH), 7.43 (1 H, t, J = 8.1 Hz, ArH), 6.01 (1H, s, ArH), 2.61 (3H, s, CH3), 2.26 (3H, s, CH3); FAB-MS 286 (M+); FAB-HRMS calcd for C11 H12CIN2O3S (MH+) 287.0257, found 287.0258
N-(5-methylisoxazol-3-yl)-4-acetamidobenzenesulphonamide (STX606, XDS01096)
White solid. Mp 220-222°C (lit [28], 225-228°C); TLC single spot at Rf 0.62 (10% ethyl acetate-DCM); HPLC purity 99% (tR 1.7 min in methanol); 1H NMR (270 MHz, DMSO- d6): δ 11.3 (1 H, s, NH), 10.4 (1 H, s, AcNH), 7.76 (4H, s, ArH), 6.12 (1 H, s, ArH), 2.29 (3H, s, CH3), 2.07 (3H, s, COCH3); FAB-MS 296 (MH+); FAB-HRMS calcd for C12H14N3O4S (MH+) 296.0705, found 296.0701
N-(5-methylisoxazol-3-yl)-3-chloro-4-acetamidobenzenesulphonamide (STX645, XDS01109)
White solid. TLC single spot at Rf 0.52 (6% methanol-DCM); HPLC purity as rotational isomers > 99% (tR 1.7 min in 4% water-methanol); 1H NMR (400 MHz, DMSO-d6): δ 11.5 (1 H, s, NH), 9.78 (1H, s, AcNH), 8.10 (1 H, d, J = 8.2 Hz, ArH), 7.90 (1 H, s, ArH), 7.77 (1 H, d, J = 8.2 Hz, ArH), 6.15 (1 H, s, ArH), 2.31 (3H, s, CH3), 2.16 (3H, s, COCH3); FAB- MS 330 (MH+); FAB-HRMS calcd for C12H13CIN3O4S (MH+) 330.0315, found 330.0321
4-Amino-N-(5-methyl-isoxazol-3-yl)-benzenesulphonamide (STX608. XDS01099)
The solution of N-(5-methylisoxazol-3-yl)-4-acetamidobenzenesulphonamid (3.4 g, 11.5 mmol) in 10% NaOH solution (15 mL) was stirred at 80°C for 1 h, cooled to rt and neutralized to pH 6 with acetic acid. The precipitate was washed with water, dried in vacuo to yield off-white solid (2,8 g, 96%). Mp167-169°C (lit [29], 168-1 1 °C); TLC single spot at Rf 0.39 (6% methanol-DCM); HPLC purity > 99% (tR 1.6 min in 4% water- methanol); 1H NMR (400 MHz, CD3OD): δ 7.54 (2H, m, ArH), 6.63 (2H, m, ArH), 6.08 (1 H, s, ArH), 2.30 (3H, s, CH3); FAB-MS 254 (MH+); FAB-HRMS calcd for C10H12N3O3S (MH+) 254.0599, found 254.0605.
4-Amino-3-chloro-N-(5-methylisoxazol-3-yl)benzenesulphonamide (STX654. XDSQ1119)
The compound was prepared as described above. White solid (370 mg, 94%) was obtained. TLC single spot at Rf 0.58 (30% ethyl acetate-DCM); HPLC purity > 99% (tR 1.7 min in methanol); 1H NMR (400 MHz, DMSO-d6): δ 11.1 (1 H. s, NH), 7.60 (1 H, d, J = 2.3 Hz, ArH), 7.45 (1 H, dd, J = 8.5, 2.3 Hz, ArH),6.82 (1 H, d, J = 8.5 Hz, ArH),), 6.23 (2H, s, NH2), 6.10 (1H, s, ArH), 2.29 (3H, s, CH3); FAB-MS 288 (MH+); FAB-HRMS calcd for C10H10CIN3O3S (MH+) 288.0210, found 288.0213.
General Method for coupling 4-Amino-N-(5-methyl-isoxazol-3-yl)-benzenesulphonamide or 4-Amino-3-chloro-N-(5-methylisoxazol-3-yl)benzenesulphonamide with arylsulphonyl chloride (STX638, 642-644. 728. 729)
To a solution arylsulphonyl chloride (1.1 eq.) in DCM were added pyridine (1.3 eq.) and catalytic amount of DMAP, followed by the amine (1 eq.). The reaction mixture was stirred at rt or 40°C under nitrogen for 24-48 h, then partitioned between DCM and water after TLC showed completion of the reaction. The organic layer was washed with 3% HCl solution and brine, dried over magnesium sulphate, and concentrated in vacuo to give crude product that was purified by recrystallization from ethyl acetate-DCM or by flash chromatography (ethyl acetate-DCM gradient elution) to give arylsulphonamide as white or off-white crystalline solid (Yield 40-80%).
N-r4-(5-Methyl-isoxazol-3-ylsulfamoyl)phenyll-3'4'-dimethoxy-benzenesulphonamide (STX638. XDS01105)
White crystalline solid. TLC single spot at Rf 0.30 (6% methanol-DCM); HPLC purity as rotational isomers > 99% (tR 1.7 min in 4% water-methanol); 1H NMR (400 MHz, DMSO- d6): δ 11.3 (1 H, s, NH), 10.8 (1 H, s, NH), 7.71 (2H, d, J = 8.2 Hz, ArH), 7.41 (1 H, m, ArH), 7.38 (3H, m, ArH), 7.07 (1 H, d, J = 8.6 Hz, ArH), 6.08 (1H, s, ArH), 3.79 (3H, s, OCH3), 3.74 (3H, s, OCH3), 2.28 (3H, s, CH3); FAB-MS 454 (MH+); FAB-HRMS calcd for Ci 8H20N3O7S2 (MH+) 454.0743, found 454.0746. N-f4-(5-Methyl-isoxazol-3-ylsulfamoyl)-phenvn-2'.3',6'-trimethyl-4'-methoxy- benzenesulfonamide (STX642, XDS01106)
White crystalline solid. TLC single spot at Rf 0.60 (10% methanol-DCM); HPLC purity as rotational isomers > 99% (tR 1.8 min in 4% water-methanol); 1H NMR (400 MHz, DMSO- d6): δ 11.3 (1 H, s, NH), 10.9 (1H, s, NH), 7.67 (2H, d, J = 9.0 Hz, ArH), 7.04 (2H, d, J = 9.0 Hz, ArH), 6.83 (1 H, s, ArH), 6.06 (1 H, s, ArH), 3.81 (3H, s, OCH3), 2.64 (3H, s, CH3), 2.27 (3H, s, CH3), 2.03 (3H, s, CH3); FAB-MS 466 (MH+); FAB-HRMS calcd for C20H24N3O6S2 (MH+) 466.1107, found 466.1109.
N-r4-(5-Methyl-isoxazol-3-ylsulfamoyl)-phenvn-2'-methyl-3'-chlorobenzenesulfonamide (STX643. XDS01107)
White crystalline solid. TLC single spot at Rf 0.56 (10% methanol-DCM); HPLC purity as rotational isomers > 99% (tR 2.0 min in 4% water-methanol); 1H NMR (400 MHz, DMSO- d6): δ 11.3 (2H, s, NH), 7.97 (1 H, d, J = 8.2 Hz, ArH), 7.75 (1 H, d, J = 8.2 Hz, ArH), 7.71 (2H, d, J = 8.6 Hz, ArH), 7.43 (1 H, t, J = 8.2 Hz, ArH), 7.21 (2H, m, ArH), 6.08 (1H, s, ArH), 2.62 (3H, s, CH3), 2.28 (3H, s, CH3); FAB-MS 442 (MH+); FAB-HRMS calcd forCi7Hi7CIN3θ5S2 (MH+) 442.0298, found 442.0297.
N-r4-(5-Methyl-isoxazol-3-ylsulfamoyl)phenvn-2',4'-dichlorobenzenesulfonamide (STX644. XDS01108)
White crystalline solid. TLC single spot at Rf 0.60 (10% methanol-DCM); HPLC purity as rotational isomers > 99% (tR 2.2 min in 10% water-methanol); 1H NMR (400 MHz, DMSO-d6): δ 11.5 (1 H, s, NH), 11.3 (1 H, s, NH), 8.12 (1 H, d, J = 8.5 Hz, ArH), 7.88 (1 H, s, ArH), 7.72 (2H, d, J = 8.6 Hz, ArH), 7.65 (1 H, d, J = 8.5 Hz, ArH), 7.23 (2H, d, J = 8.6 Hz, ArH), 6.08 (1 H, s, ArH), 2.29 (3H, s, CH3); FAB-MS 462 (MH+); FAB-HRMS calcd for C16H14CI2N3O5S2 (MH+) 461.9752, found 461.9756. 3-Chloro-4-(4-methoxybenzenesulfonylamino)-N-(5-methylisoxazol-3-yl)- benzenesulfonamide (STX728. XDS01120)
Off-white solid. TLC single spot at Rf 0.64 (30% ethyl acetate-DCM); HPLC purity > 99% (tR 1.8 min in 10% water-methanol); 1H NMR (400 MHz, DMSO-d6): δ 11.5 (1 H, s, NH), 10.4 (1 H, s, NH), 7.80 (1 H, d, J = 2.0 Hz, ArH), 7.70-7.76 (3H, m, ArH), 7.52 (1 H, d, J = 8.0 Hz, ArH), 7.08 (2H, d, J = 8.0 Hz, ArH), 6.13 (1 H, s, ArH), 3.82 (3H, s, OCH3), 2.30 (3H, s, CH3); FAB-MS 458 (MH+); FAB-HRMS calcd for C17H17CIN3O6S2 (MH+) 458.0247, found 458.0245.
3-Ch!oro-4-(34-dimethoxybenzenesulfonv!amino)-N-(5-methylisoxazol-3-vD- benzenesulfonamide (STX729, XDS01121)
White crystalline solid. TLC single spot at Rf 0.49 (25% ethyl acetate-DCM); HPLC purity as rotational isomers > 99% (tR 1.8 min in methanol); 1H NMR (270 MHz, DMSO- d6): δ 11.5 (1 H, s, NH), 10.3 (1 H, s, NH), 7.77 (1H, d, J = 2.1 Hz, ArH), 7.69 (1 H, dd, J = 8.2, 2.1 Hz ArH), 7.48 (1 H, d, J = 8.2 Hz, ArH), 7.36 (1 H, dd, J = 8.6, 2.2 Hz, ArH), 7.23 (1 H, d, J = 2.2 Hz, ArH), 7.05 (1 H, d, J = 8.6 Hz, ArH), 6.07 (1H, s, ArH), 3.77 (3H, s, OCH3), 3.67 (3H, s, OCH3), 2.25 (3H, s, CH3); FAB-MS 488 (MH+); FAB-HRMS calcd for CiδHi9ClN3θ7S2 (MH+) 488.0353, found 488.0360.
Synthesis of pyrazolyl arysulphonamides and benzopyrazole arysulphonamides
STX769, XDS01152 STX880, XDS01174 STX881 , XDS01175C STX883, XDS01178A STX882, XDS01075B
STX829, XDS01162A
STX826, XDS01159
General Method for synthesis of arylsulphonamide and N-arylsulphony arylsulphonamide (STX769. STX829-830, STX880-884)
To a solution arylsulphonyl chloride (1.05 eq.) in DCM were added pyridine (2.1 eq.) and the amine (1 eq.). The reaction mixture was stirred at rt or 40°C under nitrogen for 4-14 h, then partitioned between ethyl acetate and 5% sodium bicarbonate solution 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 that was separated by flash chromatography (ethyl acetate-DCM gradient elution) to give arylsulphonamide and N-arylsulphonyl arylsulphonamide as white or off-white solid (Yield 30-80%). N-(2-N-ethyl-2H-pyrazol-3-yl)-N-(4-n-propylphenylsulphonyl)-4-n- propylbenzenesulfonamide (STX769, XDS01152)
White powder. TLC single spot at Rf 0.71 (10% ethyl acetate-DCM); HPLC purity 99% (tR 4.1 min in 10% water-methanol); 1H NMR (400 MHz, DMSO-d6): δ 7.62-7.66 (4H, m, ArH), 7.56 (1 H, d, J = 2.3 Hz, ArH), 7.47-7.50 (4H, m, ArH), 5.93 (1H, d, J = 2.2 Hz, ArH), 3.66 (2H, q, J = 7.0 Hz, NCH2), 2.68 (4H, t J = 7.8 Hz, CH2) 1.63(4H, m, 2 x CH2), 1.18 (3H, t, J = 7.0 Hz, CH3), 0.90 (6H, t, J = 7.6 Hz, 2 x CH3); FAB-MS 476 (MH+); FAB- HRMS calcd for C23H30N3O4S2 (MH+) 476.1678, found 476.1682
N-(2.5-dimethyl-2H-pyrazol-3-yl)-3-Chloro-2-methylbenzenesulfonamide (STX880.
XDS01174)
White crystalline solid. TLC single spot at Rf 0.76 (10% methanol-DCM); HPLC purity as rotational isomers 96% (tR 2.0 min in 20% water-methanol); 1H NMR (270 MHz, DMSO- d6): δ 7.75 (2H, d, J = 8.2 Hz, ArH), 7.37 (1 H, d, J = 8.1 Hz, ArH), 5.49 (1 H, s, ArH), 3.48 (3H, s, CH3), 2.61 (3H, s, CH3), 1.98 (3H, s, CH3); APCI-MS 300 (MH+); FAB-HRMS calcd for C12H15CIN3O2S (MH+) 300.0573, found 300.0572
N-(1-Ethyl-1 H-pyrazol-3-yl)-3-Chloro-2-methylbenzenesulfonamide (STX881.
XDS01175C)
White solid. TLC single spot at Rf 0.80 (10% methanol-DCM); HPLC purity as rotational isomers 95% (tR 2.0 min in 20% water-methanol); 1H NMR (270 MHz, DMSO-d6): δ 10.5 (1 H, s, NH), 7.71 (1 H, d, J = 8.1 Hz, ArH), 7.68 (1 H, d, J = 8.1 Hz, ArH), 7.32 (1 H, t, J = 8.1 Hz, ArH), 7.24 (1 H, broad s, ArH), 5.57 (1 H, d, J = 1.8 Hz, ArH), 3.88 (2H, q, J = 7.3 Hz, CH2), 2.57 (3H, s, CH3), 1.10 (3H, t, J = 7.3 Hz, CH3); APCI-MS 300 (MH+); FAB- HRMS calcd for C12H15CIN3O2S (MH+) 300.0573, found 300.0583
N-(1-Ethyl-1 H-pyrazol-3-yl)-N-(3-chloro-2-methylphenylsulphonyl)-3-chloro-2- methylbenzenesulfonamide (STX882. XDS01175B)
Colorless oil. TLC single spot at Rf 0.82 (20% ethyl acetate-DCM); HPLC purity 98% (tR
4.5 min in 20% water-methanol); 1H NMR (270 MHz, CDCI3): δ 7.95 (2H, d, J = 8.1 Hz, ArH), 7.70 (2H, d, J = 8.1 Hz, ArH), 7.56 (1 H, d, J = 2.1 Hz, ArH), 7.32 (2H, t, J = 8.1 Hz, ArH), 6.21 (1H, d, J = 1.8 Hz, ArH), 3.71 (2H, q, J = 7.3 Hz, CH2), 2.47 (6H, s, 2 x CH3), 1.28 (3H, t, J = 7.3 Hz, CH3); APCI-MS 488 (MH+); FAB-HRMS calcd for C19H20CI2N3O4S2 (MH+) 488.0272, found 488.0263
5-(N-3-Chloro-2-methylphenylsulphonyl-r3-Chloro-2-methyl-benzenesulfonylaminol)-1- methy -1 H-pyrazole-4-carboxylic acid ethyl ester (STX883. XDS01178A)
White crystalline solid. TLC single spot at Rf 0.81 (20% ethyl acetate-DCM); HPLC purity 93% (tR 2.2 min in 15% water-methanol); 1H NMR (270 MHz, CDCI3): δ 8.09 (2H, dd, J = 8.1 , 1.5 Hz, ArH), 7.95 (1 H, s, ArH), 7.63 (2H, dd, J = 8.1 , 1.3 Hz, ArH), 7.30 (2H, t, J = 8.1 Hz, ArH), 3.88 (2H, q, J = 7.0 Hz, CH2), 3.54 (3H, s, CH3), 2.29 (6H, s, 2 x CH3), 0.96 (3H, t, J = 7.3 Hz, CH3); FAB-MS 546 (MH+); FAB-HRMS calcd for C21H22CI2N3O6S2 (MH+) 546.0327, found 546.0320
5-(3-Chloro-2-methyl-benzenesulfonylamino)-1 -methyl-1 H-pyrazole-4-carboxylic acid ethyl ester (STX884. XDS01178B)
Off-white solid. TLC single spot at Rf 0.49 (20% ethyl acetate-DCM); HPLC purity 91% (tR 2.0 min in 20% water-methanol); 1H NMR (270 MHz, CDCI3): δ 7.60 (1H, s, ArH), 7.58 (1 H, dd, J = 7.9, 2.0 Hz, ArH), 7.50 (1H, dd, J = 8.0, 2.0 Hz, ArH), 7.09 (1 H, t, J = 8.1 Hz, ArH), 3.91 (3H, s, CH3), 3.90 (2H, q, J = 7.0 Hz, CH2), 2.50 (3H, s, CH3), 1.05 (3H, t, J = 7.0 Hz, CH3); APCI-MS 358 (MH+); FAB-HRMS calcd for C14H17CIN3O4S (MH+) 358.0628, found 358.0640
3-Amino-1-(3-chloro-2-methylbenzenesulfonyl)-1 H-pyrazole-4-carboxylic acid ethyl ester (STX829, XDS01162A)
White crystalline solid. TLC single spot at Rf 0.25 (DCM); HPLC purity > 99% (tR 2.0 min in 4% water-methanol); 1H NMR (270 MHz, DMSO-d6): δ 8.62 (1H, s, ArH), 7.95 (1H, d, J = 8.2 Hz, ArH), 7.86 (1H, d, J = 8.2 Hz, ArH), 7.48 (1H, t, J = 8.2 Hz, ArH), 5.95 (2H, s, NH2), 4.17 (2H, q, J = 7.0 Hz, CH2), 2.49 (3H, s, CH3), 1.21 (3H, t, J = 7.0 Hz, CH3); APCI-MS 344 (MH+); FAB-HRMS calcd for C13H-15CIN3O4S (MH+) 344.0472, found 344.0477 5-Amino-1-(3-chloro-2-methyl-benzenesulfonyl)-1H-pyrazole-4-carboxylic acid ethyl ester (STX830, XDS01162B)
Off-white crystalline solid. TLC single spot at Rf 0.30 (DCM); HPLC purity 91% (tR 2.0 min in 4% water-methanol); 1H NMR (270 MHz, DMSO-d6): δ 7.97 (1 H, d, J = 8.0 Hz, ArH), 7.87 (1H, d, J = 8.0 Hz, ArH), 7.77 (1 H, s, ArH), 7.50 (1 H, t, J = 8.0 Hz, ArH), 7.03 (2H, s, NH2), 4.14 (2H, q, J = 6.9 Hz, CH2), 2.50 (3H, s, CH3), 1.18 (3H, t, J = 6.9 Hz, CH3); FAB-MS 344 (MH+); FAB-HRMS calcd for C13H15CIN3O4S (MH+) 344.0472, found 344.0472
N-[1-(3-Chloro-2-methylbenzenesulfonyl)-1 H-indazol-5-yll-3-chloro-2- methylbenzenesulfonamide (STX826, XDS01159)
Off-white foam. TLC single spot at Rf 0.65 (10% ethyl acetate-DCM); HPLC purity 96% (tR 2.2 min in 15% water-methanol); 1H NMR (270 MHz, DMSO-d6): δ 10.7 (1H, s, NH), 9.01 (1 H, s, 3-H), 8.05 (1H, d, J = 8.1 Hz, ArH), 7.84 (1 H, d, J = 8.0 Hz, ArH), 7.80 (1 H, d, J = 8.0 Hz, ArH), 7.62 (1 H, d, J = 8.0 Hz, ArH), 7.51 (1 H, d, J = 8.0 Hz, ArH), 7.49 (1H, t, J = 8.0 Hz, ArH), 7.29 (1 H, t, J = 8.0 Hz, ArH), 7.24 (1 H, d, J = 1.1 Hz, ArH),. 7.11 (1 H, dd, J = 8.0, 1.2 Hz, ArH), 2.36 (6H, s, 2 x CH3); APCI-MS 510 (MH+); FAB-HRMS calcd for C21 H18CI2N3O4S2 (MH+) 510.0116, found 510.0106
3-Chloro-N-(1 H-indazol-5-yl)-2-methyl-benzenesulfonamide (STX983. XDS02021 B)
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-d6): δ 10.4 (1 H, s, NH),
7.99 (1 H, s, 3-H), 7.79 (1 H, d, J = 7.9 Hz, ArH), 7.68 (1H, d, J = 7.9 Hz, ArH), 7.43 (1 H, d, J = 8.9 Hz, ArH), 7.41 (1 H, s, ArH), 7.32 (1 H, t, J = 8.0 Hz, ArH), 7.08 (1 H, d, J = 8.9
Hz, ArH),. 2.64 (3H, s, CH3); APCI-MS 322 (MH+); FAB-HRMS calcd for
C14H-13CIN3O2S (MH+) 322.0417, found 322.0417
3-Chloro-2-methyl-N-(1-methyl-1 H-indazol-5-yl)-benzenesulfonamide (STX988.
XDS02037)
Off-white solid. TLC single spot at Rf 046 (7% methanol-DCM); HPLC purity 96% (t 2.2 min in 10% water-methanol); 1H NMR (270 MHz, DMSO-d6): δ 10.4 (1 H, s, NH), 7.95 (1 H, s, 3-H), 7.78 (1H, d, J = 7.9 Hz, ArH), 7.68 (1H, d, J = 7.9 Hz, ArH), 7.52 (1 H, d, J - 9.0 Hz, ArH), 7.40 (1 H, J = 2.0 Hz, ArH), 7.31 (1H, t, J = 7.9 Hz, ArH), 7.12 (1 H, dd, J = 9.0, 2.0 Hz, ArH),. 3.97 (3H, s, NCH3), 2.65 (3H, s, CH3); APCI-MS 336 (MH+); FAB-HRMS calcd for Ci5Hi5CIN3θ2S (MH+) 336.0573, found 336.0575
Synthesis of benzamides and arysulphonamides
GS03076A
STX576, DGS03094A STX577, DGS03096A
STX770, XDS01153
N-(2-Methylbenzothiazol-5-yl)-4-propylbenzamide (STX471. DGS03074B)
To a solution of 5-amino-2-methylbenzothiazole (150 mg, 0.91 mmol) in THF (1 mL) was added triethylamine (5 mL). After stirring at rt for 15 min, 4-propylbenzoyl chloride(200 mg, 1.09 mmol) was added. The mixture was kept stirring at rt for 1 h, extracted with ethyl acetate. The organic phase was washed with brine, dried over sodium sulphate and concentrated in vacuo to yield a white solid that was recrystallized from ethyl acetate to give white needles (216 mg, 76%). mp 148-149°C; TLC single spot at Rf 0.56 (60% ethyl acetate-hexane); HPLC purity 99% (tR 3.1 min in 10% water-methanol); 1H NMR (400 MHz, DMSO-d6): δ 10.3 (1 H, s, NH), 841 (1 H, d, J = 2.0 Hz, ArH), 7.88-7.96 (3H, m, ArH), 7.50 (1 H, dd, J = 9.0, 2.3 Hz, ArH), 7.33-7.36 (1 H, m, ArH), 2.78 (3H, s, CH3), 2.63 (2H, t J = 7.4 Hz, CH2) 1.62(2H, m, CH2), 0.98 (3H, t, J = 7.4 Hz, CH3); FAB-MS 311 (MH+); FAB-HRMS calcd for C18H19N2OS (MH+) 311.1218, found 311.1227.
3,5-Dichloro-N-(2-methylbenzothiazol-5-yl)-benzamide (STX520, DGS03080A)
STX520 was synthesized as described for STX471. White crystalline solid (166 mg, 54%) was obtained, mp 194°C; TLC single spot at Rf 0.76 (60% ethyl acetate-hexane); HPLC purity 97% (tR 1.6 min in 4% water-methanol); 1H NMR (400 MHz, DMSO-d6): δ 10.6 (1 H, s, NH), 8.40 (1H, d, J = 2.0 Hz, ArH), 7.99-8.02 (3H, m, ArH), 7.89-7.90 (1H, m, ArH), 7.74 (1H, dd, J = 8.9, 2.3 Hz, ArH), 2.80 (3H, s, CH3); FAB-MS 337 (MH+); FAB-HRMS calcd for C15H11CI2N2OS (MH+) 336.9969, found 336.9972.
N-Methyl-N-(2-methylbenzothiazol-5-yl)-4-propylbenzamide (STX472, DGS03076A)
To a solution of N-(2-methylbenzothiazol-5-yl)-4-propylbenzamide (130 mg, 0.4 mmol) in DMF ( 5 mL) was added sodium hydride (17 mg, 0.44 mmol), followed by methyl iodide (85 mg, 059 mmol). The mixture was stirred at rt overnight, partitioned between water and ethyl acetate. The organic phase was washed with brine, dried over sodium sulphate and concentrated to give a residue that wad purified with flash chromatography (ethyl acetate-hexane gradient elution). A thick syrup (41 mg, 26% was obtained. 1H NMR (400 MHz, CDCI3): δ 7.61-7.79 (3H, m, ArH), 7.22-7.26 (2H, m, ArH), 6.92-7.02 (2H, m, ArH), 3.54 (3H, s, CH3). 2.81 (3H, s, CH3), 2.45 (2H, t J = 7.4 Hz, CH2) 1.52(2H, m, CH2), 0.83 (3H, t, J = 7.4 Hz, CH3); FAB-MS 325 (MH+); FAB-HRMS calcd for C19H21 N2OS (MH+) 325.1375, found 325.1992.
General Method for synthesis of arylsulphonamide (. STX576-577, . STX702, STX770)
To a solution of amine (1 eq.) in DMF was added Et3N (5 eq.), followed by corresponding sulphonyl chloride (1.2 eq.). The reaction mixture was stirred at rt under N2 overnight, poured into water after TLC showed completion of the reaction, and extracted with ethyl acetate, dried (MgS04), 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. Yield 20-80%. 4-Propyl-N-[1-(4-propylbenzenesulfonyl)-piperidin-4-ylmethyl1-benzenesulfonamide (STX576. DGS03094A)
White solid, mp 158-159°C; TLC single spot at Rf 0.59 (20% ethyl acetate-DCM); HPLC purity 99% (tR 2.0 min in 4% water-methanol); 1H NMR (400 MHz, DMSO-d6): δ 7.60- 7.66 (4H, m, ArH), 7.55 (1 H, broad, NH), 7.35-7.45 (4H, m, ArH), 3.55-3.58 (2H, dd, J = 7.2, 2.3 Hz, CH2), 2.56-2.67 (6H, m, 3 x CH2), 2.04-2.10 (2H, m, CH2), 1.54-1.65 (6H, m, 3 x CH2), 1.23 (1 H, m, CH), 1.02-1.11 (2H, m, CH2), 0.85-0.89 (6H, m, 2 x CH3); FAB- MS 479 (MH+).
N-(6-Methoxypyridin-3-yl)-4-propylbenzenesulfonamide (STX577. DGS03096A)
White solid, mp 93-94°C; TLC single spot at Rf 0.54 (20% ethyl acetate-DCM); HPLC purity 99% (tR 2.0 min in 4% water-methanol); 1H NMR (400 MHz, DMSO-d6): δ 10.0 (1 H, s, NH), 7.77 (1 H, d, J = 2.7 Hz, ArH), 7.56-7.59 (2H, m, ArH), 7.35-7.38 (3H, m, ArH), 6.72 (1H, m, ArH), 3.75 (3H, s, OCH3), 2.59 (2H, t, J = 7.4 Hz, CH2), 1.61 (2H, m, CH2), 0.85 (3H, t, J = 74 Hz, CH3); FAB-MS 307 (MH+); FAB-HRMS calcd for C15H-19N2O3S (MH+) 307.1048, found 307.1061.
4-Propyl-N-(pyridin-2-yl-methyl)-benzenesulfonamide (STX702. DGS03132A)
White crystalline solid, mp 109°C; TLC single spot at Rf 0.50 (20% ethyl acetate-DCM); HPLC purity > 99% (tR 1.8 min in 4% water-methanol); 1H NMR (400 MHz, DMSO-d6): δ 8.41 (1 H, m, ArH), 8.19 (1 H, s, NH), 7.66-7.72 (3H, m, ArH), 7.31-7.37 (3H, m, ArH), 7.22 (1 H, m, ArH), 4.06 (2H, s, CH2), 2.61 (2H, t, J = 7.0 Hz, CH2), 1.60 (2H, m, CH2), 0.88 (3H, t, J = 7.4 Hz, CH3); FAB-MS 291 (MH+); FAB-HRMS calcd for C15H19N2O2S (MH+) 291.1167, found 291.1164. N-(2,6-Dimethoxypyridin-3-yl)-4-propylbenzenesulfonamide (STX770, XDS01153)
Off-white crystalline solid. TLC single spot at Rf 0.37 (30% ethyl acetate-hexane); HPLC purity 98% (tR 2.7 min in 10% water-methanol);1H NMR (400 MHz, DMSO-d6): δ 9.39
(1 H, s, NH), 748-7.54 (2H, m, ArH), 7.42 (1 H, d, J = 8.8 Hz, ArH), 7.33 (2H, d, J = 8.6 Hz, ArH), 6.30 (1H, d, J = 8.8 Hz, ArH), 3.76(3H, s, OCH3), 345(3H, s, OCH3), 2.60 (2H, t, J = 7.8 Hz, CH2), 1.55 (2H, sextet, J = 7.8 Hz, CH2), 0.88 (3H, t, J = 7.8 Hz, CH3); FAB-MS 291 (MH+); FAB-HRMS calcd for C16H21N204S (MH+) 337.1222, found 337.1222.
Synthesis of N-Benzimidazole Aryl Amide Derivatives
STX1129, JWS01109 STX 1130, JWS01111
STX1132, JWS01116 STX1134, XDS02113 STX1133. XDS02112
General method for benzamide formation:
To a solution of substituted benzoyl chloride (1.2 eq.) in THF-triethylamine (1 : 4) was added the corresponding amine (1 eq.). The reaction mixture was stirred at rt under nitrogen for 16 h, partitioned between DCM 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 N-benzimidazole aryl amide as crystalline solid. Yield ranges from 60-80%.
Biphenyl-4-carboxylic acid (1 ,2-dimethyl-3H-benzoimidazol-6-yl)-amide (STX1129. JWS01109)
Off-white solid. Mp 270-270.3°C; TLC single spot at Rf 0.52 (10% methanol/DCM); HPLC purity 98% (tR 2.2 min in 20 % water-methanol); 1H NMR (400 MHz, DMSO): δ 10.3 (1H, s, NH), 8.05-8.10 (3H, m, ArH), 7.82 (2H, d, J = 8.2 Hz, ArH), 7.75 (2H, dd, J = 8.1 , 1.2 Hz, ArH), 7.38-7.52 (5H, m, ArH), 3.70 (3H, s, NCH3), 2.52 (3H, s, CH3); FAB- MS 342 (MH+); FAB-HRMS calcd for C22H2θN3θ (MH+) 342.1606, found 342.1615
Biphenyl-4-carboxylic acid (1 ,2-dimethyl-3H-benzoimidazol-5-yl)-amide (STX1130. JWS01112)
Off-white solid. Mp 272-274°C; TLC single spot at Rf 0.52 (10% methanol/DCM); HPLC purity 98% (tR 2.2 min in 20% water-methanol); 1H NMR (400 MHz, DMSO): δ 10.2 (1H, s, NH), 8.06 (2H, d, J = 8.2 Hz, ArH), 7.95 (1 H, d, J = 2.0 Hz, ArH), 7.82 (2H, d, J = 8.2 Hz, ArH), 7.74 (2H, dd, J = 8.2, 1.9 Hz, ArH), 748-7.56 (5H, m, ArH), 3.72 (3H, s, NCH3), 2.52 (3H, s, CH3); FAB-MS 342 (MH+); FAB-HRMS calcd for C22H20N3O (MH+) 342.1606, found 342.1611
N-(1.2-Dimethv!-1 H-benzoimidazol-5-yl)-4-propylbenzamide (STX1131. JWS01113)
Off-white solid. Mp 214-218°C; TLC single spot at Rf 0.43 (10% methanol/DCM); HPLC purity > 99% (tR 2.2 min in 20% water-methanol); 1H NMR (400 MHz, DMSO): δ 10.1 (1 H, s, NH), 7.95 (1 H, d, J = 1.5 Hz, ArH), 7.87 (2H, d, d = 8.2 Hz, ArH), 7.51 (1 H, dd, J = 8.5, 2.0 Hz, ArH), 7.39 (1 H, d, J = 8.2 Hz, ArH), 7.32 (2H, d, J = 8.5 Hz, ArH), 3.70 (3H, s, NCH3), 2.63 (2H, t, J = 7.1 Hz, CH2), 2.50 (3H, s, CH3), 1.62 (2H, sextet, J = 7.2 Hz, CH2), 0.91 (3H, t, J = 7.3 Hz, CH2); FAB-MS 308 (MH+); FAB-HRMS calcd for C19H22N3O (MH+) 308.1763, found 308.1778
2.4-Dichloro-N-(1 ,2-dimethyl-1 H-benzoimidazol-5-v!)-benzamide (STX1132. JWS01116)
light yellow solid. Mp 275-277°C; TLC single spot at Rf 0.43 (10% methanol/DCM); HPLC purity > 99% (tR 2.0 min in 10% water-methanol); 1H NMR (400 MHz, DMSO): δ 10.4 (1H, s, NH), 7.92 (1 H, d, J = 2.0 Hz, ArH), 7.74 (1 H, d, J = 1.9 Hz, ArH), 7.62 (1 H, d, J = 8.2 Hz, ArH), 7.53 (1 H, dd, J = 8.2, 2.0 Hz, ArH), 7.39-7.42 (2H, m, ArH), 3.70 (3H, s, NCH3), 2.50 (3H, s, CH3); FAB-MS 334 (MH+); FAB-HRMS calcd for CI6H14CI2N3O (MH+) 334.0514, found 334.0517 Biphenyl-4-carboxylic acid (1 ,2-dimethyl-3H-benzoimidazol-5-yl)-N-methyl-amide (STX1133. XDS02112)
White crystals. Mp 213-214.5°C; TLC single spot at R, 0.79 (10% methanol/DCM); HPLC purity 99% (tR 2.2 min in 20% water-methanol); 1H NMR (270 MHz, DMSO): δ 7.58 (2H, m, ArH), 7.30-7.50 (9H, m, ArH), 7.07 (1 H, dd, J = 8.1 , 1.9 Hz, ArH), 3.65 (3H, s, NCH3), 3.29 (3H, s, NCH3), 2.45 (3H, s, CH3); APCI-MS 356 (MH+)
2.4-Dichloro-N-(1 ,2-dimethyl-1 H-benzoimidazol-5-yl)-N-methyl-benzamide (STX1134. XDS02113)
White crystals. Mp 245-247°C; TLC single spot at Rf 0.70 (10% methanol/DCM); HPLC purity > 99% (tR 2.2 min in 10% water-methanol); 1H NMR (270 MHz, DMSO): δ 7.44 (1 H, d, J = 2.0 Hz, ArH), 7.41 (1 H, broad, W1/2 = 1.7 Hz, ArH), 7.39 (1 H, d, J = 8.2 Hz, ArH), 7.35 (1 H, d, J = 8.5 Hz, ArH), 7.22 (1 H, dd, J = 8.2, 1.9 Hz, ArH), 7.09 (1 H, dd, J = 8.5, 1.9 Hz, ArH), 3.64 (3H, s, NCH3), 3.39 (3H, s, NCH3), 2.45 (3H, s, CH3); APCI-MS 348 (MH+)
The compounds shown in the following table were synthesised in the manner described.
Synthesis of N-(2-thiopheneethyI)-benzamide derivatives
STX1377, CCM01015 STX1378, CCM01016 STX1379, CCM0101
STX1380, CCM01018 STX1381. CCM01020 STX1382, CCM01021
General method for synthesis of N-(2-thiopheneethyl)-benzamide derivatives (STX1377-1382): Method A : to a stirred solution of the amine (n mmol) in THF are added triethylamine (l.2n mmol) and the acyl chloride (l.2n mmol) at room temperature. After completion, ethyl acetate and water are added. The aqueous layer is extracted by ethyl acetate. The combined organic layers are washed with brine, dried (MgSO4), filtered and evaporated under reduce pressure. The crude product is then purified to give the amide. Method B : A solution of the acid (3n mmol) in thionyl chloride is refluxed 3 hours. Thionyl chloride is then removed under reduced pressure. The crude product is diluted in dry THF and added to a solution of the amine (n mmol) and triethylamine in THF. After completion, ethyl acetate and water are added . The aqueous layer is extracted by EtOAc. The combined organic layers are then washed with brine, dried (MgSO4), filtered and evaporated under reduce pressure. The crude product is purified to give the amide. Biphenyl-4-carboxylic acid (2-thiophen-2-yl-ethyl -amide (STX1377. CCM01015 Reaction of 2-thiopheneethylamine (50 μL, 0.42 mmol) in THF (2.5 mL) with 4- biphenylcarbonyl chloride (120 mg, 0.55 mmol) in presence of triethylamine (78 μL, 0.55 mmol) according to method A gave biphenyl-4-carboxylic acid (2-thiophen-2-yl-ethyl)- amide (70 mg, 0.23 rrrmol, 55% yield) as a grey powder after crystallisation in hexane/EtOAc.
Rf : 0.4 (EtOAc/DCM 5/95) ; M.p.:164-166 °C ; 1H NMR (270MHz, CDC13) δH 3.16 (t, 2H, J = 6.6 Hz, CH2Ar), 3.71-3.77 (m, 2H, CH2NH), 6.27 (bs, IH, NH), 6.88 (bs, IH, HAT), 6.95-6.98 (m, IH, HAT), 7.17 (d, IH, J = 4.7 Hz, HArthiophene), 7.36-7.47 (m, 3H, HAΓ), 7.57-7.64 (m, 4H, HAT), 7.77 (d, 2H, J - 8.1 Hz, HAT); 13C NMR (400MHz, CDC13) δc 30.0 (CH2), 41.4 (CH2), 124.2 (CHAΓ), 125.6 (CHAT), 127.2 (2*CHAT), 127.3 (CHAT), 127.4 (CHAT), 128.0 (CH r), 129.0 (CHAT), 133.2 (Cq), 140.0 (Cq), 141.3 (Cq), 144.3 (Cq), 167.7 (C=O); LC/MS (AP-) m/z 305.8 (M-H); tR = 2.3 min (99.6%); HRMS (FAB+) Calculated for C19H17NOS 307.1031; Found 307.1016
3.5-Dichloro-N-(2-thiophen-2-yl-ethylVbenzamide (STX1378, CCM01016
Reaction of 2-thiopheneethylamine (50 μL, 0.42 mmol) in THF (2.5 mL) with 3,5- dichlorobenzoyl chloride (120 mg, 0.57 mmol) in presence of triethylamine (78 μL, 0.55 mmol) according to method A gave 3,5-dichloro-N-(2-tl iophen-2-yl-ethyl)-benzamide (118 mg, 0.39 mmol, 92% yield) as a white powder after purification by chromatography on silica gel (eluent : EtOAc/hexane 2/8).
Rf : 0.3 (EtOAc/hexane 2/8); M.p.:100-102 °C; 1H ΝMR (270MHz, CDC13) δH 3.13 (t, 2H, J = 6.4 Hz, CH2-Ar), 3.69 (dt, 2H, J = 6.4, 6.4 Hz, CH2ΝH), 6.24 (bs, IH, NH), 6.85 (dd, IH, J = 1.0, 3.5 Hz, HArthiophene), 6.96 (dd, IH, J = 3.5, 5.2 Hz, HArthiophene), 7.18 (dd, IH, J = 1.0, 5.2 Hz, HArthiophene), 7.45 (t, IH, J = 2.0 Hz, HAT), 7.56 (d, 2H, J = 2.0 Hz, HAT); LC/MS (AP-) m/z 297.8 (M-H); tR = 2.5 min (99.3%); HRMS (FAB+) Calculated for C13HπCl2NOS 298.9938; Found 298.9934 4-Methoxy-N-(2-tMophen-2-yl-ethylVbenzamide (STX1379. CCM01017 Reaction of 2-thiopheneethylamine (50 μL, 0.42 mmol) in THF (2.5 mL) with -anisoyl chloride (74 μL, 0.57 mmol) in presence of triethylamine (78 μL, 0.55 mmol) according to method A gave 4-methoxy-N-(2-thiophen-2-yl-ethyl)-benzamid (105 mg, 0.40 mmol, 95% yield) as a white powder after purification by chromatography on silica gel (eluent : EtOAc/hexane 5/95 to 20/80).
Rf : 0.4 (EtOAc/DCM 1/9) ; M.p.: 109-111 °C ; 1H ΝMR (270MHz, CDC13) δH 3.13 (t, 2H, J = 6.4 Hz, CH2Ar), 3.69 (dt, 2H, J = 6.4, 6.4 Hz, CH2ΝH), 3.82 (s, 3H, OCH3), 6.19 (bs, IH, NH), 6.85-6.90 (m, 3H, HAT), 6.94 (dd, IH, J = 3.5, 5.4 Hz, HArthiophene), 7.16 (dd, IH, J = 1.2, 5.4 Hz, HArthiophene), 7.65-7.70 (m, 2H, HAT); LC/MS (AP-) m/z 259.9 (M-H); tR = 2.0 min (95.4%); HRMS (FAB+) Calculated for C14H15N02S 261.0824; Found 261.0826
3-Methoxy-N-(2-thiophen-2-yl-ethvD-benzamide (STX1380. CCM01018 Reaction of 2-thiopheneethylamine (50 μL, 0.42 mmol) in THF (2.5 mL) with m-anisoyl chloride (74 μL, 0.57 mmol) in presence of triethylamine (78 μL, 0.55 mmol) according to method A gave 3-methoxy-N-(2-fhiophen-2-yl-ethyl)-benzamide (100 mg, 0.38 mmol, 90% yield) as a yellow wax after purification on silica gel (eluent : EtOAc/hexane 5/95 to 20/80). Rf : 0.3 (EtOAc/DCM 5/95); *H ΝMR (270MHz, CDC13) δH 3.13 (t, 2H, J = 6.4 Hz, CH2Ar), 3.70 (dt, 2H, J = 6.4, 6.4 Hz, CH2ΝH), 3.81 (s, 3H, OCH3), 6.27 (bs, IH, NH), 6.85 (dd, IH, J = 1.2, 3.4 Hz, HArthiophene), 6.94 (dd, IH, J = 3.5, 5.2 Hz, HArthiophene), 7.00 (ddd, IH, J =1.3, 2.8, 8.2 Hz, HAT), 7.16 (dd, IH, J = 1.2, 5.2. Hz, HArthiophene), 7.19 (ddd, IH, J = 1.3, 1.3, 7.7 Hz, HAT), 7.24-7.32 (m, 2H, HAT); LC/MS (AP-) m/z 259.9 (M-H), tR = 2.0 min (99.5%); HRMS (FAB+) Calculated for C14H15N02S 261.0824; Found 261.0827
4-Propyl-N-r2-thiophen-2-yl-ethyl -benzamide (STX1381. CCM01020 Reaction of 2-thiopheneethylamine (50 μL, 0.42 mmol) in THF (2.5 mL) with 4- propylbenzoyl chloride (92 μL, 0.55 mmol) in presence of triethylamine (78 μL, 0.55 mmol) according to method A gave 4-propyl-N-(2-thiophen-2-yl-ethyl)-benzamide (100 mg, 0.36 mmol, 85% yield) as a off- white powder after purification on silica gel (eluent : EtOAc/hexane 5/95 to 20/80).
Rf : 0.4 (EtOAc DCM 5/95) ; M.p.: 97-99 °C ; 1H NMR (270MHz, CDC13) δH 0.93 (t, 3H, J = 7.4 Hz, CH3), 1.55-1.69 (m, 2H, CH2-CH3), 2.60 (t, 2H, J = 7.4 Hz, CH2Ar), 3.13 (t, 2H, J = 6.4 Hz, CH2thiophene), 3.70 (dt, 2H, J = 6.4, 6.4 Hz, CH2NH), 6.22 (bs, IH, NH), 6.85 (d, IH, J = 3.4 Hz, HArthiophene), 6.94 (dd, IH, J = 3.5, 5.0 Hz, HArthiophene), 7.16 (dd, IH, J = 1.0, 5.2. Hz, HArthiophene), 7.20 (d, 2H, J = 8.1 Hz, HAT), 7.62 (d, 2H, J = 8.1 Hz, HAT); LC/MS (AP-) m/z 211.8 (M-H); tR = 2.4 min (95.8%); HRMS (FAB+) Calculated for C16H19NOS 273.1187; Found 273.1176
2.5-Dichloro-N-(2-thiophen-2-yl-ethylVbenzamide f STX1382. CCM01021) Reaction of 2,5-dichlorobenzoic acid (164 mg, 0.85 mmol) in thionyl chloride (2 mL) then with 2-thiopheneethylamine (50 μL, 0.42 mmol) in presence of triethylamine (0.5 mL) in THF (3 mL) according to method B gave 2,5-dichloro-N-(2-thiophen-2-yl-ethyl)- benzamide (83 mg, 0.28 mmol, 66% yield) as a off-white powder after purification on silica gel (eluent : EtOAc/hexane 5/95 to 20/80).
Rf : 0.2 (EtOAc/hexane 2/8); M.p.: 99-100 °C; 1HΝMR (270MHz, CDC13) δH 3.15 (t, 2H, J = 6.6 Hz, CH2thiophene), 3.73 (dt, 2H, J = 6.6, 6.6 Hz, CH2NH), 6.33 (bs, IH, NH), 6.86 (d, IH, J = 3.4 Hz, HArthiophene), 6.94 (dd, IH, J = 3.5, 5.1 Hz, HArthiophene), 7.15 (dd, IH, J = 1.2, 5.1. Hz, HArthiophene), 7.28 (d, 2H, J = 8.1 Hz, HAT), 7.58-7.59 (m, IH, HAT); 13C NMR (400MHz, CDC13) δc 29.7 (CH2-thiophene), 41.6 (CH2NH), 124.2 (CHAT), 125.7 (CHAT), 127.2 (CHAT), 128.9 (Cq), 130.2 (CHAΓ), 131.3 (CHAT), 131.5 (CHAT), 133.3 (Cq), 136.2 (Cq), 140.9 (Cq), 165.1(C=O); LC/MS (AP-) m/z 298.0 (M- H); tR = 2.2 min (99.9%); HRMS (FAB+) Calculated for C13HnCl2NOS 298.9938; Found 298.9933 Synthesis of N-(5-indanone)-benzamide derivatives
STX1397, CCM01023 STX1398, CCM0101624 STX1399, CCM01025
STX1400, CCM01026 STX1401 , CCM01027 STX1402, CCM01028
General method for synthesis of M-(5-indanone)-benzamide derivatives (STX1397-1402):
Method A : to a stirred solution of the amine (n mmol) in THF are added triethylamine (1.2n mmol) and the acyl chloride (1.2n mrrαol) at room temperature. After completion, ethyl acetate and water are added. The aqueous layer is extracted by ethyl acetate. The combined organic layers are washed with brine, dried (MgSO4), filtered and evaporated under reduce pressure. The crude product is then purified to give the amide. Method B : A solution of the acid (3n mmol) in thionyl chloride is refluxed 3 hours. Thionyl chloride is then removed under reduced pressure. The crude product is diluted in dry THF and added to a solution of the amine (n mmol) and triethylamine in THF. After completion, ethyl acetate and water are added . The aqueous layer is extracted by EtOAc. The combined organic layers are then washed with brine, dried (MgSO4), filtered and evaporated under reduce pressure. The crude product is purified to give the amide.
2.5-Dichloro-N-(l-oxo-indan-5-yl)-benzamide (STX1397, CCM01023) Reaction of 2,5-dichlorobenzoic acid (287 mg, 1.50 mmol) in thionyl chloride (3.5 mL) then with 5-amino-indan-l-one (74mg, 0.50 mmol) in presence of triethylamine (0.5 mL) in THF (6 mL) according to method B gave 2,5-dichloro-N-(l-oxo-indan-5-yl)-benzamide (33 mg, 0.09 mmol, 35% yield) as a green powder after purification by chromatography on silica gel (eluent : DCM).
Rf : 0.4 (EtOAc/DCM 1/9) ; M.p.: 218-221 °C ; 1H NMR (270MHz, DMSO-^) δH 2.58- 2.62 (m, 2H, CH2), 3.06-3.10 (m, 2H, CH2), 7.57-7.60 (m, 4H, HAT), 7.77-7.78 (m, IH, HAT), 8.01 (s, IH, HAT), 10.94 (s, IH, NH); LC/MS (AP-) m/z 317.7 (M-H); tR = 2.1 min (98.0%)
Biphenyl-4-carboxylic acid ri-oxo-indan-5-vn-amide (SXT1398. CCM01024)
Reaction of 5-amino-indan-l-one (73 mg, 0.50 mmol) in THF (6 mL) with 4- biphenylcarbonyl chloride (140 mg, 0.65 mmol) in presence of triethylamine (90 μL, 0.65 mmol) according to method A gave biphenyl-4-carboxylic acid (l-oxo-indan-5-yl)-amide (100 mg, 0.30 mmol, 60% yield) as a brown powder after purification by washing the crude product with ethyl acetate. Rf : 0.4 (EtOAc/DCM 1/9) ; M.p.: 217-219 °C ; 1H NMR (270MHz, DMSO-dtf) δH 2.58- 2.62 (m, 2H, CH2), 3.07-3.11 (m, 2H, CH2), 7.41-7.45 (m, IH, HAT), 7.48-7.54 (m, IH, HAT), 7.63 (d, IH, J = 8.4 Hz, HAT), 7.75-7.80 (m, 3H, HAT), 7.84-7.87 (m, 2H, HAT), 8.05- 8.08 (m, 2H, HAT), 8.12 (bs, IH, HA , 10.62 (s, IH, NH); LC/MS (AP-) m/z 325.9 (M-H); tR = 2.3 min (97.4%); HRMS (FAB+) Calculated for C22H17NO2 327.1259; Found 327.1266
3.5-Dichloro-N-(l-oxo-indan-5-yl -benzamide (STX1399, CCM01025 Reaction of 5-amino-indan-l-one (73 mg, 0.50 mmol) in THF (6 mL) with 3,5- dichlorobenzoyl chloride (136 mg, 0.65 mmol) in presence of triethylamine (90 μL, 0.65 mmol) according to method A gave biphenyl-4-carboxylic acid (l-oxo-indan-5-yl)-amide (115 mg, 0.36 mmol, 72% yield) as a brown powder after purification by washing the crude product with ethyl acetate.
Rf : 0.4 (EtOAc/DCM 1/9) ; M.p.: 261-263 °C ; 1H ΝMR (270MHz, DMSO-c^) δH 2.58- 2.62 (m, 2H, CH2), 3.06-3.10 (m, 2H, CH2), 7.63 (d, IH, J = 8.4 Hz, CHAΓC-CO), 7.72 (dd, IH, J = 1.7, 8.4 Hz, Ν-C-CHAT-CHAT), 7.88 (t, IH, J = 2.0 Hz, CCI-CHAT-CCI), 7.97 (d, 2H, J = 2Hz, HA , 8.05 (d, 1H, J = 1.7 Hz, CHAT-CHAT-C-CO), 10.70 (s, IH, ΝH); 13C ΝMR (400MHz, DMSO-d6) δc 26.0 (CH2), 36.5 (CH2), 117.5 (CHAT), 120.0 (CHAT), 124.2 (CHAT), 127.1 (CH^), 131.7 (CHAT), 132.9 (Cq), 134.8 (Cq), 138.2 (Cq), 144.9 (Cq),
157.1 (Cq), 163.8 (C=0), 205.4 (C=O); LC/MS (AP-) m/z 318.0 (M-H); tR = 2.5 min (99.9%); HRMS (FAB+) Calculated for C16H11Cl2NO2 319.0167; Found 319.0156
N-fl-Oxo-indan-5-vn-4-propyl-benzamide (STX1400. CCM01026^)
Reaction of 5-amino-indan-l-one (75 mg, 0.50 mmol) in THF (6 mL) with 4- propylbenzoyl chloride (108 μL, 0.65 mmol) in presence of triethylamine (90 μL, 0.65 mmol) according to method A gave N-(l-oxo-indan-5-yl)-4-propyl-benzamide (94 mg, 0.32 mmol, 64% yield) as a white powder after purification on silica gel (eluent EtOAc/DCM 0/10 to 1/9).
Rf : 0.4 (EtOAc/DCM 1/9) ; M.p.: 181-184 °C ; 1H ΝMR (270MHz, DMSO-rftf) δH 0.88 (t, 3H, J = 7.2 Hz, CH3), 1.56-1.65 (m, 2H, CH2-CH3), 2.57-2.65 (m, 4H, 2*CH2), 3.08 (bt, 2H, J = 5.4 Hz, CH2CO), 7.35 (d, 2H, J = 8.1 Hz, HAT), 7.60 (d, IH, J = 8.4 Hz, CHAT- C-CO), 7.74 (d, IH, J = 8.4 Hz, Ν-C-CHAT-CHAT), 7.87 (d, 2H, J = 8.1 Hz, CHAT), 8.09 (d, IH, J = 8.1 Hz, CHAT-CHAΓ-C-CO), 10.50 (s, IH, ΝH); LC/MS (AP-) m/z 291.9 (M-H); tR = 2.4 min (99.1%)
4-Methoxy-N-α-oxo-indan-5-ylVbenzamide (STX1401, CCM01027
Reaction of 5-amino-indan-l-one (73 mg, 0.5O mmol) in THF (6 mL) with -anisoyl chloride (88 μL, 0.65 mmol) in presence of triethylamine (90 μL, 0.65 mmol) according to method A gave 4-methoxy-N-(l-oxo-indan-5-yl)-benzamide (107 mg, 0.38 mmol, 76% yield) as a yellow powder after purification by chromatography on silica gel (eluent EtOAc/DCM 0/10 to 2/8). Rf : 0.2 (EtOAc/DCM 1/9) ; M.p.: 237-238 °C ; 1H ΝMR (270MHz, DMSO-έfc) δH 2.57- 2.62 (m, 2H, CH2), 3.08 (bt, 2H, J = 5.5 Hz, CH2CO), 3.83 (s, 3H, OCH3), 7.07 (d, 2H, J = 8.9 Hz, HAT), 7.60 (d, IH, J = 8.4 Hz, CHAT-C-CO), 7.74 (d, IH, J = 1.6 Hz, Ν-C-CHAT- CHAT), 7.96 (d, 2H, J = 8.9 Hz, CHA , 8.08 (d, IH, 3 = 1.6 Hz, CHAT-CHAT-C-CO), 10.42 (s, IH, ΝH); 13C ΝMR (400MHz, DMSO-d6) δc 26.0 (CH2), 36.5 (CH2), 56.0 (OCH3),
114.2 (CHAT), 117.1 (CHAΓ), 119.8 (CHAT), 124.1 (CHAT), 127.0 (Cq), 130.3 (CHAT), 132.3 (Cq), 145.8 (Cq), 157.1 (Cq), 162.7 (Cq), 165.9 (Cq), 205.3 (C=O); LC/MS (AP-) m/z 279.9
(M-H); tR = 2.0 min (99.9%) 3-Methoxy-N-(l-oxo-indan-5-yl -benzamide (STX1402, CC 01028) Reaction of 5-amino-indan-l-one (73 mg, 0.50 mmol) in THF (6 mL) with m-anisoyl chloride (90 μL, 0.65 mmol) in presence of triethylamine (90 μL, 0.65 mmol) according to method A gave 3-methoxy-N-(l-oxo-indan-5-yl)-benzamide (73 mg, 0.26 mmol, 52% yield) as a yellow powder after purification by chromatography on silica gel (eluent EtOAc/DCM O/lO to l/9).
Rf : 0.2 (EtOAc/DCM 1/9) ; M.p.: 203-204 °C ; !H ΝMR (270MHz, OMSO-d6) δH 2.59- 2.63 (m, 2H, CH2), 3.09 (bt, 2H, J = 5.7 Hz, CH2CO), 3.84 (s, 3H, OCH3), 7.16-7.21 (m, IH, HAT), 7.44-7.49 (m, 2H, HAT), 7.52-7.56 (m, IH, HAT), 7.62 (d, IH, J = 8.4 Hz, CHAT- C-CO), 7.76 (dd, IH, J = 1.8, 8.4 Hz, Ν-C-CHAr-CHAr), 8.09 (d, IH, J = 1.8 Hz, CHAT- CHAT-C-CO), 10.55 (s, IH, ΝH); 13C ΝMR (400MHz, D SO-d6) δc 26.0 (CH2), 36.5 (CH2), 55.9 (OCH3), 113.6 (CH^), 117.3 (CHAT), 118.1 (CHAT), 119.9 (CHAT), 120.5 (CHAT), 124.2 (CHAT), 130.2 (CHAT), 132.5 (Cq), 136.5 (Cq), 145.4 (Cq), 157.1 (Cq), 159.7 (Cq), 166.3 (C=O), 205.3 (C=O); LC MS (AP-) m/z 280.0 (M-H); tR = 2.0 min (99.7%
Synthesis of Thiophene or Benzothiophene Arysulfonamide Derivatives
STX1317.XDS03047B STX1318, XDS03048 STX1319, XDS03049
STX1320.XDS03050 STX1321.XDS03051 STX1327, XDS03061A
STX1328.XDS03061B STX1329.XDS03062A STX1330.XDS03062B
STX1331.XDS03063A STX1332.XDS03063B
STX1334.XDS03064B STX1335.XDS03065 STX1336.XDS03066
STX1337.XDS03067 STX1338.XDS03068 STX1414.XDS03111
STX1415.XDS03112 STX1416.XDS03113 General method for synthesis of thiophene or benzothiophene arylsulphonamide derivatives (STX1317-1321, STX1327-1338, STX1414- 1416):
To a solution of arylsulphonyl chloride (1.1 eq.) in DCM was added pyridine (2.2 eq.), followed by the corresponding amine (1 eq.). The reaction mixture was stirred at ambient temperature under nitrogen for 3-6 h. After TLC showed completion of the reaction, the mixture was partitioned between ethyl acetate and 5% sodium bicarbonate solution. The organic layer was washed with brine, dried over sodium sulfate, and concentrated in vacuo to give the crude product as solid or thick syrup. The compound was then purified by flash chromatography (Ethyl acetate-hexane gradient elution) to give desired arylsulphonamide as crystalline solid or amorphous solid. Yield ranged from 35-65%.
4-Propyl-N-thiophen-2-ylmethyl-benzenesulfonamide (STX1317, XDS03047B)
White solid. TLC single spot at Rf 0.25 (20% ethyl acetate/hexane); HPLC purity > 99 % (tR 3.1 min in 30% water-acetonitrile); 1H NMR (270 MHz, DMSO): δ 8.20 (1 H, broad, NH), 7.70 (2H, d, J = 8.4 Hz, ArH), 7.37-7.40 (3H, m, ArH), 6.89 (2H, m, ArH), 4.15 (2H, s, NCH2), 2.65 (2H, t, J = 7.4 Hz, CH2), 1 .61 (2H, sextet, J = 7.4 Hz, CH2), 0.89 (3H, t, J = 7.4 Hz, CH3); FAB-MS 296 (MH+); FAB-HRMS calcd for C14Hι8NO2S2 (MH+) 296.0779, found 296.0776.
2,5-Dichloro-N-thiophen-2-ylmethyl-benzenesulfonamide (STX1318,
XDS03048) White crystalline solid. TLC single spot at Rf 0.29 (20% ethyl acetate/hexane); HPLC purity > 99 % (tR 2.8 min in 30% water-acetonitrile); 1 H NMR (270 MHz, DMSO): δ 8.73 (1 H, s, NH), 7.80 (1 H, d, J = 2.5 Hz, ArH), 7.60-7.69 (2H, m, ArH), 7.33 (1H, dd, J = 5.0, 1.5 Hz, ArH), 6.82-6.87 (2H, m, ArH), 4.36 (2H, s, NCH2); FAB-MS 322 (MH+); FAB-HRMS calcd for CnH^CIzNOzSz (MH+) 321.9530, found 321.9426. 3-Chloro-2-methyl-N-thiophen-2-ylmethyl-benzenesulfonamide (STX1319, XDS03049)
White crystalline solid. TLC single spot at Rf 0.28 (20% ethyl acetate/hexane); HPLC purity > 99 % (tR 3.1 min in 30% water-acetonitrile); 1 H NMR (270 MHz, DMSO): δ 8.58 (1 H, s, NH), 7.82 (1 H, d, J = 7.9 Hz, ArH), 7.70 (1 H, d, J = 7.9 Hz, ArH), 7.35-7.40 (2H, m, ArH), 6.84-6.87 (2H, m, ArH), 4.26 (2H, s, NCH2), 2.57 (3H, s, CH3); FAB-MS 302 (MH+); FAB-HRMS calcd forC^H^CINOzSz (MH+) 302.0076, found 301.9988.
Biphenyl-4-sulfonic acid (thiophen-2-ylmethyl)-amide (STX1320, XDS03050)
White solid. TLC single spot at Rf 0.29 (20% ethyl acetate/hexane); HPLC purity
> 99 % (tR 2.5 min in 30% water-acetonitrile); 1H NMR (270 MHz, DMSO): δ 8.32 (1H, s, NH), 7.87 (4H, s, ArH), 7.73-7.76 (2H, m, ArH), 7.39-7.55 (4H, m, ArH), 6.91 (2H, m, ArH), 4.21 (2H, s, NCH2); FAB-MS 330 (MH+); FAB-HRMS calcd for Cι7H16NO2S2 (MH+) 330.0622, found 330.0601.
4-Phenoxy-N-thiophen-2-ylmethyl-benzenesulfonamide (STX1321 XDS03051
White solid. TLC single spot at Rf 0.29 (20% ethyl acetate/hexane); HPLC purity
> 99 % (IR 2.4 min in 20% water-acetonitrile); 1H NMR (270 MHz, DMSO): δ 8.22 (1 H, s, NH), 7.75-7.80 (2H, m, ArH), 7.39-7.51 (3H, m, ArH), 7.07-7.28 (5H, m,
ArH), 6.90-6.92 (2H, m, ArH), 4.18 (2H, s, NCH2); FAB-MS 346 (MH+); FAB- HRMS calcd for Cι7H16NO3S2 (MH+) 346.0572, found 346.0574.
4-Propyl-N,N-bis-thiophen-3-ylmethyl-benzenesulfonamide (STX1327 XDS03061A)
White solid. TLC single spot at Rf 0.56 (20% ethyl acetate/hexane); HPLC purity
> 99 % (tR 4.5 min in 20% water-acetonitrile); 1H NMR (270 MHz, CDCI3): δ 7.72 (2H, dt, J = 8.4, 2.0 Hz, ArH), .7.29 (2H, dt, J = 8.5, 1.9 Hz, ArH), 7.17 (2H, dd, J = 5.0, 3.0 Hz, ArH), 6.90 (2H, dd, J = 3.0, 1.5 Hz, ArH), 6.74 (2H, dd, J = 5.0, 1.5 Hz, ArH), 4.31 (4H, s, 2 x NCH2), 2.66 (2H, t, J = 7.1 Hz, CH2), 1.67 (2H, sextet, J = 7.5 Hz, CH2), 0.95 (3H, t, J = 7.3 Hz, CH3); APCI-MS 392 (MH+); FAB-HRMS calcd for (MH+) 392.0813, found 392.0805. 4-Propyl-N-thiophen-3-ylmethyl-benzenesulfonamide (STX1328,
XDS03061B)
White solid. TLC single spot at Rf 0.29 (20% ethyl acetate/hexane); HPLC purity 92 % (fe 2.6 min in 20% water-acetonitrile); 1H NMR (270 MHz, CDCI3): δ 7.75 (2H, dt, J = 8.5, 2.0 Hz, ArH), 7.29 (2H, dt, J = 8.4, 1.8 Hz, ArH), 7.20 (1 H, dd, J = 5.2, 3.2 Hz, ArH), 7.03 (1 H, dd, J - 3.1, 1.1 Hz, ArH), 6.86 (1 H, dd, J = 5.1 , 1.1 Hz, ArH), 4.73 (1 H, t, J = 6.1 Hz, NH), 4.15 (2H, d, J = 5.9 Hz, NCH2), 2.65 (2H, t, J = 7.9 Hz CH2), 1.67 (2H, sextet, J = 8.0 Hz, CH2), 0.94 (3H, t, J = 7.3 Hz, CH3); APCI-MS 294 (M-H+); FAB-HRMS calcd for C14H18NO2S2 (MH+) 296.0779, found 296.0784.
2,5-Dichloro-N,N-bis-thiophen-3-ylmethyl-benzenesulfonamide (STX1329, XDS03062A) White crystalline solid. TLC single spot at Rf 0.50 (20% ethyl acetate/hexane); HPLC purity > 98 % (tR 4.0 min in20% water-acetonitrile); 1H NMR (270 MHz, CDCI3): δ 8.02 (1 H, m, ArH), 7.42-7.45 (2H, m, ArH), 7.23 (2H, dd, J = 5.0, 3.0 Hz, ArH), 7.01 (2H, dd, J = 3.0, 1.0 Hz, ArH), 6.82 (2H, dd, J - 5.0, 1.3 Hz, ArH), 4.41 (4H, s, 2 x NCH2); APCI-MS 418 (MH+); FAB-HRMS calcd for C16H14Cl2NO2S3 (MH+) 417.9564, found 417.9510.
2,5-Dichloro-N-thiophen-3-ylmethyl-benzenesulfonamide (STX1330,
XDS03062B)
White crystalline solid. TLC single spot at Rf 0.25 (20% ethyl acetate/hexane); HPLC purity > 99 % (tR 2.4 min in20% water-acetonitrile); 1H NMR (270 MHz, CDCI3): δ 7.99 (1 H, d, J = 2.0 Hz, ArH), 7.36-7.45 (2H, m, ArH), 7.17 (1 H, dd, J = 5.2, 3.2 Hz, ArH), 7.06 (1H, dd, J = 3.0, 1.0 Hz, ArH), 6.86 (1 H, dd, J = 5.0, 1.3 Hz, ArH), 5.25 (1 H, t, J = 6.1 Hz, NH), 4.19 (2H, d, J = 6.2 Hz, NCH2); APCI-MS 320 (M-H+); FAB-HRMS calcd for C11H10CI2NO2S2 (MH+) 321.9530, found 321.9473. 3-Chloro-2-methyl-N,N-bis-thiophen-3-yImethyl-benzenesulfonamide (STX1331, XDS03063A)
White solid. TLC single spot at Rf 0.55 (20% ethyl acetate/hexane); HPLC purity
> 98 % (IR 6.8 min in 30% water-acetonitrile); 1H NMR (270 MHz, CDCI3): δ 7.90 (1 H, dd, J = 7.8, 1.2 Hz, ArH), 7.60 (1 H, dd, J = 7.8, 1.1 Hz, ArH), 7.20-7.27 (3H, m, ArH), 7.00 (2H, dd, J = 3.0, 1.0 Hz, ArH), 6.76 (2H, dd, J = 5.0, 1.3 Hz, ArH), 4.32 (4H, s, 2 x NCH2), 2.61 (3H, s, CH3); APCI-MS 398 (MH+); FAB-HRMS calcd for Cι7H17CINO2S3 (MH+) 398.0110, found 398.0098.
3-Chloro-2-methyl-N-thiophen-3-ylmethyl-benzenesulfonamide (STX1332, XDS03063B)
White solid. TLC single spot at Rf 0.30 (20% ethyl acetate/hexane); HPLC purity
> 99 % (IR 2.5 min in 20% water-acetonitrile); 1H NMR (270 MHz, CDCI3): δ 7.91 (1 H, dd, J = 7.9, 1.0 Hz, ArH), 7.57 (1 H, dd, J = 7.8, 1.0 Hz, ArH), 7.20-7.27 (2H , m, ArH), 7.02 (1 H, m, ArH), 6.84 (2H, dd, J = 5.1 , 1.3 Hz, ArH), 4.79 (1 H, t, J = 5.9 Hz, NH), 4.16 (2H, d, J = 5.9 Hz, NCH2); 2.62 (3H, s, CH3); APCI-MS 302 (MH+); FAB-HRMS calcd for C12Hi3CINO2S2 (MH+) 302.0076, found 302.0056.
Biphenyl-4-sulfonic acid bis-thiophen-3-ylmethyl-amide (STX1333, XDS03064A)
White solid. TLC single spot at Rf 0.45 (20% ethyl acetate/hexane); HPLC purity
> 99 % (tR 4.3 min in 20% water-acetonitrile); 1H NMR (270 MHz, CDCI3): δ 7.8S (2H, dt, J = 8.6, 1.8 Hz, ArH), 7.70 (2H, dt, J = 8.5, 1.8 Hz, ArH), 7.59-7.63 (2H , m, ArH), 7.39-7.52 (3H, m, ArH), 7.19 (2H, dd, J = 5.3, 3.3 Hz, ArH), 6.95 (2H , dd, J = 3.2, 1.2 Hz, ArH), 6.80 (2H, dd, J = 5.3, 1.4 Hz, ArH), 4.36 (4H, s, 2 x NCH2); APCI-MS 426 (MH+); FAB-HRMS calcd for C22H20NO2S3 (MH+) 426.0656 , found 426.0628.
Biphenyl-4-sulfonic acid (thiophen-3-ylmethyl)-amide (STX1334, XDS03064B)
White solid. TLC single spot at Rf 0.45 (20% ethyl acetate/hexane); HPLC purity
> 99 % (tR 3.1 min in 30% water-acetonitrile); 1H NMR (270 MHz, CDCI3): δ 7.91 (2H, dt, J = 8.5, 1.7 Hz, ArH), 7.71 (2H, dt, J = 8.8, 1.8 Hz, ArH), 7.57-7.62 (2H, m, ArH), 7.42-7.52 (3H, m, ArH), 7.22 (1 H, dd, J = 5.2, 3.2 Hz, ArH), 7.08 (1 H, m, ArH), 6.90 (1 H, dd, J = 5.2, 1.2 Hz, ArH), 4.61 (1H, t, J = 6.4 Hz, NH), 4.22 (2H, d, J = 6.5 Hz, NCH2); APCI-MS 330 (MH+); FAB-HRMS calcd for Cι7Hι6NO2S2 (MH+) 330.0622, found 330.0627.
4-Propyl-N-(2-thiophen-2-yl-ethyl)-benzenesulfonamide (STX1335,
XDS03065)
White solid. TLC single spot at Rf 0.40 (25% ethyl acetate/hexane); HPLC purity > 99 % (tR 3.5 min in 30% water-acetonitrile); 1H NMR (270 MHz, CDCI3): δ 7.72 (2H, dt, J = 8.2, 1.1 Hz, ArH), 7.28 (2H, m, ArH), 7.11 (1H, dd, J = 5.2, 1.2 Hz, ArH), 6.88 (1 H, dd, J = 5.2, 3.2 Hz, ArH), 6.73 (1 H, dd, J = 3.0, 1.0 Hz, ArH), 4.78 (1H, t, J = 6.2 Hz, NH), 3.21 (2H, q, J = 6.4 Hz, CH2) 2.95 (2H, t, J = 6.8 Hz, CH2), 2.64 (2H, t, J = 6.5 Hz, CH2), 1.65 (2H, sextet, J = 6.3 Hz, CH2), 0.90 (3H, t, J = 6.5 Hz, CH3); APCI-MS 308 (M-H+).
2,5-Dichloro-N-(2-thiophen-2-yl-ethyl)-benzenesulfonamide (STX1336,
XDS03066)
Off-white crystalline solid. TLC single spot at Rf 0.37 (25% ethyl acetate/hexane); HPLC purity > 99 % (tR 3.3 min 30% water-acetonitrile); 1H
NMR (270 MHz, CDCI3): δ 8.06 (1 H, d, J = 2.5 Hz, ArH), 7.39-7.49 (2H, m, ArH),
7.16 (1H, dd, J = 5.2, 1.2 Hz, ArH), 6.91 (1 H, dd, J = 5.2, 3.2 Hz, ArH), 6.79 (1 H, dd, J = 3.2, 1.1 Hz, ArH), 5.06 (1 H, t, J = 5.8 Hz, NH), 3.24 (2H, q, J = 5.9 Hz,
CH2) 3.01 (2H, t, J = 5.8 Hz, CH2); APCI-MS 334 (M-H+).
3-Chloro-2-methyl-N-(2-thiophen-2-yl-ethyl)-benzenesulfonamide (STX1337,
XDS03067)
White crystalline solid. TLC single spot at Rf 0.34 (25% ethyl acetate/hexane);
HPLC purity > 99 % (tR 3.3 min in 30% water-acetonitrile); 1H NMR (270 MHz, CDCIs): δ 7.90 (1 H, dd, J = 7.9, 1.2 Hz, ArH), 7.57 (1 H, dd, J = 7.8, 1.2 Hz, ArH),
7.25 (1H, t, J = 7.7 Hz, ArH), 7.16 (1H, dd, J = 5.2, 1.3 Hz, ArH), 6.92 (1 H, dd, J
= 5.2, 3.3 Hz, ArH), 6.73 (1 H, dd, J = 3.2, 1.2 Hz, ArH), 4.54 (1 H, t, J = 6.5 Hz, NH), 3.24 (2H, q, J = 6.4 Hz, CH2), 2.98 (2H, t, J = 6.4 Hz, CH2), 2.50 (3H, s, CH3); APCI-MS 314 (M-H+).
Biphenyl-4-sulfonic acid (2-thiophen-2-yl-ethyl)-amide (STX1338, XDS03068) Off-white crystalline solid. TLC single spot at Rf 0.29 (25% ethyl acetate/hexane); HPLC purity > 99 % (tR 2.7 min in 30% water-acetonitrile); 1H NMR (270 MHz, CDCI3): δ 7.87 (2H, dt, J = 7.9, 1.2 Hz, ArH), 7.70 (2H, dt, J = 8.1 , 1.2 Hz, ArH), 7.58-7.62 (2H, m, ArH), 7.41-7.50 (3H, m, ArH), 7.15 (1 H, dd, J = 5.1 , 1.2 Hz, ArH), 6.91 (1 H, dd, J = 5.2, 3.2 Hz, ArH), 6.77 (1 H, dd, J = 3.0, 1.0 Hz, ArH), 4.50 (1 H, t, J = 6.5 Hz, NH), 3.29 (2H, q, J = 6.5 Hz, CH2), 3.00 (2H, t, J = 6.5 Hz, CH2); APCI-MS 342 (M-H+).
N-Benzo[b]thiophen-2-ylmethyl-4-propyl-benzenesulfonamide (STX1414, XDS03111) Off-white solid. TLC single spot at Rf 0.47 (25% ethyl acetate/hexane); HPLC purity > 99 % (tR 2.4 min in 20% water-acetonitrile); 1H NMR (270 MHz, CDCI3): δ 7.76 (2H, dt, J = 7.9, 1.7 Hz, ArH), 7.61-7.72 (2H, m, ArH), 7.24-7.32 (4H, m, ArH), 7.05 (1H, d, J = 1.2 Hz, ArH), 4.73 (1H, t, J = 6.7 Hz, NH), 4.43 (2H, d, J = 6.5 Hz, NCH2), 2.61 (2H, t, J = 7.6 Hz, CH2), 1.62 (2H, sextet, J = 7.4 Hz, CH2), 0.92 (3H, t, J = 7.3 Hz, CH3); APCI-MS 344 (M-H+).
N-Benzo[b]thiophen-2-ylmethyl-2,5-dichloro-benzenesulfonamide (STX1415, XDS03112)
Off-white solid. TLC single spot at Rf 0.46 (20% ethyl acetate/hexane); HPLC purity > 99 % (tR 2.4 min in 30% water-acetonitrile); 1H NMR (270 MHz, CDCI3): δ 7.98 (1 H, dd, J = 1.5, 0.5 Hz, ArH), 7.61-7.69 (2H, m, ArH), 7.26-7.30 (4H, m, ArH), 7.05 (1 H, d, J = 0.8 Hz, ArH), 5.41 (1 H, t, J = 5.9 Hz, NH), 4.47 (2H, d, J = 5.7 Hz, NCH2); APCI-MS 370 (M-H+). Biphenyl-4-sulfonic acid (benzo[b]thiophen-2-ylmethyl)-amide (STX1415, XDS030113)
Off-white solid. TLC single spot at Rf 0.58 (30% ethyl acetate/hexane); HPLC purity > 99 % (tR 2.4 min in 30% water-acetonitrile); 1H NMR (270 MHz, CDCI3): δ 7.92 (2H, dt, J = 7.9, 1.7 Hz, ArH), 7.61-7.72 (4H, m, ArH), 7.40-7.55 (5H, m, ArH), 7.24-7.30 (2H, m, ArH), 7.08 (1 H, d, J = 1.2 Hz, ArH), 4.81 (1 H, t, J = 5.2 Hz, NH), 4.48 (2H, d, J = 5.4 Hz, NCH2); APCI-MS 378 (M-H+).
Synthesis of 3-(2,5-Dichloro-benzenesulfonylmethyl)-thiophene
STX1339. XDS03070 STX1340. XDS03071 B
3-(2,5-Dichloro-phenylsulfanylmethyl)-thiophene (STX1339, XDS03070):
To a solution of 2,5-dichlorobenzenethiol (420 mg, 2.35 mmol) in methanol (8 mL) was added triethylamine (0.4 mL), followed by 3-chloromethyl-thiophene (266 mg, 2.00 mmol). The reaction mixture was stirred at ambient temperature under nitrogen for 4 hours, and then partitioned between ethyl acetate and water. The organic layer was washed with 1 N HCl, 5% sodium carbonate solution and brine, dried over sodium sulphate, and concentrated in vacuo to give the crude product. The compound was purified by flash chromatography (Ethyl acetate- hexane gradient elution). White crystalline solid (440 mg, yield 80%) was obtained. TLC single spot at Rf 0.49 (8% ethyl acetate/hexane); HPLC purity > 99 % (tR 7.1 min in 30% water-acetonitrile); 1H NMR (270 MHz, CDCI3): δ 7.27- 7.30 (2H, m, ArH), 7.16-7.19 (2H, m, ArH), 7.04-7.09 (2H, m, ArH), 4.16 (2H, s, CH2); FAB-MS 274 (M+); FAB-HRMS calcd for CιιHβCI2S2 (M+) 273.9444, found 273.9439. 3-(2,5-Dichloro-benzenesulfonyImethyl)-thiophene (STX1340, XDS03071B):
To a cold solution (-5°C) of 3-(2,5-DichIoro-phenylsulfanylmethyl)-thiophene (360 mg, 1.31 mmol) in DCM (10 mL) was added 3-chloroperoxy benzoic acid (800 mg, 57-86% pure). The reaction mixture was stirred at -5 - 0°C for 6 hours, and then partitioned between DCM and 5% sodium carbonate solution. The organic layer was washed with brine, dried over sodium sulphate, and concentrated in vacuo to give a yellow residue which was purified by flash chromatography (Ethyl acetate-hexane gradient elution). White crystalline solid (295 mg, 73%) was obtained. TLC single spot at Rf 0.52 (20% ethyl acetate/hexane); HPLC purity > 99 % (tR 3.0 min in 30% water-acetonitrile); 1H NMR (270 MHz, CDCI3): δ 7.68 (1 H, t, J = 1.3 Hz, ArH), 7.46 (2H, d, J = 1.2 Hz, ArH), 7.23 (1 H, m, ArH), 7.17 (1 H, m, ArH), 6.96 (1H, dd, J = 5.0, 1.2 Hz, ArH), 4.70 (2H, s, CH2); FAB-MS 307 (MH+); FAB-HRMS calcd for CHH9CI2O2S2 (MH+) 306.9421, found 306.9397
Synthesis of Thiophene or Benzothiophene Benzamide Derivatives
STX1405. XDS03101 STX1406. XDS03102 STX1407. XDS03103
STX1408. XDS03104 STX1409. XDS03105 STX1417. XDS03114
STX1418. XDS03115 STX1419. XDS03116
General method for synthesis of thiophene or benzothiophene benzamide derivatives (STX1405-1409, STX1417-1419): To a solution of substituted benzoyl chloride (1.1 eq.) in DCM was added triethylamine (2.2 eq.), followed by the corresponding amine (1 eq.). The reaction mixture was stirred at ambient temperature under nitrogen overnight. PS-Trisamine (0.2 eq.) was added and the mixture was stirred for another 3 hours at ambient temperature, filtered. The solution was concentrated in vacuo to give crude product as solid or thick syrup. The compound was then purified by flash chromatography (Ethyl acetate-hexane gradient elution) to give desired benzamide as crystalline solid. Yield ranged from 70-96%.
4-Propyl-N-thiophen-2-yImethyl-benzamide (STX1405, XDS03101)
White crystalline solid. TLC single spot at Rf 0.49 (20% ethyl acetate/hexane); HPLC purity > 99 % (tR 2.3 min in 20% water-acetonitrile); 1H NMR (270 MHz, CDCI3): δ 7.68 (2H, m, ArH), 7.20-7.25 (3H, m, ArH), 7.02 (1H, dd, J = 3.0, 1.3 Hz, ArH), 6.96 (1 H, dd, J = 5.2, 3.2 Hz, ArH), 6.37 (1 H, broad, NH), 4.80 (2H, d, J = 5.4 Hz, CH2), 2.61 (2H, t, J = 7.7 Hz, CH2), 1.61 (2H, sextet, J = 7.4 Hz, CH2), 0.92 (3H, t, J = 7.3 Hz, CH3); APCI-MS 258 (M-H+).
3,5-Dichloro-N-thiophen-2-ylmethyl-benzamide (STX1406, XDS03102)
White crystalline solid. TLC single spot at Rf 0.45 (20% ethyl acetate/hexane); HPLC purity > 99 % (tR 2.4 min in 20% water-acetonitrile); 1H NMR (270 MHz,
CDCI3): δ 7.62 (2H, d, J = 2.0 Hz, ArH), 7.46 (1 H, t, J = 1.9 Hz, ArH), 7.25 (1H, dd, J = 5.0, 1.2 Hz, ArH), 7.03 (1 H, dd, J = 3.6, 0.9 Hz, ArH), 6.96 (1 H, dd, J =
5.0, 3.5 Hz, ArH), 6.38 (1 H, broad, NH), 4.77 (2H, d, J = 5.7 Hz, NCH2); APCI-MS
284 (M-H+).
Biphenyl-4-carboxylic acid (thiophen-2-ylmethyl)-amide (STX1407,
XDS03103)
White crystalline solid. TLC single spot at Rf 0.52 (20% ethyl acetate/hexane);
HPLC purity > 99 % (tR 2.2 min in 20% water-acetonitrile); 1H NMR (270 MHz, CDCI3): δ 7.85 (2H, dt, J = 8.9, 2.0 Hz, ArH), 7.57-7.66 (4H, m, ArH), 7.35-7.48
(3H, m, ArH), 7.25 (1 H, dd, J = 5.1, 1.3 Hz, ArH), 7.06 (1 H, dd, J = 3.5, 1.0 Hz, ArH), 6.97 (1 H, dd, J = 5.0, 3.5 Hz, ArH), 6.46 (1H, broad, NH), 4.84 (2H, d, J = 5.7 Hz, NCH2); APCI-MS 294 (MH+).
4-Methoxy-N-thiophen-2-ylmethyl-benzamide (STX1408, XDS03104) White crystalline solid. TLC single spot at Rf 0.35 (20% ethyl acetate/hexane); HPLC purity > 99 % (tR 1.9 min in 20% water-acetonitrile); 1H NMR (270 MHz, CDCI3): δ 7.74 (2H, dt, J = 9.0, 2.3 Hz, ArH), 7.23 (1 H, dd, J = 5.2, 1.2 Hz, ArH), 7.02 (1 H, dd, J = 3.5, 1.0 Hz, ArH), 6.96 (1 H, dd, J = 5.1 , 3.6 Hz, ArH), 6.31 (1 H, broad, NH), 4.79 (2H, d, J = 5.7 Hz, CH2), 3.83 (3H, s, OCH3); APCI-MS 246 (M- H+).
3-Methoxy-N-thiophen-2-ylmethyl-benzamide (STX1409, XDS03105)
White crystalline solid. TLC single spot at Rf 0.35 (20% ethyl acetate/hexane); HPLC purity > 99 % (tR 2.0 min in 20% water-acetonitrile); 1H NMR (270 MHz, CDCI3): δ 7.38 (2H, dd, J = 2.3, 1.7 Hz, ArH), 7.24-7.32 (3H, m, ArH), 7.01-7.05 (2H, m, ArH), 6.97 (1 H, dd, J = 5.2, 3.4 Hz, ArH), 6.39 (1 H, broad, NH), 4.82 (2H, d, J = 5.5 Hz, CH2), 3.84 (3H, s, OCH3); APCI-MS 246 (M-H+).
N-Benzo[b]thiophen-2-ylmethyl-4-propyl-benzamide (STX1417, XDS03114) White crystalline solid. TLC single spot at Rf 0.49 (30% ethyl acetate/hexane); HPLC purity > 99 % (tR 2.5 min in 20% water-acetonitrile); 1H NMR (270 MHz, CDCI3): δ 7.77 (1 H, m, ArH), 7.69-7.73 (3H, m, ArH), 7.28-7.36 (2H, m, ArH), 7.21-7.25 (3H, m, ArH), 6.48 (1H, broad, NH), 4.88 (2H, dd, J = 5.6, 1.0 Hz, CH2), 2.61 (2H, t, J = 7.2 Hz, CH2), 1.63 (2H, sextet, J = 7.5 Hz, CH2), 0.92 (3H, t, J = 7.4 Hz, CH3); APCI-MS 308 (M-H+).
N-Benzo[b]thiophen-2-ylmethyl-3,5-dichloro-benzamide (STX1418,
XDS03115)
White crystalline solid. TLC single spot at Rf 0.45 (20% ethyl acetate/hexane); HPLC purity > 98 % (tR 2.7 min in 20% water-acetonitrile); 1H NMR (270 MHz,
CDCI3): δ 7.76-7.80 (1 H, m, ArH), 7.69-7.73 (1 H, m, ArH), 7.65 (2H, d, J = 1.7 Hz, ArH), 7.48 (1H, t, J = 1.9 Hz, ArH), 7.30-7.35 (2H, m, ArH), 7.25 (1 H, m, ArH), 6.45 (1 H, broad, NH), 4.87 (2H, d, J - 5.6 Hz, CH2); APCI-MS 334 (M-H+).
N-Benzo[b]thiophen-2-yImethyl-4-methoxy-benzamide (STX1419, XDS03116)
White crystalline solid. TLC single spot at Rf 0.52 (40% ethyl acetate/hexane); HPLC purity > 99 % (tR 2.1 min in 20% water-acetonitrile); 1H NMR (270 MHz, CDCI3): δ 7.69-7.79 (4H, m, ArH), 7.24-7.33 (3H, m, ArH), 6.92 (2H, dt, J = 8.8, 2.2 Hz, ArH), 6.43 (1H, broad, NH), 4.88 (2H, dd, J - 5.6, 1.0 Hz, CH2), 3.84 (3H, s, OCH3); APCI-MS 296 (M-H+).
Synthesis of N-(4-benzophenone)-benzamide derivatives
STX1430, CCM01029 STX1431 , CCM01031
STX1432, CCM01032 STX1433, CCM01034
STX1434, CCM01036 STX1435, CCM01037 General method for synthesis of N-(4-benzophenone)-benzamide derivatives (STX1430-1435):
Method A : to a stirred solution of the amine (n mmol) in THF are added triethylamine (1.2n mmol) and the acyl chloride (1.2n mmol) at room temperature. After completion, ethyl acetate and water are added. The aqueous layer is extracted by ethyl acetate. The combined organic layers are washed with brine, dried (MgSO4), filtered and evaporated under reduce pressure. The crude product is then purified to give the amide. Method B : A solution of the acid (3n mmol) in thionyl chloride is refluxed 3 hours. Thionyl chloride is then removed under reduced pressure. The crude product is diluted in dry THF and added to a solution of the amine (n mmol) and triethylamine in THF. After completion, ethyl acetate and water are added . The aqueous layer is extracted by EtOAc. The combined organic layers are then washed with brine, dried (MgSO4), filtered and evaporated under reduce pressure. The crude product is purified to give the amide.
Biphenyl-4-carboxylic acid (4-benzoyl-phenyl -amide (STX1430. CCM01029
Reaction of 4-aminobenzophenone (102 mg, 0.52 mmol) in THF (6 mL) with 4- biphenylcarbonyl chloride (143 mg, 0.65 mmol) in presence of triethylamine (90 μL, 0.65 mmol) according to method A gave biphenyl-4-carboxylic acid (4-benzoyl-phenyl)-amide (125 mg, 0.33 mmol, 63% yield) as a yellow powder after purification by crystallisation in DCM/EtOAc.
R : 0.2 (DCM); M.p.: 212-213 °C; 1H NMR (270MHz, DMSO-d*) δH 7.40-7.45 (m, IH, HAT), 7.49 (bs, IH, HA,), 7.52 (bs, IH, HAT), 7.54-7.59 (m, 2H, HAT), 7.64-7.68 (m, IH, HAT), 7.72 (d, IH, J = 1.54 Hz, HAT), 7.74-7.76 (m, 2H, H r), 7.77-7.79 (m, 2H, HAT), 7.81 (s, IH, HAT), 7.87 (d, 2H, J = 8.4 Hz, HAT), 8.02 (d, 2H, J = 8.4 Hz, HAT), 8.09 (d, 2H, J = 8.4 Hz, HAT),10.70 (S, IH, NH); LC/MS (AP-) m/z 376.0 (M-H); tR = 2.7 min (99.9%)
N-(4-Benzoyl-phenyl -3.5-dichloro-benzamide (STX1431. CCM0103n Reaction of 4-aminobenzophenone (100 mg, 0.51 mmol) in THF (6 mL) with 3,5- dicchlorobenzoyl chloride (136 mg, 0.65 mmol) in presence of triethylamine (90 μL, 0.65 mmol) according to method A gave N-(4-benzoyl-phenyl)-3,5-dichloro-benzamide (160 mg, 0.33 mmol, 63% yield) as a white powder after purification by crystallisation in
DCM.
Rf : 0.3 (DCM); M.p.: 219-220 °C; 1H NMR (270MHz, DMSO-d*) δH 7.52-7.58 (m, 2H,
HAT), 7.63-7.67 (m, IH, HAT), 7.69-7.71 (m, IH, B r), 7.72-7.73 (m, IH, HAT), 7.79 (d, 2H,
J = 8.6 Hz, HAT), 7.89 (t, IH, J = 2.0 Hz, CCI-CHAT-CCI), 7.94 (d, 2H, J = 8.6 Hz, HAT),
7.99 (d, 2H, J = 2.0 Hz, HAT), 10.74 (s, IH, NH); 13C NMR (400MHz, DMSO-d6) δc
120.1 (CHAT), 127.2 (CHAT), 129.0 (CHAT), 129.9 (CHAT), 131.5 (CHAT), 131.7 (CHAT), 132.7 (Cq), 132.9 (CHAT), 134.8 (Cq), 137.9 (Cq), 138.2 (Cq), 143.3 (Cq), 163.7 (C=O),
195.2 (OO); LC/MS (AP-) m/z 367.3 (M-H); tR = 3.0 min (99.9%)
N-(4-Benzoyl-phenyl -3-methoxy-benzamide (STX1432. CCM01032^) Reaction of 4-aminobenzophenone (100 mg, 0.51 mmol) in THF (6 mL) with m-anisoyl chloride (136 mg, 0.65 mmol) in presence of triethylamine (90 μL, 0.65 mmol) according to method A gave N-(4-benzoyl-phenyl)-3-methoxy-benzamide (160 mg, 0.48 mmol, 94% yield) as a white powder after purification by cliromatography on silica gel (eluent EtOAc/DCM 0/100 to 5/95.
Rf : 0.4 (EtOAc/DCM 5/95) ; M.p.: 139-141 °C ; 1H ΝMR (270MHz, CDC13) δH 3.76 (s, 3H, OCH3), 7.00 (ddd, IH, J = 1.4, 2.8, 8.0 Hz, HAT), 7.28 (t, IH, J = 7.7 Hz, HAT), 7.33 7.38 (m, 2H, H ), 7.41 (dt, 2H, J = 1.5, 7.7 Hz, HAT), 7.48-7.52 (m, IH, HAT), 7.67-7.70 (m, 2H, HAT), 7.72-7.77 (m, 4H, HA/), 8.29 (s, IH, ΝH); 13C ΝMR (400MHz, DMSO-d6) δc 55.5 (OCH3), 112.6 (CHAT), 118.4 (CHAT), 118.9 (CHAT), 119.3 (CHAT), 128.3 (CHAT), 129.9 (CHAT), 129.9 (CHAT), 131.7 (CHAΓ), 132.4 (CHAT), 133.2 (Cq), 136.0 (Cq), 137.8 (Cq), 142.1 (Cq), 160.0 (Cq), 166.0 (CO), 195.9 (C=O); LC/MS (AP-) m/z 330.0 (M-H); tR = 2.2 min (99.9%)
N-(4-Benzoyl-phenyl -2.5-dichloro-benzamide (STX1433. CCM01034 Reaction of 2,5-dichlorobenzoic acid (300 mg, 1.50 mmol) in thionyl chloride (3.5 mL) then with 4-aminobenzophenone (100 mg, 0.51 mmol) in presence of triethylamine (90 μL) in THF (6 mL) according to method B gave N-(4-benzoyl-phenyl)-2,5-dichloro- benzamide (160 mg, 0.43 mmol, 84% yield) as a off-white powder after purification by cliromatography on silica gel (eluent : EtOAc/exane 1/9 to 3/7). Rf : 0.3 (DCM); M.p.: 150-153 °C; 1H NMR (270MHz, OMSO-d6) δH 7.51-7.55 (m, IH, HAT), 7.57-7.58 (m, IH, HAT), 7.59-7.61 (m, 2H, HAT), 7.62-7.66 (m, IH, H^), 7.68-7.70 (m, IH, HAT), 7.72-7.76 (m, IH, HAT), 7.77-7.80 (m, 2H, HAT), 7.85-7.88 (m, 3H, HAT), 10.96 (s, IH, NH); LC/MS (AP-) m/z 367.8 (M-H), tR = 2.3 min (98.4%)
N-f4-Benzoyl-phenylV4-propyl-benzamide (STX1434. CCM01036 Reaction of 4-aminobenzophenone (99 mg, 0.51 mmol) in THF (6 mL) with 4- propylbenzoyl chloride (100 μL, 0.65 mmol) in presence of triethylamine (90 μL, 0.65 mmol) according to method A gave N-(4-benzoyl-phenyl)-4-propyl-benzamide (136 mg, 0.40 mmol, 78% yield) as a white powder after purification by chromatography on silica gel (eluent EtOAc/DCM 0/100 to 5/95).
Rf : 0.3 (DCM); M.p.: 119-122 °C; 1H ΝMR (270MHz, CDC13) δH 0.94 (t, IH, J = 7.2 Hz, CH3), 1.59-1.72 (m, 2H, CH2-CH3), 2.65 (t, 2H, J =7.7 Hz, CH2-Ar), 7.27-7.30 (m, 2H, HAT), 7.44-7.50 (m, 2H, HAT), 7.54-7.60 (m, IH, HAT), 7.74-7.76 (m, 2H, HAT), 7.76-7.79 (m, 3H, HAT), 7.80-7.87 (m, 3H, HAT), 8.02 (s, IH, ΝH); 13C ΝMR (400MHz, CDC13) δc 13.8 (CH3), 24.3 (CH2), 37.9 (CH2), 119.1 (CHAT), 127.2 (CHAT), 128.3 (CHAT), 129.0 (CHAT), 129.9 (CH ), 131.7 (CH ), 131.8 (Cq), 132.3 (CHAT), 133.1 (Cq), 137.9 (Cq), 142.1 (Cq), 147.7 (Cq), 165.9 (C=O), 195.7 (C=O); LC/MS (AP-) m/z 341.5 (M-H); tR = 2.8 min (99.1%)
N-(4-Benzoyl-phenyl -4-methoxy-benzamide (STX1435. CCM01037 Reaction of 4-aminobenzophenone (98 mg, 0.51 mmol) in THF (6 mL) with -anisoyl chloride (90 μL, 0.65 mmol) in presence of triethylamine (90 μL, 0.65 mmol) according to method A gave N-(4-benzoyl-phenyl)-4-methoxy-benzamide (165 mg, 0.50 mmol, 98% yield) as a white powder after purification by washing the crude product with ethyl acetate and hexane.
Rf : 0.4 (EtOAc/DCM 5/95) ; M.p.: 171-174 °C ; 1H ΝMR (270MHz, OMSO-d6) δH 3.84 (s, 3H, OCH3), 7.08 (d, 2H, J = 8.6 Hz, H ), 7.53-7.58 (m, 2H, HAT), 7.64-7.66 (m, IH, HAT), 7.71-7.79 (m, 4H, HAT), 7.97-8.01 (m, 4H, HAT), 10.45 (s, IH, ΝH); 13C ΝMR (400MHz, , DMSO-< ) δc 56.0 (OCH3), 114.2 (CHAT), 119.8 (CHAT), 127.0 (Cq), 129.0 (CHAT), 129.9 (CH ), 130.3 (CH*), 131.5 (CH*), 131.9 (Cq), 132.8 (CH*), 138.1 (Cq), 144.2 (Cq), 162.7 (Cq), 165.8 (C=O), 195.1 (C=O); LC/MS (AP-) m/z 330.0 (M-H); tR 3.6 min (97.2%)
Synthesis of N-benzothiazole benzamide, acetamide and sulfonamide derivatives
STX1355, CCM01002 STX1356, STX1357, CCM01004 CC 01003
STX1358, CCM01006 STX1363, STX1364, CCM01009 CC 01008
STX1365, CCM01010 STX1366, STX1367, CCM01012 CCM01011
STX1376, CCM01013 STX1396, CCM01022
General method for synthesis of N-benzothiazole benzamide, acetamide and sulfonamide derivatives (STX1355-1358, STX1363-1367, STX1376, STX1396):
Method A : to a stirred solution of the amine (n mmol) in THF are added triethylamine (1.2n mmol) and the acyl chloride (1.2n mmol) at room temperature. After completion, ethyl acetate and water are added. The aqueous layer is extracted by ethyl acetate. The combined organic layers are washed with brine, dried (MgSO4), filtered and evaporated under reduce pressure. The crude product is then purified to give the amide. Method B : A solution of the acid (3n mmol) in thionyl chloride is refluxed 3 hours. Thionyl chloride is then removed under reduced pressure. The crude product is diluted in dry THF and added to a solution of the amine (n mmol) and triethylamine in THF. After completion, ethyl acetate and water are added . The aqueous layer is extracted by EtOAc. The combined organic layers are then washed with brine, dried (MgSO4), filtered and evaporated under reduce pressure. The crude product is purified to give the amide.
3,5-Dichloro-Λr-(4-chloro-2-methyl-benzothiazol-5-yl)-benzamide (STX1355, CCM01002) Reaction of 4-chloro-2-methyl-benzothiazol-5-ylamine (50 mg, 0.25 mmol) in THF (1.5 mL) with 3,5-dichlorobenzoyl chloride (122 mg, 0.56 mmol) in presence of triethylamine (0.5 mL) according to method A gave 3,5-Dichloro-N-(4-chloro-2-methyl-benzothiazol-5- yl)-benzamide (66 mg, 0.15 mmol, 60% yield) as a off-white powder after crystallisation in hexane/DCM. Rf 0.3 (DCM); M.p. (°C) 252-253; 1H NMR (270MHz, OMSO-d6) 2.85 (s, 3H, CH3), 7.54 (d, IH, J = 8.7 Hz, H*benzothiazole), 7.92-7.94 (m, IH, H*), 8.03-8.04 (m, 2H, H*), 8.06 (d, IH, J = 8.7 Hz, H*benzothiazole), 10.55 (s, IH, NH); 13C NMR (400MHz, DMSO-dtf) 20.4 (CH3), 120.8 (CH*benzothiazole), 123.2 (Cq), 125.6 (CH*benzothiazole), 127.1 (CH*), 131.8 (CH*), 133.2 (Cq), 134.8 (Cq), 135.0 (Cq), 137.6 (Cq), 150.8 (Cq), 163.5 (Cq), 170.4 (OO); LC/MS (AP-) m/z 368.7 (M-H); tR=3.0 min (99.9%); HRMS (FAB+) Calculated for C15H9Cl3N2OS 369.9501; Found 369.9504
Λ/-(4-Chloro-2-methyl-benzothiazol-5-yl)-4-propyl-benzamide (STX1356,
CCM01003) Reaction of 4-chloro-2-methyl-benzothiazol-5-ylamine (50 mg, 0.25 mmol) in THF (1.5 mL) with 4-propylbenzoyl chloride (90 μL, 0.56 mmol) in presence of triethylamine (0.5 mL) according to method A gave N-(4-Chloro-2-methyl-benzothiazol-5-yl)-4-propyl- benzamide (44 mg, 0.13 mmol, 52% yield) as a orange powder after crystallisation in hexane and methanol. Rf 0.3 (DCM); M.p. 184 °C; 1H ΝMR (270MHz, DMSO-^) 0.91 (t, 3H, J = 7.3 Hz, CH2-CH3), 1.59-1.68 (m, 2H, CH2-CH3), 2.65 (t, 2H, J = 7.6 Hz, CH2-Ar), 2.85 (s, 3H, CH3), 7.36 (d, 2H, J = 8.2 Hz, H*), 7.58 (d, IH, J = 8.6 Hz, H*benzothiazole), 7.95 (m, 2H, J = 8.2 Hz, HAr), 8.03 (d, IH, J = 8.6 Hz, H*benzothiazole), 10.18 (s, IH, NH); 13C NMR (400MHz, OMSO-dδ) 14.1 (CH2-CH3), 20.4 (CH3), 24.4 (CH2-CH3), 37.5 (Ar- CH2), 120.6 (CH*benzothiazole), 125.5 (CH*benzothiazole), 128.3 (CH*), 128.9 (CH*), 131.9 (Cq), 133.9 (Cq), 134.2 (Cq), 146.9 (Cq), 150.8 (Cq), 165.9 (Cq), 170.1 (OO); LC/MS (AP-) m/z 343.0 (M-H); tR=2.9 min (98.8%); HRMS (FAB+) Calculated for C18H17ClN2OS 344.0750; Found 344.0744; IR (v, cm"1) (CC14) 3429w, 2962w, 2933w, 1692s (CO), 161 lw, 1598, 1561w, 1520s, 1505s, 1430w, 1405, 1308, 1253, 1176w, 1122w, 1097w
Biphenyl-4-carboxylic acid (4-chloro-2-methyl-benzothiazol-5-yl)-amide (STX1357, CCM01004
Reaction of 4-chloro-2-methyl-benzothiazol-5 -ylamine (50 mg, 0.25 mmol) in THF (1.5 mL) with 4-biphenylcarbonyl chloride (50 μL, 0.30 mmol) in presence of triethylamine (42 μL, 0.30 mmol) according to method A gave biphenyl-4-carboxylic acid (4-chloro-2- methyl-benzothiazol-5-yl)-amide (79 mg, 0.21 mmol, 84% yield) as a pale pink powder after crystallisation in hexane/DCM.
Rf 0.3 (DCM); M.p. 210-211 °C; 1H NMR (270MHz, OMSO-d6) 2.86 (s, 3H, CH3), 7.42- 7.47 (m, IH, H*), 7.49-7.52 (m, 2H, H*), 7.61 (d, IH, J = 8.4 Hz, H*benzothiazole), 7.77 (d, 2H, J = 7.4 Hz, H*), 7.88 (d, 2H, J = 8.4 Hz, H*), 8.04 (d, IH, J = 8.4 Hz, H*benzothiazole), 8.13 (d, 2H, J = 8.4 Hz, H*), 10.33 (s, IH, NH); 13C NMR (400MHz, OMSO-d6) 20.4 (CH3), 120.7 (CH*benzothiazole), 123.1 (Cq), 125.6 (CH*benzothiazole), 127.2 (CH*), 127.4 (CH*), 128.7 (CH*), 129.0 (CH*), 129.6 (CH*), 133.1 (Cq), 133.8 (Cq), 134.3 (Cq), 139.5 (Cq), 143.9 (Cq), 150.8 (Cq), 165.7 (Cq), 170.2 (OO); LC/MS (AP+) m/z 379.02 (M+H); tR=2.8 min (99.4%); HRMS (FAB+) Calculated for C21H15C1N20S 378.0594; Found 378.0590
2,5-Dichloro-Λ/'-(4-chloro-2-methyl-benzothiazol-5-yl)-benzamide (STX1358, CCM01006) Reaction of 2,5-dichlorobenzoic acid (145 mg, 0.76 mmol) in thionyl chloride (5 mL) then with 4-chloro-2-methyl-benzothiazol-5-ylamine (50 mg, 0.25 mmol) in presence of triethylamine (0.5 mL) in THF (2 mL) according to method B gave 2,5-dichloro-N-(4- chloro-2-methyl-benzothiazol-5-yl)-benzamide (50 mg, 0.13 mmol, 52% yield) as a white powder after purification by chromatography on silica gel (eluent : DCM). Rf 0.35 (DCM); M.p. 175 °C; 1H ΝMR (270MHz, DMSO-^) 2.85 (s, 3H, CH3), 7.61- 7.62 (m, 2H, H*), 7.66 (d, IH, J = 8.6 Hz, H*benzothiazole), 7.76 (m, IH, H*), 8.05 (d, IH, J = 8.6 Hz, H*benzothiazole), 10.52 (s, IH, NH); LC/MS (AP+) m/z 370.8 (M+H); tR=2.4 min (99.5%) HRMS (FAB+) Calculated for C15H9Cl3N2OS 369.9501; Found 369.9503
W-(4-Chloro-2-methyl-benzothiazol-5-yl)-2-(4-methoxy-phenyl)-acetamide (STX1363, CCM01008)
Reaction of 4-chloro-2-methyl-benzothiazol-5-ylamine (50 mg, 0.25 mmol) in THF (1.5 mL) with 4-methoxyphenylacetyl chloride (46 μL, 0.30 mmol) in presence of triethylamine (42 μL, 0.30 mmol) according to method A gave N-(4-chloro-2-methyl- benzothiazol-5-yl)-2-(4-methoxy-phenyl)-acetamide (50 mg, 0.14 mmol, 56% yield) as a white powder after crystallisation in hexane/DCM.
Rf 0.4 (EtOAc/DCM 1/9); M.p. 173-174 °C; 1H ΝMR (270MHz, CDC13) 2.83 (s, 3H, CH3), 3.76 (s, 2H, CH2), 3.82 (s, 3H, OCH3), 6.95 (d, 2H, J = 8.6 Hz, H*), 7.30 (d, 2H, J = 8.6 Hz, H*), 7.65 (d, IH, J = 8.9 Hz, H*benzothiazole), 7.80 (s, IH, NH), 8.40 (d, IH, J = 8.9 Hz, H*benzotlιiazole); LC/MS (AP+) m/z 347.0 (M+H); tR=2.1 min (99.2%); HRMS (FAB+) Calculated for Cι7H15ClN2O2S 346.0543; Found 346.0542
Biphenyl-4-carboxylic acid (4-chloro-2-methyl-benzothiazol-5-yl)-amide (STX1364, CCM01009) Reaction of 4-biphenylacetic acid (160 mg, 0.76 mmol) in thionyl chloride (1 mL) then with 4-chloro-2-methyl-benzothiazol-5-ylamine (50 mg, 0.25 mmol) in presence of triethylamine (0.5 mL) in THF (2 mL) according to method B gave biphenyl-4-carboxylic acid (4-chloro-2-methyl-benzothiazol-5-yl)-amide (60 mg, 0.15 mmol, 60% yield) as a pink powder after purification by chromatography on silica gel (eluent : DCM). Rf : 0.2 (DCM); M.p.:168-169 °C; 1H NMR (270MHz, CDC13) δH 2.83 (s, 3H, CH3), 3.87 (s, 2H, CH2), 7.35-7.38 (m, IH, H*), 7.41-7.48 (m, 4H, H*), 7.57-7.61 (m, 2H, H*), 7.64-7.69 (m, 3H, H*), 7.82 (s, IH, NH), 8.42 (d, IH, J = 9.1 Hz, H*benzothiazole); LC/MS (AP+) m/z 393.3; ..tR = 3.2 min (99.8%); HRMS (FAB+) Calculated for C22H17ClN2OS 392.0750; Found 392.0742 V-(4-Chloro-2-methyl-benzothiazol-5-yl)-3-methoxy-benzamide (STX1365, CCM01010)
Reaction of 4-chloro-2-methyl-benzothiazol-5-ylamine (50 mg, 0.25 mmol) in THF (1.5 mL) with m-anisoyl chloride (41 μL, 0.30 mmol) in presence of triethylamine (42 μL, 0.30 mmol) according to method A gave N-(4-chloro-2-methyl-benzothiazol-5-yl)-3- methoxy-benzamide (70 mg, 0.21 mmol, 84% yield) as a orange powder after crystallisation in hexane/DCM.
Rf : 0.4 (EtOAc/DCM 5/95); M.p.:177-179 °C; IH ΝMR (270MHz, DMSO- 5) δH 2.83 (s, 3H, CH3), 3.82 (s, 3H, OCH3), 7.17 (ddd, IH, J = 1.3, 2.7, 8.1 Hz, H*), 7.44 (dd, IH, J = 7.7, 8.1 Hz, H*), 7.54-7.55 (m, IH, H*), 7.55 (d, IH, J = 8.4 Hz, H*benzoti iazole), 7.56-7.61 (m, IH, H*), 8.02 (d, IH, J = 8.4 Hz, H*benzothiazole), 10.20 (s, IH, ΝH); LC/MS (AP+) m/z 333.2; ..tR = 2.6 min, (99.7%); HRMS (FAB+) Calculated for C16H13ClΝ2O2S 332.0386; Found 332.0383
W-(4-Chloro-2-methyl-benzothiazol-5-yl)-4-methoxy-benzamide (STX1366, CC 01011)
Reaction of 4-chloro-2-methyl-benzothiazol-5-ylamine (50 mg, 0.25 mmol) in THF (1.5 mL) with /7-anisoyl chloride (41 μL, 0.30 mmol) in presence of triethylamine (42 μL, 0.30 mmol) according to method A gave N-(4-chloro-2-methyl-benzothiazol-5-yl)-4-methoxy- benzamide (65 mg, 0.19 mmol, 76% yield) as a white powder after crystallisation in hexane/DCM.
Rf : 0.4 (EtOAc/DCM 1/9); M.p.: 182 °C IH ΝMR (270MHz, DMSO-fifc) δH 2.83 (s, 3H, CH3), 3.83 (s, 3H, OCH3), 7.06 (d, 2H, J = 8.8 Hz, H*), 7.55 (d, IH, J = 8.5 Hz, H*benzothiazole), 7.99 (d, IH, J = 8.5 Hz, H*benzothiazole), 8.00 (d, 2H, J = 8.8 Hz, H*), 10.06 (s, IH, ΝH); LC/MS (AP+) m/z 333.2;.tR = 2.6 min (99.9%); HRMS: (FAB+) Calculated for C16H13ClΝ2O2S 332.0386; Found 332.0383 W-(4-Chloro-2-methyl-benzothiazol-5-yl)-2-(3-methoxy-phenyI)-acetamide (STX1367, CCM01012)
Reaction of 4-chloro-2-methyl-benzothiazol-5-ylamine (50 mg, 0.25 mmol) in THF (1.5 mL) with 3-methoxyphenylacetyl chloride (47 μL, 0.30 mmol) in presence of triethylamine (42 μL, 0.30 mmol) according to method A gave N-(4-chloro-2-methyl- benzothiazol-5-yl)-2-(3-methoxy-phenyl)-acetamide (40 mg, 0.11 mmol, 48% yield) as a white powder after crystallisation in hexane/DCM.
Rf : 0.4 (EtOAc/DCM 1/9); M.p.:147-148 °C; 1H ΝMR (270MHz, DMSO- ) δH 2.82 (s, 3H, CH3), 3.72 (s, 2H, CH2), 3.75 (s, 3H, OCH3), 6.80-6.84 (m, IH, H*), 6.93-6.96(m, 2H, HAT), 7.25 (dd, IH, J = 8.2, 8.2 Hz, H*), 7.65 (d, IH, J = 8.6 Hz, H*benzothiazole), 7.95 (d, IH, J = 8.6 Hz, H*benzothiazole), 9.90 (s, IH, NH); LC/MS (AP+) m/z 347.2 (M+H); tR = 2.5 min, (99.3%); HRMS (FAB+) Calculated for C17H15C1N202S 346.0543; Found 346.0541
W-(4-Chloro-2-methyl-benzothiazol-5-yl)-2-(4-chloro-phenyl)-acetamide (STX1376, CC 01013)
Reaction of 4-chloro-2-methyl-benzothiazol-5 -ylamine (50 mg, 0.25 mmol) in THF (1.5 mL) with 4-cnlorophenylacetyl chloride (65 mg, 0.30 mmol) in presence of triethylamine (42 μL, 0.30 mmol) according to method A gave N-(4-chloro-2-methyl-benzothiazol-5- yl)-2-(4-chloro-phenyl)-acetamide (65 mg, 0.19 mmol, 80% yield) as a white powder after crystallisation in hexane/DCM.
Rf : 0.5 (EtOAc/DCM 1/9); M.p.:227-229 °C; 1H ΝMR (270MHz, DMSO- ) δH 2.82 (s, 3H, CH3), 3.76 (s, 2H, CH2), 7.38 (s, 4H, H*), 7.63 (d, IH, J = 8.5 Hz, H*benzothiazole), 7.95 (d, IH, J = 8.5 Hz, H*benzothiazole), 9.96 (s, IH, ΝH); LC/MS (AP-) m/z 350.8 (M-H); tR = 2.5 min, (96.5%) HRMS (FAB+) Calculated for C16Hι2Cl2Ν2OS 350.0047; Found 350.0048
W-(4-Chloro-2-methyl-benzothiazol-5-yl)-2-(2,4-dichloro-phenyl)-acetamide (STX1396, CCM01022) Reaction of 2,4-dichlorophenylacetic acid (163 mg, 0.79 mmol) in thionyl chloride (2 mL) then with 4-chloro-2-methyl-benzothiazol-5-ylamine (52mg, 0.26 mmol) in presence of triethylamine (0.5 mL) in THF (3 mL) according to method B gave N-(4-chloro-2-methyl- benzothiazol-5-yl)-2-(2,4-dichloro-phenyl)-acetamide (33 mg, 0.09 mmol, 35% yield) as a white powder after crystallisation in hexane/DCM.
Rf : 0.4 (EtOAc/DCM 1/9); M.p.:257-258 °C; 1H ΝMR (270MHz, DMSO-d*) δH 2.81 (s, 3H, CH3), 3.93 (s, 2H, CH2), 7.38-7.42 (m, IH, H*), 7.47-7.52 (m, IH, H*), 7.59-7.60 (m, IH, H*), 7.62 (d, IH, J = 8.7 Hz, H*benzothiazole), 7.95 (d, IH, J = 8.7 Hz, H*benzothiazole), 10.05 (bs, IH, ΝH); LC/MS (AP-) m/z 382.8 (M-H); tR = 2.6 min (95.1%); HRMS (FAB+) Calculated for C16HπCl3Ν2OS 383.9658; Found 383.9653
Synthesis of N-(5-tetralone)-benzamide derivatives
STX1465, CCM01044 STX142, CC 01050
STX1473, CCM01051 STX1474, CCM01052 STX1475, CCM01053
General method for synthesis of W-(5-tetraIone)-benzamide derivatives (STX1465 and STX1470-1475):
Method A : to a stirred solution of the amine (n mmol) in THF are added triethylamine (1.2n mmol) and the acyl chloride (1.2n mmol) at room temperature. After completion, ethyl acetate and water are added. The aqueous layer is extracted by ethyl acetate. The combined organic layers are washed with brine, dried (MgSO4), filtered and evaporated under reduce pressure. The crude product is then purified to give the amide. Method B : A solution of the acid (3n mmol) in thionyl chloride is refluxed 3 hours. Thionyl chloride is then removed under reduced pressure. The crude product is diluted in dry THF and added to a solution of the amine (n mmol) and triethylamine in THF. After completion, ethyl acetate and water are added . The aqueous layer is extracted by EtOAc. The combined organic layers are then washed with brine, dried (MgSO4), filtered and evaporated under reduce pressure. The crude product is purified to give the amide.
2,5-Dichloro-W-(5-oxo-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide (CCM01044, STX1465) Reaction of 2,5-dichlorobenzoic acid (300 mg, 1.50 mmol) in thionyl chloride (3.5 mL) then with 6-Amino-3,4-dihydro-2H-naphthalen-l-one (80 mg, 0.50 mmol) in presence of triethylamine (90 μL, 0.65 mmol) in TΗF (6 mL) according to method B gave 2,5- dichloro-N-(5-oxo-5,6,7,8-tetraliydro-naphthalen-2-yl)-benzamide (150 mg, 0.48 mmol, 76% yield) as a grey powder after purification by crystallisation in DCM. 1H ΝMR (270 MHz, DMSO-^) δH 1.92-1.97 (2H, m, CH2), 2.46-2.50 (2H, m CH2), 2.84 (2H, t, J= 5.7 Hz, CH2), 7.48-7.52 (3H, m, H*), 7.67 (2H, bs, H*), 7.78 (IH, d, J= 8.6 Hz, H*), 10.78 (IH, s, ΝH) ; LC/MS (AP-) tr= 2.2min (93.5%), m z 315.2 (M-H).
Biphenyl-4-carboxylic acid (5-oxo-5,6,7,8-tetrahydro-naphthalen-2-yl)-amϊde (CCM01049, STX1470)
Reaction of 6-Amino-3,4-dihydro-2H-naphthalen-l-one (80 mg, 0.50 mmol) in TΗF (6 mL) with 4-biphenylcarbonyl chloride (143 mg, 0.65 mmol) in presence of triethylamine (90 μL, 0.65 mmol) according to method A gave biphenyl-4-carboxylic acid (5-oxo- 5,6,7,8-tetrahydro-naphthalen-2-yl)-amide (150 mg, 0.43 mmol, 86% yield) as a white powder after purification by flash chromatography on silica gel (EtOAc/DCM 1/9 to 2/8). 1H ΝMR (270 MHz, OMSO-d6) δH 1.92-1.98 (2H, m, CH2), 2.44-2.51 (2H, m, CH2), 2.85 (2H, t, J= 5.9 Hz, CH2), 7.34-7.45 (3H, m, H*), 7.65-7.70 (3H, m, H*), 7.75-7.80 (4H, m, H*), 7.98 (2H, d, J= 8.6 Hz, H*), 10.48 (IH, s, NH).
3,5-Dichloro-W-(5-oxo-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide (CCM01050, STX1472) Reaction of 6-Amino-3,4-dihydro-2H-naphthalen-l-one (80 mg, 0.50 mmol) in TΗF (6 mL) with 3,5-dichlorobenzoyl chloride (136 mg, 0.65 mmol) in presence of triethylamine (90 μL, 0.65 mmol) according to method A gave 3,5-dichloro-N-(5-oxo-5,6,7,8- tetrahydro-naphthalen-2-yl)-benzamide (140 mg, 0.42 mmol, 84% yield) as a brown powder after washing by DCM.
Mp 226-228 °C ; 1H ΝMR (270 MHz, OMSO-d6) δH 1.92-1.97 (2H. m, CH2), 2.49 (2H, t, J= 6.0 Hz, CH2), 2.85 (2H, t, J = 6.0 Hz, CH2), 7.62 (IH, dd, J = 2.1;8.4 Hz, H*tetralone), 7.71 (IH, d, J= 1.7 Hz, H*), 7.79 (IH, d, J = 8.4 Hz, H*tetralone), 7.81 (IH, d, J= 2.1 Hz, H*tetralone), 7.90 (2H, d, J= 1.7 Hz, H*), 8.08 (2H, d, J = 8.4 Hz, H*), 10.61 (IH, s, ΝH) ; 13C ΝMR (50 MHz, OMSO-d6) δc 23.4 (CH2), 29.8 (CH2), 38.9 (CH2), 118.7 (CH*), 119.6 (CH*), 127.1 (CH*), 128.0 (CH*), 128.6 (Cq), 131.7 (Cq), 134.8 (CH*), 138.2 (Cq), 143.5 (Cq), 146.2 (Cq),163.6 (CO), 196.8 (CO). V-(5-Oxo-5,6,7,8-tetrahydro-naphthalen-2-yl)-4-propyl-benzamide (CCM01051, STX1473)
Reaction of 6-Amino-3,4-dihydro-2H-naphthalen-l-one (80 mg, 0.50 mmol) in TΗF (6 mL) with 4-propylbenzoyl chloride (100 μL, 0.65 mmol) in presence of triethylamine (90 μL, 0.65 mmol) according to method A gave N-(5-oxo-5,6,7,8-tetrahydro-naphthalen-2- yl)-4-propyl-benzamide (140 mg, 0.45 mmol, 90% yield) as a white powder after purification by flash chromatography on silica gel (eluent : EtOAc/hexane 2/8 to 4/6).
Mp 168-170 °C ; 1H ΝMR (270 MHz, OMSO-d6) δH 0.89 (3H, t, J = 7.4 Hz, CH3), 1.54- 1.68 (2H, m, CH2propyl), 1.97-2.07 (2H. m, CH2tetralone), 2.55 (2H, t, J= 6.5 Hz, CH2), 2.63 (2H, t, J= 7.4 Hz, CH2), 2.91 (2H, t, J = 6.5 Hz, CH2), 7.35 (2H, d, J= 8.4 Hz, H*), 7.70 (IH, dd, J= 1.7;8.7 Hz, H*tetralone), 7.83-7.89 (4H, m, H*), 10.41 (IH, s, ΝH) ; 13C ΝMR (50 MHz, OMSO-d6) δc 14.1 (CH3), 23.4 (CH2tetralone), 24.4 (CH2), 29.8 (CH2tetralone), 37.5 (CH2), 38.9 (CH2tetralone), 118.5 (CH*), 119.3 (CH*), 127.9 (CH*), 128.2 (Cq), 128.3 (CH*), 128.9 (CH*),132.5 (Cq), 144.2 (Cq), 146.2 (Cq), 147.0 (Cq),166.4 (CO), 196.8 (CO) 4-Methoxy-/V-(5-oxo-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide (CCM01052, STX1474)
Reaction of 6-Amino-3,4-dihydro-2H-naphthalen-l-one (80 mg, 0.50 mmol) in TΗF (6 mL) with/>-anisoyl chloride (90 mg, 0.65 mmol) in presence of triethylamine (90 μL, 0.65 mmol) according to method A gave 4-methoxy-N-(5-oxo-5,6,7,8-tetrahydro-naphthalen-2- yl)-benzamide (135 mg, 0.46 mmol, 92% yield) as a white powder after purification by flash chromatography on silica gel (eluent : EtOAc/DCM 0/10 to 2/8). Mp 163-166 °C ; 1H ΝMR (270 MHz, OMSO-d6) δH 1.99-2.06 (2H. m, CH2), 2.54 (2H, t, J= 6.0 Hz, CH2), 2.90 (2H, t, J= 5.8 Hz, CH2), 3.82 (3H, s, OCH3), 7.06 (2H, d, J= 8.7 Hz, H*), 7.70 (IH, dd, J= 2.0;8.7 Hz, H*tetralone), 7.81-7.84 (2H, m, H*), 7.95 (2H, d, J= 8.7 Hz, H*), 10.32 (IH, s, NH).
3- ethoxy-W-(5-oxo-5,6,7,8-tetrahydro-naphthalen-2-yl)-benzamide (CCM01053, STX1475) Reaction of 6-Amino-3,4-dihydro-2H-naphthalen-l-one (80 mg, 0.50 mmol) in TΗF (6 mL) with m-anisoyl chloride (90 μL, 0.65 rnrnol) in presence of triethylamine (90 μL, 0.65 mmol) according to method A gave 3-methoxy-N-(5-oxo-5,6,7,8-tetrahydro- naphthalen-2-yl)-benzamide (135 mg, 0.46 mmol, 92% yield) as a white powder after purification by flash cliromatography on silica gel (eluent : EtOAc/DCM 0/10 to 2/8). Mp 142-143 °C ; 1H ΝMR (270 MHz, OMSO-d6) δH 2.01-2.09 (2H. m, CH2), 2.57 (2H, t, J= 6.3 Hz, CH2), 2.93 (2H, t, J = 6.0 Hz, CH2), 3.84 (3H, s, OCH3), 7.18 (IH, ddd, J= 1.0;2.5;7.9 Hz, H*), 7.43-7.56 (3H, m, HAr), 7.73 (IH, dd, J= 2.0;8.7 Hz, H*tetralone), 7.83 (IH, d, J = 1.0 Hz, H*tetralone), 7.86 (IH, d, J = 8.7 Hz, H*tetralone), 10.32 (IH, s, ΝH). Synthesis of iV-(5-acetophenone)-benzamide derivatives
STX1462, CCM01041 STX1463, CCM01042
STX1464, CC 01043 STX1468, CCM01047 STX1469, CC 01048
General method for synthesis of Λ/-(5-acetophenone)-benzamide derivatives (STX1461-1464 and STX1468-1469):
Method A : to a stirred solution of the amine (n mmol) in THF are added triethylamine (1.2n mmol) and the acyl chloride (1.2n mmol) at room temperature. After completion, ethyl acetate and water are added. The aqueous layer is extracted by ethyl acetate. The combined organic layers are washed with brine, dried (MgSO4), filtered and evaporated under reduce pressure. The crude product is then purified to give the amide. Method B : A solution of the acid (3n mmol) in thionyl chloride is refluxed 3 hours. Thionyl chloride is then removed under reduced pressure. The crude product is diluted in dry THF and added to a solution of the amine (n mmol) and triethylamine in THF. After completion, ethyl acetate and water are added . The aqueous layer is extracted by EtOAc. The combined organic layers are then washed with brine, dried (MgS04), filtered and evaporated under reduce pressure. The crude product is purified to give the amide.
Bipherιyl-4-carboxylic acid (4-acetyl-phenyl)-amide (CCM01040, STX1461)
Reaction of 4-aminoacetophenone (75 mg, 0.55 mmol) in THF (6 mL) with 4-biphenylcarbonyl chloride (147 mg, 0.68 mmol) in presence of triethylamine (90 μL, 0.65 mmol) according to method A gave biphenyl-4-carboxylic acid (4-acetyl-phenyl)- amide (90 mg, 0.28 mmol, 50% yield) as a white powder after washing by water and ethyl acetate.
Mp 283-285 °C; 1H NMR (270 MHz, DMSO-J*) δH 2.55 (3H, s, CH3), 7.42-7.45 (IH, m, HAT), 7.48-7.54 (2H, m, H*), 7.75-7.78 (2H, m, H*), 7.86 (2H, d, J= 8.4 Hz, H*), 7.97 (4H, s, H*), 8.08 (2H, d, J= 8.4 Hz, H*), 10.61 (IH, s, NH); LC/MS (AP-) t = 2.3 min (99.2%), m/z 314.3 (M-H)
Λ/-(4-AcetyI-phenyl)-3,5-dichloro-benzamide (CCM01041, STX1462)
Reaction of 4-aminoacetophenone (76 mg, 0.56 mmol) in THF (6 mL) with 3,5- dichlorobenzoyl chloride (145 mg, 0.69 mmol) in presence of triethylamine (90 μL, 0.65 mmol) according to method A gave N-(4-acetyl-phenyl)-3,5-dichloro-benzamide (145 mg,
0.47 mmol, 84% yield) as a white powder after washing by dichloromethane.
Mp 199-200°C ; Rf. 0.3 (Ethyl acetate/hexane, 3:7) ; 1H ΝMR (270 MHz, OMSO-d6) δH
2.55 (3H, s, CH3), 7.86-7.92 (3H, m, H*), 7.97-8.00 (4H, m, H*), 10.69 (IH, s, ΝH); 13C ΝMR (50 MHz, OMSO-d6) δc 27.2 (CH3), 120.3 (CH*), 127.3 (CH*), 130.0 (CH*),
131.9 (Cq), 133.1 (Cq), 135.0 (CH*), 138.4 (Cq), 143.7 (Cq), 163.8 (CO), 197.3 (CO);
LC/MS (AP-) tr= 2.5min (99.5%), m/z 306.1 (M-H).
Λf-(4-AcetyI-phenyl)-4-propyl-benzamide (CCM01042, STX1463) Reaction of 4-aminoacetophenone (76 mg, 0.56 mmol) in THF (6 mL) with 4- propylbenzoyl chloride (100 μL, 0.65 mmol) in presence of triethylamine (90 μL, 0.65 mmol) according to method A gave N-(4-acetyl-phenyl)-4-propyl-benzamide (125 mg, 0.44 mmol, 78% yield) as a white powder after washing by dichloromethane and hexane. Mp 174-176°C ; R 0.2 (Ethyl acetate/hexane, 4:8) ; 1H ΝMR (270 MHz, OMSO-d6) δH 0.89 (3H, t, J= 7.2 Hz, CH3), 1.55-1.59 (2H, m, CH2-CH3), 2.54 (3H, s, CH3), 2.63 (2H, t, J = 7.2 Hz, ArCH2), 7.35 (2H, d, J= 8.3 Hz, H*), 7.89 (2H, d, J= 8.3 Hz, H*), 7.94- 7.98 (4H,m, H*), 10.47 (IH, s, ΝH); 13C ΝMR (50 MHz, OMSO-d6) δc 14.3 (CH3), 24.6 (CH2), 27.1 (CH3CO), 37.7 (CH2), 120.0 (CH*), 128.5 (CH*), 129.1 (CH*), 130.0 (CH*), 132.5 (Cq), 132.7 (Cq), 144.4 (Cq), 147.2 (Cq), 166.6 (CO), 197.3 (CO); LC/MS (AP-) t, = 2.4min (99.1%), m/z 280.2 (M-H). Λf-(4-Acetyl-phenyl)-2,5-dichloro-benzamide (CCM01043, STX1464)
Reaction of 2,5-dichlorobenzoic acid (300 mg, 1.50 mmol) in thionyl chloride (3.5 mL) then with 4-aminoacetophenone (85 mg, 0.63 mmol) in presence of triethylamine (90 μL, 0.65 mmol) in THF (6 mL) according to method B gave N-(4-Acetyl-phenyl)-2,5- dichloro-benzamide (150 mg, 0.48 mmol, 76% yield) as a orange powder after purification by crystallisation in DCM.
Rf. 0.35 (Ethyl acetate/hexane, 4:6) ; 1H ΝMR (270 MHz, OMSO-d6) δH 2.49 (3H, s, CH3), 7.55-7.56 (2H, m, H*), 7.73 (IH, dd, J= 1.0; 2.0 Hz, H*), 7.75-7.78 (2H, m, H*), 7.91-7.94 (2H, m, H*), 10.86 (IH, s, ΝH).
Λr-(4-Acetyl-phenyl)-4-methoxy-benzamide (CCM01047, STX1468)
Reaction of 4-aminoacetophenone (74 mg, 0.55 mmol) in THF (6 mL) with -anisoyl chloride (90 mg, 0.65 mmol) in presence of triethylamine (90 μL, 0.65 mmol) according to method A gave N-(4-acetyl-phenyl)-4-methoxy-benzamide (110 mg, 0.41 mmol, 75% yield) as a white powder after washing by dichloromethane.
Mp 222-223°C ; Rf 0.35 (Ethyl acetate/DCM, 1:9) ; 1H ΝMR (270 MHz, OMSO-d6) δH 2.53 (3H, s, CH3), 3.83 (3H, s, OCH3), 7.05-7.08 (2H, m, H*), 7.93-7.98 (6H, m, H*), 10.38 (IH, s, ΝH) ; 13C ΝMR (50 MHz, OMSO-d6) δc 27.1 (CH3CO), 56.1 (CH3), 114.4 (CH*), 120.0 (CH*), 127.2 (Cq), 130.0 (CH*), 130.5 (CH*), 132.4 (Cq), 144.5 (Cq), 162.8 (Cq), 166.0 (CO), 197.3 (CO).
Λf-(4-Acetyl-phenyl)-3-methoxy-benzamide (CCM01048, STX1469)
Reaction of 4-aminoacetophenone (72 mg, 0.53 mmol) in THF (6 mL) with m-anisoyl chloride (90 μL, 0.65 mmol) in presence of triethylamine (90 μL, 0.65 mmol) according to method A gave N-(4-acetyl-phenyl)-3-methoxy-benzamide (110 mg, 0.41 mmol, 77% yield) as a white powder after washing by dichloromethane.
Mp 152-154°C ; Rf 0.35 (Ethyl acetate/DCM, 1:9) ; 1H ΝMR (270 MHz, OMSO-d6) δH 2.49 (3H, s, CH3), 3.78 (3H, s, OCH3), 7.12 (IH, ddd, J = 1.0;2.7;8.1 Hz, H*), 7.37-7.44 (2H, m, H*), 7.49 (IH, ddd, J= 1.0;1.5;7.4 Hz, H*), 7.85 7.94 (4H, m, H*), 10.48 (IH, s, ΝH) ; 13C MR (50 MHz, DMSO-< ) δc 27.4 (CH3CO), 56.2 (OCH3), 113.9 (CH*), 118.5 (CH*), 120.4 (CH*), 120.9 (CH*), 130.2 (CH*), 130.5 (CH*), 132.9 (Cq), 136.8 (Cq), 144.4 (Cq), 160.1 (Cq), 166.6 (CO), 197.5 (CO).
DETAILED DESCRIPTION OF FIGURES Separation of Cortisone and Cortisol
Several solvent systems claiming to separate cortisone from cortisol are detailed in the literature[30, 31]. Before running an assay, 10 mg / ml solutions of cortisone and cortisol were prepared in IMS and 50 μl aliquots were spotted separately onto a silica gel TLC plate 3 cm from the bottom edge and 2.5 cm apart. The plate was run in a TLC tank in 200 ml of CH2CI2 : IMS 92 : 8 7V [30] until the solvent front reached the top of the plate. The plate was air dried and sprayed with 0.1 % Rhodamine B in IMS to visualise the spots. The Table below describes the separation obtained.
Table Separation of cortisone from cortisol by TLC
The separation was considered adequate for use in an enzyme assay. Figure 1 (Extraction efficiencies obtained with four extraction methods)
The literature details several methods of extracting cortisol from aqueous solution[30, 31]. In order to select a method for use, a 14C-labelled cortisol was obtained from NEN. A stock was prepared in PBS containing 4000 DPM in 50 μl with cold cortisol (1 μg) added as a carrier. The final ethanol concentration was 0.4 %. Aliquots of this solution were added to glass tubes (100 μl) and the following extractions were carried out: 1. 1 ml CH2CI2ι vortex and pass through phase separating filter paper (Whatman, IPS) 2. 1 ml ethyl acetate, vortex and pass through phase separating filter paper 3. 1 ml CH2CI2 and 200 μl 0.05 % CaCI2, vortex, centrifuge (500 X g for 5 min) and remove upper aqueous phase 4. 1 ml ethyl acetate and 200 μl 0.05 % CaCI2, vortex, centrifuge (500 X g for 5 min) and collect upper organic phase. The organic phases were dried and the residues were taken up in 100 μl IMS. An aliquot of this (50 μl) was spotted onto a TLC plate and run as before. Following visualisation with Rhodamine B, the spots were scraped into scintillation vials and counted on a liquid scintillation counter (TriCarb) in 5 ml Ultima gold scintillant. Extraction efficiencies were calculated and are given in Figure 1.
Assay of Human and Rat Hepatic Microsomal 11 β-HSD1 Activity using a TLC Separation of Substrate from Product
Figure 2 (Comparison of 113-HSD1 activity in rat and human hepatic microsomes)
This experiment was carried out to compare the enzyme activity in hepatic microsomes from human and rat and to assess minimum microsomal protein concentrations necessary for reasonable measurement of enzyme activity. The assay was carried out in Buffer 2 and the cortisone concentration used was 2 μM containing 0.5 μCi per incubation 3H-cortisone. Rat and human hepatic microsomes were tested at concentrations ranging from 400 μg to 50 μg microsomal protein per incubation in a final incubation volume of 100 μl in glass tubes. Buffer was substituted for microsomal protein for blanks. Samples were incubated for 1 h in a shaking water bath at 37°C and the assay was stopped by the addition of 1 ml ethyl acetate. To correct for recovery, 50 μl 14C-cortisol (approximately 4000 DPM per tube) was added to the samples followed by 200 μl 0.05 % CaCI2. The samples were vortexed and centrifuged as detailed in 2.1. The upper organic phase was removed into clean tubes and dried down. The residue was taken up in 100 μl IMS and 50 μl aliquots were spotted onto TLC plates which were run as described in 2.1. DPM were measured on a TriCarb liquid scintillation counter using a dual label programme. Recovery was determined from the DPM obtained in 50 μl 14C-cortisol solution which was counted with the samples. The results are given in Figure 2.
It had been expected that there would be higher activity in the rat microsomes but this was not the case. Using 50 μg microsomal protein per well the activities of the rat and human enzymes were quite similar, rat microsomal activity was 0.7 pmol/mg/min and human was 0.5 pmol/mg/min. Good activity was detected in the human microsomes although this was not particularly related to microsomal protein concentration. It was suspected that the concentration range examined was too high so in the next experiment a lower range was tested. Also, the dependence of apparent enzyme activity with incubation time was looked at. A linear relationship between DPM and incubation time would indicate that increases over blank were due to enzyme activity and not due to an artefact.
Figure 3 (Effect of incubation time on human microsomal 11 B-HSD1 activity) and Figure 4 (Effect of microsomal protein concentration on human microsomal 11 B-HSD1 activity)
In the next test, the same assay method was followed except that only human hepatic microsomes were examined and the concentration range of these was from 3.7 μg per sample to 100 μg per sample. The samples were incubated for 60 min, 45 min, 30 min or 15 min in a shaking water bath at 37°C and were stopped, extracted and the substrate and product were separated as detailed above. Figure 3 and Figure 4 illustrate the results:
Conclusion (Fig. 3):
• There is linearity of enzyme activity with incubation times up to 30 min with all microsomal protein concentrations tested
Conclusion (Fig. 4):
• Enzyme activity is linear with microsomal protein concentrations below 30 μg per sample
Figure 5 (Substrate (cortisone) saturation curve for human hepatic microsomal 11 β HSD1)
Substrate requirement was examined using the classical assay. The DPM in each group was kept constant (0.5 μCi / sample) and the cold cortisone was varied from 2 μM down to 43.8 nM. The assay was carried out with 10 μg microsomal protein per sample and the incubation time was 30 min at 37°C. The buffer used for this assay was Buffer 1 Figure 5 shows the data obtained. Figure 6 (Lineweaver-Burke Plot)
A double reciprocal plot of these data (Lineweaver-Burke) gives an apparent Km for cortisone of 660 nM, but it should be noted that it is unlikely that initial enzyme activity rates were measured at the lower [cortisone] over a 30 mins incubation. Figure 6 shows the Lineweaver-Burke plot obtained:
Figure 7 (ICsn determination for Glycyrrhetinic acid) and Figure 8 ( Csn determination for Carbenoxolone)
In order to reproduce the inhibition data given in [14], it was decided to use the cortisone concentration quoted in the reference (175 nM) to examine compound activity, even though Figure 5 and Figure 6 suggest that this concentration is not saturating with 10 μg microsomal protein per 30 mins incubation at 37°C. The following experiment was carried out with a lower microsomal protein concentration (5 μg) in the same buffer conditions as in the last experiment (Buffer 1) over a 30 mins incubation at 37°C in the presence of 175 nM cortisone (0.5 μCi / sample). Glycyrrhetinic acid and carbenoxolone were examined at concentrations from 3 μM to 0.012μM (DMSO concentration 1 % throughout) and the data are shown in Figure 7 and Figure 8.
The reported IC50 for carbenoxolone is 330 nM [14], which is approximately three times less active than observed in the above experiment. It appears that these assay conditions support good enzyme activity which should be measurable in a 96 well plate method.
Cortisol Immunoassay
An enzyme immunoassay kit was obtained from Assay Designs, Inc. The antibody provided in the kit is a mouse monoclonal reported to cross react 100% with cortisol (the enzyme product) and <0.1 % with cortisone (the enzyme substrate). The kit is designed for the analysis of cortisol levels in saliva, urine, serum and plasma and also in tissue culture media, not for microsomal enzyme activity. Figure 9 (11 B-HSD1 activity measured by Immunoassay)
The methodology for the enzyme assay used with the kit was based on the paper by Barf et al. [14] Human hepatic microsomes were incubated in Buffer 1 at concentrations ranging from 25 μg protein per point to 200 μg protein per point in the presence of cortisone ranging from 44 nM to 700 nM for 1 h. Also, these groups were tested in the presence and absence of 0.9 % Tween 80 since this detergent may improve the activity of enzymes involved in steroid metabolism. The basis of the assay is one of competition between the sample cortisol binding and the detector-cortisol binding. The assay detected the 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 presence of microsomes interfered with the immunoassay. Figures 9(A), 9(B) and 9(C) shows some of the data obtained.
Figure 9(A) shows the effect of protein. Data taken from 700 μM cortisone group tested in the presence of Tween-80
Figure 9(B) shows the effect of cortisone. Data taken from the 25 μg microsomal protein group tested in the presence of Tween-80
Figure 9(C) shows the effect of Tween-80. Data taken from the 25 μg microsomal protein group tested in the presence of 700 μM cortisone
Conclusions:
• Microsomal protein may interfere with the immunoassay (Figure 9(A))
• Addition of exogenous cortisone had no effect on levels of cortisol detected in the enzyme assay samples (Figure 9(B))
• Inclusion of detergent in the enzyme assay buffer had only a slight effect (Figure 9(C)) Figure 10 (Performance of the Cortisol mmunoassay: various experimental designs)
An 11 β-HSD1 assay was carried out with 24 μg microsomal protein / sample and 2 μM cortisone substrate in buffer 2 Enzyme activity was also measured in samples following the addition of steroid displacement reagent (kit component) which releases cortisol from cortisol binding protein, if present in the sample. The assay detected the cortisol in the standard curve (313 pg / ml to 1 0,000 pg / ml). Figure 10 shows the absorbance at 405 n obtained for the different groups:
Conclusions:
• The lowest and highest concentrations of the cortisol standard have been included in Figure 10 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.
Figure 11 (Effect of increasing microsomal protein on measurement of 11 β HSD1 activity detected by Assay Designs Immunoassay)
When the enzyme assay data was calculated as enzyme activity using the pg/ml cortisol indicated by the standard curve, the blank value was 47 pg/ml / min incubation and the enzyme activity was 119 pg/ml / min, a signal to noise of 2.5. Although the signal to noise obtained is rather poor, these data demonstrate that the antibody can bind the cortisolAP conjugate and that this can be displaced by cortisol. An experiment was carried out to examine the effect of slightly increasing the microsomal protein concentration in an attempt to improve the signal to noise obtained. Microsomal protein was tested from 100 μg / incubation down to 5 μg / incubation using 2 μM cortisone in buffer 2. All other conditions were identical to those detailed above. The results are shown in Figure 11.
Conclusions: • Decreasing microsomal protein from 10 μg / incubation to 5 μg / incubation results in a corresponding decrease in enzyme activity.
• Increasing microsomal protein above 10 μg / incubation results in a quenching of signal which may be due to the colour of the microsomes.
• The dynamic range of this assay cannot be improved by increasing the microsomal protein concentration.
Figure 12 (Detection of 11 B HSD1 activity by RIA using the mmunotech anti-cortisol antibody)
The next experiment was carried out to assess the Immunotech antibody. The enzyme assay was carried out in Buffer 2. The substrate (cortisone) concentration of 175 nM was chosen from the SPA method described by Barf et al. [14] with 0.5 μCi / well 3H- cortisone. The enzyme assay was carried out in a polypropylene plate in a final incubation volume of 100 μl containing 10 μg / well human hepatic microsomal protein. Blanks had either buffer substituted for microsomal protein or had 10 μl stop solution added prior to the microsomes. The assay was incubated at 37°C for 30 mins and the reaction was terminated by the addition of the stop solution to all remaining wells. The Immunotech antibody was diluted in Buffer 3 to give 25 μg / 100 μl down to 6.25 μg / 100 μl. The antibody (100 μl) was added to test wells, 100 μl Buffer 3 was added to the antibody blank wells. The remainder of the procedures followed the 96 well plate RIA protocol exactly. Results demonstrating 11 β HSD1 activity using the Immunotech are shown in Figure 12.
Conclusions: • Good enzyme activity was detected with this antibody.
• The signal to noise with 12.5 and 6.1 μg antibody per well was similar hence it may be possible to reduce the antibody concentration. Figure 13 (Effect of lowering the Immunotech antibody concentration on the signal to noise (microsome group compared to GA blank group)) and Figure 14 ( mmunotech antibody saturation curve for detection of 11 β HSD1 activity by R A)
The antibody titre was examined in the next test, investigating concentrations per well from 6.7 μg down to 0.67 μg. The usual 11 β HSD1 assay was carried out except that the microsomal protein concentration was doubled to 20 μg / well in order to get the best signal to noise. The cortisone concentration was 175 nM and the enzyme assay buffer was Buffer 2. Each antibody concentration was tested against a "no enzyme" blank (buffer substituted for microsomes) and a "GA blank" (10 μl stop solution added prior to microsomes) and a control group. The RIA was carried out exactly as indicated in the methods for assay in 96 wells. These results are shown in Figure 13 and Figure 14.
Conclusion: • The Immunotech antibody performed very well.
• The saturation curve indicates that there is no difference in the detection of enzyme activity above 1.68 μg / well.
• The antibody will be used at 1.7 μg / well.
• The signal to noise ratio (microsome CP I / microsome + GA blank CPM) with this concentration of antibody was 6 fold.
Figure 15 (Linearity of human hepatic microsornal 11 B HSD1 activity detected by RIA)
Linearity of enzyme activity with human hepatic microsomal protein concentration using RIA detection was examined in the next test. The usual 11 β HSD1 assay was carried out except that the microsomal protein concentration was varied from 40 μg / well down to 1 μg / well. The cortisone concentration was 175 nM and the enzyme assay buffer was Buffer 2. 11 β HSD1 activity was linear with protein up to concentrations of 20 μg / well confirming the results obtained with the classical enzyme assay (Figure 4). Data from these experiments are shown in Figure 15. Figure 16 (Effect of Tween 80 on detection of human hepatic microsomal 11 β HSD1 activity by RIA)
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. The data in Figure 16 are taken from the group tested with 10 μg / well microsomal protein, but the same effect was seen with all protein concentrations examined.
Conclusion:
• Including 0.05 % Tween 80 in the enzyme assay buffer increases the CPM obtained in the blanks thereby reducing the signal to noise to 3 fold (compared to 5 fold in the absence of Tween 80).
Figure 17 (Effect of buffer systems on detection of human hepatic microsomal 11 β HDS1 activity by RIA)
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 enzyme assay buffer gave similar data to the two buffers system but performing both enzyme assay and RIA in Buffer 3 appeared to improve the data slightly. These results are highlighted in Figure 17.
Figure 18 (Linearity of human hepatic microsomal 11 β HSD1 activity with incubation time detected by RIA)
Linearity of enzyme activity with incubation time was investigated. The enzyme assay was carried out exactly as indicated in the methods section in buffer 3 with 10 μg / well microsomal protein and with 175 nM cortisone. The reaction was stopped at the times indicated in Figure 18 by the addition of 10 μl stop solution. The RIA was carried out exactly as indicated in the methods section. Figure 18 illustrates these results.
Conclusion: • 30 min incubation is within the linear range of enzyme activity with 10 μg / well microsomal protein and 175 nM substrate.
It is possible that 175 nM substrate is low. The apparent Km observed in the classical 11β HSD1 assay was 660 nM (Figure 5 and Figure 6), 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 min incubation time. However, there are published Km values which suggest that the actual Km for cortisone in human hepatic microsomal 11 β HSD1 assays is in the micromolar range [31 , 32]. Even though 175 nM 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 inhibiton will fall if the substrate is increased above the concentration used in Reference [14]. (ii) Increasing the substrate will reduce the specific activity of the label, reducing the CPM and the sensitivity of the assay - this could be overcome by adding higher concentrations of 3H-cortisone
Figure 19 (Substrate saturation curve for human hepatic microsomal 11 β HDS1 activity detected bv RIA)
The substrate saturation effects were examined in the next assay. The enzyme assay was carried out exactly as indicated in the methods section in buffer 3 with 10 μg / well microsomal protein and with [cold cortisone] as indicated. 3H-cortisone was 0.5 μCi / sample throughout. The reaction was stopped after 30 min by the addition of 10 μl stop solution. The RIA was carried out exactly as indicated in the methods section. Results are shown in Figure 19.
Inspection of the data shown in Figure 19 shows that 10 μg microsomal protein is not saturated with 175 nM cortisone over an incubation period of 30 mins. The apparent Km (700 nM), determined from the Lineweaver-Burke plot of these data shown in Figure 20 is very similar to that determined in the classical 11β HSD1 assay (Figure 6, apparent Km -660 nM).
Figure 20 (Lineweaver-Burke plot)
Lowering the microsomal protein concentration or the incubation time to fit well within the linear range would partly overcome the problem, but both adjustments would decrease the assay sensitivity. All of the tests carried out so far suggest that even if increasing the microsomal protein from 10 μg / sample to 20 μg / sample does not result in a doubling of enzyme activity, decreasing it from 10 μg / sample to 5 μg / sample does result in a twofold decrease in enzyme activity. Since the purpose of the assay is to monitor inhibitory effects of compounds it is probably a better course of action to leave the assay parameters as they are.
Figure 21 (ICsn curve for inhibition of human hepatic microsomal 11 β HSD1 activity by Glycyrrhetinic acid)
In order to assess the quality of compound inhibition data obtained in this assay format, an IC50 for Glycyrrhetinic acid was determined in the next test. A 10 mM stock solution of Glycyrrhetinic acid was prepared in 100 % DMSO and was further diluted in 100 %
DMSO to 0.3 mM. This solution was serially diluted in 100 % DMSO 1 in 3 to obtain the test range and each solution was diluted in assay buffer (Buffer 3) 1 in 25. These solutions were diluted into the final enzyme reaction 1 in 4 to give assay concentrations from 3 μM down to 0.012 μM in a final [DMSO] of 1 %. Controls, NSB (no antibody) and
GA blanks (addition of 10 μl stop solution prior to the addition of microsomes) were included with and without the addition of 1 % DMSO. Human hepatic microsomal protein was tested at 10 μg / well and the substrate concentration (cortisone) was 175 nM, 0.5 μCi / well. All other procedures were as indicated in the methods section. GA inhibition data are shown in Figure 21.
The assay control and blank CPM are given in the Table below. Table. Control and blank CPM obtained in the Glycyrrhetinic acid IC50 assay showing effect of 1 % DMSO and signal to noise ratio obtained.
Conclusion: Glycyrrhetinic acid gives a concentration-related inhibition of the enzyme with reasonable fit values (r2 = 0.962) and Hillslope (Figure 21). In the classical enzyme assay (Figure 7) an IC50 of 40 nM was determined for this inhibitor, which is similar to the data given in Figure 21. • Glycyrrhetinic acid inhibition of human hepatic microsomal 11 β HSD1 using dehydro- dexamethasone as the substrate has been reported to have an IC50 of 30 nM [32] However, the compound appears more active than suggested by Barf et al., (2002) [14] Inclusion of DMSO at 1 % in the enzyme assay does not affect NSB or blank values but may slightly increase enzyme activity and the signal to noise ratio These data suggest that RIA detection of 11 β HSD1 activity can generate acceptable compound inhibition data.
Figure 22 (ICm curve for inhibition of human hepatic microsomal 11 β HSD1 activity by Glycyrrhetinic acid in the presence of 350 nM cortisone) and Figure 23 (ICgn curve for inhibition of human hepatic microsomal 11 β HSD1 activity by Carbenoxolone in the presence of 350 nM cortisone)
In the next experiment, Glycyrrhetinic acid and its hemisuccinate ester, carbenoxolone, were tested for IC50 determination.. In view of the higher than expected inhibitory activity obtained with Glycyrrhetinic acid (Figure 22) in the last test and the apparent non- saturation obtained with 175 nM cortisone (Figure 19), the substrate concentration was increased to 350 nM (0.5 μCi / well). In addition, the final DMSO concentration was reduced to 0.3 %. The assay was performed as indicated in the Methods in RIA buffer. Results with some of these compounds are shown in Figure 22 and Figure 23 .
Conclusion (Fig. 22): • Increasing the substrate concentration and decreasing the DMSO concentration has little effect on Glycyrrhetinic acid inhibitory activity.
Conclusion (Fig. 23):
• Even in the presence of a higher concentration of cortisone, carbenoxolone is about 5 times more active than is suggested Barf et al. [14]. This could be due to an effect of the buffer system used here (Buffer 3) since it differs from that Barf et al., [14] (Buffer 1). This compound was less active (IC50 of 119 nM) in the classical enzyme assay, which used the buffer described by Barf et al. [14] (Figure 8).
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.
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6. Moore CCD, Melloh SH, Murai /, Siiteri PK, Miller WL ( 993): Structure and function of the hepatic form of 11 β-HSD in the squirrel monkey, an animal model of glucocorticoid resistance. Endocrinology 133: 368-375.
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15. Matassa, Victor G. et. al. J. Med. Chem.; 33(9); 1990; 2621. 16. This compound is synthesized in the literature and the NMR spectrum is reported, however the spectrum obtained here differs from that in the literature. Baraldi, Pier
Giovanni et. al.; Bioorg. & Med. Chem. Lett; 10; 2002, 1611.
17. Horaguchi, Takaaki; Matsuda, Shinichi; Tanemura, Kiyoshi; Suzuki, Tsuneo. J.
Heterocyclic Chem.; 24; 1987; 965. 18. Pie, Patrick A., Marnett, Lawrence J.; J. Heterocyclic Chem.; 25; 1988; 1271.
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21. Elderfield, Robert C; Williamson, Thurmond A.; Gensler, Walter J.; Kremer, Chester B.; J. Org. Chem; 12; 1947; 405. 22. For 6-nitro-2,3-dimethylquinoxaline see: Bariuenga, Jose; Aznar, Fernando; Liz, Ramon; Cabal, Maria-Paz; Synthesis; 3; 1985; 313., then for 6-amino-2,3- dimethylquinoxaline: Salon, Jozef; Milata, Viktor; Pronayova, Nadezda; Lesko, Jan; Collect Czech. Chem. Commun.; 66; 11 ; 2001; 1691. 23. Klicnar, J.; and Kosek, F.; Collect. Czech. Chem. Commun.; 30; 1965; 3102. 24. Gloster, Daniel F.; Cincotta, Louis; Foley, James W.; J. Heterocyclic Chem.; 36; 1999; 25.
25. The same reduction was carried out using SnCI2 by: Case et al.; J. Amer. Chem. Soc; 81 ; 1959; 6297.
26. Modified procedure from similar compound described in US Patent 6,355,796 (Example 20)
27. Hollfelder, F.; Kirby, A. J.; Tawfik, D. S.; Kikuchi, K.; Hilvert, D.; J. Amer. Chem. Soc; 122 (6); 2000; 1022-1029
28. Fujimoto, M.; Okabe, K.; Chem.Pharm.Bull.; 10; 1962; 572-575. 29. Kawamura, T.; Yagi, N.; Sugawara, H.; Yamahata, K.; Takada, M.; Chem.Pharm.Bull.; 28; 1 ; 1980; 268-276.
30. Stewart, P.M. and Mason, J.I., (1995), Cortisol to cortisone: Glucocorticoid to mineralocortcoid. Steriods, 60,143-146. 31. Escher, G. et al., (1995), Furosemide inhibits 11 β-Hydroxysteroid Dehydrogenase in vitro and in vivo. Endocrinology, 136, 1759-1765.
32. Hult, M. et. al., (1998), Selective inhibition of human type 1 11 β-hydroxysteroid dehydrogenase by synthetic steroids and xenobiotics. FEBS Letters, 441 , 25-28.
33. Diederich S, Grossmann C, Hanke B, Quinkler , Herrmann M, Bahr V, Oelkers W (2000): In the search for specific inhibitors ofhuman 11 β-HSD: chenodeoxycholic acid selectively inhibits 11 β-HSD type 1. Europ. J. Endocrin. 142: 200-207.

Claims

1. A compound having Formula I R Z-R2 Formula I wherein Ri is an optionally substituted phenyl ring;
R2 is an optionally substituted aromatic ring; and Z is -X-Y-L- or -Y-X-L- wherein either X is selected from -S(=O)(=0)- and -C(=O)-, and Y is -NR3-; or X is selected from -S(=O)(=O)- and -S-, and Y is -C(R4)(R5)-; L is an optional linker; and R3, R4 and R5 are each independently selected from H and hydrocarbyl; and wherein when R2 comprises the following structural moiety
wherein Q is an atom selected from the group consisting of S, O, N and C; the compound is selected from compounds of the formulae
RrC(=O)-NR3-L-R2;
RrS(=O)(=O)-C(R4)(R5)-L-R2;
RrS-C(R4)(R5)-L-R2; RrNR3-S(=O)(=O)-L-R2;
R NR3-C(=O)-L-R2;
R C(R4)(R5)-S(=O)(=O)-L-R2; and
R C(R4)(R5)-S-L-R2.
2 A compound according to claim 1 wherein R- is substituted.
3. A compound according to claim 1 or 2 wherein R-i is substituted with one or more substituents selected from hydrocarbon groups, oxyhydrocarbon groups, halogens, amines and amides.
A compound according to claim 1 , 2 or 3 wherein R-j is substituted with one or more substituents selected from aromatic hydrocarbon groups, alkyl groups, oxyalkyl groups and halogens.
5. A compound according to any one of claims 1 to 4 wherein R^ is substituted with one or more substituents selected from phenyl groups, C1-5alkyl groups, oxy-C1-5-alkyl groups, chloro and bromo.
6. A compound according to any one of claims 1 to 5 wherein the optionally substituted aromatic ring of R2 is a five or six membered ring.
7. A compound according to any one of claims 1 to 6 wherein the optionally substituted aromatic ring of R2 is a heterocyclic ring.
8. A compound according to any one of claims 1 to 7 wherein the optionally substituted aromatic ring of R2 is a heterocyclic ring comprising a carbon and a hetero atom selected from 0 and N.
9. A compound according to any one of claims 1 to 8 wherein R2 is selected from the group consisting of:
10. A compound according to any one of claims 1 to 6 wherein the optionally substituted aromatic ring of R2 is a carbocyclic ring.
11. A compound according to claim 10 wherein R2 is a substituted carbocyclic ring.
12. A compound according to claim 11 wherein two or more substituents together form a ring which is fused to the carbocyclic ring of R2.
13. A compound according to claim 12 wherein the ring which is fused to the carbocyclic ring of R2 is a heterocyclic ring.
14. A compound according to any one of claims 10 to 13 wherein the carbocyclic ring is a phenyl ring.
15. A compound according to any one of claims 10 to 14 wherein R2 is selected from the group consisting of:
16. A compound according to any one of the preceding claims wherein X is -C(=O)-
17. A compound according to any one of claims 1 to 15 wherein X is -S(=O)(=O)-.
18. A compound according to any one of claims 1 to 14 wherein X is -S-
19. A compound according to any one of the preceding claims wherein Y is -NR3-.
20. A compound according to any one of claims 1 to 18 wherein Y is -C(R4)(R5)-
21. A compound according to any one of claims 1 to 15 wherein Z is -S(=O)(=O)NR3- or - NR3S(=0)(=0) -.
22. A compound according to claim 21 wherein Z is -S(=O)(=O)NH- or - NHS(=O)(=0) -.
23. A compound according to any one of claims 1 to 15 wherein Z is - S(=0)(=O)C(R4)(R5)- or -C(R4)(R5)S(=O)(=O)-.
24. A compound according to claim 23 wherein Z is -S(=O)(=O)CH2- or - CH2S(=O)(=0)-.
25. A compound according to any one of the preceding claims wherein R3 is selected from H and hydrocarbon groups.
26. A compound according to any one of the preceding claims wherein R3 is selected from H and alkyl groups.
27. A compound according to any one of the preceding claims wherein R3 is selected from H and C1-10alkyl groups.
28. A compound according to any one of the preceding claims wherein R3 is selected from H and Cι-5alkyl groups.
29. A compound according to any one of the preceding claim wherein R3 is H.
30. A compound according to any one of the preceding claims wherein R4 and R5 are independently selected from H and hydrocarbon groups.
31. A compound according to any one of the preceding claims wherein R4 and R5 are independently selected from H and alkyl groups.
32. A compound according to any one of the preceding claims wherein R4 and R5 are independently selected from H and C1-10alkyl groups.
33. A compound according to any one of the preceding claims wherein R4 and R5 are independently selected from H and C1-5alkyl groups.
34. A compound according to any one of the preceding claims wherein R4 and R5 are both H.
35. A compound according to any one of the preceding claims wherein L is present.
36. A compound according to any one of the preceding claims wherein L is selected from -C(=O)-, -S(=O)(=O)-, -S-, -NR3-, -C(R4)(R5)- and combinations thereof.
37. A compound according to any one of the preceding claims wherein L is selected from -NR3-C(=O)-C(R4)(R5)-S- and -S(=O)(=O)-NR3-NR3-C(=O)-.
38. A compound according to any one of the preceding claims wherein L is selected from -NH-C(=O)-CH2-S- and -S(=O)(=O)-NH-NH-C(=O)-.
39. A pharmaceutical composition comprising a compound according to any one of claims 1 to 38 optionally admixed with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
40. A compound according to any one of claims 1 to 38 for use in medicine.
41 . Use of a compound according to any one of claims 1 to 38 in the manufacture of a medicament for use in the therapy of a condition or disease associated with 11 β-HSD.
42. Use according to claim 41 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.
43. Use of a compound according to any one of claims 1 to 38 in the manufacture of a medicament for use in the therapy of a condition or disease associated with adverse 11 β-HSD levels.
44. Use of a compound according to any one of claims 1 to 38 in the manufacture of a pharmaceutical for modulating 11 β-HSD activity.
45. Use of a compound according to any one of claims 1 to 38 in the manufacture of a pharmaceutical for inhibiting 11 β-HSD activity.
46. A method comprising (a) performing a 11β-HSD assay with one or more candidate compounds having the formula as defined in any one claims 1 to 37; (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.
47. 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 38; (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.
48. A compound identified by the method according to claim 46 or claim 47.
49. A compound according to claim 48 for use in medicine.
50. A pharmaceutical composition comprising the compound according to claim 48 optionally admixed with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
51. Use of a compound according to claim 48 in the manufacture of a medicament for use in the therapy of a condition or disease associated with 11 β-HSD.
52. Use according to claim 51 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;
53. Use of a compound according to claim 48 in the manufacture of a medicament for use in the therapy of a condition or disease associated with adverse 11 β-HSD levels.
54. The invention of any one of claims 39 to 53 wherein 11 β-HSD is 11 β-HSD Type 1.
55. The invention of any one of claims 39 to 53 wherein 11 β-HSD is 11 β-HSD Type 2.
56. A compound as substantially hereinbefore described with reference to any one of the Examples.
57. A composition as substantially hereinbefore described with reference to any one of the Examples.
58. A method as substantially hereinbefore described with reference to any one of the Examples.
59. A use as substantially hereinbefore described with reference to any one of the Examples.
EP04791576A 2003-10-23 2004-10-22 Phenyl carboxamide and sulfonamide derivatives for use as 11-beta-hydroxysteroid dehydrogenase Withdrawn EP1675844A1 (en)

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