WO2003045381A1

WO2003045381A1 - Aminoalkylimidazole derivatives and their use in medicine

Info

Publication number: WO2003045381A1
Application number: PCT/GB2002/005329
Authority: WO
Inventors: Rachid Tazi-Ahnini; Simon Ward; Michael Cork; Gordon Duff; Joe Harrity; Claes Bavik
Original assignee: Molecular Skincare Limited
Priority date: 2001-11-27
Filing date: 2002-11-27
Publication date: 2003-06-05
Also published as: GB0128415D0; AU2002343103A1

Abstract

The present invention provides compounds of formula (I) wherein R1 is selected from phenyl, quinoline, isoquinoline and anthracene whereby each of these substituents may be optionally substituted by a group selected from halogen, C¿1-4? alkoxy, C1-4 alkyl, C1-4 dialkylamino and cyano; R?2¿ is selected from hydrogen and phenyl optionally substituted by a group selected from halogen, C¿1-4? alkoxy, C1-4 alkyl, C1-4 dialkylamino and cyano; R?3¿ is selected from halogen, C¿1-12? hydrocarbyl, phenyl optionally substituted by a group selected from halogen, C1-4 alkoxy, C1-4 alkyl, C1-4 dialkylamino and cyano, and N-acyl piperazinyl; and X is selected from CO and SO2, with the proviso that when X is CO and R?1 and R3¿ are both optionally substituted phenyl, R2 is not hydrogen or when X is CO and R?2 and R3¿ are both optionally substituted phenyl, R1 is not hydrogen; and pharmaceutically acceptable derivatives and solvates thereof, pharmaceutical formulations containing them and their use in therapy particularly as CYP24 inhibitors.

Description

AMINOALKYLIMIDAZOLE DERIVATIVES AND THEIR USE IN MEDICINE

The present invention relates to a novel class of chemical compounds. In particular, the invention concerns novel acylated aminoalkylimidazole derivatives, methods for their preparation, pharmaceutical compositions containing them and their use in medicine.

Certain acylated aminoalkylimidazole derivatives are known for treating disorders of proliferation and differentiation in Vitamin D responsive tissue, such as psoriasis. However, there remains a need for further compounds for treating such diseases.

Accordingly, the present invention provides compounds of formula (I):

wherein:

R¹ is selected from phenyl, quinoline, isoquinoline and anthracene whereby each of these substituents may be optionally substituted by a group selected from halogen, Cι. alkoxy, C₁ alkyl, Cι_ dialkylamino and cyano;

R² is selected from hydrogen and phenyl optionally substituted by a group selected from halogen, Cι_ allcoxy, Cι-₄ alkyl, Cι_ diallcylamino and cyano;

R³ is selected from halogen, Q-π hydrocarbyl, phenyl optionally substituted by a group selected from halogen, .₄ allcoxy, d.₄ alkyl, _₄ dialkylamino and cyano, and N-acyl piperazinyl; and

X is selected from CO and S0₂,

with the proviso that when X is CO and R¹ and R³ are both optionally substituted phenyl, R² is not hydrogen or when X is CO and R² and R³ are both optionally substituted phenyl, R¹ is not hydrogen; and

pharmaceutically acceptable derivatives and solvates thereof. Compounds of formula (I) contain at least one asymmetric centre, denoted by *, and thus may exist as enantiomers or diastereoisomers. It is to be understood that the invention includes each such isomer, either in substantially pure form or admixed in any proportion with one or more other isomers ofthe compounds of formula (I).

The term "pharmaceutically acceptable derivative" as used herein refers to any pharmaceutically acceptable salt, or metabolically labile derivative of a compound of formula (I), for example a derivative of an amine group, which, upon administration to the recipient, is capable of providing (directly or indirectly) a compound of formula (I). It will be appreciated by those skilled in the art that the compounds of formula (I) may be modified to provide pharmaceutically acceptable derivatives thereof at any of the functional groups in the compounds of formula (I). Such derivatives are clear to those skilled in the art, without undue experimentation, and with reference to the teaching of Burger's Medicinal Chemistry And Drug Discovery, 5th Edition, Vol 1: Principles And Practice, which is incorporated herein by reference. Preferably, the pharmaceutically acceptable derivative is a salt.

Salts and solvates of compounds of formula (I) which are suitable for use in medicine are those wherein the counterion or associated solvent is pharmaceutically acceptable. However, salts and solvates having non-pharmaceutically acceptable counterions or associated solvents are within the scope of the present invention, for example, for use as intermediates in the preparation of other compounds of formula (I) and their pharmaceutically acceptable derivatives, and solvates.

Suitable salts according to the invention include those formed with both organic and inorganic acids or bases. Pharmaceutically acceptable acid addition salts include those formed from trifluoroacetic, hydrochloric, hydrobromic, hydroiodoic, sulphuric, citric, tartaric, phosphoric, lactic, pyruvic, acetic, succinic, oxalic, fumaric, maleic, oxaloacetic, methanesulphonic, ethanesulphonic, p-toluenesulphonic, benzenesulphonic, and isethionic acids. Pharmaceutically acceptable base salts include ammonium salts, alkali metal salts such as those of sodium and potassium, alkaline earth metal salts such as those of calcium and magnesium and salts with organic bases such as dicyclohexyl amine and N-methyl-D-glucamine.

Suitable solvates according to the invention include hydrates. The term "hydrocarbyl group" as used herein denotes a radical having a carbon atom directly attached to the remainder of the molecule. Hydrocarbyl groups include the following:

(1) Aliphatic hydrocarbon groups; that is, alkyl groups such as, without limitation, methyl, ethyl, «-propyl, isopropyl, n-butyl, ø-butyl, sec-butyl, tert-butyl, neopentyl, hexyl, heptyl, octyL nonyl, decyl, undecyl and dodecyl; alkenyl groups containing a single double bond such as, without limitation, ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl and dodecenyl, and alkynyl groups containing a triple bond. All isomers of these are included, but straight chain groups are preferred.

(2) Substituted aliphatic hydrocarbon groups; that is aliphatic hydrocarbon groups as described above containing non-hydrocarbon substiruents which, in the context of this invention, do not alter the predominantly hydrocarbon character of the group. Those skilled in the art will be aware of suitable substiruents; examples include halogen, niτro, cyano, COOR' (where R' is H or Cι.₆ al yl) or a salt thereof, hydroxy and Ci-β allcoxy.

(3) Hetero hydrocarbon groups; that is, groups which, while having predominantly aliphatic hydrocarbon character within the context of this invention, contain atoms other than carbon present in a chain or ring otherwise composed of aliphatic carbon atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for example, oxygen, nitrogen and sulphur.

Preferably the hydrocarbyl group is a .₁₂ alkyl chain, more preferably a .ι₂ alkyl chain, more preferably a Gno al yl chain, more preferably a .ιo al yl chain, with Cs, C₇ and C₈ being especially preferred.

The term halogen refers to a fluorine, chlorine, bromine or iodine atom. Suitably the halogen atom is selected from chlorine, bromine or iodine, preferably chlorine or bromine. Chlorine is most preferred.

Suitably, R¹ is selected from quinoline, isoquinoline and anthracene whereby each of these substiruents may be optionally substituted by a group selected from halogen, Q.₄ alkoxy, C alkyl, Q.₄ dialkylamino and cyano. More suitably, R¹ is selected from phenyl, quinoline, isoquinoline and anthracene whereby each of these substituents are unsubstituted or substituted by halogen. More suitably, R¹ is selected from phenyl, quinoline, isoquinoline, anthracene and 4-chlorophenyl. Preferably R¹ is selected from quinoline, isoquinoline and anthracene, with anthracene being most preferred. Suitably, R² is selected from phenyl optionally substituted by a group selected from halogen, Cι.₄ allcoxy, Cι.₄ allcyl, C dialkylamino or cyano. More suitably, R² is selected from hydrogen and phenyl optionally substituted by a halogen atom. Preferably, R² is selected from hydrogen, phenyl and 4-chlorophenyl. More preferably R² is selected from hydrogen and 4-chlorophenyl, with hydrogen being most preferred.

Suitably, R³ is selected from halogen, Q.ι₂ allcyl, phenyl optionally substituted by a halogen atom and N-acyl piperazinyl. More suitably, R³ is selected from chlorine, C1-12 alkyl, 4-chlorophenyl and N-acyl piperazinyl. Preferably, R³ is selected from chlorine, C₆-ιo allcyl and N-acyl piperazinyl. More Preferably R³ is selected from C₃.10 alkyl and

N-acyl piperazinyl, with C₆-₈ allcyl being most preferred.

It is to be understood that the present invention covers all combinations of suitable and preferred groups described hereinabove.

In one embodiment the present invention provides compounds of formula (I) wherein:

R¹ is selected from phenyl, quinoline, isoquinoline and anthracene whereby each of these substituents may be optionally substituted by a group selected from halogen, C alkoxy, C allcyl, Cι_₄ dialkylamino or cyano;

R² is selected from phenyl optionally substituted by a group selected from halogen, Q.₄ alkoxy, C₁-₄ allcyl, C dialkylamino or cyano;

R³ is selected from halogen, Cι-ι₂ hydrocarbyl, phenyl optionally substituted by a group selected from halogen, Q.₄ allcoxy, . allcyl, Q. dialkylamino or cyano, and N-acyl piperazinyl; and

X is selected from CO and S0₂;

and pharmaceutically acceptable derivatives and solvates thereof.

R¹ is selected from quinoline, isoquinoline and anthracene whereby these three substituents may be optionally substituted by a group selected from halogen, Q. alkoxy, C alkyl, .₄ dialkylamino or cyano; R² is hydrogen;

R³ is selected from halogen, .ι₂ hydrocarbyl, phenyl optionally substituted by a group selected from halogen, _₄ alkoxy, .₄ alkyl, . dialkylamino or cyano, and N-acyl piperazinyl; and

X is selected from CO and S0₂;

and pharmaceutically acceptable derivatives and solvates thereof.

In one embodiment, the present invention provides compounds of formula (I) wherein:

R¹ is selected from phenyl, quinolme, isoquinoline and anthracene whereby each of these substituents may be optionally substituted by a group selected from halogen, C_M allcoxy,

C_M allcyl, C dialkylamino or cyano;

R² is selected from hydrogen and phenyl optionally substituted by a group selected from halogen, C_M alkoxy, . alkyl, C dialkylamino or cyano;

R³ is selected from halogen, -n hydrocarbyl and N-acyl piperazinyl; and

X is selected from CO and S0₂;

and pharmaceutically acceptable derivatives and solvates thereof.

R¹ is selected from phenyl, quinoline, isoquinoline and anthracene whereby each of these substituents may be optionally substituted by a group selected from halogen, C alkoxy,

CM allcyl, C dialkylamino or cyano;

R² is selected from hydrogen and phenyl optionally substituted by a group selected from halogen, C alkoxy, Cι. alkyl, CM dialkylamino or cyano; R³ is selected from halogen, Cι.]₂ hydrocarbyl, phenyl optionally substituted by a group selected from halogen, .₄ allcoxy, Q.₄ alkyl, C diallcylamino or cyano, and N-acyl piperazinyl; and

X is S0₂;

and pharmaceutically acceptable derivatives and solvates thereof.

Examples of compounds of formula (I) include:

N-[4-(4-chlorophenyl)benzoyl]-2-(lH-imidazol-l-yl)-2,2-(di-4-chlorophenyl)-l-amino ethane;

N- [4-(4-chlorophenyl)benzoyl]-2-( 1 H-imidazol- 1 -yl)-2-(isoquinol-6-yl)- 1 -amino ethane;

N- [4-(4-chlorophenyl)benzoyl]-2-( 1 H-imidazol- 1 -yl)-2-(quinol-6-yl)- 1 -amino ethane;

N-[4-(4-chlorophenyl)benzoyl]-2-(lH-imidazol-l-yl)-2-(anthracen-2-yl)-l-amino ethane;

N-[4-(oct- 1 -yl)benzoyl]-2-( 1 H-imidazol- 1 -yl)-2-phenyl- 1 -amino ethane;

N-[4-chloroberιzoyl]-2-( 1 H-imidazol- 1 -yl)-2-phenyl- 1 -amino ethane;

N-[4-(hex- 1 -yl)benzoyl]-2-( 1 H-imidazol- 1 -yl)-2 -phenyl- 1 -amino ethane;

N-[4-(hept- 1 -yl)benzoyl]-2-( 1 H-imidazol- 1 -yl)-2-phenyl- 1 -amino ethane;

N-[4-(4-acylpiperazinyl)benzoyl]-2-(l H-imidazol- 1 -yl)-2 -phenyl- 1 -amino ethane;

4-chlorophenyl- N-(2- 1 H-imidazol- 1 -yl-2-phenyl-ethyl)-sulphonamide

References herein after to compounds of the invention or to compounds of formula (I) include compounds of formula (I) and their pharmaceutically acceptable derivatives and solvates thereof.

The compounds of the present invention are inhibitors of vitamin D3 hydroxylases, particularly 25-hydroxyvitamin D3 hydroxylases, particularly CYP24. It is believed that 25-hydroxyvitamin D3 hydroxylases attack the C20-27 side chain of vitamin D3. Thus the compounds of the present invention are useful in the treatment of conditions where it is desired to inhibit the catabolism of the hormone calcitriol (1,25 [OH]₂ vitamin D3 or

1,25 dihydroxycholecalciferol) without interfering with its synthesis from its precursor 25(OH) vitamin D3, such as to increase and prolong endogenous hormone levels and therefore exert beneficial effects with respect to proliferation and differentiation, to immune function and to calcium homeostasis, such as, in hyperproliferative and inflammatory diseases such as psoriasis and eczematous diseases, in degenerative changes of connective tissue both pathologic and accompanying normal ageing and photo ageing, in the prevention of hair loss and regeneration of hair growth, in tumour suppression, in increasing tolerance against allotransplantes, in rheumatoid arthritis and in bone remodelling.

Accordingly, in a further aspect the present invention provides a method for the treatment of psoriasis, eczematous diseases, scleroderma, viteligo, degenerative changes of connective tissue both pathologic and accompanying normal ageing and photo ageing, in the prevention of hair loss and regeneration of hair growth, diseases associated with abnormal cellular proliferation such as neoplasms and cancer, in increasing tolerance against allotransplantes, in rheumatoid arthritis and in bone remodelling in a subject, preferably a human, which comprises administering to the subject an effective amount of a compound of formula (I) or pharmaceutically acceptable derivative or solvate thereof.

In the alternative, there is provided a compound of formula (I) or a pharmaceutically acceptable derivative or solvate thereof, for use in medical therapy, particularly, for use in the manufacture of a medicament for the treatment of psoriasis, eczematous diseases, scleroderma, viteligo, degenerative changes of connective tissue both pathologic and accompanying normal ageing and photo ageing, in the prevention of hair loss and regeneration of hair growth, diseases associated with abnormal cellular proliferation such as neoplasms and cancer, in increasing tolerance against allotransplantes, in rheumatoid arthritis and in bone remodelling.

Preferably the disease to be treated is psoriasis.

Furthermore we have found that, contrary to the assumptions in the reported literature, (see for example, Kirsner R.S. and Federman D., American Family Physician 52:237- 240, 1995), tachyphylaxis does in fact develop in patients undergoing chronic therapies using vitamin D and its analogues. Indeed, tachyphylaxis is a major problem in vitamin

D analogue therapy. Furthermore, we have discovered that an underlying cause of tachyphylaxis is the degradation of the drug in the patient, rather than de-sensitisation or receptor down-regulation. Thus, we have found that when a patient is exposed to a drug (for example, a vitamin D analogue) for extended periods, there is an increase in the expression of enzymes such as vitamin D3 hydroxylases (CYP24) which are capable of metabolising that drug. Therefore, our invention also provides a method for the treatment of tachyphylaxis by inhibiting such induced vitamin D3 hydroxylases.

Thus according to a further aspect, the present invention provides a method of alleviating or preventing a tachyphylactic response to vitamin D or an analogue thereof in a patient, preferably a human, which method comprises administering to the patient an effective amount of a compound of formula (I) or a pharmaceutically acceptable derivative or solvate thereof.

In the alternative, there is provided a compound of formula (I) or a pharmaceutically acceptable derivative or solvate thereof, for use in medical therapy, particularly, for use in the manufacture of a medicament for alleviating or preventing a tachyphylactic response to vitamin D or an analogue thereof.

Tachyphylaxis is a falling-off in the effects produced by a drug during continuous use or constantly repeated administration (tachyphylaxis is also known as tolerance). Thus, for example where continuous administration of a drug is taking place, it has been observed both in vivo and in vitro that with continuous administration of certain drugs, the patient develops tachyphylaxis to the drug. Where tachyphylaxis develops in response to the administration of vitamin D or an analogue thereof, this is manifested in a decrease over time of the effect (including therapeutic effect) of the drug. In other words, a given amount of vitamin D or an analogue thereof administered to a patient at a certain time will have less of an effect than the same amount of drug administered to the patient initially. The degree of therapeutic effect of an initial administration of a particular dose of a drug may be referred to as the "baseline level". Continued administration of the drug causes the therapeutic and other effects to decrease to below the baseline level. In extreme cases of tachyphylaxis, the patient fails to respond at all to the drug, and the therapeutic effect of the drug ceases altogether.

According to the present invention, a patient exhibits a tachyphylactic response to vitamin D or an analogue thereof when the effect of the drug decreases below baseline levels upon continued or repeated administration of the drug over a period of time. The effect of the drug includes therapeutic effects, for example, the effect of a vitamin D analogue may include inhibition of proliferation of epithelial cells. Other effects which may be non-therapeutic, for example, a hypercalcaemic effect are also included. Preferably, tachyphylactic response is manifested as a decrease in a therapeutic effect, most preferably a decrease in the therapeutic effect of the drug which is responsible for the disease treatment. Our methods and compositions are effective in restoring or increasing the effect of vitamin D or an analogue thereof, preferably a therapeutic effect, when administered to a tachyphylactic patient.

A tachyphylactic response may be exhibited by a patient to vitamin D or an analogue thereof which is administered continuously (for example, by infusion), or administered periodically (for example, occasional doses of a drug over time). What is important is that the administration takes place over a period of time. Preferably, the period of time the drug is administered is 7 or more days, for example, 10 days, 14 days or more, or even over a period of months, for example, a month, two months. In extreme cases of chronic therapies, the drug may be administered over the course of years. According to our invention, tachyphylaxis may be reversed, relieved, treated, or prevented by administration of one or more compounds of formula (I). Preferably, our methods and compositions are such that the therapeutic effectiveness of the drug is increased to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the baseline level. Preferably, the therapeutic effectiveness of the drug is increased to 50% of the baseline level, more preferably 70%, even more preferably 90%. Most preferably, the therapeutic effectiveness of the drug is increased to 100%) of the baseline level, in other words, complete relief of tachyphylaxis.

The effectiveness of the methods and compositions of our invention in the treatment, alleviation, relief or prevention of tachyphylaxis may be assayed in a number of ways. Such assays may conveniently be based on assays of the therapeutic effectiveness of vitamin D or an analogue thereof in treating, alleviating, preventing, etc a disease or syndrome. Such assays are known in the art, and the skilled person will readily be able to choose and if necessary design an appropriate assay to use. Likewise, the reduction of tachyphylaxis with a compound of formula (I) may be assessed by measuring the effect of treatment with the vitamin D or the analogue thereof with and without the compound of formula (I). In a patient exhibiting a tachyphylactic response to a drug which has been administered over a course of time, the therapeutic response of the patient to the drug declines (i.e., the drug is not as effective as on initial administration). Accordingly, tachyphylaxis is reduced where the therapeutic effect of the drug on the disease is elevated in the presence ofthe compound of formula (I), but not elevated in its absence.

The compound of formula (I) may be administered to a patient already exhibiting a tachyphylactic response. Alternatively, or in conjunction, the compound of formula (I) may administered simultaneously or sequentially with vitamin D or an analogue thereof.

Vitamin D analogues with which the compounds of the present invention may be administered in conjunction are, for example, those recited in US6,310,226,

US6,197,982, US5,994,332, US5,932,565, US5,716,945, US5,710,142, US5,446,034, US5,401,732, US5,401,731, US5,387,582, US5,378,695, US5,374,629, US5,206,229, US5,190,935, US4,866,048, US6,043,385, US6,008,209, US5,919,986, US5,883,124, US5,252,604, US5, 145,846, US5, 120,722, US4,906,785, US4,335,120, and U83,957,966 and those selected from the group consisting of: Calcitriol (la,25(OH)₂D₃, la,25-dihydroxyvitamin D₃, 1,25-dihyroxycholecalciferol); Calcipotriol (Dovanex; MC903) and Tacalcitol (la,24(R)-dihydroxyvitamin D₃; Curatoderm). Preferably the vitamin D analogues are selected from Calcitriol (la,25(OH)₂D₃, la,25- dihydroxyvitamin D₃, 1,25-dihyroxycholecalciferol); Calcipotriol (Dovanex; MC903) and Tacalcitol (la,24(R)-dihydroxyvitamin D₃; Curatoderm), more preferably 1,25- dihyroxycholecalciferol (Calcitriol) and Tacalcitol (Curatoderm).

Thus in a still further aspect, the present invention provides a combination of a compound of formula (I) and vitamin D or an analogue thereof.

Preferably the combination is administered as a pharmaceutical composition.

Thus, in a further aspect, the present invention provides a pharmaceutical formulation comprising a compound of formula (I) or a pharmaceutically acceptable derivative or solvate thereof and vitamin D or an analogue thereof, together with one or more pharmaceutically acceptable carriers.

According to a still further aspect of the present invention, there is provided a kit comprising in association for concomitant or sequential administration a compound of formula (I) together with vitamin D or an analogue thereof.

For human use, a compound of formula (I) can be administered as raw drug substance, but will generally be administered in admixture with a pharmaceutically acceptable carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

Accordingly, the present invention provides a pharmaceutical formulation comprising a compound of formula (I) or a pharmaceutically acceptable derivative or solvate thereof, and one or more pharmaceutically acceptable carriers.

The term pharmaceutically acceptable carrier includes substances capable of being coadministered with the compounds of formula (I), and which allow performance of the intended function, e.g., treat or prevent psoriasis or tachyphylaxis. Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone and the like. The pharmaceutical preparations can be sterilised and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilisers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colourings, flavourings and/or aromatic substances and the like which do not deleteriously react with the compounds of the invention.

The compounds of the invention may be administered via oral, parenteral or topical routes. The administration may be carried out in single or multiple doses. The compounds of the invention may be administered in a wide variety of different dosage forms, for example they may be combined with various pharmaceutically acceptable inert carriers in the form of tablets, capsules, lozenges, troches, hard candies, powders, sprays, creams, salves, suppositories, jellies, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, syrups, and the like. Such carriers include solid diluents or fillers, sterile aqueous media and various non-toxic organic solvents, etc. Moreover, oral pharmaceutical compositions may be sweetened and/or flavoured. In general, the compounds of the invention are present in such dosage forms at concentration levels ranging from about 5.0% to about 10% by weight.

For oral administration, tablets may contain various excipients such as microcrystalline cellulose, sodium citrate, calcium carbonate, dicalcium phosphate and glycine may be employed along with various disintegrants such as starch (preferably com, potato or tapioca starch), algmic acid and certain complex silicates, together with granulation binders like polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulphate and talc may be employed. Solid compositions of a similar type may also be employed as fillers in gelatin capsules; preferred materials in this connection also include lactose or milk sugar as well as high molecular weight polyethylene glycols. When aqueous suspensions and/or elixirs are desired for oral administration, the active ingredient may be combined with various sweetening or flavouring agents, colouring matter or dyes, and, if so desired, emulsifying and/or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin and various combinations thereof.

For parenteral administration (including intraperitoneal, subcutaneous, intravenous, intradermal or intramuscular injection), solutions of compounds ofthe invention in either sesame or peanut oil or in aqueous propylene glycol may be employed. The aqueous solutions may be buffered (preferably pH greater than 8) if necessary and the liquid diluent first rendered isotonic. These aqueous solutions are suitable for intravenous injection purposes. The oily solutions are suitable for intraarticular, intramuscular and subcutaneous injection purposes. The preparation of all these solutions under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art. For parenteral administration, examples of suitable preparations include solutions, preferably oily or aqueous solutions as well as suspensions, emulsions, or implants, including suppositories. Compounds of the invention may be formulated in sterile form in multiple or single dose formats. For example the compounds of the invention may be dispersed in a fluid carrier such as sterile saline or 5% saline dextrose solutions commonly used with injectables. The compounds of the invention may be administered topically for example when treating conditions of the skin. Examples of methods of topical administration include transdermal, buccal or sublingual application. For topical applications, therapeutic compounds can be suitably admixed in a pharmacologically inert topical carrier such as a gel, an ointment, a lotion or a cream. Such topical carriers may include water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, or mineral oils. Other possible topical carriers are liquid petrolatum, isopropylpalmitate, polyethylene glycol, ethanol 95%, polyoxyethylene monolauriate 5% in water, sodium lauryl sulphate 5% in water, and the like. In addition, materials such as anti-oxidants, humectants, viscosity stabilisers and the lilce also may be added if desired.

For enteral application, particularly suitable are tablets, dragees or capsules having talc and/or carbohydrate carrier binder or the lilce, the carrier preferably being lactose and/or corn starch and/or potato starch. A syrup, elixir or the lilce can be used wherein a sweetened vehicle is employed. Sustained release compositions can be formulated including those wherein the active component is protected with differentially degradable coatings, e.g., by microencapsulation, multiple coatings, etc.

In addition to treatment of human subjects, the therapeutic methods of the invention also will have significant veterinary applications, e.g. for treatment of livestock such as cattle, sheep, goats, cows, swine and the like; poultry such as chickens, ducks, geese, turkeys and the like; horses; and pets such as dogs and cats.

It will be appreciated that the actual amount of the compound of the invention used in a given therapy will vary according to the specific compound being utilised, the particular compositions formulated, the mode of application, the particular site of administration, etc. Optimal administration rates for a given protocol of administration can be readily ascertained by those skilled in the art without undue burden. For example, a suitable effective dose of one or more compounds of the invention will be in the range of from 0.01 to 100 milligrams per kilogram of body weight of recipient per day, preferably in the range of from 0.1 to 50 milligrams per kilogram body weight of recipient per day, more preferably in the range of 1 to 20 milligrams per kilogram body weight of recipient per day. The desired dose is suitably administered once daily, or several sub-doses, e.g. 2 to 5 sub-doses, are administered at appropriate intervals through the day, or other appropriate schedule.

According to a further aspect ofthe invention, there is provided a process for preparing a compound of formula (I) which comprises reacting a compound of formula (II):

(») wherein R^!, R², R³ and X are as defined previously with imidazole, preferably at elevated temperature. Compounds of formula (II) may be prepared by reacting a compound of formula (III) with a compound of formula (IV):

(Hi) (iv) wherein R¹, R², R³ and X are as defined previously and Y is a halogen atom, preferably chlorine. Preferably the reaction is conducted in a polar solvent such as dichloromethane in the presence of an activating agent such as 4-N,N-dimethylaminopyridine. In a preferred embodiment the compound of formula (II) is the crude product mixture from the reaction of a compound of formula (III) with a compound of formula (IV).

The compounds of the invention have the advantage that they may be more efficacious, show greater specificity (for CYP24), have fewer side effects, be more bioavailable by the preferred route, have a longer duration of action than similar known compounds.

Synthetic Examples:

N-r4-chlorobenzovn-2-(lH-imidazol-l-yl)-2-(phenyl -l-amino ethane To a flame dried 25 ml round bottom flask containing a magnetic stir bar was added 2- phenylaziridine (619 mg, 5.2 mmol), a few crystals of 4-N,N-dimethylaminopyridine and methylene chloride (5 ml). The reaction mixture was treated with triethylamine (1.4 ml, 10 mol) and then dropwise with 4-chlorobenzoyl chloride (0.99 ml, 7.8 mmol). The reaction mixture was stirred overnight at room temperature. The reaction mixture was treated with water and the product extracted with diethyl ether. The organic layer was washed with dilute HCI, dilute sodium bicarbonate and water and dried over magnesium sulfate. The crude product obtained on evaporation of solvent was purified by chromatography on silica gel (elution with with 5:1 hexanes:ethyl acetate) to provide 256 mg (0.99 mmol, 19%) of (4-chloro-phenyl)-(2-phenyl-aziridin-l-yl)-methanone which was used directly in the next step.

The above mixture was transferred to a 50 ml round bottom flask equiped with a stir bar and treated with imidazole (257 mg, 2.98 mmol). The mixture was capped with a glass stopper and heated for 20 h at 125 °C. Upon cooling, the residue was dissolved in methylene chloride and the organic layer washed with water. The crude product obtained on evaporation of solvent was purified by chromatography on silica gel (elution with with 4:1:7 methylene chloride:methanol:hexanes) to provide the title product as a colourless solid. The product was further purified by recrystallisation from methano hexanes to provide colourless crystals which were thoroughly dried in vacuo (5 mg, 2%). ^]H NMR (250 MHz, CDCI): 3.77-3.92 (1H, m), 4.31 (1H, dt, J = 5.2, 14.5 Hz), 5.62 (1H, dd, J = 4.4, 10 Hz), 7.17-7.43 (10H, m), 7.68 (2H, d, J = 8.5Hz).

N-[4-(hex- 1 -yl)benzoyll-2-( 1 H-imidazol- 1 -yl -2-(phenyl)- 1 -amino ethane To a flame dried 25 ml round bottom flask containing a magnetic stir bar was added 2- phenylaziridine (100 mg, 1.2 mmol), a few crystals of 4-N,N-dimethylaminopyridine and methylene chloride (5 ml). The reaction mixture was treated with triethylamine (0.36 ml, 2.62 mmol) and then dropwise with 4-«-hexylbenzoyl chloride (0.52 ml, 2.38 mmol). The reaction mixture was stirred overnight at room temperature. The reaction mixture was treated with water and the product extracted with diethyl ether. The organic layer was washed with dilute HCI, dilute sodium bicarbonate and water and dried over magnesium sulfate. The crude product obtained on evaporation of solvent was used directly in the next step.

The above mixture was transferred to a 50 ml round bottom flask equiped with a stir bar and treated with imidazole (327 mg, 4.80 mmol). The mixture was capped with a glass stopper and heated for 20 h at 125 °C. Upon cooling, the residue was dissolved in methylene chloride and the organic layer washed with water. The crude product obtained on evaporation of solvent was purified by chromatography on silica gel (elution with with 4:1:7 methylene chloride:methanol:hexanes) to provide the title product as a colourless solid. The product was further purified by recrystallisation from etheπhexanes to provide colourless crystals which were thoroughly dried in vacuo (275 mg, 61%). H NMR (250 MHz, CDC|): 0.85 (3H, br), 1.20-1.35 (6H, m), 1.45-1.60 (2H, ), 2.57 (2H, t, J=7.8 Hz), 3.77-3.87 (1H, m), 4.26 (1H, dt, J = 4.6, 14.2 Hz), 5.62 (1H, dd, J = 4.6, 10.0 Hz), 7.12 (2H, d, J=8.0 Hz), 7.15-7.40 (5H, m), 7.66 (2H, d, J = 8.0 Hz), 7.89 (1H, m).

The following examples may prepared according to procedures similar to those set out above and by the methods of Egger et al.(Egger, Helmut; Waelchli, Rudolf. Preparation and testing of azolylethylcarboxamides as aromatase inhibitors. Ger. Offen. (1988), 7 pp., DE 3740125 Al) ie by reacting the appropriate acid chloride or sulphonyl chloride with the approriate aziridine to form the corresponding amide or sulphonamide and heating the resultant amide or sulphonamide as a melt in the presence of excess imidazole.

N-[4-(4-chlorophenyl)benzoyl]-2-(lH-imidazol-l-yl)-2,2-(di-4-chlorophenyl)-l-amino ethane

N-[4-(4-chlorophenyl)benzoyl]-2-(lH-imidazol-l-yl)-2-(isoquinol-6-yl)-l-amino ethane N-[4-(4-chlorophenyl)benzoyl]-2-(lH-imidazol-l-yl)-2-(quinol-6-yl)-l-amino ethane N-[4-(4-chlorophenyl)benzoyl]-2-(lH-imidazol-l-yl)-2-(anthracen-2-yl)-l-amino ethane N-[4-(oct- 1 -yl)benzoyl]-2-( 1 H-imidazol- 1 -yl)-2-phenyl- 1 -amino ethane N-[4-chlorobenzoyl]-2-( 1 H-imidazol- 1 -yl)-2 -phenyl- 1 -amino ethane

N-[4-(hex-l-yl)benzoyl]-2-(lH-imidazol-l-yl)-2-phenyl-l-amino ethane N-[4-(hept- 1 -yl)benzoyl]-2-( 1 H-imidazol- 1 -yl)-2-phenyl- 1 -amino ethane N- [4-(4-acylpiperazinyl)benzoyl]-2-( 1 H-imidazol- 1 -yl)-2-phenyl- 1 -amino ethane 4-chlorophenyl- N-(2- 1 H-imidazol- 1 -yl-2-phenyl-ethyl)-sulphonamide

Biological Examples

The following Examples illustrate the utility of the CYP24 inhibitors of the present invention. In particular, the following examples illustrate the use of the CYP24 inhibitors: N-[4-chlorobenzoyl]-2-(lH-imidazol-l-yl)-2,2-(di-4-chlorophenyl)-l-aminoethane;

N-[4-chlorobenzoyl]-2-(lH-imidazol-l-yl)-2-phenyl-l-amino ethane ; and N-[4-(hex- 1 -yl)benzoyl] -2-( 1 H-imidazol- 1 -yl)-2 -phenyl- 1 -amino ethane in reducing cellular proliferation, for example in treating psoriasis or in tumour supression, either alone or in conjunction with a vitamin D analogue by alleviating or preventing tachyphylaxis associated with vitamin D or vitamin D analogue therapy.

Example 1 - Characterisation of CYP24 Enzyme Induction by Vitamin D Analogues In

Vitro (Cultured normal and psoriatic human epidermal keratinocvtes)

Preparation of primary keratinocyte cultures

Patient samples are collected with informed consent. Pieces of skin from normal and psoriatic donors are washed thoroughly for 30 minutes in Betadine solution (10% iodine) followed by 2 x 10 minute washes in PBS. After removal of the subcutaneous tissue and dermis with a scalpel the skin is dissected into 5 x 10 mm pieces. The skin pieces are rinsed 5 x in PBS followed by incubating in Dispase (grade II, 2.4 U/rnL, Boehringer Mannheim) for 18 hours at 4 °C. The skin pieces are then placed dermis side down in 100mm Petri dishes and irrigated with 5 L KSFM. The epidermis is peeled off with forceps and incubated in 10 mL Trypsin/EDTA solution for 10 minutes at 37 °C followed by addition of 10 mL KSFM. The sample is mechanically agitated by aspiration and ejection from a transfer pipette. The resulting cell suspension is sieved using a cell strainer (Becton Dickinson) rinsing through with KSFM. The isolated epidermal cells are then washed twice in KSFM by centrifugation at lOOg for 10 min.

Primary cultures are seeded at 3500 cells/cm² in K-SFM 25 cm² tissue culture flasks. The medium is changed (5mL per flask) the day after seeding and every other day thereafter. At 70-80% confluence the cells are subcultured by seeding 3500 cells/cm² in new 25 cm² flasks.

References

Freshney. Culture of Animal Cells a Manual of Basic Technique. 4^th ed. Wiley Liss Thesis: Psoriasis - the role of the keratinocyte. Stephen St. George-Smith. June 2000 Sheffield University.

Experiment

Human normal primary keratinocyte and human psoriatic keratinocytes are cultured in defined media (vitamin D free) in the presence or absence of 10 nanomolar of a vitamin D analogue (Calcipotriol; Tacalcitol; Calcitrol) or 0.5 micromolar 25-OH- vitamin D3. The cells are cultured for 4 days, replacing the experimental medium every other day, before harvesting the cells. RNA is isolated from the cultured cells using the RNeasy kit according to the instruction of the supplier (Qiagen Inc.). Isolated RNA is subjected to semi-quantitative RT-PCR using GeneAmp Gold RNA PCR Reagent Kit (PE Biosystems, Norwalk, Conn. USA) according to manufacturer's instructions. Primers used are specific for human CYP24 and shown in Table I.

Results

Expression of vitamin D 24-hydroxylase (CYP24, GenBank# XM 030592) mRNA is found to be increased by each of Calcipotriol, Tacalcitol, ^• Calcitrol and 25-OH-vitamin D3, as compared to controls. CYP24 is fully induced by 4 days of treatment.

Table I

Example 2 A - Characterisation of the Effect of Vitamin D Analogues and Novel CYP24

Inhibitors On Cellular Proliferation In Vitro (Cultured Normal and Psoriatic Human Epidermal Keratinocvtes)

Cell counting assay Keratinocyte cultures (isolated from donor skin as above) are seeded at 3500 cells/cm² in

K-SFM 25 cm² tissue culture flasks. The medium is changed (5 mL per flask) the day after seeding and every other day thereafter. At 70-80% confluence the cells are subcultured by seeding 3500 cells per well in 24 well cell culture plates, each well containing 500 mL of K-SFM. One well plate is seeded for each treatment. After allowing one day for settling the cells are switched to experimental medium (500 mL per well). Stock solutions of additions are as before. The medium is replaced every other day. Cells are harvested by trypsinisation. 500 mL Trypsin/EDTA is added to each well to be harvested and then aspirated off. The well plates are incubated for 5-6 min at 37 °C. 500 mL PBS is added to the wells and the cells are resuspended. The cell suspension is added to 20 mL Isoton solution in a Coulter counter "Accuvette" and the cells are counted. On each harvest, 3 wells of cells are counted for each treatment and 3 counts are made for each well. Experimental treatments

Human normal primary keratinocyte and human psoriatic keratinocytes are cultured in defined media (vitamin D free) in the presence or absence of 10 nanomolar vitamin D analogue (Calcipotriol, Tacalcitol, Calcitrol) or 0.5 micromolar 25-OH-vitamin D3 with and without addition of 10 nanomolar CYP24 inhibitors or ketoconazole.

Result

Cellular proliferation by is reduced by vitamin D analogues as compared to untreated control cultures. Proliferation is further reduced with CYP24 inhibitors adjunct to the vitamin D analogue treatment. No significant difference in viability was observed between any ofthe treatment regimes.

The concentration of Calcipotriol, Tacalcitol and l,25(OH)2D3 in the media is examined and found to be elevated in the presence of CYP24 inhibitor(s) indicating inhibition of metabolism. Furthermore, addition of 0.5 micromolar 25(OH)D3 is found to elevate the expression of the differentiation markers, as well as the concentration of l,25(OK)zD3 in the media only in the presence of CYP24 inhibitors.

Example 2B - Characterisation of the Effect of Vitamin D Analogues and Novel CYP24 Inhibitors On Cellular Proliferation In Vitro (breast cancer cell lines MCF-7 and ZR-75-

D

Cell counting assay

Breast cell cultures are seeded at 3500 cells/cm² in DMEM 25 cm² tissue culture flasks. The medium is changed (5 mL per flask) the day after seeding and every other day thereafter. At 70-80% confluence the cells are subcultured by seeding 3500 cells per well in 24 well cell culture plates, each well containing 500 mL DMEM. One well plate is seeded for each treatment. After allowing one day for settling the cells are switched to experimental medium (500 mL per well). Stock solutions of additions are as before. The medium is replaced every other day. Cells are harvested by trypsinisation. 500 mL

Trypsin/EDTA is added to each well to be harvested and then aspirated off. The well plates are incubated for 5-6 min at 37 °C. 500 L PBS is added to the wells and the cells are resuspended. The cell suspension is added to 20 mL Isoton solution in a Coulter counter "Accuvette" and the cells are counted. On each harvest, 3 wells of cells are counted for each treatment and 3 counts are made for each well.

Experimental treatments Human normal breast cells and human breast cancer cells (cell lines MCF-7 and ZR-75- 1) are cultured in defined media (vitamin D free) in the presence or absence of 10 nanomolar vitamin D analogue (Calcipotriol, Tacalcitol, Calcitrol) or 0.5 micromolar 25- OH- vitamin D3 with and without addition of 10 nanomolar CYP24 inhibitors or ketoconazole.

Result

Cellular proliferation by is reduced by vitamin D analogues as compared to untreated control cultures. Proliferation is further reduced with CYP24 inhibitors adjunct to the vitamin D analogue treatment. No significant difference in viability was observed between any of the treatment regimes.

The concentration of Calcipotriol, Tacalcitol and l,25(OH)2D3 in the media is examined and found to be elevated in the presence of CYP24 inhibitor(s) indicating inhibition of metabolism. Furthermore, addition of 0.5 micromolar 25(OH)D3 is found to elevate the expression ofthe differentiation markers, as well as the concentration of l,25(OH)2D3 in the media only in the presence of CYP24 inhibitors.

Example 3 - Characterisation of the Effect of Vitamin D Analogues and Novel CYP24 Inhibitors On Cellular Differentiation In Vitro (Cultured Normal And Psoriatic Human

Epidermal Keratinocytes)

Human normal primary keratinocyte and human psoriatic keratinocytes are isolated and cultured in defined media (vitamin D free) in 0.1 or 1.2 mM CaCt in the presence or absence of Calcipotriol, Tacalcitol, Calcitrol or 25-OH-vitamin D3 with and without addition of CYP24 inhibitors or ketoconazole as above.

Markers of keratinocyte proliferation and differentiation are examined by semi- quantitative RT-PCR on the isolated RNA using PCR primers specific for Cyclin-A, Involucrine, Kl, K5, K6, K10, K14, MDM2 essentially as for CYP24 above. We also examined if expression of vitamin D target genes are affected by the treatments. Accordingly, primers specific for vitamin D receptor (VDR), CYP24, p21Cipl/Wafl, c- Fos and b3-integrin are included among the RT-PCR's. Skin irritation is a facet of tachyphylaxis and the expression of irritation markers such as ILl-a; IL-6; IL-8; TNF-a; GM-CSF; TGF-b is examined by RT-PCR as above.

Result Skin equivalents cultured in the continuous presence of vitamin D analogues respond to the hyperproliferative challenge by increased expression of the proliferation markers Cyclin-A, K5, K6, K14, MDM2 and decreased expression of the differentiation markers Involucrine, Kl, K10. This demonstrates that the antiproliferative effect ofthe vitamin D analogues is blunted by a tachyphylactic response, up-regulation of vitamin D analogue- catabolizing CYP's. The response to the hyperproliferative challenge is abolished when CYP inhibitor(s) are co-administered with the vitamin D analogues. The expression of the vitamin D receptor is not down-regulated by the vitamin D analogue or CYP24 inhibitor treatment. This demonstrates that tachyphylaxis to vitamin D analogues can be abolished by CYP inhibitors adjunct to the vitamin D analogue regime. Furthermore, continuous vitamin D analogue treatment results in up-regulation of ILl-a; IL-6; IL-8; TNF-a; GM-CSF; TGF-b, markers of skin irritation. This response is diminished by co- administration of CYP inhibitors with the vitamin D analogues. The expression of markers for vitamin D receptor activity such as vitamin D receptor (VDR), CYP24, p21Cipl/Wafl, c-Fos and b3-integrin are increased by CYP24 inhibitors adjunct to the

VDA treatment.

Therefore, in vitro cultured human skin equivalents exposed to both vitamin D analogue and adjunct enzyme inhibitor are found to show a persistent response to vitamin D analogue treatment with lower associated irritancy and no tachyphylactic response.

Example 4 - Characterisation of CYP24 Enzyme Induction by Vitamin D Analogues In Vitro (Organotypic human skin equivalent cultures)

Organotypic skin equivalent (SE) cultures are established from human keratinocytes and fibroblasts essentially as described by Matsukova E. et al., Folia Biologica 44:59-66, 1998 and Tsunenaga M. et al., Jpn. J. Cancer Res. 85:238-44, 1994. Separate skin equivalent cultures are established and then treated with VDA: 500 ng Calcipotriol; 400 ng Tacalcitol or 150 ng Calcitrol; or vehicle control. The drugs are applied every 2 days in 10 mL vehicle and spread over the entire air-exposed surface. 9 skin equivalent cultures are exposed to each drug treatment and 3 skin equivalent cultures are withdrawn for analysis after 1, 4 and 14 days. 12 untreated skin equivalent cultures are used as controls with the first set of 3 withdrawn before treatment (day 0). At the completion of the treatment regimes RNA is isolated from the skin equivalents using the RNeasy kit according to the instruction of the supplier (Qiagen Inc.). Semi-quantitative RT-PCR, and CYP24 primers are identical to the method above. Results

Expression of CYP24 mRNA is found to be increased by each of Calcipotriol, Tacalcitol and Calcitrol, as compared to controls. CYP24 is fully induced by 4 days of treatment.

Example 5 - In Vitro model of Tachyphylaxis (Organotypic human skin equivalent cultures)

This example shows the reduction/alleviation of tachyphylaxis to vitamin D analogues by co-treatment with inhibitors of the catabolic enzyme CYP24 in organotypic skin equivalents. We verify that CYP24 inhibitors adjunct to vitamin D analogue treatment counters a tachyphylactic response in human in vitro skin equivalent. 36 skin equivalent cultures are established and treated as above with 500ng Calcipotriol. In half of the Calcipotriol-treated skin equivalent cultures the Calcipotriol treatment solution also includes an inhibitor of CYP24 (as shown in Table 2). The skin equivalent cultures are challenged on day 10 with hyperproliferative stimuli by adding O.lmM 12-0- tetradecanoylphorbol-13-acetate (Phorbol ester), ImM retinoic acid (RA) or nothing (control, 0) to the MC903 treatment solution. The skin equivalent cultures are maintained for 4 more days before analysis.

Likewise, skin equivalent cultures are also treated with 400ng Tacalcitol, 150ng Calcitrol or 500 ng 25-OH-vitamin D3 with and without CYP24 inhibitors essentially as above.

The cellular morphology of all skin equivalent cultures are assessed for signs of hyperplasia. Proliferation is assessed morphologically by microscopic inspection of histology and Ici67 immunohistochemical staining on chosen samples. On the final day of the experiments and RNA is isolated using the RNeasy kit as above.

An overview ofthe experiment

Markers of keratinocyte proliferation and differentiation are examined by semi- quantitative RT-PCR on the isolated RNA using PCR primers specific for Cyclin-A, Involucrine, Kl, K5, K6, K10, K14, MDM2 essentially as for the CYP24 above. We also examine if expression of vitamin D target genes are affected by the treatments. Accordingly, primers specific for vitamin D receptor (VDR), CYP24 p21Cipl/Wafl, c- Fos and b3-integrin are included among the RT-PCR's. Skin irritation is a facet of tachyphylaxis and the expression of irritation markers such as ILl-a; IL-6; IL-8; TNF-a; GM-CSF; TGF-b is examined by RT-PCR as above.

Results

Skin equivalent cultured in the continuous presence of vitamin D analogues respond to the hyperproliferative challenge by increased microscopic morphological markers of proliferation and increased ki67 staining as compared to untreated control. They also increased expression of the proliferation markers Cyclin-A, K5, K6, K14, MDM2 and decreased expression of the differentiation markers Involucrine, Kl, K10. This demonstrates that the antiproliferative effect of the vitamin D analogues is blunted by a tachyphylactic response, up-regulation of vitamin D analogue-catabolizing CYP's.

The response to the hyperproliferative challenge is abolished when a CYP24 inhibitor is co-administered with the vitamin D analogues. The expression of the vitamin D receptor is not down-regulated by the vitamin D analogue or CYP24-inhibitor treatment. This demonstrates that tachyphylaxis to vitamin D analogues can be abolished by CYP24 inhibitors adjunct to the vitamin D analogue regime. Furthermore, continuous vitamin D analogue treatment results in up-regulation of ILl-a; IL-6; IL-8; TNF-a; GM-CSF; TGF- b markers of skin irritation. This response is diminished by co-administration of CYP24 inhibitors with the vitamin D analogues. Therefore, in vitro cultured human skin equivalents exposed to both vitamin D analogue and adjunct CYP24 inhibitor are found to show a persistent response to vitamin D analogue treatment with lower associated irritancy and no tachyphylactic response.

Example 6 - Mouse Tail in vivo model of Tachyphylaxis

The mouse tail model is a morphometry-based, sensitive and reproducible method for the quantitative evaluation of the effects of drugs on epithelial differentiation and induction of orthokeratosis. Orthokeratosis is determined by measuring the horizontal length of the fully developed granular layer within an individual scale in relation to its total length (Sebδlc B, et al., S in Pharmacol. Appl. SkinPhysiol. 13:285-91, 2000).

The proximal half of mice are topically exposed to vitamin D analogues for 14 days with and without the CYP inhibitors according to the following protocol:

1. Vehicle alone;

2. 50 mg/g ointment Calcipotriol;

3. 40 mg/g ointment Tacalcitol; 4. 50 mg/g ointment Calcitrol;

5. 50 mg/g ointment CYP24 inhibitor alone;

6. 50 mg/g ointment Calcipotriol 4- 50 mg/g ointment CYP24 inhibitor;

7. 40mg/g ointment Tacalcitol + 50 mg/g ointment CYP24 inhibitor;

8. 50 mg/g ointment Calcitrol + 50 mg/g ointment CYP24 inhibitor; 9. 50 mg/g ointment CYP24 inhibitor treatment of mice on an 25(OH)D3 enriched diet

(4 microgram/kilo body weight/day).

Regime 5-9 is repeated with N-[4-chlorobenzoyl]-2-(lH-imidazol-l-yl)-2-phenyl-l- a ino ethane and N-[4-(hex-l-yl)benzoyl]-2-(lH-imidazol-l-yl)-2-phenyl-l-amino ethane.

At the completion of the treatment regimes RNA is isolated from the tail epidermis using the RNeasy lcit according to the instruction of the supplier (Qiagen Inc.) and used for semi-quantitative RT-PCR as above. Primers used are specific for mouse CYP24 and shown in table JJ.

Table II

Results Mouse tails exposed to both vitamin D analogues and adjunct CYP24 inhibitors are found to show an enhanced epithelial differentiation and orthokeratotic response with lower associated irritancy and no tachyphylactic response as compared to mouse tails treated with drug alone. Mouse tails treated with inhibitor alone show enhanced epithelial differentiation and orthokeratotic response compared to vehicle treated mouse tails. This response is potentiated by an 25(OH)D3 enriched diet. Expression of CYP24 mRNA is found to be increased by each of Calcipotriol, Tacalcitol and Calcitrol, as compared to controls. CYP24 is fully induced by 4 days of treatment.

Example 7 - Characterisation of the Effect of CYP24 Inhibitors On Tumor Growth In Vivo (breast cancer cells)

The severe combined immunodeficiency (SCID) mouse (scid,scid) is a strain of double mutant mouse with impaired lymphoid development and reduced natural killer cell activity. SCID mice have therefore been used as xenograft recipients to investigate the growth of implanted human tumours in vivo. The model has also been used to investigate the activity of anti-cancer therapies on the transplanted tumours. We have tested the effects of various CYP24 inhibitors on tumours transplanted into SCID mice.

Animals

The scid/scid mice used for these experiments are between the ages of 4 and 8 weeks. They are housed in microfilter cages. All cages, water and food are supplied after autoclaving. The cages are maintained in an air-conditioned and light-controlled

(12h day) room and all handling and operations are done in a laminar flow hood.

Tumour Implantation

Fresh specimens of human breast cell carcinoma are obtained shortly after surgical resection or from breast cancer cell lines MCF-7 and ZR-75-1 as in Example 2B.

Informed consent for the use of discarded tumour tissue is obtained from the patients prior to the operation. The tumour samples for experimental use are removed from the main tumour mass by a consultant pathologist in order not to compromise histopathological assessment of the resection margins of the tumour. All of the tumour procurement protocols are approved by the South Sheffield local research ethics committee. The fresh tumours are minced under sterile conditions in culture medium to 1.5 to 2.0 mm pieces and from these a single cell suspension is produced. The SCID mice are injected with 5 x 105 breast cell carcinoma cells subcutaneously into the right rear flank. Samples of the tumour cells are examined for quantification of CYP24 expression level.

CYP24 inhibition After the implanted tumours have grown to a palpable size below the skin surface (ca. 5mm³) or to a larger size above the skin surface (ca. 50mm³) the mice are injected via the tail vein with CYP24 inhibitors. A total of three injections are administered at weekly intervals and the experiment terminated six days after the last injection. A control group is injected at the same time points with the same volume of normal saline or purified antibodies from non-immunized animals. The size ofthe tumour appearing on the skin of a SCID mouse is measured in two dimensions with a caliper and the tumour volume is determined by the formula (width)² (length)/2. At the end of the experiment the mice are sacrificed, weighed and disected. The tumours are removed and weighed. The disected mice are examined for evidence of metastases. Immunohistochemistry of the resected tumours and other organs is performed.

Results

The average tumour volume in the mice injected with the CYP24 inhibitors is the same or reduced at the end of the experiment. In contrast in the control group injected with saline the average tumour volume at the end of the experiment is thirty times that at the beginning. The efficacy of the treatment correlates positively with the expression level of CYP24 in the tumor cells at time of implantation. Thus, injection of CYP24 inhibitors is capable of reducing tumour growth in vivo.

Claims

A compound of formula (I):

wherein:

R¹ is selected from phenyl, quinoline, isoquinoline and anthracene whereby each of these substituents may be optionally substituted by a group selected from halogen, C allcoxy, C allcyl, . diallcylamino and cyano;

R is selected from hydrogen and phenyl optionally substituted by a group selected from halogen, CM allcoxy, C_M allcyl, CM diallcylamino and cyano;

R³ is selected from halogen, _i₂ hydrocarbyl, phenyl optionally substituted by a group selected from halogen, _ alkoxy, C_M allcyl, . diaUcylamino and cyano, and N-acyl piperazinyl; and

X is selected from CO and S0₂,

pharmaceutically acceptable derivatives and solvates thereof.

2. A compound according to claim 1 wherein:

CM alkyl, CM diallcylamino or cyano;

R² is selected from phenyl optionally substituted by a group selected from halogen, .₄ alkoxy, CM alkyl, C dialkylamino or cyano; R³ is selected from halogen, C1-₁₂ hydrocarbyl, phenyl optionally substituted by a group selected from halogen, .₄ alkoxy, C_M allcyl, .₄ diallcylamino or cyano, and N-acyl piperazinyl; and

X is selected from CO and S0₂;

and pharmaceutically acceptable derivatives and solvates thereof.

3. A compound according to claim 1 wherein:

R¹ is selected from quinoline, isoquinoline and anthracene whereby these three substituents may be optionally substituted by a group selected from halogen, Q. allcoxy, C_M alkyl, _₄ diallcylamino or cyano;

R is hydrogen;

R³ is selected from halogen, -₁₂ hydrocarbyl, phenyl optionally substituted by a group selected from halogen, .₄ alkoxy, C_M alkyl, C_M dialkylamino or cyano, and N-acyl piperazinyl; and

X is selected from CO and S0₂;

and pharmaceutically acceptable derivatives and solvates thereof.

4. A compound according to claim 1 wherein:

R¹ is selected from phenyl, quinoline, isoquinoline and anthracene whereby each of these substituents may be optionally substituted by a group selected from halogen, C_M alkoxy, C_M allcyl, C diallcylamino or cyano;

R² is selected from hydrogen and phenyl optionally substituted by a group selected from halogen, C_M allcoxy, CM allcyl, C_M dialkylamino or cyano;

R³ is selected from halogen, C_M2 hydrocarbyl and N-acyl piperazinyl; and

X is selected from CO and S0₂; and pharmaceutically acceptable derivatives and solvates thereof.

5. A compound according to claim 1 wherein:

R¹ is selected from phenyl, quinoline, isoquinoline and anthracene whereby each of these substituents may be optionally substituted by a group selected from halogen, CM alkoxy, C allcyl, CM dialkylamino or cyano;

R² is selected from hydrogen and phenyl optionally substituted by a group selected from halogen, C allcoxy, CM allcyl, C_M diallcylamino or cyano;

R³ is selected from halogen, Cι_ι₂ hydrocarbyl, phenyl optionally substituted by a group selected from halogen, . allcoxy, Q.₄ allcyl, Q.₄ dialkylamino or cyano, and N-acyl piperazinyl; and

X is S0₂;

and pharmaceutically acceptable derivatives and solvates thereof.

6. A compound according to any of claims 1 to 5 selected from:

N-[4-(4-chlorophenyl)benzoyl]-2-(lH-imidazol-l-yl)-2,2-(di-4-chlorophenyl)-l-amino ethane; N-[4-(4-chlorophenyl)benzoyl]-2-(l H-imidazol- 1 -yl)-2-(isoquinol-6-yl)- 1 -amino ethane;

N-[4-(4-chlorophenyl)benzoyl]-2-( 1 H-imidazol- 1 -yl)-2-(anthracen-2-yl)- 1 -amino ethane;

N- [4-(oct- 1 -yl)benzoyl]-2-( 1 H-imidazol- 1 -yl)-2-phenyl- 1 -amino ethane; N-[4-chlorobenzoyl] -2-( lH-imidazol- 1 -yl)-2-phenyl- 1 -amino ethane;

N-[4-(hex-l-yl)benzoyl]-2-(lH-imidazol-l-yl)-2-phenyl-l-amino ethane;

N-[4-(hept- 1 -yl)benzoyl]-2-( 1 H-imidazol- 1 -yl)-2 -phenyl- 1 -amino ethane;

N-[4-(4-acylpiperazinyl)benzoyl]-2-(lH-imidazol- 1 -yl)-2 -phenyl- 1 -amino ethane; and

4-chlorophenyl- N-(2-lH-imidazol-l-yl-2-phenyl-ethyl)-sulphonamide.

. A method for the treatment of psoriasis, eczematous diseases, scleroderma, viteligo, degenerative changes of connective tissue both pathologic and accompanying normal ageing and photo ageing, in the prevention of hair loss and regeneration of hair growth, diseases associated with abnormal cellular proliferation such as neoplasms and cancer, in increasing tolerance against allotransplantes, in rheumatoid arthritis and in bone remodelling in a subject, preferably a human, which comprises administering to the subject an effective amount of a compound of formula (I) or pharmaceutically acceptable derivative or solvate thereof.

§ A compound of formula (I) or a pharmaceutically acceptable derivative or solvate thereof, for use in medical therapy.

9- Use of a compound of formula (I) or pharmaceutically acceptable derivative or solvate thereof for the manufacture of a medicament for the treatment of psoriasis, eczematous diseases, scleroderma, viteligo, degenerative changes of connective tissue both pathologic and accompanying normal ageing and photo ageing, in the prevention of hair loss and regeneration of hair growth, diseases associated with abnormal cellular proliferation such as neoplasms and cancer, in increasing tolerance against allotransplantes, in rheumatoid arthritis and in bone remodelling.

. A pharmaceutical formulation comprising a compound of formula (I) or a pharmaceutically acceptable derivative or solvate thereof, and one or more pharmaceutically acceptable carriers.

ii . A process for the preparation of a compound of formula (I) which comprises reacting a compound of formula

(ii) wherein R¹, R², R³ and X are as defined in claim 1 with imidazole.

A method of alleviating or preventing a tachyphylactic response to vitamin D or an analogue thereof in a patient, preferably a human, which method comprises administering to the patient an effective amount of a compound of formula (I) or pharmaceutically acceptable derivative or solvate thereof. "(3 Use of a compound of formula (I) or a pharmaceutically acceptable derivative or solvate thereof, for the manufacture of a medicament for alleviating or preventing a tachyphylactic response to vitamin D or an analogue thereof.

14- A combination of a compound of formula (I) or a pharmaceutically acceptable derivative or solvate thereof and vitamin D or an analogue thereof.

1 J, A pharmaceutical formulation comprising a compound of formula (I) or a pharmaceutically acceptable derivative or solvate thereof and vitamin D or an analogue thereof, together with one or more pharmaceutically acceptable carriers therefor.

lg», A kit comprising in association for concomitant or sequential administration a compound of formula (I) or a pharmaceutically acceptable derivative or solvate thereof together with vitamin D or an analogue thereof.