COMPOUNDS FOR TREATING DUCHENNE MUSCULAR DYSTROPHY
The present invention relates to a method of treatment of Duchenne muscular dystrophy.
Duchenne muscular dystrophy (DMD) is a common, genetic neuromuscular disease associated with the progressive deterioration of muscle function, first described over 150 years ago by the French neurologist, Duchenne de Boulogne, after whom the disease is named. DMD has been characterized as an X-linked recessive disorder that affects 1 in 3,500 males caused by mutations in the dystrophin gene. The gene is the largest in the human genome, encompassing 2.6 million base pairs of DNA and containing 79 exons. Approximately 60% of dystrophin mutations are large insertion or deletions that lead to frameshift errors downstream, whereas approximately 40% are point mutations or small frameshift rearrangements. The vast majority of DMD patients lack the dystrophin protein. Becker muscular dystrophy is a much milder form of DMD caused by reduction in the amount, or alteration in the size, of the dystrophin protein. The high incidence of DMD (1 in 10,000 sperm or eggs) means that genetic screening will never eliminate the disease, so an effective therapy is highly desirable.
A number of natural and engineered animal models of DMD exist, and provide a mainstay for preclinical studies (Allamand, V. & Campbell, K. P. Animal models for muscular dystrophy: valuable tools for the development of therapies. Hum. MoI. Genet. 9, 2459-2467 (2000).) Although the mouse, cat and dog models all have mutations in the DMD gene and exhibit a biochemical dystrophinopathy similar to that seen in humans, they show surprising and considerable variation in terms of their phenotype. Like humans, the canine (Golden retriever muscular dystrophy and German short-haired pointer) models have a severe phenotype; these dogs typically die of cardiac failure. Dogs offer the best phenocopy for human disease, and are considered a high benchmark for preclinical studies. Unfortunately, breeding these animals is expensive and difficult, and the clinical time course can be variable among litters.
The max mouse is the most widely used model due to availability, short gestation time, time to mature and relatively low cost (Bulfield, G., Siller, W. G., Wight, P. A. & Moore, K. J. X chromosome-linked muscular dystrophy (mdx) in the mouse. Proc. Natl Acad. Sci. USA 81, 1189-1192 (1984)).
Since the discovery of the DMD gene about 20 years ago, varying degrees of success in the treatment of DMD have been achieved in preclinical animal studies, some of which are being followed up in humans. Present therapeutic strategies can be broadly divided into three groups: first, gene therapy approaches; second, cell therapy; and last, pharmacological therapy. Gene- and cell-based therapies offer the fundamental advantage of obviating the need to separately correct secondary defects/ pathology (for example, contractures), especially if initiated early in the course of the disease. Unfortunately, these approaches face a number of technical hurdles. Immunological responses against viral vectors, myoblasts and newly synthesized dystrophin have been reported, in addition to toxicity, lack of stable expression and difficulty in delivery.
Pharmacological approaches for the treatment of muscular dystrophy differ from gene- and cell-based approaches in not being designed to deliver either the missing gene and/or protein. In general, the pharmacological strategies use drugs/molecules in an attempt to improve the phenotype by means such as decreasing inflammation, improving calcium homeostasis and increasing muscle progenitor proliferation or commitment. These strategies offer the advantage that they are easy to deliver systemically and can circumvent many of the immunological and/or toxicity issues that are related to vectors and cell-based therapies. Although investigations with corticosteroids and sodium cromoglycate, to reduce inflammation, dantrolene to maintain calcium homeostasis and clenbuterol to increase muscle strength, have produced promising results none of these potential therapies has yet been shown to be effective in treating DMD.
An alternative pharmacological approach is upregulation therapy. Upregulation therapy is based on increasing the expression of alternative genes to replace a defective gene and is particularly beneficial when an immune response is mounted against a previously absent protein. Upregulation of utrophin, an autosomal paralogue of dystrophin has been proposed as a potential therapy for DMD (Perkins & Davies, Neuromuscul Disord, Sl: S78-S89 (2002), Khurana & Davies, Nat Rev Drug Discov 2:379-390 (2003)). When utrophin is overexpressed in transgenic mdx mice it localizes to the sarcolemma of muscle cells and restores the components of the dystrophin-associated protein complex (DAPC), which prevents the dystrophic development and in turn leads to functional improvement of skeletal muscle. Adenoviral delivery of utrophin in the dog has been shown to prevent pathology. Commencement of increased utrophin expression shortly after birth in the mouse model can be effective and no toxicity is observed when utrophin is ubiquitously expressed, which is promising for the translation of this therapy to humans. Upregulation of endogenous utrophin to sufficient levels to decrease pathology might be achieved by the delivery of small diffusible compounds.
In our earlier applications PCT/GB2007/050055, PCT/GB2007/050056, UK Patent application No. 0617739.8, UK Patent application No. 0619282.7, UK Patent application No. 0623985.9, UK Patent application No. 0617740.6 and UK Patent application No. 0619283.5, we disclosed compounds which upregulate endogenous utrophin in predictive screens and, thus, may be useful in the treatment of DMD.
We have now found an additional group of compounds which have a similar effect and may also be useful in the treatment of this condition and therefore, in a first aspect of the present invention, there is provided a compound of general formula (I):
wherein:
X1, X2 and X3 are each independently a linker selected from a bond, -NR4", -O-, -S-, -NR4C(O)-, -NR4C(S)-, -NR4C(O)O-, -NR4SO2-, -NR4C(O)NR4-, -NR4C(S)NR4-, -NR4C(NH)NR4-, -NR4C(NH)-, -C(O)-, -C(S)-, -C(O)NR4-, -C(S)NR4-, -SO-, -SO2-, -OC(O)O-, -SO2NR4-, -OC(O)NR4- or -P(O)OR4-; each R4 is independently H or C 1-6 alkyl optionally substituted by halo;
R1, R2 and R3 are each independently hydrogen, aryl, heteroaryl, cycloalkyl, heterocyclyl, Ci -C 10 alkyl, C2-CiO alkenyl or C2-Ci0 alkynyl, any of which may optionally be substituted with halo or a group X4-R6, or when X1, X2 or X3 is a bond, R1, R2 or R3 may also be halo, NO2 or CN; or alternatively
R2 and R3 together with the atoms to which they are attached may form a 5 to 12 membered monocyclic or bicyclic aromatic or non aromatic ring system optionally containing one or more heteroatoms selected from O, S or N, optionally substituted by one or more substitutents X4R6; where: X4 is selected from a bond, -NR4", -0-, -S-,
-NR4C(O)-, -NR4C(S)-, -NR4C(O)O-, -NR4SO2-, -NR4C(O)NR4-, - NR4C(S)NR4-,
-NR4C(NH)NR4-, -NR4C(NH)-, -C(O)-, -C(S)-, -C(O)NR4-, -C(S)NR4-, -SO-, -SO2-, -OC(O)O-, -SO2NR4-, -OC(O)NR4- or -P(O)OR4-; where R4 is as defined above; and
R6 is H or Ci -CO alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl may optionally be substituted with halo or -0(CI-CO alkyl) and wherein cycloalkyl, heterocyclyl, aryl and heteroaryl groups may also be subsituted with C1-C6 alkyl; or, when X4 is a bond, R6 may also be halo; NO2 or CN;
Y is O, S or NR7;
R7 is hydrogen or Ci-Cδ alkyl; Z is N or CR8;
R8 is hydrogen or Ci-Cβ alkyl;
provided that when R1 is phenyl substituted with X4R6 and X4 is -NR4C(O)-, R6 is not phenyl; or tautomer, enantiomer or pharmaceutically acceptable salt, hydrate, solvate, complex or prodrug thereof; for use in the treatment or prophylaxis of Duchenne muscular dystrophy, Becker muscular dystrophy or cachexia.
The invention also relates to a method for the treatment or prophylaxis of Duchenne muscular dystrophy, Becker muscular dystrophy or cachexia, the method comprising administering to a patient in need of such treatment an effective amount of a compound of general formula (I) as defined above.
Some compounds of general formula (I) are known but they have not been used for the treatment of Duchenne muscular dystrophy, Becker muscular dystrophy or cachexia.
Mekonnnen et al (J. Het. Chem. 1997, 589) describe the synthesis of imidazo[2,l- bjoxazoles, which are compounds of general formula (I) in which Y is O. One of the uses of such compounds is said to be the treatment of non-bacterial inflammations. The document also refers to imidazo[2,l-b]thiazoles, wich are said to be useful as anthelmitics and as bactericides. Some imidazothiazoles are also said to be immunomodulators and the imidazo imidazole analogues are said to have immunomodulatory and anti-inflamatory activity.
Jag et al {Indian Journal of Heterocyclic Chemistry, 6(1), 21-24 (1996)) describe the synthesis and bio-activity of thiazolo [3,2-b]-s-triazoles and isomeric thiazolo [2,3- c]-s-triazoles.
In the present specification "Ci-Cβ alkyl" refers to a straight or branched saturated hydrocarbon chain having one to six carbon atoms. Examples include methyl, ethyl, n-propyl, isopropyl, t-butyl, n-hexyl.
"C1-C4 alkyl" and "Ci-Cio alkyl" have similar meanings except that they contain respectively from one to four and from one to ten carbon atoms.
"C2-Ce alkenyl" refers to a straight or branched hydrocarbon chain having from two to six carbon atoms and containing at least one carbon-carbon double bond. Examples include ethenyl, 2-propenyl, and 3-hexenyl.
"C2-Cn alkynyl" refers to a straight or branched hydrocarbon chain having from two to six carbon atoms and containing at least one carbon-carbon triple bond. Examples include ethynyl, 2-propynyl, and 3-hexynyl.
The terms "C2-CiO alkenyl" and "C2-Ci0 alkynyl" have similar meanings except that they contain from one to ten carbon atoms.
The term "C2-C6 haloalkyl" refers to a Ci_6 alkyl group as defined above substituted by one or more halogen atoms.
The term "cyclic groups" refers to cycloalkyl, heterocyclyl, aryl or heteroaryl groups.
The term "cycloalkyl" refers to a saturated 3 to 14 membered carbocyclic ring including fused bicyclic or tricyclic systems. Examples of such groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and also bridged systems such as norbornyl and adamantyl.
"heterocyclyl" refers to a saturated 3 to 14 membered ring system similar to cycloalkyl but in which at least one of the carbon atoms has been replaced by N, O or S. Examples include piperidine, piperazine, morpholine, tetrahydrofuran and pyrrolidine,
The terms "aryl" and "aromatic moiety" in the context of the present specification refer to an aromatic ring system having from 5 to 14 ring carbon atoms and containing up to three rings. Examples of aromatic moieties are benzene and
naphthalene.
The terms "heteroaryl" and "heteroaromatic moiety" refer to an aromatic ring system, which may be partially saturated and which has from 5 to 14 ring carbon atoms and containing up to three rings and at least one heteroatom selected from N, O and S. Examples include pyridine, pyrimidine, furan, thiophene, indole, iso indole, benzofuran, benzimidazole, benzimidazoline quinoline, isoquinoline, tetrahydroisoquinoline, quinazoline, thiazole, benzthiazole, benzoxazole, indazole and imidazole ring systems..
In the present specification, "halo" refers to fluoro, chloro, bromo or iodo.
Appropriate pharmaceutically and veterinarily acceptable salts of the compounds of general formulae (Ia) and (Ib) include basic addition salts such as sodium, potassium, calcium, aluminium, zinc, magnesium and other metal salts as well as choline, diethanolamine, ethanolamine, ethyl diamine and other well known basic addition salts.
Where appropriate, pharmaceutically or veterinarily acceptable salts may also include salts of organic acids, especially carboxylic acids, including but not limited to acetate, trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate, malate, pantothenate, adipate, alginate, aspartate, benzoate, butyrate, digluconate, cyclopentanate, glucoheptanate, glycerophosphate, oxalate, heptanoate, hexanoate, fumarate, nicotinate, pamoate, pectinate, 3-phenylpropionate, picrate, pivalate, proprionate, tartrate, lactobionate, pivolate, camphorate, undecanoate and succinate, organic sulfonic acids such as methanesulfonate, ethanesulfonate, 2-hydroxyethane sulfonate, camphorsulfonate, 2-naphthalenesulfonate, benzenesulfonate, p- chlorobenzenesulfonate and p-toluenesulfonate; and inorganic acids such as hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, hemisulfate, thiocyanate, persulfate, phosphoric and sulfonic acids.
Salts which are not pharmaceutically or veterinarily acceptable may still be valuable
as intermediates.
Prodrugs are any covalently bonded compounds which release the active parent drug according to general formula (I) in vivo.
If a chiral centre or another form of isomeric centre is present in a compound of the present invention, all forms of such isomer or isomers, including enantiomers and diastereoisomers, are intended to be covered herein. Compounds of the invention containing a chiral centre may be used as a racemic mixture, an enantiomerically enriched mixture, or the racemic mixture may be separated using well-known techniques and an individual enantiomer may be used alone.
In preferred compounds of general formula (I), independently or in any combination:
X1, X2 and X3 are bonds; R1 is a cycloalkyl, heterocyclyl, aryl or heteroaryl, any of which may optionally be substituted with one or more substituents selected from halo, OH, 0(CI-CO alkyl) or
NHC(O)R6, wherein R6 is Ci-C6 alkyl, optionally substituted with halo or 0(Ci-C6 alkyl);
R2 and R3 are hydrogen, Ci-C6 alkyl or halo or R2 and R3 together with the atoms to which they are attached form an aromatic ring;
Y is O or S; and
Z is CR8 where R is as defined above.
In more preferred compounds of general formula (I), independently or in any combination:
X1, X2 and X3 are bonds;
R1 is a cycloalkyl, aryl or heteroaryl group optionally substituted with one or more substituents selected from halo, OH, O(C,-C4 alkyl) or NHC(O)R6, wherein R6 is C,-
C6 alkyl optionally subsituted with halo; R2 and R3 are hydrogen, C1-C4 alkyl or halo or R2 and R3 together with the atoms to which they are attached form a phenyl ring;
Y is O or S; and
Z is CR8 where R8 is hydrogen, methyl or ethyl.
In the most active compounds of general formula (I): X1, X2 and X3 are bonds; R1 is a phenyl, naphthyl, thienyl, cyclohexyl or 3-chromene-2-onyl any or which is optionally substituted with one or more substituents selected from halo, OH, methoxy, ethoxy or NHC(O)R6, wherein R6 is Ci-C4 alkyl;
R2 and R3 are hydrogen, methyl or chloro or R2 and R3 together with the atoms to which they are attached form a phenyl ring; Y is O or S; and Z is CH.
Particularly preferred compounds of general formula (I) include:
I . 4-(imidazo[2, 1 -b]thiazol-6-yl)-N,N-dimethylaniline; 2. 6-(naphthalen-2-yl)imidazo[2,l-b]thiazole;
3. 6-(3 -chlorophenyl)imidazo [2 , 1 -b] thiazo Ie ;
4. 6-(4-chlorophenyl)-2-methylimidazo[2, l-b]thiazole;
5. N-(4-(imidazo[2,l-b]thiazol-6-yl)phenyl)acetamide;
6. 2-(thiophen-2-yl)-4H-benzimidazo[ 1 ,2-b] [ 1 ,2,4]triazole; 7. 8-methoxy-3-(imidazo[2,l-b]benzothiazol-4-yl)-2H-chromen-2-one;
8. N-(3-(imidazo[2,l-b]thiazol-6-yl)phenyl)isobutyramide;
9. N-(3-(imidazo[2,l-b]thiazol-6-yl)phenyl)-3-methylbutanamide;
10. 6-(4-chlorophenyl)-3-methylimidazo[2, l-b]oxazole;
I I. 2-(imidazo[2,l-b]thiazol-6-yl)phenol; 12. 6-(4-chlorophenyl)imidazo[2,l-b]thiazole;
13. 2-methyl-6-(naphthalen-2'-yl)imidazo[2, l-b]thiazole;
14. 6-(3-chlorophenyl)-2-methylimidazo[2,l-b]thiazole;
15. ?6-(3',4'-dichlorophenyl)-2-methylimidazo[2,l-b]thiazole;
16. 6-(4'-chlorophenyl)-3-methylimidazo[2, l-b]thiazole; 17. 6-(3',4'-dichlorophenyl)-3-methylimidazo[2,l-b]thiazole;
18. 6-(2'-naphthyl)-3 -methylimidazo[2 , 1 -bjthiazo Ie ;
19. 6-(3-chlorophenyl)-3-methylimidazo[2,l-b]thiazole;
20. 2-chloro-6-(4'-chlorophenyl)imidazo[2, 1 -b]thiazole;
21. 6-(3,4-dichlorophenyl)imidazo[2,l-b]thiazole-3-carboxylic acid;
22. 2-chloro-6-(naphthalen-2'-yl)imidazo[2, l-b]thiazole;
23. 2-chloro-6-cyclohexylimidazo[2,l-b]thiazole; 24. 3-Methyl-6-(2'-naphthyl)imidazo[2,l-b]oxazole;
25. 2-chloro-6-(5',6',7',8'-tetrahydronaphthalen-2'-yl)imidazo[2,l-b]thiazole or a tautomer, enantiomer or pharmaceutically acceptable salt, hydrate, solvate, complex or prodrug thereof.
Compounds 15 to 19 and 21 to 25 are new and therefore, in a further aspect of the invention, there is provided a compound selected from:
6-(3',4'-dichlorophenyl)-2-methylimidazo[2, 1 -bjthiazole;
6-(4'-chlorophenyl)-3-methylimidazo[2,l-b]thiazole;
6-(3',4'-dichlorophenyl)-3-methylimidazo[2, 1 -b]thiazole; 6-(2'-naphthyl)-3-methylimidazo[2, l-b]thiazole;
6-(3-chlorophenyl)-3-methylimidazo[2,l-b]thiazole;
6-(3,4-dichlorophenyl)imidazo[2,l-b]thiazole-3-carboxylic acid;
2-chloro-6-(naphthalen-2'-yl)imidazo[2, 1 -bjthiazole;
2-chloro-6-cyclohexylimidazo[2,l-b]thiazole; 3-Methyl-6-(2'-naphthyl)imidazo[2,l-b]oxazole;
2-chloro-6-(5',6',7',8'-tetrahydronaphthalen-2'-yl)imidazo[2,l-b]thiazole
or a tautomer, enantiomer or pharmaceutically acceptable salt, hydrate, solvate, complex or prodrug thereof.
In a further aspect of the invention there is provided the use of a compound of general formula (I) as defined above in the preparation of an agent for the treatment or prophylaxis of Duchenne muscular dystrophy, Becker muscular dystrophy or cachexia.
Many of the compounds of general formula (I) are commercially available and others can be synthesised from commercially available starting materials using the following method.
A compound of general formula (I) in which Z is CR8 may be prepared from a compound of general formula (II):
1
(H) wherein X1, X2, X3, R1, R2, R3, R8 and Y are as defined in general formula (I) by reaction with a Lewis acid such as titanium tetrachloride in an organic solvent such as toluene.
Preferably, the starting material is cooled to a temperature of about -15 to O0C before addition of the Lewis acid. The reaction mixture is then allowed to warm to room temperature and heated under reflux for 1 to 5 hours before the product is isolated.
An intermediate compound of general formula (II) may be prepared from a compound of general formula (III):
(HI) wherein X2, X3, R2, R3 and Y are as defined in general formula (I); by condensation with a compound of general formula (IV):
(IV) wherein X1, R1 and R8 are as defined in general formula (I) and Q is a leaving group, typically a halogen such as bromine.
The reaction may be carried out by mixing a compound of general formula (III) in a polar organic solvent such as acetonitrile and a compound of general formula (IV) in an organic solvent such as tetrahydrofuran. The reaction is most suitable carried out under anhydrous conditions and under an inert atmosphere such as nitrogen or argon. The reaction temperature is suitably from about 15 to 250C.
Compounds of general formulae (III) are known to those of skill in the art and are readily available or may be prepared by known methods, for example the methods described by Mekonnen et al, J. Heterocyclic Chem, 34, 589-599 (1997).
Compounds of general formula (III) in which X2R2 or X3R3 is optionally substituted aryl, heteroaryl, cycloalkyl, heterocyclyl, C|-Cio alkyl, C2-C10 alkenyl or C2-CiO alkynyl can be prepared from equivalent compounds of general formula (III) in which X2R2 or X3R3 is halogen, especially bromo, by reaction with a compound of general formula (IX):
R5B(OH)2 (IX) where R5 is aryl, heteroaryl, cycloalkyl, heterocyclyl, Ci-Cio alkyl, C2-CiO alkenyl or C2-C10 alkynyl, any of which may be substituted as defined for R1, R2 and R3 in general formula (I); using a palladium catalyst.
Many compounds of general formula (IV) are also known to those of skill in the art and are readily available. Alternatively, compounds of general formula (IV) may be prepared by reacting a compound of general formula V:
(V) wherein X1, R1 and R8 are as defined in general formula (I); with a halogenating agent.
For example, compounds of general formula (IV) in which Q is bromo can be prepared by reacting the appropriate compound of general formula (V) with bromine in polar organic solvent, especially an alcoholic solvent such as methanol.
Compounds of general formula (I) in which Z is CR8 and X1 is S and can be prepared from compounds of general formula (VI)
(VI) wherein X2, X3, R1, R2, R3 and Y are as defined in general formula (I); by reaction with a compound of general formula (VII):
Q-CH2R8 (VII) where R8 is as defined for general formula (I) and Q is as defined above for general formula (IV).
The reaction is typically carried out under basic conditions.
A compound of general formula (VI) can be prepared from a compound of general formula (II) as defined above by reaction with carbon disulphide followed by a compound of general formula (VIII):
R1 -Br (VIII)
wherein R1 is as defined above for general formula (I). The reaction is generally carried out under basic conditions.
Compounds of general formulae (VII) and (VIII) are well known to those of skill in the art and are either readily available or can be prepared by well known methods.
Compounds of general formula (I) in which X1, X2 or X3 are SO2 can be prepared by oxidation using standard methods from compounds of general formula (I) in which X1, X2 or X3 are S.
Compounds of general formula (I) in which Z is CR8 and X1R1 is halogen, for example chloro or bromo, may be prepared from compounds of general formula (II) as defined above in which X1R1 is OH by reaction with an appropriate phosphoryl halide.
Compounds of general formula (I) in which Z is N may be prepared following the method described by Jag et al, "Condensed bridgehead nitrogen heterocyclic systems: Synthesis and bio-activity of thiazolo [3,2-b]-s-triazoles and isomeric thiazolo [2,3-c]-s-triazoles", Indian Journal of Heterocyclic Chemistry, 6(1), 21-24 (1996).
Compounds of general formula (I) can be converted to other compounds of general formula (I). For example, compounds of general formula (I) in which X1R1, X2R2 or X3R3 is C(0)0(Ci-Cio) alkyl can be converted to the equivalent compound of general formula (I) in which X1R1, X2R2 or X3R3 is C(O)OH by hydrolysis, for example base hydrolysis using sodium hydroxide in a polar organic solvent such as tetrahydrofuran. Compounds in which X1R1, X2R2 or X3R3 is C(O)OH can in turn be converted to compounds in which X1R1, X2R2 or X3R3 is OH by standard reduction methods, for example using NMM in tetrahydrofuran at O0C, followed by reaction with aqueous sodium borohydride. The alcohols can, in turn, be converted
to numerous other functional groups, for example carbamates, phosphinates, ethers or amines, using standard methods well known to those of skill in the art.
Compounds in which X1R1, X2R2 or X3R3 is C(O)OH may in addition be converted to a range of further compounds, including amine derivatives (by a Curtius reaction), acyl derivatives and carbonyl derivatives. The methods used for these conversions are all well known and would be familiar to a skilled chemist.
In the syntheses described above, it may sometimes be necessary to use protecting groups. The necessity for using such protecting groups would be apparent to a skilled chemist who would also be capable of choosing appropriate protecting groups. Information concerning protecting groups is available in in "Protecting Groups in Organic Synthesis", Theodora W. Greene and Peter G. M. Wuts, published by John Wiley & Sons Inc.
The compounds of general formula (I) for use in the treatment of DMD will generally be administered in the form of a pharmaceutical composition and some of these pharmaceutical compositions form a further aspect of the invention.
Therefore, the invention also provides a pharmaceutical composition comprising a compound selected from:
6-(3',4'-dichlorophenyl)-2-methylimidazo[2, 1 -b]thiazole;
6-(4'-chlorophenyl)-3-methylimidazo[2,l-b]thiazole;
6-(3',4'-dichlorophenyl)-3-methylimidazo[2,l-b]thiazole; 6-(2'-naphthyl)-3-methylimidazo[2, l-b]thiazole;
6-(3-chlorophenyl)-3-methylimidazo[2,l-b]thiazole;
6-(3,4-dichlorophenyl)imidazo[2,l-b]thiazole-3-carboxylic acid;
2-chloro-6-(naphthalen-2'-yl)imidazo[2, 1 -b]thiazole;
2-chloro-6-cyclohexylimidazo[2,l-b]thiazole; 3-Methyl-6-(2'-naphthyl)imidazo[2,l-b]oxazole;
2-chloro-6-(5',6',7',8'-tetrahydronaphthalen-2'-yl)imidazo[2,l-b]thiazole; or
a tautomer, enantiomer or pharmaceutically acceptable salt, hydrate, solvate, complex or prodrug thereof; and a pharmaceutically acceptable excipient.
The pharmaceutical composition may include less than 80% w/w, more preferably less than 50% w/w, e.g. 0.1 to 20%, of a compound of general formula (I), or a pharmaceutically acceptable salt thereof, as defined above, in admixture with a pharmaceutically acceptable diluent or carrier.
We also provide a process for the production of such a pharmaceutical composition which comprises mixing the ingredients.
Examples of pharmaceutical formulations which may be used, and suitable diluents or carriers, are as follows: for intravenous injection or infusion - purified water or saline solution; for inhalation compositions - coarse lactose; for tablets, capsules and dragees - microcrystalline cellulose, calcium phosphate, diatomaceous earth, a sugar such as lactose, dextrose or mannitol, talc, stearic acid, starch, sodium bicarbonate and/or gelatin; for suppositories - natural or hardened oils or waxes.
When the compound is to be used in aqueous solution, e.g. for infusion, it may be necessary to incorporate other excipients. In particular there may be mentioned chelating or sequestering agents, antioxidants, tonicity adjusting agents, pH- modifying agents and buffering agents.
Solutions containing a compound of general formula (I) may, if desired, be evaporated, e.g. by freeze drying or spray drying, to give a solid composition, which may be reconstituted prior to use.
When not in solution, the compound of general formula (I) preferably is in a form having a mass median diameter of from 0.01 to lOμm. The compositions may also contain suitable preserving, stabilising and wetting agents, solubilisers, e.g. a water-
soluble cellulose polymer such as hydroxypropyl methylcellulose, or a water-soluble glycol such as propylene glycol, sweetening and colouring agents and flavourings. Where appropriate, the compositions may be formulated in sustained release form. The content of compound general formula (I) in a pharmaceutical composition is generally about 0.01 -about 99.9wt%, preferably about 0.1 -about 50wt%, relative to the entire preparation.
The dose of the compound of general formula (I) is determined in consideration of age, body weight, general health condition, diet, administration time, administration method, clearance rate, combination of drugs, the level of disease for which the patient is under treatment then, and other factors.
While the dose varies depending on the target disease, condition, subject of administration, administration method and the like, for oral administration as a therapeutic agent for the treatment of Duchenne muscular dystrophy in a patient suffering from such a disease is from 0.01 mg - 1O g, preferably 0.1 - 100 mg, is preferably administered in a single dose or in 2 or 3 portions per day.
The invention will now be described in more detail with reference to the following examples and the drawings in which:
FIGURE 1 shows an example of TA muscle sections stained with antibody specific for mouse utrophin. Comparison of a positive control compound, 5-amino-2-(5,6- dimethylbenzo[d]oxazol-2-yl)phenol (CPD-A) known to increase the levels of utrophin protein in dystrophin deficient mdx muscle with mdx muscle injected only with vehicle (as negative control) shows an increase in the amount of sarcolemmal bound utrophin.
FIGURE 2 Shows a Western blot of excised and processed muscles from the above treated mice stained with specific antibodies (see Figure 2). Muscle dosed with
CPD-A shows a significant increase in the overall levels of utrophin present in both
the TA leg muscle and the diaphragm. Both mice exposed to CPD-A (V2 and V3) showed increased levels of utrophin expression compared to the negative control.
In the Examples:
HPLC-UV-MS was performed on a Gilson 321 HPLC with detection performed by a Gilson 170 DAD and a Finnigan AQA mass spectrometer operating in electrospray ionisation mode. The HPLC column used is a Phenomenex Gemini Cl 8 150x4.6mm. Preparative HPLC was performed on a Gilson 321 with detection performed by a Gilson 170 DAD. Fractions were collected using a Gilson 215 fraction collector. The preparative HPLC column used is a Phenomenex Gemini Cl 8 150x 10mm and the mobile phase is acetonitrile/water.
1H NMR spectra were recorded on a Bruker instrument operating at 300 MHz. NMR spectra were obtained as CDCI3 solutions (reported in ppm), using chloroform as the reference standard (7.25 ppm) or DMSO-DO (2.50 ppm). When peak multiplicities are reported, the following abbreviations are used s (singlet), d (doublet), t (triplet), m (multiple:), br (broadened), dd (doublet of doublets), dt (doublet of triplets), td (triplet of doublets). Coupling constants, when given, are reported in Hertz (Hz).
Column chromatography was performed either by flash chromatography (40-65 μm silica gel) or using an automated purification system (SP1™ Purification System from Biotage®). Reactions in the microwave were done in an Initiator 8™ (Biotage).
The abbreviations used are DMSO (dimethylsulfoxide), HATU (O-(7- azabenzotriazol- IyI)-N ,N,N',N'-tetramethyluronium hexafluorophosphate), HCl (hydrochloric acid), MgSO4 (magnesium sulfate), NaOH (sodium hydroxide), Na2Cθ3 (sodium carbonate), NaHCU3 (sodium bicarbonate), STAB (sodium triacetoxyborohydride), THF (tetrahydrofuran).
All the compounds below were purchased from Chembridge: 4-(Imidazo[2, l-b]thiazol-6-yl)-N,N-dimethylaniline (Compound 1)
6-(Naphthalen-2-yl)imidazo[2,l-b]thiazole (Compound 2) 6-(3-Chlorophenyl)imidazo[2,l-b]thiazole (Compound 3) 6-(4-Chlorophenyl)-2-methylimidazo[2,l-b]thiazole (Compound 4) N-(4-(Imidazo[2,l-b]thiazol-6-yl)phenyl)acetamide (Compound 5) 4-(6-Methylimidazo[2,l-b]thiazol-5-yl)-N-(pyridin-2-yl)thiazol-2-amine N-Allyl-4-(6-methylimidazo[2,l-b]thiazol-5-yl)thiazol-2-amine N-(2,5-Dimethoxyphenyl)-4-(6-methylimidazo[2,l-b]thiazol-5-yl)thiazol-2-amine N-(3-(Imidazo[2, l-b]thiazol-6-yl)phenyl)isobutyramide (Compound 8) N-(3-(Imidazo[2, 1 -b]thiazol-6-yl)phenyl)-3-methylbutanamide (Compound 9) 6-(4-Chlorophenyl)imidazo[2,l-b]thiazo Ie (Compound 12)
2-Methyl-6-(naphthalen-2-yl)imidazo[2, 1 -b]thiazole (Compound 13) 6-(3-Chlorophenyl)-2-methylimidazo[2,l-b]thiazole (Compound 14) 2-(Thiophen-2-yl)-4H-benzimidazo[ 1 ,2-b][ 1 ,2,4]triazole (Compound 6); 8-Methoxy-3-(imidazo[2,l-b]benzothiazol-4-yl)-2H-chromen-2-one (Compound 7); The compound below was purchased from Bionet:
6-(4-Chlorophenyl)-3-methylimidazo[2,l-b]oxazole (Compound 10)
The compound below was purchased from Buttpark:
2-(Imidazo[2,l-b]thiazol-6-yl)phenol (Compound 11)
Example 1 - Synthesis of Compounds of General Formula (I)
The Compounds were synthesised using the following general reaction scheme:
5min
6-(3',4'-Dichlorophenyl)-2-methylimidazo[2,l-b]thiazole (Compound 15)
A solution of 5-methylthiazol-2-amine (lOOmg, 0.88mmol) and 2-bromo-l-(3,4- dichlorophenyl)ethanone (235mg, 0.88mmol) in ethanol (5mL) was heated in the
microwave at 15O0C for 5min. After cooling, the mixture was concentrated in vacuo, and dissolved in ethyl acetate. The organic layer was washed with saturated aqueous NaHCC>3 and the combined organic layers were dried over anhydrous MgSO4. The resulting solid, obtained after evaporation, was purified by column chromatography eluting with ethyl acetate/hexanes 1:2 v/v to afford 64mg (26%) of the title compound (LCMS RT= 7.50min, MH+ 282.9)
1H NMR (DMSO): 8.30 (IH, s), 8.04 (IH, d, J 2.0 Hz), 7.79 (IH, dd, J 8.4 2.0 Hz), 7.75-7.73 (IH, m), 7.63 (IH, d, J 8.4 Hz), 2.41 (3H, d, J 1.5 Hz)
AU compounds below were prepared following the same general method.
6-(4'-Chlorophenyl)-3-methylimidazo[2,l-b]thiazole (Compound 16)
LCMS RT= 6.63min, MH+ 248.9; 1H NMR (DMSO): 8.31 (IH, s), 7.87 (2H, d, J 8.6 Hz), 7.45 (2H, d, J 8.6 Hz), 6.92-6.90 (IH, m), 2.43 (3 H, d, J 1.3 Hz)
6-(3',4'-Dichlorophenyl)-3-methylimidazo[2,l-b]thiazole (Compound 17) LCMS RT= 7.26min, MH+ 282.9; 1H NMR (DMSO): 8.45 (IH, s), 8.08 (IH, d, J 2.0 Hz), 7.84 (IH, dd, J 8.4 2.0 Hz), 7.66 (IH, d, J 8.4 Hz), 6.95-6.93 (IH, m), 2.42 (3H, d, J 1.3 Hz)
6-(2'-Naphthyl)-3-methylimidazo[2,l-b]thiazole (Compound 18)
LCMS RT= 6.76min, MH+ 265.0; 1H NMR (DMSO): 8.42-8.39 (2H, m), 8.03-7.88
(4H, m), 7.53-7.45 (2H, m), 6.93-6.92 (IH, m), 2.47-2.45 (3H, m)
6-(3-Chlorophenyl)-3-methylimidazo[2,l-b]thiazole (Compound 19)
LCMS RT= 6.62min, MH+ 249.0; 1H NMR (DMSO): 8.41-8.40 (IH, m), 7.91-7.89 (IH, m), 7.83-7.80 (IH, m), 7.42 (IH, t, J 7.9 Hz), 7.31-7.28 (IH, m), 6.93-6.91 (IH, m), 2.43-2.41 (3H, m)
2-Chloro-6-(4'-chlorophenyl)imidazo[2,l-b]thiazole (Compound 10)
LCMS RT= 7.22min, MH+ 268.9; 1H NMR (DMSO): 8.33 (IH, s), 8.30 (IH, s), 7.87 (2H, d, J 8.6 Hz), 7.47 (2H, d, J 8.5 Hz)
6-(3,4-Dichlorophenyl)imidazo[2,l-b]thiazole-3-carboxylic acid (Compound 21)
LCMS RT= 4.70min, M-H+ 311.0; 1H NMR (DMSO): 8.69 (IH, s), 8.23 (IH, s), 8.20 (IH, d, J 1.5 Hz), 7.94 (IH, dd, J 8.3 1.5 Hz ), 7.66 (IH, d, J 8.34 Hz)
2-Chloro-6-(naphthalen-2'-yl)imidazo[2,l-b]thiazole (Compound 22) LCMS RT= 7.68min, MH+ 285.0; 1H NMR (DMSO): 8.39 (2H, s), 8.37 (IH, s), 7.90-8.02 (4H, m), 7.47-7.56 (2H, m)
Example 2 - Synthesis of 2-chloro-6-cyclohexylimidazo[2,l-b]thiazole (Compound 23)
A. Preparation of 2-bromo-l-cyclohexylethanone
Bromine (2mL, 39.6mmol) was added in one portion to a solution of 1- cyclohexylethanone (5g, 39.6mmol) in methanol (3OmL) at O0C. The resultant solution was allowed to stir for two hours, after which time the red colour had disappeared. Water (3OmL) was added and the reaction mixture allowed to stir for lόhours. After this time the mixture was extracted with ethyl acetate:40-60 petrol (3:1) and the combined organic extracts washed with 10% aqueous potassium carbonate solution, dried over magnesium sulphate and concentrated in vacuo to give the title compound as a pale yellow oil (7.466g, 92%). Attempted Kugelrohr distillation gave 2.447g of material of equal purity which was used directly in the subsequent reaction.
1H NMR (300MHz, CDCl3) 3.98 (2H, s), 2.68-2.77 (IH, m), 1.67-1.91 (5H, m), 1.19-1.48 (5H, m)
B. Preparation of 2-chloro-6-cyclohexylimidazo[2,l-b]thiazole 2-bromo-l-cyclohexylethanone (539mg, 2.63mmol) and 5-chloro-2-aminothiazole (354mg, 2.63mmol) were dissolved in IMS (1OmL) in a sealed vial and heated at
15O0C for lOminutes with microwave irradiation. After this time the mixture was cooled to room temperature and the solvent removed in vacuo. The resulting residue was partitioned between ethyl acetate and saturated aqueous sodium bicarbonate solution. The aqueous layer was reextracted with ethyl acetate and the combined organic extracts dried over magnesium sulphate and concentrated in vacuo. The crude reaction mixture was purified by flash column chromatography to give the title compound as a yellow solid (195mg, 31%). This material was triturated with 40-60 petrol to give a white solid (29mg, 5%) (LCMS RT=7.20min, MH+ 241). 1H NMR (300MHz, DMSO) 8.16 (IH, s), 7.45 (IH, s), 1.95 (2H, br s), 1.67-1.75 (3H, m), 1.20-1.43 (5H, m).
Example 3 - Synthesis of 3-methvI-6-(2'-naphthyl)imidazo[2,l-bloxazole (Compound 24)
See J. Heterocyclic Chem., 1997, 34, 589-599
A. Preparation of 2-imino-4-methyl-3-(2'-naphthacyl)-2,3-dihydrooxazole
A solution of 2-bromoacetylnaphthalene (889mg, 3.57mmol) in anhydrous THF (5mL) was added dropwise via cannula to a solution of 2-amino-4-methyloxazole (350mg, 3.57mmol) in anhydrous acetonitrile (5mL) and the resultant solution allowed to stir under nitrogen for 16 hours. After this time, the precipitate which had formed was filtered and washed with cold acetonitrile. The solid was suspended in ice water and the pH adjusted to pH 12 with 2M aqueous ammonium hydroxide. The resultant yellow powder was filtered and washed with water. The powder was then suspended in toluene and concentrated in vacuo before being triturated with diethyl ether to give the title compound as a peach solid (127mg, 13%) after filtration.
1H NMR (300MHz, CDCl3) 8.81 (IH, s), 8.01-8.19 (5H, m), 7.70 (2H, quintet, J7.3 Hz), 7.27 (IH, s), 5.51 (2H, s), 1.95 (3H, ά, J 1.1 Hz).
B. Preparation of 3-methyl-6-(2'-naphthyl)imidazo[2,l-b]oxazole Titanium tetrachloride (223mg, 129μL) was added dropwise to a suspension of 2- imino-4-methyl-3-(2'-naphthacyl)-2,3-dihydrooxazole (125mg, 0.469mmol) in anhydrous toluene at O0C. The reaction mixture was allowed to warm to room temperature over 20 minutes and then heated at reflux for 2 hours. After this time the reaction mixture was allowed to cool to room temperature and poured onto ice. The organic layer was separated and the organic layer rextracted with chloroform. The combined organic layers were passed through a short column of potassium carbonate, dried over magnesium sulphate and concentrated in vacuo. The crude product was purified by flash column chromatography (5:2 to 2:1 40-60 petrol:ethyl acetate) to furnish the title compound as a white solid (39mg, 33%) (LCMS RT=6.42min, MH+ 249).
1H NMR (300MHz, CDCI3) 8.34 (IH, d, J 0.7 Hz), 7.81-7.91 (4H, m), 7.42-7.51 (2H, m), 7.34 (IH, s), 7.13 (IH, q, J 1.5 Hz), 2.33 (3H, d, J 1.5 Hz).
Example 4 - Preparation of 2-chloro-6-(5',6',7\8Metrahvdronaphthalen-2'- vnimidazoβ.l-blthiazole f Compound 25)
A. Preparation of 2-bromo-l-(5',6',7',8'-tetrahydronaphthalen-2'- yl)ethanone
Bromine (0.63 mL, 12.3 mmol) was added to a stirred solution of l-(5',6',7',8'- tetrahydronaphthalen-2'-yl)ethanone (2.13 g, 12.3 mmol) in methanol (15 mL) at O0C and the resultant solution allowed to stir for 2.75 hours before the reaction mixture was diluted with water and allowed to warm to room temperature over 16 hours. After this time, the reaction mixture was extracted with ethyl acetate:40-60 petrol
(v:v 3:1, x3) and the combined organic layers washed with 10% aqueous potassium carbonate solution, dried over MgSO4 and concentrated in vacuo to furnish the crude product (2.755g, 89%).
1H NMR (DMSO) 7.68-7.71 (2H, m), 7.22 (IH, d, J 8.6Hz), 4.86 (2H, s), 2.78 (4H, br s), 1.73-1.77 (4H, m).
B. Preparation of 2-chloro-6-(5',6',7',8'-tetrahydronaphthalen-2'- yl)imidazo [2, 1-b] thiazole
2-bromo-l-(5',6',7',8'-tetrahydronaphthalen-2'-yl)ethanone (564 mg, 2.23 mmol) and 5-chloro-2-aminothiazole (300 mg, 2.23 mmol) were dissolved in IMS (10 mL) and heated at 15O0C for 10 minutes in a microwave. Volatiles were removed in vacuo and the mixture redissolved in ethyl acetate. The organic layer was washed with saturated aqueous ammonium bicarbonate solution, dried over MgSO4 and concentrated in vacuo to furnish the title compound (54 mg, 8%) as a powder after purification by column chromatography (9:1 40-60 petrol:ethyl acetate) and preparative HPLC (LCMS, RT = 8.00 minutes).
1H NMR (DMSO) 8.29 (IH, s), 8.16 (IH, s), 7.53-7.55 (2H, m), 7.08 (IH, d, J 8.4Hz), 2.73-2.77 (4H, m), 1.75-1.77 (4H, m).
Example 5
The potential activity of the compounds of general formula (I) for use in the treatment of DMD may be demonstrated in the following predictive assay and screens.
1. Luciferase reporter assay (murine H2K cells)
The cell line used for the screen is an immortalized mdx mouse H2K cell line that has been stably transfected with a plasmid containing ~5kb fragment of the Utrophin A promoter including the first untranslated exon linked to a luciferase reporter gene.
Under conditions of low temperature and interferon containing media, the cells remain as myoblasts. These are plated into 96 well plates and cultured in the
presence of compound for three days. The level of luciferase is then determined by cell lysis and reading of the light output from the expressed luciferase gene utilising a plate luminometer.
2. mdx mouse
Data obtained from the ADMET data is prioritised and the compounds with the best in vitro luciferase activity and reasonable ADMET data are tested in the mdx proof of concept study where the outcome identified is the ability to increase the levels of utrophin protein in dystrophin deficient muscle when compared to vehicle only dosed negative control animals.
Animals are injected with 10mg/kg of compound administered ip daily for 28 days plus age matched controls. Muscle samples were taken and processed for sectioning (to identify increases in sarcolemmal staining of utrophin) and Western blotting (to identify overall increases in utrophin levels).
Figure 1 shows an example of TA muscle sections stained with antibody specific for mouse utrophin. Comparison of a positive control compound, 5-amino-2-(5,6- dimethylbenzo[d]oxazol-2-yl)phenol (CPD-A) known to increase the levels of utrophin protein in dystrophin deficient mdx muscle with mdx muscle injected only with vehicle (as negative control) shows an increase in the amount of sarcolemmal bound utrophin.
Muscles from the above treated mice were also excised and processed for Western blotting and stained with specific antibodies (see Figure 2). Again using muscle dosed with CPD-A shows a significant increase in the overall levels of utrophin present in both the TA leg muscle and the diaphragm. Both mice exposed to CPD-A (V2 and V3) showed increased levels of utrophin expression compared to the negative control.
Positive upregulation data from the first 28 day study are then repeated in a further two mouse 28 day study. Such data will demonstrate the ability of the test compounds to significantly increase levels of utrophin in the mdx muscle when delivered ip. Since all the published data to date demonstrate that any increase of utrophin levels over three fold has significant functional effects on dystrophin deficient muscle, such an assay is likely to be an excellent predictor of clinical efficacy.
The H2K/mdx/Utro A reporter cell line maintenance The H2K/mdx/Utro A reporter cell line was passaged twice a week until <30% confluent. The cells were grown at 330C in the presence of 10% CO2
To remove the myoblasts for plateing, they were incubated with Trypsin / EDTA until the monolayer started to detach.
Growth Medium DMEM Gibco 41966
20% FCS
1% Pen/Strep
1 % glutamine lOmls Chick embryo extract Interferon(1276 905 Roche) Add fresh lOμl / 5OmIs medium.
Luciferase Assay for 96 Well Plates
The H2K/mdx/Utro A reporter cell line cells were plated out into 96 well plates (Falcon 353296, white opaque) at a density of approximately 5000 cells/well in 190μl normal growth medium. The plates were then incubated at 330C in the presence of 10% CO2 for 24 hrs.
Compounds were dosed by adding lOμl of diluted compound to each well giving a final concentration of lOμM. The plates were then incubated for a further 48hrs Cells were then lysed in situ following the manufacture's protocols(Promega Steady- GIo Luciferase Assay System(E2520). Then counted for 10 seconds using a plate luminometer (Victorl420).
Compound Storage
Compounds for screening were stored at -2O0C as 1OmM stocks in 100% DMSO until required.
Injection of mdx mice with compounds
Mdx from a breeding colony were selected for testing. Mice were injected daily with either vehicle or 10mg/kg of compound using the intraperitoneal route (ip). Mice were weighed and compounds diluted in 5% DMSO, 0.1% Tween™ in PBS. Mice were sacrificed by cervical dislocation at desired time points, and muscles excised for analysis
Muscle Analysis Immunohistochemistry Tissues for sectioning were dissected, immersed in OCT (Bright Cryo-M-Bed) and frozen on liquid nitrogen cooled isopentane. Unfixed 8μM cryosections were cut on a Bright Cryostat,and stored at -8O0C.
In readiness for staining, sections were blocked in 5% foetal calf serum in PBS for 30 mins. The primary antibodies were diluted in blocking reagent and incubated on sections for 1.5 hrs in a humid chamber then washed three times for 5mins in PBS.
Secondary antibodies also diluted in blocking reagent, were incubated for lhr in the dark in a humid chamber. Finally sections were washed three times 5mins in PBS and coverslip Mounted with hydromount. Slides were analysed using a Leica fluorescent microscope.
Results
Biological activity as assessed using the luciferase reporter assay in murine H2K cells, and is classified as follows: + Up to 200% relative to control
++ Between 201% and 300% relative to control +++ Between 301% and 400% relative to control
++++ Above 401% relative to control
The results are shown in Table 1, from which it can be seen that all of the exemplified compounds had had increased activity in the luciferase reporter assay relative to the control.
Table 1