WREN, Stephen, Paul (91 Milton ParkAbingdon,Oxfordshire, OX14 4RY, GB)
LECCI, Cristina (91 Milton ParkAbingdon,Oxfordshire, OX14 4RY, GB)
WYNNE, Graham, Michael,l (91 Milton ParkAbingdon,Oxfordshire, OX14 4RY, GB)
WREN, Stephen, Paul (91 Milton ParkAbingdon,Oxfordshire, OX14 4RY, GB)
LECCI, Cristina (91 Milton ParkAbingdon,Oxfordshire, OX14 4RY, GB)
Claims:
1. Use of a compound of formula (I), wherein
A 1 , A 2 , A 3 , A 4 and A 5 which may be the same or different, represent N or CR 1 ,
Y and Z, which may be the same or different, represent O, S(O) n , C=W, NR , NC(=O)R 5 and CR 6 R 7 , W is O, S 5 NR 20 , one of R 4 , R 5 , R 6 , and R 20 represents - L - R 3 , in which L is a single bond or a linker group, additionally, R 3 , R 4 , R 5 , R 6 , and R 7 , which may be the same or different, independently represent hydrogen or a substituent and R 20 represents hydrogen, hydroxyl, aryl, alkyl optionally substituted by aryl, alkoxy optionally substituted by aryl, CN, optionally substituted alkoxy, optionally substituted aryloxy, optionally substituted alkanoyl, optionally substituted aroyl, NR 30 R 31 , in which R 30 and R 31 , which may be the same or different, represent hydrogen, optionally substituted alkyl or optionally substituted aryl; additionally, one of R 30 and R 31 may represent optionally substituted alkanoyl or optionally substituted aroyl, n represents an integer from 0 to 2, in addition,
Y and Z cannot both represent O, or S, or together represent O — S, any pair of R 6 , R 7 and R 4 may when attached to adjacent atoms together represent a single bond between the adjacent atoms to which they are attached, when an adjacent pair of A 1 - A 4 each represent CR 1 , then the adjacent carbon atoms, together with their substituents may form a ring B, when Y or Z is CR 6 R 7 , R 6 and R 7 , together with the carbon atom to which they are attached may form a ring C, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the therapeutic and/or prophylactic treatment of Duchenne muscular dystrophy, Becker muscular dystrophy or cachexia.
2. Use according to claim 1, wherein
A 1 , A 2 , A 3 , A 4 and A 5 which may be the same or different, represent N or CR 1 ,
Y and Z, which may be the same or different, represent O, S(O) n , C=W, NR 4 , NC(=0)R 5 and CR 6 R 7 ,
W is O, S, NR 20 , one of R 4 , R 5 , R 6 , and R 20 represents - L - R 3 , in which L is a single bond or a linker group, additionally, R 3 , R 4 , R 5 , R 6 , and R 7 , which may be the same or different, independently represent hydrogen or a substituent and
R 20 represents hydrogen, hydroxyl, aryl, or alkyl, n represents an integer from 0 to 2, in addition,
Y and Z cannot both represent O, or S, or together represent O - S, any pair of R 6 , R 7 and R 4 may when attached to adjacent atoms together represent a single bond between the adjacent atoms to which they are attached, when an adjacent pair of A 1 - A 4 each represent CR 1 , then the adjacent carbon atoms, together with their substituents may form a ring B, when Y or Z is CR 6 R 7 , R 6 and R 7 , together with the carbon atom to which they are attached may form a ring C, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the therapeutic and/or prophylactic treatment of Duchenne muscular dystrophy, Becker muscular dystrophy or cachexia.
3. Use according to claim 1 or 2, wherein R 3 in the compound of formula 1 represents alkyl, alkoxy or aryl, each optionally substituted by one to three subsitutuents, R 2 , which may be the same or different.
4. Use according to claim 1 or 2, wherein either: one of R 5 or R 6 represents L - R 3 , and wherein L is single bond and R 3 represents: thioalkyl optionally substituted by alkyl or optionally substituted aryl, thioaryl, in which the aryl is optionally substituted, optionally substituted aryl, hydroxyl,
NO 2 ,
CN 5
NR 10 R 11 , halogen,
SO 2 R 12 ,
NR 13 SO 2 R 14 ,
C(=W)R 16 ,
OCC=W)NR 10 R 11 NR 15 C(=W)R 17 ,
R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 and R 17 , which may be the same or different, represent hydrogen, alkyl optionally substituted by optionally substituted aryl, optionally substituted aryl, in addition, R 10 and R 11 together with the nitrogen to which they are attached may form a ring,
R 12 may have the same meaning as NR 10 R 11 ,
R 16
and R 17
, which may be the same or different, may each represent alkyl substituted by one or more of halogen, alkoxy optionally substituted aryl or optionally substituted aryl, optionally substituted aryloxy,
or wherein one of R 4 or R 20 represents L - R 3 , and wherein L is single bond and R 3 represents: thioalkyl optionally substituted by alkyl or optionally substituted aryl, O-aryl or thioaryl, in which the aryl is optionally substituted, optionally substituted aryl, hydroxyl, NR 10 R 11 ,
SO 2 R 12 , NR 13 SO 2 R 14 , C(=W)R 16 , NR 15 C(=W)R 17 , R 10 , R 11 , R 12 , R 13 , R 14 , R 16 and R 17 , which may be the same or different, represent hydrogen, alkyl optionally substituted by optionally substituted aryl, optionally substituted aryl, in addition,
R 10 and R 11 together with the nitrogen to which they are attached may form a ring,
R 12 may have the same meaning as NR 10 R 11 , R 16 and R 17 , which may be the same or different, may each represent alkyl substituted by one or more of halogen, alkoxy optionally substituted aryl or optionally substituted aryl, optionally substituted aryloxy, aryl Or NR 10 R 11 , and when R 16 or R 17 represents NR 10 R 11 , one of R 10 and R 11 , may additionally represent CO alkyl optionally substituted or COaryl optionally substituted, and in addition to the definitions shared with R 17 , R 1 may represent hydroxyl.
5. Use according to claim 1 or 2, wherein either:
one of R 5 or R 6 represents L- R 3 , and wherein L is single bond and R 3 represents: thioalkyl optionally substituted by alkyl or optionally substituted aryl, thioaryl, in which the aryl is optionally substituted, optionally substituted aryl, NR 10 R 11 , SO 2 R 12 ,
N NRR 1133 SSOO 2 ; R 14 ,
C(=W)R 16 ,
OC(=W)NR 10 R π NR 15 C(=W)R 17 ,
R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 and R 17 , which may be the same or different, represent hydrogen, alkyl optionally substituted by optionally substituted aryl, optionally substituted aryl, in addition, R 10 and R 11 together with the nitrogen to which they are attached may form a ring,
R 12 may have the same meaning as NR 10 R 11 ,
R 1 and R 17 , which may be the same or different, may each represent alkyl substituted by one or more of halogen, alkoxy optionally substituted aryl or optionally substituted aryl, optionally substituted aryloxy, aryl or NR 10 R 11 , and when R 16 or R 17 represents NR 10 R 11 , one of R 10 and R 11 , may additionally represent CO alkyl optionally substituted or COaryl optionally substituted, and in addition to the definitions shared with R 17 , R 16 may represent hydroxyl;
or wherein one of R 4 or R 20 represents L - R 3 , and wherein L is single bond and R 3 represents: thioalkyl optionally substituted by alkyl or optionally substituted aryl, O-aryl or thioaryl, in which the aryl is optionally substituted, optionally substituted aryl,
NR 10 R 11 ,
SO 2 R 12 ,
NR 13 SO 2 R 14 , C(=W)R 16 ,
NR 15 C(=W)R 17 , R 10 , R 11 , R 12 , R 13 , R 14 , R 16 and R 17 , which may be the same or different, represent hydrogen, alkyl optionally substituted by optionally substituted aryl, optionally substituted aryl, in addition, R and R 11 together with the nitrogen to which they are attached may form a ring,
R 12 may have the same meaning as NR 10 R 11 ,
R 16 and R 17 , which may be the same or different, may each represent alkyl substituted by one or more of halogen, alkoxy optionally substituted aryl or optionally substituted aryl, optionally substituted aryloxy, aryl or NR 10 R 11 , and when R 16 or R 17 represents NR 10 R 11 , one of R 10 and R 11 , may additionally represent CO alkyl optionally substituted or COaryl optionally substituted, and in addition to the definitions shared with R 17 , R 16 may represent hydroxyl.
6. Use according to claim 3, in which R 1 and R 2 , which may be the same or different, may represent: alkyl optionally substituted by one or more halogen, alkoxy or optionally substituted aryl, thioaryl or aryloxy, alkoxy optionally substituted by optionally by alkyl or optionally substituted aryl, hydroxyl, OC(=W)NR 10 R π aryl, thioalkyl optionally substituted by alkyl or optionally substituted aryl, thioaryl, in which the aryl is optionally substituted,
NO 2 , CN,
NR 10 R 11 , halogen,
SO 2 R 12 , NR 13 SO 2 R 14 ,
C(=W)R 16 ,
NR 15 C(=W)R 17 ,
R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 and R 17 , which may be the same or different, represent hydrogen, alkyl optionally substituted by optionally substituted aryl, optionally substituted aryl, in addition,
NR R together with the nitrogen to which they are attached may form a ring,
R 12 may have the same meaning as NR 10 R 11 , when R 17 represents NR 10 R 11 , that NR 10 R 11 may represent hydrogen, COalkyl and CO optionally substituted aryl,
R 16 may represent hydroxy, alkoxy, Or NR 10 R 11 , and R 17 may represent alkyl substituted by one or more of halogen, alkoxy, optionally substituted aryl or NR 10 R 11 .
7. Use according to claim 1 or 2, wherein either:
one of R 5 or R 6 represents L — R 3 , in which L represents a linker group which is: O, S, NR 18 , alkylene, alkenylene, alkynylene, each of which may be optionally interrupted by one or more of O, S, NR 18 , or one or more C-C single, double or triple bonds, a -N-N- single or double bond, and R 18 represents hydrogen, alkyl, COR 16 ;
or one of R 4 or R 20 represents L — R 3 , in which L represents a linker group which is:
O, S or NR 18 , alkylene, alkenylene, alkynylene, each of which may be optionally interrupted by one or more of O, S, NR 18 , or one or more C-C single, double or triple bonds, and R 18 represents hydrogen, alkyl, COR 16 .
8. Use of a compound according to claim 1 or 2, in which R , R 5 , R , and R 7 represent hydrogen, alkyl or optionally substituted aryl. 9 Use of a compound according to claim 1 or 2 in which when any of the substituents represents alkyl, alkyl is saturated and has from 1 to 10 carbon atoms.
10. Use of a compound according to claim 1 or 2, in which aryl is an aromatic hydrocarbon or a 5 to 10 membered aromatic heterocyle containing 1 to 4 hetero atoms selected from an oxygen atom, a sulphur atom and a nitrogen atom as a ring constituent besides carbon.
11./ Use of a compound according to claim 1 or 2, in which aryl is phenyl or naphthalene.
12. Use if a compound according to claim 1 or 2, in which aryl is furan, thiophene, pyrrole or pyridine.
13. Use of a compound according to claim 1 or 2, in which ring B or ring C is a saturated or unsaturated 3 to 10 membered carbocylic or heterocyclic ring.
14. Use of a compound according to claim 1 or 2, in which ring B is benzene ring.
15. Use of a compound according to claim 1 or 2, in which ring C is a 3- 10 membered saturated or unsaturated carbocylic ring.
16. Use of a compound according to claim 1 or 2, in which at least one R 1 represents NR 15 C(=W)R 17 .
17. Use of a compound according to claim 1 or 2, in which at least one R 1 represents NR 15 C(=O)R 17 .
18. Use of a compound according to claim 1 or 2, in which at least one R 1 represents CONR 10 R 11 .
19. Use of a compound according to claim 1 or 2, in which at least one R 1 represents NHCOR 17 , wherein R 17 is selected from: alkyl C 1 - C 6 , alkyl C 1 - C 6 substituted by phenyl, alkyl C 1 - C 6 substituted by alkoxy Ci - C 6 , haloalkyl C 1 - C 6 , perfluoroalkyl C 1 - C 6 , phenyl optionally substituted by one or more of halogen, alkyl Cj - C 6 , alkoxy C 1 - C 6 , amino, (alkyl C 1 - C 6 )amino, di(alkyl C 1 - C 6 ) amino or phenyl, CH-.CH phenyl, naphthyl, pyridinyl, thiophenyl and furanyl.
20. Use of a compound according to claim 1 or 2 in which one or both of R 1 and R 2 is other than -COOH.
21. Use of a compound according to claim 1 or 2, in which at least one of R represents NR 15 CONR 10 R 11 , wherein R 10 and R 11 , which may be the same or different, are selected from optionally substituted aryl, alkyl and COaryl optionally substituted.
22. Use of a compound according to claim 1 or 2, in which at least one of R 1 represents NHCONHR 15 and R 15 is selected from phenyl, alkyl C 1 to C 6 and COphenyl optionally substituted by one or more halogen.
23. Use of a compound according to claim 1 or 2, in which at least one of R 1 represents alkyl C 1 to C 6 , optionally substituted by phenyl or a 4 to 7- membered, preferably 5 or 6-membered saturated or unsaturated heterocycle preferably containing one to two heteroatoms selected from N, S and O.
24. Use of a compound according to claim 1 or 2 in which at least one of R represents COR 16 and R 16 is alkoxy C 1 - C 6 , amino, (alkyl C 1 - C 6 )amino or di(alkyl Cj
- C 6 ) amino.
25. Use of a compound according to claim 1 or 2, in which at least one of R 1 represents:
NO 2 , halogen, amino or (alkyl C 1 - C 6 )amino or di(alkyl C 1 - C 6 ) amino in which the alkyl C 1 to C 6 is optionally substituted by phenyl or a 5 or 6 membered saturated or unsaturated heterocycle,
NHSO 2 alkyl C 1 - C 6 , NHSO 2 ρhenyl,
S0 2 alkyl C 1 - C 6 , phenyl optionally substituted by C 1 to C 6 alkoxy C 1 - C 6 , a 5 - 10 membered, saturated or unsaturated, mono- or bi-cyclic heterocycle containing from 1 - 3 heteroatoms selected from N, S and O.
26. Use of a compound according to claim 1 or 2, in which at least one of R 1 represents SO n R 12 wherein n represents 0, 1 or 2 and R 12 represents alkyl C 1 - C 6 or
NR 98 R 99 wherein R 98 and R 99 , which may be the same or different, represent hydrogen or alkyl C 1 - C 6 .
27. Use of a compound according to claim 1 or 2, in which R 3 represents aryl and is optionally substituted by one to three substituents, R 2 , which may be the same or different.
28. Use of a compound according to claim 27 in which R 3 is a 5 - 10 membered aromatic mono- or bi-cyclic system.
29. Use of a compound according to claim 28, in which the aromatic system is a hydrocarbon.
30. Use of a compound according to claim 29, in which the aromatic hydrocarbon is benzene or naphthalene.
31. Use of a compound according to claim 28, in which the aromatic system is a heterocyclic system containing up to three heteroatoms, which may be the same or different, selected from N, O and S.
32. Use of a compound according to claim 31, in which the heterocyclic system is thiophene, furan, pyridine or pyrrole.
33. Use of a compound according to claim 3, in which the substituent(s) R 2 is/are selected from is: alkyl Cj - C 6 , optionally substituted by thiophenyl or phenoxy, each optionally substituted by halogen, alkoxy Cj - C 6 , phenyl, thioalkyl C 1 - C 6 , thiophenyl, optionally substituted by halogen,
NO 2 , CN
NR 10 R 11 , in which R 10 and R 11 , which may be the same or different represent hydrogen, alkyl C 1 - C 6 , or together with the nitrogen to which they are attached form a 5 to 7 membered ring which may contain one or more additional heteroatoms selected from N, O and S, halogen,
SO 2 R 12 , in which Ri 2 represents a 5 to 7 membered ring which may contain one or more additional heteroatoms selected from N, O and S, NHCOR 17 , in which R 17 represents alkyl Ci - C 6 , optionally substituted by: phenyl or halogen, or phenyl optionally substituted by alkoxy C 1 - C 6 , carboxy, or halogen, or a 5 or 6 membered saturated or unsaturated heterocycle, phenyl or a 5 or 6 membered saturated or unsaturated heterocycle optionally substituted by halogen, alkoxy C 1 to C 6 , carboxy or a group SO 2 N NR 10 R 11 .
34. Use of a compound according to claim 33 in which NR 10 R 11 represents N- pyrrole, N-piperidine, N '(Cj - C 6 ) alkyl N piperazine or N-morpholine.
35. Use of a compound according to claim 7 in which R 5 or R 6 represents L - R 3 , in which L represents a linker group which is:
-NH.NH-, -CH=CH-, -C≡C- or
-NCOR 1 in which R 6 represents phenyl or a 5 or 6 membered saturated or unsaturated heterocycle optionally substituted by halogen, alkoxy C 1 to C 6 , carboxy.
36. Use of a compound according to claim 1 or 2 in which two of A 1 - A 4 represent nitrogen.
37. Use of a compound according to claim 1 or 2 in which one of A - A 4 represents nitrogen.
38. Use of a compound according to claim 1 or 2 in which all of A 1 - A 4 represents CR 1 .
39. Use of a compound according to any preceding claim, in which A 5 represents N.
40. Use of a compound according to claim 36, in which Y and Z together represent CR 6 =CR 6 .
41. Use of a compound according to claim 37, in which Y and Z together represent CH=CH.
42. Use of a compound according claims 1 - 38, in which A 5 represents CR 1 .
43. Use of a compound according to claim 1 or 2, in which A 5 represents CH.
44. Use of a compound according to claim 39 or 40, in which Y and Z together represent CR 6 =CR 6 .
45. Use of a compound according to claim 39 or 40, in which Y and Z together represent CH=CH.
46. Use of a compound as listed in table 1 according to claim 1. |
Treatment of 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 mdx 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 (mώc) 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 mώc 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. We have now found a group of compounds which upregulate endogenous utrophin in predictive screens and, thus, may be useful in the treatment of DMD.
According to the invention, we provide use of a compound of formula (I), wherein
A 1 , A 2 , A 3 , A 4 and A 5 which may be the same or different, represent N or CR ,
Y and Z, which may be the same or different, represent O, S(O) n , C=W, NR 4 , NCO=O)R 5 and CR 6 R 7 , W is O, S, NR 20 , one of R 4 , R 5 , R 6 , and R 20 represents - L - R 3 , in which L is a single bond or a linker group, additionally, R 3 , R 4 , R 5 , R 6 , and R 7 , which may be the same or different, independently represent hydrogen or a substituent and R 20 represents hydrogen, hydroxyl, aryl, alkyl optionally substituted by aryl, alkoxy optionally substituted by aryl, CN, optionally substituted alkoxy, optionally substituted aryloxy, optionally substituted alkanoyl, optionally substituted aroyl, NR 30 R 31 , in which R 30 and R 31 , which may be the same or different, represent hydrogen, optionally substituted alkyl or optionally substituted aryl; additionally, one
of R 30 and R 31 may represent optionally substituted alkanoyl or optionally substituted aroyl, n represents an integer from 0 to 2, in addition, Y and Z cannot both represent O, or S, or together represent O - S, any pair of R 6 , R 7 and R 4 may when attached to adjacent atoms together represent a single bond between the adjacent atoms to which they are attached, when an adjacent pair of A 1 - A 4 each represent CR 1 , then the adjacent carbon atoms, together with their substituents may form a ring B, when Y or Z is CR 6 R 7 , R 6 and R 7 , together with the carbon atom to which they are attached may form a ring C, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the therapeutic and/or prophylactic treatment of Duchenne muscular dystrophy, Becker muscular dystrophy or cachexia. Compounds of formula I may exist in tautomeric, enantiomeric and diastereomeric forms, all of which are included within the scope of the invention.
Certain compounds of formula I are novel. According to the invention, we also provide those compounds of formula I which are novel, together with processes for their preparation, compositions containing them, as well as their use as pharmaceuticals.
Some of the compounds falling within the scope of formula I are known, as such, but not as pharmaceuticals. According to the invention, we claim compounds known in the art as such, but not previously described for use as pharmaceuticals, as pharmaceuticals. AU of the compounds of formula I may be made by conventional methods.
Methods of making heteroaromatic ring systems are well known in the art. In particular, methods of synthesis are discussed in Comprehensive Heterocyclic Chemistry, Vol. 1 (Eds.: AR Katritzky, CW Rees), Pergamon Press, Oxford, 1984 and Comprehensive Heterocyclic Chemistry II: A Review of the Literature 1982-1995 The Structure, Reactions, Synthesis, and Uses of Heterocyclic Compounds, Alan R. Katritzky (Editor), Charles W. Rees (Editor), E.F.V. Scriven (Editor), Pergamon Pr, June 1996. Other general resources which would aid synthesis of the compounds of
interest include March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Wiley-Interscience; 5th edition (January 15, 2001).
Imidazopyridines of formula (I) may be synthesised in a one pot procedure as illustrated below (see Tomoda, H.; Hirano, T.; Saito, S.; Mutai, T.; Araki, K. Bull. Chem. Soc. Jpn. 1999, 72, 1327, Aslanov, L. A. et al J. Struct, Chem. (Engl. Transl.) 1983, 24, 427-434, Fisher, M. H. et al J. Med. Chem. 1972, 15, 982-985, Werbel, L. M. et al J. Heterocycl. Chem. 1965, 2, 287-290 and Jacquier et al J. Heterocycl. Chem. 1973, 10, 755-761)
II
with R = NH 2 acylation
For example, a 2-aminopyridine may be reacted with an α-bromoketone (for example at relux in dioxane : water (2 :1) for 22 h) to give imidazopyridine II. For imidazopyridines with R or R' = NO 2 standard manipulations to give amino or amide functionality may then be carried out.
Indolizidines of formula I can be synthesised as shown below.
III cyclisation
Br R' = NO 2
R
R" = H, alkyl, acyl
Pyridinium salts III, synthesised from alkylation of the corresponding pyridine, can be cyclised to give indolizidines V (see Kost, A. N.; Sagitullin, R. S.; Gromov, S. P. Khimiya Geterotsiklicheskikh Soedinenii, 1976, 7, 922-6). Nitro functionality can then be manipulated using standard techniques.
In the above processes it may be necessary for any functional groups, e.g. hydroxy or amino groups, present in the starting materials to be protected, thus it may be necessary to remove one or more protective groups to generate the compound of formula I.
Suitable protecting groups and methods for their removal are, for example, those described in "Protective Groups in Organic Synthesis" by T. Greene and P.G.M. Wutts, John Wiley and Sons Inc., 1991. Hydroxy groups may, for example, be protected by arylmethyl groups such as phenylmethyl, diphenylmethyl or triphenylmethyl; acyl groups such as acetyl, trichloroacetyl or trifluoroacetyl; or as tetrahydropyranyl derivatives. Suitable amino protecting groups include arylmethyl groups such as benzyl, (R,S)-α-phenylethyl, diphenylmethyl or triphenylmethyl, and acyl groups such as acetyl, trichloroacetyl or trifluoroacetyl. Conventional methods of deprotection may be used including hydrogenolysis, acid or base hydrolysis, or photolysis. Arylmethyl groups may, for example, be removed by hydrogenolysis in the presence of a metal catalyst e.g. palladium on charcoal. Tetrahydropyranyl groups may be cleaved by hydrolysis under acidic conditions. Acyl groups may be removed by
hydrolysis with a base such as sodium hydroxide or potassium carbonate, or a group such as trichloroacetyl may be removed by reduction with, for example, zinc and acetic acid.
The compounds of formula I, and salts thereof, may be isolated from their reaction mixtures using conventional techniques.
Salts of the compounds of formula I may be formed by reacting the free acid, or a salt thereof, or the free base, or a salt or derivative thereof, with one or more equivalents of the appropriate base or acid. The reaction may be carried out in a solvent or medium in which the salt is insoluble or in a solvent in which the salt is soluble, e.g. ethanol, tetrahydrofuran or diethyl ether, which may be removed in vacuo, or by freeze drying. The reaction may also be a metathetical process or it may be carried out on an ion exchange resin.
Pharmaceutically acceptable salts of the compounds of formula I include alkali metal salts, e.g. sodium and potassium salts; alkaline earth metal salts, e.g. calcium and magnesium salts; salts of the Group III elements, e.g. aluminium salts; and ammonium salts. Salts with suitable organic bases, for example, salts with hydroxylamine; lower alkylamines, e.g. methylamine or ethylamine; with substituted lower alkylamines, e.g. hydroxy substituted alkylamines; or with monocyclic nitrogen heterocyclic compounds, e.g. piperidine or morpholine; and salts with amino acids, e.g. with arginine, lysine etc, or an N-alkyl derivative thereof; or with an aminosugar, e.g. N-methyl-D-glucamine or glucosamine. The non-toxic physiologically acceptable salts are preferred, although other salts are also useful, e.g. in isolating or purifying the product.
Diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various optical isomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC 5 techniques. Alternatively the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation.
Substituents that alkyl may represent include methyl, ethyl, butyl, eg sec butyl.
Halogen may represent F, Cl, Br and I, especially Cl.
Examples of substituents that R 3 in the compound of formula 1 may represent include alkyl, alkoxy or aryl, each optionally substituted by one or more, preferably one to three substituents, R 2 , which may be the same or different.
Compounds that can be mentioned include those wherein
A 1 , A 2 , A 3 , A 4 and A 5 which may be the same or different, represent N or CR 1 ,
Y and Z, which may be the same or different, represent O, S(O) n , C=W, NR 4 , NC(=O)R 5 and CR 6 R 7 ,
W is O, S, NR 20 , one of R 4 , R 5 , R 6 , and R 20 represents - L — R 3 , in which L is a single bond or a linker group, additionally, R 3 , R 4 , R 5 , R 6 , and R 7 , which may be the same or different, independently represent hydrogen or a substituent and
R2 0 represents hydrogen, hydroxyl, aryl, or alkyl, n represents an integer from 0 to 2, in addition,
Y and Z cannot both represent O, or S, or together represent O - S, any pair of R 6 , R 7 and R 4 may when attached to adjacent atoms together represent a single bond between the adjacent atoms to which they are attached, when an adjacent pair of A 1 - A 4 each represent CR 1 , then the adjacent carbon atoms, together with their substituents may form a ring B, when Y or Z is CR 6 R 7 , R 6 and R 7 , together with the carbon atom to which they are attached may form a ring C, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the therapeutic and/or prophylactic treatment of Duchenne muscular dystrophy, Becker muscular dystrophy or cachexia.
In addition, compounds that may be mentioned include those wherein either:
one of R 5 or R 6 represents L - R 3 , and wherein L is single bond and R 3 represents: thioalkyl optionally substituted by alkyl or optionally substituted aryl,
O-aryl or thioaryl, in which the aryl is optionally substituted, optionally substituted aryl, hydroxyl,
NO 2 ,
CN,
NR 10 R 11 , halogen,
SO 2 R 12 ,
NR 13 SO 2 R 14 ,
C(=W)R 16 ,
OC(^W)NR 10 R 1 J NR 15 C(=W)R 17 ,
R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 and R 17 , which may be the same or different, represent hydrogen, alkyl optionally substituted by optionally substituted aryl, optionally substituted aryl, in addition, R 10 and R 11 together with the nitrogen to which they are attached may form a ring,
R 12 may have the same meaning as NR 10 R 11 ,
R 16 and R 17 , which may be the same or different, may each represent alkyl substituted by one or more of halogen, alkoxy optionally substituted aryl or optionally substituted aryl, optionally substituted aryloxy, aryl OrNR 10 R 11 , and when R 16 or R 17 represents NR 10 R 11 , one of R 10 and R 11 , may additionally represent CO alkyl optionally substituted or COaryl optionally substituted, and in addition to the definitions shared with R 17 , R 16 may represent hydroxyl;
or wherein one of R 4 or R 20 represents L- R 3 , and wherein L is single bond and R 3 represents:
thioalkyl optionally substituted by alkyl or optionally substituted aryl,
O-aryl or thioaryl, in which the aryl is optionally substituted, optionally substituted aryl, hydroxyl, NR 10 R 11 ,
SO 2 R 12 ,
NR 13 SO 2 R 14 ,
C(=W)R 16 ,
NR 15 C(=W)R 17 , R 10 , R 11 , R 12 , R 13 , R 14 , R 16 and R 17 , which may be the same or different, represent hydrogen, alkyl optionally substituted by optionally substituted aryl, optionally substituted aryl, in addition,
R 10 and R 11 together with the nitrogen to which they are attached may form a ring,
R 12 may have the same meaning as NR 0 R 11 ,
R 16 and R 17 , which may be the same or different, may each represent alkyl substituted by one or more of halogen, alkoxy optionally substituted aryl or optionally substituted aryl, optionally substituted aryloxy, aryl Or NR 10 R 11 , and when R 16 or R 17 represents NR 10 R 11 , one of R 10 and R 11 , may additionally represent CO alkyl optionally substituted or COaryl optionally substituted, and in addition to the definitions shared with R 17 , R 16 may represent hydroxyl.
In addition, compounds that can be mentioned include those wherein either:
one of R 5 or R 6 represents L - R 3 , and wherein L is single bond and R 3 represents: thioalkyl optionally substituted by alkyl or optionally substituted aryl, thioaryl, in which the aryl is optionally substituted, optionally substituted aryl, NR 10 R 11 ,
SO 2 R 12 ,
NR 13 SO 2 R 14 ,
C(=W)R 16 ,
OCC=W)NR 10 R 11 NR 15 C(=W)R 17 ,
R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 and R 17 , which may be the same or different, represent hydrogen, alkyl optionally substituted by optionally substituted aryl, optionally substituted aryl, in addition, R 10 and R 11 together with the nitrogen to which they are attached may form a ring,
R 12 may have the same meaning as NR 10 R 1 ',
R 16 and R 17 , which may be the same or different, may each represent alkyl substituted by one or more of halogen, alkoxy optionally substituted aryl or optionally substituted aryl, optionally substituted aryloxy,
3TyI Or NR 10 R 11 , and when R 16 or R 17 represents NR 10 R 11 , one of R 10 and R 11 , may additionally represent CO alkyl optionally substituted or COaryl optionally substituted, and in addition to the definitions shared with R 17 , R 16 may represent hydroxyl;
or wherein one of R 4 or R 20 represents L - R 3 , and wherein L is single bond and R 3 represents: thioalkyl optionally substituted by alkyl or optionally substituted aryl, O-aryl or thioaryl, in which the aryl is optionally substituted, optionally substituted aryl,
NR 10 R 11 ,
SO 2 R 12 ,
NR 13 SO 2 R 14 , C(=W)R 16 ,
NR 15 CC=W)R 17 ,
R 10 , R 11 , R 12 , R 13 , R 14 , R 16 and R 17 , which may be the same or different, represent hydrogen, alkyl optionally substituted by optionally substituted aryl, optionally substituted aryl, in addition, R 10 and R 11 together with the nitrogen to which they are attached may form a ring,
R 12 may have the same meaning as NR 10 R 11 ,
R , which may be the same or different, may each represent alkyl substituted by one or more of halogen, alkoxy optionally substituted aryl or optionally substituted aryl, optionally substituted aryloxy, aryl Or NR 10 R 11 , and when R 16 or R 17 represents NR 10 R 11 , one of R 10 and R 11 , may additionally represent CO alkyl optionally substituted or COaryl optionally substituted, and in addition to the definitions shared with R 17 , R 16 may represent hydroxyl.
Examples of substituents that R 1 and R 2 , which may be the same or different, may represent: alkyl optionally substituted by one or more halogen, alkoxy or optionally substituted aryl, thioaryl or aryloxy, alkoxy optionally substituted by optionally by alkyl or optionally substituted aryl, hydroxyl,
OC(=W)NR 10 R π aryl, thioalkyl optionally substituted by alkyl or optionally substituted aryl, thioaryl, in which the aryl is optionally substituted,
NO 2 ,
CN, NR 10 R 11 , halogen,
SO 2 R 12 ,
NR 13 SO 2 R 14 ,
C(=W)R 16 , NR 15 C(=W)R 17 ,
R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 and R 17 , which may be the same or different, represent hydrogen, alkyl optionally substituted by optionally substituted aryl, optionally substituted aryl, in addition,
NR 10 R 11 together with the nitrogen to which they are attached may form a ring, R 12 may have the same meaning as NR 10 R 11 , when R 17 represents NR 10 R 11 , that NR 10 R 11 may represent hydrogen, COalkyl and CO optionally substituted aryl,
R 16 may represent hydroxy, alkoxy, or NR 10 R 11 , and R 17 may represent alkyl substituted by one or more of halogen, alkoxy, optionally substituted aryl or NR 10 R 11 .
Compounds that may be mentioned include those wherein either: one of R 5 or R 6 represents L - R 3 , in which L represents a linker group which is:
O, S, NR 18 , alkylene, alkenylene, alkynylene, each of which may be optionally interrupted by one or more of O, S, NR 18 , or one or more C-C single, double or triple bonds, a -N-N- single or double bond, and R 18 represents hydrogen, alkyl, COR 16 ;
or one of R 4 or R 20 represents L - R 3 , in which L represents a linker group which is:
O, NR 18 , alkylene, alkenylene, alkynylene, each of which may be optionally interrupted by one or more of O, S, NR 18 , or one or more C-C single, double or triple bonds, and R 18 represents hydrogen, alkyl, COR 16 .
Although the scope for variation of R 4 , R 5 , R 6 , and R 7 is large, preferably R 4 , R 5 , R 6 , and R 7 represent hydrogen, alkyl or optionally substituted aryl.
R 4 , R 5 , R 6 , or R 20 may represent -L-R 3 .
Alkyl may represent any alkyl chain. Alkyl includes straight and branched, saturated and unsaturated alkyl, as well as cyclic alkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. However, preferably, when any of the substituents represents alkyl, alkyl is saturated, linear or branched and has from 1 to 10 carbon atoms, preferably from 1 to 8 carbon atoms and more preferably from 1 to 6 carbon atoms. When any of the substituents represents alkyl, a particularly preferred group is cycloalkyl, for example cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
Aryl may represent any aromatic system. Preferably, in the compounds of formula I, aryl is an aromatic hydrocarbon or a 5 to 10 membered aromatic heterocycle containing 1 to 4 hetero atoms selected from an oxygen atom, a sulfur atom and a nitrogen atom as a ring constituent besides carbon. We prefer heterocycles which contain one or two heteroatoms. Aromatic heterocycles that may be mentioned include furan, thiophene, pyrrole, pyridine.
Particularly preferably, when aryl is an aromatic hydrocarbon, aryl represents a 6 to 10 membered monocyclic or bicyclic system, for example phenyl or naphthalene.
Saturated and unsaturated heterocycles that may be mentioned include those containing 4 to 7 ring atoms, preferably 5 or 6 ring atoms, preferably containing one to two heteroatoms selected from N, S and O. Heterocycles that may be mentioned include pyrrolidine, piperidine, tetrahydrofuran, piperazine and morpholine. N- containing heterocycles are particularly preferred, eg when NR 10 Rn forms a heterocyclic ring.
As detailed above, when an adjacent pair of A 1 — A 4 each represent CR 1 , the adjacent carbon atoms, together with their substituents may form a ring ring B. Also, when Y or Z is CR 6 R 7 , R 6 and R 7 , together with the carbon to which they are attached may form a ring C. Preferably ring B and/or ring C is a saturated or unsaturated 3 to 10 membered carbocylic or heterocyclic ring.
Particularly preferably ring B is benzene ring.
Particularly preferably ring C is a 3- 10 membered saturated or unsaturated carbocylic ring.
We particularly prefer compounds in which at least one R 1 represents NR 15 C(=W)R 17 , more particularly the group NR 15 COR 17 .
We also prefer compounds in which at least one R 1 represents CONR 10 R 11 .
For one group of particularly preferred compounds at least one Ri represents an amide group NHCOR 17 ; wherein R 17 is selected from: alkyl C 1 - C 6 , alkyl C 1 - C 6 substituted by phenyl alkyl C 1 - C 6 substituted by alkoxy Ci - C 6 , haloalkyl Ci - C 6 , perfluoroalkyl Ci - C 6 , phenyl optionally substituted by one or more of halogen, alkyl C] — C 6 , alkoxy C 1 - C 6 , amino, (alkyl Ci - C 6 )amino, di(alkyl C 1 - C 6 ) amino or phenyl,
CH:CH phenyl, naphthyl, pyridinyl, thiophenyl and fur any 1.
We prefer compounds in which one or both of R and R are other than - COOH.
For another group of particularly preferred compounds at least one R 1 represents a group NR 15 CONR 10 R 11 , then in which R 10 and R 11 , which may be the same or different, are selected from optionally substituted aryl, alkyl and COaryl optionally substituted. A particularly preferred group which at least one of R 1 may represent is NHCONHR 15 and R 15 is selected from phenyl, alkyl Ci to C 6 and COphenyl optionally substituted by one or more halogen.
For another group of particularly preferred compounds at least one R 1 represents alkyl C 1 to C 6 , optionally substituted by phenyl or a 5 or 6- membered saturated or unsaturated heterocycle containing one to two heteroatoms selected from N, S and O.
For another group of preferred compounds at least one of R 1 represents SO 2 R 12 wherein R 12 represents alkyl C 1 - C 6 or NR 98 R 99 wherein R 98 and R 99 , which may be the same or different, represent hydrogen or alkyl C 1 - C 6 .
For another group of particularly preferred compounds at least one R 1 represents COR 16 and R 16 is alkoxy C 1 - C 6 , amino, (alkyl C 1 - C 6 )amino or di(alkyl C 1 - C 6 ) amino.
For another group of particularly preferred compounds at least one R 1 represents:
NO 2 , halogen, amino or (alkyl C 1 - C 6 )amino or di(alkyl C 1 - C 6 ) amino in which the alkyl C 1 to C 6 is optionally substituted by phenyl or a 5 or 6 membered saturated or unsaturated heterocycle,
NHSO 2 alkyl C 1 - C 6 , NHSO 2 phenyl,
S0 2 alkyl C 1 - C 6 , phenyl optionally substituted by C 1 to C 6 alkoxy Cl - C6, a 5 — 10 membered, saturated or unsaturated, mono- or bi-cyclic heterocycle containing from 1 — 3 heteroatoms selected from N, S and O.
There is also wide scope for variation of the group R . Preferably R represents aryl an idd iiss ooppttiioonnally substituted by one to three substituents, R , which may be the same or different.
Particularly preferably, R is a 5 - 10 membered aromatic mono- or bi-cyclic system, especially a hydrocarbon 5 - 10 membered aromatic mono- or bi-cyclic system, for example benzene or naphthalene.
Alternatively, the 5 - 10 membered aromatic mono- or bi-cyclic system, may be a heterocyclic system containing up to three heteroatoms selected from N, O and S, for example a thiophene, furan, pyridine or pyrrole.
Preferably the substituent(s) R 2 is/are selected from: alkyl C 1 - C 6 , optionally substituted by thiophenyl or phenoxy, each optionally substituted by halogen, alkoxy C 1 - C 6 phenyl, thioalkyl C 1 - C 6 thiophenyl, optionally substituted by halogen, NO 2 , CN NR 10 R 11 , in which R 10 and R 11 , which may be the same or different represent hydrogen, alkyl C 1 - C 6 , or together with the nitrogen to which they are attached form a 5 to 7 membered ring which may contain one or more additional heteroatoms selected from N, O and S, halogen SO 2 R 12 , in which R 12 represents a 5 to 7 membered ring which may contain one or more additional heteroatoms selected from N, O and S NHCOR 17 , in which R 17 represents alkyl C 1 — C 6 , optionally substituted by: phenyl or halogen, or phenyl optionally substituted by alkoxy C 1 - C 6 , carboxy or halogen, or a 5 or 6 membered saturated or unsaturated heterocycle, phenyl or a 5 or 6 membered saturated or unsaturated heterocycle optionally substituted by halogen, alkoxy C 1 to C 6 , carboxy or a group SO 2 NR 10 R 11 ,
Particularly preferably when R 2 represents NR 10 R 11 , NR 10 R 11 represents N- pyrrole, N-piperidine, N^C 1 - C 6 ) alkyl N piperazine or N-morpholine.
Compounds that may be mentioned include those wherein R 5 or R 6 represents L - R , in which L represents a linker group which is:
-NH.NH-,
-CH=CH-, -C≡C- or
-NCOR 16 in which R 16 represents phenyl or a 5 or 6 membered saturated or unsaturated heterocycle optionally substituted by halogen, alkoxy C 1 to C 6 , carboxy.
A 1 - A 4 may represent N or CR 1 . Consequently, the six membered ring may contain 1, 2, 3 or 4 nitrogen atoms. Embodiments of the invention exist in which two of A 1 - A 4 represent nitrogen, one of A 1 - A 4 represents nitrogen and in which all of A 1 — A represents CR 1 .
Compounds that may be mentioned include those in which A 5 represents N, and
Y and Z together represent CR 6 =CR 6 , for example CH=CH.
Compounds that may be mentioned include those in which A 5 represents CR 1 , for example CH, and in which Y and Z together represent CR 6 =CR 6 , for example CH=CH.
In a particularly preferred group of compounds:
A 1 , A 2 , A 3 and A 4 which may be the same or different, represent N or CR 1 , and either:
A 5 represents N, and Y and Z together represent CR 6 =CR 6 , for example CH=CH, or A 5 represents CR 1 , for example CH, and in which Y and Z together represent CR 6 =CR 6 , for example CH=CH, additionally, each R 6 which may be the same or different, indepenendently represent hydrogen or a substituent, optionally one of R 6 represents - L -R 3 , in which L is a single bond and wherein L is single bond and R 3 represents:
thioalkyl optionally substituted by alkyl or optionally substituted aryl, thioaryl, in which the aryl is optionally substituted, optionally substituted aryl, hydroxyl, NO 2 ,
CN,
NR 10 R 11 , halogen,
SO 2 R 12 , NR 13 SO 2 R 14 ,
C(=W)R 16 ,
OC(=W)NR 10 R n
NR 15 C(=W)R 17 ,
R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 and R 17 , which may be the same or different, represent hydrogen, alkyl optionally substituted by optionally substituted aryl, optionally substituted aryl, in addition,
R 10 and R 11 together with the nitrogen to which they are attached may form a ring, R 12 may have the same meaning as NR 10 R 11 ,
R 16 and R 17 , which may be the same or different, may each represent alkyl substituted by one or more of halogen, alkoxy optionally substituted aryl or optionally substituted aryl, optionally substituted aryloxy, aryl Or NR 10 R 11 , and when R 16 or R 17 represents NR 10 R 11 , one of R 10 and R 11 , may additionally represent CO alkyl optionally substituted or COaryl optionally substituted, and in addition to the definitions shared with R 17 , R 16 may represent hydroxyl, and R 1 and R 2 , which may be the same or different, may represent: alkyl optionally substituted by one or more halogen, alkoxy or optionally substituted aryl, thioaryl or aryloxy,
alkoxy optionally substituted by optionally by alkyl or optionally substituted aryl, hydroxy.,
OC(=W)NR 10 R π aryl, thioalkyl optionally substituted by alkyl or optionally substituted aryl, thioaryl, in which the aryl is optionally substituted,
NO 2 ,
CN, NR 10 R 11 , halogen,
SO 2 R 12 ,
NR 13 SO 2 R 14 ,
C(=W)R 16 , NR 15 C(=W)R 17 ,
R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 and R 17 , which may be the same or different, represent hydrogen, alkyl optionally substituted by optionally substituted aryl, optionally substituted aryl, in addition, NR 10 R 11 together with the nitrogen to which they are attached may form a ring,
R 12 may have the same meaning as NR 10 R 11 , when R 17 represents NR 10 R 11 , that NR 10 R 11 may represent hydrogen, COalkyl and CO optionally substituted aryl,
R 16 may represent hydroxy, alkoxy, or NR 10 R 11 , and R 17 may represent alkyl substituted by one or more of halogen, alkoxy, optionally substituted aryl or NR 10 R 11 , and in addition, when an adjacent pair of A 1 - A 4 each represent CR 1 , then the adjacent carbon atoms, together with their substituents may form a ring B, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the therapeutic and/or prophylactic treatment of Duchenne muscular dystrophy, Becker muscular dystrophy or cachexia.
The compounds of formula I for use in the treatment of DMD will generally be administered in the form of a pharmaceutical composition.
Thus, according to a further aspect of the invention there is provided a pharmaceutical composition including preferably less than 80% w/w, more preferably less than 50% w/w, e.g. 0.1 to 20%, of a compound of formula I 5 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 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 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 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 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 potential activity of the compounds of 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 mώc 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 (see figure 1).
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.
Example of pharmacological dose response of compounds in the assay is shown in figure 2.
2. mdx mouse
Data obtained from the ADMET data was prioritised and the compounds with the best in vitro luciferase activity and reasonable ADMET data were prioritised for testing in the mdx proof of concept study where the outcome was to identify whether any of the compounds had the ability to increase the levels of utrophin protein in dystrophin deficient muscle when compared to vehicle only dosed control animals.
There were two animals 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 3 shows an example of TA muscle sections stained with antibody specific for mouse utrophin. Comparison to the mdx muscle only injected with vehicle 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 (figure 4). 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 control.
Positive upregulation data from the first 28 day study were then repeated in a further two mouse 28 day study. A total of three different compounds have shown in duplicate the ability to increase the level of utrophin expression in the mdx mouse when delivered daily by ip for 28 days. This data demonstrates the ability of the compound when delivered ip causes a significant increase in the levels of utrophin found in the mdx muscle and therefore gives us the confidence that this approach will ameliorate the disease as all the published data to date demonstrates that any increase of utrophin levels over three fold has significant functional effects on dystrophin deficient muscle.
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 33 0 C in the presence of 10% CO 2
To remove the myoblasts for platting, they were incubated with Trypsin / EDTA until the monolayer stalled 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 33 0 C in the presence of 10% CO 2 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-Glo Luciferase Assay System(E2520). Then counted for 10 seconds using a plate luminometer (Victor 1420).
Compound Storage
Compounds for screening were stored at -2O 0 C as 1OmM stocks in 100% DMSO until required.
Injection of max 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 Irnmunohistochemistry 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 -8O 0 C
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
Table 1:
Example Chemical Name Activity number
1. 2,3-dimethyl-6-phenylimidazo[1 ,2-b][1 ,2,4]triazine +
2. 3,7-diphenylimidazo[1 ,2-b][1 ,2,4]triazine ++
3. N-(imidazo[1,2-a]pyridin-2-ylmethyi)-4-iodoaniline +
4. 2-(4-chlorophenyl)imidazo[1,2-a]pyridine ++++
5. 2-(3-(imidazo[1,2-a]pyridin-2-yl)phenylcarbamoyl)-6-nitroben zoic acid
6. N-(3-(imidazo[1,2-a]pyridin-2-yl)phenyl)-4-nitrobenzamide 7. N-(3-(imidazo[1 ,2-a]pyridin-2-yl)phenyl)-3-nitrobenzamide + 8. 6-amino-2-((3-chloroimidazo[1 ,2-a]pyridin-2- + yl)methylthio)pyrimidin-4-ol
9. 3-(6,8-dibromoimidazo[1 ,2-a]pyridin-2-yl)-4-methyl-1 ,2,5- oxadiazole
10. 2-(4-fluorophenyl)-7-methylimidazo[1 ,2-a]pyridine +++ 11. 2-(4-fluorophenyl)-6-methylimidazo[1 ,2-a]pyridine ++ 12. N-(3-(imidazo[1,2-a]pyridin-2-yl)phenyI)-3~methoxybenzamide + 13. N-(3-(imidazo[1 ,2-a]pyridin-2-yl)phenyl)propionamide 14. 4-(imidazo[1 ,2-a]pyridin-2-yl)benzene-1 ,2-diol 15. N-(4-(imidazo[1,2-a]pyridin-2-yl)phenyl)-2-methoxybenzamide + 16. N-(3-(imidazo[1 ,2-a]pyridin-2-yl)phenyl)pentanamide + 17. N-(3-(7-methylimidazo[1,2-a]pyridin-2-yl)phenyl)benzamide 18. N-(3-(imidazo[1,2-a]pyridin-2-yl)phenyl)-4- methoxybenzenesulfonamide
19. 2-(3-bromo-4-methoxyphenyl)imidazo[1,2-a]pyridine ++++ 20. N-(4-(imidazo[1 ,2-a]pyridin-2-y))phenyl)propionamide + 21. N-(3-(imidazot1,2-a]pyridin-2-yl)phenyl)thiophene-2-carboxam ide + 22. N-(4-(imidazo[1 ,2-a]pyridin-2-yl)phenyl)-4- methylbenzenesulfonamide
23. N-(4-(imidazo[1 ) 2-a]pyridin-2-yl)pheny!)-4-methoxybenzamide 24. N-(3-(imidazo[1 ,2-a]pyridin-2-yl)phenyl)-4-methoxybenzamide 25. 3-(6-chloroimidazo[1 ,2-a]pyridin-2-yl)-2H-chromen-2-one ++ 26. 3-(imidazo[1,2-a]pyrimidin-2-yl)-6-methoxy-2H-chromen-2-one + 27. 8-methoxy-3-(8-methylimidazo[1 ,2-a]pyridin-2-yl)-2H-chromen-2- one
28. 6-chloro-3-(8-methylimidazo[1,2-a]pyridin-2-yl)-2H-chromen-2 -one
29. N,N-diethyl-4-(imidazo[1 ,2-a]pyridin-2-yl)benzenesulfonamide +
30. 7-methyl-2-(4-(piperidin-1-ylsulfonyl)phenyl)imidazo[1,2-a]p yridine +
31. 6-chloro-2-(4-(piperidin-1-ylsulfonyl)phenyl)imidazo[1,2-a]p yridine +
32. N,N-diethyl-4-(8-methylimidazo[1,2-a]pyridin-2- + yl)benzenesulfonamide
33. 4-(4-(6-methylimidazo[1 ,2-a]pyridin-2-yl)phenylsulfoπyl)morpholine +
34. 2-(3-fluoro-4-methoxyphenyl)imidazo[1 ,2-a]pyridine ++++
35. N,N-diethyl-4-(7-methylimidazo[1 ,2-a]pyridin-2- + yl)benzenesulfonamide
36. 4-(6-chloroimidazo[1,2-a]pyridin-2-yl)-N,N- + diethylbenzenesulfonamide
37. 2-(3-fluoro-4-methoxyphenyl)-7-methylimidazo[1 ,2-a]pyridine ++++
38. 2-(4-(azepan-1-ylsulfonyl)phenyl)imidazo[1,2-a]pyridine +
39. 2-(3-fluoro-4-methoxyphenyl)-6-methylimidazo[1 ,2-a]pyridine ++++
40. 4-(6-chloroimidazo[1,2-a]pyridin-2-yl)-N,N- + dimethylbenzenesulfonamide
41. 2-(4-fluorophenyl)-8-methylimidazo[1 ,2-a]pyridine ++++
42. 4-(4-(7-methylimidazo[1 ,2-a]pyridin-2-yl)phenylsulfonyl)morpholine +
43. N,N-dimethyl-4-(6-methylimidazo[1 ,2-a]pyridin-2- + yl)benzenesulfonamide
44. N-(4-(imidazo[1 ,2-a]pyiϊdin-2-yl)phenyl)-4-methy)benzamide +
45. N-(4-(imidazo[1 ,2-a]pyridin-2-yl)phenyl)methanesulfonamide +
46. N-(3-(imidazo[1 ,2-a]pyridin-2-yl)phenyl)methanesulfonamide +
47. N-(4-(7-methylimidazo[1 ,2-a]pyridin-2- + yl)phenyl)benzenesulfonamide
48. 2-fluoro-N-(4-(imidazo[1 ,2-a]pyridin-2-yl)phenyl)benzamide +
49. 2-chloro-N-(4-(imidazo[1 ,2-a]pyridin-2-yl)phenyl)-4-nitrobenzamide +
50. N-(3-(7-methylimidazo[1 ,2-a]pyridin-2- + yl)phenyl)benzenesulfonamide
51. 2-chloro-N-(3-(7-methylimidazo[1 ,2-a]pyridin-2-yl)phenyl)-4- + nitrobenzamide
52. 3,4-dinnethoxy-N-(4-(7-methylimidazo[1 ,2-a]pyridin-2- + yl)phenyl)benzenesulfonamide
53. 2-fluoro-N-(3-(7-methylimidazo[1 ,2-a]pyridin-2- + yl)phenyl)benzamide
54. 4-(3-(8-methylimidazo[1 ,2-a]pyridin-2-yl)phenylsulfonyl)morpholine +
55. N,N-dimethyl-3-(8-methylimidazo[1 ,2-a]pyridin-2- + yl)beπzenesulfonamide
56. 3-(6-chloroimidazo[1 ,2-a]pyridin-2-yl)-N,N- + dimethylbenzenesulfonamide
57. 4-bromo-N-(imidazo[1,2-a]pyridin-2-ylmethyl)aniline +
58. 4-chloro-N-(imidazo[1 ,2-a]pyridin-2-ylmethyl)aniline +
59. 2-((8-methylimidazo[1 ,2-a]pyridin-2-yl)methylthio)aniline ++
60. 2-((4-chlorophenoxy)methyl)imidazo[1,2-a]pyridine +
61. N-((8-methylimidazo[1,2-a]pyridin-2-yl)methyl)aniline +
62. 2-(4-methoxyphenyl)imidazo[1 ,2-a]pyridin-3-amine +
63. N-(4-(imidazo[1 ,2-a]pyrimidin-2-yl)phenyl)-3,4- + dimethoxybenzenesulfonamide
64. N-(3-(imidazo[1 ,2-a]pyridin-2-yl)phenyl)furan-2-carboxamide +
65. N-(4-(imidazo[1 ,2-a]pyridin-2-yl)phenyl)pivalamide +
66. 2-(4-methoxyphenyl)-N-(3-(8-methylimidazo[1 ,2-a]pyridin-2- + yl)phenyl)acetamide
67. 2-(3,4-dimethoxyphenyl)-N-(4-(8-methylimidazo[1 ,2-a]pyridin-2- +
yl)phenyl)acetamide
68. 3-fluoro-N-(3-(imidazo[1 ,2-a]pyridin-2-yl)phenyl)benzamide +
69. 3,4-dimethoxy-N-(4-(7-methylimidazo[1 ,2-a]pyridin-2- + yl)phenyl)benzamide
70. N-(4-(8-methylimidazo[1 ,2-a]pyridin-2-yl)phenyl)pivalamide +++
71. 2-(imidazo[1,2-a]pyridin-2-ylmethylthio)benzo[d]oxazole ++
72. N-(5-(imidazof1 ,2-a]pyridin-2-yl)-2-methylphenyl)furan-2- + carboxamide
73. N-(2-methyl-5-(7-methylimidazo[1 ,2-a]pyridin-2- + yl)phenyl)propionamide
74. N-(5-(imidazo[1 ,2-a]pyridin-2-yl)-2-methylphenyl)acetamide +
75. N-(5-(imidazo[1 ,2-a]pyridin-2-yl)-2~methylphenyl)thiophene-2- + carboxamide
76. N-(5-(imidazo[1 ,2-a]pyridin-2-yl)-2-methylphenyl)pivalamide +
77. 2-fluoro-N-(5-(imidazo[1 ,2-a]pyridin-2-yl)-2- + methylphenyl)benzamide
78. 2-(4-ch!orophenyl)imidazo[1 ,2-a]pyridin-6-amine ++++
79. N-(2-(4-chlorophenyl)imidazo[1 ,2-a]pyridin-6-yl)isobutyramide ++
80. 2-(4-bromophenyl)-6-nitroimidazo[1,2-a]pyridine . +++
81. N-(2-(3,4-dichlorophenyl)imidazo[1 ,2-a]pyridin-6-yl)isobutyramide ++
82. 2-(3,4-dichlorophenyl)imidazo[1 ,2-a]pyridin-6-amine ++++
83. N-(2-(4-chlorophenyl)imidazo[1 ,2-a]pyridin-6-yl)acetamide ++++
84. N-(2-(3,4-dichlorophenyl)imidazo[1 ,2-a]pyridin-6-yl)acetamide ++++
85. ethyl 2-(3,4-dichlorophenyl)imidazo[1 ,2-a]pyridin-6-ylcarbamate +
86. N-(2-(3,4-dichlorophenyl)imidazo[1,2-a]pyridin-6- ++ ytycyclopropanecarboxamide
87. 2-(3,4-dichlorophenyl)-N-methylimidazo[1 ,2-a]pyridin-6-amine +
88. N-(2-(4-fluorophenyl)imidazo[1 ,2-a]pyridin-6-yl)acetamide +++
89. ethyl 2-(3,4-dichloropheny!)imidazo[1,2-a]pyridin-6- ++ yl(methyl)carbamate
90. 2-(3,4-dichlorophenyl)-N,N-dimethylimidazo[1 ,2-a]pyridin-6-amine +
91. 6-(ethylthio)-2-(4-iodophenyl)imidazo[1 ,2-a]pyridine +
92. 6-(ethylsulfinyl)-2-(4-iodophenyl)imidazo[1 ,2-a]pyridine +
93. N-(3-(3,4-dichlorophenyi)imidazo[1 ,2-a]pyridin-δ-yl)thiophene-2- + carboxamide
94. N-(3-(3,4-dichlorophenyl)imidazo[1,2-a]pyridin-8-yl)-3- + (trifluoromethyl)benzamide
95. 2,4-dichloro-N-(3-(4-chlorophenyl)imidazo[1 ,2-a]pyridin-8- + yl)benzamide
96. N-(3-(4-chlorophenyl)imidazo[1 ,2-a]pyridin-8-yl)-2- + fluorobenzamide
97. 2-(4-chlorophenyl)-6-(ethylthio)imidazo[1 ,2-a]pyridine ++++
98. 2-(4-chlorophenyl)-6-(ethylsulfonyl)imidazo[1 ,2-a]pyridine ++++
99. 2-(4-chlorophenyl)-6-(ethylsulfinyl)imidazo[1 ,2-a]pyridine ++++
100. 2-(4-chlorophenyl)-7-methylimidazo[1,2-a]pyridine ++++
101. 2-(4-ch]orophenyl)-6-methylimidazo[1 ,2-a]pyridine ++++
102. 3-(4-chlorophenyl)-N-isopropylimidazo[1 ,2-a]pyridin-8-amine +
103. N-(2-(naphthalen-2-yl)imidazo[1 ,2-a]pyridin-6-yl)acetamide ++++
104. N-(3-(3,4-dichlorophenyl)imidazo[1 ,2-a]pyridin-8-yl)acetamide +++
105. N-(2-(3,4-dichlorophenyl)imidazo[1,2-a]pyridin-6-yl)butyrami de ++++
106. N-(2-(3,4-dichlorophenyl)imidazo[1 ,2-a]pyridin-6-yl)-N- ++++ methylbutyramide
107. 6-(ethylthio)-2-(naphthalen-2-yl)imidazo[1 ,2-a]pyridine +
108. 6-(ethylsulfonyl)-2-(naphthalen-2-yl)imidazo[1 ,2-a]pyridine ++++
109. 6-(ethylsulfinyl)-2-(naphthalen-2-yl)imidazo[1 ,2-a}pyιϊdine +
110. 2-(3,4-dichiorophenyl)-6-(ethylthio)imidazo[1 ,2-a]pyridine ++++
111. 2-(3,4-dichlorophenyl)-6-(ethylsulfonyl)imidazo[1 ,2-a]pyridiπe ++++
112. 2-(4-chlorophenyl)-6-(trifluoromethyl)imidazo[1 ,2-a]pyridine ++++
113. 2-(naphthalen-2-yl)-6-(trifluoromethyl)imidazo[1,2-a]pyridin e +
114. 2-(4-fluorophenyl)imidazo[1 ,2-a]pyridine-6-carboxylic acid +
115. 2-(4-(methylsulfonyl)phenyl)imidazo[1,2-a]pyridine-6-carboxy lic + acid
116. 2-(6-nitroimidazo[1 ,2-a]pyridin-2-yl)benzo[d]thiazole ++
Experimental
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 150x10mm and the mobile phase is acetonitrile/water. 1 H NMR spectra were recorded on a Bruker instrument operating at 300 MHz. NMR spectra were obtained as CDCl 3 solutions (reported in ppm), using chloroform as the reference standard (7.25 ppm) or DMSO-D 6 (2.50 ppm). When peak multiplicities are reported, the following abbreviations are used s (singlet), d (doublet), t (triplet), m (multiplet), 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 (SP 1™ Purification System from Biotage ® ). Reactions in the microwave were done in an Initiator 8™ (Biotage). The abbreviations used are DMSO (dimethylsulfoxide), HCl (hydrochloric acid), MgSO 4 (magnesium sulfate), NaOH (sodium hydroxide), Na 2 CO 3 (sodium carbonate), NaHCO 3 (sodium bicarbonate), THF (tetrahydrofuran).
O 4
, Ethanol : Water 16h
O
A, Cl pyridine, rt, 18h Method 2
Method 1 ; Compounds I
2-(4-ChlorophenyI)imidazo[l,2-a]pyridin-6-amine
2,5-Diaminopyridine dihydrochloride (2.0Og, l l.Ommol), 2-bromo-l-(4- chlorophenyl)ethanone (2.56g, l l.Ommol) and potassium carbonate (3.03g, 22.0mmol) in dioxane:water (40ml:20mL) were heated to reflux for 30 minutes. The remaining portion of potassium carbonate (2.27g, 16.45mmol) was then added at room temperature and the resulting mixture was refluxed for 22 hours. After cooling, concentrated hydrochloric acid (15mL) was added and the insoluble solid was filtered off. The filtrate was then basified with IM sodium hydroxide to reach pH 14. The aqueous layer was extracted with dichloromethane (3x200mL). The combined organic layers were washed with brine until pH 7 was reached, dried over anhydrous MgSO 4 and evaporated. The brown solid was then purified by column chromatography on silica gel eluting using a gradient (dichloromethane to dichloromethane/methanol 93:7 v/v) to afford 562mg (21%) of the title compound as a brown solid (LCMS RT= 5.67min, MH + 244.0)
1 H NMR (DMSO): 8.20 (IH 5 s), 7.90 (2H 5 d 5 J 8.5 Hz) 5 7.67-7.66 (IH 5 m), 7.45 (2H 5 d 5 J 8.6 Hz) 5 7.34 (IH, d 5 J 9.0 Hz), 6.85 (IH, dd, J9.5 1.9 Hz) 5 4.94 (2H 5 br)
2-(4-Bromophenyl)-6-nitroimidazo[l,2-a]pyridine was purchased from Specs : LCMS RT= 6.71min, MH + 317.9; 1 H NMR (DMSO): 9.84-9.83 (IH 5 m) 5 8.65 (IH, s) 5 8.00-7.93 (3H, m), 7.75 (IH 5 d, J9.8 Hz), 7.71-7.66 (2H, m)
All compounds below were prepared following the same general procedure.
2-(3,4-Dichlorophenyl)imidazo[l,2-a]pyridin-6-amine
LCMS RT= 5.98min, MH + 277.9; 1 H NMR (DMSO): 8.31 (IH, s), 8.10 (IH, d, J2.0 Hz), 7.85 (IH, dd, J 8.4 2.0 Hz), 7.66-7.64 (2H, m), 7.35 (IH 5 d, J 8.6 Hz) 5 6.87 (IH, dd, J9.5 2.1 Hz), 4.99 (2H 5 br)
2-(4-Chlorophenyl)-6-(ethylthio)imidazo [1 ,2-a] pyridine
LCMS RT= 7.58min, MH + 289.1; 1 H NMR (DMSO): 8.61 (IH 5 q, J 0.9 Hz), 8.37 (IH, d, J 0.4 Hz), 7.97 (2H 5 d, J 8.7 Hz), 7.57 (IH 5 dt, J 9.7 0.8 Hz), 7.51 (2H, d, J 8.7 Hz), 7.30 (IH, dd, J 9.4 1.8 Hz), 2.95 (2H, q, J 7.3 Hz), 1.22 (3H, t, J 7.4 Hz)
2-(4-Chlorophenyl)-7-methylimidazo[l,2-a]pyridine
LCMS RT= 6.70min, MH + 243.1; 1 H NMR (DMSO): 8.41 (IH 5 dd, J 7.0 0.4 Hz), 8.33 (IH, d, J 0.5 Hz), 7.96 (2H, d, J 8.7 Hz) 5 7.48 (2H, d 5 J 8.7 Hz), 7.35 (IH, br), 6.76 (IH, dd, J 6.9 1.6 Hz), 2.36 (3H 5 d, J 0.8 Hz)
2-(4-Chlorophenyl)-6-methylimidazo[l,2-a]pyridine
LCMS RT= 6.83min, MH + 243.1; 1 H NMR (DMSO): 8.34-8.32 (2H 5 m), 7.97 (2H 5 d, J 8.6 Hz) 5 7.51-7.46 (3H, m), 7.12 (IH, dd, J9.2 1.7 Hz) 5 2.28 (3H 5 d, J0.8 Hz)
6-(Ethylthio)-2-(naphthalen-2-yl)imidazo[l ,2-a] pyridine LCMS RT= 7.34min, MH + 305.1; 1 H NMR (DMSO): 8.66-8.65 (IH, m), 8.53 (IH, br), 8.47 (IH, s), 8.09 (IH 5 dd, J 8.6 1.7 Hz), 8.03-7.90 (3H, m), 7.60 (IH 5 d, J 9.3 Hz), 7.55-7.50 (2H, m), 7.32 (IH, dd, J 9.3 1.8 Hz) 5 2.96 (2H, q, J 7.3 Hz) 5 1.23 (3H, t, J 7.3 Hz)
2-(3,4-DichIorophenyI)-6-(ethylthio)imidazo[l,2-a]pyridin e
LCMS RT= 8.12min, MH + 323.1; 1 H NMR (DMSO): 8.61-8.60 (IH 5 m), 8.46 (IH, s), 8.18 (IH, d, J 2.0 Hz), 7.93 (IH, dd, J 8.4 2.0 Hz), 7.70 (IH, d, J 8.4 Hz), 7.58 (IH, d, J9.4 Hz), 7.32 (IH, dd, J9.5 1.9 Hz), 2.96 (2H, q, J7.3 Hz), 1.22 (3H, t, J7.4 Hz)
2-(4-Chlorophenyl)-6-(trifluoromethyl)imidazo[l,2-a]pyrid ine
LCMS RT= 6.93min, MH + 297.1; 1 H NMR (DMSO): 9.25 (IH, s), 8.57 (IH, s), 8.02
(2H, d, J 8.7 Hz), 7.82 (IH, d, J9.3 Hz), 7.59-7.53 (3H, m)
2-(Naphthalen-2-yl)-6-(trifluoromethyl)imidazo[l,2-a] pyridine
LCMS RT= 7.19min, MH + 313.1; 1 H NMR (DMSO): 9.27-9.24 (IH, m), 8.64 (IH, s),
8.60-8.58 (IH, m), 8.12 (IH, dd, J 8.6 1.7 Hz), 8.06-7.99 (2H, m), 7.97-7.91 (IH, m),
7.82 (IH, dd, J 9.4 0.7 Hz), 7.59-7.49 (3H, m)
Method 2 : Compounds II
N-(2-(4-ChlorophenyI)imidazo [1 ,2-a] py ridin-6-yl)isobutyramide
To a stirred solution of 2-(4-chlorophenyl)imidazo[l,2-a]pyridin-6-amine (200mg, 0.82mmol) in pyridine (2OmL) was added isobutyryl chloride (103μL, 0.99mmol) at room temperature. The resulting mixture was stirred at room temperature overnight.
The solvent was then removed in vacuo to afford a brown solid, which was purified by column chromatography on silica gel eluting using a gradient (dichloromethane to dichloromethane/methanol 70:30 v/v). The resulting solid was washed with acetonitrile, and the filtrate evaporated to afford 90.9mg (35%) of the title compound (LCMS RT=
6.28min, MH + 314.1)
1 H NMR (DMSO): 9.97 (IH, s), 9.22-9.21 (IH, m), 8.47 (IH, s), 7.94 (2H, d, J 8.6
Hz), 7.55 (IH, d, J 9.5 Hz), 7.49 (2H, d, J 8.6 Hz), 7.20 (IH, dd, J 9.6 2.0 Hz), 2.69-
2.58 (lH,m), 1.13 (6H, d, J6.8 Hz)
AU compounds below were prepared following the same general procedure and using the appropriate electrophilic coupling reagent.
N-(2-(4-Chlorophenyl)imidazo [1 ,2-a] py ridin-6-y I)acetamide LCMS RT= 5.65min, MH + 286.0; 1 H NMR (DMSO): 10.11 (IH, s), 9.20-9.19 (IH, m), 8.50 (IH, s), 7.93 (2H 5 d, J 8.5 Hz) 3 7.55 (IH, d, J 9.5 Hz), 7.49 (2H, d, J 8.6 Hz), 7.16 (IH, dd, J9.6 2.0 Hz) 5 2.10 (3H, s)
N-(2-(3,4-Dichlorophenyl)imidazo[l,2-a]pyridin-6-yl)isobutyr amide LCMS RT= 6.67min 5 MH + 348.0; 1 H NMR (DMSO): 10.01 (IH 5 s), 9.22 (IH, s), 8.56 (IH 5 s), 8.15 (IH, d, J 1.8 Hz), 7.91 (IH 5 dd, J 9.4 1.8 Hz), 7.73 (IH 5 d, J 8.4 Hz) 5 7.57 (IH 5 d, J9.4 Hz) 5 7.22 (IH 5 dd, J9.6 1.8 Hz), 2.63 (lH, t, J6.7 Hz), 1.13 (6H 5 d, J6.7 Hz)
N-(2-(3,4-DichIorophenyl)imidazo[l,2-a]pyridin-6-yl)aceta mide
LCMS RT= 6.11min 5 MH + 319.9; 1 H NMR (DMSO): 10.12 (IH, s), 9.20 (IH 5 s), 8.58 (IH 5 s), 8.14 (IH 5 d, J 1.8 Hz) 5 7.89 (IH, dd, J 8.4 1.9 Hz), 7.70 (IH, d, J 8.4 Hz) 5 7.57 (IH 5 d 5 J 9.4 Hz) 5 7.18 (IH 5 dd, J 9.6 1.9 Hz) 5 2.10 (3H, s)
Ethyl 2-(3,4-dichlorophenyl)imidazo[l,2-a]pyridin-6-ylcarbamate
LCMS RT= 6.82min 5 MH + 350.0; 1 H NMR (DMSO): 9.78 (IH 5 s) 5 8.84 (IH 5 s), 8.56 (IH 5 s), 8.14 (IH 5 d, J 1.8 Hz) 5 7.89 (IH 5 dd, J 8.4 1.9 Hz), 7.69 (IH 5 d 5 J 8.3 Hz), 7.56 (IH 5 d, J 9.4 Hz) 5 7.22 (IH 5 dd 5 J 9.4 1.9 Hz), 4.16 (2H, q, J 7.0 Hz) 5 1.27 (3H 5 1, J 7.0 Hz)
N-(2-(3,4-Dichlorophenyl)imidazo[l,2-a]pyridin-6-yI)cyclo propanecarboxamide
LCMS RT= 6.67min, MH + 346.1; 1 H NMR (DMSO): 10.37 (IH 5 s) 5 9.17 (IH 5 s), 8.55 (IH 5 s), 8.14 (IH 5 d 5 J 2.0 Hz), 7.90 (IH 5 dd, J 8.4 2.0 Hz) 5 7.69 (IH 5 d 5 J 8.4 Hz), 7.58 (IH, d, J 9.5 Hz), 7.21 (IH 5 dd, J 9.6 1.9 Hz) 5 1.85-1.77 (IH, m) 5 0.84 (4H 5 d 5 J 6.1 Hz)
N-(2-(4-FIuorophenyl)imidazo[l,2-a]pyridin-6-yl)acetamide LCMS RT= 5.43min 5 MH + 270.1; 1 H NMR (DMSO): 10.09 (IH 5 s), 9.19 (IH, s), 8.45 (IH 5 s), 7.98-7.92 (2H 5 m) 5 7.55 (IH 5 d, J9.6 Hz), 7.27 (2H, t, J 9.0 Hz), 7.14 (IH, dd, J 9.6 2.0 Hz), 2.09 (3H, s)
N-(2-(Naphthalen-2-yl)imidazo[l,2-a]pyridin-6-yl)acetamid e
LCMS RT= 5.85min, MH + 302.2; 1 H NMR (DMSO): 10.11 (IH, s), 9.24-9.21 (IH, m), 8.60 (IH, s), 8.49 (IH 5 s), 8.08-7.91 (4H, m), 7.61-7.47 (3H, m), 7.18 (IH, dd, J9.6 2.0 Hz), 2.11 (3H, s)
N-(2-(3,4-Dichlorophenyl)imidazo[l,2-a]pyridin-6-yl)butyr amide
LCMS RT= 8.43mm, MH + 348.1; 1 H NMR (DMSO): 10.06 (IH, s), 9.22 (IH 5 s), 8.57 (IH, s) 5 8.14 (IH, d, J 1.9 Hz), 7.90 (IH, dd, J 8.3 2.0 Hz), 7.69 (IH, d, J 8.4 Hz), 7.57 (IH, d, J 9.6 Hz), 7.20 (IH, dd, J 9.6 2.0 Hz) 5 2.34 (2H 5 1 5 J 7.3 Hz) 5 1.71-1.58 (2H 5 m), 0.94 (3H, t, J7.3 Hz)
Method 3 III R=OEt
NaH, Mel, DMF rt, 18h
Method 4
IV R=OEt
Method 3 : Compounds III
2-(3,4-Dichlorophenyl)-N-methylimidazo[l,2-a]pyridin-6-am ine
To a stirred suspension of lithium aluminium hydride (85mg, 2.24mmol) in tetrahydrofuran (1OmL) 5 was slowly added a solution of ethyl 2-(3,4- dichlorophenyl)imidazo[l 5 2-a]pyridin-6-ylcarbamate (156.7mg, 0.45mmol) in tetrahydrofuran (5mL). The resulting solution was stirred at 7O 0 C for 18h. The crude mixture was then poured into diethyl ether (10OmL), and quenched with water (85 μL), 15% aqueous sodium hydroxide solution (85μL) and water again (155μL). The resulting solid was filtered off and washed with methanol. The filtrate obtained was evaporated in vacuo. The brown solid was purified by column chromatography on
silica gel eluting using a gradient (dichloromethane to dichloromethane/methanol 60:40 v/v) and then purified by reverse phase HPLC to afford 3mg (3%) of the title product (LCMS RT= 6.63min, MH + 292.0)
1 H NMR (DMSO): 8.29 (IH, s), 8.10 (IH, d, J 1.7 Hz), 7.85 (IH, dd, J 8.6 2.0 Hz), 7.65 (IH, d, J 8.4 Hz), 7.46 (IH, d, J 2.0 Hz) 5 7.36 (IH, d 5 J 9.6 Hz), 6.88 (IH, dd, J 9.6 2.0 Hz), 5.63 (IH, d, J4.8 Hz), 2.67 (3H, d, J 5.1 Hz)
Method 4 : Compounds IV
Ethyl 2-(3,4-dichlorophenyl)imidazo[l,2-a]pyridin-6-yl(methyl)carb amate
A solution of ethyl 2-(3,4-dichlorophenyl)imidazo[l,2-a]pyridin-6-ylcarbamate (240mg, 0.69mmol) in dimethylformamide (5mL) was added dropwise via a cannula to a stirred suspension of sodium hydride (32.9mg, 0.82mmol) in dimethylformamide (5mL) at room temperature. The resulting solution was stirred at room temperature for 30min. Methyl iodide (0.13mL, 2.06mmol) was then added dropwise at room temperature and the resulting solution was stirred for 18h. Water (15mL) was added, and the reaction mixture was partitioned between dichloromethane (15OmL) and water (10OmL). The aqueous layer was extracted twice with dichloromethane (2x10OmL). The combined organic layers were dried over anhydrous MgSO 4 and evaporated. The brown solid was then purified by column chromatography eluting using a gradient (dichloromethane to dichloromethane/methanol 85:15 v/v) to afford 120mg (48%) of the title compound (LCMS RT= 6.43min, MH + 364.1)
1 H NMR (DMSO): 8.66-8.63 (IH, m), 8.51 (IH, s), 8.21 (IH, s), 7.96 (IH, d, J 8.3 Hz), 7.71 (IH, d, J 8.3 Hz), 7.59 (IH, d, J 9.6 Hz), 7.31 (IH, dd, J 9.6 1.1 Hz), 4.09 (2H, q, J 6.9 Hz), 3.25 (3 H, s), 1.17 (3H, t, J 7.0 Hz)
The compound below was prepared following the same general procedure.
N-(2-(3,4-Dichlorophenyl)imidazo[l,2-a]pyridin-6-yl)-N-methy lbutyramide LCMS RT= 6.73min, MH + 362.0; 1 H NMR (DMSO): 8.72 (IH, s), 8.53 (IH, s), 8.23 (IH, d, J 1.8 Hz), 7.98 (IH, dd, J 8.5 1.9 Hz), 7.74-7.64 (2H, m), 7.30 (IH, d, J 9.4 Hz), 3.18 (3H, s), 2.13-2.05 (2H, m), 1.54-1.42 (2H, m), 0.82-0.74 (3H, m)
Method 5 : Compounds V
2-(3,4-Dichlorophenyl)-N,N-dimethylimidazo[l,2-a]pyridin- 6-amine
To a stirred suspension of lithium aluminium hydride (61mg, l.όOmmol) in tetrahydrofuran (1OmL) was slowly added a solution of ethyl 2-(3,4- dichlorophenyl)imidazo[l,2-a]pyridin-6-yl(methyl)carbamate (194.4mg, 0.53mmol) in tetrahydrofuran (5mL). The resulting solution was stirred at reflux for 18 h. The crude mixture was then poured into diethyl ether (10OmL), and quenched with water (61 μL), 15% aqueous sodium hydroxide solution (61μL) and further water (183μL). The resulting solid was filtered off and the filtrate obtained was evaporated in vacuo. The brown oil was purified by column chromatography eluting using a gradient (dichloromethane to dichloromethane/methanol 50:50 v/v) and by reverse phase HPLC to afford 0.6mg (0.4%) of the title product (LCMS RT= 7.32min, MH + 306.0) 1 H NMR (DMSO): 8.34 (IH, s), 8.13 (IH 5 d, J 1.9 Hz), 7.88 (IH, dd, J 8.4 1.9 Hz), 7.77 (IH, d, J 2.0 Hz), 7.64 (IH, d, J 8.4 Hz), 7.46 (IH, d, J 9.8 Hz), 7.25 (IH, dd, J 9.8 2.3 Hz), 2.85 (6H, s)
Method 6 : Compounds VI and VII
To a stirred suspension of trifluoroacetic anhydride (0.36mL, 2.57mmol) in dichloromethane (3mL) was added hydrogen peroxide (35% w/w in water, 146μL) followed by 2-(4-chlorophenyl)-6-(ethylthio)imidazo[l,2-a]pyridine (400mg, 1.39mmol) in dichloromethane (5mL). The resulting mixture was refluxed for 3h. After cooling, the mixture was diluted with dichloromethane, transferred into a separating
funnel and washed with saturated aqueous NaHCO 3 (10OmL). The aqueous layer was extracted twice with dichloromethane (2x10OmL) and the organic layer was washed with water until pH 7 was reached. The combined organic layers were dried over anhydrous MgSO 4 and evaporated. The solid was then purified by column chromatography on silica gel eluting using a gradient (ethyl acetate/hexanes 0:100 v/v to ethyl acetate/hexanes 100:0 v/v) to afford 273.7mg (65%) of 2-(4-chlorophenyl)-6- (ethylsulfinyl)imidazo[l,2-a]pyridine (LCMS RT= 5.75min, MH + 305.1; 1 H NMR (DMSO): 8.82 (IH, q, J 0.8 Hz), 8.59 (IH, d, J 0.6 Hz) 5 8.00 (2H, d, J 8.7 Hz), 7.76 (IH, d, J 9.4 Hz), 7.53 (2H, d, J 8.6 Hz), 7.46 (IH, dd, J 9.5 1.8 Hz), 3.21-3.08 (IH, m), 3.00-2.88 (IH, m), 1.08 (3H, t, J7.3 Hz)). Further elution gave 64.6mg (15%) of 2- (4-chlorophenyl)-6-(ethylsulfonyl)imidazo[l,2-a]pyridine (LCMS RT= 6.18min, MH + 321.2; 1 H NMR (DMSO): 9.22 (IH, q, J 0.9 Hz), 8.64 (IH, d, J 0.4 Hz), 8.01 (2H, d, J 8.6 Hz), 7.80 (IH, d, J 9.5 Hz), 7.61-7.53 (3H, m), 3.42 (2H, q, J 7.3 Hz), 1.18 (3H, t 5 J 7.3 Hz))
The compound below was prepared following the same general procedure.
6-(Ethylsulfinyl)-2-(4-iodophenyl)imidazo[l,2-a]pyridine
LCMS RT= 5.92min, MH + 397.0; 1 H NMR (DMSO): 8.82 (IH, q, J 0.8 Hz), 8.59 (IH, s), 7.85-7.73 (5H, m), 7.46 (IH, dd, J 9.4 1.8 Hz), 3.20-3.08 (IH, m), 3.00-2.88 (IH, m), 1.08 (3H, t, J7.2 Hz)
6-(EthyIsulfonyl)-2-(naphthalen-2-yl)imidazo[l,2-a]pyridi ne
LCMS RT= 6.28min, MH + 337.2; 1 H NMR (DMSO): 9.27-9.25 (IH, m), 8.74 (IH, s), 8.59 (IH, s), 8.13-7.93 (4H, m), 7.83 (IH, d, J 9.5 Hz), 7.61 (IH, dd, J 9.5 1.9 Hz), 7.57-7.53 (2H, m), 3.44 (2H, q, J7.4 Hz), 1.19 (3H, t, J7.4 Hz)
6-(Ethylsulfinyl)-2-(naphthalen-2-yl)imidazo [1 ,2-a] pyridine
LCMS RT= 5.87min, MH + 321.1; 1 H NMR (DMSO): 8.87-8.86 (IH, m), 8.69 (IH, s), 8.56 (IH, s), 8.11 (IH, dd, J 8.7 1.7 Hz), 8.05-7.92 (3H, m), 7.79 (IH, d, J 9.4 Hz), 7.58-7.52 (2H, m), 7.48 (IH, dd, J 9.4 1.8 Hz), 3.22-3.10 (IH, m), 3.02-2.90 (IH, m), 1.10 (3H, t, J7.3 Hz)
2-(3,4-DichIorophenyl)-6-(ethylsulfonyl)imidazo[l,2-a]pyr idine
LCMS RT= 6.59min, MH + 355.0; 1 H NMR (DMSO): 9.23-9.21 (IH, m), 8.72 (IH, s), 8.22 (IH, d, J2.0 Hz), 7.97 (IH, dd, J 8.3 1.9 Hz) 5 7.81 (IH 5 d, J 9.5 Hz) 5 7.75 (IH, d 5 J 8.5 Hz), 7.61 (IH 5 dd, J 9.5 1.9 Hz), 3.43 (2H 5 q, J7.4 Hz), 1.18 (3H, t, J 7.3 Hz)
VlI
Method 7 : Compounds VII
N-(3-(3,4-Dichlorophenyl)imidazo[l,2-a]pyridin-8-yl)aceta mide
To a stirred solution of 3-(3,4-dichlorophenyl)imidazo[l 5 2-a]pyridin-8-amine (300mg, 1.08mmol) in pyridine (1OmL) was added acetyl chloride (92μL, 1.29mmol) at room temperature. The resulting mixture was stirred at room temperature overnight. The mixture was then diluted with dichloromethane and extracted twice with water (2x10OmL). The aqueous layer was extracted with dichloromethane (2x50mL). The combined organic layers were washed with water, dried over anhydrous MgSO 4 and evaporated. The solid was purified by column chromatography on silica gel eluting using a gradient (dichloromethane/methanol 100:0 v/v to dichloromethane/methanol 92:8 v/v), and was then precipitated from diethyl ether to afford 51.6mg (15%) of the title compound (LCMS RT= 6.93min, MH + 320.1)
1 H NMR (DMSO): 9.85 (IH, s) 5 8.55 (IH, s), 8.32 (IH 5 d, J 1.8 Hz) 5 8.24 (IH, d 5 J 6.7 Hz) 5 8.02-7.95 (2H, m), 7.73 (IH 5 d, J 8.4 Hz) 5 6.88 (IH 5 1 5 J7.3 Hz), 2.26 (3H, s)
Method 8 : Compounds VIII
3-(4-Chlorophenyl)-N-isopropyIimidazo[l,2-a]pyridin-8-ami ne
To a stirred solution of 3-(4-chlorophenyl)imidazo[l,2-a]pyridin-8-amine (300mg, 1.23mmol) and acetone (2mL) in tetrahydrofuran/dichloromethane 1:1 v/v (2OmL) was added sodium triacetoxyborohydride (1.04g, 4.92mmol). The resulting solution was heated at reflux for 18h. After cooling, the mixture was then diluted with dichloromethane and water and transferred into a separating funnel. Brine (10OmL) was added and the organic layer separated, the aqueous layer was extracted with dichloromethane (2x5 OmL) and the combined organic layers were dried over anhydrous MgSO 4 and evaporated. The resulting oil was purified by column chromatography on silica gel eluting using a gradient (ethyl acetate/hexanes 0:100 v/v to ethyl acetate/hexanes 10:90 v/v) to afford 184.3mg (32%) of the title compound (LCMS RT= 7.94min, MH + 286.2)
1 H NMR (DMSO): 8.30 (IH, s), 7.99 (2H, d, J 8.7 Hz), 7.76 (IH, dd, J 6.6 0.9 Hz), 7.49 (2H, d, J 8.6 Hz), 6.71 (IH, dd, J 7.3 6.8 Hz), 6.14 (IH, d, J 7.4 Hz), 5.42 (IH, d, J8.4 Hz), 3.82-3.71 (IH, m), 1.26 (6H, d, J6.4 Hz)
Method 9 2-(6-nitroimidazo[l,2-a]pyridin-2-yl)benzo[d]thiazole
A solution of 2-amino-5-nitropyridine (480mg, 3.45mmol, leq) and l-(l-3- benzothiazol-2-yl)-2-bromo-l-ethanone (973mg, 3.798mmol, l.leq) in acetone was stirred at room temperature for 48 hours. The mixture was then concentrated in vacuo, the residue was treated with sat. aq. NaHCO 3 . The title compound (yellow solid) was collected by filtration and purified by recrystallisation from ethanol (22% Yield). LCMS RT= 6.24min, MH + = 297.1, 100% UV 1 H NMR (CDCl 3 ): 9.34 (IH, s), 8.57 (IH 5 s), 8.06 (3H, m, J 6.11 Hz), 7.79 (IH, d, J 9.87Hz), 7.52 (2H, dt, J 8.77 Hz).
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