Arylpiperazines and arylpiperidines and their use as metalloproteinase inhibiting agents Number:7,153,857 from the United States Patent and Trademark Office (PTO) owispatent Compounds of the formula I ##STR00001## useful as metalloproteinase inhibitors, especially as inhibitors of MMP 13.<H metalloproteinase, arylpiperidines, Arylpiperazines, 7153857.html, Patent, pto, 7153857

Title: Arylpiperazines and arylpiperidines and their use as metalloproteinase inhibiting agents

Abstract: Compounds of the formula I ##STR00001## useful as metalloproteinase inhibitors, especially as inhibitors of MMP 13.

Patent Number: 7,153,857 Issued on 12/26/2006 to Finlay, et al.

Inventors: Finlay; Maurice Raymond Verschoyle (Macclesfield, GB), Tucker; Howard (Macclesfield, GB), Waterson; David (Macclesfield, GB)
Assignee: AstraZeneca AB (Sodertalje, SE)

Appl. No.: 10/485,409

Filed: August 8, 2002

PCT Filed: August 08, 2002

PCT No.: PCT/SE02/01436

371(c)(1),(2),(4) Date: January 28, 2004

PCT Pub. No.: WO03/014111

PCT Pub. Date: February 20, 2003

Foreign Application Priority Data


Aug 09, 2001 [GB]			0119474.5

Current U.S. Class: 514/252.12 ; 514/247; 514/252.1; 514/252.13; 544/224; 544/336; 544/358; 544/359; 546/184; 546/192; 546/207; 546/210

Current International Class: A61K 31/496 (20060101); C07D 241/04 (20060101)

Field of Search: 544/224,336,358,359 546/184,192,207,210 514/247,252.1,252.12,252.13

References Cited [Referenced By]

U.S. Patent Documents


5202324	April 1993	Miyake et al.
5506242	April 1996	MacPherson et al.
5552419	September 1996	MacPherson et al.
5646167	July 1997	MacPherson et al.
5672615	September 1997	MacPherson et al.
5817822	October 1998	Nantermet et al.
5998412	December 1999	Broka et al.
6057336	May 2000	Duan et al.
6100266	August 2000	Montana et al.
6130220	October 2000	Broka et al.
6143744	November 2000	Broka et al.
6235786	May 2001	Dai et al.
6294573	September 2001	Curtin et al.
6376506	April 2002	Broka et al.
6479502	November 2002	Martin
6482827	November 2002	Alpegiani et al.
6492367	December 2002	DeCrescenzo et al.
6495568	December 2002	Dack et al.
6511993	January 2003	Dack et al.
6610731	August 2003	Duan et al.
6734183	May 2004	Barlaam et al.
2002/0037900	March 2002	Hannah et al.
2003/0050310	March 2003	Martin
2003/0216404	November 2003	Hannah et al.

Foreign Patent Documents


198 02 350	Jul., 1998	DE
0 381 132	Aug., 1990	EP
WO-96/00214	Jan., 1996	WO
WO-96/40101	Dec., 1996	WO
WO-98/05635	Feb., 1998	WO
WO-98/16514	Apr., 1998	WO
WO-98/32748	Jul., 1998	WO
WO-99/02510	Jan., 1999	WO
WO-99/18074	Apr., 1999	WO
WO-99/29667	Jun., 1999	WO
WO-99/38843	Aug., 1999	WO
WO-00/12477	Mar., 2000	WO
WO-00/12478	Mar., 2000	WO
WO-00/75108	Dec., 2000	WO
WO-01/62742	Aug., 2001	WO
WO-01/62750	Aug., 2001	WO
WO-01/62751	Aug., 2001	WO
WO-01/87870	Nov., 2001	WO
WO-03/014092	Feb., 2003	WO
WO-03/014098	Feb., 2003	WO

Other References

US 6,387,931, 05/2002, Dack et al. (withdrawn) cited by other.

Primary Examiner: Shameem; Golam M. M.
Attorney, Agent or Firm: Fish & Neave IP Group Ropes & Gray LLP

Claims

What we claim is:

1. A compound of the formula I or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof, ##STR00020## wherein B is selected from H, C.sub.1-6alkyl, up to C12 cycloalkyl, up to C12 aryl, and up to C12 heteroaryl; wherein B is optionally substituted by up to three groups independently selected from OH, NO.sub.2, CF.sub.3, CN, halogen, SC.sub.1-4alkyl, SOC.sub.1-4alkyl, SO.sub.2C.sub.1-4alkyl, C.sub.1-4alkyl, and C.sub.1-4alkoxy; L.sub.1 and L.sub.2 are each independently selected from a direct bond and C.sub.1-6alkyl; M.sub.1, M.sub.2, M.sub.3, M.sub.4 and M.sub.5 are each independently selected from N and C; R1 is the group --X--Y; X is C.sub.1-6alkyl; and Y is selected from up to C10 cycloalkyl, up to C10 aryl, and up to C10 heteroaryl; wherein Y is optionally substituted by up to three groups independently selected from OH, NO.sub.2, CF.sub.3, CN, halogen, SC.sub.1-4alkyl, SOC.sub.1-4alkyl, SO.sub.2C.sub.1-4alkyl, C.sub.1-4alkyl, and C.sub.1-4alkoxy.

2. A compound of the formula II or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof, ##STR00021## wherein B is selected from H, C.sub.1-6alkyl, up to C12 cycloalkyl, up to C12 aryl, and up to C12 heteroaryl; wherein B is optionally substituted by up to three groups independently selected from OH, NO.sub.2, CF.sub.3, CN, halogen, SC.sub.1-4alkyl, SOC.sub.1-4alkyl, SO.sub.2C.sub.1-4alkyl, C.sub.1-4alkyl, and C.sub.1-4alkoxy; L.sub.1 and L.sub.2 are each independently selected from a direct bond and C.sub.1-6alkyl; M.sub.1, M.sub.2, M.sub.3, M.sub.4 and M.sub.5 are each independently selected from N and C; R1 is the group --X--Y; X is C.sub.1-6alkyl; and Y is selected from up to C.sub.10 cycloalkyl, up to C.sub.10 aryl, and up to C.sub.10 heteroaryl; wherein Y is optionally substituted by up to three groups independently selected from OH, NO.sub.2, CF.sub.3, CN, halogen, SC.sub.1-4alkyl, SOC.sub.1-4alkyl, SO.sub.2C.sub.1-4alkyl, C.sub.1-4alkyl, and C.sub.1-4alkoxy.

3. A compound as claimed in claim 1 or claim 2 or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof, wherein B is selected from H, C.sub.1-6alkyl, C6 aryl, and up to C6 heteroaryl.

4. A compound as claimed in claim 3 or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof, wherein B is selected from H, C.sub.2-4alkyl, C6 aryl, and up to C6 heteroaryl.

5. A compound as claimed in claim 4 or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof, wherein B is up to C6 heteroaryl.

6. A compound as claimed in claim 1 or claim 2 or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof, wherein B is not substituted or B is substituted by at least one group selected from CF.sub.3, CN, halogen, and C.sub.1-4alkyl.

7. A compound as claimed in claim 1 or claim 2 or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof, wherein at least one of L.sub.1 and L.sub.2 is a direct bond.

8. A compound as claimed in claim 7 or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof, wherein each of L.sub.1 and L.sub.2 is a direct bond.

9. A compound as claimed in claim 1 or claim 2 or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof, wherein M.sub.1 is N.

10. A compound as claimed in claim 1 or claim 2 or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof, wherein at least one of M.sub.2, M.sub.3, M.sub.4, and M.sub.5 is C, and at least one of M.sub.2, M.sub.3, M.sub.4, and M.sub.5 is N.

11. A compound as claimed in claim 10 or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof, wherein each of M.sub.4 and M.sub.5 is C, and at least one of M.sub.2 and M.sub.3 is N.

12. A compound as claimed in claim 11 or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof, wherein M.sub.2 is C or N and M.sub.3 is N.

13. A compound as claimed in claim 1 or claim 2 or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof, wherein X is C.sub.2-5alkyl.

14. A compound as claimed in claim 13 or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof, wherein X is C.sub.2-3alkyl.

15. A compound as claimed in claim 1 or claim 2 or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof, wherein Y is C6 aryl or C6 heteroaryl.

16. A compound as claimed in claim 15 or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof, wherein Y is selected from phenyl, pyridyl, pyrimidinyl, or pyrazinyl.

17. A compound as claimed in claim 1 or claim 2 or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof wherein Y is not substituted or Y is substituted by at least one halogen group.

18. A compound as claimed in claim 1 or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof, wherein the compound is selected from hydroxy{-1-[({4-[5-(pyridin-2-ylethynyl)pyridin-2-yl]piperazin-1-yl}sulfo- nyl)methyl]-4-pyrimidin-2-ylbutyl}formamide, hydroxy[4-pyrimidin-2-yl-1-({[4-(5-{[5-(trifluoromethyl)pyridin-2-yl]ethy- nyl}pyridin-2-yl)piperazin-1-yl]sulfonyl}methyl)butyl]formamide, hydroxy{(3S)-3-phenyl-1-[({4-[4-(phenylethynyl)phenyl]piperazin-1-yl}sulf- onyl)methyl]butyl}formamide, hydroxy[(3S)-3-phenyl-1-({[4-(4-ethynylphenyl)piperazin-1-yl]sulfonyl}met- hyl)butyl]formamide, hydroxy{4-pyrimidin-2-yl-1-[({4-[5-(thien-2-ylethynyl)pyridin-2-yl]pipera- zin-1-yl}sulfonyl)methyl]butyl}formamid, 1-{[(4-{5-[(4-fluorophenyl)ethynyl]pyridin-2-yl}piperazin-1-yl)sulfonyl]m- ethyl}-4-pyrimidin-2-ylbutyl(hydroxy)formamide, hydroxy{(1S)-1-[({4-[5-(pyridin-2-ylethynyl)pyridin-2-yl]piperazin-1-yl}s- ulfonyl)methyl]-4-pyrimidin-2-ylbutyl}formamide, hydroxy {1-[({4-[5-(pyridin-2-ylethynyl)pyrimidin-2-yl]piperazin-1-yl}sulfonyl)me- thyl]-4-pyrimidin-2-ylbutyl}formamide, and hydroxy{(1S)-1-[({4-[5-(pyridin-2-ylethynyl)pyrimidin-2-yl]piperazin-1-yl- }sulfonyl)methyl]-4-pyrimidin-2-ylbutyl}formamide.

19. A pharmaceutical composition which comprises a pharmaceutically acceptable carrier and a compound of claim 1 or claim 2 or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. 371 of International Application No. PCT/SE02/01038, filed May 30, 2002, which claims priority from United Kingdom Patent Application No. 0101980.1, filed Jun. 1, 2001, the specification of which is incorporated by reference herein. International Application No. PCT/SE02/01038 was published under PCT Article 21(2) in English.

FIELD OF THE INVENTION

The present invention relates to compounds useful in the inhibition of metalloproteinases and in particular to pharmaceutical compositions comprising these, as well as their use. In particular, the compounds of this invention are inhibitors of matrix metalloproteinase 13 (MMP13), known also as collagenase 3.

Metalloproteinases are a superfamily of proteinases (enzymes) whose numbers in recent years have increased dramatically. Based on structural and functional considerations these enzymes have been classified into families and subfamilies as described in N. M. Hooper (1994) FEBS Letters 354:1 6. Examples of metalloproteinases include the matrix metalloproteinases (MMPs); the reprolysin or adamalysin or MDC family which includes the secretases and sheddases such as TNF converting enzymes (ADAM10 and TACE); the astacin family which include enzymes such as procollagen processing proteinase (PCP); and other metalloproteinases such as aggrecanase, the endothelin converting enzyme family and the angiotensin converting enzyme family.

Metalloproteinases are believed to be important in a plethora of physiological disease processes that involve tissue remodelling such as embryonic development, bone formation and uterine remodelling during menstruation. This is based on the ability of the metalloproteinases to cleave a broad range of matrix substrates such as collagen, proteoglycan and fibronectin. Metalloproteinases are also believed to be important in the processing, or secretion, of biological important cell mediators, such as tumour necrosis factor (TNF); and the post translational proteolysis processing, or shedding, of biologically important membrane proteins, such as the low affinity IgE receptor CD23 (for a more complete list see N. M. Hooper et al., (1997) Biochem J. 321:265 279).

Metalloproteinases have been associated with many diseases or conditions. Inhibition of the activity of one or more metalloproteinases may well be of benefit in these diseases or conditions, for example: various inflammatory and allergic diseases such as, inflammation of the joint (especially rheumatoid arthritis, osteoarthritis and gout), inflammation of the gastro-intestinal tract (especially inflammatory bowel disease, ulcerative colitis and gastritis), inflammation of the skin (especially psoriasis, eczema, dermatitis); in tumour metastasis or invasion; in disease associated with uncontrolled degradation of the extracellular matrix such as osteoarthritis; in bone resorptive disease (such as osteoporosis and Paget's disease); in diseases associated with aberrant angiogenesis; the enhanced collagen remodelling associated with diabetes, periodontal disease (such as gingivitis), corneal ulceration, ulceration of the skin, post-operative conditions (such as colonic anastomosis) and dermal wound healing; demyelinating diseases of the central and peripheral nervous systems (such as multiple sclerosis); Alzheimer's disease; extracellular matrix remodelling observed in cardiovascular diseases such as restenosis and atheroscelerosis; and chronic obstructive pulmonary diseases, COPD (for example, the role of MMPs such as MMP12 is discussed in Anderson & Shinagawa, 1999, Current Opinion in Anti-inflammatory and Immunomodulatory Investigational Drugs, 1(1): 29 38).

The matrix metalloproteinases (MMPs) are a family of structurally-related zinc-containing endopeptidases which mediate the breakdown of connective tissue macro-molecules. The mammalian MMP family is composed of at least twenty enzymes, classically divided into four sub-groups based on substrate specificity and domain structure [Alexander & Werb (1991) in Hay, E. D. ed. "Cell Biology of the Extracellular Matrix", New York, Plenum Press, 255 302; Murphy & Reynolds (1993) in Royce, P. M. & Steinman, B. eds. "Connective Tissue and its Heritable Disorders", New York, Wiley-Liss Inc., 287 316; Birkedal-Hansen (1995) Curr. Opin. Cell Biol. 7:728 735]. The sub-groups are the collagenases (such as MMP1, MMP8, MMP13), the stromelysins (such as MMP3, MMP10, MMP11), the gelatinases (such as MMP2, MMP9) and the membrane-type MMPs (such as MMP14, MMP15, MMP16, MMP17). Enzyme activity is normally regulated in vivo by tissue inhibitors of metalloproteinases (TIMPs).

Because of their central role in re-modelling connective tissue, both as part of normal physiological growth and repair and as part of disease processes, there has been substantial interest in these proteins as targets for therapeutic intervention in a wide range of degenerative and inflammatory diseases, such as arthritis, atherosclerosis, and cancer (Whittaker et al (1999) Chem. Rev. 99:2735 2776).

A number of MMP inhibitor compounds are known and some are being developed for pharmaceutical uses (see for example the review by Beckett & Whittaker (1998) Exp. Opin. Ther. Patents, 8(3):259 282). Different classes of compounds may have different degrees of potency and selectivity for inhibiting various MMPs. Whittaker M. et al (1999, Chem. Rev. 99:2735 2776) review a wide range of known MMP inhibitor compounds. They state that an effective MMP inhibitor requires a zinc binding group or ZBG (functional group capable of chelating the active site zinc(II) ion), at least one functional group which provides a hydrogen bond interaction with the enzyme backbone, and one or more side chains which undergo effective van der Waals interactions with the enzyme subsites. Zinc binding groups in known MMP inhibitors include hydroxamic acids (--C(O)NHOH), reverse hydroxamates (--N(OH)CHO), thiols, carboxylates and phosphonic acids.

We have discovered a new class of compounds that are inhibitors of metalloproteinases and are of particular interest in inhibiting MMP13. The compounds of this invention have beneficial potency and/or pharmacokinetic properties. In particular they show selectivity for MMP13.

MMP13, or collagenase 3, was initially cloned from a cDNA library derived from a breast tumour [J. M. P. Freije et al. (1994) Journal of Biological Chemistry 269(24):16766 16773]. PCR-RNA analysis of RNAs from a wide range of tissues indicated that MMP13 expression was limited to breast carcinomas as it was not found in breast fibroadenomas, normal or resting mammary gland, placenta, liver, ovary, uterus, prostate or parotid gland or in breast cancer cell lines (T47-D, MCF-7 and ZR75-1). Subsequent to this observation MMP13 has been detected in transformed epidermal keratinocytes [N. Johansson et al., (1997) Cell Growth Differ. 8(2):243 250], squamous cell carcinomas [N. Johansson et al., (1997) Am. J. Pathol. 151(2):499 508] and epidermal tumours [K. Airola et al., (1997) J. Invest. Dermatol. 109(2):225 231]. These results are suggestive that MMP13 is secreted by transformed epithelial cells and may be involved in the extracellular matrix degradation and cell-matrix interaction associated with metastasis especially as observed in invasive breast cancer lesions and in malignant epithelia growth in skin carcinogenesis.

Recent published data implies that MMP13 plays a role in the turnover of other connective tissues. For instance, consistent with MMP13's substrate specificity and preference for degrading type II collagen [P. G. Mitchell et al., (1996) J. Clin. Invest. 97(3):761 768; V. Knauper et al., (1996) The Biochemical Journal 271:1544 1550], MMP13 has been hypothesised to serve a role during primary ossification and skeletal remodelling [M. Stahle-Backdahl et al., (1997) Lab. Invest. 76(5):717 728; N. Johansson et al., (1997) Dev. Dyn. 208(3):387 397], in destructive joint diseases such as rheumatoid and osteo-arthritis [D. Wernicke et al., (1996) J. Rheumatol. 23:590 595; P. G. Mitchell et al., (1996) J. Clin. Invest. 97(3):761 768; O. Lindy et al., (1997) Arthritis Rheum 40(8): 1391 1399]; and during the aseptic loosening of hip replacements [S. Imai et al., (1998) J. Bone Joint Surg. Br. 80(4):701 710]. MMP13 has also been implicated in chronic adult periodontitis as it has been localised to the epithelium of chronically inflamed mucosa human gingival tissue [V. J. Uitto et al., (1998) Am. J. Pathol 152(6):1489 1499] and in remodelling of the collagenous matrix in chronic wounds [M. Vaalamo et al., (1997) J. Invest. Dermatol. 109(1):96 101].

U.S. Pat. No. 6,100,266 and WO-99/38843 disclose compounds of the general formula B--X--(CH.sub.2).sub.m--(CR.sup.1R.sup.2).sub.n--W--COY for use in the manufacture of a medicament for the treatment or prevention of a condition associated with matrix metalloproteinases. Specifically disclosed is the compound N-{1S-[4-(4-Chlorophenyl)piperazine-1-sulfonylmethyl]-2-methylpropyl}-N-h- ydroxyformamide.

WO-00/12478 discloses arylpiperazines that are matrix metalloproteinase inhibitors, including compounds with an hydroxamic acid zinc binding group and compounds with a reverse hydroxamate zinc binding group.

We have now discovered compounds that are potent MMP13 inhibitors and have desirable activity profiles.

In a first aspect of the invention we now provide a compound of the formula I

##STR00002##

wherein

B is selected from H, C.sub.1-6alkyl, up to C.sub.1-2 cycloalkyl, up to C.sub.1-2 aryl, and up to C.sub.1-2 heteroaryl;

B is optionally substituted by up to three groups independently selected from OH, NO.sub.2, CF.sub.3, CN, halogen, SC.sub.1-4alkyl, SOC.sub.1-4alkyl, SO.sub.2C.sub.1-4alkyl, C.sub.1-4alkyl, C.sub.1-4alkoxy;

L.sub.1 and L.sub.2 are each independently selected from a direct bond and C.sub.1-6alkyl;

M.sub.1, M.sub.2, M.sub.3, M.sub.4 and M.sub.5 are each independently selected from N and C;

R1 is the group --X--Y;

X is C.sub.1-6alkyl;

Y is selected from up to C10 cycloalkyl, up to C10 aryl, and up to C10 heteroaryl; Y is optionally substituted by up to three groups independently selected from OH, NO.sub.2, CF.sub.3, CN, halogen, SC.sub.1-4alkyl, SOC.sub.1-4alkyl, SO.sub.2C.sub.1-4alkyl, C.sub.1-4alkyl, C.sub.1-4alkoxy;

Any alkyl group outlined above may be straight chain or branched;

Any heteroaryl group outlined above is an aromatic ring containing one or more heteroatoms independently selected from N, O, S.

In a further aspect of the invention we provide compounds of the formula II

##STR00003## wherein B, L.sub.1, L.sub.2, M.sub.1, M.sub.2, M.sub.3, M.sub.4, M.sub.5, R1, X and Y are as defined above for the compound of formula I.

Preferred compounds of the formulae I or II are those wherein any one or more of the following apply:

B is selected from H, C.sub.1-6alkyl, C6 aryl, and up to C6 heteroaryl; preferably B is H, C.sub.2-4alkyl, C6 aryl, and up to C6 heteroaryl; most preferably B is up to C6 heteroaryl;

B is not substituted or is substituted by at least one group selected from CF.sub.3, CN, halogen (preferably fluoro or chloro), C.sub.1-4alkyl;

At least one of L.sub.1 and L.sub.2 is a direct bond; preferably each of L.sub.1 and L.sub.2 is a direct bond;

M.sub.1 is N;

At least one of M.sub.2, M.sub.3, M.sub.4, M.sub.5 is C, and at least one of M.sub.2, M.sub.3, M.sub.4, M.sub.5 is N; preferably each of M.sub.4 and M.sub.5 is C, and at least one of M.sub.2 and M.sub.3 is N; most preferably each of M.sub.4 and M.sub.5 is C, M.sub.2 is C or N and M.sub.3 is N;

X is C.sub.2-5alkyl; preferably X is C.sub.2-3alkyl;

Y is C6 aryl or C6 heteroaryl; preferably Y is phenyl, pyridyl, pyrimidinyl, or pyrazinyl; most preferably Y is pyrimidinyl;

Y is not substituted or is substituted by at least one halogen group (preferably fluoro or chloro).

For example, preferred compounds of the invention include those wherein B is phenyl, pyridyl, pyrimidinyl, or thienyl.

Other preferred compounds include those wherein L.sub.1 is a direct bond, L.sub.2 is a direct bond, M.sub.1 is N, M.sub.2 is C or N, M.sub.3 is N, M.sub.4 is C, and M.sub.5 is C.

Other preferred compounds include those wherein R1 is 3- or 4-chlorophenylethyl, 3- or 4-chlorophenylpropyl, 2- or 3-pyridylethyl, 2- or 3-pyridylpropyl, 2- or 4-pyrimidinylethyl (optionally monosubstituted by fluoro or chloro), 2- or 4-pyrimidinylpropyl (optionally monosubstituted by fluoro or chloro), 2-(2-pyrimidinyl)ethyl (optionally monosubstitued by fluoro or chloro), 2-(2-pyrimidinyl)propyl (optionally monosubstitued by fluoro or chloro). Particularly preferred compounds include those wherein R1 is 2-pyrimidinylpropyl, 2-pyrimidinylethyl or 5-fluoro-2-pyrimidinylethyl.

It will be appreciated that the particular substituents and number of substituents on B and/or R1 are selected so as to avoid sterically undesirable combinations.

Each exemplified compound represents a particular and independent aspect of the invention.

Where optically active centres exist in the compounds of formulae I or II, we disclose all individual optically active forms and combinations of these as individual specific embodiments of the invention, as well as their corresponding racemates.

It will be appreciated that the compounds according to the invention can contain one or more asymmetrically substituted carbon atoms. The presence of one or more of these asymmetric centres (chiral centres) in a compound of formulae I or II can give rise to stereoisomers, and in each case the invention is to be understood to extend to all such stereoisomers, including enantiomers and diastereomers, and mixtures including racemic mixtures thereof.

Where tautomers exist in the compounds of formulae I or II, we disclose all individual tautomeric forms and combinations of these as individual specific embodiments of the invention.

As previously outlined the compounds of the invention are metalloproteinase inhibitors, in particular they are inhibitors of MMP13. Each of the above indications for the compounds of the formulae I or II represents an independent and particular embodiment of the invention. Whilst we do not wish to be bound by theoretical considerations, the compounds of the invention are believed to show selective inhibition for any one of the above indications relative to any MMP1 inhibitory activity, by way of non-limiting example they may show 100 1000 fold selectivity over any MMP1 inhibitory activity.

The compounds of the invention may be provided as pharmaceutically acceptable salts. These include acid addition salts such as hydrochloride, hydrobromide, citrate and maleate salts and salts formed with phosphoric and sulphuric acid. In another aspect suitable salts are base salts such as an alkali metal salt for example sodium or potassium, an alkaline earth metal salt for example calcium or magnesium, or organic amine salt for example triethylamine.

They may also be provided as in vivo hydrolysable esters. These are pharmaceutically acceptable esters that hydrolyse in the human body to produce the parent compound. Such esters can be identified by administering, for example intravenously to a test animal, the compound under test and subsequently examining the test animal's body fluids. Suitable in vivo hydrolysable esters for carboxy include methoxymethyl and for hydroxy include formyl and acetyl, especially acetyl.

In order to use a compound of the formulae I or II or a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof for the therapeutic treatment (including prophylactic treatment) of mammals including humans, it is normally formulated in accordance with standard pharmaceutical practice as a pharmaceutical composition.

Therefore in another aspect the present invention provides a pharmaceutical composition which comprises a compound of the formulae I or II or a pharmaceutically acceptable salt or an in vivo hydrolysable ester and pharmaceutically acceptable carrier.

The pharmaceutical compositions of this invention may be administered in standard manner for the disease or condition that it is desired to treat, for example by oral, topical, parenteral, buccal, nasal, vaginal or rectal adminstration or by inhalation. For these purposes the compounds of this invention may be formulated by means known in the art into the form of, for example, tablets, capsules, aqueous or oily solutions, suspensions, emulsions, creams, ointments, gels, nasal sprays, suppositories, finely divided powders or aerosols for inhalation, and for parenteral use (including intravenous, intramuscular or infusion) sterile aqueous or oily solutions or suspensions or sterile emulsions.

In addition to the compounds of the present invention the pharmaceutical composition of this invention may also contain, or be co-administered (simultaneously or sequentially) with, one or more pharmacological agents of value in treating one or more diseases or conditions referred to hereinabove.

The pharmaceutical compositions of this invention will normally be administered to humans so that, for example, a daily dose of 0.5 to 75 mg/kg body weight (and preferably of 0.5 to 30 mg/kg body weight) is received. This daily dose may be given in divided doses as necessary, the precise amount of the compound received and the route of administration depending on the weight, age and sex of the patient being treated and on the particular disease or condition being treated according to principles known in the art.

Typically unit dosage forms will contain about 1 mg to 500 mg of a compound of this invention.

Therefore in a further aspect, the present invention provides a compound of the formulae I or II or a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof for use in a method of therapeutic treatment of the human or animal body. In particular we disclose use in the treatment of a disease or condition mediated by MMP13.

In yet a further aspect the present invention provides a method of treating a metalloproteinase mediated disease or condition which comprises administering to a warm-blooded animal a therapeutically effective amount of a compound of the formulae I or II or a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof. Metalloproteinase mediated diseases or conditions include arthritis (such as osteoarthritis), atherosclerosis, chronic obstructive pulmonary diseases (COPD).

In another aspect the present invention provides a process for preparing a compound of the formulae I or II or a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof which process is outlined below.

A compound of the formula I can be prepared from a compound of the formula III by reaction with hydroxylamine followed by formylation.

##STR00004##

A compound of the formula III can be prepared from a compound of the formula IV and a compound of the formula V or from a compound of the formula VI and a compound of the formula VII. A compound of the formula V may be prepared from a compound of the formula VII.

##STR00005##

A compound of the formula VII may be prepared from a compound of the formula VIII by reaction with a suitable aldehyde (R1CHO) or a suitable ester (R1COOR). A compound of formula VIII may be convieniently prepared from a compound of formula IX. A compound of the formula IX can be prepared conveniently from a compound of the formula X and a compound of the formula XI (where P is hydrogen or a suitable protecting group and M.sub.1' is hydrogen or a suitably reactive group).

##STR00006## A compound of the formula III can also be prepared from a compound of the formula XII by reaction with a suitable aldehyde (R1CHO) or a suitable ester (R1COOR). A compound of the formula XII can be prepared conveniently from a compound of the formula XIII and a compound of the formula XIV

##STR00007##

A compound of the formula II may be prepared from a compound of the formula XV by similar methodology to that described for compound I above.

##STR00008##

It will be appreciated that many of the relevant starting materials are commercially available or may be made by any convenient method as described in the literature or known to the skilled chemist or described in the Examples herein.

The compounds of the invention may be evaluated for example in the following assays:

Isolated Enzyme Assays

Matrix Metalloproteinase Family Including for Example MMP13.

Recombinant human proMMP13 may be expressed and purified as described by Knauper et al. [V. Knauper et al., (1996) The Biochemical Journal 271:1544 1550 (1996)]. The purified enzyme can be used to monitor inhibitors of activity as follows: purified proMMP13 is activated using 1 mM amino phenyl mercuric acid (APMA), 20 hours at 21.degree. C.; the activated MMP13 (11.25 ng per assay) is incubated for 4 5 hours at 35.degree. C. in assay buffer (0.1M Tris-HCl, pH 7.5 containing 0.1M NaCl, 20 mM CaCl2, 0.02 mM ZnCl and 0.05% (w/v) Brij 35 using the synthetic substrate 7-methoxycoumarin-4-yl)acetyl.Pro.Leu.Gly.Leu.N-3-(2,4-dinitrophenyl)-L-2- ,3-diaminopropionyl.Ala.Arg.NH.sub.2 in the presence or absence of inhibitors. Activity is determined by measuring the fluorescence at .lamda.ex 328 nm and .lamda.em 393 nm. Percent inhibition is calculated as follows: % Inhibition is equal to the [Fluorescence.sub.plus inhibitor-Fluorescence.sub.background] divided by the [Fluorescence.sub.minus inhibitor-Fluorescence.sub.background].

A similar protocol can be used for other expressed and purified pro MMPs using substrates and buffers conditions optimal for the particular MMP, for instance as described in C. Graham Knight et al., (1992) FEBS Lett. 296(3):263 266.

Adamalysin Family Including for Example TNF Convertase

The ability of the compounds to inhibit proTNF.alpha. convertase enzyme may be assessed using a partially purified, isolated enzyme assay, the enzyme being obtained from the membranes of THP-1 as described by K. M. Mohler et al., (1994) Nature 370:218 220. The purified enzyme activity and inhibition thereof is determined by incubating the partially purified enzyme in the presence or absence of test compounds using the substrate 4',5'-Dimethoxy-fluoresceinyl Ser.Pro.Leu.Ala.Gln.Ala.Val.Arg.Ser.Ser.Ser.Arg.Cys(4-(3-succinimid-1-yl)- -fluorescein)-NH.sub.2 in assay buffer (50 mM Tris HCl, pH 7.4 containing 0.1% (w/v) Triton X-100 and 2 mM CaCl.sub.2), at 26.degree. C. for 18 hours. The amount of inhibition is determined as for MMP13 except .lamda.ex 490 nm and .lamda.em 530 nm were used. The substrate was synthesised as follows. The peptidic part of the substrate was assembled on Fmoc-NH-Rink-MBHA-polystyrene resin either manually or on an automated peptide synthesiser by standard methods involving the use of Fmoc-amino acids and O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) as coupling agent with at least a 4- or 5-fold excess of Fmoc-amino acid and HBTU. Ser.sup.1 and Pro.sup.2 were double-coupled. The following side chain protection strategy was employed; Ser.sup.1(But), Gln.sup.5(Trityl), Arg.sup.8,12(Pmc or Pbf), Ser.sup.9,10,11(Trityl), Cys.sup.13(Trityl). Following assembly, the N-terminal Fmoc-protecting group was removed by treating the Fmoc-peptidyl-resin with 20% piperidine in DMF. The amino-peptidyl-resin so obtained was acylated by treatment for 1.5 2 hr at 70.degree. C. with 1.5 2 equivalents of 4',5'-dimethoxy-fluorescein-4(5)-carboxylic acid [Khanna & Ullman, (1980) Anal Biochem. 108:156 161) which had been preactivated with diisopropylcarbodiimide and 1-hydroxybenzotriazole in DMF]. The dimethoxyfluoresceinyl-peptide was then simultaneously deprotected and cleaved from the resin by treatment with trifluoroacetic acid containing 5% each of water and triethylsilane. The dimethoxyfluoresceinyl-peptide was isolated by evaporation, trituration with diethyl ether and filtration. The isolated peptide was reacted with 4-(N-maleimido)-fluorescein in DMF containing diisopropylethylamine, the product purified by RP-HPLC and finally isolated by freeze-drying from aqueous acetic acid. The product was characterised by MALDI-TOF MS and amino acid analysis.

Natural Substrates

The activity of the compounds of the invention as inhibitors of aggrecan degradation may be assayed using methods for example based on the disclosures of E. C. Amer et al., (1998) Osteoarthritis and Cartilage 6:214 228; (1999) Journal of Biological Chemistry, 274 (10), 6594 6601 and the antibodies described therein. The potency of compounds to act as inhibitors against collagenases can be determined as described by T. Cawston and A. Barrett (1979) Anal. Biochem. 99:340 345.

Inhibition of Metalloproteinase Activity in Cell/Tissue Based Activity Test as an Agent to Inhibit Membrane Sheddases Such as TNF Convertase

The ability of the compounds of this invention to inhibit the cellular processing of TNF.alpha. production may be assessed in THP-1 cells using an ELISA to detect released TNF essentially as described K. M. Mohler et al., (1994) Nature 370:218 220. In a similar fashion the processing or shedding of other membrane molecules such as those described in N. M. Hooper et al., (1997) Biochem. J. 321:265 279 may be tested using appropriate cell lines and with suitable antibodies to detect the shed protein.

Test as an Agent to Inhibit Cell Based Invasion

The ability of the compound of this invention to inhibit the migration of cells in an invasion assay may be determined as described in A. Albini et al., (1987) Cancer Research 47:3239 3245.

Test as an Agent to Inhibit Whole Blood TNF Sheddase Activity

The ability of the compounds of this invention to inhibit TNF.alpha. production is assessed in a human whole blood assay where LPS is used to stimulate the release of TNF.alpha.. Heparinized (10 Units/mL) human blood obtained from volunteers is diluted 1:5 with medium (RPMI1640+bicarbonate, penicillin, streptomycin and glutamine) and incubated (160 .mu.l) with 20 .mu.l of test compound (triplicates), in DMSO or appropriate vehicle, for 30 min at 37.degree. C. in a humidified (5% CO.sub.2/95% air) incubator, prior to addition of 20 .mu.l LPS (E. coli. 0111:B4; final concentration 10 .mu.g/mL). Each assay includes controls of diluted blood incubated with medium alone (6 wells/plate) or a known TNF.alpha. inhibitor as standard. The plates are then incubated for 6 hours at 37.degree. C. (humidified incubator), centrifuged (2000 rpm for 10 min; 4.degree. C.), plasma harvested (50 100 .mu.l) and stored in 96 well plates at -70.degree. C. before subsequent analysis for TNF.alpha. concentration by ELISA.

Test as an Agent to Inhibit in vitro Cartilage Degradation

The ability of the compounds of this invention to inhibit the degradation of the aggrecan or collagen components of cartilage can be assessed essentially as described by K. M. Bottomley et al., (1997) Biochem J. 323:483 488.

Pharmacodynamic Test

To evaluate the clearance properties and bioavailability of the compounds of this invention an ex vivo pharmacodynamic test is employed which utilises the synthetic substrate assays above or alternatively HPLC or Mass spectrometric analysis. This is a generic test which can be used to estimate the clearance rate of compounds across a range of species. Animals (e,g. rats, marmosets) are dosed iv or po with a soluble formulation of compound (such as 20% w/v DMSO, 60% w/v PEG400) and at subsequent time points (e.g. 5, 15, 30, 60, 120, 240, 480, 720, 1220 mins) the blood samples are taken from an appropriate vessel into 10 U heparin. Plasma fractions are obtained following centrifugation and the plasma proteins precipitated with acetonitrile (80% w/v final concentration). After 30 mins at -20.degree. C. the plasma proteins are sedimented by centrifugation and the supernatant fraction is evaporated to dryness using a Savant speed vac. The sediment is reconstituted in assay buffer and subsequently analysed for compound content using the synthetic substrate assay. Briefly, a compound concentration-response curve is constructed for the compound undergoing evaluation. Serial dilutions of the reconstituted plasma extracts are assessed for activity and the amount of compound present in the original plasma sample is calculated using the concentration-response curve taking into account the total plasma dilution factor.

In vivo Assessment

Test as an Anti-TNF Agent

The ability of the compounds of this invention as ex vivo TNF.alpha. inhibitors is assessed in the rat. Briefly, groups of male Wistar Alderley Park (AP) rats (180 210 g) are dosed with compound (6 rats) or drug vehicle (10 rats) by the appropriate route e.g. peroral (p.o.), intraperitoneal (i.p.), subcutaneous (s.c.). Ninety minutes later rats are sacrificed using a rising concentration of CO.sub.2 and bled out via the posterior vena cavae into 5 Units of sodium heparin/mL blood. Blood samples are immediately placed on ice and centrifuged at 2000 rpm for 10 min at 4.degree. C. and the harvested plasmas frozen at -20.degree. C. for subsequent assay of their effect on TNF.alpha. production by LPS-stimulated human blood. The rat plasma samples are thawed and 175 .mu.l of each sample are added to a set format pattern in a 96 well plate. Fifty .mu.l of heparinized human blood is then added to each well, mixed and the plate is incubated for 30 min at 37.degree. C. (humidified incubator). LPS (25 .mu.l; final concentration 10 .mu.g/mL) is added to the wells and incubation continued for a further 5.5 hours. Control wells are incubated with 25 .mu.l of medium alone. Plates are then centrifuged for 10 min at 2000 rpm and 200 .mu.L of the supernatants are transferred to a 96 well plate and frozen at -20.degree. C. for subsequent analysis of TNF concentration by ELISA.

Data analysis by dedicated software calculates for each compound/dose: Percent inhibition of TNF.alpha.=Mean TNF.alpha.(Controls)-Mean TNF.alpha.(Treated).times.100 Test as an Anti-arthritic Agent

Activity of a compound as an anti-arthritic is tested in the collagen-induced arthritis (CIA) as defined by D. E. Trentham et al., (1977) J. Exp. Med. 146,:857. In this model acid soluble native type II collagen causes polyarthritis in rats when administered in Freunds incomplete adjuvant. Similar conditions can be used to induce arthritis in mice and primates.

Test as an Anti-cancer Agent

Activity of a compound as an anti-cancer agent may be assessed essentially as described in I. J. Fidler (1978) Methods in Cancer Research 15:399 439, using for example the B16 cell line (described in B. Hibner et al., Abstract 283 p75 10th NCI-EORTC Symposium, Amsterdam Jun. 16 19 1998).

The invention will now be illustrated but not limited by the following Examples:

EXAMPLE 1

hydroxy{-1-[({4-[5-(pyridin-2-ylethynyl)pyridin-2-yl]piperazin-1-yl}sulfon- yl)methyl]-4-pyrimidin-2-ylbutyl}formamide

##STR00009##

To formic acid (1.5 mL, 39 mmol) at 0.degree. C. was added acetic anhydride (375 .mu.L, 3.9 mmol) and the mixture was stirred at RT for 10 minutes. The reaction was then recooled to 0.degree. C., and a solution of 2-[4-(hydroxyamino)-5-({4-[5-(pyridin-2-ylethynyl)pyridin-2-yl]piperaz- in-1-yl}sulfonyl)pentyl]pyrimidine (403 mg, 0.79 mmol) in THF (8 mL) was then added. The reaction was brought to RT and stirred for one hour. Volatiles were then removed in vacuo, and the residue azeotroped with toluene (2.times.5 mL). The residue was then dissolved in MeOH (10 mL) and heated to 40.degree. C. for one hour. The solution was then cooled to RT and concentrated in vacuo. The residue was then purified by flash chromatography (silica gel, 10% MeOH in EtOAc) to give the title compound as a pale yellow foam (231 mg, 0.43 mmol, 54%).

.sup.1H NMR (DMSO-D6, 373K): 9.41 (br s, 1H), 8.65 (d, 2H), 8.56 (d, 1H), 8.36 (d, 1H), 8.11 (br s, 1H), 7.79 (m, 1H), 7.71 (dd, 1H), 7.55 (d, 1H), 7.32 (m, 1H), 7.28 (dd, 1H), 6.88 (d, 1H), 3.72 (m, 4H), 3.45 (dd, 1H), 3.32 (m, 4H), 3.17 (dd, 1H), 2.92 (m, 3H), 1.77 (m, 4H)

MS (ESI): 536.45 (MH.sup.+)

The starting material was prepared as follows:

To a stirred solution of 2-chloro-5-iodo-pyridine (CAS number 69045-79-0, 10.52 g, 43.9 mmol) and diisopropylethylamine (11.5 mL, 65.9 mmol) in DMA (200 mL) was added piperazine (15.14 g, 0.176 mol). The mixture was then heated to 120.degree. C. for 20 hours. The solid precipitate was then filtered off, and the filtrate evaporated in vacuo. The residue was partitioned between EtOAc (100 mL) and water (100 mL) and the layers were separated. The aqueous layer was then extracted with EtOAc (2.times.100 mL). The combined organic extracts were then dried, (MgSO.sub.4), filtered and concentrated in vacuo, to give 1-(5-iodopyridin-2-yl)piperazine as a yellow solid (10.42 g, 36 mmol, 82%).

.sup.1H NMR (CDCl.sub.3): 8.30 (d, 1H), 7.65 (dd, 1H), 6.45 (d, 1H), 3.46, (t, 4H), 2.97 (t, 4H).

MS (ESI): 290.29 (MH.sup.+)

To a stirred solution of 1-(5-iodopyridin-2-yl)piperazine (CAS number 219635-89-9, 10.42 g, 36 mmol), in CH.sub.2Cl.sub.2 (150 mL) at 0.degree. C. was added a solution of BOC--O--BOC (7.87 g, 26 mmol). The reaction was allowed to warm to RT and was stirred for 18 hours. Volatiles were removed in vacuo, and the residue was dissolved in EtOAc (200 mL). The solution was washed with water (100 mL), and the aqueous layer was extracted with EtOAc (2.times.100 mL). The combined organic extracts were then dried, (MgSO.sub.4), filtered and concentrated in vacuo, to give tert-butyl 4-(5-iodopyridin-2-yl)piperazine-1-carboxylate as a yellow solid (14 g, 36 mmol, 99%).

.sup.1H NMR (CDCl.sub.3): 8.31 (d, 1H), 7.08 (dd, 1H), 6.46 (d, 1H), 3.50 (s, 8H), 1.48 (s, 9H).

MS (ESI): 390.37 (MH.sup.+)

To a stirred solution of tert-butyl 4-(5-iodopyridin-2-yl)piperazine-1-carboxylate (4.087 g, 10.5 mmol) in DMA (75 mL) at RT was added 2-ethynylpyridine, (CAS number 1945-84-2, 1.17 mL, 11.5 mmol), triethylamine (4.4 mL, 31.5 mmol), CuI (800 mg, 4.2 mmol) and Pd(PPh.sub.3).sub.4 (1.21 g, 1.05 mmol). After stirring for 30 minutes, the DMA was removed in vacuo, and the residue diluted with EtOAc (100 mL). The dark solution was then washed with water (100 mL). The layers were separated and the aqueous phase extracted with EtOAc (3.times.50 mL). The combined organic extracts were then dried, (MgSO.sub.4), filtered and concentrated in vacuo. The residue was then purified by flash chromatography (silica gel, 50% EtOAc in hexanes) to give tert-butyl 4-[5-(pyridin-2-ylethynyl)pyridin-2-yl]piperazine-1-carboxylate as a brown oil (3.8 g, 10.5 mmol, 99%).

.sup.1H NMR (CDCl.sub.3): 8.62 (m, 1H), 8.43 (d, 1H), 7.68 (m, 2H), 7.49 (d, 1H), 7.21 (dd, 1H), 6.58 (d, 1H), 3.61 (m, 8H), 1.51 (s, 9H).

MS (ESI): 364.94 (MH.sup.+)

To a stirred solution of tert-butyl 4-[5-(pyridin-2-ylethynyl)pyridin-2-yl]piperazine-1-carboxylate (3.8 g, 10.4 mmol) in CH.sub.2Cl.sub.2 (35 mL) at 0.degree. C. was added trifluoroacetic acid (17.2 mL). The reaction was brought to room temperature and stirred for 30 minutes. Volatiles were removed in vacuo, and the residue was azeotroped with toluene (2.times.10 mL). The residue was then dissolved in CH.sub.2Cl.sub.2 and washed with aqueous sodium hydroxide solution (2M, 2.times.20 mL). The layers were then separated and the aqueous phase extracted with CH.sub.2Cl.sub.2 (3.times.10 mL). The combined organic extracts were then dried, (MgSO.sub.4), filtered and concentrated in vacuo. The residue was dissolved in CH.sub.2Cl.sub.2 (50 mL), and cooled to 0.degree. C. and treated sequentially with triethylamine (1.5 mL, 16.7 mmol) and methanesulfonyl chloride (1.1 mL, 13.5 mmol). The reaction was brought to RT and stirred for 1 hour. The reaction was then quenched with water (50 mL). The layers were separated and the aqueous phase extracted with CH.sub.2Cl.sub.2 (3.times.20 mL). The combined organic extracts were then dried, (MgSO.sub.4), filtered and concentrated in vacuo. The residue was then purified by flash chromatography (silica gel, 10% MeOH in EtoAc) to give 1-(methylsulfonyl)-4-[5-(pyridin-2-ylethynyl)pyridin-2-yl]piperazine as a brown solid (3.5 g, 10.2 mmol, 98%).

.sup.1H NMR (CDCl.sub.3): 8.60 (d, 1H), 8.46 (d, 1H), 7.69, (m, 2H), 7.52 (d, 1H), 7.23 (m, 1H), 6.65 (d, 1H), 3.79 (m, 4H), 3.35 (m, 4H), 2.83 (s, 3H).

MS (ESI): 342.83 (MH.sup.+)

To a stirred suspension of 1-(methylsulfonyl)-4-[5-(pyridin-2-ylethynyl)pyridin-2-yl]piperazine (456 mg, 1.33 mmol) in THF (10 mL) at -20.degree. C., was added dropwise a solution of LiHMDS in THF (3.0 mL, 11.0M solution, 3.0 mmol). The resulting suspension was stirred at -20.degree. C. for 30 minutes before being treated with diethyl chlorophosphate (212 .mu.L, 1.46 mmol). The solution was then maintained at -20.degree. C. for 15 minutes before being treated with a solution of 4-pyrimidin-2-ylbutanal (220 mg, 1.46 mmol, CAS number 260441-10-9) in THF (3 mL). The solution was stirred at -20.degree. C. for a further 20 minutes before being quenched with saturated aqueous ammonium chloride solution (10 mL). The layers were separated and the aqueous phase extracted with ethyl acetate (3.times.10 mL). The combined organic extracts were then dried, (MgSO.sub.4), filtered and concentrated in vacuo to give -[(4E/Z)-5-({4-[5-(pyridin-2-ylethynyl)pyridin-2-yl]piperazin-1-yl}sulfon- yl)pent-4-enyl]pyrimidine. This material was used crude in the next step.

MS (ESI): 474.97 (MH.sup.+)

To a stirred solution of 2-[(4E/Z)-5-({4-[5-(pyridin-2-ylethynyl)pyridin-2-yl]piperazin-1-yl}sulfo- nyl)pent-4-enyl]pyrimidine (crude from previous step), in THF (15 mL) at RT was added a solution of hydroxylamine (3 mL, 50% aqueous solution in water). The reaction was stirred for 3 hours at RT before being quenched with saturated aqueous ammonium chloride solution (5 mL). The layers were separated and the aqueous phase extracted with ethyl acetate (3.times.10 mL). The combined organic extracts were then dried, (MgSO.sub.4), filtered and concentrated in vacuo. The residue was then purified by flash chromatography (silica, 10% MeOH in ethyl acetate) to give 2-[4-(hydroxyamino)-5-({4-[5-(pyridin-2-ylethynyl)pyridin-2-yl]piperazin-- 1-yl}sulfonyl)pentyl]pyrimidine (403 mg, 0.79 mmol).

MS (ESI): 508.22 (MH.sup.+)

EXAMPLE 2

hydroxy[4-pyrimidin-2-yl-1-({[4-(5-{[5-(trifluoromethyl)pyridin-2-yl]ethyn- yl}pyridin-2-yl)piperazin-1-yl]sulfonyl}methyl)butyl]formamide

##STR00010##

To formic acid (0.36 mL, 9.5 mmol) at 0.degree. C. was added acetic anhydride (90 .mu.L, 0.95 mmol) and the mixture was stirred at RT for 10 minutes. The reaction was then recooled to 0.degree. C., and added to a solution of 2-(4-(hydroxyamino)-5-{[4-(5-{[5-(trifluoromethyl)pyridin-2-yl]ethynyl}py- ridin-2-yl)piperazin-1-yl]sulfonyl}pentyl)pyrimidine (403 mg, 0.79 mmol) and formic acid (0.36 mL, 9.5 mmol) in THF (3 mL) at 0.degree. C. The reaction was brought to RT and stirred for one hour. Volatiles were then removed in vacuo, and the residue azeotroped with toluene (2.times.5 mL). The residue was then dissolved in MeOH (10 mL) and heated to 40.degree. C. for one hour. The solution was then cooled to RT and concentrated in vacuo. The residue was then purified by flash chromatography (silica gel, 10% MeOH in EtOAc) to give the title compound as a pale yellow foam (66 mg, 0.11 mmol, 58%).

.sup.1H NMR (DMSO-D6, 373K): 9.38 (br s, 1H), 8.90 (d, 1H), 8.67 (d, 2H), 8.42 (d, 1H), 8.15 (dd, 1H), 8.14 (br s, 1H), 7.73 (m, 2H), 7.27 (t, 1H), 6.90 (d, 1H), 3.71 (m, 4H), 3.42 (dd, 1H), 3.30 (m, 4H), 3.16 (dd, 1H), 2.88 (m, 3H), 1.76 (m, 3H), 1.66 (m, 1H)

MS (ESI): 604.39 (MH.sup.+)

The starting material was prepared as follows:

To a stirred solution of tert-butyl 4-(5-iodopyridin-2-yl)piperazine-1-carboxylate (1.0 g, 2.57 mmol, prepared as in Example 1), trimethylsilyl acetylene (0.73 mL, 5.14 mmol), triethylamine (1.11 mL, 7.71 mmol), and cuprous iodide (196 mg, 1.03 mmol) in DMA (20 mL) at 25.degree. C. was added tetrakistriphenylphosphine palladium (0) (149 mg, 5 mol %). The reaction was stirred for 10 minutes. Volatiles were removed in vacuo. The residue was then dissolved in ethyl acetate (250 mL) and washed with water (100 mL). The layers were then separated and the aqueous phase extracted with ethyl acetate (250 mL). The combined organic extracts were then dried, (MgSO.sub.4), filtered and concentrated in vacuo. The residue was then purified by flash chromatography (silica gel, 10% EtOAc in hexanes) to give tert-butyl 4-{5-[(trimethylsilyl)ethynyl]pyridin-2-yl}piperazine-1-carboxylate as a pale brown solid (652 mg, 1.81 mmol, 70%).

.sup.1H NMR (CDCl.sub.3): 8.28 (d, 1H), 7.50 (dd, 1H), 6.50 (d, 1H), 3.57 (m, 8H), 1.49 (s, 9H), 0.25 (s, 9H).

MS (ESI): 360.51 (MH.sup.+)

To a stirred solution of tert-butyl 4-{5-[(trimethylsilyl)ethynyl]pyridin-2-yl}piperazine-1-carboxylate (637 mg, 1.77 mmol), in THF (8 mL) at 25.degree. C. was added tetrabutylammonium fluoride (1.77 mL, 1.0M in THF). The reaction was stirred for 1 hour. Water (5 mL) was then added and product extracted with ethyl acetate (2.times.5 mL). The combined organic extracts were then dried, (MgSO.sub.4), filtered and concentrated in vacuo to give tert-butyl 4-(5-ethynylpyridin-2-yl)piperazine-1-carboxylate as an orange solid (509 mg, 1.77 mmol, 100%).

.sup.1H NMR (CDCl.sub.3): 8.30 (d, 1H), 7.54 (dd, 1H), 6.52 (d, 1H), 3.56 (m, 8H), 3.07 (s, 1H), 1.49 (s, 9H).

To a stirred solution of tert-butyl 4-(5-ethynylpyridin-2-yl)piperazine-1-carboxylate (509 mg, 1.77 mmol), 2-Bromo-5-trifluoromethylpyridine (400 mg, 1.77 mmol), triethylamine (0.74 mL, 5.31 mmol), and cuprous iodide (135 mg, 0.71 mmol) in DMA (20 mL) at 25.degree. C. was added tetrakistriphenylphosphine palladium (0) (102 mg, 5 mol %). The reaction was then stirred for 1 hour. Volatiles were removed in vacuo, and the residue was azeotroped with toluene (2.times.10 mL). The residue was then dissolved in CH.sub.2Cl.sub.2 (10 mL) and washed with water (10 mL). The layers were then separated and the aqueous phase extracted with CH.sub.2Cl.sub.2 (2.times.100 mL). The combined organic extracts were then dried, (MgSO.sub.4), filtered and concentrated in vacuo. The residue was then purified by flash chromatography (silica gel, 25% EtOAc in hexanes) to give tert-butyl 4-(5-{[5-(trifluoromethyl)pyridin-2-yl]ethynyl}pyridin-2-yl)piperazine-1-- carboxylate as a yellow solid (729 mg, 1.68 mmol, 95%).

.sup.1H NMR (CDCl.sub.3): 8.83 (d, 1H), 8.42 (d, 1H), 7.88, (dd, 1H), 7.67 (dd, 1H), 7.56 (d, 1H), 6.59 (d, 1H), 3.60 (m, 4H), 3.53 (m, 4H), 1.50 (s, 9H).

MS (ESI): 433.49 (MH.sup.+)

To a stirred solution of tert-butyl 4-(5-{[5-(trifluoromethyl)pyridin-2-yl]ethynyl}pyridin-2-yl)piperazine-1-- carboxylate (724 mg, 1.67 mmol) in CH.sub.2Cl.sub.2 (8 mL) at 0.degree. C. was added trifluoroacetic acid (4 mL). The reaction was brought to room temperature and stirred for 30 minutes. Volatiles were removed in vacuo. The residue