U.S. patent application number 10/379092 was filed with the patent office on 2003-09-04 for phenylalanine derivatives.
Invention is credited to Archibald, Sarah Catherine, Head, John Clifford, Porter, John Robert, Warrellow, Graham John.
Application Number | 20030166691 10/379092 |
Document ID | / |
Family ID | 26313188 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030166691 |
Kind Code |
A1 |
Archibald, Sarah Catherine ;
et al. |
September 4, 2003 |
Phenylalanine derivatives
Abstract
Phenylalanine derivatives of formula (1) are described: 1
wherein R is a carboxylic acid or a derivative thereof; L.sup.1 is
a linker atom or group; Ar is an optionally substituted aromatic
group; and the salts, solvates, hydrates and N-oxides thereof. The
compounds are able to inhibit the binding of alpha4 integrins to
their ligands and are of use in the prophylaxis and treatment of
immune or inflammatory disorders.
Inventors: |
Archibald, Sarah Catherine;
(Maidenhead, GB) ; Head, John Clifford;
(Maidenhead, GB) ; Warrellow, Graham John;
(Northwood, GB) ; Porter, John Robert; (Chinnor,
GB) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP
ONE LIBERTY PLACE, 46TH FLOOR
1650 MARKET STREET
PHILADELPHIA
PA
19103
US
|
Family ID: |
26313188 |
Appl. No.: |
10/379092 |
Filed: |
March 3, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10379092 |
Mar 3, 2003 |
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09258522 |
Feb 26, 1999 |
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6555562 |
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Current U.S.
Class: |
514/355 ;
514/341; 546/268.1; 546/315 |
Current CPC
Class: |
C07D 213/61 20130101;
C07C 235/60 20130101; C07C 233/87 20130101; A61P 29/00 20180101;
A61P 43/00 20180101; A61P 37/02 20180101; C07C 237/30 20130101;
C07C 323/62 20130101; C07D 213/81 20130101 |
Class at
Publication: |
514/355 ;
514/341; 546/268.1; 546/315 |
International
Class: |
C07D 43/02; C07D 213/46;
A61K 031/455; A61K 031/4439 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 26, 1998 |
GB |
9804161.9 |
Dec 3, 1998 |
GB |
9826668.7 |
Claims
1. A compound of formula (1): 7wherein R is a carboxylic acid or a
derivative thereof; R.sup.1 is a hydrogen atom or a hydroxyl,
straight or branched alkoxy or optionally substituted
cycloaliphatic, polycycloaliphatic, heterocycloaliphatic,
polyheterocycloaliphatic, aromatic or heteroaromatic group;
Alk.sup.1 is an optionally substituted aliphatic or heteroaliphatic
chain; L.sup.1 is a linker atom or group; r and s, which may be the
same or different, is each zero or an integer 1 provided that when
r is zero R.sup.1 is an optionally substituted cycloaliphatic,
polycycloaliphatic, heterocycloaliphatic, polyheterocycloaliphatic,
aromatic or heteroaromatic group; R.sup.a and R.sup.b, which may be
the same or different is each an atom or group
-L.sup.2(CH.sub.2).sub.pL.sup.3(R.sup.c).sub.q in which L.sup.2 and
L.sup.3 is each a covalent bond or a linker atom or group, p is
zero or the integer 1, q is an integer 1, 2 or 3 and R.sup.c is a
hydrogen or halogen atom or a group selected from straight or
branched alkyl, OR.sup.d [where R.sup.d is a hydrogen atom or an
optionally substituted straight or branched alkyl group],
--SR.sup.d, --NR.sup.dR.sup.e, [where R.sup.e is as just defined
for R.sup.d and may be the same or different], --NO.sub.2, --CN,
--CO.sub.2R.sup.d, --SO.sub.3H, --SO.sub.2R.sup.d,
--OCO.sub.2R.sup.d, --CONR.sup.dR.sup.e, --OCONR.sup.dR.sup.e,
--CSNR.sup.dR.sup.e, --COR.sup.d, --N(R.sup.d)COR.sup.e,
N(R.sup.d)CSR.sup.e, --SO.sub.2N(R.sup.d)(R.sup.e),
--N(R.sup.d)SO.sub.2R.sup.e, --N(R.sup.d)CONR.sup.eR.sup.f [where
R.sup.f is a hydrogen atom or an optionally substituted straight or
branched alkyl group], --N(R.sup.d)CSNR.sup.eR.sup.f or
--N(R.sup.d)SO.sub.2NR.sup- .eR.sup.f; Alk.sup.2 is a straight or
branched alkylene chain; m is zero or an integer 1; R.sup.2 is a
hydrogen atom or a methyl group; R.sup.3 is a hydrogen atom or a
straight or branched alkyl group; Ar is an optionally substituted
aromatic group; and the salts, solvates, hydrates and N-oxides
thereof for use in modulating cell adhesion.
2. A compound of formula (1a): 8wherein R is a carboxylic acid or a
derivative thereof; R.sup.1 is an optionally substituted
cycloaliphatic, polycycloaliphatic, heterocycloaliphatic,
polyheterocyclialiphatic, aromatic or heteroaromatic group;
Alk.sup.1 is an optionally substituted aliphatic or heteroaliphatic
chain; L.sup.1 is a linker atom or group; r and s, which may be the
same or different, is each zero or an integer 1; R.sup.a and
R.sup.b, which may be the same or different is each an atom or
group -L.sup.2(CH.sub.2).sub.pL.sup.3(R.sup.c).sub.q in which
L.sup.2 and L.sup.3 is each a covalent bond or a linker atom or
group, p is zero or the integer 1, q is an integer 1, 2 or 3 and
R.sup.c is a hydrogen or halogen atom or a group selected from
straight or branched alkyl, OR.sup.d [where R.sup.d is a hydrogen
atom or an optionally substituted straight or branched alkyl
group], --SR.sup.d, --NR.sup.dR.sup.e, [where R.sup.e is as just
defined for R.sup.d and may be the same or different], --NO.sub.2,
--CN, --CO.sub.2R.sup.d, --SO.sub.3H, --SO.sub.2R.sup.d,
--OCO.sub.2R.sup.d, --CONR.sup.dR.sup.e, --OCONR.sup.dR.sup.e,
--CSNR.sup.dR.sup.e, --COR.sup.d, --N(R.sup.d)COR.sup.e,
N(R.sup.d)CSR.sup.e, --SO.sub.2N(R.sup.d)(R.sup.e),
--N(R.sup.d)SO.sub.2R.sup.e, --N(R.sup.d)CONR.sup.eR.sup.f [where
R.sup.f is a hydrogen atom or an optionally substituted straight or
branched alkyl group], --N(R.sup.d)CSNR.sup.eR.sup.f or
--N(R.sup.d)SO.sub.2NR.sup- .eR.sup.f; Alk.sup.2 is a straight or
branched alkylene chain; m is zero or an integer 1; R.sup.2 is a
hydrogen atom or a methyl group; R.sup.3 is a hydrogen atom or a
straight or branched alkyl group; Ar is an optionally substituted
aromatic group; and the salts, solvates, hydrates and N-oxides
thereof.
3. A compound according to claim 2 wherein Ar is an optionally
substituted phenyl group.
4. A compound according to claim 2 wherein R is a --CO.sub.2H
group.
5. A compound according claim 2 wherein Alk.sup.2 is a --CH.sub.2--
chain and m is an integer 1.
6. A compound according to claim 2 wherein each of R.sup.2 and
R.sup.3 is a hydrogen atom.
7. A compound according to claim 2 wherein R.sup.1 is an optionally
substituted aromatic or heteroaromatic group.
8. A compound according to claim 7 wherein R.sup.1 is an optionally
substituted phenyl, pyridyl or pyrimidinyl group.
9. A compound according to claim 2 wherein
-(Alk.sup.1).sub.r(L.sup.1).sub- .s is a --CH.sub.2O--,
--SO.sub.2NH--, --C(O)O-- or --CON(R.sup.4)-- group.
10. A compound according to claim 9 wherein
-(Alk.sup.1).sub.r(L.sup.1).su- b.s is a --CONH-- group.
11. A compound according to claim 2 which has the formula (1b):
9wherein --W.dbd. is --CH.dbd. or --N.dbd., R.sup.9 and R.sup.10,
which may be the same or different is each a
-L.sup.2(CH.sub.2).sub.pL.sup.3(R.sup.c).sub.- q atom or group, and
the salts, solvates, hydrates and N-oxides thereof.
12. A compound according to claim 11 wherein Ar is an optionally
substituted phenyl group.
13. A compound which is:
(N-2,6-Dimethoxybenzoyl)-[O-(3,5-dichloro-4-pyrid-
inyl)methyl]-L-tyrosine;
2-Carboxybenzoyl-(N'-3,5-dichloroisonicotinoyl)-L-
-4-aminophenylalanine;
(N-2,6-Dimethoxybenzoyl)-(N'-3,5-dichloroisonicotin-
oyl)-L-4-aminophenylalanine;
(N-3-Carboxybenzoyl)-(N'-3,5-dichloroisonicot-
oninoyl)-L-4-aminophenyalanine;
(N-4-Carboxybenzoyl)-(N'-3,5-dichloroisoni-
cotinoyl)-L-4-aminophenylalanine;
(N-2-t-Butoxycarbonylbenzoyl)-(N'-3,5-di-
chloroisonicotinoyl)-L-4-aminophenylalanine;
(N-3-Cyanobenzoyl)-(N'-3,5-di-
chloroisonicotinoyl)-L-4-aminophenylalanine;
[N-3-(1-H-Tetrazol-5-yl)]-(N'-
3,5-dichloroisonicotinoyl)-L-4-aminophenylalanine;
[N-(3-Methoxycarbonylbe-
nzoyl)]-(N'-3,5-dichloroisonicotinoyl)-L-4-aminophenylalnine; and
the salts, solvates, hydrates and N-oxides thereof.
14. A pharmaceutical composition comprising a compound according to
claim 1 together with one or more pharmaceutically acceptable
carriers, excipients or diluents.
Description
[0001] This invention relates to a series of phenylalanine
derivatives, to compositions containing them, to processes for
their preparation, and to their use in medicine.
[0002] Over the last few years it has become increasingly clear
that the physical interaction of inflammatory leukocytes with each
other and other cells of the body plays an important role in
regulating immune and inflammatory responses [Springer, T A.
Nature, 346. 425, (1990); Springer, T. A. Cell 76, 301, (1994)].
Many of these interactions are mediated by specific cell surface
molecules collectively referred to as cell adhesion molecules.
[0003] The adhesion molecules have been sub-divided into different
groups on the basis of their structure. One family of adhesion
molecules which is believed to play a particularly important role
in regulating immune and inflammatory responses is the integrin
family. This family of cell surface glycoproteins has a typical
non-covalently linked heterodimer structure. At least 14 different
integrin alpha chains and 8 different integrin beta chains have
been identified [Sonnenberg, A Current Topics in Microbiology and
Immunology, 184, 7, (1993)]. The members of the family are
typically named according to their heterodimer composition although
trivial nomenclature is widespread in this field. Thus the integrin
termed .alpha..sub.4.beta..sub.1 consists of the integrin alpha 4
chain associated with the integrin beta 1 chain, but is also widely
referred to as Very Late Antigen 4 or VLA4. Not all of the
potential pairings of integrin alpha and beta chains have yet been
observed in nature and the integrin family has been subdivided into
a number of subgroups based on the pairings that have been
recognised [Sonnenberg, A. ibid].
[0004] The importance of cell adhesion molecules in human leukocyte
function has been further highlighted by a genetic deficiency
disease called Leukocyte Adhesion Deficiency (LAD) in which one of
the families of leukocyte integrins is not expressed [Marlin, S. D.
et al J. Exp. Med. 164, 855 (1986)]. Patients with this disease
have a reduced ability to recruit leukocytes to inflammatory sites
and suffer recurrent infections which in extreme cases may be
fatal.
[0005] The potential to modify adhesion molecule function in such a
way as to beneficially modulate immune and inflammatory responses
has been extensively investigated in animal models using specific
monoclonal antibodies that block various functions of these
molecules [e.g. Issekutz, T. B. J. Immunol. 3394, (1992); Li, Z. et
al Am. J. Physiol. 263, L723, (1992); Binns, R. M. et al J.
Immunol. 157, 4094, (1996)]. A number of monoclonal antibodies
which block adhesion molecule function are currently being
investigated for their therapeutic potential in human disease.
[0006] One particular integrin subgroup of interest involves the
.alpha.4 chain which can pair with two different beta chains
.beta.1 and .beta.7 [Sonnenberg, A. ibid]. The
.alpha..sub.4.beta..sub.1 pairing occurs on many circulating
leukocytes (for example lymphocytes, monocytes and eosinophils)
although it is absent or only present at low levels on circulating
neutrophils. .alpha..sub.4.beta..sub.1 binds to an adhesion
molecule (Vascular Cell Adhesion Molecule-1 also known as VCAM-1)
frequently up-regulated on endothelial cells at sites of
inflammation [Osborne, L. Cell, 62, 3, (1990)]. The molecule has
also been shown to bind to at least three sites in the matrix
molecule fibronectin [Humphries, M. J. et al. Ciba Foundation
Symposium, 189, 177, (1995)]. Based on data obtained with
monoclonal antibodies in animal models it is believed that the
interaction between .alpha..sub.4.beta..sub.1 and ligands on other
cells and the extracellular matrix plays an important role in
leukocyte migration and activation [Yednock, T. A. et al, Nature,
356, 63, (1992); Podolsky, D. K. et al. J. Clin. Invest. 92, 373,
(1993); Abraham, W. M. et al. J. Clin. Invest. 93, 776,
(1994)].
[0007] The integrin generated by the pairing of .alpha.4 and
.beta.7 has been termed LPAM-1 [Holzmann, B and Weissman, I. EMBO
J. 8, 1735, (1989)] and like .alpha..sub.4.beta..sub.1, binds to
VCAM-1 and fibronectin. In addition, .alpha..sub.4.beta.7 binds to
an adhesion molecule believed to be involved in the homing of
leukocytes to mucosal tissue termed MAdCAM-1 [Berlin, C. et al,
Cell, 74, 185, (1993)]. The interaction between
.alpha..sub.4.beta..sub.7 and MAdCAM-1 may also be important at
sites of inflammation outside of mucosal tissue [Yang, X-D. et al,
PNAS, 91, 12604 (1994)].
[0008] Regions of the peptide sequence recognised by
.alpha..sub.4.beta..sub.1 and .alpha..sub.4.beta..sub.7 when they
bind to their ligands have been identified.
.alpha..sub.4.beta..sub.1 seems to recognise LDV, IDA or REDV
peptide sequences in fibronectin and a QIDSP sequence in VCAM-1
[Humphries, M. J. et al, ibid] whilst .alpha..sub.4.beta..sub.7
recognises a LDT sequence in MAdCAM-1 [Briskin, M. J. et al, J.
Immunol. 156, 719, (1996)]. There have been several reports of
inhibitors of these interactions being designed from modifications
of these short peptide sequences [Cardarelli, P. M. et al J. Biol.
Chem. 269, 18668, (1994); Shroff, H. N. Bioorganic. Med. Chem.
Lett. 6, 2495, (1996); Vanderslice, P. J. Immunol. 158, 1710,
(1997)]. It has also been reported that a short peptide sequence
derived from the .alpha..sub.4.beta..sub.1 binding site in
fibronectin can inhibit a contact hypersensitivity reaction in a
trinitrochlorobenzene sensitised mouse [Ferguson, T. A. et al, PNAS
88, 8072, (1991)].
[0009] Since the alpha 4 subgroup of integrins are predominantly
expressed on leukocytes their inhibition can be expected to be
beneficial in a number of immune or inflammatory disease states.
However, because of the ubiquitous distribution and wide range of
functions performed by other members of the integrin family it is
very important to be able to identify selective inhibitors of the
alpha 4 subgroup.
[0010] We have now found a group of compounds which are potent and
selective inhibitors of .alpha.4 integrins. Members of the group
are able to inhibit .alpha.4 integrins such as
.alpha..sub.4.beta..sub.1 and/or .alpha..sub.4.beta..sub.7 at
concentrations at which they generally have no or minimal
inhibitory action on a integrins of other subgroups. The compounds
are thus of use in medicine, for example in the prophylaxis and
treatment of immune or inflammatory disorders as described
hereinafter.
[0011] Thus according to one aspect of the invention we provide a
compound of formula (1) 2
[0012] wherein
[0013] R is a carboxylic acid or a derivative thereof;
[0014] R.sup.1 is a hydrogen atom or a hydroxyl, straight or
branched alkoxy or optionally substituted cycloaliphatic,
polycycloaliphatic, heterocycloaliphatic, polyheterocycloaliphatic,
aromatic or heteroaromatic group;
[0015] Alk.sup.1 is an optionally substituted aliphatic or
heteroaliphatic chain;
[0016] L.sup.1 is a linker atom or group;
[0017] r and s, which may be the same or different, is each zero or
an integer 1 provided that when r is zero R.sup.1 is an optionally
substituted cycloaliphatic, polycycloaliphatic,
polyheterocycloaliphatic, aromatic or heteroaromatic group;
[0018] R.sup.a and R.sup.b, which may be the same or different is
each an atom or group
-L.sup.2(CH.sub.2).sub.pL.sup.3(R.sup.c).sub.q in which L.sup.2 and
L.sup.3 is each a covalent bond or a linker atom or group, p is
zero or the integer 1, q is an integer 1, 2 or 3 and R.sup.c is a
hydrogen or halogen atom or a group selected from straight or
branched alkyl, -OR.sup.d [where R.sup.d is a hydrogen atom or an
optionally substituted straight or branched alkyl group],
--SR.sup.d, --NR.sup.dR.sup.e, [where R.sup.e is as just defined
for R.sup.d and may be the same or different], --NO.sub.2, --CN,
--CO.sub.2R.sup.d, --SO.sub.3H, --SO.sub.2R.sup.d,
--OCO.sub.2R.sup.d, --CONR.sup.dR.sup.e, --OCONR.sup.dR.sup.e,
--CSNR.sup.dR.sup.e, --COR.sup.d, --N(R.sup.d)COR.sup.e,
N(R.sup.d)CSR.sup.e, --SO.sub.2N(R.sup.d)(R.sup.e)- ,
--N(R.sup.d)SO.sub.2R.sup.e, --N(R.sup.d)CONR.sup.eR.sup.f [where
R.sup.f is a hydrogen atom or an optionally substituted straight or
branched alkyl group], --N(R.sup.d)CSNR.sup.eR.sup.f or
--N(R.sup.d)SO.sub.2NR.sup.eR.sup.f;
[0019] Alk.sup.2 is a straight or branched alkylene chain;
[0020] m is zero or an integer 1;
[0021] R.sup.2 is a hydrogen atom or a methyl group;
[0022] R.sup.3 is a hydrogen atom or a straight or branched alkyl
group;
[0023] Ar is an optionally substituted aromatic group;
[0024] and the salts, solvates, hydrates and N-oxides thereof, for
use in modulating cell adhesion.
[0025] The compounds of formula (1) are potent and selective
inhibitors of .alpha.4 integrins. The ability of the compounds to
act in this way may be simply determined by employing tests such as
those described in the Examples hereinafter. In particular
compounds of the invention are advantageously selective
.alpha..sub.4.beta..sub.1 inhibitors
[0026] The compounds of formula (1) are thus of use in modulating
cell adhesion and in particular are of use in the prophylaxis and
treatment of diseases or disorders involving inflammation in which
the extravasation of leukocytes plays a role. The invention extends
to such a use and to the use of compounds of formula (1) for the
manufacture of a medicament for treating such diseases or
disorders. Diseases or disorders of this type include inflammatory
arthritis such as rheumatoid arthritis vasculitis or
polydermatomyositis, multiple sclerosis, allograft rejection,
diabetes, inflammatory dermatoses such as psoriasis or dermatitis,
asthma and inflammatory bowel disease.
[0027] For the prophylaxis or treatment of disease the compounds of
formula (1) may be administered as pharmaceutical compositions, and
according to a further aspect of the invention we provide a
pharmaceutical composition which comprises a compound of formula
(1) together with one or more pharmaceutically acceptable carriers,
excipients or diluents, for use in modulating cell adhesion,
particularly in the prophylaxis and treatment of diseases or
disorders involving inflammation as just described.
[0028] Pharmaceutical compositions for use according to the
invention may take a form suitable for oral, buccal, parenteral,
nasal, topical or rectal administration, or a form suitable for
administration by inhalation or insufflation and the invention
extends to the use of a compound of formula (1) in the manufacture
of such formulations.
[0029] For oral administration, the pharmaceutical compositions may
take the form of, for example, tablets, lozenges or capsules
prepared by conventional means with pharmaceutically acceptable
excipients such as binding agents (e.g. pregelatinised maize
starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose);
fillers (e.g. lactose, microcrystalline cellulose or calcium
hydrogen phosphate); lubricants (e.g. magnesium stearate, talc or
silica); disintegrants (e.g. potato starch or sodium glycollate);
or wetting agents (e.g. sodium lauryl sulphate). The tablets may be
coated by methods well known in the art. Liquid preparations for
oral administration may take the form of, for example, solutions,
syrups or suspensions, or they may be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents,
emulsifying agents, non-aqueous vehicles and preservatives. The
preparations may also contain buffer salts, flavouring, colouring
and sweetening agents as appropriate.
[0030] Preparations for oral administration may be suitably
formulated to give controlled release of the active compound.
[0031] For buccal administration the compositions may take the form
of tablets or lozenges formulated in conventional manner.
[0032] The compounds for formula (1) may be formulated for
parenteral administration by injection e.g. by bolus injection or
infusion. Formulations for injection may be presented in unit
dosage form, e.g. in glass ampoule or multi dose containers, e.g.
glass vials. The compositions for injection may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilising,
preserving and/or dispersing agents. Alternatively, the active
ingredient may be in powder form for constitution with a suitable
vehicle, e.g. sterile pyrogen-free water, before use.
[0033] In addition to the formulations described above, the
compounds of formula (1) may also be formulated as a depot
preparation. Such long acting formulations may be administered by
implantation or by intramuscular injection.
[0034] For nasal administration or administration by inhalation,
the compounds for use according to the present invention are
conveniently delivered in the form of an aerosol spray presentation
for pressurised packs or a nebuliser, with the use of suitable
propellant, e.g. dichlorodifluoromethane, trichloro-fluoromethane,
dichlorotetrafluoroetha- ne, carbon dioxide or other suitable gas
or mixture of gases.
[0035] The compositions may, if-desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack or dispensing device may
be accompanied by instructions for administration.
[0036] The quantity of a compound of formula (1) required for the
prophylaxis or treatment of a particular condition will vary
depending on the compound chosen, and the condition of the patient
to be treated. In general, however, effective daily dosages may
range from around 100 ng/kg to 100 mg/kg e.g. around 0.01 mg/kg to
40 mg/kg body weight for oral or buccal administration, from around
10 ng/kg to 50 mg/kg body weight for parenteral administration and
around 0.05 mg to around 1000 mg e.g. around 0.5 mg to around 1000
mg for nasal administration or administration by inhalation or
insufflation.
[0037] Particular compounds of formula (1) form a further feature
of the invention and in a further aspect we therefore provide a
compound of formula (1a): 3
[0038] wherein
[0039] R is a carboxylic acid or a derivative thereof;
[0040] R.sup.1 is an optionally substituted cycloaliphatic,
polycycloaliphatic, heterocycloaliphatic, polyheterocyclialiphatic,
aromatic or heteroaromatic group;
[0041] Alk.sup.1 is an optionally substituted aliphatic or
heteroaliphatic chain;
[0042] L.sup.1 is a linker atom or group;
[0043] r and s, which may be the same or different, is each zero or
an integer 1;
[0044] R.sup.a and R.sup.b, which may be the same or different is
each an atom or group
-L.sup.2(CH.sub.2).sub.pL.sup.3(R.sup.c).sub.q in which L.sup.2 and
L.sup.3 is each a covalent bond or a linker atom or group, p is
zero or the integer 1, q is an integer 1, 2 or 3 and R.sup.c is a
hydrogen or halogen atom or a group selected from straight or
branched alkyl, OR.sup.d [where R.sup.d is a hydrogen atom or an
optionally substituted straight or branched alkyl group],
--SR.sup.d, --NR.sup.dR.sup.e, [where R.sup.e is as just defined
for R.sup.d and may be the same or different], --NO.sub.2, --CN,
--CO.sub.2R.sup.d, --SO.sub.3H, --SO.sub.2R.sup.d,
--OCO.sub.2R.sup.d, --CONR.sup.dR.sup.e, --OCONR.sup.dR.sup.e,
--CSNR.sup.dR.sup.e, --COR.sup.d, --N(R.sup.d)COR.sup.e,
N(R.sup.d)CSR.sup.e, --SO.sub.2N(R.sup.d)(R.sup.e)- ,
--N(R.sup.d)SO.sub.2R.sup.e, --N(R.sup.d)CONR.sup.eR.sup.f [where
R.sup.f is a hydrogen atom or an optionally substituted straight or
branched alkyl group], --N(R.sup.d)CSNR.sup.eR.sup.f or
--N(R.sup.d)SO.sub.2NR.sup.eR.sup.f;
[0045] Alk.sup.2 is a straight or branched alkylene chain;
[0046] m is zero or an integer 1;
[0047] R.sup.2 is a hydrogen atom or a methyl group;
[0048] R.sup.3 is a hydrogen atom or a straight or branched alkyl
group;
[0049] Ar is an optionally substituted aromatic group;
[0050] and the salts, solvates, hydrates and N-oxides thereof.
[0051] It will be appreciated that compounds of formulae (1) and
(1a) may have one or more chiral centres. Where one or more chiral
centres is present, enantiomers or diastereomers may exist, and the
invention is to be understood to extend to all such enantiomers,
diasteromers and mixtures thereof, including racemates. Formulae
(1) and (1a) and the formulae hereinafter are intended to represent
all individual isomers and mixtures thereof, unless stated or shown
otherwise.
[0052] In the compounds of formulae (1) and (1a), derivatives of
the carboxylic acid group R include carboxylic acid esters and
amides. Particular esters and amides include --CO.sub.2Alk.sup.4
and --CON(R.sup.4).sub.2 groups as described herein.
[0053] When in the compounds of formulae (1) and (1a) L.sup.1 is
present as a linker atom or group it may be any divalent linking
atom or group. Particular examples include --O-- or --S-- atoms or
--C(O)--, --C(O)O--, --C(S)--, --S(O)--, --S(O).sub.2--,
--N(R.sup.4)-- [where R.sup.4 is a hydrogen atom or a straight or
branched alkyl group], --CON(R.sup.4)--, --OC(O)N(R.sup.4)--,
--CSN(R.sup.4)--, --N(R.sup.4)CO--, --N(R.sup.4)C(O)O--,
--N(R.sup.4)CS--, --S(O)N(R.sup.4)--, --S(O).sub.2N(R.sup.4)--,
--N(R.sup.4)S(O)--, --N(R.sup.4)S(O).sub.2--,
--N(R.sup.4)CON(R.sup.4)--, --N(R.sup.4)CSN(R.sup.4)--,
--N(R.sup.4)SON(R.sup.4)-- or --N(R.sup.4)SO.sub.2N(R.sup.4)--
groups. Where the linker group contains two R.sup.4 substituents,
these may be the same or different.
[0054] Alk.sup.2 in the compounds of formulae (1) and (1a) may be
for example a straight or branched C.sub.1-3alkylene chain.
Particular examples include --CH.sub.2--, --CH(CH.sub.3)--,
--C(CH.sub.3).sub.2-- and --(CH.sub.2).sub.2--.
[0055] When R.sup.3 and/or R.sup.4 in the compounds of formula (1)
is a straight or branched alkyl group it may be a straight or
branched C.sub.1-6 alkyl group, e.g. a C.sub.1-3 alkyl group such
as a methyl or ethyl group.
[0056] When Alk.sup.1 in compounds of formula (1) is an optionally
substituted aliphatic chain it may be an optionally substituted
C.sub.110 aliphatic chain. Particular examples include optionally
substituted straight or branched C.sub.1-6 alkylene, C.sub.2-6
alkenylene, or C.sub.2-6 alkynylene chains.
[0057] Heteroaliphatic chains represented by Alk.sup.1 include the
aliphatic chains just described but with each chain additionally
containing one, two, three or four heteroatoms or
heteroatom-containing groups. Particular heteroatoms or groups
include atoms or groups L.sup.4 where L.sup.4 is as defined above
for L.sup.1 when L.sup.1 is a linker atom or group. Each L.sup.4
atom or group may interrupt the aliphatic chain, or may be
positioned at its terminal carbon atom to connect the chain to the
atom or group R.sup.1.
[0058] Particular examples of aliphatic chains represented by
Alk.sup.1 include optionally substituted --CH.sub.2--,
--CH.sub.2CH.sub.2--, --CH(CH.sub.3)--, --C(CH.sub.3).sub.2--,
--(CH.sub.2).sub.2CH.sub.2--, --CH(CH.sub.3)CH.sub.2--,
--(CH.sub.2).sub.3CH.sub.2--, --CH(CH.sub.3)CH.sub.2CH.sub.2--,
--CH.sub.2CH(CH.sub.3)CH.sub.2--, --C(CH.sub.3).sub.2CH.sub.2--,
--(CH.sub.2).sub.4CH.sub.2--, --(CH.sub.2).sub.5CH.sub.2--,
--CHCH--, --CHCHCH.sub.2--, --CH.sub.2CHCH--,
--CHCHCH.sub.2CH.sub.2--, --CH.sub.2CHCHCH.sub.2--,
--(CH.sub.2).sub.2CHCH--, --CC--, --CCCH.sub.2--, --CH.sub.2CC--,
--CCCH.sub.2CH.sub.2--, --CH.sub.2CCCH.sub.2--, or
--(CH.sub.2).sub.2CC-- chains. Where appropriate each of said
chains may be optionally interrupted by one or two atoms and/or
groups L.sup.4 to form an optionally substituted heteroaliphatic
chain. Particular examples include optionally substituted
-L.sup.4CH.sub.2--, --CH.sub.2L.sup.4CH.sub.2--,
-L.sup.4(CH.sub.2).sub.2--, --CH.sub.2L.sup.4(CH.sub.2).sub.2--,
--(CH.sub.2).sub.2L.sup.4CH.sub.2--, -L.sup.4(CH.sub.2).sub.3-- and
--(CH.sub.2).sub.2L.sup.4(CH.sub.2).sub.2-- chains.
[0059] The optional substituents which may be present on aliphatic
or heteroaliphatic chains represented by Alk.sup.1 include one,
two, three or more substituents selected from halogen atoms, e.g.
fluorine, chlorine, bromine or iodine atoms, or hydroxyl,
C.sub.1-6alkoxy, e.g. methoxy or ethoxy, thiol, C.sub.1-6alkylthio
e.g. methylthio or ethylthio, amino or substituted amino groups.
Substituted amino groups include --NHR.sup.4 and --N(R.sup.4).sub.2
groups where R.sup.4 is a straight or branched alkyl group as
defined above. Where two R.sup.4 groups are present these may be
the same or different. Particular examples of substituted chains
represented by Alk.sup.1 include those specific chains just
described substituted by one, two, or three halogen atoms such as
fluorine atoms, for example chains of the type --CH(CF.sub.3)--,
--C(CF.sub.3).sub.2--CH.sub.2CH(CF.sub.3)--,
--CH.sub.2C(CF.sub.3).sub.2--, --CH(CF.sub.3)-- and
--C(CF.sub.3).sub.2CH.sub.2.
[0060] Alkoxy groups represented by R.sup.1 in compounds of formula
(1) include straight of branched C.sub.1-6alkoxy groups such as
methoxy and ethoxy groups.
[0061] When R.sup.1 is present in compounds of formulae (1) and
(1a) as an optionally substituted cycloaliphatic group it may be an
optionally substituted C.sub.3-10 cycloaliphatic group. Particular
examples include optionally substituted C.sub.3-10cycloalkyl, e.g.
C.sub.3-7cycloalkyl, C.sub.3-10cycloalkenyl e.g.
C.sub.3-7cycloalkenyl or C.sub.3-10cycloalkynyl e.g.
C.sub.3-7cycloalkynyl groups.
[0062] Optionally substituted heterocycloaliphatic groups
represented by R.sup.1 include the optionally substituted
cycloaliphatic groups just described for R.sup.1 but with each
group additionally containing one, two, three or four heteroatoms
or heteroatom-containing groups L.sup.2 as just defined.
[0063] Optionally substituted polycycloaliphatic groups represented
by R.sup.1 include optionally substitued C.sub.7-10 bi- or
tricycloalkyl or C.sub.7-10bi- or tricycloalkenyl groups.
Optionally substituted polyheterocycloaliphatic groups represented
by R.sup.1 include the optionally substituted polycycloalkyl groups
just described, but with each group additionally containing one,
two, three or four L.sup.2 atoms or groups.
[0064] Particular examples of R.sup.1 cycloaliphatic,
polycycloaliphatic, heterocycloaliphatic and
polyheterocycloaliphatic groups include optionally substituted
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,
2-cyclobuten-1-yl, 2-cyclopenten-1-yl, 3-cyclopenten-1-yl,
adamantyl, norbornyl, norbornenyl, pyrroline, e.g. 2- or
3-pyrrolinyl, pyrrolidinyl, pyrrolidinone, oxazolidinyl,
oxazolidinone, dioxolanyl, e.g. 1,3-dioxolanyl, imidazolinyl, e.g.
2-imidazolinyl, imidazolidinyl, pyrazolinyl, e.g. 2-pyrazolinyl,
pyrazolidinyl, thiazolinyl, thiazolidinyl, pyranyl, e.g. 2- or
4-pyranyl, piperidinyl, piperidinone, 1,4-dioxanyl, morpholinyl,
morpholinone, 1,4-dithianyl, thiomorpholinyl, piperazinyl,
1,3,5-trithianyl, oxazinyl, e.g. 2H-1,3-, 6H-1,3-, 6H-1,2-, 2H-1,2-
or 4H-1,4-oxazinyl, 1,2,5-oxathiazinyl, isoxazinyl, e.g. o- or
p-isoxazinyl, oxathiazinyl, e.g. 1,2,5 or 1,2,6-oxathiazinyl, or
oxadiazinyl e.g. 1,3,5-oxodiazinyl groups.
[0065] The optional substituents which may be present on the
R.sup.1 cycloaliphatic, polycycloaliphatic, heterocycloaliphatic or
polyheterocycloaliphatic groups include one, two, three or more
substituents represented by R.sup.5 in which R.sup.5 is selected
from halogen atoms, e.g. fluorine, chlorine, bromine or iodine
atoms, or C.sub.1-6alkyl, e.g. methyl or ethyl, haloC.sub.1-6alkyl,
e.g. halomethyl or haloethyl such as difluoromethyl or
trifluoromethyl, hydroxyl, C.sub.1-6alkoxy, e.g. methoxy or ethoxy,
haloC.sub.1-6alkoxy, e.g. halomethoxy or haloethoxy such as
difluoromethoxy or trifluoromethoxy, thiol, C.sub.1-6alkylthio e.g.
methylthio or ethylthio, --N(R.sup.4).sub.2, --CN,
--CO.sub.2R.sup.4, --NO.sub.2, --CON(R.sup.4).sub.2,
--CSN(R.sup.4).sub.2, --COR.sup.4, --CSN(R.sup.4).sub.2,
--N(R.sup.4)COR.sup.4, --N(R.sup.4)CSR.sup.4,
--SO.sub.2N(R.sup.4).sub.2, --N(R.sup.4)SO.sub.2R.sup.4,
--N(R.sup.4)CON(R.sup.4).sub.2, --N(R.sup.4)CSN(R.sup.4) and
--N(R.sup.4)SO.sub.2N(R.sup.4).sub.2 groups. In these substituents
the group R.sup.4 when present is a hydrogen atom or a straight or
branched alkyl group as defined above. Where more than one R.sup.4
group is present in a substituent each group may be the same or
different. The substituent may be present on any available carbon
atom or where appropriate any nitrogen atom, in the R.sup.1
group.
[0066] In the compounds of formulae (1) and (1 a), optionally
substituted aromatic groups represented by the group R.sup.1
include for example monocyclic or bicyclic fused ring C.sub.6-12
aromatic groups, such as phenyl, 1- or 2-naphthyl, 1- or
2-tetrahydronaphthyl, indanyl or indenyl groups, optionally
substituted by one, two, three or more
L.sup.2(CH.sub.2).sub.pL.sup.3(R.sup.c).sub.q atoms or groups,
where L.sup.2, L.sup.3, p and q are as previously defined and
R.sup.c is as previously defined but is other than a hydrogen atom
when L.sup.2 and L.sup.3 is each a covalent bond and p is zero.
[0067] Optionally substituted heteroaromatic groups, represented by
the group R.sup.1 in compounds of formulae (1) and (1a) include for
example optionally substituted C.sub.1-9 heteroaromatic groups
containing for example one, two, three or four heteroatoms selected
from oxygen, sulphur or nitrogen atoms. In general, the
heteroaromatic groups may be for example monocyclic or bicyclic
fused ring heteroaromatic groups. Monocyclic heteroaromatic groups
include for example five- or six-membered heteroaromatic groups
containing one, two, three or four heteroatoms selected from
oxygen, sulphur or nitrogen atoms. Bicyclic heteroaromatic groups
include for example nine- to thirteen-membered fused-ring
heteroaromatic groups containing one, two or more heteroatoms
selected from oxygen, sulphur or nitrogen atoms.
[0068] Particular examples of heteroaromatic groups of these types
include optionally substituted pyrrolyl, furyl, thienyl,
imidazolyl, N--C.sub.1-6aimidazolyl, oxazolyl, isoxazolyl,
thiazolyl, isothiazolyl, pyrazolyl, 1,2,3-triazolyl,
1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl,
1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,3,4-thiadiazole, pyridyl,
pyrimidinyl, pyridazinyl, pyrazinyl, 1,3,5-triazinyl,
1,2,4-triazinyl, 1,2,3-triazinyl, benzofuryl,
[2,3-dihydro]-benzofuryl, benzothienyl, benzotriazolyl, indolyl,
isoindolyl, benzimidazolyl, imidazo[1,2-a]pyridyl, benzothiazolyl,
benzoxazolyl, benzopyranyl, [3,4-dihydro]benzopyranyl,
quinazolinyl, naphthyridinyl, pyrido[3,4-b]pyridyl,
pyrido[3,2-b]pyridyl, pyrido[4,3-b]pyridyl, quinolinyl,
isoquinolinyl, tetrazolyl, 5,6,7,8-tetrahydroquinolinyl,
5,6,7,8-tetrahydroisoquinolinyl, and imidyl, e.g. succinimidyl,
phthalimidyl, or naphthalimidyl such as 1,8-naphthalimidyl.
[0069] Optional substituents which may be present on R.sup.1
heteroaromatic groups include one, two, three or more
-L.sup.2(CH.sub.2).sub.pL.sup.3(R.sup.c).sub.q atoms or groups as
just defined.
[0070] Examples of the substituents represented by R.sup.a and
R.sup.b in compounds of formula (1) and which may be present on
aromatic or heteroaromatic groups represented by R.sup.1 include
atoms or groups -L.sup.2(CH.sub.2).sub.pLR.sup.c,
-L.sup.2(CH.sub.2).sub.pR.sup.c, -L.sup.2R.sup.c,
--(CH.sub.2).sub.pR.sup.c and R.sup.c wherein L.sup.2,
(CH.sub.2).sub.p, L and R.sup.c are as defined above. Particular
examples of such substituents include
L.sup.2CH.sub.2L.sup.2R.sup.c, -L.sup.2CH(CH.sub.3)L.sup.3R.sup.c,
L.sup.2(CH.sub.2).sub.2L.sup.3R.sup.c- , L.sup.2CH.sub.2R.sup.c,
L.sup.2CH(CH.sub.3)R.sup.c, -L.sup.2(CH.sub.2).sub.2R.sup.c,
--CH.sub.2R.sup.c, --CH(CH.sub.3)R.sup.c and
--(CH.sub.2).sub.2R.sup.c groups.
[0071] Thus each of R.sup.a and R.sup.b and, where present,
substituents on R.sup.1 and aromatic or heteroaromatic groups in
compounds of the invention may be for example selected from a
hydrogen atom, a halogen atom, e.g. a fluorine, chlorine, bromine
or iodine atom, or a C.sub.1-6alkyl, e.g. methyl, ethyl, n-propyl,
i-propyl, n-butyl or t-butyl, C.sub.1-6alkylamino, e.g. methylamino
or ethylamino, C.sub.1-6hydroxyalkyl, e.g. hydroxymethyl,
hydroxyethyl or --C(OH)(CF.sub.3).sub.2, carboxyC.sub.1-6alkyl,
e.g. carboxyethyl, C.sub.1-6alkylthio e.g. methylthio or ethylthio,
carboxyC.sub.1-6alkylthi- o, e.g. carboxymethylthio,
2-carboxyethylthio or 3-carboxypropylthio, C.sub.1-6alkoxy, e.g.
methoxy or ethoxy, hydroxyC.sub.1-6alkoxy, e.g. 2-hydroxyethoxy,
haloC.sub.1-6alkyl, e.g. --CF.sub.3, --CHF.sub.2, CH.sub.2F,
haloC.sub.1-6alkoxy, e.g. --OCF.sub.3, --OCHF.sub.2, --OCH.sub.2F,
C.sub.1-6alkylamino, e.g. methylamino or ethylamino, amino
(--NH.sub.2), aminoC.sub.1-6alkyl, e.g. aminomethyl or aminoethyl,
C.sub.1-6dialkylamino, e.g. dimethylamino or diethylamino,
C.sub.1-6alkylaminoC.sub.1-6alkyl, e.g. ethylaminoethyl,
C.sub.1-6dialkylaminoC.sub.1-6alkyl, e.g. diethylaminoethyl,
aminoC.sub.1-6alkoxy, e.g. aminoethoxy,
C.sub.1-6alkylaminoC.sub.1-6alkox- y, e.g. methylaminoethoxy,
C.sub.1-6dialkylaminoC.sub.1-6alkoxy, e.g. dimethylaminoethoxy,
diethylaminoethoxy, isopropylaminoethoxy, or dimethylaminopropoxy,
nitro, cyano, amidino, hydroxyl (--OH), formyl [HC(O)--], carboxyl
(--CO.sub.2H), --CO.sub.2R.sup.12, C.sub.1-6 alkanoyl e.g. acetyl,
thiol (--SH), thioC.sub.1-6alkyl, e.g. thiomethyl or thioethyl,
sulphonyl (--SO.sub.3H), C.sub.1-6alkylsulphonyl, e.g.
methylsulphonyl, aminosulphonyl (--SO.sub.2NH.sub.2),
C.sub.1-6alkylaminosulphonyl, e.g. methylaminosulphonyl or
ethylaminosulphonyl, C.sub.1-6dialkylaminosulphonyl, e.g.
dimethylaminosulphonyl or diethylaminosulphonyl,
phenylaminosulphonyl, carboxamido (--CONH.sub.2),
C.sub.1-6alkylaminocarbonyl, e.g. methylamino-carbonyl or
ethylaminocarbonyl, C.sub.1-6dialkylaminocarbonyl- , e.g.
dimethylaminocarbonyl or diethylaminocarbonyl,
aminoC.sub.1-6alkylaminocarbonyl, e.g. aminoethylaminocarbonyl,
C.sub.1-6dialkylaminoC.sub.1-6alkylaminocarbonyl, e.g.
diethylaminoethylaminocarbonyl, aminocarbonylamino,
C.sub.1-6alkylaminocarbonylamino, e.g. methylaminocarbonylamino or
ethylamino-carbonylamino, C.sub.1-6dialkylaminocarbonylamino, e.g.
dimethylamino-carbonylamino or diethylaminocarbonylamino,
C.sub.1-6alkylaminocabonylC.sub.1-6alkylamino, e.g.
methylaminocarbonylmethylamino, aminothiocarbonyl-amino,
C.sub.1-6alkylaminothiocarbonylamino, e.g.
methylaminothiocarbonyl-amino or ethylaminothiocarbonylamino,
C.sub.1-6dialkylaminothiocarbonyl-amino, e.g.
dimethylaminothiocarbonylamino or diethylaminothiocarbonyl-amino,
C.sub.1-6alkylaminothiocarbonylC.sub.1-6alkylamino, e.g.
ethylaminothio-carbonylmethylamino, C.sub.1-6alkylsulphonylamino,
e.g. methylsulphonylamino or ethylsulphonylamino,
C.sub.1-6dialkylsulphonylami- no, e.g. dimethylsulphonyl-amino or
diethylsulphonylamino, aminosulphonylamino (--NHSO.sub.2NH.sub.2),
C.sub.1-6alkylaminosulphonyla- mino, e.g.
methylamino-sulphonylamino or ethylaminosulphonylamino,
C.sub.1-6dialkylaminosulphonylamino, e.g.
dimethylaminosulphonylamino or diethylaminosulphonylamino,
C.sub.1-6alkanoylamino, e.g. acetylamino,
aminoC.sub.1-6alkanoylamino e.g. aminoacetylamino,
C.sub.1-6dialkylaminoC.sub.1-6alkanoylamino, e.g.
dimethylaminoacetylamin- o, C.sub.1-6alkanoylaminoC.sub.1-6alkyl,
e.g. acetylaminomethyl, C.sub.1-6alkanoylaminoC.sub.1-6alkylamino,
e.g. acetamidoethylamino, C.sub.1-6alkoxycarbonylamino, e.g.
methoxycarbonylamino, ethoxycarbonylamino or t-butoxycarbonylamino
group.
[0072] Aromatic groups represented by the group Ar in compounds of
formulae (1) and (1a) include optionally substituted monocyclic of
bicyclic fused ring C.sub.16 aromatic groups. Particular examples
include optionally substituted phenyl, 1- or 2-naphthyl, 1- or
2-tetrahydronaphthyl, indanyl or indenyl groups.
[0073] Optional substituents present on the aromatic groups
represented by Ar include one, two, three or more substituents,
each selected from an atom or group R.sup.6 in which R.sup.6 is
--R.sup.6a or -Alk.sup.3(R.sup.6a).sub.m, where R.sup.6a is a
halogen atom, or an amino (--NH.sub.2), substituted amino, nitro,
cyano, amidino, hydroxyl (--OH), substituted hydroxyl, formyl,
carboxyl (--CO.sub.2H), esterified carboxyl, thiol (--SH),
substituted thiol, --COR.sup.7 [where R.sup.7 is an
-Alk.sup.3(R.sup.6a).sub.m, aryl or heteroaryl group], --CSR.sup.7,
--SO.sub.3H, --SO.sub.2R.sup.7--SO.sub.2NH.sub.2,
--SO.sub.2NHR.sup.7 SO.sub.2N(R.sup.7).sub.2, --CONH.sub.2,
--CSNH.sub.2, --CONHR.sup.7, --CSNHR.sup.7, --CON[R.sup.7].sub.2,
--CSN(R.sup.7).sub.2, --N(R.sup.4)SO.sub.2R.sup.7,
--N(SO.sub.2R.sup.7).sub.2, --NH(R.sup.4)SO.sub.2NH.sub.2,
--N(R.sup.4)SO.sub.2NHR.sup.7,
--N(R.sup.4)SO.sub.2N(R.sup.7).sub.2, --N(R.sup.4)COR.sup.7,
--N(R.sup.4)CON(R.sup.7).sub.2, --N(R.sup.4)CSN(R.sup.7).sub.2,
--N(R.sup.4)CSR.sup.7, --N(R.sup.4)C(O)OR.sup.7,
--SO.sub.2NHet.sup.1 [where --NHet.sup.1 is an optionally
substituted C.sub.5-7cyclicamino group optionally containing one or
more other --O-- or --S-- atoms or --N(R.sup.4)--, --C(O)-- or
--C(S)-- groups], --CONHet.sup.1, --CSNHet.sup.1,
--N(R.sup.4)SO.sub.2NHet.sup.1, --N(R.sup.4)CONHet.sup.1,
--N(R.sup.4)CSNHet.sup.1, --SO.sub.2N(R.sup.4)Het.sup.2 [where
Het.sup.2 is an optionally substituted monocyclic
C.sub.5-7carbocyclic group optionally containing one or more --O--
or --S-- atoms or --N(R.sup.4)--, --C(O)-- or --C(S)-- groups],
--CON(R.sup.4)Het.sup.2, --CSN(R.sup.4)Het.sup.2,
--N(R.sup.4)CON(R.sup.4)Het.sup.2,
--N(R.sup.4)CSN(R.sup.4)Het.sup.2, aryl or heteroaryl group;
Alk.sup.3 is a straight or branched C.sub.1-6alkylene,
C.sub.2-6alkenylene or C.sub.2-6alkynylene chain, optionally
interrupted by one, two or three --O-- or --S-- atoms or
--S(O).sub.n [where n is an integer 1 or 2] or --N(R.sup.8)--
groups [where R.sup.8 is a hydrogen atom or C.sub.1-6alkyl, e.g.
methyl or ethyl group]; and m is zero or an integer 1, 2 or 3. It
will be appreciated that when two R.sup.4 or R.sup.7 groups are
present in one of the above substituents, the R.sup.4 or R.sup.7
groups may be the same or different.
[0074] When in the group -Alk.sup.3(R.sup.6a).sub.m m is an integer
1, 2 or 3, it is to be understood that the substituent or
substituents R.sup.6a may be present on any suitable carbon atom in
-Alk.sup.3. Where more than one R.sup.6a substituent is present
these may be the same or different and may be present on the same
or different atom in -Alk.sup.3. Clearly, when m is zero and no
substituent R.sup.6a is present the alkylene, alkenylene or
alkynylene chain represented by Alk.sup.3 becomes an alkyl, alkenyl
or alkynyl group.
[0075] When R.sup.6a is a substituted amino group it may be for
example a group --NHR.sup.7 [where R.sup.7 is as defined above] or
a group --N(R.sup.7).sub.2 wherein each R.sup.7 group is the same
or different.
[0076] When R.sup.6a is a halogen atom it may be for example a
fluorine, chlorine, bromine, or iodine atom.
[0077] When R.sup.6a is a substituted hydroxyl or substituted thiol
group it may be for example a group --OR.sup.7 or a --SR.sup.7 or
--SC(.dbd.NH)NH.sub.2 group respectively.
[0078] Esterified carboxyl groups represented by the group R.sup.6a
include groups of formula --CO.sub.2Alk.sup.4 wherein Alk.sup.4 is
a straight or branched, optionally substituted C.sub.1-8alkyl group
such as a methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl,
s-butyl or t-butyl group; a C.sub.6-12arylC.sub.1-18alkyl group
such as an optionally substituted benzyl, phenylethyl,
phenylpropyl, 1-naphthylmethyl or 2-naphthylmethyl group; a
C.sub.6-12aryl group such as an optionally substituted phenyl,
1-naphthyl or 2-naphthyl group; a C.sub.6-12aryloxyC.sub.1-8alkyl
group such as an optionally substituted phenyloxymethyl,
phenyloxyethyl, 1-naphthyl-oxymethyl, or 2-naphthyloxymethyl group;
an optionally substituted C.sub.1-8alkanoyloxyC.sub.1-8alkyl group,
such as a pivaloyloxymethyl, propionyloxyethyl or
propionyloxypropyl group; or a C6-12aroyloxyC.sub.1-8alkyl group
such as an optionally substituted benzoyloxyethyl or
benzoyloxy-propyl group. Optional substituents present on the
Alk.sup.4 group include R.sup.6a substituents described above.
[0079] When Alk.sup.3 is present in or as a substituent it may be
for example a methylene, ethylene, n-propylene, i-propylene,
n-butylene, i-butylene, s-butylene, t-butylene, ethenylene,
2-propenylene, 2-butenylene, 3-butenylene, ethynylene,
2-propynylene, 2-butynylene or 3-butynylene chain, optionally
interrupted by one, two, or three --O-- or --S--, atoms or
--S(O)--, --S(O).sub.2-- or --N(R.sup.8)-- groups.
[0080] Aryl or heteroaryl groups represented by the groups R.sup.6a
or R.sup.7 include mono- or bicyclic optionally substituted
C.sub.6.sub.12 aromatic or C.sub.1-9 heteroaromatic groups as
described above for the groups R.sup.1 and Het. The aromatic and
heteroaromatic groups may be attached to the remainder of the
compound of formula (1) by any carbon or hetero e.g. nitrogen atom
as appropriate.
[0081] When --NHet.sup.1 or -Het.sup.2 forms part of a substituent
R.sup.6 each may be for example an optionally substituted
pyrrolidinyl, pyrazolidinyl, piperazinyl, morpholinyl,
thiomorpholinyl, piperidinyl or thiazolidinyl group. Additionally
Het.sup.2 may represent for example, an optionally substituted
cyclopentyl or cyclohexyl group. Optional substituents which may be
present on --NHet.sup.1 or -Het.sup.2 include those R.sup.5
substituents described above.
[0082] Particularly useful atoms or groups represented by R.sup.6
include fluorine, chlorine, bromine or iodine atoms, or
C.sub.1-6alkyl, e.g. methyl, ethyl, n-propyl, i-propyl, n-butyl or
t-butyl, optionally substituted phenyl, pyridyl, pyrrolyl, furyl,
thiazolyl, or thienyl, C.sub.1-6alkylamino, e.g. methylamino or
ethylamino, C.sub.1-6hydroxyalkyl, e.g. hydroxymethyl or
hydroxyethyl, carboxyC.sub.1-6alkyl, e.g. carboxyethyl,
C.sub.1-6alkylthio e.g. methylthio or ethylthio,
carboxyC.sub.1-6alkylthio, e.g. carboxymethylthio,
2-carboxyethylthio or 3-carboxypropylthio, C.sub.1-6alkoxy, e.g.
methoxy or ethoxy, hydroxyC.sub.1-6alkoxy, e.g. 2-hydroxyethoxy,
optionally substituted phenoxy, pyridyloxy, thiazolyoxy, phenylthio
or pyridylthio, C.sub.5-7cycloalkoxy, e.g. cyclopentyloxy,
haloC.sub.1-6alkyl, e.g. trifluoromethyl, haloC.sub.1-6alkoxy, e.g.
trifluoromethoxy, C.sub.1-6alkylamino, e.g. methylamino or
ethylamino, amino (--NH.sub.2), aminoC.sub.1-6alkyl, e.g.
aminomethyl or aminoethyl, C.sub.1-6dialkylamino, e.g.
dimethylamino or diethylamino, C.sub.1-6alkylaminoC.sub.1-6alkyl,
e.g. ethylamino ethyl, C.sub.1-6dialkylaminoC.sub.1-6alkyl, e.g.
diethylaminoethyl, aminoC.sub.1-6alkoxy, e.g. aminoethoxy,
C.sub.1-6alkylaminoC.sub.1-6alkox- y, e.g. methylaminoethoxy,
C.sub.1-6dialkylaminoC.sub.1-6alkoxy, e.g. dimethylaminoethoxy,
diethylaminoethoxy, isopropylaminoethoxy, or dimethylaminopropoxy,
imido, such as phthalimido or naphthalimido, e.g.
1,8-naphthalimido, nitro, cyano, amidino, hydroxyl (--OH), formyl
[HC(O)--], carboxyl (--CO.sub.2H), --CO.sub.2Alk.sup.4 [where
Alk.sup.4 is as defined above], C.sub.1-6 alkanoyl e.g. acetyl,
optionally substituted benzoyl, thiol (--SH), thioC.sub.1-6alkyl,
e.g. thiomethyl or thioethyl, --SC(.dbd.NH)NH.sub.2, sulphonyl
(--SO.sub.3H), C.sub.1-6alkylsulphonyl, e.g. methylsulphonyl,
aminosulphonyl (--SO.sub.2NH.sub.2), C.sub.1-6alkylaminosulphonyl,
e.g. methylaminosulphonyl or ethylaminosulphonyl,
C.sub.1-6dialkylaminosulphon- yl, e.g. dimethylaminosulphonyl or
diethylaminosulphonyl, phenylaminosulphonyl, carboxamido
(--CONH.sub.2), C.sub.1-6alkylaminocarb- onyl, e.g.
methyl-aminocarbonyl or ethylaminocarbonyl,
C.sub.1-6dialkylaminocarbonyl, e.g. dimethylaminocarbonyl or
diethylaminocarbonyl, aminoC.sub.1-6alkylaminocarbonyl, e.g.
aminoethylaminocarbonyl,
C.sub.1-6dialkylaminoC.sub.1-6alkylaminocarbonyl- , e.g.
diethylaminoethylaminocarbonyl, aminocarbonylamino,
C.sub.1-6alkylaminocarbonylamino, e.g. methylaminocarbonylamino or
ethylamino-carbonylamino, C.sub.1-6dialkylaminocarbonylamino, e.g.
dimethylamino-carbonylamino or diethylaminocarbonylamino,
C.sub.1-6alkylaminocabonylC.sub.1-6alkylamino, e.g.
methylaminocarbonylmethylamino, aminothiocarbonyl-amino,
C.sub.1-6alkylaminothiocarbonylamino, e.g.
methylaminothiocarbonyl-amino or ethylaminothiocarbonylamino,
C.sub.1-6dialkylaminothiocarbonyl-amino, e.g.
dimethylaminothiocarbonylamino or diethylaminothiocarbonyl-amino,
C.sub.1-6alkylaminothiocarbonylC.sub.1-6alkylamino, e.g.
ethylaminothio-carbonylmethylamino, --CONHC(.dbd.NH)NH.sub.2,
C.sub.1-6alkylsulphonylamino, e.g. methylsulphonylamino or
ethylsulphonylamino, C.sub.1-6dialkylsulphonylamino, e.g.
dimethylsulphonylamino or diethylsulphonylamino, optionally
substituted phenylsulphonylamino, aminosulphonylamino
(--NHSO.sub.2NH.sub.2), C.sub.1-6alkylaminosulphonylamino, e.g.
methylaminosulphonyl-amino or ethylaminosulphonylamino,
C.sub.1-6dialkylaminosulphonylamino, e.g.
dimethylaminosulphonylamino or diethylaminosulphonylamino,
optionally substituted morpholinesulphonylamino or
morpholinesulphonylC.sub.1-6alkyl- amino, optionally substituted
phenylaminosulphonylamino, C.sub.1-6alkanoylamino, e.g.
acetylamino, aminoC.sub.1-6alkanoylamino e.g. aminoacetylamino,
C.sub.1-6dialkylaminoC.sub.1-6alkanoyl-amino, e.g.
dimethylaminoacetylamino, C.sub.1-6alkanoylaminoC.sub.1-6alkyl,
e.g. acetylaminomethyl, C.sub.1-6alkanoylaminoC.sub.1-6alkylamino,
e.g. acetamidoethylamino, C.sub.1-6alkoxycarbonylamino, e.g.
methoxycarbonylamino, ethoxycarbonylamino or t-butoxycarbonylamino
or optionally substituted benzyloxy, pyridylmethoxy,
thiazolylmethoxy, benzyloxycarbonylamino,
benzyloxycarbonylaminoC.sub.1-6alkyl e.g.
benzyloxy-carbonylaminoethyl benzothio, pyridylmethylthio or
thiazolylmethylthio groups.
[0083] Where desired, two R.sup.6 substituents may be linked
together to form a cyclic group such as a cyclic ether, e.g. a
C.sub.1-6alkylenedioxy group such as methylenedioxy or
ethylenedioxy.
[0084] It will be appreciated that where two or more R.sup.6
substituents are present, these need not necessarily be the same
atoms and/or groups. In general, the substituent(s) may be present
at any available ring position in the heteroaromatic group
represented by Het.
[0085] The presence of certain substituents in the compounds of
formula (1) may enable salts of the compounds to be formed.
Suitable salts include pharmaceutically acceptable salts, for
example acid addition salts derived from inorganic or organic
acids, and salts derived from inorganic and organic bases.
[0086] Acid addition salts include hydrochlorides, hydrobromides,
hydroiodides, alkylsulphonates, e.g. methanesulphonates,
ethanesulphonates, or isothionates, arylsulphonates, e.g.
p-toluenesulphonates, besylates or napsylates, phosphates,
sulphates, hydrogen sulphates, acetates, trifluoroacetates,
propionates, citrates, maleates, fumarates, malonates, succinates,
lactates, oxalates, tartrates and benzoates.
[0087] Salts derived from inorganic or organic bases include alkali
metal salts such as sodium or potassium salts, alkaline earth metal
salts such as magnesium or calcium salts, and organic amine salts
such as morpholine, piperidine, dimethylamine or diethylamine
salts.
[0088] Particularly useful salts of compounds according to the
invention include pharmaceutically acceptable salts, especially
acid addition pharmaceutically acceptable salts.
[0089] R in compounds of the invention is preferably a --CO.sub.2H
group.
[0090] When present, the aliphatic chain reprsented by Alk.sup.1 in
compounds of the invention is preferably a --CH.sub.2-- chain.
[0091] Alk.sup.2 in compounds of formula (1) is preferably a
--CH.sub.2-- chain and m is preferably an integer 1. In compounds
of this type, the carbon atom to which Alk.sup.2 and R are attached
forms a chiral centre and is preferably in the L configuration.
[0092] R.sup.2 in compounds of formula (1) is preferably a hydrogen
atom.
[0093] R.sup.3 in compounds of the invention is preferably a
hydrogen atom.
[0094] In general in compounds of the invention
-(Alk.sup.1).sub.n(L.sup.1- ).sub.s-is preferably --CH.sub.2O--,
--SO.sub.2NH, --C(O)O-- or --CON(R.sup.4)-- and is especially
--CONH--.
[0095] In general in compounds of the invention the group R.sup.1
is preferably an optionally substituted aromatic or heteroaromatic
group. Particularly useful groups of these types include optionally
substituted phenyl, pyridyl or pyrimidinyl groups, particularly
those in which the substituent when present is an atom or group
-L.sup.2(CH.sub.2).sub.pL.su- p.3(R.sup.c).sub.q as described
above. Each substituent may be present on any available ring carbon
or nitrogen atom.
[0096] The aromatic group represented by Ar in compounds of
formulae (1) and (1a) is preferably on optionally substituted
phenyl group. Each optional substituent when present is preferably
an atom or group R.sup.6 as defined above.
[0097] A particularly useful class of compounds according to the
invention has the formula (1b) 4
[0098] wherein --W=is --CH=or --N.dbd., R.sup.9 and R.sup.10, which
may be the same or different, is each a
-L.sup.2(CH.sub.2).sub.pL.sup.3(R.sup.c)- .sub.q atom or group as
generally and particularly defined above, and Alk.sup.1, r,
L.sup.1, s, R.sup.a, R.sup.b, R and Ar are as generally and
particularly defined above, and the salts, solvates, hydrates and
N-oxides thereof.
[0099] It will be appreciated that the various preferences stated
above in relation to groups present in compounds of formulae (1)
and (1a) apply equally to the same groups when present in compounds
of formula (1b).
[0100] Additionally, in compounds of formula (1b)
-(Alk.sup.1).sub.r(L.sup- .1).sub.r-is preferably a --CH.sub.2O or
--CON(R.sup.4)-- group and is especially a --CONH-- group.
[0101] Ar is preferably an optionally substituted phenyl group.
Particularly useful compounds of formula (1b) are those wherein Ar
is a 2-, 3- or 4-monosubstituted or a 2,6-disubstituted phenyl
group.
[0102] One of R.sup.9 or R.sup.10 in compounds of formula (1a) may
be for example a hydrogen atom and the other a substituent
L.sup.2(CH.sub.2).sub.pL.sup.3(R.sup.c).sub.q in which R.sup.c is
not a covalent bond and p is zero, but preferably each of R.sup.9
and R.sup.10 is a substituent
-L(2CH.sub.2).sub.pL.sup.3(R.sup.c).sub.q where R.sup.c is as just
defined. Particularly useful R.sup.9 or R.sup.10 substituents
include a hydrogen atom or halogen atom, especially fluorine or
chlorine atoms, or a methyl, ethyl, methoxy, ethoxy, --CF.sub.3,
--OH, --CN, --NO.sub.2, --NH.sub.2, --NHCH.sub.3,
--N(CH.sub.3).sub.2, --COCH.sub.3, --SCH.sub.3, --CO.sub.2H or
--CO.sub.2CH.sub.3 group.
[0103] Particularly useful compounds according to the invention
include the following:
[0104]
(N-2,6-Dimethoxybenzoyl)-[O-(3,5-dichloro-4-pyridinyl)methyl]-L-tyr-
osine;
[0105]
2-Carboxybenzoyl-(N'-3,5-dichloroisonicotinoyl)-L-4-aminophenylalan-
ine;
[0106]
(N-2,6-Dimethoxybenzoyl)-(N'-3,5-dichloroisonicotinoyl)-L-4-aminoph-
enylalanine;
[0107]
(N-3-Carboxybenzoyl)-(N'-3,5-dichloroisonicotoninoyl)-L-4-aminophen-
yalanine;
[0108]
(N-4-Carboxybenzoyl)-(N'-3,5-dichloroisonicotinoyl)-L-4-aminophenyl-
alanine;
[0109]
(N-2-t-Butoxycarbonylbenzoyl)-(N'-3,5-dichloroisonicotinoyl)-L-4-am-
inophenylalanine;
[0110] (N-3-Cyanobenzoyl)-(N'-3,
5-dichloroisonicotinoyl)-L-4-aminophenyla- lanine;
[0111]
[N-3-(1-H-Tetrazol-5-yl)]-(N'-3,5-dichloroisonicotinoyl)-L-4-aminop-
henylalanine;
[0112]
[N-(3-Methoxycarbonylbenzoyl)]-(N'-3,5-dichloroisonicotinoyl)-L-4-a-
minophenylalnine;
[0113] and the salts, solvates, hydrates and N-oxides thereof.
[0114] The compounds of formulae (1), (1a) and (1b) may be prepared
by a number of processes as generally described below and more
specifically in the Examples hereinafter. In the following process
description, the symbols R.sup.1--R.sup.3, L.sup.1, Alk.sup.1,
R.sup.a, R.sup.b, Alk.sup.2, m, r, s and Ar when used in the
formulae depicted are to be understood to represent those groups
described above in relation to formula (1) unless otherwise
indicated. In the reactions described below, it may be necessary to
protect reactive functional groups, for example hydroxy, amino,
thio or carboxy groups, where these are desired in the final
product, to avoid their unwanted participation in the reactions.
Conventional protecting groups may be used in accordance with
standard practice [see, for example, Green, T. W. in "Protective
Groups in Organic Synthesis", John Wiley and Sons, 1991]. In some
instances, deprotection may be the final step in the synthesis of
the desired compound and the processes described hereinafter are to
be understood to extend to such removal of protecting groups. For
convenience, the processes described below all refer to the
preparation of a compound of formula (1) but clearly the
description applies equally to the preparation of compounds of
formulae (1a) and (1b).
[0115] Thus a compound of formula (1) may be obtained by hydrolysis
of an ester of formula (2): 5
[0116] where R.sup.11 is an alkyl group.
[0117] The hydrolysis may be performed using either an acid or a
base depending on the nature of R.sup.9, for example an organic
acid such as trifluoracetic acid or an inorganic base such as
lithium hydroxide optionally in an aqueous organic solvent such as
an amide, e.g. a substituted amide such as dimethylformamide, an
ether, e.g. a cyclic ether such as tetrahydrofuran or dioxane or an
alcohol, e.g. methanol at around ambient temperature. Where
desired, mixtures of such solvents may be used.
[0118] Esters of formula (2) may be prepared by coupling an amine
of formula (3): 6
[0119] (where R.sup.11 is as just described) or a salt thereof with
an acid of formula (4):
ArCO.sub.2H (4)
[0120] or an active derivative thereof.
[0121] Active derivatives of acids of formula (4) include
anhydrides, esters and halides. Particular esters include
pentafluorophenyl or succinyl esters.
[0122] The coupling reaction may be performed using standard
conditions for reactions of this type. Thus for example the
reaction may be carried out in a solvent, for example an inert
organic solvent such as an amide, e.g. a substituted amide such as
dimethylformamide, an ether, e.g. a cyclic ether such as
tetrahydrofuran, or a halogenated hydrocarbon, such as
dichloromethane, at a low temperature, e.g. around -30.degree. C.
to around ambient temperature, optionally in the presence of a
base, e.g. an organic base such as an amine, e.g. triethylamine,
pyridine, or dimethyl-aminopyridine, or a cyclic amine, such as
N-methylmorpholine.
[0123] Where an acid of formula (4) is used, the reaction may
additionally be performed in the presence of a condensing agent,
for example a diimide such as
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or
N,N'-dicyclo-hexylcarboduimide, advantageously in the presence of a
catalyst such as a N-hydroxy compound e.g. a N-hydroxytriazole such
as 1-hydroxy-benzotriazole. Alternatively, the acid may be reacted
with a chloroformate, for example ethylchloroformate, prior to
reaction with the amine of formula (3).
[0124] Intermediates of formulae (2), (3) and (4), or compounds of
formula (1), may be manipulated to introduce substituents to
aromatic or heteroaromatic groups or modify existing substituents
in groups of these types. Typically, such manipulation may involve
standard substitution approaches employing for example alkylation,
arylation, heteroarylation, acylation, thioacylation, halogenation,
sulphonylation, nitration, formylation or coupling reactions.
Alternatively, exisitng substituents may be modified for example by
oxidation, reduction or cleavage reactions. Particular examples of
such reactions are given below. Where these are paticularly
described in relation to the generation of the group
R.sup.1(Alk.sup.1).sub.r(L.sup.1).sup.s-, it will be appreciated
that each reaction may also be used to introduce or modify R.sup.5
and/or R.sup.6 substituents in for example Ar groups as
appropriate.
[0125] Thus in one example, a compound wherein R.sup.1
(Alk.sup.1).sub.r(L.sup.1).sub.s-is a -L.sup.1H group may be
alkylated, arylated or heteroarylated using a reagent
R.sup.1(Alk.sup.1).sub.rX in which R.sup.1 is other than a hydrogen
atom and X is a leaving atom or group such as a halogen atom, e.g.
a fluorine, bromine, iodine or chlorine atom or a sulphonyloxy
group such as an alkylsulphonyloxy, e.g.
trifluoro-methylsulphonyloxy or arylsulphonyloxy, e.g.
p-toluenesulphonyloxy group.
[0126] The reaction may be carried out in the presence of a base
such as a carbonate, e.g. caesium or potassium carbonate, an
alkoxide, e.g. potassium t-butoxide, or a hydride, e.g. sodium
hydride, in a dipolar aprotic solvent such as an amide, e.g. a
substituted amide such as dimethylformamide or an ether, e.g. a
cyclic ether such as tetrahydrofuran.
[0127] In another example, a compound where R.sup.1
(Alk.sup.1).sub.r(L.sup.1).sub.s is a -L.sup.1H group is a hydrogen
atom may be functionalised by acylation or thioacylation, for
example by reaction with a reagent R.sup.1(Alk.sup.1).sub.rL.sup.1X
[wherein L.sup.1 is a --C(O)--, C(S)--, --N(R.sup.4)C(O)-- or
N(R.sup.4)C(S)-- group], in the presence of a base, such as a
hydride, e.g. sodium hydride or an amine, e.g. triethylamine or
N-methylmorpholine, in a solvent such as a halogenated hydrocarbon,
e.g. dichloromethane or carbon tetrachloride or an amide, e.g.
dimethylformamide, at for example ambient temperature, or by
reaction with R.sup.1 (Alk.sup.1).sub.rCO.sub.2H, R.sup.1
(Alk).sub.4COSH or an activated derivative thereof, for example as
described above for the preparation of esters of formula (2).
[0128] In a further example a compound may be obtained by
sulphonylation of a compound where R.sup.1
(Alk.sup.1).sub.r(L.sup.1).sub.s is an --OH group by reaction with
a reagent R.sup.1(Alk.sup.1).sub.rL.sup.1 Hal [in which L.sup.1 is
--S(O)-- or --SO.sub.2-- and Hal is a halogen atom such as chlorine
atom] in the presence of a base, for example an inorganic base such
as sodium hydride in a solvent such as an amide, e.g. a substituted
amide such as dimethylformamide at for example ambient
temperature.
[0129] In another example, a compound where
R.sup.1(Alk.sup.1).sub.r(L.sup- .1).sub.s is a -L.sup.1H group, may
be coupled with a reagent R.sup.1 OH (where R.sup.1 is other than a
hydrogen atom) or R.sup.1Alk.sup.1OH in a solvent such as
tetrahydrofuran in the presence of a phosphine, e.g.
triphenylphosphine and an activator such as diethyl, diisopropyl-
or dimethylazodicarboxylate to yield a compound containing a
R.sup.1 (Alk.sup.1).sub.rO-- group.
[0130] In a further example, ester groups --CO.sub.2R.sup.4 or
--CO.sub.2Alk.sup.4 in compounds of formula (1) may be converted to
the corresponding acid [--CO.sub.2H] by acid- or base-catalysed
hydrolysis depending on the nature of the grousp R.sup.4 or
Alk.sup.4. Acid- or base-catalysed hydrolysis may be achieved for
example by treatment with an organic or inorganic acid, e.g.
trifluoroacetic acid in an aqueous solvent or a mineral acid such
as hydrochloric acid in a solvent such as dioxane or an alkali
metal hydroxide, e.g. lithium hydroxide in an aqueous alcohol, e.g.
aqueous methanol.
[0131] In a second example, --OR.sup.7 [where R.sup.7 represents an
alkyl group such as methyl group] groups in compounds of formula
(1) may be cleaved to the corresponding alcohol --OH by reaction
with boron tribromide in a solvent such as a halogenated
hydrocarbon, e.g. dichloromethane at a low temperature, e.g. around
-78.degree. C.
[0132] Alcohol [--OH] groups may also be obtained by hydrogenation
of a corresponding --OCH.sub.2R.sup.7 group (where R.sup.7 is an
aryl group) using a metal catalyst, for example palladium on a
support such as carbon in a solvent such as ethanol in the presence
of ammonium formate, cyclohexadiene or hydrogen, from around
ambient to the reflux temperature. In another example, --OH groups
may be generated from the corresponding ester [--CO.sub.2Alk.sup.4
or CO.sub.2R.sup.4] or aldehyde [--CHO] by reduction, using for
example a complex metal hydride such as lithium aluminium hydride
or sodium borohydride in a solvent such as methanol.
[0133] In another example, alcohol --OH groups in compounds of
formula (1) may be converted to a corresponding --OR.sup.3 group by
coupling with a reagent R.sup.7OH in a solvent such as
tetrahydrofuran in the presence of a phosphine, e.g.
triphenylphosphine and an activator such as diethyl-, diisopropyl-,
or dimethylazodicarboxylate.
[0134] Aminosulphonylamino [--NHSO.sub.2NH.sub.2] groups in
compounds of formula (1) may be obtained, in another example, by
reaction of a corresponding amine [--NH.sub.2] with sulphamide in
the presence of an organic base such as pyridine at an elevated
temperature, e.g. the reflux temperature.
[0135] In a further example amine (--NH.sub.2) groups may be
alkylated using a reductive alkylation process employing an
aldehyde and a borohydride, for example sodium triacetoxyborohyride
or sodium cyanoborohydride, in a solvent such as a halogenated
hydrocarbon, e.g. dichloromethane, a ketone such as acetone, or an
alcohol, e.g. ethanol, where necessary in the presence of an acid
such as acetic acid at around ambient temperature.
[0136] In a further example, amine [--NH.sub.2] groups in compounds
of formula (1) may be obtained by hydrolysis from a corresponding
imide by reaction with hydrazine in a solvent such as an alcohol,
e.g. ethanol at ambient temperature.
[0137] In another example, a nitro [--NO.sub.2] group may be
reduced to an amine [--NH.sub.2], for example by catalytic
hydrogenation using for example hydrogen in the presence of a metal
catalyst, for example palladium on a support such as carbon in a
solvent such as an ether, e.g. tetrahydrofuran or an alcohol e.g.
methanol, or by chemical reduction using for example a metal, e.g.
tin or iron, in the presence of an acid such as hydrochloric
acid.
[0138] Aromatic halogen substituents in compounds of the invention
may be subjected to halogen-metal exchange with a base, for example
a lithium base such as n-butyl or t-butyl lithium, optionally at a
low temperature, e.g. around -78.degree. C., in a solvent such as
tetrahydrofuran and then quenched with an electrophile to introduce
a desired substituent. Thus, for example, a formyl group may be
introduced by using dimethylformamide as the electrophile; a
thiomethyl group may be introduced by using dimethyldisulphide as
the electrophile.
[0139] In another example, sulphur atoms in compounds of the
invention, for example when present in the linker group L.sup.1 may
be oxidised to the corresponding sulphoxide using an oxidising
agent such as a peroxy acid, e.g. 3-chloroperoxybenzoic acid, in an
inert solvent such as a halogenated hydrocarbon, e.g.
dichloromethane, at around ambient temperature.
[0140] Intermediates of formulae (3) and (4), R.sup.1
(Alk.sup.1).sub.rX, R.sup.1 (Alk.sup.1).sub.rL.sup.1X,
R.sup.1(Alk.sup.1).sub.rCO.sub.2H, R.sup.1OH and R.sup.1Alk.sup.1OH
for use in the above processes are either known compounds or may be
prepared from known starting materials by use of analogous
processes to those used for the preparation of the known compounds
and/or by treating known compounds by one or more of the
alkylation, acylation and other manipulations described herein,
such as particularly described for the preparation of the
Intermediates in the exemplification section hereinafter.
[0141] N-oxides of compounds of formula (1) may be prepared for
example by oxidation of the corresponding nitrogen base using an
oxidising agent such as hydrogen peroxide in the presence of an
acid such as acetic acid, at an elevated temperature, for example
around 70.degree. C. to 80.degree. C., or alternatively by reaction
with a peracid such as peracetic acid in a solvent, e.g.
dichloromethane, at ambient temperature.
[0142] Salts of compounds of formula (1) may be prepared by
reaction of a compound of formula (1) with an appropriate base in a
suit able solvent or mixture of solvents e.g. an organic solvent
such as an ether e.g. diethylether, or an alcohol, e.g. ethanol
using conventional procedures.
[0143] Where it is desired to obtain a particular enantiomer of a
compound of formula (1) this may be produced from a corresponding
mixture of enantiomers using any suitable conventional procedure
for resolving enantiomers.
[0144] Thus for example diastereomeric derivatives, e.g. salts, may
be produced by reaction of a mixture of enantiomers of formula (1)
e.g. a racemate, and an appropriate chiral compound, e.g. a chiral
base. The diastereomers may then be separated by any convenient
means, for example by crystallisation and the desired enantiomer
recovered, e.g. by treatment with an acid in the instance where the
diastereomer is a salt.
[0145] In another resolution process a racemate of formula (1) may
be separated using chiral High Performance Liquid Chromatography.
Alternatively, if desired a particular enantiomer may be obtained
by using an appropriate chiral intermediate in one of the processes
described above.
[0146] The following Examples illustrate the invention. All
temperatures are in .degree. C. The following abbreviations are
used:
1 EDC 1-(3-dimethylaminopropyl)3-ethycarbodiimide; DMF
dimethylformamide; HOBT 1-hydroxybenzotriazole; NMM
N-methylmorpholine; Ph phenyl; EtOAc ethyl acetate; Ar aryl;
Et.sub.2O diethylether; DMSO dimethylsulphoxide; THF
tetrahydrofuran; DCM dichloromethane Boc tert-butoxycarbonyl; MeOH
methanol; Me methyl; EtOH ethanol;
[0147] Intermediate 1
[0148] N-(2,6-Dimethoxybenzoyl)-O-benzyl)-L-tyrosine Methyl
Ester
[0149] EDC (212 mg, 1.1 mmol) was added to a solution of
(O-benzyl)tyrosine methyl ester hydrochloride (322 mg, 1 mmol),
2,6-dimethoxybenzoic acid (182 mg, 1 mmol), HOBT (149 mg, 1.1 mmol)
and NMM (242 .mu.l, 2.2 mmol) in DCM (10 ml). The mixture was
stirred at room temperature overnight, then diluted with DCM (100
ml), washed with 10% citric acid (20 ml), saturated sodium
bicarbonate solution (20 ml) and brine (20 ml), dried
(Na.sub.2SO.sub.4) and evaporated under reduced pressure. The
residue was purified by column chromatography (SiO.sub.2;
EtOAc/hexane, 60:40) to give the title compound as a colourless gum
(392 mg, 87%). .delta. H (CDCl.sub.3) 7.44-7.31 (5H, m,
OCH.sub.2Ph), 7.29 (1H, t, J 8.4 Hz, Ar(OMe) H), 7.14 (2H, d, J 8.7
Hz, CH.sub.2Ar(O) H), 6.88 (2H, d, J 8.7 Hz, CH.sub.2Ar(O) H), 6.55
(2H, d, J 8.4 Hz, Ar(OMe) H), 6.36 (1H, br d, J 7.4 Hz, CONH), 5.11
(1H, dt, J 7.5, 5.2 Hz, CH.alpha.tyr) 5.02 (2H, s, OCH.sub.2Ph),
3.80 (6H, s, 2.times.ArOMe), 3.72 (3H, s, CO.sub.2Me), 3.29 (1H,
dd, J 5.5, 14.0 Hz, CHCH.sub.AH.sub.BAr) and 3.16 (1H, dd, J 4.9,
14.0 Hz, CHCH.sub.AH.sub.BAr); m/z (ESI, 15V) 450 (Mh.sup.+).
[0150] Intermediate 2
[0151]
2-Thio(S-4-methylphenyl)benzoyl-(O-2,6-dichlorobenzyl)-L-tyrosine
Methyl Ester
[0152] A solution of (O-2,6-dichlorobenzyl)-L-tyrosine methyl ester
hydrochloride (0.39 g, 1 mmol), 2-thio(S-4-methylphenyl)benzoic
acid (0.24 g, 1 mmol), EDC (0.21 g, 1.1 mmol), HOBT (0.16 g, 1.2
mmol) and NMM (0.28 ml; 0.25 g, 2.5 mmol) in DMF (5 ml) was stirred
at room temperature for 16 h. The solvent was evaporated in vacuo
and the residue partitioned between 10% hydrochloric acid (10 ml)
and DCM (20 ml). The aqueous layer was extracted with DCM (20 ml)
and the combined organic layers washed with NaHCO.sub.3 solution
(25 ml), dried over MgSO.sub.4 and the solvent removed to give a
brown oil that was purified by column chromatography (SiO.sub.2;
2:5 EtOAc/hexane) to give an off-white solid that was
recrystallised from EtOAc/hexane (1:2) to give the title compound
as a white solid (0.24 g, 41%). .delta. H (CDCl.sub.3) 7.53-6.90
(15H, m), 6.81 (1H, d, J 7.3 Hz), 5.22 (2H, s), 5.05 (1H, m), 3.77
(3H, s), 3.27 (1H, dd, J 5.9, 14.0 Hz), 3.18 (1H, dd, J 5.2, 14.0
Hz) and 2.35 (3H, s).
[0153] Intermediate 3
[0154] Methyl Phthalate
[0155] Solid sodium methoxide (5.4 g, 0.1 mmol) was added to a
suspension of phthalic anhydride (14.8 g, 0.1 mmol) in MeOH (100
ml) and the reaction mixture stirred for 2 h at room temperature.
The solvent was evaporated in vacuo, and the residue partitioned
between DCM and 1.0M hydrochloric acid. The aqueous layer was
extracted with DCM (2.times.20 ml), and the combined organic layers
dried over MgSO.sub.4. The solvent was removed to give the title
compound as a white solid (17.3 g, 96%). .delta. H (CDCl.sub.3)
7.94-7.91 (1H, m), 7.70-7.63 (1H, m), 7.61-7.54 (2H, m) and 3.92
(3H, s).
[0156] Intermediate 4
[0157] a)
2-Methoxycarbonyl)benzoyl-[N-3,5-dichloroisonicotinoyl)-L-4-amin-
ophenylalanine Methyl Ester
[0158] Intermediate 3 (0.681 g, 3.78 mmol) was dissolved in dry DMF
(20 ml) and (N-3,5-dichloroisonicotinoyl)-L-4-aminophenylalanine
methyl ester hydrochloride (4 mmol; prepared from
3,5-dichloro-4-picoloyl chloride and N-Boc-L-4-aminophenylalanine
methyl ester) added in one portion. To the mixture was added NMM
(0.896 ml, 8.16 mmol), HOBT.H.sub.2O (0.551 g, 4.08 mmol) and EDC
(0.783 g, 4.08 mmol). The reaction mixture was stirred under argon
at room temperature for 2.5 h, poured into 50% saturated sodium
bicarbonate solution (100 ml) and extracted with EtOAc (2.times.50
ml).
[0159] The organic layer was washed with 50% saturated sodium
bicarbonate solution (1.times.50 ml), 10% citric acid (1.times.50
ml), brine (1.times.50 ml), and dried over MgSO.sub.4. The solvent
was evaporated to give a white foam which was purified by
chromatography (SiO.sub.2; gradient elution DCM to DCM/MeOH (99:1))
to give the title compound as a white foam (1.0 g, 51%). .delta. H
(DMSO-d.sup.6) 10.86 (1H, s), 8.92 (1H, d, J 7.9 Hz), 8.79 (2H, s),
7.70 (1H, dd, J 7.29, 1.3 Hz), 7.63-7.52 (4H, m), 7.37 (1H, dd, J
7.5, 1.3 Hz), 7.30 (2H, d, J 8.0 Hz), 4.66-4.58 (1H, m), 3.67 (3H,
s), 3.363 (3H, s) and 3.15-2.95 (2H, m).
[0160] The following compound was prepared in a similar manner
using 3-cyanobenzoic acid in place of Intermediate 3:
[0161] b)
(N-3-Cyanobenzoyl)-(N'-3,5-dichloroisonicotinoyl)-L-4-aminopheny-
lalanine Methyl Ester
[0162] .delta. H (CDCl.sub.3) 8.55 (2H, s), 8.1-7.5 (7H, m), 7.12
(2H, d, J 8.5 Hz), 6.7 (1H, d, J 8.5 Hz), 5.15-5.05 (1H, m), 3.82
(3H, s) and 3.35-3.18 (2H, m). m/z (ES) 497 (MH.sup.+).
[0163] Using a similar procedure to Intermediate 4a) employing the
phenylalanine t-butyl ester in place of the corresponding methyl
ester, the following compound was prepared:
[0164] c)
[N-3-Methoxycarbonyl)benzoyl]-(N'-3,5-dichloroisonicotinoyl)-L-4-
-aminophenylalanine t-Butyl Ester
[0165] .delta. H (CDCl.sub.3), 8.44 (2H, s), 7.92-7.2 (9H, m), 6.53
(1H, d, J 7.8 Hz), 4.99-4.93 (1H, m), 3.85 (3H, s), 3.23 (2H, d, J
5.6 Hz) and 1.46 (9H, s). m/z (ES) 572 (MH.sup.+).
[0166] Intermediate 5
[0167]
Benzyl-4-{2-[(diphenylmethylene)amino]-3-ethoxy-3-oxopropyl}benzoat-
e
[0168] A mixture of benzyl-4-(bromomethyl)benzoate (7.61 g, 24.9
mmol), N-(diphenylmethylene)glycine ethyl ester (6.66 g, 24.9 mmol)
and potassium carbonate (6.9 g, 50 mmol) in acetonitrile (300 ml)
were heated to reflux for 20 h. The reaction was concentrated in
vacuo, and the residue partitioned between EtOAc (100 ml) and water
(100 ml). The aqueous layer was extracted with EtOAc (100 ml) and
the combined organic layers were dried (Na.sub.2SO.sub.4), and
concentrated in vacuo to give the title compound as a pale straw
coloured oil (13.0 g) which was used without further purification.
.delta. H (CDCl.sub.3) 7.90 (2H, d, J 8.3 Hz, Ar--H), 7.58 (2H, d,
J 7.3 Hz, Ar--H), 7.50-7.25 (13H, m, Ar--H), 7.14 (2H, d, J 8.1 Hz,
Ar--H), 6.65 (2H, d, J 6.8 Hz, CH.sub.2Ph), 5.33 (2H, m,
CHCH.sub.2Ar), 4.30-4.15 (3H, m, CHCH.sub.2Ar) and
CH.sub.2CH.sub.3) and 1.26 (3H, t, J 7.1 Hz, CH.sub.2CH.sub.3). m/z
(ESI, 60V) 492 (MH.sup.+).
[0169] Intermediate 6
[0170] 4-(Benzyloxycarbonyl)phenylalanine Ethyl Ester
Hydrochloride
[0171] A solution of Intermediate 5 (13.0 g, 25 mmol) in THF (200
ml) was treated with hydrochloric acid (2.0M solution, 20 ml, 40
mmol) and stirred at room temperature for 2 h. then concentrated in
vacuo and triturated with Et.sub.2O to give a white solid which was
purified by recrystallistion from EtOH/EtOAc to give the title
compound as a white solid (4.24 g, 47%). .delta. H (DMSO-d.sup.6)
7.96 (2H, dd, J 6.6, 1.7 Hz, Ar--H), 7.48-7.37 (7H, 6m, Ar--H),
5.35 (2H, s, CH.sub.2Ph), 4.29 (1H, dd, J 7.7, 5.0 Hz, CH.alpha.),
4.11 (2H, q, J 7.1 Hz, CH.sub.2CH.sub.3), 3.27 (1H, dd, J 14.0, 5.9
Hz, CHCH.sub.AH.sub.BAr), 3.15 (1H, dd, J 14.0, 7.7 Hz,
CHCH.sub.AH.sub.BAr) and 1.08 (3H, t, J 7.1 Hz, CH.sub.2CH.sub.3),
m/z (ESI, 60V) 328 (MH.sup.+),
[0172] Intermediate 7
[0173] (N-2,6-Dimethoxybenzoyl)-4-(benzyloxycarbonyl)phenylalanine
Ethyl Ester
[0174] A solution of Intermediate 6 (2.75 g, 8.4 mmol) and NMM
(1.25 mg, 11.2 mmol) in DCM (50 ml) was treated with
2,6-dimethoxybenzoyl chloride (2.25 g, 11.2 mmol) and stirred for
16 h at room temperature. The solution was concentrated in vacuo
and partitioned between dilute HCl (50 ml) and EtOAc (100 ml). The
aqueous layer was extracted with EtOAc (2.times.50 ml) and the
combined organic layers washed with water (100 ml) and NaHCO.sub.3
solution (100 ml), dried (Na.sub.2SO.sub.4) and concentrated in
vacuo to give the title compound as a pale amber glass (3.36 g,
82%) which was used without further purification. .delta. H
(CDCl.sub.3) 7.96 (2H, d, J 8.4 Hz, Ar--H), 7.45-7.28 (8H, m,
Ar--H), 6.56 (2H, d, J 8.4 Hz, Ar--H), 6.37 (1H, d, J 7.2 Hz, NH),
5.34 (2H, s, COCH.sub.2Ph), 5.13 (1H, m, CH.alpha.), 4.16 (2H, q, J
7.1 Hz, CH.sub.2CH.sub.3), 3.80 (6H, s, OMe), 3.42 (1H, dd, J 13.8,
5.7 Hz, CHCH.sub.AH.sub.bAr), 3.26 (1H, dd, J 13.8, 4.9 Hz,
CHCH.sub.AH.sub.BAr) and 1.25 (3H, t, J 7.1 Hz, CH.sub.2CH.sub.3).
m/z (ESI, 60V) 492 (MH.sup.+).
[0175] Intermediate 8
[0176] (N-2,6-Dimethoxybenzoyl)-4-carboxyphenylalanine Ethyl
Ester
[0177] A solution of Intermediate 7 (3.35 g, 6.8 mmol) in EtOH (300
ml) was treated with 10% palladium on charcoal (400 mg) and stirred
under an atmosphere of H.sub.2 for 20 h. The catalyst was removed
by filtration through Celite.RTM. and the solution concentrated in
vacuo to give the title compound as a glass (2.78 g, 100%). .delta.
H (CDCl.sub.3) 7.99 (2H, d, J 8.2 Hz, Ar--H), 7.36 (2H, d, J 8.2
Hz, Ar--H), 7.27 (1H, t, J 8.4 Hz, Ar--H), 6.55 (2H, d, J 8.4 Hz,
Ar--H), 6.42 (1H, d, J 7.3 Hz, NH), 5.15 (1H, m, CH.alpha.), 4.18
(2H, q, J 7.1 Hz, CH.sub.2CH.sub.3), 3.81 (6H, s, OMe), 3.45 (1H,
dd, J 13.7, 5.6 Hz, CHCH.sub.AH.sub.BAr), 3.28 (1H, dd, J 13.7, 6.9
Hz, CHCH.sub.AH.sub.BAr) and 1.26 (3H, t, J 7.1 Hz,
CH.sub.2CH.sub.3). m/z (ESI, 60V) 402 (MH.sup.+).
[0178] Intermediate 9
[0179]
(N-2,6-Dimethoxybenzoyl)-4-(benzylaminocarbonyl)phenylalanine Ethyl
Ester
[0180] A solution of Intermediate 8 (0.73 g, 1.82 mmol) in DCM (40
ml) was treated with oxalyl chloride (5 ml) and DMF (1 drop) and
stirred at room temperature for 2 h. The solvent was removed in
vacuo and the residue azeotroped with toluene, dissolved in DCM (40
ml), and treated with benzylamine (1.0 ml) and stirred at room
temperature for 3 h. The reaction was concentrated in vacuo and
partitioned between dilute HCl (1.0M, 30 ml) and EtOAc (50 ml). The
aqueous layer was extracted with EtOAc (3.times.50 ml) and the
combined organic layers washed with NaHCO.sub.3 solution
l(2.times.50 ml) and brine (50 ml), dried (Na.sub.2SO.sub.4) and
concentrated in vacuo to give the title compound as a white solid
(1.07 g) which was used without further purification. .delta. H
(CDCl.sub.3) 7.75 (1H, br s, NH), 7.69 (2H, d, J 8.3 Hz, ArH),
7.38-7.24 (8H, m, Ar--H), 6.65 (2H, d, J 8.4 Hz, Ar--H), 6.36 (1H,
d, J 6.9 Hz, Ar--H), 5.12 (1H, m, CH.alpha.), 4.63 (2H, d, J 5.6
Hz, NHCH.sub.2Ph), 4.16 (2H, q, J 7.1 Hz, CH.sub.2CH.sub.3), 3.80
(6H, s, OMe), 3.40 (1H, dd, J 13.8, 5.8 Hz, CHCH.sub.AH.sub.BAr),
3.25 (1H, dd, J 13.8, 4.9 Hz, CHCH.sub.AH.sub.BAr) and 1.25 (3H, t,
J 7.1 Hz, CH.sub.2CH.sub.3). m/z (ESI, 60V) 491 (MH.sup.+).
[0181] Intermediate 10
[0182]
[N-3-(-1-H-Tetrazol-5-yl)benzyl]-(N'-3,5-dichloroisonicotinoyl-L-4--
aminophenylalanine Methyl Ester
[0183] To Intermediate 4b) (0.424 g, 0.85 mmol) in dry toluene (10
ml) was added trimethylsilyl azide (0.22 ml) and di-n-butyl tin
oxide (21 mg). The solution was refluxed overnight, cooled and then
evaporated. Chromatography on silica gel eluting with EtOAc hexane
50.50 afforded the title compound as a foam (0.214 g, 46%). .delta.
H (CD.sub.3OD), 8.6 (2H, s), 8.4-7.42 (6H, m), 7.3 (2H, d, J 8.5
Hz), 4.92 (1H, m), 4.8 (3H, s) and 3.18-3.09 (2H, m). m/z (ES) 497
(MH.sup.+).
[0184] Inermediate 11
[0185] 3,5-Dichloro-4-hydroxymethylpyridine
[0186] A solution of 3,5-dichloropyridine-4-carboxaldehyde (1.34 g,
7.6 mmol) in MeOH (10 ml) was treated with NaBH.sub.4 (0.29 g, 7.6
mmol) and stirred at room temperature for 2 h. The reaction was
quenched with water (5 ml) and concentrated in vacuo. The residue
was partitioned between EtOAc (20 ml) and 10% HCl (10 ml). The
aqueous layer was extracted with EtOAc and the combined organic
extracts, washed with 10% NaHCO.sub.3 solution, dried (MgSO.sub.4)
and concentrated in vacuo to give the title compound as a white
solid (1.05 g, 78%). .delta. H (CDCl.sub.3) 8.52 (2H, s, pyr-H),
4.94 (2H, br s, CH.sub.2OH) and 2.28 (1H, br s, OH).
[0187] Intermediate 12
[0188] 3,5-Dichloro-4-bromomethylpyridine
[0189] A solution of Intermediate 11 (0.50 g, 2.80 mmol) in DCM (10
ml) was treated with thionyl bromide (3.51 g, 1.32 ml, 16.9 mmol)
and heated to reflux for 3 h. The reaction was quenched with 10%
NaHCO.sub.3 solution (10 ml) and extracted wth DCM (25 ml). The
organic layer was dried (MgSO.sub.4) and concentrated in vacuo to
give the title compound as a yellow oil that solidified on standing
(0.65 g, 96%) and was used without further purification. .delta. H
(CDCl.sub.3) 8.50 (2H, s, pyr-H), 4.63 (2H, s, CH.sub.2Br). m/z
(ESI, 60V) 242 (MH.sup.+).
EXAMPLE 1
[0190] N-(2,6-Dimethoxybenzoyl)-(O-benzyl)-L-tyrosine
[0191] Lithium hydroxide monohydrate (4.2 mg, 1 mmol) was added to
a solution of Intermediate 1 (385 mg, 0.857 mmol) in THF (10 ml)
and water (10 ml). The mixture was stirred for 1 h at room
temperature. The THF was removed under reduced pressure, the
aqueous residue acidified (dilute HCl aq.) and extracted with DCM
(3.times.50 ml). The extracts were dried (Na.sub.2SO.sub.4) and
evaporated under reduced pressure. The residue was freeze dried
from a mixture of MeOH and water to give the title compound as a
white fluffy solid (365 mg, 98%). .delta. H (DMSO-d.sup.6) 12.53
(1H, br s, CO.sub.2H), 8.13 (1H, d, J 7.9 Hz, CONH), 7.45-7.30 (5H,
m, OCH.sub.2Ph), 7.25 (1H, t, J 8.4 Hz, Ar (OMe)H), 7.18 (2H, d, J
8.6 Hz, CH.sub.2Ar(O)H), 6.92 (2H, d, J 7.5 Hz, CH.sub.2Ar(O)H),
6.61 (2H, d, J 8.4 Hz, Ar(OMe)H), 5.07 (2H, s, OCH.sub.2Ph), 4.51
(1H, dt, J 5.1, 8.3 Hz, CH.alpha.tyr), 3.63 (6H, s,
2.times.Ar(OMe)), 3.00 (1H, dd, J 5.1, 14.0 Hz,
CHCH.sub.AH.sub.BAr) and 2.87 (1H, dd, J 8.6, 14.0 Hz,
CHCH.sub.AH.sub.BAr); m/z (ESI, 15V) 436 (MH.sup.+).
EXAMPLE 2
[0192] a)
2-Thio(S-4-methylphenyl)benzoyl-(O-2,6-dichlorobenzyl)-L-tyrosin-
e
[0193] A solution of Intermediate 2 (0.24 g, 0.41 mmol) in THF (7.5
ml) and water (4.5 ml) was treated with lithium hydroxide
monohydrate (26 mg, 0.62 mmol) and stirred at room temperature for
16 h. The reaction was acidified to pH 1 with 10% hydrochloric acid
and extracted with DCM (2.times.20 ml). The combined organic layers
were dried over MgSO.sub.4 and the solvent evaporated in vacuo to
give a white solid which was triturated with EtOAc/hexane (1:1) to
give the title compound (176 mg, 76%). .delta. H (CDCl.sub.3) 7.52
(1H, m), 7.34-7.14 (11H, m), 7.05 (1H, m), 6.90 (2H, m), 5.18 (2H,
s), 5.04 (1H, m), 3.34 (1H, dd, J 5.6, 14.2 Hz), 3.24 (1H, dd, J
5.7, 14.2 Hz) and 2.34 (3H, s). m/z (ES+, 60V), 566 (MH.sup.+).
[0194] The following compounds were prepared in a similar manner
from the corresponding esters produced in a similar manner to
Intermediate 2:
[0195] b) (N-2-Chlorobenzoyl)-(O-2,6-dichlorobenzyl)-L-tyrosine
[0196] .delta. H (DMSO-d.sup.6), 8.71 (1H, d, J 8.2 Hz), 7.7-7.3
(7H, m), 7.23 (2H, d, J 8.4 Hz), 6.97 (2H, d, J 8.4 Hz), 4.58 (1H,
m), 3.2-2.8 (2H, m). m/z 478 (MH.sup.+)
[0197] c)
(N-2.4.6-Trimethylbenzoyl)-(O-2,6-dichlorobenzyl)-L-tyrosine
[0198] .delta. H (DMSO-d.sup.6), 8.46 (1H, d, J 7.3 Hz), 7.65-7.4
(3H, m), 7.25 (2H, d, J 8.6 Hz), 6.95 (2H, d, J 8.6 Hz), 6.76 (2H,
s), 5.19 (2H, s), 4.65 (1H, m), 3.3-3.05 (1H, m), 2.95-2.75 (1H,
m), 2.19 (3H, s) and 1.93 (6H, s); m/z (ESI, 60V) 486
(MH.sup.+).
[0199] d)
(N-2,6-Dimethoxybenzoyl)-(O-3,5-dichloro-4-pyridinyl)methyl]-L-t-
yrosine
[0200] The tyrosine starting material for coupling with acid to
produce the required ester for hydrolysis was obtained by reaction
of Intermediate 12 with N-Boc-L-tyrosine methyl ester in the
presence of sodium hydride.
[0201] .delta. H (DMSO-d.sup.6), 12.55 (1H, br s, CO.sub.2H), 8.72
(2H, s, pyr H), 8.15 (1H, d, J 7.8 Hz, NH), 7.29-7.22 (3H, m,
Ar--H), 6.98 (2H, d, J 8.6 Hz, Ar--H), 6.63 (2H, d, J 8.6 Hz,
Ar--H), 5.21 (2H, s, CH.sub.2O), 4.56 (1H, m, CH.alpha.), 3.65 (6H,
s, OMe), 3.30-2.90 (2H, m, CHCH.sub.2Ar); m/z (ESI, 60V) 505
(MH.sup.+).
[0202] e)
N-2-Carboxybenzoyl-[O-(3,5-dichloro-4-pyridinyl)methyl]-L-tyrosi-
ne
[0203] .delta. H (DMSO-d.sup.6), 12.8 (1H, br s, CO.sub.2H), 8.72
(2H, s, pyr-H), 8.63 (1H, d, J 8.0 Hz, NH), 7.72-7.69 (1H, m,
Ar--H), 7.57-7.47 (2H, m, Ar--H), 7.31 (1H, m, Ar--H), 7.24 (2H, d,
J 8.6 Hz, Ar--H), 6.97 (2H, d, J 8.6 Hz, Ar--H), 5.20 (2H, s,
CH.sub.2O), 4.55 (1H, m, CH.alpha.), 3.06-2.98 (2H, m,
CHCH.sub.2Ar); m/z (ESI, 60V) 489 (MH.sup.+).
EXAMPLE 3
[0204] a)
2-Carboxybenzoyl-(N'-3,5-dichloroisonicotinoyl)-L-4-aminophenyla-
lanine
[0205] Intermediate 4 (0.5 g, 0.94 mmol) was treated with lithium
hydroxide monohydrate (0.095 g, 2.26 mmol) in dioxane/H.sub.2O (30
ml, 2:1). After 30 min. glacial acetic acid was added (1 ml) and
the solvent removed in vacuo. Water was added to the residue and
the white solid filtered, washed well with H.sub.2O and dried in
vacuo to give the title compound as a white solid (0.18 g, 38%).
.delta. H (DMSO-d.sup.6) 10.84 (1H, s), 8.78 (2H, s), 8.68 (1H, d,
J 7.2 Hz), 7.71 (1H, dd), 7.58-7.50 (4H, m), 7.36-7.29 (3H, m),
4.61-4.56 (1H, m) and 3.13-2.98 (2H, m).
[0206] The following compounds were prepared in a similar manner
from the corresponding ester. Each ester was prepared using a
similar procedure to that described for the preparation of
Intermediate 4, using
(N-3,5-dichloroisonicotinoyl)-L-4-aminophenylalanine methyl ester
hydrochloride [see Intermediate 4] and the appropriate activated
acid:
[0207] b)
[N-(6-Carboxy-2-methoxyybenzoyl]-(N-3,5-dichloroisonicotinoyl)-L-
-4-aminophenylalanine
[0208] .delta. H (DMSO-d.sup.6), 10.85(1H, s, CO.sub.2H), 8.77 (2H,
s, ArCl.sub.2H), 8.22 (2H, d, J 7.8 Hz), 7.50-7.35 (2H, m),
7.35-7.20 (3H, m), 4.70-4.60 (1H, m, CH), 3.70 (3H, s, OMe), and
3.01 (2H, d, J 6.9 Hz, CH.sub.2); m/z (ESI, 60V) 532
(MH.sup.+).
[0209] c)
[N-(2-Phenoxy-6-carboxy)benzoyl]-(N'-3,5-dichloroisonicotinoyl)--
L-4-aminophenylalanine
[0210] .delta. H (DMSO-d.sup.6), 10.82 (1H, S, CO.sub.2H), 8.77
(2H, s, ArCl.sub.2H), 8.52 (1H, d, J 7.8 Hz), 7.64 (1H, d, J 7.7
Hz), 7.44 (2H, t, J 7.4 Hz), 7.36 (2H, t, J 7.9 Hz), 7.19 (2H, d, J
8.4 Hz), 7.12 (2H, t, J 7.13 Hz), 7.01 (2H, d, J 8.4 Hz), 4.68-4.61
(1H, m, CH) and 2.96 (2H, d, J 6.2 Hz, CH.sub.2); m/z (ESI, 60V)
594 (MH.sup.+).
[0211] d)
(N-2-Phenoxybenzoyl)-(N'-3,5-dichloroisonicotinoyl)-L-4-aminophe-
nylalanine
[0212] .delta. H (DMSO-d.sup.6), 10.78 (1H, brs, CO.sub.2H) 8.77
(2H, s, ArCl.sub.2H), 8.35 (1H, d, , 5.9 Hz), 7.92 (1H, dd, J 7.8,
1.8 Hz), 7.94-7.34 (6H, m, ArH), 7.21 (1H, t, J 7.5 Hz), 7.15 (1H,
t, J 7.2 Hz), 7.02 (2H, d, J 8.6 Hz), 6.95 (2H, d, J 8.6 Hz), 6.84
(1H, d, J 8.2 Hz), 4.20-4.13 (1H, m, CH), 3.16 (1H, dd, J 13.0, 5.3
Hz, CH.sub.AH.sub.B) and 3.01 (1H, dd, J 13.0, 4.3 Hz,
CH.sub.AH.sub.B); m/z (ESI, 60V) 550 (MH.sup.+).
[0213] e)
[N-(2-Carboxy-6-methyl)benzoyl]-(N'-3,5-dichloroisonicotinoyl)-L-
-4-aminophenylalanine
[0214] .delta. H (DMSO-d.sup.6), 8.78 (2H, s, ArCl.sub.2H), 8.49
(1H, d, J 7.6 Hz), 7.69 (1H, d, J 7.0 Hz), 7.55 (2H, d, J 8.3 Hz),
7.40-7.20 (4H, m), 4.72-4.60 (1H, m, CH), 3.11-2.93 (3H, m, Me) and
2.10 (2H, s, CH.sub.2); m/z (ESI, 60V) 516 (MH.sup.+).
[0215] f)
(N-2,6-Dimethoxybenzoyl)-(N'-3,5-dichloroisonicotinoyl)-L-4-amin-
ophenylalanine
[0216] .delta. H (DMSO-d.sup.6), 12.60 (1H, br s, NH), 10.86 (1H,
s, NH), 8.79 (2H, s, pyr-H), 8.21 (1H, d, J 8.1 Hz, Ar--H), 7.58
(2H, d, J 8.5 Hz, Ar--H), 7.29 (2H, d, J 8.4 Hz,Ar--H), 6.63 (2H,
d, J 8.4 Hz, Ar--H), 4.59 (1H, m, CH.alpha.), 3.66 (6H, s, OMe),
3.07 (1H, dd, J 13.8, 5.0 Hz, CHCH.sub.AH.sub.BAr) and 2.92 (1H,
dd, J 13.8, 9.0 Hz, CHCH.sub.AH.sub.BAr); m/z (ESI, 60V) 518
(MH.sup.+).
[0217] g) (N-3-Carboxybenzoyl)-N'-3,5-dichloroisonicotoninoyl)-4
aminophenyalanine
[0218] .delta. H (DMSO-d.sup.6), 12.95 (1H, br s, CO.sub.2H) 10.83
(1H, s, NH), 8.95 (1H, d, J 8.1 Hz, Ar--H), 8.77 (2H, s, pyr-H),
8.43 (1H, t, J 1.6 Hz, Ar--H), 8.06 (2H, m, Ar--H), 7.60 (1H, m,
Ar--H), 7.55 (2H, d, J 8.5 Hz, Ar--H), 7.32 (2H, d, J 8.5 Hz,
Ar--H), 4.64 (1H, m, CH.alpha.) and 3.10 (2H, m, CHCH.sub.2Ar); m/z
(ESI, 160V) 502 (MH.sup.+).
[0219] h)
(N-4-Carboxybenzoyl)-(N'-3,5-dichloroisonicotinoyl)L-4-aminophen-
ylalanine
[0220] .delta. H (DMSO-d.sup.6, 350K), 12.62 (1H, br s, CO.sub.2H),
10.60 (1H, s, NH), 8.72 (2H, s, pyr-H), 8.56 (1H, d, J 7.9 Hz, NH),
8.01 (2H, d, J 8.4 Hz, Ar--H), 7.88 (2H, d, J 8.4 Hz, Ar--H), 7.55
(2H, d, J 8.4 Hz, Ar--H), 7.31 (2H, d, J 8.4 Hz, Ar--H), 4.70 (1H,
m, CH.alpha.), 3.22 (1H, dd, J 14.0, 5.0 Hz, CHCH.sub.AH.sub.BAr)
and 3.11 (1H, dd, J 14.0, 9.5 Hz, CHC.sub.HAH.sub.BAr); m/z (ESI,
160V), 502 (MH.sup.+).
[0221] i)
(N-2-Carboxy-6-hydroxybenzoyl)-(N'-3,5-dichloroisonicotinoyl)-L--
4-aminophenylalanine
[0222] .delta. H (DMSO-d.sup.6), 10.84 (1H, s, NH), 8.87 (2H, s,
pyr-H), 8.52 (1H, d, J 8.0 Hz, Ar--H), 7.56 (2H, d, J 8.3 Hz,
Ar--H), 7.29-7.26 (3H, m, Ar--H), 6.94 (1H, d, J 8.3 Hz, Ar--H),
6.85 (1H, d, J 8.3 Hz, Ar--H), 4.454 (1H, m, CH.alpha.), 3.16-2.96
(2H, m, CHCH.sub.2Ar); m/z (ESI, 60V) 518 (MH.sup.+).
[0223] j)
(N-2-t-Butoxycarbonylbenzoyl)-(N'-3,5-dichloroisonicotinoyl)-L-4-
-aminophenylalanine
[0224] .delta. H (DMSO-d.sup.6), 10.84 (1H, s, NH), 8.78 (2H, s,
pyr-H), 8.62 (1H, m, NH), 7.64-7.44 (5H, m, Ar--H), 7.39-7.20 (3H,
m, Ar--H), 4.56 (1H, m, CH.alpha.), 3.16 (1H, m,
CHCH.sub.AH.sub.BS), 3.09-2.94 (1H, m, CHCH.sub.AH.sub.BAr) and
1.36 (9H, s, tBu); m/z (ESI, 60V) 580 (MH.sup.+).
EXAMPLE 4
[0225] a)
(N-2,6-Dimethoxybenzoyl)-4-(carboxamidobenzyl)phenylalanine A
solution of Intermediate 9 (1.07 g, 1.82 mmol) in 1,4-dioxane (100
ml) was treated with lithium hydroxide (168 mg, 4.0 mmol) and water
(20 ml). The reaction was stirred at room temperature for 2 h then
THF (50 ml) was added and stirring continued for a further 1 h. The
reaction was concentrated in vacuo and the residue partitioned
between EtOAc (50 ml) and water (50 ml). The aqueous layer was
washed with EtOAc.(50 ml) and the combined organic layers extracted
with water (100 ml). The combined aqueous layers were acidified to
pH 1 with 2.0M hydrochloric acid and extracted with EtOAc
(3.times.50 ml). The combined organic layers were dried
(Na.sub.2SO.sub.4) and concentrated in vacuo to give a white solid
(0.96 g) which was purified by crystallisation from
EtOAc/diisopropyl ether to give the title compound as a white solid
(0.56 g, 66%). .delta. H (DMSO-d.sup.6) 8.99 (1H, t, J 6.0 Hz, NH),
8.24 (1H, d, J 8.0 Hz, NH), 7.84 (2H, d, J 8.3, Ar--H), 7.38 (2H,
d, J 8.3 Hz, Ar--H), 7.33-7.30 (5H, m, Ar--H), 7.26 (1H, t, J 8.4
Hz, Ar--H), 6.63 (2H, d, J 8.4 Hz, Ar--H), 4.62 (1H, m, CH.alpha.),
4.48 (2H, d, J 6.0 Hz, NHCH.sub.2Ph), 3.64 (6H, s, OMe), 3.13 (1H,
dd, J 14.0, 4.9 Hz, CHCH.sub.AH.sub.BAr) and 2.99 (1H, dd, J 14.0,
9.0 Hz, CHCH.sub.AH.sub.BAr). m/z (ESI, 60V) 463 (MH.sup.+).
[0226] The following compounds were prepared in a similar manner to
the compound of Example 4a) by hydrolysis of the corresponding
ester. Each ester was prepared by reaction of Intermediate 8 with
the amine shown using a similar procedure to that described for the
preparation of Intermediate 9.
[0227] b)
(N-2,6-Dimethoxybenzoyl)-4-[carboxamido(3,5-dichlorobenzyl)]-phe-
nylalanine
[0228] from Intermediate 8 and 3,5-dichloroaniline. .delta. H
(DMSO-d.sup.6, 390K), 10.46 (1H, s, NH), 8.27; (1H, d, J 8.0 Hz,
NH), 7.91 (2H, d, J 1.9 Hz, Ar--H), 7.89 (2H, d, J 8.3 Hz, Ar--H),
7.46 (2H, d, J 8.3 Hz, Ar--H), 7.31 (1H, t, J 1.9 Hz, Ar--H), 7.26
(1H, t, J 8.4 Hz, Ar--H), 6.63 (2H, d, J 8.4 Hz, Ar--H), 4.64 (1H,
m, CH.alpha.), 3.65 (6H, s, OMe), 3.17 (1H, dd, J 14.0, 5.1 Hz,
CHCH.sub.AH.sub.BAr), and 3.03 (1H, dd, J 14.0, 9.0 Hz,
CHCH.sub.AH.sub.BAr). m/z (ESI, 60V) 518 (MH.sup.+).
[0229] c)
(N-2,6-Dimethoxybenzoyl)-4-(carboxamidomorpholyl)-phenylalanine
[0230] from Intermediate 8 and morpholine. .delta. H (DMSO-d.sup.6,
390K), 8.26 (1H, d, J 7.9 Hz, NH), 7.34 (4H, m, Ar--H), 7.26 (1H,
t, J 8.4 HZ), 6.62 (2H, d, J 8.4 Hz, Ar--H), 4.61 (1H, m,
CH.alpha.), 3.81-3.40(8H, m, CH.sub.2), 3.64(6H, s, OMe), 3.11 (1H,
dd, J 13.9, 4.75 Hz, CHCH.sub.AH.sub.BAr) and 2.98 (1H, dd, J 13.9,
9.0 Hz, CHCH.sub.AH.sub.BAr). m/z (ESI, 60V) 443 (MH.sup.+)
[0231] d)
(N-2,6-Dimethoxybenzoyl)-4-(carboxamidodiethyl)phenylalanine
[0232] from Intermediate 8 and diethylamine .delta. H
(DMSO-d.sup.6), 8.24 (1H, d, J 8.0 Hz, NH), 7.32 (5H, m, Ar--H),
6.63 (2H, d, J 8.4 Hz, Ar--H), 4.61 (1H, m, CH.alpha.), 4.11 (4H,
m, CH.sub.2CH.sub.3), 3.65 (6H, s, OMe), 334-2.99 (2H, m,
CHCH.sub.2Ar) and 1.08 (6H, t, J 7.1 Hz, CH.sub.2CH.sub.3). m/z
(ESI, 60V) 429 (MH.sup.+)
EXAMPLE 5
[0233]
[N-(4-Phenyl)benzoyl]-[O-(3,5-dichloro-4-pyridinyl)methyl]-L-tyrosi-
ne
[0234] To 4-phenylbenzoyl-L-tyrosine (0.75 g, 2 mmol) and DMF (20
ml) in a flask under N.sub.2 was added 2.1 equivalents of a 60%
dispersion of NaH (0.168 g).
[0235] This mixture was stirred at room temperature for 20 minutes
followed by the addition of Intermediate 12 (0.484 g, 2 mmol).
Stirring was continued for 2.5 h. The reaction was then quenched
with aqueous NaHCO.sub.3, the DMF was evaporated, EtOAc and water
were added and a white solid appeared which was filtered and washed
with water and Et.sub.2O. The white solid was passed through a
Dowex-56WX4-400 ion exchange resin, eluting with THF/acetonitrile
[50:50] and freeze dried to afford the title compound as a white
solid (0.3 g). .delta. H (DMSO-d.sup.6), 8.7 (2H, s), 7.95-7.35
(10H, m), 7.1 (2H, d, J 8.5 Hz), 6.85 (2H, d, J 8.5 Hz), 5.15 (2H,
s), 4.15 (1H, br s) and 3.31-3.05 (2H, m). m/z 521 (MH.sup.+)
EXAMPLE 6
[0236]
(N-3-Cyanobenzoyl)-(N'-3,5-dichloroisonicotinoyl)-L-4-aminophenylal-
anine
[0237] To Intermediate 4b) (0.174 g, 0.35 mmol) in THF (5 ml) and
water (5 ml) was added lithium hydroxide monohydride (22 mg). The
reaction was stirred at room temperature for 3 h. The THF was
evaporated, the pH adjusted to 4 and the solution extracted with
EtOAc, dried over MgSO.sub.4, filtered and evaporated to afford the
title compound as an off-white solid (0.137 g, 81%). .delta. H
(CD.sub.3OD), 8.55 (2H, s), 8.15-7.35 (6H, m), 7.25 (2H, d, J 7.02
Hz), 4.97 (1H, m) and 3.25-3.13 (2H, m). m/z (ES) 483
(MH.sup.+).
EXAMPLE 7
[0238]
[N-3-(1-H-Tetrazol-5-yl)]-(N'3.5-dichloroisonicotinoyl)-L-4-aminoph-
enylalanine
[0239] To Intermediate 10 (0.214 g, 0.396 mmol) in MeOH (5 ml) and
water (5 ml) was added lithium hydroxide monohydrate (25 mg) and
the reaction stirred at room temperature for 3 h. The meOH was
evaporated and the pH was adjusted to 6.5 using 1 M hydrochloric
acid. A white solid appeared, which was filtered and washed with
EtOAc. The solid was then passed through a Dowex-50WX4-400 ion
exchange column eluting with CH.sub.3CN/water (50:50) to afford the
title compound as a white solid (0.088 g, 42%). .delta. H
(CD.sub.3OD) 8.60 (2H, s), 8.40 (1H, s), 8.15 (1H, d, J 7.8 Hz),
7.9 (1H, d, J 7.8 Hz), 7.68-7.51 (3H, m), 7.3 (2H, d, J 8.5 Hz),
4.95-4.85 (1H, m) and 3.39-3.08 (2H, m). m/z (ES) 526
(MH.sup.+).
EXAMPLE 8
[0240]
[N-(3-Methoxycarbonylbenzoyl)]-(N'-3,5-dichloroisonicotinoyl)-L-4-a-
minophenylalnine
[0241] To Intermediate 4c) (0.39 g) in dry DCM (5 ml) was added
trifluoroacetic acid (1 ml) and the reaction stirred for 3 h.
Further trifluoroacetic acid (1.5 ml) and DCM (5 ml) were added and
the reaction stirred for a total of 6 h. The mixture was
evaporated, acetonitrile/water was added and freeze drying afforded
the title compound as a white solid (0.257 g, 60%). .delta. H
(CD.sub.3OD), 8.61 (2H, s), 7.85-7.03 (8H, m), 4.89 (1H, m), 3.69
(3H, s) and 3.1-3.0 (2H, m). m/z (ES) 516 (MH.sup.+).
[0242] The following assays can be used to demonstrate the potency
and selectivity of the compounds according to the invention. In
each of these assays an IC.sub.50 value was determined for each
test compound and represents the concentration of compound
necessary to achieve 50% inhibition of cell adhesion where
100%=adhesion assessed in the absence of the test compound and
0%=absorbance in wells that did not receive cells.
[0243] .alpha..sub.4.beta..sub.1 Integrin-Dependent Jurkat Cell
Adhesion to VCAM-Ig
[0244] 96 well NUNC plates were coated with F(ab).sub.2 fragment
goat anti-human IgG Fc.gamma.-specific antibody [Jackson Immuno
Research 109-006-098: 100 .mu.l at 2 .mu.g/ml in 0.1M NaHCO.sub.3,
pH 8.4], overnight at 40. The plates were washed (3.times.) in
phosphate-buffered saline (PBS) and then blocked for 1 h in PBS/1%
BSA at room temperature on a rocking platform. After washing
(3.times. in PBS) 9 ng/ml of purified 2d VCAM-Ig diluted in PBS/1%
BSA was added and the plates left for 60 minutes at room
temperature on a rocking platform. The plates were washed (3.times.
in PBS) and the assay then performed at 37.degree. for 30 min in a
total volume of 200 .mu.l containing 2.5.times.10.sup.5 Jurkat
cells in the presence or absence of titrated test compounds.
[0245] Each plate was washed (2.times.) with medium and the
adherent cells were fixed with 100 .mu.l methanol for 10 minutes
followed by another wash. 100 .mu.l 0.25% Rose Bengal (Sigma R4507)
in PBS was added for 5 minutes at room temperature and the plates
washed (3.times.) in PBS. 100 .mu.l 50% (v/v) ethanol in PBS was
added and the plates left for 60 min after which the absorbance
(570 nm) was measured.
[0246] .alpha..sub.4.beta..sub.7 Integrin-Dependent JY Cell
Adhesion to MAdCAM-Ig
[0247] This assay was performed in the same manner as the
.alpha..sub.4.beta..sub.1 assay except that MAdCAM-Ig (150 ng/ml)
was used in place of 2d VCAM-Ig and a sub-line of the .beta.-lympho
blastoid cell-line JY was used in place of Jurkat cells. The
IC.sub.50 value for each test compound was determined as described
in the .alpha..sub.4.beta..sub.1 integrin assay.
[0248] .alpha..sub.5.beta..sub.1 Integrin-Dependent K562 Cell
Adhesion to Fibronectin
[0249] 96 well tissue culture plates were coated with human plasma
fibronectin (Sigma F0895) at 5 .mu.g/ml in phosphate-buffered
saline (PBS) for 2 hr at 37.degree. C. The plates were washed
(3.times. in PBS) and then blocked for 1 h in 100 .mu.l PBS/1% BSA
at room temperature on a rocking platform. The blocked plates were
washed (3.times. in PBS) and the assay then performed at 37.degree.
C. in a total volume of 200 .mu.l containing 2.5.times.10.sup.5
K562 cells, phorbol-12-myristate-13-acetate at 10 ng/ml, and in the
presence or absence of titrated test compounds. Incubation time was
3b minutes. Each plate was fixed and stained as described in the
.alpha..sub.4.beta..sub.1 assay above.
[0250] .alpha..sub.M.beta..sub.2-Dependent Human Polymorphonuclear
Neutrophils Adhesion to Plastic
[0251] 96 well tissue culture plates were coated with RPMI 1640/10%
FCS for 2 h at 37.degree. C. 2.times.10.sup.5 freshly isolated
human venous polymorphonuclear neutrophils (PMN) were added to the
wells in a total volume of 200 .mu.l in the presence of 10 ng/ml
phorbol-12-myristate-13-a- cetate, and in the presence or absence
of test compounds, and incubated for 20 min at 37.degree. C.
followed by 30 min at room temperature. The plates were washed in
medium and 100 .mu.l 0.1% (w/v) HMB (hexadecyl trimethyl ammonium
bromide, Sigma H5882) in 0.05M potassium phosphate buffer, pH 6.0
added to each well. The plates were then left on a rocker at room
temperature for 60 min. Endogenous peroxidase activity was then
assessed using tetramethyl benzidine (TMB) as follows: PMN lysate
samples mixed with 0.22% H.sub.2O.sub.2 (Sigma) and 50 .mu.g/ml TMB
(Boehringer Mannheim) in 0.1M sodium acetate/citrate buffer, pH 6.0
and absorbance measured at 630 nm.
[0252] .alpha.IIb/.beta..sub.3-Dependent Human Platelet
Aggregation
[0253] Human platelet aggregation was assessed using impedance
aggregation on the Chronolog Whole Blood Lumiaggregometer. Human
platelet-rich plasma (PRP) was obtained by spinning fresh human
venous blood anticoagulated with 0.38% (v/v) tri-sodium citrate at
220.times.g for 10 min and diluted to a cell density of
6.times.10.sup.8/ml in autologous plasma. Cuvettes contained equal
volumes of PRP and filtered Tyrode's buffer (g/liter: NaCl 8.0;
MgCl.sub.2.H.sub.2O 0.427; CaCl.sub.2 0.2; KCl 0.2; D-glucose 1.0;
NaHCO.sub.3 1.0; NaHPO.sub.40.2H.sub.2O 0.065). Aggregation was
monitored following addition of 2.5 .mu.M ADP (Sigma) in the
presence or absence of inhibitors.
[0254] In the above assays the compounds of the invention generally
have IC.sub.50 values in the .alpha..sub.4.beta..sub.1 and
.alpha..sub.4.beta..sub.7 assays of 1 .mu.M and below. Compounds of
the Examples typically had IC.sub.50 values of 100 nM and below in
these assays and demonstrated selective inhibition of
.alpha..sub.4.beta..sub.1- . In the other assays featuring at
integrins of other subgroups the same compounds had IC.sub.50
values of 50 .mu.M and above thus demonstrating the potency and
selectivity of their action against .alpha..sub.4 integrins.
* * * * *