U.S. patent application number 09/964161 was filed with the patent office on 2002-03-21 for phenylalanine derivatives.
This patent application is currently assigned to Celltech Therapeutis, Limited. Invention is credited to Archibald, Sarah Catherine, Head, John Clifford, Porter, John Robert, Warrellow, Graham John.
Application Number | 20020035127 09/964161 |
Document ID | / |
Family ID | 26313022 |
Filed Date | 2002-03-21 |
United States Patent
Application |
20020035127 |
Kind Code |
A1 |
Head, John Clifford ; et
al. |
March 21, 2002 |
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; Het is an optionally substituted
heteroaromatic 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: |
Head, John Clifford;
(Maidenhead, GB) ; Archibald, Sarah Catherine;
(Maidenhead, GB) ; Warrellow, Graham John;
(Northwood, GB) ; Porter, John Robert; (Chinnor,
GB) |
Correspondence
Address: |
Francis A. Paintin, Esq.
WOODCOCK WASHBURN KURTZ MACKIEWICZ & NORRIS
46th Floor
One Liberty Place
Philadelphia
PA
19103
US
|
Assignee: |
Celltech Therapeutis,
Limited
|
Family ID: |
26313022 |
Appl. No.: |
09/964161 |
Filed: |
September 26, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09964161 |
Sep 26, 2001 |
|
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09237060 |
Jan 26, 1999 |
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Current U.S.
Class: |
514/317 ;
514/255.01; 514/423; 544/386; 546/226; 548/530 |
Current CPC
Class: |
A61P 37/02 20180101;
C07D 209/42 20130101; A61P 43/00 20180101; A61P 7/02 20180101; C07D
231/14 20130101; C07D 277/56 20130101; C07D 213/81 20130101; C07D
409/12 20130101; C07D 241/24 20130101; C07D 215/50 20130101; C07D
207/416 20130101; C07D 213/82 20130101; C07D 401/12 20130101; A61P
29/00 20180101 |
Class at
Publication: |
514/317 ;
514/255.01; 514/423; 544/386; 546/226; 548/530 |
International
Class: |
A61K 031/495; A61K
031/445; A01N 043/40; A61K 031/53 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 27, 1998 |
GB |
9801674.4 |
Dec 3, 1998 |
GB |
9826669.5 |
Claims
1. A compound of formula (1) 6wherein 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, heterocyclo-aliphatic,
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; Het is an optionally substituted
heteroaromatic group; and the salts, solvates, hydrates and
N-oxides thereof.
2. A compound according to claim 1 wherein Het is an optionally
substituted C.sub.3-5 monocyclic heteroaromatic group containing
one, two or three heteroatoms selected from oxygen, sulphur or
nitrogen atoms.
3. A compound according to claim 2 wherein Het is an optionally
substituted pyrrolyl or pyridyl group.
4. A compound according to claim 1 wherein R is a --CO.sub.2H
group.
5. A compound according to claim 1 wherein Alk.sup.2 is a
--CH.sub.2-- chain and is an integer 1.
6. A compound according to claim 1 wherein each of R.sup.2 and
R.sup.3 is a hydrogen atom.
7. A compound according to claim 1 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 1 wherein
--(Alk.sup.1).sub.r(L.sup.1).su- b.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).s- ub.s is a --CONH-- group.
11. A compound according to claim 1 which has the formula (1a):
7wherein --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 as
generally and particularly defined above, and Alk.sup.1, r,
L.sup.1, s, R.sup.a, R.sup.b, R and Het are as generally and
particularly defined above, and the salts, solvates, hydrates and
N-oxides thereof.
12. A compound according to claim 11 wherein Het is an optionally
substituted pyrrolyl or pyridyl group.
13. A compound according to claim 1 which is:
2-Thio(S-2,5-dimethoxyphenyl-
)nicotinoyl-(N-2,6-dichlorobenzoyl)-L-4-aminophenylalanine;
2-Thio(S-2,5-dimethoxyphenyl)nicotinoyl-(N-2,6-dichlorobenzoyl)-L-4-amino-
phenylalanine;
N-(3,5-Dichloro-4-picolyl)-N'-(3,5-dichloro-4-picolyl)-L-4--
aminophenylalanine;
N-(2-Chloronicotinoyl)-N'-(3,5-dichloro-4-picolyl)-L-4-
-amino-phenylalanine;
O-(2,6-dichlorobenzyl)-N-(4-acetyl-1,2,5-trimethyl-3-
-pyrroyl)-L-tyrosine;
(N'-3,5-Dichloroisonicotinoyl)-N-{([3-pyridinylmethy-
l]thio)isonicotinoyl}-L-4-aminophenylalanine;
N-(4-Acetyl-1,2,5-trimethyl--
1H-pyrrole-3-carbonyl)-N'-(3,5-dichloro-4-picolyl)-L-4-aminophenylalanine;
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.4.beta.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.4.beta.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.4.beta.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.4.beta.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.4.beta.1, binds to VCAM-1 and
fibronectin. In addition, .alpha.4.beta.7 binds to an adhesion
molecule believed to be involved in the homina of leukocytes to
mucosal tissue termed MAdCAM-1 [Berlin, C. et al. Cell, 74, 185,
(1993)]. The interaction between .alpha.4.beta.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.4.beta.1 and .alpha.4.beta.7 when they bind to their ligands
have been identified. .alpha.4.beta.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.4.beta.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.4.beta.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.4.beta.1
and/or .alpha.4.beta.7 at concentrations at which they generally
have no or minimal inhibitory action on .alpha. 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,
heterocycloaliphatic, 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] Het is an optionally substituted heteroaromatic group;
[0024] and the salts, solvates, hydrates and N-oxides thereof.
[0025] It will be appreciated that compounds of formula (1) 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. Formula (1)
and the formulae hereinafter are intended to represent all
individual isomers and mixtures thereof, unless stated or shown
otherwise.
[0026] In the compounds of formula (1), derivatives of the
carboxylic acid group R include carboxylic acid esters and amides.
Particular estes and amides include --CO.sub.2Alk.sup.4 and
--CON(R.sup.4).sub.2 groups as described herein.
[0027] When in the compounds of the invention 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).sup.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.
[0028] Alk.sup.2 in the compounds of the invention 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--.
[0029] 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.1-6alkyl group, e.g. a C.sub.1-3alkyl group such as
a methyl or ethyl group.
[0030] When Alk.sup.1 in compounds of formula (1) is an optionally
substituted aliphatic chain it may be an optionally substituted
C.sub.1-10 aliphatic chain. Particular examples include optionally
substituted straight or branched chain C.sub.1-6alkyl, C.sub.2-6
alkenyl, or C.sub.2-6 alkynyl chains.
[0031] 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.
[0032] 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. 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 groupss.
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.
[0033] Alkoxy groups represented by R.sup.1 in compounds of the
invention include straight of branched C.sub.1-6alkoxy groups such
as methoxy and ethoxy groups.
[0034] When R.sup.1 is present in compounds of formula (1) 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.
[0035] 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.
[0036] Optionally substituted polycycloaliphatic groups represented
by R.sup.1 include optionally substituted 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.
[0037] 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, isoxazinyl, oxathiazinyl, e.g. 1,2,5 or
1,2,6-oxathiazinyl, or 1,3,5-oxadiazinyl groups.
[0038] 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 tfifluoromethoxy, 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
[0039] In the compounds of formula (1), optionally substituted
aromatic groups represented by the group R.sup.1 include for
example monocyotic 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.sub.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.
[0040] Optionally substituted heteroaromatic groups, represented by
the group R.sup.1 or Het in compounds of formula (1) 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.
[0041] 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.
[0042] 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 on groups as
just defined.
[0043] 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.sub.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, -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.
[0044] Thus each of R.sup.a and R.sup.b and, where present,
substituents on R.sup.1 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.1-6alkylamino.sub.1-6alkyl, e.g. ethylaminoethyl,
C.sub.1-6dialkylaminoC.sub.1-6alkyl, e.g. diethylaminoethyl,
aminoC.sub.1-6alkoxy. e.g. amino ethoxy,
C.sub.1-6alkylaminoC.sub.1-6alko- xy, 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.1-6alkanoyl 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. methylaminocarbonyl 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
ethylaminocarbonylamino, C.sub.1-6dialkylaminocarbonylamino, e.g.
dimethylaminocarbonylamino or diethylaminocarbonylamino,
C.sub.1-6alkylaminocarbonylC.sub.1-6alkylamino, e.g.
methylaminocarbonylmethylamino, aminothiocarbonylamino,
C.sub.1-6dialkylaminothiocarbonylamino, e.g.
methylaminothiocarbonylamino or ethylaminothiocarbonylamino,
C.sub.1-6dialkylaminothiocarbonylamino, e.g.
dimethylaminothiocarbonylamino or diethylaminothiocarbonylamino,
C.sub.1-6alkylaminothiocarbonylC.sub.1-6alkylamino, e.g.
ethylaminothiacarbonylmethylamino, 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. methylaminosulphonylamino
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.
[0045] Optional substituents present on the heteroaromatic groups
represented by Het 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.rm, 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 heteoaryl 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, --N H(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.sub.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.
[0046] 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.
[0047] 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.
[0048] When R.sup.6a is a halogen atom it may be for example a
fluorine, chlorine, bromine, or iodine atom.
[0049] 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.
[0050] 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.1-8alkyl 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 C.sub.6-12aroyloxyC.sub.1-8alkyl group such as an optionally
substituted benzoyloxyethyl or benzoyloxypropyl group. Optional
substituents present on the Alk.sup.4 group include R.sup.6a
substituents described above.
[0051] 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.
[0052] Aryl or heteroaryl groups represented by the groups R.sup.6a
or R.sup.7 include mono- or bicyclic optionally substituted
C.sub.6-12 aromatic or C.sub.1-9 heteroaromatic groups as described
above for the groups R.sup.1 and Het.
[0053] 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.
[0054] 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.
[0055] 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, thiazolyloxy,
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. 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,
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.1-6alkanoyl 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,
optionally substituted phenylsulphonyl, aminosulphonyl
(--SO.sub.2NH.sub.2), C.sub.1-6alkylaminosulphonyl, e.g.
methylaminosulphonyl or ethylaminosulphonyl,
C.sub.1-6dialkylaminosulphonyl, e.g. dimethylaminosulphonyl or
diethylaminosulphonyl, phenylamino-sulphonyl, carboxamido
(--CONH.sub.2), C.sub.1-6alkylaminocarbonyl, e.g.
methylaminocarbonyl 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
ethylaminocarbonylamino, C.sub.1-6dialkylaminocarbonylamino, e.g.
dimethylaminocarbonylamino or diethylaminocarbonylamino,
C.sub.1-6alkylaminocabonylC.sub.1-6alkylamino, e.g.
methylaminocarbonylmethylamino, aminothiocarbonylamino,
C.sub.1-6alkylaminothiocarbonylamino, e.g.
methylaminothiocarbonylamino or ethylaminothiocarbonylamino,
C.sub.1-6dialkylaminothiocarbonylamino, e.g.
dimethylaminothiocarbonylamino or diethylaminothiocarbonylamino,
C.sub.1-6alkylaminothiocarbonylC.sub.1-6alkylamino, e.g.
ethylaminothiocarbonylmethylamino, --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.
benzyloxycarbonylaminoethyl, benzylthio, pyridylmethyithio or
thaizolylmethylthio groups.
[0056] In the above groups of particularly useful R.sup.6
substituents, the reference to optional substitution is intended to
relate primarily to the aromatic or heteroaromatic portions of the
groups described. Thus for example such groups may be optionally
mono-, di- or tri-substituted by those particular atoms or groups
described above for each of R.sup.a and R.sup.b.
[0057] 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.
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] Particularly useful salts of compounds according to the
invention include pharmaceutically acceptable salts, especially
acid addition pharmaceutically acceptable salts.
[0063] R in compounds of the invention is preferably a --CO.sub.2H
group.
[0064] When present, the aliphatic chain represented by Alk.sup.1
in compounds of the invention is preferably a --CH.sub.2--
chain.
[0065] 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.
[0066] R.sup.2 in compounds of formula (1) is preferably a hydrogen
atom.
[0067] R.sup.3 in compounds of the invention is preferably a
hydrogen atom.
[0068] In general in compounds of the invention
-(Alk.sup.1).sub.r(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--.
[0069] In general in compounds of the invention the group R.sup.1
is preferably an optionally substituted aromatic or heteroaromate
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.
[0070] The heteroaromatic group represented by Het in compounds of
formula (1) is preferably on optionally substituted C.sub.3-5
monocyclic heteroaromatic group containing one, two or three
heteroatoms selected from oxygen, sulphur or nitrogen atoms.
Particularly useful groups of this type include optionally
substituted pyrrolyl and pyridyl groups. Especially useful
heteroaromatic groups represented by Het include optionally
substituted 3- or 4-pyridyl groups, particularly 2-monosubstituted
3- or 4-pyridyl or 2,6-disubstituted 3- or 4-pyridyl groups. In
these, and in general in the group Het, the optional substituent
when present is preferably an atom or group R.sup.6 as defined
above.
[0071] A particularly useful class of compounds according to the
invention has the formula (1a) 3
[0072] wherein --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.su- p.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 Het are as generally and
particularly defined above, and the salts, solvates, hydrates and
N-oxides thereof.
[0073] It will be appreciated that the various preferences stated
above in relation to groups present in compounds of formula (1)
apply equally to the same groups when present in compounds of
formula (1).
[0074] Additionally, in the compounds of formula (1a)
-(Alk.sup.1).sub.r(L.sup.1).sub.s- is preferably a --CH.sub.2O or
--CON(R.sup.4)-- group and is especially a --CONH-- group. Het is
preferably an optionally substituted pyrrolyl or especially an
optionally substitued pyridyl group.
[0075] Particularly useful compounds of formula (1a) are those
wherein Het is a 2-monosubstituted 3- or 4-pyridyl group or a
2,6-disubstituted 3- or 4-pyridyl group.
[0076] 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(.sup.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.2,
--N(CH.sub.3).sub.2, --COCH.sub.3, --SCH.sub.3, --CO.sub.2H or
--CO.sub.2CH.sub.3 group.
[0077] Particularly useful compounds according to the invention
include the following:
[0078]
2-Thio(S-2,5-dimethoxyphenyl)nicotinoyl-(N-2,6-dichlorobenzoyl)-L-4-
-aminophenylalanine;
[0079]
2-Thio(S-2,5-dimethoxyphenyl)nicotinoyl-(N-2,6-dichlorobenzoyl)-L-4-
-aminophenylalanine;
[0080]
N-(3,5-Dichloro4-picolyl)-N'-(3,5-dichloro-4-picolyl)-L-4-aminophen-
ylalanine;
[0081]
N-(2-Chloronicotinoyl)-N'-(3,5-dichloro-4-picolyl)-L-4-amino-phenyl-
alanine;
[0082]
O-(2,6-dichlorobenzyl)-N-(4-acetyl-1,2,5-trimethyl-3-pyrroyl)-L-tyr-
osine;
[0083]
(N'-3,5-Dichloroisonicotinoyl)-N-{([3-pyridinylmethyl]thio)isonicot-
inoyl}-L-4-aminophenylalanine;
[0084]
N-(4-Acetyl-1,2,5-trimethyl-1H-pyrrole-3-carbonyl)-N'-(3,5-dichloro-
-4-picolyl)-L-4-aminophenylalanine; and the salts, solvates,
hydrates and N-oxides thereof.
[0085] Compounds according to the invention 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, such as the compounds of
formula (1a) herein, the compounds are advantageously selective
.alpha.4.beta.1 inhibitors.
[0086] The compounds are 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 and the invention extends to such a use and
to the use of the compounds for the manufacture of a medicament for
treating such diseases or disorders.
[0087] 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.
[0088] For the prophylaxis or treatment of disease the compounds
according to the invention 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.
[0089] Pharmaceutical compositions 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.
[0090] 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.
[0091] Preparations for oral administration may be suitably
formulated to give controlled release of the active compound.
[0092] For buccal administration the compositions may take the form
of tablets or lozenges formulated in conventional manner.
[0093] 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.
[0094] 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.
[0095] 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, trichlorofluoromethane,
dichlorotetrafluoroethan- e, carbon dioxide or other suitable gas
or mixture of gases.
[0096] The compositions may, it 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.
[0097] The quantity of a compound of the invention 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, 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.
[0098] The compounds of the invention 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, R.sup.1-R.sup.3, R.sup.a, R.sup.b, L.sup.1, Alk.sup.1,
Alk.sup.2, m, r, s and Het when used in the formulas 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 a compound
of formula (1) and the processes according to the invention
described hereinafter are to be understood to extend to such
removal of protecting groups.
[0099] Thus according to a further aspect of the invention, a
compound of formula (1) may be obtained by hydrolysis of an ester
of formula (2): 4
[0100] where R.sup.11 is an alkyl group.
[0101] The hydrolysis may be performed using either an acid or a
base depending on the nature of R.sup.11, 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.
[0102] Esters of formula (2) may be prepared by coupling an amine
of formula (3): 5
[0103] (where R.sup.11 is as just described) or a salt thereof with
an acid of formula (4):
HetCO.sub.2H (4)
[0104] or an active derivative thereof.
[0105] Active derivatives of acids of formula (4) include
anhydrides, esters and halides. Particular esters include
pentafluorophenyl or succinyl esters.
[0106] 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 dimethylaminopyridine, or a cyclic amine, such as
N-methylmorpholine.
[0107] 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'-dicyclohexylcarbodiimide, advantageously in the presence of a
catalyst such as a N-hydroxy compound e.g. a N-hydroxytriazole such
as 1-hydroxybenzotriazole. Alternatively, the acid may be reacted
with a chloroformate, for example ethylchloroformate, prior to
reaction with the amine of formula (3).
[0108] 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, existing substituents may be modified for example by
oxidation, reduction or cleavage reactions. Particular examples of
such reactions are given below. Where these are described in
relation to the generation of the group
R.sup.1(Alk.sup.1).sub.r(L.sup.1).sub.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 as appropriate.
[0109] 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.
trifluoromethylsulphonyloxy or arylsulphonyloxy, e.g.
p-toluenesulphonyloxy group.
[0110] 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.
[0111] 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).
[0112] 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.
[0113] 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.1OH (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.
[0114] 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 dioxan or an alkali metal
hydroxide, e.g. lithium hydroxide in an aqueous alcohol, e.g.
aqueous methanol.
[0115] 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.
[0116] 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 aluminum hydride or
sodium borohydride in a solvent such as methanol.
[0117] 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.
[0118] 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.
[0119] 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.
[0120] 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.
[0121] 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. tetrhydrofuran 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.
[0122] 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 electrophil, 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.
[0123] 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.
[0124] 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
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
selection hereinafter.
[0125] 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.
[0126] 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.
[0127] 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.
[0128] 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.
[0129] 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.
[0130] 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; TFA -
trifluoroacetic acid; DCM - dichloromethane; BOC -
tert-butoxycarbonyl; MeOH - methanol; tyr - tyrosine; HetAr -
heteroaryl; thiopro - thioproline; Me - methyl; DMSO -
dimethylsulphaxide; THF - tetrahydrofuran; NMM -
N-methylmorpholine; Ph - phenyl; EtOAc - ethyl acetate; LDA -
lithium diisoprapylamide Ar - aryl: pyr - pyridine; Bu - butyl; app
- apparent
[0131] INTERMEDIATE 1
[0132] 2-Chloronicotinoyl-O-(2,6-dichlorobenzyl)-L-tyrosine methyl
ester
[0133] A solution of O-(2,6-dichlorobenzyl)-L-tyrosine methyl ester
hydrochloride, (1.11 g, 2.84 mmol), 2-chloronicotinic acid (0.45 g,
2.84 mmol), EDC (0.60 g, 3.13 mmol), HOBT (0.46 g, 3.41 mmol) and
NMM (0.467 ml, 0.43 g, 4.26 mmol) in DCM (25 ml) was stirred at
room temperature for 24 h. The reaction mixture was partitioned
between DCM (50 ml) and 10% NaHCO.sub.3 solution (30 ml). The
organic layer was separated, dried over MgSO.sub.4 and the solvent
removed under vacuum to give a pale yellow solid that was
recrystalised from EtOAc/hexane to give the title compound as an
off white solid (1.14 g, 81%). .delta.H (CDCl.sub.3) 8.46 (1H, dd,
J 2.0, 4.71z), 8.05 (1H, dd, J 2.0, 7.6Hz), 7.34 (2H, m), 7.25 (2H,
m), 7.11 (2H, m), 6.96 (3H, m), 5.24 (2H, s), 5.06 (1H, m), 3.80
(3H, s) and 3.24 (2H, m).
[0134] INTERMEDIATE 2
[0135]
2-Thio(S-2,5-dimethoxyphenyl)nicotinoyl-O-(2,6-dichlorobenzyl)-L-ty-
rosine methyl ester
[0136] A solution of O(2,6-dichlorobenzyl)-L-tyrosine methyl ester
hydrochloride (0.78 g, 2.0 mmol), EDC (0.42 g, 2.2 mmol) HOBT (0.32
g, 2.4 mmol) and NMM (0.55 ml, 0.50 g, 5.0 mmol) in DMF (10 ml) was
treated with a solution of 2-thio(2,5-dimethoxyphenyl)nicotinic
acid (0.58 g, 2.0 mmol) in DMF (2 ml) and stirred for 16 h at room
temperature. Solvent was removed in vacuo and the residue was
partitioned between EtOAc (50 ml) and 10% hydrochloric acid (25
ml). The organic layer was separated, washed with 10% NaHCO.sub.3
solution (30 ml), dried over MgSO.sub.4 and the solvent removed in
vacuo to give a yellow oil which was purified by chromatography
(SiO.sub.2; EtOAc/hexane 1:1) to give the title compound as a white
foam (1.14 g, 86%). .delta.H (CDCl.sub.3) 8.39 (1H, dd, J 1.9,
4.7Hz), 7.85(1H, dd, J 1.9, 7.7Hz), 7.47 (1H, d, J 7.6HJz),
7.37-6.81 (11H, m), 5.18 (2H, s), 5.09 (1H, m), 3.78 (6H, s), 3.53
(3H, s) and 3.25 (2H, m).
[0137] INTERMEDIATE 3
[0138] 2-Mercaptonicotinoyl-O-(2,6-dichlorobenzyl)-L-tyrosine
methyl ester
[0139] A solution of O-(2,6dichlorobenzyl)-L-tyrosine methyl ester
hydrochloride (2.50 g, 6.4 mmol), 2-mercaptonicotinic acid (0.99 g,
6.4 mmol) and NMM (1.41 ml, 1.29 g, 12.8 mmol) in DMF (10 ml) was
stirred at room temperature for 64 h. Solvent was removed in vacuo
and the residue partitioned between DCM (30 ml) and water (25 ml).
The aqueous layer was extracted with DCM (30 ml) and the combined
organic layers were washed with 10% NaHCO.sub.3 solution (30 ml),
dried over MgSO.sub.4 and the solvent removed in vacuo to give a
brown oil which was purified by chromatography (SiO.sub.2; gradient
elution, 4:1 EtOAc/hexane to 100% EtOAc) to give the title compound
as a yellow foam, (2.92 g, 93%). .delta.H (CDCl.sub.3) 8.71 (1H,
dd, J 1.8, 7.6Hz), 8.05 (1H, s), 7.61 (1H, dd, J 1.8, 6.1Hz), 7.35
(2H, m), 7.33-7.19 (2H, m), 6.94 (2H, d, J 8.7Hz), 5.22 (2H, s),
4.97 (1H, m), 3.74 (3H, s) and 3.21 (2H, m).
[0140] INTERMEDIATE 4
[0141]
2-Thio(S4-picolinyl)nicotinoyl-O-(2,6-dichlorobenzyl)-L-tyrosine
methyl ester
[0142] A solution of Intermediate 3 (0.50 g, 1.0 mmol) and
4-picolyl chloride hyrochloride (0.17 g, 1.0 mmol) in DCM (10 ml)
was treated with 1,8-diazabicyclo[5.4.0]undec-7-ene (0.31 ml, 0.31
g, 2.0 mmol) and stirred at room temperature for 5 h. The reaction
was partitioned between water and DCM, the organic layer separated,
dried over MgSO.sub.4 and the solvent removed in vacuo to give a
yellow gum that was purified by chromatography (SiO.sub.2, EtOAc),
to give a pale yellow solid, which was recrystallised from
EtOAc/hexane (1:1) to give the title compound as an off white solid
(0.30 g, 52%). .delta.H (CDCl.sub.3) 8.47(2H, m), 7.69 (1H, dd, J
1.8, 77Hz), 7.38-7.22 (6H, m), 7.06 (3H, M), 6.90 (2H, m), 6.62
(1H, d, J 7.5Hz), 5.23 (2H, s), 5.03 (1H, m), 4.40 (2H, m), 3.79
(3H, s), 3.28 (1H, dd, 15.8, 14.1Hz) and 3.19 (1H, dd, J 5.4, 14.1
Hz).
[0143] INTERMEDIATE 5
[0144]
2-Thio(S-2,5-dimethoxyphenyl)nicotinoyl-L-4-aminophenylalanine
methyl ester
[0145] A solution of 4-amino-L-phenylalanine methyl ester
dihydrochloride (0.53 g, 2.0 mmol), EDC (0.42 g, 2.2 mmol) HOBT
(0.32 g, 2.4 mmol) and NMM (0.66 ml, 0.61 g, 6.0 mmol) in DMF (10
ml) was treated with a solution of
2-thio(2,5-dimethoxyphenyl)nicotinic acid (0.58 g, 2.0 mmol) in DMF
(2 ml) and stirred for 64 h at room temperature. The solvent was
removed in vacuo, and the residue partitioned between DCM (30 ml)
and water (20 ml). The organic layer was separated, washed with 10%
NaHCO.sub.3 (20 ml) solution, dried over MgSO.sub.4 and the solvent
evaporated in vacuo to give a brown gum which was purified by
chromatography (SiO.sub.2; EtOAc) to give the title compound as a
yellow foam (0.67 g, 72%). .delta.H (CDCl.sub.3) 8.35 (1H, dd, J
1.8, 4.8Hz), 7.81 (1H, dd, J 1.9, 7.7Hz), 7.41 (1H, d, J 7.6Hz),
7.12 (1H, d, J 3.0Hz), 7.07-6.81 (5H, m), 6.49 )2H, d, J 8.4Hz),
5.00 (1H, m), 3.76 (3H, s), 3.74 (3H, s, 3.55 (3H, s) and 3.14 (2H,
m).
[0146] INTERMEDIATE 6
[0147]
2-Thio(S-2,5-dimethoxyphenyl)nicotinoyl-(N-2,6-dichlorobenzoyl)-L-4-
-aminophenylalanine methyl ester
[0148] A solution of Intermediate 5 (0.68 g, 1.5 mmol) and NMM
(0.53 ml, 0.49 g, 4.8 mmol) in DCM (20 ml) was treated with
2,6-dichlorobenzoyl chloride (0.23 ml, 0.33 g, 1.6 mmol) and the
reaction stirred for 16 h at room temperature, then partitioned
between DCM (50 ml) and 10% NaHCO.sub.3 solution (30 ml). The
organic layer was separated, dried over MgSO.sub.4 and the solvent
evaporated in vacuo to give an off-white solid that was triturated
with EtOAc/diethyl ether (2:1) to give the title compound as an
off-white solid (0.44 g, 46%). .delta.H (MeOH-d.sub.4) 8.28 (1H,
dd, J 1.8, 4.9Hz), 7.70 (1H, dd, J 1.8, 7.6Hz). 7.58 (2H, d, J
8.6Hz), 7.48-7.38 (3H, m), 7.30 (2H, d, J 8.6Hz), 7.27 (1H, dd, J
4.9, 7.7Hz), 7.01 (1H, dd, J 1.1, 2.3Hz), 6.93 (2H, m), 4.87 (1H,
m), 3.77 (3H, s), 3.75 (3H, s), 3.60 (3H, s), 3.32 (1H, m) and 3.13
(H, dd, J 8.6, 14.0Hz).
[0149] INTERMEDIATE 7
[0150]
O(2,6-dichlorobenzyl)-N-(4-acetyl-1,2,5-trimethyl-3-pyrroyl)-L-tyro-
sine methyl ester
[0151] NMM (155 mg, 169 .mu.l, 1.54 mmol), HOBT (227 mg, 1.68
mmol), 4-acetyl-1,2,5-trimethylpyrrole-3-carboxylic acid (300 mg,
1.54 mmol) and EDC (295 mg, 1.54 mmol) were added sequentially to a
stirred solution of O(2,6-dichlorobenzyl)-L-tyrosine methyl ester
hydrochloride (546 mg, 1.40 mmol) in dry DMF (15 ml). The reaction
was stirred at room temperature under N.sub.2 for 18 h. The solvent
was removed in vacuo and the residue partitioned between EtOAc (50
ml), and 10% aqueous Na.sub.2CO.sub.3 (40 ml). The phases were
separated and the queous phase extracted with EtOAc (2.times.25
ml). The combined organic extracts were washed consecutively with
5% aqueous hydrochloric acid (20 ml), 10% aqueous Na.sub.2CO.sub.3
(20 ml) and brine (10 ml), dried (Na.sub.2SO.sub.4) and evaporated
in vacuo. The obtained orange foam (0.6 g) was chromatographed
(silica; 50% EtOAc/Hexane.fwdarw.100% EtOAc) affording the title
compound as a white foam (380 mg, 51%); .sup.1Hnmr (d.sup.6DMSO)
8.53 (1H, d, J 8Hz, NH), 7.57-7.43 (3H, m, s, aryl-H), 7.22 (2H, d,
J 8.5Hz), aryl-H), 6.95 (2H, d, J 8.5Hz, aryl-H), 5.18 (2H, br s,
CH.sub.2--O), 4.67 (1H, m, .alpha.-tyr-H), 3.66 (3H, s, Me--O),
3.34 (3H, s, Me--N), 3.12 (1H, dd, J 4.1, 13.8Hz,
CH.sub.AH.sub.BAr), 2.90 (1H, dd, J 11.3, 13.8Hz,
CH.sub.AH.sub.BAr), 2.32 (3H, s, MeCO), 2.0 (3H, s, pyrrole-Me) and
1.94 (3H, s, pyrrole-Me). m/z (ES+60V) 531 (MH.sup.+, 100), 533
(MH.sup.+, 75) 553 (MNa.sup.+, 15%).
[0152] INTERMEDIATE 8
[0153]
2-Chloronicotinyl-(N-2,6-dichlorobenzoyl)-L-4-aminophenylalanine
methyl ester
[0154] EDC (270 mg, 1.5 mmol) was added to a stirred solution of
(N-2,6-dichlorobenzoyl)-L-4-aminophenylalanine methyl ester (500
mg, 1.3 mmol), 2 chloronicotinic acid (200 mg, 1.3 mmol), HOBT (100
mg, 1.5 mmol) and NMM (423 .mu.l, 3.9 mmol) in anhydrous DMF (2 ml)
at 0.degree.. The DMF solution was stirred overnight at room
temeprature then the DMF was evaporated in vacuo. The residue was
taken up in DCM (50 ml), washed with water (3.times.10 ml),
saturated aqueous NaHCO.sub.3 (2.times.10 ml) and water (2.times.10
ml), dried (Na.sub.2SO.sub.4) and evaporated in vacuo. The residue
was purified by chromatography (SiO.sub.2; 1:1 EOAc:hexane) to give
the title compound as a white foam (450 mg, 80%). .delta.H
(CDCl.sub.3), 8.50 (1H, m, pyr H), 8.05 (1H, d, J 9.2Hz, pyrH),
7.58 (2H, d, J 9.5Hz, ArH), 7.46 (1H, brs, NH), 7.36-7.24 (3H, m,
2ArH, 1pyrH), 7.21 (2H, d, J 8.5Hz, 2 ArH), 7.12 (1H, d, J 6.6Hz,
NH), 5.05-5.18 (1H, m, CH.alpha.tyr), 3.81 (3H, s, CO.sub.2Me) and
3.39-3.18 (2H, m, CH.sub.2Ar).
[0155] INTERMEDIATE 9
[0156]
Methyl-2-thio(S-acetate)nicotinoyl-O-(2,6-dichlorobenzyl)-L-tyrosin-
e methyl ester
[0157] A solution of Intermediate 3 (370 mg, 0.76 mmol) in
anhydrous DMF (2 ml) was added to a suspension of sodium hydride
(60% in oil, 33 mg, 0.83 mmol) in anhydrous DMF (3 ml) at
0.degree.. The mixture was stirred for 10 min at room temperature,
then recooled to 0.degree.. Methyl bromoacetate (115 mg, 0.76 mmol)
was added dropwise, then the mixture was stirred overnight at room
temperature. The mixture was quenched with water (0.5 ml) and the
DMF evaporated in vacuo. The residue was dissolved in EtOAc, washed
with water (3.times.10 ml), dried (Na.sub.2SO.sub.4) and evaporated
in vacuo. The residue was purified by chromatography (SiO.sub.2;
EtOAc/hexane 1:1) to give the title compound as a white solid (360
mg, 85%). .delta.H (CDCl.sub.3) 8.45 (1H, m, pyrH), 7.71 (1H, d, J
7.7, pyrH), 7.35 (2H, d, J 7.3Hz, 2ArH), 7.28-6.9 (6H, m, 5ArH,
1pyrH), 6.71 (1H, d, J 7.5Hz, NH), 5.24 (2H, s, OCH.sub.2Ar),
5.08-5.03 (1H, m, CH.alpha. tyr), 3.96 (2H, s, SCH.sub.2), 3.78
(3H, s, CO.sub.2CH).sub.3, 3.71 (3H, s, CO.sub.2CH.sub.3), 3.31
(1H, dd, J 14, 5.4Hz, CH.sub.AH.sub.BAr) and 3.21 (1H, dd, J 14,
5.2Hz, CH.sub.AH.sub.BAr).
[0158] INTERMEDIATE 10
[0159] 2-Thio(S-methyl)nicotinoyl-O-(2,6dichlorobenzyl)-L-tyrosine
methyl ester
[0160] EDC (540 mg, 3 mmol) was added to a stirred solution of
(O-2,6-dichlorobenzyl)-L-tyrosine methyl ester (1 g, 2.6 mmol), 2
methylmercaptonicotinic acid (433 mg, 2.6 mmol), HOBT (364 mg, 2.6
mmol) and NMM (846 .mu.l, 7.8 mmol) in anhydrous DMF (4 ml) at
0.degree.. The DMF solution was stirred overnight at room
temperature, then the DMF was evaporated in vacuo. The residue was
taken up in DCM (70 ml), washed with water (3.times.15 ml),
saturated aqueous NaHCO.sub.3 (2.times.15 ml) and water (2.times.15
ml), dried (Na.sub.2SO.sub.4) and evaporated in vacuo. The residue
was purified by chromatography (SiO.sub.2; EtOAc) to give the title
compound as a white solid (1.2 gm, 92%). .delta.H (CDCl.sub.3) 8.42
(1H, dd, J 1.7, 4.8, 1pyrH)(, 7.67 (1H, dd, J 1.7, 7.6, 1pyrH),
7.31 (2H, d, J 8.5, ArH), 7.18 (1H, dd, J 2.3, 8.5, ArH), 7.09 (3H,
d, plus broad peak, J 8.6, 2ArH, 1NH), 6.97 (1H, m, 1pyrH), 6.88
(2H, d, J 8.6, 2ArH), 5.18 (2H, s, OCH.sub.2Ar), 4.96 (1H, m,
CH.alpha.tyr), 3.72 (3H, s, OCH.sub.3), 3.25-3.04 (2H, m,
CH.sub.2Ar) and 2.47 (3H, s, SCH.sub.3). m/z (ESI, GOU) 505
(MH.sup.+).
[0161] INTERMEDIATE 11
[0162] Ethyl
3-(4-{[(4-methoxybenzyl)oxy]carbonyl}phenyl)-2-[(diphenylmeth-
ylene)amino]proganoate
[0163] N-(Diphenylmethylene) glycine ethyl ester (8.6 g, 24.6 mmol)
and potassium carbonate (6.8 g, 49 mmol) were added to a solution
of 4-methoxybenzyl-4-(bromomethyl)benzoate (8.2 g, 24.6 mmol) in
acetonitrile (200 ml). The mixture was heated at reflux ovenight,
then filtered and the solvent removed in vacuo to give the title
compound as a yellow oil (13.55 g). .delta.H (CDCl.sub.3, 300 MHz)
7.8 (2H, d, J 9.0Hz), 7.5 (10H, m), 7.3 (2H, d, J 9.0Hz), 6.9 (2H,
d), 6.6 (2H, m), 5.23 (2H, s), 4.1 (3H, m), 3.7 (3H, s), 3.2 (2H,
m) and 1.2 (3H, m); m/z (ESI) 522 (MH.sup.+).
[0164] INTERMEDIATE 12
[0165] Ethyl 2-amino-3-(4{[(4-methoxybenzyl)oxy]carbonyl}phenyl)
propanoate
[0166] Hydrochloric acid (2M, 15.83 ml, 1.5 eq) was added to a
solution of Intermediate 11 (11.0 g, 21.12 mmol) in THF (30 ml).
After 20 min the reaction mixture was basified to pH7 with
NaHCO.sub.3 and the solvent removed in vacuo. The residue was taken
up in EtOAc (300 ml) and washed with water (200 ml) and brine (200
ml), dried (MgSO.sub.4) and evaporated in vacuo. Chromatography
(SiO.sub.2; EtOAc) gave the title compound as a yellow oil (4.91 g,
65%). .delta.H (CDCl.sub.3, 300MHz) 7.88 (2H, d, J 8.0Hz), 7.4 (4H,
dd), 7.0 (2H, d, J 8.0Hz), 5.25 (2H, s), 4.05 (2H, q), 3.7 (3H, s),
3.57 (1H, t), 2.87 (2H, m) and 1.1 (3H, t), m/z (ESI) 358
(MH.sup.+).
[0167] INTERMEDIATE 13
[0168]
Ethyl-2-{[(2-chloro-3-pyridinyl)carbonyl]amino}-3-(4-{[(4-methoxybe-
nzyl)oxy]carbonyl}phenyl)propanoate
[0169] EDC;HCl (591 mg, 3.08 mmol) and HOBT (416 mg, 3.08 mmol)
were added to a solution of Intermediate 12 (1.0 g, 2.8 mmol),
2-chloronicotinic acid (450 mg, 2.86 mmol) and NMM (370 .mu.l, 3.36
mmol) in DMF (30 ml). The reaction mixture was stirred overnight at
room temperature. The solvent was removed in vacuo and the residue
partitioned between EtOAc (300 ml) and NaHCO.sub.3 solution (300
ml). The organic phase was washed with citric acid (10%,
2.times.200 ml), NHCO.sub.3 solution (200 ml) and brine (300 ml),
dried (MgSO.sub.4) and concentrated in vacua to give the title
compound as a yellow oil (1.36 g, 98%). .delta.H (CDCl.sub.3,
300MHz) 9.12 (1H, d, J 8.0Hz), 8.47 (1H, m), 7.91 (2H, d, J 8.0Hz),
7.6 (1H, dd), 7.45 (5H, m), 6.95 (2H, d, J 8.0Hz), 4.67 (1H, m),
4.15 (2H, m), 3.75 (3H, s), 3.31 (1H, m), 3.23 (1H, m) and 1.17
(3H, s); m/z (ESI) 497 (MH.sup.+).
[0170] INTERMEDIATE 14
[0171] Ethyl
2-{[(2-chloro-3-pyridinyl)carbonyl]amino}-3-[4-(carboxyl)phen-
yl]propanoate
[0172] TFA (20 ml) was added to a solution of Intermediate 13 (1.36
g, 2.75 mmol) in toluene (20 ml). The reaction mixture was stirred
for 30 min at room temperature. The white solid obtained was
recrystallised (EtOAc/hexane) to give the title compound (1.04 g,
100%). .delta.H (CDCl.sub.3, 300MHz) 12.84 (1H, br s), 9.1 (1H, d,
J 8.0Hz), 8.47 (1H, m), 7.87 (2H, d, J 8.0HZ), 4.1 (2H, m), 3.2
(1H, m), 3.07 (1H, m) and 1.17 (3H, m); m/z (ESI) 377
(MH.sup.+).
[0173] INTERMEDIATE 15
[0174] Ethyl
2-{[(2-chloro-3-pyridinyl)carbonyl]amino}-3-(4-{(2,6-dichloro-
anilino)carbonyl}phenyl)propanoate
[0175] Carbon tetrachloride (5 ml) was added to a suspension of
Intermediate 14 (1.04 g, 2.76 mol) and triphenylphosphine (0.87 g,
3.31 mmol) in acetonitrile. The reaction mixture was stirred for 2
h at room temperature. 2,6-Dichloroaniline (0.89 g, 5.52 mmol) and
NMM (455 .mu.l, 4.14 mmol) were added and the mixture stirred for a
further 48 h at room temperature. The solvent was removed in vacuo
and the residue partitioned between water and EtOAc. The aqueous
layer was extracted with EtOAC (.times.2) and the combined organic
extracts washed with water (.times.2) and saturated aqueous
NaHCO.sub.3 (.times.2), dried (Na.sub.2SO.sub.4) and concentrated
in vacuo. Chromatography (SiO.sub.2; EtOAc/hexane 50:50) gave the
title compound (924 mg) .delta.H (CDCl.sub.3, 300MHz) 8.46 (1H, s),
8.05 (1H, d, J 7.4Hz), 7.89 (2H, d, J 7.9Hz), 7.69-7.10 (8H, m),
5.11 (1H, dt, J 6.7, 5.8Hz), 4.25 (2H, q, J 71Hz), 3.42 (1H, dd, J
13.9. 5.8Hz), 3.31 (1H, dd, J 13.9, 5.8Hz) and 1.30 (3H, t, J 7.1
Hz).
[0176] INTERMEDIATE 16
[0177] Methyl 4-[2(2,6-dichlorophenyl)-2-hydroxylethyl]benzoate
[0178] A solution of methyl 4-(bromomethyl)benzoate (2.0 g, 8.7
mmol) in THF (4.4 ml) was added slowly to cut zinc foil (683 mg,
10.44 mmol) which had been activated with 1,2-dibromoethane (80
mg). After 3 h of stirring at room temperature 2 ml of the solution
was transferred to a solution of copper cyanide (396 mg, 4.4 mmol)
and lithium chloride (356 mg, 8.4 mmol) in THF (4 ml) cooled to
-78.degree.. This solution was warmed to -20.degree. and then
cooled back to -78.degree.. Boron trifluoride etherate (983 .mu.l,
8 mmol) was then added followed by 2,6-dichlorobenzaldehyde (0.56
g, 3.2 mmol) in THF (1 ml). The reaction was stirred for 2 h and
then allowed to warm slowly to room temperature. Water (20 ml) was
then added and the reaction mixture extracted into EtOAc
(3.times.25 ml) and the combined organics dired (Na.sub.2SO.sub.4)
and evaporated. Purification by column chromatography (SiO.sub.2;
hexane:EtOAc. 4:1) gave the title compound as a colourless oil (863
mg, 83%). .delta.H (CDCl.sub.3) 7.95 (2H, m, ArH), 7.93-7.26 (4H,
m, ArH), 7.17-7.12 (1H, m, ArH), 5.73-5.65 (1H, m, CH), 3.91 (3H,
s, CO.sub.2Me), 3.43 (1H, dd, J 13.5, 8.4Hz, CH.sub.AH.sub.B) and
3.28 (1H, dd, J 13.5, 6.3Hz, CH.sub.AH.sub.B), m/z (ESI, 60V) 325
(MH.sup.+).
[0179] INTERMEDIATE 17
[0180] Methyl
4-[2-{[(tert-butyl(dimethyl)silyl]oxy}-2-(2,6-dichlorophenyl- )
ethyl]benzoate
[0181] To a solution of Intermediate 16 (20 g, 6.15 mmol) in DCM
(10 ml) cooled to 0.degree. was added 2,4,6-collidine (2.03 ml,
15.39 mmol). After 15 min
tert-butyldimethylsilyltrifluoromethanesulphonate (2.12 ml, 9.23
mmol) was added. The reaction mixture was stirred overnight at room
temperature then diluted with DCM (100 ml) and washed with 1M
hydrochloric acid (50 ml), water (50 ml) and brine (50 ml), dried
(Na.sub.2SO.sub.4) and evaporated under reduced pressure.
Purification by column chromatography (SiO.sub.2; hexane:EtOAc,
5:1) gave the title compound as a pale pink oil (2.67 g, 100%).
.delta.H (CDCl.sub.3) 7.94 (2H, d, J 6.5Hz, ArH), 7.33, 7.21 (4H,
m, ArH), 7.13-7.07 (1H, m, ArH), 5.58 (1H, dd, J 9.4, 4.6Hz, CH),
3.90 (1H, s, CO.sub.2Me), 3.46 (1H, dd, J 13.1, 9.4Hz,
CH.sub.AH.sub.B), 3.04 (1H, dd, J 13.1, 4.6Hz, CH.sub.AH.sub.B),
0.74 (9H, s, Si.sup.tBu), -0.31 (3H, s, SiMe) and -0.32 (3H, s,
SiMe); m/z (ESI, 60V) 361 (MH.sup.+).
[0182] INTERMEDIATE 18
[0183]
4-[2-{[Tert-butyl(dimethyl)silyl]oxy}-2-(2,6-dichlorophenyl)ethyl]b-
enzylalcohol
[0184] Lithium aluminium hydride (1M solution in THF, 6.46 ml, 6.46
mmol) was added to an ice cold solution of Intermediate 17 (2.67 g,
6.15 mmol) in THF (20 ml). The reaction mixture was stirred for 1 h
then quenched with the addition of water and extracted into DCM
(3.times.50 ml), dried (Na.sub.2SO.sub.4) and evaporated under
reduced pressure. Purification by column chromatography (SiO.sub.2;
hexane:EtOAc, 4:1) gave the title compound as a colourless oil (2.
g, 87%) .delta.H 7.32-7.06 (7H, m, ArH), 5.56 (1H, dd, J 9.3,
4.7Hz, CH.sub.2CH), 4.65 (2H, d, J 5.9Hz, CH.sub.2O), 3.39 (1H, dd,
J 13.2, 9.3Hz, CH.sub.AH.sub.B), 3.01 (1H, dd, J 13.2, 4.7Hz,
CH.sub.AH.sub.B), 0.75 (9H, s, Si.sup.tBu), -0.29 (3H, s, SiMe) and
-0.31 (3H, s, SiMe); m/z (ESI, 60V) 433 (MH.sup.+).
[0185] INTERMEDIATE 19
[0186]
4-[2-{[Tert-butyl(dimethyl)silyl]oxy}-2-(2,4-dichlorophenyl)ethyl]b-
enzylbromide
[0187] A solution of triphenylphosphine (843 mg, 3.21 mmol) in DCM
(2 ml) was added to a solution of carbon tetrabromide (1.42 g, 3.73
mmol) and Intermediate 18 (1.10 g, 2.67 mmol) in DCM (3 ml) and
stirred at room temperature for 24 h. Ether (100 ml) was added and
the solid precipitate formed removed by filtration. The filtrate
was evaporated under reduced pressure and the residue purified by
column chromatography (SiO.sub.2; 8:1, hexane:EtOAc) to give the
title compound (1.20 g, 95%). .delta.H (CDCl.sub.3) 7.32-7.07 (7H,
m, ArH0, 5.54 (1H, dd, J 9.5, 4.4Hz, CH.sub.2CH), 4.49 (2H, s,
CH.sub.2Br), 3.39 (1H, dd, J 13.2, 9.5Hz, CH.sub.AH.sub.B), 2.96
(1H, dd, J 13.2, 4.4Hz, CH.sub.AH.sub.B), 0.73 (9H, s, Si.sup.tBu),
-0.31 (3H, s, SiMe) and -0.32 (3H, s, SiMe); m/z (ESI, 60V) 474
(MH.sup.+).
[0188] INTERMEDIATE 20
[0189]
4-[2-{[Tert-butyl(dimethyl)silyl]oxy}-2-(2,6-dichlorophenyl)ethyl]p-
henylalanine ethyl ester
[0190] To a solution of ethyl N(diphonylmethylene)glycinate (2.63
g, 9.81 mmol) in THF (50 ml) cooled to -78.degree. was added
lithium diisopropylamine (2M in heptane/THF/ethylbenzene, 5.64 ml,
11.28 mmol). The solution was stirred for 45 min. Intermediate 19
(4.20 g, 8.92 mmol) in THF (20 ml) was then added dropwise. The
reaction mixture was stirred for 2 h at -78.degree. and then warmed
to room temperature. EtOAc (100 ml) was added and the mixture
washed with water (75 ml) and brine (75 ml), dried
(Na.sub.2SO.sub.4) and evaporated under reduced pressure. The
residue was then taken up in acidic ethanol and stirred for 10 min.
The volatiles were then removed and the residue partitioned between
EtOAc (150 ml) and saturated aqueous Na.sub.2CO.sub.3 (100 ml). The
aqueous layer was extracted several times with EtOAc and the
combined organics dried (Na.sub.2SO.sub.4) and evaporated under
reduced pressure. The remaining residue was purified by column
chromatography (SiO.sub.2; EtOAc) to give the title compound as a
colourless oil (3.95 g, 95%). .delta.H (CDCl.sub.3) 7.32-7.05 (7H,
m, ArH), 5.52 (1H, dd, J 9.4, 4.6Hz, CHOSi), 4.18 (2H, q, J 7.1Hz,
CO.sub.2CHCH.sub.3), 3.68 (1H, dd, J 7.9, 5.1Hz, CHNH.sub.2), 3.35
(1H, dd, J 13.2, 9.4Hz, CHOSiCH.sub.AH.sub.B), 3.07 (1H, dd, J
13.5, 5.1Hz, CHNH.sub.2CH.sub.AH.sub.B), 2.95 (1H, dd, J 13.2,
4.6Hz, CHOSiCH.sub.AH.sub.B), 2.95 (1H, dd, J 13.2, 4.6Hz,
CHOSiCH.sub.AH.sub.B), 2.82 (1H, dd, J 13.5, 7.9Hz,
CHNH.sub.2CH.sub.AH.sub.B), 1.53 (2H, br s, NH.sub.2), 1.27 (3H, t,
J 7.1 Hz, CO.sub.2CH.sub.2CH.sub.3), 0.74 (9H, s, Si.sup.tBu) and
-0.32 (6H, s, SiMe.sub.2); m/z (ESI, 60V) 496 (MH.sup.+).
[0191] INTERMEDIATE 21
[0192]
{4-[2-{[Tert-butyl(dimethyl)silyl]oxy}-2-(2,6-dichlorophenyl)ethyl}-
-(N-2-chloronicotinoyl) phenylalanine ethyl ester
[0193] To a solution of Intermediate 20 (1.50 g, 3.2 ommol) and
2-chloronicotinic acid (504 mg, 3.20 mmol) in DCM (75 ml) at room
temperature was added NMM (386 .mu.l, 3.53 mmol), EDC (675 mg, 3.53
mmol) and HOBT (477 mg, 3.53 mmol). The reaction mixture was
stirred overnight at room temperature and then diluted with DCM (50
ml) and washed with saturated aqueous Na.sub.2CO.sub.3 (50 ml),
water (50 ml) and brine (50ml), dried (Na.sub.2SO.sub.4) and
evaporated under reduced pressure. The residue was purified by
column chromatography (SiO.sub.2; EtOAc) to give the title compound
as a white solid (1.72 g, 85%). .delta.H (CDCl.sub.3) 8.41-8.38
(1H, m, NH), 8.01-7.96 (1H, m, ArH), 7.29-6.97 (9H, m, ArH), 5.47
(1H, dd, J 9.2, 4.7Hz, CHOSi), 5.24-4.96 (1H, m, CHNH), 4.19 (2H,
qd, J 7.1, 1.1 Hz, CO.sub.2CH.sub.2CH.sub.3), 3.34-3.14 (4H, m,
CH.sub.2.times.2), 1.27 (3H, td, J 7.1, 1.3Hz,
CO.sub.2CH.sub.2CH.sub.3), 0.69 (s) and 0.66 (s); together (9H,
Si.sup.tBu) and -0.38 (s) and -0.40 (s); together (6H, SiMe.sub.2);
659 (M+Na.sup.+).
[0194] INTERMEDIATE 22
[0195]
(N-2-Chloronicotinoyl)-4-[2-(2,6-dichlorophenyl)-2-hydroxyethyl]phe-
nylalanine ethyl ester
[0196] Tetrabutylammonium fluoride (1M in THF, 4.7 ml, 4.70 mmol)
was added to a solution of Intermediate 21 (1.50 g, 2.35 mmol) in
THF (75 ml) at room temperature. The reaction mixture was stirred
for 2 h and then the THF removed and the residue partitioned
between EtOAc and water. The layers were separated and the aqueous
layer extracted with EtOAc. The combined organics were dried
(Na.sub.2SO.sub.4) and evaporated under reduced pressure.
Purification of the residue by column chromatography (SiO.sub.2;
MeOH:DCM, 5:95) gave the title compound as a pals brown oil (1.03
g, 84%) .delta.H (CDCl.sub.3) 8.31-8.29 (1H, m, NH), 7.86-7.82 (1H,
m, ArH), 7.25-6.96 (9H, m, ArH), 5.54-5.49 (1H, m, CH), 4.94-4.88
(1H, m, CH), 4.11 (2H, qd, J 7.1, 2.0Hz, CO.sub.2CH.sub.2CH.sub.3),
3.28-3.03 (4H, m, 2.times.CH.sub.2) and 1.19 (3H, td, J 7.1, 1.1Hz,
CO.sub.2CH.sub.2CH.sub.3); m/z (ESI, 60V) 521 (MH.sup.+).
[0197] INTERMEDIATE 23
[0198]
(N-2-Chloronicotinoyl)-{4-[2-(2,6-dichlorophenyl)-2-oxoethyl]}pheny-
lalanine ethyl ester
[0199] To a solution of Intermediate 22 (300 mg, 0.58 mmol) in
acetone (20 ml) was added Jones' Reagent dropwise until an orange
colour persisted. i-Propyl alcohol was added to use up excess
reagent and then the solution was basified by the addition of
saturated aqueous Na.sub.2CO.sub.3 solution. The solution was then
decanted from the solids and the acetone removed in vacuo. The
remaining aqueous solution was then extracted with ether (.times.2)
and the combined organics dried (Na.sub.2SO.sub.4) and evaporated
under reduced pressure. The residue was purified by column
chromatography (SiO.sub.2; hexane:EtOAc, 3:2) to give the title
compound as a colourless oil (200 mg, 67%). .delta.H (CDCl.sub.3)
8.40 (1H, dd, J 4.8, 2.0Hz), 7.94 (1H, dd, J 7.7, 2.0Hz), 7.38-7.11
(8H, m, ArH), 7.02 (1H, d, J 7.5Hz, ArH), 4.19 (2H, q, J 7.2Hz,
CO.sub.2CH.sub.2CH.sub.3), 4.09 (2H, s, CH.sub.2CO), 3.28 (1H, dd,
J 14.0, 5.9Hz, CH.sub.AH.sub.B), 3.18 (1H, dd, J 14.0, 6.1Hz,
CH.sub.AH.sub.B) and 1.26 (5H, t, J 7.2Hz,
CO.sub.2CH.sub.2CH.sub.3); m/z (ESI, 60V) 519 (MH.sup.+).
[0200] INTERMEDIATE 24
[0201] Methyl 4-[(E)-2-(2,6-dichlorophenyl)ethenyl]benzoate
[0202] A solution of Intermediate 16 (2.0 g, 6.15 mmol) in toluene
(25 ml) containing p-toluenesulphonic acid (100 mg) was heated to
reflux in a Dean-Stark apparatus for 4 h. Toluene was then removed
under reduced pressure and the residue purified by column
chromatography (SiO.sub.2; hexane:EtOAc, 5:1) to give the title
compound as an off white solid (1.64 g, 87%); .delta.H (CDCl.sub.3)
8.05 (2H, d, J 8.3Hz), 7.60 (2H, d, J 8.3Hz, ArH), 7.36 (2H, d, J
8.0Hz), 7.21 (2H, d, J 1.7Hz), 7.15-7.10 (1H, m), and 3.93 (3H, s,
CO.sub.2CH.sub.3); m/z (ESI, 60V) 329 (MH.sup.+).
[0203] INTERMEDIATE 25
[0204] 4-[(E)-2-(2,6-Dichlorophenylethenyl]benzyl alcohol
[0205] To an ice cold solution of Intermediate 24 (1.56 g, 5.08
mmol) in THF (20 ml) was added lithium aluminium hydride (1M in
THF, 5.34 ml, 5.34 mmol). The reaction mixture was stirred for 30
min and then quenched by the addition of water (10 ml). The
resulting biphasic solution was filtered through Celite.RTM. and
then extracted with DCM (2.times.50 ml). The combined organics were
dried (Na.sub.2SO.sub.4) and evaporated under reduced pressure to
give the title compound as a colourless oil which solidified on
standing (1.5 g, 99%). .delta.H (CDCl.sub.3) 7.55 (2H, d, J 8.2Hz),
7.42-7.33 (4H, m), 7.20-7.08 (3H, m) and 4.75 (2H, d, J 5.3Hz,
CH.sub.2); m/z (ESI, 60V) 301 (M.sup.++Na.sup.+).
[0206] INTERMEDIATE 26
[0207] 4-[(E)-2-(2,6-Dichlorophenyl)ethenyl]benzyl bromide
[0208] A solution of triphenylphosphine (1.58 h, 6.04 mmol) in DCM
(10 ml) was added to a solution of Intermediate 25 (1.40 g, 5.03
mmol) and carbon tetrabromide (2.33 g, 7.04 mmol) in DCM (10 ml).
The resulting solution was stirred for 1 h and then diluted with
ether (150 ml) and the resulting solid removed by filtration. The
filtrate was then evaporated under reduced pressure and the
resulting residue purified by column chromatography (SiO.sub.2;
hexane: EtOAc, 6:1) to give the title compound as a colourless oil
(1 g, 49%). .delta.H (CDCl.sub.3) 7.55 (2H, d, J 8.3Hz), 7.44-7.32
(3H, m), 7.23-7.09 (4H, m) and 4.53 (2H, s, CH.sub.2); m/z (ESI,
60V) 342 (MH.sup.+)
[0209] INTERMEDIATE 27
[0210] 4-[(E)-2-(2,6-Dichlorophenyl)ethenyl]phenyalanine ethyl
ester
[0211] To a solution of ethyl N-(diphenylmethylene)glycinate (860
mg, 3.21 mmol) in THF (20 ml) cooled to -78.degree. was added
lithium diisopropylamine (2M in heptane/THF/ethylbenzene, 1.68 ml,
3.36 mmol). The solution was stirred for 45 min. Intermediate 26
(10 g, 2.92 mmol) in THF (2 ml) was added and the resulting
reaction mixture stirred for 2 h at -78.degree. and then warmed to
room temperature. EtOAc (100 ml) was added and the mixture washed
with water (75 ml) and brine (75 ml), dried (Na.sub.2SO.sub.4) and
evaporated under reduced pressure. The residue was then taken up in
acidic ethanol and stirred for 1 min. The volatiles were then
removed and the residue partitioned between EtOAc (150 ml) and
saturated aqueous Na.sub.2CO.sub.3 (100 ml). The aqueous layer was
extracted several times with EtOAc and the combined organics dried
(Na.sub.2SO.sub.4) and evaporated under reduced pressure. The
remaining residue was purified by column chromatography (SiO.sub.2;
EtOAc) to give the title compound as a colouress oil (860 mg, 81%).
.delta.H (CDCl.sub.3) 7.47 (2H, d, J 8.1 Hz), 7.32 (2H, d, J
8.1Hz), 7.21 (2H, d, J 8.2Hz), 7.19-6.98 (3H, m), 4.18 (2H, q, , J
7.1Hz, CO.sub.2CH.sub.2CH.sub.3), 3.71 (1H, dd, J 7.8, 5.3Hz, CH),
3.10 (1H, dd, J 13.5, 5.3Hz, CH.sub.AH.sub.B), 2.88 (1H, dd, J
13.5, 7.8Hz, CH.sub.AH.sub.B) and 1.26 (3H, t, J 7.1Hz,
CO.sub.2CH.sub.2CH.sub.3); m/z (ESI, 60V) 364 (MH.sup.+)
[0212] INTERMEDIATE 28
[0213]
(N-2-Chloronicotinoyl)-4-[(E)-2-(2,6-dichlorophenyl)ethenyl]phenyla-
lanine ethyl ester
[0214] To a solution of Intermediate 27 (860 mg, 2.36 mmol) and
2-chloronicotinic acid (372 mg, 2.36 mmol) in DCM (25 ml) was added
NMM (285 .mu.l, 2.60 mmol), EDC (498 mg, 2.60 mmol) and HOBT (352
mg, 2.60 mmol). The resulting solution was stirred for 3 h and then
diluted with DCM (50 ml) and brine (50 ml), dried
(Na.sub.2SO.sub.4) and evaporated under reduced pressure. The
residue was purified by column chromatography (SiO.sub.2; EtOAc) to
give the title compound as a pale yellow oil (1.1 g, 93%). .delta.H
(CDCl.sub.3), 8.43 (1H, dd, J 4.8, 2.0Hz), 8.02 (1H, dd, J 7.6,
2.0Hz), 7.46 (2H, d, J 8.2Hz), 7.34-6.92 (9H, m), 5.10-5.03 (1H, m,
CH), 4.24 (2H, q, J 7.1Hz, CO.sub.2CH.sub.2CH.sub.3), 3.33 (1H, dd,
J 13.9, 5.8Hz, CH.sub.AH.sub.B), 3.24 (1H, dd, J 13.9, 5.8Hz,
CH.sub.AH.sub.B) and 1.30 (3H, t, J 7.1 Hz,
CO.sub.2CH.sub.2CH.sub.3); m/z (ESI, 60V) 503.5 (MH.sup.+).
[0215] INTERMEDIATE 29
[0216] N-(2-Chloronicotinoyl)-L-tyrosine methyl ester
[0217] EDC.HCl (2.11 g, 11 mmol) was added to a mixture of
L-tyrosine methyl ester hydrochloride (2.32 g, 10 mmol),
2-chloronicotinic acid (1.58 g, 10 mmol), HOBT (1.49 g, 11 mmol)
and NMM (2.31 ml, 21 mmol) in DMF (50 ml). The mixture was stirred
overnight at room temperature. The solvent was removed in vacuo,
the residue dissolved in EtOAc (300 ml) and washed with dilute HCl
(100 ml), saturated aqueous NaHCO.sub.3 (100 ml), water
(3.times.100 ml) and brine (50 ml), dried (Na.sub.2SO.sub.4) and
solvent removed in vacuo to give the title compound as a yellow gum
(3.27 g, 9.8%). .delta.H (DMSO-d.sub.6, 300MHz) 9.21 (1H, s, OH),
9.03 (1H, d, J 7.9Hz, CONH), 8.45 (1H, dd, J 4.8, 1.9Hz, pyrH),
7.67 (1H, dd, J 7.4, 1.9Hz, pyrH), 7.47 (1H, dd, J 7.5, 4.8Hz,
pyrH), 7.05 (2H, d, J 8.5Hz, ArH), 6.67 (2H, d, J 8.5Hz, ArH), 4.58
(1H, ddd, J 9.6, 7.9, 5.4Hz, CH.alpha.), 3.65 (3H, s, CO.sub.2Me),
3.01 (1H, dd, J 13.9, 5.4Hz, CH.sub.AH.sub.BAr) and 2.85 (1H, dd, J
13.9, 9.6Hz, CH.sub.AH.sub.BAr), m/z (ESI, 60V) 335 (MH.sup.+).
[0218] INTERMEDIATE 30
[0219] N-(2-Chloronicotinoyl)-O-(2,6-dichlorobenzoyl)-L-tyrosine
methyl ester
[0220] A solution of Intermediate 29 (919 mg, 2.75 mmol) in DMF (10
ml) was added to a suspension of sodium hydride (60% in mineral
oil, 3.03 mmol, 121 mg) in DMF (20 ml) at 0.degree.. After 15 min,
2,6-dichlorobenzoyl chloride (414 .mu.l, 2.89 mmol) was added and
the mixture stirred for 2 h at room temperature. Water (.about.5
ml) was added and the solvent removed in vacuo. The residue was
dissolved i nEtOAc (150 ml), washed with water (3.times.50 ml) and
brine (50 ml), dried (Na.sub.2SO.sub.4) and evaporated in vacuo.
Chromatography (SiO.sub.2; DCM/MeOH, 98:2) gave the title compound
as a white foam (1.10 g, 79%). .delta.H (DMSC-d.sub.6, 300MHz) 9.12
(1H, d, 17.6Hz, CONH), 8.45 (1H, dd, J 4.8, 2.0Hz, pyrH), 7.69-7.58
(4H, m, pyrH+Cl.sub.2ArH.sub.3), 7.46 (1H, dd, J 7.5, 4.9Hz, pyrH),
7.43 (2H, d, J 8.4Hz, ArH.sub.2), 7.23 (2H, d, J 8.5Hz, ArH.sub.2),
4.73 (1H, m, CH.alpha.), 3.68 (3H, s, CO.sub.2Me), 3.22 (1H, dd, J
13.9, 5.2Hz, CH.sub.AH.sub.BAr) and 3.02 (1H, dd, J 13.9, 10.1Hz,
CH.sub.AH.sub.BAr); m/z (ESI, 60V) 507 (MH.sup.+).
[0221] INTERMEDIATE 31
[0222]
N-(2-Chloronicotinoyl)-N-methyl-N'-(3,5-dichloro-4-picolyl)-L-4-ami-
nophenylalanine methyl ester
[0223] 2-Chloronicotinoyl chloride (132 mg, 0.75 mmol) was added to
N-methyl-N'-(3,5-dichloroisonicotinyl)-L-4-aminophenylalanine
methyl ester [prepared from
N-Boc-N'-phthaloyl4-amino-L-phenylalanine methyl ester and methyl
iodide, followed by treatment with hydazine monohydrate and
reaction with 3,5-dichloroisonicotinyl choride with subsequent
removal of the Boc group] and NMM (165 .mu.l, 1.5 mmol) in DCM (10
ml). The mixture was stirred for 1 h at room temperature then
diluted with DCM (100 ml) and washed with dilute HCl (30 ml), dried
(Na.sub.2SO.sub.4) and evaporated in vacuo. Chromatography
(SiO.sub.2; EtOAc/hexane, 10:90) gave the title compound as a
colourless gum (380 mg, 97%). .delta.H (DMSO-d.sub.6, 300MHz, 405K)
10.35 (1H, br s, CONH), 8.87 (2H, s, Cl.sub.2pyrH), 8.42 (1H, t, J
3.4Hz, ClpyrH), 7.55 (2H, br d, J 7.2Hz, ArH), 7.45-7.15 (4H, v br
m, ArH+ClpyrH), 5.3 (1H, v br s, CH.alpha.), 3.74 (3H, s,
CO.sub.2Me), 3.4-3.3 (1H, br m, CH.sub.AH.sub.BAr), 3.16 (1H, dd, J
14.4, 9.6Hz, CH.sub.AH.sub.BAr) and 2.73 (3H, br s, NMe): m/z (ESI,
60V) 521 (MH.sup.+).
[0224] INTERMEDIATE 32
[0225]
[(S-2,5-dimethoxyphenyl)sulphonyl]nicotinoyl-O-(2,6-dichlorobenzyl)-
-L-tyrosine methyl ester
[0226] A solution of Intermediate 2 (0.79 g, 1.26 mmol) in DCM (50
ml) was treated with 3-chloroperoxybenzoic acid (2.17 g, 12.6 mmol)
and stored at 4.degree. for 48 h. The reaction was partitioned
between DCM (20 ml) and NaHCO.sub.3 solution (20 ml). The aqueous
layer was extracted with DCM (25 ml) and the combined organic
layers washed with 10% aqueous Na.sub.2SO.sub.3 (50 ml), dried
(MgSO.sub.4) and the solvent removed in vacuo to give a yellow foam
that was purified by chromatography (SiO.sub.2; EtOAc/hexane 3:1)
to give the title compound as a white foam, (0.50 g, 60%). .delta.H
(CDCl.sub.3) 8.58 (1H, dd, J 4.7, 1.7Hz, pyr-H), 7.95 (1H, dd, J
7.8, 1.7Hz, pyr-H), 7.70 (1H, d, J 3.2Hz, Ar--H), 7.50 (1H, dd, J
7.8, 4.7Hz, pyr-H), 7.38-7.11 (6H, m, Ar--H), 7.01 (1H, d, J 7.6Hz,
NH), 6.92-6.84 (3H, m, Ar--H), 5.21 (2H, s, CH.sub.2O), 5.10 (1H,
m, CH.alpha.), 3.85 (3H, s, OMe), 3.74 (3H, s, OMe), 3.49 (3H, s,
CO.sub.2Me) and 3.27 (2H, m, CHCH.sub.2Ar). m/z (ESI, 60V) 659
(MH.sup.+).
EXAMPLE 1
[0227] 2-Chloronicotinoyl-O-(2,6-dichlorobenzyl)-L-tyrosine
[0228] A solution of Intermediate 1 (0.20 g, 0.41 mmol) in THF (5
ml) and water (5 ml) was treated with lithium hydroxide monohydrate
(25 ml, 0.61 mmol. 1.5 equiv.) and stirred at room temperature for
1.5 h. The reaction was acidified to pH1 with 10% hydrochloric acid
to give a white precipitate which was isloated by filtration,
washed with water (5 ml) and diethyl ether (5 ml) and dried under
vacuum to give the title compound as a white powder (0.14 g, 72%).
.delta.H (DMSO-d.sup.6) 8.93 (1H, d, J 8.1Hz), 8.45 (1H, dd, J 2.0,
4.8Hz), 7.66 (1H, dd, J 2.0, 7.5Hz), 7.56 (2H, m), 7.47 (2H, m),
7.24 (2H, d, J 8.6Hz), 6.99 (2H, d, J 8.6Hz), 5.20 (2H, s), 4.60
(1H, d), 3.13 (1H, ABX, J 4.7, 13.9Hz) and 2.90 (1H, ABX, J 10.0,
13.9Hz). m/z (ES+, 60V) 479, 481 (MH.sup.+).
EXAMPLE 2
[0229]
2-Thio(S-2,5-dimethoxyphenyl)nicotinoyl-O-(2,6-dicholorobenzyl)-L-t-
yrosine hydrochloride
[0230] A solution of Intermediate 2 (0.35 g, 0.56 mmol) in THF (15
ml) and water (7.5 ml) was treated with lithium hydroxide
monohydrate (28 mg, 0.67 mmol) and stirred at room temperature for
16 h. The reaction was acidified to pH1 with 10% hydrochloric acid,
extracted with DCM (2.times.30 ml), and the combined organic layers
were dried over MgSO.sub.4, the solvent removed in vacuo to give a
gummy residue, which was purified by chromatography (SiO.sub.2;
7.5% MeOH/DCM) to give a gum which was dissolved in acetonitrile
(20 ml) and water (20 ml) and lyophylised to give the title
compound as a white powder (0.26 g, 71%). .delta.H (DMSO-d.sup.6)
8.71 (1H, m), 8.29 (1H, dd, J 1.7, 4.8Hz), 7.75 (1H, d, J 6.0Hz),
7.56-6.94 ( 11H, m), 5.16 (2H, s), 4.56 (1H, m) 3.68 (3H, s), 3.60
(3H, s) and 3.21-1.96 (2H,m). m/z (ES+60V) 613, 615 (MH.sup.+).
EXAMPLE 3
[0231] a)
2-Thio(S-4-picolinyl)nicotinoyl-O-(2,6-dichlorobenzyl)-L-tyrosin-
e
[0232] A solution of Intermediate 4 (0.30 g, 0.52 mmol) in THF (7.5
ml) and water (5 ml) was treated with lithium hydroxide monohydrate
(33 mg, 0.7 mmol) and stirred at room temperature for 16 h. The pH
was adjusted to 6.5-7 with 10% hydrochloric acid to give a yellow
precipitate which was isolated by filtration, washed with water and
dried in vacuo to give the title compound as a yellow powder (0.28
g, 95%). .delta.H (DMSO-d.sup.6) 8.83 (1H, d, J 8.0Hz), 8.50 (3H,
m), 7.75 (1H, d, J 7.6Hz), 7.56-7.20 (8H, m), 6.96 (2H, d, J
8.5Hz), 5.17 (2H, s), 4.54 (1H, m), 4.37 (2H, s), 3.12 (1H, m) and
2.97 (1H, m). m/z (ES+, 60V) 568, 570 (MH.sup.+).
[0233] The following compouns were prepared in a similar manner by
hydrolysis of the corresponding methyl ester. Each ester starting
material was obtained either by alkylation of Intermediate 3 or
alterntive mercaptopyridine using the reagents shown using a
similar procedure to that described for Intermediate 4:
[0234] b)
2-Thio-S-benzyl-nicotinoyl-(0-2,6-dichlorobenzyl)-L-tyrosine
[0235] ester from Intermediate 3 and benzyl chloride. .delta.H
(DMSO-d.sub.6) 8.78 (1H, d, J 8.1 Hz, pyr-H), 8.54 (1H, dd, J 4.8,
1.Hz, pyr-H), 7.69 (1H, dd, J 7.7, 1.7Hz, pyr-H), 7.68-7.17 (11H,
m, Ar--H), 6.94 (2H, d, J 8.6, Ar--H), 5.16 (2H, s, CH.sub.2O),
4.53 (1H, m, CH.alpha.), 4.34 (2H, s, CH.sub.2Ar), 3.10 (1H, dd, J
13.9, 4.6Hz, CHCH.sub.AH.sub.BAr), 2.94 (1H, dd, J 13.9, 10.2Hz,
CHCH.sub.AH.sub.BAr); m/z (ESI, 60V) 567 (MH.sup.+).
[0236] c)
2-Thio(S-4-Methylphenyl)-nicotinoyl-(0-2,6-dichorobenzyl-L-tyros-
ine
[0237] ester from Intermediate 3 and 4-methylbenzyl chloride.
.delta.H (DMSO-d.sub.6) 8.87 (1H, d, J 8.1Hz, pyr-H), 8.31 (1H, dd,
J 4.7, 1.8Hz, pyr-H), 7.70 (1H, dd, J 7.6, 1.7Hz, pyr-H), 7.54 (2H,
m, Ar--H), 7.45 (1H, m, Ar--H), 7.32-7.17 (7H, m, Ar--H), 6.98 (2H,
d, J 8.6Hz, Ar--H), 5.17 (2H, s, CH.sub.2O), 4.58 (1H, m,
CH.alpha.), 3.16 (1H, dd, J 14.6, 14.0Hz, CHCH.sub.AH.sub.BAr),
2.98 (1H, dd, J 14.0, 10.1 Hz, CHCH.sub.AH.sub.BAr) and 2.32 (3H,
s, Me); m/z (ESI, 60V) 569 (MH.sup.+).
[0238] d)
2-Thio-S-(3-picolyl)-nicotinoyl-(0-2,6-dichlorobenzyl)-L-tyrosin-
e
[0239] ester from Intermediate 3 and 3-picolyl chloride. .delta.H
(DMSO-d.sub.6) 9.04-8.54 (5H, m, Ar--H), 7.95-7.84 (2H, m, Ar--H),
7.58-7.43 (3H, m, Ar--H), 6.96 (2H, d, J 8.3Hz, Ar--H), 5.17 (2H,
s, CH.sub.2O), 4.55 (1H, m, CH.alpha.), 4.48 (2H, s, CH.sub.2pyr),
3.05 (2H, m, CHCH.sub.2Ar); m/z (ESI, 60V) 569 (MH.sup.+).
[0240] e)
N-[2-thio(S-3-picolinyl)nicotinoyl]-0-,2,6-dichlorobenzyl-L-tyro-
sine
[0241] ester from Intermediate 3 and 3-picoyl chloride using DBU as
base. .delta.H (DMSO-d.sub.6) 8.8 (1H, br d), 8.57 (1H, m), 8.50
(1H, m), 7.72 (2H, m), 7.55 (2H, d), 7.5-7.4 (2H, m), 7.3-7.15 (4H,
m), 6.95 (2H, d), 5.15 (2H, s), 4.55-4.45 (3H, m), 3.2-3.1 (1H, m),
3.0-2.9 (1H, m); m/z (ESI, 60V) 568 (MH.sup.+).
[0242] f)
N-2-Thio(S-4-butanoate)nicotinoyl]-(O-2,6-dichlorobenzyl)-L-tyro-
sine
[0243] ester from Intermediate 3 and methyl-4-chlorobutyrate using
K.sub.2CO.sub.3 as base. .delta.H (DMSO-d.sub.6) 8.76 (1H, d, J
8.1Hz), 8.49 (1H, dd, J 4.8, 1.7Hz), 7.61 (1H, dd, J 7.7, 1.7Hz),
7.55 (2H, d, L 8.9Hz), 7.45 (1H, m), 7.23 (2H, d, J 8.5Hz), 7.2
(1H, m), 6.96 (2H, d, J 8.5Hz), 5.18 (2H, s), 4.53 (1H, m),
3.17-2.87 (4H, m), 2.40-2.30 (2H, t, J 7.3Hz), 1.83 (2H, m); m/z
(ESI, 60V) 563 (MH.sup.+).
[0244] g)
(N'-3,5-Dichloroisonicotinoyl)-N-{([3-pyridinylmethyl]thio)
isonicotinoyl}-L-4-aminophenylalanine
[0245] ester from
2-mercaptoisonicotinoyl-(N'-3,5-dichloroisonicotinoyl)-L-
-4-aminophenylalanine and 3-picolyl chloride. .delta.H
(DMSO-d.sub.6) 10.86 (1H, s, CO.sub.2H), 8.92-8.75 (3H, m, ArH),
8.68-8.52 (2H, m), 8.39 (1H, br s), 7.75 (2H, t, J 7.5Hz), 7.56
(2H, d, J 6.3Hz), 7.39-7.18 (4H, m), 4.60-4.47 (1H, m, CH) and
3.25-2.92 (2H, m, CH.sub.2); m/z (ESI, 60V) 582 (MH.sup.+).
EXAMPLE 4
[0246] a)
2-Thio(S-2,5-dimethoxyphenyl)nicotinoyl-(N-2,6-dichlorobenzoyl)--
L-4-aminophenylalanine
[0247] A solution of Intermediate 6 (0.44 g, 0.69 mmol) in THF (7.5
ml) and water (5 ml) was treated with lithium hydroxide monohydrate
(43 mg, 1.0 mmol) and stirred at room temperature for 16 h, then
acidified to pH1 with 10% hydrochloric acid. The mixture was
extracted with DCM (2.times.30 ml) and the solvent evaporated in
vacuo to give an off-white solid that was triturated with boiling
MeOH to give the title compound as a white solid (210 mg, 49%).
.delta.H (DMSO-d.sup.6) 11.18 (1H, br s, CO.sub.2H), 10.66 (1H, s,
NH), 8.89 (1H, d, J 8.0Hz), 8.30 (1H, dd, J 1.6, 4.7Hz), 7.77 (1H,
dd, J 1.6, 7.6Hz), 7.62-7.45 (5H, m), 7.32 (2H, d, J 8.5Hz), 7.17
(1H, dd, 4.8, 7.6Hz), 6.96 (3H, m), 4.62 (1H, m), 3.70 (3H, s),
3.60 (3H, s) and 3.20-3.00 (2H, m). m/z (ES+) 626, 628
(MH.sup.+).
[0248] The following compounds were prepared in a similar manner to
the compound of Example 4a) by hydrolysis of the corresponding
methyl ester. Each ester was obtained by coupling the starting
materials shown according to the method described for Intermediate
6:
[0249] a)
2-Thio(S-2,5-dimethoxyphenyl)nicotinoyl-(N-2,6-dichlorobenzoyl)--
L-4-aminophenylalanine
[0250] from (N-2,6dichlorobenzoy)-L-4-aminophenylalanine methyl
ester and 3,5-dichloropyridyl-4-carbonyl chloride.
.delta.H(DMSO-d.sub.6) 8.64 (2H, s), 7.60-7.46 (5H, m), 7.24 (2H,
d, J 8.5Hz), 4.78-4.65 (1H, m), 3.22-2.85 (2H, m); .delta.H m/z
(ESI, 60V) 528 (MH.sup.+).
[0251] c)
(N'-2,4-Dimethylnictinoyl)-(N-2,6-dichlorobenzoyl)-L-4-aminophen-
ylalanine
[0252] from (N-2,6-dichlorobenzoyl)-L-4-aminophenylalanine methyl
ester and 2,4-dimethylpyridyl-4-carbonyl chloride. .delta.H
(DMSO-d.sub.6) 8.39 (1H, br d), 8.23 (1H, d, J 5.0Hz), 7.59-7.45
(6H, m), 7.26 (2H, d, J 8.4Hz), 7.01 (1H, d, J 5.1Hz), 4.65-4.52
(1H, m), 3.26-3.18 (1H, m), 2.95-2.84 (1H, m), 2.17 (3H, s), 2.01
(3H, s); m/z (ESI, 60V) 486 (MH.sup.+).
[0253] d)
N-(2,6-Dichloroisonicotinoyl)-O-(2,6-dichlorobenzyl)-L-tyrosine
[0254] from O-(2,6-dichlorobenzyl)-L-tyrosine methyl ester
hydrochloride and 2,6-dichloropyridyl-4-carbonyl chloride. .delta.H
(DMSO-d.sub.6) 9.2 (1H, d), 7.81 (2H, s), 7.5-7.3 (3H, m), 7.21
(2H, d, J 8.5Hz), 6.93 (2H, d, J 8.5Hz), 5.15 (2H, s), 4.65 (1H,
m), 3.28-3.15 (1H, m), 3.05-2.95 (1H, m); m/z (ESI, 60V) 513
(MH.sup.+).
[0255] e) N'-(2-nicotinoyl)-O-2,6-dichlorobenzyl)-L-tyrosine
[0256] from O-(2,6-dichlorobenzyl)-L-tyrosine methyl ester
hydrochloride and nicotinoyl chloride using triethylamine as base.
.delta.H (DMSO-d.sub.6) 9.0-8.85 (2H, m), 8.7 (1H, m), 8.14 (1H,
m), 7.55-7.41 (4H, m), 7.24 (2H, d, J 8.6Hz), 6.95 (2h, d, J
8.6Hz), 5.16 (2H, s), 4.59 (1H, m), 3.17-3.12 (1H, m), 3.04-2.96
(1H, m); m/z (ESI, 60V) 445 (MH.sup.+).
[0257] f)
N-(3,5-Dichloro-4-picolyl)-N'-(3,5-dichloro-4-picolyl)-L-4-amino-
phenylalanine
[0258] from (N-3,5-dichloro-4-picolyl)-L-4-aminophenylalanine
methyl ester and 3,5-dichlorophridyl-4-carbonyl chloride. .delta.H
(DMSO-d.sub.6, 300MHz) 9.26 (1H, d, J 8.3Hz), 8.79 (2H, s), 8.65
(2H, S), 7.57 (2H, d, J 8.4Hz), 7.30 (2H, d, J 8.4Hz), 4.70 (1H,m),
3.15 (1H, dd, J 14.1, 5.2Hz) and 2.93 (1H, dd, J 14.0, 9.3Hz); m/z
(ESI, 160V) 527 (MH.sup.+).
[0259] g)
N-(2-Chloronicotinoyl)-N'-(3,5-dichloro-4-picolyl)-L-4-aminophen-
ylalanine
[0260] from (N-3,5-dichloro-4-picolyl)-L-4-aminophenylalanine
methyl ester and 2-chloro-nicotinoyl chloride. .delta.H
(DMSO-d.sub.6, 300MHz)12.85 (1H, br s), 10.88 (1H,s), 8.97 (1H, d,
J 8.1 Hz), 8.79 (2H, s), 8.46 (1H, dd, J 4.8, 1.8Hz), 7.70 (1H, dd,
J 7.5, 1.8Hz), 7.59 (2H, d, J 8.4Hz), 7.48 (1H, dd, J 7.5, 4.8Hz),
7.30 (2H, d, J 8.4Hz), 4.63 (1H, m), 3.16 (1H, dd, J 13.9, 4.7Hz)
and 2.95 (1H, dd, J 13.8, 9.8Hz) m/z (ESI, 160V) 493
(MH.sup.+).
EXAMPLE 5
[0261] a)
O-(2,6-dichlorobenzyl)-N-(4-acetyl-1,2,5-trimethyl-3-pyrroyl)-L--
tyrosine
[0262] Intermediate 7 (360 mg, 0.68 mmol) was treated with
LiOH.H.sub.2O (34 mg, 0.81 mmol) in dioxane (6 ml), water (6 ml)
and MeOH (4 ml) at room temperature for 2 h. The solvent was
removed in vacuo and the obtained residue taken up in water. The pH
was made acidic by addition of a few drops of acetic acid and the
obtained precipitate filtered off with water washing affording the
title compound as a white amorphous powder (245 mg, 70%). .delta.H
(d.sup.6-DMSO) 8.37 (1H, d, J 8.2Hz, NH), 7.57-7.43 (3H, m's,
aryl-H), 7.23 (2H, d, J 8.6Hz, aryl-H), 6.95 (2H, d, J 87.6Hz,
aryl-H, 5.18 (2H, br s, CH.sub.2--O), 4.62 (1H, m, .alpha.tyr-H),
3.33 (3H, s, MeN, 3.13 (1H, dd, J 4.1, 13.8Hz, CH.sub.AH.sub.BAr),
2.32 (3H, s, MeCO), 2.01 (3H, s, pyrrole-Me) and 1.92 (3H, s,
pyrrole-Me). m/z (ES+, 60V), 517 (MH.sup.+, 100), 519 (MH.sup.+,
70).
[0263] The following compounds were prepared in a similar manner to
the compound of Example 5a) by hydrolysis of the corresponding
methyl ester. Each ester was obtained by coupling the starting
materials shown according to the method described for Intermediate
7:
[0264] b)
O-(2,6-dichlorobenzyl)-N-(4-actyl-3,5-dimethyl-2-pyrroyl)-L-tyro-
sine
[0265] from O-(2,6-dichlorobenzyl)-L-tyrosine methyl ester
hydrochloride and 3,5-dimethyl-4-acetylpyrrote2-carboxylic acid.
Freeze drying afforded the title compound as a light cream
amorphous solid (550 mg). .delta.H (DMSO-d.sub.6) 11.56, (1H, s),
7.61 (1H, d, J 7.8Hz), 7.51 (2H, d, J 8.0Hz), 7.41 (1H, t, J
8.0Hz), 7.21 (2H, d, J 85Hz), 6.96 (2H, d, J 8.5Hz), 5.16 (2H, s),
4.69-4.55 (1H, m), 3.12 (1H, dd, J 13.7, 4.7Hz), 2.99 (1H, dd, J
13.7, 9.1Hz), 2.43 (3H,s), 2.38 (3H, s), 2.31 (3H, s); m/z (ESI,
60V) 503 (MH.sup.+).
[0266] c)
O-(2,6-dichlorobenzyl)-N-(4-acetyl-2,5-dimethyl-3-pyrrolyl)-L-ty-
rosine
[0267] from O-(2,6-dichlorobenzyl)-L-tyrosine methyl ester
hydrochloride and 2,5-dimethyl-4-acetyl-pyrrole-3-carboxylic acid.
Feeze drying afforded the title compound as a white amorphous solid
(203 mg). .delta.H (DMSO-d.sub.6) 11.2 (1H, s), 8.83 (1H, d, J
8.0Hz), 7.56 (2H, app.d. J 8.0Hz), 7.45 (1H, app.t, J 8.0Hz), 7.21
(2H, d, J 8.5Hz), 6.95 (2H, d, J 8.5Hz), 5.17 (2H, s), 4.61-4.52
(1H, m), 3.11 (1H, dd, J 13.8, 4.3Hz), 2.86 (1H, dd, J 13.8,
10.4Hz), 2.31 (3H, s), 2.08 (3H, s), 2.07 (3H, s); m/z (ESI, 60V)
503 and 505 (MH.sup.+).
[0268] d)
O-(2,6-dichlorobenzyl)-N-(1-methyl-2-indolyl)-L-tyrosine
[0269] from O-(2,6-dichlorobenzyl)-L-tyrosine methyl ester
hydrochloride and 1-methylindole-2-carboxylic acid. Freeze drying
afforded the title compound as a white amorphous solid (220 mg).
.delta.H (DMSO-d.sub.6) 11.7 (1H, br s), 8.66 (1H, d, J 8.3Hz),
7.65 (1H, d, J 7.9MHz), 7.53 (2H, app.d, J 8l1Hz), 7.26 (1H,
obscured m), 7.09 (2H, app.t, J 7.5Hz), 6.96 (2H, d, J 8.5Hz), 5.16
(2H, s), 4.64-4.54 (1H, m), 3.89 (3H, s), 3.16 (1H, dd, J 13.8,
4.3Hz) and 3.00 (1H, dd, J 13.8, 10.4Hz); m/z (ESI, 60V) 497 and
499 (MH.sup.+).
[0270] e)
O-(2,6-dichlorobenzyl)-N-[2-(4-chlorophenyl)-3-(trifluoromethyl)-
-4-pyrazoyl]-L-tyrosine
[0271] from O-(2,6-dichlorobenzyl)-L-tyrosine methyl ester
hydrochloride and
2-(4-chlorophenyl)-3-(trifluoromethyl)-pyrazole-4-carboxylic acid.
The title compound was isolated as an off-white solid (220 mg).
.delta.H (DMSO-d.sub.6) 11.17 (1H, br s), 8.82 (1H, d, J 8.2Hz),
8.08 (1H, s), 7.65 (2H, d, J 8.1 Hz), 7.54 (4H, app. d, J 8.1Hz),
7.45 (1H, app. t, J 8.0Hz), 7.24 (2H, d, J 8.3Hz), 6.98 (2H, d, J
8.3Hz), 5.18 (2H, s), 4.61-4.51 (1H, m), 3.17 (1H, dd, J 13.8,
4.5Hz) and 2.94 (1H, dd, J 13.8, 9.0Hz); m/z (ESI, 60V) 527 and 529
(MH.sup.+).
[0272] f) O-(2,6-dichlorobenzyl)-N-(2-phenyl-4-thiazoyl)-L-tyrosine
from O-(2,6-dichlorobenzyl)-L-tyrosine methyl ester hydrochloride
and 2-phenyl-thiazole-4-carboxylic acid. Freeze-drying afforded the
title compound as a pale yellow amorphous solid (340 mg). .delta.H
(DMSO-d.sub.6) 11.2 (1H, br s), 8.40 (1H, d, J 8.1Hz), 8.31 (1H, s)
8.05-8.02 (2H, m), 7.55-7.51 (5H, m), 7.46-7.40 (1H, m), 7.23 (2H,
d, J 8.0Hz), 6.97 (2H, d, J 7.8Hz), 5.16 (2H, s), 4.71-4.64 (1H,
m), 3.18 (2H, app. d, J 6.6Hz); m/z (ESI, 60V) 527 and 529
(MH.sup.+).
[0273] g)
(N'-1-Methyl-5-nitropyrazolyl)-(N-2,6-dichlorobenzoyl)-L-4-amino-
phenylalanine
[0274] from (N-2,6-dichlorobenzoyl)-L-4-aminophenylalanine methyl
ester with N-methyl-5-nitropyrazole-4-carboxylic acid. .delta.H
(DMSO-d.sub.6) 10.64-(1H, s), 8.68 (1H, br d), 7.80 91H, s),
7.64-7.48 (6H, m), 7.19 (2H, d, J 8.5Hz), 4.59-4.49 (1H, m), 4.05
(3H, s), 3.18-2.9 (2H, m); m/z (ESI, 60V) 506 (MH.sup.+).
[0275] h) N-(2-Methylnicotinoyl)-O-(2,6-dichlorobenzyl)
L-tyrosine
[0276] from O-(2,6-dichlorobenzyl)-L-tyrosine methyl ester
hydrochloride and 2-methylnicotinic acid. .delta.H (DMSO-d.sub.6)
8.73 (1H, br d), 8.48 (1H, m), 7.7-7.46 (4H, m), 7.4-7.23 (3H, m),
7.00 (2H, d, J 8.4Hz), 5.2) (2H, s), 4.60 91H, m), 3.27-3.12 (1H,
m), 3.0-2.82 (1H, m); m/z (ESI, 60V) 509 (MH.sup.+).
[0277] i)
(N'-2-Chloronicotinoyl)-(N-benzoyl)-L-4-aminophenylalanine
[0278] from (N-benzoyl)-L-4-aminophenylalanine methyl ester and
2-chloronicotinic acid. .delta.H (DMSO-d.sub.6) 10.19 (1H, s), 8.96
(1H, d, J 8.2Hz), 8.46 (1H, dd, J 4.8, 1.9Hz), 7.96 (2H, dd, J 6.7,
1.7H), 7.75-7.65 (3H, m), 7.62-7.45 (4H, m), 7.25 (2H, d, J 8.5Hz),
4.6 (1H, m), 3.2-3.12 (1H, m), 3.0-2.89 (1H, m), m/z (ESI, 60V) 424
(MH.sup.+).
[0279] j)
N'-(Quinoline-4-carbonyl)-O-(2,6-dichlorobenzyl)-L-tyrosine
[0280] from O-(2,6-dichlorobenzyl)-L-tyrosine methyl ester
hydrochloride and 4-quinoline carboxylic acid. .delta.H
(DMSO-d.sub.6) 9.0 (1H, d), 8.94 (1H, d, J 4.3), 8.06 (1H, d, J
8.2Hz), 7.79 (2H, m), 7.62-7.42 (4H, m), 7.34 (1H, d, J 4.3Hz),
7.34 (2H, d, J 8.7Hz), 7.00 (2H, d, 48.7Hz), 5.22 (2H, s), 4.72
(1H, m), 3.29-3.19(1H, m), 3.0-2.88 (1H, m), m (ESI, 60V) 495
(MH.sup.+).
[0281] k)
N'-(2-Phenoxynicotinoyl)-O-(2,6-dichlorobenzyl)-L-tyrosine
[0282] from O-(2,6-dichlorobenzyl)-L-tyrosine methyl ester
hydrochloride and 2-phenoxynicotinic acid. .delta.H (DMSO-d.sub.6)
8.55 (1H, d), 8.18 (2H, m), 7.6-7.4 (5H, m), 7.3-7.15 (6H, m), 6.71
(2H, d), 5.1 (2H, s), 4.65 (1H, m), 3.22-3.0 (2H, m); m/z (ESI,
60V) 537 (MH.sup.+).
[0283] l)
N-(Pyridine-2-carbonyl)-O-(2,6-dichlorobenzyl)-L-tyrosine
[0284] from O-(2,6-dichlorobenzyl)-L-tyrosine methyl ester
hydrochloride and 2-picolinic acid. .delta.H (DMSO-d.sub.6) 8.8-8.6
(2H, m), 8.0 (2H, m), 7.7-7.4 (4H, m), 7.14 (2H, d, J 8.7Hz), 6.92
(2H, d, J 8.7Hz), 5.15 (2H, s), 4.72 (1H, m), 3.17 (2H, m); m/z
(ESI, 60V) 445 (MH.sup.+).
[0285] m)
N-(Pyridine-4-carbonyl)-O-(2,6-dichlorobenzyl)-L-tyrosine
[0286] from O-(2,6-dichlorobenzyl)-L-tyrosine methyl ester
hydrochloride and isonicotinic acid. .delta.H (DMSO-d.sub.6) 8.68
(2H, dd, J 4.5, 1.6Hz), 8.4 (1H, d, J 7.2Hz), 7.63 (2H, dd, J 4.5,
1.6Hz), 7.6-7.4 (3H, m), 7.15 (2H, d, J 8.6Hz), 6.87 (2H, d, J
8.6Hz), 5.14 (2H, s), 4.31 (1H, m), 3.2-2.96 (2H, m); m/z (ESI,
60V) 445 (MH.sup.+).
[0287] n)
N-(2-Hydroxynicotinoyl)-O-(2,6-dichlorobenzyl)-L-tyrosine)
[0288] from O-(2,6-dichlorobenzyl)-L-tyrosine methyl ester
hydrochloride and 2-hydroxynicotinic acid. .delta.H (DMSO-d.sub.6)
10.15 (1H, d, J 7.4Hz), 8.31 (1H, dd, J 7.1, 2.0Hz), 7.71 (1H, br
s), 7.65-7.45 (3H, m), 7.16 (2H, d, J 8.4Hz), 6.97 (2H, d, J
8.4Hz), 6.46 (1H, t, J 6.8Hz), 5.17 (2H, s), 4.68 (1H, m), 3.2-3.0
(2H, m); m/z (ESI, 60V) 461 (MH.sup.+).
[0289] o)
(N'-2-Aminonicotinoyl)-(N-2,6-dichlorobenzoyl)-L-4-aminophenylal-
anine
[0290] from (N-2,6-dichlorobenzoyl)-L-4aminophenylalanine methyl
ester and 2-aminonicotinic acid. .delta.H (DMSO-d.sub.6) 10.64 (1H,
s), 8.55 (1H, d), 8.06 (1H, m), 7.8 (1H, d), 7.7-7.4 (4H, m), 7.28
(2H, d, J 8.5Hz), 6.9 (2H, br s), 6.5 (1H, m), 4.52(1H, m),
3.2-2.9(2H, m); m/z (ESI, 60V) 473 (MH.sup.+).
[0291] p)
(N'-2-Hydroxynicotinoyl)-(N-3,5-dichloroisonicotinoyl)-L-4-amino-
phenylalanine
[0292] from (N-3,5dichloroisonicotinoyl)-L-4-aminophenylalanine
methyl ester and 2-hydroxynicotinic acid. .delta.H (DMSO-d.sub.6)
10.14 (1H, d, J 7.6Hz), 8.78 (2H, s), 8.31 (1H, dd, J 7.2, 2.2Hz),
7.70(1H, br m), 7.57 (2H, d, J 8.5Hz), 7.20 (2H, d, J 8.5Hz), 6.48
(1H, m), 4.69 (1H, m), 3.20-2.95 (2H, m); m/z (ESI, 60V) 475
(MH.sup.+).
[0293] q)
(N'-2-Methylnicotinoyl)-(N-3,5-dichloroisonicotinoyl)-L-4-aminop-
henylalanine
[0294] from (N-3,5-dichloroisonicotinoyl)-L-4-aminophenylalanine
and 2-methylnicotinic acid. .delta.H (DMSO-d.sub.6) 8.78 (2H, s),
8.60 (1H, br d), 8.48 (1H, dd, J 4.8, 1.6Hz), 7.7-7.5 (3H, m),
7.4-7.2 (3H, m), 4.5 (1H,), 3.3-2.85 (2H, m), 2.34 (3H, s); m/z
(ESI, 60V) 473 (MH.sup.+).
[0295] r)
(N'-2,6-Dichlorobenzoyl)-(N-2-phenoxynicotinoyl)-L-4-aminophenyl-
alanine
[0296] from (N-2,6-dichlorobenzoyl)-L-4-aminophenylalanine methyl
ester and 2-phenoxynicotinic acid. .delta.H (DMSO-d.sub.6) 10.61
(1H, s), 8.56 ( H, d, J 7.5Hz), 8.22-8.17 (2H, m), 7.59-7.39 (8H,
m), 7.27-7.11 (7H, m), 4.72-4.84 (1H, m, CH), 3.16 (1H, dd, J 13.7,
4.9Hz, CH.sub.AH.sub.B) and 3.00 (1H, dd, J 13.7, 7.8Hz,
CH.sub.AH.sub.B); m/z (ESI, 60V) 550 (MH.sup.+).
[0297] s)
(N'3,5-Dichloroisonicotinoyl)-(N-2-phenoxynicotinoyl)-L-4-aminop-
henylalanine
[0298] from (N-3,5-dichloroisonicotinoyl)-L-4-aminophenylalanine
methyl ester and 2-phenoxynicotinic acid. .delta.H (DMSO-d.sub.6)
10.79 (1H, s), 8.78 (2H, s, ArH), 8.57 (1H, d, J 7.6Hz), 8.25-8.13
(2H, m), 7.46-7.35 (4H, m), 7.29-7.05 (6H, m), 4.73-4.62 (1H, m,
CH), 3.17 (1H, dd, J 13.7, 4.7Hz, CH.sub.AH.sub.B) and 3.09 (1H,
dd, J 13.7, 7.8Hz, CH.sub.AH.sub.B); m/z (ESI, 60V) 551
(MH.sup.+).
[0299] t)
N-(4-Acetyl-1,2,5-trimethyl-1H-pyrrole-3-carbonyl)-N'-(3,5-dichl-
oro-4-picolyl)-L-4-aminophenylalanine
[0300] from (N-3,5-dichloro-4-picolyl)-L-4-aminophenylalanine
methyl ester and 4-acetyl-1,2,5-trimethyl-1H-pyrrole-3-carboxylic
acid. .delta.H (DMSO-d.sub.6, 300MHz) 400K) 12.72 (1H, brs), 10.83
(1H,s), 8.80 (2H, s), 8.42 (1H, d, J 8.4Hz), 7.54 (2H, d, J 8.5Hz),
7.30 (2H, d, J 8.4Hz), 4.66 (1H, m), 3.34 (3H, s), 3.16 (1H, dd, J
13.8, 4.3Hz), 2.89 (1H, dd, J 13.8, 11.0Hz), 2.32 (3H, s), 2.05
(3H, s) and 1.90 (3H, s); m/z (ESI, 60V) 531 (MH.sup.+).
[0301] u)
N-(4-(Carboxynicotinoyl)-N'-(3,5-dichloro-4-picolyl)-L-4-aminoph-
enylalanine
[0302] from (N-3,5-dichloro-4-picolyl)-L-4-aminophenylalanine
methyl ester and 4-(methoxycarbonyl)nicotinic acid. .delta.H
(DMSO-d.sub.6, 300MHz) 10.98 (1H, s), 9.01 (1H, d, J 8.0Hz), 8.77
(2H, s), 8.63 (1H, s), 7.66 (1H, d, J 5.1Hz), 7.59 (2H, d, J
8.5Hz), 7.32 (2H, d, J 8.5Hz), 4.63 (1H, m), 3.14 (1H, dd, J 13.9,
5.4Hz) and 3.03 (1H, dd, J 13.9, 8.8Hz); m/z (ESI, 160V) 503
(MH.sup.+).
[0303] v)
(2-Acetyl-3-thienyl)carbonyl-(N-3,5-dichloro-4-picolinyl)-4-amin-
ophenylalanine
[0304] from (N-3,5-dichloro4-picolyl)-L-4-aminophenylalanine methyl
ester and 2-acetyl-thiophene-3-carboxylic acid. .delta.H
(DMSO-d.sub.6) 9.17 (1H, d, J 8.2Hz, NH), 8.77 (2H, s, pyr-H), 7.99
(1H, d, J 5.1Hz, thiophene H-5), 7.57 (2H, ABd, J 8.5Hz, Ar--H),
7.31 (2H, ABd, 2H, 4 8.5Hz, Ar--H), 7.07 (1H, d, J 5.1Hz, thiophene
H-4), 5.38 (1H, m, CH.alpha.) 3.18 (1H, dd, J 13.8, 4.6Hz,
CHCH.sub.AH.sub.BAr), 2.93 (1H, dd, J 13.8, 10.3Hz,
CHCH.sub.AH.sub.BAr) and 2.26 (3H, s, COMe); m/z (ESI, 60V) 506
(MH.sup.+).
EXAMPLE 6
[0305] a)
2-Chloronicotinoyl-(N-2,6-dichlorobenzoyl)-L-4-aminophenylalanin-
e
[0306] Lithium hydroxide monohydrate (109 mg, 2.5 mmol) was added
to a solution of Intermediate 8 (450 mg, 1 mmol) in a mixture of
THF (10 ml) and water (10 ml). The mixture was stirred for 2 h at
room temperature, then the THF was evaporated in vacuo. The aqueous
residue was neutralised (1M hydrochloric acid), and the precipitate
isolated by filtration, washed with water and dried to give the
title compound (300 mg, 61%). .delta.H (DMSO-d.sup.6, 400K) 8.90
(1H, d, J 9.1Hzm 1NH), 8.48 (1H, m, Py H), 7.69 (1H, m, Py H),
7.64-7.45 (7H, m, 4ArH, 1 NH, 1 PyH), 7.29 (2H, d, J 8.3Hz, 2 ArH),
4.66-4.53 (1H, m, CH.alpha.tyr), 3.18 (1H, dd, J 14, 5.2Hz
CH.sub.AH.sub.BAr), and 2.91 (1H, dd, J 9.6. 14Hz,
CH.sub.AH.sub.BAr). m/z (ESI, 60V) 491 (MH.sup.+).
[0307] The following compounds were prepared in a similar
manner
[0308] b)
N-(2-Chloronicotinoyl-O-(2,6-dichlorobenzoyl)-L-tyrosine
[0309] from Intermediate 30 to give the title compound as a white
solid. .delta.H (DMSO-d.sub.6, 300MHz) 12.91 (1H, br s, CO.sub.2H),
8.98 (1H, d, H 8.3Hz, CONH), 8.44 (1H, dd, J 4.8, 1.9Hz, PyH),
7.69-7.58 (4H, m, Cl.sub.2ArH.sub.3+PyH), 7.45 (1H, dd, J 7.5,
4.9Hz, PyH), 7.43 (2H, d, J 8.7Hz, ArH), 7.22 (2H, d, J 8.5Hz,
ArH), 4.67 (1H, ddd, J 10.0, 8.2, 4.7Hz, CH.alpha.), 3.24 (1H, dd,
J 14.0, 4.0Hz, CH.sub.AH.sub.BAr) and 2.99 (1H, dd, J 13.9, 10.2Hz,
CH.sub.AH.sub.BAr); m/z (ESI, 60V) 493 (MH.sup.+).
[0310] c)
N-(2-Chloronicotinoyl)-N-methyl-N'-(3,5-dichloro-4-picolyl)-L-4--
aminophenylalanine
[0311] from Intermediate 31 to give the title compound as a white
solid .delta.H (DMSO-d.sub.6, 300MHz, 405K) 10.36(1H, br s, CONH),
8.67 (2H, s, Cl.sub.2PyH), 8.42-8.39 (1H, m, ClPyH), 7.54-7.15 (6H,
br m, 4.times.ArH+2.times.ClPyH), 5.30 (1H, v br s, CH.alpha.),
3.4-2.6 (5H, br m, NMe+CHCH.sub.2Ar) (Acid proton not observed at
405K, at 300K .delta.H 13.06 (1H, br s, CO.sub.2H)): m/z (ESI, 70V)
507 (MH.sup.+).
[0312] d)
[(S-2,5-dimethoxyphenyl)sulphonyl]nicotinoyl-O-(2,6-dichlorobenz-
yl)-L-tyrosine
[0313] from Intermediate 32 .delta.H (DMSO-d.sub.6) 8.57 (2H, m,
pyrH, NH), 7.83 (1H, d, J 7.8Hz, pyr-H), 7.69 (1H, dd, J 7.8,
4.7Hz, pyr-H), 7.55 (4H, m, Ar--H), 7.28-7.22 (3H, m, Ar--H), 7.07
(1H, d, J 9.1 Hz, Ar--H), 6.95 (2H, d, J 8.5Hz, Ar--H), 5.18 (1H,
s, CH.sub.2O), 4.55 (1H, m, CH.alpha.), 3.81 (3H, s, OMe), 3.40
(3H, s, OMe) and 3.07 (2H, m, CHCH.sub.2Ar). m/z (ESI, 30V) 645
(MH.sup.+).
[0314] e)
2{[(2-Chloro-3-pyridinyl)carbonyl]amino}-3-(4-{(2,6-dichloroanil-
ino)carbonyl}phenyl)propanoic acid
[0315] from Intermediate 15 to give the title compound as an off
white solid .delta.H (DMSO-d.sub.6, 300K) 11.06 (1H, br s), 10.23
(1H, br s), 9.00 (1H, d, J 7.8Hz), 8.46 (1H, br d), 7.95 (2H, d, J
7.4Hz), 7.69 (1H, d, J 7.1Hz), 7.59 (2H, d, J 8.0Hz), 7.50-7.36
(3H, m), 4.71 (1H, br), 3.26 (1H) and 3.06 (1H, dd, J 13.8,
10.0Hz); m/z (ESI, 60V) 492 (MH.sup.+).
[0316] f)
2-{[(2-Chloro-3-pyridinyl)carbonyl]amino}-3-(4-{[(3,5-dichloro-4-
-pyridinyl)amino]carbonyl}phenyl propionic acid
[0317] from the corresponding intermediate ester prepared in a
similar way to Intermediate 15 to give title compounds as an
offwhite solid. .delta.H (DMSO-d.sub.6, 300MHz) 12.92 (1H, br s),
10.57 (1H, s), 9.00 (1H, d, J 8.2Hz), 8.75 (2H, s), 8.45 (1H, dd, J
4.7, 1.7Hz), 7.96 (2H, d, J 8.15Hz), 7.69 (1H, d, J 7.3, 1.7Hz),
7.49 (2H, d, J 8.0Hz), 7.48 (1H, 4.71 (1H, br m), 3.28 (1H, dd, J
13.9, 4.7Hz) and 3.06 (1H, dd, J 13.9, 10.0Hz); m/z (ESI, 60V) 483
(MH.sup.+).
EXAMPLE 7
[0318] 2-Thio(S-acetic
acid)nicotinoyl-O-(2,6-dichlorobenzyl)-L-tyrosine
[0319] Lithium hydroxide monohydrate (75 mg, 1.8 mmol) was added to
a solution of Intermediate 9 (360 mg, 0.6 mmol) in a mixture of THF
(13 ml) and water (10 ml). The mixture was stirred for 2 hr at room
temperature, then the THF was evaporated in vacuo. The aqueous
residue was neutralised (1M hyrochloric acid), and the precipitate
isolated by filtration, washed with water and dried to give the
title compound (200 mg, 58%). .delta.H (DMSO-d.sub.6), 400K),
8.5-8.35 (2H, m, pyrH, 1NH), 7.71 (1H, dd, J 1.7, 7.6, pyrH), 7.57
(2H, d, J 8.9, 2ArH), 7.45 (1H, m, 1ArH), 7.22 (2H, d, J 8.5,
2ArH), 7.18 (1H, m, 1pyrH), 6.97 (2H, d, J 8.5, 2ArH), 5.18 (2H, s,
OCH.sub.2Ar), 4.43 (1H, m, CH.alpha.tyr), 3.82 (2H, S,
SCH.sub.2CO.sub.2H), 2.94-3.23 (2H, m, CH.sub.2Ar); m/z (ESI, GOU)
535 (MH.sup.+).
EXAMPLE 8
[0320]
2-Thio(S-methyl)nicotinoyl-O-(2,6-dichlorobenzyl)-L-tyrosine
[0321] Lithium hydroxide monohydrate (140 mg, 3.3 mmol) was added
to a solution of Intermediate 10 (1.4 gm, 2.7 mmol) in a mixture of
THF (10 ml) and water (10 ml). The mixture was stirred for 2 hr at
room temperature then the THF was evaporated in vacuo. The aqueous
residue was neutralised (1M hydrochloric acid), and the precipitate
isolated by filtration, washed with water and dried to give the
title compound (1.1 gm, 81%). .delta.H (DMSO-d.sub.6, 400K) 8.73
(1H, d, J 8.1, NH), 8.52 (1H, dd, J 1.7, 4.8, 1pyrH), 7.67 (1H, dd,
J 1.7, 7.6, 1pyrH), 7.55 (2H, d, J 8.9, 2ArH), 7.45 (1H, dd, J 2.3,
8.9, 1ArH), 7.24 (2H, d, J 8.6, 2ArH), 7.16 (1H, m, 1pyrH), 6.99
(2H, d, J 8.6, 2ArH), 5.18 (2H, s, OCH.sub.2Ar), 4.55 (1H, m,
CH.sub.2tyr), 3.16-2.95 (2H, m, CH.sub.2Ar) and 2.38 (3H, s,
SCH.sub.3); m/z (ESI, GOU) 491 (MH.sup.+).
EXAMPLE 9
[0322]
(N-2-Chloronicotinoyl)-4-[(E)-2-(2,6-dichlorophenyl)ethenyl]phenyla-
lanine
[0323] To a solution of Intermediate 28 (1.0 g, 1.99 mmol) in THF
(5 ml) and water (5 ml) was added lithium hydroxide monohydrate (88
mg 2.09 mmol). The reaction mixture was stirred for 1 h. The THF
was then removed in vacuo and the remaining aqueous solution
acidified to pH6 with 1M hydrochloric acid. The resulting
precipitate was collected and washed with water and ether and
finally freeze dried. The resulting compound contained an impurity
so a small amount was purified by preparavie HPLC (98 mg). .delta.H
(DMSO-d.sub.6) 12.90 (1H, dr s, CO.sub.2H), 8.98 (1H, d, J 8.0Hz),
8.45 (1H, d, J 3.4Hz), 7.70-7.03 (9H, m), 4.72-4.60 (1H, m, CH),
3.20 (1H, dd, J 14.0, 4.5Hz, CH.sub.AH.sub.B) and 3.01 (1H, dd, J
14.0, 9.9Hz, CH.sub.AH.sub.B); m/z (ESI, 60V) 475 (MH.sup.+).
EXAMPLE 10
[0324]
(N-2-Chloronicotinoyl)-4-[2-(2,6-dichlorophenyl)-2-hydroxyethyl]phe-
nylalanine
[0325] Lithium hydroxide monohydrate (13 mg) was added to a
solution of Intermediate 22 (150 mg, 0.29 mmol) in THF (5 ml) and
H.sub.2O (5 ml). The solution was stirred for 1 h and then the THF
removed in vacuo and the remaining aqueous solution acidified to
pH6 with 1M hydrochloric acid. The solid precipitate formed was
collected by filtration, washed with copious quantities of water
and finally freeze dried to give the title compund as a fluffy
white solid (70 mg, 49%). .delta.H (DMSO-d.sub.6) 12.78 (1H, br s,
CO.sub.2H), 8.90 (1H, d, J, 8.0Hz), 8.45 (1H, d, J 4.8Hz),
7.63-7.58 (1H, m, ArH), 7.49-7.45 (1H, m, ArH), 7.34-7.07 (7H, m,
ArH), 5.50-5.45 (1H, m, CH), 4.61-4.51 (1H, m, CH) and 3.30-2.35
(4H, m, 2.times.CH.sub.2); m/z (ESI, 60V) 493 (MH.sup.+).
EXAMPLE 11
[0326]
(N-2-Chloronicotinoyl)-{4-[2-(2-dichlorophenyl)-2-oxoethyl]}phenyla-
lanine
[0327] Lithium hydroxide monohydrate (36 mg, 0.85 mmol) was added
to a solution of Intermediate 23 (400 mg, 0.77 mmol) in THF (5 ml)
and water (5 ml). The reaction mixture was stirred for 3 h and then
the THF was removed invacuo. The remaining aqueous solution was
acidified with 1M hydrochloric acid. The resulting white
precipitate was collected and washed well with water. Further
purification by column chromatography (SiO.sub.2; acetic acid:
MeOH:DCM, 2:8:90) gave the title compound as a white solid (78 mg,
19%). .delta.H (DMSO-d.sub.6) 8.45 (1H, dd, J 4.8, 2.0 Hz), 8.03
(1H, dd, J 7.7, 2.0Hz), 7.36-7.20 (8H, m, ArH), 6.98 (1H, d, J
7.2Hz, ArH), 5.14-5.05 (1H, m, CH), 4.11 (2H, s, CH.sub.2C.dbd.O)m,
3.39 (1H, dd, J 14.1, 5.6Hz, CH.sub.AH.sub.B) and 3.25 (1H, dd, J
14.1, 6.2Hz, CH.sub.AH.sub.B); m/z (ESI, 60V) 491 (MH.sup.+).
[0328] .alpha..sub.4.beta..sub.1 Integrin-dependent Jurkat cell
adhesion to VCAM-Ig
[0329] 96 well NUNC plates were coated with F(ab).sub.2 fragment
goat anti-human IgG Fc.gamma.-specific antibody [Jackson Immuno
Research 109006-098: 100 .mu.l at 2 .mu.g/ml in 0.1M NaHCO.sub.3,
pH 8.4]. overnight at 4.degree.. 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.
[0330] 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.
[0331] .alpha..sub.4.beta..sub.7 Integrin-dependent JY cell
adhesion to MAdCAM-Ig
[0332] 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.
[0333] .alpha..sub.5.beta..sub.1 Integrin-dependent K562 cell
adhesion to fibronectin
[0334] 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
30 minutes. Each plate was fixed and stained as described in the
.alpha..sub.4.beta..sub.1 assay above.
[0335] .alpha..sub.m.beta..sub.2-dependent human polymorphonucler
neutrophils adhesion to plastic
[0336] 96 well tissue culture plates were coated with RPMI 1640/10%
FCS for 2h 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 0.1 M sodium acetatelcitrate buffer, pH
6.0 and absorbance measured at 630 nm.
[0337] .alpha.llb/.beta..sub.3-dependent human platelet
aggregation
[0338] 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.4.2H.sub.2O 0.065). Aggregation was
monitored following addition of 2.5 .mu.M ADP (Sigma) in the
presence or absence of inhibitors.
[0339] 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. Thus
compounds of the Examples typically had IC.sub.50 values of 100 nM
and below in these assays and demonstrated selective inhibition of
.alpha.4.beta.1. In the other assays featuring .alpha. 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.
* * * * *