U.S. patent application number 11/815928 was filed with the patent office on 2008-10-23 for ligands for g-protein coupled receptors.
Invention is credited to David John Fox, David J. Grainger.
Application Number | 20080261948 11/815928 |
Document ID | / |
Family ID | 34356201 |
Filed Date | 2008-10-23 |
United States Patent
Application |
20080261948 |
Kind Code |
A1 |
Grainger; David J. ; et
al. |
October 23, 2008 |
Ligands for G-Protein Coupled Receptors
Abstract
The invention provides compounds of general formulae I-IV or
pharmaceutically acceptable salts thereof: ##STR00001## The
invention also provides methods of preparing the compounds,
pharmaceutical compositions comprising the compounds and use of the
compounds for the preparation of medicaments intended to modulate
the activity of one or more members of the G-protein coupled
receptor (GPCR) class. Compounds of the invention may be used to
create a compound library for use in screening for agents which
modulate signalling through GPCRs.
Inventors: |
Grainger; David J.;
(Cambridge, GB) ; Fox; David John; (Coventry,
GB) |
Correspondence
Address: |
SCHWEGMAN, LUNDBERG & WOESSNER, P.A.
P.O. BOX 2938
MINNEAPOLIS
MN
55402
US
|
Family ID: |
34356201 |
Appl. No.: |
11/815928 |
Filed: |
February 10, 2006 |
PCT Filed: |
February 10, 2006 |
PCT NO: |
PCT/GB06/00475 |
371 Date: |
April 15, 2008 |
Current U.S.
Class: |
514/211.03 ;
506/15; 506/23; 506/7; 514/218; 540/544; 540/575 |
Current CPC
Class: |
A61P 9/12 20180101; A61P
25/18 20180101; A61P 3/04 20180101; C07D 281/06 20130101; A61P
37/00 20180101; A61P 9/10 20180101; A61P 25/00 20180101; A61P 11/06
20180101; A61P 37/02 20180101; C07D 243/08 20130101; A61P 25/28
20180101; A61P 43/00 20180101 |
Class at
Publication: |
514/211.03 ;
540/544; 540/575; 514/218; 506/15; 506/7; 506/23 |
International
Class: |
A61K 31/554 20060101
A61K031/554; A61K 31/551 20060101 A61K031/551; C07D 281/06 20060101
C07D281/06; C40B 30/00 20060101 C40B030/00; A61P 9/10 20060101
A61P009/10; A61P 3/04 20060101 A61P003/04; A61P 25/00 20060101
A61P025/00; A61P 11/06 20060101 A61P011/06; C40B 50/00 20060101
C40B050/00; C40B 40/04 20060101 C40B040/04; C07D 243/08 20060101
C07D243/08 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 11, 2005 |
GB |
0502914.5 |
Claims
1. A compound of general formula (I) ##STR00032## wherein X is
--CO--(Y).sub.k-(Z).sub.n or SO.sub.2--(Y).sub.k-(Z).sub.n; k is 0
or 1 Y is a cycloalkyl or polycyloalkyl group; or Y is a
cycloalkenyl or polycycloalkenyl group; each Z is independently
selected from hydrogen or an alkyl, haloalkyl, alkoxy, haloalkoxy,
alkenyl, alkynyl, alkylamino, alkylaminoalkyl, alkylaminodialkyl,
charged alkylaminotrialkyl or charged alkylcarboxylate radical of 1
to 20 carbon atoms; or each Z is independently selected from
fluoro, chloro, bromo, iodo, hydroxy, oxyalkyl, amino, aminoalkyl,
aminodialkyl, charged aminotrialkyl, or carboxylate radical; and n
is any integer from 1 to m, where m is the maximum number of
substitutions permissible on the cyclo-group Y; or alternatively Z
may be selected from a peptido radical; and R.sup.3 and R.sup.4
independently represent any variable substituent.
2. A compound of general formula (II) ##STR00033## wherein X is
--CO--(Y).sub.k-(Z).sub.n or SO.sub.2--(Y).sub.k-(Z).sub.n; k is 0
or 1 Y is a cycloalkyl or polycyloalkyl group; or Y is a
cycloalkenyl or polycycloalkenyl group; each Z is independently
selected from hydrogen or an alkyl, haloalkyl, alkoxy, haloalkoxy,
alkenyl, alkynyl, alkylamino, alkylaminoalkyl, alkylaminodialkyl,
charged alkylaminotrialkyl or charged alkylcarboxylate radical of 1
to 20 carbon atoms; or each Z is independently selected from
fluoro, chloro, bromo, iodo, hydroxy, oxyalkyl, amino, aminoalkyl,
aminodialkyl, charged aminotrialkyl, or carboxylate radical; and n
is any integer from 1 to m, where m is the maximum number of
substitutions permissible on the cyclo-group Y; or alternatively Z
may be selected from a peptido radical; and R.sup.3 and R.sup.4
independently represent any variable substituent.
3. A compound of general formula (III) ##STR00034## wherein X is
--CO--(Y).sub.k-(Z).sub.n or SO.sub.2--(Y).sub.k-(Z).sub.n; k is 0
or 1 Y is a cycloalkyl or polycyloalkyl group; or Y is a
cycloalkenyl or polycycloalkenyl group; each Z is independently
selected from hydrogen or an alkyl, haloalkyl, alkoxy, haloalkoxy,
alkenyl, alkynyl, alkylamino, alkylaminoalkyl, alkylaminodialkyl,
charged alkylaminotrialkyl or charged alkylcarboxylate radical of 1
to 20 carbon atoms; or each Z is independently selected from
fluoro, chloro, bromo, iodo, hydroxy, oxyalkyl, amino, aminoalkyl,
aminodialkyl, charged aminotrialkyl, or carboxylate radical; and n
is any integer from 1 to m, where m is the maximum number of
substitutions permissible on the cyclo-group Y; or alternatively Z
may be selected from a peptido radical; and R.sup.2, R.sup.3 and
R.sup.4 independently represent any variable substituent.
4. A compound of general formula (IV) ##STR00035## wherein X is
--CO--(Y).sub.k-(Z).sub.n or SO.sub.2--(Y).sub.k-(Z).sub.n; k is 0
or 1 Y is a cycloalkyl or polycyloalkyl group; or Y is a
cycloalkenyl or polycycloalkenyl group; each Z is independently
selected from hydrogen or an alkyl, haloalkyl, alkoxy, haloalkoxy,
alkenyl, alkynyl, alkylamino, alkylaminoalkyl, alkylaminodialkyl,
charged alkylaminotrialkyl or charged alkylcarboxylate radical of 1
to 20 carbon atoms; or each Z is independently selected from
fluoro, chloro, bromo, iodo, hydroxy, oxyalkyl, amino, aminoalkyl,
aminodialkyl, charged aminotrialkyl, or carboxylate radical; and n
is any integer from 1 to m, where m is the maximum number of
substitutions permissible on the cyclo-group Y; or alternatively Z
may be selected from a peptido radical; and R.sup.2 and R.sup.3
independently represent any variable substituent.
5. A pharmaceutical composition comprising, as active ingredient, a
compound of general formula (I), as defined in claim 1, or a
pharmaceutically acceptable salt thereof, and at least one
pharmaceutically acceptable excipient and/or carrier.
6. A pharmaceutical composition comprising, as active ingredient, a
compound of general formula (II), as defined in claim 2, or a
pharmaceutically acceptable salt thereof, and at least one
pharmaceutically acceptable excipient and/or carrier.
7. A pharmaceutical composition comprising, as active ingredient, a
compound of general formula (III), as defined in claim 3, or a
pharmaceutically acceptable salt thereof, and at least one
pharmaceutically acceptable excipient and/or carrier.
8. A pharmaceutical composition comprising, as active ingredient, a
compound of general formula (IV), as defined in claim 4, or a
pharmaceutically acceptable salt thereof, and at least one
pharmaceutically acceptable excipient and/or carrier.
9-18. (canceled)
19. A method of treatment, amelioration or prophylaxis of the
symptoms of disease or condition selected from the group consisting
of hypertension, atherosclerosis, asthma, obesity,
neurodegenerative disorders, autoimmune disorders or psychopathic
disorders by the administration to a patient of an effective amount
of a compound, as claimed in any of claims 1 to 4.
20. A library composed of, or enriched in, diverse library elements
which are compounds according to any of claims 1 to 4, where said
compounds have diverse substitutions as the variable
substituents.
21. A method which involves the use of a library according to claim
20 in an assay for the purpose of screening to identify agent(s)
which modulate signalling through GPCRs comprising screening the
library according to claim 20 to identify agent(s) which modulate
signalling through GPCRs.
22. A method according to claim 21, where the agent(s) identified
are antagonists at one or more GPCRs.
23. A method according to claim 21 where the agent(s) identified
are agonists at one or more GPCRs.
24. A method according to claim 21 where the GPCR is selected from
the group consisting of adrenalin receptors, endothelin receptors,
chemokine receptors, EDG receptors, VIP/PECAP receptors, dopamine
receptors, serotonin receptors, purine receptors, metabotropic
gluatmate receptors, acetyl choline receptors, C5a receptors, fMLP
receptors, glucagon or GLP receptors, NPY receptors, MSH receptors,
glycoprotein hormone receptors, protease activated receptors
(PARs), somatostatin receptors, angiotensin receptors,
cholecystokinin receptors or melatonin receptors.
25. A method according to claim 21 further comprising synthesising
a carbon analogue of said compound, wherein the heteroatom in the
lactam ring which is not part of the "ideal" GPCR substrate motif
(at position 1) is replaced by --CH.sub.2--; and providing said
carbon analogue (or a pharmaceutically acceptable salt thereof) in
isolated and purified form.
26. A pharmaceutical composition comprising, as active ingredient,
a compound produced according to step (d) of claim 25, or a
pharmaceutically acceptable salt thereof, and at least one
pharmaceutically acceptable excipient and/or carrier.
27. The use of a compound produced according to step (d) of claim
25, or a pharmaceutically acceptable salt thereof, for the
preparation of a medicament intended to modulate the activity of
one or more members of the G-protein coupled receptor (GPCR)
class.
28. Use of a compound according to claim 27 wherein the GPCR to be
modulated is selected from the group consisting of adrenalin
receptors, endothelin receptors, chemokine receptors, EDG
receptors, VIP/PECAP receptors, dopamine receptors, serotonin
receptors, purine receptors, metabotropic gluatmate receptors,
acetyl choline receptors, C5a receptors, FMLP receptors, glucagon
or GLP receptors, NPY receptors, MSH receptors, glycoprotein
hormone receptors, protease activated receptors (PARs), somatostain
receptors, angiotensin receptors, cholecystokinin receptors or
melatonin receptors.
29. A method of treatment, amelioration or prophylaxis of the
symptoms of disease or condition selected from the group consisting
of hypertension, atherosclerosis, asthma, obesity,
neurodegenerative disorders, autoimmune disorders or psychopathic
disorders by the administration to a patient of an effective amount
of a compound produced according to claim 25.
30. A method for preparing a compound of formula (I) as defined in
claim 1, which method comprises the steps of; (a) selective
acylation of the N-.alpha.-amino group of cysteine, S-alkyl
cysteine or a 7-membered lactam ring derived from an S-alkyl
cysteine; (b) S-alkylation of cysteine or N-acyl cysteine; and (c)
cyclisation of S-alkyl-cysteine or S-alkyl-N-acyl-cysteine where
the steps are performed in any appropriate order, using selective
protecting groups as required.
31. A method for preparing a compound of formula (II) as defined in
claim 2, which method comprises the steps of; (a) selective
acylation of the N-.alpha.-amino group of penicillamine, S-alkyl
penicillamine or a 7-membered lactam ring derived from an S-alkyl
penicillamine; (b) S-alkylation of penicillamine or N-acyl
penicillamine; and (c) cyclisation of S-alkyl penicillamine or
S-alkyl-N-acyl penicillamine where the steps are performed in any
appropriate order, using selective protecting groups as
required.
32. A method for preparing a compound of formula (III) as defined
in claim 3, which method comprises the steps of; (a) selective
acylation of the N-.alpha.-amino group of 3-aminoalanine,
N-.beta.-alkyl 3-aminoalanine, N-.beta.-aryl 3-aminoalanine,
N-.beta.-dialkyl 3-aminoalanine or a 7-membered lactam ring derived
from an N-.beta.-alkyl 3-aminoalanine, N-.beta.-aryl-N-.beta.-alkyl
3-amoinoalanine, N-.beta.-dialkyl 3-aminoalanine; (b)
N-.beta.-alkylation or N-.beta.-arylation or N-.beta.-sulfonation
of 3-aminoalanine, N-.beta.-alkyl 3-aminoalanine or a 7-membered
lactam ring derived from an N-.beta.-alkyl 3-aminoalanine, all with
or without prior N-.alpha.-acyl modification; (c)
N-.beta.-alkylation using a protected .beta.-amino alcohol of
3-aminoalanine, N-.beta.-alkyl 3-aminoalanine or N-.beta.-aryl
3-aminoalanine, all with or without prior N-.alpha.-acyl
modification; and (d) cyclisation of N-.beta.-alkyl 3-aminoalanine,
N-.beta.-dialkyl 3-aminoalanine or N-.beta.-aryl-N-.beta.-alkyl
3-aminoalanine, all with or without prior N-.alpha.-acyl
modification where the steps are performed in any appropriate
order, using selective protecting groups as required.
33. A method for preparing a compound of formula (IV) as defined in
claim 4, which method comprises the steps of; (a) selective
acylation of the N-.alpha.-amino group of 3-aminoalanine,
N-.beta.-alkyl 3-aminoalanine, N-.beta.-aryl 3-aminoalanine,
N-.beta.-alkyl-N-.beta.-acyl 3-aminoalanine,
N-.beta.-aryl-N-.beta.-acyl 3-aminoalanine or a 7-membered lactam
ring derived from an N-.beta.-acyl 3-aminoalanine,
N-.beta.-aryl-N-.beta.-acyl 3-amoinoalanine,
N-.beta.-alkyl-N-.beta.-acyl 3-aminoalanine; (b)
N-.beta.-alkylation or N-.beta.-arylation of 3-aminoalanine,
N-.beta.-acyl 3-aminoalanine or a 7-membered lactam ring derived
from an N-.beta.-acyl 3-aminoalanine, all with or without prior
N-.alpha.-acyl modification; (c) N-.beta.-acylation using a
protected .alpha.-amino acid of 3-aminoalanine, N-.beta.-alkyl
3-aminoalanine or N-.beta.-aryl 3-aminoalanine, all with or without
prior N-.alpha.-acyl modification; and (d) cyclisation of
N-.beta.-acyl 3-aminoalanine, N-.beta.-acyl-N-.beta.-aryl
3-aminoalanine or N-.beta.-acyl-N-.beta.-alkyl 3-aminoalanine, all
with or without prior N-.alpha.-acyl modification where the steps
are performed in any appropriate order, using selective protecting
groups as required.
34. A method according to claim 30, for preparing a compound of
formula (I), which method uses a synthetic route selected from the
group consisting of (i) Scheme 1A; (ii) Scheme 1B; and (iii) Scheme
1C.
35. A method according to claim 31, for preparing a compound of
formula (II), which method uses a synthetic route selected from the
group consisting of (i) Scheme 2A; (ii) Scheme 2B; and (iii) Scheme
2C.
36. A method according to claim 32, for preparing a compound of
formula (III), which method uses a synthetic route selected from
the group consisting of (i) Scheme 3A; (ii) Scheme 3B; (iii) Scheme
3C; (iv) Scheme 3D; (v) Scheme 3E; and (vi) Scheme 3F.
37. A method according to claim 33, for preparing a compound of
formula (IV), which method uses a synthetic route selected from the
group consisting of (i) Scheme 4A; (ii) Scheme 4B; and (iii) Scheme
4C.
38. A method for preparing a library of compounds enriched in GPCR
antagonists and/or GPCR agonists wherein the library is prepared by
reacting a diversity of .alpha.-amino acids and/or .beta.-amino
alcohols with a "suitable" acylamino acid; a "suitable" acylamino
acid being defined as any acylamino acid which generates an
acylaminolactam when reacted with an .alpha.-amino acid or a
.beta.-amino alcohol.
39. A compound of formula (II) according to claim 2 where R.sup.3
is not carboxylate; or a carboxylate ester (with the carboxylate
attached directly to the acylaminolactam ring); or a carboxylate
amide (with the carboxylate attached directly to the
acylaminolactam ring); or a carboxylate thioester (with the
carboxylate attached directly to the acylaminolactam).
40. A compound of formula (IV) according to claim 4 wherein the
compound is not R.sup.3=benzyl or R.sup.3.dbd.CH.sub.2CH.sub.2COR'
where R' is variable.
41. The compound according to claim 1, 2, 3 or 4 wherein Y is an
adamantyl, adamantanemethyl, bicyclooctyl, cyclohexyl, or
cyclopropyl group.
42. The compound according to claim 1, 2, 3 or 4 wherein the
peptide radical has from 1 to 4 peptidic moieties linked together
by peptide bonds.
43. The compound according to claim 42 where the peptido radical
has 1 to 4 amino acid residues.
44. The compound according to claim 1, 2, 3 or 4 wherein the
R.sup.1 radical has a key carbon which is di-substituted with the
same or different groups selected from: alkyl, haloalkyl, alkoxy,
haloalkoxy, alkenyl, alkynyl and alkylamino radicals.
45. The compound according to claim 44 wherein the key carbon is
chiral.
46. The compound according to claim 44 wherein the key carbon has
sp3 hybridised bonds.
47. The compound according to claim 44 wherein the key carbon has
essentially tetrahedral bond angles.
48. The compound according to claim 1, 2, 3 or 4 wherein the ring
or rings of Y constrain the bond angles at the key carbon to be
essentially tetrahedral.
49. A method to modulate the activity of one or more members of the
G-protein coupled receptor (GPCR) class, comprising: administering
to a patient an effective amount of a compound of general formula
(I), (II), III) or (IV), or a pharmaceutically acceptable salt
thereof, wherein formula (I) is ##STR00036## wherein X is
--CO--(Y).sub.k-(Z).sub.n or SO.sub.2--(Y).sub.k-(Z).sub.n; k is 0
or 1 Y is a cycloalkyl or polycyloalkyl group; or Y is a
cycloalkenyl or polycycloalkenyl group; each Z is independently
selected from hydrogen or an alkyl, haloalkyl, alkoxy, haloalkoxy,
alkenyl, alkynyl, alkylamino, alkylaminoalkyl, alkylaminodialkyl,
charged alkylaminotrialkyl or charged alkylcarboxylate radical of 1
to 20 carbon atoms; or each Z is independently selected from
fluoro, chloro, bromo, iodo, hydroxy, oxyalkyl, amino, aminoalkyl,
aminodialkyl, charged aminotrialkyl, or carboxylate radical; and n
is any integer from 1 to m, where m is the maximum number of
substitutions permissible on the cyclo-group Y; or alternatively Z
may be selected from a peptido radical; and R.sup.3 and R.sup.4
independently represent any variable substituent; formula (II) is
##STR00037## wherein X is --CO--(Y).sub.k-(Z).sub.n or
SO.sub.2--(Y).sub.k-(Z).sub.n; k is 0 or 1 Y is a cycloalkyl or
polycyloalkyl group; or Y is a cycloalkenyl or polycycloalkenyl
group; each Z is independently selected from hydrogen or an alkyl,
haloalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, alkylamino,
alkylaminoalkyl, alkylaminodialkyl, charged alkylaminotrialkyl or
charged alkylcarboxylate radical of 1 to 20 carbon atoms; or each Z
is independently selected from fluoro, chloro, bromo, iodo,
hydroxy, oxyalkyl, amino, aminoalkyl, aminodialkyl, charged
aminotrialkyl, or carboxylate radical; and n is any integer from 1
to m, where m is the maximum number of substitutions permissible on
the cyclo-group Y; or alternatively Z may be selected from a
peptido radical; and R.sup.3 and R.sup.4 independently represent
any variable substituent; formula (III) is ##STR00038## wherein X
is --CO--(Y).sub.k-(Z).sub.n or SO.sub.2--(Y).sub.k-(Z).sub.n; k is
0 or 1 Y is a cycloalkyl or polycyloalkyl group; or Y is a
cycloalkenyl or polycycloalkenyl group; each Z is independently
selected from hydrogen or an alkyl, haloalkyl, alkoxy, haloalkoxy,
alkenyl, alkynyl, alkylamino, alkylaminoalkyl, alkylaminodialkyl,
charged alkylaminotrialkyl or charged alkylcarboxylate radical of 1
to 20 carbon atoms; or each Z is independently selected from
fluoro, chloro, bromo, iodo, hydroxy, oxyalkyl, amino, aminoalkyl,
aminodialkyl, charged aminotrialkyl, or carboxylate radical; and n
is any integer from 1 to m, where m is the maximum number of
substitutions permissible on the cyclo-group Y; or alternatively Z
may be selected from a peptido radical; and R.sup.2, R.sup.3 and
R.sup.4 independently represent any variable substituent, or
formula (IV) is ##STR00039## wherein X is --CO--(Y).sub.k-(Z).sub.n
or SO.sub.2--(Y).sub.k-(Z).sub.n; k is 0 or 1 Y is a cycloalkyl or
polycyloalkyl group; or Y is a cycloalkenyl or polycycloalkenyl
group; each Z is independently selected from hydrogen or an alkyl,
haloalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, alkylamino,
alkylaminoalkyl, alkylaminodialkyl, charged alkylaminotrialkyl or
charged alkylcarboxylate radical of 1 to 20 carbon atoms; or each Z
is independently selected from fluoro, chloro, bromo, iodo,
hydroxy, oxyalkyl, amino, aminoalkyl, aminodialkyl, charged
aminotrialkyl, or carboxylate radical; and n is any integer from 1
to m, where m is the maximum number of substitutions permissible on
the cyclo-group Y; or alternatively Z may be selected from a
peptido radical; and R.sup.2 and R.sup.3 independently represent
any variable substituent.
50. The method according to claim 49 wherein the GPCR to be
modulated is selected from the group consisting of adrenalin
receptors, endothelin receptors, chemokine receptors, EDG
receptors, VIP/PECAP receptors, dopamine receptors, serotonin
receptors, purine receptors, metabotropic gluatmate receptors,
acetyl choline receptors, C5a receptors, FMLP receptors, glucagon
or GLP receptors, NPY receptors, MSH receptors, glycoprotein
hormone receptors, protease activated receptors (PARs), somatostain
receptors, angiotensin receptors, cholecystokinin receptors and
melatonin receptors
51. The method according to claim 1, 2, 3 or 4 wherein the R.sup.1
radical has a key carbon which is di-substituted with the same or
different groups selected from: alkyl, haloalkyl, alkoxy,
haloalkoxy, alkenyl, alkynyl and alkylamino radicals.
52. The method according to claim 51 wherein the key carbon is
chiral.
53. The method according to claim 51 wherein the key carbon has sp3
hybridised bonds.
54. The method according to claim 51 wherein the key carbon has
essentially tetrahedral bond angles.
55. The method according to claim 1, 2, 3 or 4 wherein the ring or
rings of Y constrain the bond angles at the key carbon to be
essentially tetrahedral.
56. A method of treatment, amelioration or prophylaxis of the
symptoms of disease or condition selected from the group consisting
of hypertension, atherosclerosis, asthma, obesity,
neurodegenerative disorders, autoimmune disorders or psychopathic
disorders by the administration to a patient of an effective amount
of pharmaceutical composition comprising, as active ingredient, a
compound of general formula (I), (II), or (IV), or a
pharmaceutically acceptable salt thereof, and at least one
pharmaceutically acceptable excipient and/or carrier, wherein
formula (I) is ##STR00040## wherein X is --CO--(Y).sub.k-(Z).sub.n
or SO.sub.2--(Y).sub.k-(Z).sub.n; k is 0 or 1 Y is a cycloalkyl or
polycyloalkyl group; or Y is a cycloalkenyl or polycycloalkenyl
group; each Z is independently selected from hydrogen or an alkyl,
haloalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, alkylamino,
alkylaminoalkyl, alkylaminodialkyl, charged alkylaminotrialkyl or
charged alkylcarboxylate radical of 1 to 20 carbon atoms; or each Z
is independently selected from fluoro, chloro, bromo, iodo,
hydroxy, oxyalkyl, amino, aminoalkyl, aminodialkyl, charged
aminotrialkyl, or carboxylate radical; and n is any integer from 1
to m, where m is the maximum number of substitutions permissible on
the cyclo-group Y; or alternatively Z may be selected from a
peptido radical; and R.sup.3 and R.sup.4 independently represent
any variable substituent; formula (II) is ##STR00041## wherein X is
--CO--(Y).sub.k-(Z).sub.n or SO.sub.2--(Y).sub.k-(Z).sub.n; k is 0
or 1 Y is a cycloalkyl or polycyloalkyl group; or Y is a
cycloalkenyl or polycycloalkenyl group; each Z is independently
selected from hydrogen or an alkyl, haloalkyl, alkoxy, haloalkoxy,
alkenyl, alkynyl, alkylamino, alkylaminoalkyl, alkylaminodialkyl,
charged alkylaminotrialkyl or charged alkylcarboxylate radical of 1
to 20 carbon atoms; or each Z is independently selected from
fluoro, chloro, bromo, iodo, hydroxy, oxyalkyl, amino, aminoalkyl,
aminodialkyl, charged aminotrialkyl, or carboxylate radical; and n
is any integer from 1 to m, where m is the maximum number of
substitutions permissible on the cyclo-group Y; or alternatively Z
may be selected from a peptido radical; and R.sup.3 and R.sup.4
independently represent any variable substituent; formula (III) is
##STR00042## wherein X is --CO--(Y).sub.k-(Z).sub.n or
SO.sub.2--(Y).sub.k-(Z).sub.n; k is 0 or 1 Y is a cycloalkyl or
polycyloalkyl group; or Y is a cycloalkenyl or polycycloalkenyl
group; each Z is independently selected from hydrogen or an alkyl,
haloalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, alkylamino,
alkylaminoalkyl, alkylaminodialkyl, charged alkylaminotrialkyl or
charged alkylcarboxylate radical of 1 to 20 carbon atoms; or each Z
is independently selected from fluoro, chloro, bromo, iodo,
hydroxy, oxyalkyl, amino, aminoalkyl, aminodialkyl, charged
aminotrialkyl, or carboxylate radical; and n is any integer from 1
to m, where m is the maximum number of substitutions permissible on
the cyclo-group Y; or alternatively Z may be selected from a
peptido radical; and R.sup.2, R.sup.3 and R.sup.4 independently
represent any variable substituent, or formula (IV) is ##STR00043##
wherein X is --CO--(Y).sub.k-(Z).sub.n or
SO.sub.2--(Y).sub.k-(Z).sub.n; k is 0 or 1 Y is a cycloalkyl or
polycyloalkyl group; or Y is a cycloalkenyl or polycycloalkenyl
group; each Z is independently selected from hydrogen or an alkyl,
haloalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, alkylamino,
alkylaminoalkyl, alkylaminodialkyl, charged alkylaminotrialkyl or
charged alkylcarboxylate radical of 1 to 20 carbon atoms; or each Z
is independently selected from fluoro, chloro, bromo, iodo,
hydroxy, oxyalkyl, amino, aminoalkyl, aminodialkyl, charged
aminotrialkyl, or carboxylate radical; and n is any integer from 1
to m, where m is the maximum number of substitutions permissible on
the cyclo-group Y; or alternatively Z may be selected from a
peptido radical; and R.sup.2 and R.sup.3 independently represent
any variable substituent.
Description
[0001] The invention relates to the generation of a library of
compounds enriched in agonists and antagonists for members of the
G-protein coupled class of receptors (GPCRs).
[0002] Members of the G-protein coupled receptor (GPCR) class of
membrane proteins (also known as seven-transmembrane spanning or
7TM receptors and serpentine receptors) mediate cellular signalling
in response to a very wide variety of extracellular signals,
including hormones, neurotransmitters, cytokines and even
environmental substances such as odours and tastes. In response to
the ligand interacting with the extracellular portion of the
receptor (most usually the N-terminal tail of the receptor
protein), the receptor is converted temporarily to an activated
state (this conversion is usually designated R+L.fwdarw.R*L where R
is the inactive receptor, R* is the activated receptor and L is the
ligand).
[0003] The activated (or R*) conformation of the receptor is then
able to interact with a member of the G-protein family. The
G-proteins are a large family of trimeric intracellular proteins
which bind guanine nucleotides. On interacting with the activated
receptor (probably by a mechanism called "collisional coupling")
the G-protein exchanges a bound guanosine diphosphate (GDP) for a
guanosine triphosphate (GTP). In this GTP-bound form the G-protein
trimer dissociates, yielding a free G.alpha. subunit, and a
.beta..gamma. dimer. Both the G.alpha. and .beta..gamma. subunits
can then participate in further signalling cascades. For example,
the G.alpha. subunit can activate the adenylate cyclase (AC)
enzyme, which generates cyclic adenosine monophospate (cAMP) from
adenosine triphosphate. The .beta..gamma. subunit can activate
members of the PI-3-kinase family of enzymes. Ultimately, these
signals can result in modulation of almost every aspect of cell
behaviour, from contraction to motility, metabolism to further
signalling.
[0004] The signal, once activated, is then slowly turned off by a
number of mechanisms. The GTP associated with the G.alpha. subunit
is hydrolysed back to GDP, resulting in the reassociation of the
G.alpha. and .beta..gamma. subunits to form the inactive trimeric
GDP-bound G-protein. The GPCR itself also becomes phosphorylated on
the intracellular C-terminus, preventing further interaction with
G-proteins. Eventually, the bound ligand may also dissociate.
[0005] This generic signalling pathway is so central and ubiquitous
in mammalian physiology that as many as 40% of licensed
pharmaceuticals have a GPCR among their molecular targets.
Similarly, bacteria have evolved to target G-protein signalling in
order to disrupt host physiology and immunity: Vibrio cholerae (the
organism responsible for cholera), for example, makes a protein
known as cholera toxin which irreversibly inhibits the G.alpha.
subunit of a widely distributed G-protein called G.sub.s.
Similarly, Bordetella pertussis (the organism responsible for
Whooping Cough) makes a protein known as Pertussis toxin which has
a similar effect on a different G-protein, G.sub.i.
[0006] One approach to identifying pharmaceuticals which will
modulate GPCR signalling has been to screen very large random
compound libraries for the ability to interfere with ligand binding
to membrane preparations containing recombinant or purified GPCRs.
In such high throughput screens, various methods have been adopted
to facilitate the detection of binding. For example, in
scintillation proximity assays, the binding of a radiolabelled
ligand to the receptor brings the radionuclide into proximity with
a scintillant molecule bound to the receptor--as the nuclide
decays, light is emitted which can be detected and quantified.
Alternatively, the ligand can be fluorescently labelled and the
binding detected by fluorescence polarisation (dependent on the
reduced rotational degrees of freedom of the fluorescent tag when
the ligand is immobilised on binding to the receptor).
[0007] While these techniques have been successful in some
instances, and yielded lead compounds which have subsequently been
developed as human pharmaceuticals (for example, the 5HT.sub.3
receptor antagonist Ondansetron, used to treat migraine headaches),
there remain large numbers of GPCRs for which few, if any, suitable
non-peptide agonist or antagonist compounds have been identified,
despite intensive screening across the pharmaceutical industry. For
example, there are few specific non-peptide antagonists for the
chemokine receptor family of GPCRs, and no agonists. Since
chemokines play a central role in immune regulation, such molecules
would be expected to be extremely valuable pharmaceuticals with
immunomodulatory properties useful in treating a wide range of
diseases with an inflammatory component.
[0008] Two factors limit the likely success of random screening
programmes: firstly, there is a very large compound space to be
screened, and even with the best available high throughput
technology and the best combinatorial chemistry approaches to
generating diverse libraries, only a small fraction of all possible
molecular structures can be investigated. Secondly, even when leads
have been successfully identified the core pharmacophores are often
not suitable for use in vivo--the lead compound and its analogs may
be simply too toxic.
[0009] Another major problem with such "negative screening"
paradigms (where you detect the ability of the test library to
block binding of a labelled ligand) is that most of the leads
identified are receptor antagonists. Few of the leads have any
agonist activity (as expected--agonist activity requires the
ability to bind to and then convert the receptor to the activated
conformation, whereas antagonist actively merely requires the
ability to bind to the receptor or ligand in such a way as to
prevent their interactions) and generating analogs of the initial
antagonist leads to convert them to agonists is a "hit and miss"
affair with very low success rates.
[0010] One approach to circumventing this problem would be to
replace the random compound library with a library of molecular
structures preselected to contain a high proportion of GPCR binding
compounds. Such a library would also ideally include both agonists
and antagonists in similar proportion so that either could be
readily located. Ideally, also, the basic molecular structures used
in the library would be non-toxic.
[0011] Whether or not real libraries can be constructed which
approximate these ideal properties is not at all clear. If they do,
it will require the existance of a putative "ideal" GPCR substrate
which would interact with many different GPCRs irrespective of
their natural ligand preferences. By varying the substitution of
this idealised substrate it may then be possible to impart
selectivity for one receptor in the class over all the others.
[0012] Here we describe an example of such an "ideal" GPCR
substrate. Based on this "ideal" substrate, we provide a range of
related skeletons which can be variously substituted to generate
agonists and/or anatagonists at a range of different GPCRs. The
invention also provides for the preparation of libraries of said
substituted compounds as well as their application in a screening
process in order to generate GPCR ligands with any prescribed set
of specificities. In this way, it is now possible to "dial up" a
GPCR ligand with a known set of properties (for example, a ligand
which has agonist activity at dopamine D2 receptors at the same
time as antagonist activity at serotonin 5HT.sub.1a (receptors)).
In contrast, identifying such mixed ligands serendipitously from
random libraries is a very rare event.
[0013] The requirements for the generation of said libraries are:
(i) the identification of the molecular motif necessary for binding
to GPCRs (ii) a skeleton incorporating this molecular motif so as
to retain GPCR binding, but also providing low intrinsic toxicity
and good stability, pharmacokinetics and/or pharmacodynamics (iii)
a facile synthetic route to generate diverse substitution of the
skeleton.
[0014] The present invention is based on the ideal substrate
below:
##STR00002##
where X is R--CO-- or R--SO.sub.2-- and R represents diverse
substitution. The dotted line indicates constraint of the bond
angles within the molecular motif, for example by cyclisation.
[0015] The skeletons which are provided have been designed using
the wide availability of .alpha.-amino acids as starting materials
in order to generate the diversity of substitution (see, for
example, Unnatural Amino Acids: Tools for Drug Discovery;
Sigma-Aldrich ChemFiles Vol 4 No. 6). Each of the skeletons is
synthesised from the combination of two .alpha.-amino acids. The
first may be selected from cysteine, penicillamine or
3-aminoalanine. Any amino acid is then paired with the first amino
acid to generate the requisite diversity.
[0016] The invention provides compounds and salts thereof of
general formula (I), representing the synthetic product of
reactions using cysteine and a second amino acid as the starting
materials:
##STR00003##
[0017] wherein
[0018] X is --CO--(Y).sub.k-(Z).sub.n or
SO.sub.2--(Y).sub.k-(Z).sub.n;
[0019] k is 0 or 1
[0020] Y is a cycloalkyl or polycyloalkyl group (such as an
adamantyl, adamantanemethyl, bicyclooctyl, cyclohexyl, cyclopropyl
group);
[0021] or Y is a cycloalkenyl or polycycloalkenyl group;
[0022] each Z is independently selected from hydrogen or an alkyl,
haloalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, alkylamino,
alkylaminoalkyl, alkylaminodialkyl, charged alkylaminotrialkyl or
charged alkylcarboxylate radical of 1 to 20 carbon atoms;
[0023] or each Z is independently selected from fluoro, chloro,
bromo, iodo, hydroxy, oxyalkyl, amino, aminoalkyl, aminodialkyl,
charged aminotrialkyl, or carboxylate radical; and
[0024] n is any integer from 1 to m, where m is the maximum number
of substitutions permissible on the cyclo-group Y.
[0025] Alternatively Z may be selected from a peptido radical, for
example having from 1 to 4 peptidic moieties linked together by
peptide bonds (for example a peptido radical of 1 to 4 amino acid
residues)
[0026] R.sup.3 and R.sup.4 represent the diverse substitutions
which, together with Z, distinguish one library element from
another.
[0027] This class of compounds, 6-acylamino-[1,4]thiazepan-5-ones,
are described as "Thiofoxins". The key structural features of the
molecules are the lactam amide in a ring system, with an amino
group attached to the carbon atom next to the lactam carbonyl group
(the 6-position, termed the .alpha.-carbon), and sulfur at the 1
position and R.sup.3 and R.sup.4 (variable) at the 3 and 2
positions of the lactam ring, respectively.
[0028] The invention also provides compounds and salts thereof of
general formula (II), representing the synthetic product of
reactions using penicillamine and a second amino acid as the
starting materials:
##STR00004##
[0029] wherein
[0030] X is --CO--(Y).sub.k-(Z).sub.n or
SO.sub.2--(Y).sub.k-(Z).sub.n;
[0031] k is 0 or 1
[0032] Y is a cycloalkyl or polycyloalkyl group (such as an
adamantyl, adamantanemethyl, bicyclooctyl, cyclohexyl, cyclopropyl
group);
[0033] or Y is a cycloalkenyl or polycycloalkenyl group;
[0034] each Z is independently selected from hydrogen or an alkyl,
haloalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, alkylamino,
alkylaminoalkyl, alkylaminodialkyl, charged alkylaminotrialkyl or
charged alkylcarboxylate radical of 1 to 20 carbon atoms;
[0035] or each Z is independently selected from fluoro, chloro,
bromo, iodo, hydroxy, oxyalkyl, amino, aminoalkyl, aminodialkyl,
charged aminotrialkyl, or carboxylate radical; and
[0036] n is any integer from 1 to m, where m is the maximum number
of substitutions permissible on the cyclo-group Y.
[0037] Alternatively Z may be selected from a peptido radical, for
example having from 1 to 4 peptidic moieties linked together by
peptide bonds (for example a peptido radical of 1 to 4 amino acid
residues)
[0038] R.sup.3 and R.sup.4 represent the diverse substitutions
which, together with Z, distinguish one library element from
another. R.sup.3 and R.sup.4 may be independently selected as any
substituent group, except that R.sup.3 may not be --COOH, --COOR',
--COSR', or --CONR'R'' where R' and R'' independently are any
substituent and either or both of R' and R'' can be H.
[0039] This class of compounds,
6-acylamino-7,7-dimethyl[1,4]thiazepan-5-ones, are described as
"Dimethylthiofoxins". The key structural features of the molecules
are the lactam amide in a ring system, with an amino group attached
to the carbon atom next to the lactam carbonyl group (the
6-position, termed the .alpha.-carbon), and sulfur at the 1
position and R.sup.3 and R.sup.4 (variable) at the 3 and 2
positions of the lactam ring, respectively.
[0040] The invention also provides compounds and salts thereof of
general formula (III), representing the synthetic product of
reactions using 3-aminoalanine and a second amino acid as the
starting materials:
##STR00005##
[0041] wherein
[0042] X is --CO--(Y).sub.k-(Z).sub.n or
SO.sub.2--(Y).sub.k-(Z).sub.n;
[0043] k is 0 or 1
[0044] Y is a cycloalkyl or polycyloalkyl group (such as an
adamantyl, adamantanemethyl, bicyclooctyl, cyclohexyl, cyclopropyl
group);
[0045] or Y is a cycloalkenyl or polycycloalkenyl group;
[0046] each Z is independently selected from hydrogen or an alkyl,
haloalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, alkylamino,
alkylaminoalkyl, alkylaminodialkyl, charged alkylaminotrialkyl or
charged alkylcarboxylate radical of 1 to 20 carbon atoms;
[0047] or each Z is independently selected from fluoro, chloro,
bromo, iodo, hydroxy, oxyalkyl, amino, aminoalkyl, aminodialkyl,
charged aminotrialkyl, or carboxylate radical; and
[0048] n is any integer from 1 to m, where m is the maximum number
of substitutions permissible on the cyclo-group Y.
[0049] Alternatively Z may be selected from a peptido radical, for
example having from 1 to 4 peptidic moieties linked together by
peptide bonds (for example a peptido radical of 1 to 4 amino acid
residues)
[0050] R.sup.2, R.sup.3 and R.sup.4 represent the diverse
substitutions which, together with Z, distinguish one library
element from another.
[0051] This class of compounds, 6-acylamino-[1,4]diazepan-5-ones,
are described as "Azafoxins". The key structural features of the
molecules are the lactam amide in a ring system, with an amino
group attached to the carbon atom next to the lactam carbonyl group
(the 6-position, termed the .alpha.-carbon), and nitrogen at the 1
position and R.sup.3 and R.sup.4 (variable) at the 3 and 2
positions of the lactam ring, respectively. Optionally, further
diversity can be generated by substitution with R.sup.2 (variable)
at the N1 position.
[0052] The invention also provides compounds and salts thereof of
general formula (IV), representing an alternative synthetic product
of reactions using 3-aminoalanine and a second amino acid as the
starting materials:
##STR00006##
[0053] wherein
[0054] X is --CO--(Y).sub.k-(Z).sub.n or
SO.sub.2--(Y).sub.k-(Z).sub.n;
[0055] k is 0 or 1
[0056] Y is a cycloalkyl or polycyloalkyl group (such as an
adamantyl, adamantanemethyl, bicyclooctyl, cyclohexyl, cyclopropyl
group);
[0057] or Y is a cycloalkenyl or polycycloalkenyl group;
[0058] each Z is independently selected from hydrogen or an alkyl,
haloalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, alkylamino,
alkylaminoalkyl, alkylaminodialkyl, charged alkylaminotrialkyl or
charged alkylcarboxylate radical of 1 to 20 carbon atoms;
[0059] or each Z is independently selected from fluoro, chloro,
bromo, iodo, hydroxy, oxyalkyl, amino, aminoalkyl, aminodialkyl,
charged aminotrialkyl, or carboxylate radical; and
[0060] n is any integer from 1 to m, where m is the maximum number
of substitutions permissible on the cyclo-group Y.
[0061] Alternatively Z may be selected from a peptido radical, for
example having from 1 to 4 peptidic moieties linked together by
peptide bonds (for example a peptido radical of 1 to 4 amino acid
residues)
[0062] R.sup.2 and R.sup.3 represent the diverse substitutions
which, together with Z, distinguish one library element from
another.
[0063] This class of compounds,
6-acylamino-[1,4]diazepan-2,5-diones, are described as
"Amidofoxins". The key structural features of the molecules are the
lactam amide in a ring system, with an amino group attached to the
carbon atom next to the lactam carbonyl group (the 6-position,
termed the .alpha.-carbon), nitrogen at the 1 position, a carbonyl
group at the 2 position with R.sup.3 (variable) at the 3 position
of the lactam ring. Optionally, further diversity can be generated
by substitution with R.sup.2 (variable) at the N1 position.
[0064] The .alpha.-carbon of these Thiofoxins, Dimethylthiofoxins,
Azafoxins, and Amidofoxins is asymmetric and consequently, the
compounds according to the present invention have two possible
enantiomeric forms, that is, the "R" and "S" configurations. The
present invention encompasses the two enantiomeric forms and all
combinations of these forms, including the racemic "RS" mixtures.
With a view to simplicity, when no specific configuration is shown
in the structural formulae, it should be understood that the two
enantiomeric forms and their mixtures are represented.
[0065] The compounds of general formulae (I), (II), (III) and (IV)
are all N-substituted on the exocyclic amine group. The
N-substitutent is either a carbon amide or a sulfonamide. The
geometry of the carbon atom next to the carbonyl of the carbon
amide or the sulfoyl group of the sulfonamide (the "key" carbon)
may be important for the bioactivity of the molecule. The nature of
this N-substituent may be such that the ring or rings of Y
constrain the bond angles at the "key"-carbon to be essentially
tetrahedral (i.e. sp3 hybrid bonds). Any substituent Z may be a
substituent at any permissible position on the ring or rings of the
cyclo-group Y. In particular it is to be noted that the invention
includes compounds in which the "key carbon" is both part of the
cyclo group and is itself substituted. The definition of (Z).sub.n
encompasses compounds of the invention with no substitution (i.e.
Z=hydrogen), compounds of the invention with mono substitution
(i.e. Z is not hydrogen and n=1), and also multiple substitution
(i.e. at least two Z groups are not hydrogen and n=2 or more).
[0066] One major advantage of the compounds of the invention is
that the diverse library elements may be readily synthesised from
widely available starting materials. Thiofoxins,
Dimethylthiofoxins, Azafoxins and Amidofoxins each represent a
diverse class of compounds (with diversity at the Z, R.sup.2 (if
applicable), R.sup.3 and (if applicable) R.sup.4 positions) which
can be readily synthesised from two different .alpha.-amino acids.
.alpha.-amino acids represent an ideal starting material for
diversity-oriented synthesis since a wide range of .alpha.-amino
acids (differing only in the nature of the R.sup.3 substituent) are
known, and are commercially available. For the synthesis of
Thiofoxins, Dimethylthiofoxins and Azafoxins the .alpha.-amino
acids are readily reduced to yield .beta.-amino alcohols (using
protecting groups to retain the structure of the R.sup.3 moiety if
required). The diverse .beta.-amino alcohols are then coupled to
cysteine to yield "Thiofoxamines", to penicillamine to yield
"Dimethylthiofoxamines", or to 3-aminoalanine to yield
"Azafoxamines. These .alpha.-aminolactams can then be coupled to an
appropriate acyl side chain by conventional amide coupling
reactions (again protecting the R.sup.3 substituent if required),
yielding Thiofoxins, Dimethylthiofoxins or Azafoxins
respectively.
##STR00007##
[0067] In contrast, Amidofoxins are generated by reacting the
.alpha.-amino-acid (without first reducing it to the
.beta.-aminoalcohol) with 3-aminoalanine. It will be noticed that
Amidofoxins are 7-membered ring analogs of the well-studied
diketopiperazines generated from .alpha.-amino acid dimers, but
generated from a dimer of a .beta.-amino acid (3-aminoalanine) and
an .alpha.-amino acid.
##STR00008##
[0068] The resulting Thiofoxins, Dimethylthiofoxins, Azafoxins and
Amidofoxins have the same range of R.sup.3 substituents as the
original collection of available .alpha.-amino acids. Furthermore,
general synthetic routes for generating .alpha.-amino acids are
well known in the art (for example, see R. M. Williams, Synthesis
of Optically Active a-Amino Acids (Pergamon, New York) 1989),
allowing even greater diversity to be generated as required.
[0069] Alternatively or additionally, use of directly available
.beta.-amino alcohols, such as ephidrine, with variable
2-substitution allow the introduction of additional diversity as a
variable R.sup.4 group at the 2-position of the ring in Thiofoxins,
Dimethylthiofoxins and Azafoxins.
[0070] Alternatively or additionally, a prior reductive alkylation
of the 3-aminoalanine moiety allows introduction of diversity at
the N1 position in Azafoxins or Amidofoxins (the variable R.sup.2
group).
[0071] A particular feature of the present invention is the facile
stereocontrol of the synthesis. The exemplified routes use cheap
and readily available L-cysteine, L-penicillamine and
L-3-aminoalanine to couple with diverse .beta.-amino alcohols (or
directly with .alpha.-amino acids in the case of Amidofoxins). This
results in a diverse series of .alpha.-aminolactams which are of
the (S)-configuration. Alternatively, D-cysteine, D-penicillamine
and D-3-aminoalanine are also readily available, and can be coupled
with the same diverse .beta.-amino alcohols (or .alpha.-amino
acids) to yield .alpha.-aminolactams of the (R)-configuration. In
the same way, by selecting the appropriate enantiomerically pure
.alpha.-amino acid starting material, and then using a synthetic
route which retains stereochemistry (such as the routes exemplified
below), then Thiofoxamines, Dimethylthiofoxamines, Azafoxamines
and/or Amidofoxamines and therefore Thiofoxins, Dimethylthiofoxins,
Azafoxins and/or Amidofoxins with the appropriate configuration at
the carbon bearing the R.sup.3-substituent can readily be
synthesised. For example, performing the Thiofoxin synthesis with
L-alanine and L-cysteine yields
(6R)-amino-(3S)-methyl-[1,4]thiazapan-5-one. In contrast,
performing the synthesis with D-alanine and L-cysteine yields
(R,R)-6-amino-3-methyl-[1,4]thiazapan-5-one, using L-alanine and
D-cysteine yields (S,S)-6-amino-3-methyl-[1,4]thiazapan-5-one, and
using D-alanine and D-cysteine yields
(6S)-amino-(3R)-methyl-[1,4]thiazapan-5-one. Alternatively, racemic
mixtures of one or both starting materials may be selected,
yielding mixed stereoisomers of the Thiofoxamine,
Dimethylthiofoxamine, Azafoxamine and/or Amidofoxamine and hence
Thiofoxin, Dimethylthiofoxin, Azafoxin, and/or Amidofoxin
products.
[0072] Importantly, it is possible to carry out the steps of the
synthetic reactions in different orders as required. When
generating a diverse library through combinatorial synthesis, it is
important to generate as much of the diversity as possible late in
the synthesis. Ideally, the last step should introduce the greatest
diversity into the library. Since diversity may be introduced at
several steps in the synthesis of the compounds of the invention
(with variable Z, R.sup.2 (for Azafoxins and Amidofoxins). R.sup.3
and R.sup.4 (for Thiofoxins, Dimethylthiofoxins and Azafoxins), it
is possible to introduce diversity equally at each step (for
example to have 8 different Z groups, R.sup.2 groups and R.sup.3
groups in an Amidofoxin library, thereby yielding 512 compounds) or
to introduce greater diversity at one of the steps (for example to
have 2 different Z and R.sup.2 groups, but 128 different R.sup.3
groups in an Amidofoxin library, thereby yielding 512 compounds).
In such cases where diversity is introduced to a greater extent in
one particular step it is advantageous to have this step as late as
possible in the synthetic route. One advantage of the invention
provided here is that the synthetic routes are well suited to
changes in the order of the reaction steps. Specific examples of
synthetic routes to yield Thiofoxins, Dimethylthiofoxins,
Azafoxins, and Amidofoxins are provided in the examples below. For
each of the classes, examples are given where the reaction steps
are performed in different orders. However, It should be stressed
that other synthetic routes exist which could alternatively be
used, and would fall under the scope of the present invention.
[0073] It is envisaged that synthesis of library elements may be
carried out using parallel synthesis methods well known in the art.
For example, the synthesis may be performed using resins or other
solid-phase supports to simplify the introduction of diversity and
to facilitate the purification, or partial purification, of the
library element products. Application of such solid phase, or other
parallel synthesis, methodologies, whether manual, semi-automated
or automated, to generate a library of Thiofoxins,
Dimethylthiofoxins, Azafoxins or Amidofoxins falls under the scope
of the present invention.
[0074] The invention also provides pharmaceutical compositions
comprising, as active ingredient, a compound of general formula
(I), (II), (III) or (IV), or a pharmaceutically acceptable salt
thereof, and at least one pharmaceutically acceptable excipient
and/or carrier.
[0075] By pharmaceutically acceptable salt is meant in particular
the addition salts of inorganic acids such as hydrochloride,
hydrobromide, hydroiodide, sulphate, phosphate, diphosphate and
nitrate or of organic acids such as acetate, maleate, fumarate,
tartrate, succinate, citrate, lactate, methanesulphonate,
p-toluenesulphonate, palmoate and stearate. Also within the scope
of the present invention, when they can be used, are the salts
formed from bases such as sodium or potassium hydroxide. For other
examples of pharmaceutically acceptable salts, reference can be
made to "Salt selection for basic drugs", Int. J. Pharm. (1986),
33, 201-217.
[0076] The pharmaceutical composition can be in the form of a
solid, for example powders, granules, tablets, gelatin capsules,
liposomes or suppositories. Appropriate solid supports can be, for
example, calcium phosphate, magnesium stearate, talc, sugars,
lactose, dextrin, starch, gelatin, cellulose, methyl cellulose,
sodium carboxymethyl cellulose, polyvinylpyrrolidine and wax. Other
appropriate pharmaceutically acceptable excipients and/or carriers
will be known to those skilled in the art.
[0077] The pharmaceutical compositions according to the invention
can also be presented in liquid form, for example, solutions,
emulsions, suspensions or syrups. Appropriate liquid supports can
be, for example, water, organic solvents such as glycerol or
glycols, as well as their mixtures, in varying proportions, in
water.
[0078] The invention may also provide the use of a compound of
general formula (I), (II), (III) and/or (IV), or a pharmaceutically
acceptable salt thereof, for the preparation of a medicament
intended to modulate the activity of one or more members of the
G-protein coupled receptor (GPCR) class.
[0079] The invention provides compounds, compositions and uses of
the compounds of general formula (I), (II), (III) and (IV) or their
pharmaceutically acceptable salts, wherein the R.sup.1 radical has
a "key" carbon which is di-substituted with the same or different
groups selected from: alkyl, haloalkyl, alkoxy, haloalkoxy,
alkenyl, alkynl and alkylamino radicals.
[0080] The invention provides compounds, compositions and uses
wherein the "key" carbon is chiral.
[0081] The invention provides compounds, compositions and uses
wherein the "key" carbon has sp3 hybridised bonds.
[0082] The invention provides compounds, compositions and uses
wherein the "key" carbon has essentially tetrahedral bond
angles.
[0083] The compounds of general formula (I), (II), (III) or (IV)
when used in the invention, or their salts, may be such that the
ring or rings of Y constrain the bond angles at the "key" carbon to
be essentially tetrahedral (i.e. sp3 hybrid bonds).
[0084] The invention also provides the sulfonamide analogues of the
exemplified compounds: i.e. the
sulfonyl-.alpha.-aminolactam-derived Thiofoxin, Dimethylthiofoxin,
Azafoxin or Amidofoxin equivalents of the compounds of Formula (I),
(II), (III) and (IV) respectively.
[0085] The invention includes compounds, compositions and uses
thereof as defined, wherein the compound is in hydrated or solvated
form.
[0086] The amide and sulfonamide Thiofoxins, Dimethylthiofoxins,
Azafoxins and Amidofoxins described here are likely to be
functional GPCR agonists and antagonists. The core consisting of
the "key" carbon, the carbonyl or sulfonyl group, the .alpha.-amino
group and the Thiofoxin, Dimethylthiofoxin, Azafoxin or Amidofoxin
ring represents an example of a GPCR ligand. By varying the
substitution of this core, particularly at the position bearing the
R.sup.3 substituent, it is possible to generate GPCR agonists and
antagonists with a wide range of desirable properties much more
readily than by screening random compound libraries.
[0087] As a result, the invention also provides for a library
consisting of two or more members of the class of compounds
designated by general formula (I), (II), (III) and/or (IV), such
that the library may be screened to identify a molecule with a
particular desirable set of properties with regard to modulating
signalling at one (or more) GCPRs. The said library would then be
screened for antagonist or agonist activity at the said GPCR(s)
using methods well known in the art. For example, the library may
be screened for the ability of individual library elements to block
the binding of a radiolabelled GPCR ligand to a membrane
preparation containing recombinant or purified GPCR. Alternatively,
the library may be screened for the ability of individual library
elements to stimulate cAMP production in cells expressing a
recombinant GPCR.
[0088] Any Thiofoxin, Dimethylthiofoxin, Azafoxin or Amidofoxin
compound according to the invention which exhibits desirable
properties can be used as a template for synthesis of the analogous
"Carbofoxin" (which has a carbon group rather than sulfur at the 1
position in Thiofoxins or Dimethylthiofoxins, or nitrogen at the 1
position in Azafoxins or Amidofoxins), for example by using
ring-closing metathesis synthetic routes which are well known in
the art (such as Truka, T. M.; Grubbs, R. H. Acc. Chem. Res. 2001,
34, 18). Suitable ring closing-metathesis routes have also been
exemplified which could be used to synthesise "Carbofoxin" analogs
of Azafoxins or Amidofoxins where the N at position 1 is
substituted (i.e. R.sup.2.noteq.H), such as the method of Del Valle
R. R. & Goodman M. J. Org. Chem. 2004, 69.8946. Such
"Carbofoxin" compounds are expected to be useful GPCR agonists or
antagonists.
[0089] The invention also provides a method of treatment,
amelioration or prophylaxis of the symptoms of disease or condition
selected from the group consisting of hypertension,
atherosclerosis, asthma, obesity, neurodegenerative disorders,
autoimmune disorders or psychopathic disorders by the
administration to a patient of an effective amount of a compound,
composition or medicament of the invention designed to modulate
GPCR activity.
DEFINITIONS
[0090] The term "about" refers to an interval around the considered
value. As used in this patent application, "about X" means an
interval from X minus 10% of X to X plus 10% of X, and preferably
an interval from X minus 5% of X to X plus 5% of X.
[0091] The use of a numerical range in this description is intended
unambiguously to include within the scope of the invention all
individual integers within the range and all the combinations of
upper and lower limit numbers within the broadest scope of the
given range. Hence, for example, the range of 1 to 20 carbon atoms
specified in respect of (inter alia) formula I is intended to
include all integers between 1 and 20 and all sub-ranges of each
combination of upper and lower numbers, whether exemplified
explicitly or not.
[0092] As used herein, the term "comprising" is to be read as
meaning both comprising and consisting of: Consequently, where the
invention relates to a "pharmaceutical composition comprising as
active ingredient" a compound, this terminology is intended to
cover both compositions in which other active ingredients may be
present and also compositions which consist only of one active
ingredient as defined.
[0093] The term "peptidic moieties" used herein is intended to
include the following 20 naturally-occurring proteogenic amino acid
residues:
TABLE-US-00001 SYMBOL: MEANING Ala Alanine Cys Cysteine Asp
Aspartic Acid Glu Glutamic Acid Phe Phenylalanine Gly Glycine His
Histidine Ile Isoleucine Lys Lysine Leu Leucine Met Methionine Asn
Asparagine Pro Proline Gln Glutamine Arg Arginine Ser Serine Thr
Threonine Val Valine Trp Tryptophan Tyr Tyrosine
[0094] Modified and unusual amino acid residues, as well as
peptido-mimetics, are also intended to be encompassed within the
definition of "peptidic moieties".
[0095] Unless otherwise defined, all the technical and scientific
terms used here have the same meaning as that usually understood by
an ordinary specialist in the field to which this invention
belongs. Similarly, all the publications, patent applications, all
the patents and all other references mentioned here are
incorporated by way of reference (where legally permissible).
[0096] The following examples are presented in order to illustrate
the invention and should in no way be considered to limit the scope
of the invention.
EXAMPLES
[0097] In each of the following examples, a range of protecting
groups are used. The functional properties of the required
protecting groups are specified (that is, in certain steps it is
required that two different protecting groups are used which are
removed by different reaction condtions--i.e. orthogonal protecting
groups), but the molecular composition is not specified. Any
suitable protecting group well known in the art may be substituted.
Consequently, variable elements of such protecting groups are
designated R.sup.5, R.sup.6, R.sup.7 and/or R.sup.8 in the
following examples. The protecting groups (and hence the R.sup.5,
R.sup.6, R.sup.7 and R.sup.8 substituents) are not themselves part
of the products falling under the scope of the invention.
Example 1
Synthesis of Thiofoxins
[0098] The Thiofoxins are products of the coupling of cysteine with
a .beta.-amino alcohol (possibly derived from the reduction of an
.alpha.-amino acid). In the first scheme (Scheme 1A below), the
R.sup.1 group is introduced, then the R.sup.3/R.sup.4 group, and
then the compounds are cyclised. Such a route would be optimal if
greater diversity was to be introduced at R.sup.3/R.sup.4 than
R.sup.1.
##STR00009##
[0099] In the first step the R.sup.1-containing acyl (or
sulfonamide) substituent is introduced by an appropriate amide
coupling route, several of which are well known in the art, e.g.
DCC coupling.
[0100] Separately, one or more .beta.-amino alcohols are obtained
(e.g. ephedrine) or synthesised from .alpha.-amino acids, In each
case, the amino group is protected (e.g. by addition of a Boc
group, or by obtaining the appropriately protected .alpha.-amino
acid from a commercial supplier). Boc-protected amino acids are
converted to amino alcohols by reduction, and the Boc-protected
.beta.-amino alcohol (whether purchased or obtained by reduction of
an amino acid) is modified so as to provide a suitable leaving
group to allow alkylation of the side-chain heteroatom (in this
case, the sulfur of cysteine), for example the amino-protected
.beta.-amino alcohol could be mesylated. Several suitable methods
for reduction and mesylation exist, and are well known in the art
(e.g. Synthesis (1992) 1359 or Synthesis (1996) 1223).
[0101] In the next step, the acylcysteine is alklylated at the
sulfur centre using any of several appropriate methods which are
well known in the art (e.g. J Med Chem. (1987) 30:1984). Each
.beta.-amino alcohol is reacted separated with an appropriate
acylcysteine to yield individual library elements with variable
R.sup.1, R.sup.2 and R.sup.3 depending on the selection of
acylcysteines and .beta.-amino alcohols available. The nitrogen
introduced at this step is hereafter termed the .omega.-amine
group.
[0102] In the final step, the S-alkyl acylcysteines are cyclised.
The .omega.-amine is selectively deprotected and condensed with the
carboxy ester (or after selective hydrolysis the corresponding
carboxylic acid) to form a seven-membered ring, yielding Thiofoxin
library elements. Similar methods are well known in the art (e.g.
J. Med. Chem (1987) 30:1984).
[0103] In the second scheme (Scheme 1B below), the R.sup.3/R.sup.4
groups are introduced, then the compounds are cyclised and finally
the R.sup.1 group is introduced. Such a route would be optimal if
greater diversity was to be introduced at R.sup.1 than
R.sup.3/R.sup.4
##STR00010##
[0104] In the first step, cysteine is selectively protected at the
.alpha.-amine, and is then alkylated on S with an N-protected
.beta.-amino-alcohol in which the alcohol has been activated to
form a leaving group suitable for nucleophilic substitution with
inversion of stereochemistry at carbon. The introduced nitrogen is
here-on called the .omega.-amine group (for similar see
Tetrahedron, 1999, 55, 10155).
[0105] One or more .beta.-amino alcohols suitable for this
alkylation are obtained (e.g. ephedrine) or synthesised from
.alpha.-amino acids, In each case, the amino agroup is protected
(e.g. by addition of a Boc group, or by obtaining the appropriately
protected .alpha.-amino acid from a commercial supplier).
Boc-protected amino acids are converted to amino alcohols by
reduction, and the Boc-protected .beta.-amino alcohol (whether
purchased or obtained by reduction of an amino acid) is mesylated.
Several suitable methods for reduction and mesylation exist, and
are well known in the art (e.g. Synthesis (1992) 1359 or Synthesis
(1996) 1223).
[0106] In the next step, the .omega.-amine is selectively
deprotected and condensed with the carboxy ester (or after
selective hydrolysis the corresponding carboxylic acid) to form a
seven-membered ring (according to J. Med. Chem., 1987, 30,
1984).
[0107] In the final step, following cyclisation, the .alpha.-amine
is selectively deprotected and acylated as required to introduce
diversity at the R.sup.1 position, using an appropriate peptide
coupling reagent, several of which are well known in the art.
[0108] In the third scheme (Scheme 1C below), the R.sup.3/R.sup.4
groups are introduced, then the R.sup.1 group, and finally the
compounds are cyclised. Such a route might be useful if greater
diversity was to be introduced at R.sup.1 than R.sup.3/R.sup.4.
##STR00011##
[0109] This route is similar to Scheme 1B, except that the
acylation of the .alpha.-amine of cysteine is performed prior to
the cyclisation (for similar see Tetrahedron, 1999, 55, 10155).
[0110] These three schemes illustrate the facile nature of the
synthesis of a library composed of one or more Thiofoxin elements.
In particular, the ability to perform the steps of the reaction in
various orders in order to allow greater diversity to be introduced
into different regions of the molecule while keeping the synthesis
practicable is illustrated.
[0111] A number of related structures are already known in the
literature, including two examples described as GPCR ligands.
##STR00012##
[0112] Compound (V) is a histamine H2 or gastrin receptor ligand
(depending on the nature of R and X) described in Bioorg. Med. Chem
(1997) 5:1411 and compound (VI) is a Neuropeptide Y receptor ligand
described in Bioorg. Med. Chem, (1999) 7:1703. Although clearly
related in structure to Thiofoxins, neither compound is a
Thiofoxin, nor falls under the scope of the present invention
because of the presence of substitution at the N4 position.
However, the existance of these compounds underlines the high
density of GPCR ligands likely to be found in libraries composed
of, or enriched in, Thiofoxins.
Example 2
Synthesis of Dimethylthiofoxins
[0113] The Dimethylthiofoxins are analagous to the Thiofoxins,
except that penicillamine is used in place of cysteine.
Dimethylthiofoxins are then products of the coupling of
penicillamine with a .beta.-amino alcohol (possibly derived from
the reduction of an .alpha.-amino acid). In the first scheme
(Scheme 2A below), the R.sup.1 group is introduced, then the
R.sup.3/R.sup.4 group, and then the compounds are cyclised. Such a
route would be optimal if greater diversity was to be introduced at
R.sup.3/R.sup.4 than R.sup.1.
##STR00013##
[0114] In the first step the R.sup.1-containing acyl (or
sulfonamide) substituent is introduced onto pencillamine by an
appropriate amide coupling route, several of which are well known
in the art, e.g. DCC coupling.
[0115] Separately, one or more .beta.-amino alcohols are obtained
(e.g. ephedrine) or synthesised from .alpha.-amino acids, In each
case, the amino agroup is protected (e.g. by addition of a Boc
group, or by obtaining the appropriately protected .alpha.-amino
acid from a commercial supplier). Boc-protected amino acids are
converted to amino alcohols by reduction, and the Boc-protected
.beta.-amino alcohol (whether purchased or obtained by reduction of
an amino acid) is mesylated. Several suitable methods for reduction
and mesylation exist, and are well known in the art (e.g. Synthesis
(1992) 1359 or Synthesis (1996) 1223).
[0116] In the next step, the acylpenicillamine is alklylated at the
sulfur centre using any of several appropriate methods which are
well known in the art (e.g. J Med Chem. (1987) 30:1984). Each
.beta.-amino alcohol is reacted separately with an appropriate
acylpenicillamine to yield individual library elements with
variable R.sup.1, R.sup.2 and R.sup.3 depending on the selection of
acylcysteines and .beta.-amino alcohols available. The nitrogen
introduced at this step is hereafter termed the
.omega.-nitrogen.
[0117] In the final step, the S-alkyl acylpencillamines are
cyclised. The .omega.-amine is selectively deprotected and
condensed with the carboxy ester (or after selective hydrolysis the
corresponding carboxylic acid) to form a seven-membered ring,
yielding Dimethylthiofoxin library elements. Similar methods are
well known in the art (e.g. J. Med. Chem (1987) 30:1984).
[0118] In the second scheme (Scheme 2B below), the R.sup.3/R.sup.4
groups are introduced, then the compounds are cyclised and finally
the R.sup.1 group is introduced. Such a route would be optimal if
greater diversity was to be introduced at R.sup.1 than
R.sup.3/R.sup.4
##STR00014##
[0119] In the first step, penicillamine is selectively protected at
the .alpha.-amine, and is then alkylated on S with an N-protected
.alpha.-amino-alcohol in which the alcohol has been activated to
form a leaving group suitable for nucleophilic substitution with
inversion of stereochemistry at carbon. The introduced nitrogen is
here-on called the .omega.-amine group (for similar see
Tetrahedron, 1999, 55, 10155).
[0120] One or more .beta.-amino alcohols suitable for this
alkylation are obtained (e.g. ephedrine) or synthesised from
.alpha.-amino acids, In each case, the amino agroup is protected
(e.g. by addition of a Boc group, or by obtaining the appropriately
protected .alpha.-amino acid from a commercial supplier).
Boc-protected amino acids are converted to amino alcohols by
reduction, and the Boc-protected .beta.-amino alcohol (whether
purchased or obtained by reduction of an amino acid) is mesylated.
Several suitable methods for reduction and mesylation exist, and
are well known in the art (e.g. Synthesis (1992) 1359 or Synthesis
(1996) 1223).
[0121] In the next step, the .omega.-amine is selectively
deprotected and condensed with the carboxy ester (or after
selective hydrolysis the corresponding carboxylic acid) to form a
seven-membered ring (according to J. Med. Chem., 1987, 30,
1984).
[0122] In the final step, following cyclisation, the .alpha.-amine
is selectively deprotected and acylated as required to introduce
diversity at the R.sup.1 position, using an appropriate peptide
coupling reagent, several of which are well known in the art.
[0123] In the third scheme (Scheme 2C below), the R.sup.3/R.sup.4
groups are introduced, then the R.sup.1 group, and finally the
compounds are cyclised. Such a route might be useful if greater
diversity was to be introduced at R.sup.1 than R.sup.3/R.sup.4.
##STR00015##
[0124] This route is similar to Scheme 2B, except that the
acylation of the .alpha.-amine of penicillamine is performed prior
to the cyclisation (for similar see Tetrahedron, 1999, 55,
10155).
[0125] These three schemes illustrate the facile nature of the
synthesis of a library composed of one or more Dimethylthiofoxin
elements. In particular, the ability to perform the steps of the
reaction in various orders in order to allow greater diversity to
be introduced into different regions of the molecule while keeping
the synthesis practicable is illustrated.
[0126] Many compounds related in structure to the Dimethylthioxoins
are known in the public domain (see for example J. Chem. Soc.,
Chem. Commum., 1993, 1599 and Liebigs Ann. Recl. 1997, 1711),
because cyclisation products between penicillamine and other
.alpha.-amino acids are analogs or biosynthetic intermediates of
well-studied .beta.-lactam antibiotics such as penicillin. However,
these penicillin analogs have carboxylate or the ester, thioester
or amide derivative of carboxylate as the substituent at the
3-position of the ring.
Example 3
Synthesis of Azafoxins
[0127] The Azafoxins are the products of the coupling of
3-aminoalanine with a .beta.-amino alcohol (possibly derived from
the reduction of an .alpha.-amino acid). Unlike the
sulfur-containing lactams (Thiofoxins and Dimethylthiofoxins), the
introduction of a further nitrogen into the lactam ring in
Azafoxins allows for the possibility of further substitution (and
hence diversity) at the ring heteroatom. In the first scheme
(Scheme 3A below), the R.sup.1 group is introduced, then the
R.sup.2 group (substitution at nitrogen) and then the
R.sup.3/R.sup.4 group, before finally the compounds are cyclised.
Such a route would be optimal if greater diversity was to be
introduced at R.sup.3/R.sup.4 than R.sup.2 with least diversity at
R.sup.1.
##STR00016## ##STR00017##
[0128] In the first step, a protected 3-aminoalanine is
synthesised, for example by the Hoffman degradation of
N-.alpha.-carbamate ester protected asparagine (according to J.
Org. Chem., 1997, 62, 6918), and the carboxylic acid is esterified
((according to J. Med. Chem., 1998, 41, 2786).
[0129] In order to introduce substitution at the .alpha.-amine, the
free .beta.-amine is then orthogonally protected and the
.alpha.-amine selectively deprotected by removal of the carbamate
ester. The free .alpha.-amine is then acylated as in the previous
examples to introduce the R.sup.1 functionality.
[0130] Next, the R.sup.2 functionality is introduced, for example
by mono-alkylation, mono-arylation or reductive alkylation, or by
acylation with sulfonyl chloride, using reaction conditions well
known in the literature.
[0131] The .beta.-amine is then alkylated with an N-protected
.beta.-amino-alcohol in which the alcohol has been activated to
form a leaving group suitable for nucleophilic substitution with
inversion of stereochemistry at carbon (as described for Thiofoxins
and Dimethylthiofoxins). Alternatively the .beta.-amine can be
condensed with an N-protected .alpha.-amino-aldehyde in the
presence of a reducing agent forming an amine (according to J. Org.
Chem., 2002, 67, 4017). The introduced nitrogen is here-on called
the .omega.-amine group. This step introduces the R.sup.3/R.sup.4
functionality, depending on the selected .beta.-aminoalcohol
(possibly derived from an .alpha.-amino acid).
[0132] In the final step (as for Thiofoxins and
Dimethylthiofoxins), the .omega.-amine is selectively deprotected
and condensed with the carboxy ester (or after selective hydrolysis
the corresponding carboxylic acid) to form a seven-membered ring
(according to J. Med. Chem., 1987, 30, 1984).
[0133] In the second scheme (Scheme 3B below), the R.sup.1 group is
introduced, then the R.sup.3/R.sup.4 group(s) are introduced and
the compounds are cyclised, and then finally the R.sup.2 group
(substitution at nitrogen) is introduced. Such a route would be
optimal if greater diversity was to be introduced at R.sup.2 than
R.sup.3/R.sup.4 with least diversity at R.sup.1.
##STR00018## ##STR00019##
[0134] In this scheme, protected 3-aminoalanine is synthesised, for
example by Hoffman degradation of N-.alpha.-protected asparagine,
and is then orthogonally protected on the .beta.-amine, deprotected
and acylated at the .alpha.-amine and esterified at the carboxylic
acid all as described for Scheme 3A.
[0135] Then, following selective deprotection of the .beta.-amine,
it is alkylated on nitrogen with an N-protected
.beta.-amino-alcohol in which the alcohol has been activated to
form a leaving group suitable for nucleophilic substitution with
inversion of stereochemistry at carbon (as described for Thiofoxins
and Dimethylthiofoxins). Alternatively the .beta.-amine can be
condensed with an N-protected .alpha.-amino-aldehyde in the
presence of a reducing agent forming an amine (according to J. Org.
Chem., 2002, 67, 4017). The introduced nitrogen is here-on called
the .omega.-amine group. This step introduces the R.sup.3/R.sup.4
functionality, depending on the selected .beta.-aminoalcohol
(possibly derived from an .alpha.-amino acid).
[0136] The .omega.-amine is then selectively deprotected and
condensed with the carboxy ester (or after selective hydrolysis the
corresponding carboxylic acid) to form a seven-membered ring
(according to J. Med. Chem., 1987, 30, 1984).
[0137] Finally, the R.sup.2 functionality is introduced, for
example by mono-alkylation, mono-arylation or reductive alkylation,
or by acylation with sulfonyl chloride, using reaction conditions
well known in the literature.
[0138] In the third scheme (Scheme 3C below), the R.sup.2 group is
introduced, then the R.sup.1 group and then the R.sup.3/R.sup.4
group(s), before the compounds are finally cyclised. Such a route
would be optimal if greater diversity was to be introduced at
R.sup.3/R.sup.4 than R.sup.1 with least diversity at R.sup.2.
##STR00020## ##STR00021##
[0139] In this scheme, protected 3-aminoalanine is synthesised, for
example by Hoffman degradation of N-.alpha.-protected asparagine,
and the carboxylic acid esterified (as in Scheme 3A, for example),
but the .beta.-amine is then mono-alkylated, mono-arylated or
sulfonated as required to introduce the R.sup.2 functionality,
using methods well known in the literature. Thereafter, the
secondary .beta.-amine is protected orthogonally to the
.alpha.-amine.
[0140] In the next step, the .alpha.-amine is selectively
deprotected and acylated as described above to introduce the
R.sup.1 functionality.
[0141] Then, following deprotection of the .beta.-amine, it is
alkylated on nitrogen with an N-protected .beta.-amino-alcohol in
which the alcohol has been activated to form a leaving group
suitable for nucleophilic substitution with inversion of
stereochemistry at carbon (as described for Thiofoxins and
Dimethylthiofoxins). Alternatively the .beta.-amine can be
condensed with an N-protected .alpha.-amino-aldehyde in the
presence of a reducing agent forming an amine (according to J. Org.
Chem., 2002, 67, 4017). The introduced nitrogen is here-on called
the .omega.-amine group. This step introduces the R.sup.3/R.sup.4
functionality, depending on the selected .beta.-aminoalcohol
(possibly derived from an .alpha.-amino acid).
[0142] The .omega.-amine is then selectively deprotected and
condensed with the carboxy ester (or after selective hydrolysis the
corresponding carboxylic acid) to form a seven-membered ring
(according to J. Med. Chem., 1987, 30, 1984).
[0143] In the fourth scheme (Scheme 3D below), the R.sup.2 group is
introduced, then the R.sup.3/R.sup.4 group(s). The compounds are
next cyclised, and finally the R.sup.1 group is introduced. Such a
route would be optimal if greater diversity was to be introduced at
R.sup.1 than R.sup.3/R.sup.4 with least diversity at R.sup.2.
##STR00022##
[0144] In this scheme, protected 3-aminoalanine is synthesised, for
example by Hoffman degradation of N-.alpha.-protected asparagine,
and the carboxylic acid esterified. The .beta.-amine is then
mono-alkylated, mono-arylated or sulfonated as required to
introduce the R.sup.2 functionality, using methods well known in
the literature, all as in Scheme 3C.
[0145] In the next step, the secondary .beta.-amine, is alkylated
on nitrogen with an N-protected .beta.-amino-alcohol in which the
alcohol has been activated to form a leaving group suitable for
nucleophilic substitution with inversion of stereochemistry at
carbon (as described for Thiofoxins and Dimethylthiofoxins).
Alternatively the .beta.-amine can be condensed with an N-protected
.alpha.-amino-aldehyde in the presence of a reducing agent forming
an amine (according to J. Org. Chem., 2002, 67, 4017). The
introduced nitrogen is here-on called the .omega.-amine group. This
step introduces the R.sup.3/R.sup.4 functionality, depending on the
selected .beta.-aminoalcohol (possibly derived from an
.alpha.-amino acid).
[0146] The .omega.-amine is then selectively deprotected and
condensed with the carboxy ester (or after selective hydrolysis the
corresponding carboxylic acid) to form a seven-membered ring
(according to J. Med. Chem., 1987, 30, 1984).
[0147] Finally, the .beta.-amine is selectively deprotected and
acylated as described above to introduce the R.sup.1
functionality.
[0148] In the fifth scheme (Scheme 3E below), the R.sup.3/R.sup.4
group(s) are introduced first, followed by cyclisation. The R.sup.1
group and then the R.sup.2 group are then introduced. Such a route
would be optimal if greater diversity was to be introduced at
R.sup.2 than R.sup.1 with least diversity at R.sup.3/R.sup.4.
##STR00023## ##STR00024##
[0149] In this scheme, protected 3-aminoalanine is synthesised, for
example by Hoffman degradation of N-.alpha.-protected asparagine,
and the carboxylic acid esterified. Next, the .beta.-amine is
alkylated on nitrogen with an N-protected .beta.-amino-alcohol in
which the alcohol has been activated to form a leaving group
suitable for nucleophilic substitution with inversion of
stereochemistry at carbon (as described for Thiofoxins and
Dimethylthiofoxins). Alternatively the .beta.-amine can be
condensed with an N-protected .alpha.-amino-aldehyde in the
presence of a reducing agent forming an amine (according to J. Org.
Chem., 2002, 67, 4017). The introduced nitrogen is here-on called
the .omega.-amine group. This step introduces the R.sup.3/R.sup.4
functionality, depending on the selected .beta.-aminoalcohol
(possibly derived from an .alpha.-amino acid).
[0150] The .omega.-amine is then selectively deprotected and
condensed with the carboxy ester (or after selective hydrolysis the
corresponding carboxylic acid) to form a seven-membered ring
(according to J. Med. Chem., 1987, 30, 1984). The secondary
.beta.-amine (now in the ring) is subsequently protected
orthogonally to the .alpha.-amine.
[0151] Next, the .alpha.-amine is selectively deprotected and
acylated as described above to introduce the R.sup.1
functionality.
[0152] Finally the .beta.-amine is deprotected and mono-alkylated,
mono-arylated or sulfonated as required to introduce the R.sup.2
functionality, using methods well known in the literature.
[0153] In the sixth scheme (Scheme 3F below), the R.sup.3/R.sup.4
group(s) are introduced first, followed by cyclisation. The R.sup.2
group and then the R.sup.1 group are then introduced. Such a route
would be optimal if greater diversity was to be introduced at
R.sup.1 than R.sup.2 with least diversity at R.sup.3/R.sup.4.
##STR00025##
[0154] This scheme is very similar to Scheme 3E above, except that
following the cyclisation step, the secondary .beta.-amine (now in
the ring), is alkylated, arylated or sulfonated, using methods well
known in the literature, rather than being orthogonally protected.
This introduces the R.sup.2 functionality.
[0155] In the final step, the .alpha.-amine is then deprotected and
acylated as described above to introduce the R.sup.1
functionality.
[0156] These six schemes illustrate the facile nature of the
synthesis of a library composed of one or more Azafoxin elements.
In particular, the ability to perform the steps of the reaction in
various orders in order to allow greater diversity to be introduced
into different regions of the molecule while keeping the synthesis
practicable is illustrated.
Example 4
Synthesis of Amidofoxins
[0157] The Amidofoxins are products of the coupling of
3-aminoalanine with an .alpha.-amino acid (as opposed to the
coupling of 3-aminoalanine with a .beta.-amino alcohol, possibly
derived from an .alpha.-amino acid, which yields Azafoxins as
described above). As with the Azafoxins, but not Thiofoxins and
Dimethylthiofoxins, substitution at the ring heteroatom is possible
(R.sup.2) to introduce further diversity. In the first scheme
(Scheme 4A below), the R.sup.1 group is introduced, followed by the
R.sup.2 group (substitution on nitrogen), then the R.sup.3 group,
followed by cyclisation. Such a route would be optimal if greater
diversity was to be introduced at R.sup.3 than R.sup.2 with least
diversity at R.sup.1.
##STR00026## ##STR00027##
[0158] As for Azafoxins, the first step is to synthesise a
protected 3-amino alanine, for example by subjecting an
N-.alpha.-carbamate ester protected asparagine to Hoffman
degradation (according to J. Org. Chem., 1997, 62, 6918). The
carboxylic acid group is then esterified, and the .beta.-amine
group protected orthogonally to the .alpha.-amine group.
[0159] In the next step, the .alpha.-amine is selectively
deprotected and acylated as described above for Thiofoxins (for
example in Scheme 1A). This introduces the R.sup.1
functionality.
[0160] Next, the .beta.-amine is also selectively deprotected, and
mono-alkylated or mono-arylated as required, using methods well
known in the literature, in order to introduce the R.sup.2
functionality.
[0161] The secondary .beta.-amine is then acylated with an
N-protected .alpha.-amino-acid. The introduced nitrogen is here-on
called the .omega.-amine group. It will be obvious than any of a
range of suitable peptide coupling methods, well known in the
literature, could be used to perform this step. This introduces the
R.sup.3 functionality, depending on the selection of the
.alpha.-amino acid used in the reaction.
[0162] Finally, the .omega.-amine is selectively deprotected and
condensed with the carboxy ester (or after selective hydrolysis the
corresponding carboxylic acid) to form a seven-membered ring (for
example, according to J. Med. Chem., 1987, 30, 1984).
[0163] In the second scheme (Scheme 4B below), the R.sup.1 group is
introduced, followed by the R.sup.1 group (substitution on
nitrogen), then the R.sup.3 group, followed by cyclisation. Such a
route would be optimal if greater diversity was to be introduced at
R.sup.3 than R.sup.1 with least diversity at R.sup.2.
##STR00028## ##STR00029##
[0164] As in Scheme 4A, the first step is to synthesise a protected
3-aminoalanine, for example by subjecting an N-.alpha.-carbamate
ester protected asparagine to Hoffman degradation (according to J.
Org. Chem., 1997, 62, 6918). The carboxylic acid group is then
esterified, but the .beta.-amine is mono-alkylated or
mono-arylated, using methods well known in the literature, prior to
protecting the resulting secondary the .beta.-amine group
orthogonally to the .alpha.-amine. This introduces the R.sup.2
functionality.
[0165] In the next step, the .alpha.-amine is selectively
deprotected and acylated as described above. This introduces the
R.sup.1 functionality.
[0166] The secondary .beta.-amine is then selectively deprotected
and acylated with an N-protected .alpha.-amino-acid. The introduced
nitrogen is here-on called the .omega.-amine group. It will be
obvious than any of a range of suitable peptide coupling methods,
well known in the literature, could be used to perform this step.
This introduces the R.sup.3 functionality, depending on the
selection of the .alpha.-amino acid used in the reaction.
[0167] Finally, the .omega.-amine is selectively deprotected and
condensed with the carboxy ester (or after selective hydrolysis the
corresponding carboxylic acid) to form a seven-membered ring (for
example, according to J. Med. Chem., 1987, 30, 1984).
[0168] In the third scheme (Scheme 4C below), the R.sup.2 group is
introduced, followed by the R.sup.3 group. Following cyclisation,
the R.sup.1 group is then introduced. Such a route would be optimal
if greater diversity was to be introduced at R.sup.1 than R.sup.3
with least diversity at R.sup.2.
##STR00030##
[0169] As in Scheme 4B, the first step is to synthesise a protected
3-aminoalanine, for example by subjecting an N-.alpha.-carbamate
ester protected asparagine to Hoffman degradation (according to J.
Org. Chem., 1997, 62, 6918). The carboxylic acid group is then
esterified, and the .beta.-amine is mono-alkylated or
mono-arylated, using methods well known in the literature,
introducing the R.sup.2 functionality.
[0170] The secondary .beta.-amine is then acylated with an
N-protected .alpha.-amino-acid. The introduced nitrogen is here-on
called the .omega.-amine group. It will be obvious than any of a
range of suitable peptide coupling methods, well known in the
literature, could be used to perform this step. This introduces the
R.sup.3 functionality, depending on the selection of the
.alpha.-amino acid used in the reaction.
[0171] Next, the .omega.-amine is selectively deprotected and
condensed with the carboxy ester (or after selective hydrolysis the
corresponding carboxylic acid) to form a seven-membered ring (for
example, according to J. Med. Chem., 1987, 30, 1984).
[0172] Finally, the .alpha.-amine is selectively deprotected and
acylated as described above. This introduces the R.sup.1
functionality.
[0173] These three schemes illustrate the facile nature of the
synthesis of a library composed of one or more Amidofoxin elements.
In particular, the ability to perform the steps of the reaction in
various orders in order to allow greater diversity to be introduced
into different regions of the molecule while keeping the synthesis
practicable is illustrated.
[0174] A small number of members of the Amidofoxin class are
already known. For example, see J. Org Chem. (2003) 68:7893.
However, these examples are restricted to R.sup.3=benzyl or
CH.sub.2CH.sub.2COR' (where R' is variable), and they were not
tested for activity as GPCR ligands." Other related structures in
the public domain, such as the antibiotic TAN1067C (VII), are not
Amidofoxins and would not fall under the scope of the present
invention because of the absence of a 3-acylamino or 3-sulfonamide
substituent on the ring as well as the presence of substitution at
N1.
##STR00031##
Pharmacological Study of the Products of the Invention
Principle of the Assays
A: GPCR Antagonism
[0175] In principle, a compound of the invention can be tested for
antagonist activity at a given GPCR by exposing the receptor to a
labelled ligand under appropriate conditions for binding, in the
absence and presence of various concentrations of the test
compound. The amount of label associated with the receptor is then
quantitated. If the test compound is able to compete with the
labelled ligand for binding then the amount of label associated
with the receptor will decrease with increasing concentration of
the test compound. From the plot of ligand bound against test
compound concentration it is possible to estimate the binding
affinity of the test compound to the receptor.
[0176] Such an assay therefore requires:
[0177] (1) A source of the GPCR of interest. The sequence of every
member of the GPCR superfamily from humans is now available from
the human genome sequence. Such sequences can be cloned into a
suitable vector and expressed in a suitable cell type (for example,
Jurkat T cells which are already known to express virtually no
endogenous GCPRs with the exception of the chemokine receptor
CXCR4). After selection using an antibiotic appropriate to the
vector used, stable cell lines expressing high levels of the chosen
GPCR can be established.
[0178] Membrane fractions from cell lines expressing the chosen
GPCR can be prepared using a range of methods well known in the
art. For example, according to Kuo et al. (Proc. Natl. Acad. Sci.
USA (1980) 77:7039), the cells may be resuspended in 25 mM HEPES
buffer pH7.5 containing 0.25M sucrose, 2.5 mM MgCl.sub.2, 2.5 mM
EGTA and 50 mM .beta.-mercaptoethanol, as well as protease
inhibitors such as PMSF and leupeptin and split open using a Dounce
homogeniser. The suspension is then subjected to centrifugation at
120.times.g to pellet unbroken cells and large cellular fragments,
and the supernatant containing small membrane fragments and
cytosolic components is retained. This supernatant is then
subjected to ultracentrifugation at 100,000.times.g, producing a
pellet of membrane fragments enriched in the chosen GPCR. The
pellet is resuspended in an appropriate binding buffer, and the
total protein concentration determined using, for example, a
commercially available protein assay such as Coomassie Plus
(Pierce). The membrane preparation can be adjusted in volume to
yield a standardised total protein concentration, for example of 1
mg/ml. The standardised preparation can be stored at -85.degree. C.
in aliquots until required.
[0179] (2) A labelled ligand with high affinity for the chosen
GPCR. Suitable ligands for most GPCRs are well known in the
literature. Such ligands may be the natural ligand for the receptor
(for example, dopamine) or it may be a pharmacological tool (such
as domperidone). A list of suitable ligands for a wide range of
commonly investigated GPCRs is provided in Table 1, but it will be
obvious to those skilled in the art that other suitable ligands
exist for many of these receptors. Ligands most useful for this
purpose will have an affinity constant for binding to the chosen
receptor of at least 1 .mu.M, and preferably less than 100 nM, and
more preferably less than 10 nM.
TABLE-US-00002 TABLE 1 Conc Receptor Radioligand Conc (nM)
Competitor (.mu.M) Adenosine A.sub.1 [.sup.3H]DPCPX 1 DPCPX 1
Adenosine A.sub.2 [.sup.3H]CGS 21680 6 NECA 10 Adenosine A.sub.3
[.sup.125I]AB-MECA 0.1 IB-MECA 1 .alpha..sub.1-adrenoceptor
[.sup.3H]prazosin 0.25 prazosin 0.5 .alpha..sub.2-adrenoceptor
[.sup.3H]RX 821002 0.5 (-)-epinephrine 100
.beta..sub.1-adrenoceptor [.sup.3H](-)-CGP 12177 0.15 alprenolol 50
.beta..sub.2-adrenoceptor [.sup.3H](-)-CGP 12177 0.15 alprenolol 50
Angiotensin AT.sub.1 [.sup.125I][sar.sup.1,ile.sup.8]-AII 0.05
angiotensin II (AII) 10 Angiotensin AT.sub.2 [.sup.125I]CGP 42112A
0.05 angiotensin II (AII) 1 Central BZD [.sup.3H]flunitrazepam 0.4
diazepam 3 Peripheral BZD [.sup.3H]PK 11195 0.2 PK 11195 10
Bombesin (ns) [.sup.125I][Tyr.sup.4]bombesin 0.01 bombesin 1
Bradykinin B.sub.2 [.sup.3H]bradykinin 2 bradykinin 1 CGRP receptor
[.sup.125I]hCGRP.alpha. 0.03 hCGRP.alpha. 1 Cannabinoid CB.sub.1
[.sup.3H]WIN 55212-2 2 WIN 55212-2 10 Cholecystekinin A
[.sup.125I]CCK-8 0.08 CCK-8 1 Cholecystekinin B [.sup.125I]CCK-8
0.025 CCK-8 1 Dopamine D1 [.sup.3H]SCH 23390 0.3 SCH 23390 1
Dopamine D2s [.sup.3H]spiperone 0.3 (+)-butaclamol 10 Dopamine D3
[.sup.3H]spiperone 0.3 (+)-butaclamol 10 Dopamine D4.4
[.sup.3H]spiperone 0.3 (+)-butaclamol 10 Dopamine D5 [.sup.3H]-SCH
23390 0.3 SCH 23390 10 Endothelin ET.sub.A [.sup.125I]endothelin-1
0.03 endothelin-1 0.1 Endothelin ET.sub.B [.sup.125I]endothelin-1
0.03 endothelin-1 0.1 GABA (ns) [.sup.3H]-GABA 10 GABA 100 Galanin
GAL1 [.sup.125I]galanin 0.03 galanin 1 Galanin GAL2
[.sup.125I]galanin 0.05 galanin 1 IL8RB (CXCR2) [.sup.125I]IL-8
0.025 IL-8 0.3 CCR1 [.sup.125I]MIP1.alpha. 0.03 MIP1.alpha. 0.1
Histamine H.sub.1 [.sup.3H]pyrilamine 3 pyrilamine 1 Histamine
H.sub.2 [.sup.125I]APT 0.2 tiotidine 100 MC4
[.sup.125I]NDP-.alpha.-MSH 0.05 NDP-.alpha.-MSH 1 Melatonin
ML.sub.1 [.sup.125I]iodomelatonin 0.025 melatonin 1 Muscarinic
M.sub.1 [.sup.3H]pirenzepine 2 atropine 1 Muscarinic M.sub.2
[.sup.3H]AF-DX 384 2 atropine 1 Muscarinic M.sub.3 [.sup.3H]4-DAMP
0.2 atropine 1 Muscarinic M.sub.4 [.sup.3H]4-DAMP 0.2 atropine 1
Muscarinic M.sub.5 [.sup.3H]4-DAMP 0.2 atropine 1 Neurokinin
NK.sub.1 [.sup.125I][sar.sup.9,met.sup.11]-SP 0.15
[sar.sup.9,met.sup.11]-SP 1 Neurokinin NK.sub.2 [.sup.125I]NKA 0.1
[nle.sup.10]-NKA(4-10) 10 Neurokinin NK.sub.3 [.sup.3H]SR 142801
0.2 SB 222200 10 Neuropeptide Y.sub.1 [.sup.125I]peptide YY 0.05
NPY 1 Neuropeptide Y.sub.2 [.sup.125I]peptide YY 0.015 NPY 1
Neurotensin NT.sub.1 [.sup.125I][Tyr.sup.3]- 0.02 neurotensin 1
neurotensin .delta. opioid (.delta..sub.2) [.sup.3H]DADLE 0.5
naltrexone 10 .kappa. opioid [.sup.3H]U 69593 0.7 naloxone 10 .mu.
opioid [.sup.3H]DAMGO 0.5 naloxone 10 ORL1 opioid
[.sup.3H]nociceptin 0.2 nociceptin 1 PACAP [.sup.125I]PACAP(1-27)
0.02 PACAP(1-27) 0.1 Purine P2X [.sup.3H].alpha.,.beta.-MeATP 3
.alpha.,.beta.-MeATP 10 Purine P2Y [.sup.35S]dATP.alpha.S 10
dATP.alpha.S 10 Serotonin 5HT.sub.1A [.sup.3H]8-OH-DPAT 0.5
8-OH-DPAT 10 Serotonin 5HT.sub.1B [.sup.125I]CYP 0.1 serotonin 10
Serotonin 5HT.sub.2A [.sup.3H]ketanserin 0.5 ketanserin 1 Serotonin
5HT.sub.2C [.sup.3H]mesulurgine 1 SB 242084 10 Serotonin 5HT.sub.3
[.sup.3H]BRL 43694 0.5 MDL 72222 10 Serotonin 5HT.sub.5A
[.sup.3H]LSD 1 serotonin 100 Serotonin 5HT.sub.6 [.sup.3H]LSD 2
serotonin 100 Serotonin 5HT.sub.7 [.sup.3H]LSD 4 serotonin 10 Sigma
receptor (ns) [.sup.3H]DTG 8 haloperidol 10 Somatostatin (ns)
[.sup.125I][Tyr.sup.11]-sst14 0.05 sst14 0.3 Vasopressin VIP.sub.1
[.sup.125I]VIP 0.04 VIP 0.3 Abbreviation used: (ns) =
non-selective
[0180] Once the ligand has been selected, it will likely be
necessary to label to the ligand to that subsequently the amount
bound to the chosen GPCR can be determined (although it may be
possible to perform an assay without labelling the ligand,
providing that a sensitive and accurate method of determining the
amount of unbound ligand is available--for example it may be
possible to use an ELISA assay to measure unbound ligand, and by
inference calculate the amount of bound ligand). Appropriate
methods of labelling the ligand vary depending on the nature of the
ligand: small molecules may be most readily labelled with a
radionuclide such as .sup.3H, .sup.14C or .sup.35S; peptides may be
most readily labelled with a co-synthetic biotin (and subsequently
with labelled streptavidin), with fluorescent tags (such as
fluorescein isothiocyanate) or with radionuclides (such as
.sup.125I-iodination of tyrosine residues in the peptide); proteins
may be most readily labelled with fluorescent tags (such as
fluorescein isothiocyanate) or with radionuclides (such as
.sup.125I-iodination of tyrosine residues in the protein).
[0181] The extent of the labelling (that is, the proportion of
molecules in the sample bearing the label) must be sufficient that
the amount of ligand binding to the receptor can be conclusively
quantitated.
[0182] With these two components it is then possible to test
whether the compounds of the invention modulate ligand binding to
any given GPCR, using methods well known in the art. For example,
in a series of tubes the membrane preparation is mixed with the
radioligand at a concentration near to the affinity constant for
the binding of the ligand to the chosen GPCR. In some tubes, the
compound of the invention is also added at various concentrations.
In yet other tubes a positive control inhibitor is added (which may
be a large excess of the same ligand as the radioligand but in the
absence of the radionuclide tag). Typically, three tubes would be
prepared under each set of conditions. The tubes are then
incubated, typically at between 4.degree. C. and 37.degree. C.,
more typically at room temperature for a period of time to allow an
equilibrium to be reached between free and bound radioligand.
Typically, this will take from between 20 minutes and 4 hours, and
the period required for any given set of reaction conditions can be
determined by methods well known in the art (for example, by
performing a time-course experiment). Once equilibrium is achieved,
it is necessary to determine the amount of radioliagnd bound. For
example, the membrane-bound receptor (plus any bound radioligand)
can be separated from free radioligand in solution by filtration
through filters (such as GF/C filters treated with 1%
polyethyleneimine). The filters may then be air-dried and subjected
to scintillation counting to determine the fraction of the
radioligand added which is now bound to the receptor.
[0183] Alternatively, the compounds of invention may be subjected
to screening using commercially available receptor screening
procedures (for example, the services offered by Cerep, 128 Rue
Danton, Paris, France). Such services readily identify members of a
library, such as the library provided for in the invention, which
modulate ligand binding to one or more GPCRs.
[0184] Compounds identified as modulating ligand binding to one or
more GPCRs using the methods outlined above will usually be full
antagonists. However, it is necessary to perform functional assay
in order to confirm the antagonist properties of the compound. For
example, depending on the GPCR and/or the ligand used certain
second messenger signals will be stimulated (or inhibited) in order
to transduce the signal that the ligand is present. Cells may show
and increase (or a decrease) in the cellular concentration of
cyclic adenosie monophosphate (cAMP), various phosphorylated
inositol-containing compounds (including I(1,4,5)P3 and
I(1,3,5)P3), calcium ions, polyadenosine or other intracellular
messengers known in the art, in response to presentation of the
ligand. Full antagonists will abrogate the change in intracellular
messengers caused by the natural ligand(s), and have no effect in
the absence of natural ligand. In marked contrast, full agonists
will have no effect when added with the natural ligand(s), but
mimic the changes in intracellular messengers caused by the natural
ligand(s) when added in the absence of natural ligand. Some
compounds, including compounds of the invention may be partial
antagonists, partial agonists or mixed agonist/anatgonists
depending on the pattern of effects on intracellular messengers.
Despite the complex pharmacological definition of such compounds,
they may have useful therapeutic properties in certain diseases,
and a number of well established human pharmaceuticals are known to
be partial agonists, partial antagonists or mixed
agonist/antagonists at one or more GPCRs.
B: GPCR Agonism
[0185] It is inherently considerably more difficult to test for
agonist activity than antagonist activity, particularly using high
throughput screening techniques. The compounds of the invention
are, therefore, likely to be particularly useful in the search for
agonists than general lead discovery libraries because of the
higher incidence of GPCR agonists among the library elements.
[0186] A test for a GPCR agonist, in principle, requires a cell or
organ culture system which responds to a natural ligand of the
chosen GPCR(s) with a desirable biochemical or physiological
response. Examples of such a response include, but are not limited
to, changes in intracellular messengers (such as cAMP, IP(1,4,5)P3,
calcium ions or polyadenosine), changes in enzyme activity (such as
activation of protein kinases, phosphatases, metabolic enzymes or
transport proteins), changes in gene expression patterns, altered
phagocytosis, altered protein secretion, altered rate of
proliferation, contraction of muscle cells/tissue,
neurotransmission and so forth. Since responses such as these are
inherently more complex to measure than the binding of natural
ligand(s) to chosen GPCRs, this is why assays for GPCR agonists are
more challenging than for antagonists.
[0187] The general method required to test whether a compound of
the invention is an agonist at one or more chosen GPCRs is well
established in the art. Cells are exposed to various concentrations
of the test compound, for example, by addition of the compound in a
suitable vehicle (such as DMSO, ethanol or methanol) at various
concentrations (for example, from about 0.1 nM to about 10 mM) in
the cell culture medium for period of time (for example, from 1
minute to 48 hours, depending on the timecourse of the response to
be measured), typically at 37.degree. C. In parallel cells are also
exposed to the natural ligand, and left unexposed to any additional
compound(s) (as control cells). At the end of the incubation
period, a response known in the art to occur in response to the
natural ligand binding to the chosen GPCR(s) is measured. If the
compound of the invention is an agonist at the chosen GPCRs, then
the responses to the test compound (at certain concentrations) will
be qualitatively similar to the response to the natural ligand.
[0188] Examples of suitable assay systems for agonists at
particular GPCRs follow:
[0189] Somatostatin is an agonist at the sstr2 and sstr5 receptors
such that it inhibits the secretion of growth hormone by isolated
pituitary cells. To determine whether compounds of the invention
are agonists at sstr2 and/or sstr5, rat pituitary cells are
isolated and placed into culture. The cells are then incubated
alone, or in the presence of somatostatin at 33 nM, or in the
presence of the test compound(s) at various concentrations from
about 0.1 nM to about 10 mM at 37.degree. C. for 24 hours. At the
end of the experiment, the cell culture medium is removed,
clarified by centrifugation and subjected to an assay for growth
hormone (GH), for example by performing a commercially available
ELISA or radioimmunoassay. The cells exposed to somatostatin will
have produced between 30% and 90% less GH than cells incubated
alone. If the compound of the invention is an agonist at the
somatostatin receptors, then the level of GH will be lower in the
medium from cells exposed to the test compound (at least at certain
concentrations) than in the medium from cells incubated alone.
Typically, medium is collected from three replicate wells
containing cells treated identically under each of the conditions
of the experiment, so that an appropriate statistical test (such as
an ANOVA or unpaired Student's t-test) can be used to demonstrate
that the test compound produced a statistically significant
reduction in GH secretion, and therefore likely possesses agonist
activity at the chosen receptors, sstr2 and/or sstr5.
[0190] Endothelin-1 is a peptide which signals through the ET-A
and/or ET-B receptor to cause vasoconstriction. To determine
whether compounds of the invention are agonists at ET-A and/or
ET-B, rings of human aorta (obtained from transplant donor hearts)
can be put into organ culture. Rings are then exposed either to
increasing concentrations of Endothelin-1 (from 0.01 nM to 100 nM),
or to increasing concentrations of the test compound(s) (from about
0.1 nM to about 10 mM) at 37.degree. C., raising the concentration
of the appropriate agent approximately every 5 minutes. Throughout
the experiment the contraction of the aortic ring is measured by a
strain gauge designed and commercially available for such a
purpose. The rings exposed to endothelin-1 will contract as the
concentration of endothelin-1 is increased, so that by the time the
top concentration is reached the force exerted on the strain gauge
will be significantly higher than prior to addition of
endothelin-1. If the compound of the invention is an agonist at the
endothelin receptors, then the force exerted on the strain gauge
will also be higher (at least at certain concentrations) than prior
to addition of the test compound. Typically, three or more separate
aortic rings are treated with increasing concentrations of the same
agent under identical experimental conditions, so that an
appropriate statistical test (such as an ANOVA or unpaired
Student's t-test) can be used to demonstrate that the test compound
produced a statistically significant increase in aortic
contraction, and therefore likely possesses agonist activity at the
chosen receptors, ET-A and/or ET-B
[0191] The chemokine SDF-1a is a peptide which signals through the
CXCR4 receptor to cause leukocyte migration. To determine whether
compounds of the invention are agonists at CXCR4 cultured human
immortalised T-cells (Jurkat T cells, for example), are placed in
the top well of a purpose-built commercially available transwell
migration apparatus. Replicate wells are then exposed to lower
chambers containing only culture medium, or to lower chambers
containing SDF-1a at 75 nM, or to lower chambers containing various
concentrations of the test compound(s) (from about 0.1 nM to about
10 mM) and incubated for a period of time (typically between 30
minutes and 3 hours) at 37.degree. C. At the end of the incubation,
the number of cells present in the lower chamber is a measure of
the amount of migration occurring. The number of cells in the lower
chamber may be counted by direct visualisation, or by various
well-known methods such as incubation with MTT dye which is
converted to an insoluble blue formazan product in proportion to
the number of cells present. In wells exposed to a lower chamber
containing SDF-1a, the number of cells in the lower chamber will be
between 2-fold and 10-fold higher than the number of cells in lower
chambers containing culture medium alone. If the compound of the
invention is an agonist at CXCR4, then the number of cells in the
lower chambers containing the test compound(s) will also be higher
(at least at certain concentrations) than in the lower chambers
containing medium alone. Typically, three or more separate chambers
are treated identically under each of the experimental conditions,
so that an appropriate statistical test (such as an ANOVA or
unpaired Student's t-test) can be used to demonstrate that the test
compound produced a statistically significant increase in leukocyte
migration, and therefore likely possesses agonist activity at the
chosen receptor, CXCR4.
[0192] The bioactive amine adrenalin increases the intracellular
concentration of cAMP in vascular smooth muscle cells. To determine
whether compounds of the invention are agonists at
.beta.-adrenoreceptors, rat vascular smooth muscle cells from
thoracic aorta are isolated and placed into culture. The cells are
then incubated alone, or in the presence of the adrenalin agonist
salbutamol at 33 nM, or in the presence of the test compound(s) at
various concentrations from about 0.1 nM to about 10 mM at
37.degree. C. for 15 minutes. At the end of the experiment, the
cell culture medium is removed, the cells are washed three times in
cold buffer and then lysed in an appropriate lysis buffer, prior to
measurement of the intracellular concentration of cAMP, for example
by performing a commercially available ELISA or radioimmunoassay.
The cells exposed to salbutamol will have an intracellular cAMP
concentration between 15% and 150% higher than cells exposed to
medium alone. If the compound of the invention is an agonist at the
.beta.-adrenoreceptors, then the intracellular concentration of
cAMP will be higher in the cells exposed to the test compound (at
least at certain concentrations) than in the cells incubated alone.
Typically, cell lysate is prepared from three replicate wells
containing cells treated identically under each of the conditions
of the experiment, so that an appropriate statistical test (such as
an ANOVA or unpaired Student's t-test) can be used to demonstrate
that the test compound produced a statistically significant
increase in intracellular cAMP concentration, and therefore likely
possesses agonist activity at the chosen
.beta.-adrenoreceptors.
[0193] It will be obvious that assays such as the examples above
will identify agonists at the chosen GPCRs, and distinguish the
compounds of the invention from inactive compounds and from
compounds with antagonist or partial antagonist activity at the
chosen GPCR, but will not necessarily uniquely identify the chosen
GPCR as the molecular target of the compound of the invention. For
example, a compound of the invention demonstrated to elevate cAMP
in vascular smooth muscle cells to the same extent as the
.beta.-adrenoreceptor agonist salbutamol, may be an agonist at the
.beta.-adrenoreceptor GPCRs, or it may be an agonist at another
GPCR which also elevates cAMP (such as dopamine D2 receptor).
Alternatively, a compound of the invention which stimulates the
migration of leukocytes to a similar extent to SDF-1a may be an
agonist at CXCR4, or it may be an agonist at another GPCR which
stimulates leukocyte migration (such as the C5a receptor).
Validation of the molecular target GPCR at which compounds of the
invention act as an agonist will require the performance of
additional experiments using specific antagonists already
identified against the chosen GPCR, or the use of recombinant cell
lines expressing only the chosen GPCR. For example, if the
leukocyte migration induced by a compound of the invention were
inhibited by the addition of the CXCR4-specific antagonist AMD3100
at an appropriate concentration, then it would be reasonable to
conclude that CXCR4 was the molecular target of the compound of the
invention. Similarly, if the leukocyte migration induced by a
compound of the invention was observed using a cell line expressing
CXCR4, but absent in the same cell line not expressing CXCR4, then
it would be reasonable to conclude that CXCR4 was the molecular
target of the compound of the invention.
* * * * *