U.S. patent application number 12/193943 was filed with the patent office on 2009-08-13 for cyclic agonists and antagonists of c5a receptors and g protein-coupled receptors.
This patent application is currently assigned to THE UNIVERSITY OF QUEENSLAND. Invention is credited to David FAIRLIE, Angela Monique Finch, Stephen Maxwell Taylor, Allan Wong.
Application Number | 20090203760 12/193943 |
Document ID | / |
Family ID | 3801840 |
Filed Date | 2009-08-13 |
United States Patent
Application |
20090203760 |
Kind Code |
A1 |
FAIRLIE; David ; et
al. |
August 13, 2009 |
CYCLIC AGONISTS AND ANTAGONISTS OF C5a RECEPTORS AND G
PROTEIN-COUPLED RECEPTORS
Abstract
The present invention relates to novel cyclic or constrained
acyclic compounds which modulate the activity of G protein-coupled
receptors and are useful in the treatment of conditions mediated by
G protein-coupled receptors, for example, inflammatory
conditions.
Inventors: |
FAIRLIE; David; (Springwood,
AU) ; Taylor; Stephen Maxwell; (Bellbird Park,
AU) ; Finch; Angela Monique; (Narangba, AU) ;
Wong; Allan; (Bundaberg, AU) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
THE UNIVERSITY OF
QUEENSLAND
Brisbane
AU
|
Family ID: |
3801840 |
Appl. No.: |
12/193943 |
Filed: |
August 19, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10937852 |
Sep 10, 2004 |
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12193943 |
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09446109 |
Apr 21, 2000 |
6821950 |
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PCT/AU98/00490 |
Jun 25, 1998 |
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10937852 |
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Current U.S.
Class: |
514/413 ;
540/460 |
Current CPC
Class: |
A61P 25/00 20180101;
A61P 29/00 20180101; A61P 17/06 20180101; C07K 7/56 20130101; A61P
37/06 20180101; A61P 1/02 20180101; A61P 25/28 20180101; A61P 19/02
20180101; C07K 14/472 20130101; A61P 9/10 20180101; A61K 38/00
20130101; C07K 2299/00 20130101; A61P 11/00 20180101 |
Class at
Publication: |
514/413 ;
540/460 |
International
Class: |
A61K 38/12 20060101
A61K038/12; C07D 245/00 20060101 C07D245/00; A61P 25/28 20060101
A61P025/28 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 25, 1997 |
AU |
PO 7550 |
Claims
1-32. (canceled)
33. A method of treating Alzheimer's disease mediated by a C5a
receptor, comprising administering to a mammal in need thereof, a
compound in amount effective to treat Alzheimer's disease, which
compound has antagonist activity against a C5a receptor, has no
agonist activity against a C5a receptor, and has the general
formula II: ##STR00009## where A is H, alkyl, aryl, NH.sub.2,
NHalkyl, N(alkyl).sub.2, NHaryl or NHacyl; B is an alkyl, aryl,
phenyl, benzyl, naphthyl or indole group, or the side chain of a D-
or L-amino acid selected from the group consisting of
phenylalanine, homophenylalanine, tryptophan, homotryptophan,
tyrosine, and homotyrosine; C is the side chain of a D-, L- or
homo-amino acid selected from the group consisting of proline,
alanine, leucine, valine, isoleucine, arginine, histidine,
aspartate, glutamate, glutamine, asparagine, lysine, tyrosine,
phenylalanine, cyclohexylalanine, norleucine, tryptophan, cysteine
and methionine; D is the side chain of a D- or L-amino acid
selected from the group consisting of cyclohexylalanine,
homocyclohexylalanine, leucine, norleucine, homoleucine,
homonorleucine and tryptophan; E is the side chain of a D- or
L-amino acid selected from the group consisting of tryptophan and
homotryptophan; F is the side chain of a D- or L-amino acid
selected from the group consisting of arginine, homoarginine,
lysine and homolysine or is one of the following side-chains
##STR00010## or another mimetic of an arginine side chain, where X
is NCN, NNO.sub.2, CHNO.sub.2 or NSO.sub.2NH.sub.2; n is an integer
from 1 to 4, and R.sup.1 is H or an alkyl, aryl, CN, NH.sub.2, OH,
--CO--CH.sub.2CH.sub.3, --CO--CH.sub.3,
--CO--CH.sub.2CH.sub.2CH.sub.3, --CO--CH.sub.2Ph, or --CO-Ph; and
X.sup.1 is --(CH.sub.2).sub.nNH-- or (CH.sub.2).sub.r--S--,
--(CH.sub.2).sub.2O--, --(CH.sub.2).sub.3O--, --(CH.sub.2).sub.3--,
--(CH.sub.2).sub.4--, or --CH.sub.2COCHRNH--, where R is the side
chain of any common or uncommon amino acid, and where n is an
integer of from 1 to 4.
34. The method according to claim 33, which is a compound selected
from the group consisting SEQ ID NOS: 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27 and 28.
35. The method according to claim 32, in which n is 2 or 3.
36. The method according to claim 32, in which F is one of the
following side-chains ##STR00011## or another mimetic of an
arginine side chain; where X is NCN, NNO.sub.2, CHNO.sub.2 or
NSO.sub.2NH.sub.2; n is an integer from 1 to 4, and R.sup.1 is H or
an alkyl, aryl, CN, NH.sub.2, OH, --CO--CH.sub.2CH.sub.3,
--CO--CH.sub.3, --CO--CH.sub.2CH.sub.2CH.sub.3, --CO--CH.sub.2Ph,
or --CO-Ph; B is an indole, indole methyl, benzyl, phenyl,
naphthyl, naphthyl methyl, cinnamyl group, or any other derivative
of the aromatic group; and C is D- or L-cyclohexylalanine (Cha),
leucine, valine, isoleucine, phenylalanine, tryptophan or
methionine.
37. The method according to claim 32, which has the formula
TABLE-US-00007 Ac-Phe-[Lys-Pro-(dCha)-Trp-Arg] or
Ac-Phe-[Orn-Pro-(dCha)-Trp-Arg].
38. The method according to claim 32, in which A is L-arginine.
39. The method according to claim 33, in which F is a L-amino
acid.
40. The compound according to claim 39, in which F is
L-arginine.
41. The method according to claim 32, wherein the compound is
administered together with a pharmaceutically-acceptable carrier or
excipient.
42. The method according to claim 41, wherein the mammal is a
human.
43. The method according to claim 32, wherein the mammal is a
human.
44. A method of treating Alzheimer's disease, comprising
administering to a mammal in need thereof, a compound in amount
effective to treat Alzheimer's disease, which compound is an
agonist of the C5a receptor, and has the formula IV: ##STR00012##
where A is any common or uncommon, basic, charged amino acid side
chain which serves to position a positively charged group in this
position; B is a non-aromatic amino acid, and C is any common or
uncommon, hydrophobic amino acid side chain which serves to
position any alkyl, aromatic or other group in this position; and D
is any common or uncommon, aromatic amino acid which serve to
position an aromatic side-chain in this position, and has the
structure: ##STR00013## where Z is indole, indole methyl, benzyl,
benzene, naphthyl, naphthyl methyl, or a derivative thereof; and R
is H or an alkyl, aromatic, acyl or aromatic-acyl group; E is any
amino acid other than tryptophan and homotryptophan, and F is the
side chain of a D- or L-amino acid selected from the group
consisting of arginine, homoarginine, lysine and homolysine.
45. A method of claim 44, wherein the compound is administered
together with a pharmaceutically acceptable carrier or
excipient.
46. The method of claim 44, wherein the mammal is a human.
47. A method of treating Alzheimer's disease, comprising
administering to a mammal in need thereof, a compound in amount
effective to treat Alzheimer's disease, which compound has the
formula ##STR00014##
48. The method of claim 47, wherein the compound is administered
together with a pharmaceutically acceptable carrier or
excipient.
49. The method of claim 47, wherein the mammal is a human.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present invention is a continuation application of U.S.
application Ser. No. 10/937,852 filed Sep. 10, 2004, abandoned,
which is a continuation of U.S. application Ser. No. 09/446,109
filed Apr. 21, 2000, now U.S. Pat. No. 6,821,950, which is a 371
application of PCT/AU98/00490 filed Jun. 25, 1998.
[0002] This invention relates to novel cyclic compounds which have
the ability to modulate the activity of G protein-coupled
receptors. The invention provides both agonists and antagonists. In
preferred embodiments, the invention provides cyclic peptidic and
cyclic or non-cyclic non-peptidic antagonists or agonists of C5a.
The compounds of the invention are both potent and selective, and
are useful in the treatment of a variety of inflammatory
conditions.
BACKGROUND OF THE INVENTION
[0003] Activation of human complement, a system of plasma proteins
involved in immunological defense against infection and injury,
contributes significantly to the pathogenesis of numerous acute and
chronic diseases. In particular, the complement protein C5a has
been extensively investigated. For general reviews, see Whaley
(1987), and Sim (1993). Table 1 provides a summary of known roles
of C5a in disease.
[0004] During host defense, the complement system of plasma
proteins initiates inflammatory and cellular immune responses to
stimuli such as infectious organisms (bacteria, viruses,
parasites), chemical or physical injury, radiation or neoplasia.
Complement is activated through a complex cascade of interrelated
proteolytic events that produce multiple bioactive peptides, some
of which (eg. anaphylatoxins C3a and C5a) interact with cellular
components to propagate inflammatory processes. Complement
activation, either by the classical pathway, after antigen-antibody
(Ag/Ab) binding, or by the antibody-independent alternate pathway,
ends with a terminal sequence in which protein C5 is
proteolytically cleaved by C5 convertase to C5a and C5b. The latter
facilitates assembly of a "membrane attack complex" that punches
holes in membranes of target cells such as bacteria, leading to
leakage, lysis and cell death. Steps in the cascade are tightly
regulated to avoid stepwise amplification of proteolysis by
sequentially formed proteases. If these regulatory mechanisms
become inefficient, protracted activation of complement can result,
causing enhanced inflammatory responses as in autoimmune
diseases.
[0005] Although the broad features of the complement system and its
activation are known, mechanistic details remain poorly understood.
A principal and very potent mediator of inflammatory responses is
the plasma glycoprotein C5a, which interacts with specific surface
receptors (C5aR) on mast cells, neutrophils, monocytes,
macrophages, non-myeloid cells, and vascular endothelial cells
(Gerard and Gerard, 1994). C5aR is a G protein-coupled receptor
with seven transmembrane helices (Gerard and Gerard, 1991). This
receptor is one of the rhodopsin superfamily of GTP-linked binding
proteins, but differs from rhodopsin receptors in that the receptor
and G protein are linked prior to activation.
[0006] G protein-coupled receptors are prevalent throughout the
human body, comprising approximately 80% of known cellular receptor
types, and mediate signal transduction across the cell membrane for
a very wide range of endogenous ligands. They participate in a
diverse array of physiological and pathophysiological processes,
including, but not limited to those associated with cardiovascular,
central and peripheral nervous system, reproductive, metabolic,
digestive, immunoinflammatory, and growth disorders, as well as
other cell-regulatory and proliferative disorders. Agents, both
agonists and antagonists, which selectively modulate functions of G
protein-coupled receptors have important therapeutic
applications.
[0007] C5a is one of the most potent chemotactic agents known, and
recruits neutrophils and macrophages to sites of injury, alters
their morphology; induces degranulation; increases calcium
mobilisation, vascular permeability (oedema) and neutrophil
adhesiveness; contracts smooth muscle; stimulates release of
inflammatory mediators (including histamine, TNF-.alpha., IL-1,
IL-6, IL-8, prostaglandins, leukotrienes) and lysosomal enzymes;
promotes formation of oxygen radicals; and enhances antibody
production (Gerard and Gerard, 1994). Overexpression or
underregulation of C5a is implicated in the pathogenesis of
immunoinflammatory conditions such as rheumatoid arthritis, adult
respiratory distress syndrome (ARDS), systemic lupus erythematosus,
tissue graft rejection, ischaemic heart disease, reperfusion
injury, septic shock, psoriasis, gingivitis, atherosclerosis,
Alzheimer's disease, lung injury and extracorporeal post-dialysis
syndrome, and in a variety of other conditions, as summarised in
Table 1.
TABLE-US-00001 TABLE 1 The Role of C5a in Disease C5a C5aR
Condition/disease levels expression Details allergy ++ allergen
challenge leads to nasal symptoms and increased C5a levels
Alzheimer's disease ++ ++ up-regulation of the receptor in reactive
astrocytes, microglia and endothelial cells in the CNS, complement
system activated by .beta.- amyloid ARDS/respiratory ++ distress
Behcet's disease ++ levels highest just prior to ocular attack
bronchial asthma ++ capillary leak ++ syndrome chronic lung ++
Increased C5a levels in pulmonary effluent disease fluid from
mechanically ventilated infants with chronic lung disease
Churg-Strauss hypersensitivity of granulocytes to C5a cystic
fibrosis generation of C5a/effects on PMNs decompression ++
increased C5a levels during saturation diving stress diabetes type
I ++ C5a generated during onset; circulating monocytes in newly
diagnosed type 1 diabetes patients are activated Familial lack of
C5a inactivator Mediterranean fever Guillain-Barre ++ CSF levels
elevated ischaemic disease migration of monocytes into myocardium
after states/myocardial reperfusion. Damage prevented with sCR1
infarct Kimura's disease humoral factor up-regulates the response
of PMNs to C5a Multiple Sclerosis ++ increased expression of the
receptor on foamy macrophages in acute and chronic MS and fibrous
astrocytes in chronic MS Meningitis C5a induces experimental
meningitis; PMN accumulation seen in the CSF pancreatitis ++
post-dialysis ++ - C5a generated via complement activation by
syndrome tubing material, C5aR levels decreased on PMNs &
monocytes in chronic state preeclampsia/HELLP ++ C5a levels
elevated at delivery psoriasis ++ C5a levels high in scales
reperfusion injury ++ inhibited by C5 antibody retinitis ++ C5a
detected in vitreous humor Rheumatoid ++ elevated concentration of
C5a found in arthritis synovial fluid (5-fold) and plasma (3-fold)
Severe congenital - neutropenia transplant/graft ++ monoclonal
antibodies block the damage rejection seen with xenogenic
transplant; increased levels of C5a seen in the plasma and urine of
patients with renal graft rejection
[0008] New agents which limit the pro-inflammatory actions of C5a
have potential for inhibiting chronic inflammation, and its
accompanying pain and tissue damage. For these reasons, molecules
which prevent C5a binding to its receptors are useful for treating
chronic inflammatory disorders driven by complement activation.
Importantly, such compounds provide valuable new insights to
mechanisms of complement-mediated immunity.
[0009] In another context, agonists of C5a receptors or other G
protein-coupled receptors may also be found to have therapeutic
properties in conditions either where the G protein-coupled
receptor can be used as a recognition site for drug delivery, or
where triggering of such receptors can be used to stimulate some
aspect of the human immune system, for example in the treatment of
cancers, viral or parasitic infections.
[0010] One approach to the development of agonists or antagonists
of C5a is through receptor-based design, using knowledge of the
three-dimensional structures of C5a, its receptor C5aR, and the
interactions between them. The structure of the receptor is
unknown. The solution structure of human C5a, a 74 amino acid
peptide that is highly cationic and N-glycosylated with a 3 kDa
carbohydrate at Asn64, has been determined and is essentially a
4-helix bundle. The C-terminal end (residues 65-74, C5a.sub.65-74)
was found to be unstructured (Zuiderweg et al, 1989) and this
conformational flexibility in the C-terminus has made
structure-function studies extremely difficult to interpret.
[0011] C5a has a highly ordered N-terminal core domain (residues
1-64; C5a.sub.1-64), consisting of a compact antiparallel 4-helix
bundle (residues 4-12, 18-26, 32-39, 46-63) connected by loops
(13-17, 27-31, 40-45), and further stabilised by 3 disulphide bonds
(C21-Cys47, Cys22-Cys54, Cys34-Cys55).
[0012] Although the structure of the C5a receptor, C5aR, is
unknown, the C5a-binding subunit of human monocyte-derived C5aR has
been cloned and identified as a G protein-coupled receptor with
transmembrane helices (Gerard and Gerard, 1991). Interactions
between C5a and C5aR have been the subject of many investigations
which, in summary, suggest that C5a binds via a two-site mechanism
in which the N-terminal core domain of C5a is involved in
receptor-recognition and binding, while the C-terminus is
responsible for receptor activation. This mechanism is illustrated
schematically in FIG. 1. The C-terminal "effector" region alone
possesses all the information necessary for signal transduction,
and is thought to bind in the receptor's interhelical region
(Siciliano et al, 1994; deMartino et al, 1995).
[0013] An N-terminal interhelical positively-charged region of C5a
is responsible for receptor recognition and binding, and binds to a
negatively-charged extracellular domain of C5aR (site 1), while the
C-terminal "effector" region of C5a is thought to bind with the
interhelical region of the receptor (site 2), and is responsible
for receptor activation leading to signal transduction (Siciliano
et al, 1994).
[0014] Numerous short peptide derivatives of the C-terminus of C5a
have been found to be agonists of C5a (Kawai et al, 1991; Kawai et
al, 1992; Kohl et al, 1993; Drapeau et al, 1993; Ember et al, 1992;
Sanderson et al, 1994; Sanderson et al, 1995; Finch et al, 1997;
Tempero et al, 1997; Konteatis et al, 1994; DeMartino et al, 1995).
The structures of some of these agonists are shown in Table 2 below
(compounds 1-6). High molecular weight polypeptide inhibitors of
the action of C5a at its receptor, such as monoclonal antibodies to
the C5a receptor, are also known (Morgan et al, 1992).
[0015] A small molecule,
N-methylphenylalanine-lysine-proline-D-cyclohexylalanine-tryptophan-D-arg-
inine (7, MeF--K--P-dCha-W--R), is a full antagonist of the C5a
receptor, with no agonist activity when tested on isolated cellular
membranes (Konteatis et al, 1994) or intact whole cells. This
hexapeptide was developed by modifications of the agonist
Nme-F--K--P-dCha-L-r, in which the molecule was progressively
substituted at leucine residues with substituents of increasing
size (Cha, F, Nph and W). This had the effect of reducing agonist
activity. Receptor-binding assays, performed on isolated human
neutrophil membranes, showed that the antagonist had only 0.04%
relative affinity of C5a for the receptor (Konteatis et al, 1994).
A key feature of these reports is the definition of the binding of
7 to the C5a receptor. These authors state that the C-terminal
arginine is essential for receptor binding and antagonist activity.
This is also the case in all the reports of agonist activity by
small peptide analogues of the C-terminus of C5a. However, for the
antagonist 7, the authors go further and state that [0016] "the
C-terminal carboxylate is an essential requirement for antagonist
activity and receptor binding."
[0017] They proposed that the requirement of the carboxylate is
probably the result of its specific interaction with an arginine
(Arg 206) in the receptor (De Martino et al, 1995). This idea was
supported by a great reduction in receptor-affinity for an analogue
of 7 in which the D-arginine
(NH.sub.2--CH(CO.sub.2H)--(CH.sub.2).sub.3NHC(:NH)NH.sub.2) was
replaced by agmatine
(NH.sub.2--CH.sub.2--(CH.sub.2).sub.3NHC(:NH)NH.sub.2). In summary,
De Martino et al claim that the D-arginine interacts via its
guanidinium side chain with a negatively-charged amino acid side
chain in the receptor. A second interaction between the
negatively-charged C-terminal carboxylate of 7 and a
positively-charged side chain residue in the receptor is also
thought to occur.
[0018] We have now determined the solution structure of this
hexapeptide 7 and several analogues, and have surprisingly found
that in fact a terminal carboxylate group is not required for
binding to C5aR or for antagonist activity, and that instead an
unusual hitherto unrecognised structural feature, a turn
conformation, is responsible for C5a antagonist or agonist binding
and activity. The hexapeptide and several new structurally related
antagonists have been examined for both their receptor-binding
affinities and antagonist activity, using intact polymorphonuclear
(PMN) cells. Our results show the hitherto unknown specific
structural requirement for the binding of C5a antagonists or
agonists to the C5a receptor, which we believe to be common to
ligands for the G protein-coupled receptor family. Our
establishment of this specific structural requirement has enabled
us to design and develop improved molecular probes of the
complement system and of C5a-based drugs, and to design small
molecules that target other G protein-coupled receptors, which are
becoming increasingly recognised as important drug targets due to
their crucial roles in signal transduction (G protein-coupled
Receptors, IBC Biomedical Library Series, 1996).
[0019] Thus our results have enabled us to design constrained
structural templates which enable hydrophobic groups to be
assembled into a hydrophobic array for interaction with a G
protein-coupled receptor, for example at Site 2 of the C5a receptor
illustrated in FIG. 1. Such templates or scaffolds, which may be
cyclic or acyclic, have not heretofore been suggested for
modulators of the activity of C5a receptors or other G
protein-coupled receptors.
SUMMARY OF THE INVENTION
[0020] The invention provides cyclic and non-cyclic modulators of
the activity of G-protein-coupled receptors.
[0021] According to a first aspect, the invention provides a
compound which is an antagonist, of a G protein-coupled receptor,
which has no agonist activity, and which has a cyclic or
constrained acyclic structure adapted to provide a framework of
approximate dimensions as follows:
##STR00001##
[0022] where the numerals refer to distances between C.sub..alpha.
carbons of amino acids or their analogues or derivatives, and A, B,
C and D are not necessarily on adjacent amino acids, or analogues
or derivatives thereof; and
[0023] where the critical amino acid side chains are designated by
A, B, C and D, or are as defined below;
[0024] A is any common or uncommon, basic, charged amino acid side
chain which serves to position a positively charged group in this
position, including, but not limited to the following side chains
and other mimetics of arginine side chains:
##STR00002##
where
[0025] X is NCN, NNO.sub.2, CHNO.sub.2 or NSO.sub.2NH.sub.2;
[0026] n is an integer from 1 to 4, and
[0027] R is H or an alkyl, aryl, CN, NH.sub.2 or OH group
[0028] B is any common or uncommon aromatic amino acid side chain
which serves to position an aromatic side-chain group in this
position, including but not limited to the indole, indole methyl,
benzyl, phenyl, naphthyl, naphthyl methyl, cinnamyl group, or any
other derivatives of these aromatic groups;
[0029] C is any common or uncommon hydrophobic amino acid side
chain which serves to position any alkyl, aromatic or other group
in this position, including, but not limited to D- or L-cyclohexyl
alanine (Cha), leucine, valine, isoleucine, phenylalanine,
tryptophan, or methionine
[0030] D is any common or uncommon aromatic amino acid which serves
to position an aromatic side-chain in this position, and has the
structure:
##STR00003##
where Z includes but is not limited to indole, indole methyl,
benzyl, benzene, naphthyl, naphthyl methyl, or any other
derivatives of these aromatic groups, and
[0031] R.sup.1 is H or any alkyl, aromatic, acyl or aromatic-acyl
group including, but not limited to methyl, ethyl, propyl, butyl,
--CO--CH.sub.2CH.sub.3, --CO--CH.sub.3,
--CO--CH.sub.2CH.sub.2CH.sub.3, --CO--CH.sub.2Ph, or --CO-Ph.
[0032] Preferably the G protein-coupled receptor is a C5a
receptor.
[0033] Other cyclic or constrained acyclic molecules, which may be
peptidic or non-peptide in nature, can similarly be envisaged to
support groups such as A, B, C and D for interaction with a C5a
receptor or other G protein-coupled receptor.
[0034] In one preferred embodiment, the compound has antagonist
activity against C5aR, has no C5a agonist activity, and has the
general formula:
##STR00004##
where A is H, alkyl, aryl, NH.sub.2, NHalkyl, N(alkyl).sub.2,
NHaryl or NHacyl; OH, Oalkyl, Oaryl.
[0035] B is an alkyl, aryl, phenyl, benzyl, naphthyl or indole
group, or the side chain of a D- or L-amino acid selected from
phenylalanine, homophenylalanine, tryptophan, homotryptophan,
tyrosine, and homotyrosine;
[0036] C is the side chain of a D-, L- or homo-amino acid selected
from the group consisting of proline, alanine, leucine, valine,
isoleucine, arginine, histidine, aspartate, glutamate, glutamine,
asparagine, lysine, tyrosine, phenylalanine, cyclohexylalanine,
norleucine, tryptophan, cysteine and methionine;
[0037] D is the side chain of a D- or L-amino acid selected from
the group consisting of cyclohexylalanine, homocyclohexylalanine,
leucine, norleucine, homoleucine, homonorleucine and
tryptophan;
[0038] E is the side chain of a D- or L-amino acid selected from
the group consisting of tryptophan and homotryptophan;
[0039] F is the side chain of a D- or L-amino acid selected from
the group consisting of arginine, homoarginine, lysine and
homolysine; and
[0040] X.sup.1 is --(CH.sub.2).sub.nNH-- or (CH.sub.2).sub.n--S--,
where n is an integer of from 1 to 4, preferably 2 or 3,
--(CH.sub.2).sub.2O--, --(CH.sub.2).sub.3O--, --(CH.sub.2).sub.3--,
--(CH.sub.2).sub.4--, or --CH.sub.2COCHRNH--, where R is the side
chain of any common or uncommon amino acid.
[0041] For the purposes of this specification, the term "alkyl" is
to be taken to mean a straight, branched, or cyclic, substituted or
unsubstituted alkyl chain of 1 to 6, preferably 1 to 4 carbons.
Most preferably the alkyl group is a methyl group. The term "acyl"
is to be taken to mean a substituted or unsubstituted acyl of 1 to
6, preferably 1 to 4 carbon atoms. Most preferably the acyl group
is acetyl. The term "aryl" is to be understood to mean a
substituted or unsubstituted homocyclic or heterocyclic aryl group,
in which the ring preferably has 5 or 6 members.
[0042] A "common" amino acid is a L-amino acid selected from the
group consisting of glycine, leucine, isoleucine, valine, alanine,
phenylalanine, tyrosine, tryptophan, aspartate, asparagine,
glutamate, glutamine, cysteine, methionine, arginine, lysine,
proline, serine, threonine and histidine.
[0043] An "uncommon" amino acid includes, but is not restricted to,
D-amino acids, homo-amino acids, N-alkyl amino acids, dehydroamino
acids, aromatic amino acids (other than phenylalanine, tyrosine and
tryptophan), ortho-, meta- or para-aminobenzoic acid, ornithine,
citrulline, norleucine, .gamma.-glutamic acid, aminobutyric acid
and .alpha.,.alpha.-disubstituted amino acids.
[0044] For the purposes of this specification it will be clearly
understood that the word "comprising" means "including but not
limited to", and that the word "comprises" has a corresponding
meaning.
[0045] According to a second aspect, of the invention provides a
compound which is an agonist of G protein-coupled receptors, and
which has structure III
##STR00005##
[0046] where the numerals refer to distances between C.sub..alpha.
carbons of amino acids or their analogues or derivatives, and A, B,
C and D are not necessarily on adjacent amino acids, or analogues
or derivatives thereof; and
[0047] where B is a non-aromatic amino acid, and is preferably the
D- or L-form of alanine, leucine, valine, norleucine, glutamic
acid, aspartic acid, methionine, cysteine, isoleucine, serine or
threonine,
[0048] and A, C and D are as defined above.
[0049] Preferably the compound is of structure IV,
##STR00006##
[0050] where E is any amino acid other than tryptophan and
homotryptophan, for example D- or L-forms of alanine, leucine,
valine, norleucine, phenylalanine, glutamic acid, aspartic acid,
methionine, cysteine, isoleucine, serine, threonine, and F and
X.sup.1 are as defined in Structure II. Preferably the compound is
an agonist of C5a.
[0051] According to a third aspect, the invention provides a
composition, comprising a compound according to the invention
together with a pharmaceutically-acceptable carrier or
excipient.
[0052] The compositions of the invention may be formulated for oral
or parenteral use, but oral formulations are preferred. It is
expected that most if not all compounds of the invention will be
stable in the presence of digestive enzymes. Such stability can
readily be tested by routine methods known to those skilled in the
art.
[0053] Suitable formulations for administration by any desired
route may be prepared by standard methods, for example by reference
to well-known textbooks such as Remington; The Science and Practice
of Pharmacy, Vol. II, 1995 (19.sup.th edition), A. R. Gennaro (ed),
Mack Publishing Company, Easton, Pa., or Australian Prescription
Products Guide, Vol. 1, 1995 (24.sup.th edition) J. Thomas (ed),
Australian Pharmaceutical Publishing Company Ltd, Victoria,
Australia.
[0054] In a fourth aspect, the invention provides a method of
treatment of a pathological condition mediated by a G
protein-coupled receptor, comprising the step of administering an
effective amount of a compound of the invention to a mammal in need
of such treatment.
[0055] Preferably the condition mediated by a G protein-coupled
receptor is a condition mediated by a C5a receptor, and more
preferably involves overexpression or underregulation of C5a. Such
conditions include but are not limited to rheumatoid arthritis,
adult respiratory distress syndrome (ARDS), systemic lupus
erythematosus, tissue graft rejection, ischaemic heart disease,
reperfusion injury, septic shock, psoriasis, gingivitis,
atherosclerosis, Alzheimer's disease, lung injury and
extracorporeal post-dialysis syndrome.
[0056] While the invention is not in any way restricted to the
treatment of any particular animal or species, it is particularly
contemplated that the compounds of the invention will be useful in
medical treatment of humans, and will also be useful in veterinary
treatment, particularly of companion animals such as cats and dogs,
livestock such as cattle, horses and sheep, and zoo animals,
including large bovids, felids, ungulates and canids.
[0057] The compounds may be administered at any suitable dose and
by any suitable route. Oral administration is preferred because of
its greater convenience and acceptability. The effective dose will
depend on the nature of the condition to be treated, and the age,
weight, and underlying state of health of the individual treatment.
This will be at the discretion of the attending physician or
veterinarian. Suitable dosage levels may readily be determined by
trial and error experimentation, using methods which are well known
in the art.
BRIEF DESCRIPTION OF THE FIGURES
[0058] FIG. 1 shows a diagrammatic representation of the two-site
model for binding of C5a to its G protein-coupled receptor, C5aR.
The black rods represent .alpha.-helical regions, and the open
cylinders represent the transmembrane helices. Sites 1 and 2 are
indicated on the figure.
[0059] FIG. 2 shows stacked plots of .sup.1H-NMR spectra, showing
time-dependent decay of amide NH resonances for Trp (8.10 ppm) and
D-Cha (7.90 ppm) residues of 7 in d.sub.6-DMSO containing D.sub.2O
after 10 minutes (bottom plot) and then 25, 40, 55, 70, 130, 190,
250, 385 and 520 minutes.
[0060] FIG. 3 shows backbone C, N, O atoms of twenty lowest energy
minimized NMR structures of 7 in d.sub.6-DMSO at 24.degree. C.)
[0061] FIG. 4 shows a schematic representation of H-bonding in the
structure of 7 from proton NMR spectra in d.sub.6-DMSO.
[0062] FIG. 5a shows (a) receptor binding, as indicated by
inhibition of binding of .sup.125I--C5a to human PMNs by 7
(.cndot.); 8 (.DELTA.); 9 (.tangle-solidup.); 12
(.largecircle.).
[0063] FIG. 5b shows C5a antagonist potency as inhibition of
myeloperoxidase (MPO) release from human PMNs by: 7 (.box-solid.,
n=9) and 12 (.tangle-solidup., n=4). FIG. 5c shows C5aR binding and
antagonist potencies of 7, 15 and 17.
[0064] A-C show the effect of increasing concentrations (top to
bottom) of C5a antagonists inhibiting myeloperoxidase release in
human PMNs (n=3 in A-C).
[0065] A: 7 at 0, 0.1, 0.3, 1.0 .mu.M (top to bottom)
[0066] B: 15 at 0, 0.1, 0.03, 0.1 .mu.M (top to bottom)
[0067] C: 17 at 0, 0.01, 0.03, 0.1 .mu.M (top to bottom)
[0068] D: Comparative affinities for PMN C5qR receptor. Inhibition
of binding of .sup.125I--C5a to human PMNs by 7 (top), 15 (middle),
17 (bottom). All data are means.+-.SEM.
[0069] FIG. 6 shows receptor binding of cyclic C5a antagonists, as
shown by inhibition of binding of .sup.125I--C5a to human PMNs
(n=5).
[0070] FIG. 7 shows superimposed structures of 7 (light, NMR
structure) and 12 (dark, computer modelled structure). Phe and Trp
side chains are omitted from 12 for clarity.
[0071] FIG. 8 shows inhibition of C5a-induced neutropenia in Wistar
rats by the cyclic antagonist F-[OPdChaWR] given i.v. at 1 mg/kg.
Results shown from n=3 in each group, *P<0.05 compared to
C5a-treated group only. Results are expressed as mean.+-.SEM.
[0072] FIG. 9 shows inhibition of LPS-induced neutropenia and
changes in hematocrit induced by the cyclic antagonist
F--[OPdChaWR] (0.03-10 mg/kg, i.v., 10 min prior to
lipopolysaccharide [LPS]) in Wistar rats. Abscissa: time after LPS
(1 mg/kg i.v. injection). Ordinate: percent change in hematocrit
(A) value or level of circulating polymorphonuclear (PMN)
leukocytes (B) compared to time zero.
[0073] FIG. 10 shows inhibition of carrageenan-induced (Wistar) rat
paw oedema by cyclic antagonist (3D35) AcF-[OPdChaWr] (1 mg/kg
single dose i.p. given 30 min prior to carrageenan). Results shown
from 4 rats/group, mean.+-.SEM. Ordinate: percent change in paw
volume. Abscissa: time (mins) after carrageenan injection
DETAILED DESCRIPTION OF THE INVENTION
[0074] The invention will now be described by way of reference only
to the following general methods and experimental examples, and to
the figures. Abbreviations used herein are as follows: [0075] BOP
benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium
hexafluorophosphate [0076] D-Cha D-cyclohexylamine [0077] DIPEA
diisopropylethylamine [0078] DMF dimethylformamide [0079] DMSO
dimethylsulphoxide [0080] HBTU O-benzotriazole
N',N',N',N'-tetramethyluronium hexafluorophosphate; [0081] LPS
lipopolysaccharide [0082] PMN polymorphonuclear granulocyte [0083]
RMSD root mean square deviation [0084] RP-HPLC reverse phase-high
performance liquid chromatography [0085] TFA trifluoroacetic
acid;
[0086] Throughout the specification conventional single-letter and
three-letter codes are used to represent amino acids.
General Methods
[0087] Protected amino acids and resins were obtained from
Novabiochem. TFA, DIPEA and DMF (peptide synthesis grade) were
purchased from Auspep. All other materials were reagent grade
unless otherwise stated. Preparative scale reverse-phase HPLC
separations were performed on a Vydac C18 reverse-phase column
(2.2.times.25 cm), and analytical reverse-phase HPLC separations
were performed on a Waters Delta-Pak PrepPak C18 reverse-phase
column (0.8.times.10 cm), using gradient mixtures of solvent
A=water/0.1% TFA and solvent B=water 10%/acetonitrile 90%, 0.09%
TFA. The molecular weight of the peptides was determined by
electrospray mass spectrometry recorded on a triple quadrupole mass
spectrometer (PE SCIEX API III), as described elsewhere (Haviland
et al, 1995). .sup.1H-NMR spectra were recorded on either a Bruker
ARX 500 MHz or a Varian Unity 400 spectrometer. Proton assignments
were determined by 2D NMR experiments (DFCOSY, TOCSY, NOESY).
[0088] Non-peptidic compounds were synthesized using conventional
organic chemical methods. Compounds were analysed by .sup.1H-NMR
spectroscopy and by mass spectrometry.
Peptide Synthesis
[0089] Some representative peptide syntheses are now given. Linear
peptide sequences were assembled by manual step-wise solid-phase
peptide synthesis with HBTU activation and DIEA in situ
neutralisation. Boc chemistry was employed for temporary
N.sup..alpha.-protection of amino acids with two 1 min treatments
with TFA for Boc group removal. The peptides were fully deprotected
and cleaved by treatment with liquid HF (10 ml; p-cresol (1 ml);
-5.degree. C.; 1-2 hr). Analytical HPLC (gradient; 0% B to 50% B
over 40 min): 7, Rt=32.0 min, [M+H].sup.+ (calc.)=900.5,
[M+H].sup.+ (exper.)=900.7; 8, Rt=32.2 min, [M+H].sup.+
(calc.)=899.6, [M+H].sup.+ (exper.)=899.7; 9, Rt=30.0 min,
[M+H].sup.+ (calc.)=900.5, [M+H].sup.+ (exper.)=900.7; 10, Rt=23.8
min, [M+H].sup.+ (calc.)=860.5, [M+H].sup.+ (exper.)=860.5.
[0090] Structures for the peptides are shown in Table 4 below.
a) Synthesis of Cycle 11
[0091] This is a general method used for the synthesis of a wide
range of cyclic antagonists covered by this patent. For example, in
the case of cycle 11, its linear precursor peptide was synthesised
by Fmoc chemistry using HBTU/DIEA activation on an
Fmoc-D-Arg(Mtr)-Wang resin. Fmoc group removal was effected using
two 1 min treatments with 50% piperidine/DMF. Cleavage and
deprotection using 95% TFA/2.5% TIPS/2.5% H.sub.2O gave the
Mtr-protected peptide, which was purified by RP-HPLC. Cyclization
of the protected, purified peptide using 3 eq BOP and 10 eq DIEA at
a 1 mM concentration in DMF stirring for 15 hr gave the cyclised
product, which was fully deprotected using 1M TMSBr in TFA. A final
RP-HPLC purification gave the desired peptide in yields of 50% for
the cyclisation. Rt=37.7 min, [M+H].sup.+ (calc.)=910.5,
[M+H].sup.+ (exper.)=910.7.
b) Synthesis of Cycle 12
[0092] Cyclization of the cleaved and fully deprotected peptide was
achieved by stirring a 1 mM solution in DMF with 3 eq BOP and 10 eq
pyridine as base for 15 hr. A final RP-HPLC purification gave the
desired peptide in yields of 22% for the cyclization. Rt=37.3 min,
[M+H].sup.+ (calc.)=896.5, [M+H].sup.+ (exper.)=896.5.
NMR Structure Determination
[0093] .sup.1H-NMR spectra were recorded for compound 7 (3 mg in
750 .mu.l d.sub.6-DMSO, .delta. 2.50) referenced to solvent on a
Varian Unity 400 spectrometer at 24.degree. C. Two dimensional
.sup.1H-NMR NOESY (relaxation delay 2.0 s, mix time 50-300 ms),
DFQ-COSY and TOCSY (mixing time 75 ms) experiments were acquired
and recorded in phase sensitive mode. Acquisition times=0.186 s,
spectral width=5500 Hz, number of complex points (t.sub.1
dimension)=1024 for all experiments. Data was zero-filled and
Fourier transformed to 1024 real points in both dimensions.
[0094] NMR data was processed using TRIAD software (Tripos Assoc.)
on a Silicon Graphics Indy work station. 2D NOE cross peaks were
integrated and characterised into strong (1.8-2.5 .ANG.), medium
(2.3-3.5 .ANG.) and weak (3.3-5.0 .ANG.). Preliminary
three-dimensional structures were calculated from upper and lower
distance limit files using Diana 2.8 (69 distance constraints,
including 27 for adjacent residues and 6 further away) with the
redundant dihedral angle constraints (REDAC) strategy. Upper and
lower distance constraints were accurately calculated using
MARDIGRAS. At this stage the peptide was examined for possible
hydrogen bonds, and these were added as distance constraints. The
50 lowest energy Diana structures were subjected to restrained
molecular dynamics (RMD) and energy minimisation (REM). Initially,
REM consisted of a 50 step steepest descent followed by 100 step
conjugate gradient minimisation. RMD was performed by simulated
heating of the structures to 300K for 1 ps, followed by 500K for 1
ps. The temperature was gradually lowered to 300K over 2 ps and
finally for 2 ps at 200K. REM was performed again with a 50 step
steepest descent, 200 step conjugate gradient followed by a 300
step Powell minimisation. The final structures were examined to
obtain a mean pairwise rms difference over the backbone heavy atoms
(N, C.alpha. and C). Twenty of the 50 structures had a mean
rmsd<0.5 .ANG. for all backbone atoms (O, N, C).
Molecular Modelling
[0095] A model of cycle 12, shown in FIG. 7, was created from the
NMR structure of 7 by deleting all NMR constraints, fusing the
ornithine side chain amine to the C-terminal carboxylate of d-Arg
to form an amide, and minimising using Powell forcefield (1000
iterations). The modelled structure was then superimposed on the
NMR structure with an rmsd 0.224 .ANG..
Receptor-Binding Assay
[0096] Assays were performed with fresh human PMNs, isolated as
previously described (Sanderson et al, 1995), using a buffer of 50
mM HEPES, 1 mM CaCl.sub.2, 5 mM MgCl.sub.2, 0.5% bovine serum
albumin, 0.1% bacitracin and 100 .mu.M phenylmethylsulfonyl
fluoride (PMSF). In assays performed at 4.degree. C., buffer,
unlabelled human recombinant C5a (Sigma) or peptide, Hunter/Bolton
labelled .sup.125I--C5a (.about.20 pM) (New England Nuclear, MA)
and PMNs (0.2.times.10.sup.6) were added sequentially to a
Millipore Multiscreen assay plate (HV 0.45) having a final volume
of 200 .mu.L/well. After incubation for 60 min at 4.degree. C., the
samples were filtered and the plate washed once with buffer.
Filters were dried, punched and counted in an LKB gamma counter.
Non-specific binding was assessed by the inclusion of 1 mM peptide
or 100 nM C5a which typically resulted in 10-15% total binding.
[0097] Data was analysed using non-linear regression and statistics
with Dunnett post test.
Myeloperoxidase Release
[0098] Cells were isolated as previously described (Sanderson et
al, 1995) and incubated with cytochalasin B (5 .mu.g/mL, 15 min,
37.degree. C.). Hank's Balanced Salt solution containing 0.15%
gelatin and peptide was added on to a 96 well plate (total volume
100 .mu.L/well), followed by 25 .mu.L cells (4.times.10.sup.6/mL).
To assess the capacity of each peptide to antagonise C5a, cells
were incubated for 5 min at 37.degree. C. with each peptide,
followed by addition of C5a (100 nM) and further incubation for 5
min. Then 50 .mu.L of sodium phosphate (0.1M, pH 6.8) was added to
each well, the plate was cooled to room temperature, and 25 .mu.L
of a fresh mixture of equal volumes of dimethoxybenzidine (5.7
mg/mL) and H.sub.2O.sub.2 (0.51%) was added to each well. The
reaction was stopped at 10 min by addition of 2% sodium azide.
Absorbances were measured at 450 nm in a Bioscan 450 plate reader,
corrected for control values (no peptide), and analysed by
non-linear regression.
In Vivo Assays of Anti-Inflammatory Activity
[0099] The following well-known in vivo assay systems may be used
to assess the anti-inflammatory activity of compounds of the
invention. All assay data are analysed using non-linear regression
analysis and Student's t-test, analysis of variance, with p<0.05
as the threshold level of significance.
(a) Carrageenan Paw Oedema
[0100] Anaesthetised (i.p. ketamine & xylazine) Wistar rats
(150-200 g) or mice were injected with sterilised air (20 ml day 1,
10 ml day 4) into the subcutaneous tissue of the back. The cavity
can be used after 6 days, whereupon carrageenan (2 ml, 1% w/w in
0.9% saline) was injected into the air pouch and exudate was
collected after 10 hr. Test compounds are administered daily after
Day 6 and their anti-inflammatory effects assayed by differential
counting of cells in the air-pouch exudate. Animals were killed at
appropriate times after injection and 2 ml 0.9% saline was used to
lavage the cavity, lavage fluids were transferred to heparinised
tube and cells were counted with a haemocytometer and Diff-Quik
stained cytocentrifuged preparation.
[0101] Alternatively, a routine carrageenan paw oedema was
developed in Wistar rats by administering a pedal injection of
carrageenan to elicit oedema which is visible in 2 h and maximised
in 4 h. Test compounds are given 40 min before inflammagen and
evaluated by microcaliper measurements of paws after 2 & 4 hr.
See Fairlie, D. P. et al (1987). Also see Walker and Whitehouse
(1978).
(b) Adjuvant Arthritis.
[0102] Adjuvant arthritis was induced in rats (3 strains) either
microbially (injection of heat-killed Mycobacterium tuberculosis)
or chemically (with pyridine) by inoculation with the arthritogenic
adjuvant co-administered with oily vehicles (Freund's adjuvants) in
the tail base. (See Whitehouse, M. W., Handbook of Animal Models
for the Rheumatic Diseases, Eds. Greenwald, R. A.; Diamond, H. S.;
Vol. 1, pp. 3-16, CRC Press)
[0103] Within 13 days the adjuvant arthritis is manifested by local
inflammation and ulceration in the tail, gross swelling of all four
paws, inflammatory lesions in paws and ears, weight loss and fever.
These symptoms, which are similar to those of inflammatory disease
in humans (Winter and Nuss, 1966), can be alleviated by agents such
as indomethacin or cyclosporin which also show beneficial effects
in man (eg. Ward and Cloud, 1966). Without drug treatment at Day
14, arthritic rats had hypertrophy of the paws, reduced albumin but
raised acute phase reaction proteins in serum, and depressed
hepatic metabolism of xenobiotics as indicated by prolonged
barbiturate-induced sleeping times.
[0104] To assess activity, compounds were administered for 4 days
orally (.ltoreq.10 mg/kg/day) or i.p. from Days 10-13 following
inoculation with arthritogen (Day 0). The inflammation was either
not visible or very significantly reduced in rear or front paws as
assessed by microcaliper measurements of paw thickness and tail
volume, as well as by gross inspection of inflammatory lesions.
Animals are sacrificed by cervical dislocation on Day 18 unless
arthritis signs are absent, whereupon duration of observations is
continued with special permission from the Ethics committees.
Experiments are staggered to maximise throughput and allow early
comparisons between compounds. This routine assay is well-accepted
as identifying anti-inflammatory agents for use in humans.
Example 1
Structure-Activity Relationship of C5a Agonists
[0105] We have focussed on the C-terminal residues of C5a, in order
to explore structure-activity relationships in the search for
peptide sequences with potent agonist activity. Many of these
peptides are full agonists relative to C5a, but have markedly lower
potency (Sanderson et al, 1994, 1995; Finch et al, 1997). Our
initial structure-activity investigations have been particularly
informative. Mutating the decapeptide C-terminus of C5a (1,
C5a.sub.6-74, ISHKDMQLGR) twice with I.sub.65Y and H.sub.67F (eg.
2) led to enhancement of agonist potency by about 2 orders of
magnitude. These results are summarised in Table 2. Analyses of
Ramachandran plots and 2D NMR spectra for compound 2 suggested that
certain structural features, namely a twisted "helix-like" backbone
conformation for residues 65-69 and a .beta.-turn for residues
71-74, might be responsible for activity. These preliminary results
provided some insight to structural requirements for tight binding
to a C5a receptor.
TABLE-US-00002 TABLE 2 Pharmacological Activity of C5a Agonist
Analogues* PMN Enzyme Binding Fetal Artery Release EC.sub.50
Affinity Peptide No. Peptide EC.sub.50 (.mu.M) (.mu.M) IC.sub.50
(.mu.M) 1 C5a.sub.65-74 (ISHKDMQLGR) >1000 >1000 >1000 2
YSFKDMQLGR 9.6 92 1.3 3 YSFKDMPLaR 0.5 72 3.7 4 YSFKPMPLaR 0.2 4.1
6.0 5 C5a.sub.37-46-ahxYSFKPMPLaR 0.06 5.9 0.7 6
C5a.sub.12-20-ahxYSFKPMPLaR 0.08 0.7 0.07 C5a 0.02 0.03 0.0006
*Finch et al, 1997
[0106] Compounds 4, 5 and 6 in Table 2 are the highest affinity
small C5a agonists so far known, with up to 25% C5a potency in
human fetal artery, 5% C5a potency in human PMN enzyme release
assays and 1% C5a affinity for PMN C5aR (Finch et al., 1997). For
the PMN receptor, these compounds have up to 100-fold higher
apparent affinity than any small molecule previously described in
the literature.
[0107] The "high" affinities (70 nM-6 .mu.M) of these agonist
analogues for C5aR in intact PMN cells have enabled us to identify
a common topographical feature in peptide agonists that correlates
with expression of spasmogenic activities and enzyme-release assays
in human PMNs. This preferred backbone conformation is a type II
.beta.-turn.
[0108] The small size of these agonist peptides makes them amenable
to synthetic modification to optimise their affinities, activities,
and bioavailabilities, and hence useful as mechanistic probes of
receptor activation.
Example 2
NMR Structure of C5a Antagonist
[0109] We used two dimensional nuclear magnetic resonance
spectroscopy to determine the three dimensional structure of 7 and
found that while there is no discernible structure in water, there
is evidence of a stable gamma-turn structure in
dimethylsulfoxide.
[0110] The 1D .sup.1H-NMR spectrum of peptide 7 in d.sub.6-DMSO at
24.degree. C. shows 4 distinct resonances for amide-NH protons, as
summarized in Table 3. To establish their possible involvement in
intramolecular hydrogen bonds, a deuterium exchange experiment was
performed by adding a 10-fold excess of D.sub.2O to the solution.
Two of the amide-NH doublets disappeared immediately, along with
resonances attributable to the N-terminal methylamine protons.
However, the other two amide NH resonances, as well as a broad
resonance at approximately 8.05 ppm, persisted for up to 6.5 hours
(FIG. 2). These three slowly-exchanging protons are assigned to the
amide NHs of Trp and d-Cha and the side chain amine of Lys, the
slow exchange behaviour being characteristic of hydrogen-bonding.
The amine assignment was established from the TOCSY spectrum where
cross peaks were observed between the protonated amine and the
.epsilon., .delta. and .gamma. CH.sub.2 protons. A temperature
dependence study (20-60.degree. C.) of the amide-NH chemical shifts
(.DELTA..delta./T=2.5 ppb/deg, dCha-NH; 6 ppb/deg, Trp-NH; 6.5 ppb,
Lys-NH; 8.7 ppb, Arg-NH) unambiguously confirmed the involvement of
the dCha-NH only in intramolecular hydrogen bonding.
TABLE-US-00003 TABLE 3 .sup.1H-NMR Assignments.sup.a for 7 in
d.sub.6-DMSO Residue .sup.bHN H.alpha. H.beta. H.gamma. Others
MePhe -- 4.06 3.09, 3.06 -- .sup.c7.17, 7.29; .sup.d2.46;
.sup.f8.98 Lys 8.83 4.54 1.74, 1.55 1.32 .sup.e1.51; .sup.f2.74,
.sup.g7.76 (NH.sub.2) Pro -- 4.30 2.084, 1.74 1.88, 1.78
.sup.e3.61, .sup.f3.48 d-Cha 7.91 4.35 1.19, 1.06 0.76 .sup.e1.43,
1.08; .sup.f1.61, 1.58; 0.73 Trp 8.01 4.65 3.11, 2.94 --
.sup.c6.97, 7.06, 7.13, 7.32, 7.65; .sup.g10.80 d-Arg 8.44 4.20
1.73, 1.58 1.42 .sup.e3.08; .sup.g7.60 .sup.aReferenced to residual
d.sub.5-DMSO at 2.50 ppm. .sup.bAmide NHs, .sup.3J.sub.NH--CaH
values (Hz): 7.91 (Lys), 7.77 (d-Arg), 8.34 (Trp), 8.53 (d-Cha).
.sup.cAromatics .sup.dN-Me. .sup.eH.delta.. .sup.fH.epsilon.
.sup.gNH/NH.sub.2 amine.
[0111] A series of 2D .sup.1H-NMR spectra were measured for 7 at
24.degree. C. in d.sub.6-DMSO to determine the three-dimensional
structure. TOCSY and DFQ-COSY experiments were used to identify
residue types, while sequential assignments were made from analysis
of NOESY data. From a series of 100 structures generated from NOESY
data, fifty of the lowest energy structures were subjected to
restrained molecular dynamics (200K-500K) and energy minimised. A
set of 20 calculated structures with a root mean square deviation
(rmsd)<0.5 .ANG. (backbone atoms) are superimposed in FIG. 3,
and clearly depict a turn conformation.
[0112] In combination, the NMR constraint data,
.sup.3J.sub.NH--C.alpha.H values, deuterium exchange and
temperature dependence data establish an unusual turn structure for
hexapeptide 7 which is constrained by up to three hydrogen bonds,
as shown in FIG. 4. The evidence is very strong for one
intramolecular hydrogen bond from dCha-NH . . . OC-Lys (2.72 .ANG.,
N--H . . . O angle 157.degree., C.dbd.O . . . H angle 84.degree.),
forming a 7-membered ring that defines an inverse .gamma.-turn. The
dChaNH--O-TrpNH angle is 56.4.degree.. The deuterium exchange data
and NMR constraint data together point to a second intramolecular
hydrogen bond Trp-NH . . . OC-Lys (3.31 .ANG., N--H . . . O angle
159.degree., CO . . . H angle 137.3.degree.) forming a 10-membered
ring characteristic of a .beta.-turn. The .phi. and .psi. angles
(.phi..sub.2=-58.4.degree., .psi..sub.2=62.0.degree.;
.phi.=96.6.degree., .psi..sub.3=16.6.degree.) most closely match a
type II .beta.-turn (Bandekar, 1993; Hutchinson and Thornton, 1994)
which is distorted by the presence of the .gamma.-turn wholly
within the .beta.-turn.
[0113] To our knowledge this is the first example of an
intramolecular hydrogen bond between residues within a .beta.-turn,
although there are many examples of hydrogen bonds between a
residue within the "10 membered ring" of a .beta.-turn and a
residue outside of it (Bandekar, 1993). A third hydrogen bond (2.76
.ANG., N--H . . . O angle 160.3.degree.), between the side-chain
amine of Lys and the C-terminal carboxylate, is suggested by the
NMR constraint data, by slow NH/ND exchange and by detection of a
weak NOE between Lys-NH . . . Trp-.alpha.CH.sub.2. This may further
constrain the molecule into the observed turn conformation. Such
ion-pairing is common in dipolar aprotic solvents such as
dimethylsulphoxide and may also be relevant in a hydrophobic
protein environment.
[0114] NMR solution structures have also been determined for
several of the cyclic antagonists described in the following
examples, and show that in each case the type II .beta.-turn is
preserved and stabilized by the cyclic structure.
[0115] The constraining .beta. and .gamma. turns proposed in the
linear peptide 7 have parallels in cyclic peptides. We have
previously detected overlapping .beta. and .gamma. turns in a
cyclic octapeptide from ascidiacyclamide (Abbenante et al, 1996).
Combinations of a .beta.- and .gamma.-turn have also been found in
the backbones of cyclic penta- and hexapeptides, particularly those
containing alternating D- and L-amino acids (Marraud and Aubry
1996; Fairlie et al, 1995; Kessler et al, 1995; Stradley et al,
1990). For example a type II .beta.-turn and an inverse
.gamma.-turn have been identified in cyclic antagonists
c-(D-Glu-Ala-D-allo-Ile-Leu-D-Trp] (Ihara et al, 1991; Coles et al,
1993; Ihara et al, 1992; Bean et al, 1994) and
c-(D-Asp-Pro-D-Val-Leu-D-Trp) (Bean et al, 1994) for endothelin
receptors, and in members of the rhodopsin family of G
protein-coupled receptors with seven transmembrane domains (X.-M.
Cheng et al, 1994). In the latter case, as in 7, an inverse
.gamma.-turn forms between residues (Asp-CO . . . Val-NH, Lys-CO .
. . dCha-NH) that flank the proline.
Example 3
Structure-Activity Relationships In Vitro
[0116] We also examined the receptor-binding and antagonist
activity of the hexapeptide 7 for comparison with our new
compounds. The previous report by Konteatis et al (1994) concerned
the ability of 7 to compete with C5a binding to receptors on
isolated PMN membranes (IC.sub.50 70 nM), which is not necessarily
physiologically relevant. We examined competition between 7 and C5a
using intact PMN cells, and found that, under these conditions, 7
binds with much lower receptor affinity of IC.sub.50 1.8 .mu.M. We
confirmed that 7 is a full antagonist with no agonist properties.
These results are summarized in FIG. 5a and Table 4. The relative
affinity (ratio) of 7 for the C5aR in intact PMNs in our assays was
similar to that previously reported for isolated PMN membranes.
[0117] We have also found that 7 shows antagonist activity against
both C5a (FIG. 5b) and a C-terminal agonist decapeptide analogue 4
(YSFKPMPLaR) (Finch et al, 1997) of the C-terminus C5a.sub.65-74,
suggesting that it acts on site 2 of the receptor. Compounds 7 and
4 have similar .mu.M affinity for the receptor C5aR on intact
polymorphonuclear leukocytes, as shown in Table 4.
[0118] A new discovery from the data in Table 4 is the linear
correlation between the log of binding affinities and the log of
antagonist potencies for these Site 2 antagonists (compounds 7-12,
Table 4). The importance of this linear relationship is that since
receptor affinity and antagonist activity are directly
proportional, the experimentally simpler approach of measuring
receptor binding may be used to estimate the antagonist activity
for such small compounds, provided that there is no evidence of
agonist activity.
TABLE-US-00004 TABLE 4 Receptor-Binding Affinities.sup.a and
Antagonist Activities.sup.b in Human PMNs Antag- Receptor onist
Affinity.sup.a Potency.sup.b Agonist Compound IC.sub.50 (.mu.M)
IC.sub.50 (.mu.M) Activity.sup.c SEQ. ID NO:7 MeFKP (dCha) Wr 1.8
(15) 0.085 (9) No SEQ. ID NO:8 MeFKP (dCha) Wr-CONH.sub.2 14 (5)
0.5 (3) No SEQ. ID NO:9 MeFKP (dCha)WR 11 (5) 0.7 (3) No SEQ. ID
NO:10 MeFKPLWR 144 (1) >1000 (3) nd SEQ. ID NO:11 Ac-F-[KP
(dCha) Wr 3.2 (40 0.090 (5) No SEQ. ID NO:12 Ac-F-[Op (dCha) Wr
0.28 (6) 0.012 (4) No SEQ. ID NO:4 YSFKPMPLaR 6.0.sup.d -- Yes SEQ.
ID NO:1 C5a.sub.65-74, ISHKDMQLGR >1000.sup.e -- -- C5a 0.0008
(9) -- Yes Number of experiments in parenthesis. Corrected for
amino acid content. Square brackets indicate cyclic portion. nd =
not determined .sup.a50% reduction in binding of .sup.125I-C5a to
intact human PMNs .sup.b50% reduction in myeloperoxidase secretion
from human PMNs mediated by 100 nM C5a .sup.cAgonist activity in
dose range 0.1 nM-1 mM Finch et al, 1997; .sup.eKawai et al,
1991
[0119] It has previously been proposed that the C-terminus of C5a
and of agonist peptides is essential for activity, due to its
interaction with a positively-charged Arg206 of the receptor
(DeMartino et al, 1995). We confirm here that the C-terminal
carboxylate is indeed important for activity (8 vs. 7), but
wondered whether the origin of this effect might be due to hydrogen
bonding between the carboxylate anion and the positively charged
amine side chain of Lys. Conversion to the amide (8) certainly
reduces both receptor-affinity and antagonist activity
approximately 5-fold. Changing chirality of the Arg-C.alpha. (9 vs.
7) causes a similar reduction in activity, and replacing dCha with
the less bulky Leu residue (10) is also detrimental to receptor
binding. However, potency is recovered for cyclic compounds 11 and
12, in which an amide bond is tolerated at the C-terminus,
consistent with the structural interpretation above that the
advantage of the carboxylate in 7 may be associated with
intramolecular hydrogen bonding. The replacement of this hydrogen
bond in 7 with a covalent amide bond in 11 and 12 more effectively
stabilizes the turn conformation.
[0120] FIG. 5C compares C5aR binding and antagonist potency in
vitro on human PMNs for compounds 15 and 17 with those for compound
7. Both 15 and 17 are potent inhibitors at nM concentrations of the
action of C5a and the binding of .sup.125I--C5a to its receptor
(e.g. 4, K.sub.b=1.4 nM). Their cyclic nature and the acetylation
at the N-terminal phenylalanine both protect against the
proteolytic degradation typically encountered by peptides, making
such cyclic compounds more suitable than acyclic peptides as drug
candidates. The results are shown in Table 5.
TABLE-US-00005 TABLE 5 Receptor Binding and Antagonist Activity of
Cyclic Molecules ##STR00007## ##STR00008## Receptor Affinity
Agonist Compound n R Isomer* .mu.M Activity 13 1 H S- 9 No 14 R- 34
No 15 2 H S- 0.3 No 16 R- 3.7 No 17 3 Ac S- 0.3 No 11 Ac R- 38 No
18 4 Ac S- 3.2 No 12 Ac R- 51 No *Refers to stereochemistry of Arg
side chain.
Example 4
Cyclic Antagonists of C5a
[0121] Some examples of these cyclic antagonists and their apparent
receptor-binding affinities and antagonist potencies are given in
Tables 4, 5 and 6 as well as in FIGS. 5 and 6. In the tables the
single letter code for amino acids is used.
TABLE-US-00006 TABLE 6 PEPTIDE pD.sub.2 .+-. SE.sup.a IC.sub.50
(M).sup.a (n) pD.sub.2 .+-. SE.sup.b IC.sub.50 (M).sup.b (n) Effect
of Cyclisation on Antagonist Binding Affinity and Antagonist
Potency SEQ. ID NO: 11 AcF--[KPdChaWr] 5.49 .+-. 0.22 3.2 4 7.07
.+-. 0.29 0.09 5 SEQ. ID NO: 12 AcF--[OPdChaWr] 6.44 .+-. 0.14* 0.4
9 7.30 .+-. 0.09 0.05 9 SEQ. ID NO: 19 [FWPdChaWr] 4.37 .+-. 0.36*
43 3 nd SEQ. ID NO: 20 AcF--[KMdChaWr] 4.81 .+-. 0.06 15 2 nd SEQ.
ID NO: 21 AcF--[KKdChaWr] 3.94 .+-. 0.4 116 3 4.88 13 1 Effect of
length of linker in cycle on antagonist binding affinity and
antagonist potency -- SEQ ID NO: 22 AcF--[XPdChaWr] 5.02 .+-. 0.07
9.5 3 4.71 .+-. 0.23 20 3 SEQ ID NO: 23 AcF--[X.sup.2PdChaWr] 4.77
.+-. 0.14* 17 3 6.09 .+-. 0.08* 0.8 4 SEQ ID NO: 12 AcF--[OPdChaWr]
4.60 .+-. 0.06* 16 4 6.42 .+-. 0.10 0.4 4 SEQ ID NO: 24
AcKF--[OPdChaWr] 4.96 .+-. 0.03 11 3 6.73 0.2 1 PEPTIDE pD.sub.2
.+-. Se.sup.a IC.sub.50 (.mu.M).sup.a (n) pD.sub.2 .+-. SE.sup.b
IC.sub.50 (.mu.M).sup.b (n) SEQ. ID NO: 14 F--[XPdChaWr] 4.39 .+-.
0.10* 41 3 nd SEQ. ID NO: 16 F--[X.sup.2PdChaWr] 5.42 .+-. 0.05 3.8
3 6.70 .+-. 0.04 0.4 3 SEQ. ID NO: 25 F--[OPdChaWr] 5.51 .+-. 0.07
3.1 3 5.79 .+-. 0.34* 1.6 3 SEQ. ID NO: 26 F--[KPdChaWr] 5.09 .+-.
0.08 8.1 3 5.55 .+-. 0.57* 2.8 3 Effect of L-Arg on antagonist
binding affinity and antagonist potency SEQ. ID NO: 17
AcF--[OPdChaWR] 6.57 .+-. 0.05* 0.3 3 7.91 .+-. 0.17* 0.01 3 SEQ.
ID NO: 13 F--[XPdChaWR] 4.98 .+-. 0.05 10 3 5.63 .+-. 0.13* 2.4 3
SEQ. ID NO: 15 F--[X.sup.2PdChaWR] 6.50 .+-. 0.04* 0.3 5 7.36 .+-.
0.13 0.04 3 SEQ. ID NO: 27 F--[OPdChaWR] 7.21 .+-. 0.01* 0.06 3
7.41 .+-. 0.14 0.04 3 SEQ. ID NO: 28 F--[KPdChaWR] 6.50 .+-. 0.12*
0.3 4 6.69 .+-. 0.04 0.2 3 .sup.apD.sub.2/IC.sub.50; concentration
of peptide resulting in 50% inhibition in the binding of
[.sup.125I]C5a to intact PMNs. The IC50 is the antilog of the mean
pD2 value .sup.bpD.sub.2/IC.sub.50; concentration of peptide
resulting in 50% inhibition in the ability of C5a (100 nM)to cause
the release of MPO from PMNs X = (CH.sub.2)--NH.sub.2 X.sup.2 =
(CH.sub.2).sub.2--NH.sub.2 pD2 values are expressed as mean .+-. SE
n represents the number of experiments performed *Significant
change in affinity/potency compared to NMeFKPdChaWR (p < 0.05)
.sup.#indicates isomer number
[0122] These results demonstrate:
[0123] (1) that the cyclic molecules have higher apparent receptor
affinity and may be more potent antagonists than acyclic (linear)
peptides,
[0124] (2) that one of the two possible cyclic diastereomers is
consistently favoured for binding to the C5a receptor, and it is
surprisingly the opposite stereochemistry (L-arginine) to that
favoured in the linear compounds (D-arginine)
[0125] (3) that the cycles have an optimum ring size for
receptor-binding,
[0126] (4) that there is a pseudo-linear relationship between log
(antagonist potency) and log (receptor affinity).
[0127] Tables 5 and 6 list the C5a receptor affinities of some
examples of cyclic antagonists of C5a, and their ability to bind
to, and inhibit, binding of C5a to human PMNs is illustrated in
FIG. 6. Surprisingly these data show that the L-arginine is
preferred over the D-arginine, in contrast to the linear compound 7
in which the D-arginine confers higher affinity for the receptor
than does L-arginine. The data also show that the size of the
macrocycle is optimal when n=2 or 3, the smaller cycle where n=1
and the larger cycle when n=4 being clearly less active. This
requirement for a tightly constrained cycle is probably due to the
need to correctly position the attached side chain residues of, for
example, Trp, dCha, Arg and Phe for interaction with the
receptor.
Example 5
Computer Modelling of Antagonist Structures
[0128] FIG. 7 compares the computer-modelled structure of the
cyclic antagonist 12 with the NMR solution structure for the
acyclic antagonist 7. These backbone structures are strikingly
similar, and strongly suggest that the receptor-binding
conformations of these molecules involve the same turn structure.
Compound 12, a more potent antagonist than 11, also has a shorter
linker, which tightens the turn and slightly alters the
conformational space accessible to the key side chains of Phe,
dCha, Trp and Arg. The conformational limitations placed on the
hexapeptide derivative 12 by the cycle are responsible for a
.gtoreq.10.sup.4 increase in receptor-binding affinity over the
conformationally flexible decapeptide C-terminus of C5a (1, Table
2).
[0129] There is a correlation between binding affinities and
antagonist potency for the site 2 antagonists (compounds 7-12,
Table 2). It thus appears that antagonist potency is dependent upon
changes that occur at site 2 alone. Without wishing to be bound by
any proposed mechanism, we believe that this may be because the
mechanism of antagonism is related to conformational change to a
turn conformation induced by 7 at site 2 of the receptor.
Example 6
Characterisation of C5aRs on Different Cells
[0130] Currently there is no information about different types of
C5aRs. We have previously shown marked differences in the
responsiveness of different cells containing functional C5aRs to
agonists (Sanderson et al, 1994, 1995; Finch et al, 1997) and we
can now provide more information by examining potency and efficacy
of selective agonists and antagonists relative to human recombinant
C5a. For agonists, the tissue or cell selectivity may reveal
functionally different receptors. Binding assays using human PMNs,
U937 cells, or circulating monocytes are used to determine
affinities for C5aRs. Selectivity for different C5aRs is
ascertained by differential antagonism. This combined approach
allows pharmacological characterisation of new agonists or
antagonists, and may lead to a potential functional classification
of C5aRs on different cells.
Example 7(a)
Neutropenia and C5a Antagonism In Vivo
[0131] Compounds were evaluated in an acute model of C5a-induced
neutropenia. Transient neutropenia maximises 5 min after i.v. C5a
and is profound, with >90% of circulating neutrophils
disappearing from circulation at effective doses of C5a, as shown
in FIG. 8. The neutropenia is due to transient adherence of
circulating neutrophils to the vascular endothelium. Preliminary
data show that neutropenia caused by i.v. C5a is blocked by a C5a
antagonist. For example, F--[OPdChaWR], (1 mg/kg), given prior to 2
.mu.g C5a i.v., inhibits C5a-induced neutropenia in vivo (FIG.
8).
Example 7(b)
Inhibition of Lipopolysaccharide-Induced Effects by C5a
Antagonists
[0132] LPS causes rapid neutropenia in rats. If this effect of LPS
is blocked by C5a antagonists, then C5a may be of major importance
in the acute effects of LPS, and the results shown in FIG. 9 were
in agreement with this hypothesis. C5a antagonists were injected
(bolus i.v.) 10 min prior to challenge with LPS. Rats were
anaesthetised, and blood samples (0.3 ml) were taken for
measurements of PMNs. PMNs are isolated and quantified. Preliminary
results show that F--[OPdChaWR], (1 mg/kg), given prior to i.v.
LPS, inhibits neutropenia.
[0133] The results also indicate that the C5a antagonist inhibits
the increase in hematocrit caused by LPS, showing that vascular
leakage of serum caused by LPS is also inhibited.
[0134] These results demonstrate that C5a receptor antagonists,
such as those described in this invention, may have therapeutic
utility in septicaemic individuals. The ability to inhibit the
adherence of PMNs to vascular endothelium, and to inhibit the
vascular leakage to LPS as shown by the reduction of hematocrit
values, indicates powerful anti-inflammatory effects of these
compounds against proinflammatory stimuli activating the complement
system, such as endotoxin or LPS.
Example 8
In Vivo Activity of Cyclic C5a Antagonists
[0135] Preliminary experiments in rats have revealed that the
cyclic antagonists summarized in Table 5 are active at less than 20
mg/kg as anti-inflammatory agents in suppressing the onset of
either carrageenan-induced paw oedema or adjuvant-induced
polyarthritis. The maximally effective dosages for even
moderately-effective antagonists are 10 mg/kg or less, given i.p.
or p.o. Many anti-inflammatory drugs currently used in humans were
initially evaluated in such assays, and also showed activity in
these rat models of inflammation. These preliminary indications of
efficacy in vivo indicate that C5a antagonists have therapeutic
potential in human inflammatory conditions.
[0136] Using the rat carageenan paw oedema assay, we found that a
compound, AcF--[O--P-dCha-W-r], which is 100 times less active than
17 in vitro as a C5a antagonist in PMNs, has some in vivo activity
in rats given 1 mg/kg of the compound I.P, 30 min prior to the
carageenan injection. Paw swelling was measured for up to 4.5 hr.
The results, shown in FIG. 10, suggest that even this weak C5a
antagonist significantly inhibits development of the oedema after
180 and 270 min. This anti-inflammatory activity suggests that C5a
receptor antagonists, such as those described in this invention,
may have therapeutic activity in diseases involving vascular
leakage following inflammatory stimuli.
[0137] In recent years there have been many attempts to mimic
.beta.- and .gamma.-turn peptides that represent bioactive protein
surfaces, resulting in notable mimetics for RGD
(arginine-glycine-aspartate) peptides, somatostatin and opioid
peptides, to name a few derived through structure-activity
relationships (see for example Marraud and Aubry, 1996; Fairlie et
al, 1995). Most of these examples preserve a turn structure through
cyclisation of the peptide. On the other hand, there are
comparatively few short acyclic peptides that have been found to
have substantial turn structure in solution (Dyson et al, 1988;
Rizo and Gierasch, 1992; Pracheur et al, 1994). It is usually
argued that short acyclic peptides adopt a myriad of solution
structures that may include small populations of turn structures
that are responsible for bioactivity.
[0138] This invention describes a series of
conformationally-constrained turn-containing molecules that are
preorganized for binding to the same G protein-coupled receptor(s)
of human cells that are targeted by human C5a. The invention is
applicable to other G protein-coupled receptors.
[0139] The principal feature of the compounds of the invention is
the preorganized arrangement, which brings at least three
hydrophobic groups and a charged group into neighbouring space,
creating a hydrophobic surface `patch`. These results enable the
design and development of even more potent
conformationally-constrained, small molecule antagonists of
C5a.
[0140] In the light of the aforementioned prior art, it was
surprising to find that a C-terminal carboxylate was not necessary
in our compounds in order to obtain good receptor-binding or
antagonist activity. The cyclic antagonists have an amide bond at
the `C-terminal` arginine position. The replacement of the
carboxylate in 7 with a covalent amide bond effectively stabilises
the required turn conformation.
[0141] Cyclic and non-peptidic antagonists have several important
advantages over peptides as drugs. The cycles described in this
invention are stable to proteolytic degradation for at least
several hours at 37.degree. C. in human blood or plasma, or in
human or rat gastric juices or in the presence of digestive enzymes
such as pepsin, trypsin and chymotrypsin. In contrast, short
peptides composed of L-amino acids are rapidly degraded to their
component amino acids within a few minutes under these conditions.
A second advantage lies in the constrained single conformations
adopted by the cyclic and non-peptidic molecules, whereas acyclic
or linear peptides are flexible enough to adopt several structures
in solution other than the required receptor-binding structure.
Thirdly, cyclic and non-peptidic compounds such as those described
in this invention are usually more lipid-soluble and more
pharmacologically bioavailable as drugs than peptides, which can
rarely be administered orally. Fourthly, the plasma half-lives of
cyclic and non-peptidic molecules are usually longer than those of
peptides.
[0142] It will be apparent to the person skilled in the art that
while the invention has been described in some detail for the
purposes of clarity and understanding, various modifications and
alterations to the embodiments and methods described herein may be
made without departing from the scope of the inventive concept
disclosed in this specification.
[0143] References cited herein are listed on the following pages,
and are incorporated herein by this reference.
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Sequence CWU 1
1
24110PRTArtificial Sequencesynthetic peptide 1Ile Ser His Lys Asp
Met Gln Leu Gly Arg1 5 10210PRTArtificial Sequencesynthetic peptide
2Tyr Ser Phe Lys Asp Met Gln Leu Gly Arg1 5 10310PRTArtificial
Sequencesynthetic peptide 3Tyr Ser Phe Lys Asp Met Pro Leu Xaa Arg1
5 10410PRTArtificial Sequencesynthetic peptide 4Tyr Ser Phe Lys Pro
Met Pro Leu Xaa Arg1 5 10521PRTArtificial Sequencesynthetic peptide
5Arg Ala Ala Arg Ile Ser Leu Gly Pro Arg Xaa Tyr Ser Phe Lys Pro1 5
10 15Met Pro Leu Xaa Arg 20620PRTArtificial Sequencesynthetic
peptide 6Lys Tyr Lys His Ser Val Val Lys Lys Xaa Tyr Ser Phe Lys
Pro Met1 5 10 15Pro Leu Xaa Arg 2076PRTArtificial Sequencesynthetic
peptide 7Phe Lys Pro Xaa Trp Arg1 586PRTArtificial
Sequencesynthetic peptide 8Phe Lys Pro Xaa Trp Arg1
596PRTArtificial Sequencesynthetic peptide 9Phe Lys Pro Xaa Trp
Arg1 5106PRTArtificial Sequencesynthetic peptide 10Phe Lys Pro Leu
Trp Arg1 5116PRTArtificial Sequencesynthetic peptide 11Phe Lys Pro
Xaa Trp Arg1 5126PRTArtificial Sequencesynthetic peptide 12Phe Xaa
Pro Xaa Trp Arg1 5135PRTArtificial Sequencesynthetic peptide 13Phe
Pro Xaa Trp Arg1 5145PRTArtificial Sequencesynthetic peptide 14Phe
Pro Xaa Trp Arg1 5155PRTArtificial Sequencesynthetic peptide 15Phe
Pro Xaa Trp Arg1 5165PRTArtificial Sequencesynthetic peptide 16Phe
Pro Xaa Trp Arg1 5176PRTArtificial Sequencesynthetic peptide 17Phe
Xaa Pro Xaa Trp Arg1 5186PRTArtificial Sequencesynthetic peptide
18Phe Xaa Pro Xaa Trp Arg1 5196PRTArtificial Sequencesynthetic
peptide 19Phe Trp Pro Xaa Trp Arg1 5206PRTArtificial
Sequencesynthetic peptide 20Phe Lys Met Xaa Trp Arg1
5216PRTArtificial Sequencesynthetic peptide 21Phe Lys Lys Xaa Trp
Arg1 5225PRTArtificial Sequencesynthetic peptide 22Phe Pro Xaa Trp
Arg1 5235PRTArtificial Sequencesynthetic peptide 23Phe Pro Xaa Trp
Arg1 5247PRTArtificial Sequencesynthetic peptide 24Lys Phe Xaa Pro
Xaa Trp Arg1 5
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