The invention relates to the generation of a library of compounds enriched in agonist and antagonists for members of the G-protein coupled class of receptors (GPCRs).
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 temporally to an activated state (this conversion is usually designated R + L -> R*L where R is the inactive receptor, R* is the activated receptor and L is the ligand).
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 Ga subunit, and a Py dimer. Both the Ga and Py subunits can then participate in further signalling cascades. For example, the Ga subunit can activate the adenylate cyclase (AC) enzyme, which generates cyclic adenosine monophospate (cAMP) from adenosine triphosphate. The Py 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.
The signal, once activated, is then slowly turned off by a number of mechanisms. The GTP associated with the Ga subunit is hydrolysed back to GDP, resulting in the reassociation of the Ga and Py 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.
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 chokrae (the organism responsible for cholera), for example, makes a protein known as cholera toxin which irreversibly inhibits the Got subunit of a widely distributed G-protein called Gs. 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, Gj.
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 radionucleide into proximity with a scintillant molecule bound to the receptor - as the nucleide 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 degress of freedom of the fluorescent tag when the ligand is immobilised on binding to the receptor).
While these techniques have been successful in some instances, and yielded lead compounds which have subsequently been developed as human pharmaceuticals (for example, the 5HT3 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.
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 highthroughput 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.
Another major problem with such "negative screening" paradigms (where you detect the abilioty 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.
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.
Whether or nor real libraries can be constructed which approximate these ideal properties is not at all clear. If they do, it will require the existence 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.
Here we describe an "ideal" GPCR substrate which can be used as-a three-dimensional skeleton that can be variously substituted to generate agonists and/or anatagonists at a range of different GPCRs. The invention also provides for the preparation of a library of said substituted compounds to be applied 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 5HTla (receptors). In contrast, identifying such mixed ligands serendipitously from random libraries is a very rare event.

Claims
1. A compound of general formula (I)
(Formula Removed)
wherein
y is any integer from 1 to 8;
z is any integer from 0 to 8 with the proviso that y and z cannot simultaneously be 1:
X is -CO-(Y)K-(R:)r. or SO2 C(Y)k-(R1),:
k is 5 or 1
Y is a cycloalkyl or polycyloalkvl group (such as an adamantyl, adamantanemethyl. bicyclooctyl, cyclohexyl, cyclopropyl group):
or Y is a cycloalkenyl or polycycloalkenyl group;
each R: is independently selected Srom hydrogen or an alkyl. haloalkyl, alkoxy. haloalkoxy. alkenyl, alkynyl, alkylarr-ino, alkylaminoalkyl, alkylarninodialkyl, charged alkylamiriotrialkyl or charged alkyicarboxyiate radical of 1 to 20 carbon atoms:
or each R: is independently selected rom f.uoro. 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 R2 may be selected from a peptido radical, for example having from 1to 4 peptidic moieties linked together by peptide bonds (for example a peptido radical of 1 to 4 amino acid residues).
2. A pharmaceutical composition comprising, as active ingredient, a compound of general formula (I), or a pharmaceuticaily acceptable salt thereof, and at least one pharmaceutically acceptable excipient and/or carrier:
(Formula Removed)
wherein
is any integer from 1 to 8;
z. :s ai:\ imeger from 0 to 8 with the proviso that y and z cannot simultaneously be 1;
X :s -CO-(Y)k-(R1)4 or SO2-(Y)k-(R1)2;
k is 0 or 1
Y is a cycloalkyl or polycyloalkyl group (such as an adamantyl, adamantanemethyl. bicyclooctyl, cyclohexyl, cyciopropyl group);
or Y is a cycloalkenyl or polycycloalkenyl group;
each R is independently selected from hydrogen or an alkyl. haloalkyl. alkoxy. haloalkoxy. alkenyl, alkynyl, alkylamino. alkylaminoalkyl, alkylaminocialkyl. charged aiky'aminotrialkyl or charged alkylcarboxylaie radical of 1 to 20 carbon atoms:
or each R2 is independently selected from fluoro, chloro, bromo, iodo, hydroxy, oxyalkyl. amino, aminoalkyl, aminodialkyl. charged aminotrialkyl, or carboxylate radical: and

n is any in:eger from 1 to m, where m is the maximum number of substitutions permissible on the cyclo-group Y: or
alternatively R4 may be selected from a peptido radical, for example having from 1 to 4 peptidie moieties linked together by peptide bonds (for example a peptido radical of 1 to 4 amino acid residues).
3. The use of a compound of general formula (I), or a phamaceutically 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:
(Formula Removed)
wherein
2 is an integer from 1 :o 8;
z is any integer iron 0 to 8 with the proviso that y and z cannot simultaneously be 1
X is - CO-(YX-(R1')2 or S02-(Y)k-(R:)2;
k is 0 or 1
Y is a cycloalkyl or polycyloalkyl group (such as an adamantyl, adamantanemethyl. hicyclooctyl. cyclohexyl, cyclopropyl group):
or Y is a cycloalkenyl or poiycycloalkenyl group;
each R' is independently selected from hydrogen or an alkyl, haloalkyl. alkoxy. haloalkoxy. alkenyl, alkynyl, alkylamino, alkylaminoalkyl, aikylarninodialkyl. charged alkylarninoirialkyl or charged alkylcarboxylate radical of 1 to 20 carbon atoms;

or each R' is independently selected from fiuoro, chloro, bromo, iodo. hydroxy, oxyalkyl. amino, aminoalkyl. aminodialkyl. charged aminotrialkyl, or carboxylate radical; and
r. is any integer from 1 to m, where m is the maximum number of substitutions permissible on the cyclo-group Y; or
alternatively R- may be selected from a peptido radical, for example having from 1 to 4 pepricic moieties linked together by peptide bonds (for example a peptido radical of 1 to - amino acid residues).
4. Compounds, compositions and uses according to any preceding claim wherein the R: radical has a "key" carbon which is di-substituted with the same or different groups -elected fro—: aikyl, haloalkyl, alkoxy. haloalkoxy, alkenyl, aUcynl and alkylammc r..cica. s.
5. Compounds, compositions and uses according to claim 4 wherein the "key" carbon is chirai.
6. Compounds, compositions and uses according to claim 4 wherein the "key*' carbon h:is sp5 hybridised bonds.
7. Compounds, compositions and uses according to claim 4 wherein the "key" carbon has essentially tetrahedral bond angles.
8. Compounds, compositions and uses of the compounds of general formula (I), or their pharmaceutically acceptable salts, according to any preceding claim, wherein the ring or rings of Y constrain the bond angles at the "key" carbon to be essentially tetrahedral (i.e. sp3 hybrid bonds).
9. A compound according to claim 1. or a pharmaceutical composition according to
claim 2, or a use according to claim 3. wherein general formula (I) is modified such that
the C3-C7 alky! bridge -(CH2)y- is replaced by a bridging group independently selectable
from the group consisting of alkenyl, haloalkyl, alkylarnino. alkylaminoalkyl.
alklarninodialkyl, charged alkylaminotrialkyl, charged alkylcarboxylate and
aIkylhydroxy moieties having a carbon chain length of from 1 to S.

10. A compound, composition or use according to any preceding claim wherein y and z are the same integer, whereby the a-aminobicyclolactam ring is non-chiral.
11. A compound, composition or use according to any of claims 1 to 9 wherein y and z arc not the same integer, whereby the a-aminobicyclolactam ring is chiral.
12. A compound, composition or use according to claim 11 wherein z is 3 and y is 1 or 2 or 4-8. whereby the compound contains a lactam ring which is seven membered.
13. A compound, composition or use according to claim 11 wherein z is 2 and y is 1 or 3-i>. whereby the compound contains a lactam ring which is 6 membered.

14. Use of a compound of formula (I) according to claim 3 or 9 wherein the GPCR to be modulated is selected from the group consisting of adrenalin receptors, endothelir. 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, XPY receptors. MSK receptors, glycoprotein hormone receptors, protease activated receptors (PARs), somatcstain receptors, angiotensin receptors, cnolecystokinin receptors or melatonin receptors.
15. 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 designed to modulate GPCR activity as claimed in any of claims 1 to 13.
16. A library composed of, or enriched in, library elements which are compounds according to any of claims 1 to 13.
17. A method which involves the use of a library according to claim 16 in an assay for the purpose o: screening to identify agent(s) which modulate signalling through GPCRs.
18. A method according to claim 17, where the agent(s) identified are antagonists at one or more GPCRs
19. A method according to claim 17 where the agent(s) identified are agonists at one or more GPCRs

20. A method according to claim 17 where the GPCR is selected from ±e group consisting of adrenalin receptors, endothelir. 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 honnone receptors, protease activated receptors (PARs). somatostain receptors, angiotensin receptors, cholecystokinin receptors or melatonin receptors.