Classes of compounds that interact with GPCRs

This application is the US national phase of international application PCT/AU2003/001347 filed 10 Oct. 2003 which designated the U.S. and claims priority of AU 2002951995, filed 11 Oct. 2002, the entire contents of each of which are hereby incorporated by reference.

The invention provides classes of biologically active compounds that interact in a pharmaceutically significant manner with G-Protein Coupled Receptors (GPCRs), pharmaceutical compositions containing such compounds and methods of treatment of humans suffering from a disorder which can be at least partially overcome by the compounds or compositions.

The drug discovery landscape has been transformed by the genomics revolution. Advances in the understanding of biomolecular pathways and the roles they play in disease will lead to vast numbers of targets for therapeutic intervention. GPCRs represent the most important collection of therapeutic targets available.

GPCRs are proteins that tranduce signals across a cell membrane. They consist of a single polypeptide chain that threads back and forth seven times across the phospholipid bilayer that forms the cell membrane. The polypeptide chain has a portion inside the cell which form a G-protein coupling domain, and a receptor portion outside or in the cell wall. A signal molecule interacts with the receptor which sends the signal through the membrane wall and the signal causes the G-protein coupling domain to interact with a G protein.

Over 50% of marketed drugs target GPCRs. Whilst the druggable extent of GPCRs numbers some 450 receptors only some 200 GPCRS have been matched with their ligands. Orphan receptors suitable for drug targeting may therefore number in excess of 200 receptors. These are receptors with less than approximately 45% sequence identity to known GPCRs for which ligands have not been identified.

The targets of current GPCR drugs include, pain and inflammation, cancer, metabolic and gastrointestinal, cardiovascular and central nervous system disorders.

There is a continuing demand for new therapeutics, especially as our understanding of biological processes expands from the genomics revolution. The aforementioned GPCRs are suitable targets for therapeutic intervention due to their roles in such disorders as cancers, obesity and erectile dysfunction.

Considering the rate of generation and nature of the targets currently being deconvoluted by biologists, there is a need for the development of drug candidates, designed in a rational manner to purposely interact with selected targets, such as the GPCRs.

From a drug discovery perspective, carbohydrate pyranose and furanose rings and their derivatives are well suited as templates. Each sugar represents a three-dimensional scaffold to which a variety of substituents can be attached, usually via a scaffold hydroxyl group, although occasionally a scaffold carboxyl or amino group may be present for substitution. By varying the substituents, their relative position on the sugar scaffold, and the type of sugar to which the substituents are coupled, numerous highly diverse structures are obtainable.

An important feature to note with carbohydrates, is that molecular diversity is achieved not only in the type of substituents, but also in the three dimensional presentation. The different stereoisomers of carbohydrates that occur naturally, offer the inherent structural advantage of providing alternative presentation of substituents.

Employing a related methodology, Hirschmann et al (Hirschmann, R., et. al., J. Am. Chem. Soc., 1992, 114, 9217-9218, U.S. Pat. No. 5,552,534, WO 97/28172, WO 95/11686) synthesised several compounds designed as somatostatin analogues and integrin binders. The methodology employed by Hirschmann relied on protracted, linear, non-combinatorial syntheses, employed exclusively non-aminated pyranoses, and did not exploit any epimerisation chemistry to allow greater access to structural diversity. Consequently, these compounds and methods are manifestly distinct from this present invention.

We have developed a system that allows the chemical synthesis of highly structurally and functionally diverse derivatised carbohydrate and tetrahydropyran structures, of both natural and unnatural origin. The diversity accessible is particularly augmented by the juxtaposition of both structural and functional aspects of the molecules.

Using the axioms of this drug discovery methodology, we synthesised several novel classes of chemotypes in an effort to develop drug candidates against GPCR targets.

It is a general object of the invention to provide compounds that interact with GPCRs in a biologically significant manner,

It is an optional object of the invention to provide a pharmaceutical formulation comprising at least one compound as described herein or a pharmaceutically acceptable salt thereof, together with one or more pharmaceutically acceptable carriers, diluents or excipients.

In one aspect the invention provides for compounds of general formula I, that interact with GPCRs in a biologically significant manner,

Wherein the ring may be of any configuration;

Z is sulphur, oxygen, CH 2 , C(O), C(O)HNR A , NH, NR A or hydrogen, in the case where Z is hydrogen then R 1 is not present, R A is selected from the set defined for R 1 to R 5 , X is oxygen or nitrogen providing that at least one X of General Formula I is nitrogen, X may also combine independently with one of R 1 to R 5 to form an azide, R 1 to R 5 are independently selected from the following definition which includes but is not limited to H or an alkyl, acyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, arylalkyl or heteroarylalkyl substituent of 1 to 20 atoms, which is optionally substituted, and can be branched or linear. Typical substituents include but are not limited to OH, NO, NO 2 , NH 2 , N 3 , halogen, CF 3 , CHF 2 , CH 2 F, nitrile, alkoxy, aryloxy, amidine, guanidiniums, carboxylic acid, carboxylic acid ester, carboxylic acid amide, aryl, cycloalkyl, heteroalkyl, heteroaryl, aminoalkyl, aminodialkyl, aminotrialkyl, aminoacyl, carbonyl, substituted or unsubstituted imine, sulfate, sulfonamide, phosphate, phosphoramide, hydrazide, hydroxamate, hydroxamic acid, heteroaryloxy, aminoaryl, aminoheteroaryl, thioalkyl, thioaryl or thioheteroaryl, which may optionally be further substituted, and R 6 and R 7 are hydrogen, or may combine to form a carbonyl function.

In one embodiment the invention provides for compounds of general formula II that interact with GPCRs in a biologically significant manner,

Wherein R 1 , R 2 , R 3 , R 5 , Z and X are defined as in General Formula I.

In a second embodiment the invention provides for compounds of general formula III that interact with GPCRs in a biologically significant manner,

Wherein A is defined as hydrogen, SR 1 , or OR 1 where R 1 is defined as in General Formula I, and

X and R 2 to R 5 are defined as in General Formula I.

In a preferred embodiment the invention provides for compounds of General Formula IV that interact with GPCRs in a biologically significant manner,

Wherein R 1 -R 3 are defined as in General Formula I.

In a second preferred embodiment the invention provides for compounds of General Formula V that interact with GPCRs in a biologically significant manner,

Where in R 1 , R 2 and R 5 are defined as in General Formula I.

In a third preferred embodiment the invention provides for compounds of General Formula VI that interact with GPCRs in a biologically significant manner,

Wherein R A is H or combines with R 2 to form an azide, and

R 3 , R 3 and R 5 are defined as in General Formula I.

In a fourth preferred embodiment the invention provides for compounds General Formula VII that interact with GPCRs in a biologically significant manner of,

Wherein, R 2 , R 3 and R 5 are defined as in General Formula I.

In a fifth preferred embodiment the invention provides for compounds of General Formula VIII that interact with GPCRs in a biologically significant manner,

Wherein R 1 to R 3 are defined as in General Formula I.

In a sixth preferred embodiment the invention provides for compounds of General Formula IX that interact with GPCRs in a biologically significant manner,

Wherein R 2 and R 5 are defined as in General Formula I.

In a seventh preferred embodiment the invention provides for compounds of General Formula X that interact with GPCRs in a biologically significant manner,

Wherein R 2 and R 5 are defined as in General Formula I.

In an eighth preferred embodiment the invention provides for compounds of General Formula XI that interact with GPCRs in a biologically significant manner,

Wherein R 2 and R 3 are defined as in General Formula I.

In a ninth preferred embodiment the invention provides for compounds of General Formula XII that interact with GPCRs in a biologically significant manner,

Wherein R 2 and R 3 are defined as in General Formula I.

The compounds of the invention may be mixed with a pharmaceutical acceptable carrier, adjuvant, or vehicle which may comprise a-toxic carrier, adjuvant, or vehicle that may be administered to a patient, together with a compound of this invention, and which does not destroy the pharmacological activity thereof.

The pharmaceutical derivative may comprise a salt, ester, salt of an ester or other derivative of a compound of this invention which, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound of this invention, although no limitation is meant thereby.

Compounds of the invention may be administered orally such as by means of a tabled, powder, liquid, emulsion, dispersion and the like; by inhalation; topically such as by means of a cream, ointment, salve etc; and as a suppository, although no limitation is meant thereby.

Substituents per Example Libraries 1-14

Assay Conditions

GPCR Radioligand Binding (RLB) Assays

Recombinant human receptors expressed in HEK 293 cells were used for all experiments. Receptor membrane preparations were purchased from Perkin Elmer BioSignal. The labelled ligand used in somatostatin GPCR RLB assays was [ 125 I]SST-14 and in melanocortin assays was [ 125 I]NDP-αMSH. All assays were done in a 96-well plate format using either glass fiber filter mats or filter plates. All reagents purchased were of the highest quality.

Specific assay buffer, incubation and washing conditions were optimized for each receptor however they all followed the same general format. The procedures for both filter mat and filter plate formats are based on the receptor manufacturers recommendations or those described extensively in the literature. The procedures are briefly outlined below.

In assays where filter mats are used we incubate receptor membranes, assay buffer and [ 125 I] labelled ligand in 96 well microplates. Add compounds to incubation mixture and continue incubation for optimized period. Presoak Filter mat GF/B in 0.5% PEI for ˜2 hr at 4° C. On completion of assay mixture incubation add additional 100 μL/well of assay buffer immediately prior to filtration. Filter the assay mixture onto the GF/B filter mat using a cell harvester. Dry the filter mats prior to sealing them into a scintillation counting bag with scintillant. Radioactivity in each well is detected by liquid scintillation counting.

In assays where filter plates are used Multiscreen glass fiber filter plates (Millipore, Cat No MAFCNOB10) are precoated with 0.5% PEI for ˜2 hr at 4° C. All wells are then washed with 200 □l/well assay buffer and filtered using the Multiscreen Separation System. Subsequently receptor membranes, assay buffer and labelled ligands are added to the wells and equilibrated. Compounds for testing are then added to the mixture and incubation is continued for an optimized time. Plates are then put into the Multiscreen Separation System and the assay mixture is filtered through the plate under vacuum. Each well is then washed several times with assay buffer. Plates are then dried prior to putting sealing tape onto the bottom of the plate. Scintillant is added to each well and radioactivity measured by liquid scintillation counting.

Comparison of Assay Conditions for 2 Different Assays

MC4 SST5 Volume □L Receptor membranes 20 (1:40 40 (1:40 dilution dilution of of stock) stock) labelled ligand (~80000 cpm) 10 40 unlabelled ligand — — mQH 2 O — — Compounds 10 20 assay buffer 10 100  Total volume (□L) 50 200

Data Analysis

Raw data was analysed according to standard methods using either GraphPad Prism software or IDDBS ActivityBase software.

Key for Assay Results Libraries 1-14

“+” Indicates inhibition greater than . . . 50%

“−” Indicates inhibition less than . . . 50%

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It should be appreciated that various other changes and modifications can be made to any embodiment described without departing from the spirit and scope of the invention.