1
Selective Inhibitors
Field of the invention.
5 The invention relates to a method of identifying compounds with selective
biologically activities, and libraries of compounds.
Background.
Small molecules involved in molecular interactions with a biological target, be it
10 enzyme or receptor, are often described in terms of binding elements or
pharmacophore groups which directly interact with the target, and non-binding
components which form the framework of the bioactive molecule. In the case of
peptide ligands or substrates for instance, a number of amino acid side chains
usually form direct interactions with their receptor or enzyme, whereas specific
15 folds of the peptide backbone (and other amino acid residues) provide the structure
or scaffold that controls the relative positioning of these side chains. In a
peptidomimetic approach to drug discovery, the side chains of important amino
acids may be systematically modulated to identify better binding interactions. This
is referred to as a scanning approach. Unfortunately, the side chains of peptides
20 are rarely independent, such that each interaction cannot be optimised without
consideration of the others.
One way to overcome this problem is to construct diversity libraries.
25 So far, approaches for creating universal diversity have largely focused on the
combination of substituents aspects. When it comes to creating diversity in

2
presentation of these substituents, pharmaceutical companies generally turn to the
known heterocyclic scaffolds, with an emphasis on the so-called 'privileged
structures'. Creating structural diversity in libraries has been highly desired but has
been limited by the lack of structural diversity in the chemically useful scaffolds.
5
Monosaccharides provide an excellent sugar scaffold to design molecular diversity
by appending desired substituents at selected positions around the sugar scaffold.
The monosaccharide-based scaffold contains five chiral, functionalized positions,
enabling attachment of various substituents at each position. This provides a
10 unique opportunity to create libraries of structurally diverse molecules, by varying
the pharmacophoric groups, the scaffold and the positions of attachment of the
pharmacophoric groups in a systematic manner. A pharmacophoric group in the
context of these libraries is an appended group or substituent, or part thereof,
which imparts pharmacological activity to the molecule.
15
Molecular diversity could be considered as consisting of diversity in
pharmacophoric group combinations (diversity in substituents) and diversity in the
way these pharmacophoric groups are presented (diversity in shape). Libraries of
compounds in which either diversity of substituents, or diversity of shape, or both of
20 these parameters are varied systematically are said to scan molecular diversity.
There is a need for methods to improve the development of drug candidates that
purposely interact with selected targets, and not with other targets, in order to
minimize side effects. Selectivity profiles are determined by biological assays,
25 either in vitro or in vivo, in which compounds exhibit a specific response in each

3
assay. The panel of specific responses represents the selectivity profile across the
selected assays. The profile distinguishes actives against non-actives in each
assay. Methods to improve the identification of selectivity profiles overcome or at
least partially ameliorate this problem.
5
In previous applications (WO2004014929 and WO2003082846) we demonstrated
that arrays of novel compounds could be synthesized in a combinatorial manner.
The libraries of molecules described in these inventions were synthesized in a
manner such that the position, orientation and chemical characteristics of
10 pharmacophoric groups around a range of chemical scaffolds, could be modified
and/or controlled.
In a later application (WO2004032940), we demonstrated that classes of
molecules from the above cited applications exhibited biological activity when
15 screened against melanocortin and somatostatin GPCRs. Classes of molecules
from the applications WO2004014929 and WO2003082846 were also tested
against integrin receptors (Australian patent Application No. 2003900242).
Selections of these molecules were also demonstrated to display activity against
this class of receptors.
20
We have now found that libraries of molecules described in the applications
WO2004014929 and WO2003082846 can be used to scan molecular diversity.
This diversity approach provides an improved method, for effectively identifying
selectivity profiles.
25
It will be clearly understood that, if a prior art publication is referred to herein, this
reference does not constitute an admission that the publication forms part of the
common general knowledge in the art in Australia or in any other country.
30

4
Summary of the invention.
In one aspect the invention provides a method of identifying biologically active
5 compounds with defined selectivity profile(s) comprising:
(a) designing a library of compounds of formula 1 to scan molecular diversity;
and
(b) assaying the library of compounds in at least two different biological assays;
wherein formula 1 represents:

Formula I
wherein the ring may be of any configuration;
Z is sulphur, oxygen, CH2, C(O), C(O)NRA, NH, NRA or hydrogen, in the case
15 where Z is hydrogen then R1 is not present, RA is selected from the set defined for
R1 to R5, or wherein Z and R1 together form a heterocycle,
X is oxygen or nitrogen, when X is nitrogen, each X may combine independently
with the corresponding R2 to R5 to form an azide, or wherein each X may also
combine independently with any one of corresponding R2-R5 to form a heterocycle;
20 R1 to R5 are independently selected from the group which includes but is not
limited to H or an C1 to C20 alkyl or acyl; C2 to C20 alkenyl, alkynyl, heteroalkyl;
C5 to C20 aryl, heteroaryl, arylalkyl or heteroarylalkyl, which is optionally
substituted, and can be branched or linear.
25 In a preferred embodiment the invention relates to a library of compounds selected
from compounds of formula 1 when used according to first said method.
In a preferred embodiment, the invention relates to first said method wherein at
least one X is nitrogen.
30

5
In a preferred embodiment, the invention relates to first said method wherein two of
X is nitrogen.
In a preferred embodiment, the invention relates to first said method wherein X and
5 R2 combine to form heterocycle.
In a preferred embodiment, the invention relates to first said method wherein R1-
R5 optional substituents are selected from OH, NO, NO2, NH2, N3, halogen, CF3,
CHF2, CH2F, nitrile, alkoxy, aryloxy, amidine, guanidiniums, carboxylic acid,
10 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
15 further substituted.
The term "halogen" denotes fluorine, chlorine, bromine or iodine, preferably
fluorine, chlorine or bromine.
20 The term "alkyl" used either alone or in compound words such as"optionally
substituted alkyl","optionally substitutedcycloalkyl,"arylalkyl'or"heteroarylalkyl",
denotes straight chain, branched or cyclic alkyl, preferably C1-20 alkyl or
cycloalkyl. Examples of straight chain and branched alkyl include methyl, ethyl,
propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, amyl, isoamyl, sec-amyl, 1,2-
25 dimethylpropyl,1, 1-dimethylpropyl, hexyl, 4-methylpentyl, 1-methylpentyl, 2-
methylpentyl, 3methylpentyl,1, 1-dimethylbutyl, 2,2-dimethylbutyl,
3,3dimethylbutyl,1, 2-dimethylbutyl, 1,3-dimethylbutyl, 1,2,2trimethylpropyl,1, 1,2-
trimethylpropyl, heptyl, 5methylbexyl, 1-methylhexyl, 2,2-dimethypentyl, 3,3

6
dimethylpentyl, 4,4-dimethylpentyl, 1,2-dimethylpentyl, 1,3-dimethylpentyl, 1,4-
dimethylpentyl, 1,2,3trimethylbutyl,1, 1,2-trimethylbutyl, 1,1,3-trimethylbutyl, octyl,
6-methylheptyl, 1-methylheptyl, 1,1,3,3 tetramethylbutyl, nonyl,1-, 2-, 3-, 4-, 5-, 6-or
7-methyloctyl,1-, 2-, 3-, 4-or 5-ethylheptyl, 1-, 2-or 3propylhexyl, decyl,1-, 2-, 3-, 4-,
5 5-, 6-, 7-or 8methylnonyl,1-, 2-, 3-, 4-, 5-or 6-ethyloctyl, 1-, 2-, 3or 4-propylheptyl,
undecyl1-, 2-, 3-, 4-, 5-, 6-, 7-, 8or 9-methyldecyl,1-, 2-, 3-, 4-, 5-, 6-or 7-
ethylnonyl,1-, 2-, 3-, 4-or 5-propyloctyl,1-, 2-or 3-butylheptyl,1-pentylhexyl,
dodecyl,1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9or 10-methylundecyl,1-, 2-, 3-, 4-, 5-, 6-, 7-or
8ethyldecyl,1-, 2-, 3-, 4-, 5-or 6-propylnonyl, 1-, 2-, 3or 4-butyloctyl, 1-2
10 pentylheptyl and the like. Examples of cyclic alkyl include mono-or polycyclic alkyl
groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,
cyclooctyl, cyclononyl, cyclodecyl and the like.
The term "alkylene" used either alone or in compound words such as "optionally
15 substituted alkylene" denotes the same groups as "alkyl" defined above except that
an additional hydrogen has been removed to form a divalent radical. It will be
understood that the optional substituent may be attached to or form part of the
alkylene chain.
20 The term "alkenyl" used either alone or in compound words such as "optionally
substituted alkenyl" denotes groups formed from straight chain, branched or cyclic
alkenes including ethylenically mono-, di-or polyunsaturated alkyl or cycloalkyl
groups as defined above, preferably C2-6 alkenyl. Examples of alkenyl include
vinyl, allyl,1-methylvinyl, butenyl, iso-butenyl, 3-met.hyl-2but.enyl, 1-pentenyl,

7
cyclopentenyl, 1-methyl-cyclopentenyl, 1-hexenyl, 3-hexenyl, cyclohexenyl,1-
heptenyl, 3-heptenyl, 1-octenyl, cyclooctenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl,1-
decenyl, 3-decenyl, 1,3-butadienyl, 1,4-pentadienyl, 1,3 cyclopentadienyl, 1,3-
hexadienyl, 1,4-hexadienyl, 1,3cyclohexadienyl, 1,4-cyclohexadienyl, 1,3-
5 cycloheptadienyl, 1,3,5-cycloheptatrienyl and 1,3,5,7-cyclooctatetraenyl.
The term "alkynyl" used either alone or in compound words, such as "optionally
substituted alkynyl" denotes groups formed from straight chain, branched, or
mono-or poly-or cyclic alkynes, preferably C2-6 alkynyl.
10
Examples of alkynyl include ethynyl,1-propynyl, 1-and 2butynyl, 2-methyl-2-
propynyl, 2-pentynyl, 3-pentynyl, 4pentynyl, 2-hexynyl, 3-hexylnyl, 4-hexynyl, 5-
hexynyl, 10undecynyl,4-ethyl-l-octyn-3-yl, 7-dodecynyl, 9-dodecynyl, 10-
dodecynyl,3-methyl-1-dodecyn-3-yl, 2-tridecynyl, 11-tridecynyl, 3-tetradecynyl, 7-
15 hexadecynyl, 3-octadecynyl and the like.
The term "alkoxy" used either alone or in compound words such as "optionally
substituted alkoxy" denotes straight chain or branched alkoxy, preferably C 1-7
alkoxy. Examples of alkoxy include methoxy, ethoxy, npropyloxy, isopropyloxy and
20 the different butoxy isomers.
The term "aryloxy" used either alone or in compound words such as "optionally
substituted aryloxy" denotes aromatic, heteroaromatic, arylalkoxy or heteroaryl

8
alkoxy, preferably C6-13 aryloxy. Examples of aryloxy include phenoxy,
benzyloxy, 1 -napthyloxy, and 2-napthyloxy.
The term "acyl" used either alone or in compound words such as "optionally
5 substituted acyl "or" heteroarylacyl" denotes carbamoyl, aliphatic acyl group and
acyl group containing an aromatic ring, which is referred to as aromatic acyl or a
heterocyclic ring which is referred to as heterocyclic acyl. Examples of acyl include
carbamoyl; straight chain or branched alkanoyl such as formyl, acetyl, propanoyl,
butanoyl, 2-methylpropanoyl, pentanoyl, 2,2-dimethylpropanoyl, hexanoyl,
10 heptanoyl, octanoyl, nonanoyl, decanoyl, undecanoyl, dodecanoyl, tridecanoyl,
tetradecanoyl, pentadecanoyl, hexadecanoyl, heptadecanoyl, octadecanoyl,
nonadecanoyl, and icosanoyl; alkoxycarbonyl such as methoxycarbonyl,
ethoxycarbonyl, t butoxycarbonyl, t-pentyloxycarbonyl and heptyloxycarbonyl;
cycloalkylcarbonyl such as cyclopropylcarbonyl cyclobutylcarbonyl,
15 cyclopentylcarbonyl and cyclohexylcarbonyl; alkylsulfonyl such as methylsulfonyl
and ethylsulfonyl; alkoxysulfonyl such as methoxysulfonyl and ethoxysulfonyl; aroyl
such as benzoyl, toluoyl and naphthoyl; aralkanoyl such as phenylalkanoyl (e. g.
phenylacetyl, phenylpropanoyl, phenylbutanoyl, phenylisobutyl, phenylpentanoyl
and phenylhexanoyl) and naphthylalkanoyl (e. g. naphthylacetyl, naphthlpropanoyl
20 and naphthylbutanoyl); aralkenoyl such as phenylalkenoyl (e. g. phenylpropenoyl,
phenylbutenoyl, phenylmethacrylyl, phenylpentenoyl and phenylhexenoyl and
naphthylalkenoyl (e. g. naphthylpropenoyl, naphthylbutenoyl and
naphthylpentenoyl); aralkoxycarbonyl such as phenylalkoxycarbonyl (e. g.
benzyloxycarbonyl); aryloxycarbonyl such as phenoxycarbonyl and

9
naphthyloxycarbonyl; aryloxyalkanoyl such as phenoxyacetyl and
phenoxypropionyl; arylcarbamoyl such as phenylcarbamoyl; arylthiocarbamoyl
such as phenylthiocarbamoyl; arylglyoxyloyl such as phenylglyoxyloyl and
naphthylglyoxyloyl; arylsulfonyl such as phenylsulfonyl and naphthylsulfonyl;
5 heterocycliccarbonyl; heterocyclicalkanoyl such as thienylacetyl, thienylpropanoyl,
thienylbutanoyl, thienylpentanoyl, thienylhexanoyl, thiazolylacetyl, thiadiazolylacetyl
and tetrazolylacetyl; heterocyclicalkenoyl such as heterocyclicpropenoyl,
heterocyclicbutenoyl, heterocyclicpentenoyl and heterocyclichexenoyl; and
heterocyclicglyoxyloyl such as thiazolylglyoxyloyl and thienyglyoxyloyl.
10
The term "aryl" used either alone or in compound words such as "optionally
substituted aryl", "arylalkyl "or "heteroaryl" denotes single, polynuclear, conjugated
and fused residues of aromatic hydrocarbons or aromatic heterocyclic ring
systems. Examples of aryl include phenyl, biphenyl, terphenyl, quaterphenyl,
15 phenoxyphenyl, naphthyl, tetrahydronaphthyl, anthracenyl, dihydroanthracenyl,
benzanthracenyl, dibenzanthracenyl, phenanthrenyl, fluorenyl, pyrenyl, indenyl,
azulenyl, chrysenyl, pyridyl, 4-phenylpyridyl, 3-phenylpyridyl, thienyl, furyl, pyrryl,
pyrrolyl, furanyl, imadazolyl, pyrrolydinyl, pyridinyl, piperidinyl, indolyl, pyridazinyl,
pyrazolyl, pyrazinyl, thiazolyl, pyrimidinyl, quinolinyl, isoquinolinyl, benzofuranyl,
20 benzothienyl, purinyl, quinazolinyl, phenazinyl, acridinyl, benzoxazolyl,
benzothiazolyl and the like. Preferably, the aromatic heterocyclic ring system
contains 1 to 4 heteroatoms independently selected from N, O and S and
containing up to 9 carbon atoms in the ring.

10
The term "heterocycle" used either alone or in compound words as "optionally
substituted heterocycle" denotes monocyclic or polycyclic heterocyclyl groups
containing at least one heteroatom atom selected from nitrogen, sulphur and
oxygen. Suitable heterocyclyl groups include N-containing heterocyclic groups,
5 such as, unsaturated 3 to 6 membered heteromonocyclic groups containing 1 to 4
nitrogen atoms, for example, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl,
pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl or tetrazolyl; saturated to 3 to 6-
membered heteromonocyclic groups containing 1 to 4 nitrogen atoms, such as,
pyrrolidinyljmidazolidinyl, piperidin or piperazinyl; unsaturated condensed
10 heterocyclic groups containing 1 to 5 nitrogen atoms, such as, indolyl, isoindolyl,
indolizinyl, benzimidazoyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl or
tetrazolopyridazinyl; unsaturated 3 to 6-membered heteromonocyclic group
containing an oxygen atom, such as, pyranyl or furyl; unsaturated 3 to 6-membered
heteromonocyclic group containing 1 to 2 sulphur atoms, such as, thienyl;
15 unsaturated 3 to 6-membered heteromonocyclic group containing 1 to 2 oxygen
atoms and 1 to 3 nitrogen atoms, such as, oxazolyl, isoxazolyl or oxadiazolyl;
saturated 3 to 6-membered heteromonocyclic group containing 1 to 2 oxygen
atoms and 1 to 3 nitrogen atoms, such as, morpholinyl; unsaturated condensed
heterocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, such
20 as, benzoxazolyl or benzoxadiazolyl; unsaturated 3 to 6-membered
heteromonocyclic group containing 1 to 2 sulphur atoms and 1 to 3 nitrogen atoms,
such as, thiazolyl or thiadiazolyl; saturated 3 to 6-membered heteromonocyclic
group containing 1 to 2 sulphur atoms and 1 to 3 nitrogen atoms, such as
thiazolidinyl; and unsaturated condensed heterocyclic group containing 1 to 2

11
sulphur atoms and 1 to 3 nitrogen atoms, such as, benzothiazolyl or
benzothiadiazolyl.
In a preferred embodiment, the invention relates to first said method comprising a
5 library of compounds selected from compounds of formula II,

Formula II
10
wherein R1 R2, R3, R5, Z and X are defined as in Formula I.
In a preferred embodiment the invention relates to a library of compounds selected
from compounds of formula II
15
In a preferred embodiment, the invention relates to first said method comprising a
library of compounds selected from compounds of formula III,

20
Formula III
wherein A is defined as hydrogen, SR1, or OR1 where R1 is defined as in Formula I,
and
25 X and R2 to R5 are defined as in Formula I.

12
In a preferred embodiment the invention relates to a library of compounds selected
from compounds of formula III.
5 In a preferred embodiment, the invention relates to first said method comprising a
library of compounds selected from compounds of formula IV,

10 Formula IV
wherein R1 R2, R3 and R5 are defined as in Formula I.
In a preferred embodiment the invention relates to a library of compounds selected
15 from compounds of f formula IV.
In a preferred embodiment, the invention relates to first said method comprising a
library of compounds selected from compounds of formula V,

Formula V
wherein R1, R2, R3 and R5 are defined as in Formula I.
25

13
In a preferred embodiment the invention relates to a library of compounds selected
from compounds of formula V.
In a preferred embodiment, the invention relates to first said method comprising a
5 library of compounds selected from compounds of formula VI,

Formula VI
10
wherein R1, R2, R3 and R5 are defined as in Formula I.
In a preferred embodiment the invention relates to a library of compounds selected
from compounds of formula VI
15
In a preferred embodiment, the invention relates to first said method comprising a
library of compounds selected from compounds of formula VII,

Formula VI
wherein R1,, R2, R3 and R5 are defined as in Formula I.
25 In a preferred embodiment the invention relates to a library of compounds selected
from compounds of formula VII.

14
In a preferred embodiment, the invention relates to first said method comprising a
library of compounds selected from compounds of formula VIII,

Formula VIII
wherein R1, R2, R3 and R5 are defined as in Formula I.
10
In a preferred embodiment the invention relates to a library of compounds selected
from compounds of formula VIII.
In a preferred embodiment, the invention relates to first said method comprising a
15 library of compounds selected from compounds of formula IX,

Formula IX
20
wherein R2, R3 and R5 are defined as in Formula I.
In a preferred embodiment the invention relates to a library of compounds selected
from compounds of formula IX.
25

15
In a preferred embodiment, the invention relates to said methods wherein
biological assays involve Peptide Ligand class of GPCRs.
In a preferred embodiment, the invention relates to first said method wherein
5 biological assays involve opioid, melanocortin, melanin-concentrating hormone,
neurokinin, neuropeptide and urotensin receptors.
In a preferred embodiment, the invention relates to first said method wherein
biological assays involve 8-opioid (DOP), K-Opioid (KOP), Melanocortin MC3,
10 Melanocortin MC4, Melanocortin MC5, Melanin-Concentrating Hormone (MCH1),
μ-opioid (MOP), Neurokinin (NK1), Neuropeptide Y (NPY-Y1), Opioid (ORL1) and
urotensin (UR2) receptors.
In another aspect the invention provides a compound according to formula 1 in
15 which at least one X is nitrogen, and said X is combined with the corresponding R2-
R5 to form a heterocycle.
In a preferred embodiment, the invention provides a compound according to
formula 1 wherein X and R2 combine to form a heterocycle.
20
In a preferred embodiment, the invention provides a compound according to
formula 1 wherein the heterocycle is heteroaryl, including triazoles,
25 benzimidazoles, benzimidazolone, benzimidazolothione, imidazole, hydantoine,
thiohydantoine and purine
30 Detailed Description of the invention
Embodiments of the invention will be described with reference to the following
examples. Where appropriate, the following abbreviations are used.
Ac Acetyl

16
DTPM 5-Acyl-1,3-dimethylbarbiturate
Ph Phenyl
TBDMS t-Butyldimethylsilyl
TBDPS t-Butyldiphenylsilyl
5 Bn benzyl
Bz benzoyl
Me methyl
DCE 1,2-dichloroethane
DCM dichloromethane, methylene chloride
10 Tf trifluoromethanesulfonyl
Ts 4-methylphenylsulfonyl, p-toluenesulfonyl
DMF N,N-dimethylformamide
DMAP N,N-dimethylaminopyridine
α,α-DMT α,α-dimethoxytoluene, benzaldehyde dimethyl acetal
15 DMSO dimethylsulfoxide
DTT dithiothreitol
DMTST Dimethyl(methylthio)sulphoniumtrifluoro-methanesulphonate
TBAF tetra-n-butylammonium fluoride
20 Selectivity profiles are determined by biological assays, either in vitro or in vivo, in
which compounds exhibit a specific response in each assay. The panel of specific
responses represents the selectivity profile across the selected assays. The
selectivity profile may be determined by testing compounds against (a) a series of
commercially available assays, and/or (b) self-designed assays. The profile
25 distinguishes actives against non-actives in each assay, as indicated in Table 3
below.

17
The designing of libraries is based on methods known in the art, including
designing to scan for molecular diversity using molecular modeling. The libraries
may be designed by using molecular modeling techniques as described by Thanh
Le et al (Drug Discovery Today 8, 701-709 (2003)).
5
Part A: Preparation of building blocks:
In order to fully enable the invention, we detail below methods for the preparation
of certain building blocks used in the preparation of the compounds of the
invention. The building blocks described are suitable for both solution and solid
10 phase synthesis of the compounds of the invention.
Compounds of the library as presented exhibit different selectivity profiles. It is also
apparent from these relationships that new compounds with different selectivity
profiles may be designed.
15 Example A: Synthesis of a 2,4 dinitrogen containing Galactopyranoside Building
Block

Conditions: (i) α,α-dimethoxytoluene (α,α-DMT), p-toluenesulphonic acid (TsOH),
20 acetonitrile (MeCN), 76°C, 85%; (ii) Benzoylchloride (BzCI), triethylamine; DCM,

18
99%; (iii) methanol (MeOH)/MeCN/water, TsOH, 75°C, 98%; (iv) t-
butyldiphenylsilylchloride (TBDPS-CI), imidazole, pyridine, 120°C, 99% ; (v) Tf2O,
pyridine, DCM, 0°C, 100%;(b) NaN3, DMF, 16hr, RT, 99%.
5 Example B: Synthesis of a 3-nitroqen containing Gulopyranoside Building Block

Conditions: (i) (a) trifluoromethanesulfonic anhydride (Tf2O), pyridine, -20°C,
dichloromethane (DCM), 1 hour, 100%, (b) sodium azide (NaN3), N,N-
10 dimethylformamide (DMF), 50°C, 5 hours, quantitative; (ii) TsOH, MeCN/
MeOH/water (12:3:1), 90°C, 6 hours, 88%(iii) TBDPSCI, DMAP, pyridine, 120°C,
12 hours, 93%

19
Example C: Synthesis of a 2.6-dinitroqen substituted Glucopyranoside Building
Block

5 Conditions: (i) (a) Tosylchlodride, pyridine, RT, 24 hours, 33%(b) NaN3, DMF,
RT, 168 hours.
Example D: Synthesis of a 2-nitrogen containing Tallopyranoside Building Block

Conditions: (i) TBDPSCI, imidazole, 1,2-DCE, reflux; (ii) NaOMe/MeOH; (iii) (a)
Tf2O, pyridine, -20°C, DCM, 1 hour, (b) NaN3, DMF, 50°C, 5 hours; (iv) TsOH,
15 MeCN/MeOH/water; (v) benzoylchloride, DMAP, 1,2-DCE, -20°C.

20
Example E: Synthesis of two 3-nitroqen containing Altropyranoside Building Block

5 Conditions: (i) cyclohexanone dimethylacetal, TsOH, MeCN; (ii) p-
methoxybenzaldehyde dimethylacetal, TsOH, MeCN; (iii) DIBAL, -78°C, diethyl
ether; (iv) (a) Tf2O, pyridine, -20°C, DCM, 1 hour, (b) NaN3, DMF, 50°C, 5 hours;
(v) TsOH, MeCN/MeOH/water; (vi) TBDPSCI, DMAP, 1,2-DCE; (vii) (a) CAN, (b)
BzCI, DMAP, 1,2-DCE, (c) TsOH, MeCN/MeOH/water; (viii) TBDPSCI, DMAP, 1,2-
10 DCE.

21
Example F: Synthesis of a 2-nitroqen containing Glucopyranoside Building Block

Conditions: (i) α,α-DMT, TsOH, MeCN; (ii) 1,2-DCE, BzCI, DMAP; (iii) TsOH,
5 MeOH/MeCN; (iv) TBDPS-CI, DMAP, 1,2-DCE.

Conditions: (i) TBDPSCI, DMAP, pyridine, 120°C, 0.5 hours, 81%; (ii) a.
(Bu)2SnO, MeOH; b. Benzoylchloride, RT, 24 hour;

22
Example G: Synthesis of a 2-nitrogen containing Allopyranoside Building Block

Conditions: (i) DCM/pyridine, MsCI, DMAP, O°C; (ii) sodium benzoate,
5 dimethylsulphoxide (DMSO), 140°C; (iii) TsOH, MeOH/MeCN/water; (iv) TBDPS-
Cl, imidazole, DCM, 1 hour, reflux.
Part B: Biological Assays Experimental Method
10 Cloned receptor membrane preparations from Perkin Elmer Biosignal™ were used
in radioligand binding assays.
Membranes (A1 - A11 = Codes for Table 3: Results).
A1 Human 5-opioid (DOP), A2 Human K-Opioid (KOP), A3 Human Melanocortin
15 (MC3), A4 Human Melanocortin (MC4), A5 Human melanocortin (MC5), A6
Human melanin-concentrating hormone (MCH1), A7 Human |j.-opioid (MOP), A8
Human neurokinin (NK1), A9 Human neuropeptide Y (NPY-Y1), A10 Human opioid
(ORL1) A11 Mouse urotensin (mUR2)

23
Materials and Methods
Screening experiments were performed in either a 50 μl filtration or 25 μl
FlashPlate assay format using the following protocol:
5
Table 1: Assay format, radioligands and reference ligands
Assay Final cone. Reference Final
Receptor format Radioligand (nM) ligand cone. (μM)
25 μl
MCH1 FlashPlate [125]]-S36057 0.1 MCH 1
50 μl [125I]-NDP-
MC3 FlashPlate αMSH 0.25 NDP-αMSH 10
25μl [125I]-NDP-
MC4 FlashPlate αMSH 0.25 NDP-αMSH 10
25 μl [125I]-NDP-
MC5 FlashPlate αMSH 0.25 NDP-aMSH 10
50 μl [125l]-Substance
NK1 Filtration P 0.1 L703.606 10
25 μl
NPY-Y1 FlashPlate [125I]-PYY 0.35 BIBP 10
25 μl
ORL1 FlashPlate [125l]-Nociceptin 0.22 Nociceptin 1
25 μl
[μ-opioid FlashPlate [3H]-Naloxone 3 Naltrexone 10
50 μl [3H]-
K-opioid Filtration Diprenorphine 1 nor-BNI 1
25 μl [3H]-
6-opioid FlashPlate Bremazocine 3 Naltrindole 1
25 μl [125l]-Urotensin
UR2 FlashPlate II 0.3 Urotensin II 10

24
Table 2: Assay buffers
Receptor Buffer
MCH1 25 mM HepespH7.0, 10mMMgCI2, 1 mM EDTA and 0.5% BSA
MC3 25 mM Tris-HCI PH 7.4, 1 mM MgCI2, 1.5 mM CaCI2,1mM NaCI and 0.2% BSA
25 mM Tris-HCI pH 7.4, 1 mM MgCI2, 1.5 mM CaCI2 1 mM NaCI and 0.2%
MC4 BSA
25 mM Tris-HCI PH 7.4, 1 mM MgCI2, 1.5 mM CaCI2,1 mM NaCI and 0.2%
MC5 BSA
40 mM Hepes pH 7.4, 5 mM MgCI2,1 mM EDTA, 0.5% BSA, 0.025%
NK1 bacitracin and 25 |μM phosphoramidon
50 mM Tris-HCI pH 7.4, 5 mM KCI, 1 mM MgCI2, 2 mM CaCI2, 120 mM NaCI,
NPY-Y1 0.5% BSA and 50 |μM thiorphan
ORL1 50 mM Tris-HCI PH 7.4, 10mMMgCI 2, 1 mM EDTA and 0.5% BSA
50 mM Tris-HCI PH 7.4, 10mMMgCI 2, 1 mM EDTA, 0.5% BSA and 0.01%
μ-opioid bacitracin
K-opioid 50 mM Tris-HCI PH 7.4
XXX-opioid 50 mM Tris-HCI pH 7.4, l0mMMgCI 2, 1 mM EDTA and 0.5% BSA
UR2 50 mM Tris-HCI pH 7.4, 10 mM MgCI2, 1 mM EDTA and 0.5% BSA
5 Format 1: FlashPlate Assay Volumes
19.5 μl buffer, 0.5 μl of compound diluted in DMSO, 5 μl of radioligand diluted in
binding buffer.
Format 2: Filtration Assay Volumes
10 44 μl membranes diluted in buffer, 1 μl of compound diluted in DMSO, 5 μl of
radioligand diluted in binding buffer.
Compound handling and dilutions
The day prior to performing the experiment 50 μl DMSO was added to each well of
15 the compound plates to yield compounds at a final concentration of 10 mM.
Daughter plates were then created by diluting the compounds further in DMSO to a
concentration of 0.5 mM. The mother plates were frozen immediately.

Protocols:
Filtration
Thaw membranes on ice then dilute membranes in binding buffer at a
5 concentration of 1 Unit per well. Dilute radio-ligand to 10 times the final
concentration in binding buffer. Add 44 ul of diluted membranes to each well of the
deep-well plate. Add 1 ul of DMSO (total value, 5 wells), reference ligand (non-
specific value, 3 wells) or compound to the corresponding wells in the deep-well
plate. Initiate the reaction by adding 5 ul of radioligand to each well and vortex
10 gently. Incubate at room temperature for 1 hour. During incubation, pre-incubate
the Multiscreen Harvest plates in 0.3% PEL Filter over pre-soaked Multiscreen
Harvest plates using a Tomtec Harvester. Wash 9 times with 500 jal of cold 50 mM
Tris-HCI pH 7.4 at 4°C and air-dry for 30 minutes at room temperature under a
fume hood. Apply a bottom seal to the Multiscreen Harvest plates. Add 25 μl of
15 MicroScint-0 to each well. Apply TopSeal-A to the plate. Count for 30 seconds per
well on TopCount Microplate Scintillation and Luminescence Counter
(PerkinElmer) using a count delay of 60 seconds.
FlashPlate
20 Immobilize membranes into FlashPlate microplates using PerkinElmer BioSignal's
proprietary coating procedure. Dilute radioligand to 5x the final concentration in
binding buffer. Add 19.5 ul buffer to each well of the FlashPlate. Add 0.5 ul of
DMSO (total value, 5 wells), reference ligand (non-specific value, 3 wells) or
compound to the corresponding wells in the FlashPlate microplate. Initiate the
25 reaction by adding 5 ul of radioligand to each well. Apply TopSeal-A onto
FlashPlate microplates. Incubate at room temperature for 1 hour in the dark. Count
for 30 seconds per well on TopCount Microplate Scintillation and Luminescence
Counter (PerkinElmer) using a count delay of 60 seconds.

26
Data Analysis
Percentage inhibition was calculated using the following formula:
% inhibition = (compound - Total) x 100
5 Non Specific - Total
Key to Blocks for Table 3: Results.


27
Table 3: Radioligand Binding Results


28


29


30


31


32


33


34


35


36


37


38


39
Key to Table 3: Results
"+" indicates greater than 50% inhibition at 10 μM,"-" indicates less than 50%
5 inhibition at 10μM "P" indicates precipitation
X1 - X30 are sidearms selected from the figure below.
Figure 1: Sidearms for Table 3.


40
Throughout the specification and the claims (if present), unless the context
requires otherwise, the term "comprise", or variations such as "comprises" or
"comprising", will be understood to apply the inclusion of the stated integer or
group of integers but not the exclusion of any other integer or group of integers.
5
Throughout the specification and claims (if present), unless the context requires
otherwise, the term "substantially" or "about" will be understood to not be limited to
the value for the range qualified by the terms.
10 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.

41
5 CLAIMS as amended under Article 34
1. A method of identifying biologically active compounds with defined
selectivity profile comprising:
(a) designing a library of compounds of formula 1 to scan molecular
io diversity; and
(b) assaying the library of compounds in at least two different biological
targets;
wherein formula 1 represents:

15 Formula I
wherein the ring may be of any configuration;
Z is oxygen, CH2, C(O), C(O)NRA, NH, NRA or hydrogen, in the case
where Z is hydrogen then Ri is not present, RA is selected from the set
20 defined for R1 to R5, or wherein Z and R1 together form a heterocycle;
X is oxygen or nitrogen;
(i) When X is oxygen, R1 to R5 are independently selected from the
group which includes but is not limited to H or an C1 to C20 alkyl or
2 5 acyl; C2 to C20 alkenyl, alkynyl, heteroalkyl; C5 to C20 aryl, heteroaryl,
arylalkyl or heteroarylalkyl, which is optionally substituted, and can be
branched or linear wherein R1-R5 optional substituents are selected
from the group consisting of OH, NO, NO2, NH2, N3, halogen, CF3,
CHF2, CH2F, nitrile, alkoxy, aryloxy, amidine, guanidiniums, aryl,
3 0 cycloalkyl, heteroalkyl, heteroaryl, aminoalkyl, aminodialkyl,

42
aminotrialkyl, aminoacyl, carbonyl, substituted or unsubstituted imine,
sulfate, sulfonamide, phosphate, phosphoramide, hydrazide,
hydroxamate, hydroxamic acid, heteroaryloxy, aminoaryl,
aminoheteroaryl, thioalkyl, thioaryl or thioheteroaryl, which may be
5 further substituted; or
(ii) when X is nitrogen, each X may combine independently with the
corresponding R2 to R5 to form an azide, or wherein each X may also
combine independently with any one of corresponding R2-R5 to form a
heterocycle, wherein R1 to R5 are independently selected from the
10 group which includes but is not limited to H or an C1 to C20 alkyl or
acyl; C2 to C20 alkenyl, alkynyl, heteroalkyl; C5 to C20 aryl, heteroaryl,
arylalkyl or heteroarylalkyl, which is optionally substituted, and can be
branched or linear wherein R1-R5 optional substituents are selected
from the group consisting of OH, NO, NO2, NH2, N3, halogen, CF3,
15 CHF2, CH2F, nitrile, alkoxy, aryloxy, amidine, guanidiniums, aryl,
cycloalkyl, heteroalkyl, heteroaryl, aminoalkyl, aminodialkyl,
aminotrialkyl, aminoacyl, carbonyl, substituted or unsubstituted imine,
sulfate, sulfonamide, phosphate, phosphoramide, hydrazide,
hydroxamate, hydroxamic acid, heteroaryloxy, aminoaryl,
20 aminoheteroaryl, thioalkyl, thioaryl or thioheteroaryl, which may be
further substituted.
2. The method according to claim 1 wherein at least one X is
nitrogen.
25
3. The method according to claim 1 wherein two of X is nitrogen.
4. The method according to claim 1 wherein X and R2 combine to
form a heterocycle.
30
5. The method of claim 1 wherein R1-R5 optional substituents are
selected from the group consisting of OH, NO, NO2, NH2, N3, halogen,
CF3, CHF2, CH2F, nitrile, alkoxy, aryloxy, amidine, guanidiniums, aryl,

44

Formula III
5 wherein A is defined as hydrogen, or OR1
R1 to R5 are independently selected from the group which
includes H or an C1 to C20 alkyl or acyl; C2 to C20 alkenyl, alkynyl,
heteroalkyl; C5 to C20 aryl, heteroaryl, arylalkyl or heteroarylalkyl,
10 which may be substituted, and can be branched or linear,
X is oxygen or nitrogen, when X is nitrogen, each X may
combine independently with the corresponding R2 to R5 to form an
azide, or wherein each X may also combine independently with any
15 one of corresponding R2-R5 to form a heterocycle.
20 8. The method according to claim 1, wherein the library of
compounds is selected from compounds of formula IV,

25 Formula IV
wherein R1, R2, R3 and R5 are independently selected from the group
which includes H or an C1 to C20 alkyl or acyl; C2 to C20 alkenyl,

45
alkynyl, heteroalkyl; C5 to C20 aryl, heteroaryl, arylalkyl or
heteroarylalkyl, which may be substituted, and can be branched or
linear.
5
9. The method according to claim 1, wherein the library of
compounds is selected from compounds of formula V,

10
Formula V
wherein R1, R2, R3 and R5 are independently selected from the group
which includes H or an C1 to C20 alkyl or acyl; C2 to C20 alkenyl,
15 alkynyl, heteroalkyl; C5 to C20 aryl, heteroaryl, arylalkyl or
heteroarylalkyl, which may be substituted, and can be branched or
linear.
20
10. A method according to claim 1 wherein the library of
compounds is selected from compounds of formula VIII,

25
Formula VIII
wherein R1, R2, R3 and R5 are independently selected from the group

46
which includes H or an C1 to C20 alkyl or acyl; C2 to C20 alkenyl,
alkynyl, heteroalkyl; C5 to C20 aryl, heteroaryl, arylalkyl or
heteroarylalkyl, which may be substituted, and can be branched or
linear.
5
11. The method according to claim 1, wherein the library of
compounds is selected from compounds of formula IX,

Formula IX
wherein R2, R3 and R5 are independently selected from the group
which includes H or an C1 to C20 alkyl or acyl; C2 to C20 alkenyl,
15 alkynyl, heteroalkyl; C5 to C20 aryl, heteroaryl, arylalkyl or
heteroarylalkyl, which may be substituted, and can be branched or
linear.
12. The method according to claim 1 wherein the biological assays
20 involve Peptide Ligand class of GPCRs.
13. The method according to claim 12 wherein biological assays
involve opioid, melanocortin, melanin-concentrating hormone,
neurokinin, neuropeptide and urotensin receptors.
2 5
14. The method according to claim 13 wherein biological assays
involve 5-opioid (DOP), k-Opioid (KOP), Melanocortin MC3,
Melanocortin MC4, Melanocortin MC5, Melanin-Concentrating
Hormone (MCH1),μ-opioid (MOP), Neurokinin (NK1), Neuropeptide Y
30 (NPY-Y1), Opioid (ORL1) and urotensin (UR2) receptors.

47
15. A library of compounds selected from compounds of formula 1,
when used according to claim 1.
5 16. A library of compounds selected from compounds of formula II,
when used according to claim 6.
17. A library of compounds selected from compounds of formula III,
when used according to claim 7.
10
18. A library of compounds selected from compounds of formula
IV, when used according to claim 8.
19. A library of compounds selected from compounds of formula V,
15 when used according to claim 9.
20. A library of compounds selected from compounds of formula
VIII, when used according to claim 10.
20
21. A library of compounds selected from compounds of formula
IX, when used according to claim 11.
22. A biologically active compound identified by the method of claim
25 1.
23. A compound according to formula 1 in which at least one X is
nitrogen, and said X is combined with the corresponding R2-R5 to form
a heterocycle.
30
24. A compound according to claim 23 wherein X and R2 combine
to form a heterocycle.

48
25. A compound according to claim 24, wherein the heterocycle is
heteroaryl.
26. A compound according to claim 25, wherein the heteroaryl is
5 selected from triazoles, benzimidazoles, benzimidazolone,
benzimidazolothione, imidazole, hydantoine, thiohydantoine and
purine.

A method of identifying biologically active compounds with defined selectivity
profile comprises; ( c ) designing a library of compounds of formula (1) to scan
molecular diversity; and (d) assaying the library of compounds in at least two
different biological assays.