Skip to main content

Full text of "USPTO Patents Application 10555896"

See other formats


(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(19) World Intellectual Property Organization 

International Bureau 

(43) International Publication Date 
9 January 2003 (09.01.2003) 




Hill 



PCT 



(10) International Publication Number 

WO 03/003012 Al 



(51) International Patent Classification 7 : 

C12Q 1/02, C07C 59/64 



G01 N 33/53, David [AU/AU] ; 2/1 6 Conyngham Street, Glenside, South 

Australia 5065 (AU). 



(21) International Application Number: PCT/AU02/00856 



(22) International Filing Date: 28 June 2002 (28.06.2002) 



(25) Filing Language: 

(26) Publication Language: 

(30) Priority Data: 

PR 5986 



English 



English 



29 June 2001 (29.06.2001) AU 



(74) Agent: PHILLIPS ORMONDE & FITZPATRICK; 367 

Collins Street, Melbourne, VIC 3000 (AU). 

(81) Designated States (national): AE, AG, AL, AM, AT, AU, 
AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU, 
CZ, DE, DK, DM, DZ, EC, EE, ES, H, GB, GD, GE, GH, 
GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, 
LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, 
MX, MZ, NO, NZ, OM, PH, PL, PT, RO, RU, SD, SE, SG, 
SI, SK, SL, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, 
VN, YU, ZA, ZM, ZW. 



(71) Applicant (for all designated States except US): MEDI- 
MOLECULAR PTY. LTD. [AU/AU]; Science Park Ade- 
laide, Bedford Park, South Australia 5042 (AU). 

(72) Inventors; and 

(75) Inventors/Applicants (for US only): JAMES, Robert 

[AU/AU]; 23 A Carunta Street, Wattle Park, South Australia 
5066 (AU). EDDIE, Lawrence [AU/AU]; 35 Highland 
Drive, Bellevue Heights, South Australia 5050 (AU). 
KAZENWADEL, Jan [AU/AU]; 4 Thorngate Drive, 
Belair, South Australia 5052 (AU). O'CONNOR, Susan 
[AU/AU]; 3 Clifton Street, Prospect, South Australia 5082 
(AU). RAZZINO, Pasquale [AU/AU]; 191 Greenhill 
Road, Parkside, South Australia 5063 (AU). WARD, 



(84) Designated States (regional): ARIPO patent (GH, GM, 
KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW), 
Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), 
European patent (AT, BE, CH, CY, DE, DK, ES, FI, FR, 
GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OAPI patent 
(BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, ML, MR, 
NE, SN, TD, TG). 

Published: 

— with international search report 

For two-letter codes and other abbreviations, refer to the "Guid- 
ance Notes on Codes and Abbreviations" appearing at the begin- 
ning of each regular issue of the PCT Gazette. 



< 



(54) Title: IDENTIFICATION OF INTERACTING MOLECULES 
w 

2 (57) Abstract: The present invention provides a method for identifying a protein capable of binding to a specific target molecule. 

The method involves allowing candidate proteins to bind to the target molecule in the presence of a second molecule which is struc- 
Q turally similar to the non-nucleic acid target molecule, but deficient in a desired activity of the target molecule, and isolating the 

proteins that bind to the target molecule. The invention also provides analogues of flurbiprofen and sulindac as target molecules for 
^ use in the methods of the invention. 



PCT/AU02/00856 

WO 03/003012 

-1- 

IDENTIFICATION OF INTERACTING MOLECULES 
PiPlfjnfthft Invention 

, The present — reiates ,o — <or iden^a* = s - 

proteins identified using such methods, ana » y 
to identify candidate proteins. 

efficacy. 

analyses may also revaal the identity of interacting protems. 

30 fc m o,eou,es to separate * "^-^JJ^ re, on 
mixture of molecules over the solid support, wash,ng the solid supp 



WO 03/00301 2 PCT/AU02/00856 

-2^ 

remove molecules that do not bind, and isolating the molecules that remain 
bound to the immobilised target. Molecules that remain bound to the target are 
generally ones that bind to the target molecule with a higher affinity, and are 
therefore more likely to be of biological relevance. The lower affinity binding 
5 molecules that are removed by the selected washing treatment are usually less 
likely to be of biological relevance. 

A variety of molecules may be used as targets in affinity purification systems. 
Such targets include small organic molecules, cofactors, nucleic acids, small 
10 peptides, proteins, oligosaccharides and lipids. 

Affinity purification methods may generally be used to isolate any type of 
molecule that interacts with a target molecule of interest. As many biologically 
active molecules exert their effects by binding to proteins, affinity purification 
15 methods have been used to isolate proteins that bind to a specific target. In such 
cases, the candidate binding protein may often need to be isolated from a 
complex mixture of proteins. 

Methods have become available whereby very complex pools of candidate 
20 binding molecules may be generated for the purposes of screening. This may be 
important in cases where a candidate binding molecule is likely to be present at 
a very low abundance. In such a case, the likelihood of finding an appropriate 
binding molecule will increase with increasing complexity of the mixture to be 
screened. Methods for generating complex mixtures of potential binding 
25 molecules include, for example, the chemical synthesis of short random peptides 
and the chemical synthesis of short random nucleic acid molecules (aptamers). 

Complex mixtures of proteins may be generated by the cloning of a large number 
of individual cellular DNAs into expression systems to form a library of proteins 
30 expressed from the cloned DNA molecules. For example, a pool of 
complementary DNAs (cDNAs) may be isolated from mRNAs isolated from a 
particular source, the cDNAs cloned into an expression vector and the proteins 
encoded by each of the cDNAs expressed. The proteins expressed from such 



PCT/AU02/00856 

WO 03/003012 

-3- 

pools or libraries may be used as the source of potential binding proteins for 
screening by affinity purification. 

One method for screening complex mixtures of proteins using an affinity 
5 purification approach is the method referred to as "phage display". In this 
method, various DNAs are cloned in a viral nucleic vector. The DNAs are 
inserted into a viral gene that normally expresses a protein that is found on the 
surface of the virus. When viral particles are produced, the protein expressed 
from the particular DNA inserted into the virus DNA will be displayed on the 
10 surface of the virus. If a library of DNA molecules is cloned into the viral vector, 
each virus produced will display a different protein from the library on its surface. 

The complex mixture of viral particles so produced may be passed over a target 
molecule immobilised to a solid support. To identify candidate proteins that may 

15 bind to the target, the solid support is washed and the viral particles that remain 
bound are collected. Phage display methods are useful for the screening of 
potential candidate binding proteins, because once the viral particles binding to 
the target are isolated, the viral particles may be allowed to infect new cells and 
produce a new enriched population of viral particles. The resultant new mixture 

20 of viral particles may then be re-applied to the immobilised target molecule and 
the process reiterated. In this way, a population of viral particles may be 
successively enriched for those viral particles expressing candidate binding 
proteins on their surface. 

25 However, a deficiency with the use of affinity purification methods such as phage 
display has been the inability to readily distinguish between the binding of bona 
fide candidate molecules and the binding of other molecules that are not 
biologically relevant. This may occur for a number of reasons. Many proteins 
may bind to the target molecule with high affinity, but the binding may be for 

30 reasons that are unrelated to the biological activity of the target molecule. 
Alternatively, the washing regime selected may not be sufficiently capable of 
discriminating between the binding of bona fide candidate proteins and the 
binding of other proteins with reduced affinity for the target. Additionally, some 



WO 03/00301 2 PCT/AU02/00856 

-4- 

interactions with other proteins may also interfere with the binding of bona fide 
candidate proteins. 

The present invention relates to an improved method for isolating proteins from 
5 complex mixtures, by providing means to improve the likelihood that the proteins 
identified are biologically relevant to the desired activity or function of the target 
molecule. 

Summary of the Invention 

10 

The present invention provides a method for identifying a protein capable of 
binding to a target molecule, the method including the steps of: 

(a) providing a pool of candidate proteins; 

(b) providing a non-nucleic acid target molecule, wherein the non-nucleic 
15 acid target molecule is coupled to a selectable moiety; 

(c) providing a second molecule which is structurally similar to the non- 
nucleic acid target molecule, wherein the second molecule is deficient 
in a desired activity of the target molecule; 

(d) allowing one or more of the candidate proteins to bind the non-nucleic 
20 acid target molecule in the presence of the second molecule; 

(e) isolating a protein bound to the target molecule; and 

(f) identifying the binding protein. 

The present invention provides a method for identifying a protein capable of 
25 binding to a target molecule, the method including the steps of: 

(a) providing a pool of candidate proteins, wherein each candidate protein 
is displayed on the surface of a viral particle; 

(b) providing a non-nucleic acid target molecule, wherein the non-nucleic 
acid target molecule is coupled to a selectable moiety; 

30 (c) providing a second molecule which is structurally similar to the non- 

nucleic acid target molecule, wherein the second molecule is deficient 
in a desired activity of the target molecule; 



PC T7AU02/00856 

WO 03/003012 

h andidate proteins to bind to the non- 
id) allowing one or more of to ^ molecule . 

„ ud eic acid m* "**«* " ** T^. ^ m „ te0Ul e; 

in amplifying the viral particles a 
)re«era«rtg steps (a)*,ough«; and 

(h) identifying the binding protein. 

a method for identity a P-otein capable of 
The Present — provides ^ teps0 , 
bi ndingtotargetmoleouto>em* 

to (a)P^ aW ^l^Smo,ec,le,«he,e,«heno^ 

(b) providing a non-nucleic acid 9 

add target ™~* is coupled to as* ^ ^ ^ „„, 

(c) a aecona m^:;;, ^nd mo^cule is decent 
nucleic acid target molecule,* 

,n a desired acMy of the «- in „. w pool to bind 

to the non-nucleic acd target moie 

molecule; . t tne target molecule; 

20 ffloompanngtheleveloffhepro , protei „ s; an* 

(g) identifying a protein that is dine 
and second pools. 

a method for identifying a protein capable of 
25 Th9 presenf invention pro^ ^ * 

iWingtoatarge.mo.ecu^em* 

W pro.ding.irst and wherei „ t He non-nu*,c 



WO03/0fl30t2 PCT/AU02/00S56 

-6- 

(d) allowing one or more of the candidate proteins in the first pool to bind 
to the non-nucleic acid target molecule in the presence of the second 
molecule; 

(e) isolating one or more proteins in the first pool that bind to the target 
5 molecule; 

(f) allowing one or more of the candidate proteins in the second pool to 
bind to the non-nucleic acid target molecule in the presence of the 
second molecule; 

(g) isolating one or more proteins in the second pool that bind to the target 
10 molecule; and 

(h) comparing one or more proteins isolated from each of the first and 
second pools to identify a protein that is differentially represented 
between the first and second pools. 



15 The present invention relates to a method for identifying proteins that are able to 
bind to a target molecule, the target molecule preferentially being a biologically 
active molecule. The identification of proteins that are able to bind to such a 
target molecule may be important for a number of reasons. For example, it may 
allow the identification of the proteins that the target molecule binds to in order to 

20 exert its biological effect. It may also allow the identification of new proteins that 
themselves may serve as drugs to inhibit or augment the biological activity of the 
target molecule. 



It has been determined by the applicant that when screening a large number of 
25 candidate proteins for their ability to bind to a target molecule, the presence in 
the binding reaction of a second molecule, which is structurally similar to the 
target molecule but deficient in a biological activity of the target molecule, may 
allow improved detection of proteins that are involved in a biologically relevant 
interaction with the target molecule. 

30 

This improved effect is particularly apparent when the candidate binding proteins 
are screened by a method in which the proteins are displayed on the surface of a 
viral particle. It also appears that reiterated cycles of binding the protein to the 



WO 03/003012 

PCT/AU02/00856 

that bind to the target in a way that may be biologically important. 

OifferenMy represented hereon poote o, candidate pnoteins. 

in a preterm torn, of the invent the target moiecule is e«he, of ft. non 

~ ««~y ^ofen or sulindao su,„de or ^Z, 

Z 091,63 °' ^ °' ' n,hfe,om - •» mention ££Z 

" 7 te m "* - — r A^profen or sulindao 

■naclve or has a reduced biological acMy re , ative to the ^ " 



WO 03/003012 



-8- 



PCT/AU02/00856 




or a salt thereof, wherein: 

- R 1 is selected from hydrogen and lower alkyl (C1 to C8); 

5 - R 2 is YX 2 ((CH 2 ) m X 2 ) n -, wherein m is 2 to 4, n is 1 to 6, X 2 is selected 

from O, S and N, and Y is independently selected from hydrogen, 
lower alkyl, or a suitable heteroatom protecting group; 

- R 3 is selected from one or more of hydrogen, alkyl, aryl, halogen, 
hydroxy, alkoxy, aryloxy, amino (unsubstituted and substituted) and 

10 caroboxy; 

- R 4 is selected from one or more of hydrogen, alkyl, aryl, halogen, 
hydroxy, alkoxy, aryloxy, amino (unsubstituted and substituted) and 
caroboxy; 

- X is selected from fluoro, chloro, bromo and iodo; 

15 - M is selected from hydroxy, alkoxy, aryloxy, amino, alkylamino (mono- 

and di-), arylamino (mono- and di-), N-morpholino, hydroxyalkylamino, 
dialkylaminoalkylamino, aminoalkylamino, polyhydroxyamino, and 
salts of any of the aforementioned. 



20 Alternatively or in addition, the present invention provides an analogue of 
sulindac sulfide, the analogue having the formula (II): 



PCT/AU02/00856 



WO 03/M5012 




00 



. H i b selected from hydrogen, hydroxy (when 

sulfoxide), and lower alky! (C1 to C8); , 

^^.o-asu^eheteroa^P^^ )no 

alkylamino (mono- and di-), aryiamin v 
^sled^hydrogenend^raM^oC^ 

salts of any of the aforementioned. 

t , m or fln may be used as target molecules or second 

. rrrm - — 

respectively- 
25 some of these terms will now be defined. 



WO 03/003012 



PCT/AU02/00856 



-10- 



The term "non-nucleic acid target molecule" as used throughout the specification 
is to be understood to mean any molecule to which a protein may bind, but which 
is not a nucleic acid molecule. For example, the target molecule may include 
5 drug molecules, proteins, peptides, polypeptides, polysaccharides, glycoproteins, 
hormones, receptors, lipids, small molecules, metabolites, cofactors, transition 
state analogues and toxins. 

In terms of the present invention, the second molecule will be a molecule that is 
10 structurally similar to the non-nucleic acid target molecule. The term "structurally 
similar" as used throughout the specification is to be understood to mean a 
molecule that is similar to the non-nucleic acid target molecule in terms of its 
three dimensional structure. 

15 For example, structurally similar molecules may include isomeric molecules such 
as isomers, geometric isomers, enantiomers, conformers, stereoisomers, 
structural isomers, molecules that substitute one or more chemical groups in a 
molecule with other chemical groups, or molecules that are substantially similar 
in the three dimensional structure of one or more parts of the molecule. 

20 

The term "protein" as used throughout the specification is to be understood to 
mean any polypeptide consisting of two or more constituent amino acids. The 
polypeptide may also contain one or more side chains derived from a modified 
amino acid. 

25 

The term "viral particle" as used throughout the specification is to be understood 
to mean any virus with a protein coat, wherein the virus genome contains a gene 
for a coat protein that will allow the display of a subject protein on the surface of 
the protein, when the DNA encoding the subject protein is inserted into the gene 
30 for an appropriate coat protein. For example, the viral particle according to the 
present invention may include bacteriophage particles. 



• mrouflh out the specification is to be 

5 saoeaiSMoi^st^^ 

th nds of the present invention may be 

^^eculeto^ohaptoteinn^ybrt drug molecule s, 

Ziecuie. For exampie, the W J ^ hormones, 

receptors, lipids, small 

analogues and toxins. ^ 

^ moiecu* - - J", ^i'SiS 
20 nerein to as 'a desired acWy" »< «- - J mo(e ^ e «ects o« the 
n.iecoie may be an act* -"-J* ^ the ^ ac* o< the 
w , moieouie on a « t0 contro, the P— n - 

target molecule may be assoc.* 
neoplastic cells. 



WO 03/003012 



-12- 



PCT/AU02/00856 



An example of an analogue of flurbiprofen that may be used as an active target 
molecule is the (^-stereoisomer of a molecule with the following chemical 
formula: 

5 

R2Q 




(0 



or a salt thereof, wherein: 

- R 1 is selected from hydrogen and lower alkyl (C1 to C8); 

10 - R 2 is YX 2 ((CH 2 ) m X 2 ) n -, wherein m is 2 to 4, n is 1 to 6, X 2 is selected 

from O, S and N, and Y is independently selected from hydrogen, 
lower alkyl, or a suitable heteroatom protecting group; 

- R 3 is selected from one or more of hydrogen, alkyl, aryl, halogen, 
hydroxy, alkoxy, aryloxy, amino (unsubstituted and substituted) and 

15 caroboxy; 

- R 4 is selected from one or more of hydrogen, alkyl, aryl, halogen, 
hydroxy, alkoxy, aryloxy, amino (unsubstituted and substituted) and 
caroboxy; 

- X is selected from fluoro, chloro, bromo and iodo; 

20 - M is selected from hydroxy, alkoxy, aryloxy, amino, alkylamino (mono- 

and di-), arylamino (mono- and di-), N-morpholino, hydroxyalkylamino, 
dialkylaminoalkylamino, aminoalkylamino, polyhydroxyamino, and 
salts of any of the aforementioned. 

25 X is preferably fluoro and most preferably substituted meta to the 
alkylcarboxylate group. 



Preferably, R 1 is a lower alkyl group, and is most preferably a methyl group. 



WO 03/003012 



-13- 



PCT/AU02/00856 



Preferably, R is an alkyleneoxy or polyoxyalkylene chain, more preferably 
having between 1 and 4 alkyleneoxy repeating units. Suitable alkyleneoxy 
repeating units include ethyleneoxy and propyleneoxy. In one particularly 
5 preferred form of the invention, R 2 is a Methylene glycol group. 

Preferably, the R 2 0- group is substituted at a position para to the aryl 
substituent. 

10 Preferably, both R 3 and R 4 are hydrogen. 
M is preferably hydroxy or a salt thereof. 

From the above, it will be evident that in one particularly preferred form, the 
15 present invention provides a compound of formula (III): 



Compounds of formula (I) and (III) are available as (R)- and (^-stereoisomers 
20 and the present invention contemplates the production and/or use of either 
pure (R)- or pure (^-stereoisomers, as well as racemic mixtures or mixtures 
enriched with either stereoisomer. The (R)-stereoisomer of compounds of 
formula (I) or (III) may be active and therefore suitable as a target molecule in 
the methods of the present invention. Conversely, the (S)-stereoisomer of 
25 compounds of formula (I) or (III) may be inactive or have a reduced activity 
relative to the (^-stereoisomer and therefore the (S)-stereoisomer may be 
suitable as a second molecule in the methods of the present invention. 




(Ill) 



WO 03/003012 



-14- 



PCT/AU02/00856 



The stereoisomers of compounds of formula (I) may be separated by any of 
the techniques used for that purpose in the art, including chromatography for 
example. 

5 Compounds of formula (I) may be produced by any suitable synthetic method, 
including those known methods for the production of flurbiprofen. An example 
of a suitable synthetic method is shown in Scheme 1, in which the method 
includes the steps of hydroxylation of the unsubstituted phenyl ring in 
flurbiprofen, followed by substitution of the free phenol with the triethylene 
10 glycol group. 




Scheme 1 



15 An example of an analogue of sulindac sulfide that may be used as an active 
target molecule is a molecule with the following chemical formula: 



PCT/AU02/00856 



WO 03/003012 

-15- 




10 



15 



20 



(ID 



or a salt thereof, wherein: 

X 1 is selected from sulfide, sulfone and sulfoxide; 
. R i is selected from hydrogen, hydroxy (when X 1 is sulfone or 
sulfoxide), and lower alkyl (C1 to C8); ^ 
R 2 is YX 2 ((CH 2 ) m XV wherein m is 2 to 4, n is 1 to 6, X is selected 
from O, S and N, and Y is independently selected from hydrogen, 
lower alkyl. or a suitable heteroatom protecting group; 
R 3 is selected from hydrogen, halogen, alkyl, alkoxy, acyloxy, am.no, 
alkylamino (mono- and di-), arylamino (mono- and di-), nitro, cyano, 
carboxyl; 

R> is selected from hydrogen and lower alkyl (C1 to C8); and 
M is selected from hydroxy, alkoxy, arylcxy, amino, alkylamino (mono- 
and di-), arylamino (mono- and di-), N-morpholino, hydroxyalkylamrno, 
dialkylaminoalkylamino, aminoalkylamino, polyhydroxyamrno, and 
salts of any of the aforementioned. 

X' is preferably efther a sulfone or a sulfide, and is most preferably a suKide. 

Preferably, ft is a lower alkyl group, and is most preferably a methyl group. 

Preferably, R 2 is an alkyleneoxy or polyoxyalkylene chain, more preferably 
to*, between 1 and 4 alkyleneoxy repeating units. Surtabte alkyleneoxy 



WO 03/003012 



PCT/AU02/00856 



-16- 

repeating units include ethyleneoxy and propyleneoxy. In one particularly 
preferred form of the invention, R 2 is a triethylene glycol group. 

Preferably, R 3 is a halogen group (iodo-, bromo-, chloro- or fluoro-), more 
5 preferably a fluoro group, and most preferably a fluoro group ortho to the 
hydroxy group. 

Preferably, R 4 is a lower alkyl group and is most preferably methyl. 

10 Mis preferably hydroxy or a salt thereof. 

Compounds of formula (II) are available as (£) or (2) geometric isomers. The 
compounds may be used as a mixture of (£) and (2) isomers (whether a 1:1 
mixture or some other ratio), or the geometric isomers may be separated using 
15 any of the standard techniques that are used for that purpose, such as 
chromatography. 

From the foregoing it will be evident that in one preferred form, the invention 
provides a compound of formula (IV), or a salt thereof: 

20 



It will be appreciated that compounds of formula (II) and (IV) may be formed by 
any one of a number of synthetic routes. However, in one form the present 
25 invention also provides a process for the preparation of compounds of formula 
(II), the process including the steps of: 




MeS 



(IV) 



WO 03/003012 



PCT/AU02/00856 



17- 



intramolecular cyclisation of acyl donor (a) to form ketone (b); 
i. enolate addition to (b) followed by dehydration to form indene (c); and 
addition of (c) to aldehyde (d) to form indene (e). 



F IXX 

(a> 




C0 2 R' 



(b> 







0 


-* 

CHO 




<> 










R"X 








C0 2 R' 



(d) 



10 



The second molecule according to the methods of the present invention is any 
molecule that is structurally similar to the target molecule, but which does not 
show a desired activity associated with the target molecule. 



Structurally similar molecules include isomeric molecules such as isomers, 
geometric isomers, enantiomers, conformers, stereoisomers, structural isomers, 
molecules that substitute one or more chemical groups in a molecule with other 
chemical groups, or molecules that are substantially similar in the three 
1 5 dimensional structure of one or more parts of the molecule. 



WO 03/003012 



PCT/AU02/00856 



-18- 



For example, the second molecule may include a drug analogue that shows 
reduced activity as compared to the biologically active drug. The second 
molecule may be an analogue of a known drug, such as flurbiprofen or sulindac 
sulfide. The second molecule may also be a molecule that is structurally similar 
5 to the target molecule, but which has been altered in a region (or regions) that is 
of interest in the target molecule. 

An example of an analogue of flurbiprofen that may be utilised as a second 
molecule in the methods of the present invention is the molecule with the 
1 0 following chemical formula: 



This molecule is the (^-stereoisomer. The (/^-stereoisomer shows similar 
15 activity to flurbiprofen, while the (^-stereoisomer is inactive. 

The source of the pool of candidate proteins in the methods of the present 
invention may be any source of proteins, including proteins derived from cellular 
or viral extracts, proteins displayed on the surface of a viral particle by cloning 

20 one or more DNAs into a suitable viral vector, the protein products of one or 
more DNAs cloned into a protein expression vector and expressed in a suitable 
expression system, the products of in vitro translation of different mRNAs, or 
chemically synthesized polypeptides or proteins. The pool of candidate proteins 
may also contain post-translation modifications. For example, the pool of 

25 candidate proteins may include one or more glycosylated proteins. 

Preferably, the source of the pool of candidate proteins is a pool of proteins 
expressed from suitable DNA molecules inserted into a viral genome. More 
preferably, the source of the pool of candidate proteins is a pool of viral particles 




WO 03/003012 PCT/AU02/00856 

-19- 

wherein each of the candidate proteins in the pool is displayed on the surface of 
a viral particle. 

In the case of a pool of candidate proteins derived from cellular extracts, the 
5 source of the pools of candidate proteins includes cellular extracts derived from 
cell populations, group of cells, tissues or organs. Cellular extracts may be 
prepared by suitable methods known in the art. Cellular extracts may be derived 
from any prokaryotic or eukaryotic organism, including animals or humans. 

10 In the case of cellular extracts derived from tissues, the cellular extract may be 
derived from cells selected from one or more of the following types of tissue: 
colorectal tissue, breast tissue, cervical tissue, uterine tissue, renal tissue, 
pancreatic tissue, esophageal tissue, stomach tissue, lung tissue, brain tissue, 
liver tissue, bladder tissue, bone tissue, prostate tissue, skin tissue, ovary tissue, 

1 5 testicular tissue, muscle tissue or vascular tissue. 

These tissues may further contain cells that are normal (non-cancerous), pre- 
cancerous (having acquired some but not all of the cellular mutations required 
for a cancerous genotype) or cancerous cells (malignant or benign). Such 

20 tissues may contain cells that are normal, pre-cancerous or cancerous, any 
combination of cells that are normal, pre-cancerous or cancerous, or any other 
form of diseased cell. As will be readily appreciated, there are numerous 
methods well known in the art for determining whether cells are normal, pre- 
cancerous, cancerous or diseased, including histopathology and other 

25 phenotypic and genotypic methods of identifying cells. 

As stated above, the source of the pool of proteins may also be a pool of 
proteins expressed from one or more DNAs inserted into a viral genome and 
which are displayed on the surface of the viral particle when expressed. The 
30 DNAs inserted may be complementary DNAs (cDNAs), DNA fragments derived 
from genomic or viral DNAs, or chemically synthesized DNAs. The DNAs 
inserted into the viral genome may also make up a library of DNAs, including 
libraries of cDNAs produced by reverse transcription of cellular mRNAs and 



WO 03/00301 2 PCT/AU02/00856 

-20- 

genomic DNA fragments. The DNAs inserted into the viral genome may also 
include one or more random DNA sequences, resulting in the expression of 
random polypeptides. For example, the random DNAs so inserted may be 
chemically synthesized DNAs. 

5 

Suitable viruses for cloning of DNA so as to express proteins on the surface of 
the viral particle include bacteriophage viruses such as those derived from T7, 
T4, lambda, lambdoid phage, or filamentous phage, including M13, f1 and fd. 
The DNAs may be cloned into the vectors derived from these phage by suitable 
10 methods known in the art. The insertion of the DNA to be expressed into an 
appropriate coat protein gene allows the protein encoded by the DNA to be 
displayed on the surface of a viral particle. 

Viruses displaying proteins on their surface may then be produced by infection of 
15 a suitable host and preparation of viral extracts by suitable methods that are 
known in the art. 

The source of the pool of proteins may also be a pool of proteins expressed from 
one or more DNAs inserted into a vector (plasmid vector or viral genome) and 

20 expressed in a suitable expression system. Once again, the DNAs inserted may 
be complementary DNAs (cDNAs), DNA fragments derived from genomic or viral 
DNAs, or chemically synthesized DNAs. The DNAs inserted into the vector may 
also make up a library of DNAs, including libraries of cDNAs and genomic DNA 
fragments. The DNAs inserted into the vector may also include one or more 

25 random DNA sequences, resulting in the expression of random polypeptides. For 
example, the random DNAs so inserted may be chemically synthesized DNAs. 

The selectable moiety coupled to the non-nucleic acid target molecule according 
to the methods of the present invention is any moiety that allows the target 
30 molecule to be substantially purified away from other molecules, including the 
pool of candidate proteins. For example, the selectable moiety may be a 
chemical group such as an activated carbonate group that allows the target 
molecule to be covalently linked to a solid support. For protein molecules, 



WO 03/003012 



-21 - 



PCT/AU02/WI856 



covalent coupling of the protein molecule to the solid support also includes 
coupling to the solid support via primary amines or cysteines by suitable methods 
that are known in the art. 

5 In addition, a chemical moiety such as a biotin containing group may be coupled 
to the target molecule allowing the target molecule to be captured by an avidin or 
streptavidin group coupled to a solid support. 

Further examples of methods of immobilisation of the target molecule include 
10 coupling of an antigen to the target molecule and capture by an immobilised 
antibody. Immobilisation of the target molecule may also utilise capture of 
glutathion-S-transferase-fusion proteins by anti GST antibodies, capture of 6xHis- 
fusion proteins by anti 6xHis antibodies or a nickel-chelating surface, capture of 
cAMP or cGMP binding proteins by cAMP or cGMP immobilised on a solid 
15 support. In these cases, the target molecule may be coupled to a protein 
molecule to be captured, or alternatively, the target molecule may be a protein 
engineered to be able to be captured by one of these methods. 

The binding of the pool of candidate proteins to the non-nucleic acid target 
20 molecule in the presence of the second molecule in the methods of the present 
invention may be achieved under conditions suitable to the particular pool of 
candidate proteins being used. For example, the temperature and solution may 
be selected depending upon the properties of the pool of candidate proteins 
being used. Preferably, the temperature of binding may be within the range from 
25 4°C to 42°C, and the binding achieved in a suitable buffer, including the use of 
tris-based and/or phosphate buffered solutions. Such solutions may further 
include appropriate amounts of further components, including salts, detergents 
and other agents depending upon the properties of the particular pool of 
candidate proteins being used. 

30 

The target molecule may be free in solution when binding occurs (and then 
subsequently captured), or alternatively be immobilised to a solid support during 



WO 03/003012 PCT/AU02/00856 

-22- 

the binding reaction. In a similar fashion, the second molecule may be free in 
solution, or alternatively, be immobilised to a solid support. 

Preferably, the binding of the candidate proteins to the target molecule in the 
5 presence of the second molecule is performed under conditions where the 
second molecule is present in a molar excess to the target molecule. Most 
preferably the second molecule is in a molar excess of at least one hundred fold. 

The ratio of the second molecule to the target molecule may also be selected so 
10 as to obtain binding proteins that bind to the target molecule with varying affinity. 
The greater the ratio of the second molecule to the target molecule in the binding 
reaction, the greater the affinity of proteins that bind to the target is likely to be. 

In the methods of the present invention, the proteins bound to the target 
15 molecule may be isolated by a suitable means. If the target molecule is coupled 
to a fixed solid support, the proteins bound to the target molecule may be 
isolated by washing the solid support in a suitable buffer to remove any proteins 
that do not bind to the target molecule. If the target molecule is coupled to a solid 
support such as beads (for example paramagnetic beads), the beads may first 
20 be isolated and the proteins that do not bind removed by washing the beads in a 
suitable buffer. Alternatively, the target molecule may be captured and thus 
immobilised on the solid support, and proteins that do not bind them removed by 
washing. 

25 The removal of proteins bound to the target molecule may also be achieved by 
eluting in a suitable buffer containing free target molecule in substantial molar 
excess. Preferably, the free target molecule is in a molar excess of greater than 
one thousand fold. 



30 In removing proteins that are bound to the target molecule, the eluates may be 
isolated at different times during the washing procedure to allow for the 
identification of binding proteins that have different dissociation rates. 



PCT/AU02/00856 

WO 03/003012 _ ^ _ 

u „mods of the present invention may be subject 
^ pro .eins so isolated in the methods of me ^ w exampte , « 

t0 m etheds to *w tbeir idenb.rca.on an* dtt ^ & ^ 

— - be detemVmed * a 

pr o,ein isolated accord,ng to <he P re*nt 
6 Lftod that includes determine.™ of theem.no 
massspeotrometry may be performed. 

^nrtirtate Drotein on their surface, the 
iden «y of the expressed protem may be d ter ^ ^ jn 

10 „ DNA sequence o, the ^ L will be appreeiated, 

expression o, -^^i. - predion o, fhe amino - 
aetermination of the on me surtace of me viral pan*, 

sequence of the protein that is display 

t . j ,„ mo taraet molecule may be re- 

r^r.:^-— 

the target molecule is achieved. 

20 nation - the ^^^^^ 
pres en. invenbon to bind to t*W* — punfied ^ the n 

known in the art. For example, the proteir , my by 

^binding ™*^.^™^Z *-» «"•"» " 
. suable means, in.ud.ns - ^ ^ of . phage part* 

25 atomic fore* microscopy. be detcmlin od by a suable 



WO 03/003012 



-24- 



PCT/AU02/0085r> 



acid target molecule is coupled to a selectable moiety; 
(c) providing a second molecule which is structurally similar to the non 
nucleic acid target molecule, wherein the second molecule is deficient 
in a desired activity of the target molecule; 
5 (d) allowing one or more of the candidate proteins to bind to the non 

nucleic acid target molecule in the presence of the second molecule; 

(e) isolating one or more proteins bound to the target molecule; 

(f) amplifying the viral particles encoding the isolated binding proteins; 

(g) reiterating steps (a) through (f); and 
1 0 (h) identifying the binding protein. 

In this form of the present invention, the source of the pool of proteins is a pool 
of proteins expressed from one or more DNAs inserted into a viral genome, the 
proteins so expressed being displayed on the surface of the viral particle. 
15 Suitable viruses for cloning of DNA so as to express proteins on the surface of 
the viral particle include bacteriophage viruses such as those derived from T7, 
T4, lambda, lambdoid phage, or filamentous phage, including M13, f1 and fd. 
The DNAs may be cloned into vectors derived from these phage by suitable 
methods known in the art. 

20 

The DNAs inserted into such viral vectors may be complementary DNAs 
(cDNAs), DNA fragments derived from genomic or viral DNAs, or chemically 
synthesized DNAs. The DNAs inserted into the viral genome may also make up 
a library of DNAs, including libraries of cDNAs (for example obtained by the 
25 reverse transcription of cellular mRNAs) and genomic DNA fragments. The 
DNAs inserted into the viral genome may also include one or more random DNA 
sequences, resulting in the expression of random polypeptides. For example, the 
random DNAs so inserted may be chemically synthesized DNAs. 

30 Viruses displaying proteins on their surface may then be produced by infection of 
a suitable host and preparation of viral extracts by methods that are well known 
in the art. In this way, a pool of .candidate proteins in which each candidate 
protein is displayed on the surface of the viral particle may be produced. 



WO 03/003012 



-25- 



PCT/AU02/00856 



The amplification of viral particle encoding the isolated proteins may be achieved 
by re-infecting a competent viral host with the isolated viral particles by a suitable 
procedure. The viral particles so concentrated may be concentrated by a suitable 
5 means, so as to allow the process of binding and isolating the proteins that bind 
to the target molecule to be reiterated. 



Preferably, a proportion of the DNA inserts that make up the viral population after 
amplification may be characterised for insert size and their DNA sequence. For 
10 example, the DNA inserted into each viral particle may be isolated by obtaining a 
pure viral population by way of an isolated plaque and isolating the DNA inserted 
in that particular viral DNA by polymerase chain reaction using appropriate 
primers. The size of the DNA inserts may be determined by a suitable method. 
The DNA sequence of the DNA insert may be determined by a suitable method. 

15 

The reiteration step of this form of the present invention is continued until a 
desired level of representation of the DNA inserts is reached in the viral 
population. The representation of the DNA inserts in the viral population may be 
determined by a suitable method. Preferably five or more reiterations are 
20 performed. 



The identity of the binding protein may then be determined by determination of 
the DNA sequence of the DNA inserted into the viral genome that results in 
expression of the protein on the surface of the virus. As will be appreciated, 
25 determination of the DNA sequence will allow the prediction of the amino acid 
sequence of the protein expressed on the surface of the viral particle. 



The present invention also provides a method for identifying a protein capable of 
binding to target molecule, the method including the steps of: 
30 (a) providing a first pool of candidate proteins; 

(b) providing a non-nucleic acid target molecule, wherein the non-nucleic 
acid target molecule is coupled to a selectable moiety; 



WO 03/003012 



-26- 



PCT/AU02/00856 



(c) providing a second molecule which is structurally similar to the non- 
nucleic acid target molecule, wherein the second molecule is deficient 
in a desired activity of the target molecule; 

(d) allowing one or more of the candidate proteins in the first pool to bind 
5 to the non-nucleic acid target molecule in the presence of the second 

molecule; 

(e) isolating a protein in the first pool that binds to the target molecule; 

(f) comparing the level of the protein in the first pool of candidate proteins 
with the level of the protein in a second pool of proteins; and 

10 (g) identifying a protein that is differentially represented between the first 

and second pools. 

In this form of the present invention, the identification of proteins that bind to a 
target molecule and which are differentially represented between two pools of 
15 candidate proteins may be achieved. 

The determination of the level of a protein in the first pool may be achieved by a 
suitable procedure known in the art, including the determination of the 
concentration by methods that include the use of antibodies to detect the binding 
20 protein. For example, the concentration of the binding protein in the first pool 
may be achieved with an antibody raised to the binding protein, and the 
subsequent use of the antibody to visualise the protein by Western analysis or 
the use of the antibody to immunoprecipitate the protein. 

25 The level of the binding protein in a second pool of candidate proteins may then 
be determined in a similar fashion. In this way, proteins isolated from the first 
pool of candidate proteins may be compared with a second pool of candidate 
proteins, so as to identify proteins that are differentially represented between the 
two pools of binding proteins. 

30 

For example, the first pool of proteins may be derived from a tissue that contain 
cells that are normal (non-cancerous), and the second pool of proteins may be 
derived from cells that are pre-cancerous (having acquired some but not all of 



WO 03/003012 

PCT/AU02/00856 

-27- 

the cellular mutations required for a cancerous genotype) or cancerous ceils 
(malignant or benign). Any differences in the level of a binding protein between 
the pool of proteins derived from a normal tissue and another tissue allows the 
identification of binding proteins that are differentially expressed between the two 
different pools of proteins. 

The present invention also provides a method for identifying a protein capable of 
binding to target molecule, the method including the steps of: 

(a) providing first and second pools of candidate proteins; 

(b) providing a non-nucleic acid target molecule, wherein the non-nucleic 
acid target molecule is coupled to a selectable moiety; 

(c) providing a second molecule which is structurally similar to the non- 
nucleic acid target molecule, wherein the second molecule is deficient 
in a desired activity of the target molecule; 

(d) allowing one or more of the candidate proteins in the first pool to bind 
to the non-nucleic acid target molecule in the presence of the second 
molecule; 

(e) isolating one or more proteins in the first pool that bind to the target 
molecule; 

(f) allowing one or more of the candidate proteins in the second pool to 
bind to the non-nucleic acid target molecule in the presence of the 
second molecule; 

(g) isolating one or more proteins in the second pool that bind to the target 
molecule; and 

(h) comparing the level of one or more proteins isolated from each of the 
first and second pools to identify a protein that is differentially 
represented between the first and second pools. 

In this form of the present invention, the identification of proteins that bind to a 
target molecule and which are differentially represented between two pools of 
candidate proteins may also be achieved. 



WO 03/003012 PCT/AU02/00856 

-28- 

Preferably, the reactions utilising the first and second pools are performed in 
parallel experiments under exactly the same conditions. 

The determination of the level of a protein isolated from the first pool may be 
5 achieved by a suitable procedure, including the determination of the 
concentration by methods that include the use of antibodies to detect the binding 
protein. For example, the concentration of the binding protein isolated from the 
first pool of candidate proteins may be achieved with an antibody raised to the 
binding protein, and the subsequent use of the antibody to visualise the protein 
10 by Western analysis or the use of the antibody to immunoprecipitate the protein. 

The level of the binding protein isolated from a second pool of candidate proteins 
may then be determined in a similar fashion. In this way, proteins isolated from 
the first pool of candidate proteins may be compared with those isolated from a 
15 second pool of candidate proteins, so as to identify isolated proteins that are 
differentially represented between the two pools of binding proteins. 

For example, the first pool of proteins may be derived from a tissue that contain 
cells that are normal (non-cancerous), and the second pool of proteins may be 

20 derived from cells that ate pre-cancerous (having acquired some but not all of 
the cellular mutations required for a cancerous genotype) or cancerous cells 
(malignant or benign). Any differences in the level of an isolated binding protein 
between the protein isolated from a normal tissue and that isolated from another 
tissue allows the identification of binding proteins that are differentially 

25 represented between the two different pools of proteins. 

Description of the Preferred Embodiments 



The present invention will now be described in relation to various examples of 
30 preferred embodiments. However, it must be appreciated that the following 
description is not to limit the generality of the above description. 



WO 03/003012 PCT/AU02/00856 

-29- 

Example 1 - Synthesis ofanafogues ofsulindac sulfide and attachment to a solid 
phase 

Sulindac sulfide is a drug that acts to decrease the number of precancerous 
5 lesions (adenomas) in the colon both in animals and in humans. 

The structure of sulindac sulfide is as follows: 

-C0 2 H 




MeS 



10 

An analogue of sulindac sulfide, designated as compound (IV), was prepared 
from 2-fluoroanisole as shown in Scheme 2. 



WO 03/003012 



-30- 



PCT/AU02/00856 




(28) 




C0 2 H 



MeS 



Scheme 2 



PCT/AU02/00856 



WO 03/003012 31 



^^^^ 

5 Anhydrous jlT^"* 
2 . flu oroanisole (16.78 9, 0.133 mol), P ^ ^ 

» 1 h ,he Mrogen onto* *-» ho mogeneous. Upon 

for a Mrther 3 h. Duhng this » ^ 
10 cooiing, — (SO -3, and ^^^^.A* 

N.-^-*-:; [ZZ1 ad-s 2M sodium hydro* 
combined toluene extracts were wa ^ 

(2x75 cm3) and 

— - s *- (M C' n ; ™ ^ 30 o «* . « * 

15 (11) as a colourless oil (22-68 &»'*) 
3H); 4.53 (a, 2H), 6.92 (m,lH); 7.11 (m.2H). 

me tftylpfopane-1,Wtoa»(12) 

, tB( 27 87 9 0.160 mO l)v.asadded«oas«r f edso.u«onot 
melhy-malonale (27.87 9, ^ „ M 

sodium epoxide (0.160 mol) in an^dro ^ ^ ^ 

25 cooling and removal o. most of* ^ fc ^ 

(100 om3) and ether (100 A The ,W» ^ ^ ^ 

U washed -t more elher. The com «d* ^ ^ ^ 
seated auueous sodium oh,onde. The there, ^ ^ 

..eredandovaporatedrnvacuotosrvethdr^eO) i4(si 
M 8 , 9 1 % ,lH.N M R(COa3,300MHz): 8 1.25M7H,6H), 

L);3.85 ( s,3H,;4,9«,, J 7H Z ,4H,;6.8a(m,3H). 



WO 03/003012 



-32- 



PCT/AU02/00856 



Example 1.3 - Synthesis of 3-(3-tiuoro-4-methoxyphenyl)-2-methylpropanoic 
acid (13) 

The diester (12) was added to a solution of potassium hydroxide (40.00 g, 0.713 
5 mol) in water (60 cm 3 ) and ethanol (50 cm 3 ) and the mixture refluxed for 16 h. 
Upon cooling, a mixture of concentrated sulfuric acid (70 g) and water (50 cm 3 ) 
was added carefully and reflux recommenced. After 15 h, the reaction mixture 
was cooled and extracted with ether three times. The combined ether washings 
were shaken with saturated aqueous sodium chloride then dried (MgS04), 
10 filtered and evaporated in vacuo to give the acid (13) as a pale yellow oil (27.13 
g, 89%). 1H-NMR (CDCI3, 300 MHz): 8 1.78 (d, J 7 Hz, 3H); 2.68 (m, 2H); 2.98 
(dd, J 6 Hz, 1 3 Hz, 1 H); 3.87 (s, 3H); 6.90 (m, 3H). 

Example 1.4 - Synthesis of5-fluoro-6-methoxy-2-methylindan-1-one (14) 

15 

The acid (13) (26.95 g, 0.127 mol) was added to polyphosphoric acid (270 g) at 
room temperature. The mixture was swirled by hand in an oil bath heated to 90° 
until homogeneity was achieved. The viscous solution was stirred magnetically 
for a further 2 h at 90'. The hot mixture was then poured on to crushed ice (500 
20 g), ether (100 cm 3 ) was added and the mixture stirred at room temperature for 
15 h. The layers were separated and the aqueous layer washed with more ether 
(2x50 cm 3 ). These ethereal washings were combined with the original ether 

layer and washed with saturated aqueous sodium bicarbonate (50 cm 3 ) followed 
by saturated aqueous sodium chloride. Drying (MgS04), filtration and removal of 

25 solvent gave the indanone as a white solid (23.32 g, 95%). 1 H-NMR (CDCI3, 300 
MHz): 8 1.30 (d, J 7.5 Hz, 3H); 2.69 (m, 2H); 3.31 (dd, J 7.5 Hz, 17 Hz, 1H); 3.91 
(s, 3H); 7.12 (dt, J 10.5 Hz, 1 Hz, 1H); 7.29 (d, J 8 Hz, 1H). 

Example 1.5 - Synthesis of5-fluoro-6-hydroxy-2-methylindan-1-one (15) 

30 

The methoxyindanone (14) (23.00g, 0.118 mol) and tetrabutylammonium 
bromide (3.80 g, 11.8 mmol) were dissolved in 48% aqueous hydrogen bromide 



WO 03/00301 2 PCT/AU02/00856 

-33- 

(130 ml) and the stirred solution was heated at 115° for 5.5 h. Upon cooling, 
water (300 cm 3 ) and ether (200 cm 3 ) were added and the resultant layers 
separated. The aqueous layer was washed with more ether (2x50 cm 3 ) and 
these washings were combined with the original ether layer and extracted with 
5 5% aqueous sodium hydroxide (2x100 cm 3 ). The ether layer was discarded and 
the alkaline aqueous layer acidified to pH 1 with 50% aqueous sulfuric acid. 
Extraction with ether, drying (MgS04), filtration and evaporation of the ether in 
vacuo afforded the crude hydroxyindanone (20.55g) as a dark brown solid. "Dry 
Column" Flash Chromatography (13 cm diameter sintered glass funnel, 7 cm 

10 depth of flash silica, hexane/ethyl acetate gradient) gave the pure 
hydroxyindanone (15) (15.28 g, 72%) as a pale yellow solid. 1 H-NMR (CDCI3, 
300 MHz): 5 1.30 (d, J 7.5 Hz, 3H); 2.70 (m, 2H); 3.31 (dd, J 7.5 Hz, 16.5 Hz, 
1H); 5.98 (broad s, 1H); 7.14 (d, J 10 Hz, 1H); 7.38 (d, J 8 Hz, 1H). Later 
fractions yielded the hydroxyindanone in lower purity: 2.37g (11%) of yellow solid 

15 pure enough (1 H-NMR ) for the next step and finally 2.33 g (11%) of a dark 
brown oil insufficiently pure ( 1 H-NMR ) for further use. 

Example 1.6 - Synthesis of 5-fluoro-2-methyl-6-(tert-butyldimethylsilyloxy)indan- 
1-one (16) 

20 

The hydroxyindanone (15) (9.01 g, 50.0 mmol) and imidazole (8.51 g, 0.125 mol) 
were dissolved in dry dimethylformamide (40 cm 3 ). Terf-Butyldimethylsilyl 
chloride (9.04 g, 60 mmol) was added to this solution and stirring was continued 
at room temperature for 16 h. The reaction mixture was combined with 5% 

25 aqueous sodium bicarbonate (210 cm 3 ) and extracted with hexane (75 cm 3 then 
2x30 cm 3 ). The combined hexane extracts were dried (MgS04), filtered and the 
solvent evaporated in vacuo to yield a pale yellow oil (15.30 g) which solidified 
upon standing at room temperature. 1 H-NMR analysis indicated that the product 
was a mixture containing the desired silyl ether (16) (ca. 86 mole %) and two 

30 unidentified components with just te/f-butyldimethylsilyl 1 H-NMR signals (total 
ca. 14 mole %). 1 H-NMR of the silyl ether (CDCI3, 300 MHz): 8 0.20 (s, 6H); 



WO 03/003012 



-34- 



PCT/AU02/00856 



1.00 (s, 9H); 1.29 (d, J 7 Hz, 3H); 2.68 (m, 2H); 3.31 (dcf, J 7.5 Hz, 16.5 Hz, 1H); 
7.10 (d, J 10 Hz, 1H); 7.25 (d, J 8 Hz, 1H). Without purification, this crude 
product was successfully used in the next step. 

5 Example 1.7 - Synthesis of ethyl 2-[5-fluoro-1-hydroxy-2-methyl-6-(tert- 
butyldimethylsilyloxy)indanyl]acetate (1 7) 

A 1.0 M solution of lithium hexamethyldisilazide in tetrahydrofuran (50 cm 3 , 50 
mmol) was cooled to -75' by stirring in a dry ice/acetone bath. Ethyl acetate (4.9 

10 cm 3 , 50 mmol) was added over a period of 3 minutes and stirred at -75' for a 
further 15 minutes. A solution of the crude ketone (16) (15.30 g, ca. 50 mmol) in 

dry tetrahydrofuran (30 cm 3 ) was added to the lithium enolate at a rate slow 
enough to keep the internal reaction temperature below -60' (ca. 20 minutes). 

After stirring at -75' for a further 5 minutes, 20% hydrochloric acid (10 cm 3 ) was 
15 added. After the mixture had warmed to room temperature, the tetrahydrofuran 
was removed and ether (100 cm 3 ) and water (50 cm 3 ) were added. The layers 
were separated and the aqueous phase washed with more ether (50 cm 3 ). The 
combined ether solutions were washed with saturated aqueous sodium chloride. 
Drying (MgSCH), filtration and removal of solvent gave the alcohol (17) (18.52 g, 

20 97%) as a yellow-orange oil. 1 H-NMR (CDCI3, 300 MHz): 8 0.1 7 (s, 6H); 0.99 (s, 
9H); 1.08 (d, J 6.5 Hz, 3H); 1.28 (t, J 7 Hz, 3H); 2.38 (m, 1H); 2.56 (m, 1H); 2.72 
(m, 2H); 2.91 (dd, J 7.5 Hz, 15.5 Hz, 1H); 4.20 (q, J 7 Hz, 2H); 6.87 (m, 2H). 

Example 1.8 - Synthesis of ethyl 2-[6-fluoro-2-methyl-5-(tert- 
25 butyldimethylsilyloxy)inden-3-ylJacetate (18) 

Sicapent™ (Merck, 17.8 g; 80% diphosphorus pentoxide, 13.4 g, 94.2 mmol) 
was added to a solution of the alcohol (17) (18.00 g, 47.1 mmol) in benzene. The 
mixture was refluxed for 0.5 h, cooled and filtered through flash silica. Elution 

30 with more benzene (50 cm 3 ) was followed by elution with ether until the eluate 
was colourless. The benzene and ether solutions were pooled and evaporated in 
vacuo to give a mixture of dehydration products (16.29 g, 95%) as a yellow- 



I 

WO 03/003012 PCT/AU02/00856 

-35- 

orange oil. 1 H-NMR indicated that the indene (18) was the major product (ca. 
75%). (CDCI3, 300 MHz): 8 0.18 (s, 6H); 1.01 (s, 9H); 1.24 (t, J 7 Hz, 3H); 2.09 
(s, 3H); 3.25 (s, 2H); 3.45 (s, 2H); 4.13 (q, J7 Hz, 2H); 6.80 (d, J8 Hz, 1H); 7.05 
(d, J 11 Hz, 1H). The remaining 25% of the mixture consisted of the (£) and (2) 
5 isomers of the corresponding exo alkene. Acid catalysed desilylation in the next 
step also isomerised this material to the desired indene. 

Example 1.9 - Synthesis of 2-(6-fluoro-5-hydroxy-2-methylinclen-3-yl)acetic acid 
(19) 

10 

The silyl ether (18) was added to ethanol (200 cm 3 ) which had been pre-treated 
with acetyl chloride (4.3 g, 55 mmol) and the solution refluxed for 3 hours. Upon 
cooling, the volatile components of the mixture were removed in vacuo and the 
residue was taken up into ether (100 cm 3 ). The ether solution was extracted with 

15 1 M aqueous sodium hydroxide solution (50 cm 3 then 25 cm 3 ) and then 
discarded. Acidification of the aqueous washings with 25% w/w aqueous sulfuric 
acid was followed by extraction with ether (1x70 cm 3 then 2x30 cm 3 ). The 
combined ether washings were dried (MgS04), filtered and the ether evaporated 
in vacuo to give the phenolic carboxylic acid (19) (9.12 g, 94%) as a beige solid. 

20 1 H-NMR (CDCI3, 300 MHz): 5 2.09 (s, 3H); 3.26 (s, 2H); 3.50 (s, 2H); 6.88 (d, J 
8 Hz, 1H);7.08(d, J10Hz,1H). 

Example 1.10 - Synthesis of ethyl 2-(6-fluoro-5-hydroxy-2-methylinden-3- 
yl)acetate (20) 

25 

The acid (19) (9.12 g, 41.0 mmol) was added to a solution of concentrated 
sulfuric acid (2.00 g, 20.4 mmol) in ethanol (100 cm 3 ). The solution was refluxed 
for 3.5 h, cooled and the ethanol removed in vacuo. The residue was taken up 
into ether (100 cm 3 ) and the solution washed with water (3x50 cm 3 ) followed by 
30 saturated aqueous sodium chloride. The ether solution was dried (MgS04), 
filtered and the ether evaporated in vacuo to give the ester (20) (9.75 g, 95%) as 



WO 03/003012 PCT/AU02/00856 

-36- 

a beige solid. 1 H-NMR (CDCI3, 300 MHz): 8 1.25 (t, J 7 Hz, 3H); 2.09 (s, 3H); 
3.25 (s, 2H); 3.46 (s, 2H); 4.14 (q, J 7 Hz, 2H); 6.89 (d, J 8 Hz, 1H); 7.07 (d, J 10 
Hz, 1H). 

5 Example 1.11 - Synthesis of 2-{2-[2-(triphenylmethoxy)ethoxy]ethoxy}ethan-1-ol 
(21) 

A solution of trityl chloride (13.94 g, 50.0 mmol) in dichloromethane (25 cm 3 ) 
was added to a stirred solution of tri(ethylene glycol) (15.02 g, 0.100 mol) and 

10 triethylamine (7.69 g, 75.0 mmol) in dichloromethane (50 cm 3 ) at 0*. Stirring at 
0° was continued for 2 h and then at room temperature for a further 14 h. The 

mixture was washed with 1 M hydrochloric acid (50 cm 3 ), followed by water (50 
cm 3 ) and saturated aqueous sodium chloride (50 cm 3 ). Drying (MgS04), 
filtration and evaporation of dichloromethane in vacuo gave an orange-brown oil 
15 which was purified by "Dry Column" Flash Chromatography (13 cm diameter 
sintered glass funnel, 7 cm depth of flash silica, hexane/ethyl acetate gradient) 

gave pure mono-tritylated material (21) (10.91 g, 56%) as a colourless oil. 1 H- 
NMR (CDCI3, 300 MHz): 8 3.30 (t, J 5 Hz, 2H); 3.71 (m, 10H); 7.31 (m, 9H); 7.52 
(m, 6H). 

20 

Example 1.12 - Synthesis of ethyl 2-[6-fluoro-2-methyl-5-(2-{2-[2- 
(triphenylmethoxy)ethoxy]ethoxy}ethoxy)inden-3-yl]acetate (22) 

Diethyl azadicarboxylate (3.05 g, 17.5 mmol) was added slowly to a stirred 
25 solution of the alcohol (21) (6.26 g, 15.9 mmol), the phenol (20) (3.99 g, 15.9 
mmol) and triphenylphosphine (4.60 g, 17.5 mmol) in dry tetrahydrofuran (70 

cm 3 ) at 0*. Stirring was continued at 0° for 1 h and then for a further 66 h at 
room temperature, at which time TLC analysis indicated all of the phenolic 
starting material had been consumed. The tetrahydrofuran was removed in 

30 vacuo and the residue taken up into dichloromethane (50 cm 3 ) and loaded on to 
a bed of flash silica (6 cm depth, 10 cm diameter glass sinter funnel). "Dry 
Column" Flash Chromatography (hexane/ethyl acetate gradient) gave the pure 



WO 03/003012 PCT/AUOZ/00856 

-37- 

ether (22) (9.35 g, 94%) as a colourless oil. 1H-NMR (CDCI 3) 300 MHz): 5 1.26 
(t, J 7 Hz, 3H); 2.14 (s, 3H); 3.29 (t, J 5 Hz, 2H); 3.29 (s, 2H); 3.50 (s, 2H); 3.78 
(m, 6H); 3.95 (t, J 5 Hz, 2H); 4.16 (q, J 7 Hz, 2H); 4.26 (t, J 5 Hz, 2H); 6.96 (d, J 
7.5 Hz, 1H); 7.12 (d, J11 Hz, 1H); 7.30 (m, 9H); 7.51 (m, 6H). 

5 

Example 1.13 - Synthesis of 2-[6-fluoro-2-methyl-1-[(4- 
methylthiophenyl)methylene]-5-(2-{2-[2- 
(triphenylmethoxy)ethoxy]ethoxy}ethoxy)inden-3-yl]acetic acid (23) 

10 A ca. 0.5M solution of sodium methoxide in methanol was made by adding 
sodium (229 mg, 9.52 mmol) to dry methanol (20 cm3). The indene (22) (2.98 g, 
4.76 mmol) and 4-methylthiobenzaldehyde (797 mg, 5.24 mmol) were dissolved 
in this solution by swirling of the flask by hand. The resultant bright purple 
solution was refluxed for 1 h. During this time the solution turned orange and an 
15 orange oil separated out. Water (20 cm 3 ) was added and reflux continued for a 
further 0.5 h and the mixture became homogeneous. Water (250 cm 3 ) and ether 
(100 cm 3 ) were added to the cooled orange solution and the resultant emulsion 
broken by the addition of sodium chloride. The ether layer was discarded and 
the orange oil which had precipitated from the aqueous phase was dissolved 
20 with hot water. The aqueous solution was acidified with acetic acid (4 cm 3 , 70 
mmol) and extracted with ether (1x100 cm 3 then 2x50 cm 3 ). The combined 
ether washings were dried (Na2S04), filtered and evaporated to dryness to give 
2.60 g (75%) of the product (23) as a viscous orange oil. 1 H-NMR (CDCI3, 300 
MHz) revealed the presence of two geometric isomers in a ca. 5:1 ratio. Data for 
25 the major isomer (2): 5 2.21 (s, 3H); 2.59 (s, 3H); 3.29 (t, J 5 Hz, 2H); 3.50 (s, 
2H); 3.76 (m, 6H); 3.92 (t, J 5 Hz, 2H); 4.29 (t, J 5 Hz, 2H); 6.94 (d, J 8 Hz, 1H); 
7.14 (s, 1H); 7.30 (m, 12H); 7.51 (m, 8H). 



Example 1.14 - Synthesis of 2-(6-fluoro-5-{2-[2-(2- 
30 hydroxyethoxy)ethoxy]ethoxyh2-methyl-1-[(4-methylthiophenyl)meth 
3-yl)acetic acid (IV) 



WO 03/003012 PCT/AU02/00856 

-38- 

The trityl ether (23) (2.60 g, 3.56 mmol) was dissolved in a mixture of formic acid 
(40 cm 3 ) and ether (40 cm 3 ). After 16 h at room temperature, the solvents were 
evaporated in vacuo and the residue was taken up into ether (50 cm 3 ) and 0.2 M 
aqueous sodium hydroxide (50 cm 3 ). The layers were separated and the 
5 aqueous phase extracted with more ether (2x30cm 3 ), then acidified with 1 M 

hydrochloric acid. The acidified mixture was extracted with ether (3x50cm 3 ) and 
the combined ether extracts were dried (Na2S04), filtered and the ether 
evaporated in vacuo to give the crude product (1.56 g) as an orange solid. 
Recrystallisation from ethyl acetate/hexane gave the pure (2) isomer (IV) (0.823 

10 g, 45%) as orange needles. 1 H-NMR (CDCI3, 600 MHz): 8 2.18 (s, 3H); 2.55 (s, 
3H); 3.58 (s, 2H); 3.63 (t, J 5 Hz, 2H); 3.68 (m, 4H); 3.76 (t, J 5 Hz, 2H); 3.81 (t, 
J 5 Hz, 2H); 4.30 (t, J 5 Hz, 2H); 7.00 (d, J 8 Hz, 1H); 7.10 (s, 1H); 7.18 (d, J 
12.5 Hz, 1H); 7.30 (d, J 8 Hz, 2H); 7.43 (d, J 8 Hz, 2H). 

15 This compound was used as an example of the chemistry that may be used to 
attach a drug to a solid phase. 

To attach analogue (IV) to a solid phase, the alcohol group was converted to the 
corresponding 4-nitrophenyl carbonate. This active carbonate was reacted with 
20 TentaGel S-NH2 to yield the polymer-supported derivative (27). 

Example 1.15 - Synthesis of methyl 2-(6-fluoro-5-{2-[2-(2- 
hydroxyethoxy)ethoxy]ethoxy}-2-methyl-1-[(4-methylthiophenyl)methylene]inden- 
3-yl)acetate (25) 

25 

Material isolated from the mother liquor of recrystallisation of the acid (IV) (1 .23 

g, 2.52 mmol) was dissolved in methanol (40 cm 3 ). Sulfuric acid (200 mg, 2.04 
mmol) was added and the solution was refluxed for 3 h. Upon cooling, the 

methanol was removed, ether (30 cm 3 ) and water (30 cm 3 ) were added and the 
30 phases separated. The ether phase was washed with saturated aqueous sodium 
bicarbonate solution (2x20 cm 3 ) then saturated aqueous sodium chloride 
solution (20 cm 3 ). Drying (Na2S04), filtration and removal of the solvent in 



WO 03/003012 

PCT/AU02/00856 

- 39 ' 

vacuo gave 1.22 g of crude product. This material was purified by radial 
chromatography ("Chromatotron") using an ethyl acetate/hexane gradient to give 
539 mg of an orange solid which still contained some of the minor (£) isomer. 
Recrystallisation (ethyl acetate/hexane) gave the pure (2) isomer (25) (296 mg, 
5 23%) as orange plates. 1 H-NMR (CDCI 3( 300 MHz): 5 2.17 (s, 3H); 2.55 (s, 3H); 
3.56 (s, 2H); 3.66 (m, 2H); 3.72-3.82 (m, 6H); 3.74 (s, 3H); 3.88 (t, J 5 Hz, 2H); 
4.23 (t, J 5 Hz, 2H); 6.84 (d, J 8 Hz, 1H); 7.10 (s, 1H); 7.24 (d, J 12 Hz, 1H);' 7.29 
(d, J 8 Hz, 2H); 7.43 (d, J 8 Hz, 2H). 

10 Example 1.16 - Synthesis of methyl 2-[6-fluoro-2-methyl-1-[(4- 
methylthiophenyl)methylene]-5-(2-{2-[2-(4- 
nitrophenoxy(^rbonyloxy)ethoxy]ethoxy}ethoxy)ind^ 

4-Nitrophenyl chloroformate (45 mg, 0.220 mmol) was added in one portion to a 
15 stirred solution of the alcohol (25) (98 mg, 0. 1 95 mmol), /V-methylmo/pholine (30 
mg, 30 mmol) and dimethylaminopyridine (1.2 mg, 10 mmol) in dichloromethane 
(5 cm3) at 0'. The ice bath was removed and the mixture was stirred for 15 h. 
The dichloromethane was removed in vacuo and ethyl acetate and 1 M 
hydrochloric acid were added. The layers were separated and the organic phase 
20 washed with more 1 M hydrochloric acid, followed by saturated aqueous sodium 
hydrogen carbonate and finally with saturated aqueous sodium chloride. Drying 
(MgS0 4 ), filtration and removal of solvent in vacuo gave the crude product which 
was purified by Flash Chromatography (3:2 ethyl acetate/hexane) to give the 
pure active carbonate (26) (98 mg, 75%) as an orange oil. 1 H-NMR (CDCI 3 , 300 
MHz): 5 2.17 (s, 3H); 2.55 (s, 3H); 3.55 (s, 2H); 3.73 (s, 3H); 3.77 (m, 4H); 3.83 
(m, 2H); 3.89 (t, J 5 Hz, 2H); 4.23 (t, J 5 Hz, 2H); 4.43 (m, 2H); 6.82 (d, J 8 Hz, 
1H); 7.10 (s, 1H); 7.19 (d, J12 Hz, 1H); 7.29 (d, J8 Hz, 2H); 7.45 (d, J9.5 Hz,' 
2H); 7.44 (d, J 8 Hz, 2H); 8.24 (d, J 9.5 Hz, 2H). 



25 



30 



Example 1.17 - Synthesis of TentaGel bound methyl 2-(6-fluoro-$J2-[2-(2- 

hydroxyethoxy)ethoxylethoxy}-2-methyl-1-[(^ 

3-yl)acetate (27) 



WO 03/003012 



PCT/AU02/00856 



-40- 



TentaGe! S-NH2 (Fluka, ca. 0.45 mmol N/g resin, particle size 150-200 mm; 50 
mg) was added to a solution of the carbonate (26) (20 mg, 30 mmol) and A/- 

methylmorpholine (30 mg, 0.30 mmol) in dimethylformamide (1 cm 3 ). The 
5 mixture was shaken at room temperature for 16 h and filtered. The resin was 
washed with dimethylformamide (3x) followed by methanol (3x) then dried in 
vacuo to give 61 mg of the resin (27). IR (potassium bromide disc): v 1734, 1718 

cm" 1 . 

10 Example 1.18 - Synthesis of TentaGel bound 2-(6'fiuoro-5'{2-l2'(2' 
hydroxyethoxy)ethoxy]ethoxyy2-methyl-1-[(4-m^ 
3-yl)acetic Acid (28) 

The resin (27) (58 mg) was added to a 0.25 M solution of sodium hydroxide in 

15 2:1 ethanol water (4 cm 3 ). The mixture was shaken at room temperature for 4 h 
and filtered. The resin was washed with water (3x) then 1 M hydrochloric acid 
(3x), water (3x) and finally methanol (3x). Drying in vacuo gave 58 mg of the 

modified resin. IR (potassium bromide disc): v 1718 cm"" 1 . 

20 Alternatively, the sulindac analogue (IV) was conjugated with glycine and the 
amine thus produced was coupled to a commercially available biotin derivative to 
give the biotin labelled compound (29). The biotin derivative was then attached 
to a solid support by way of the biotin moiety using a biotin:streptavidin coupling 
technique. 



25 



0 

X 

HN NH 
H4-4-H 




(29) 



PCT/AU02/0A856 



WO 03/003012 



■ 41- 



• . ran of sulindac sulfide was also produced by 



biotin derivative. 




HN^NH 

If 
0 



(30) 



«nri (32^ were produced via the same 

rrr^r.-- 



10 coupling. 



Me-! 




cT 7 



(3D 



H 
NH 




(32) 



15 



WO 03/003012 PCT/AU02/00856 

-42- 

Example 2 - Synthesis of analogues of flurbiprofen and attachment to a solid 
phase 

Flurbiprofen is a drug that acts to decrease the number of precancerous lesions 
5 (adenomas) in the colon in animals. The structure of flurbiprofen is as follows: 




10 

Compounds of formula (III) were produced by following the method provided in 
Scheme 3. 



WO 03/003012 



-43- 



PCT/AU02/00856 




WO 03/003012 



-44- 



PCT/AU02/00856 



Example 2.1 - Synthesis of methyl (2R,S)-(3-fluoro-4-phenylphenyl)propanoate 
(41) 

5 

Methanolic hydrogen chloride was generated by the careful addition of thionyl 

chloride (0.57 g, 4.7 mmol) to methanol (16 cm 3 ) at 5*. (2fl,S)-(3-fluoro-4- 
phenylphenyl)propanoic acid (Flurbiprofen; 1 .00 g, 4.09 mmol) was added and 
the solution stirred at 5° for 4 h, followed by stirring for a further 15 h at room 
10 temperature. The methanol was removed in vacuo to give the methyl ester (41) 

(1.06 g, quantitative) as a colourless oil. 1 H-NMR (CDCI3, 200 MHz): 5 1.54 (d, J 
7 Hz, 3H); 3.69 (s, 3H); 3.75 (q, J 7 Hz, 1 H); 7.0-7.6 (m, 8H). 

Example 2.2 - Synthesis of methyl (2R,S)-[4-(4-acetylphenyl)-3- 
15 fluorophenylpropanoate (42) 

A solution of methyl ester (41) (1.00 g, 3.87 mmol) and acetyl chloride (0.58 g, 
7.4 mmol) in dry dichloromethane (5 cm 3 ) was added slowly to a stirred slurry of 

aluminium chloride (1.40 g, 10.5 mmol) and dry dichloromethane (5 cm 3 ) at 0°. 
20 The mixture was stirred at room temperature for 2 h and then poured on to ice 
(25 g). The phases were separated and the aqueous phase was washed twice 
with more dichloromethane. All of the organic washings were combined and 
dried (MgS04). Filtration and removal of the solvents in vacuo gave the ketone 

(42) (0.976 g, 84%) as a yellow oil. 1 H-NMR (CDCI3, 200 MHz): 8 1.52 (d, J 7 
25 Hz, 3H); 2.62 (s, 3H); 3.69 (s, 3H); 3.76 (q, J 7 Hz, 1H); 7.10 (m, 2H); 7.40 (t, J 8 
Hz, 1H); 7.61 (m, 2H); 8.00 (m, 2H). 

Example 2.3 - Synthesis of methyl (2R,S)-[4-(4-acetyloxyphenyl)-3- 
fluorophenylpropanoate (43) 

30 

Mefa-Chloroperoxybenzoic acid (Aldrich 77%; 1 .495 g contained a maximum of 
1.151 g, 6.67 mmol of peracid) was added in one portion to a stirred solution of 



WO 03/003012 



-45- 



PCT/AU02/00856 



the ketone (42) (1 .00 g, 3.33 mmol) in dry dichloromethane (7 cm 3 ). The mixture 
was cooled in an ice bath and trifluoroacetic acid (379 mg, 3.32 mmol) was 
added over a 20 min period. Removal of the ice bath was followed by stirring at 
room temperature for 3 days, at which time tic. analysis indicated that 
5 consumption of starting material was complete. The mixture was diluted with 

more dichloromethane (20 cm 3 ) and washed with 10% aqueous sodium sulphite 

(10 cm 3 ), followed by saturated aqueous sodium carbonate (10 cm 3 ) and water 

(2x10 cm 3 ). The dichloromethane solution was dried (MgS04), filtered and 

evaporated in vacuo to give the diester (43) (754 mg, 72%) as a white solid. 1 H- 
10 NMR (CDCI3, 200 MHz): 5 1 .59 (d, J 7 Hz, 3H); 2.39 (s, 3H); 3.76 (s, 3H); 3.82 
(q, J 7 Hz, 1H); 7.20 (m, 4H); 7.28 (t, J 8 Hz, 1H); 7.60 (m, 2H). 

Example 2.4 - Synthesis of methyl (2R,S)-[3-fluoro-4-(4- 
hydroxyphenyl)phenyl]propanoate (44) 

15 

Water (30 cm 3 ) and saturated aqueous sodium bicarbonate (23 cm 3 ) were 
added to a solution of the phenolic acetate (43) (1.00 g, 3.16 mmol) in ethanol 

(100 cm 3 ). The mixture was stirred at room temperature for 15 h then its pH was 
adjusted to 4 with 10% hydrochloric acid. Most of the ethanol was removed in 

20 vacuo and the residue extracted with ethyl acetate (3x200 cm 3 ). The combined 
organic extracts were dried (MgS04), filtered and the solvent evaporated in 
vacuo. The crude product was purified by Flash Chromatography (30% ethyl 
acetate/70% hexane) to give the pure phenol (44) (613 mg, 71%) as pale yellow 

crystalline solid. 1H-NMR (Acetone-d6, 200 MHz): 8 1.47 (d, J 7 Hz, 3H); 3.64 (s, 
25 3H); 3.83 (q, J 7 Hz, 1H); 6.91 (m, 2H); 7.16 (m, 2H); 7.41 (m, 3H); 8.53 (s, 1H). 

Example 2.5 - Synthesis of methyl (2R,S)-{3-fluoro-4-[4-(2-{2-[2- 
(triphenylmethoxy)ethoxy]ethoxy}ethoxy)phenyl]phenyl}propanoate (45) 

30 Diethyl azadicarboxylate (404 mg, 2.32 mmol) was added slowly to a stirred 
solution of the alcohol (41) (909 mg, 2.32 mmol), the phenol (44) (600 mg, 2.19 



WO 03/003012 PCT/AU02/00856 

-46- 

' mmol) and triphenylphosphine (608 mg, 2.32 mmol) in dry tetrahydrofuran (30 

cm 3 ) at 0°. Stirring was continued at 0" for 3 h and then for a further 16 h at 
room temperature. The tetrahydrofuran was removed in vacuo and the residue 
taken up into a minimal amount of 2:3 ethyl acetate/hexane. Filtration removed 
5 the resultant white precipitate and the dissolved crude product was purified by 
Flash Chromatography (2:3 ethyl acetate/hexane) to give the pure phenolic ether 

(45) (895 mg, 63%) as pale yellow oil. 1H-NMR (CDCI3, 200 MHz): 5 1.51 (d, J 7 
Hz, 3H); 3.23 (t, J 5 Hz, 2H); 3.67 (s, 3H); 3.72 (m, 7H); 3.88 (t, J 5 Hz, 2H); 4.14 
(t, J 5 Hz, 2H); 6.93 (m, 2H); 7.10 (m, 2H); 7.30 (m, 10H); 7.44 (m, 8H). 

10 

Example 2.6 - Synthesis of methyl (2R,S)-[3-fiuoro-4-(4-{2-[2-(2- 
hydroxyethoxy)ethoxy]ethoxy}phenyl)phenyl]propanoate (46) 

A solution of the trityl ether (45) (6.94 g, 10.7 mmol) in ether (21 cm 3 ) and formic 

1 5 acid (21 cm 3 ) was stirred at room temperature for 7 min. The mixture was diluted 

with ether (100 cm 3 ) and washed with saturated aqueous sodium chloride 
solution. Washing with saturated aqueous sodium bicarbonate solution was 
repeated until carbon dioxide was no longer evolved. After washing again with 
saturated aqueous sodium chloride solution, the ether solution was dried 

20 (MgS04) and filtered. Evaporation of the ether in vacuo gave a crude product 
which was purified by Flash Chromatography (ethyl acetate) to give the pure 
alcohol (46) (3.14 g, 72%) as a colourless oil. ^H-NMR (CDCI3, 200 MHz): 8 
1.51 (d, J 7 Hz, 3H); 3.61 (m, 2H); 3.68 (s, 3H); 3.71 (m, 7H); 3.87 (t, J 5 Hz, 
2H); 4.17 (t, J 5 Hz, 2H); 6.97 (m, 2H); 7.09 (m, 2H); 7.34 (t, J 8 Hz, 1H); 7.44 

25 (m, 2H). 

Example 2.7 - Synthesis of (2R,S)-[3-fluoro-4-(4-{2-[2-(2- 
hydroxyethoxy)ethoxy]ethoxy}phenyl)phenyl]propanoic acid (III) 

30 The methyl ester (46) (1.60 g, 3.94 mmol) was dissolved in a solution of sodium 

hydroxide (520 mg, 13.0 mmol) in water (6.5 cm 3 ) and ethanol (13 cm 3 ). The 
solution was stirred at room temperature for 2 h and then adjusted to pH 7 with 



PCT/AU02/00856 

WO 03/003012 

-47- 

hydrochloric acid. The ethanol was removed in vacuo and the residue extracted 
with ether. The ether solution was dried (MgS0 4 ), filtered and the solvent 
removed to yield the acid (1.05 g, 68%) as an off-white solid. The aqueous layer 
was acidified to pH 1 and again extracted with ether; processing of the ether 
5 solution as above yielded more of the acid (III) (389 mg, 25%) as a white solid. 
Both samples of the acid gave identical 1 H-NMR spectra. 1 H-NMR (Acetone-d 6l 
200 MHz): d 1.49 (d, J 7 Hz, 3H); 3.56 (m, 2H); 3.67 (m, 7H); 3.84 (t, J 5 Hz, 
2H); 4.18 (t, J 5 Hz, 2H); 7.16 (m, 2H); 7.23 (m, 2H); 7.44 (t, J 8 Hz, 1H); 7.51 
(m, 2H). 



10 



Attachment of compound (III) to the solid phase was achieved by conversion to 
the corresponding 4-nitrophenyl carbonate, followed by reaction with TentaGel 
S-NH2 to give compound (50). 

15 Example 2.8 - Synthesis of methyl (2R,S)-{3-mon>4-l4-(2-{2-[2-(^ 
nitrophenoxy(^rbonyloxy)ethoxy]ethoxy}ethoxy)phenyl]pheny^ 

4-Nitrophenyl chloroformate (209 mg, 1.04 mmol) was added in one portion to a 
stirred solution of the alcohol (III) (211 mg, 0.519 mmol), N-methylmorpholine 
20 (158 mg, 1.56 mmol) and dimethylaminopyridine (6.3 mg, 52 mmol) in 
dichloromethane (5 cm3) at 0\ The ice bath was removed and the mixture was 
stirred for 15 h. The dichloromethane was removed in vacuo and ether and 1 M 
hydrochloric acid were added. The layers were separated and the ether phase 
washed with more 1 M hydrochloric acid, followed by saturated aqueous sodium 
25 hydrogen carbonate and finally with saturated aqueous sodium chloride. Drying 
(MgS04), filtration and removal of solvent in vacuo gave the crude product which 
was purified by radial chromatography ("Chromatotron") using an ethyl 
acetate/hexane gradient to give the pure active carbonate (48) (192 mg, 65%) as 
a colourless oil. 1 H-NMR (CDCI3, 200 MHz): 8 1.53 (d, J 7 Hz, 3H); 3.70 (s, 3H); 
30 3.76 (m, 5H); 3.82 (m, 2H); 3.90 (m, 2H); 4.17 (m, 2H); 4.45 (m, 2H); 6.97 (m, 
2H); 7.09 (m, 2H); 7.3-7.5 (m, 3H); 7.37 (d, J 9 Hz, 2H); 8.26 (d, J 9 Hz, 2H). 



WO 03/003012 



-48- 



PCT/AU02/00856 



Example 2.9- Synthesis of TentaGel bound methyl (2R,S)-[3-fluoro-4-(4-{2-[2- 
(2-hydroxyethoxy)ethoxy]ethoxy}phenyl)phenyl]propanoate (49) 

TentaGel S-NH2 (Fluka, ca. 0.45 mmol N/g resin, particle size 150-200 mm; 106 
5 mg) was added to a solution of the carbonate (48) (68 mg, 120 mmol) and N- 

methylmorpholine (100 mg, 0.99 mmol) in dimethylformamide (5 cm 3 ). The 
mixture was shaken at room temperature for 16 h and filtered. The resin was 
washed with a 5% (v/v) solution of diisopropylethylamine in dimethylformamide 
(5x) followed by dimethylformamide (3x) and methanol (3x), then dried in vacuo 
10 to give 114 mg of the modified resin (49). IR (potassium bromide disc): v 1734, 
1718 cm-1. 

Example 2.10 - Synthesis of TentaGel bound (2R,S)-[3-fluoro-4-(4-{2-[2-(2- 
hydroxyethoxy)ethoxy]ethoxy}phenyl)phenyl]propanoic acid (50) 

15 

The resin (49) (80 mg) was added to a 0.50 M solution of sodium hydroxide in 

2:1 ethanol water (3 cm 3 ). The mixture was shaken at room temperature for 4 h 
and filtered. The resin was washed with water (3x) then 1 M hydrochloric acid 
(3x), water (3x) and finally methanol (3x). Drying in vacuo gave 78 mg of the 

20 resin (50). IR (potassium bromide disc): v 1718 cm" 1 . 
Example 3 - Phage display library construction 

Phage display libraries were constructed using standard protocols for directional 
25 cloning of cDNA. The bacteriophage phage display lambda vectors were 
prepared as outlined below. The vectors used were T7 Select 1-1b, T7 Select 
10-3, MooDc and Xvsx.1. cDNA libraries were made using RNA isolated from 
human adenomas and normal colonic tissue. The use of two different cDNA 
libraries not only allows the identification of proteins in a particular sample that 
30 bind to a target molecule in the presence of a second molecule, but also allows 
the identification of any binding proteins that may be differentially represented 
between the two populations of cells. 



WO 03/003012 



-49- 



PCT/AU02/00856 



CDNA for cloning into the T7 Select veotor was synthesised using standard 
procedures with mg ^ primens ^ £coWHM|| ^ ^ for 

cloning into XfooDc was synthesised using Ecofli random primers «■ 
5 TCNNNNNN 3') and HMWEcM linkers (5' ATTCMGCTTGAAT 3'). 

cDNA for cloning Into Xvsx.1 was synthesised using Nd random primers (5' 
GCNNNNNN 3') and Ecofll/Afotl linkers (5' GGCCGCGAATTCGCGGCC 3). 

10 Size selecfion of cDNA was performed on Size^ep 400 columns according to 
standard protocols. 

Vector arms and cDNA were ligated overnight a. 16-C, at a cDNA to arms ratio 
of OSpgSSng in a volume of 10pj or less. Ligations were packaged according to 
15 standard protocols. The libraries were titred, amplified and stored using standard 



:o 



The preparation of vectors Xvsx.1 and XfooDc were as foBows: bacteriophage 
DNA was isolated by the plate lysate method, by piating 20 x 150mm dishes for 
confluent lysis (4 x 10'pfu/dish) using LE392MP as host cells. The phage was 
eluted in SM buffer <10ml/dish) for 6 hours, halted and 1% v/v CHCI added 
The eluate was spun at 3000rpm for 10 minutes and the supernatant recovered' 
The DNase I concentration was adjusted to 2 pg/ml and incubated at 37°C for 60 
mrnutes. The mixture was adjusted to 10% w/v PEG8000, 1CCmM NaCI by 
drssolution and phage precipitated at 4°C overnight. The mixlure was spun at 
4500rpm for 20 minutes and the phage pellet resuspended in 4ml SM buffer and 
.mastered to 1.5ml mlcrofuge lubes. A spin of 2 minutes a, room temperature 
was performed, supernatant pooled and TEAE-cellulose pre-equilibrated as 
follows was added: 

Wash 1 .5 g dry weight resin in 3 x 1 0 ml SM buffer 
Adjust to 80% slurry 
Rotate for 10 minutes 

Spin at 3000rpm for 10 minutes and filter supernatant (0.45 urn). 



WO 03/003012 



-50- 



PCT/AU02/0085f> 



Proteinase K and EDTA were added to a final concentration of 50jxg/ml and 
20mM respectively, incubated at 45°C for 30 minutes and 0.05 volumes 
5%CTAB/0.5M NaCI added. This was incubated at 68°C for 10 minutes, chilled 
5 on ice for 2 minutes, spun in a microfuge at room temperature for 10 minutes the 
pellet resuspended in 1.2M NaCI and add 2.5 volumes EtOH added, spun 10 
minutes at room temperature and the pellet resuspended in TE pH 8.0. The 
mixture was extracted with buffered phenol/ CHCI 3 and the aqueous phase 
recovered. DNA was precipitated with 0.1 volumes 3M NaOAc and 2.5 volumes 
10 EtOH, resuspended in TE pH 8.0 and concentration adjusted to 0.5 \ig/\i\ 

The cos ends were ligated as follows: 
Mix 42|xlADNA(0.5jig/fil) 

5^lI 10 x Ligation Buffer (including DTT and ATP) 
15 10 units T4 DNA Ligase 

H 2 0 to 50pJ 

The reaction was incubates at room temperature overnight (reaction becomes 
very viscous) 

20 

Vector Arms were prepared as follows: 
Xvsx.1 Digest with EcoR\ and Wofl 
XfooDc Digest with Hindltt and EcoR\ 

25 Ligation reactions were adjusted to 1 x Ecofll buffer in a final volume of 200 
and included 40 units each of the appropriate enzymes (also include 0.1mg/ml 
BSA in the Xvsx.1 digest). Reactions were incubated at 37°C overnight. A further 
10 units of each enzyme was added and incubated overnight at 37°C. 

30 Vectors were then treated with alkaline phosphatase to reduce background of 
non-recombinants in the library. Ligation/digestion was monitored by agarose gel 
electrophoresis. The vector was extracted with buffered phenol/ CHCI 3 and the 
aqueous phase recovered. DNA was precipitated with 0.1 volumes 3M NaOAc 



PCT/AU02/00856 

WO 03/003012 

-51 - 

and 2.5 volumes E.OH and resuspend in TE pHS.O. Concentrator, was adiusfed 
to o.5ng/^l. 

Exampfe 4 ■ Ubrary screening for pro>eins that b/nd fo a ape* drug coupled to 
5 a solid support, in the presence of a free drug analogue 

Binding of proteins displayed on the surface o, a phage to the (Renter of the 
following molecule in the presence of the inactive (SHsomer: 




CO2H 



10 



Resin alone with linker attached, and drug covalenty coupled ,0 coated res n. 
were hlooked with 2% skim milk in binding buffer by constan. mixing for 1 hour a 
1m temperature. The resins we. washed 5 times with , .5 m, of b,nd,ng buffer 
15 (20 mM Tris HCI pH 7.5, 0.25 Ml NaCI, 0.t%Tween 20). 

10 « phage pa«es <T7 Select 1-tb, T7 Select 10-3, HooDc or Xvsxf) 
containing cloned adenoma and nonna, colon cDNA librartea were ^ 
with resin (25 mg) plus linker in binding buffer with constant moong for 1 hou 
20 room temperature and the phage containing supernatant recovered, ,n order .0 
remove any phage that may bind to the resin/linker alone. 

The resultant phage supernatant was then incubated wiUi drugged resin (20 
m ) a. room temperature with constant mixing for 2 hours in the presence of 0.5 
25 ,0 5 mM inactive drag analogue, being fhe (S)-isomer 0. the above molecule. 

The drug-coated resin was washed 5 times with 1.5 ml of binding buffer. Tne 
pound phage were eluted from .he resin by washes w«h increasing 
concentrations of free drug from 10 nM-100 * using ten fold increments in 
30 binding buffer over a total period 0. 2-16 hours at room temperature w«h 



WO 03/003012 PCT/AU02/00856 

-52- 

constant mixing. For T7 phage a further elution of the resin with 1% SDS for 5 
minutes at room temperature was required. 

For re-use of the resin for subsequent steps, the resin is stripped with 5% SDS, 
5 washed 5 times with binding buffer, and stored at 4°C for next round of selection. 

Eluted phage were titred, amplified by re-infection of a competent host and the 
newly enriched pool phage titred using standard protocols. 

10 Twenty plaques so isolated were picked and amplified by PCR using synthetic 
oligonucleotide primers that flank the vector polyiinker site. The size of inserts 
was determined by agarose gel electrophoresis and the sequence of inserts was 
determined by standard procedures using an Applied Biosystems 310 automatic 
DNA sequencing machine. 

15 

Cycles of the above process of binding phage to the solid support with coupled 
drug, in the presence of the analogue, were reiterated until multiple 
representations of the same insert were present in the purified phage pool. 
Typically 5-10 rounds of selection were required. 

20 

The oligonucleotides used for amplifying inserts from T7 vectors are 
commercially available. The specific oligonucleotides used for amplifying inserts 
from XfooDc are: 

S'-GACCGTTGGGCCAATTGTC and S'-TAAAACGACGGCCAGTGCC 
25 Oligonucleotides used for amplifying inserts from Xvsx.1 are: 

5'-AMTTACCGTCACCGCCAGT and 5'-mGATGCCTGGCAGTTCC 

The aim of this screening strategy is to not only identify within the one 
experiment phage that may bind to the drug, but also to identify phage that may 
30 bind with differing affinities. 

Example 6 - Library screening for proteins that bind to a specific biotinylated 
drug in the presence of a drug analogue 



WO 03/003012 



-53- 



PCT/ATJ02/00856 



10 



Binding of proteins displayed on the surface of a phage to the (R)-isomer of the 
following drug molecule in the presence of the inactive (S)-isomer: 




C0 2 H 



10 phage particles (T7 Select Mb, 17 Select 10-3, XfooDc or Xvsx.1) with the 
adenoma and normal colon cloned cDNA libraries inserted were bound to a 
biotinylated version of the above drug in the range of concentrations from 10 nM 
- 50 uM and in the presence of 0.5 - 5 mM of the (S)-isomer analogue, and the 
complexes captured with 0.25 ml of paramagnetic streptavidin particles (Dynal). 

The virus particles were washed 5 times with binding buffer (5x volume) at room 
temperature and phage eluted with 20Q drug in binding buffer for 2-16 hours 
15 at room temperature. Alternatively, the streptavidin particles may be saturated 
wrth biotinylated drug, 0.45 ml of 2 M M drug per 0.1 ml of particles. 

The bound phage were then eluted from the paramagnetic particles by washes 
wrth .ncreasing concentrations of free drug in binding buffer from 10 nM-100 uM 
20 us.ng ten fold increments over a total period of 2-16 hours at room temperature 
with constant mixing. 



Eluted phage were titred, amplified by re-infection of a competent host and the 
new ennched pool phage titred using standard protocols 

25 

Twenty plaques so isolated were picked and amplified by PGR using synthetic 
oligonucleotide primers that flank the vector polylinker site. The size of inserts 
was determined by agarose gel electrophoresis and the sequence of inserts was 
determined by standatd procedures using an Applied Biosyatems 310 automatic 
30 DNA sequencing machine. 



WO 03/003012 



-54- 



PCT/AU02/00856 



Cycles of the above process of binding phage to the solid support with coupled 
drug, in the presence of the analogue, were reiterated drug until multiple 
representations of the same insert were present in the purified phage pool. 
Typically 5-10 rounds of selection were required. 

5 

The oligonucleotides used for amplifying inserts from T7 vectors are 
commercially available. For the other vectors, the following oligonucleotides are 
suitable: 

10 

The specific oligonucleotides used for amplifying inserts from XfooDc are: 
5'-GACCGTTGGGCCAATTGTC and 5-TAAAACGACGGCCAGTGCC 
Oligonucleotides used for amplifying inserts from Xvsx.1 are: 
5-AAATTACCGTCACCGCCAGT and S'-TTTGATGCCTGGCAGTTCC 

15 

The aim of this screening strategy was to not only identify within the one 
experiment phage that may bind to the drug, but also to identify phage that may 
bind with differing affinities. 

20 Finally, it is to be understood that various other modifications and/or alterations 
may be made without departing from the spirit of the present invention as 
outlined herein. 



WO 03/003012 



-55- 



PCT/AU02/00856 



10 



Claims: 

1 A method for identifying a protein capable of binding to a target 
molecule, the method including the steps of: 
(*) oroviding a pool of candidate proteins; 

acid targe, molecule is coupled to a eelecteble moiety; 
' « ^ng a second moieoule which is etructurally similar to the no„ 
"'ira.d.rgetmo.e.le.wher.n.esecondm.ecu.eisdeh.en, 

in a desired activity of the target molecule; 

(d ) allowing one or more of the candidate proteins to tad .0 ft. non- 
W S Ldfargefmoteculeinthe presence o, .he second moiecule; 

(e) isolaflng a protein bound to the targe, molecule; and 

(f) identifying the binding protein. 

5 2 A method according to claim 1 . wherein the non-nucleic acid 

Lecule reelected from the group consieung of drug motels protem, 
potysacchahdes, glycoproteins, hormones, receptors ..prds, sma» 

20 3 . A mettrod according to claim 2, wherein the non-nudefc acid targe. 
molecule is a drug molecule. 

4 A method according to any one of claims 1 to 3, wherein the second 
target molecule. 

A method according .o claim 3, wherein me drug molecule is 
30 flurbiprofen. 

6 A m ethod according .0 Cairn 3, herein the targe, molecule k .he (fl)- 
stereoieomer of a molecule wito me following formula: 



WO 03/003012 



-56- 



PCT/AU02/00856 




7. A method according to claim 5, wherein the second molecule is an 
5 isomer, geometric isomer, enantiomer, conformer, stereoisomer, structural 

isomer or a chemically substituted derivative of flurbiprofen. 

8. A method according to claim 6, wherein the second molecule is the (S)- 
stereoisomer of a molecule with the following formula: 

10 




9. A method according to claim 3, wherein the drug molecule is suldinac 
sulfide. 

15 

10. A method according to claim 9, wherein the second molecule is a 
isomer, geometric isomer, enantiomer, conformer, stereoisomer, structural 
isomer or a chemically substituted derivative of sulindac sulfide. 

20 11. A method according to any one of claims 1 to 10, wherein the second 
molecule is present in a molar excess to the target molecule. 

12. A method according to claim 1 1 , wherein the second molecule is present 
in a molar excess of one hundred fold or greater relative to the target molecule. 

25 



r. 



WO 03/003012 PCT/AU02/00856 

-57- 

13. A method according to any one of claims 1 to 12, wherein the pool of 
candidate proteins is expressed from DNA molecules inserted into a viral 
genome. 

5 14. A method according to claim 13, wherein each of the candidate proteins 
is displayed on the surface of a viral particle. 

15. A method according to claim 14, wherein the viral particle is derived from 
a bacteriophage. 

10 

16. A method according to claim 1.5, wherein the bacteriophage is selected 
from the group consisting of 77, 74, lambda, lambdoid phage, or filamentous 
phage. 

15 17. A method according to any one of claims 1 to 16, wherein the selectable 
moiety is a biotin containing group or an activated carbonate group. 

18. A method for identifying a protein capable of binding to a target 
molecule, the method including the steps of: 
20 (a) providing a pool of candidate proteins, wherein each candidate protein 

is displayed on the surface of a viral particle; 

(b) providing a non-nucleic acid target molecule, wherein the non-nucleic 
acid target molecule is coupled to a selectable moiety; 

(c) providing a second molecule which is structurally similar to the non- 
25 nucleic acid target molecule, wherein the second molecule is deficient 

in a desired activity of the target molecule; 

(d) allowing one or more of the candidate proteins to bind to the non- 
nucleic acid target molecule in the presence of the second molecule; 

(e) isolating one or more proteins bound to the target molecule; 

30 (f) amplifying the viral particles encoding the isolated binding proteins; 

(g) reiterating steps (a) through (f); and 

(h) identifying the binding protein. 



WO 03/003012 



-58- 



PCT/AU02/00856 



19. A method according to claim 18, wherein the non-nucleic acid target 
molecule is selected from the group consisting of drug molecules, proteins, 
peptides, polysaccharides, glycoproteins, hormones, receptors, lipids, small 
molecules, metabolites, cofactors, transition state analogues and toxins. 



20. A method according to claim 19, wherein the non-nucleic acid target 
molecule is a drug molecule. 

21. A method according to any one of claims 18 to 20, wherein the second 
10 molecule is an isomer, geometric isomer, enantiomer, conformer, stereoisomer, 

structural isomer or a chemically substituted derivative of the non-nucleic acid 
target molecule. 

22. A method according to claim 20, wherein the drug molecule is 
15 flurbiprofen. 

23. A method according to claim 20, wherein the target molecule is the (fi)- 
stereoisomer of a molecule with the following formula: 



24. A method according to claim 20, wherein the second molecule is an 
isomer, geometric isomer, enantiomer, conformer, stereoisomer, structural 
isomer or a chemically substituted derivative of flurbiprofen. 



25. A method according to claim 23, wherein the second molecule is the (S)- 
stereoisomer of a molecule with the following formula: 



5 



20 




25 



WO 03/003012 



-59- 



PCT/AU02/00856 




26. 

sulfide, 



H-nn to claim 20 wherein the drug molecule is suldinac 
A method according to claim ^u, w 



10 



* t n Haim 26 wherein the second molecule is a 

28 A meth od according .0 any one - d*. 1 • • " — ' * 
lo,ecu,e i sp,esen ti na m .arexoe 8 3 t o t he t ar 8 et m o,eou l , 

" 30. A method according to any one of claims 18 to 29, wherein the viral 
particle is derived from a bacteriophage. 

, „ im ™ wherein the bacteriophage is selected 

3 , A method tST«-« «-<* °< — 

20 from the group consisting of 17, T4. lamoa 

phage. 

25 * • . nrotein capable of binding to target molecule, 

33. A method for identifying a protein capaoie 

the method including the steps of: 

• — — rrrri^enon^, 

ffl providing a non-nucleic acid larger mo , 

acidfargetmoleouleiscoupledfoaselecablemo*; 



WO 03/00301 2 PCT/AU02/00856 

-60- 

(k) providing a second molecule which is structurally similar to the non- 
nucleic acid target molecule, wherein the second molecule is deficient 
in a desired activity of the target molecule; 

(I) allowing one or more of the candidate proteins in the first pool to bind 
5 to the non-nucleic acid target molecule in the presence of the second 

molecule; 

(m)isolating one or more proteins in the first pool that bind to the target 
molecule; 

(n) allowing one or more of the candidate proteins in the second pool to 
10 bind to the non-nucleic acid target molecule in the presence of the 

second molecule; 

(o) isolating one or more proteins in the second pool that bind to the target 
molecule; and 

(p) comparing one or more proteins isolated from each of the first and 
15 second pools to identify a protein that is differentially represented 

between the first and second pools. 

34. A method according to claim 33, wherein the non-nucleic acid target 
molecule is selected from the group consisting of drug molecules, proteins, 

20 peptides, polysaccharides, glycoproteins, hormones, receptors, lipids, small 
molecules, metabolites, cofactors, transition state analogues and toxins. 

35. A method according to claim 34, wherein the non-nucleic acid target 
molecule is a drug molecule. 

25 

36. A method according to any one of claims 33 to 35, wherein the second 
molecule is an isomer, geometric isomer, enantiomer, conformer, stereoisomer, 
structural isomer or a chemically substituted derivative of the non-nucleic acid 
target molecule. 

30 

37. A method according to claim 35, wherein the drug molecule is 
flurbiprofen. 



WO 03/003012 



PCT/AU02/00856 



-61 - 

38. A method according to claim 35, wherein the target molecule is the (fl)- 
stereoisomer of a molecule with the following formula: 



39. A method according to claim 37, wherein the second molecule is an 
isomer, geometric isomer, enantiomer, conformer, stereoisomer, structural 
isomer or a chemically substituted derivative of flurbiprofen. 

10 40. A method according to claim 38, wherein the second molecule is the (S)- 
stereoisomer of a molecule with the following formula: 



15 41. A method according to claim 35, wherein the drug molecule is suldinac 
sulfide. 

42. A method according to claim 41, wherein the second molecule is a 
isomer, geometric isomer, enantiomer, conformer, stereoisomer, structural 

20 isomer or a chemically substituted derivative of sulindac sulfide. 

43. A method according to any one of claims 33 to 42, wherein the second 
molecule is present in a molar excess to the target molecule. 

25 44. A method according to claim 43, wherein the second molecule is present 
in a molar excess to the target molecule of one hundred fold or greater. 




C0 2 H 




C0 2 H 



WO 03/003012 PCT/AU02/00856 

-62- 

45. A method according to any one of claims 33 to 45, wherein the pool of 
candidate proteins is expressed from DNA molecules inserted into a viral 
genome. 

5 46. A method according to claim 45, wherein each of the candidate proteins 
is displayed on the surface of a viral particle. 

47. A method according to claim 46, wherein the viral particle is derived from 
a bacteriophage. 

10 

48. A method according to claim 47, wherein the bacteriophage is selected 
from the group consisting of T7, T4, lambda, lambdoid phage, or filamentous 
phage. 

15 49. A method according to any one of claims 33 to 48, wherein the 
selectable moiety is a biotin containing group or an activated carbonate group. 

50. A method according to any one of claims 33 to 49, wherein the first pool 
of candidate proteins is derived from a cellular extract from non-cancerous cells 

20 and the second pool of candidate proteins is derived from a cellular extract from 
cancerous cells. 

51 . A method according to any one of claims 33 to 49, wherein the first pool 
of candidate proteins is derived from a cellular extract from non-cancerous cells 

25 and the second pool of candidate proteins is derived from a cellular extract from 
pre-cancerous cells. 

52. A method according to any one of claims 33 to 49, wherein the first pool 
of candidate proteins is derived from a cellular extract from pre-cancerous cells 

30 and the second pool of candidate proteins is derived from a cellular extract from 
cancerous cells. 



PCT/AU02/00856 

WO 03/003012 

-63- 

53 . A method according » any one of claims 50 to 52, wherein the csllufcr 
extracts are derived from a human cell. 

54 a method according to claim 53. wherein the human cell is derived from 
5 colorectal tissue, hreas, «ssue, cervical «ssue, utenne tissue, renal hssue, 
pancreafc tissue, oesophageal tissue, stomach tissue, lung hssue, brarn ^ 
Mr issue, bladder tissue, bone fcsue, prostate tissue, sldn hssue, ovary tissue, 
testicular tissue, muscle tissue or vascular tissue. 

10 55. A method for identifying a protein capable of binding to target molecule, 
the method including the steps of: 

(a) providing a first pool of candidate proteins; 

0 providing a non-nucleic acid targe, molecule, wherein the non-nuclerc 
acid target molecule is coupled to a selectable moiety; 
15 (c) providing a second molecule which is struck similar to the ^on- 

nucleic add targe, molecule, wherein the second molecule is decent 
in a desired activity of the target molecule; 
(d ) allowing one or more of the candidate proteins in the firs, pool to b,nd 
,o the non-nucleic acid target molecule in the presence of the second 

on molecule; 

(elMatingaproteininthefirstpoolthatbindstothetargetmolecule; 

A comparing the level of the protein in the first pool of candidate prote,ns 

with the level of the protein in a second pool of proterns; and 
(g, identifying a protein that is differential represented between the M 
25 and second pools. 



56 A method according to claim 55, wherein the non-nucleic acid target 
mo ,ecule is selected from .he group consisting of drug molecules, protons 
30 paptos, polysacchandes, gtycoproteins, hormones, receptors, l,p*s, small 
molecules, metabolites, cofactors, transition state analogues and toons. 



WO 03/003012 



PCT/AU02/00S56 



-64- 

57. A method according to claim 56, wherein the non-nucleic acid target 
molecule is a drug molecule. 

58. A method according to any one of claims 1 to 3, wherein the second 
5 molecule is an isomer, geometric isomer, enantiomer, conformer, stereoisomer, 

structural isomer or a chemically substituted derivative of the non-nucleic acid 
target molecule. 

59. A method according to claim 57, wherein the drug molecule is 
10 flurbiprofen. 

60. A method according to claim 57, wherein the target molecule is the (fl)- 
stereoisomer of a molecule with the following formula: 



61. A method according to claim 59, wherein the second molecule is an 
isomer, geometric isomer, enantiomer, conformer, stereoisomer, structural 
isomer or a chemically substituted derivative of flurbiprofen. 



62. A method according to claim 60, wherein the second molecule is the (S)- 
stereoisomer of a molecule with the following formula: 



63. A method according to claim 57, wherein the drug molecule is suldinac 





25 



sulfide. 



WO 03/005012 



-65- 



PCT/AU02/00856 



5 



64 A method according to claim 63, wherein the second molecule is a 
isomer, geometric isomer, enantiomer, conformer, stereoisomer, structural 
isomer or a chemically substituted derivative of sulindac sulfide. 

65. A method according to any one of claims 55 to 64, wherein the second 
molecule is present in a molar excess to the target molecule. 

66. A method according to claim 65, wherein the second molecule is present 
10 in a molar excess to the target molecule of one hundred fold or greater. 

67 A method according to any one of claims 55 to 66, wherein the pool of 
candidate proteins is expressed from DNA molecules inserted into a viral 



15 



genome. 

68. A method according to claim 67, wherein each of the candidate proteins 
is displayed on the surface of a viral particle. 

69. A method according to claim 68, wherein the viral particle is derived from 
20 a bacteriophage. 

70 A method according to claim 69, wherein the bacteriophage is selected 
from the group consisting of T7, T4, lambda, lambdoid phage, or filamentous 



phage. 



25 



71. A method according to any one of claims 55 to 70, wherein the 
selectable moiety is a biotin containing group or an activated carbonate group. 

72 A method according to any one of claims 55 to 71 , wherein the first pool 
30 of candidate proteins is derived from a cellular extract from non-cancerous cells 
and the second pool of candidate proteins is derived from a cellular extract from 
cancerous cells. 



WO 03/00301 2 PCT/AU02/00856 

-66- 

73. A method according to any one of claims 55 to 71 , wherein the first pool 
of candidate proteins is derived from a cellular extract from non-cancerous cells 
and the second pool of candidate proteins is derived from a cellular extract from 
pre-cancerous cells. 

5 

74. A method according to any one of claims 55 to 71 , wherein the first pool 
of candidate proteins is derived from a cellular extract from pre-cancerous cells 
and the second pool of candidate proteins is derived from a cellular extract from 
cancerous cells. 

10 

75. A method according to any one of claims 72 to 74, wherein the cellular 
extracts are derived from a human cell. 

76. A method according to claim 75, wherein the human cell is derived from 
15 colorectal tissue, breast tissue, cervical tissue, uterine tissue, renal tissue, 

pancreatic tissue, oesophageal tissue, stomach tissue, lung tissue, brain tissue, 
liver tissue, bladder tissue, bone tissue, prostate tissue, skin tissue, ovary tissue, 
testicular tissue, muscle tissue or vascular tissue. 

20 77. A protein identified according to the method of any one of claims 1 to 1 7. 

78. A protein identified according to the method of any one of claims 18 to 
32. 

25 79. A protein identified according to the method of any one of claims 33 to 
54. 

80. A protein identified according to the method of any one of claims 55 to 
76. 

30 

81 . A compound with the following chemical formula: 



WO 03/003012 



-67- 



PCT/AU02/00856 



R2Q 




or a salt thereof, wherein: 

- R 1 is selected from hydrogen and lower alkyl (C1 to C8); 

5 - R 2 is YX 2 ((CH 2 ) m X 2 ) n -, wherein m is 2 to 4, n is 1 to 6, X 2 is selected 

from 0, S and N, and Y is independently selected from hydrogen, 
lower alkyl, or a suitable heteroatom protecting group; 

- R 3 is selected from one or more of hydrogen, alkyl, aryl, halogen, 
hydroxy, alkoxy, aryloxy, amino (unsubstituted and substituted) and 

10 caroboxy; 

- R 4 is selected from one or more of hydrogen, alkyl, aryl, halogen, 
hydroxy, alkoxy, aryloxy, amino (unsubstituted and substituted) and 
caroboxy; 

- X is selected from fluoro, chloro, bromo and iodo; 

15 - M is selected from hydroxy, alkoxy, aryloxy, amino, alkylamino (mono- 

and di-), arylamino (mono- and di-), N-morpholino, hydroxyalkylamino, 
dialkylaminoalkylamino, aminoalkylamino, polyhydroxyamino, and 
salts of any of the aforementioned. 

20 82. A compound as in claim 81 wherein X is fluoro. 

83. A compound as in claim 81 wherein the fluoro group is substituted meta 
to the alkylcarboxylate group. 

25 84. A compound as in claim 81 wherein R 1 is a lower alkyl group. 
85. A compound as in claim 84 wherein R 1 is a methyl group. 



WO 03/003012 



PCT/AU02/00856 



-68- 

86. A compound as in claim 84 wherein R 2 is an alkyleneoxy or 
polyoxyalkylene chain. 

87. A compound as in claim 86 wherein the alkyleneoxy or polyoxyalkylene 
5 chain has between 1 and 4 alkyleneoxy repeating units. 

88. A compound as in claim 87 wherein R 2 is a triethylene glycol group. 

89. A compound as in claim 86 wherein the R 2 0- group is substituted at a 
1 0 position para to the aryl substituent. 

90. A compound as in claim 89 wherein both R 3 and R 4 are hydrogen. 

91 . A compound as in claim 90 wherein M is hydroxy or a salt thereof. 



15 



92. 



A compound with the following chemical formula: 



R20. 




C0 2 M 



R 1 -X 1 



(II) 



20 or a salt thereof, wherein: 

- X 1 is selected from sulfide, sulfone and sulfoxide; 

- R 1 is selected from hydrogen, hydroxy (when X 1 is sulfone or 
sulfoxide), and lower alkyl (C1 to C8); 



WO 03/003012 PCT/AU02/00856 

•69- 

- R 2 is YX 2 ((CH2) m X 2 ) n -, wherein m is 2 to 4, n is 1 to 6, X 2 is selected 
from O, S and N, and Y is independently selected from hydrogen, 
lower alkyl. or a suitable heteroatom protecting group; 

- R 3 is selected from hydrogen, halogen, alkyl, alkoxy, acyloxy, amino, 
5 alkylamino (mono- and di-), arylamino (mono- and di-), nitro, cyano, 

carboxyl; 

- R 4 is selected from hydrogen and lower alkyl (C1 to C8); and 

- M is selected from hydroxy, alkoxy, aryloxy, amino, alkylamino (mono- 
and di-), arylamino (mono- and di-), N-morpholino, hydroxyalkylamino, 

10 dialkylaminoalkylamino, aminoalkylamino, polyhydroxyamino, and 

salts of any of the aforementioned. 

93. A compound as in claim 92 wherein X 1 is either a sulfone or a sulfide. 

1 5 94. A compound as in claim 93 wherein X 1 is a sulfide. 

95. A compound as in claim 94 wherein R 1 is a lower alkyl group. 

96. A compound as in claim 95 wherein R 1 is a methyl group. 

20 

97. A compound as in claim 95 wherein R 2 is an alkyleneoxy or 
polyoxyalkylene chain. 

98. A compound as in claim 97 wherein the alkyleneoxy or polyoxyalkylene 
25 chain has between 1 and 4 alkyleneoxy repeating units. 

99. A compound as in claim 98 wherein R 2 is a triethylene glycol group. 

1 00. A compound as in claim 97 wherein R 3 is a halogen group. 

30 

101. A compound as in claim 1 00 wherein R 3 is a fluoro group. 



WO 03/00301 2 PCT/AU02/00856 

-70- 

102. A compound as in claim 101 wherein the fiuoro group is ortho to the 
hydroxy group. 

1 03. A compound as in claim 1 00 wherein R 4 is a lower alkyl group. 

5 

1 04. A compound as in claim 1 03 wherein R 4 is methyl. 

1 05. A compound as in claim 1 03 wherein M is hydroxy or a salt thereof. 



10 



THIS PAGE BLANK (usptoj