AU691296B2 - Combinatorial dihydrobenzopyran library - Google Patents

Description

WO 95/30642 PCT/US95/05940 TITLE OF THE INVENTION COMBINATORIAL DIHYDROBENZOPYRAN LIBRARY BACKGROUND OF THE INVENTION There is interest in methods for the synthesis of large numbers of diverse compounds which can be screened for various possible physiological or other activiLes. Techniques have been developed in which one adds individual units sequentially as part of the chemical synthesis to produce all or a substantial number of the possible compounds which can result from all the different choices possible at each sequential stage of the synthesis. For these techniques to be successful, it is necessary for the compounds to be amenable to methods by which one can determine the structure of the compounds so made.

Brenner and Lerner (PNAS USA 81: 5381-83 (1992)) and WO 93/20242, for example, describe a synthesis wherein oligonucleotides are produced in parallel with and are chemically linked as genetic tags to oligopeptides as the compounds of interest. WO 93/06121 teaches methods for particle-based synthesis of random oligomers wherein identification tags on the particles are used to facilitate identification of the oligomer sequence synthesized. A detachable tagging system is described in Ohlmeyer et al., Proc. Natl. Acad. Sci. USA, 9QJ10922- 10926, Dec. 1993.

SUMMARY OF THE INVENTION The present invention relates to combinatorial chemical libraries of compounds encoded with tags and to the use of these libraries in assays to discover biologically active compounds. The present invention also relates to libraries containing dihydrobenzopyrans and using these libraries to identify biologically active members by screening for inhibition of carbonic anhydrase isozymes. The present invention also relates to members of the library which interact as agonists or antagonists) with a adrenergic receptors, dopamine receptors, o-opiate receptors, and K+ channels. In particular, the present invention also relates to members of the library

I

I

which are inhibitors of carbonic anhydrase. The invention also relates to methods for their preparation, intermediates, and to methods and pharmaceutical formulations for using these dihydrobenzopyrans in the treatment of mammals, especially humans, Because of their activity as inhibitors of carbonic anhydrase isozymes, compounds of the present invention are useful in the treatment of such diseases as glaucoma.

Detailed Description of the Invention The combinatorial libraies of the present invention are represented by Formula I: (T r -C(O)-II I wherein: 10 is a solid support; is an identifier residue wherein T' is a tag and L is a first linker; q is chosen from 3-30; and is a linker/ligand residue wherein L' is a second linker and II' is an attached ligand represented by s

R

3 0 R' O~ 2 R C: ~R2 wherein: RI is chosen from -(CH2)12-0-, -C(O)-C11 2 and and when L' terminates in N(CH 2 1

R

1 7 Rla may additionally be chosen from and -C(O)-4-Phe-

CH

2

R

2 is chosen from H and lower alkyl;

R

3 is chosen from H, alkyl, aryl and arylalkyl;

R

4 and R 5 are each independently chosen from H, lower alkyl, and substituted lower alkyl wherein the substituents are 1-3 alkoxy, aryl, substituted aryl, carboalkoxy, carboxamido or diloweralkylamido, or R 4 and R 5 taken together is chosen from -(CH12)n-,

-(CH

2 2

-O-(CH

2 2

-CH

2

-O-(CH

2 3

-(CH

2 2

-NR

8

-(CH

2 2

-CH

2

-NR

8

-(CH

2 )mn-

-(CI-H

2 2

CH(NHR

8

)(CH

2 2 -(CI-1 2 2 2

-(CIH

2 2 and

-CH

2 CH(N-loweralkyl)(CI 2 2 CI I-1 2 one of RG and R 7 is H and the other is chosen from H, OH, and N(CH 2 1 6

R'

I

4 1 5 or R6 (NAtIHAIO0525 SSC

IM

and R 7 taken together is chosen from 11S S\ 0S\/ mid S

R

8 is chosen from COOR 9 CONHRIO, CSNFLR' 1 C0R 12 S0 2

R

1 3 lower alkyl, aryl lower alkyl, heteroaryl, and hieteroaryl lower alkyl, wherein aryl is optionally substituted with 1-3 substituents selected from lower alkyl, lower alkoxy, halo, CN, NFL 2 COOH-, CONH 2 carboalkoxy, and mono- or di-lower alkylamino and wherein hieteroaryl is a mono- or bicyclic heteroaromatic ring system of 5 to 10 members including 1 to 3 heteroatoms selected from 0, N, onil S and 0-3 substituents selected from halo, amino, cyano, lower alkyl, carboalkoxy, CONIFL 2 and S-lower alkyl;

R

9 is chosen from lower alkyl, aryl, aryl lower alkyl, hecteroaryl, aryl substituted by 1-3 substituents selected from alkyl, alkenyl, alkoxy, methylene dioxy, and halo, and a to 6-memibered heterocyclic ring wherein the hetero atom is 0 or N, wherein heteroaryl is a hieteroaromatic ring of 5 to 6 members including 1 to 2 heteroatoms selected from 0, N, and S and 0-2 substituents selected from lower alkyl, dialkylamino, lower alkoxy, and halo;

R

10 and RII are each independently chosen from lower alkyl, aryl, aryl lowver alkyl, *V and aryl substituted by 1-3 substituents selected from lower alkyl., halo, alkoxy and lilolk l haoaR12 is chosen from lower alkyl, aryl, heteroaryl, aryl lower alkyl, heteroaryl lower alkyl, a 5- or 6-memibered heterocyclic ring containing 1-2 heteroatoms selected from 0, o 5, and N, a 5- or 6-membered heterocyclic ring c ontaining 1-2 heteroatoms selected from 0, S, and N lower alkyl, and aryl substituted with 1-3 substituents selected. from lower alkyl, alkoxy, halo, sulfamoyl, lower alkyl sulfamoyl, cyano, and phenyl;

R

13 is chosen from lower alkyl, aryl, and aryl substituted with 1-3 substituents 25 selected from lower alkyl, alkoxy, halo, CN, and haloalkyl;

R

14 is chosen from FL, alkyl, alkyl substituted by 1-3 alkoxy, S-loweralkyl, .0 sulfamoyl, halo, alkylsulphonamido, or arylsulphonamido, alkenyl, alkynyl, aryl, substituted aryl, leteroaryi, substituted heteroaryl, heterocycloalkyl, -CFL 2 N R 16

C(O)R

1 6

-C(O)NR

16

R

16

-CFL

2 00(O)R 16 and -CFL 2

SC(O)RI

6

R

15 is chosen from alkyl, and -C(NCN)NR 3

R

3

R

16 is chosen from lower alkyl, substituted lower alkyl, aryl, and substituted aryl;

R

17 is chosen from alkyl, alkyl substituted by 1-3 alkoxy, S-loweralkyl, sulfamoyl, halo, alkylsuiphonamido, or arylsulphonamido, alkenyl, alkyny I, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycloalkyl, -CFL 2

NR

1 6

C(O)R

1 6

-C(O)NR

16

RI

6

-CFL

2

OC(O)R

1 6 and -CH 2

SC(O)R

1 6 X is chosen from alkyl, aryl, arylalkyl, 0-loweralkyl, and -NR 3

R

3 IN ILMfA1OO525 SSC Z is chosen from -(CH 2 1 6 optionally substituted with 1-3 lower alkyl, -CFIR 2 -Phe-CI{ 2 wherein Phe is optionally mono-substituted with halogen, lower alkyl, or alkoxy, and hieteroarylene-(CH- 2 III is chosen from 2 and 3; and 11 is chosen from 4-9.

Preferred compounds of Formula 1 are those wherein: is of the Formula: (012~)n O~13~ -Ar wherein n 3 =12 when Ar is pentachlorophienyl and n 3-6 when Ar is 2,4,6-trichlorophenyl; q is 4-12; and IN *tUI02 SS I I WO 95/30642 PCT/US95/05940 is 0 (f 0 B- V 'NO 2 Olz B

OB-

S/'^NO

(b) wherein the left-hand bond as shown is the point of attachment to the solid support and the right hand bond is the point of attachment to the ligand, and B is O or N(CH2)1-6R 17 with the proviso that in when B is N(CH2)1-6R 17 the ligand is attached to B through a carbonyl group.

Other preferred compounds of Formula I are those of Formulae Ia, Ib, or Ic wherein is: RK,

R

(CH

2 1

R'

7

(CH

2

.R

1 7

I

WO 95/30642 ~VO 9530642PCTILIS95/05940) -4- Depending on the ch-oice of L' (see Table the ligands of Formula 11 may be detached by photolytic, oxidative, or other cleavage techniques. For example, when is and B is 0 (or N(CH2)1-6R 17 photolytic detachment may be represented by: I C(O)-L"I 111OH (B 0) or II'Nirl(CH, 2

X

6

R'

7 (B N(CH7) 1 6

R

17 wherein U is the residue from L' and IPOH (or l1'NH(CH2)1..6R 17 )is

IL.

Therefore, compounds of the present invention are also represented by Formula 11 R 6 R 7 3 R~ 0 i wherein: R Ais Ris

R

3 is

R

4 and R 5 OH, O(CH2)I-20H, OCH2CO2H, CQ2H, O-Z-C(O)N'H(CH2)l1*6RI 7 or OCH2-4-Phe-C(O)NH(CH2) l-6R 1 7 H or lower alkyl; H, alkyl, aryl, or aryialkyl; is each independently H1, lower alkyl, or substituted lower alkyl. where the substituents are 1-3 atkoxy, aryl, substituted aryl, carboalkoxy, carboxamido, or diloweralkylamido; or R 4 and R 5 taken together are -CI-2-0-(CH2)3-, -(CH2)2-NR 8 (CH2)2-, -CH2-NR 8 -(CH2)2CH(NHR 8 -(CH2)2-S 2 ,or

-CH

2 CH(N-loweralkyl) (CH HCH,one of R 6 and R 7 is H and the other is H, OH, or N(CH 2 )1- 6

R

14

R

15 or

R

6 and R 7 taken together are /--7R2 S S O S 0 0 S\ O or S with the proviso that when R 1 is OH and R 2 is H, R 6 and R 7 taken together is not 0

O\/O;

R

8 is H, COOR 9

CONHR

10

CSNHR

11

COR

12

SO

2

R

13 lower alkyl, aryl lower alkyl, heteroaryl, or heteroaryl lower alkyl, wherein aryl is optionally substituted with 1-3 substituents selected from lower alkyl, lower alkoxy, halo, CN, NH 2 COOH, CONH 2 carboalkoxy, and mono- or di- lower alkylamino and wherein heteroaryl is a mono- or bicyclic heteroaromatic ring system of 5 to 10 members including 1 to 3 heteroatoms selected from O, N, and S and 0-3 substituents selected from halo, amino, cyano, lower 0o S alkyl, carboalkoxy, CONH 2 and S-lower alkyl; 15 R 9 is lower alkyl, aryl, aryl lower alkyl, heteroaryl, aryl substituted by 1-3 substituents selected from alkyl, alkenyl, alkoxy, methylene dioxy, and halo, or a 5 to 6i membered heterocyclic ring wherein the hetero atom is O or N, wherein heteroaryl is a heteroaromatic ring of 5 to 6 members including 1 to 2 heteroatoms selected from 0, N, and S and 0-2 substituents selected from lower alkyl, dialkylamino, lower alkoxy, and 20 halo;

R

10 and R 11 is each independently lower alkyl, aryl, aryl lower alkyl, or aryl substituted by 1-3 substituents selected from lower alkyl, halo, alkoxy, and haloalkyl;

R

12 is lower alkvl arvl. heteroarvl arvl lower alkvl. heteroarvl lower alkvl. a 5- or 6-me.ered heterocyclc ring ctaig 1-2 eroatoms selcted from 0, S, and N a 6-membered heterocyclic ring containing 1-2 heteroatoms selected from O, S, and N, a or 6-membered heterocyclic ring containing 1-2 heteroatoms selected from 0, S, and N lower alkyl, or aryl substituted with 1-3 substituents selected from lower IN ILIBA100525 SSC

II

WO 95/30642 WO 9530642PCT1U595105940 -6alkyl, alkoxy, halo, sulfamoyl, lower alkyl sulfamoyl, cyano, and phenyl;

R

13 is lower alkyl, aryl, or aryl substituted with 1-3 substituents selected from lower alkyl, alkoxy, halo, CN, and haloalkyl;

R

14 is H; alkyl substituted by 1-3 alkoxy, S-loweralkyl, sulfamoyl, halo, alkylsulphonamido, or arylsulphonamido; alkenyl; alkynyl; aryl; substituted aryl; heteroaryl; substituted heteroaryl; heterocycloalkyl; -CH2NR1 6 C(O)Rl 6 -C(O)NR16Rlh; -CH2OC(O)R 1 6 or -CH2SC(O)R' 6

R

15 is H, alkyl, or -C(NCN)NR 3

R

3

R

16 is lower alkyl, substituted lower alkyl, aryl, or substituted aryl;

R

17 is H; alkyl substituted by 1-3 alkoxy, S-loweralkyl, sulfamoyl, halo, alkylsulphonamido, or arylsulphonamido, alkenyl; alkynyl; aryl; substituted aryl; heteroaryl; substituted heteroaryl; heterocycloalkyl; -CH2NR' 6

C(O)R

1 6

-C(O)NR

16

R

1 6 -CH2OC(O)R' 6 or -CR2SC(O)RI 6; X is alkyl, aryl, arylalkyl, 0-loweralkyl, or NR 3

R

3 Z is optionally substituted with 1-3 lower alkyl;

CHR

2 Phe-CH2-, where Phe is optionally monosubstituted with halogen, lower alkyl, or alkoxy; or heteroarylene-(CH2)-; m is 2 or3; n is 4-9; or a pharmaceutically acceptable salt thereof.

Preferred compounds of Formula 11 are those wherein R 12 is sulfamoylphenyl, most preferably p-sulfamoylphenyl.

A preferred embodiment of the invention is a compound of Formula 11 wherein: RI is OH, OCH,2C(O)NH(CH2)1-6R', or OCH2-4-Phe- C(O)NH(CH2)) 1-6R' 4

R

2 is H or lower alkyl; WO95/30642 PCT/IlS95/05940 -7-

R

4 and R 5 is each lower alkyl; or R 4 and R 5 taken together are -(CH2) 5 -(CH2)2-O-(CH2)2-, -(CH2)2.NR 8 -(CH2)2-, -(CH2)2-CH(NHRS)(CH 2 -(CH2)2-S-(CH2)2- or

-CH

2

CH(NCH

3

)(CH),HCHCH

2 I

R

6

/R

7 are H/OH, or -S(CHM2)2S-;

R

8 is H, COOR 9

CONHR

10

CSNHR

11

COR

12 S02R 13 lower alkyl, aryl lower alkyl, heteroaryl wherein the ring members include 1 to 3 N atoms and the substituents are halo or amino, heteroaryl lower alkyl wherein heteroaryl is 6-membered and the heteroatoms are N, or aryl lower alkyl substituted with 1 substituent selected from lower alkyl, alkoxy, and ialo;

R

9 is lower alkyl, aryl 'cwer alkyl, aryl, tetrahydrofuranyl, tetrahydropyranyl, or aryl substituted by 1 to 2 substituents selected from lower alkyl, alkenyl, alkoxy, methylene dioxy, and halo;

R

10 and R 11 is each independently aryl, aryl lower alkyl, or aryl substituted by 1 substituent selected from lower alkyl, halo, alkoxy, trifl romethyl, and pentafluoroethyl;

R

12 is lower alkyl, aryl, aryl lower alkyl, heteroaryl lower alkyl wherein the heteroatoms are N, a 5- or 6-membered heterocyclic ring containing 1-2 heteroatoms selected from S and N lower alkyl, or aryl substituted with I substituent selecred from lower alkyl, alkoxy, halo, sulfamoyl, cyano, or phenyl;

R

13 is lower alkyl, aryl, or aryl substituted with 1 substituent selected from lower alkyl, alkoxy, and halo; or a pharmaceutically acceptable salt thereof.

Most preferred compounds of the invention are represented by the formula: WO 95/30642 WO 951f)642PCTCS95OS94) wherein: RI is R" is

R

4 and R5 one of R 6

R

8 is

R

9 is 6- or 7-OH; H or lower alkyl; is each methyl; or R 4 and R 5 taken together are -(CH2)2-0-(CH2)2-, -(CH2)2-NR 8 -(CH2)2-, -CH2-NR 8 -CH2-NR 8 or -(CH2)2-CH(NHR 8 )(CH2)2-; and R 7 is H and the other is OH or R 6 and R 7 taken together are =0 or -S(CH2)2S-; H, COOR 9

CONHR

10 CSNHRII, CORI", S02)RI 3 benzyl, -CH2-Ph-4-F, -CH2-Ph-4-OCH3, -CHl2-4-Py, n-butyl, -CH2-c-propyl, NDCl N Cl Nl TN, or N N-flNH 2 N C l i-propyl, phenyl, phenethyl, t-butyl, 0-C RIO is RII is phenyl, p-chlorophenyl, or p-tn fluoromethyiphenyl; phenyl, benzyl, or 1-naphthyl; WO 95/30642 WO 9530642PCTIUS95/05940

R

12 is 'Kly CO<SH 0 NH

-H

H H SO 0 CH 2 N N -CH-LIi H

H

d-o, {j&S 2

NH

2 and

R

13 is 1- or 2-naphthyl, phenyl, 4-chlorophenyl, 4methyiphenyl, 4-t-butylphenyl, n-butyl, or i-propyl; or a pharmaceutically acceptable salt thereof.

Most proferred. compounds of the invention are also represented by the formula: wherein: RI is 6- or 7-OH when R 2 is H; RI is 7-OH when R 2 is CH3;

R

4 and R 5 is each methyl; or R 4 and R 5 taken together are -(CH2)2-O-(CH2)2-, -(CH2)2-NR 8 -(CH2)2-, -CH2)-NR 8 -GH2-NR 8 or -(CH2)2-

CH(NHR

8 )(CH2)2-; one of R 6 and R 7 is H and the other is OH or R 6 and R 7 taken together are =0 or -S(CH2)2S-; and

R

8 is WO 95/30642 0141- 100

CH

2

CHI

200 ~0 PCTUS95IO5940 WO 95/30642 WO 9530642PCTUS95105940 -11- WO 95/30642 PCT[US95/05940 -12- Most preferred compounds of the invention are represented by the formula: wherein: RI is R1 is

R

4 and R 5 is 6- or 7-OCH2C(O)NH(CH2)l-6R17, or 6- or 7-OCH2-4- Phe-C(O)NH(CH2)1..6RI 7 when R' is H; 7-OCH2C(O)NH(CH 2 1-6R 1 7, or 7-OCH2-4-Phe- C(O)NH(CH2) l-6R' 7 when R 2 is CH 3 each methyl; or R 4 and R 5 taken together are -(CH2)2-O-(CH2)2-, -(CH2)2-NR 8 -(CH2)2-,

-(CH

2 )2-CH(NHR 8

)(CH

2 2 -(CH2)2-S-(CH2)2- or

-CH

2 7CH(NCH 3

)(CH

2 )2,CHCH 2 'I -I or R 4 is methyl and R 5 is CH2OCH3 or -(CH2-)3N(Et)2; one of R 6 and R 7 is H and the other is OH; or R 6 and R 7 taken together are =0 or -S(CH2)2S-; or one of R 6 and R 7 is H and the other is NAB, where A is methyl, 2-methoxyethyl, 2-phenylethy 1, 4-methoxybenzy 1, 2- tetra hydrofuranylmethyl, 2(3 ,4-dimethoxyphenyl)ethyl, or 2,2-diphenylIethyl and B is H, -SO 2

CHI,

0 YK 0 0 0 Y (CH 2 4 NH2

NH

2 0

NH

<Uk (CH 2 5 N Nl12 WO 95/30642 W095/0642PCTIUS95/05940) -13- NCN 0 AN(n-propyl) 2

NHCH

3 0 A NH~h

S

NHCH

3 or

S

<UA NHPh

R

8 is H, CONHCH3, SO,"h, (CH2)3CH3, CO(CHo2)2CH3, benzyl, -C(0)-(4-Phe)-SO2NH2, or

R

13 is H, -SO 2

CH

3 0 <k0 0 0 <K(Y (CH 2 4

NH

2

NH

2 0

NH

k (CH 2 5 N)k NH 2 NCN 0 0 N(n-propyl) 2

,NHCH

3 NHPh

S

<1%NHCH 3

S

<KNHPh (GH2) 1-6R 14 is methyl, n-butyl, 3-methoxy-n-propyl, CH2-c-Propy1, or -(CH2) 1-3-phenyl; and (CH2) 1-6R 17 is methyl, 2-methoxyethyl, 2-phenylethyl, 4-methoxybenz~yl, rnethyl-2-tetrahydrofurany 1, 2(3,4- dimetho xyphenyl)ethyl, or 2 ,2-diphenylethyl; or a .pharmaceutically acceptable salt thereof.

WO 95/30642 PCT/US95/05940 -14- Especially preferred. as inhibitors of carbonic anhydrase, are compounds of formulae lib, l1c, and id:.

R 6 R 7 5 6 R 3 3

NHR'

whereit R1 is

R

2 is

R

8 is

R

6 and 6- or 7-OH, 6- or 7-OCH2C(O)NH(CH2)1..6RI 7 or 6- or 7-OCH24-Ph-C(O)N1(CH2)l -6R 1 7; H or CH3; -CO-Ph-p-SO2NH2; and

R

7 together are =0 or -SCH2CH2S-.

Most preferred of these are the following compounds: For-mula Formula Ilc Formula Ild 11b R1 7-OH 6-OH 6-OH1 6-OH 7-OH R2 H H H MH

R

6 1R 7 -SCH2C2 S -SCH2CH2S- -SCH2CH2S-

R

14 and R 17 may each be any pharmacologically relevent orgaic radical, such as those derived by removal of H2NCH2- fromn the following compounds: WO095/30642 PCT/US95/05940 WO 95/30642 PTU9/54 PCT/US9510594(i

H

2

C-CH

2

H

2

N-CH

2

-CH

2

CH

2 -N \0

H

2 C -CH 2 1 i

H

2

N-CH

2

-CH

2 t

H

2

N-CH

2

H

2

N-CH

2 2 5 ci

H

2

N-CH

2

-CH

2 0H 2

N-CH

2 cil -N

H

2 N-CH 2 0

H

2

N-CH

2 0 0

H

3

C

H

2

N

CH

2 H N-CH 2

N

2 2 -co

H

3

C-CH

2 -0.-a WO 95/30642 PC'r/US951594) -17- WO095/30642 PTU9IS4 PCTIIJS95/05940 -18- H CH 0

H

3 0 -NH 2

CH

3

H

3

C-C-CH

2

-NH

2 H 3 0-CH 2 NH 2

C-H

3

NH

2

CH

2 H 2 N-0H 2

CH

2

-F

H

3 C

CH

3

NH

2

CH

2 "IC I Hl~lH 2 N-CH 2 -CH 2

-NH

CH

2

CH

2 3

NHHC

0CH o

H

2

/CNH,

CH C 2C

CH

3 CHNHH

I

WO 95/30642 PTU9IS4 PCT/US95/05940 -19.*

H

3

C

0

H

3 C- NH-- CH 2

-CH

2

NH

2

H

3 C-O-

CH

2

-CH

2

-NH

2

H

3

C-CH

2

-CH

2

-NH-CH

2

-CH

2

-NH

2 H 2 NCH 2

CH

2 Q -Br

H

3

C-O-CH

2

-CH

2

-NH

2 H 2

N-CH

2

-CH

2

F

H2 N-CH 2 -CH c HNC2C2P

H

3 C-0

-CH

2

-CH

2

-NH

2 0

H

3

C

ICH

2

CH

2

-CH

2

-NH

2 WO 95/30642 WO 9530642PCT/US95/05940

H

3 0 k1 2

N-CH

2

-C-CH

H

3 C LH C 2

CH

2 CH2

NH

H

2

N-CH

2

-CH-CH

2

H

3

C-CH

2

-O-CH

2

-CH

2

-CH

2

-NH

2

CH

3

H

3

L-CH

2

-CH

2

-NH

2

H

2

N-CH

2

-CH

2

CH

2 1

C-H

3 0 CH2

H

2

N-CH

2 -CH 2 -C 0

H

3

C

CH~H

3

C-CH

2 -CH -CH -CH -NH 2

H

3 C C 2H 2

H

3 0 :-CH 2

-CH

2

-NH

2

HC-CH

2

-CH

2

-CH

2

-CH

2

-CH

2

-NH

2 WO 95/30642 PTU9154 PCT/US95/05940 -21-

F

H

3

C-CH

2

-CH

2

CH

2

-CH

2

-CH

2

'-CH

2

-NH

2

H

2

N-CH

2

D

F

F

H2N -CH 2-CH 2 2N2

H

2

N-CH

2 cX

-F

H

3

C

F

HN H CH 22 o N H 2

N-CH

2

C-F

F

NH

2 F-C--F

F

0/F C -r F F

F

F 11 CH3 c C 4R 2 o I/9A 0i

H

2

N-CH

2 bF F0 0

H

3 C -C H 2

-CH

2 C H 2

-NH

2 ICH 2

F

H

2

NI

WO 95/30642 PCT/US95/059410 -22-

H

2

C-CH

2

QH

CH

2 H~.CH2 Q

H

2

N-CH

2

-CH

2 c /H 2

HC-CH

2

CH

3 CH2 0 N CH CH CH 2 Q2N QH-- CH2.

HNH

B0 Q0o~(~ CH2

I

H

2

NCH

2 CH2 PCTUS95IOS940 WO 95/30642 -23-

H

2

N,

CH

2 Br0 0

H

3

C

H2 CH 2 0\ C=0

CH

2

H

2

N

0- 0 H t'LCH 2 0 C'CH

H

2

H

3 0 H N-CH -C Br /cR

CH

2

H

2

N

NH

2

CH

2

H

2

N-CH

2 I-- F

H

2 N, CH2 0-

H

3 C \c C 2 0

H

2

N

WO 95/30642 W09510642PCrtUS95IO5940 -24-

H

2

N-CH

2

-CH

2 H2-H2 22-CH 1 0 2/H- H2 -0

CH

2 0N

CH

2 CH 2

H

3 C

CH

2 0

NH

2 H2C2

CH

2 HC-cH 2

-S-CH

2

-CH

2

-NH

2

H

2

NCH

2 CHiCH 2 N I

H

2 C

H

H

2

N-CH

2 C CH

H

3 C

CH

2

-NH

2 0 ~F F F" 11 ~1 1 1

ICH

2 0C- 2 H2 I2-H- H.C

N

2 F F F H N-C-H -CH -CH--Br-Bn 0uu WO095/30642 PCTIUS95/05940 WO 95/30642

H

H,

H

2

N

PCT/1JS95/05940 -26- WO 9530642PCT/US95105940 -2CH2 C--NHC CHr 2 0 H2N2 H3CO Q 00 HC Q0 0 0 CH 2

-CH

2 100 CH( CH2 H2H

NHN

H

2

HOH

2

CH

2 0 -t 2

N

H

3 C-O 0 0 0 N O 0 0 200

CH

2

CH

2 0 H; N-CH 2

CH

2 cC,\ TN

C

/227C PcTIUS9SIO5940 WO 95/30642 -28- 0 0 C H 2 C H 2 H -C 2 0NH 2

H

3

C

00 O c H2

H

2 N-CH2

CH

2

CHN/

~H

2 CC2 CH0 0 0CH 2 N c~NN

~NH

2

L

H

2 200 11 HCH

CH

2

H

3 C CH H" HI-C lC 2 H NH 2 H 3 C- t4 L o 0 ~CH 2

DH

r-4 WO 95/30642 WO 9530642PCT/US95/0594) -29- !CH2-NH2

H

2

N-CH

2

-CH

2

CN

H

3

C-CH

2

CH

2

CH

2

CH

2

CH

2

CH

2

CH

2

NH

2 0 H C-C -0 C2-NHC-NH 0

H

3

-C

2 -CH 2

-CH

2 CHN

H

2 0 0

H

2

N

H

3

C-CH

2

-CH

2

-CH

2

-CH

2

-CH

2

-NH

2 H 4-CH 2

-CHCH

0 0

CH

3 Cl

H

3 C CH C 2I H NH 2 H 2 N C 2c

H

3 CH IH 2 0

CH

3 H2 H2 CH2- H2 CH2-CH2- H -C

CH

3

H

2 N- C, 2 l

CH

3 01 pCTIUS95IOS94) WO 95/30642

ICA

NH I- -C

CH

2 "4

VIA

~NNH

2

I

0-/v0 00 C H

NH

2 0M 0 042 C=

H

3 1 K c

OH

2 ,H3

CH

3

COH

PCT/US95/05940 WO095/30642 -31-

H

3 C -CH 2

CH

2

CH

2

-CH

2

NH

2

H

2

N-CH

2

H

2

N-CH

2 Br H 3

CCH

2

CH

2

CH

2 00H 2

-CH

2

-NH

2 Cl

H

2

N-CH

2 -pc 2-H-H

H

3

C

H

2

N--CH

2 I H, 2

N-CH

2

-CH

2 ZI~ 0 CH NH2 CH H ~II7I 0H 3

C

CH2

I

WO 95/30642 W095/0642PCTIUS95105940 -32-

\C-NH-CH

2

CZCH

2

-CH

2

C

2

C

2 2

OH

2

C

o OH 2

C

H

3 Cc /C

NCHF

HC

CH

2

H

3

C-S-CH

2

CH

2

-H

2 NH 2 CH 3 H N

NH

2 CH 2 Ci HC CH 2 0 H2,CH 2

CH

3 c 0I H 2 N-CH 2 (II

CH

3 F

CH

3 F F I> I H3j- 1 CH 2

-NH

2 H 2 N-CH 2

-C-C-F

CH 3 1H F F F F F F F 1 tl~~ 2

N-H

2

-C-C-C-C-C-C-CF

~CHN I I I I I I OH 2

CH

2 F F F F F F F

H

3 C NH 2 WO 95/30642 WO 9530642PCTIUS95/05940) -33clH 2 N CH CH2

H

2

N-CH

2 Q F H 2

N-CH

2

-CH

2 0 0 ci CH H Hj\I-CH 2

-CH

2

-S-CH

2

Q

0 2 -CH2 N H 2-

CH

3 I Cl H3CNCH2 1-N-CH 2

-CH

2

-S-CH

2 0 0

CH

3 ci 0

H

3 C- 0

CH

2

NH

2

H

3

C

CH

3

NH

2

H

3 C CH 2 NH2

CH

2 (II)H 3 C-o0 (7

-CH-

3 WO 95/30642 WO 9530642PCTUS9SIOS94O -34- 0 NH~ Q 0H oz:/ NH 0 I ,C 2 C 0CH 2 C-H 2

FCH

2 32 0 NH 2 CCN 6>

HCH

2 F/H2 CHH2 00

HN/

CNHH 2 NH 00N K -C C6-H 3 -CH2 C2C:0 ci -i H Nc H 2 CH 2 CH2

H

0 0-1N

H

3 C-O 0 CH 2

-CH

2

-NH

2 0 H2 N-CH-C0 WO 95/30642 WO 9530642PCT/US95/05940 1 HNQ

-CH

Do-C

NH

2 NH 2

H

2

~C

2

H

2 HH 2N

CH

H 3

C-CH

2

-H

2 -2NNH H2NC/ 2 G2-H 2

H

CHC

HH3 2

C

2

~H

C H 2 L H 2 C 2 H C H

H

2

N

HN-CH 2

-CH

2

CH--\

H2 2C2 l HZN-CH 2 0

C-

0 H3C-O- O O-CH 2

-CH

2

-NH

2 F F I I

H

2

N-CH

2 CC- F

I

WO 95/30642 PCJ'lUS95105940 -36- WO 95/30642 P1IJ9I54 PCT/US95/05940 -37- 0 N

I

H C CP C8 2 -NH2

NH

2 0 cl

NH

HNCCH CHCH 2 c /0 CNH-CH 2 0 0 Q /H 2 K 0

H

2

N

1Yz C 0C 0

/N

Q

I

CH

3 0 1H

H

3 C uCH 2

H

II N 0

CH

2 0 0 0

NH

2 H2-CH 2

-CH

2

-CH

2

-CH

2 -NH- ~NC

H

0 00

CH

3 WO095/30642

H

2 r PCT/US9510594() -38- WO 95/30642 PTU9I54 PCT/US95/05940 -39lHC 1CH 2

H

CI0 CH 2

H

2

N-CH

2

-CH

2 D( ClI 0

CHH

F

F NH 2 2 \2 cCH 2

C\

F

F

F

NH

2 F

CH

2

F

H

2 N-CH U L- WO 95/30642 PTU9154 PC'r/US95/05940 ci

H

2

N-CH

2

-CH

2 0 Cl 0 0

CH,-

HN-CH2- 0 0

SQN

H

2

N-CH

2

-CH

2

NH

CH-CH,-CH,-CH, -CH 2

-NH

2

H

2

C-NH-C

0

CH

2

CH

2

NH

2

CH

3 IIIC H",H,0-CH2CH 2

-NH

2

ICH

2

CH

2

H

3 C

NH

2

HC

ICH

3 HN F

C=

CH0

CH

2

F

.CH,

CH

3

FH

F

WO 95/30642 W095/0642PCT/US95/05940 -41- CH -MCF[-,CH 2 2-NH 2 H, CH 2 CNHci

CH-CH

2 0 H2N-CH FCR 3

CH

2

-CH

2

-NH

2

CH

3 F 0 0 00 O 2 N-CH C I

H

2 N- CH 2

CH

2

N

3 C~ N 00 CH, 3 C CH 2

-NH

2 -2 CH2

CCH,

NH

2 W0 95/30642 PCJIYUS95O59U0 -42-

F

H

2

N,

CH

2 C0\F NCHY -CHZ 0 Hac

I

00 oc 0 0 CH -C CH2 C CH 2

H

2 N- 2

HC

sO N /0

NH,

H

2

N-CH

2 0 CH2,Vi

NH

2 WO 95/30642 ,PC'T1US95/05940 -43-

H

3 C H 0

H

3

C-CH

2 -O CH2CH2NH2 0\

CH

3 0 00

H

2

N-CH,-

2 OIIl- H C-CH 2

-CH

2

-CH

2

-NH

2 If 0- 00 0 H3CC NHCH

CH,

HC I CH-CH 2 NH2 HH2 2 0 0

C

H

3 3C 0~ 0 0 Hj'I-CH 2

-CH

2 0 H 2

N-CH

2

-CH

2

CH

2 -S 0IZ WO 95/30642 W095/0642PCT1US9510594) -44- N N

NH

2

N

CH

2 0F

H

3 -O--CH

-CH

2 -CH -CH -NH 2 0 0-C-F 0 H N- 0 H2N CH 2 -CH

CH

2

-N

C-CHH

2

NCH

2 2

CH

2

H

2

C-

2

H

2

C,

Hoc

CH

2 0 H3C s-NH-CH 2

-CH

2

-NH

2

C

2 2-H2C 0

H

3 C C2-NH2 H2N-CH 2

-C-CH-CH

2

-CH

2 2 2 Q I CH 2

H

2

N

0 0

H

2

C-CH

2 c

H

2

N-CH

2

-CH

2

-CH

2

CH,

Hr-cH'

H

2 C -CH,

CH

2 ___Nli WO 95/30642 W095/0642 C'rUS95/05940

S

14A H2N-CH2 CI O-CH 2 0D

CH

2 ~2C

H

2

N

S

NH

2

CH

2

CH

2

CH

CHH3 HH H 2 C H CH, H 3

C-CH

2 -CH 2

-O-CH

2

-H

2

-CH

2 -NH 2

H

2 c C H2c S NHI C1 zs"- N H 2-C2\0 0 sOH 2 l, l

H

NH 6H N CH2 WO 95/30642 P~ISSO9( PCT/US95/05940 -46- N3 0 0 C-NH-CH 2

-CH

2

-CH

2

-NH

2

H

2

N-CH

2

CH

2 -0 Q0 0 -CH 3

H

3 C 0 0C CH2 NHH2C O H 2- CH H2N C .CH 2 CH2 H 0

F

CH

3

H

3 C0 H C 0N

H

2 -C 2 N H -1 3 c H H 2 N- CH 2 I Ej N 0H 3 C N Cl N F

H

2

N-CH

2

-CH

2 -S I 2 -H-CH -0

F

Cl S-CH, 0IDF

CH

2 H 2

N-CH

2

-CH

2

/H

2

N

C, 0 0 \CHM.-

NH-CH

2 -CH 2

-NH

2 MC-O0

CH

2

-CM

2

-NM

2 r

CH

CF

H,

A

WO 95/30642 WO 95/0642 cT/us95/0594t) -47-

H

2

NN

CHH2 0

H

2 N-CHi 2 01CH 2 F H2H 3

C

ci2 CH 0HC 0 CH2NCH2CH 2 0 0 0

H

3 0 H3-CH-CH-NH 0H2 CH 0 S-F, 0HL,

H

2

N-HCHH

2 C)2 0H-N 0 N0 1CIUS95OS940 WO 95/30642

H.

-48- 1- CH 2 )i 5

CH

3 3 0-(0H 2 7 CHCH (0H 2 8 -N H 2

H

3 0-(CH' 2 9 -N H 2

H

3 0-(0H 2 12

-NH

2

H

3 0-(C H 2 15 -N H 2

H

3 0-(0H 2 )lrNH 2 0 WO 95/30642 WO 9530642PCTICUS95/05940 -49-

S

HNC2 CH 0

H

2 N- CH2-H-_

CH

3

H

3 C 10

H

NH

2

CH

2 0 N H 2

N-CH

2 -0H 2

O

0

CH

3 cNH 2 M NH -CH CH 2ZH

CHCH

CH 2 CM\

H

3 Co%-c

H

3 0WCH2- CM 2

CH

2

NH.

2

C

3 0 H 3 C-C-H 3 0 NH 2 F F 00 0F H2 H, C H2 0F o= c j

CH

2 2 WO 95/30642 PCTIUS95/059410

H

3 C

C

CH

3

F

H3C "INH CH 2 H2N-.CH2 b -B cNH 2 Q0 B

CH

3 0 11 0 F* 0 lia NH. 3 0H v Br

CH

3

(~NI

1)

CH

2

ICH

2

F

H N

NH

2 0 H H 3

CH-CH

2

H

F

~~~CH

2 3 2 2N NH 2

F

Br

F

WO 95/30642 WO 9530642PCT/US95/05940 -51- NQ2 0 0 z O/ \\Os 0 0 0

H

2

N-GH

2 -C NH 00H.~ H H 00CH-CH 2 -n CH-C2CH 2 NH-C'

H

3 C 2

NH

2 0- H 0 CH2CH2- 0 C oCH2

~.CH

2 C2

CH

2 H CH 2H 3 C..CH2 2H

H

2

N

011 -I~uH 3

-CH

CH3 0 C 0 CH2 2 0 0 0> CH3..CCH C H 2 WO 95/30642 PCTIUS95IO594O0 -52- WO 95130642PCIS9I94 013 0 \C-NH 0, 01, CH/ 0C2CH 2

NHH

0 00 0104 040 150 03 0 9 0 0

HN

043H 2

N-CH

2

-CH

2 s-~ I I 0 N 0

H

3 C 0 0

K

3

C

Ti Cw2 1111r 7 1\ w C 2 M 4 C H 2 H 'C 2 HC9 WO 95/30642 PCT/US95/05940 -54f H 3

C

NH

H

3 0-(0H 2 11

-NH

2

CH

2

H

2

N

0 (CH 2 3

NH

2 C IH I. CCH cl

\HC

H

3 C 02 1 3 ~J

H

H

2

N

H

(0H 3 3 NH2

I

WO 95/30642 PCT/US95/05940 One embodiment of the invention is the use of the combinatorial library of Formula I in assays to discover biologically active compounds (ligands) of Formula II. Thus, an aspect of the invention is a method of identifying a compound having a desired characteristic which comprises synthesizing a combinatorial library of Formula I and testing the compounds of Formula I and the ligands of Formula II, either attached to the solid support or detached therefrom, in an assay which identifies compounds having the desired characteristic. A further embodiment of the invention is determining the structure of any compound so identified.

Another embodiment of the invention is a process for preparing a compound of the formula:

HO

9 C 0 3

O

OMe

NO

2

R

2

OH

where R 2 is H or lower alkyl; which comprises a) reacting allyl or methyl 4-(hydroxymethyl)-3nitrobenzoate with a compound of the formula: 0 HO- Me HOO

R

2

OH

in the presence of triphenylphosphine, toluene, and DEAD and stirring the mixture at room temperature to produce

RO

2 CO 0 o-^I Me

NO

2

R

2

OH

where R is allyl or methyl and b) when R is allyl reacting said compound with methylene chloride, tetrakistriphenylphosphine palladium(0), a .d pyrrolidine and stirring IBIIAC WO 95/30642 PCTIUS95/05940 -56the mixture at O°C, or when R is methyl reacting said compound with dilute NaOH and THF and stirring the mixture at 0°C.

For this reaction, R=allyl is preferable to the t-butyl or methyl esters since the milder conditions would not induce aldol type condensation of the acetophenone portion of the molecule.

Another embodimrnt of the invention is a method for identifying compounds that are inhibitors of carbonic anhydrase which comprises preparing a mixture of 20-300 pmol test compound and aqueous solutions (total volume: 25-100, preferably about 50, pL) of 0.03-0.12, preferably about 0.06, pM carbonic anhydrase and 0.04- 0.16, preferably about 0.08, piv dansylamide, exposing said mixture to U.V. (preferably 274 nm) light, and determining the amount of emitted U.V. (preferably 454 nm) light.

Another embodiment of the invention is a method for identifying compounds that are enzyme inhibitors which is a lawn assay which comprises contacting a colloidal matrix containing enzyme, which matrix has embedded therein a mono-layer of solid supports with attached ligands, with a layer of fluorogenic substrate-containing material, eluting said ligands by exposure to U.V. light, and detecting zones of inhibition in the colloidal matrix produced thereby. A preferred such lawn assay comprises contacting an agarose matrix containing bovine carbonic anhydrase with a fluorescein diacetatecontaining layer of agarose.

Another embodiment of the invention is a compound of the formula: Br 0 NO 2

R

H N(CH) N

R

-(CH2,R 1 wherein: WO 95/30642 WO 9530642PCTUS95IO5940 -57- ®''is R is

R

16 is R1 7 is Y is a solid suppoit; H or alkyl; lower alkyl, substituted lower alkyl, aryl, or substituted aryl; H; alkyl substituted by 1-3 alkoxy, S-loweralkyl, sulfamoyl, halo, alkylsuiphonamido, or arylsuiphonamido; alkenyl; alkynyl; aryl; substituted aryl; heteroaryl; substituted heteroaryl; heterocycloalkyl; -CH2NR 16

C(O)R'

6

-C(O)NR

16

RI

6 -CH2OC(O)R' 6 or -CH2SC(O)R 16 and aryl. or heteroaryl.

Compounds of formula 14 are useful as intermediates in the construction of combinatorial libraries and are especially useful in automated or batch mode syntheses thereof.

Definitions The following abbreviations have the indicated meaning: Boc

DEAD

DBU

DCM

DIC

DMAP

DMF

DMSO

DVB

EDT

equiv.

Et

FACS

Fmoc

GC

HOBt hr t-butyloxycarbonyl cyclo -diethylazodicarboxylIate I,8-diazabicyclo[5,4,0]undec-7-ene dichioromethane methylene chloride diisopropylcarbodiimide N,N-dimethy lam inopyridine N,N-dimethylformamide dimethyl sulfoxide 1 ,4-divinylbenzene 1 ,2-ethanedithiol equivalent ethyl fluorescence activated cell sorting 9-fluorenylmethoxycarbonyl gas chromatography N-hydroxybenzotriazole hour, hours I I_ ~1_ WO 95/30642 PCT/US95/05940 -58im imidazole in indole m- meta Me methyl Mtr 4-methoxy-2,3,6-trimethylbenzenesulfonyl n- normal Naph naphthyl p- para PEG polyethylene glycol Ph phenyl Phe phenylene Pmc 2,2,5,7,8-pentamethylchroman-6-sulfamoyl Py pyridyl r.t. room temperature sat'd saturated s- secondary t- tertiary t-Boc t-butyloxycarbonyl TFA trifluoroacetic acid THF tetrahydrofuran "Alkyl" is intended to include linear, branched, or cyclic structures and combinations thereof of from 1 to 20 carbon atoms.

"Lower alkyl" includes alkyl groups of from 1 to 8 carbon atoms.

Examples of lower alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, s- and t-butyl, pentyl, hexyl, octyl, c-propyl, c-butyl and the like. "Lower cycloalkyl" includes cycloalkyl groups of from 3 to 8 carbon atoms. Examples of lower cycloalkyl groups include cpropyl, c-butyl, c-pentyl, 2-methylcyclopropyl, cyclopropylmethyl, and the like.

"Alkenyl" is C2-C6 alkenyl of a linear, branched, or cyclic

(C

5

-C

6 configuration and combinations thereof. Examples of alkenyl groups include allyl, isopropenyl, pentenyl, hexenyl, c-hexenyl, 1propenyl, 2-butenyl, 2-methyl-2-butenyl, and the like.

"Alkynyl" is C2-C6 alkynyl of a linear, branched, or cyclic

(C

5

-C

6 configuration and combinations thereof. Examples of alkynyl groups include ethynyl, propargyl, 3-methyl-l-pentynyl, 2-heptynyl WO 95/30642 PCT/US95/05940 -59isopropynyl, pentynyl, hexynyl, c-hexynyl, 1-propynyl, 2-butynyl, 2methyl-2-butynyl, and the like.

"Alkoxy" means alkoxy groups of from 1 to 6 carbon atoms of a straight, branched, or cyclic configuration. Examples of alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy, cyclohexyloxy, and the like.

"Substituted loweralkyl" means lower alkyi substituted with 1-3 alkoxy, carboalkoxy, carboxamido, di-loweralkylamino, aryl, substituted aryl, or heteroaryl.

"Aryl" means phenyl or naphthyl.

"Substituted aryl" means aryl substituted with 1-3 halo, loweralkyl, alkoxy, aryl, S-loweralkyl, alkylsulphonamido, arylsulphonamido, or sulfamoyl.

"Heteroaryl" means a 5 or 6 membered aromatic ring containing 1-3 hetero atoms selected from O, N, and S.

"Substituted heteroaryl" means heteroaryl substituted with 1-3 halo, loweralkyl, alkoxy, aryl, S-loweralkyl, alkylsulphonamido, arylsulphonamido, or sulfamoyl.

"Heterocycloalky" means lower cycloalkyl containing 1-3 hetero atoms selected from O, N, and S.

Halogen includes F, CI, Br, and 1.

L and L' are depicted in Table 1, which also shows cleavage reagents. In designing a synthetic scheme, L and L' are chosen such that they are orthogonally reactive; they must allow for removal of either T or II (where T T'-OH) without removal of the other since simultaneous cleavage of both T and II from the solid support is disadvantageous. In the structures as shown, the left-hand bond is the point of attachment to the solid support and the right-hand bond is the point of attachment to either T or II.

'111 WO 95/306d2 WO 9530642PCT/US95/05940 TABLE I LINKER GROUPS Linker Group Cleavage Reagent

NO

2 hv 1. \CH 2 or

CH

2 O-lLBhv 2. 0 2 N-

CH

2

O-

OR Ce(NH 4 )2(N0 3 )6 4 RO ~Ce(NH4)2(N03)6

-CH=CH(CH

2 2 03, OSO4/IO4-, or KMnO 4 6. -CH-CHCH 2 03, 0s041104-, or KMnO4 7. -CH 2 CH=CH- 03, 0s041104-, or KMnO4 80 0- 1) 02 or Br2, MeOH 2) 9. -CH--CHCH 2 O- (Ph3P)3RhC1(H) Br Li, MgF, or BuLi 11 Hg+ 2 12 XC 2 O Zn or Mg 1. OH Oxidation, Pb(OAc)4 13 1

CH

2 or H5106 R H or lower alkyl X electron withdrawing group such as Br, Cl, and I.

m WO 95/30642 PCT/US95/05940 -61- The tags of this invention, T, are chemical entities which possess several properties: they must be detachable from the solid supports, preferably by photolysis or oxidation; they must be individually differentiable, and preferably separable; they must be stable under the synthetic conditions; they must be capable of being detected at very low concentrations, 10- 18 to 10- 9 mole; they should be identifiable with readily-available equipment which does not require sophisticated technical capabilities to operate; and they should be relatively economical. The tags may be structurally related or unrelated, a homologous series, repetitive functional groups, related members of the Periodic Chart, different isotopes, combinations thereof, and the like. At the end of the combinatorial synthesis, to each solid support, there will usually be attached at least 0.01 femtomol, usually 0.001-50 pmol, of each tag. The tags may be aliphatic, alicyclic, aromatic, heterocyclic, or combinations thereof.

Distinguishing features may be the number of repetitive units, such as methylene groups in an alkyl moiety; alkyleneoxy groups in a polyalkyleneoxy moiety; halo groups in a polyhalo compound; aand/or P-substituted ethylene groups where the substituents may be alkyl groups, oxy, carboxy, amino, halo, or the like; isotopes; etc.

The materials upon which the combinatorial syntheses of the invention are performed are referred to as solid supports, beads, and resins. These terms are intended to include: a) beads, pellets, disks, fibers, gels, or particles such as cellulose beads, controlled pore-glass beads, silica gels, polystyrene beads optionally cross-linked with divinylbenzene and optionally grafted with polyethylene glycol and optionally functionalized with amino, hydroxy, carboxy, or halo groups, grafted co-poly beads, polyacrylamide beads, latex beads, dimethylacrylamide beads optionally cross-linked with N,N'-bis-acryloyl ethylene diamine, glass particles coated with hydrophobic polymer, etc., material having a rigid or semi-rigid surface; and b) soluble supports such as low molecular weight noncross-linked polystyrene.

I I I WO 95/30642 PCT/US95/05940 -62- It is intended that the definitions of any substituent or symbol R 3 in a particular molecule be independent of its definitions elsewhere in the molecule. Thus, NR 3

R

3 represents NH2, NHCH3, N(CH3)2, etc.

Optical Isomers Diastereomers Geometric Isomers Tautomers Some of the compounds described herein contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms which may be defined in terms of absolute stereochemistry as or The present invention is meant to comprehend all such possible diastereomers as well as their racemic and optically pure forms and mixtures thereof. Optically active and forms may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended to include both E and Z geometric isomers. Likewise, all tautomeric forms are intended to be included.

Salts The pharmaceutical compositions of the present invention comprise a compound of Formula II as an active ingredient or a pharmaceutically acceptable salt thereof, and may also contain a pharmaceutically acceptable carrier and, optionally, other therapeutic ingredients. The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic acids or bases including organic and inorganic acids or bases.

When a compound of the present invention is acidic, salts may be prepared from pharmaceutically acceptable non-toxic bases.

Salts derived from all stable forms of inorganic bases include aluminum, ammonium, calcium, copper, iron, lithium, magnesium, manganese, potassium, sodium, zinc, etc. Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion-exchange resins such as arginine, betaine, caffeine, _1 I WO 95/30642 PCT/US95/05940 -63choline, N,N'-dibenzylethylenediamine, diethylamine, 2diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, isopropylamine, lysine, methylglucosamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, etc.

When a compound of the present invention is basic, salts may be prepared from pharmaceutically acceptable non-toxic acids.

Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic, etc. Particularly preferred are citric, hydrobromic, maleic, phosphoric, sulfuric, and tartaric acids.

In the discussion of methods of treatment herein, reference to the compounds of Formula II is meant to also include the pharmaceutically acceptable salts thereof.

Utilities The ability of the compounds of Formula II to interact with a adrenergic receptors indicates that the compounds are useful to treat, prevent, or ameliorate hypertension and benign prostate hypertrophy in mammals, especially in humans.

The ability of the compounds of Formula II to interact with dopamine receptors indicates that the compounds are useful to treat, prevent, or ameliorate Alzheimer's disease and depression in humans.

The ability of the compounds of Formula II to interact with o-opiate receptors indicates that the compounds are useful to treat, prevent, or ameliorate schizophrenia in mammals, especially in humans.

The ability of the compounds of Formula II to interact with K+ channels indicates that the compounds are useful to treat, prevent, or ameliorate hypertension, asthma, and pulmonary insufficiency in mammals, especially in humans.

The ability of certain compounds of Formula II to inhibit carbonic anhydrase isozymes makes them useful for preventing or reversing the symptoms induced by these enzymes in a mammal. This pss~ WO 95/30642 PICTUS95105940 -64enzyme inhibition indicates that the compounds are useful to treat, prevent, or ameliorate ocular diseases, particularly glaucoma in mammals, especially in humans.

Dose Ranges The magnitude of the prophylactic or therapeutic dose of the compounds of Formula II will vary with the nature and severity of the condition to be treated and with the particular compound of Formula II and its route of administration. In general, the daily dose range for anti-enzymic use lies in the range ot 20 to 0.001 mg/kg body weight of a mammal, preferably 10 to 0.01 mg/kg, and most preferably to 0.1 mg/kg, in single or divided doses. In some cases, it may be necessary to use doses outside these ranges.

When a composition for intravenous administration is employed, a suitable daily dosage range is from about 10 to 0.0005 mg (preferably 5 to 0.01 mg) compound of Formula II per kg body weight.

When a composition for oral administration is employed, a suitable daily dosage range is from about 20 to 0.001 mg (preferably to 0.01 mg) compound of Formula II per kg body weight.

When a composition for ophthalmic administration is employed, a suitable daily dosage range is from about 10-0.01% (preferably 5.0-0.5% compound of Formula II, typically prepared as a 2.0-0.1% by weight solution or suspension of a compound of Formula II in an acceptable ophthalmic formulation.

The compounds of Formula II may also be used in combination with other pharmaceutically active ingredients. For example, a typical oc',lar formulation may comprise the compound alone or in combination with a P-adrenergic blocking agent such as timolol maleate or a parasympathomimetic agent such as pilocarpine.

When used in combination, the two active ingredients are present in approximately equal parts.

Pharmaceutical Compositions Any suitable route of administration may be employed for providing a mammal, especially a human, with an effective dosage of a compound of Formula II. For example, oral, rectal, topical, parenteral, ocular, pulmonary, nasal, etc. routes may be employed. Dosage forms WO 95/306,42 PCT/US95/05940 include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, and the like.

The pharmaceutical compositions of the present invention comprise a compound of Formula II, or a pharmaceutically acceptable salt thereof, as an active ingredient, and may also contain a pharmaceutically acceptable carrier and, optionally, other therapeutically active ingredients.

The compositions include compositions suitable for oral, rectal, topical (including tansdermal devices, aerosols, creams, ointments, lotions, and dusting powders), parenteral (including subcutaneous, intramuscular, and intravenous), ocular (ophthalmic), pulmonary (nasal or buccal inhalation), or nasal administration; although the most suitable route in any given case will depend largely on the nature and severity of the condition being treated and on the nature of the active ingredient. They may be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.

A compound of Formula II may be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the nature of thle preparation desired for administration, oral, parenteral, etc. In preparing oral dosage forms, any of the usual pharmaceutical media may be used, such as water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like in the case of oral liquid preparations suspensions, elixirs, and solutions); or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, etc. in the case of oral solid preparations such as powders, capsules, and tablets. Solid oral preparations are preferred over liquid oral preparations. Because of their ease of administration, tablets and capsules are the preferred oral dosage unit form. If desired, capsules may be coated by standard aqueous or non-aqueous techniques.

In addition to the dosage forms described above, the compounds of Formula II may be administered by controlled release means and devices such as those described in U.S.P. Nos. 3,536,809; C- II WO 95130642 PCTIUS95105940 -66- 3,598,123; 3,630,200; 3,845,770; 3,916,899; and 4,008,719, which are incorporated herein by reference.

Pharmaceutical compositions of the present invention suitable for oral administration may be prepared as discrete units such as capsules, cachets, or tablets each containing a predetermined amount of the active ingredient in powder or granular form or as a solution or suspension in an aqueous or nonaqueous liquid or in an oil-in-water or water-in-oil emulsion. Such compositions may be prepared by any of the methods known in the art of pharmacy. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers, finely divided solid carriers, or both and then, if necessary, shaping the product into the desired form. For example, a tablet may be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as powder or granule optionally mixed with a binder, lubricant, inert diluent, or surface active or dispersing agent. Molded tablets may b6 made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. Ophthalmic inserts are made from compression molded films which are prepared on a Carver Press by subjecting the powdered mixture of active ingredient and HPC to a compression force of 12,000 lb. (gauge) at 149°C for 1-4 min. The film is cooled under pressure by having cold water circulate in the platen. The inserts are then individually cut from the film with a rod-shaped punch. Each insert is placed in a vial, which is then placed in a humidity cabinet (88% relative humidity at 30*C) for 2-4 days. After removal from the cabinet, the vials are capped and then autoclaved at 121'C for 0.5 hr.

The following are representative pharmaceutical dosage forms of the compounds of Formula Ii: -I WO 95/33642 -67- I.M. Injectable Suspension Compound of Formula II Methylcellulose Tween 80 Benzyl alcohol Benzalkonium chloride Water for injection to a total volume of 1 mL PCT/US95/05940 Tablet Compound of Formula II Microcrystalline cellulose Povidone Pregelatinized starch Magnesium stearate Capsule Compound of Formula II Lactose powder Magnesium stearate mg/mL 9 1 mg/tablet 415 14 43.5 500 mg/capsule 573.5 600 Per canister 24 mg 1.2 mg 4.025 gm 12.15 gm mg/mL 1 9.38 28.48 1 mg/g 1 Aerosol Compound of Formula II Lecithin, NF liquid concentrate Tnchlorofluoromethane, NF Dichlorodifluoromethane, NF Ophthalmic Solution Compound of Formula II Monobasic sodium phosphate2H20 Dibasic sodium phosphate 12H20 Benzalkonium chloride Water for injection to a total volume of 1 mL Ophthalmic Suspension Compound of Formula II Petrolatum liquid to a total weight of 1 g

M

I I- WO 95/30642 PCT/US95/05940 -68- Ophthalmic Insert mg/insert Compound of Formula II 1 Hydroxypropylcellulose 12 These compounds of Formulae I and II may also be used as libraries for discovering new lead structures by evaluation across an array of biological assays, including the discovery of selective inhibition patterns across isozymes. These libraries are thus tools for drug discovery; as a means to discover novel lead compounds by screening the libraries against a variety of biological targets and to develop structure-activity relationships in large families of related compounds. The libraries may be tested with the ligands attached to the solid supports as depicted in Formula I or the individual compounds II may be detached prior to evaluation. With the compounds of Formula 1, screening assays such as FACS sorting, bead lawn assays, and cell lawn assays may be used. When a compound is detached prior to evaluation, its relationship to its solid support is maintained, for example, by location within the grid of a standard 96-well plate or by location of activity on a lawn of cells. The solid support associated with bioactivity or the solid support related to the detached ligand may then be decoded to reveal the structural or synthetic history of the active compound (Ohlmeyer et al., Proc. Natl. Acad. Sci. USA, 90, 10922-10926, Dec.

1993).

Assays for Determining Biological Activity The compounds of the present invention may be tested by assays well known in the art for interaction with a adrenergic receptors, interaction with dopamine receptors, interaction with oopiate receptors, interaction with K+ channels, and carbonic anhydrase inhibition. For example, representative references teaching carbonic anhydrase inhibition assays are: Carbonic Anhydrase Inhibition Maren and Couto, "The Nature of Anion Inhibition of Human Red Cell Carbonic Anhydrases", Archiv. of Biochem. and Biophy., 196, No. 2, Sept., 501-510 (1979).

I1II -L 1-- WO 95/30642 PCT/US95/05940 -69- Carbonic Anhvdrase Inhibition Ponticello et al., "Thienothiopyran-2-sulfonamides: A Novel Class of Water-Soluble Carbonic Anhydrase Inhibitors", J. Med. Chem., 30, 591-597 (1987).

Carbonic Anhvdrase Inhibition It has now been found that the use of very low initial concentrations (0.04-1.6, preferably about 0.6, pM) of dansylamide and (0.03-1.2, preferably about 0.3, pM) of carbonic anhydrase to assay test compounds for carbonic anhydrase inhibition not only allows the use of very small total volumes (approx.

25-100, preferably about, 50 pjL) per assay but also allows one to distinguish high-affinity from low-affinity compounds without either re-elution or re-synthesis of the test compound. By increasing the concentration of dansylamide from -0.1 jpM to -200 pM directly in the assay sample, relatively weak inhibitors can be distinguished from relatively strong inhibitors on the same aliquot of test compound. The small total volume advantageously permits high throughput assaying of small quantities of test compounds, for example, in 96-well plates, and the reduced concentration of dansylamide advantageously permits the detection of test compounds that have a wide range (<500 nM) of characteristic dissociation constants. The following materials are used: 100. mM sodium phosphate buffer, pH 7.4 0.6 pM dansylamide (Sigma D-3882) 0.3 pM bovine carbonic anhydrase (Sigma C-3934) inhibitor Reactions are carried out in 50 pL total volume in 96-well plates, preferably, Dynatech MicroFluor plates, white with bottom, containing the test compounds. The assay mix is prepared immediately before use, and 50 pL of the assay solution is pipetted into each well of plates in which the test compounds are previously dried. The plates are spun briefly in a tabletop centrifuge before reading fluorescence.

Fluorescence is read in a Perkin-Elmer I S 50B spectrofluorimeter fitted with a Well Plate Reader Accessory using an excitation wavelength of 274 nm (2.5 nm slit) and an emission wavelength of 454 nm (20 nm slit), with a 390 nm cutoff filter in place. Fluorescence measurements are averaged over 1 sec for each well. To identify wells in which inhibitors are present, first a plate with no exogenous WO 95/30642 PCTIUS95/05940 inhibitors is read, which typically gives a fluorescence reading of 2.6- 3.1 (typical standard deviation 0.06) for a given assay solution. In plates containing inhibitor candidates, active inhibitors cause a decrease in the fluorescence signal of greater than 5 times the standard deviation.

To distinguish high-affinity from low affinity candidates, pL of a 2 mM stock of dansylamide in DMSO is added to the above test solution, and the assay repeated as above. Typical readings are 7.5 to 8.5±0.4 (standard deviation) among previously identified inhibitors.

High-affinity compounds lower the signal by greater than 3 standard deviations). Thus, the increased concentration of dansylamide is sufficient to displace relatively weak inhibitors chlorothiazide, Ki 75 nM) without displacing relatively strong inhibitors acetazolamide, Ki 7.5 nM).

Bead Lawn Assay (General Method). An enzyme of interest is incorporated into a gellable gum such as silica gel, agar, agarose, pectin, polyacrylamide, gelatin, starch, and gellan gum, preferably a low melting-temperature agaroe gel wt./vol.), which is layered on top of a lawn, no greater than one bead in thickness, of solid supports with attached ligands. The detection of an active combinatorial library member is accomplished by photoeluting the ligands from the beads in situ by exposure to U.V. light. To minimize premature photoelution, the beads are preferably protected from ambient light sources prior to U.V. exposure. The beads are evaluated by placing a second layer, preferably low-melt agarose gel, containing a substrate on top of the one containing the enzyme and the photoreleased library members, and allowing enzymic conversion of substrate into product by diffusion of the substrate into the enzyme-containing gel. The substrate is preferably one that produces a photometric change upon conversion into product; the generation of a colored product, a fluorescent product, or a chemiluminescent reaction (where one of the products is a photon). The second layer may comprise a gellable gum such as silica gel, agar. agarose, pectin, polyacrylamide, gelatin, starch, and gellan gum, or a solid material such as a matrix containing an array of fluorogenic-pellets. Inhibition of the enzyme by a library member results in a difference in appearance in the vicinity of the attached bead and allows for selection of the bead and the identifiers which encode for WO 95/30642 PCTIUS95/05940 -71the inhibitor. This technique may for example: Acid Phosphatase Activated Protein C Alkaline Phosphatase Aminopeptidases B M Amyloid A4-Generating Enzyme Angiotensinase Aryl Sulfatase 3-Galactosidase P-Glucosidase P-Glucuronidase Calpains I II Cathepsins B, C, D, G Cholinesterase Chymotrypsin Collagenase Dipeptidyl Peptidases I- IV Elastase Endothelin Converting Enzyme Factor Xa Factor XIa Factor XIIa Df-Protease be used with a variety of enzymes, Furin y-Glutamyltranspeptidase Granzymes A B HIV Protease IL-1B Convertase Kallikrein Lysozyme Mast Cell Protease Peroxidase Plasmin Prohormone Convertase rANP Precursor Processing Enzyme Renin Spleen Fibrinolytic Proteinase Staphylocoagulase Thrombin Tissue Plasminogen Activator Trypsin Tryptase Urokinase A bead lawn assay for testing carbonic anhydrase inhibition preferably comprises agarose for both layers, bovine carbonic anhydrase, and fluorescein diacetate.

Bead Lawn Assay (Carbonic Anhydrase). Beads to be tested are arrayed in a minimal amount of methanol in a 60 mm polystyrene tissue culture dish and then all the methanol allowed to evaporate. A 2.5% (wt./vol.) mixture of agarose (SeaPlaque, FMC BioProducts, Rockland, ME) in 20 mM sodium phosphate buffer (pH 7.4) is heated on a hot plate until the agarose dissolves and then is equilibrated to 37 0 C in a water bath. A separate stock of the same buffer is also equilibrated to 37 0 C. The enzyme layer is prepared as follows: 100 pL of a bovine carbonic anhydrase stock (0.5 mg/mL or 53 pM based on absorbance at 280 nm, Sigma #C-3934) is added to 2.15 mL of buffer, and 1.25 mL agarose solution is added to the mixture. The agarose/enzyme solution is poured onto the dish

I

_1_ WO 95/30642 PCTIUS95/05940 -72containing the beads and the agarose is allowed to solidify at r.t. for min. To identify zones of inhibition, the compounds, which are optionally photoeluted by exposure to 4.7-6 mW/cm 2 365 nm UV light for 5 sec. to 1 hr., are overlayed with fluorescein diacetate (FLDA, Molecular Probes, Eugene, OR), which is prepared as follows: to 2.25 mL phosphate buffer is added 10 pL FLDA stock (10 mM in DMF at and 1.25 mL agarose (final FLDA concentration: 30 pM). The solution is mixed thoroughly then poured over the enzyme layer in the dish. Zones of inhibition appear after 1-2 min. and intensify over 45 min. They are dark against a yellow-green background when illuminated by short-wave UV light (Xmax 254 nm).

Bead Lawn Assay (Inositol Monophosphatase). The assay is similar to that for carbonic anhydrase, with the following substitutions: The buffer used is 20 mM Tris, 1 mM EGTA, pH 7.8.

The enzyme layer contains 1 mg/mL recombinant human inositol monophosphatase (purified from E. coli) and 10 mM MgCI2. Three alternative substrates are used: methylumbelliferyl phosphate (Sigma, M-8883), a fluorogenic substrate, detected using filters around Xex 388 nm and Xem 420 nm; or CSPD or CDP-Star (chemiluminescent substrates for alkaline phosphatase, Tropix, Bedford MA), detected directly without requiring filters. The preferred substrate is CSPD.

I

WO 95/30642 PCT/US95/05940 -73- Methods of Synthesis The compounds of the present invention can be prepared according to the following methods. At each step in the synthesis each solid support upon which a compound is being synthesized is uniquely tagged to define the particular chemical event(s) occurring during that step. The tagging is accomplished using identifiers such as those of Formula IV, which record the sequential events to which the support is exposed during the synthesis, thus providing a reaction history for the compound produced on each support. The identifiers are used in combination with one another to form a binary or higher order encoding scheme permitting a relatively small number of identifiers to encode a relatively large number of reaction products. For example, when used in a binary code, N identifiers can encode up to 2N different compounds and/or conditions. By associating each variable or combination of variables at each step of the synthesis with a combination of identifiers which uniquely define the chosen variables such as reactant, reagent, reaction conditions, or combinations of these, one can use the identifiers to define the reaction history of each solid support.

In carrying out the syntheses, one begins with at least 103, desirably at least 104, and generally not exceeding 1015 solid supports.

Depending on the pre-determined number of R 1

/R

2 choices for the first step, one divides the supports accordingly into as many containers. The appropriate reagents and reaction conditions are applied to each container and the combination of identifiers which encode for each R1/R 2 choice is added and attached. Depending on the chemistries involved, the tagging may be done prior to, concomitantly with, or after the reactions which comprise each choice. As a control, sample supports may be picked at any stage and a portion of their tags detached and decoded to verify that the correct tags are bound to the sample supports. As needed, one may wash the beads free of any excess reagents or by-products before proceeding. At the end of each step, the supports are usually combined, mixed, and again divided, this time into as many containers as pre determined for the number of choices for the second step in the synthesis. This procedure of dividing, reacting, WO 95/306412 PCT/US95/05940 -74tagging, and remixing is repeated until the combinatorial synthesis is completed.

Scheme 1 Functionalized supports such as amino-functionalized or hydroxy-terminating PEG grafted polystyrene beads are divided into a pre-determined number of reaction vessels and are reacted with a cleavable linker/ligand element 3, which has been pre-formed, to generate 4. Unique tagging of the supports in each reaction vessel is achieved with combinations of identifiers encoded in a binary scheme, as depicted in Table 1-1 for three choices of R 1 and R 2 The identifiers are attached by adding a solution of the identifiers (in a wt./wt. identifier:solid support ratio) to a batch of supports suspended in CH2C12 and shaking the mixture for 30 min. A dilute solution of rhodium trifluoroacetate dimer is added and the mixture is immediately shaken 4 hr and washed in CH2C12. The procedure is repeated and the mixture shaken for 14 hr and then washed in DMF/DCM.

Scheme 2 The compounds 4 are pooled, mixed, and divided into a pre-determined number of reaction vessels, each of which is treated with one reagent corresponding to ligand element =CR 4

R

5 in the presence of pyrrolidine to produce 5a, 5b, and 5c. Unique tagging of the supports in each reaction vessel is achieved with combinations of additional identifiers encoded in a binary scheme, as depicted in Table 1-2 for seven choices of R 4

R

5 Scheme 3 The compounds 5c, where R 4

/R

5 /X represents the residue of piperidine, pyrrolidine, or aminocyclohexane, are pooled, mixed, and then divided into a pre-determined number of reaction vessels. The supports in each reaction vessel are uniquely tagged with combinations of additional identifiers encoded in a binary scheme, as depicted in Table 1-3 for 30 choices of R 8 and in Table 1-5 for six choices of R 14 and four choices of heteroaryl groups. After removal of any N- I -r I i- WO 95/30642 PCT/US95/05940 protecting Boc group in R 4

R

5 each reaction vessel is treated with one reagent corresponding to ligand element R 8 in the presence of solvents such as CH2C12, DMF, or EtOH and, when required, bases such as triethylamine or 2,6-lutidine to produce 6 having an R 8 substituent at C-2 and a ketone at C-4, when R 6

R

7 together are 0. In Scheme 3,

R

14 is benzyl, -CH2-Ph-4-F, -CH2-Ph-4-OCH3, -CH2-4-Py, n-pentyl, or-CH2-c-propyl; and heteroaryl is C1 Cl Cl N H N C CI C l NH2 Scheme 4 A portion of the compounds 5a, 5b, 5c, and6 may be pooled, mixed, and then divided into a pre-determined number of reaction vessels where they may be uniquely tagged with combinations of additional identifiers encoded in a binary scheme, as in Table 1- 4 for three choices of R 6

/R

7 Each vessel is treated with sodium borohydride to yield 7 as an alcohol at C-4 or is treated with 1,2dithioethane and a Lewis acid such as BF3*Et20 to yield 8 as a dithiolane at C-4, or is treated with an appropriate non-beta branched primary amine in the presence of NaCNBH3 in MeOH, optionally with acetic acid, to yield secondary amine 9, or is left untreated.

Compounds 5a, 5b, 5c, d, 6, 7, 8 ,and 9 are then exposed to UV light (-360 nm) in polar solvents such as DMSO, H20, or a lower alkanol such as MeOH to cleave the compounds of Formula II from the support/linker complex.

Scheme TentaGel resin may be modified with bis-Boc Lysine to increase the available reaction sites for ligand attachment Bis-Boclysine in DMF, HOBt, and DIC are shaken at r.t. and then dry TentaGel resin is added. The mixture is shaken at r.t. for 17 hr and then washed alternately with methanol and DCM and then with THF and dried under vacuum. To deprotect the resin, DCM is added, followed by a TFA solution in DCM (100 mL). The vessel is shaken at room temperature for 15 min. before adding neat TFA.. The vessel is shaken L WO 95/30642 PCTIUS95/05940 -76at room temperature for 2.5 hr at which time the resin is washed with DCM, then treated with a solution of 10% triethylamine in DCM, then washed with DCM and DMF.

For purposes of simplicity, the schemes do not show the use of this bis modification.

Scheme 6 Functionalized supports such as amino-functionalized or hydroxy-terminating PEG grafted polystyrene beads are divided into a pre-determined number of reaction vessels and are reacted with a cleavable linker/ligand element 10, which has been pre-formed, to generate 11. Unique tagging of the supports in each reaction vessel is achieved with combinations of identifiers encoded in a binary scheme, as depicted in Table 2-1 for seven choices of -(CH2) 1-6R 17 The identifiers are attached by adding a solution of the identifiers (in a 7% wt./wt. identifier:solid support ratio) to each batch of supports suspended in EtOAc and shaking the mixture for 1 hr. A dilute solution of rhodium trifluoroacetate dimer in DCM is added and the mixture is shaken 15 hr and washed with DCM (4X) and EtOAc The procedure is repeated for each identifier.

To deprotect the encoded resin, it is suspended in DCM and then agitated with a TFA solution in DCM. The resin is then washed with DCM followed by treatment with triethylamine in DCM and then washed with DCM.

Scheme 7 The compounds 12 are pooled, mixed, and divided into a pre-determined number of reaction vessels, each of which is treated with one acetophenone reagent corresponding to ligand element R 2 in the presence of DIC, HOBt, and DMF to produce Unique tagging of the supports in each reaction vessel is achieved with combinations of additional identifiers encoded in a binary scheme analogous to that in Table 2-1.

Scheme 8 The compounds 4' are mixed, pooled, and divided into a predetermined number of reaction vessels, each of which is treated with II~ PIIISPU~ l CIII I WO 95/30642 PCT/US95/05940 -77and aldehyde or ketone element corresponding to R 4

/R

5 in the presence o pyrrolidine in methanol at 75°C to produce the compounds 5a', and 5c'. Unique tagging of the supports in each reaction vessel is achieved with combinations of additional identifiers encoded in a binary scheme analogous to that in Table 2-1.

Scheme 9 The compounds 5c, where R 4

/R

5 /X represents the residue of t-Boc protected piperidine, t Boc protected aminocyclohexane, or other amine functionalized molecules aic mixed, pooled, and divided into a predetermined number of reaction vessels. The supports in each reaction vessel are uniquely tagged with combinations of additional identifiers encoded in a binary scheme analogous to that in Table 2-1.

After removal of any N-protecting group in R 4

/R

5 each vessel is treated with one reagent such as a chloroformate, isocyanate, thioisocyanate, carboxylic acid, alkyl or aryl sulfonyl halide, aldehyde, or a haloheteroaromatic compound corresponding to ligand element R 8 in the presence of solvents such as CH2C12, DMF, EtOH, or methanol.

When required, bases such as triethylamine, DBU, or 2,6-lutidine and/or other reagents or combinations of reagents such as DIC, NaCNBH3, HOBt, and acetic acid are added to produce having an R 8 substituent at C-2 and a ketone at C-4, i.e. when R 6

R

7 together are 0.

Scheme A portion of compounds 5a 5c', and 6' may be pooled, mixed, and then divided into a pre-determined number of reaction vessels where they may be uniquely tagged with combinations of additional identifiers encoded in a binary scheme analogous to that in Table 2-1. Each vessel is treated with 1) sodium borohydride in methanol to yield 7' as an alcohol at C-4; 2) 1,2-dithioethane and a Lewis acid such as boron trifluoride etherate to yield 8' as the dithiolane at C-4; 3) an unhindered primary amine along with NaCNBH3 in acetic acid/methanol solvent at ca. 75°C to yield 9' as an amine at C-4; or 4)is left untreated.

WO 95/30642 PCT/US95105940 -78- Scheme 11 The compounds 9 or 9' are divided into a predetermined number of reaction vessels. Each vessel is treated with one reagent such as a chloroformate, isocyanate, thioisocyanate, carboxylic acid, alkyl or aryl sulfonyl halide, aldehyde, or a haloheteroaromatic compound corresponding to ligand element R 15 in the presence of solvents such as CH2C12, DMF, EtOH, or methanol. When required, bases such as triethylamine, DBU, or 2,6-lutidine and/or other reagents or combinations of reagents such as DIC, NaCNBH3, HOBt, and acetic acid are added to produce the corresponding compound 1 or 13'.

Scheme 12 Functionalized supports such as amino-functionalized or hydroxy-terminating PEG grafted polystyrene beads are placed into a reaction vessel and are reacted with a cleavable linker/ligand element 10, which has been pre-formed, to generate 11', To deprotect the resin, it is suspended in DCM and then agitated with a TFA solution in DCM.

The resin is then washed with DCM followed by treatment with triethylamine in DCM and then washed with DCM to yield 12'.

In an appropriately sized synthesis vessel is placed HOBt (3 equiv.) and the carboxylic acid Q (X OH) (3 equiv.) in a solvent such as DMF. DIC (3 equiv.) is added and the vessel agitated for 15 min.

before adding the amino resin 12' (1 equiv. of amino sites). The resin is agitated for 5 hrs., then washed with alternating DCM and MeOH each) and then with THF (2X) to yield 14.

In an appropriately sized synthesis vessel is placed the amino resin 121 (1 equiv. of amino sites). A solvent such as DCM is added, followed by an organic base such as triethylamine, pyridine, Hiinig's base (di-isopropylethylamine), or 2,6-lutidine (10 equiv.). The resin is agitated for 15 min. before adding the acid halide Q (X Cl, Br) (5 equiv.) as a dilute solution in a solvent such as DCM. The resin is agitated for 4 hrs. and then washed with DCM and MeOH (5X each) to yield 14.

WVO95/30642 PTU9IS.( PCVUS95105940 -79- SCHEME 1 LINKER/i s LIGAND ELEMENT t-B u09C.

OH

NO

2 Me R? OH IPPh 3 toluene 2 C 0 0- M

N

2 2

OH

0

M

C N 0 H 0 Me NO, R 2

OH

DCM/TFA

2 33 TentaGel-NH 2 DIC, HOBt, DMF 1 (x IdentifiersX x is 1-30, depending on the binary code for the selected solid support PcT/lIS95/05940 WO 95/30642 SCHEME 2 ADDITION OF R 4 1R R'0 O= 0N-1 Pyrrolidiie, Lower alkanol Identifiers 1vx 5am( NOI

(I)

NO

2 R2 (X is =CH 2 or =0) km(I NOl (X is =N(t-Boc) or =CHNH(t-Boc)) WO 95/30642 WO 9530642PCTIUS95/05940 -81- S"CHEME 3 ADDITION OF R' ClC0 2

R'

2,6 lutidine Identifiers/ yI

OCNR

10

SCNR"

HO R1 2

DMF

C IS0 3

R'

3 Et 3

N

H(O)CR

14 NaCNBH ICI-heteroaryl organ~ic base 6 (l where Y is 43 4R5 or -NR

NHR'

I

PCTI1S95IOS94(I WO 95/30642 -82- SCHEME 4 ADDITION OF R 6 /R7 )0 OH

NO.,

NaBH 4 Jldentifiers Identifiers/

H

2

N(CH

2 16 R 1 4 NaGNBH 3 MeOR

-R'

201)

HS(CH

2 2

SH/

BF

3 ether/ Identifiers cDS /v 360 nrn UV/alkanol 5a,5b,5c,5d.6,7..8,9 pCT!US95/0594() WO095/30642 -83- SCHEME B IS-LINKER

KFFACHMENT

S )NH 2 HOIC' NHBoc DIC, HOBt, DMF NHBoc

N'

CD-H

NHBoc TFA, CH 2

CH

2 NHBoc 0 N NH 2

H

NH

2 PCT1ItS95/05940 WO095/30642 -84- SCHEME 6 CLEAVABLE LINKER/i s LIGAND ELEMENT aNH 2 HO N. 2 1

R

7 0 HOBt, DMF, DIC Identifiers S N NO0-\ 1 H (CH 2 1 -6' L~x1) TFA/DCM 2) NEt 3

IDCM

y 0 NO 2 H N

HN(CH,)

1

,RR

7 12 IVx WO 95/30642 TIS/09( PCTfUS95/05940 SCHEME 7 ATTACHMENT OF HYDROXYACETOPHENONES H0 2 C

M

12

OH

0-1 DIC, HOBt, DMF

IDENTIFIERS

0 -0

(CH

2 1 .6R' S N

N

H7 0 0 "I Me 0v1 OH

I

PCT/US95/05940 WO 95/30642 -86- SCHEME 8 ADDITION OF R 4 /R 0 LR 4 NO,) 0 HN c Qs-N (CH 2 7) 1 O1 0-Il 0 (X CH 2 0, S, N-Me) 00 (XS -NHBco HN~-o 00 NO2 H N 0-

NN

PCTUS95IOS940 WO 95/30642 -87- SCHEME 9 ADDITION OF R'

CCO

2

R?

Identifiers/

%TFAJDCM

-d i) 2,6 lutid

OCNR'

0

SCNR"

HO0 2 R 1 2 ine DIC, DMF CIS0 3 R 13 NaCNBH 3

NH

Ivx I where Y is ~QR,>C9NHR o Me 1 -(CH),C(OP _.NR' NVO 95/30642 P'CTIUS95/05940 -88-, SCHEME ADDITION OF R 6 /R7 HN (CR 2 HV, N 0- Identifiers/NaBH 4

OH

"IR'

Jdentifiers/H 2 N(CH2)j6R 14 NaCNBH 3 MeOH 6' Lvx

NO

2 Identifiers/

HS(CH

2 2

S

B F 3 -ether

S

N

H

Ivx R*=(CH 2 I R' 4

(CH

2 16 R 1 7

N

F-

0 N S S 0~ 5b'. 5c,. 5d', 7' 9' 360 nm UV/alkanol 11 PCTIUS95/05940 WO 95/30642 -89- SCHEME 11 ADDITION OF R" Reagents as in Scheme 9 Jvx 4

R

NO

2 /oN Ivx 0

N

H

-I(CH

2 1 -6R 1

N

NO2 N 1 (H 1

R

4 1~R' 13'1 f 9 PcTIUS95IOS94() WO 95/30642 SCHEME 12 COMBINATORIAL

SYNTHONS

NH

2

HO.

(I)

(CHI) R 17 HOBt, DMF, DIC 0 NO 2 0 N

N-

1 -6R' I1) TFA/DCM 2) NEt 3

IDCM

0

NO

2 S -N II R1 12' H Br 0 X (Y) 0

I

0

N

-H

-Q

B r 0

R

O17 X =OH, Cl, Br Y aryl, heteroaryl R alkyl

I

WO 95/30642 PCT/US95105940 -91- Table 2 illustrates compounds of Formula 11 which are representative of the present invention:

R

6

R

7 TABLE 2 REPRESENTATIVE COMPOUNDS RI R 2

IR

4

R

5 I 6 I R 7 6-OH 8-CH3 1C2H5 C2H45 OH H 7-OH 8-CH3 1 CH3 CR3 H OH 7-CH2H5 H CH2H5 NH2 H 6-O-(CH 2 )20H H _C3H47 ICR3 =0 7-OCH2CO2H H -(CH2)4- H morpholino 8-O-(CH2)20B- H -(CH2)5- N(CH3)2 H 6-CO2H 8-CH3 -(CH2)6- S(CH2)25- 6-OH H -(CH2)20(CH2)2- =0 7-OH 8-CH3 CH3 CR3 -S(CH2)2S- 6-OH H -(CH2)5- =0 t WO 95/30642 rcrIUS95J05940 Table 3 il1lustrates additional compounds orrnula 11 representative of the present invention-, TABLE 3 REPRESENTATIVE

COMPOUNDS

6-OHH 7-OH 5-O(CH2)20H 6-OH 7-OH E6-OH 6-OH 8-CH3 7-C2H5

H

H

H

H

8-CH3

R

3

/R

4 -(CH2)2NR 8 (CH2)2-- -CH2NR 8 (CH2)3- -(CH2)2NR 8 CH2- -(CH2)2NR 8 (CH2)2- -(CH2)2CH-

(NR

8 (CH2)2- -(CH2)2NR 8 (CH2)2- -(CH2)2NR 8 CH2- -(CH2)2N

R

8

R

6

OH

-N SO 2

-ND

R7OH-h-4CF

H

H

-S(CH2)2S- -0 -S(CH2)2S- S(CH2)2S- -SQ2-2-Naph -CSNH-Ph -CO-Ph-4-SO2NH2 -CO-Ph-4-SO.2NH2 GOCH 2Ph -C0 2 -2-Py -CO-Ph-4-S01.NH., CO-Ph-4-SO2NH2 6-OH H -(CH2)2NR 8 -S(CH2)2S-

CH?,-

PCTIUS95/05940 WO095/30642 -93- Table 3 (Cont.) 7-OHF 6-OH 7-OH S-O(CH2)20H 6-OH 7-OH 6-OH 6-OH 7-OH 6-OH 7-OH1 -CH3 7-

H

-(CH2)2N

R

8 (CH2)2- -(CH2)2NR 8 (CH2)2- -(CH'))2NR 8 CH2- -(CH2)2NR 8 -CO-Ph-4-SO2N H2

I.

OH

N(CH

3 )2

-SCH

2

CH-

(CHi)S- =0o

LH

H

H

77

H

H

S-CH3

H

H

CH2- -(CH2)2NR 8 (CH2)- -(CH2)2NR 8 (CH2)2- -(CH2)2NR 8 (CH-2)2- -(CH2)2NR 8 CH2- -(CH2)2NR 8 (CH2)2- -(CH2)2NR 8 CH2- -S (CH2)2S =0 -S(CH2)2S- -S(CH2)2S- -S(CH2)2S CONH-Ph-4-CF3 .502-2--Naph -CSNH-Ph -CO-Ph-4-SO2NH2 -CO-Ph-4-SO2N H2 COCH2Ph -CO2-2-Py -CO-Ph-4--SO2NH-2 -CO-Ph-4-SO2NH2 -CO-Ph-4-SO2NH2 -(CH2)2NR 8 _=0 (GH2)2- t WO 95/30642 PCT/US95/05940 -94- The invention is further defined by reference to the following examples, which are intended to be illustrative and not limiting.

PREPARATION 1

IDENTIFIERS

Twelve compounds of the general formula:

N

2 O (CH 2 )n-O-Ar OCH3

IV

0 O wherein: n 3-12 and Ar is pentachlorophenyl or n 5-6 and Ar is 2,4,6-trichlorophenyl were prepared according to Scheme 13 and the following illustrative example.

a) Methyl vanillate (0.729 g, 4.0 mmol), 1-hydroxy-9- (2,3,4,5,6-pentachlorophenoxy)nonane (1.634 g, 4.0 mmol) and triphenylphosphine (1,258 g, 4.8 mmol) were dissolved in 20 mL dry toluene under argon. DEAD (0.76 mL, 0.836 g, 4.8 mmol) was added dropwise and the mixture was stirred at 25*C for one hr. The solution was concentrated to half volume and purified by flash chromatography eluting with DCM to give 1.0 g (1.7 mmol, 43%) of the product as a white crystalline solid.

b) The methyl ester from Step (1.0 g, 1.7 mmol) was dissolved in 50 mL THF, 2 mL water was added, followed by LiOH (1.2 g, 50 mmol). The mixture was stirred at 25'C for one hr. then refluxed for 5 hr. After cooling to 25'C, the mixture was poured onto ethyl acetate (200 mL) and the solution was washed with 1 M HCI (3x mL) then sat'd aq. NaCI (Ix 50 mL) and dried over sodium sulfate.

The solvent was removed and the crude acid azeotroped once with toluene. The crude material was dissolved in 100 mL toluene, 10 mL (1.63 g, 14 mmol) thionyl chloride was added, and the mixture was refluxed for 90 min. The volume of the solution was reduced to

I

WO 95/30642 PCT/US95/05940 approx. 30 mL by distillation, then the remaining toluene was removed by evaporation.

c) The crude acid chloride from Step was dissolved in mL dry DCM and cooled to -70C under argon and a solution of approx. 10 mmol diazomethane in 50 mL anhydrous ether was added.

The mixture was warmed to r.t. and stirred for 90 min. Argon was bubbled through the solution for 10 min., then the solvents were removed by evaporation and the crude material was purified by flash chromatography, eluting with 10-20% ethyl acetate in hexane. The diazoketone (0.85 g, 1.4 mmol, 82% yield over three steps) was obtained as a pale yellow solid.

In alternate Step there is a change to the final diazomethylation step, whereby the a-'d chloride is reacted with (trimethylsilyl)diazomethane and triethylamine to give the identifier, which can then be used without further purification. With this alternate step, the identifier can be obtained in high yield with no chloromethylketone byproduct. Also, purification by flash chromatography is no longer necessary, which in some cases has resulted in significant acid-catalyzed o :omposition of the identifier.

Alternate Step To a solution of the acid chloride (3.8 mmol, 1.00 equiv.) and 1.85 mL (13.3 mmol, 3.50 equiv.) of triethylamine in anhydrous THF/acetonitrile at 0°C under argon was added 5.7 mL (11.4 mmol, 3.00 equiv.) of a 2.0 M solution of (trimethylsilyl)diazomethane in hexanes. The resulting orange solution was :irred at 0OC for 2 hr, then at 25'C for 17 hr. (If a precipitate formed immediately upon addition of (trimethylsilyl)diazomethane,

CH

2 C1 2 was added until the precipitate redissolved). EtOAc was added (250 mL), and the organic layer washed with saturated aq. NaHCO 3 (100 mL) and H 2 0 (100 mL), then dried (anhydrous MgSO4). Removal of the volatiles in vacuo gave the product as yellow crystals in 60-100% yield.

The other 11 identifiers of Formula IV were prepared by analogous synthetic routes, steps and I I WO95/30642 PCT/US95/05940 -96- In the synthesis of Example 1, the 12 identifiers were used to encode the combinatorial library. In Step 1, pentachlorophenyl identifiers where n 11-12 (abbreviated CI C15 and C12C15 were used in the following binary encoding scheme: 01 (n 12) and 10 (n 11). In Step 2, pentachlorophenyl identifiers where n 8-10 (abbreviated C8C15, C9C15, and C10C15) were used and encoded as follows: 001 (n 10), 010 (ri and 100 (n In Step 3, pentachlorophenyl identifiers where n 3-7 (abbreviated C3C15, C4C15, C5C15, C6C15, and C7C15) were used and encoded as follows: 00001 00010 (n 00100 (n 01000 (n 4), and 10000 (n In Step 4, trichlorophenyl identifiers where n 5-6 (abbreviated C5C13 and C 6 C1 3 were used and encoded as follows: 01 and 10=(n=5).

Thus, in Step 1 reagent 3 (Table 1-1) is encoded "11" which represents tagging this choice in the synthesis with the two pentachloro-phenyl identifiers where n 11 and 12.

r WNO 95/30642 CTS9/54 PCTIUS95/05940 -97- SCHEME 13

IDENTIFIERS

~OH

Me Oy'a Me 0 H0-(CH 2 PPh 3 DEAD,Toluene

I

Y

-HA,-0-Ar MeO OMe 0 1. LiOH, THF/MeOH 2. SOCd 2 toluene reflux TMS -CHN 9 7 Et 3

N

0- (CH 2 )n-.0-Ar OMe

CH

2

N

2

DCM

Et 2 0 0 0 C, THF/MeCN 1)

(CH

2 )n-0-Ar OMe

I

WO 95/30642 PCT/US95/05940 -98- PREPARATION 2 t-BUTYL 4-(HYDROXYMETHYL)-3-NITROBENZOATE t-Butyl 4-(acetoxymethyl)-3-nitrobenzoate was prepared as described by Barany and Albericio, J. Am. Chem. Soc. 1985, 107, 4936-4942. The reference's final procedure for hydrazinolysis of the acetate using hydrazine hydrate in CHC13 at 25*C produces only trace amounts of the desired hydroxynmethyl final product, which is the tbutyl ester pre-cursor of the photocleavable linker used herein.

However, hydrazinolysis using hydrazine hydrate in MeOH at 25 OC produces t-butyl 4-(hydroxymethyl)-3-nitrobenzoate in high yield.

Using MeOH as solvent, only the desired final product is obtained in near quantitative yield t-Butyl 4-(hydroxymethyl)-3-nitrobenzoate: To a solution of 14.1 g (47.7 mmol, 1.00 equiv.) of t-butyl 4-(acetoxymethyl)-3-nitrobenzoate in MeOH (200 mL) was added 27.0 mL (477 mmol, 10.0 equiv.) of hydrazine hydrate (55% hydrazine). The resulting yellow solution was stirred at 25*C for 4 hr. EtOAc (250 imL) and saturated aq. NaCI mL) were added, and the organic layer collected after shaking. The organic layer was washed further with saturated aq. NaCI (2 x 85 mL), and then dried (MgSO4). Removal of volatiles in vacuo gave the product in 93% yield as yellow crystals.

PREPARATION 3 ALLYL 4-(HYDROXYMETHYL)-3-N!TROBENZOATE In a 100 mL round bottom flask was placed 4-hydroxymethyl- 3-nitrobenzoic acid (1.97 g, 10 mmol). Allyl alcohol (20 mL) was added, followed by p-toluenesulfonic acid (0.190 g, 1 mmol). The mixture was heated to reflux for 24 hr., at which time all the volatiles were removed in vacuo. The residue was taken up in EtOAc and washed with sat'd KHCO3. The organic layer was dried over MgSO4 and concentrated to afford the title compound as a cream colored solid; 2.4 g (100%).

I

WO 95/30642 PCT/US95/05940 -99- PREPARATION 4 METHYL 4-(HYDROXYMETHYL)-3-NITROBENZOATE Following the procedure of Preparation 3, but using methanol instead of allyl alcohol, the title compound was prepared in 57% yield.

PREPARATION BIS-LINKER MODIFIED RESIN Step 1 Addition of bis-Boc lysine In a 250 mL synthesis vessel was placed bis-Boc-(L)-lysine (7.71g, 22.2 mmol) as a solution in DMF (150 mL). HOBt (2.84g, 21.0 mmol) was added followed by DIC (3.25 mL, 21.0 mmol) and the solution shaken at r.t for 15 min. before adding TentaGel resin (25.8 g, approximately 7.2 mmol amino sites). The mixture was shaken at r.t.

for 17 hr and then washed alternately with methanol and DCM each) and then with THF (2X) and dried under vacuum.

Step 2 Deprotection Into each of seven 250 mL synthesis vessel was placed modified TentaGel resin (8.0 g, approx. 4.5 mmol of N-Boc amine sites). DCM (75 mL) was added followed by a 30 TFA solution in DCM (100 mL). The vessel was shaken at room temperature for min before adding neat TFA (15 mL). The vessel was shaken at room temperature for 2.5 hr at which time the resin was washed with DCM The resin was then treated with a solution of 10% triethylamine in DCM (2X150 mL) shaking for 20 min. each time. The resin was then washed with DCM (4X) and DMF (IX).

PREPARATION 6 t-Boc-PROTECTED AMINO ACID In a 1 L flask was placed 3-nitro-4-(bromomethyl) benzoic acid (20.03 g, 77.0 mmol). THF (300 mL) was added followed by 4methoxybenzylamine (10.0 mL, 77.0 mmol) and triethylamine (35 mL).

The resulting clear solution was stirred at r.t. for 17.5 hr. Solid ditert-butyl dicarbonate (16.8 g, 77.0 mmol) was added, followed by DMF (100 mL) and the resulting suspension stirred at r.t. for 72 hr.

LII___ WO 95/30642 PCTIUS95/05940 -100- The reaction mixture was concentrated in vacuo and the residue taken up in ethyl acetate, washed with 1 N HCI dried (Na2SO4), filtered and concentrated to afford a dark brown oil. Purification via flash chromatography (ethyl acetate:hexane) resulted in a yellow foam which was triturated with acetonitrile to give the expected protected amino acid (Table 2-1, compound 4) as a fine white powder (9.91g, 31%).

PREPARATION 7 t-Boc-PROTECTED AMINO ACID Substantially following the procedure of Preparation 6, but substituting the appropriate amine for 4-methoxybenzylamine, the remaining compounds of Table 2-1 are prepared.

EXAMPLE 1 1299 COMPOUND LIBRARY Step 1 a) R1/R 2 To a solution of t-butyl 4-hydroxymethyl-3-nitrobenzoate (2 g, 7.89 mmol, 1 equiv.), 2,4-dihydroxyacetophenone (1.20 g, 7.89 mmol, 1 equiv.), and triphenylphosphine (2.69 g, 10.26 mmol, 1.3 equiv.) in toluene (20 mL) was added dropwise DEAD (1.79 g, 10.26 mmol, 1.3 equiv.). After addition was complete the mixture was stirred for 16 hours at room temperature. The solvent was removed in vacuo and the residue was purified by flash chromatography (Si0 2 eluted with 10% ethyl acetate in hexanes) affording 1.47 g of the product (48% yield).

The t-butyl ester (500 mg, 1.29 mmol, 1 equiv.) above was dissolved/suspended in DCM (8 mL) and treated with TFA (3 mL).

The mixture was stirred at room temperature for 8 hours. The DCM and TFA were removed in vacuo affording a white solid. This was azeotroped once with toluene then dried in vacuo affording 427 mg (100% yield) of the carboxylic acid.

The acid (636 mg, 1.92 mmol, 1.5 equiv.) prepared above was dissolved in DMF (40 mL) and added to divinylbenzene-crosslinked, polyethyleneglycol-grafted polystyrene beads (TentaGel® S i I WO 95/30642 PCT/US95/05940 -101- NH2, Rapp Polymere) (4.0 g, 0.32 mmol/g, 1.28 mmol, 1 equiv.) in a Merrifield reaction vessel. The resin was suspended by agitation, then HOBt (259 mg, 1.92 mmol, 1.5 equiv.) and DIC (0.31 mL, 1.92 mmol, equiv.) were added in that order. The resin was agitated at room temperature for 7 hours at which time it gave a negative Kaiser test.

The resin was filtered and washed (DMF 3x50 mL, DCM 3x50 mL) then dried in vacuo.

The two other dihydroxyacetophenones were attached to the resin via the photocleavable linker in an analogous manner using the reagents of Table 1-1.

Alternate a) In an analogous fashion the allyl and methyl esters were prepared from allyl 4-hydroxymethyl-3-nitrobenzoate (Preparation 3) and methyl 4-hydroxymethyl-3-nitrobenzoate (Preparation 4).

In a 10 mL flask was placed the allyl ester (110 mg. 0.3 mmol). Methylene chloride (2 mL) was added followed by tetrakistriphenylphospine palladium(0) (11.5 mg, 0.01 mmol) and the mixture cooled to 0°C. Pyrrolidine (50 mL, 0.6 mmol) was added and the reaction stirred at 0*C for 45 min. The mixture was diluted with 2J EtOAc (10 mL) and washed with 3.5N HC1. The organic layer was dried (MgSO4), filtered, and concentrated to afford a yellow solid; 90.6 mg.

In an analogous manner the methyl ester was deprotected by basic hydrolysis using a mixture of dilute NaOH and THF.

b) Encoding of Step 1 Quantities of the three resin batches (2.5 g) from Step l(a) were placed in separate synthesis vessels and each was suspended in DCM (20 mL). The three appropriate binary coding mixtures (Table 1-1) for each batch of resin were prepared by dissolving the appropriate choice (37.5 mg) or choices (37.5 mg of each) of C 12 C1 and CluC1 5 -linker diazoketone (Preparation 1) in DCM (1 mL for each solution). These solutions were added to the appropriate synthesis vessel and the resin was agitated for 30 mins.

Rhodium trifluoroacetate dimer (1 mL of a 1 mg/mL solution in DCM) was added to each of the vessels and the resin was WO 95/30642 PCT/US95/05940 -102agitated at room temperature for 4 hours. Each batch of resin was then filtered and washed with DCM (2x20 mL) then each was resuspended in DCM (20 mL) and treated a second time with the appropriate binary encoding mixture as described above. The resin was again agitated for 30 mins before addition of the rhodium triflouroacetate dimer. The same quantities of catalyst and diazoketone compounds were used in the second coupling step as in the first. The resin was agitated for 14 hours. Each resin batch was then washed with DCM (5x20 mL) then the batches were combined and the entire library (three compounds) was washed with DCM (10x50 mL).

Step 2.

a) Cyclocondensation Reactions The dried resin from Step 1(b) was divided into four batches of 1.5 g (ca. 0.42 mmol) and three additional batches of 0.2, (ca. 0.056 mmol). The 1.5g batches were placed into 25 mL roundbottomed flasks and the 0.2 g batches were placed into 5 mL roundbottomed flasks. The portions of resin were suspended in methanol mL in the four flasks with 1.5 g of resin, 2 mL in the three flasks with 0.2 g of resin) and pyrrolidine (0.6 mL, 7.2 mmol, ca. 15 equiv. in the flasks with 1.5 g of the resin; 0.08 mL, 0.96 mmol, ca. 15 equiv. in the flasks with 0.2 g of resin) was added to each flask. The reaction vessels were then allowed to stand for 5 min. to allow mixing of the reagents.

The appropriate ketone (>10 equiv.) was then added to the vessels. The four BOC protected aminoketones were added to the flasks containing 1.5 g of resin and the other ketones were added to the flasks containing the 0.2 g of resin. The mixtures were heated at 75°C for 16 hr. The flasks were then cooled to room temperature and each batch of resin was poured into a separate sintered funnel and washed thoroughly with DMF (3x20 mL) and DCM (3x20 mL).

b) Encoding of Step 2 Each batch of resin from Step 2(a) was placed into a separate synthesis vessel and was suspended in DCM (5 mL for the batches of 1.5 g of resin, 1 mL for the batches containing 0.2 g of resin). The seven appropriate binary coding mixtures (see Table 1-2) for each batch of resin were prepared by dissolving the appropriate

M

WO 95/30642 PCT/US95/05940 -103choice (22.5 mg if added to a batch of 1.5 g of resin; 3.0 mg if added to a batch of 0.2 g of resin) or choices (22.5 mg of each if added to a batch of 1.5 g of resin; 3.0 mg of each if added to a batch of 0.2 g of resin) of CloC1 5

C

9

C

5 and C 8 C1 5 linker-diazoketone (Preparation 1) in DCM (1 mL for each solution). These solutions were added to the appropriate synthesis vessel and the resin was agitated for 30 mins.

Rhodium trifluoroacetate dimer (1 mL of a 1 mg/mL solution in DCM) was added to each of the vessels and the resin was agitated at room temperature for 4 hr. Each batch of resin was then filtered and washed with DCM (2x20 mL) then each was resuspended in DCM (5 mL for the batches of 1.5 g of resin, 1 mL for the batches of 0.2 g of resin) and treated a second time with the appropriate binary encoding mixture as described above. The resin was again agitated for thirty mins before addition of the rhodium trifluoroacetate dimer. (The same quantities of catalyst and diazoketone compounds were used in the second coupling step as in the first.) The resin was then agitated for 16 hr. Each resin batch was then washed with DCM (5x20 mL). The four batches of 1.5 g of resin were combined and washed with DCM (10x50 mL). These combined batches were then reacted further in Step 3.

The three batches of 0.2 g of resin were combined and washed with DCM (10x20 mL). These combined batches were not used in Step 3 but were saved for Step 4.

Step 3 a) Encoding of Step 3 The four batches of 1.5 g of resin which had been combined in Step 2(b) were now divided into thirty lots of 170 mg each in 1 dram shell vials (Fisher Scientific) and each was suspended in DCM (2 mL). The thirty appropriate binary coding mixtures (see Table 1-3) for each batch of resin were prepared by dissolving the appropriate choice (3 mg) or choices (3 mg of each) of C 7

CI

5

C

6 sC 5

C

5 Cls, C 4 C1 5 and C 3 C1 5 linker-diazoketone (Preparation 1) in DCM (1 mL for each solution). These solutions were added to the appropriate synthesis vessel and the resin was agitated for 30 mins.

Rhodium trifluoroacetate dimer (1 mL of a 1 mg/mL solution in DCM) was added to each of the vessels and the resin was agitated at room temperature for 4 hr. The supernatant solution was WO 95/30642 I'CT/US95/05940 -104then decanted away from the resin with a Pasteur pipette. The resin was washed twice with DCM (3 mL) and the washings removed by Pasteur pipette. Each batch of resin was resuspended in DCM (2 mL) and treated a second time with the appropriate binary encoding mixture as described above. The resin was again agitated for thirty minutes before addition of the rhodium trifluoroacetate dimer. (The same quantities of catalyst and diazoketone compounds were used in the second coupling step as in the first.) The batches of resin were then agitated for 16 hr. Each resin batch was then transferred to a small Merrifield reaction vessel and washed with DCM (3x15 mL), DMF (2x15 mL), and DCM again (2x15 mL).

b) Deprotection Each batch of resin was treated with a 50% solution of TFA in DCM (6 mL:6 mL). The resin was agitated for 2 hr and then filtered and washed with DCM (3x15 mL). The resin was then treated with a 10% solution of triethylamine in DCM (1 mL:9 mL) and agitated for 10 mins. This treatment was repeated once. The resin was filtered and washed with DCM (4x10 mL).

c) Addition of R 8 To each of the first six flasks was added DCM (5 mL) and the resin was agitated for 10 mins. 2,6-Lutidine (0.11 mL, 20 equiv.) was .iked to each flask followed by a solution of the appropriate chloroformate (fable 1-3) in DCM (5 mL) and the resin was agitated for 4 hr. Except for isopropylchloroformate (Aldrich), the chloroformates were prepared from the appropriate alcohols by treating the alcohols (0.1 g) with a solution of phosgene in toluene mL of a 1.8 M solution) for 1 hr, then evaporating to dryness in vacuo ,and then redissolving in DCM (5 mL).

To flasks 8, 9, and 10 was added ethanol (10 mL) and the appropriate isocyanate (Table 1-3) (0.1 mL, ca. 10 equiv.) and the resin was agitated for 4 hr.

To flasks 11, 12, and 13 was added ethanol (10 mL) and the appropriate isothiocyanate (0.1 mL, 0.1 g of the naphthaleneisothiocyanate, ca. 10 equiv.) and the resin was agitated for 4 hr, WO 95/30642 PC'j'US9/05940 -105- To flasks 7 and 14-22 was added DMF (10 mL) and the appropriate carboxylic acid (ca. 10 equiv.) and HOBt (0.103 g, ca. equiv.). The flasks were agitated for 30 mins then DIC (0.12 mL, ca.

equiv.) was added to each flask and the resin was agitated for 4 hr.

To flasks 23-30 was added DCM (10 mL) and triethylamine (0.15 mL, ca. 15 equiv.) and the resin was agitated for mins. The appropriate sulfonyl chloride (ca. 10 equiv.) was added to the reaction vessels and the resin agitated for 4 hr.

The flasks were filtered and the resin washed with DCM (3x10 mL). All of the resin was combined in one large synthesis vessel and was washed with DCM (3x50 mL), DMF (3x50 mL), and DCM again (3x50 mL). The resin was dried in vacuo.

Alternative Step 3 a) Encoding of Alternative Step 3 The remaining 900 mg of resin from the four combined batches of 1.5 g from Step 2(b) which had not been used in Step 3 was divided into ten portions of 90 mg, and each portion placed in a separate 1 dram shell vial (Fisher Scientific). The ten appropriate binary coding mixtures (see Table 1-5) for each batch of resin were prepared by dissolving the appropriate choice (1.5 mg) or choices mg each) of C7C15, C5C15, C4Cl5, and C 3 C1 5 linker-diazoketone (Preparation 1) in DCM (1 mL for each solution). These solutions were added to the appropriate synthesis vessels and the resin was agitated for 30 min.

Rhodium trifluoroacetate dimer (1 mL of a 1 mg/mL solution in DCM) was added to each of the vessels and thu resin was agitated at room temperature for 4 hr. The supernatant solution was then decanted from the resin. The resin was washed (DCM 2x 3 mL) and the washings removed by Pasteur pipette. The resin was then treated a second time with solutions of the appropriate binary coding mixtures and agitated for 30 min. before the addition of the rhodium trifluoroacetate dimer. The same quantities of catalyst and diazoketone compounds were used in the second coupling as in the first. The batches of resin were then agitated for 16 hr. The resin was then transferred into small Merrifield synthesis vessels and washed (DCM 6x mL).

I

WO 95/30642 PCT/US95/05940 -106b) Deprotection.

Each batch of resin was treated with a solution of TFA in DCM (4 mL:4 mL). The resin was agitated for 1 hr, then filtered and washed with DCM (2x 15 mL). The resin was then treated with a solution of piperidine in DCM (4 mL:4 mL) and agitated for 15 min.

This treatment was repeated once. Each batch of beads was washed with methanol (2x 15 mL) and DCM (4x 15 mL). Flasks 1-4 were washed with THF (3x 15 mL).

c) Heteroarylation Reactions The resin in flasks 1-4 was suspended in THF (6 mL).

Flasks 1-3 were then treated with DBU (190 pL, Ca. 40 equiv.) followed by the appropriate heteroaryl chloride (ca. 20 equiv.). Flasks 1 and 2 were heated at 55 *C for 16 hr. Flask 3 was heated at reflux for 16 hr. Flask 4 was treated with triethylamine (700 pL) and the appropriate heteroaryl chloride (ca. 20 equiv.). The resin was shaken at r.t. for 16 hr. Each batch of resin was then washed in THF (2x mL) and dried in vacuo.

d) Reductive Alkylations The resin in flasks 5-10 was suspended in DMF (8 mL) and the appropriate aldehyde (ca. 67 equiv.) added. Acetic acid (160 pL) was added to each of the flasks followed by sodium cyanoborohydride (ca. 67 equiv.). Flasks 5, 6, 7, 9, and 10 were shaken at r.t. for 16 hr.

Flask 8 was heated to 55 *C for 16 hr. Each batch of resin was filtered and washed with DMF (3x 15 mL). Each of the reductive alkylation reactions was repeated under the same conditions. The batches of resin were washed with DMF (2x 15 mL), methanol (3x 15 mL), and DCM (3x 15 mL). The resin was then mixed, washed with DCM (2x 20 mL), and dried in vacuo.

This part of the library did not undergo further elaboration.

Step 4 a) Encoding of Step 4 To the combined resin from Step 3(c) was added 45 mg of resin from each of the seven flasks from Step 2(b) and the resin was washed and mixed thoroughly with DCM (3x50 mL). From this

I

WO 95/30642 PCT/US95/05940 -107mixture was weighed out three portions of 800 mg of resin and these were placed into three separate Merrifield synthesis vessels and suspended in DCM (10 mL). The three appropriate binary coding mixtures (see Table 1-4) for each batch of resin were prepared by dissolving the appropriate choice (24 mg) or choices (24 mg of each) of the C 6 C1 3 and C5Cl 3 linker-diazoketone compound in DCM (1 mL for each solution). These solutions were added to the appropriate synthesis vessel and the resin was agitated for 30 mins.

Rhodium trifluoroacetate dimer (1 mL of a 1 mg/mL solution in DCM) was added to each of the vessels and the resin was agitated at room temperature for 4 hr. Each batch of resin was then filtered and washed with DCM (2x20 mL) then each was resuspended in DCM (10 mL) and treated a second time with the appropriate binary encoding mixture as described above. The resin was again agitated for 30 mins before addition of the rhodium trifluoroacetate dimer. The same quantities of catalyst and diazoketone compounds were used in the second coupling step as in the first. The resin was agitated for 14 hr.

Each resin batch was then washed with DCM (3x20 mL) and then filtered.

b) Carbonyl Reaction (addition of R 6 and R 7 The resin in flask 1 was resuspended in DCM (6 mL) and 1,2-ethanedithiol (1 mL) and boron trifluoride etherate (1 mL) were added. The flask was shaken at room temperature for 6 hr. The resin was then washed with DCM (20 mL) and then resuspended in DCM (6 mL) and treated once more with ethanedithiol (1 mL) and boron trifluoride etherate (1 mL). The resin was agitated at room temperature for 14 hr. The resin was then filtered and washed with DCM (5x20 mL).

The resin in flask 2 was suspended in methanol (5 mL) and solid sodium borohydride (200 mg) was added cautiously. The flask was vented and allowed to shake gently for 1 hr. The resin was filtered and resuspended in methanol and the reduction process repeated a total of 5 times at 1 hr intervals using 200 mg of sodium borohydride each time. After the final cycle the resin was washed with methanol (3x20 mL) and DCM (3x20 mL).

The resin in flask 3 was not reacted further.

I

WO 95/30642 PCT/US95/05940 -108- The resin from the three flasks was combined and washed with DCM (5x50 mL) and then dried in vacuo. A portion (500 mg) of the resin was suspended in DCM (5 mL) and TFA (5 mL) and shaken for 2 hr. The resin was then treated twice with a 10% solution of triethylamine in DCM (10 mL) and washed with DCM (5x20 mL). The resin was then dried in vacuo.

d) Decoding Procedure A bead was placed in a 1.3 mm diameter pyrex capillary with 2 pL of acetonitrile. Ceric ammonium nitrate solution (2 pL of a 0.1 M aq.. solution) and hexane (3 pL) were added and the two-phase mixture centrifuged briefly. The tube was sealed and left at 35 "C for 16 hrs, then opened. The organic layer was removed by syringe and mixed with 1 aL of N,O-bis(trimethylsilyl)acetamide. The silated tag solution (1 pL) was analyzed by GC with electron capture (EC) detection.

The GC analysis was performed with a Hewlett Packard 5890 plus gas chromatograph. On column injection into a 5 m, 0.32 mm retention gap connected to a 25 m, 0.2 mm crosslinked phenylmethyl silicone column was used. The temperature program was set at 200 "C for 1 min and then increased at a rate of 15°C/min from 200-320 The pressure program was set at 20 psi for 1 min, then increased at 2 psi/min to 40 psi with a total run time of 10 min. The EC detector was maintained at 400 °C and the auxiliary gas was set at psi.

WO 95/30642 W09510642PCT1US95/05940 -109- Table 1-1 R1/R2 Reagents and Encoding Scheme

NO

2 00 00 1. 01 2. 0

NO

2 O H HO i. Me 3. 11 0 Table 1-2 R4/R~i Reagents and Encoding Scheme WO 95/30642 WO 9530642PCT[US95/0594) -110- Table 1-3 RB Reanents and Encodine Scheme WO 95/30042 PTU9/54 PCT/US95/05940 -111- Table 1-4 RN/RI Reagents and Encoding Scheme HS(CH2) 2 SW/BF3-OEt 2 NaBH4 No reaction 1. 01 2. 10 3. 11 Table R~-and Heteroarv I Encoding Scheme -N ClI

N-H

1.200 2. 0010 Cla N C N N 3. 0011 0100 25F--CR0

H

3 CO CR0 4 Ai WO 95/30642 PCT/US95/05940 -112- EXAMPLE 2 87.906 COMPOUND LIBRARY Step 1 a) Addition of (CH2)1-6R 17 In a 250 mL synthesis vessel was placed the deprotected modified TentaGel resin (8.0 g, approx. 4.5 mmol of amine sites) from Preparation 5. HOBt (1.81g, 13.4 mmol) was added followed by the N- Boc-p-methoxybenzylamino acid (Table 2-1, compound 4) (5.60g, 13.4 mmol) and DMF (150 mL). The mixture was shaken at r.t. for 10 min.

before adding DIC (2.1 mL, 13.4 mmol). The mixture was shaken at r.t. for 16 hr and then washed alternately with methanol and DCM (4X each) and then with EtOAc Analysis of the resin via the standard Kaiser ninhydrin test indicated that the coupling reaction was complete.

In six separate vessels, analogous couplings were carried out with the six other Boc-protected amino acids listed in Table 2-1.

All coupling reactions were repeated until satisfactory Kaiser ninhydrin test results were obtained (in all cases either one or two couplings).

b) Encoding of Step 1 While still in their separate 250 mL synthesis vessels, resin batches number 4, 5, 6, and 7 from Step 1 were suspended in EtOAc (100 mL). Into each of these four vessels was placed the C15C7-linker diazoketone (0.56 g) and the mixtures agitated for 1 hr. To each of the four vessels was then added rhodium trifluoroacetate dimer (6 mL of a 1 mg/mL solution in DCM) and the resin was agitated for 15 hr. The resin was then washed with DCM (4X) and EtOAc (2X).

In turn, the C15C8-linker diazoketone was applied to resin batches numbered 2, 3, 6, and 7 and the C15C11-linker diazoketone was applied to resin batches 1, 3, 5, and 7. Application of each tagging molecule was done separately and in analogous fashion to that of the Cl5C7-linker diazoketone outlined above. The seven batches of encoded resin were all combined in a 2 L Erlenmeyer flask along with THF (1 L) and mixed thoroughly by swirling and stirring gently with a glass rod. The resin was then recovered by filtration and vacuum dried.

WO 95/30642 PCT/US95/05940 -113- Step 2: Addition of R 2 a) Deprotection In a 250 mL synthesis vessel is placed mixed, encoded resin from Step 1 (9 g) along with DCM (just enough to suspend resin). TFA (75 mL of a 30 solution in DCM) is added and the resin agitated for hr. The resin is then washed with DCM (2X) followed by treatment with 10% triethylamine in DCM (2X 20 min. each) and then washed with DCM (4X).

b) Coupling The deprotected resin from Step 2(a) (9 g) is suspended in DMF (7 mL). HOBt (2.04 g, 15 mmol) is added followed by the acetophenone acid (Table 2-2, compound 31 (3.36g, 15 mmol) and the mixture agitated for 15 min. DIC (2.3 mL, 15 mmol) is added and the mixture agitated for 21 hr. The resin is washed alternately with DCM and methanol (5X each) and then with EtOAc (4X).

In five separate vessels, analogous couplings are carried out with the five other acetophenone acids listed in Table 2-2.

c) Encoding of Step 2 The six batches of resin from Step 2 are binarily encoded in a fashion analogous to that described above for encoding of Step 1 The six batches of encoded resin are combined in a 2 L Erlenmeyer flask along with THF (1 L) and mixed thoroughly by swirling and stirring gently with a glass rod. The resin is then recovered by filtration and vacuum dried.

Step 3 Addition of R 4 a) Cyclocondensation reactions The mixed resin from Step 2 is divided into three batches of 14.4 g (ca. 8.1 mmol) and seven additional batches of 1.5 g (ca. 0.84 mmol). The 14.4 g batches are placed into 250 mL round bottom flasks and the 1.5 g batches are placed in 25 mL round bottom flasks. The portions of resin are suspended in methanol (150 mL in the three flasks with 14.4 g resin, 15 mL in the seven flasks with 1.5 g resin) and pyrrolidine (10.1 mL, 121 mmol, ca. 15 equiv. in the flasks with 14.4 g resin; 1.0 mL, 12.6 mmol, ca. 15 equiv. in the flasks with 1.5 g resin) is -r I I I WO 95/30642 PCT/US95/05940 added to each flask. The reaction vessels are then allowed to stand for min. to allow mixing of the reagents. The appropriate ketone (5 to equiv.) is then added to the vessels. The three Boc-protected aminoketones from Table 2-3 are added to the flasks containing 14.4 g of resin and the seven other ketones, from Table 2-4, are added to the flasks containing 1.5 g of resin. The mixtures are heated at 75 0 C for 16 hr. The flasks are then cooled to r.t. and each batch of resin is poured into a separate synthesis vessel of appropriate size and washed thoroughly with DCM, DMF, and methanol (alternating: 5X each).

b) Encoding of Step 3 Each of the ten batches of resin from Step 3(a) is binarily encoded in a fashion analogous to that described for encoding Step 1.

c) Mixing and dividing The seven 1.5 g batches of encoded resin are combined in a 500 mL Erlenmeyer flask along with THF (250 mL) and mixed thoroughly by swirling and stirring gently with a glass rod. The resin is then recovered by filtration and vacuum dried. This combined resin is kept separate from the three 14.4 g batches of resin and is not subjected to the reaction conditions of Step 4, but rather re-divided into three 0.2 g portions and seven 1.4 g portions and saved to be used in Step 5 and alternate Step 5. The three 14.4 g batches of encoded resin are combined in a 2 L Erlenmeyer flask along with THF (1 L) and mixed thoroughly by swirling and stirring gently with a glass rod. The resin is then recovered by filtration, vacuum dried, and used in Step 4.

Step 4 a) Deprotection Into each of seven 250 mL synthesis vessels is placed mixed, encoded resin from the three combined 14.4 g batches from Step 3 (6 g) along with DCM (just enough to suspend the resin). TFA mL of a 30 solution in DCM) is added and the resin agitated for hr. The resin is then washed with DCM (2X) followed by treatment with 10% triethylamine in DCM (2X 20 min. each) and then washed with DCM (4X).

I A WO 95/30642 PCT/US95/05940 -115b) Nitrogen elaboration In the first of the seven 250 mL synthesis vessels containing deprotected resin from Step 4(a) (6 g, ca. 3.4 mmol) is placed DCM (150 mL) and triethylamine (15 equiv.). Phenylsulfonyl chloride (ca.

10 equiv.) is added and the resin agitated for 4 hr. The resin is washed with alternating DCM and methanol (5X each) and then with EtOAc (2

X).

In the second of the seven 250 mL synthesis vessels containing deprotected resin from Step 4(a) (6 g, ca. 3.4 mmol) is placed DCM (150 mL) and triethylamine (15 equiv.). Butryl chloride (ca. 10 equiv.) is added and the resin agitated for 4 hr. The resin is washed with alternating DCM and methanol (5X each) and then with EtOAc (2X).

In the third of the seven 250 mL synthesis vessels containing deprotected resin from Step 4(a) (6 g, ca. 3.4 mmol) is placed DMF (150 mL) and HOBt (ca. 15 equiv.). 4-Carboxybenzenesulfonamide (ca. 10 equiv.) is added and the resin agitated for min. DIC (ca. 10 equiv.) is added and the resin agitated for 4 hr.

The resin is washed with alternating DCM and methanol (5X each) and then with EtOAc (2X).

In the fourth of the seven 250 mL synthesis vessels containing deprotected resin from Step 4(a) (6 g, ca. 3.4 mmol) is placed DMF (150 mL) and acetic acid (3 mL). Benzaldehyde (ca. equiv.) is added and the resin agitated for 30 min. Sodium cyanoborohydride (ca. 50 equiv.) is added and the resin agitated for 16 hr. The resin is washed with alternating DCM and methanol (5X each) and then with EtOAc (2X).

In the fifth of the seven 250 mL synthesis vessels containing deprotected resin from Step 4(a) (6 g, ca. 3.4 mmol) is placed DMF (150 mL) and acetic acid (3 mL). Butyraldehyde (ca. equiv.) is added and the resin agitated for 30 min. Sodium cyanoborohydride (ca. 50 equiv.) is added and the resin agitated for 16 hr. The resin is washed with alternating DCM and methanol (5X each) and then with EtOAc (2X).

The resin from the sixth of the seven 250 mL synthesis vessels containing deprotected resin from Step 4(a) (6 g, ca. 3.4 mmol) WO 95/30642 PCTIUS95/05940 -116is transferred to a 250 mL round bottom flask. THF (150 mL) is added followed by DBU (ca 40 equiv.). 2-Chloropyrimidine (ca equiv.) is added. The mixture is heated to 55°C for 16 hr. The resin is transferred back to a 250 mL synthesis vessel, washed with alternating DCM and methanol (5X each), and then with EtOAc (2X).

In the seventh of the seven 250 mL synthesis vessels containing deprotected resin from Step 4(a) (6 g, ca. 3.4 mmol) is placed absolute ethanol (150 mL). Methyl isocyanate (ca 15 equiv.) is added and the resin agitated for 12 hr. The resin is washed with alternating DCM and methanol (5X each) and then with EtOAc (2X).

c) Encoding of Step 4 Each of the seven batches of resin from Step 4(b) are binarily encoded in a fashion analogous to that described for the encoding of Step 1.

The seven batches of encoded resin are combined in a 2 L Erlenmeyer flask along with THF (1 L) and mixed thoroughly by swirling and stirring gently with a glass rod. The resin is then recovered by filtration and vacuum dried. This resin is then divided into three batches of 0.7 g each and seven batches of 5.7 g each. The seven 5.7 g. batches are subjected to Step 5. The three 0.7 g batches are subjected to alternate Step Step a) Encoding Each of the seven 5.7 g. batches of resin from Step 4(c) and the seven 1.4 g batches from Step 3(b) are binarily encoded in a fashion analogous to that described for the encoding of Step 1.

b) Reductive amination The seven encoded 5.7 g. batches of resin from Step are placed in 200 mL round bottom flasks. The seven encoded 1.4 g.

batches from Step 3(c) are placed in 50 mL round bottom flasks. To each of the fourteen flasks is added a solution of 10% glacial acetic acid in methanol (60 mL in the 200 mL flasks, 15 mL in the 50 mL flasks).

The appropriate amine from Table 2-6 (ca. 40 equiv.) is added followed by sodium cyanoborohydride (ca. 40 equiv.). Condensers are attached WO 95/30642 PCTIUS95/05940 -117and the mixtures are heated to 75°C for 48 hr. The resin is washed with alternating DCM and methanol (5X each) and then with EtOAc (2X).

Alternate Step a) Thioketalization One of the three 0.7 g. batches of resin from Step 4(c) and one of the 0.2 g batches of resin from Step 3(c) are placed in two separate 30 mL synthesis vessels. To each is added DCM (6 mL), followed by 1,2-ethanedithiol (1 mL) and boron trifluoride etherate (1 mL). The resin is agitated at r.t. for 6 hr. The resin is washed with DCM (IX) and then treated once more with ethanedithiol (1 mL) and boron trifluoride etherate (1 mL). The resin is agitated at r. t. for 14 hr. The resin is then filtered and washed with alternating DCM and methanol (5X each) and then with EtOAc (2X).

b) Reduction One of the three 0.7 g. batches of resin from Step 4(c) and one of the 0.2 g batches of resin from Step 3(c) are placed in two separate 30 mL synthesis vessels. To each is added methanol (6 mL) and (cautiously) solid sodium borohydride (200 mg). The flasks are vented and allowed to gently shake for 1 hr. The resin is filtered and resuspended in methanol (6 mL) and the reduction process repeated a total of 5 times at 1 hr. intervals using 200 mg portions of sodium borohydride each time. After the final cycle, the resin is washed with alternating DCM and methanol (5X each) and then with EtOAc (2X).

c) One of the three 0.7 g. batches of resin from Step 4(c) and one of the 0.2 g batches of resin from Step 3(c) is left unaltered.

Step 6 a) Mixing The seven 5.7 g batches of encoded resin from Step are combined in a 2 L Erlenmeyer flask along with THF (1 The seven 1.4 g batches of encoded resin from Step 5(b) are combined in a 500 mL Erlenmeyer flask. Each batch of resin is mixed thoroughly by WO 95/30642 PCT/US95/05940 -118swirling and stirring gently with a glass rod. The resin from each flask is recovered by filtration, vacuum dried, and kept separate.

b) Nitrogen elaboration.

The mixed and dried resin from the combined 5.7 g.

batches in Step 6(a) (total of ca, 32.5 is divided into ten 3.2 g.

batches and placed in 100 mL synthesis vessels. The mixed and dried resin from the combined 1.4 g. batches in Step 6(a) (total of ca. 9.8 g.) is divided into ten 0.98 g. batches and placed in 30 mL synthesis vessels.

These vessels are paired up into ten sets of two where each set has one 100 mL vessel and one 30 mL vessel, Both members of earh set are subjected to the same reaction conditions as outlined below.

In the first set of vessels is placed N,N'-bis Boc-(L)-lysine (ca. 10 equiv.) as a solution in DMF (60 mL in the larger vessel, 15 mL in the smaller). HOBt (ca. 15 equiv.) is added and the resin agitated for 15 min. DIC (ca. 10 equiv.) is added and the resin agitated for 4 hr.

The resin is washed with DCM (2X) and then treated with TFA solution in DCM) (1.5 hrs.). The resin is then washed with DCM (2X) and treated with 10% triethylamine in DMF (2X. 30 min. each). The resin is washed with alternating DCM and methanol (5X each) and then with EtOAc (2X).

In the second set of vessels is placed N.-a-Fmoc-N-co-Pmc- (L)-arginine (ca. 10 equiv.) as a solution in DMF (60 mL in the larger vessel, 15 mL in the smaller). HOBt 15 equiv.) is added and the resin agitated for 15 min.. DIC (ca. 10 equiv.) is added and the resin agitated for 4 hr. The resin is washed with DCM (2X) and then treated with TFA (50% solution in DCM) (1.5 hrs.). The resin is then washed with DCM (2X) and treated with 50% piperidine in DMF (2X, 30 min.

each). The resin is washed with alternating DCM and methanol ach) and then with EtOAc (2X).

In the third set of vessels is placed DCM (60 mL in the larger vessel, 15 mL in the smaller). N,N-di-n-propyl-N'-cyanoethylthioformamidine (ca. 15 equiv.) is added, followed by triethylamine (ca. 20 equiv.) and the resin agitated for 12 hrs. The resin is washed with alternating DCM and MeOH (5X each) and then with EtOAc (2X).

WO 95/30642 PCT/US95/05940 -119- In the fourth set of vessels is placed absolute ethanol mL in the larger vessel, 15 mL in the smaller). Methyl isocyanate (ca equiv.) is added and the resin agitated for 12 hr. The resin is washed with alternating DCM and methanol (5X each) and then with EtOAc (2X).

In the fifth set of vessels is placed absolute ethanol (60 mL in the larger vessel, 15 m:L in the smaller). Methyl isothiocyanate (ca..

equiv.) is added and the resin agitated for 12 hr. The resin is washed with alternating DCM and methanol (5X each) and then with EtOAc (2X).

In the sixth set of vessels is placed absolute ethanol (60 mL in the larger vessel, 15 mL in the smaller). Phenyl isocyanate equiv.) is added and the resin agitated for 12 hr. The resin is washed with alternating DCM and methanol (5X each) and then with EtOAc (2X).

In the seventh set of vessels is placed absolute ethanol mL in the larger vessel, 15 mL in the smaller). Phenyl isothiocyanate 15 equiv.) is added and the resin agitated for 12 hr. The resin is washed with alternating DCM and methanol (5X each) and then with EtOAc (2X).

In the eighth set of vessels is placed DCM (60 mL in the larger vessel, 15 mL in the smaller) and 2,6-lutidine 20 equiv.).

Isopropyl chloroformate (ca. 15 equiv.) is added and the resin agitated for 4 hr. The resin is washed with alternating DCM and methanol each) and then with EtOAc (2X).

In the ninth set of vessels is placed DCM (60 mL in the larger vessel, 15 mL in the smaller) and triethylamine (15 equiv.).

Isobutryl chloride 10 equiv.) is added and the resin agitated for 4 hr. The resin is washed with alternating DCM and methanol (5X each) and then with EtOAc (2X).

In the tenth set of vessels is placed DCM (60 mL in the larger vessel, 15 mL i. the smaller) and triethylamine (15 equiv.).

Methanesulfonyl chloride 10 equiv.) is added and the resin agitated for 4 hr. The resin is washed with alternating DCM and methanol each) and then with EtOAc (2X).

WO 95/30642 WO 95/0642 CT/US95/05940 -120- Table 2-1 (CH2116R-i--Reagents and Encoding Scheme

NO

9 0 \7 HO M e 0 C1 5 C I 5

HO

N0 2 0KOx 1-~ 2

NO

2 HO

I

0 1C N1 0 OMe HO

I~

0 C1 5

C

7

CISC

8 0NL Oe OMe 3 NO 2 0 X NO, HO

HO

0 0- C511CISC 8 C1 5 GC15C 1N 4

NO

2 N0 Oi HO 1 0 C1 5

C

7 OMe WO 95/30642PC1S509( PCT/US95/05940 -121- Table 2-2 Substituted Hydroxyacetophenone Reapgents Table 2-3 RWR Step 3Rea ents WO 95/30642 WO 9530642PCT1US95/05940 -122- Table 2-4 Step 3 Alt. Reagents 1 5 0=C7> 2 JQ0 Me 6 0KQ7S 0 F- Me- N 3 N 7 Table Ra Reagents WO 95/30042 PTU9/54 PCT/US95/05940 -123- Table 2-6

(CH

2 1 1 6 R Reagents

H

3 C- NH., NH 2 1 NH2 NH 2 6 MeG N11- 7

NH

2 3 Table 2-7 R.WR2Reag~ents WO 95/30642 WO 95/0642 CTIUS95/05940 -124- Table 2-8 Rf3 Reagents NHBOC C6 HO f-(CH,)-NHBO6

NC

NHFmoc

NH

HOY' (C2-NNHM 7 2 HH NPM\/N

S

N' CN 0 3 EtS N2 4 Me-N=C=O 0 9>-Lc Me-NCS 110 Me-SO 2

CI

Claims (23)

1. A combinatorial library comprised of a plurality of compounds of Formula 1: wherein: is a solid support; is an identifier residue wherein T' is a tag and L is a first linker; q is chosen from 3-30; and is a linker/ligand residue wherein L' is a second linker and IF' is an attached ligand represented by 3 RL 04 R2 8 R wherein: RI is chosen from -(CH 2 1 2 -C(O)-CH 2 and and when L' terminates inN(CH) 1 l 6 Rl 7 Ria may additionally be chosen from and -C(O)-4-Phe- CH 2 15 R 2 is chosen from H and lower alkyl.; R 3 is chosen from H, alkyl, aryl and arylalkyl; R 4 and R 5 are each independently chosen from H, lower alkyl, and substituted lower alkyl wherein the substituents are 1-3 alkoxy, aryl, substituted aryl, carboalkoxy, or diloweralkylamido, or R 4 and R 5 taken together is chosen from -(CI- 2

2-(CH 2 2 -0-(CH 2 2 -CH 2 -0-(CH 2 3 -(CH- 2 2 -NR 8 -(CH 2 2 -CH 2 -NR 8 2 )in-, -(CI-1 2 2 CH(NHR 8 )(CH 2 2 -(CH- 2 2 -S(0) 0 2 -(CH 2 2 and -CHCI-(N-loweralkyl)(C- 2 2 C1-lCH 2 one of R 6 and R 7 is H and the other is chosen fromi H, OH, and N(CIT 2 1 6 R 1 4 R 1 5 or IN.XUGA00625:SSC -126- R 6 and R 7 taken together is chosen from R R2 R 2 n n> II o s o /O s, and s8 s R 8 is chosen from H, COOR 9 CONHR 1 0 CSNHR"', COR 2 SO 2 R' 3 lower alkyl, aryl lower alkyl, heteroaryl, and heteroaryl lower alkyl, wherein aryl is optionally substituted with 1-3 substituents selected from lower alkyl, lower alkoxy, halo, CN, NH 2 COOH, CONH 2 carboalkoxy, and mono- or di-lower alkylamino and wherein heteroaryl is a mono- or bicyclic heteroaromatic ring system of 5 to 10 members including 1 to 3 heteroatoms selected from O, N, and S and 0-3 substituents selected from 1 0 halo, amino, cyano, lower alkyl, carboalkoxy, CONH 2 and S-lower alkyl; R 9 is chosen from lower alkyl, aryl, aryl lower alkyl, heteroaryl, aryl substituted by 1-3 substituents selected from alkyl, alkenyl, alkoxy, methylene dioxy, and halo, and a 5- to 6-membered heterocyclic ring I: wherein the hetero atom is O or N, wherein heteroaryl is a heteroaromatic 15 ring of 5 to 6 members including 1 to 2 heteroatoms selected from 0, N, and S and 0-2 substituents selected from lower alkyl, dialkylamino, lower alkoxy, and halo; Ro 1 and R" are each independently chosen from lower alkyl, aryl, aryl lower alkyl, and aryl substituted by 1-3 substituents selected from lower alkyl, halo, 20 alkoxy and haloalkyl; R12 is chosen from lower alkyl, aryl, heteroaryl, aryl lower alkyl, heteroaryl lower alkyl, a 5- or 6-membered heterocyclic ring containing 1-2 heteroatoms selected from 0, S, and N, a 5- or 6-membered heterocyclic ring containing 1-2 heteroatoms selected from 0, S, and N lower alkyl, and 2 5 aryl substituted with 1-3 substituents selected from lower alkyl, alkoxy, halo, sulfamoyl, lower alkyl sulfamoyl, cyano, and phenyl; RP 3 is chosen from lower alkyl, aryl, and aryl substituted with 1-3 substituents selected from lower alkyl, alkoxy, halo, CN, and haloalkyl; Li o^ i -127- R 14 is chosen from H, alkyl, alkyl substituted by 1-3 alkoxy, S-loweralkyl, sulfainoyl, halo, alkylsuiphonamido, or arylsulphonamido, alkenyl, alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycloalkyl, 1C 2 R 6 C( 1R 6 -C(O)NR1 6 R 1 6 -CH 2 OC(0)R 16 and -CH 2 SC()R 16 R 1 5 is chosen from H, alkyl, and -C(NCN)NRR 3 R 1 6 is chosen from lower alkyl, substituted lower alkyl, aryl, and substituted aryl; R 17 is chosen from H, alkyl, alkyl substituted by 1-3 alkoxy, S-loweralkyl, sulfamoyl, halo, alkylsulphonamido, or arylsuiphonamido, alkenyl, alkynyl, alyl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycloalkyl, -CH 2 NR1 6 C(O)R 1 6 -C(0)NR1 6 R 1 6 -CH- 2 OC(0)R1 6 and -CH 2 SC(0)R1 6 X is chosen from alkyl, aryl, arylalkyl, 0-1oweralkyl, and -NR 3 R 3 Z is chosen from -(CH 2 1 6 optionally substituted with 1-3 lower alkyl, -CHR 2 -Phe-CH- 2 wherein Phe is optionally mono-substituted with halo gen, lower alkyl, or alkoxy, and heteroarylene-(CH-; in is chosen from 2 and 3; and 9 n is chosen from 4-9. 0*2. The combinatorial library according to claim 1, wherein: is of Formula III: 00 wheei n0-coe fo -2wenA spnAr hoohn n 0 0" B- B- NO 2 NO 2 is chosen from and (b) 4Y) I -128- wherein the left-hand bond as shown is the point of attachment to the solid support and the right-hand bond is the point of attachment to the ligand, and Bis chosen from 0 and N(CH 2 1 6 R' 7 with the proviso that in whati B is N(CH 2 )1. 6 R' 7 the ligand is attached to B through a carbonyl group.

3. The combinatorial library according to claim 1, wherein -C(O)-L'IlT is represented by at least one of:- GO' ON 0. 00O 55 0 soG 0055 06 O a Ge 0 s o a 0 0 5 IC R4

4. A compound represented by Formula 11 R 6 R 7 3 wherein: RI is chosen from OH-, 0(CH 2 1 2 0H, OCF1 2 CO 2 1H, CO 2 H, 0-Z-C(0)NH-(CH 2 16 R1 7 and OCI- 2 -4-Phe-C(O)NH-(CI-1 2 1 6 11 7 -129- R' is chosen from H and lower alkyl; R3 is chosen from H, alkyl, aryl, and arylalkyl; R 4 and R1 are each independently chosen from H, lower alkyl, and substituted lower alkyl wherein the substituents are 1-3 alkoxy, aryl, substituted aryl, carboalkoxy, carboxamido, and di-loweralkylamido, or R 4 and R5 taken together is chosen from -(CH 2 -(CH' 2 2 -O-(CH 2 2 -CH 2 -O-(CH 2 3 -(CH2)2-NR!-(CH1)2-, -CH2-NR8-(CH2),-, -(C_2)2CH(NHR1)(CH_2)2-> -(CI-H 2 2 -S(O)o. 2 -(CH 2 2 and -CH 2 CH(N-loweralkyl)(CH 2 2 2CI--; one of R and R7 is I-I and the other is chosen from H, OH, and N(CH 2 1 6 R'R's or R 6 and R 7 taken together is chosen from 0 R-<R R2 II o,0S O\ 0 S,O and /S with the proviso that when R' is OH and R2 is H, R 6 and R 7 taken together is not 0\ 'o RI is chosen from H, COOR 9 CONHRIo, CSNHR", COR' 2 SO 2 R' 3 lower :alkyl, aryl lower alkyl, heteroaryl, and heteroaryl lower alkyl, wherein aryl is optionally substituted with 1-3 substituents selected from lower alkyl, lower alkoxy, halo, CN, NH 2 COOH, CONH 2 carboalkoxy, and mono- or di-lower alkylamino and wherein heteroaryl is a mono- or bicyclic heteroaromatic ring system of 5 to 10 members including 1 to 3 heteroatoms selected from O, N, and S and 0-3 substituents selected from halo, amino, cyano, lower alkyl, carboalkoxy, CONH 2 and S-lower alkyl; is chosen from lower alkyl, aryl, aryl lower alkyl, heteroaryl, aryl substituted by 1-3 substituents selected from alkyl, alkenyl, alkoxy, methylene dioxy, and halo, and a 5- to 6-membered heterocyclic ring wherein the hetero atom is O or N, wherein heteroaryl is a heteroaromatic ring of 5 to 6 members including 1 to 2 heteroatoms selected from 0, N, and S and 0-2 substituents selected from lower alkyl, dialkylamino, lower alkoxy, and halo; IC-r -r -130- R'o and R" are each independently chosen from lower alkyl, aryl, aryl lower alkyl, and aryl substituted by 1-3 substituents selected from lower alkyl, halo, alkoxy and haloalkyl; R' 2 is chosen from lower alkyl, aryl, heteroaryl, aryl lower alkyl, heteroaryl lower alkyl, a 5- or 6-membered heterocyclic ring containing 1-2 heteroatoms selected from O, S, and N, a 5- or 6-membered heterocyclic ring containing 1-2 heteroatoms selected from 0, S, and N lower alkyl, and aryl substituted with 1-3 substituents selected from lower alkyl, alkoxy, halo, sulfamnoyl, lower alkyl sulfamoyl, cyano, and phenyl; R 13 is chosen from lower alkyl, aryl, and aryl substituted with 1-3 substituents selected from lower alkyl, alkoxy, halo, CN, and haloalkyl; R 14 is chosen from H, alkyl, alkyl substituted by 1-3 alkoxy, loweralkyl, sulfamoyl, halo, alkylsulphonamido, or arylsulphonamido, alkenyl, alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycloalkyl, -CHNR' 6 C(O)R' 6 -C(0)NR' 6 R' 6 -CH 2 OC(O)R'6 and -CH 2 SC(O)R 1 6 R1 5 is chosen from H, alkyl, and -C(NCN)NRR; R' 16 is chosen from lower alkyl, substituted lower alkyl, aryl, and substituted aryl; SR' 7 is chosen from H, alkyl, alkyl substituted by 1-3 alkoxy, loweralkyl, sulfamoyl, halo, alkylsulphonamrnido, or aylsulphonamido, alkenyl, alkynyl, 2£9 aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycloalkyl, -CH 2 NR' 6 C(O)R' 6 -C(0)NR 1 6 R'16 -CI-1 2 0C(0)R' 6 and -CH2I-ISC(0)R' 6 X is chosen from alkyl, aryl, arylalkyl, O-loweralkyl, and -NR 3 R 3 Z is chosen from 1 6 optionally substituted with 1-3 lower alkyl, -CHR 2 -Phe-CH 2 where Phe is optionally mono-substituted with halogen, lower alkyl, or alkoxy, and heteroarylene-(CH 2 S m is chosen from 2 and 3; n is chosen from 4-9; or a pharmaceutically acceptable salt thereof A compound according to claim 4, wherein R' 2 is sulfamoylphenyl. -V, -131-

6. A compound according to claim 4, wherein R 1 2 is p-sulfamoylphenyl.

7. A compound according to claim 4, wherein: R' is chosen from OH, OCH 2 C(O)NH(CH),. 6 R' 4 and OCH 2 -4-Phe-C(O)NH(CH 2 ),.iPR4; R 2 is chosen from H and lower alkyl; R 4 and R S are each lower alkyl or, R 4 and R 5 taken together is chosen from -(CH 2 -(CH 2 2 -O-(CH 2 2 -(CH 2 2 -NR'-(CH 2 2 -(CH 2 2 -CH(NHR 8 )(CH 2 2 (CH 2 2 -S-(CH 2 2 and -CH 2 CNCI- 3 )(CH)CHCH 2 one ofR 6 and R 7 are H and the other is OH or, R 6 and R 7 taken together is cho-.en from =0 and -S(CH 2 2 S-; R 8 is chosen from H, COOR 9 CONHR 0 CSNHR 1 COR 2 S0 2 R 1 3 lower alkyl, aryl lower alkyl, heteroaryl wherein the ring members include 1 to 3 N atoms and the substituents are halo or amino, heteroaryl lower alkyl wherein heteroaryl is 6-membered and the heteroatoms are N, and aryl lower alkyl substituted with 1 substituent selected from lower alkyl, alkoxy, and halo; R 9 is chosen from lower alkyl, aryl lower alkyl, aryl, tetrahydrofuranyl, tetrahydropyranyl, and aryl substituted by 1 to 2 substituents selected from lower alkyl, alkenyl, alkoxy, methylene dioxy, and halo; R 1 0 and R" are each independently chosen from aryl, aryl lower alkyl, and aryl substituted by 1 substituent selected from lower alkyl, halo, alkoxy, trifluoromethyl, and pentafluoroethyl; R 1 2 is chosen from lower alkyl, aryl, aryl lower alkyl, heteroaryl lower alkyl wherein the heteroatoms are N, a 5- or 6-membered heterocyclic ring containing 1-2 heteroatoms selected from S and N lower alkyl, and aryl .25 substituted with 1 substituent selected from lower alkoxy, halo, sulfamoyl, cyano, and phenyl; R 1 3 is chosen from lower alkyl, aryl, or aryl substituted with 1 substituent selected from lower alkyl, alkoxy, and halo; or a pharmaceutically acceptable salt thereof. v [v -132-

8. A compound according to claim 4, represented by Formula Ila R' J R 0 4 R 2 RIla wherein: R' is chosen from 6- and 7-OH; W 2 is chosen from H and lower alkyl; R' and Wi are each methyl. or, or RI and RI taken together are chosen from -(C-1 2 5 -(CH 2 2 0-(CH- 2 2 -(CI- 2 2 NR 8 -(CH- 2 2 -CH 2 -NR 8 -(CTT2)- -CH 2 NR-(CH 2 2 and -(CI- 2 2 -CH(NH-R 8 )(CH 2 2 cne of Wi and R 7 is H and the other is OH or, R 6 and R 7 taken to,, Aher are chosen from =0 and -S(CH 2 2 S-; R6 is chosen from H, COOR 9 CONHR' 0 CSNH-R 1 C0R 2 S02R 13 benzyl, -CH 2 -Ph-4-F, -CH 2 -Ph-4-OCH 3 -CH 2 -4-Py, r-luutyl, -CH 2 -c-propyl, C1 N 4 N C1N-~ iD/NH 2 N and 0 CI. R'B is chosen from i-propyl, phenyl, phenethyl, t-butyl, :-CH 2 0 I ~and -C R1 0 is chosen from phenyl, p-chlorophienyl, or p-trifluoromethylpheniyl; R 1 is chosen from phenyl, benzyl, and 1-naplithyl; 0 N ON" NH 1_CH LN N 'ZO R 2 is chosen from, H I -133- -CH 2 O \a N 0 N 0NH N -O 2 Nj O HH -O H 2 ,S N -C H H NH NZ and -0N2; and R 1 3 is chosen from 1- or 2-naplithyl, phenyl, 4-chiorophenyl, 4-methyiphenyl, 4-t-butylphienyl, n-butyl, and i-propyl; or a pharmaceutically acceptable salt thereof.

9. A compound according to claim. 4, reprt.sented by Formula Ila *6 7 R. I la whierein:P. a 6 R' is chosen from 6- and 7-OI- when R1 is HI; R 1 is 7-OH- when R' is CH 3 R' and R' are each methyl or, R 4 and R' taken together are chosen from -(CH 2 2 2 2 -(CH- 2 2 ,NR 8 -(CH- 2 2 -CI4 2 -NR 8 -(CI -CH 2 .NR 8 -(CF 2 2 and -(CH 2 2 -CH-INHRW)(CH 2 2 1 5 one of R' and R' is H and the other is OH or, W. and P. 7 taken together are chosen from =0 and -S(CH 2 2 and -134- R 8 is chosen from CH 2 CHGH 2 S 0 -135- sa 0* S S S S SS S p So 55 S S S NN 'x0 -J CI2- NN N >/LLL t~u\~f an I 73 omm r136- A compound according to claim 4, represented by Formula Ila RG :3 R ~R 5 Ila 0 R 2 0R4 wherein: R' is chosen from 6- or 7-OC 2 C(O)NH(CR 2 1 6 R', 6- or 7-C24PeCO)HC-216' when R 2 is H; RI is chosen from 7-OCH- 2 C(O)NTR(C 2 1 -6R and 7-OCH 2 -4-Phe-C(O)N1-i(C 2 )j6R'7 when R2 is CR- 3 R' and R' are each methyl or, RI is methyl and RI is chosen from -CI{ 2 0C 3 and -(CR2) 3 N(Et) 2 or, RI and RI taken together are chosen from -(CR 2 5 -(CH2) 2 -O-(CR2) 2 -(CH2) 2 -NR 8 -(0R 2 )0 2 -(CH 2 2 -CHqNuipy)(C- 2 2 -(CI-12) 2 -S-(cR2) 2 and -C{2CH(NCH- 3 )(CH 2 )oIrCI{1 2 one of R6 and R7 is HRand the other is OH- or, R1 and R7 taken together are chosen :fronm.=0 and -S(CII2) 2 S- or, one of R 6 and R 7 is H- and the other is NAB, 9 wheeinA ischosn fom methyl, 2 -lnethoxYethyl, 2 -phienylethyl, 4 -methoxybenzyl, 2-tetrahydro-furanylmethyl, 2-(3 ,4- dimethioxyphenyl)ethyl, and 2 2 -diphenylethlyl and B is chosen from 000 H H -SO 2 CH 3 AI a a)NH 99 9 )NH 2 0 N NH NON 0 (CH 2 5 N-kNH 2 \'N(n-proPY) 2 ~'NHCH3 09 9 9NHPh NHCH 3 and LL.L NHPh R8 is chosen from Hi, CONRCH 3 SO 2 Phe, (CI-1 2 3 CH 3 CO(C-12) 2 CH 3 11 bein'-1, C(O)-(4-Phe)-SO 2 H 2 and N~ 2 and -137- (CH 2 1 -6R' 7 is chosen from methyl, 2-methoxyethyl, 2-phenylethyl, 4-methoxybenzyl, methyl-2-tetrahydrofuranyl, 2-(3,4-dimethoxyphenyl)ethyl, or 2,2-diphenylethyl; or a pharmaceutically acceptable salt thereof.

11. A compound according to claim 4, represented by one of Formulas l~b, Ic and Ild 11b NHR 8 a a a a. a a a *a ~5 a a a a a wherein: R1 is chosen from 6- or 7-OH, 6- or 7-OCH- 2 C(O)NH(CH 2 16 R1 7 and 6- or 7-OCH 2 -4-Phe-C(O)NH(CH- 2 16 R' 7 P3 is chosen from H- or CH- 3 P3is -CO-Phe-p-SO 2 NH 2 and W3 and W? taken together are chosen from =0 and -SCH 2 CH 2 S-.

12. A compound according to claim 11, wherein R 1 R3, and R3 6 P7are as follows: Formula Ilb Formula Ile Formula Ild R1 7-OH 6-OH 6-OH 6-OH 7-OH- R 2 H H H H Cl-I 3 1 6R1-0- -SCH 2 GHS- -SCH 2 CH 2 S- -SCH 2 CH 2 S- -138- roo 0 0. 0. 1 0 5 S. S

13. A compound according to claim 4, represented by Formula II R R 7 3 R J R %28 14 if wherein R2, R 4 I, RS, R' and 7 are as follows: RR2 J R 4 R' RR' 6-OH I-CI-13 C21I 5 C21 5 OHl 1I 7-OH 8-C- 3 C-IH 3 CI- 3 H Oil f 7-C 2 H 5 1- C- 2 II NH 2 Hl 6-OH-(GH 2 2 0H H C31 7 ICH 3 =0 7-OC- 2 C0 2 H I7-CHFI -(CI 4 H morpholino 8-O-(CH 2 2 0H IH -(CJ 5 N(CI-3)2 H 6-CO 2 1- I8-C-1 3 -S(CI-D2)2S- 6-OHJ H -(CH 2 2 0(CI 2 2 7-OH 8-C13CI -s(cID2s- 6-O-1 H I_ 'iP iCI p 'h'b, 1 I; L -139-

14. A compound according to claim 4, represented by Formula 11 6 R 7 3 R R wherein RI, R 5 P, W7 and R' are as follows: 6-01- 7-OH 2 2 0H 6-OH 7-OH 6-OH 6-OH 7-OH 6-01I-1 H 8-CH 3 7-C 2 H 5 H H H H 8-CH 3 R' -(CH 2 2 NR 8(CH 2 2 -CH 2 NR8 (Cl- 2 3 -(CI- 2 2 NR 8 CH 2 -(CH 2 2 NR 8 (CH4 2 2 -(CH 2 2 CH(NR 8)(CH 2 2 -(CH 2 2 NR 8 (CH 2 2 -(CI- 2 2 NR 3 CH 2 -(CH 2 2 NR 8 (CI) 2 R 6 OH N'S02 R 7 H -ND~ H =0 -S(CH 2 2 S- -0 -S (CH2-I -S(CH 2 2 S- R 8 -CONH-Ph-4-CI' 3 -S0 2 -2-Niph CSNH--Ph -CO-Ph-4-SO 2 NH 2 -CO-Ph-4-SO 2 NH 2 -COCH 2 Ph -C0 2 -2-Py -CO-Ph-4-50 ,NH- 2 *99* 0* 9 9 .9 *4 9 9 9 9 9 I* 9 99 V 9' 4 9 9. 9 9 9 9 *9 9* 9 9 99 9 9 H I -(CH 2 2 NR 8 CH 2 I-S(CH 2 2 S- 'I-CO-Ph-4-S0 2 NHi 2 -140- 1(CH2)- I ISCiJS I 7-OHH S(CI)2S- -CO-Ph-4-S NH 2 6-OH H -(CH 2 2 R- =0 COCI-JPh 6-OH H -(CFI) 2 NR- -S(CII) 2 S- -Cq-2-Py CH 2 7-01- 8-GCl 3 -(CH 2 2 NR 8 -S(ClT) 2 S- -CO-Ph-4-SW~H 2 (Cl-I 2 2 6-OH H -(CH 2 2 NR- -S(CH4) 2 S- -CO-Ph-4-SQNH 2 7-OH H -(CH- 2 2 NR 8 =0 -CO-Phi-4-SQNH 2 (GCl 2 2 A.hraetclcmoiincmrsn hrpuial efetv amun ofacmonrpesne yFrua R6 R7 RI 15. Ahse phromacuia copoito comprising atherapeuticall a( R 7 7 7 2l6 an 5 -24Pe-()HC 21 loerly whereintesbttet r loy rl usiue rl Z 1 i hsnfo 11 (H) 2 H C 2 OI1 OH -141- carboalkoxy, carboxamido, and di-loweralkylamido, or R 4 and Rs taken together are chosen from -(CH 2 -(CH 2 2 -O-(CI- 2 2 -CH 2 -O-(CH) 3 -(CH 2 2 -NR 8 (CI- 2 2 -CH 2 -NR8-(CI-H 2 -(CH 2 2 CH(NHRB)(CH 2 2 -(CH 2 2 -S(0)o 0 2 -(CH 2 2 and -CH 2 CH(N-loweralkyl)(CH 2 2 CHICH 2 one of R 6 and R 7 is H and the other is chosen from H, OH, and N(CH 2 16 R' 4 R 5 or R 6 and R7 taken together is chosen from o r-<R R(1 s 0 so II S ,O and S, S R 8 is chosen from H, COOR 9 CONHRO, CSNHR 1 COR' 2 SO 2 R 13 lower alkyl, aryl lower alkyl, heteroaryl, and heteroaryl lower alkyl, wherein aryl is optionally substituted with 1-3 substituents selected from lower alkyl, lower alkoxy, halo, CN, NH 2 COOH, CONH,, carboalkoxy, and mono- or di-lower alkylamino and wherein heteroaryl is a mono- or bicyclic heteroaromatic ring system of 5 to 10 members including 1 to 3 heteroatoms selected from O, N, and S and 0-3 substituents selected from halo, amino, cyano, lower alkyl, carboalkoxy, CONI-H 2 and S-lower alkyl; R is chosen from lower alkyl, aryl, aryl lower alkyl, heteroaryl, aryl substituted by 1-3 cubstituents selected from alkyl, alkenyl, alkoxy, methylene dioxy, and halo, and a 5- to 6-membered heterocyclic ring wherein the hetero atom is O or N, wherein heteroaryl is a heteroaromatic ring of 5 to 6 members including 1 to 2 heteroatoms selected from O, N, and S and 0-2 substituents selected from lower alkyl, dialkylamino, lower alkoxy, and halo; R 10 and R" are each independently chosen from lower alkyl, aryl, aryl lower alkyl, and aryl substituted by 1-3 substituents selected from lower alkyl, halo, alkoxy and haloalkyl; S R 12 is chosen from lower alkyl, aryl, heteroaryl, aryl lower alkyl, heteroaryl lower alkyl, a 5- or 6-membered heterocyclic ring containing 1-2 heteroatoms selected from 0, S, and N, a 5- or 6-membered heterocyclic ring containing 1-2 heteroatoms selected from 0, S, and N lower alkyl, and aryl substituted with 1-3 substituents selected from lower alkyl, alkoxy, -142- halo, sulfamoyl, lower alkyl sulfamoyl, cyano, and phenyl; R' 3 is chosen from lower alkyl, aryl, and aryl substituted with 1-3 substituents selected from lower alkyl, alkoxy, halo, CN, and haloalkyl; R' 4 is chosen from H, alkyl, alkyl substituted by 1-3 alkoxy, loweralkyl, sulfamoyl, halo, alkylsulphonamido, or arylsulphonamido, alkenyl, alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycloalkyl, -CH 2 NR' 1 6 C(O)R" 6 -C(O)NR'"R' 6 -CH 2 OC(O)R'6 and -CH 2 SC(O)R' 6 R' 5 is chosen from H, alkyl, and -C(NCN)NR 3 R 3 R 16 is chosen from lower alkyl, substituted lower alkyl, aryl, and substituted aryl; R17 is chosen from H, alkyl, alkyl substituted by 1-3 alkoxy, loweralkyl, sulfamoyl, halo, alkylsulphonamido, or arylsulphonamido, alkenyl, alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycloalkyl, -CHNR 6 C(O)R' 6 -C(O)NR' 6 R' 6 -CH20C(O)R' 6 and -CH2SC(O)R' 6 X is chosen from alkyl, aryl, arylalkyl, O-loweralkyl, and -NR 3 R 3 Z is chosen from -(CH 2 1 optionally substituted with 1-3 lower alkyl, -CHR 2 -Phe-CH 2 where Phe is optionally mono-substituted with halogen, lower alkyl, or alkoxy, and heteroarylene-(CH 2 Sm is chosen from 2 and 3; n is chosen from 4-9; S..20O or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable :o: *I carrier.

16. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to claim 9 and a pharmaceutically acceptable carrier. 25 17. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to claim 10 and a pharmaceutically acceptable carrier. I, -143-

18. A pharmaceutical composition for inhibiting carbonic anhydrase in a mammal, said composition comprising a therapeutically effective amount of a compound according to claim 11 and a pharmaceutically acceptable carrier.

19. A method of inhibiting carbonic anhydrase isozymes in a mammal which comprises administering to said mammal an effective amount of a compound according to claim 11. A method of treating glaucoma in a mammal which comprises administering to a mammal in need of such trea'ment an effective amount of a compound according to claim 11.

21. A method of identifying a ligand having a desired characteristic which comprises: synthesizing a combinatorial library according to claim 1; and testing the compounds of said library in an assay that identifies compounds having the desired characteristic. 0 *library are those wherein -C(O)-L-II is represented by Formula Ia. R 4 I "NO 2

23. The method according to claim 21 further comprising: determining the structure of any compound so identified. 0*

24. The method according to claim 21 wherein said desired characteristic is carbonic anhydrase inhibition. R 0 7 '1 L 24 Th metho acodn-ocam2 weensieie chrcersi iscroi U yrs niiin g r 1L-- i- ii -144- A method of identifying a ligand having a desired characteristic which comprises: synthesizing a combinatorial library according to claim 1; detaching the ligands from the solid supports in said library; and testing said detached ligands in an assay that identifies ligands having the desired characteristic.

26. The method according to claim25 further comprising: determining the structure of any ligand so identified.

27. The use of divinylbenzene-cross-linked, polyethyleneglycol-grafted polystyrene beads as the solid supports for constructing a combinatorial library So according to claim 1.

28. The method according to claim 27, wherein the ligands are detached from said solid supports by photolysis. S**29. A process for preparing a compound represented by 0* *~N0 CO O Me wherein R 2 is H or lower alkyl, said method comprising: reacting allyl or methyl 4-(hydroxymethyl)-3-nitrobenzoate with a ecompound represented by 0 HO HMe OH R2 in the presence of tripherylphosphine, toluene, and DEAD and stirring the mixture at room temperature to produce p I 1 -145- RO2CO 0 NO 2 OH 2 R wherein R is allyl or methyl; and when R is allyl, reacting said compound with methylene chloride, tetrakistriphenylphospine palladium and pyrrolidine and stirring the mixture at 0°C or, when R is methyl, reacting said compound with dilute NaOH and THF and stirring the mixture at 0°C. A compound represented by Br O NOz 2 0 N (CH2)1-6R 7 14 wherein is a solid support; S: 1'0 R 6 is chosen from lower alkyl, substituted lower alkyl, aryl, and substituted aryl; R 17 is chosen from H, alkyl, substituted heterocycloalkyl, alkyl substituted by 1-3 alkoxy, S-loweralkyl, sulfamoyl, halo, alkylsulphonamido, or arylsulphonamido, alkenyl, alkynyl, aryl, substituted aryl, heteroaryl, l k Ssubstituted heteroaryl, heterocycloalkyl, -CHjSC(O)R', CHzNR' 6 C(O)R 6 -C(O)NR 1 6 R 16 and -CH 2 0C(O)R' 6 R is chosen from H and alkyl; and Y is chosen from aryl and heteroaryl.

31. A combinatorial dihydrobenzopyran library, substantially as hereinbefore described with reference to any one of the Examples.

32. A method of identifying a ligand having a desired characteristic, substantially as hereinbefore described with reference to any one of the Examples. 146 Dated 16 March, 1998 Pharmacopela, Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON t~ C C C CC C 0* CC C I. C C C. C C *C C C. C C C C C. C C C C C. C C C C CC C. CC C CCC... JNAL1BA1OO525:SSC INTERNATIONAL SEARCH REPORT In, .tional ipplicauti No. '05940 A. CLASSIFICATION OF SUB.JECT MIATTEi.R IPC(6) Please See Extra Sheet. US CL Please See Extra Sheet. According to International Patent Classification lIPC, or to both national classification and IPC B. FIELDS SEARCHED MIinimum documentation searched (classification sytem followed by classification symbols) US 5621434; 549114. 30, 398. 5481300.1, 5461I; 5141189, 222.2, 247, 433, 439, 456 Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched Electronic data base consulted during the international search tname of data base and. ".here practicable. search terms used) CAS/STN, APS C. DOCUMENTS CONSIDERED) TO BE RELEWANT Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. A US, A, 4,499,299 (BERNSTEIN ET AL) 12 FEBRUARY 1985. 1-3, 5-6 A J. Med. Chem. Volume 32, Number 4, issued April 1989, 1-3, 5-6 Brown et al., "Hydroxyacetophenone-Cerived antagonists of the Peptidoleukotrienes", pages 807-826, see entire document. X Abstract, Mitsui Petrochemical Industries, Ltd, WO, A, 4, 7-10, 14-17, 8700840 February 1987, abstract 236509b, see entire abstract. D Further documents are listed in the continuation of Box C. D See patent family annex. Speciai categories of cited documents; T" later docunent pubtished sfter the initnaional riling date or priority date wnd not in confice with the application but cited to understand the docuxnentdfef'ning the general state or the ant which is not considered piriciple or theory underlying the invention to be of particular relevance eaiW otneiL o ratr~ document of particular relevance. thte clsune. invention cannot be eale ouetpublished ooratrthe internxationai riling daeconitidered novel or cannot be considere to involve an tnvenive stp *LU documeni which may throw doubts on priorit claimts) or which is whets the document is taken atone cied to establish the publication date or another citation or tither special reason (as specified) doctuneni of particular relevance; the climed invenion cannot be considered to involve an inventivec step %%hen the document us .0 document referrig io an orai disclosure, use, exhibition or other combined wids oat or mare other such documents, su~h cointation means being obvious to a person skilled in the art P. document published prior to the internatiot 31 iling date but later than document member of the same potent fiuniiy the priority date claimed Date of the actual completion of the international search Date of' mailing of the international search report 21 AUGUST 1995 05SEPI1995 Name and mailing address or the ISA/US Authorized officer Commissioner of Psicnts and Trademarkst t 1 Bos Pr JOSEPH M. CONRAD Ill Washington. DC. 20231 Facsimile No. (703) 305-3230 Telephone No. (703) 308-1235 Form PCT/ISA/210 (second shect)(July 1992)w INTERNATIONAL SEARCH REPORT Int ,tional application No PCTIUS95/05940 Box I Observations where certain claims were found unsearchable (Continuation of item I of first sheet) This international report has not been established in respect of certain claims under Article 17(2)(a) for the following reasons: 1. FO- Claims Nos.: Sbecause they relate to subject matter not rcquired to be searched by this Authority. namely" 2. OF Claims Nos.: Sbecausethey relate to parts of the nternational application that do not comply witi the prescribed requirements to such an extent that no meaningful international search can be carred out, specifically: 3. O Claims Nos.: because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a). Box II Observations where unity of invention is lacking (Continuation of item 2 of first sheet) This International Searching Authority found multiple inventions in this international application, as iollows: Please See Extra Sheet. 1. r7 As all required additional search fees were timely paid by the applicant, this international search report covers all searchable claims. 2. As all searchable claims could be searched without effort justifying an additional fee, this Authority did not invite payment of any additional fee. 3. F- As only some of the required additional search fees were timely paid by the applicant, this international search report covers only those claims for which fees were paid, specifically claims Nos.: 4. D No required additional search fees were timely paid by the applicant. Consequently, this international search report is restricted to the invention first mentioned in the claims: it is covered by claims Nos.: Remark on Protest rl The additional scach fees were accompanied by the applicant's protest. E] No protest accompanied the payment of additional search fees. Form PCT/ISA/210 (continuation of first shcct(l))(July 1992)* INTERNATIONAL SEARCH REPORT In.. .ional application No PCT/US95105940 A. CLASSIFICATION OF SUBJECT MATTER: IPC C07C 205/06; C07D 311/04, 279/10, 275/02, 207/00; A61K 31/555, 31154, 31/50, 311385. 31/35 A. CLASSIFICATION OF SUBJECT MATTER: US CL 562/434; 549/14. 30, 398; 546/1; 548/300 1, 514/188. 222.2, 247, 433, 439. 456 BOX II. OBSERVATIONS WHERE UNITY OF INVENTION WAS LACKING This ISA found multiple inventions as follows: This application contains the following inventions or groups of inventions which are not so linked as to form a single inventive concept under PCT Rule 13,1. In order for all inventions to be examined, the appropriate additional examination fees must be paid. Group lClaims 1-3, 5-6 and 21-24. drawn to the compound of formula 1 and a method of use for said compound. Group II.Claims 4, 7-20, drawn to a compound of formula II, composition thereof and first method of use. Group Ill.Claims 25-26. drawn to a screening method for the identification of carbonic anhydrase inhibitors. Group IV. Claims 27-28, drawn to a 2nd screening method for enzyme inhibitors. Group V.Claims 29-30, drawn to a 3rd screening method for ligands. Group VI. Claims 31-32, drawn to a 2nd method of use for the compound of formula I. Group VII. Claim 33, drawn to a method for making the compound of formula 14. Group VIII.Claim 34, drawn to the compound of formula 3, The inventions listed as Groups 1-VIII do not relate to a single inventive concept under PCT Rule 13.1 because, under PCT Rule 13.2, they lack the same or corresponding special technical features for the following reasons: a) Groups I, II, VII and VIII are drawn to different compounds and subsequently different methods of use and or methods of making, b) screening methods from Groups III, IV and V are different in the manner in which they are earned out, c) Group VI is drawn to a 2nd method of use for the compound of formula 1, which is distinct from that claimed in Group I, Form PCT/ISA/210 (extra sheet)(July 1992)*