AU705844B2 - Combinatorial 1,4-benzodiazepin-2, 5-dione library - Google Patents

Description

Combinatorial 1,4-benzodiazepin-2,5-dione 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 activities. 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. See eg., Still et al., W094/08051. For techniques such as these to be successful, numerous solid state chemical reactions must be developed.

Ellman et al., ('Progress Toward the Synthesis of a Library of 1,4 ACS National Meeting Anaheim, CA, April 2-6, 1995, Abstr. ORGN264) have reported a solid-phase synthesis of 1,4-benzodiazepin-2,5-diones. The Ellman et al. method limits the diversity of the scaffold because attachment of the scaffold to the solid support during synthesis is through the benzene ring, a residuum remaining on said ring after detachment of the from the solid support. Solution-phase synthesis of 1,4-benzodiazepin-5ones via intramolecular aza-Wittig reaction has been disclosed by Egushi et al. (SYNLETT, 295-6, April 1992).

It is also desirable for compounds produced by combinatorial synthesis 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 W093/20242, 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 particles-based synthesis of random oligomers wherein identification tags on the particles are used to facilitate identification of the oligomer sequence synthesised. A detachable tagging system is described in Ohlmeyer et al., Proc. Natl. Acad. Sci. USA, 90, 10922-10926, Dec. 1993.

Summary of the Invention The present invention relates to a method of synthesising a combinatorial library of 1,4benzodiazepin-5-ones on solid supports via an aza-Wittig ring closure, said compounds optionally encoded with tags, and to the use of this library in assays to discover biologically active compounds, and, optionally, to cleave 1,4-benzodiazepin-2,5-diones therefrom.

30 Detailed Description of the Invention The combinatorial chemical library which may be synthesised by the method of the present invention is represented by Formula I: wherein: is a solid support; is an identifier residue; is a linker/ligand residue; q is 0-30; and II'is Libc/03289 wherein: Ri is H, lower alkyl, c-lower alkyl, or (CH2)mR4, or Ri and R 2 together with the atoms to which they are attached, join to form a or 6-membered heterocyclic ring, optionally monosubstituted with OH, alkoxy, or arylalkoxy; R2 is H, loweralkyl, arylRsR7R8, or heteroarylR 6

R

7 Rs, or R 1 and R 2 together with the atoms to which they are attached. join to form a 5- or 6-membered heterocyclic ring, optionally monosubstituted with OH, alkoxy, or arylalkoxy; R 3 is H or loweralkyl, R 4 is aryl, substituted aryl, heteroaryl, substituted heteroaryl, NR 3

R

5 C0 2

R

3

CONR

3

R

3 or OH; R 5 is H, lower alkyl, -C(=NR3)NHR3, or -C(O)R 3 Re, R 7 and R 8 are each, independently, H, lower alkyl, lower alkoxy, halogen, aryl, lower alkylthio, X-aryl, X-substituted aryl, lower alkylaryl, C(hal)3, (CH 2 )mNR 3

R

5 or-X-CH(C0 2

R

3 2 or R 6 and R 7 together with the atoms to which they are attached. join to form a 5- or 6-membered heterocyclic ring: X is O or S; and Xi and X 2 are independently chosen from hydrogen, loweralkyl, loweralkoxy, loweralkylthio, hydroxy, cyano, nitro, phenoxy, benzyloxy, halo, aryl,

NH(C=O)R

3 and carboxy, or Xi and X 2 taken together represent a fused benzene ring substituted with hydrogen, loweralkyl, loweralkoxy. loweralkylthio, hydroxy, cyano, nitro, phenoxy, benzyloxy, halo, aryl, NH(C=O)R 3 or carboxy.

Preferred libraries of Formula I are those wherein is of the formula III -H2C o \-Ar 0 I I 0 O CH 3 wherein: n 3-12; Ar is halophenyl; and q is 3-12.

More preferred libraries of Formula I are those wherein in Formula III: 1) n=3-12 and Ar is pentachlorophenyl; or 2) n=5-6 and Ar is 2,4,6-trichlorophenyl.

Depending on the choice of L' (see Table the ligands of formula II may be detached by photolytic, oxidative, acidic, basic, or other cleavage techniques. For example, when is acidic cleavage may be represented by: o o wherein L" is the residue from L' and 'OH is II, in its tautomeric amde fOH 25 wherein L" is the residue from L'and Il'OH is 11, in its tautomeric amide form: Libc/03289 wherein the symbols are as defined above for formula II'.

According to a first embodiment of the invention there is provided a process of synthesising a 1,4which comprises: a. attaching an amino-protected a-amino acid or an amino protected N-alkyl a-amino acid to a solid support via its carboxyl group to form a resin linked amino-protected a-amino acid or aminoprotected N-alkyl a-amino acid; b. cleaving an amino protecting group from said resin linked amino-protected a-amino acid or amino-protected N-alkyl a-amino acid to provide a resin linked a-amino acid or N-alkyl a-amino acid; c. acylating said resin linked a-amino acid or N-alkyl a-amino acid with a 2-azidobenzoyl chloride to form a resin linked N-(2 azidobenzoyl)amino acid; and d. cyclising said resin linked N-(2-azidobenzoyl)amino acid via aza-Wittig ring closure to provide a resin linked 1,4-benzodiazepin-2,5 dione.

According to a second embodiment of the invention there is provided a process of synthesising a S. 15 1,4-benzodiazepin-2,5-dione which comprises: a. attaching an amino-protected a-amino acid to a solid support via its carboxyl group to form a S* resin linked amino-protected a-amino acid; b. cleaving an amino protecting group from said resin linked amino-protected a-amino acid to provide a resin linked a-amino acid; 20 c. reductively alkylating said linked a-amino acid with an aldehyde and a reducing agent to provide a resin linked N-alkyl a-amino acid; d. acylating said resin linked N-alkyl a-amino acid with a 2-azidobenzoyl chloride to form a resin linked N-(2-azidobenzoyl)amino acid; and e. cyclising said resin linked N-(2-azidobenzoyl)amino acid via aza-Wittig ring closure to provide a resin linked 1,4-benzodiazepin-2,5-dione.

According to a third embodiment of the invention there is provided a process of synthesising, 1,4of the formulae: [N:\LIBFF]0373:SSD which comprises: a) reacting a resin-linked a-amino ester of the formula: wherein is a solid support and R 1 is hydrogen or benzyloxy, suspended in an aprotic, polar s solvent and an excess of a soluble organic base with an excess of a substituted 2-azidobenzoyl chloride of the formula: a

I

wherein X 1 and X 2 are hydrogen or methoxy, to produce a resin-linked N-(2-azidobenzoyl)amino ester; b) suspending said resin-linked ester in an involatile solvent and treating said suspension with an excess of a trivalent phosphorus reagent at 80-140 0 C for 2-24h to produce a resin-linked 1,4of the formula: 0 a:i

R..

and c) suspending said resin-linked 1, 4 -benzodiazepin-5-one in an acidic solution at room temperature for 1-24h to produce a 1,4-benzodiazepin-2,5-dione of the formula: O-Bz NO.N O ON N HQ O H O H/

O

SCH

3

CH

3 or According to a fourth embodiment of the invention there is provided a process of synthesising a r 1, 4 -benzodiazepin-2,5-dione of the formula: 7x,. IN:\LIBFFI0373:SSD r o_ which comprises: a) reacting a resin-linked c-amino ester of the formula: O s

CH

3 a.

a a 0 .0 *500

S

a wherein is a solid support, suspended in an aprotic, polar solvent and an excess of a soluble organic base with an excess of a substituted 2-azidobenzoyl chloride of the formula: Cl

N

3 O xl x2 wherein X 1 and X 2 are hydrogen, to produce a resin-linked N-(2-azidobenzoyl)amino ester; b) suspending said resin-linked ester in an involatile solvent and treating said suspension with an 10 excess of a trivalent phosphorus reagent at 80-140°C for 2-24h to produce a resin-linked 1,4of the formula: a..

a a a v and c) suspending said resin-linked 1,4-benzodiazepin-5-one in an acidic solution at room temperature for 1-24h to produce a 1,4-benzodiazepin-2.5-dione of the formula: [N:\LIBFF]0373:kml According to a fifth embodiment of the invention there is provided a compound of the formula:

S

S

S.

S

S

S

According to a sixth embodiment of the invention there is provided a compound of the formula:

*S.S

S

S

S

S.

S

wherein is a solid support; Rl is hydrogen or benzyloxy; and X, and X 2 are hydrogen or methoxy.

Disclosed herein is the solid phase synthesis of 1,4-benzodiazepin-2,5-diones via aza-Wittig ring closure. The process comprises: 10 a) attaching a set of suitably protected a-amino acids or N-alkyl-a-amino acids to solid supports to form resin linked N-alkyl-a-amino acids; or b) attaching a set of suitably protected N-unsubstituted-a-amino acids to solid supports to form resin linked N-unsubstituted-a-amino acids and reductively alkylating said resin linked amino acids with a set of aldehydes to form resin linked N-arylalkyl or heteroarylalkyl-a-amino acids; c) acylating the resin linked N-alkyl-a-amino acids or the N-arylalkyl or heteroarylalkyl-a-amino acids of steps or with a set of 2-azidobenzoyl chlorides to form resin linked N-(2azidobenzoyl)amino esters; d) cyclising the resin linked N-(2-azidobenzoyl)amino esters of step via aza-Wittig ring closure to form resin linked benzodiazepines; and, optionally, 'g e) cleaving the resin linked benzodiazepines of step to form 1,4-benzodiazepin-2,5-diones.

[N:\LIBFF]O373:SSD iiy -o C i i 6a Also disclosed is the solid phase synthesis of 1,4-benzodiazepin-2,5-diones via aza-Wittig ring closure, wherein the process comprises: a) reacting a set of suitably protected a-amino acids of the formula: R R HO N3Fmoc o H in the presence of DMF and DMAP with solid supports suspended in methylene chloride to form resin linked amino acids of the formula: RR3 O N3Fmoc 0 H b) reacting the resin linked amino acids of step suspended in DMF and acetic acid, with a set of aldehydes of the formula HC(O)R 2 in HOAc/DMF and sodium cyanoborohydride in THF to form resin linked N-alkyl-a-amino acids of the formula: 3 :O N R 2 O H O H c) reacting the resin linked N-alkyl-a-aminoacids of step in methylene chloride and diisopropylethylamine, with 2-azidobenzoyl chlorides of formula: Cl

:N

3 0 to form resin linked N-(2-azidobenzoyl)amino esters of formula:

_R

2 0 X* X 2 d) treating the resin linked N-(2-azidobenzoyl)amino esters of step suspended in an involatile solvent a non-protic, organic solvent with a boiling point of 80-140'C) such as toluene, xylene, or chlorobenzene, with an excess of a trivalent phosphorus reagent such as triphenylphosphine or tributylphosphine at 80-150C and then cooling said mixture to room temperature to form resin linked benzodiazepines of formula: temperature to form resin linked benzodiazepines of formula: IN:\LIBFF]O373:SSD and, optionally, e) suspending the resin linked benzodiazepines of step in TFA/water at room temperature for 1-24 hours to form 1,4-benzodiazepin-2,5-diones of formula:

R

3 S/N2

N

wherein the symbols are as defined above for formula II'.

There is further disclosed the solid phase synthesis of 1,4-benzodiazepin-2,5-diones and 1,3cyclo-1,4-benzodiazepin-2,5-diones via aza-Wittig ring closure, wherein the process comprises: a) reacting a set of suitably protected a-amino acids of the formula:

R

3 2

O

0 Fmoc in the presence of DMF and DMAP with solid supports suspended in methylene chloride to form resin linked amino acids of the formula:

**R

R R2 1 01 0

N

0 Fmoc b) reacting the resin linked N-alkyl-a-amino acids of step in methylene chloride and diisopropylethylamine, with 2 azidobenzoyl chlorides of formula: ci

N

3

-O

x, x2 to form resin linked N-(2-azidobenzoyl)amino esters of formula: IN:\LIBFF]O373:SSD

SR

3 R R R2

N

X, X c) treating the resin linked N-(2-azidobenzoyl)amino esters of step suspended in an involatile solvent a non-protic, organic solvent with a boiling point of 80-140°C) such as toluene, xylene, or chlorobenzene, with an excess of a trivalent phosphorus reagent such as triphenylphosphine or tributylphosphine at 80-150 0 C and then cooling said mixture to room temperature to form resin linked benzodiazepines of formula: O-R2

R

3 Ri' 2

N

/N

N 0 X- 'X2 and, optionally d) suspending the resin linked benzodiazepines of step in TFA/water at room temperature for 1-24 hours to form 1,4-benzodiazepin-2,5-diones of formula: H 0

R

3

RX

2

N

H0 X X2 10 wherein the symbols are as defined above for formula II'.

Also disclosed 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 synthesising a combinatorial library of Formula I and testing the library of Formula I, either attached to the solid support or detached therefrom, in an assay which identifies compounds of Formula II having the desired characteristic. Also disclosed is a method of identifying a compound having a desired characteristic which comprises testing the library of Formula I, either attached to the solid support or detached therefrom, in an assay which identifies compounds of Formula II having the desired characteristic.

Further, there is disclosed the determining the structure of any compound so identified.

The determination of the structures of compounds having the desired characteristic can be accomplished by decoding the tags (represented by in Formula I) or, alternatively, by deconvolution of the library (Smith et al., BioMed. Chem. Lett., 4, 2821 (1994); Kurth et al., J. Org.

Chem., 59, 5862 (1994); Murphy et al., J. Am. Chem. Soc., 117, 7029 (1995); Cambell et al., J. Am.

[N:\LIBFF]0373:SSD 6d Chem. Soc., 117, 5381 (1995); and Erb etal., Proc. Nat. Acad Sci. USA, 91, 1 1422 (1994)). In the latter case, q 0 and the library of the present invention may be represented by Formula I' -K0 L'-l' wherein the symbols are as defined for Formula I.

Divinylbenzene-cross-linked, polyethyleneglycol-grafted polystyrene beads optionally functionalised with amino groups (for example, TentaGel® S NH 2 Rapp Polymere) may be used as the solid supports for constructing a combinatorial library of Formula I or I'.

Definitions The following abbreviations have the indicated meaning: Bn benzyl BnOH benzyl alcohol Boc t-butyloxycarbonyl Bz benzoyl c- =cyclo ,15 DEAD diethylazodicarboxylate 0 0 0* 00* *0 [N:\LIBFF]O373:SSD DCM dichloromethane methylene chloride DIC diisopropylcarbodiimide DMAP 4-N,N-dimethylaminopyridine DMF N,N-dimethylformamide equiv. equivalent Et ethyl FACS fluorescence activated cell sorting Fmoc 9-fluorenylmethoxycarbonyl Fmoc-OSu 9-fluorenylmethylsuccinimidyl carbonate GC gas chromatography h hour, hours m- meta Me methyl p- para PEG polyethylene glycol Ph phenyl r.t. room temperature sat'd saturated secondary t- tertiary t-Boc t-butyloxycarbonyl TFA trifluoroacetic acid THF tetrahydrofuran Thy thienyl 25 TsOH p-toluenesulfonic acid Alkyl is intended to include linear, branched, or cyclic hydrocarbon structures and combinations thereof. 'Lower alkyl" means 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, cyclopropylethyl, and the like. 'Lower cycloalkyl" includes cycloalkyl groups of from 3 to 8 carbon atoms. Examples of lower cycloalkyl groups include c-propyl, c-butyl, c-pentyl, 2-methylcyclopropyl, cyclopropylmethyl, norbornyl, and the like.

"Alkenyl" is C 2

-C

8 alkenyl of a linear, branched, or cyclic (C5-C 6 configuration and combinations thereof. Examples of alkenyl groups include vinyl, allyl, isopropenyl, pentenyl, hexenyl, c-hexenyl, 1propenyl, 2-butenyl, 2-methyl-2-butenyl, and the like.

35 "Alkynyl" is C 2

-C

8 alkynyl of a linear or branched configuration and combinations thereof.

Examples of alkenyl groups include ethynyl, propynyl, butynyl, pentynyl, 3-methyl-l-butynyl, 3,3 dimethyl-1-butynyl, and the like.

"Alkoxy" means alkoxy groups of from 1 to 8 carbon atoms of a straight, branched, or cyclic configuration and combinations thereof. Examples of alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy, cyclohexyloxy, and the like.

Libc/03289 "Acylamino" means acylamino groups of from 1 to 8 carbon atoms of a straight, branched or cyclic configuration and combinations thereof. Examples of acylamino groups are acetylamino, butylamino, cyclohexylamino, and the like.

Hal means halogen, which includes F, Cl, Br, and I.

"Halophenyl" means phenyl substituted by 1-5 halogen atoms. Halophenyl includes pentachlorophenyl, pentafluorophenyl, and 2,4,6-trichlorophenyl.

"Aryl" and "heteroaryl" mean a 5- or 6-membered aromatic or heteroaromatic ring containing 0- 3 heteroatoms selected from O, N, and S; a bicyclic 9- or 10-membered aromatic or heteroaromatic ring system containing 0-3 heteroatoms selected from O, N, and S; or a tricyclic 13- or 14-membered aromatic or heteroaromatic ring system containing 0-3 heteroatoms selected from O, N, and S; each of which rings is optionally substituted with 1-3 substituents selected from lower alkyl, alkenyl, alkynyl, substituted lower alkyl, substituted alkenyl, substituted alkynyl, NO 2 halogen, hydroxy, alkoxy, cyano, NR 3

R

3 acylamino, phenyl, benzyl, phenoxy, benzyloxy, heteroaryl, and heteroaryloxy, each of said phenyl, benzyl, phenoxy, benzyloxy, heteroaryl, and heteroaryloxy is optionally substituted with 1- 3 substituents selected from lower alkyl, alkenyl, alkynyl, halogen, hydroxy, alkoxy, C(hal) 3 cyano, phenyl, phenoxy, benzyl, benzyloxy, caboxamido, heteroaryl, heteroaryloxy, N02, and NR 3

R

3 The aromatic 6 to 14-membered carbocyclic rings include benzene, naphthalene, indane, tetralin, and fluorene and the 5- to 10 membered aromatic heterocyclic rings include imidazole, pyridine, indole, thiophene, benzopyranone, thiazole, furan, benzimidazole, quinoline, isoquinoline, quinoxaline, pyrimidine, pyrazine, tetrazole, and pyrazole.

"Substituted" alkyl, alkenyl, or alkynyl means alkyl, alkenyl, or alkynyl wherein up to three H atoms on each C therein are replaced by halogen, hydroxy, loweralkoxy, carboxy, carboalkoxy, carboxarnido, cyano, carbonyl, N02, NR3R3, alkylthio, sulfoxide, sulfone, acylamino, amidino, phenyl, benzyl, heteroaryl, phenoxy, benzyloxy, heteroaryloxy, and substituted phenyl, benzyl, heteroaryl, 25 phenoxy, benzyloxy, and heteroaryloxy.

:It is intended that the definitions of any substituent or symbol R 3 m, etc.) in a particular molecule be independent of its definitions elsewhere in the molecule. Thus, "NR 3

R

3 represents NHH,

NHCH

3 N(CH3)2, etc.

The linkers may be any component capable of being selectively cleaved to release both T and II from the solid support. See, eg., Greene and Wuts, "Protective Groups in Organic Synthesis", 2nd ed., Wiley, 1991. Specific linkers L' are depicted in Table 1 (note that or -CH 2 which also shows cleavage reagents. In designing a synthetic scheme, L and L' are chosen such that they are orthogonally reactive, ie., 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 S 35 disadvantageous. In the structures as shown, the left-hand bond is the point of attachment to the solid support (via for L' and or -CH2C(O)- for L) and the right-hand bond is the point of attachment to either T or II.

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 Libc/03289 9 be capable of being detected at very low concentrations, eg., 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, eg., 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 0.001-50pmol, 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; a and/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. fibres, gels, or particles such as cellulose beads, pore-glass beads, silica gels, polystyrene beads optionally cross-linked with divinylbenzene and optionally grafted with polyethylene glycol and optionally functionalised with amino, hydroxy, carboxy, or halo groups, grafted co-poly beads, poly-acrylamide beads, latex beads, dimethylacrylamide beads optionally cross-linked with N,N'-bis-acryloyl ethylene diamine, glass particles coated with hydrophobic polymer, etc., ie., material having a rigid or semi-rigid surface; b) soluble supports such as low molecular weight non cross-linked polystyrene; and c) derivatised forms thereof such as O Suitable amino acid protecting groups are well known in the art and include Fmoc, Alloc (allyloxycarbonyl), etc.

Table 1 25 Linker Groups I -i -1I a ft* Linker Group Cleavage Keagent

NO

2 hv

NO

2 6

O-B-

B- O 1 or 2 N hv 02

O-

2 O-R Ce(NH 4 2 (N03)6 3 Libc/03289 Linker Group Cleavage Reagent Ce(NH4) 2 (NO3)6 R O -0

O

-CH=CH(CH2)2- 03, Os04/10 4 or KMnO4 6 -CH=CHCH 2 03, Os0 4 /10 4 or KMn04 7 -CH 2 CH=CH- 03, Os04/104-, or KMn04 0 H 02 or Br2, MeOH 2) 8 9 -CH=CHCH 2 0- (Ph 3

P)

3 RhCI(H) Br Li, Mg, or BuLi

O-

11 -S-CH2-O- Hg 2 x 0- Zn or Mg 12 HO O- Oxidation, eg., 13 Pb(OAc) 4 or H 5 10 6 13 o Q

H

3 0

O-

14 or OQ O- 0-

O-CH

3 R H or lower alkyl; B O or NH; and X electron withdrawing group such as Br, CI, and I.

Optical Isomers Diastereomers- Geometric Isomers Some of the compounds described herein contain one or more asymmetric centres and may thus give rise to enantiomers, diastereomers, and other stereoisometric forms which may be defined in terms of absolute stereochemistry as or or as or for amino acids. The present invention is meant to include all such possible diastereomers as well as their racemic and optically pure forms. Optically active and or (D and isomers 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 centres 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.

*r

S

S

Libc/03289 Utility The library of the present invention is useful as a screening tool for discovering new lead structures by evaluation across an array of biological assays, including the discovery of selective inhibition patterns across isozymes. The library is thus a tool for drug discovery; ie., as a means to discover novel lead compounds by screening the library against a variety of biological targets and to develop structure-activity relationships (SAR) in large families of related compounds. The library may be tested with the ligands attached to the solid supports as depicted in Formula I or or the compounds II may be detached prior to evaluation. With the compounds of Formula I or screening assays such as EACS sorting 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. Whether the compounds are tested attached or detached from the solid supports, the tags attached to solid support associated with bioactivity 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 and Still et al, Complex Combinatorial Chemical Libraries Encoded with Tags, W094/08051) or, alternatively, the structures may be determined by deconvolution. The usefulness of such a library as a screening tool is demonstrated by Burbaum et al., Proc. Natl. Acad. Sci. USA, 92, 6027-6031, June 1995, who describe the assaying of encoded combinatorial libraries for, eg., carbonic anhydrase inhibition. Even if no compounds are found to be active in a given screen, such lack of activity often provides useful SAR information.

Assays for Determining Biological Activity *Assays for evaluating the compounds of the present invention are well known in the art.

Although one usually does not know a priori in which specific assays a particular compound or group of library compounds will have activity, a useful system for screening libraries of the format of that S 25 described in the present invention, to identify activities with respect to a wide variety of enzymes and molecular targets, is disclosed in US. 08/553,056, filed November 3, 1995.

Methods of Synthesis S* 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 synthesised may be 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 S •or higher order encoding scheme permitting a relatively small number of identifiers to encode a 35 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.

Libc/03289 In carrying out the syntheses, one begins with at least 10 4 desirably at least 10 7 and generally not exceeding 1015 solid supports. Depending on the pre-determined number of 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 step 1 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 byproducts before proceeding. At the end of each step, the supports are 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, tagging, and remixing is repeated until the combinatorial synthesis is completed.

As an example of the synthesis via ring closure for the preparation of four compounds of Formula I, but excluding the tagging steps, resin-linked a-amino ester of the formula: o O OH 0- aQo N' Ra' 1 is suspended in an aprotic, polar solvent such methylene chloride, DMF, THF or ethyl acetate.

An excess of a soluble organic base such as triethylamine, N,N-diisopropylethylamine, or pyridine is *added to the suspended resin. This mixture is treated with an excess of an appropriately substituted 2-azidobenzoyl chloride of the formula: X2 N3 and agitated at room temperature to produce a resin linked N-(2-azidobenzoyl)amino ester of the formula: X2 0 N3

R

a The resin is filtered and washed and then suspended in an involatile solvent a non-protic, organic solvent with a boiling point of 80-140°C) such as toluene, xylene, or chlorobenzene and treated with an excess of a trivalent phosphorus reagent such as triphenylphosphine or Libc/03289 13 tributylphosphine. This mixture is agitated and heated to 80-140°C for 2-24h, then cooled to produce a resin-linked 1,4-benzodiazepin-5-one of the formula:

X,

X

2

-N

Ra which is washed and then is suspended in an acidic solution and agitated at room temperature for 1-24h. The resin is filtered and washed and the filtrate and washings are combined and H X2

N

0

N

R wherein Xi, R 2 and Ra are selected such that formula D represents compounds of the formulae: CH3 N O-CH H H 0 O o* N/ N N H 0 H N N N 0 B. 0 0

SOB

z 3 and 4 8 11 16 Compounds 4, 8, 11, and 16 have been synthesised by the aza-Wittig method of the present invention. Compound 4 is a known intermediate useful in the synthesis of antitumour antibiotics (Kaneko et al., Tet. Lett., 1983, p. 5165; Kaneko et al., J. Med. Chem., 1985, p. 388). Compound 8 is a novel compound useful as an intermediate in the synthesis of the antibiotic (Kamal et al, Bioorg. Med. Chem. Lett., 3, p. 743, 1993) and abbeymycin. Compound 11 is a novel compound useful as an intermediate in the synthesis of antitumour antibiotics (Hurley et al., Chem.

Res. Toxicol, 1, p. 258, 1988. Compound 16 is a natural product isolated from Penicilleum cyclopium with potential antibiotic properties (Framm et al., Eur. J. Biochem, 37, p. 78, 1973.) Scheme I Resin-linked ax-amino ester, A, is suspended in an aprotic, polar solvent such methylene jii> chloride, DMF, TEIF or ethyl acetate. An excess (2-50 equivalents) of a soluble organic base such as S '2 triethylamine, N,N-diisopropylethylamine, or pyridine is added to the suspended resin. Optionally, an S'/Libc/03289 14 acylation catalyst such as 4-dimethylaminopyridine may be added. This mixture is treated with an excess (2-10 equivalents) of an appropriately substituted 2-azidobenzoyl chloride and agitated at room temperature for 2-24h. The resin is filtered and washed multiple times with an appropriate solvent such as methylene chloride to remove excess reagents and byproducts. Resin linked N-(2azidobenzoyl)amino ester, B, is suspended in an involatile solvent such as toluene, xylene, or chlorobenzene and treated with an excess (2-10 equivalents) of a trivalent phosphorus reagent such as triphenylphosphine or tributylphosphine. This mixture is agitated and heated to 80-140oC for 2-24h, then cooled and the resin-linked 1,4-benzodiazepin-5-one, C, is washed multiple times with appropriate solvents such as methylene chloride or toluene to remove excess reagents and byproducts. Resin-linked 1,4-benzodiazepin-5-one, C, is suspended in an acidic solution such as trifluoroacetic acid/methylene chloride (50-90% TFA/CH 2

CI

2 or hydrogen chloride/dioxane (1-4M HCI/dioxane) and agitated at room temperature for 1-24h. The resin is filtered and washed with appropriate solvents such as methylene chloride or dioxane. The filtrate and washings are combined and evaporated to give the crude 1,4-benzodiazepin-2,5-dione, D, which may be purified and characterised by standard techniques.

Scheme 1

X

2 N Nz

H

C l O O X

PR

3 Ra B a s e Ra,2 e S* A B A B N

N

N 0 N -o o o o Ra"" H+ R 20 D

C

The invention is further defined by reference to the following examples, which are intended to be illustrative and not limiting.

O o

S

Preparation 1 Identifiers 25 Twelve compounds of the general formula: 0 0

*A

o/CH3 0 O wherein: n 3-12 and Ar is pentachlorophenyl or n 54-6 and Ar is 2,4,6-trichlorophenyl were prepared according to Scheme 6 and the following illustrative example.

Libc/03289 a) Methyl vanillate (0.729g, 4.0mmole), 1-hydroxy-9-(2,3,4,5,6-pentachlorophenoxy)nonane (1.634g, 4.0mmole) and triphenylphosphine (1258g, 4.8mmole) were dissolved in 20mL dry toluene under argon. DEAD (0.76mL, 0.836g, 4.8mmole) was added dropwise and the mixture was stirred at 250C for 1h. The solution was concentrated to half volume and purified by flash chromatography eluting with DMC to give 1.0g (1.7mmole, 43%) of the product as a white crystalline solid.

b) The methyl ester from Step (1.0g, 1.7mmole) was dissolved in 50mL THF, 2mL water was added, followed by LiOH (1.2g, 50mmole). The mixture was stirred at 250C for one hr, then refluxed for 5h. After cooling to 25°C, the mixture was poured onto ethyl acetate (200mL) and the solution was washed with 1M HCI (3x 50mL) then sat'd aq. NaCI (1x 50mL) and dried over sodium sulfate. The solvent was removed and the crude acid azeotroped once with toluene.

c) The crude material from Step was dissolved in 100mL toluene, 10mL (1.63g, 14mmole) thionyl chloride was added, and the mixture was refluxed for 90 min The volume of the solution was reduced to approx. 30mL by distillation, then the remaining toluene was removed by evaporation. The crude acid chloride was dissolved in 20mL dry DCM and cooled to -70oC under argon and a solution of approx 10mmole diazomethane in 50mL 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 ethyl acetate in hexane. The diazoketone (0.85g, 1.4mmole, 82% yield over three steps) was obtained as a pale yellow solid.

An improvement was made to the final diazomethylation step, whereby the acid chloride was reacted with (trimethylsilyl) diazomethane and triethylamine to give the identifier, which was then used without further purification. This was a significant improvement over the original reaction with diazomethane, as the identifier was now obtained in high yield with no chlorometylketone byproduct.

Also, purification by flash chromatography was no longer necessary, which in some cases had 25 resulted in significant acid-catalysed decomposition of the identifier.

Alternate Step c) To a solution of the acyl chloride (3.8mmol, 1.00eq.) and 1.85mL (13.3mmol, 3.50eq.) of triethylamine in anhydrous THF/acetonitrile at 0°C under argon was added 5.7mL (11.4mmol, 3.00eq.) of a 2.0M solution of (trimethylsilyl) diazomethane in hexane. The resulting orange solution was stirred at 0°C for 2h, then at 25°C for 17h. (If a precipitate formed immediately upon addition of (trimethylsilyl)diazomethane, CH 2

CI

2 was added until the precipitate redissolved).

EtOAc was added (250mL), and the organic layer washed with saturated aq. NaHCO 3 (100mL) and

H

2 0 (100mL), 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 35 and In the synthesis of Example 5, the 12 identifiers were used to encode the combinatorial library.

In Step 1, pentachlorophenyl identifiers where n 7-12 (abbreviated C7C15, C8C15, C12C05) were used in the following binary encoding scheme: 000001 (n 12), 000100 (n 11) though 100000 (n In Step 2, pentachlorophenyl identifiers where n 6-9 (abbreviated C6CI5, C7C5, C8CI5, and C 9 were used and encoded as follows: 000001 (n 000010 (n 000100 (n and 00100 Libc/03289 Also in Step 2, trichlorophenyl identifiers where n 4-6 (abbreviated C 4 1 3

C

5 01 3 and

C

6 C1 3 were used and encoded as follows: 01000 (n Step 3 was not encoded.

Thus, in Step 1 reagent 3 is encoded '011" which represents tagging this choice in the synthesis with the two pentachlorophenyl identifiers where n 11 and 12. Likewise, in Step 3 reagent 30 is encoded '0110" which represents tagging this choice in the synthesis with the pentachlorophenyl identifiers where n 3-6 and the trichlorophenyl identifier where n 6.

Scheme 6 Identifiers 0-Ar HC 0OH H3 -(4

H

3 C- -C H 3

HO-(CH

2 )n-O-Ar 3 Y aCH 3 0 PPh 3 DEAD, Toluene0 I1. LOH, THF/MeOH 2. S001 2 toluene reflux O-Ar 0-Ar O-g ~TMS -CHN 2 Et 3 N N2 0 0 C, THF/MeCN (1:1) ra O-CH 3

CH

2

N

2 DCM, Et 2 O M 0-OH 3 0 _0 Preparation I N-Fmoc-cis-4-benzoyloxy-L-proline-p-alkoxybenzyI Resin N-Fmoc-cis-4-benzoyloxy-L-proline p-alkoxybenzyl resin, was prepared by routine methods as 15 outlined below.

0 0.

0 H 0 Fmnoc Ho-" ON' BnOH, TsOH, C 6

H

6 reflux n- N 2. Fmoc-05u, acetonitrile 3H BzOH, DEAD, PPh 3 tolueneOz 1. 1 ,3-cyclohexadienr, Pd/C, toIuene 2. p-alkoxybenzyl alcohol resin, 0 DMAP, methylene chloride 0 Fmnoc OBz Libc/03289 17 Preparation 2 Bis-Linker Attachment TentaGel resin may be modified with bis-Fmoc lysine to increase the available reaction sites for ligand attachment. For purposes of simplicity, the schemes elsewhere herein do not show the use of this modification with lysine.

1) Preparation of 4-acetoxymethylphenoxy acetic acid: A solution of 4hydroxymethylphenoxyacetic acid (9.9g, 55mmol) in pyridine (200mL) was treated with acetic anhydride (22.15g, 218mmol, 4eq), and the reaction mixture was stirred 25°C under argon for 48h.

The reaction mixture was concentrated in vacuo to ~25mL, then diluted with 200mL of EtOAc, and placed in a separatory funnel. The resulting suspension was treated with ca. 50mL of 1N aq. HCI, and shaken. The pH of the aqueous layer was checked, and adjusted to pH2 with portionwise addition of conc. HCI and shaking. The layers were separated. The organic layer was washed with brine, then dried (MgSO4), and concentrated in vacuo to afford crude product as a reddish-brown oil. This material was purified by flash chromatography, eluting with ethyl acetate/hexanes (1 followed by ethyl acetate/hexanes to afford 7.0g of 4-acetoxymethylphenoxy acetic acid as an offwhite solid.

TLC: Rf=0.2, silica gel, 100% EtOAc (UV).

1 H-NMR: (CD30D) 2.0 3H), 4.6 2H), 5.0 2H), 6.80 2H), 7.20 2H).

2) Preparation of resin support A: A 300mL synthesis vessel was charged with Tentagel-S-NH2 resin (25g, 0.30mmol/g capacity, (7.5mmol)), and the beads were washed with 2 x 150mL methylene chloride. A solution of N-a-N-s-di-Fmoc-lysine (13.3g, 22.5mmol, 3eq) in 100mL of DMF/methylene chloride was added to the vessel. The resulting mixture was treated with 4-dimethylamino pyridine (92mg, 0.75mmol, 0.1eq), followed by N,N'diisopropylcarbodiimide (4.73g, 37.5mmol, and the reaction mixture was shaken at ambient temperature. After 6h, the solvent was removed by 25 filtration, and the beads were washed successively with 5 x 150mL DMF, and 5 x150 mL methylene Schloride. A small portion of the resin was checked by the Kaiser test for disappearance of free NH 2 and found to be negative.

The resin was treated with a 30% solution of piperidine in DMF (100mL), and shaken at for 1h. The resin was filtered and washed successively with 5 x 150mL DMF and 5 x 150mL methylene chloride A small portion of the resin was checked with the Kaiser test to assure removal of the Fmoc groups, and found to be positive.

A suspension of 4-acetoxymethylphenoxy acetic acid (20.2g, 90mmol, 6eq) in 60mL of methylene chloride was treated with DMF dropwise until all solid went into solution. This solution was "2 then added to resin above (nom. 15mmol) in a 300mL synthesis vessel. The resulting suspension was i 35 treated with 4-dimethylaminopyridine (366mg, 3.0mmol, 0.2eq), followed by N,N'diisopropylcarbodiimide (18.9g, 150mmol, 10eq), and the reaction mixture was shaken at 25°C for 16h. The solvent was removed by filtration, and the resin was washed successively with 5 x 150mL DMF and 5 x 150mL CH 2 C1 2 to afford the acetate protected resin. A small aliquot of the resin was checked for disappearance of free NH2 with the Kaiser test, and found to be negative.

Libc/03289 18 The acetate protected resin from above was treated with a solution of 10% hydrazine hydrate in methanol (lO0mL-), and shaken at ambient temperature. After 6h., the solvent was removed by filtration, and the resin was washed successively with 5 x l5OmL- MeOH and then re-treated with hydrazine hydrate in MeCH (lO0mL-) and shaken at 25 0 C for 16h. The solvent was removed by filtration, and the resin was washed successively with 5 x l5OmL- DMF and 5 x l5OmL- 0H 2

CI

2 to afford the hydroxy resin A.

Bis-Linker Atachment 0 H I N 0

MA

H 0 1. N-a-N-c-di-Fmoc-L-lysine R I DIG, cat. DMAP H N N MA 2. 30% piperidine/DMF Ni 3. 4-acetoxymethylphenoxyacetic acid 0 H A R NH- 2 DIC, cat. DMAP hydrazi ne hydrate/methanoI~ 0

N

0H0

.A

Libc/03289 Example 1 (11 aS)-1,2,3,10,11,11 a-Hexahydro-5H-pyrrolo[2,1 -c](1,4)-benzodiazepine-5,11 -dione 1. piperidine/DMF

N

0 Fmoc 2. 0 O 01o-^° ,Nlr' Cl -N- 1 2

PPH

3 toluene, 6 O 50% TFA/CH 2 Cl2 O 4 3 N-Fmoc-L-proline p-alkoxybenzyl resin, 1, (Bachem) (0.34mmole/g, 2.0g, 0.68mmol) was suspended in 30mL DMF, then filtered. The resin was then shaken with 50% piperidine/DMF for 2h then filtered and washed with DMF (5 x 30mL) and methylene chloride (10 x 30mL). The resin was suspended in 20mL methylene chloride, and 2mL (14mmol) triethylamine was added, followed by 8eq) 2-azidobenzoyl chloride. The mixture was shaken at 25 0 C for 3h and then the resin was washed with methylene chloride (10 x 30mL) followed by toluene (10 x 30mL). The resin, 2, was S suspended in 20mL toluene and 0.3g (1.14mmol, 1.7eq) triphenylphosphine was added and the 15 mixture was shaken until the triphenylphosphine had dissolved. The mixture was shaken and heated to 80°C for 3h then cooled and washed with methylene chloride (10 x 30mL). The pale brown resin, 3, was dried under vacuum.

The resin, 3 (2.0g, 0.68mmol), prepared as described above, was suspended in methylene chloride and 20mL trifluoroacetic acid was added. The resin was shaken at 25 0 C for 20 min then filtered and washed with methylene chloride (2 x 20mL). The filtrate and washings were collected and combined, then evaporated to give the crude product. The benzodiazepine was purified by flash chromatography, eluting with 60% ethyl acetate/hexane to give the product, 4, as a white solid.

1 H NMR (300MHz, COC13): 6 2.03 3H), 2.77 1H), 3.62 3.80 1H), 4.08 (m, 25 1H), 7.07 1H), 7.26 1H), 7.48 8.00 1H, J 7.7 Hz), 9.07 (br s, 1H, ND; 13C NMR (125MHz, CDCI3): 8 23.45, 26.12, 47.26, 56.66, 121.81, 124.90, 127.03, 131.02, 132.34,135.44, 165.41,171.40; CIMS: 217 (MH+) Libc/03289 Example 2 (2S,11 aS)-2-Benzoyloxy-1,2,3,10,11,11 a-hexahydro-5H-pyrrolo-[2.1 -c](1,4)-benzodiazepine-5,1 1dione

N

3 0 Fmoc 1. piperidine/DMF 0 j O O N 2. a N OBz N 3 OBz 5 6

PPH

3 toluene, 0 0 H-N O N O O N OOQ N OBz 50% TFA/CH 2

CI

2 OBz 8 7 N-Fmoc-cis-4-benzoyloxy-L-proline p-alkoxybenzyl resin, 5, (400mg) was suspended in DMF, then filtered. The resin was then shaken with 50% piperidine/DMF, 10mL, for 2h then filtered and washed with DMF (5 x 10mL) and methylene chloride (10 x 10mL). The resin was suspended in methylene chloride, 0.4mL (~3mmol) triethylamine was added, followed by 0.2g (1.1mmol) 2azido-benzoyl chloride. The mixture was shaken at 25 0 C for 12h and then the resin was washed with methylene chloride (10 x 10mL), followed by toluene (10 x 10mL). The resin, 6, was suspended in 15 50mL toluene and 0.20g (0.8mmol) triphenylphosphine was added and the mixture was shaken until the triphenylphosphine had dissolved. The mixture was shaken and heated to 80 0 C for 2h then cooled and washed with toluene (5 x 10mL) and methylene chloride (10 x 10mL). The resin, 7, was dried under vacuum.

The resin, 7, prepared as described above, was suspended in 5mL methylene chloride and 20 5mL trifluoroacetic acid was added. The resin was shaken at 25 0 C for 30 min then filtered and washed with methylene chloride (2 x 10mL). The filtrate and washings were collected and combined, then evaporated to give the crude product which was purified by flash chromatography eluting with 50% ethyl acetate/hexane to give the product, 8, as a white solid.

1 H NMR (300MHz, CDC13): 6 2.41 1H), 3.31 1H, J 14.4Hz),3.94(m, 25 1H),4.16(m,2H),5.58(m, 1H),6.84(d, 1H,J=8.1Hz), 7.07 2H), 7.20-7.37 3H), 6.47 1H), 7.87 1H, J 8.1Hz), 8.10 1H, J 7.71Hz), 9.94 (brs, 1H, NH); 13C NMR (125MHz, CDC13): 8 31.64, 53.18, 56.06, 71.93, 120.88, 124.67, 125.75, 127.87, 129.33, 129.50, 131.06, 132.73, 133.25, 135.61,165.86, 165.95, 171.93; Libc/03289 CIMS: 337 (MH+) Example 3 (11 aS)-7,8-dimethoxy-1,2,3,1 0,11,11 a-hexahydro-5H-pyrrolo[2,1-c](1,4)-benzodiazepine-5,11 -dione MeO O moc 1. piperidine/DMF N 3

M

MeO c

O

1 9

PPH

3 toluene, MeO MeO MeO OMe MeO OMe H-N o

N

O N [O O/ t50% TFA/CH 2 ClI 2 11 N-Fmoc-L-proline p-alkoxybenzyl resin, 1, (0.34mmoleg, 5.0g, 1.8mmol) was suspended in 50mL: DMF, then filtered. The resin was then shaken with 50% piperidine/DMF for 2h then filtered and washed with DMF (5 x 50mL) and methylene chloride (10 x 50mL). The resin was suspended in methylene chloride, 3mL (21mmol) triethylamine was added, followed by 0.75g (3mmol, 1.7eq) 2o. azido-4,5-dimethoxybenzoyl chloride as a solid. The mixture was shaken at 25°C for 12h and then the resin was washed with methylene chloride (10 x 50mL), followed by toluene (10 x 50mL). The resin, 9, 15 was suspended in 50mL toluene and 1.26g (4.8mmol 2.6eq) triphenylphosphine was added and the mixture was shaken until the triphenylphosphine had dissolved. The mixture was shaken and heated to 80°C for 2h then cooled and washed with toluene (10 x 50mL), alternately with methanol and methylene chloride (5 x 50mL each) and finally methylene chloride (5 x 50mL). The pale brown resin, 10, was dried under vacuum.

20 A 1.0g (-0.34mmol) portion of the resin, 10, prepared as described above, was suspended in methylene chloride and 20mL trifluoroacetic acid was added. The resin was shaken at 5°C for 30 min then filtered and washed with methylene chloride (2 x 20mL). The filtrate and washings were collected and combined, then evaporated to give the crude product. The benzodiazepine was purified by flash chromatography eluting with ethyl acetate to give the product, 11, as an off white solid.

25 1 H NMR (300MHz, CDCI 3 6 2.03 3H), 2.75 1H). 3.61 1H), 3.78 1H), 3.91 (s, 3H), 3.93 3H), 4.04 1H, J 6Hz), 6.47 1H), 7.46 1H), 8.30 (br s, 1H, NH); 13C NMR (125MHz, CDCI 3 8 23.49, 26.11, 47.26, 56.05, 56.11, 56.80, 103.85, 112.01, 119.16, 129.79, 146.27, 152.20, 165.97, 171.13; Libc/03289 CIMS: 277 (MHW) Cyclopeptin Example 4 IFmnoc OH I O-Cr-- DIG, DMAP, CH 2

CI

2 12 pipeddine/DMF 0 NEt 3

CH

2

C

2

PPH

3 toluene,

TFA/CH

2 01 2

S

*5

S

S

S.

S

S

*55*

S

*5S* p-Alkoxybenzyl alcohol resin, 12, 2.5g (1.Ommol/g) was suspended in 30mL methylene chloride. N-Fmoc-N-methyl-Lphenylalanine, 3.0g (7.5mmol, 3eq), DMAP, 1.0g (0.82mmol, 0.3eq), and DIC I .l4mL (1 .26g, I Ommol, 4eq) were added and the mixture was agitated for 2h The resin was 15 filtered and washed with methylene chloride, (10 x 30mL), then dried.

The N-Fmoc-N-methyl-L-phenylalanine p-alkoxybenzyl ester resin, 13, 1.0g was suspended in piperidine/DMF, 30mL, and agitated for 2h, then filtered and washed with DMF (5 x 30mL-) and methylene chloride (10 x 3OmL). The resin was suspended in 2OmL methylene chloride; 1.OmL (-0 7mmol) triethylamine was added followed by 0.6g (3.3mmol) 2-azido-benzoyl chloride. The mixture was shaken at 25 0 C for 1h and then the resin was washed with methylene chloride (10 x 3OmL) followed by toluene (10 x 3OmL). The resin, 14, was suspended in 2OmL- toluene and 0.52g Libc/03289 23 (2.Ommol) triphenylphosphine was added and the mixture was shaken until the triphenyiphosphine had dissolved. The mixture was shaken and heated to 90 0 C for 3h then cooled and washed with toluene (5 x 2OmQ) and methylene chloride (10 x 2OmL-) to give the resin linked 1 one, The resin, 15, was suspended in l0mL- methylene chloride and l0mL trifluoroacetic acid was added. The resin was shaken at 25'C for 2h, then filtered and washed with methylene chloride (2 x 2OmL). The filtrate and washings were collected and combined, then evaporated to give the crude product which was purified by flash chromatography eluting with 50% ethyl acetate/hexane to give the product, 16.

1 H NMR (300MHz, DMSO-d6, 100 0 OC): 6 2.91-3.06 (in, 2H), 2.95 3H), 4.30 1 H, J 7.9Hz), 7.13-7.28 (in, 7H), 7.48 (in, 1 7.80 1 H, J 7.7Hz), 10.22 (br s, 1 H, NH); CIMS: 281 (MH+) ExampIl Synthesis of 1,4-Benzodiazepin-2,5-dione Library a-amino acids (Reagent Set 1) RI R 3 HO"X NFmc R, R 3 E.moc 0 H DIG, cat. OMVAP, CH 2 CLJDMF 0 H

B

OH

1. 30% piperidine R R 1

R

2. Aldehydes (Reagent Set 2) kR 0 H>-2 N X 3' R 2 NaBH 3 CN, I%HOAc/DMF 0 H

C

2-azidobenzoyl chloride (Reagent Set 3)

CI

N0 X, X N-iPr 2 Et, CH 2

CI

2

S

S

S

*5

S.

S

X{N 7X 2 30% TFA/CH 2

CI

2

F

a-amino acids (Reagent Set 1)

R

3 R R 2

IHOX

kOH DIG, cat. DMAP, CH 2

CL

2

IDMF

_PBU

3 xylene, heat 2-azidobenzoyl chloride (Reagent Set 3)

CI

Ne

X-~

2 t 2 tLbc/03289 PBu 3 xylene, heat

R

3 R 2

R

3

RIR

2 N No X X 2 30% TFA/CH 2

CI

2 X X2 E D A 1.2g (0.53mmol/g, 0.64mmol OH) portion of p-alkoxybenzyl resin A, was placed in each of 46 separate 100mL synthesis vessels (vessels 1-46). The resin in each vessel was suspended in methylene chloride, agitated for 5 min and then filtered. The solvated resin was resuspended in methylene chloride. Solutions of 46 protected a-amino acids (Reagent Set 1, 4.3mmole, ~7eq), prepared in 20mL 1:1 methylene chloride:DMF, were added to the vessels, one solution per vessel, containing the suspended resin. The vessels were agitated for 5 min, then 2mL of 45mg/mL DMAP (90mg, 0.73mmol, 1.2eq) solution in methylene chloride was added to each vessel and the mixtures were agitated for 5 min. DIC, 1.lmL (91mg, 7.2mmol, 11eq), was added to each vessel and the mixtures were agitated for 14h. Then the resin batches, B, were filtered and washed with 5 x methylene chloride.

The 46 resin batches were encoded with six tags as follows.

The resin batches were suspended in 30mL methylene chloride. Aliquots, 2mL of tag precursor/vessel by mass of resin), C 1 2C1 5 tag precursor solution in methylene chloride were added to the appropriate vessels and the vessels were shaken for 2 min. Aliquots, 2mL of 45mg/mL (90mg tag precursor/vessel -7.5 by mass of resin), of CllCI5 tag precursor solution in methylene chloride were added to the appropriate vessels and the vessels were shaken for 2h.

20 Rhoiium (II) trifluoroacetate dimer, 2mL of 1mg/mL in methylene chloride, was added to each vessel in Sturn with ~30s agitation of the vessels after each addition. The resin batches, tag precursor and catalyst were agitated for 12h, then filtered and washed with 5 x 40mL methylene chloride.

(ii) The procedure from was repeated for tags CloC5 and C9C15.

(iii) The procedure from was repeated for CsCIs and C 7 C1 5 25 The resin batches, encoded 000001-101110, were combined in a separatory funnel and washed with 5 x 300mL methylene chloride. The resin was then filtered and dried for 12h in vacuo.

The resin above was divided into two equal batches of 1.75g which were placed into two S* separate 100mL synthesis vessels (vessels 1 and 2) and sixty equal batches of 0.85g which were placed into sixty separate 100mL synthesis vessels (vessels 3-62).

The 62 resin batches were encoded with tags as follows.

(iv) The resin batches were suspended in 30mL methylene chloride. Aliquots, 2mL of 37mg/mL (74mg tag precursor/vessel by mass of resin), of C6CI5 tag precursor solution in methylene chloride were added to the appropriate vessels from 3 to 62 and the vessels were shaken for 2 min. A 3mL aliquot of 37mg/mL C6CI5 tag precursor solution was added to vessel 1 (111mg tag precursor in Libc/03289 vessel 1, 6.3% by mass of resin). Aliquots, 2mL of 37mg/mL (74mg tag precursor/vessel by mass of resin), of CC5ls tag precursor solution in methylene chloride were added to the appropriate vessels from 3 to 62 and the vessels were shaken for 2 min. A 3mL aliquot of 37mg/mL CsCI5 tag precursor solution was added to vessel 2 (111mg tag precursor in vessel 2, 6.3% by mass of resin).

Rhodium (II) trifluoroacetate dimer, 2mL of 1mg/mL in methylene chloride, was added to each vessel in turn with -30 sec agitation of the vessels after each addition. The resin batches, tag precursor and catalyst were agitated for 12h, then filtered and washed with 5 x 40mL methylene chloride.

The procedure from (iv) was repeated for tags C 4 C0 5 and (vi) The procedure from (iv) was repeated for C6C13 and C5Cl3 with the modification that 4mL aliquots 37mg/mL C6C13 and C5013 tag precursor solutions were used and also a 4mL aliquot of 37mg/mL C 4 01 3 tag precursor solution was added to vessel 32.

The resin batches, encoded 000001-111110, were suspended in 30mL DMF, agitated for 5 min and then filtered. A solution of 30% piperidine in DMF was added to each vessel, the mixtures were agitated for 30 min and then filtered. The resin batches were washed with 2 x 30mL DMF, then 2 x 30mL 1% acetic acid in DMF and then filtered. The resin batches were resuspended in 20mL 1% acetic acid in DMF, and 2.4mmol (-5eq) of the appropriate aldehydes (Reagent Set was added, as a solution in 10mL 1% HOAc/DMF, to each of the vessels 3-62. The mixtures were agitated for 2h then 5mL of 1M sodium cyanoborohydride (5mmol, 10eq) in THF was added to each of the vessels.

The quantity of aldehyde, and volume of sodium cyanoborohydride solution was doubled for vessels 1 and 2. The mixtures were shaken for a further 90 min, then filtered and washed with 2 x 30mL DMF and 5 x 30mL methylene chloride to give resin batches C.

A collection of 51 319 1,4-benzodiazepine-2,5-diones was prepared as follows. Resin batches 3C-62C were combined and mixed thoroughly in a separatory funnel, then divided as a slurry in methylene chloride into nineteen equal portions 1.4mmol) in 100mL synthesis vessels. The 25 resin was suspended in 30mL methylene chloride and diisopropylethylamine, 2.5mL (1.8g, 14mmol, 10eq), was added followed by 5mmol of the appropriate o-azidobenzoyl chloride (Reagent Set The mixtures were shaken at room temperature for 16h and then filtered and washed with 5 x methylene chloride and 2 x 30mL xylene to give resin batches D. Each resin batch was transferred as a slurry into separate 50mL flasks and the suspensions were sparged with argon for 5 min, then 30 sealed with a septum. Tributylphosphine, 1.8mL (1.45g, 7.2mmol, 5eq), was added to each flask and the mixtures were heated to 140-150°C for 6h, then cooled, filtered, and washed with 2 x toluene and 5 x 30mL methylene chloride to give the resin linked benzodiazepines, E.

The product 1,4-benzodiazepine-2,5-diones, F, were cleaved from the resin support by suspending the resin, 20 beads, in 100pL of 70 TFA/water for 4h and filtering the solution.

A second collection of 1170 1,4-benzodiazepine-2,5-diones was prepared as follows. Resin batches 1C and 2C were combined and mixed thoroughly in a separatory funnel. The resin was dried in vacuo and divided into thirteen equal portions (-0.27g, 0.14mmol) then placed in 20mL synthesis vessels. The resin was suspended in 10mL methylene chloride and diisopropylethylamine, 0.25mL (0.18g, 1.4mmol, 10eq), was added followed by 0.5mmol of thirteen o-azidobenzoyl chlorides. The mixtures were shaken at room temperature for 16h and then filtered and washed with 5 x Libc/03289 26 methylene chloride and 2 x 10mL xylene to give resin batches D. Each resin batch was transferred as a slurry into separate 25mL flasks and the suspensions were sparged with argon for 5 min, then sealed with a septum. Tributylphosphine, 0.14mL (0.11g, 0.55mmol, 4eq), was added to each flask and the mixtures were heated to 140-150oC for 6h, then cooled, filtered, and washed with 2 x toluene and 5 x 10mL methylene chloride to give the resin batches E, from which the product 1,4- F, may be cleaved as described above.

Example 6 Decoding Procedure A bead is placed in a 1.3mm diameter pyrex capillary with 2pL of acetonitrile. Ceric ammonium nitrate solution (2iL of a 0.1M aq. solution) and hexane (3tL) are added and the two-phase mixture centrifuged briefly. The tube is sealed and left at 35°C for 16h, then opened. The organic layer is removed by syringe and mixed with 1IL of N,O-bis(trimethylsilyl)acetamide. The silated tag solution (1 L) is analysed by GC with electron capture (EC) detection.

The GC analysis is performed with a Hewlett Packard 5890 plus gas chromatograph. On column injection into a 5m, 0.32mm retention gap connected to a 25m, 0.2mm crosslinked phenylmethyl silicone column is used. The temperature and pressure programs for the analysis are 200-3200C, 15°C/min, then 320°C for 10 min and 20-40psi at 2psi/min, then 40psi for 10 min. The EC detector is maintained at 400°C and the auxiliary gas is set at 35 psi.

The identity of the library compound attached to the bead is ascertained based on the reagents utilised in the synthesis of such compound, which are readily determined from the binary codes associated, respectively, with each of the identifiers for such reagents, as characterised through the above procedure.

S.

S

Libc/03289 27 The claims defining the invention are as follows: 1. A process of synthesising a 1,4-benzodiazepin-2,5-dione which comprises: a. attaching an amino-protected a-amino acid or an amino protected N-alkyl a-amino acid to a solid support via its carboxyl group to form a resin linked amino-protected a-amino acid or aminoprotected N-alkyl a-amino acid; b. cleaving an amino protecting group from said resin linked amino-protected a-amino acid or amino-protected N-alkyl a-amino acid to provide a resin linked a-amino acid or N-alkyl a-amino acid; c. acylating said resin linked a-amino acid or N-alkyl a-amino acid with a 2-azidobenzoyl chloride to form a resin linked N-(2 azidobenzoyl)amino acid; and d. cyclising said resin linked N-(2-azidobenzoyl)amino acid via aza-Wittig ring closure to provide a resin linked 1,4-benzodiazepin-2,5 dione.

2. A process according to claim 1 additionally comprising the step of cleaving said resin linked 1,4-benzodiazepin-2,5-dione from said resin to provide a 1,4-benzodiazepin-2,5-dione.

3. A process of synthesising a 1,4-benzodiazepin-2,5-dione which comprises: a. attaching an amino-protected a-amino acid to a solid support via its carboxyl group to form a resin linked amino-protected a-amino acid; S: b. cleaving an amino protecting group from said resin linked amino-protected a-amino acid to provide a resin linked a-amino acid; c. reductively alkylating said linked a-amino acid with an aldehyde and a reducing agent to 20 provide a resin linked N-alkyl a-amino acid; d. acylating said resin linked N-alkyl a-amino acid with a 2-azidobenzoyl chloride to form a resin linked N-(2-azidobenzoyl)amino acid; and e. cyclising said resin linked N-(2-azidobenzoyl)amino acid via aza-Wittig ring closure to provide a resin linked 1,4-benzodiazepin-2,5-dione.

25 4. A process according to claim 3 additionally comprising the step of cleaving said resin linked 1,4-benzodiazepin-2,5-dione from said resin to provide a 1,4-benzodiazepin-2,5-dione.

A process according to claim 1 which comprises: reacting a suitably protected a-amino acid of the formula:

R

1

R

3 N HO Fmoc O H in the presence of DMF and DMAP with a solid support suspended in methylene chloride to form a resin linked N-protected amino acid of the formula:

RR

O Fmoc 0 H cleaving the Fmoc from said resin linked N-protected amino acid to provide a resin linked amino acid; ,7 [N:\LIBFF]0373:SSD y

Claims (7)

1. 6. A process according to claim 5 additionally comprising the step of: suspending the resin linked benzodiazepine of step in TFA/water at room temperature for 1- 24 hours to form a 1,4-benzodiazepin-2,5-dione of formula: R, R 3 HN 0
2. 7. A process according to claim 5 wherein the involatile solvent is toluene, xylene, or chlorobenzene and the trivalent phosphorus reagent is triphenylphosphine or tributylphosphine.
3. 8. A process according to claim 1 which comprises: reacting a suitably protected a-amino acid of the formula: HO R 1 R 3 HO Fmoc N S R 2 in the presence of DMF and DMAP with a solid support suspended in methylene chloride to form a 15 resin linked amino acid of the formula: 000R, R 3 0 L- Fmoc S0R 2 r09 o cleaving the Fmoc from said resin linked N-protected amino acid to provide a resin linked amino acid; reacting the resin linked N-alkyl-a-amino acid of step in methylene chloride and diisopropylethylamine with a 2-azidobenzoyl chloride of formula: Cl N 3 0 to form a resin linked N-(2-azidobenzoyl)amino ester of formula: A >[N:\LIBFF]0373:SSD treating the resin linked N-(2-azidobenzoyl)amino ester of step suspended in an involatile solvent, with an excess of a trivalent phosphorus reagent at 80-150°C and then cooling said mixture to room temperature to form a resin linked benzodiazepine of formula: R N R 2 N O xleX wherein: R 1 is H, lower alkyl, c-lower alkyl, or (CH2)mR4, or R 1 and R 2 together with the atoms to which they are attached, join to form a or 6-membered heterocyclic ring, optionally monosubstituted with OH, alkoxy, or arylalkoxy; R 2 is H, loweralkyl, arylR 6 R 7 R 8 or heteroarylR6R7R8, or R 1 and R 2 together with the atoms to which they are attached, join to form a 5- or 6-membered heterocyclic ring, optionally monosubstituted with OH, alkoxy, or arylalkoxy; R 3 is H or loweralkyl; R4 is aryl, substituted aryl, heteroaryl, substituted heteroaryl, NR 3 R 5 CO 2 R 3 CONR 3 R 3 or OH; R 5 is H, lower alkyl, -C(=NR 3 )NHR 3 or -C(O)R 3 R 6 R 7 and R8 is each, independently, H, lower alkyl, lower alkoxy, halogen, aryl, lower alkylthio, X-aryl, X-substituted aryl, lower alkylaryl, C(hal)3, -(CH 2 )mNR 3 R 5 or -X-CH(C0 2 R3) 2 or Re and R 7 together with the atoms to which they are attached, join to form a 15 or 6-membered heterocyclic ring; X is O or S; Xi and X 2 are independently chosen from hydrogen, loweralkyl, loweralkoxy, loweralkylthio, hydroxy, cyano, nitro, phenoxy, benzyloxy, halo, aryl, NH(C=O)R 3 and carboxy, or Xi and X 2 taken together represent a fused benzene ring substituted with hydrogen, loweralkyl, loweralkoxy, loweralkylthio, hydroxy, cyano, nitro, phenoxy, benzyloxy, halo, aryl, -NH(C=O)R 3 or carboxy, and is a derivatised solid support. 20 9. A process according to claim 8 additionally comprising the step of: suspending the resin linked benzodiazepine of step in TFA/water at room temperature for 1- 24 hours to form a 1,4-benzodiazepin-2,5-dione of formula: R, HNx H-A Libc103289 process of claim 8 wherein the involatile solvent is toluene, xylene, or chlorobenzene and the trivalent phosphorus reagent is triphenylphosphine or tributylphosphine. 11 .A process of synthesising, 1,4-benzodiazepin-2,5-diones of the formulae: O-Bz I I CH 3 CH 3 which comprises: a) reacting a resin-linked a-amino ester of the formula: wherein is a solid support and Rli is hydrogen or benzyloxy, suspended in an aprotic, polar solvent and an excess of a soluble organic base with an excess of a substituted 2-azidobenzoyl chloride of the formula: 4 4. 4 4 4 4* 4 4 4. 4444 4* 4. 4. 44 4 4444 4444 wherein Xi and X 2 are hydrogen or methoxy, to produce a resin-linked N-(2-azidobenzoyl)amino ester; b) suspending said resin-linked ester in an involatile solvent and treating said suspension with an excess of a trivalent phosphorus reagent at 80-140 0 C for 2-24h to produce a resin-linked 1,4- benzodiazepin-5-one of the formula: *444 4 4 and c) suspending said resin-linked 1,4-benzodiazepin-5-one in an acidic solution at room temperature for 1-24h to produce a 1,4-benzodiazepin-2,5-dione of the formula: Libc/03289 or
4. 12.A process of synthesising a 1,4-benzodiazepin-2,5-dione of the formula: 9 .2:22: S *o f *o *o go o go g which comprises: 5 a) reacting a resin-linked a-amino ester of the formula: s CH 3 wherein is a solid support, suspended in an aprotic, polar solvent and an excess of a soluble organic base with an excess of a substituted 2-azidobenzoyl chloride of the formula: Cl wherein X 1 and X 2 are hydrogen, to produce a resin-linked N-(2-azidobenzoyl)amino ester; b) suspending said resin-linked ester in an involatile solvent and treating said suspension with an excess of a trivalent phosphorus reagent at 80-140°C for 2-24h to produce a resin-linked 1,4- of the formula: [N:\LIBFF0]373:kml _11and c) suspending said resin-linked 1 ,4-benzodiazepin-5-one in an acidic solution at room temperature for 1 -24h to produce a 1 ,4-benzodiazepin-2.5-dione of the formula:
5. 13.A process of synthesising a 1,4-ben zod iazepin-2,5-d ion e derivative, substantially as hereinbefore described with reference to any one of the examples. 14,A 1 ,4-benzodiazepin-2,5-dione derivative, synthesised by the process of any one of claims 1 to 1 5.A compound of the formula: S S. 9 .9 .9 9 9 9 9. S 9 S. *9 9 9 9 5 V. 999 S 9 p. 99 9. 9 S 9 5995 9*95 1 6.A compound of the formula: ibc/03289 wherein G)is a solid support; R 1 is hydrogen or benzyloxy; and X, and X 2 are hydrogen or methoxy.
6. 17. A process of synthesising a 1 ,4-benzod iazepin-2,5-d ion e, which process is substantially as herein described with reference to Example
7. 18. A 1 ,4-benzodiazepin-2,5-dione prepared by the process of any one of claims 1-13 or 17. Dated 19 March 1999 PHARMACOPEIA, INC. Patent Attorneys for the Applicant/Nominated Person SPRUSON&FERGUSON [N:\LIBFF10373:SSD Combinatorial 1,4-benzodiazepin-2,5-dione Library Abstract A method has now been found of synthesising a combinatorial library of I ,4-benzodiazepin-2,5- diones on solid supports via an aza-Wittig ring closure, said compounds optionally encoded with tags, and to the use of this library in assays to discover biologically active compounds, and optionally to cleave 1 ,4-benzodiazepin-2,5-diones therefrom. 0 s 0e 0@ *to. 6s -tes: 6 Libc03289