CA2225914A1 - Combinatorial 1,4-benzodiazepin-2,5-dione library - Google Patents

Abstract

A method has now been found of synthesizing a combinatorial library of 1,4-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.

Description

W O 97/01560 PCTrUS96/11070 TITLE OF THE INVENTION
COMBINATORIAL 1,4-BENZODIAZEPIN-2,5-DIONE LIBRARY
CROSS REFERENCE
This application claims the benefit of provisional S application Ser. No. 60/000,657, June 29, 1995.
Lawn Assay for Compounds That Affect Enzyme Activity or Bind to Target Molecules, U.S. Ser. No. 08/553,056, filed November 3, 1995, is incorporated herein by reference.
All patents and other ler~ ces cited herein are hereby incorporated by reference.
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 seq~lçnti~lly as part of the chemical synthesis to produce all or a subs~nti~l number of the possible compounds which can result from all the dirre~ t choices possible at each seqllenti~l stage of the synthesis. See e.g., Still et al., PCT Appli.
WO 94/08051. For techniques such as these to be successful, numerous solid state chemical reactions must be developed.
Fllm~n et aL, ("Progress Toward the Synthesis of a Library of 1,4 Benzodiazepin-2,5-diones" ACS National Meeting, Anaheim, CA, April 2-6, 1995, Abstr. ORGN 264) have reported a solid-phase synthesis of 1,4 benzodiazepin-2,5-diones. The F11m~n et aL method limits the diversity of the benzodiazepin-2,5-dione scaffold because attachment of the scaffold to the solid support during synthesis is through the ben7çne ring, a re~ 1-m rem~inin~ on said ring after detachment of the benzodiazepin-2,~-dione from the solid support.
Solution-phase synthesis of 1,4 benzodiazepin-5-ones via intramolecular aza-Wittig reaction has been disclosed by Egushi et aL (SYNLEl'r, 295-6, April 1992.
It is also 11e~ hle for compounds produced by combinatorial syn~hPsis to be ~ n~kle to methods by which one can determine the structure of the compounds so made. Brenner and CA 022259l4 l997-l2-29 W O 97/01560 PCTrUS96/11070 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 5 particles-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, 90, 10922-10926, Dec.
1993.

The present invention relates to a method of synthesizing a combinatorial library of 1,~benzodiazepin-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 15 active compounds, and, optionally, to cleave 1,4-benzodiazepin-2,5-diones therefrom.
DETAILED DESCRIPTION OF THE INVENTION
The combinatorial chemical library which may be synthesized by the method of the present invention is represented by 20 Formula I:
(T'-L)q- ~_J -C(O)-L'-II' I
wherein:
~ is a solid support;
T'-L- is an identifier residue;
-L'-II' is a linker/ligand residue;
q is 0-30; and II' is , O~ N
N~
x~l~ X2 W O 97/01560 PCT~US96/11070 wherein:
R1 is H, lower alkyl, c-lower alkyl, -or (CH2)mR4, or Rl and R2, together with the atoms to which they are attached, join to form a 5-, or 6-membered heterocyclic ring, optionally S monosubstituted with OH, alkoxy, or arylalkoxy;
R2 is H, loweralkyl, arylR6R7R8, or heteroarylR6R7R8, or Rl and ~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, R3 is H or loweraLkyl;
R4 is aryl, substituted aryl, heteroaryl, substituted heteroaryl, NR3R5, Co2R3, CoNR3R3, or OH;
R5 is H, lower alkyl, -CNHR3R3, or -C(o)R3;
R6, R7, and R8 is each, independently, H, lower alkyl, lower alkoxy, halogen, aryl, lower alkylthio, X-aryl, X-substituted aryl, lower alkylaryl, C(hal)3,-(CH2)mNR3RS, or -X-CH(Co2R3)2~ or R6 and R7, together with the atoms to which they are attached, join to form a 5- or ~membered heterocyclic ring; and X is O or S.
Preferred libraries of Formula I are those wherein T'-L- is of the Formula ~ O--(CH2)n l \ III
-C~2~ OCH3 wherein:
n = 3-12;
Ar is halophenyl; and q is 3-12.
More-preferred libraries of Formula I are those wherein in Folmula III: 1) n = 3-12 and Ar is pentachlorophenyl; or 2) n = 5-6 30 and Ar is 2,4,6-trichlorophenyl.
Depen~lin~ on the choice of L' (see Table 1), the li~n~l~ of Formula II may be detached by photolytic, oxidative, acidic, basic, or CA 022259l4 l997-l2-29 W O 97/01560 PCTrUS96/11070 other cleavage techniques. For example, when -L'- is (a), acidic cleavage may be represented by:
(T '-L)q~C~O)-L'I + II'OH
wherein L" is the residue from L' and II'OH is II, in its tautomeric 5 amide form:

~ 3 ~0 HN~
x2 xl wherein the symbols are as defined above for formula II'.
An embodiment of the invention is the solid phase synthesis of 1,4-benzodiazepin-2,5-diones via aza-Wittig ring closure. The 10 process comprises:
a) attaching a set of suitably protected a-aminoacids or N-alkyl-a-aminoacids to solid supports to form resin linked N-alkyl-a-aminoacids; or b) attaching a set of suitably protected N-unsubstitued-a-15 aminoacids to solid supports to form resin linked N-unsubstitued-a-aminoacids and reductively alkylating said resin linked aminoacids with a set of aldehydes to form resin linked N-arylalkyl or heteroarylalkyl-a-aminoacids;
c) acylating the resin linked N-alkyl-a-aminoacids or the 20 N-arylalkyl or heteroarylalkyl-a-aminoacids of steps (a) or (b) with a set of 2-azidobenzoyl chlorides to form resin linked N-(2-azidobenzoyl)amino esters;
d) cyclizing the resin linked N-(2-azidobenzoyl)amino esters of step (c) via aza-Wittig ring closure to form resin linked 25 benzodiazepines; and, optionally, e) cleaving the resin linked benzodiazepines of step (d) to form 1,4-benzodiazepin-2,5-diones.
A preferred embodiment of the invention is the solid phase synthesis of 1,4-benzodiazepin-2,5-diones via aza-Wittig ring closure, 30 wherein the process comprises:

CA 022259l4 l997-l2-29 PCTrUS96/11070 a) reacting a set of suitably protected a-aminoacids of the formula:

O
in the presence of DMF and DMAP with solid supports suspended in S methylene chloride to form resin linked aminoacids of the formula:

O H
b) reacting the resin linked aminoacids of step (a), suspended in DMF and acetic acid, with a set of aldehydes of the formula HC(O)R2 in HOAc/DMF and sodium cyanoborohydride in THF
10 to form resin linked N-alkyl-a-aminoacids of ~e formula:
Q lR~R R2 O H
c) reacting the resin linked N-alkyl-a-aminoacids of step ~b), in methylene chloride and diisopropylethyl~mine, with 2-azidobenzoyl chlorides of formula:
Cl N3~
~x2 X
to form resin linked N-(2-azidobenzoyl)amino esters of formula:

R3 ~R2 d~ N~f o N3~X2 xl d) treating the resin linked N-(2-azidobenzoyl)amino esters of step (c), suspended in an involatile solvent (i.e., a non-protic, organic 20 solvent with a boiling point of 8~140 C) such as toluene, xylene, or W O 97/01560 PCTrUS96/11070 chlorobenzene, with an excess of a trivalent phosphorus reagent such as triphenylphosphine or tributylphosphine at 80-150~C and then cooling said mixture to room temperature to fo~n resin linked benzodiazepines of formula:
Q ~/ N
N~O
~;,~, X2 Xl ; and, optionally, e) suspending the resin linked benzodiazepines of step (d) in TFA/water at room temperature for 1-24 hours to form 1,~
benzodiazepin-2,5-diones of formula:
R2 o x2 J~X
O H
wherein the symbols are as defined above for formula II'.
A second preferred embodiment of the invention is the solid phase synthesis of 1,4-benzodiazepin-2,5-diones and 1,3-cyclo-1,~
benzodiazepin-2,5-diones via aza-Wittig ring closure, wherein the process comprises:
a) reacting a set of suitably protected a-aminoacids of the formula:
3R Rl--R
HO~ 2 O Fmoc in the presence of DMF and DMAP with solid supports suspended in methylene chloride to form resin linked aminoacids of the formula:

(~3 3~Rl--R
Fmoc b) reacting the resin linked N-alkyl-a-aminoacids of step (b), in methylene chloride and diisopropylethylamine, with 2-azidobenzoyl chlorides of formula:

CA 022259l4 l997-l2-29 W O 97/01560 PCTrUS96/11070 N3 ~ 0 l~X2 to form resin linked N-(2-azidobenzoyl)amino esters of formula:
~ R2 ~ N O

xl c) treating the resin linked N-(2-azidobenzoyl)amino esters 5 of step (b), suspended in an involatile solvent (i.e., a non-protic, organic solvent with a boiling point of 80-140 C) such as toluene, xylene, or chlorob~n~ene, with an excess of a trivalent phosphorus reagent such as triphenylphosphine or tributylphosphine at 80-150~C and then cooling said mixture to room temperature to form resin linked benzodiazepines 10 of formula:
Q~R1---- R2 N~O
~X2 Xl ; and, optionally d) suspen(lin~ the resin linked benzodiazepines of step (c) in TFA/water at room temperature for 1-24 hours to form 1,~
benzodiazepin-2,5-diones of formula:
R2' ~ x2 lR

3R~N
O H
wherein the symbols are as defined above for formula II'.
Another embodiment of the invention is the use of the combinatorial library of Formula I in assays to discover biologically ~ active compounds (lig~n~1.c) of Formula II. Thus, an aspect of the CA 0222~914 1997-12-29 W O 97/01560 PCTr~S96tllO70 invention is a method of identifying a compound having a desired characteristic which comprises synthesizing 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. Another embodiment of the invention 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 i~l~ntifies compounds of Formula II having the desired characteristic. A further embodiment of the invention is dete~ in~ the structure of any compound so identified.
It is within the scope of the present invention that the deterrnination of the structures of compounds having the desired characteristic can be accomplished by decoding the tags (represented by T'-L- 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., ~9, 5862 (1994); Murphy et al., J. Am. Chem. Soc., 1 1 7, 7029 (1995); Cambell et aL J. Am. Chem. Soc., 1 1 7, 5381 (1995); and Erb et ah, Proc. Nat. Acad. Sci. USA, 91, 11422 (1994)).
In the latter case, q = 0 and the library of the present invention may be represented by Formula I' ~)-C(O)-L'-II' I' wherein the symbols are as defined for Formula I.
Another embo~1iment of the invention is the use of divinylben7ene-cross-linked, polyethyleneglycol-grafted polystyrene beads optionally functionalized with amino groups (for example, TentaGel(~) S NH2, Rapp Polymere) as the solid supports for constructing a combinatorial library of Formula I or I'.
Definitions The following abbreviations have the indicated mt~nin~: -Bn = benzyl BnOH = benzyl alcohol Boc = t-butyloxycarbonyl Bz = benzoyl c- = cyclo CA 022259l4 l997-l2-29 W O 97/01560 PCTrUS96/11070 _9_ DEAD = diethylazodicarboxylate DCM = dichloromethane = methylene chloride DIC = diisopropylcarbofliimide DMAP = 4-N,N-dimethylaminopyridine , S DMF = N,N-dimethylformamide equiv. = equivalent Et = ethyl FACS = fluorescence activated cell sorting Fmoc = 9-fluorenylmethoxycarbonyl Fmoc-OSu = 9-fluorenylmethylsuccinimidyl carbonate GC = gas chromatography hr = hour, hours m- = meta Me = methyl p- = para PEG = polyethylene glycol Ph = phenyl r.t. = room temperature sat'd = saturated s- = secondary t- = tertiary t-Boc = t-butyloxycarbonyl TFA = trifluoroacetic acid THF = tetrahydrofuran Thy = thienyl 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. Fx~mples of lower 30 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.
Px~mples of lower cycloalkyl groups include c-propyl, c-butyl, c-pentyl, 2-methylcyclopropyl, cyclopropylmethyl, norbornyl, and the 35 like.
"Alkenyl" is C2-C8 aLkenyl of a linear, branched, or cyclic (Cs-C6~ configuration and combinations thereof. Fx~mI)les of aLkenyl groups include vinyl, allyl, isopropenyl, pentenyl, hexenyl, c-hexenyl, 1-propenyl, 2-butenyl, 2-methyl-2-butenyl, and ~e like.
-CA 0222~9l4 l997-l2-29 W O 97/01560 PCTrUS96/11070 "Alkynyl" is C2-C8 alkynyl of a linear or branched configuration and combinations thereof. Examples of alkenyl groups include ethyne, propyne, butyne, pentyne, 3-methyl-1-butyne, 3,3-dimethyl-1-butyne, and the like.
S "Alkoxy" means alkoxy groups of from 1 to 8 carbon atoms of a straight, branched, or cyclic configuration and combinations thereof. F~mples of aLkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy, cyclohexyloxy, and the like.
"Acylamino" means acylamino groups of from 1 to 8 carbon atoms of a straight, branched or cyclic configuration and combinations thereof. F~mples of acylamino groups are acetyl~mino, 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 cont~inin~ 0-3 heteroatoms selected from O, N, and S; a bicyclic 9- or 10-membered aromatic or heteroaromatic ring system cont~inin~ 0-3 heteroatoms selected from O, N, and S; or a tricyclic 13- or 14-membered aromatic or heteroaromatic ring system cont~ining 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, =O, NO2, halogen, hydroxy, alkoxy, cyano, NR3R3, acyl~mino, 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, aL~enyl, alkynyl, halogen, hydroxy, alkoxy, C(hal)3, cyano, phenyl, phenoxy, benzyl, benzyloxy, caboxamido, heteroaryl, heteroaryloxy, NO2, and NR3R3;
The aromatic ~ to 14-membered carbocyclic rings include benzene, naphthalene, in~l~ne, tetralin, and fluorene and the 5- to 1 membered aromatic heterocyclic rings include imi~l~7ole, pyridine, indole, thiophene, benzopyranone, thiazole, furan, ben7imidazole, W O 97/01560 PCTrUS96/11070 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 5 replaced by halogen, hydroxy, loweralkoxy, carboxy, carboalkoxy, carboxamido, cyano, carbonyl, NO2, NR3R3, aLkylthio, sulfoxide, sulfone, acylamino, amidino, phenyl, benzyl, heteroaryl, phenoxy, benzyloxy, heteroaryloxy, and substituted phenyl, benzyl, heteroaryl, phenoxy, benzyloxy, and heteroaryloxy.
It is intended that the de~mitions of any substituent or symbol (e.g., R3, m, etc.) in a particular molecule be indepen~ent of its definitions elsewhere in the molecule. Thus, "NR3R3" represents NHH, NHCH3, N(CH3~2, etc.
The linkers may be any component capable of being l5 selectively cleaved to release both T and II from the solid support. See, e.g., Greene and Wuts, "Protective Groups in Organic Synthesis", 2nd ed., Wiley, 1991. Specific linkers L' are depicted in Table 1 (note that -L- = -C(O)L'- or -CH2-C(O)L'-), which also shows cleavage reagents. In desi~nin~ a synthetic scheme, L and L' are chosen such 20 that they are orthogonally reactive, i.e., 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 (via -C(O)- for L' and -C(O)-25 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 l,r~cllies: they must be detachable from the solid supports, preferably by photolysis or oxidation; they must be 30 individually diLrclcllliable, and plercldbly separable; they must be stable under the synthetic conditions; they must be capable of being detected at very low concentrations, e.g., 10-18 to 10-9 mole, they should be identifi~ble with readily-available equipment which does not require sophisticated technical capabilities to operate; and they should 35 be relatively economical. The tags may be structurally related or unrelated, e.g., a homologous series, repetitive functional groups, CA 0222~9l4 l997-l2-29 W O 97/01560 PCTrUS96/11070 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, 5 alicyclic, aromatic, heterocyclic, or combinations thereof.
Disting~ hin~ 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 ~-substituted ethylene groups where the substituents may be 10 alkyl groups, oxy, carboxy, amino, halo, or the like; isotopes; etc.
The materials upon which the combinatorial syntheses of the invention are p~lrolll~ed 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 15 cellulose beads, pore-glass beads, silica gels, polystyrene beads optionally cross-linked with divinylben~enç and optionally grafted with polyethylene glycol and optionally functionalized with amino, hydroxy, carboxy, or halo groups, grafted co-poly beads, poly-acrylamide beads, latex beads, dimethylacrylamide beads optionally cross-linked with 20 N,N'-bis-acryloyl ethylene t1i~min~, glass particles coated with hydrophobic polymer, etc., i.e., material having a rigid or semi-rigid surface;
b) soluble supports such as low molecular weight non-cross-linked polystyrene; and.
Q

c) derivatized forms thereof such as OH.
Suitable aminoacid protecting groups are well known in the art and include Fmoc, Alloc (allyloxycarbonyl), etc.

, CA 022259l4 l997-l2-29 W O 97/01560 PCTrUS96/11070 LINKER GROUPS
Linker Group Cleavage Reagent NO~ h~
~ CH2B-~r 1. NO2 0 CH2(~B-~{~ hv 2. 02N~ CH20-~ OR Ce(NH4)2(N03)6 3. g~O-4. RO~O Ce(NH4)2(N03)6 5. --CH=CH(CH2)2- 03, Os04tI04-, or KMnO4 6 --CH=CHCH2- 03, Os04/IO4-, or KMnO4 7. --CH2CH=CH- 03, Os04/I04-, or KMnO4 ~0 o 1) 02 or Br2, MeOH

8- ~ 2) H30+

9. --CH=CHCH20- (Ph3P)3RhCl(H) 10. ~Bo Li, Mg, or BuLi 11. --S-CH2-O- Hg+2 X Zn or Mg 12. I CH2-O-OH Oxidation, e.g., Pb(OAc)4 13. I CH2-O- or HsI06 14. ~0- or H30+

~OCH3 R = H or lower alkyl; B = O or NH; and X = electron withdrawing group such as Br, Cl, and I.

CA 0222~9l4 l997-l2-29 W O 97/01560 PCTrUS96/11070 -1~

Optical Isomers - Diastereomers - Geometric Isomers Some of the compounds described herein contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisometric forms which may be defined 5 in terms of absolute stereochemistry as (R) or (S), or as (D) or (L) 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 (R) and (S), or (D and L), isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional 10 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.
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 libra~ is thus a tool for drug discovery;
i.e., as a means to discover novel lead compounds by screening the 20 library against a variety of biological l~ el~ and to develop structure-activity relationships (SAR) in large families of related compounds.
The library may be tested with the li~nc1s attached to the solid supports as depicted in Formula I or I', or the compounds II may be detached prior to evaluation. With the compounds of Formula I or I', screening 25 assays such as FACS sorting and cell lawn assays may be used. When a compound is detached prior to evaluation, its relationship to its solid support is m~int~ined, 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 30 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. Aca~ Sci. USA, 90, 10922-10926, Dec. 1993 and Still et aL, Complex Combinatorial Chemical Libraries Encoded with Tags, WO 94/08051) or, 35 alternatively, the structures may be determined by deconvolution. The W O 97/01560 PCTrUS96/11070 usefulness of such a library as a screening tool is demonstrated by Burbaum et ah, Proc. Natl. Acad. Sci. USA, 92, 6027-6031, June 1995, who describe the assaying of encoded combinatorial libraries for, e.g., carbonic anhydrase inhibition. Even if no compounds are found to be S active in a given screen, such lack of activity often provides useful SAR
information.
Assays for Determinin~ Biolo~ical Activity Assays for evaluating the compounds of the present invention are well known in the art. Although one usually does not 10 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 described in the present invention, to identify activities with respect to a wide variety of enzymes and molecular ~;els, is disclosed in U.S. 08/553,0~6, filed November 3, 15 1995.
Methods of Synthesis The compounds of the present invention can be ~r~ed according to the following methods. At each step in the synthesis each solid support upon which a compound is being synthesized may be 20 uniquely tagged to define the particular chemical event(s) occurring during that step. The t~ggin~ 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 25 are used in combination with one another to form a binary or higher order encoding scheme permittin~ a relatively small number of i~lentifiers to encode a relatively large number of reaction products.
For example, when used in a binary code, N identifers can encode up to 2N different compounds and/or conditions. By associating each 30 variable or combination of variables at each step of the synthesis with a combination of identifiers which uniquely define the chosen variables such as re~ct~nt, reagent, reaction conditions, or combinations of these, one can use the identifi~,rs to define the reaction history of each solid support.

CA 0222~914 1997-12-29 W O 97/01560 PCTrUS96/11070 In carrying out the syntheses, one begins with at least 104, desirably at least 107, and generally not exceeding 101~ solid supports.
Depending on the pre-determined number of choices for the first step, one divides the supports accordingly into as many containers. The 5 ap~r~pliate reagents and reaction conditions are applied to each cont~in~r and the combination of identifiers which encode for each step 1 choice is added and attached. Depending on the chemistries involved, the t~g~in~ may be done prior to, concomitantly with, or after the reactions which comprise each choice. As a control, sample supports 10 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 combined, mixed, and again divided, this time into as many containers 15 as pre-determined for the number of choices for the second step in the synthesi~. 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 20 steps, resin-linked a-amino ester of the formula:
Qo ~

is suspended in an aprotic, polar solvent such methylene chloride, DMF, THF or ethyl acetate. An excess of a soluble organic base such as triethyl~min~, N,N-diisopropylethyl~mine, or pyridine is added to the 2~ suspended resin. This mixtllre is treated with an excess of an a~l~.pliately substituted 2-azidobenzoyl chloride of the formula:
xl O
r\~Cl X2~ N3 and ~it~ted at room tempel~ e to produce a resin linked N-(2-azidobenzoyl)amino ester of the formula:

CA 022259l4 l997-l2-29 W O 97/01560 PCTrUS96/11070 Q o~f ', ~
B Ra The resin is filtered and washed and then suspended in an involatile solvent (i.e., a non-protic, organic solvent with a boiling point of 8~140 C) such as toluene, xylene, or chlorobenzene and treated with an 5 excess of a trivalent phosphorus reagent such as triphenylphosphine or tributylphosphine. This mixtllre is agitated and heated to 80-140 C for 2-24 hr, then cooled to produce a resin-linked 1,4-benzodiazepin-5-one of the formula:

Nl ~o2 Q o ~ ~r,.

10 which is washed and then is suspended in an acidic solution and agitated at room temperature for 1-24 hr. The resin is filtered and washed and the filtrate and w~hin~.c are combined and evaporated to give a 1,~
benzodiazepin-2,5-dione of the formula:
x2 0 ~\~N ", X N~
H O
D

15 wherein Xl, ~, and Ra are selected such that formula D represents compounds of the formulae:

CA 022259l4 l997-l2-29 W O 97/01560 PCTrUS96/11070 H3~ H,~ H,~ ~H

4 8 OBz 11 and 16 Compounds 4, 8, 11, and 16 have been synthesized by the aza-Wittig method of the present invention. Compound 4 is a known intermediate useful in the synthesis of alllilulnor 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 5-thioabbeymycin (K~m~l et aL, Bioorg. Med. Chem. Lett., 3, p. 743, 1993) and abbeymycin. Compound 11 is a novel compound useful as an int~rme~ te in the synthe~is of anlilulnor antibiotics (Hurley et aL, Chem. Res. Toxicol, 1, p. 258, 1988. Compound 16 is a natural product isolated from Penicilli~m cyclopium with potential antibiotic properties (Framm et aL, Eur. J. Biochem, 3 7, p. 78, 1973.) Scheme 1 Resin-linked a-amino ester, A, is suspended in an aprotic, polar solvent such methylene chloride, DMF, THF or ethyl acetate. An excess (2-50 equivalents) of a soluble organic base such as triethyl~minP, N,N-diisopropylethyl~mine, or pyridine is added to the suspended resin. Optionally, an acylation catalyst such as ~
~1imPthyl~minopyridine may be added. This mixture is treated with an excess (2-10 equivalents) of an a~pr~pliately substituted 2-azidobenzoyl chloride and agitated at room temperature for 2-24 hr. The resin is ~lltered and washed multiple times with an app~ )liate solvent such as methylene chloride to remove excess reagents and byproducts. Resin linked N-(2-azidoben-oyl)~minQ ester, B, is suspended in an involatile solvent such as toluene, xylene, or chloroben7ene and treated with an excess (2-10 equivalents) of a trivalent phosphorus reagent such as triphenylphosphine or tributylphosphine. This mixture is agitated and he~t~l to 80-140 C for 2-24 hr, then cooled and the resin-linked 1,4-W O 97/01560 PCTrUS96/11070 benzodiazepin-5-one, C, is washed multiple times with ~plvpriate solvents such as methylene chloride or toluene to remove excess reagents and byproducts. Resin-linked 1,~benzodiazepin-5-one, C, is suspended in an acidic solution such as trifluoroacetic acid/methylene 5 chloride (50-90% TFAlcH2cl2) or hydrogen chloride/dioxane (1-4M
HCl/dioxane) and agitated at room temperature for 1-24 hr. The resin is filtered and washed with a~r~l;ate solvents such as methylene chloride or dioxane. The filtrate and w~hin~ are combined and evaporated to give the crude 1,4-benzodiazepin-2,5-dione, D, which 10 may be purified and characterized by standard tec~niques.

OJ~ ~ ,\ X~N~
A Base N,~3_x2 Q ~f oJ~N\ o Heat B Ra ~0 H+ H N~, Ra C D

=
CA 022259l4 l997-l2-29 W O 97/OlS60 PCTrUS96/11070 The invention is further defined by reference to the following examples, which are inten-l~d to be illustrative and not limiting.

S IDE~ l~RS
Twelve compounds of the general formula:

(~ ~ (CH2)n-O-Ar wherein:
10 n = 3-12 and Ar is pentachlorophenyl or n = 54-6 and Ar is 2,4,6-trichlorophenyl were ~r~ared according to Scheme 6 and the following illustrative example.
a) Methyl v~nill~te (0.729 g, 4.0 mmole), l-hydroxy-9-(2,3,4,5,6-pentachlorophenoxy)nonane (1.634 g, 4.0 mmole) and triphenylphosphine (1,258 g, 4.8 mmole) were dissolved in 20 mL dry toluene under argon. DEAD (0.76 m~, 0.836 g, 4.8 mmole) 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 cllr~ atography eluting with DMC to give 1.0 g (1.7 mmole, 43%) of the product as a white cryst~lline solid.
b) The methyl ester from Step (a) (1.0 g, 1.7 mmole) was dissolved in 50 mL THF, 2 mT . water was added, followed by LiOH
(1.2 g, 50 mmole). 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 HCl (3x 50 mL) then sat'd a~. NaCl (1x 50 mL) and dried over sodium sulfate.
The solvent was removed and the crude acid azeotroped once with toluene.
c) The crude material from Step ~b) was dissolved in 100 mL toluene, 10 mL (1.63 g, 14 mmole ) thionyl chloride was added, and the mixhlre was refluxed for 90 min. The volume of the solution was reduced to approx. 30 mL by di~till~tion, then the rem~ining CA 022259l4 l997-l2-29 W O 97/01560 PCTrUS96/11070 toluene was removed by evaporation. The crude acid chloride was dissolved in 20 mL dry DCM and cooled to -70~C under argon and a solution of approx. 10 mmole diazomethane in 50 mL anhydrous ether was added. The mixture was warmed to r.t. and stirred for 90 min.
5 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 mmole, 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 triethyl~mine to give the identifier, which was then used without further purification. This was a significant improvement over the ori~in~l 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 resulted in significant acid-catalyzed decomposition of the identifier.
Altemate Step c) To a solution of the acyl chloride (3.8 mmol, 1.00 eq.) and 1.85 mL (13.3 mmol, 3.50 eq.) of triethyl~mine in anhydrous THF/acelollillile (1:1) at 0~C under argon was added 5.7 mL
(11.4 mmol, 3.00 eq.) of a 2.0 M solution of (trimethylsilyl)-diazomethane in hexanes. The resulting orange solution was stirred at 0~C for 2 hr, then at 25~C for 17 hr. (If a precipitate formed imme~ tely upon addition of (trimethylsilyl)diazomethane, CH2C12 was added until the precipitate redissolved). EtOAc was added (250 mL), and the organic layer washed with saturated aq. NaHCO3 (100 mL) and H2O (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 pr~altd by analogous synthetic routes, steps (a), (b), and (c).
In the synthesi~ of Example 5, the 12 identifiers were used to encode the combinatorial library. In Step 1, pentachlorophenyl 35 identifiers where n = 7-12 (abbreviated C7C15, C8C15, , C12C15) were used in the following binary encoding scheme: 0000Q1 = (n = 12), CA 0222~9l4 l997-l2-29 W O 97/01560 PCT~US96/11070 000100 = (n = 1 l) though 100000 = (n = 7). In Step 2, pentachlorophenyl identifiers where n = 6-9 (abbreviated C6Cl5, C7Cl5, C8Cl5, and CgCl5) were used and encoded as follows: 000001 =
(n = 6), 000010 = (n = 5), 000100 = (n = 4), and 00100 = (n = 3).
S Also in Step 2, trichlorophenyl identifiers where n = 4-6 (abbreviated C4Cl3, C5Cl3, and C6cl3) were used and encoded as follows: 01000 =
(n = 6). 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 pentachloro-10 phenyl identifiers where n = 11 and 12. Likewise, in Step 3 reagent 30is encoded "01110" which represents tagging this choice in the synthesis with the pentachlorophenyl identifiers where n = 3-6 and the trichlorophenyl identifier where n = 6.

W O 97101560 PCTrUS96/11070 IDEl!l l l~ KS

~ OH HO-(CH2)n-O-Ar MeO ~
)rMe PPh3, DEAD,Toluene ~ ,~0--(CH2)n-O-Ar MeO~" OMe 1. LiOH, THF/MeOH
2. SOCI2, toluene reflux ~,0--(CH2)n-O-Ar Cl~
TMS - cHN2 n OMe Et3N / O

~~C, THF/MeCN (1:1) DHC2M 2 Et20 N~O--(CH2)n-O-Ar O OMe IV

W O 97/01560 PCT~US96/11070 N-Fmoc-cis-4-benzoyloxy-L-proline-p-alkoxybenzyl Resin N-Fmoc-cis~benzoyloxy-L-proline p-alkoxybenzyl resin, 5, was prepared by routine methods as outlined below.
~ ~ 1. BnOH, TsOH, C6H6, reflux ~ Fmoc HoJ~ - N ' BnO~N
~ 2. Fmoc-OSu, acetonitrile ~
OH 3. BzOH, DEAD, PPh3, toluene OBz 1. 1,3-cyclohexadiene, Pd/C, toluene 2. p-Alkoxybenzyl alcohol resin, DIC, DMAP, methylene chloride.
Il Fmoc (~OJ~ ~
OB~

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) I~a,dtion of 4-acetoxymethylphenoxy acetic acid: A
15 solution of ~hydroxymethyl-phenoxy acetic acid ~9.9 g, 55 mmol) in pyridine (200 mL) was treated with acetic anhydride (22.15 g, 218 mmol, 4 equiv.), and the reaction mixture was stirred 25~C under argon for 48 hr. The reaction mixtllre was concentl~ted in vacuo to ~25 mL, then diluted with 200 mL of EtOAc, and placed in a separatory funnel.
2Q The resulting suspension was treated with ca. 50 mL of lN aq. HCl, and sh~ken The pH of the aqueous layer was checked, and adjusted to pH 2 with portionwise addition of conc. HCl and ~h~king The layers were W O 97101560 PCTrUS96/11070 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 ~1:9), followed by 5 ethyl acetate/hexanes (1:1) to afford 7.0 g (57%) of 4-acetoxymethyl-phenoxy acetic acid as an off-white solid.
TLC: Rf=0.2, silica gel, 100% EtOAc (UV).
lH-NMR: (CD30D) 2.0 (s, 3H), 4.6 (s, 2H), 5.0 (s, 2H), 6.80 (d, 2H), 7.20 (d, 2H).
2) ~aMtion of resin support A: A 300 mL synthesis vessel was charged with Tentagel-S-NH2 resin (25 g, 0.30 mmol/g capacity, (7.5 mmol)), and the beads were washed with 2 x 150 mL methylene chloride. A solution of N-a-N-~-diFmoc-lysine (13.3 g, 22.5 mmol, 3 equiv.) in 100 mL of DMF/methylene chloride (1:1), was added to the 15 vessel. The resulting mixture was treated with 4-dimethylamino-pyridine (92 mg, 0.75 mmol, 0.1 equiv.), followed by N,N'-diisopropylcarbo-liimicl~ (4.73 g, 37.5 mmol, 5 equiv.), and ~e reaction mixture was .sh~k~n at ambient tempeldlule. After 6 hours, the solvent was removed by filtration, and the beads were washed successively with 20 5 x 150 mL DMF, and 5 x 150 mL methylene chloride. A small portion of the resin was checked by the Kaiser test for disappearance of free NH2, and found to be negative.
The resin was treated with a 30% solution of piperidine in DMF
(100 mL), and ~h~ken at 25~C for 1 hr. The resin was filtered and 25 washed successively with 5 x 150 mL DMF and 5 x 150 mL 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 ~aceto~cymethylphenoxy acetic acid (20.2 g, 90 mmol, 6 equiv.) in 60 mL of methylene chloride was treated with DMF
30 dr~ /vise until all solid went into solution. This solution was then added to resin above (nom. 15 mmol) in a 300 mL synthesis vessel. The resulting suspension was treated with ~1imethylaminopyridine (366 mg, 3.0 mmol, 0.2 equiv.), followed by N,N'-diisopropylcarbo-liimi~le (18.9 g, 150 mmol, 10 equiv.), and the reaction mixture was shaken at 35 25 C for 16 hr. The solvent was removed by filtration, and the resin CA 022259l4 l997-l2-29 W O 97/01560 PCTrUS96/11070 was washed successively with 5 x 150 mL DMF and 5 x 150 mL
CH2C12 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.
S The acetate protected resin from above was treated with a solution of 10% hydrazine hydrate in methanol (100 mL), and ~h~ken at ambient temperature. After 6 hr., the solvent was removed by filtration, and the resin was washed successively with 5 x 150 mL
MeOH and then re-treated with 10% hydrazine hydrate in MeOH (100 mL) and shaken at 25 C for 16 hr. The solvent was removed by filtration, and the resin was washed successively with 5 x150 mL DMF
and 5 x 150 mL CH2Cl2 to afford the hydroxy resin A.

W O 97/01560 PCTrUS96/11070 Bis-Linker Attachment 1. N-a-N-e-diFmoc-L-lysine, DIC, cat. DMAP
(~ 2. 30% Piperidine/DMF
2 3 4-Acetoxymethyl-phenoxy acetic acid DIC, cat DMAP

HNJ~, O~
~,OAc Q, ~ O 10% hydrazine hydrate/methanol H OAc HNJ~, ~~1 ~,OH
~ N~N~O~ Q OH
O H OH A

W O 97/01560 PCTrUS96/11070 (1 laS~-1,2,3,10,11,1 la-Hexahydro-5H-pyrrolo[2,1-c](1,4)-benzo-diazepine-5. 1 1 -dione "~Fmoc 1 P~ dine/DMF

~CI

,~, 3 ~~
0 PPh3, Toluene, 80~C

~ ~ 0 50% ~ A/C~H~C12 ~0~~~

H. N~
0~

N-Fmoc-L-proline p-alkoxybenzyl resin, 1, (Bachem) (0.34 mmole/g, 2.0 g, 0.68 mmole) was suspended in 30 mL DMF, then filtered. The resin was then .~h~ken with 50% piperidine/DMF for 2 hr.
10 then filtered and washed with DMF (5 x 30 mL) and methylene chloride (10 x 30 mL). The resin was suspended in 20 mL methylene chloride, and 2 mL (1~ mmole~ triethyl~mine was ~l(lç-l, followed by 1.0 g (5.5 mmole, 8 equiv.) 2-azidobenzoyl chloride. The mixture was ~h~ken at , CA 022259l4 l997-l2-29 W O 97/01560 PCT~US96/11070 25~C for 3 hr. and then the resin was washed with methylene chloride (10 x 30 mL) followed by toluene (10 x 30 mL). The resin, 2, was suspended in 20 mL toluene and 0.3 g (1.14 mmole, 1.7 equiv.) triphenylphosphine was added and the mixture was shaken until the S tnphenylphosphine had dissolved. The mixture was ~h~k~n and heated to 80~C for 3 hr. then cooled and washed wi~ me~ylene chloride (10 x 30 mL). The pale brown resin, 3, was dried under vacuum.
The resin, 3 (2.0 g, 0.68 mmole), prepared as described above, was suspended in 20 mL methylene chloride and 20 mL
10 trifluoroacetic acid was added. T~e resin was shaken at 25~C for 30 min then filtered and washed with methylene chloride (2 x 20 mL).
The filtrate and w~hin~.~ 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 15 product, 4, as a white solid.
H NMR (300 MHz, CDC13): o 2.03 (m, 3H), 2.77 (m, lH), 3.62 ~m, lH), 3.80 (m, lH), 4.08 (m, lH), 7.07 (m, lH), 7.26 (m, lH), 7.48 (m, lH), 8.00 (d, lH, J = 7.7 Hz), 9.07 (br s, lH, NO;
13C NMR (125 MHz, CDC13): o 23.45, 26.12, 47.26, 56.66, 121.81, 20 124.90, 127.03, 131.02, 132.34, 135.44, 165.41, 171.40;
CIMS: 217 (MH+) (2S, 1 laS)-2-Ben~oyloxy-1,2,3,10,11,1 la-hexahydro-SH-pyrrolo-r2.1-c~ 4)-benzo-diazepine-5~11-dione ,¢f ,~>Fmoc I Piperidine/DMF

OBz G~ Cl CA 022259l4 l997-l2-29 W O 97/01560 PCTrUS96/11070 N3~
O ~ PPh3, Toluene, 80~C
Qo~~~ ~
6 OBz 50% TFA/CH.CI. H~

OBz 8 OBz N-Fmoc-cis-4-benzoyloxy-L-proline p-alkoxybenzyl resin, 5, (400 mg) was suspended in 10 mL DMF, then filtered. The resin S was then shaken with 50% piperidine/DMF, 10 mL, for 2 hr. then filtered and washed with DMF (S x 10 mL) and methylene chloride (10 x 10 mL). The resin was suspended in 10 mL methylene chloride, 0.4 mL (~3 mmole) triethylamine was added, followed by 0.2 g (1.1 mmole) 2-azido-benzoyl chloride. The mixture was sh~k~n at 25~C for 12 hr. and then the resin was washed with methylene chloride (10 x 10 mL), followed by toluene (10 x 10 mL). The resin, 6, was suspended in S0 mL toluene and 0.20 g (0.8 mmole) triphenylphosphine was added and the mixture was shaken until the triphenylphosphine had dissolved.
The mixture was ~h~ken and heated to 80~C for 2 hr. then cooled and lS washed with toluene (S x 10 mL) and methylene chloride (10 x 10 mL).
The resin, 7, was dried under vacuum The resin, 7, ~lcpalcd as described above, was suspended in 5 mL methylene chloride and S mL trifluoroacetic acid was added.
The resin was ~h~k~.n at 25~C for 30 min then filtered and washed with 20 methylene chloride (2 x 10 mL). The filtrate and w~hing~ were coll~Gte~l and combined, then evaporated to give the crude product which was purified by flash chromatography eluting with 50% ethyl acetate/he~cane to give the product, 8, as a white soli~

CA 0222~9l4 l997-l2-29 W O 97/01560 PCTrUS96/11070 lH NMR ~300 MHz, CDCl3): ~ 2.41 (m, lH), 3.31 (d, lH, J = 14.4 Hz), 3.94 (m, lH), 4.16 (m, 2H), 5.58 (m, lH), 6.84 (d, lH, J = 8.1 Hz), 7.07 (m, 2H), 7.20-7.37 (m, 3H), 6.47 (s, lH), 7.87 (d, lH, J = 8.1 Hz), 8.10 (d, lH, J = 7.71 Hz), 9.94 (br s, lH, N~;
13C NMR (125 MHz, CDC13): ~ 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;
CIMS: 337 (MH+) (11aS)-7,8-Dimethoxy-1,2,3,10,11,1 la-hexahydro-SH-pyrrolo[2,1-c]-~1.4)-benzo-diazepine-S.l l -dione o 1. Piperidine/DMF
Il Fmoc Q ~f o~ 2 O

MeO N3 OMe N3~0Me o r PPh3,Toluene,8~ C

QoJ~ ~

MeO OMe ~ 50%~FA/CH2CI~ l~OMe lS N-Fmoc-L-proline p-alkoxybenzyl resin, 1, (0.34 mmole/g, 5.0 g, 1.8 mmole) was suspended in 50 mL DMF, then filtered. The resin was then shaken with 50% piperidine/DMF for 2 hr. then filtered and CA 0222~914 1997-12-29 W O 97/01560 PCTrUS96/11070 washed with DMF (5 x 50 mL) and methylene chloride (10 x 50 mL).
The resin was suspended in 50 mL methylene chloride, 3 mL (21 mmole) triethyl~mine was added, followed by 0.75 g (3 mmole, 1.7 equiv.) 2-azido-4,5-dimethoxybenzoyl chloride as a solid. The mixture S was ~h~ken at 25~C for 12 hr. and then the resin was washed wi~h methylene chloride (10 x 50 mL), followed by toluene (10 x 50 mL).
The resin, 9, was suspended in 50 mL toluene and 1.26 g (4.8 mmole 2.6 equiv.) triphenylphosphine was added and the mixture was shaken until the triphenylphosphine had dissolved. The mixture was shaken and heated to 80~C for 2 hr. then cooled and washed with toluene (10 x 50 mL), alternately with methanol and methylene chloride (5 x 50 mL
each) and finally methylene chloride (5 x 50 mL). The pale brown resin, 1 0, was dried under vacuum.
A 1.0 g (~0.34 mmole) portion of the resin, 1 0, prepared as described above, was suspended in 20 mL methylene chloride and 20 mL trifluoroacetic acid was ~flde~. The resin was shaken at 25~C for 30 min then filtered and washed wi~ methylene chloride (2 x 20 rnT 3.
The filtrate and w~hinE~ 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, 1 1, as an off white solid.
1H NMR (300 MHz, CDCl3): ~ 2.03 (m, 3H), 2.75 (m, lH), 3.61 (m, lH), 3.78 (m, lH), 3.91 (s, 3H), 3.93 (s, 3H), 4.04 (d, lH, J = 6 Hz), 6.47 (s, lH), 7.46 (s, lH), 8.30 (br s, lH, N~;
13C NMR (125 MHz, CDCl3): ~ 23.49, 26.11, 47.26, 56.05, 56.11, 56.80, 103.85, 112.01, 119.16, 129.79, 146.27, 152.20, 165.27, 171.13;
CIMS: 277 (MH+) Cyclopeptin CA 022259l4 l997-l2-29 W O 97/01560 PCTrUS96/11070 HOJ~'N' Fmoc Q ~f OH 13J

12 DIC, DMAP, CH ,Ch O CH3 I. Piperidine/DMF
Q ~ ' Fmoc 2. O

13 NEt3, CH2CI~

N~ PPh3, Q ¢~f ~ N,--~o Toluene, 90 C

l ~0 50% TFA/CHzCl2 ~,3 W O 97/01560 PCT~US96/11070 p-Alkoxybenzyl alcohol resin, 12, 2.5 g (1.0 mmole/g~ was suspended in 30 mL methylene chloride. N-Fmoc-N-methyl-L-phenyl~l~nint?, 3.0 g ~7.5 mmole, 3 equiv.), DMAP, 1.0 g (0.82 mmole, 0.3 equiv.), and DIC 1.74 mL (1.26 g, 10 mmole, 4 equiv.) were added and the mixture was ~it~ted for 2 hr. The resin was filtered and washed with methylene chloride, (10 x 30 mL), then dried.
The N-Fmoc-N-methyl-L-phenyl~l~nine p-alkoxybenzyl ester resin, 13, 1.0 g was suspended in 50% piperidine/DMF, 30 mL, and ~it~ted for 2 hr, then filtered and washed with DMF (5 x 30 mL) and methylene chloride (10 x 30 mL). The resin was suspended in 20 mL methylene chloride; 1.0 mL (~0 7 mmole) triethyl~mine was added followed by 0.6 g (3.3 mmole) 2-azido-benzoyl chloride. The mixture was sh~ken at 25 C for 1 hr and then the resin was washed with methylene chloride (10 x 30 mL) followed by toluene (10 x 30 mL).
The resin, 14, was suspended in 20 mL toluene and 0.52 g (2.0 mmole) triphenylphosphine was added and the mixture was shaken until the triphenylphosphine had dissolved. The mixtllre was ~h~ken and heated to 90~C for 3 hr then cooled and washed with toluene (5 x 20 mL) and methylene chloride (10 x 20 mL) to give the resin linked 1,4-benzodiazepine-5-one, 15.
The resin, 15, was suspended in 10 mT. methylene chloride and 10 mT trifluoroacetic acid was added. The resin was shaken at 25 C for 2 hr, then filtered and washed with methylene chloride (2 x 20 mL). The filtrate and w~.chin~ were collected and combined, then evaporated to give the crude product which was purified by flash chromatography eluting with 50% ethyl acetate/~exane to give the product, 16.
lH NMR (300 MHz, DMS~d6, 100 C): ~ 2.91-3.06 (m, 2H), 2.95 (s, 3H), 4.30 (t, lH, J = 7.9 Hz), 7.13-7.28 (m, 7H), 7.48 (m, lH), 7.80 (d, lH, J = 7.7 Hz), 10.22 (br s, lH, N~, CIMS: 281 (MH+) Synthesis of 1.4-Benzodiazepin-2~5-dione Library CA 022259l4 l997-l2-29 W O 97/01560 PCTrUS96/11070 a-Amino acids (Reagent Set 1), (~ OH ~ O~ FFmoc A DIC, cat. DMAP, CH2Ck/DMF o H

1. 30% Piperidine/DMF

2. Aldehydes(ReagentSet2), (~-o~N3 O~R2 C O H
H

NaBH3CN, 1% HOAc/DMF

2-Azidobenzoyl chloride (Reagent Set 3) X~X O~ oR2 C > N3~ x2 NiPr2Et, CH2Cl2 D Xl PBu3, xylene, heat Q~ N 30 % TFA/CH2C12 D > N~
1~ X2 X R2N ~ x2 R~
Rl xl - O H F

W O 97/01560 PCTr~S96/11070 a-Amino acids (Reagent Set 1), (~ OH ~ Fmoc Qo~ Nl A DIC, cat. DMAP, CH2Cl2/DMF o Fmoc 2-Azidobenzoyl chloride (Re~gent Set 3) ?~ Q 2 B , N
3 ~x2 NiPr2Et, CH2CI2 D Xl C 3' Y , 1~ 30 ~ TFA/CH2Cl2 X~

CA 022259l4 l997-l2-29 W O 97/01560 PCTrUS96/11070 A 1.2 g (0.53 mmole/g, 0.64 mmole OH) portion of p-alkoxybenzyl resin A, was placed in each of 46 separate 100 mL
synthesis vessels (vessels 1~6). The resin in each vessel was suspended in 30 mL methylene chloride, agitated for S min and then filtered. The 5 solvated resin was resuspended in 20 mL methylene chloride. Solutions of 46 protected a-amino acids (Reagent Set 1, 4.3 mmole, ~ 7 equiv), prepared in 20 mL 1:1 methylene chloride:DMF, were added to the vessels, one solution per vessel, Cont~inin~ the suspended resin. The vessels were agitated for 5 min, then 2 mL of 45 mg/mL DMAP (90 10 mg, 0.73 mmole, 1.2 equiv~ solution in methylene chloride was added to each vessel and the mixtures were agitated for 5 min. DIC, 1.1 mL
(91 mg, 7.2 mmole, 11 equiv), was added to each vessel and the mixtures were ~pit~ted for 14 hr. Then the resin batches, B, were filtered and washed with S x 40 mL methylene chloride.
The 46 resin batches were encoded with six tags as follows.
(i) The resin batches were suspended in 30 mL methylene chloride. Aliquots, 2 mL of 45 mg/mL (90 mg tag precursor/vessel ~7.5 % by mass of resin), C12Cl5 tag precursor solution in methylene chloride were added to the a~ r~liate vessels and the vessels were shaken for 2 min. Aliquots, 2 mL of 45 mg/mL (90 mg tag precursor/vessel ~7.5 % by mass of resin), of C1 1Cl5 tag precursor solution in methylene chloride were added to the a~l~liate vessels and the vessels were ~h~ken for 2 hr. Rhodium (II) trifluoroacetate dimer, 2 mL of 1 mg/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 ~it~ted for 12 hr, then filtered and washed with S x 40 mL methylene chloride.
(ii) The procedure from (i) was repeated for tags C1oC15 and CgCl5~
(iii) The procedure from (i) was repeated for CgC15 and The resin batches, encoded 000001-101110, were combined in a seperatory funnel and washed with 5 x 300 mL methylene chloride.
The resin was then filtered and dried for 12 hr in vacuo.
The resin above was divided into two equal batches of 1.75 g which were placed into two s~dle 100 mL synthesis vessels ~vessels CA 0222~914 1997-12-29 W O 97/01560 PCTrUS96/11070 1 and 2) and sixty equal batches of 0.85 g which were placed into sixty separate 100 mL synthesis vessels (vessels 3-62).
The 62 resin batches were encoded with six tags as follows.
(iv) The resin batches were suspended in 30 mL methylene chloride.
Aliquots, 2 mL of 37 mg/mL (74 mg tag precursor/vessel ~8.7 % by mass of resin), of C6Cl5 tag precursor solution in methylene chloride were added to the al,~r~,liate vessels from 3 to 62 and the vessels were shaken for 2 min. A 3 mL aliquot of 37 mg/mL C6Cl5 tag precursor solution was added to vessel 1 (111 mg tag precursor in vessel 1, 6.3%
by mass of resin). Aliquots, 2 mL of 37 mg/mL (74 mg tag precursor/vessel ~8.7 % by mass of resin), of C5Cl5 tag precursor solution in methylene chloride were added to the ~ iate vessels from 3 to 62 and the vessels were shaken for 2 min. A 3 mL aliquot of 37 mg/mL C5Cl5 tag precursor solution was added to vessel 2 (111 mg tag precursor in vessel 2, 6.3% by mass of resin). Rhodium (II) trifluoroacetate dimer, 2 mL of 1 mg/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 ~git~ted for 12 hr, then filtered and washed with 5 x 40 mL methylene chloride.
(v) The procedure from (iv) was repeated for tags C4Cl5 and C3C15.
~vi) The procedure from (iv) was repeated for C6Cl3 and C5Cl3 with the modification that 4 mL aliquots 37 mg/mL C6cl3 and C5C13 tag precursor solutions were used and also a 4 mL aliquot of 37 mg/mL C4Cl3 tag precursor solution was added to vessel 32.
The resin batches, encoded 000001-111110, were suspended in 30 mL DMF, ~git~ted for S min and then filtered. A
solution of 30% piperidine in DMF was added to each vessel, the mixtures were ~it~tçd for 30 min and then filtered. The resin batches were washed with 2 x 30 mL DMF, then 2 x 30 mL 1% acetic acid in DMF and then filtered. The resin batches were resuspended in 20 mL
1% acetic acid in DMF, and 2.4 mmole ~~ 5 equiv) of the ~lo~liate aldehydes (Reagent Set 2), was ?~ddç~l, as a solution in 10 mL 1%
HOAc~DMF, to each of the vessels 3-62. The mixtures were ~h~ted for 2 hr then 5 mL of lM sodium cyanoborohydride (5 mmole, 10 equiv) CA 022259l4 l997-l2-29 W O 97/01560 PCT~US96/11070 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 shakèn for a further 90 min, then filtered and washed with 2 x 30 mL DMF and S x 30 mL methylene chloride to S 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 seperatory funnel, then divided as a slurry in methylene chloride into nineteen equal portions (~2.7 g, 1.4 mmole) in 100 mL
synth~qsi~ vessels. The resin was suspended in 30 mL me~ylene chloride and diisopropylethyl~mine, 2.5 mL (1.8 g, 14 mmole, 10 equiv~, was added followed by S mmole of the a~ ,iate o-azidobenzoyl chloride (Reagent Set 3). The mixtures were ~h~kt~n at room temperature for 16 hr and then filtered and washed with 5 x 30 mL methylene chloride and 2 x 30 mL xylene to give resin batches D.
Each resin batch was transferred as a sluITy into separate 50 mL flasks and the suspensions were sparged with argon for 5 min, then sealed with a septum. Tributylphosphine, 1.8 mL (1.45 g, 7.2 mmole, 5 equiv), was added to each flask and the mixtures were heated to 140-150~C for 6 hr, then cooled, filtered, and washed with 2 x 30 mL
toluene and S x 30 mL 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 100 ~uL of 70 % TFA/water for 4 hours and filtering the solution.
A second collection of 1 170 1,4-benzodiazepine-2,5-diones was ~r~a~ed as follows. Resin batches lC and 2C were combined and mixed thoroughly in a seperatory funnel. The resin was dried in vacuo and divided into thirteen equal portions (~0.27 g, 0.14 mmole) then placed in 20 mL synthesis vessels. The resin was suspended in 10 mL
methylene chloride and diisopropylethyl~mine, 0.25 mL (0.18 g, 1.4 mmole, 10 equiv), was added followed by 0.5 mmole of thirteen o-azidobenzoyl chlorides. The mixtures were ~h~kPn at room temperature for 16 hr and then filtered and washed with 5 x 10 mL
methylene chloride and 2 x 10 mL xylene to give resin batches D. Each resin batch was tra~srell~d as a slurIy into separate 25 mL flasks and W O 97/01560 PCT~US96/11070 -40-the suspensions were sparged with argon for 5 min, then sealed with a septum. Tributylphosphine, 0.14 mL (0.11 g, 0.55 mmole, 4 equiv), was added to each flask and the mixtures were heated to 140-150~C for 6 hr, then cooled, filtered, and washed with 2 x 10 mL toluene and 5 x 5 10 mL methylene chloride to give the resin batches E, from which the product 1,~benzodiazepine-2,5-diones, F, may be cleaved as described above.

Decodin~ Procedure A bead is placed in a 1.3 mm diameter pyrex capillary with 2 ,uL of acetonitrile. Ceric ammonium nitrate solution (2 ,uL of a 0.1 M aq. solution) and hexane (3 ,uL) are added and the two-phase mixture centrifuged briefly. The tube is sealed and left at 35~C for 16 hrs, then opened. The organic layer is removed by syringe and mixed with 1 ,uL
15 of N,O-bis(trimethylsilyl)acet~mit1e. The silated tag solution (1 ,uL) is analyzed 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 5 m, 0.32 mm retention gap connected to a 25 m, 0.2 mm crosslinked 5%
20 phenylmethyl silicone column is used. The temperature and pressure programs for the analysis are 200-320~C, 15~C/min, then 320~C for 10 min and 2040 psi at 2 psi/min, then 40 psi for 10 min. The EC
detector is m~int~insd at 400~C and the auxiliarv gas is set at 35 psi.
The identity of the library compound attached to the bead 25 is ascertained based on the reagents utilized in the synthesis of such compound, which are readily deterrnined from the binary codes associated, respectively, with each of the i-lenti~1ers for such reagents, as char~cteri~ed through the above procedure.

Claims (9)

WHAT IS CLAIMED IS:

1. A method of synthesizing 1,4-benzodiazepin-2,5-diones which comprises:
a) attaching a set of suitably protected .alpha.-aminoacids or N-alkyl-.alpha.-aminoacids to solid supports to form resin linked N-alkyl-.alpha.-aminoacids; or b) attaching a set of suitably protected N-unsubstitued-.alpha.-aminoacids to solid supports to form resin linked N-unsubstitued-.alpha.-aminoacids and reductively alkylating said resin linked aminoacids with a set of aldehydes to form resin linked N-arylalkyl or heteroarylalkyl-.alpha.-aminoacids;
c) acylating the resin linked N-alkyl-.alpha.-aminoacids or the N-arylalkyl or heteroarylalkyl-.alpha.-aminoacids of steps (a) or (b) with a set of 2-azidobenzoyl chlorides to form resin linked N-(2-azidobenzoyl)amino esters;
d) cyclizing the resin linked N-(2-azidobenzoyl)amino esters of step (c) via aza-Wittig ring closure to form resin linked benzodiazepines; and, optionally, e) cleaving the resin linked benzodiazepines of step (d) to form 1,4-benzodiazepin-2,5-diones.

2. A method of Claim 1 which comprises:
a) reacting a set of suitably protected .alpha.-aminoacids of the formula:
in the presence of DMF and DMAP with solid supports suspended in methylene chloride to form resin linked aminoacids of the formula:
b) reacting the resin linked aminoacids of step (a), suspended in DMF and acetic acid, with a set of aldehydes of the formula HC(O)R2 in HOAc/DMF and sodium cyanoborohydride in THF
to form resin linked N-alkyl-.alpha.-aminoacids of the formula:
;
c) reacting the resin linked N-alkyl-.alpha.-aminoacids of step (b), in methylene chloride and diisopropylethylamine, with 2-azidobenzoyl chlorides of formula:
to form resin linked N-(2-azidobenzoyl)amino esters of formula:
;
d) treating the resin linked N-(2-azidobenzoyl)amino esters of step (c), 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 resin linked benzodiazepines of formula:
; and, optionally, e) suspending the resin linked benzodiazepines of step (d) in TFA/water at room temperature for 1-24 hours to form 1,4-benzodiazepin-2,5-diones of formula:

wherein:
R1 is H, lower alkyl, c-lower alkyl, -or (CH2)mR4, or R1 and R2, 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;
R2 is H, loweralkyl, arylR6R71R8, or heteroarylR6R7R8, or R1 and R2, 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;
R3 is H or loweralkyl;
R4 is aryl, substituted aryl, heteroaryl, substituted heteroaryl, NR3R5, CO2R3, CONR3R3, or OH;
R5 is H, lower alkyl, -CNHR3R3, or -C(O)R3;
R6, R7, and R8 is each, independently, H, lower alkyl, lower alkoxy, halogen, aryl, lower alkylthio, X-aryl, X-substituted aryl, lower alkylaryl, C(hal)3, -(CH2)mNR3R5, or -X-CH(CO2R3)2, or R6 and R7, together with the atoms to which they are attached, join to form a 5- or 6-membered heterocyclic ring; and X i O or S.

3. A method of Claim 2 wherein the involatile solvent is toluene, xylene, or chlorobenzene and the trivalent phosphorus reagent is triphenylphosphine or tributylphosphine.

4. A method of Claim 1 which comprises:
a) reacting a set of suitably protected .alpha.-aminoacids of the formula:

in the presence of DMF and DMAP with solid supports suspended in methylene chloride to form resin linked aminoacids of the formula:
b) reacting the resin linked N-alkyl-a-aminoacids of step (b), in methylene chloride and diisopropylethylamine, with 2-azidobenzoyl chlorides of formula:
to form resin linked N-(2-azidobenzoyl)amino esters of formula:

c) treating the resin linked N-(2-azidobenzoyl)amino esters of step (b), 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 resin linked benzodiazepines of formula:

; and, optionally d) suspending the resin linked benzodiazepines of step (c) in TFA/water at room temperature for 1-24 hours to form 1,4-benzodiazepin-2,5-diones of formula:

wherein:
R1 is H, lower alkyl, c-lower alkyl, -or (CH2)mR4, or R1 and R2, 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;
R2 is H, loweralkyl, arylR6R7R8, or heteroarylR6R7R8, or R1 and R2, 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;
R3 is H or loweralkyl;
R4 is aryl, substituted aryl, heteroaryl, substituted heteroaryl, NR3R5, CO2R3, CONR3R3, or OH;
R5 is H, lower alkyl, -CNHR3R3, or -C(O)R3;
R6, R7, and R8 is each, independently, H, lower alkyl, lower alkoxy, halogen, aryl, lower alkylthio, X-aryl, X-substituted aryl, lower alkylaryl, C(hal)3, -(CH2)mNR3R5, or -X-CH(CO2R3)2, or R6 and R7, together with the atoms to which they are attached, join to form a 5- or 6-membered heterocyclic ring; and X is O or S.

5. A method of Claim 4 wherein the involatile solvent is toluene, xylene, or chlorobenzene and the trivalent phosphorus reagent is triphenylphosphine or tributylphosphine.

6. A method of synthesizing 1,4 benzodiazepine-2,5-diones of the formulae:

and which comprises:
a) reacting a resin-linked .alpha.-amino ester of the formula:

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:
to produce resin-linked N-(2-azidobenzoyl)amino ester of the formula:

;

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 2-24 hr to produce a resin-linked 1,4-benzodiazepin-5-one of the formula:

and c) suspending said resin-linked 1,4-benzodiazepin-5-one in an acidic solution at room temperature ~ -24 hr. to produce a 1,4-benzodiazepin-2,5-dione of the formula:

7. A compound of the formula:

8. A compound of the formula:

9. A compound of the formula: