WO1996016333A1

WO1996016333A1 - METHODS FOR SYNTHESIZING DIVERSE COLLECTIONS OF β-LACTAM COMPOUNDS

Info

Publication number: WO1996016333A1
Application number: PCT/US1995/015261
Authority: WO
Inventors: Mark A. Gallop; Eric Gordon; Beatrice Ruhland
Original assignee: Affymax Technologies NV
Current assignee: Affymax Technologies NV
Priority date: 1994-11-23
Filing date: 1995-11-22
Publication date: 1996-05-30
Anticipated expiration: 1997-05-23
Also published as: AU4368896A

Abstract

Disclosed are methods for synthesizing very large collections of diverse β-lactam compounds on solid supports and synthetic compound libraries comprising β-lactam groups prepared by such methods.

Description

METHODS FOR SYNTHESIZING DIVERSE COLLECTIONS

OF β-LACTAM COMPOUNDS

BACKGROUND OF THE INVENTION

Field of the Invention

This invention is directed to methods for synthesizing very large collections of diverse β- lactam (2-azetidinones) compounds on solid supports. This invention is further directed to methods for identifying and isolating β-lactam compounds with useful and diverse activities from such collections. This invention is still further directed to the incorporation of identification tags in such collections to facilitate identification of compounds with desired properties.

References

The following publications, patents and patent applications are cited in this application as superscript numbers: ¹ International Patent Application Publication No. WO 93/06121

² U.S. Patent Application Serial No. 07/946,239 ³ U.S. Patent No. 5,143,854, issued September 1, 1992

⁴ Firestone, et al., Tetrahedron, 4^:2255 (1990) ⁵ Doherty, et al., Proc. Natl . Acad. Sci . USA, 90:8727 (1993) 261

-- 2 --

Burnett , et al . , J. Med. Chem . , 32: 1733 ( 1994 )

Georg and Ravikumar , Chapter 6 , pp . 295-368 , " Stereocontrolled J.etene-1-τιirιe Cycloaddi tion

Reactions" in "The Organic Chemistry of β- Lactamε" (1992)

⁸ Carey, et al., Advanced Organic Chemistry (Part B) , page 178, Plenum Publishing Company, New

York, New York (1977)

⁹ Evans and Sjogren, Tet. Lett . , 2£:3783 (1985)

10 Cooper, et al. , Pure and Applied Chem . , .51:485 (1987)

11 Georg and Wu Tetrahedron Lett . , 35:381-384 ¹² OOjjiimmaa,, pppp.. 119977--225555,, in " The Organic Chemistry ofβ-Lactams" (1993)

All of the above publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

State of the Art

Compounds having biological activity can be identified by screening diverse collections of compounds (i.e., libraries of compounds) produced through either molecular biological or synthetic chemical techniques. Such screening methods include methods wherein each member of the library is tagged with a unique identifier tag to facilitate identification of compounds having biological activity^1,2 or where the library comprises a plurality of compounds synthesized at specific locations on the surface of a solid substrate wherein a receptor is __ 3 __

appropriately labeled to identify binding to the compound, e.g., fluorescent or radioactive labels. Correlation of the labelled receptor bound to the substrate with its location on the substrate identifies the binding compound.

Central to these methods is the screening of a multiplicity of compounds in the library and the ability to identify the structures of the compounds which have a requisite biological activity.

Preferably, in order to facilitate synthesis and identification, the compounds in the library are typically formed on solid supports wherein the compound is covalently attached to the support via a cleavable or non-cleavable linking arm. In this regard, libraries of diverse compounds are prepared and then screened to identify "lead compounds" having good binding affinity to the receptor.

Pharmaceutical drug discovery relies heavily on studies of structure-activity relationships wherein the structure of "lead compounds" is typically altered to determine the effect of the alteration on activity. Alteration of the structure of the lead compounds permits evaluation of the effect of the structural alteration on activity. Thus libraries of compounds derived from a lead compound can be created by including derivatives of the lead compound and repeating the screening procedures.

Ideally, the compounds are synthesized in si tu on the solid support so that the support can be tagged to identify the synthetic steps employed and/or the derivative incorporated onto the support. However, relatively simple synthetic methods to produce a diverse collection of such derivatives on the supports are often not available. ---- 4 ----

One particular class of compounds which would be useful for inclusion in screening libraries are β- lactam compounds. These compounds form the basis of an important class of compounds having diverse pharmaceutical and chemical properties. In addition to their great clinical success achieved as antibacterial agents, β-lactams have also been found to possess other diverse biological activities, including mechanism-based inactivators of serine proteases such as elastase ' and inhibition of acyl- CoA:cholesterol acyltransferase (ACAT) . β-lactams are also valuable chiral starting materials for the synthesis of other natural and unnatural products.

A variety of solution phase techniques have been developed to prepare β-lactams ' . Generally, a ketene precursor (e.g. a carboxylic acid chloride) is in si tu converted to a ketene in the presence of a base and then, in the presence of an imine, undergoes a [2+2] cycloaddition to provide for a β-lactam.

However, the incorporation of a multiplicity of β-lactam derivatives on solid supports is not previously known. The ability to synthesize a multiplicity of β-lactam derivatives on a solid support or on different solid supports would enhance the structural variation of a library and provide important structure-activity information.

SUMMARY OF THE INVENTION

This invention is directed to general synthetic methods for incorporating a β-lactam group onto a solid support which methods can be employed in conjunction with known stochastic methods for preparing libraries of compounds comprising one or __ 5 __

more β-lactam groups. In one embodiment, the β-lactam compounds generated on the solid support via the methods described below can be further derivatized thereby elaborating the structure of these compounds .

In another embodiment, the β-lactam compounds generated on the solid support via the methods of this invention have a substituent at the 3 and/or 4- position thereof which substituent comprises a ketene precursor group or a group convertible to an imine, imidate, thioimidate or amidine functionality thereby permitting synthesis of polymeric β-lactam compounds or the appendage of a heterocyclic compound.

Solid supports containing such β-lactam groups preferably comprise a linking arm which links the solid support to the group. The linking arm can be either cleavable or non-cleavable and when cleavable, can be used to prepare a library of soluble β-lactam compounds. The library of β-lactam compounds, whether soluble or insoluble, can be screened to isolate individual compounds that possess some desired biological activity. In a preferred embodiment, each compound in the library is unique.

In another embodiment, the invention provides for methods of synthesizing N-unsubstituted-β-lactams.

These compounds are important building blocks for the preparation of both monocyclic and bicyclic antibiotics. Moreover, N-unsubstituted-β-lactams are useful precursors of chiral β-amino acids.

Accordingly, in one of its method aspects, this invention is directed to a method for synthesizing a β-lactam group covalently attached to a solid support which method comprises: -- 6 --

(a) providing a ketene precursor compound;

(b) converting the ketene precursor compound to a ketene;

(c) contacting the ketene produced in (b) above with a complementary compound comprising an imine, imidate, thioimidate or amidine functionality under conditions effective to provide for a β-lactam group wherein the ketene precursor compound and/or the complementary compound is covalently attached to a solid support.

Preferably, the ketene precursor compound is converted to the ketene in si tu in the presence of the complementary compound. In this embodiment, the ketene precursor compound and the complementary compound are combined into a reaction mixture and the precursor compound is in si tu converted to the ketene compound which then undergoes [2+2] cycloaddition with the complementary compound to provide for the β- lactam.

In a particularly preferred embodiment, the ketene precursor compound is a carboxylic acid halide (e.g., acid chloride or acid bromide) or an activated carboxylic acid ester and the ketene precursor is converted in si tu to the ketene by reaction with a base.

In one embodiment, the ketene precursor compound and the complementary compound are not coupled to the same compound and β-lactam formation is by intermolecular [2+2] cycloaddition.

In another embodiment, the ketene precursor compound and the complementary compound are coupled to the same compound having at different sites thereon both a ketene precursor functionality and __ 7 ---.

functionality selected from the group consisting of imine, imidate, thioimidate or amidine functional groups. In this embodiment, β-lactam formation can proceed by intramolecular [2+2] cycloaddition.

The solid supports prepared in the methods described above can be used, for example, in creating libraries of compounds in the manner described in International Patent Application Publication No. WO 93/06121 or in creating solid supports such as those described in U.S. Patent No. 5,143,854², to screen said compounds for biological activity. The disclosures of International Patent Application Publication No. WO 93/06121 and U.S. Patent No. 5,143,854 are incorporated herein by reference in their entirety.

Accordingly, in one of its composition aspects, this invention is directed to a library of diverse β- lactam structures comprising a plurality of solid supports having a plurality of covalently bound β- lactams, wherein the β-lactams bound to each of said supports is substantially homogeneous and further wherein the β-lactam bound on one support is different from the β-lactams bound on selected other supports.

In another of its method aspects, this invention is directed to a method for preparing a synthetic β- lacta compound library produced by synthesizing on each of a plurality of solid supports a single compound wherein each compound comprises at least one β-lactam group, which library is synthesized in a process comprising: a) apportioning the supports comprising a covalently bound ketene precursor group or a covalently bound complementary group comprising an imine, imidate, thioimidate or amidine functionality ---- ₈ ----

or a group convertible to an imine, imidate, thioimidate or amidine functionality among a plurality of reaction vessels, and b) exposing the supports in each reaction vessel under conditions wherein the ketene precursor group or the complementary group is converted to a β-lactam group wherein said β-lactam group is different for each of the reaction vessels.

In a preferred embodiment, this method further comprises pooling the supports produced in part b) .

In still another of its method aspects, this invention is directed to a method for preparing a synthetic polymeric β-lactam compound library produced by synthesizing on each of a plurality of solid supports a single compound wherein each compound comprises from 2 to 5 β-lactam groups, which library is synthesized in a process comprising: a) apportioning the supports comprising a covalently bound ketene precursor group or a covalently bound complementary group comprising an imine, imidate, thioimidate or amidine functionality or a group convertible to an imine, imidate, thioimidate or amidine functionality among a plurality of reaction vessels, b) exposing the supports in each reaction vessel under conditions wherein the ketene precursor group or the complementary group is converted to a β-lactam group wherein said β-lactam group is different for each of the reaction vessels; c) pooling the supports; d) repeating procedures a) through c) up to about 4 times; with the proviso that the β-lactam group produced in each run of procedures a) through c) above, except only optionally in the last run, comprises at least __ 9 __

one substituent at the 3 or 4 position thereof having a ketene precursor group or a group convertible to an imine, imidate, thioimidate or amidine functionality.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates the synthesis of imine 3. on a solid support, followed by reaction of imine 3_. with a ketene (not shown) formed from ketene precursor compound 4_. by reaction with triethylamine (NEt₃) 5_. in dichloromethane (CH₂C1₂) to provide for a β-lactam, as a mixture of stereoisomers 6_. and 1_, bound to the solid support via a cleavable linking arm. Figure 1 further illustrates cleavage of the β-lactam isomers, 6_. and 1_, from the solid support to provide for soluble β-lactam compounds 8. and S_.

Figure 2 illustrates the stereoselective synthesis of β-lactam __ bound to a solid support via a cleavable linking arm and subsequent cleavage of this linking arm to provide for soluble β-lactam 16.

Figure 3 illustrates the stereoselective synthesis of β-lactam 2_ bound to a solid support via a cleavable linking arm and subsequent cleavage of this linking arm to provide for soluble β-lactam 23.

Figure 4 illustrates the stereoselective synthesis of β-lactam 2_ bound to a solid support via a cleavable linking arm and subsequent cleavage of this linking arm to provide for soluble β-lactam 30.

Figure 5 illustrates the stereoselective synthesis of β-lactam 3_6 and subsequent conversion of this compound to carboxyalkyl amine __ , which is an enalapril type metalloprotease inhibitor. 61

-- 10 --

Figure 6 illustrates the synthesis of a β-lactam 47 having an amino group at the 3-position thereof which amino group can be converted to an imine and reacted with a ketene to provide for a β-lactam dimer

51 or can be converted to heterocycles ___ and 57.

Figures 7A-7D illustrates several cleavable linking arms for covalently linking compounds comprising at least one β-lactam group to the solid support.

Figure 8 illustrates several photocleavable linking arms for covalently linking compounds comprising at least one β-lactam group to the solid support.

Figure 9 illustrates the synthesis of imine 0 on a solid support from immobilized photolinker 58, followed by reaction of imine joO with a ketene (not shown) formed from phthalimido acetyl chloride by reaction with triethylamine (NEt₃) in dichloromethane (CH₂C1₂) to provide for a β-lactam .61, bound to the solid support via a cleavable linking arm. Figure 9 further illustrates the photolytic cleavage of the β- lactam jS_l from the solid support to provide for soluble β-lactam compound 62.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to synthetic methods for preparing β-lactam groups in si tu on solid supports and the use of these methods to incorporate β-lactam groups in large synthetic compound libraries.

Prior to discussing this invention in further detail, the following terms will first be defined: The term "substrate" or "solid support" refers to a material having a rigid or semi-rigid surface which contain or can be derivatized to contain reactive functionality which covalently links a compound to the surface thereof. Such materials are well known in the art and include, by way of example, silicon dioxide supports containing reactive Si-OH groups, polyacrylamide supports, polystyrene supports, polyethyleneglycol supports, and the like. Such supports will preferably take the form of small beads, pellets, disks, or other conventional forms, although other forms may be used. In some embodiments, at least one surface of the substrate will be substantially flat. A particularly preferred solid support is the acid-labile Sasrin resin.

The term "halogen" refers to fluorine, chlorine, bromine and iodine and preferably chlorine.

The term "ketene precursor" refers to any group, substituent or functionality which is convertible to a ketene group. Such precursors are known in the art and include, by way of example only, carboxyl acid halides and activated carboxyl acid esters each having a methine or methylene hydrogen atom α to the carbonyl atom of the carboxyl acid halide or the activated carboxyl acid ester and the like.

The term "an activated carboxyl acid ester having a methine or methylene hydrogen atom α to the carbonyl atom of the carboxyl group" refers to carboxyl groups of the formula -CH_nC(0)OR where R is any ester functionality which facilitates conversion to a ketene as compared to the carboxyl group in the absence of such an ester and n is an integer equal to 1 or 2. Such activated carboxyl acid esters are well known in 61

-- 12 --

the art and are described, for example, by Georg and Ravikumar which is incorporated herein by reference in its entirety.

The term "a complementary compound (group or functionality) comprising an imine, imidate, thioimidate or amidine functionality" refers to those compounds, groups and functionalities having an imine, imidate, thioimidate or amidine moiety which is reactive with ketenes to form β-lactam compounds.

Compounds comprising an imine, imidate, thioimidate or amidine functionality are known per se in the art and the particular compound employed is not critical . For example, imines can be prepared from a primary amine (e.g., an amino acid) and an aldehyde or ketone; imidates and thioimidates and amidines are conveniently prepared from imino chlorides by reaction with alkoxides or aryl oxides, thiolates or primary and secondary amines respectively. Imino chlorides, in turn, can be prepared from amides by reaction with phosphorus pentachloride. See, for example, Patai, The Chemistry of Amidines and Imidates, Wiley, New York, New York (1975) .

The term "a complementary compound having a group convertible to an imine, imidate, thioimidate or amidine functionality" refers to those compounds which contain a functional group which is convertible to an imine, imidate, thioimidate or amidine functionality by methods known in the art. For example, ketone and aldehyde groups are known to react with primary amines to form imines. Accordingly, compounds having a ketone, an aldehyde, or a primary amine contain a group convertible to an imine. Similarly, hydroxyl groups can be oxidized to provide for a ketone or an aldehyde group and carboxyl groups can be converted to ketones via reaction with an alkyl lithium reagent . /US95/15261

-- 13 —

Accordingly, hydroxyl and carboxyl groups comprise groups convertible to an imine. Other groups convertible to imidate, thioimidate or amidine functionalities are well known in the art. Examples of groups convertible to imidate, thioimidate and amidine functionality are recited above. The particular complementary compound and ketene precursor compound employed in the methods described herein are not critical.

Particularly preferred groups convertible to an imine functionality are amino acids wherein the amine group of the amino acid is employed to form an imine with the carbonyl group of a ketone or aldehyde.

In one embodiment, the complementary compound comprising an imine, imidate, thioimidate or amidine functionality is covalently attached directly to the solid support or is attached to the support via a linking arm. In this embodiment, subsequent cycloaddition of the complementary compound to a ketene will provide for covalent attachment of the resulting β-lactam to the solid support regardless of whether the ketene compound is initially bound to the solid support.

In another embodiment, the ketene precursor compound is covalently attached directly to the solid support or is attached to the support via a linking arm. In this embodiment, subsequent conversion of this precursor compound to the ketene followed by cycloaddition with the complementary compound comprising an imine, imidate, thioimidate or amidine functionality will provide for covalent attachment of the resulting β-lactam to the solid support regardless of whether the complementary compound is initially bound to the solid support. Linking arms are well known in the art and include, by way of example only, conventional linking arms such as those comprising ester, amide, carbamate, ether, thio ether, urea, amine groups and the like. The linking arm can be cleavable or non-cleavable. "Cleavable linking arms" refer to linking arms wherein at least one of the covalent bonds of the linking arm which attaches the compound comprising the β-lactam group to the solid support can be readily broken by specific chemical reactions thereby providing for compounds comprising β-lactam groups free of the solid support ("soluble compounds") . The chemical reactions employed to break the covalent bond of the linking arm are selected so as to be specific for bond breakage thereby preventing unintended reactions occurring elsewhere on the compound. The cleavable linking arm is selected relative to the synthesis of the compounds to be formed on the solid support so as to prevent premature cleavage of this compound from the solid support as well as not to interfere with any of the procedures employed during compound synthesis on the support.

Suitable cleavable linking arms are well known in the art and Figures 7A-7D illustrates several embodiments of such linking arms. Specifically, Figure 7A illustrates a cleavable Sasrin resin comprising polystyrene beads and a cleavable linking arm as depicted therein which linking arm is cleaved by strong acidic conditions such as trifluoroacetic acid. Cleavage results in breakage at the wavy line interposed between the oxygen and carbonyl moieties of the ester so as to provide for a compound terminating in a carboxylic acid. Figures 7B and 7C illustrate cleavable TentaGel AC and TentaGel PHB resins respectively, each comprising a polystyrene bead and the cleavable linking arm depicted therein both of which are cleaved by strong acidic conditions such as trifluoroacetic acid. Cleavage results in breakage at the wavy line interposed between the oxygen and carbonyl moieties of the ester so as to provide for a compound terminating in a carboxylic acid.

Figure 7D illustrates a cleavable TentaGel RAM resin comprising a polystyrene bead and a cleavable linking arm depicted therein which is cleaved by strong acidic conditions such as trifluoroacetic acid. Cleavage results in breakage at the wavy line interposed between the nitrogen and the benzhydryl carbon of the linking arm so as to provide for a compound terminating in an amide group. In this case, this linking arm facilitates formation of the amide bond by stabilizing the intermediate carbonium ion on the carbon atom between the two aromatic groups . Such stabilization permits selective bond cleavage as compared to bond cleavage for other amide groups of the compound comprising a β-lactam group.

The linker may be attached between the tag and/or the molecule and the support via a non-reversible covalent cleavable linkage. For example, linkers which can be cleaved photolytically can be used. Preferred photocleavable linkers of the invention include 6- nitroveratryloxycarbonyl (NVOC) and other NVOC related linker compounds (see PCT patent publication Nos. WO 90/15070 and WO 92/10092; see also U.S. patent application Serial No. 07/971,181, filed 2 Nov. 1992, incorporated herein by reference) ; the ortho- nitrobenzyl-based linker described by Rich (see Rich and Gurwara (1975) J. Am. Chem. Soc. 97:1575-1579; and Barany and Albericio (1985) J. Am. Chem. Soc. 107 : 4936-4942) and the phenacyl based linker discussed by Wang, (see Wang (1976) J. Orσ. Chem. 41:3258: and Bellof and Mutter (1985) Chimia 22:10) .

Other particularly preferred photocleavable linkers are described in copending patent application U.S. Serial No. 08/493,8755, filed June 23, 1995 which is a continuation in part application of U.S. Serial No. 08/374,492, filed January 17, 1995, each of which is incorporated herein by reference, and are shown in Figure 8.

"Non-cleavable linking arms" refer to linking arms wherein one or more of the covalent bonds linking the compound comprising a β-lactam group to the solid support can only be cleaved under conditions which chemically alters unintended parts of the structure of the compound attached thereto.

The term "β-lactam" or "2-azetidinone" refers to a saturated 4-member ring heterocyclic compound containing one (1) ring nitrogen atom which can be depicted as follows:

Substituents to the β-lactam group can occur at any of the 1, 3 and 4 positions thereof including the nitrogen atom in the manner depicted above. Such substituents are governed solely by the reagents employed thereby providing flexibility in preparing a large library of β-lactam compounds. Preferred substituents at the 3 and 4 positions include, by way of example only: alkyl groups of from 1 to 10 carbon atoms optionally substituted with 1 or more (typically up to 5) substituents selected from the group consisting of hydroxyl, halo, cyano, amino, mono- and di-alkylamines of from 1 to 10 carbon atoms in each alkyl group, alkoxy of from 1 to 10 carbon atoms, -SH, -SR where R is alkyl of from 1 to 10 carbon atoms, carboxyl, carboxyl esters of from 1 to 10 carbon atoms in the ester moiety, -NRC(0)R where R and R are independently selected from the group consisting of hydrogen and alkyl of from 1 to 10 carbon atoms, heterocycles having from 2 to 6 carbon atoms and 1 to 3 ring hetero atoms selected from the group consisting of nitrogen, oxygen and sulfur, aryl groups of from 6 to 10 carbon atoms optionally substituted with from 1 to 3 substituents on the aryl moiety selected from the group consisting of halo, hydroxyl, amino, cyano, carboxyl, nitro, alkyl of from 1 to 10 carbon atoms and alkoxy of from 1 to 10 carbon atoms, alkoxy of from 1 to 10 carbon atoms optionally substituted with 1 or more (typically up to 5) substituents selected from the group consisting of hydroxyl, halo, cyano, amino, mono- and di-alkylamines of from 1 to 10 carbon atoms in each alkyl group, alkoxy of from 1 to 10 carbon atoms, -SH, -SR where R is alkyl of from 1 to 10 carbon atoms, carboxyl, carboxyl esters of from 1 to 10 carbon atoms in the ester moiety,

-NR¹C(0)R² where R¹ and R² are independently selected from the group consisting of hydrogen and alkyl of from 1 to 10 carbon atoms, heterocycles having from 2 to 6 carbon atoms and 1 to 3 ring hetero atoms selected from the group consisting of nitrogen, oxygen and sulfur, aryl groups of from 6 to 10 carbon atoms optionally substituted with from 1 to 3 substituents on the aryl moiety selected from the group consisting of halo, hydroxyl, amino, cyano, carboxyl, nitro, alkyl of from 1 to 10 carbon atoms, and alkoxy of from 1 to 10 carbon atoms, amino, alkyl and dialkylamino of from 1 to 10 carbon atoms in each alkyl group,

-NR¹C(0)-X-R² where R¹ and R² are independently selected from the group consisting of hydrogen and alkyl of from 1 to 10 carbon atoms, aryl groups of from 6 to 10 carbon atoms optionally substituted with from 1 to 3 substituents on the aryl moiety selected from the group consisting of halo, hydroxyl, amino, cyano, carboxyl, nitro, alkyl of from 1 to 10 carbon atoms, and alkoxy of from 1 to 10 carbon atoms, and X is selected from the group consisting of a bond, 0, and NR¹ where R is as defined above, halo, hydroxyl,

-OC(0)Y-R where R is selected from the group consisting of alkyl of from 1 to 10 carbon atoms, aryl groups of from 6 to 10 carbon atoms optionally substituted with from 1 to 3 substituents on the aryl moiety selected from the group consisting of halo, hydroxyl, amino, cyano, carboxyl, nitro, alkyl of from 1 to 10 carbon atoms, and alkoxy of from 1 to 10 carbon atoms, and Y is selected from the group consisting of a bond and NR where R is as defined above, vinyl (=CH₂), alkenyl groups of from 2 to 10 carbon atoms, alkynyl groups of from 3 to 10 carbon atoms, carboxyl groups, carboxyl ester groups wherein the ester group comprises from 1 to 10 carbon atoms, heterocycles having from 2 to 6 carbon atoms and 1 to 3 ring hetero atoms selected from the group consisting of nitrogen, oxygen and sulfur optionally substituted with 1 to 3 substituents selected from the group consisting of hydroxyl, halo, cyano, amino, mono- and di-alkylamines of from 1 to 10 carbon atoms in each alkyl group, alkoxy of from 1 to 10 carbon atoms, -SH, -SR where R is alkyl of from 1 to 10 carbon atoms, carboxyl, carboxyl esters of from 1 to 10 carbon atoms in the ester moiety, -NR¹C(0)R² where R¹ and R² are independently selected from the group consisting of hydrogen and alkyl of from 1 to 10 carbon atoms, aryl groups of from 6 to 10 carbon atoms optionally substituted with from 1 to 3 substituents on the aryl moiety selected from the group consisting of halo, hydroxyl, amino, cyano, carboxyl, nitro, alkyl of from 1 to 10 carbon atoms, and alkoxy of from 1 to 10 carbon atoms,

R-C(O)- groups where R is hydrogen or an alkyl group of from 1 to 10 carbon atoms optionally substituted on the alkyl group with 1 or more (typically up to 5) substituents selected from the group consisting of hydroxyl, halo, cyano, amino, mono- and di-alkylamines of from 1 to 10 carbon atoms in each alkyl group, alkoxy of from 1 to 10 carbon atoms, -SH, -SR where R is alkyl of from 1 to 10 carbon atoms, carboxyl, carboxyl esters of from 1 to 10 carbon atoms in the ester moiety, -NRC(0)R where R¹ and R² are independently selected from the group consisting of hydrogen and alkyl of from 1 to 10 carbon atoms, heterocycles having from 2 to 6 carbon atoms and 1 to 3 ring hetero atoms selected from the group consisting of nitrogen, oxygen and sulfur, aryl groups of from 6 to 10 carbon atoms optionally substituted with from 1 to 3 substituents on the aryl moiety selected from the group consisting of halo, hydroxyl, amino, cyano, carboxyl, nitro, alkyl of from 1 to 10 carbon atoms, and alkoxy of from 1 to 10 carbon atoms, aryl groups of from 6 to 10 carbon atoms optionally from 1 to 3 substituents on the aryl moiety selected from the group consisting of halo, hydroxyl, amino, cyano, carboxyl, nitro, alkyl of from 1 to 10 carbon atoms, and alkoxy of from 1 to 10 carbon atoms.

Disubstitution at the 3 or 4 positions is also permitted and preferred substituents are those where each substitution is independently selected from the substituents recited above.

Suitable substituents at the 1 position include, by way of example only: alkyl groups of from 1 to 10 carbon atoms optionally substituted with 1 or more (typically up to 5) substituents selected from the group consisting of hydroxyl, halo, cyano, amino, mono- and di-alkylamines of from 1 to 10 carbon atoms in each alkyl group, alkoxy of from 1 to 10 carbon atoms, -SH, -SR where R is alkyl of from 1 to 10 carbon atoms, carboxyl, carboxyl esters of from 1 to 10 carbon atoms in the ester moiety, -NRC(0)R where R and R are independently selected from the group consisting of hydrogen and alkyl of from 1 to 10 carbon atoms, heterocycles having from 2 to 6 carbon atoms and 1 to 3 ring hetero atoms selected from the group consisting of nitrogen, oxygen and sulfur, aryl groups of from 6 to 10 carbon atoms optionally substituted with from 1 to 3 substituents on the aryl moiety selected from the group consisting of halo, hydroxyl, amino, cyano, carboxyl, carboxyl esters of from 1 to 10 carbon atoms in the ester, nitro, alkyl of from 1 to 10 carbon atoms, and alkoxy of from 1 to 10 carbon atoms, alkenyl groups of from 2 to 14 carbon atoms, alkynyl groups of from 3 to 14 carbon atoms, heterocycles having from 2 to 6 carbon atoms and 1 to 3 ring hetero atoms selected from the group consisting of nitrogen, oxygen and sulfur optionally substituted with 1 to 3 substituents selected from the group consisting of hydroxyl, halo, cyano, amino, mono- and di-alkylamines of from 1 to 10 carbon atoms in each alkyl group, alkoxy of from 1 to 10 carbon atoms, -SH, -SR where R is alkyl of from 1 to 10 carbon atoms, carboxyl, carboxyl esters of from 1 to 10 carbon atoms in the ester moiety, -NR¹C(0)R² where R¹ and R² are independently selected from the group consisting of hydrogen and alkyl of from 1 to 10 carbon atoms, aryl groups of from 6 to 10 carbon atoms optionally substituted with from 1 to 3 substituents on the aryl moiety selected from the group consisting of halo, hydroxyl, amino, cyano, carboxyl, carboxyl esters of from 1 to 10 carbon atoms in the ester, nitro, alkyl of from 1 to 10 carbon atoms, and alkoxy of from 1 to 10 carbon atoms, aryl groups of from 6 to 10 carbon atoms optionally from 1 to 3 substituents on the aryl moiety selected from the group consisting of halo, hydroxyl, amino, cyano, carboxyl, carboxyl esters of from 1 to 10 carbon atoms in the ester, nitro, alkyl of from 1 to 10 carbon atoms, and alkoxy of from 1 to 10 carbon atoms, and the like.

The term "substantially homogeneous" refers to collections of molecules wherein at least 80%, preferably at least about 90% and more preferably at least about 95% of the molecules are a single compound or stereoisomer thereof.

The term "stereoisomer" refers to a chemical compound having the same molecular weight, chemical composition, and constitution as another, but with the atoms grouped differently. That is, certain identical chemical moieties are at different orientations in space and, therefore, when pure, have the ability to rotate the plane of polarized light. However, some pure stereoisomers may have an optical rotation that is so slight that it is undetectable with present instrumentation. The compounds described herein may have one or more asymmetrical carbon atoms and therefore include various stereoisomers. All stereoisomers are included within the scope of the invention.

The term "removable protecting group" or "protecting group" refers to any group which when bound to a functionality such as hydroxyl, amino, or carboxyl groups prevents reactions from occurring at these functional groups and which protecting group can be removed by conventional chemical or enzymatic steps to reestablish the functional group. The particular removable protecting group employed is not critical.

Methods for Preparing β-Lactam Groups on Solid Supports The synthesis of a β-lactam group on the solid support is effected by reaction of a ketene with a complementary compound comprising an imine, imidate, thioimidate or amidine functionality. In turn, the ketene is generated from a ketene precursor compound by methods well known in the art for solution chemistry. Surprisingly, it has been found that these known methods can be conducted on solid supports wherein at least one of ketene or the complementary compound is covalently bound to the solid support thereby providing methods for generating libraries of compounds containing β-lactam groups on solid supports. The generation of a ketene from a precursor molecule via solution chemistry is documented in the art . However, one particularly preferred method is the treatment of a carboxylic acid halide (e.g., the chloride or bromide) having a methylene or methine hydrogen atom α to the carbonyl group with a base which converts the acid halide, in situ, to a ketene. The resulting ketene is then reacted with a complementary compound comprising an imine, imidate, thioimidate or amidine functionality to provide for the β-lactam.

An example of the entire reaction process is depicted in Figure 1 which figure illustrates formation of imine 2. as the complementary compound having imine functionality (derived from benzaldehyde

2 and

L-alanine bound to a solid support through the carboxyl group 1 ) , optionally in the presence of a dehydrating agent.

Figure 1 further illustrates the formation of imine compound 3., which serves as the complementary compound for reaction with the ketene, by conventional methods from a suitable aldehyde 2. (ketones can also be used) and amine . The reaction is typically conducted by contacting an amine with a 10-15 fold molar excess of an alkyl, aromatic, or (X,β-unsaturated aldehyde. The reaction is conducted in an inert solvent under conditions which eliminate water thereby forming imine 3..

Preferably, the water of reaction is removed from the reaction system to facilitate reaction completion. One means for effecting water removal is the use of molecular sieves (e.g., 4A molecular seives) in the reaction medium. Another means is to employ a solvent that will form an azeotrope with water so that water generated during reaction can be readily removed. Such preferred solvents include by way of example, benzene, toluene, etc. A preferred means to effect water removal is to use a dehydrating solvent such as trimethyl orthoformate to effect imine formation.

Imine 2 is then contacted with a ketene precursor compound, phenyloxyacetyl chloride 4_., optionally in the presence of a base (for example, triethylamine) to in si tu generate the ketene (PhO-CH=C=0) , not shown, which thereupon reacts with imine 3. to provide for isomeric β-lactams 6. and 1_ bound to a solid support.

The particular base employed is not critical and is selected relative to its ability to extract the methine or methylene hydrogen atom alpha to the carbonyl group of the ketene precursor thereby generating the ketene and to be compatible with both the starting materials and the products formed therefrom. Suitable bases include, by way of example only, triethylamine, trimethylamine, pyridine, and the like.

Each procedure in this reaction sequence is preferably conducted in a single reaction medium employing an inert diluent or a mixture of inert diluents. The inert diluent employed in the reaction is not critical and suitable diluents include, by way of example only, acetonitrile, benzene, toluene, methylene chloride, chloroform, carbon tetrachloride, tetrahydrofuran and the like.

The reaction is typically conducted by contacting the ketene precursor compound with at least a stoichiometric amount of a suitable base to form the ketene. Preferably, a large molar excess of ketene (relative to imine) at high concentrations to effect the cycloaddition reaction. Typically, the ketene precursor is present at a concentration of about 0.5 to 5 M, preferably, at about 0.5 to 1.5 M, and more preferably, at about 1 M. Typically, the base is present at a concentration of about 0.5 to 5 M, preferably, at about 0.7 to 2 M, and more preferably, at about 1.5 M.

This reaction is conducted under conditions sufficient to effect formation of the ketene and is preferably conducted at a temperature of from about -78°C to about 100°C, and preferably from about 0°C to about room temperature, for a period of from about 4 to about 24 hours and preferably for from about 5 to about 15 hours.

In a particularly preferred embodiment, ketene formation is preferably conducted in the presence of the complementary compound comprising an imine, imidate, thioimidate or amidine functionality which, upon in si tu ketene formation, reacts therewith to form a β-lactam under the conditions recited above.

The resulting β-lactam isomeric compounds 6. and 1_ are recovered by conventional methods, i.e., filtration, centrifugation, etc. Confirmation that the resin (i.e., solid support) contains the desired β-lactam compound can be accomplished by cleaving the β-lactam compound from a small portion of the treated resins (if a cleavable linking arm is employed) and subjecting this product to conventional analysis, e.g., nuclear magnetic resonance spectroscopy ( H, C, etc.), high performance liquid chromatography, and the like. Alternatively, the reaction can be monitored by use of appropriate resins using gel-phase C¹³-nuclear magnetic resonance spectroscopy. Suitable resins for this use include those illustrated in Figures 7B-7D attached. For example, the imine carbon of Schiff bases derived from the condensation of benzaldehyde labeled with a l^C at the carbonyl carbon with various resin-bound amino acids resonates at 160-165 ppm, and upon cycloaddition becomes C-4 of the β-lactam ring with a corresponding resonance at 63-65 ppm.

Alternatively, - ^" ^C labeled amino acids, such as alanine or valine, can be used. Using this method, one can monitor the disappearance of mine and formation of the β-lactam. Moreover, the method is sufficiently sensitive to monitor for the presence of isomers.

According to one embodiment, the ketene precursor comprises phthalimidoacetyl chloride or 4- nitrophthalimido glycine. Treatment of the corresponding α-phthalimido-β-lactam derivative with hydrazine or methyl hydrazine in an appropriate solvent yields the corresponding 3-amino-2- azetidinone. Preferably, the reaction is conducted in with about 10% to about 50% methylhydrazine in DMF or ethanol. More preferably, about 20% methyl hydrazine in DMF or ethanol (4M) is used.

Thus, the phthalimido group serves as a latent amino functionality which can be further modified, for example, through alkylation, acetylation, addition of a carbobenzoyl group, formation of a succinimide group ( see, e . g. , Wollenberg et al . U.S. Patent No. 4,612,132), etc., using techniques well known in the art. For example, the free 3-amino functionality can be further modiified through acylation. Acylation with a variety of amino acids, including valine and phenylalanine, using standard peptide coupling reagents proceeds smoothly to afford the desired 3- amido-2-azetidinones in excellent yield and purity upon cleavage from the resin.

Other means for forming ketenes from suitable ketene precursor compounds are well known in the art and include by way of example, treatment of activated esters of carboxylic acids having a methylene or methine hydrogen atom α to the carbonyl group with a base and photochemical conversion from metal carbenes. Suitable activating esters and metal carbenes are well known in the art and are described, for example, by Georg and Ravikumar . The particular ketene precursor compound and the method employed to convert this precursor to the ketene employed in the methods of this invention is not critical.

In the case of alkyl ketenes (derived from alkyl carboxylic acid chlorides) , it is contemplated that the use of very reactive imines like imino esters or imines derived from phenylglyoxal can be employed to readily prepare β-lactams having 3-alkyl substituents.

A preferred embodiment utilizes the amide- generating photolinker shown below

to form the complementary compound wherein the linker is tethered to the solid support via the phenolic hydroxyl, typically through a spacer. More specifically, the amino group of the linker can participate directly in Schiff base formation and subsequent [2+2] cycloaddition, as exemplified by the synthesis of two 3-phthalimido-2-azetidinones derivatives. See Figure 9. Photocleavage from the support provides a convenient synthesis of N- unsubstituted β-lactams, important precursors of both monocyclic and bicyclic antibiotics.¹² In addition, the use of a photolabile support offers opportunities to configure antimicrobial assays in which compounds are photochemically released from the solid supports directly onto a confluent lawn of a test organism.

Procedures for the synthesis of other complementary compounds such as those comprising an imidate, thioimidate and amidine functionality are also conventional and well known in the art as are ketene precursor compounds .

While Figure 1 illustrates formation of imine 3_. from an amine covalently attached to a solid support, it is understood, however, that aldehyde 2 (or a ketone) could likewise be covalently attached to the solid support and that a soluble amine (i.e., not attached to the solid support) could be employed to form the covalently bound imine.

It is further understood that the ketene precursor compound could be covalently attached to the solid support which can then be reacted with a soluble complementary compound (not attached to the support) to provide for a β-lactam group covalently attached to the support. In such a case, this reaction is conducted in the manner to that described above. The stereochemical course of the cycloaddition reaction has been extensively studied for solution chemistry and the stereochemical outcome is dependent on the nature of both the imine and ketene components. As depicted in Figure 1, the reaction of the ketene derived from phenoxyacetyl chloride with the imine 3_. derived from immobilized L-alanine 1 and benzaldehyde 2. affords a nearly equimolar mixture of cis diastereomer in complete analogy with solution phase chemistry. Typically, the cis diastereomeric β- lactams are formed in ratios from about 1:1 to about 3:2, depending on the steric bulk of the amino acid substituent.

Methods for asymmetric syntheses have been developed in homogeneous solution, and those that employ chiral ketene precursors have proven especially effective. ' Surprisingly, asymmetric syntheses conducted on solid phase resins also give predominantly single products even when sterically hindered amines (e.g., valine) are used. Such asymmetric syntheses are illustrated in Figures 2-4 which syntheses provide for selective preparation of resin bound β-lactams 1J5/ 2_2, and 2_ respectively. Cleavage of these β-lactams from the resin provides for soluble β-lactams __, 23_. and __ respectively.

The choice of suitable reagents among the ketene precursor compounds and the reagents employed to prepare the complementary compound having an imine, amidate, thioamidate or amidine group provides facile means for preparing β-lactams having a variety of substituents at the 1, 3 and 4 positions of the β- lactam. Moreover, chemical derivation on the substituents so formed leads to still other substituents. For example, O-protected hydroxyketenes and N-protected aminoketenes will lead to 3-hydroxy and 3-amino β-lactams after cycloaddition and deprotection. In turn, each group can be further modified by conventional methods to further elaborate this substituent. Hydroxy groups, for example, can be acylated or oxidized and amine groups can be alkylated or converted to amides by conventional methods . Figure 5 illustrates the formation of a 3-benzyloxy β- lactam 3_ from a benzyloxyprotected hydroxyketene precursor __ . After β-lactam _6 formation, the protecting benzyloxy group is removed with hydrogen (H₂) and subsequently oxidized with phosphorus pentaoxide (P₂0₅) in dimethylsulfoxide (DMSO) to provide for the 3-keto β-lactam _1_ . Subsequent ring expansion with m-chloroperbenzoic acid in methylene chloride (CH₂C1₂) leads to N-carboxyanhydride .38 which can be ring opened via either hydroxyl anion to provide for carboxyalkyl amine __ or via an amine to provide amide 40.

Figure 6 illustrates a general procedure for the formation of a β-lactam __ having a 3-protected amino group (Pg is a removable protecting group) which is converted to the free amino compound .47 and subsequently employed to prepare a β-lactam dimer 51. Alternatively, the 3-amino group of compound _J_ can be used to prepare heterocyclic compounds __\ and ___ . For example, the 3-amino group can be incorporated into a thiazolidinone or metathiazonone, as described in U.S. Serial No. 08/ , filed July 27, 1995 (Attorney Docket No. 1059.1), which application claims priority from PCT Application Serial No. PCT/US95/07988, filed June 23, 1995 and is a continuation in part of U.S. Serial No. 08/265,090, filed June 23, 1995, each of which is incorporated here in by reference in its entirety. The β-lactam with a free amino group is treated with a carbonyl compound, preferably an aldehyde, and a thiol compound, preferably, a ercapto carboxylic acid, to yield the corresponding thiazolidinone __l or metathiazanone.

Preferred substituents at the 1, 3 and 4 positions of the β-lactam which are prepared via the methods described herein from starting materials either known per se in the art or which can be prepared by art recognized methods are described above.

As illustrated in Figure 6, the 3-amino group on the β-lactam also serves as a basis for the preparation of β-lactam dimers and higher polymers by conversion of this amino group to an imine by reaction with a suitable aldehyde or ketone which in turn can be reacted with a ketene in the manner described above to prepare a β-lactam dimer. Similarly, substituents at the 3 or 4-positions comprising a ketene precursor functionality can be employed to prepare β-lactam dimers.

Method for Producing Larαe Synthetic Libraries of β- Lactam Compounds The above described synthetic methods can be incorporated into one or more reaction procedures in the stochastic methods described in International Patent Application Publication No. 93/06121 to prepare synthetic libraries of β-lactam compounds on solid supports. This application is incorporated herein by reference in its entirety. In such libraries, each solid support will preferably contain a single compound which compound is different to the compounds found on the other solid supports but each compound will also comprise a β-lactam group. It is understood, however, that the term "single compound" as used herein includes different regio and stereoisomers of that compound. Also, the term "single compound" does not mean that only one copy of that compound is attached to each support. Rather, multiple copies of that compound can be included on the support.

In general, such methods comprise apportioning the supports comprising a covalently bound ketene precursor or a covalently bound complementary compound comprising an imine, imidate, thioimidate or amidine group among a plurality of reaction vessels; exposing the supports in each reaction vessel under conditions wherein the ketene precursor or the complementary compound is converted to a β-lactam group wherein said β-lactam group is different for each of the reaction vessels; and then preferably pooling the supports.

In one embodiment, the ketene precursor is converted to a β-lactam group by first converting the ketene precursor to the ketene followed by reaction of the ketene with a complementary compound comprising an imine, imidate, thioimidate or amidine group.

In another embodiment, the complementary group comprising an imine, imidate, thioimidate or amidine group is converted to a β-lactam group by reaction with a ketene.

Preferably, the library will contain at least about 10² compounds, more preferably from about 10² to about 10¹ compounds and still more preferably from about 10³ to about 10 compounds.

In another preferred aspect of this embodiment, each solid support is tagged with an identifier tag that can be easily decoded to report the compounds formed on the solid support. The tag can be directly attached either to the solid support or the tag can be included on the compound itself. In this latter embodiment, cleavage of the compound from the solid support will still permit identification of the compound. Each of these embodiments is disclosed in International Patent Application Publication No. WO 93/06121 or WO 95/12608, each of which is corporated herein by reference in its entirety. Alternatively, a portion of the same compounds attached to a single support is cleaved and subjected to mass spectroscopy, nuclear magnetic resonance spectroscopy and/or other forms of direct structural analysis so as to identify the compound on the support.

Still another method for incorporating a tag with the solid support is disclosed in U.S. Patent Application Serial No. 08/146,886 and entitled "METHOD OF SYNTHESIZING DIVERSE COLLECTIONS OF COMPOUNDS" which application is incorporated herein by reference in its entirety.

In still another embodiment, the β-lactam group can be incorporated into each compound in a library of different compounds all of which are covalently linked to the same solid support in the manner described in U.S. Patent No. 5,143,854. Such a library of different compounds can be simultaneously screened for receptor binding or some other activity. U.S. Patent No. 5,143,854 is incorporated herein by reference in its entirety.

Additionally, libraries of compounds attached to solid supports can be used for a variety of additional uses as set forth in International Patent Application Publication No. WO 93/06121. EXAMPLES

The following examples are set forth to illustrate the claimed invention and are not to be construed as a limitation thereof.

Unless otherwise stated, all temperatures are in degrees Celsius. Also, in these examples, unless otherwise defined below, the abbreviations employed have their generally accepted meaning:

FMOC = fluorenyl ethyl oxycarbonyl

H-nmr = proton nuclear magnetic resonance HPLC = high performance liquid chromatography mL = milliliter mmol = millimol

NMP = N-methylpyrrolidone

TFA = trifluoroacetic acid THF = tetrahydrofuran μL = microliters

Additionally, the Sasrin resin described herein is commercially available from Bache Biosciences and the TentaGel Ac resin, TentaGel PHB resin and TentaGel RAM resin are commercially available from Rapp Polymere, Tubigen, Germany. Each of these resins is depicted in Figures 7A-7D respectively.

Example 1 illustrates a typical procedure for effecting solid phase synthesis of a β-lactam.

Example 2 illustrates the synthesis of a chiral ketene precursor compound suitable for use in preparing a chiral ketene which induces asymmetry to the ketene- imine cycloaddition reaction. Examples 3-10 illustrate asymmetric ketene-imine cycloaddition reactions using the chiral ketene precursor of Example 2. Examples 11 and 12 illustrate the formation of 3- phthalimido-2-azetidinone compounds wherein the phthalimido group serves as a latent amino functionality. Examples 13-27 illustrate the synthesis of a 25 compound library of different β- lactam compounds. Examples 28 and 29 illustrate the synthesis of 3-vinyl-2-azetidones. Example 30 illustrates the synthesis of 3-benzyloxy-2- azetidinone. Example 31 illustrates the use of a photolinker. Example 32 illustrates the use of a free amino group to form a heterocyclic compound.

EXAMPLE 1 Synthesis of N- (1-carboxyethyl) 3-phenoxy-4-phenyl-2-azetidinone

A. Procedure I

Commercially available FMOC-Ala-Sasrin (200 mg, 0.68 mmol loading) was deprotected by treating the resin with 30% piperidine/NMP (5 mL) for 45 minutes. The resin was filtered, washed (4 x 5 mL methylene chloride, 2 x 5 mL methanol, 2 x 5 mL diethyl ether) and dried under high vacuum for 15-20 minutes. The resin was poured into a 10 mL screw capped vial and suspended in methylene chloride (4 mL) . Benzaldehyde (276 μL, 2.72 mmol) and molecular sieves (4A, 10 pellets) were added to the vial and the mixture was heated to 40° to 42°C for 3 hours by placing the vial into a dry heating block. The resin was transferred into a centrifuge filter unit and washed (4 x 2 mL methylene chloride, 2 x 2 mL methanol, 2 x 2 mL diethyl ether) and dried under high vacuum for 30 minutes . The resulting support bound imine was poured in a 10 mL screw capped vial and suspended in methylene chloride (4 mL) . Triethylamine (379 μL, 2.72 mmol) was added to the vial and the mixture was cooled to -78°C. To the suspension was added phenoxyacetyl chloride (283 μL, 2.04 mmol) dropwise and the mixture was kept for 10 minutes at -78°C and placed on a shaker table and agitated for 15 hours at 25°C. The resin was transferred into a centrifuge filter unit, washed (4 x 2 mL methylene chloride, 2 x 2 mL methanol, 3 x 2 mL diethyl ether) and dried under high vacuum for 30 minutes. The resin was placed in a vial and treated with 2% TFA in methylene chloride (3 mL) for 45 minutes. The resin was filtered and the filtrate was rotary evaporated to dryness to give the title compound.

B. Procedure II

Sasrin resin pre-loaded with an N-Fmoc-protected amino acid (0.165 mmol, 0.3 g of resin, loading 0.55 mmol/g) was treated with a solution of 30% piperidine in N-methylpyrrolidone (NMP) for 45 minutes. The resin was rinsed with NMP or DMP, methylene chloride, methanol, and ether, and dried under reduced pressure.

The resin was suspended in a mixture of methylene chloride (1.5 mL) and trimethylorthoformate (1.5 mL) and the aldehyde (2.3 mmol) was added. After agitating for 3 hours, the resin was rinsed with methylene chloride, methanol, and ether and dried under reduced pressure.

The resin was transfered to a glass vial, suspended in methylene chloride (3 mL) and cooled to 0°C. To the suspension was added triethylamine (3.3 mmol) followed by a slow addition of the acid chloride (2.5 mmol) . The reaction mixture was left at 0°C for 5 minutes and then agitated overnight at room temperature. The resin was filtered, rinsed with DMF, methylene chloride, methanol and ether and dried under reduced pressure.

The product was cleaved from the support by treating the resin with a solution of 3% (v/v) TFA/methylene chloride for 45 minutes. For products derived from amino acids having acid-labile side chain protection, the crude material was subjected to a second TFA treatment (50% TFA/methylene chloride) to remove the protecting groups. The solution was filtered, and after removal of the solvent, the crude product was purified by preparative HPLC.

C. Examples Following the procedures set forth above, the following compounds have been prepared: cis-1- ( (S) -2-propionic acid) -3-phenoxy-4-phenyl- azetidin-2-one; cis-1- ( (S) -2-propionic acid) -3- (1-methylvinyl) - 4- (2,2-diphenylvinyl)azetidin-2-one; cis-1- ( (S) -2-propionic acid) -3-vinyl-4- (2,2- diphenylvinyl)azetidin-2-one; cis-1- [ (S) -2- (6-aminohexanoic acid) ] -3-phenoxy- 4-phenylazetidin-2-one; cis-1- ( (S) -2-succinic acid) -3-phenoxy-4- phenylazetidin-2-one; cis-1- [ (S) -2- (3-hydroxy)butyric acid] -3-phenoxy- 4-phenylazetidin-2-one; cis-1- [ (S) -2- (3-methyl)butanoic acid] -3-phenoxy- 4-phenylazetidin-2-one; cis-1- [ (S) -2- (3-methyl)butanoic acid] -3-phenoxy- 4- (2-furyl) -azetidin-2-one; cis-1- [ (S) -2- (3-methyl)butanoic acid] -3-phenoxy- 4- (2-thiophenyl) -azetidin-2-one; cis-1- [ (S) -2- (3-methylJbutanoic acid] -3-phenoxy- 4- (2-pyridyl) -azetidin-2-one; cis-1- [ (S) -2- (3-methyl)butanoic acid] 3-phenoxy- 4- (2, 2-diphenylvinyl) -azetidin-2-one,- and cis-1- [ (S) -2-propionic acid] -3-phenoxy-4- (cyclohexyl) -azetidin-2-one. EXAMPLE 2 Synthesis of (4S-Phenyl Oxazolidinyl) Acetyl Chloride

Ethyl bromoacetate (1 equivalent) was added to 4S-phenyloxa-zolidinone (1 equivalent) and sodium hydride (1 equivalent) in THF and the mixture was stirred at 0°C for 2 hours and the resulting ester saponified (NaOH, H₂0-THF, 1 hour, 25°C) to provide 4 (s)-phenyloxazolidinyl acetic acid. Transformation of the carboxylic acid group of this compound to the acid chloride was achieved with oxalyl chloride (1.5 equivalents, toluene, 60°C) . The unpurified acid chloride was employed in subsequent reactions.

Examples 3-10

Synthesis of β-lactams from Amino Acids and (4S-Phenyl Oxazolidinyl) Acetyl Chloride

The following examples illustrate asymmetric ketene-imine cycloaddition reactions using the chiral ketene precursor of Example 2.

Specifically, using the procedure set forth in Example 1, the (4S-phenyl oxazolidinyl) acetyl chloride of Example 2 and the appropriate amino acid, the β-lactams of the Table 1 below were prepared, after cleavage of the cleavable linking arm with 1 to 3% TFA in dichloromethane.

TABLE 1

In each case, only one cis isomer was recovered with great purity. The "alanine-furane" derived β- lactam was contaminated with 13% of the corresponding methyl ester formed during cleavage. In the presence of TFA and methanol, the β-lactam opens to give the corresponding methyl ester. This can be resolved by avoiding a methanol rinse of the resin after cleavage.

These results on solid supports are in accordance with the literature for solution chemistry. The chiral center in the imine does not have any significant influence on the asymmetric induction and no appreciable double asymmetric induction is observed. Only the chiral center in the ketene plays a key role in the asymmetric induction. After removing the chiral auxiliary, this process provides an effective route to chiral 3-amino- 2-azetidones.

EXAMPLES 11-12 Synthesis of β-lactams from Amino Acids and Phthalimido Acetyl Chloride

The following examples illustrate the formation of 3-phthalimido-2-azetidinone compounds. The phthalimido group serves as a latent amino functionality which can, at the appropriate point in the synthesis, be easily removed with N-methyl hydazine without disrupting the β-lactam ring.

Phthalimido acetyl chloride was prepared in the manner consistent with Example 2 above and was converted to the phthalimido ketene with triethylamine, dichloromethane at -78°C in the manner of Example 1 above and then treated with solid phase imines to form β-lactam compounds. The resulting β- lactam was cleaved by use of TFA (2%) in dichloromethane. The crude product was purified by preparative HPLC and each fraction analyzed by ^""Ή-NMR and mass spectroscopy thereby providing the expected cis β-lactam compounds as set forth in Table 2 below. These compounds were, however, contaminated with the corresponding methyl ester which contamination could be obviated by eliminating the methanol rinse. TABLE 2

Ex. R Stereochemistry Purity

No.

11 -CH3 2 cis β-lactams 27% 2 cis Me esters (alanine) 45%

12

-CH(CH3⁾2 2 cis β-lactams 54% 2 cis Me esters (valine) 30%

Following the procedures set for above, the following compounds were also prepared: cis-1- [ (S) -2- (3-methylJbutanoic acid] -3- phthalimido-4- (2-furyl) -azetidin-2-one; cis-1- [ (S) -2- (3-methyl)butanoic acid] -3- phthalimido-4- (2-pyridyl) -azetidin-2-one; cis-1- [ (S)-2- (3-methyl)butanoic acid]-3- phthalimido-4- (2,2, -diphenylvinyl) -azetidin-2-one;

General Procedure for Deprotection

A. Procedure I

To a suspension of the phthalidmido-β-lactam prepared as in Example 8 above (360 mg) in dichloromethane (20 ml) was added methyl hydrazine (10 equivalents) . The reaction was stirred overnight and filtered. The resin was washed with methanol and diethyl ether and dried to yield the desired 3-amino- 2-azetidinone. Acylation of the 3-amino-2-azetidinone prepared above was accomplished by treating a suspension of the 3-amino-2-azetidinone (80 mg) in NMP with an Fmoc- protected amino acid (Fmoc-valine, 5 equivalents) and then with HOBt (5 equivalents) and DIC (5 eqivalents) . The reaction was stirred overnight, washed with NMP, methanol, and ether, and dried. Treatment of the resin with 2% TFA in dichloromethane (2 ml) yielded 55% of the desired soluble dipeptide.

B. Procedure II

Methyl hydrazine (0.11 mL, 2.1 mmol) was added to a suspension of Sasrin resin bearing the 3- phthalimido-azetidin-2-one (0.14 mmol based on the initial loading of the resin) in methylene chloride (1.5 mL) at room temperature. After agitating the suspensin overnight, the resin was filtered, washed with DMF, methylene chloride, methanol, and ether and dried under reduced pressure. The product was then suspended in DMF (1 mL) and treated with an N-FMoc- protected amino acid (0.69 mmol), HOBt (0.75 mmol) and DIC (0.75 mmol) . The reaction mixture was agitated for 6 hours, filtered, and washed with DMF, methylene chloride, methanol, and ether. The resin was dried and thea resulting β-lactam was cleaved from the support by exposure to 5% TFA/methylene chloride for 1 hour.

Following the above procedures, the following compounds were prepared: cis-1- [ (S) -2- (3-methylJbutanoic acid] -3- (N-Fmoc- L-valinamido) -4-styryl-azetidin-2-one; and cis-1- [ (S) -2- (3-methyl)butanoic acid]-3- (N-Fmoc- L-valinamido) -4- (2-furyl) -azetidin-2-one. EXAMPLES 13-27

Synthesis of a Library of β-Lactam Compounds on Solid Supports

The purpose of these examples is to illustrate the formation of a library of β-lactam compounds formed via known stochastic methods.

Specifically, this library was formed as follows. FMOC-Val-Sasrin was deprotected with 30% piperidine in NMP. The resin was filtered, rinsed (dichloromethane, diethyl ether) , dried and split equally in 5 flasks and suspended in dichloromethane. To each vial was added an excess of one aldehyde, R₂- CHO where Rj is as defined in Table 3 below, (15 equivalents) and a small amount of molecular sieves (4A) . This mixture was shaken for 3 hours and the resins were filtered, rinsed, dried and then each reaction vessel was equally divided into three aliquots to provide a total of 15 reaction vessels the resins for each of which were suspended in dichloromethane. To each aliquot was added an excess of triethylamine (20 equivalents) and the mixture was cooled to -78°C, treated with one of three acid chlorides, R₂-CH₂C(0)C1 where R₂ is as defined in

Table 4 below (prepared as in Example 1 above) , and left at 25°C for 15 hours. The resins were filtered, rinsed, dried, and cleaved with 1-2% TFA in dichloromethane.

The resulting 15 products are shown in Table 5 below and were analyzed by H-NMR and mass spectroscopy. It is important to note that even by using a sterically hindered amino acid like valine, these cycloaddition reactions gave the desired products. TABLE 3 Ri-CHO

TABLE 4

10 R₂-CH₂-C(0)C1

15 TABLE 5

Example No. Rl R2

13 A X

14 A Y

15 A z

16 B X

17 B Y

18 B z

19 C X

20 C Y

21 C z

22 D X

23 D Y

24 D z

25 E X

26 E Y

27 E z

In Table 5, A, B, C, D, and E correspond to the R_x substituents recited in Table 3 whereas X, Y and Z correspond to the R₂ substituents listed in Table 4. As in Examples 11-12, the use of phthalimido provided for 2 cis isomers whereas the use of 4S-phenyl oxazolidinyl provided for one cis isomer regardless of the aldehydes used.

Additionally, these stochastic methods could be extended to include the use of different primary amine compounds in place of valine used in this example. Such different primary amine compounds include, by way of example only, different naturally occurring amino acids, D-isomers of naturally occurring amino acids, ω-amino acids, and the like. -- 46 —

In a preferred embodiment, the library of β- lactam compounds can be prepared as above provided that at least one of the following conditions is satisfied 1) at least two different ketene precursor groups are used to produce the β-lactam group; 2) at least two different complementary groups are used to produce the β-lactam group; and 3) at least two different sets of reaction conditions are used to produce the β-lactam group.

EXAMPLES 28 and 29 Synthesis of 3-vinyl-2-azetidones

The following examples illustrates the synthesis on solid supports of substituents having unsaturation at the 3-position of the β-lactam.

Specifically, cycloaddition reactions were conducted in a manner similar to that described above using crotonyl chloride and dimethylacryloyl chloride, an imine derived from aldehyde E of Table 3 and alanine, and triethylamine. In both cases, the expected cis β-lactams were formed with purity greater than 80% which β-lactams contained a 3 and 4 substituent having unsaturation.

EXAMPLE 30 Synthesis of 3-benzyloxy-2-azetidinone

3-Benzyloxy substituents on the β-lactam can be obtained by condensing benzyloxyacetyl chloride and an imine derived from alanine (attached to a solid support) and benzaldehyde in the manner described above. The desired β-lactam is recovered in the manner described above. EXAMPLE 31 Imine formation on photolinker and subsequent cycloaddition A. General Procedure

TentaGel S H2 resin (0.29 mmol) was supended in DMF (3 mL) and treated with 4-(4-(l-(9- fluorenylmethoxycarbonylamino)ethyl)-2-methoxy-5- nitrophenoxy)butanoic acid (452 mg, 0.87 mmol), HOBt (129 mg, 0.955 mmol) and DIC (148 mg, 0.955 mmol) . After agitating the reaction mixture at room temperature overnight, the resin was filtered, washed with DMF, methylene chloride, methanol, and ether and dried. The resin (300 mg, 0.08 mmol) was treated with 30% piperidine in DMF (1 mL) for 30 minutes, filtered, washed with DMF, methylene chloride, methanol, and ether and dried under high vacuum. The free amino aci resin was then suspended in trimethylorthoformate (1 mL) and treated with aldehyde (1.7 mmol) for 3 hours at room temperature to form resin-bound imine. The resin was washed several times with methanol and ether and dried under high vacuum.

The imine resin was suspended in methylene chloride (1 mL) and cooled to 0°C. Triethylamine (1.2 mmol) was added followed by slow addition of phthalimidoacetyl chloride (1.0 mmol) . The reaction mixture was agitated overnight at room temperature, then filtered, washed with DMF, methylene chloride, methanol, and ether and dried. The b-lactam product was cleaved from the resin by photolysis in DMSO at 365 run for 6 hours.

B. Alternate Procedure To a suspension of the deprotected photolinker (35 mg) in tri ethyl orthoformate (1 ml) was added t- butyl glyoxalate (50 mg) . The reaction was heated at 70°C for 3 hours with frequent shaking. The reaction mixture was cooled, filtered, washed, and dried to yield the corresponding imine.

The 3-phthalimido-2-azetidinone was prepared by treating the imine with phthalimido acetyl chloride

(25 equivalents) and triethylamine (30 equivalents) using the procedures set forth in Examples 7-8 above.

The reaction was conducted at 0°C.

The β-lactam was cleaved from the resin through photolysis in ethanol or DMSO using a 500 W Hg ARC lamp fitted with a 350-450 nm dichroic mirror at a 10 mW/cπ.2 power level measured at 365 nm. After photolysis, the samples were filtered and the filtrate was collected. The samples were analyzed by HPLC, mass spectroscopy and NMR and the data indicated that the desired β-lactam was produced in high purity. This reaction sequence is shown schematically in Figure 9.

C. Compounds

Following the procedures set forth above, the following compounds have been made: cis-3-phthalimido-4- (tert-butoxycarbonyl) - azetidin-2-one; cis-3-phthalimido-4- (2, 2-diphenylvinyl) -azetidin- 2-one; and cis-3-phthalimido-4- (2-pyridyl) -azetidin-2-one.

EXAMPLE 32

Utilization of the 3-amino group in the production of a heterocyclic compound

The corresponding imine was produce from cinnamaldehyde and valine on Sasrin resin as described above. The β-lactam was formed from the imine and phthalimido acetyl chloride as described above. The b-lactam was deprotected with methyl hydrazine in methylene chloride as described above.

The resulting amine was treated with benzaldehyde (20 eq. , 0.75 M) and mercaptoacetic acid (35 eq. , 2M) in a 1:1 trimethylorthoformate:THF mixture at 70°C for two hours. Alternatively, the reaction was conducted in THF with molecular sieves at 70°C. Both sets of conditions resulted in the production of a thiazolidinone coupled to the b-lactam. The adduct was cleaved from the resin by treatment with 10% TFA in methylente chloride.

HPLC showed four peaks representing four isomers, two for each β-lactam (two cis isomers) . The structures of the compounds were elucidated by NMR and mass spectroscopy.

Claims

---- so —WHAT IS CLAIMED IS

1. A library of diverse β-lactam structures comprising a plurality of solid supports having a plurality of covalently bound β-lactams, wherein the β-lactams bound to each of said supports is substantially homogeneous and further wherein the β- lactam bound on one support is different from the β- lactams bound on the other supports.

2. The library according to Claim 1 wherein said covalently bound β-lactams are bound to the solid support via a linking arm.

3. The library according to Claim 2 wherein said linking arm is non-cleavable.

4. The library according to Claim 2 wherein said linking arm is cleavable.

5. The library according to Claim 1 wherein each of said solid supports further comprises a surface bound tag which identifies the molecule attached thereto.

6. The library according to Claim 5 wherein said tag is an oligonucleotide.

7. A method for synthesizing a β-lactam group covalently attached to a solid support which method comprises: (a) providing a ketene precursor compound; (b) converting the ketene precursor compound to a ketene; (c) contacting the ketene produced in (b) above with a complementary compound comprising an imine, imidate, thioimidate or amidine functionality under conditions effective to provide for a β-lactam group wherein the ketene precursor compound and/or the complementary compound is covalently attached to a solid support.

8. The method according to Claim 7 wherein said ketene precursor is converted to said ketene in the presence of said complementary compound so that upon ketene formation, it in si tu reacts with said complementary compound to provide for a β-lactam.

9. The method according to Claim 8 wherein the ketene precursor is a carboxylic acid halide or an activated carboxylic acid ester and the ketene precursor is converted in situ to the ketene by reaction with a base.

10. The method according to Claim 7 wherein the ketene precursor compound and the complementary compound are different from each other and β-lactam formation proceeds via intermolecular [2+2] eye1oaddition.

11. The method according to Claim 7 wherein the ketene precursor compound and the complementary compound are the same compound having at different sites thereon both a ketene precursor functionality and functionality selected from the group consisting of imine, imidate, thioimidate or amidine functional groups and β-lactam formation proceed via intramolecular [2+2] cycloaddition.

12. A method for preparing a synthetic β-lactam compound library produced by synthesizing on each of a plurality of solid supports a single compound wherein each compound comprises at least one β-lactam group, which library is synthesized in a process comprising: a) apportioning the supports comprising a covalently bound ketene precursor group or a covalently bound complementary group comprising an imine, imidate, thioimidate or amidine functionality or a group convertible to an imine, imidate, thioimidate or amidine functionality among a plurality of reaction vessels, and b) exposing the supports in each reaction vessel under conditions wherein the ketene precursor group or the complementary group is converted to a β-lactam group provided that at least one of the following conditions is satisfied: 1) at least two different ketene precursor groups are used to produce the β-lactam group; 2) at least two different complementary groups are used to produce the β-lactam group; and 3) at least two different sets of reaction conditions are used to produce the β-lactam group.

13. The method according to Claim 12 wherein each reaction vessel contains a different compound.

14. The method according to Claim 12 which further comprises pooling the supports.

15. The method according to Claim 12 wherein said ketene precursor group or said complementary group are covalently bound to the solid support via a linking arm.

16. The method according to Claim 15 wherein said linking arm is non-cleavable.

17. The method according to Claim 15 wherein said linking arm is cleavable.

18. The method according to Claim 12 wherein said solid support further comprises a surface bound tag which identifies the molecule attached thereto.

19. The method according to Claim 18 wherein said tag is an oligonucleotide.

20. The method according to Claim 12 wherein each of said supports contains a compound having a different β-lactam group.

21. The method according to Claim 12 wherein said supports comprise a ketene precursor compound which is converted to a β-lactam group by conversion to the ketene followed by reaction with a complementary compound comprising an imine, imidate, thioimidate or amidine functionality.

22. The method according to Claim 21 wherein the ketene precursor compound is a carboxylic acid halide or an activated carboxylic acid ester and the ketene precursor is converted to the ketene by reaction with a base.

23. The method according to Claim 22 wherein said ketene precursor is converted to said ketene in the presence of said complementary compound so that upon ketene formation, it in si tu reacts with said complementary compound to provide for a β-lactam.

24. The method according to Claim 12 wherein said supports comprise a complementary compound comprising an imine, imidate, thioimidate or amidine functionality which is converted to a β-lactam group by reaction with a ketene compound.

25. The method according to Claim 24 wherein said complementary compound comprises an imine functionality.

26. A method for preparing a synthetic polymeric β-lactam compound library produced by synthesizing on each of a plurality of solid supports a single compound wherein each compound comprises from 2 to 5 β-lactam groups, which library is synthesized in a process comprising: a) apportioning the supports comprising a covalently bound ketene precursor group or a covalently bound complementary group comprising an imine, imidate, thioimidate or amidine functionality or a group convertible to an imine, imidate, thioimidate or amidine functionality among a plurality of reaction vessels, b) exposing the supports in each reaction vessel under conditions wherein the ketene precursor group or the complementary group is converted to a β-lactam group wherein said β-lactam group is different for each of the reaction vessels; c) pooling the supports; d) repeating procedures a) through c) up to about 4 times; with the proviso that the β-lactam produced in each repeat of procedure a) through c) above, except only optionally in the last repeat, comprises at least one substituent at the 3 or 4 position thereof having a ketene precursor group or a group convertible to an imine, imidate, thioimidate or amidine functionality.

27. A compound comprising a solid support having a plurality of covalently bound β-lactams, wherein the β-lactams bound to said support are substantially homogeneousa.