NZ207346A

NZ207346A - Preparation of carbapenem derivatives

Info

Publication number: NZ207346A
Application number: NZ207346A
Authority: NZ
Inventors: P Dextraze
Original assignee: Bristol Myers Co
Priority date: 1983-03-08
Filing date: 1984-03-01
Publication date: 1987-01-23
Also published as: IT8419945A0; HUT34980A; DK140584D0; IL71149A; SE8901162D0; DK140584A; YU49486A; IE840549L; AT387968B; FR2542316A1; AU7592487A; KR840008157A; IT1178465B; ES537349A0; HU193895B; PT78211B; IE57098B1; SG35490G; CA1284324C; YU42084A

Description

<div class="application article clearfix" id="description"> New Zealand Paient Spedficaiion for Paient Number £07346 10 DRAWINGS NEW ZEALAND PATENTS ACT, 1953 No.: Date: COMPLETE SPECIFICATION 2 °7346 j Priority Qate(s): ?. " gmplete Specification Filed: ?. Class: C.TlPMh • .C.??M. ■ 73?A . Xf.?.?>.•? 7.?.. . AGQ9.4.*7. o, n E3 JAN 1987 Publication Date: Pf P.O. Journal, No: 1 ~,M/t T>r> -• .-<j4 CARBAPENEM PROCESS iI We, BRISTOL-MYERS COMPANY, a State of Delaware corporation having its offices at 345 Park Avenue, New York, New York 10154, United States of America; hereby declare the invention for which Ic/ we pray that a patent may be granted to me/us, and the method by which it is to be performed, to be particularly described in and by the following statement:- - 2 - 207346 BACKGROUND OF THE INVENTION The present invention is directed to a new process for the preparation of carbapenem derivatives having a 2-substituent of the formula -s-a-r14 wherein a represents cyclopentylene, cyclohexylene or C2~C6 alkylene optionally substituted by one or more C,-C. alkyl groups 14 14 and r represents a quaternized nitrogen-containing aromatic or non-aromatic heterocycle attached to a throuah a quaternary nitrogen atom. The carbapenem derivatives prepared by the process of the present invention are disclosed and claimed in U.S. Patent Specifications Nos. 4,536,335 and 4,552,696; the entire disclosure of each of these specifications is incorporated herein by reference. U.S. Patent Specification No. 4,552,696 discloses preparation of carbapenem antibiotics of the formula coor IA 8 1 wherein R is hydrogen and R is selected from the group consistino of hydrogen; substituted and unsubstituted: alkyl, alkenyl and alkynyl, having from 1-10 carbon atoms; cycloalkyl and cycloalkyl- alkyl, having 3-6 carbon atoms in' the cycloalkyl ring and 1-6 carbon atoms in the alkyl moieties; phenyl; aralkyl, aralkenyl; and aralkynyl wherein the aryl moiety is phenyl and the aliphatic portion has 1-6 carbon atoms; heteroaryl, heteroaralkyl, hetero-' cyclyl and heterocyclylalkyl wherein the hereto atom or atoms in the above-named heterocyclic moieties are selected from the group *->• Jrt?. / c - 3 - consisting of 1-4 oxygen, nitrogen or sulfur atoms ana the alkyl moieties associated with said heterocyclic moieties have 1-6 carbon atoms; wherein the substituent or substituents relative to the above-named radicals are selected from the group consisting of Ci-C6 alkyl optionally substituted by •amino, halo, hydroxy or carboxyl halo -OR3 " 3 4 -OCNR R 0 " 3 4 -CNR R 3 4 -NR R 3 4 -S02NR R 0 II 3 4 -NHCNR R 0 3 P 4 R CNR - -C02R3 =0 0 « 3 -OCR -SR3 0 II 5 -SR* 0 II e -SR II O -CK 20 - 4 -3 -oso.r J 3 -0S02R -NR"5S02R4 3 4 -nrjc=nr . i? 3 4 -nr cojr -N02 wherein, relative to the above-named substituents, the croups r3 and R.^ are independently selected from hydroaen; aDcyl, alXenvl ana aDcynyl, having from 1-10 carbon atoms; cycloalkyl,' cycloalkylalkyl and alkylcycloalkyl, having 3-5 carbon atoms r"*-. in the cvcloalkyl rinc and 1-6 carbon atoms in "the aDcyl moieties; phenyl; aralkyl, aralkeny1 and aralkynyl wherein the aryl moiety is phenyl and the aliphatic portion has 1-6 carbon atoms; and heteroaryl, heteroaralky1, beterocyclyl and heterocyclylalkv1 wherein the hetero atoa or a tons in the above-named heterocyclic moieties are selected from the croup consisting of 1-4 oxygen, nitrogen or sulfur atoms and the alkyl moieties associated with said heterocyclic moieties have 2 4 1-6 carbon atoms, or R and R taken together vith the nitrogen to which at least one is attached may form a 5-or 5-membered 9 ^ nitrooea-containing heterocyclic ring; R is as defined for R" "1 R except that it may not be hydrogen; or wherein R~ and R taken_ together.represent C2-C10 alkyliaene or C2-C10 alkyliaene ' substituted by hydroxy; A is cyclopentylene, cyclohexylene or C2~Cg alkylene optionally substituted by one or more cj_~C4 groups; is hydrogen, an anionic charge or a conventional - 5 - readily removable carboxyl protecting croup, providing that when R is hydrogen or a protecting group, there is also present a counter anion; and e -N ft' represents a substituted or unsubstituted mono-, bi- or polycyclic aromatic heterocyclic radical containing at least one nitrogen in the ring and attached to A through a ring nitrogen, thereby forming a quaternary ammonium group; and pharmaceutically acceptable salts thereof by the process shown in the following reaction scheme: O R8 H n COOR diphenyl chlorophosphate — > III R8 B rv o •O?(OC6H5)2 COOR2' HS-A-OH > A - alkylene or Cc-Cr cvcloalkylene 5 o COOR methanesulfonyl .chloride r> - 6 - ) n7 '346 h8 h N s-a-oso2ck3 "coor 3® VI Q e8 e 3-a-i 0^~N NW' n Ag ,©'P = counter anion) ii r8 h n. ■s-A-H^J coor & i'a o h N © s-a-n coor' ia 207346 7 U.S. Patent Specification No. 4,536,335 discloses preparation of carbapenem antibiotics of the formula R 1 0 IB 8 1 wherein R is hydrogen and R is selected from the group consisting of hydrogen; substituted and unsubstituted: alkyl, alkenyl and alkynyl, having from 1-10 carbon atoms; cycloalkyl and cycloalkyl-alkyl, having 3-6 carbon atoms in the cycloalkyl ring and 1-6 carbon atoms in the alkyl moieties; phenyl; aralkyl, aralkenyl and aralkynyl wherein the aryl moiety is phenyl and the aliphatic portion has 1-6 carbon atoms; heteroaryl, heteroaralkvl, hetero-cyclyl and heterocyclylalkyl wherein the hetero atom or atoms in the above-named heterocyclic moieties are selected from the group consisting of 1-4 oxygen, nitrogen or sulfur atoms and the alkyl moieties associated with said heterocyclic moieties have 1-6 carbon atoms; wherein the substituent or substituents relative to the above-named radicals are independently selected from the group consisting of o _ -.wy 2 0 /: 3 4 6 I - 8 - i- C^-Cg alkyl optionally substituted by amino, halo, hydroxy or carboxyl halo -or3 0 l) 3_4 -ocnr r 0 " 3 * n -cnr r' 3 4 -nr r - nr3 < nr3r4 3 4 -so2nrjr* 0 " 3 4 -nhcnr r 0 3 if 4 r cnr -co2r3 =0 0 -ocr3 -sr3 O H g -sr 0 II g -sr 'w 0 -cn -N 3 3 -oso3r -oso,r3 3 i -nr s02r - 9 - 207 3 4 -NR C=NR P • 3 4 -NR C02R -N02 ' wherein, relative'to 'the above-named substituents, the grouc-s R^ and R4 are independently selected from hydrogen; alkyl, • alkenyl and alkynyl, having ■ from' 1-10 carbon atoms; cycloalkyl, cycloalkylalkyl and alkyleycloaIky1, having 3-6carbon atons in the cycloalkyl ring and 1-6 carbon atoms' in the alkyl moieties? " . phenyl; aralkyl, aralkenyl and aralkynyl wherein the aryl moiety is phenyl and the aliphatic portion has 1-6 carbon atoms; and heteroaryl, heteroaralkyl, heterocvclyl and heterocyclylal^iv.;:'.' • wherein the hetero-atom or atoms in the above-named heterocyclic moieti-es are selected from the" croup consisting. cf 1-4 oxygen, nitrogen or sulfur 'atoms and the-alkyl moieties associated with 3 4 said heterocyclic moieties have 1-6'carbon atoms, or R and R taken together with the nitrogen to which at least one is attached may form a 5-or■6-membered nitrogen-containing heterocyclic ring; 9 3 R is as defined for R except that it may not be hydrogen; or 1 8 " wherein' R and R taken together represent Cj-C^p alkyliaene or Cj-C,0 alkyliaene substituted by hydroxy; R^ is. selected from the group consisting of substituted'and unsubstituted: alkyl, alkenyl and alkynyl, having from 1-10 carbon, atoms; cycloalkyl and cyclo- • alky lalkyl, having .3-6 carbon atoms in the cycloalkyl ring and 1-6 carbon atoms in the alkyl moieties; phenyl; aralkyl, aralkenyl and aralkynyl wherein the aryl moiety .is phenyl and the aliphatic portion has 1-6 carbon ■ atoms; heteroaryl, heteroaralkyl, hetero-^..' cycly 1 and heterocyclylalkyl wherein the hetero atcm or atoms in the above-named heterocyclic moieties are selected from the group consisting of 1-4 oxygen, nitrogen or sulfur atoms and the alkyl moieties associated with said heterocyclic moieties have 1-6 carbon atoms; wherein the above-named R^ radicals are optionally substituted by 1-3 substituents independently selected from: I o U 2073 i & - 10 - C^-Cg alkyl optionally substituted by snino, fluoro, chlcro, carboxv'l, hydroxy or carbamoyl; fluoro, chloro or bromo; -or3 ; -0c02r3 ; -ocor3 ; -oconr3r4 ; ' . -0S02R2 ; ' ,_y -OXC ; —NR R r3conr4- ; -NR3C02R4 ; 3 3 4 -nr conr r 3 4 -kr s02r ; -sr3 ; Q -S-R ; 0 0 1 9 -s-r ; O "S03e3 ! .-co2s3 ; -conr3r4 -CN; or phenyl optionally substituted by 1-3 fluoro, 3 ' 4 chloro, bromo, C,-Cc alkyl, -OR , -NR~R , *5 ^ ^ ^ "5 ^ -S0,R , -CO,R or -CONR R , wherein R , R and 9 2 R in such'R substituents are'as defined tbove; - 11 - or R may represent a divalent phenylene or C^-C^ alkylene croup joined to the ring so as to form a bridged polycyclic group; A is cyclopentylene, or cyclohexvlene or C~-Cc alkylene optionally substituted by one " 2 b 2 or more C^-C^ alkyl croups; R is hydrogen., an anionic charge or a.conventional readily removable carboxyl protecting croup, provid-ing that when R is hydrogen or a protecting group, there is also present a counter ion; and - *o represents a substituted or unsubstituted mono-, bi- or polycyclic non-aromatic heterocyclic radical containing at least one nitrogen in the ring and attached to A through a ring nitrogen, thereby forming a quaternary ammonium group; and pharmaceutically acceptable salts thereof, by the process shown in the following reaction scheme: >8 H diphenyl chlorophosphate COOR III - 12 - 20^346 .8 H (°C6H5)2 COOR IV HS—A OH > A=alkylene or C^-Cg cycloalkylene n .8 H (5^- N £—A—OH 2' COOR methanesulfonyl chloride n V R8 H N :S-A-0S02CH3 COOR .6 VI R8 H -N -S-A-I COOR n Ag~ X6 ^ II ,8 H /r~ 0 N I 'B S\ S-A— "N COOR ,6 optional de-blocking - 13 - 207346 IB n To elaborate on the prior art scheme, starting material III is reacted in an inert organic solvent with diphenyl chlorophosphate in the presence of a base to give intermediate IV. Intermediate IV is then reacted with a mercaptan reagent of the formula HS-A-OH in an inert organic solvent and in the presence of a base to give intermediate V. Intermediate V is then acylated with methanesulfonyl chloride in an inert organic solvent and in the presence of base to give intermediate VI which is reacted with a source of iodide ions in an inert organic solvent to give intermediate II. Intermediate II is reacted with the desired amine in an inert organic solvent and in the presence of silver ion to produce the quaternized product I'A or I'B which may then be deblocked to give the corresponding de-blockec carbapenem of Formula IA or IE. The above-described process has several disadvantages. Thus, for example, the process involves several steps which ' advantageously could be reduced in number. The overall reaction yield is also quite low and the quaternization step is performed on the entire carbapenem compound. It would be desirable to have a new process for producing compounds of Formula IA or IB which (1) involves fewer reaction steps, (2) gives higher yields, (3) allows / the quaternized amine to be formed first and then attached to the carbapenem nucleus at a later'stage in the synthesis and (4) can be used to more easily form quaternary amine products with a wide variety of amines, i.e. amines with steric hindrance and those with low pK^ values. - 14 - The present invention provides a novel process for preparation of carbapenem derivatives of the formula // \ 2 0 COOR I 8 1 wherein R is hydrogen and R is selected frora the group consisting of hydrogen; substituted and unsubstituted: alkyl, alkenyl and alkynyl, having from 1-10 carbon atoms; cycloalkyl and cycloalkylalkyl, having 3-6 carbon atoms in the cycloalkyl ring and 1-6 carbon atoms in the alkyl moieties; phenyl; .aralkyl, aralkenyl and aralkynyl wherein the aryl moiety is phenyl and the aliphatic portion has 1-6 carbon atoms; heteroaryl, heteroaralkyl, heterocyclyl and heterocyclylalkyl wherein the hetero atom or atoms in the above-named heterocyclic moieties are selected from the group consisting of 1-4 oxygen, nitrogen or sulfur atoms and the alkyl moieties associated with said heterocyclic moieties have 1-6 carbon atoms; wherein the substituent or substituents relative to the above-named radicals are selected from the group consisting of - 15 - G C^-Cg alkyl optionally substituted by amino, halo, hydroxy or carboxyl halo -or3 H 34 -ocnr r " 3 4 -cnr r •u -s02nrjr4 o "3 A -nhcnr r 0 3H 4 r cnr - -c02rj =0 0 h 3 -ocr -sr3 0 // 9 -sr 0 II 9 -sr . jj O — cn - 16 n -OSO..R3 i 3 -OSC^R -Na3£02R4 ■5 4 -Nir^=NR R3 3 4 -HR C02R -N02 wherein, relative to the above-named substituents, the croups R3 and R^ are independently selected from hydrogen; alkyl, alkenyl ad alkynyl, havir.c from 1-10 carbon atoms; cycloalkyl, cycloalkylalkyl and alkyIcycloalky 1, having 3-6 carbon atcns in the cycloalkyl ring and 1-6 carbon atoms in the alkyl moieties; phenyl; aralkyl, aralkenyl a_nd aralkynyl vherein the aryl moiety is phenyl ana the aliphatic portion has 1-5 carbcn atoms; ana heteroaryl, heteroaralkyl, heterocyclyl and heterocyclylalkyl wherein the hetero atom or atoms in the above-named heterocyclic moieties are selected from the croup consisting of 1-4 oxygen, nitrogen or sulfur atoms a^jc the alkyl moieties associated vith said heterocyclic moieties have 3 4 1-5 carbon atoms, .or R .anc R taken together vith the nitrogen to which at least one is attached may form a 5-or 6-membered 5 . 2 nitronen-containing heterocyclic ring; R is as defined for R" 1 C except that it may not be hydrogen; or vherein R~ and R taken together represent C2-ClQ alkylidene or C2-Cl0 alkyliaene substituted by hydroxy; A is cyclopentylene, cyclohexylene or Calkylene- optionally substituted by one or more alkyl groups; R^ is hydrogen, an anionic charge or a conventional readily removable 2 carboxyl protecting group, providing that when R is hydrogen or a protecting group, there is also present a counter o - 17 - 14 anion; and R is a quaternized nitrogen-containing aromatic or non-aromatic heterocycle attached to A through a ring nitrogen, thereby forming a quaternary ammonium group, or a pharmaceuticallv acceptable salt thereof, which process comprises reacting an intermediate of the formula COOR 18 21 wherein R and R are as defined above, R is a conventional, readily removable carboxyl protecting group and L is a conventiona leaving groupj with a thiol compound of the formula HS-A-R' 14 ,e VII 14 0 wherein A and R are as defined above and X is a counter anion in an inert solvent and in the presence of ba.seJ to produce a carbapenem product of the formula .8 H S-A-R COOR 14 ,9 .8 14 0 A, R and X are as defined above and, if wherein R , R", R desired, removing the carboxyl protecting group R4 to give the corresponding de-blocked compound of formula I, or a pharmaceutical^ acceptable salt thereof. - 18 - 207346 The carbapenem compounds of Formula I are potent antibacterial agents or intermediates useful in the preparation of such agents. The compounds of general Formula I above contain the carbapenem nucleus 1 O 4 and may thus be named as l-carba-2-penem-3-carboxylic acid derivatives. Alternatively, the compounds may be considered to have the basic structure ^ and named as 7-oxo-l-azabicyclo(3.2.0)hept-2-ene-2-carboxylic acid derivatives. While the present invention includes compounds wherein the relative stereochemistry of the 5,6-protons is cis as well as trans, the preferred compounds have the 5R,6S (trans) stereochemistry as in the case of thienamycin. The compounds of Formula I may be unsubstituted in the 6-position or substituted by .substituent groups previously disclosed for other carbapenem derivatives. More specifically, - 19- - 8 1 R may be hydrogen and R may be hydrogen or a non-hydrogen substituent disclosed, for example, in European Patent Application 8 1 38,869 (see definition of R-) . Alternatively, R and R taken o together may be C2~C10 a^^y^-^^ene or C2-(~10 substituted, •for exainple, by hydroxy. 1 8 To elaborate on the definitions for R and R : (a) The aliphatic "alkyl", "alkenyl" and "alkynyl" groups may be straight or branched chain having 1-10 carbon atoms; preferred are 1-6, most preferably 1-4, carbon groups; J when part of another substituent, e.g. as in cycloalkyl-alkyl, or heteroaralkyl or aralkenyl, the alkyl, alkenyl and alkynyl group preferably contains 1-6, most preferably 1-4, carbon atoms. (b) "heteroaryl" includes mono-, bi- and polycyclic aromatic heterocyclic groups containing 1-4 0, N or S atoms; preferred are 5- or 6- raembered heterocyclic rings such as thienyl, furyl, thiadiazolyl, oxadiazolyl, triazolyl, isothiazolyl, thiazolyl, imidazolyl, isoxazolyl, tetrazolyl, • oxazolyl, pyridyl, pyrazinyl, pyrimidiny1, pyridazinyl, pyrrolyl, pyrazolyl, etc. ^ (c) "heterocyclyl" includes mono-, bi- and polycyclic saturated or unsaturated non-aromatic heterocyclic groups containing 1-4 0, N or S atoms; preferred are 5- or 6-membered heterocyclic rings such as morpholinyl, piperazinyl, piperidyl, pyrazolinyl, pyrazolidinvl, imidazolinyl, imidazolidiny1, pyrrolinyl, pyrrolidiny1, etc. (d) "halo" includes chloro, bromo, fluoro and iodo and is preferably chloro or bromo. / - 20 - The term "conventional readily removable carboxyl protecting group" refers to a known ester group which has been employed to block a carboxyl group during the chemical reaction steps described below and which can be removed, if desired, by methods which do not result in any appreciable destruction of the remaining portion of the molecule, e.g. by chemical or enzymatic hydrolysis, treatment with chemical reducing agents under mild conditions, irradiation with ultraviolet light or catalytic hvdrogenation. Examples of such ester protecting groups include benzhydryl, p-nitrobenzyl, 2-naphthylmethy1, allyl, benzyl, trichloroethyl, silyl such as trimethylsilyl, phenacyl, p-methoxybenzvl, acetonyl, o-nitrobenzyl, 4-pyridyl-methyl and C^-C^ alkyl such as methyl, ethyl or t-butyl. Included within such protecting groups are those which are hydrolyzed under physiological conditions such as pivaloyloxy-methyl, acetoxymethyl, phthalidyl, indanyl and methoxvmethyl. Particularly advantageous carboxyl protecting groups are p-nitrobenzyl which may be readily removed by catalytic hydro-genolysis and allyl which can be removed by Pd(P0^)^-catalyzed reaction. above include the nontoxic acid addition salts, e.g. salts with mineral acids such as hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, etc. and salts with organic acids such as maleic, acetic, citric, succinic, benzoic, tartaric, fumaric, mandelic, ascorbic, lactic, gluconic and malic. Compounds of Formula I in the form of acid addition salts may be written as The pharmaceutically acceptable salts referred to R 8 H R 1 X 0 N 2 'COOR 2 R = H or protecting group r\ 207346 - 21 - 0 Q where X represents the acid anion. The counter anion X may be selected so as to provide pharmaceutically acceptable salts for therapeutic administration but, in the case of 0 intermediate compounds of Formula I, X may also be a toxic .anion. In such a case the ion can be subsequently removed or substituted by a pharmaceutically acceptable anion to form active end product for therapeutic use. When acidic or basic 1 14 groups are present in the R group or on the quaternized R radical, the present invention may also include suitable base or acid salts of these functional groups, e.g. acid addition salts in the case of a basic group and metal salts (e.g. sodium, potassium, calcium and aluminum), the ammonium salt and salts with nontoxic amines (e.g. . trialkylamines, procaine, dibenzylamine, 1-ephenamine, N-benzyl-S-phenethylamine, N,N1 - dibenzylethylenediamine, etc.) in the case of an acidic group. 2 Compounds of Formula I wherein R is hydrogen, an anionic charge or a physiologically hydrolyzable ester group together with pharmaceutically acceptable salts thereof are useful as antibacterial agents. The remaining compounds of Formula I are valuable intermediates which can be converted into the above-mentioned biologically active compounds. A preferred embodiment of the present invention impounds c is hydrogen, CH^CI^- 8 1 comprises compounds of Formula I wherein R is hydrogen and R CH, CH OH OH v3 v3 i i CHT" CH CH-, C- or CH3CH- Among this subclass, the preferred compounds are those in which R^" is OH I CH^CH-, most preferably compounds having the absolute configuration 5R, 6S, 8R. Another preferred embodiment comprises compounds of I in which R s radical of the formula 1 8 Formula I in which R and R taken together form an alkylidene HOCH- \2 C= / CH, The alkylene or cycloalkylene radical A in the compounds of Formula I may be cyclopentylene cyclohexylene or C2~"Cg alkylene optionally substituted by one or more C1~C4 alkyl substituents. Preferred A substituents are cyclopentylene cyclohexylene or alkylene of the formula R10 R12 I I -C C- R11 R13 in which R^, R^, R^"2 and R^"3 are each independently hydrogen o C1-C4 alkyl. A preferred embodiment comprises those compounds o Formula I in which" substituent A is -CH2CH2-, -CHCH^-/ iH3 or -CH-CH- . 2 I CH3 In the case of certain compounds of Formula I having a cycloalkylene or branched alkylene A substituent, one or more -additional assymmetric carbon atoms may be created which result in formation of diastereoisomers. The present invention includes mixtures of such diastereoisomers as well as the individual Durified diastereoisomers. - 23 - 14 The quaternized R substituent may be an optionally substituted mono-, bi- or polycyclic aromatic or non-aromatic heterocyclic radical containing at least one nitrogen in the ring and attached to A through a ring nitrogen, thereby forming a quaternary ammonium group. 14 One preferred class of R substituents may be represented by the general formula which is meant to define a substituted or unsubstituted mono-, bi- or polycyclic heteroaryl radical containing at least one nitrogen in the ring and attached to a carbon atom.of substituent A through a ring nitrogen, thereby forming a quaternary ammonium group. The heteroaryl radical may be optionally substituted by such substituents as C^-C^ alkyl, alkvl substituted by hydroxy, amino, carboxy or halo, C^-Cg cycloalkyl, C^-C^'alkoxy, C^-C^ alkylthio, amino, C2.-C"4 al^y!311^110' (C]_~C4 alkyl) amino, halo, C^-C^ alkanoylamino, alkanoyloxy, carboxy, 0 II -C-OC1~C4 alkyl, hydroxy, amidino, guanidino, trifluoromethvl, phenyl, phenyl substituted by one, two or three amino, halo, hydroxy1, trifluoro-methyl, alkyl or alkoxy groups, heteroaryl and hetero aralkyl' in which the hetero atom or atoms in the above-named heterocyclic moieties .are selected from the group consisting of 1-4 O, N or S atoms and the alkyl moiety associated with heteroaralkyl has 1-6 carbon atoms. The heteroaryl radical attached' to substituent A is preferably a 5- or 6- membered aromatic heterocyclic radical containing a quaternized nitrogen atom (which is directly bonded to a carbon atom of the alkylene or cycloalkylene radical) and, optionally, one or more additional hetero atoms selected from 0,' N or S. While, in general, any heteroaryl radical bonded to A - 24 - via a quaternized nitrogen atom is found to produce biologically active carbapenem derivatives, a preferred embodiment comprises compounds in which represents a radical selected from the group consisting of (a) NV>< wherein , R® and are independently selected from'hydrogen; C1~C4 alkyl; alkyl substituted by hydroxy, amino, carboxy or halo; C^-Cg cycloalkyl; C^-C^ alkoxy; alkylthio; amino; C^-C4 alkylamino; di(C^-C4 alkyl)amino; halo; Cj_-C4 alkanoylamino C^-C4 alkanoyloxy; carboxy; 0 II , -C-OC^-C4 alkyl; hydroxy, amidino; guanidino; trifluoromethyl; phenyl; phenyl substituted by one, two or three amino, halo, hvdroxyl, trif luoromethyl, C]_~C4 alkyl or C^-C4 alkoxy groups; and heteroaryl and heteroaralkyl in which the hetero atom or atoms in the above-named heterocyclic moieties are selected from the group consisting of 1-4 oxygen, nitrogen or sulfur atoms and the alkyl moiety associated with said hetero- Lr J aralkyl moiety has 1-6 carbon atoms; or wherein two of R^, or R' taken together may be a fused saturated carbocyclic ring, a fused aromatic carbocyclic ring, a fused saturated heterocyclic ring or a fused heteroaromatic ring, (b) ;n. or N' - 25 - optionally substituted on a carbon atom by one or more substituents independently selected from alkyl; alkyl substituted by hydroxy, amino, carboxy or halogen; C-^-Cg cycloalkyl; alkoxy; C^-C^ alkylthio; amino; C^-C^ alkylamino; di(C^-C4 alkyl) amino; halo; Cj_-C4 alkanoylamino; alkanoyloxy; 0 II carboxy; -C-OC^-C^ alkyl; hydroxy; amidino; guanidino; trifluoro-methyl; phenyl; phenyl substituted by one, two or three amino, halo, hydroxyl, trifluoromethyl, alkyl or C^-C^ alkoxy groups; and heteroaryl or heteroaralkyl in which the heteroatom or atoms in the above-named heterocyclic moieties are selected from the group consisting of 1-4 oxygen, nitrogen or sulfur atoms and the alkyl moiety associated with said heteroaralkyl moiety has 1-6 carbon atoms, or optionally substituted so as to form a fused carbocyclic or heterocyclic ring; (c) ©. N- N H N IS N or e4 N optionally substituted on a carbon atom by one or more substituents independently selected from alkyl; C^-C^ alkyl substituted by hydroxy, amino, carboxy or halogen; C^-Cg cycloalkyl; C^-C^ . alkoxy; alkylthio; amino; C^-C^ alkylamino; di(C^-C4 alkyl) - amino; halo; c*l~c4 alkanoylamino; C-^-C^ alkanoyloxy; carboxy; - 1? ' -C-OC^-C^ alkyl; hydroxy; amidino; guanidino; trifluoromethyl; phenyl; phenyl substituted by one, two or three amino, halo, - 26 - hydroxyl, trifluoromethyl, C^-C^ alkyl or C^-C4 alkoxy groups; and heteroaryl or heteroaralkyl in which the hetero atom or atoms in the above-named heterocyclic moieties are selected from the group consisting of 1-4 oxygen, nitrogen or sulfur atoms and the alkyl moiety associated with said heteroaralkyl .moiety has 1-6 carbon atoms, or optionally substituted so as to form a fused carbocyclic or heterocyclic ring; (d) -N X ll N N" ©I N N. N 01 ✓N N N or © N ^ j optionally substituted on a carbon atom by one or more substituen independently selected from alkyl; C^-C4 alkyl substituted by hydroxy, amino, carboxy or halogen; C^-Cg cycloalkyl; alkoxy; C^-C^ alkylthio; amino; C^-C4 alkylamino; difC^-C^ alkyl) amino; halo; C^-C'4 alkanoylamino; cj_~c4 alkanoyloxy; carboxy? O II -C-OC^-C4 alkyl; hydroxy; amidino; guanidino; trifluoromethyl; phenyl; phenyl substituted by one, two or three amino, halo, hydroxyl, trifluoromethyl, C^-C4 alkyl or C^-C4 alkoxy groups; and heteroaryl or heteroaralkyl in which the hetero atom or atoms in the above-named heterocyclic moieties are selected from the group consisting of 1-4 oxygen, nitrogen or sulfur atoms and the alkyl moiety associated with said heteroaralkyl moiety has 1-6 carbon atoms , or optionally substituted so as to form a fused -• carbocyclic or heterocyclic ring; "k. wherein X is 0, S or NR'in which R is C-^-C^ alkyl or phenyl, said radical being optionally substituted on a carbon atom by one or more substituents independently selected from alkyl; C., -C^ aLkyl substituted by hydroxy, amino, carboxy or halocen; "C,-C, cycloalkvl; C,-C. alkoxv; C.-C. alkvlthio; arino; C.-C, 3 6 14 *14" 14 alkylamino; di(C^-C4 alkyl)amino; halo; Cj_-C4 alkanoylamino; C^-C^ alkanoyloxy; carboxy; O II -C-OC^-C^ alkyl; hydroxy; amidino; guanidino; trifluoromethyl; phenyl; phenyl substituted by one, two or three amino, halo, hydroxyl, trifluoromethyl, aUcyl or alkoxy groups; and heteroaryl or heteroaralkyl in which the hetero atom or atoms in the above-named heterocyclic moieties are selected from the group consisting of 1-4 oxygen, nitrogen or sulfur atoms-anc the alkyl moiety associated with said heteroaralkyl moiety has 1-6 carbon atoms, or optionally substituted so as to form a fused carbocyclic or heterocyclic ring; •90S 9 ( f) r. A r. N- [l ^ © fi \v/N" ' %< 9 6 X N.- N N- - 28 - wherein X is O, S or NR in which R is C^-C4 alkyl or phenyl, said radical being optionally substituted on a carbon atom by one or more substituents indeoendently selected from C.-C. 1 4 alkyl; alkyl substituted by hydroxy, amino, carboxy or halogen; C^-Cg cycloalkyl; C^-C^ alkoxy; C^-C4 alkylthio; amino; C^-C4 alkylamino; di(C^-C4 alkyl)amino; halo; C^-C4 alkanoylamino C^-C4 alkanoyloxy; carboxy; 0 II -C-OC^-C4 alkyl; hydroxy; amidino; guanidino; trifluoromethyl; phenyl; phenyl substituted by one, two or three amino, halo, hydroxyl, trifluoromethyl, C^-C^ alkyl or C^-C4 alkoxy groups; and heteroaryl or heteroaralkyl in which the hetero atom or atoms in the above-named heterocyclic moieties are selected from the group consisting of 1-4 oxygen, nitrogen or sulfur atoms and the alkyl moiety associated with said heteroaralkyl moiety has 1-6 carbon atoms; and 0 N-zr-N- ^N~N / \ / \ M , n ' N . N-R ' v^/N-R N =■ N £ N-R J \ ' !' X © XT T? N N-R ft - 4 2 0 7 - 29 - wherein R is alkyl or phenyl, said radical being optionally substituted on the carbon atom by a substituent selected from C^-C^ alkyl; alkyl substituted by hydroxy; amino, carboxy or halogen;- C^-Cg cycloalkyl; alkoxy; alkylthio; amino; alkylamino; di(C^-C4 alky1)amino; alkanoylamino; carboxy; 0 II -C-OC-^-C^ alkyl; hydroxy; amidino; guanidino; trifluoromethyl; phenyl, phenyl substituted by one, two or three amino, halo, 'hydroxyl, trif luoromethyl, a^yl or C^-C^ alkoxy groups; and heteroaryl or heteroaralkyl in which the hetero atom or atoms in the above-named heterocyclic moieties are selected from the group consisting of 1-4 oxygen, nitrogen or sulfur atoms and the alkyl moiety associated with said heteroaralkyl moiety has 1-6 carbon atoms. Within the above subclass, the preferred compounds are those in which substituent A is -^HCHj" , / \ o: CH , .1 . .a „8 3 CH3 HOCH2 -CH^CH- and wherein either (a) R - and R taken together represeni 2J, ^ C= CH3 8 1 or (b) R is hydrogen and R represents hydrogen, CH2CH2-/ CH CH OH . . ^,?H 1 CH- , C- or CH3CH- . C^C CH3 g Particularly preferred are the compounds wherein R is hydrogen and R"*" is . OH CH3CH-, preferably compounds having the absolute configuration 5R, 6S, 8R. c - 30 - A particularly preferred embodiment of the present invention comprises preparation of compounds wherein -O represents a radical of the formula R7 5 6 7 in which R , R and R are independently selected from the group consisting of hydrogen, C^-C4 alkyl, ci~C4 alkoxy, alkyl substituted by a hydroxy group, C^-C4 alkylthio, amino, carboxy and carbamoyl. Within this subclass, the preferred compounds are those wherein substituent A is -CH2CH2~, -CHCH^- , /\ or -CK2CH- ch3 -\J ia3 and wherein either (a) R and R taken together represent HOCH- C= of3 8 1 or (b) R is hydrogen and R represents hydrogen, CH3CH2~, CH, CH, OH OH ^ i I CH- , ^C- or CH3CH- CH3 off Q Particularly preferred are the compounds wherein R is hydrogen and R^" is T CH3cH-, preferably compounds having the absolute configuration 5R, 6S, 8R. ( ! I 20?346 - 31 - A most preferred embodiment of the present invention comprises preparation of compounds wherein represents a radical of the formula R7 in which R^, R^ and R7 are independently selected from the group consisting of hydrogen, C^-C^ alkyl, alkoxy, alkyl substituted by a hydroxy group, C^-C4 alkylthio and amino. Within this subclass, the preferred compounds are those wherein substituent A is -CH2CH2~, -chchj • ,/ \ or -ch2ch- ch, \ / ch, 1 8 / and wherein either (a) R and R taken together represent hoch. c= / ch , 8 1 or (b) R is hydrogen and R represents hydrogen, CH^CH^-/ ch , ch „ oh oh I ch- , c- or ch3ch- ch3 ch3 8 Particularly preferred are the compounds wherein R is hydrogen and is oh CH3CH~, preferably compounds having the absolute configuration 5R, 6S, 8R. Another preferred embodiment of the present invention comprises preparation of compounds wherein - 32 - represents a radical of the formula © Within this subclass, the preferred compounds are those wherein substituent A is -CHCH2~f -CH2CH2~ , / or -CH^CR- ch3 \ / ch3 C= CH. 18 and wherein either (a) R and R taken together represent HOCH. 8 1 or (b) R is hydrogen and R represents hydrogen, CH3CH2~, CH, CH, OH OH ^ ^ 1 1 ^;ch- \ C- or CH3CH- CH3 CH3 o Particularly preferred are the compounds wherein R is hydrogen and R^" is OH I CH^CH-, preferably compounds having the absolute configuration 5R, 6S, 8R. Another preferred embodiment of the present invention comprises preparation of compounds wherein represents a pyridinium radical. Within this subclass, the preferred compounds are those wherein substituent A is -CHCH2~, ch3 -CH2CH2- ^ \ / or -CII2CH- and wherein either (a) ' CH, 18 R and R taken together represent - 33 - >07346' HOCH. X CH. C= 3 8 1 or (b) R is hydrogen and R represents hydrogen, CH^O^-, CH CH, OH O I CH- C- OH or CH3CH- CH. CH. .8 Particularly preferred are the compounds wherein R is hydrogen OH and R^" is CH^CH-, preferably compounds having the absolute configuration 5R, 6S, 8R. A most preferred embodiment of the present invention comprises the preparation of compounds of the formula OH H (r) wherein represents - 34 - (i) -ch2 (3) -CH2 ( 5) -CH2CH2^>—CH3 e // \\ (7) - ch 2 cs2 n y ch 2 oh och3 (9) -CE2CH2eN ^ (2) (4) (5) ( 8 ) (10 ) (11) -CHCH2 _ CH3 \-=/ (12 ) R or S diastereoisomers nh. (13) -CH.CH ®N^ 2 2 w or (14) ^SC23 -C52CH29^^ ch. -CH2CK2eN-=T sch. R,R or S ,S diastereoisomers at two assymmetr carbons of the cyclohexyl group 2 073461 - 35 - 2 and R is hydrogen, an anionic charge or a conventional readily 2 removable carboxyl protecting group, providing that when R is hydrogen or a protecting group, there is also present a counter anion, and pharmaceutically acceptable acid addition salts thereof. 14 Another preferred class of quaternized R substituents may be represented by the general formula is in which R is selected from the group consisting of substituted and unsubstituted: alkyl, alkenyl and alkynyl, having from 1-10 carbon atoms; cycloalkyl and cycloalkylalkyl, having 3-6 carbon atoms in the cycloalkyl ring and 1-6 carbon atoms in the alkyl moieties; phenyl; aralkyl, aralkenyl and aralkynyl wherein the aryl moiety is phenyl and the aliphatic portion has 1-6 carbon atoms; heteroaryl, heteroaralkyl, heterocyclyl and heterocyclylalkyl wherein the hetero atom or atoms in the above-named heterocyclic moieties are selected from the group consisting of 1-4 oxygen, nitrogen or sulfur atoms and the alkyl moieties associated' with said heterocyclic moieties have 1-6 carbon atoms; wherein the above-named R^ radicals are-optionally substituted by 1-3 substituents independently selected from: I : / /" -N I - ... ... v ' 2 207346 — 35 - C^-Cg alkyl optionally substituted by arir.o, fluoro, chloro, carboxyl, hydroxy or carbajocy 1; fluoro, chloro or bromo; -OR3 ; r -0C02R ; -OCOR3 ; 3 4 -OCONR R A/ ~CS02R ; -oxo' ; -NR3R4 ; R3CONR4- ; -NR3C02R4 ; 3 3 --NR CCNR R 3 4 -NR S02R -SR3 7 O -r o -S-R* ; 0 O -S-R3 ; -S03R3 y -C02R3 ; 3 4 -CONR 5 O -CN; or phenyl optionally substituted by 1-3 fluoro, chloro, bromo, C. -Cc alxyl, -O?.3, -NR3R4, 3 3 ~ 3" 4 . 3 4 • • -SO,R , -CO_R ■ or -CONR R , whfereia R , R_...ar.c " 9 " iS R in such'R substituents are'as defined above; v - 37 - £ * 5 A(- % or R^® may represent a divalent phenylene or alkylene group joined to the ring so as to form a bridged polycyclic group and o represents a substituted or unsubstituted mono-, bi- or polycyclic 'non-aromatic (which may be fused to another aromatic or non-aromatic ring) heterocyclic radical containing at least one nitrogen in the ring and attached to A through a ring nitrogen, thereby forming a quaternary ammonium group. The heterocyclic radical may be saturated or unsaturated (with 1-2 double bonds) and may contain up to two additional hetero atoms in addition to the quaternary nitrogen, such additional hetero atoms being selected from 0, S(0) , N, NR15 or NR17R18 wherein m is 0, 1 or 2, R15 is hydrogen, optionally substituted C,-Cc alkyl or optionally substitute 17 18 " phenyl and R and R are each independently optionally substituted C^-Cg alkyl or optionally substituted phenyl. ! r In a preferred embodiment - vJ; ! represents a non-aromatic 4-7 membered, preferably 5- or 6-membered, N-containing heterocyclic ring containing 0.-2 double bonds and 0-2 additional hetero-atoms selected from 0, S (0) , N, 151718 IS m NR or NR R wherein m is 0, 1 or 2, R is hydrogen, C^-Cg alkyl optionally substituted by 1-2 substituents indeoencentlv selected -*.- 3 3 4 3 1 rrom -OR , -NR R , -CO_R , oxo, ohenyl, fluoro, chloro, bromo, 3 3 4 -SO^R and -CONR R or phenyl optionally substituted by 1-3 substituents independently selected from C^Cg alkyl, -OR3, -NR3R4, fluoro, chloro, bromo, -S03R3, -C02R2 and'-CONR3R4 and R17 and R13 are each independently C^-Cg alkyl optionally substituted by 1-2 substituents independently selected from -OR3, -NR3R4, -c62R3, oxo, phenyl, fluoro, chloro, bromo, -S03R3 and -CONR3R4 or phenyl J - 38 - ootionally substituted by 1-3 substituents independently selected — A a ^ frora C,-C^ alkyl, -OR , -NR R , fluoro, chloro, bromo, -SO.R , -lb, A 3 a .15 -CO_R and -CONR R , wherein R and R in such heterocyclic NR or 2 2.6 nr. groups are as defined above in connection with the R substituent. In such preferred embodiment the -O ring may be optionally substituted by 1-3 substituents independently selected from (a) C^-Cj. alkyl optionally substituted by 1-2 substituents * 3 independently selected from fluoro, chloro, bromo, -or , -ocor3, -oconr3r4, oxo, -nr3r4, -nr3cor4, -nr3conr3r4, -nr3s02r4, -sr3, -s03r3, -c02r3 and -conr3r4; (b) c--c- alkenyl optionally substituted by 1-2 substituents 3 independently selected from fluoro, chloro, bromo, -or , -ocor3, -oconr3r4, oxo, -nr3r4, -nr3cor4, -nr3conr3r4, -nr3s02r4, -sr3, -s03r3, -c02r3 and -conr3r4; (c) C2~Cg alkynyl optionally substituted by 1-2 substituents independently selected from fluoro, chloro, bromo, -or3/ -ocor3, -0c0nr3r4, oxo, -nr3r4, -nr3cor4, -nr3conr3r4, -nr3s02r4, -sr3, -s03r3, -c02r3 and -conr3r4; (d) C3~C6 cycloalkyl optionally substituted by 1-2 substituents independently selected from fluoro, chloro, bromo, -or3/ -ocor3, -oconr3r4, oxo/ -nr3r4, -nr3cor4, -nr3conr3r4, -nr3s02r4, -sr3, -s03r3, -c02r3 and -conr3r4; (e) cycloalkylalkyl having 3-S carbon atoms in the cycloalkyl ring and 1-6 carbon atoms in the alkyl moiety, optionally substituted by 1-2 substituents independently selected from fluoro, chloro, bromo, -OR3, -OCOR3, -OCONR3R4/ oxo, -NR3R4, -nr3cor4, -nr3conr3r4, -nr3so-r4, -sr3, -so.r3, -co-r3 a.nd 3 4 -conr r : A •"*' ■v - 39 - (f) heteroaryl wherein the hetero.atom or atoms are selected from the group consisting of 1-5 oxygen, nitrogen or sulfur atoms, optionally substituted by 1-2 substituents 3 independently selected iron fluoro, chloro, brcmo, -or , -ocor3, -oconr3r4, oxo, -nr3r4 , -nr3c0r4 ,• -nr3conr3r4, ■ -nr3s02r4, -sr3, -so-jr3, -c02r3 and -conr3r4; preferred heteroaryl radicals are 5- or 6-raembered aromatic hetero-O' cyclic, rings; (g) heteroaralkyl■wherein the hetero atom or atoms are selected from the group consisting of 1-4 oxygen, nitrogen or sulfur atoms and the alkyl moiety has 1-6 carbon atoms, optionally • substituted by 1-2 substituents independently selected from 3 3 3 4 fluoro, chloro, bromo, -or , -ocor , -oconr r , oxo, 34 34 3 3 4 34 3 3 -nr r , -nr cor , -nr conrjr , -nr so,r , -sr , -so,r , 3 3 4 ■ -c02r and -conr r ; preferred heteroaralkyl are those in which the heteroaryl radical is a 5- or S-membered aromatic heterocyclic ring and the alkyl moiety has 1-2 carbon atoms; (h) heterocyclyl wherein the hetero atom or atoms are selected from the group consisting of 1-4 oxygen, nitrogen or sulfur atoms, optionally substituted by 1-2 substituents independently selected from fluoro, chloro, bromo, -or3, -ocor3, -oconr3r4, oxo, -nr3r4, -nr3cor4, -nr3conr3r4,' -nr3s02r4, -sr3, -s03r3, -c02r3 and -conr3r4; preferred heterocyclyl are 5- or 6-membered saturated or unsaturated rings; - (i) heterocyclylalkyl wherein the hetero atom or -atoms are selected from the group consisting of 1-4 oxygen, nitrogen or sulfur a.toms and the alkyl moiety has 1-6 carbon atoms, optionally substituted by 1-2 substituents independently 3 *3 3 4 selected from fluoro, chloro, bromo, -or , -ocor , -oconr r , oxo, -nr3r4, -nr3cor4, -nr3conr3r4, -nr3so.,r4, -sr3, -sq7r3, 3 3 4 -c02r and -conr r ; preferred heterocyclylalkyl are those in which the heterocyclyl moiety is a 5- or 6-membered saturated or unsaturated ring; -ii'.'.;'/ ' rv - 40 - w?- % o © (j) (k> (1) (m) (n) (o) (p) (q) (r) (s) (t) (u) (v) (w) fluoro, chloro or bromo; -or3 ; -OCOjR3 ; -OCOR3 ; -OCONR3R4 ; -0S02R3 ; oxo ; -NR3R4 ; 3 4 R CONR -; 3 4 -NR C02R ; 3 3 4 -NR CONR R ; 3 4 -nr sojr ; -sr3 ; 0 * 9 -s-r ; (x) 0 O ? q -s-r (y) (z) -S03R" -C02R 7 (aa) -C0NR3R4 ; (bb) -CN; or (cc) phenyl optionally substituted by 1-3 fluoro, chloro, brozao, C^Cg alkyl, -OR3, -NR3R4, -S03R3, -C02R3 or -C0NR3R4. 3 •"" * 4 9 The R , "R and R substituents mentioned above are as defined in connection with substituent R"*". The -Q ^0/346' - 41 - ring as defined above is a non-aromatic heterocycle group. This ring, however, may be fused to another ring which may be a saturated or unsaturated' carbocyclic ring, preferably a carbocyclic ring, a phenyl ring, a 4-7 membered heterocyclic (saturated or unsaturated) ring containing 1-3 hetero atoms 1C 17 18 selected from O, N, S(0) , NR ■ or NR R or a 5-6 membered m heteroaromatic ring containing 1-3 hetero atoms selected 0, S(0)m, N, NR^ or NR^R^8 in which m, R^, and R^® are as defined above. The R^ substituent of the non-aromatic R^"4 radical may be either (a) an optionally substituted C^-Cg alkyl, C2"C10 alkenyl, C2-C^Q alkynyl, C^-Cg cycloalkyl, C^-Cg cycloalkyl-C^-Cg alkyl, phenyl, phenyl-C^-Cg alkyl, phenyl-C^-Cg alkenyl, phenyl-C^-Cg alkynyl, heteroaryl, heteroaralkyl in which the alkyl moiety has 1-6 carbon atoms, heterocyclyl or hetero- ' cyclylalkyl in which the alkyl moiety has 1-6 carbon atoms or (b) a divalent phenylene or alkylene group joined to the - 42 - c- ring so as to form a bridged ring polycyclic group, e.g. a quinuclidine group. The heteroaryl (or heteroaryl portion of heteroaralkyl) substituent may be a mono-, bi- or polycyclic aromatic heterocyclic group containing 1-4 O, N or S atoms; preferred are 5- or 6-membered heterocyclic rings such as thienyl, furyl, thiadiazolv1, oxadiazolyl, triazolyl, isothiazolyl, thiazolyl, imidazolyl, isoxazolyl, tetrazolyl, oxazolyl, pvridyl, pyrazinyl, pyrimidinvl, pyridazinyl, pyrrolyl and pyrazolyl. The heterocyclyl (or heterocyclyl portion of heterocyclylalkyl) substituent may be a mono-, bi- or polycyclic saturated or unsaturated non-aromatic heterocyclic group containing 1-4 0, N or S atoms; preferred are 5- or 6-membered heterocyclic rings such as morpholinyl, piperazinyl, piperidyl, pyrazolinvl, pvrazolidinyl, imidazolinyl, imidazolidinyl, pyrrolinyl and pyrrolidinyl. In the case where "the R^'® substituent is an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, phenyl,, phenyl- ' alkyl, phenylalkeny1, pheny1alkynyl, heteroaryl, heteroaralkyl, heterocyclyl or heterocyclylalkyl group, such groups may be optionally substituted by 1-3 substituents independently selected from: (a) C-^-Cg alkyl optionally substituted by, preferably 1-3, amino, fluoro, chloro, carboxyl, hydroxy or carbamoyl groups; •(b) fluoro, chloro or bromo; (c) -OR3 ; (d) -OC02R3 ; (e) -OCOR3 ; (f) -OCONR3R4 ; (g) -0S02R3 ? . (h) -oxo ; (i) -NR3R4 ; (j) R3C0NR4- ; (k) -NR3C02R4 (1) -NR3C0NR3R4 ; / - 43 - (m) (n) 3 4 -NR SO,R -SR3 ; (o) -SOR5 ; (p) (q) (r) -SC7R9 ; ^ 3 -SO_R ; 3 -C02R-* ; (s) 3 4 -CONR R (t) -CN ; or (u) phenyl op cXoT- 34* fo dependently selected from fluoro, chloro, bromo, C^-Cg alkyl, -OR3, -NR3R4, -SO^R3, -C02R3 or -CONR3R4, wherein, 16 relative to the above-named R substituents, the groups ^ A R and R are independently selected from hydrogen; alkyl, alkenyl and alkynyl, having 1-10 carbon atoms; cycloalkyl, cycloalky lalkyl and alky Icycloalkyl, having 3-6 carbon atoms in the cycloalkyl ring and 1-6" carbon atoms in the alkyl moieties; phenyl; aralkyl, aralkenyl and aralkynyl wherein the aryl moiety is phenyl and the aliphatic portion has 1-5 carbon atoms; and heteroaryl, heteroaralkyl, heterocyclyl and heterocyclylalkyl wherein the heteroaryl and heterocyclyl group or portion of a group is as defined above for R"^ and the alkyl moieties associated with said heterocyclic moieties have 1-6 3 4 carbon atoms; or R and R taken together with the nitroce: to which at least one is attached may form a 5- or 6-membered nitrogen-containing heterocyclic (as defined 5 9 3 above for R ) ring;-' and R is as defined above for R 16 except that it may not be hydrogen. A most preferred R substituent is C.-C- alkvl, especially methvl. i (C ' ' In the case where Rx is a divalent phenylene or C^_Cg alkylene group,, such group is bonded to another atom of the -O ring so as to form a bridged polycyclic ring, e.g. a quaternized cuinuclidine rinc of the formula i e 2 **£•> ,o j - 44 - A particularly preferred embodiment of the present invention coniDrises oreoaration of compounds of Formula I wherein RlfV—S CH —N .0 v CH" CH. V -N® S \ / \3f^- —N NH w *fr CH, e z ■N -I—CH. V CH \ 5? s~> 0 ®\ / 9 ■n n; \_y -CH. or vj wherein Y is hydrogen, C^-Cg alkyl,'hydroxy, -SC^-Cg alkyl, carboxyl, carbamoyl, chloro, bromo, iodo/ fluoro or phenyl. Within this subclass, the preferred compounds are those \ i - 45 - 5 wherein A is -CHCH_-I 1 CH, , -CH^H- or ch- -(CH-,) - in which n is 2, 3 or 4, more oreferablv those in which A ^ n ■ is -CH2CK2-, -CH2CK2CH2", -chch2- CH. or -ch-ch-2 I ch3 and most preferably those in which A is -CH-CH--, and wherein either 18 • (a) R and R taken together represent HOCH-, ch3 c= 8 1 or (b) R is hydrogen, and R represents hydrogen, CE3CH2-, OH CH V y CH * OH C- or CH3CH- . CH. CH. Particularly preferred are the compounds wherein R is hydrogen and R^" is ' OH CH3CH- especially .compcuncs having the absolute configuration 5R, 6S, 8R. . D A still more preferred embodiment of the present invention comprises preparation of compounds of Formula I wherein represents "O yr\ \ / / )7 * # - 46 - \ \3/ —-N S">° e V7 CH -*S 3/\ n-ch- ©" \ / Sx 9 J*3 N' -ch. r3 C> CH \ / ch 3 / \ — N® NH w or Within this preferred subclass, the preferred compounds are those wherein A is -CHCH2~ , CH, , -CH2CH- or CH, — (CH2)n~ in which A is 2, 3 or 4, more preferably those in which or -CH,CH- , and A is -CH2CH2-j -CH,CH,CH,-, -CHCH,- , 2^2 I CH. k2T CH. t preferably those in which A is -CS_CH,-, and wherein either T fl it mos 1 3 (a) R and R taken together represent Vj HOCH 2\ C= CH. o CH 8 1 ■r (b) R is hydrogen and R represents hydrogen, CH-jC^-r OH CH. 3\ CH- / CT3 ch3 oh or CHjCH- O 8 Particularly preferred are the compounds wherein R is hydrogen and R^" is OH I CH^CE— , especially compounds having the absolute configuration 5R, 5S, 8R. A still more preferred embodiment of the present invention comprises preparation of compounds of Formula I wherein r) - 47 - represents in which Y is hydrogen, C^-Cg aDcyl, hydroxy, -S-C^-Cg alkyl, carboxyl, carbamoyl, chloro, bromo, iodo, fluoro or phenyl. Within this preferred subclass, the preferred compounds are those wherein A is -(CH.,) — in which n is 2, 3 or 4 , most c n preferably those in which A is -CH7CK,- and wherein either 18 (a) R and R taken together represent hoce2 *^C= 8 1 or (b) R is hydrogen and.R represents hydrogen, CH^CH^-, AU CH, CH, , OH 3^ ~ . 3vJ j CH- '/ C- or CH. CH- . CH3 CH3 8 Particularly preferred are the compounds wherein R is hydrogen and R^" is OH I CH3CH-, especially compounds: having the absolute configuration 5?., 6S, 8R. 2 07 34-6 - 48 - A most preferred embodiment of the present invention comprises preparation of compounds of Formula I wherein O ->£ ) represents ° Within this preferred subclass, the preferred compounds are those wherein A is -(C5.,)- in which n is 2, 3 or 4, most ■ z n ' preferably those in which A is -CH-CE-- and wherein either T g (a) R~ and R taken together represent HOCH- CH^ 8 1 or (b) R is hydrogen and R represents hydrogen, CH^C^-, O C\ =3^ ?S CK- , C- or CH^CH- ch3^ g Particularly preferred are the compounds wherein R is hydrogen and R^" is OH I CH 3CH—, especially compounds having the absolute configuration 5R, 6S, SR. 20" 346 49 - , v C A most preferred embodiment of the present invention comprises preparation of the compounds of the formula S-CH2CH2-^N wherein "^COOR" represents C> V/ CH N-CH. \3 © V et \ s^o \ / \ (both a- arid 3-diastereoisomers) CH _ N V "^CH. and R is hydrogen, an anionic charge or a conventional readily 2 removable carboxyl protecting group, providing that when R is hydrogen or a protecting group, there is also present a counter ion, and pharmaceutically acceptable acid addition salts thereof. It will be appreciated that certain products, within the scope of formula I may be formed as optical isomers as well as epimeric mixtures thereof. It is intended that the present invention include within its scope all such .optical isomers and epimeric mixtures. For example,.when the 6-substituent is hydroxyethyl, such substituent may be in either the R cr -S configuration and the resulting isomers as well as epimeric" mixtures thereof are encompassed by the present invention, 31.. - 50 - V • The process of the present invention utilizes the intermediate of the formula IV which has been disclosed, for example, in European Patent Application 38,869 and which may be prepared by the general methods described therein. L represents a conventional leaving group (defined as "X" in European Patent Application 38,869) such as chloro, bromo, iodo, benzenesulfonyloxy, p-toluene-sulfonyloxy, p-nitrobenzenesulfonyloxy, methanesulfonyloxy, trifluoromethanesulfonyloxy, diphenoxyphosphinvloxy or di(tri-chloroethoxy)phosphinyloxy. The preferred leaving group is diphenoxyphosphinyloxy. Intermediates of Formula IV are generally formed in situ by reacting an intermediate of the formula // N "~~-COOR2' III 18 21 wherein R , R and R are as defined above with a suitable O acylating agent R -L. The preferred intermediate IV where L is diphenoxyphosphinyloxy may be prepared by reacting keto ester III in an inert organic solvent such as methylene chloride, acetonitrile or dimethylformamide with about an equimolar amount of diphenyl chlorophosphate in the presence of a base such as diisopropylethylamine, triethylamine, 4-dimethylaminopyridine or the like at a temperature of from about -20°C to +40°C, most preferably at about 0°C. Intermediate IV may be isolated, if desired, but is conveniently used as the starting material for the process of the present invention without isolation or purification. - 51 - 207346 In the present process, carbapenem intermediate IV is reacted with a quaternary amine thiol compound of the formula HS—A—R14 X® VII wherein A is cyclopentylene, cyclohexylene or C2-Cg alkylene optionally substituted by one or more alkyl groups, most preferably cyclopentylene, cyclohexylene or R10 R12 II--C C- i11 in which R^0, R^, R"1"2 and R13 are each independently hydrogen cr 0 C^-C^ alkyl, X is a counter anion associated with a strong such as Cl~, Br', CH^SO^ , CF^SO^ or CK3 y—S03 and R"*"4 is a quaternized nitrogen-containing aromatic or non-'S^gfcfetxe heterocycle as defined above. The reaction is carried out in an inert solvent such as acetonitri le, acetonitrile-dimethylformamide, tetrahydrofuran, tetrahydrofuran-H20, acetonitrile-KjO or acetone in the presence of base. The nature of the base is not critical. Best results, however, have been obtained when a non-nucleophilic tertiary ' amine base such as diisopropylethylamine, 1,8-diazabicyclo-[5.4.0]undec-7-ene, 1,5-diazabicyclo[4.3.0]non-5-ene or a tri(C^-C4) alkylamine such as trie thy lamine, tributy lamine or tripopylaiaine is employed. Reaction of intermediate IV with thiol VII may be carried out over a wide temperature range, e.g. -15°C up to room temperature, but is preferably done at a temperature in the range of from -15°C to substantially +15°C, most preferably at around 0°C. The carbapenem product produced by reaction of the quaternary amine thiol VII with intermediate IV will have a Q g counter anion associated with it Je .g . (CgH^O^PC^ , CI or the -anion associated with the quaternary thiol) which may at this stage be substituted by a different counter anion, e.g. one which \ . - , "V ^ ^ - 52 - ,:4 O ' . .. "J ■** is more pharmaceutically acceptable, by conventional procedures. Alternatively, the counter anion may be removed during the subsequent de-blocking step. Where the quaternized carbapenem . compound and counter anion form an insoluble product, the product may crystallize out as it is formed and be collected pure by filtration. Following formation of the desired carbapenem product, the 2 1 carboxyl protecting group R of Compound I' may be optionally removed by conventional procedures such as solvolysis, chemical C: reduction or hydrogenation. Where a protecting group such as p-nitrobenzyl, benzyl, benzhydryl or 2-naphthylmethyl is used which can be removed by catalytic hydrogenation, intermediate I' in a suitable solvent such as dioxane-water-ethanol, tetrahydrofuran- diethylether-buffer, tetrahydrofuran-acueous dipotassium hydrogen phosphate-isopropanoi or the like may be treated under a hydrogen pressure of from 1 to 4 atmospheres in the presence of a hydrogenation catalyst such as palladium on charcoal, palladium hydroxide, platinum oxide or the like at a temperature of from 0 to 50°C for from about 2 1 * 0.24 to 4 hours. When R is a group such as o-nitrobenzvl, photolysis may also be used, for deblocking. Protecting groups such as 2,2,2-trichloroethy1 may be removed by mild zinc reduction. The allyl protecting group may be removed by using a catalyst comprising a mixture of a palladium compound and triphenyl phosphine in a suitable aprotic solvent such as tetrahydrofuran, methylene chloride or diethyl ether. Similarly/other conventional carboxyl protecting groups may be removed by methods known to those skilled in the art. Finally, as mentioned above, compounds of formula I' 2' where R is a physiologically hvdrolyzable ester such as acetoxy-methyl, phthalidyl, indanyl, pivaloyloxyrnethv 1, methoxymethyl, etc. may be administered directly to the host without de-blocking since such esters are hvdrolyzed in vivo under physiological ! conditions. , T Q It will be understood that where the R and/or R i 14 substituent or the quaternized nucleophile R attached to substituent A contain a functional group which might i j interfere with the intended course of reaction, such group j may be protected by a conventional blocking group and then : subsequently de-blocked to regenerate the desired functional i group. Suitable blocking croups and procedures for intro ducing and removing such groups are well known to those - 53 - skilled in the art. As in the case of other S-lactam antibiotics, compounds of general formula I may be converted by known procedures to pharmaceutically acceptable salts which, for purposes of the present invention, are substantially equivalent to the non-salted compounds. Thus, for example, one may dissolve a compound of 2 Formula I wherein R is an anionic charge in a suitable inert solvent and then add an equivalent of a pharmaceutically acceptable acid. The desired acid addition salt may be recovered by conventional procedures, e.g. solvent precipitation, lyophiliza-tion, etc. Where other basic or acidic functional groups are present in the compound of Formula I, pharmaceutically acceptable base addition salts and acid addition salts may be similarly prepared by known methods. 2 A compound of Formula I where R is hydrogen or an anionic charge, or a pharmaceutically acceptable salt thereof may • •• also be converted by conventional procedures to a corresponding 2 compound where R is a physiologically hydrolyzable ester group, or a compound of Formula I wherein R2 is a conventional carboxyl protecting group may be converted to the corresponding compound 2 * • where R is hydrogen, an anionic charge or a physiologically hydrolyzable ester group, or a pharmaceutically acceptable salt thereof. Certain of the thiol intermediates of Formula VII may be ^ prepared, for example, by reacting a sulfide of the formula or ; Villa VII lb :G s R10—-C — C—R12 ! / \ | R11 R13 • I VIIIc wherein R^, R^, R"'"2 and R^-3 are each independently hydrogen or C,-C, alkyl with a heteroaromatic amine (as defined above) of the formula 207346 or a non-aromatic heterocyclic amine (as defined above) of the formula and a strong acid; The reaction may be carried out in the presence or absence of an inert organic solvent which is preferably a non-polar organic solvent such as methylene chloride, benzene, xylene, toluene or the like. Where the amine and sulfide reagents are liquids or where a solid amine is soluble in a liquid sulfide reagent, it is preferred to carry out the reaction without use of an additional solvent. The particular strong acid used in the reaction is not critical and may be, for example, such strong inorganic or organic acids as hydrochloric, hydrobromic , • methanesulf onic, p-toluenesulfonic, trifluoromethanesulfonic, etc. Formation of the quaternary amine thiol intermediate VII may be carried out at a temperature in the range of from substantially -20°C to substantially 100°C. Preferred temperatures are generally in the range of about 50-70°C. The sulfide reagent, aromatic amine and acid are preferably employed so that the sulfide and acid are used in approximately equinolar amounts with the amine being used in excess, e.g. two to three moles of amine per mole of sulfide or acid. The quaternary amine thiol intermediate will have a counter anion associated with it which will be determined by the particular acid employed. It is, 'of course, possible to substitute at this point a different counter anion by conventional procedures for use in the subsequent reaction with carbapenem intermediate IV. . <- , a , ' n*> - V?,. - 55 - 2 0' 3 46 The carbapenem derivatives of general Formula I 2 wherein R is hydrogen, an anionic charge or a physiologically hydrolyzable carboxyl protecting group, or the pharmaceutically acceptable salts thereof, are potent antibiotics active against various gran-positive and gran-negative bacteria and they may be used, for example, as animal feed additives for promotion of gro*. as preservatives in food, as bactericides in industrial applications, for example in waterbased paint and in the white water of paper mills to inhibit the growth of harmful bacteria and as disinfectants for destroying or inhibiting the growth of harmful bacteria on medical and cental equipment. They are especially useful, however, in the treatment of infectious disease in humans ■ and other animals caused by gram-positive or gram-negative bacteria. The pharmaceutically active compounds provided by the novel process of this invention may be used alone or' formulated as pharmaceutical" compositions comprising, in addition to the active carbapenem ingredient, a pharmaceutically acceptable carrier or diluent. The compounds may be administered by a variety of means; those of principal interest include: orally, topically or parenterally (intravenous or intramuscular injection] , The pharmaceutical compositions may be in solid form such as capsules, tablets, powders, etc., or in liquid form such as solutions, suspensions or emulsions. Compositions for injection, the preferred route of delivery, may be prepared in unit dose form in ampules or in multidose containers and raav contain formulatory agents such as suspending, stabilizing and dispersing agents. The compositions may be in ready to use form or in powder form for reconstitution at the time of delivery with a suitable vehicle such as sterile water. 20734 - 56 - The dosage to be administered depends to a large extent on the particular compound being used, the particular composition formulated, the route of administration, the nature and condition of the host and the particular situs and organism being treated. Selection of the particular preferred dosage-and route of application, then, is left to the discretion of the therapist. In general, however, the compounds may be administered parenteral or orally to mammalian hosts in an amount of from about 5 to 200 mg/kg/day. Administration is generally carried out in divided doses, e.g. three to four times a day. The following examples illustrate but do not limit the scope of the present invention. CZt% r-v. £xj - 57 - Exaaple 1 Preparation of 3-( 2-( l-pyridiniun) et'nylthio )- $g-(H.2)-hydroxyethyl)-7-oxo-l-a:a'bicyclo(3-2.0)-hept-2-ene-2-carboxylate A. l-( 2-aercaotoethyl )nyri dinius se t h ane s ulf on at e A ♦ ,0 ♦ To a suspension of pyridinius sethanesulfonate in pyridine prepared by the dropv/ise addition of nethejiesulfonic acid CI-95 cL, 0.03 aol) to pyridine (S.O ml, '0.099 sol) with" cooling, was added' ethylene sulfide (1.96 si, 0.033 sol) . The resulting aixture was stirred at 55#C for 16 h and concentrated under reduced pressure to a thick syrup which was nixed with few si, of water. The solution was poured on top of a column (40 x IS cs) of U-bondapak C-18 which was eluted with water. Lyophylization of the appropriate fractions gave a colorless syrup 6.5 g (91%), ir (fils) v : 2300-2600 far, SH •. sax 1635 (pyridinius), 1490, 1200 (sulfonate), 1068, 1060, 1045, 791, 780 cs"1, 1ftnr.(DMS0 "O 0= 2.32 (3H, s, CH SO ~) ,' 2. 61, 2. 70 , 2.73, 2.82 C1H, 3 part of A^ system, SH) , 3.07 (2K, a [with DO, '3.03 (2H J=6.5 Hz)], CH2S), 4.76 (2H, t, J=6.5 Hz, CH N+) , 8.19 (2H, m, Hs of pyridinius), 8.6 (1H, m, Ho of pyridinius) , 9-08 (2H, ad, £=6.8 H-,~ J-1.4 Hz, Ho of pyridinius), uv (K,0) X : 206 (E5230) , 258 (£3760) — 2 sax. Ms OH. 55"C, 16 h HS •»A + OsO METHOD A £•' 1- (2-raercaptoethyl) pyridi.-iua chloride HsO ?emutit S-l CI An aqueous solution of crude 1— (2-aercaptoethyl)pyr"idir methanesulfonate (9.4 g, 0.04 rol) was poured on top of collars (2.5 x 41 cm) of perautit S-l CI - The colirrji was eluted with water at a rate of 0.5 ai per ain and the appropriate'fractions, were combined ana lyophylized giving a yellowish syrup 7.0 g (100%) which was used as it was for the next step, ^-iar (D^O) os 3.22 (2H, m, CT-^S) , 4.88 (a, 'C3 N +) , 8.13 (22, =; Ha of'pyridi.i: 8.7 (1H, a/ Hp of pyridiniun) , 9.0 ppra (2H, a, Ho of pyridir-iua). METHOD 3 A N + 'J - CI CI To a precooled (ice bath) pyridine (5.6 mL, 70 innol) was added pyridine hydrochloride (4.05 g, 35 mmol) and ethylene sulfide (2.1 mL, 35 mmol). The mixture was heated at 65°C and scirred for 75 min to give a two phases system. The lighter phase was removed. The remaining oil was washed with ether (5 x 10 mL) and pumped under high vacuum to give the title conpound (90-100%) which was used as such for the next steo. - 59 - 0 . Parar.itrobenzy 1 3- [2- (l-oyridiniu.'n) ethyl thiol -6c- f I~ f ?■) -hvdrcyvethvl ] -7-cxo-l-azabicyclo(3.2.0)heor-2-ene-2-carboxvlate'chloride A solution of p-nitrobenzyl Z-j.~ [1- (&) -hydroxyethyl] -3,7-dicxo-l-asabicyclo (3.2. 0) heptane-'-carboxyiate" (6.09 S> IT• 5 =eo1) in acetonitrile (20 mL) vas cooled to +5CC under a nitrogen atmosphere and treated successively with diisopropylethylaziine (3.65 riL, 21.0 ssol] and diphenyl chlorophosphate (4.34 nL, 21.0 r=nolJ . The resulting nixture vas stirred for 30 min at 5cC, cooled to -5°C and treated successively with a solution of crude 1- (2-nercaptoethyl)pyridinius chloride (4.3 g, 24 raol) in N, N-dimethylfor7naru.de (1.0 r.L) and cropvise with diisopropylethylamine (3.65 nL, 21.0 ctoI) , The reaction fixture was stirred at 0°C for 1 b, cooled to -30°C and stirred for 15 nin. aore. The solid was filtered off and washed with cold (-30°C) acetcr.itrile, 5.77 g (65*), ir (nujol) V : 3300 (OH), 1775 (C=0 of 0-lactaa) , itu8. x. 1690 (C=0 of ?N3 ester), 1530 (pyridinius) , 1605 (phenyl of ?K3 ester), 1515 (N02), 1335 cn'1 (N02), ' (OMSO-tf )5: 1.17 (3H, d, £=5.1 S=, CH3CH0H)f 3.2-3.75 <5H,E-U » H-6# CH2S) , 3.75-4.5 (2H, H-5,' Cr. CjOK) .. 4.92 (2H, brt, £=6.5 Hz, CH2N+), 5.18 (1H, d, £=4.9 Hz,OH), 5.37 (center of A3q, J =14.2 Hz, CH„ of ?N3) , 7.69 (2H, d, 3=8.1 Hz, Ho — £ / O * 1 " m of ?N3) , 8.24 (d, J=8.7 Hz, Hn of ?JCS) ,8.0-5.4 (4H, Hn of PN^ H= ' / 2 n - U s 4 &* - 60 - of pyridiniua), 8.66 (Hi, a, Hp of pyridiniuni) 9.17 (2E, brd, J-5.5. Kr, So of oyridi.ai.un) . The filtrate and washing vere corjined 2nd diluted with ether (150 sL) . ■ The supernatant was decanted ar.d the gun was dissolved in water (40 aL) containing enough acetonitrile to have a solution-.which was poured on top of a.colusn (3 x 10 era) of V-bondapajc C-1S. The colra. was eluted with 10* acetonitrile -90* water (150 clL) and 50* acetonitrile - 50* water (100 aL) mixtures. The appropriate fractions were combined and.lyophylized aftex the acetonitrile has been removed under vacuum giving a yellowish powder. An of, it showed the presence of the title conpound. mixed with scsie p-ni trobenzyl 3- [2- (l-pyri'dir.iu=0 ethylthio]-6c- [1- (B)'-hydroxy ethyl] -7—exo-1-azibicyclo(3.2.0)hept-2-ene-2-carboxylate diphenylphosphate (2:1) . The powder was dissolved in- water" (minimum amount) and passed through a column (1.5 x 21 ca) of 'pemutit S-1C1 with water. Lyophylizaticn-of the appropriate fractions gave 1.3 g (20%) of the title compound. - 61 - yaranitrobencyl 3- [2- (1-pyridir.ius) ethyl thio}-6::- [1- (g)-hydrcxyethyl]-7-oxc— 1-azabicyclo(3.2.0)hept-2-ene-2-carboxylate diphenylphosphate. 0 • COOPN3 1) NBt(i?r), 2) Cll>(0?h) 31 M0" 4) N-t(iPr) • 2 00PK3 (?h0)2?0 A solution of'.p-nitrobenzy! 6c-[1-(3)-hydroxyethyl]- 3,7-dioxo-l-arabicyclo(3.2.D)b5ptane-2-carl2oxylats (O.tjk g, 0.50 rsaol) in acetonitrile (2 aL) was' cooled to 0®C under a nitrogen atmosphere aj^d treated .successively vith diisopropylethylaaine (0.105 a 0.60 aaol)-and diphenyl chlor opho sphate (0.124 a!., 0-60 zr.-nol) . The resulting solution was stirred for 30 siin at 0®C and treated successively with a solution of 1- (S-nercaptoethyDpyridiniua methanesulfsnare (0.170 g, 0.72 jtctoI) in acetonitrile (0.6'si) ajid diisopropylethylasir.e (0.105 raL, 0.50 iraol) . The reaction fixture was stirred at 0°C for 15 nin, diluted with cold (0°C) water (7 aL) and poured on top of a coluan (1.5 x 6.4 ca) of y-bondapaJ; C-18. The coluan was eluted'with a mixture of acetonitrile (25*-50%) in water (75%-50%). The apprcpriat fractions were combined and lyophylized after the acetonitrile has been removed under vacuus giving a yellowish powder 0.33 g (92*), ir (K3r) V : 3600-3000 (OH), 1765 (C=0 of S-lactajn) , 1690 (C=0 cf ?N3 ester), max ■ 1625 (pyridinius), 158 5 (phenyl), 1510 (N02) , 1330 (N02) , 835 tea"1. (NO) V' ■ - >• , \ 7 P -? ^ W" 4' - 62 - !Hnr (Df-iSO-dL) 6: 1.16 (3H, d, £=5'.'2 Hz, CH^CHOH), 4.37 (2H, brt, J=6.S Hz, CH'S), 5.37 (center, of A3q, J .=14.*3 Hz, CH of »N3) * . . 2 6.7-7.5 (phenyl), 7.63 (d, J=8;S Hz, Ho of .PN3) 8.23 (d,' J=8.3 & Ha of ?K3) , 8.0-8.3 (si, ■ Ha of pyridinius), 3.4-3.3 (1H, Hp of . pyridiniua), 9.09 (23, dd; J-6.7-H2, J~1.3Kz, Ho of pyridinius). 3- [2- (1-pyridi.iiua) ethylthio)-6e- [1- (?.) -hydroxyethyl]-7-oxo-l-azabicyclo (3.2.0)hept-2-ene-2-carboxylate; Method A ■ OH 10* ?d/C, H. THT, IXher \ • , ? . COOPN3 (?k'0) 2^° To a solution of p-nitrobenzy 1 3-[2-(1-pyridiniua)ethylthio] -6c- [1- (R) -hydroxyethyl] -7-oxo-l-azabicyclo (3.2.0) hept-2-ene-2-carboxvla . diphenylphosphate (0.16 g, 0.22 raol) in wet tetrahydrofuran (10 &L) was added ether (10 aL), potassium phosphate raonobasic-sodiua hydroxide buffer pH 7.4 (IS aL, 0.05 M) and 10* palladiua on charcoal (0.16 g). The resulting aixture was hydrogenated under 40 psi for 1 h at 2S*C. The two phases were separated and the organic phase was extracted with water (2x3 aL). The aqueous solutions were combined, washed -with ether'(2 x 10 nL) and poured on top of a coluan (1.5 x 6.2 ca) of y-boncapak C-18 after the traces of organic solvents have, been reao'v - 63 - under vacua. Elution .of the colunn vith water gave after lyophylizati of the appropriate fractions a yellowish powder 0.0S2 g (84*), ir (K3r) V : 3700-3000 (OH), 1755 (C=0 of B-lactan) , 1630 (ovridiniua) , 1530 nsx en"1 (carboxylate) , lHar (D20) : 1.22 (3H, d, J=6.4 Hz, CH CH0H), 2.92 (d, £=9.1 Hz, E-10 , 2.97 (d, £=9.1 Hz, s_l») , 3.20 (dd, £=2.5 Hz, £=S.l H-o), 3.44 ft,"£=6.0 Hz, CH S), 3.93 (dd, £=9.1 Hz, £= 2.5 Hz, H-5) , 4.82 (t, £-6.0 Hz, N+) , 8.04 (a, Ha of pyridir.iua) , 8.5 (a, Hp of pyridiniua), 8.82 (dd, £=3.2.Hz, £=1.1 Hz, Ho of pyridiniua), uv (320) X ' i-259 (£5800) , :29S -(c7030) ay, =13.5 h (measured at a SULX 2 concentration of 10 ^ H in phosohate buffer pH 7.4 at 3S.3*C). 'Method 3 ,TrSr- ether, buffer 7.2 COO To a solution of p-nitrcbenzyl 3- [2- (1-pyriciniua) ethylthio] 6g-ti-(3)-hydroxyethyl]-7-oxo-l-a:abicyclo (3.2.0)hept-2-ene-2-car-boxylate chloride (5.77 g, 11.4 amol) in potassium phosphate-monobasic .sodiua hydroxide buffer (170 aL, 0.2 H, pH 7.22) was added tetrahydro-furan (30 aL) , ether (30 ciL) and 10% palladium or. charcoal (5.7 g) . The resulting mixture was hydrogenated at 22®C under 40 psi for 1 h and filtered on a Celite pad. The pad was washed, with water (2 x 15 izli) . The filtrate and washings were combined and diluted with ether ( 100 aL). The aqueous phase was separated, washed vith ether (3 x 100 aL) and poured on top of a column (4.5 x 20 ca) of y-bondapaT< C-13 after the organic solvants have been removed, under vacuus. Elution of the column with water followed by a mixture of 1* acetonitrile in water gave after lyophvlization of the appropriate fractions 2.43 g (65%) of the title compound as a yellowish powder. The analytical data were identical to those reported .for the compound prepared in the method A. - 65 - Sxamole le 2 Preparation of 3-[2-(1-(3,5-dimethvloyridinium) ethv 1 thiol] -6c - [ 1- (Jg) -hvdroxyeiihvl) -7-oxo-l-azabicyclo(3.2.0.)he?t-2-ens-2-carboxvlate 1- (2-mercaotoethyl)-3,5-dimethylpyridiniun roethanesulfonate A. + N + Ms OH MsO To a suspension of 3,5-lutidir.ium aethaaesulfonate in 3,5-lutidine prepared by.the addition of methanesulfonic acid (0.65 =L, 0.010 nol) to cold 3,5-lutidine (2.51 mL, 0.022 aol) was'added ethylene sulfide (0.655 mL, 0.011 mol). The resulting mixture was stirred under a nitrogen atmosphere at 55"C for 24 h, cooled to 23®C and diluted vith water (5 mL) and ether (5 mL).. The organic layer was separated and the aqueous solution was washed with,.ether (6x4 mL) . The traces of ether were removed under vacuum and the solution was applied on top of a column (2.5 x 6.0 cm) of p-bondapak C-18. The column was elated with water and lycphilization of the 207345 - 66 - appropriate fractions gave a colourless syrrjp 2.4 g (91*); — (flln) /j V : 2520 (SH), 1523 (pyridinius), 1500, 1495, 1325, 1305, 12S3, 1200 max (sulfonate) , 1040, 938, 755, 6S0 cnf1; "(2MS0 d.) 5: 2.31 (3E, 6 s, C:\jS0 ~) , 2.47 (6H,s, CH3 on pyridini.-J=) , 2.57, 2l6S, 2.53, 2.78 (IK, 3 .part of A3 system, SH) , 3.06 (2 H, r. [vi th D.O added (2H, t, £=5.5 Kz) ] , CH^S) , 4.55 (2H, £=5.5 H=, C-: N ), 8-34 flH, s, Hp of pyridinium), 8.79 (2H, s, Ho of pyridi.ni-c.—) ; uv (H O) X : 271 2. sax (S4S60) ir.y. Anal, calcd. for C, QH,..NO S^-0.5H O: C44.09, H 6.55, N 5.14, S 23.54; found: C 44.25, H 5.49, N 5.17^ S 24.IS. B . ?arani.trcber.::y 1 3- [2- (1- (3 . 5-d 1 rr.etr.1 o^r 1 dir.l'-rr,)) ethylthio']-5c- fl- (?.) hydroxy ethyl] -7-oxo- l-anabi ;y:lo(3- 2■3 ;;:sp~-2-ene-2-:Lsr~::c:cyla~; ohcsrhate COO?N3 1) NZt(i?r) 2) Cl?(0?h) ■ OH To a cold (0°C) solution of p-nitrobenzyl 6c-(1-(B)-hydroxyethyl)-3,7-dioxo-l-£;a"=icyclo(3.2.0) hep^ane-2-carboxylate (0.523 £, 1-50 —ol) ir. acetonitrile (6.0 zL) kept under a nitrogen atmosphere vas added diisopropylethyl- acine (0.31- -1', 1.6 racl) follovec by diphenyl chlorophosphate * ( 0. 37 3 mL, 1.8 imol) . The reaction mixture was stirred for 30 min and treated vith a solution of 1-( 2-cer c apt oe thy 1)-3 , 5-di^e th;^!^- pyridiniun se t han e s ul f on at e (0.1;93 St 1-87 -sol) in acetc^niNVr:Ll£v"\ - 67 - 20734£ n (1.9 zsZ) followed by diisopropylethylamine (0.314 aL, 1.8 irool). The reaction siixfure was stirred at 0°C for 1 h,diluted vi^h cold (0°C) vatsr (2S at) and poured on.top of a cclu.-=n (7.0 x 3.5 e=) of y-bor.dapak' C-IS. Elution of the colur-n with 25-50* acetonitrile - 75-5C* vatsr raixture cave after lyophiliraticn of the appropriate fractions 1.01 g (90%) of the title conpound as yellowish powder, ir (X3r) : 3700— 3100 (OH)', 177S (C=0 of g-lactam)', 1700 (C=0 of ?N3 ester) , 1S35 (pyri- diniua) , 1595 (phenyl), 1521 (N02> , 1335 (NO ) , 395 era"1 (NO ), 'k.t.t (CMSO d ) o: 1.15 (3H, d, J=S.l Hz, C»CH0HJ, 2.43 (s, G-, on pyridini—) , 6 . "" —3 > 3 4.75 (2H, =, C= N**") , 5.38 (center of A3c, J ', *=14.3 ?.=, CH„ of ?H3) , 6.5-7.5 (10H, :r., phenyl), 7.70 (2E, d, £=8-7 -=, Ho of ?.V3), 8.0-3.5 (3H, a. Hp of pyridir-iu=s, Hn of ?K3) , 3.32' (2H, s, Ho or pyric^r-ius) , uv (H20) 270 (£11570) , .306 (C7343) ni'j. Anal, calcd. fcr C H „N 0 S?- H_0: • C 58.03, H 5.25, N 5.43, S 4.13; found: C 57.93, 3/ 33 3 10 2 ' H 5.05, N 5.22, S 4.34. o I n dC / - 68 - C. 3- f 2- (1- f3, S-dinethyloyriciniurQ ) ethylthio]-6c- [I- (B) -hydroxyethyl] 7-oxo-l-anabicyclo (3.2. 0)hept-2-ene-2-carboxylate 10% ?d/C, H2 THJ, ether, buffer COO To.a solution of p-nitrobenzyl 3-[2- (1- (3, 5-diaethylpyridiri.ua) ) i ethylthio]-6c- [1- (3) -hydroxyethyl]-7-oxo-l-£nibicyclo (3. 2. 0) h.ept-2-ers-2-carboxylate diphenylphosphate (0.600 g, 0.80 roiol)" ia'wet tetrahydrc-furan (36 nL) was added ether (36 bL) , -potassium phosphate monobasic- ■ • sodium hydroxide buffer (0.05>i, pH 7.4, 44 aL)-and 10% palladium.on charcoal (0.60 g). The resulting mixture vas hydrogenated under 40 psi. at 23*C for 1.25 h. The organic layer vas separated and extracted with buffer (2x5 &L) . Water layers were combined, filtered through a Celite pad, washed with ether (40 mL) , pulped to eliminate traces of organic solvents and poured on top of a column (2.5 xlO.O c=) of .» « )J-bondapak C-IS. Elution of tfie column vith water and lyophilizatioa of the appropriate fractious gs.T9 "hh«'title compound. 0.186 g [6k%) as a yellowish powder, ir. (X3r) V 3700-3100 (OH), 1760 (C=0 of B-lactaa) , max 1595 cm -1 (carboxylate) , 5 Kmr (D20)' 6: 1.21 (3H, d, J=6.3 Hz,. G CHOs) , 2.45 (6K, s, CH on pyridinium), 2.81 (d, J=S.2 Hz, H-U ), 2.96 (d, J=9.2 Hz, K-h), 3.22 [cd, J=2.6 Hz, J=6.2 Hz, H-6J, 3.40 (t, J-S.2'Hs, CH2S), 3.84 (cd, J=9.2 Hz, J = 2.6 Hz, H-5), 4.15 (m, CK CHOK) , 4.71 .itsttw. ■' (t, _J=6.2 CH^N )/ 8.21 (1H, s, Hp of pyridiniura), 8.46 (2H, s, 2 i Ho of pyridinium), w (^O) 279 (£8345), 296 (£7714) n , [{X] sa + 40.7 (£0.53, H^O), — 16.9 h (aeasured at a concentration of -4 . 10 H in phosphate buffer pH 7.4 at 36.8°C). Exasole 3 Preparation of f53, Eg-)-3- f [2- (3-hydroxyaethyljyridinio)et'nvl]thio)-S-(1- (S) -hydrc*yethyl) -7-oxc-l-azabicyclo [3 .'2. 0]hePt-2-ene-2-ca.rboxylatg i 207346 - 70 - A. 3-Hvdroxymethyl-l- (2-nercaptoethyl)pyridiniura triflucromethanesul.fc.-.ate rr CH2 OH C?3so3H A. 4 HSCH2CH2N Trifluoromethanesulfonic acid (1.327 riL, 0.015 aol) vas . added dropvise to 3-pyridirenethanol (2.91 aL, 0.030 =aol), followed by ethylene sulfide (0.89 mL, -0.015 mol) . The resulting homogeneous mixture was heated (oil bath) at 50-70°C under h. The' reaction mixture was then taken up in K2<0 (15 nL) and extracted with O^Cl^ (5x5 'mL) . The a<rueo\:s phase was concentrated -Ln vacuo ar.d then aoplied to a C, _ reverse-Dhase coIhtji. Slution with H_0 followed ■ IS 2 by evaporation of the relevant fractions gave a pale yellow oil. Th^s material was rechronatographed to give a nearly colourless oil. drying -Ul vacuo afforded the product (4.50 c, 94%) as a -i viscous oil. ir (film) v : 3450 (s, OH), 2560 (w, SH) a \jfcnr sax (d -acetone) 5: 9.10-8.05 (m, 4H, aromatic), 5.01 (t, J=5.5 Hz, 2H, N-CH„), 6 — 2 4.93 (s, 2H, -CH2OH), 4.43 (br S, 1H, -OH), 3.43-3.18 (a, 2H, S-CH ) , 2.34-2.10 (ra, 1H, SH). 207346 - 71 - p- N i t-T c b e r. z y 1 { 5jR, SS) - 3- f 2-(3-hydroxyaethylpyridinic) ethyl thio]-5- [ 1— (?.) -hydroxys~zliy 13 — 7-oxo-1 -azahicyclo [3.2 . 0] hs?t- 2-ar,.e-2-carb'Oxvia~ ; cishe.-ivlohosohate To a solution of p-r.itrober.zyl (5H, 65_)-6- [1-(RJ -hydroxyethyl] 3, 7-dioxc—l-p.-abicyclo 13.2. 0] heptane-2-car boxy late [0.114. g,.0.50 nzol) in 2 aL cf-dry acetonitrile was added ciisoprcpylethylaair.e fO.CSo aL, 0.55 raol)- at 0°C under N^. Dipher.yl. chlcrop'r.csphate. (0.114 aL, 0.55 — was then added drorwise and the reacticn mixture was stixred at 0°C for 3D min. A solution of 3-hydroxynethyl-l-.(2-aercaptoethyl)pyridiniua triflucre-"aethanesulfonate (0.223 c, 0.7C —.cl) in 0.50 aL of acetonitrile was . then adced, followed by diiscpropylethylaaine (0.122 =.L, 0.70 a^-.cl) After-being kept at 0°C for 30 air. the reaction mixture was concentrated vcLcin and the residual yellow gua was taken up ir. H^O (enough acetonitrile was added to aid in dissolving the cua) . This solution . was applied to a C1 s reverse-phase coluan which vas eluted with 15H acetoriitrile-n^O- Lycphilizaticn cf the relevant fractions afforded the oroduct (0.305 g, 81^)' as a beice-colourec solid. ir (>3r) \> : r.ix 34 20 (br, CH) , 1775 (8-lactam CO) 1SS5 (-CO ?>3) c^"1; ^-tar (d - 2 6 °Aceione) 0: 9.44-7.72 (rn, SH, arcsatic) , 7.22-6.51 10H, dipher.yl-phosphate) , 5.53, 5.27 (A3q, J=14Ke,2H, benz'ylic) , 5.04 [t. J=7.4 Hz, 2n, N-CH ), 4.75 (s, 2H, CH OH) . 4.5-3.1 (m, EH), 1.21 (d, 0=6.3 Hz, 2°734S - 72 - C. (SR, 6S)-3- [ 2- (3-hycroxymethylpyridinio) ethyl] thio]-6- [ 1-(H) -hydroxy- ■ ethyl]-7-oxo-l-azabicvclo (3.2.0]he?t-2-ene-2-carbcxvlate <"w> To a solution of p-nitrobenzyl (5R, 6S)-3- [ 2- (3-hydroxymethyi-pyridinio) ethyl thio] -6- [ 1- (R).-hydroxy ethyl] -7-oxo-l-azabicyclo [3.2.0]-hept-2-ene-2-carboxylate diphenylphosphate (0.145 g, 0.194 r=mol) in 10 nL of TKF containing 5 drops of was added 6.0 mL of phosphate ■ buffer (0.05 M, pH 7.4), 0.145 g of 10%. palladiu^on-charcoal and 10 n\L of ether. The -mixture was hydrogenated (Parr) at 40 psi' fcr 1 h and then filtered through a pad of Celite. The filter cake vas vashed vith a little K„0 and ether and .the acueous ohase was seoarated 2 - • and extracted vith ether (3x). The aqueous solution vas then cooled at 0°C and the pH vas adjusted to 7.0 vith pH 7.4 buffer. After.removing residual volatiles -Ul vacuo the acueous solution vas aoolied to a C. _ • * " 18 reverse-phase column which vas eluted vith Lycphilisation of the relevant fractions gave the product (36 ag,'51%) as a light yellov solid. Further purification by reverse-phase hplc gave the pure product (31 mg, 41*) as a solid. ir (K3r) V : 3300 (br, OH), 1755 (S-lactam CO), 1590 (-C02~) cm"1; JHnar (DO) 6: 8.7S-7.94 (m, 4H, aromatic), 4. S3 (t, J=6.0 Hz, 2H, N-Ci'^) , 4.83 (s, 2n, CH^CH) , rv - 73 - l*.l6 (d of q, J=J'=6.2 Hz, 1H , H-l'), 3.98 (d, of t, J = 9-l Hz, J'=2.6 Hz, 1H, H-5), 3.75-3.20 (a, 3H), 3-20-2.65 (m, 2H), 1.22 (d, J=6.U Hz, 3H, CHMe); Uv (H„0) X : 29^ (e76lU), 266 (e 69 36) — d max aa; t, (dH 7- U, 3o.8°C) lk.0 h. C 15 Preparation of ( 5g., 6S_) - 3- [ 2-( U-hy droxynethylpyri dial 0 ) ethyl thio]-6-[l-(R)-hydroxyethyl]-7-oxo-l-2.zabicyclo[3.2.0]he?t-^ = 2-ene-2-c arboxylat e 2 ij / "s /"■' / ■ W I „ ! * V - 74 - ■k* 4-Hydroxy7ne thyl-1- (2-rne reap toe thy 1) oyridiniua trifluoroaethanesulfonate »J^J)-cs2o= + cr3so3a + To a solution of 4-pyridineaeths_3ol (1.625 g, 0.015 aol) in 10 aL of CH^Cl^, at 0°C under N^, vas added drcp-*ise trifluoro- ' methanesulfor.ic acid (1.327 aL, 0.015 aol) . ' A yellcv-brovn oil rap'idly separated out. An. additional equivalent of 4-pyridine- aethanol (1.635 g, 0.015 aol) was added to this, ai-xture and the .. solvent vas reaoved under reduced pressure to give an oil. . To this cil vas added ethylene sulfide (0.8S1 aL, 0.015 aol) and the ■ resulting homogeneous aixture vas heated (oil bath) at about 60°C for 3 h. The reaction aixrure vas then taken up in 15 mL of H^O and the acueous solution vas vashed vith CH^C^ (5x5 aL) . After removing residual organic solvent jjr» vc.cu.0 the aqueous solution vas applied to a C reverse-phase coluan. Elution vith H O and 1® " 2 subsequent evaporation of the relevant fractions afforded an cil.vhich vas further dried Jjl ~*JCCUC over '2^5 ?-ve product (4.64 c, 97%) as a colourless oil. ir (film) v :.3455 (s, OH), 2565, (w, Sii) ca~*; IT»2LX 3iinar (d -acetone) 0: 9.07, 8.18 (ABq, _J=S.3 Hr, 4K, aroaatic) , "5.03 • © (s, 2H, C-^OK) , 4.96 (t, J=6-5 Hz, 2H, N-CH^, 4.09 (br s, 1H, -OH), 3.5-3.1 (a, 2H, S-G2), 2.25 (brs, 1H, -SH) . C-"Xt-y V f V. /> ' r ; JtcA? x , -- • < r 75 - 3 • p-Nitrobenzyl (5R,6S)-3-[ 2- (4-hydrcxymethylpyridinio)ethyl thio]-5- .I(.E ^-hydroxy ethyl ] - 7-oxo-l-azabicyclo T3 J 2. 0]hept-2-ene-2-carboxylate ciphenylphosphate • . ■ OH A N. c02?nb • To a solution of p-ni trobenzyl (5£, 6S) -6- [ 1~ (R) -hydroxyethyl]- 3 ,7-dio*o-l-azabicyclo[3.2. 0]heptane-2-carboxylate (0.348 g, 1.0 =al) in 5 iaL of dry acetonitrile, at 0°C under vas added dropvise • diisopropylethylaaine (0.191 mL, 1.1 raol) followed by diphehyl chlorophosphate (0.228 ml., 1.1 n=nol) . The resulting golden-yellow* solution was stirred at 0°C for 40 nin. To this, solution was added a solution .of 4-hvdroxyroethyl-l- (2-nercaptoethyl)py"ridiniu=i triflucrc- xnethanesulfonate '(0.447 g, 1.4 eedoI) in'1 xaL" of acetonitrile, followed by diisopropylethylaaine (0.191 nL, 1.1 nsirol). A reddish-black- gira separated froa the reaction laixture. 'After 20 nin at 0°C the reaction mixture was filtered and concentrated -il\ vacuo. The residue was taken up in a ainiraua'volune of acetonitrile-H^O (1:1) and applied to a C, _ reverse-phase colimn. Elution with 25% 18 acetonitrile-H^O ana subsequent lyophilization of the relevant fractions gave the product (0.3S3 g, 47%) .as a . ere an:-coloured solid. ir (X3r) V : 3240 (br, OH), 1775 (S-lactam CO) , 1695 (-CO ?N3) ess"1-sax 2. ' lHrcnr (d -acetone) 6: 9.24-7.84 (n, 8H , aromatic), 7.4-6.9 (ia,-l0H# 6 • ciphenylphcsphate), 5.52, 5.24 (ABo, J=14 Hz, 2H, benzylic), 5.15- 4 . £0 (n, 4H) , 4 .45-3.CS (in, 7H), 1.35 (c, J=6.6 Hz, 3H, GCMe) . 2 . V - '■a' - 76 - C. (SR, SS) - 3- [ 2- (4-hydr3xyr»ethylpyridinio) e thy 1 thio] -6- [ 1~ (R)-hydroxy-ethy1) -7-oxo-l-a~abicyclo [3-2.0] heot- 2-ene-2-carboxylate OH ^ Xjx>- c-:2oz \^o CO 2 o A mixture cf p^r-itrobenzyl (5R, 6S)-3-[ 2- (4-hydrcxyaethyl-pyricinio) ethyl thio]-6-[ 1-(R)-hydrcxyetbyl]-7-cxo-l-£~abicyclo — - • I3.'2.0]hept-2-e.ne-2-carboxylate diphenylphosphate (0.34B g, "0.465 rrrol) and 10% palladiua-oa-charcoal (0.35 g) ia 11 aL cf phosphate buffer (0.05 y~r pH 7.4), 5 aL" cf TSF and 10 aL of ether vas hydrocer.itedit 40 psi for 1.25 h. The fixture was then filtered through s. Celite pad and the acueous phase vas vashed vith ether (3 x) . The pH cf the acueous solution vas then adjusted to 7.0 using additional . pH 7.4 buffer- After removing residual volatiles-ir. vacuo the acueous solution i--as applied to-a C^g reverse-phase column. Elution vith 2% acetonitrile-E^O and subsequent lyophilization gave a yellov-brovr. solid. This material vas rechrcmatocraphed (C a reverse-phase/H 0) Id. * ~ Z to give the desired product (0.060.g, 36%) as a light yellow solid, ir (K2r) V___^: 3400 (br, OH), 1755 (5-lactaa CO) , 1590 (-C02~) ca"1; 3Hnmr (D O)' 0: 8.73, 7.96 (A3q, 3= 6.8 Kz, 4K, aror.atic) , 4.93 (s, 2K, CH OK) , 4.77 (t, J=6.0 Hz, 2H, K-C^) , 4.15 (d of c, J=J'-6.3"K=, IK, H-l1 ) , 3.96 (d of t, J=9.2 Hz, J,-«2.6 Hz, IK, K-5) , 3.65-3.20 (a, 3K) , 3.13-2.62 (ra, 2K) , 1.21 (d, J=6.3 Hz, 3K, CXMe); uv (K 0) >. : 295 (C6S50), 256 (C55SS), 224 (cSUl) nr.; t- (pH 7.4, 26.£eC) 14.5 h ~ty. Z - 77 - Ex ant Is Preparation of 3- f 2-(1- ( 2-roe thy loyrldir.ium)) ethylthio]-6 c-f1-(R3-hvcroxvethy 11 -7-oxo-l-azabicyclo (3.2 . 0) herst-2-ene-carboxylate /y ~>i A. 1- ( 2-mercaptoe thyl) -2-methylpyridinium methanesulf on ate A ' *'* + Ms OK 55"C, 21 h -MsO To a suspension, of 2-methylpyridini'iri methanesulfcnate in 2-methylpyridine prepared by- the addition of methanesulfosic . acid (0.65 mL, 0.010'mol) to.cold 2-nethylpyridine (2.17 mL, 0.022-do1)' vas added ethylene sulfide (0.655-mL, 0.011 mol). S:e reaction mixture vas stiLrred under a nitrogen atmosphere at 55"C for 21 h, cooled to 22°C and diluted vith water (5 nZ) . The aqueous solution vas washed with ether (5x4 mL) pumped to resove traces of organic solvents and poured -on top cf-a column (2.5 x 10.0 cm) • ^*3SL' 20 7 ^ a i* yj' - 78 - of p-bondapax C-18. "Die coluzm -was eluted with water and lyophili- zation of the appropriate fra'ctions gave 2.13 g (85%) of the title- conoound, if (film) V : 2520 (SH) , 1623 (oyridiniun) 1574, 1512, 1435, najc 1412, 1195 (sulfonate), 1038 en"1, 1H=r (EM SO-d + 0^ 5: 2.37 (35, ■ s, C33S03~) 2.83 (3H,.s, CH3 on pyridir-i.ua) , 3.09 (2K, J= 6.9 Hz, ' CH2S), 4.71 {2H, t, J.=6.9 Hz, CH N+) , 7.93 (2?., n, Ha of pyridinius) , 8.44 (1H, a, Hp of pyridiniua)', 8.89 (1H, a, Ho of pyridiniun), uv (HO) X : 266 (G3550) my. ' 2 max 3 . Par an i trobenzyl 3— [2— (1— (2— nethylpyridiniua) ) ethylthio) —62— fl— (jR) — hydroxyethyl] -7-oxo-l-azabicyclo-(3.2.0) he?t-.2-ene-2-carboxylate diphenylphosphate o 1) NStfiPr) ? ? fo?> 2- 2) Cl?(0?h) COOPK3 ' ©"> MsO~ ' . (?h0)^ To a cold (0CC) solution of p-nitrobenzyl 6a- [1- (3) -bydrcxy-ethyl]-3, 7-dioxo-l-azabicyclo (3 . 2. 0)he?tane-2-carocxylate (0.523 g, 1.50 r^rol) in acetonitrile (6 ml.) kept "under.a nitrogen atmosphere was added diisopropylethylaaine (0.314 mL, 1.80 r^sol) followed by diphenyl chlorophosphate (0-.373 nL, 1.80 r^^ol). The reaction nixture was stirred for 30 rain at 0*C and-treated with solution of 1-(2-nercapto-ethyl)-2-methylpyridinium methanesulfonate (0.530 g, 2.16 rrrol) ir. acetonitrile (18 irJL) followed by diisopropylethyia^-ine (0.314 —, ■ ry - 79 - 1.8 rjnol) . The reaction mixture was stirred at 0®C for 1 h diluted. ■ with cold (0°C) .water (26 aL) and poured on top of a coluan (3.5 x 7.0 c of p-boncapak C-18. Elution of the coluan with 25% acetonitrile - 75% water and with 50% acetonitrile -. 50% water gave after lycphiliza'tion of the appropriate fractions 1.06 g, (96%) of the title compound as a. yellowish oowder, ir (KBr) v> : 3650-3100 (OH), 1770 (C=0 of 5-lactaa), max 1695 and 1690 (C=0 of PN3 ester) , 1530 (pyridiniua) , 1595 (phenyl), 1518 (N0_), 1335 (NOJ, 890 a"1 (NO,), 1 Har (DMSO, d ) 0: 1.15 (2H, * 2 . 2 © d', J=f.l Hs, CH CHOK), 2.87 (s, CH3 on pyridiniua), 3.5-4.4 (2H, a, h-5, . CH3C30H), 4.75 (2H, a, CH2N+j, 5.37 (center of A3q, J=14 Hs, C?>2 of ?N2) , 6.5-7.4 (10 H, a, phenyl), 7.70 (2H, d,.J=8.8 Hs, Ho of PK3) , 8.0 .(2H, a, # Ha of pyridiniua) , .8.24 (23, d, 'J=8.8. Hz, Ha of PK3) , 8.50 (12, n. Hp of pyridiniua) , .8.95 (IK, brd, £=6.1 Hz, Ho of pyridiniua), rv (2 0) * * 255 (£11990), 314 (e3020)=JJ TV .X C. .3- [2- (1- (2-aethvlpyridir.iua) ) ethylthio] -6c:- [1- (S) -hydroxyethyl] -7—oxc-1-azabicyclo(3.2. 0)heat-2-ene-carboxylate COO? >3 10% ?d/C, H OH a; ———2—> ri THT, ether, buffer ^ COO 207346 f i - 80 - To a solution of p-nitrobenzyl 3- [2- (1- (2-aethylpyridiniua)) ethylthio] -6a- [1- (S)-hydroxyethyl] -7-oxo-l-azabicyclo (3.2.0)hept-2-ene-2-carboxylate diphenylphosphate (0.66 g, 0.90-aaol) in vet tetrahydro-furan (34 aL) was added ether'(34 aL) , potassiua phosphate xaonobasic-sodiua hydroxide buffer (0.15 M, 16.5 aL, pK 7.22) and 10% palladiua on charcoal (0.66 g). The resulting aixture was hydrogenated tinder 40 psi at 23"C for 1.25 h. The organic layer was separated and extracted with buffer (2x6 aL) . Water layers were coabined, filtered ' . through a Celite pad, washed with ether (40 aL) , pimped to eliair.ate traces of organic solvents and poured'on top of a coluan (2.5 x 10 ca.) of y-bondapak C-18. Slution of the coluan with water and lyophylizaticn of the appropriate fractions gave the. title conpound 0,093 g (31%) as a yellowish powder, ir (K3r) V- : 3650-3100 (OH),' 1755 (C=0 of 'S-lact«a), PSX 1630 (pyridiniua) , 1595 ca ^ (carboxyl2te)., *Har (D^O) c: 1.20 (3H, d, J=6;3 Hz, CK CHOH) , 2.83 (s, C-?3 on pyridiniua) , 2.7-3.1 (S3, K-4, CH on pyridiniua), 3.1-3.7 (3H, a, CH2S, H-6),.3.90 (cd, J=9.1 ^ . J=2.6 Hz, H-5) , 3.1 (a, CH CXOK) , 4.78 (t, £=6.2 Hz, ) , 7.S (2H, a. Ha of pyridiniua), 8.3 (IK, a, Hp of pyridiniua)., S.65 (1H, a, Ho ■ of pyridiniua), uv • (3,0) A : 268 (£9350), 296 (£8840) ay, 1=] *3 2 ' R2.X . D ; +41° (c 0.5, H,0), Ti « 15.0 h (measured at a concentration of 10 ^ M ! • ~ 2 . 2 . . iO in phosphate buffer pH 7.4 at 36.8*C). \ " I -C W - 81 - Zxannle 6 Preparation of 3-[2-(1-(4-methvlpyridiniuin) ) ethyl thio] -6a- (KB) -hydroxy e thy I ] -7-oxo-l-azablcyclo(3.2.0)heot-2-ene-2-carboxy late coo A. 1- (2-mercaptoethyl) -4-niethvlpvridinium methanesulfonate A . +.Mscy 55°C, 24 h HS- Hs 0 To a suspension of 4-picoliniur> methanesulf onate in 4--picoline prepared by the addition of aeth&nesulfonic acid (0.65 0.010 aol) -to 4-picoline (2-14 cl, 0.022 nol) in cooling was acded ethylene sulfide (0. 655 bI,, "0.011-raol). The reaction nixture was stirred under a nitrogen atmosphere- at 55°C for 24 h, cooled to 23"C and diluted with water (5 inL) and ether (10 nS).' The organic layer was separated and the acueous layer was washed with ether (5x5 nL) 'and applied on top of a column (2.5 x 10 era) of p-bondapak C-18-after traces of ether have been removed under reduced pressure. Zlution of the column with 15% acetonitrile 85% water, rcixture cave after' lyophylization of the appropriate fractions a colorless - 82 - 207346 syrup 2.66 g (100%) , ir (filn) \> : 2500 (SH) , 1=40 (pyridi.-.i\=) , max 1572, 1520, 1473, 1200 (sulfonate), 1040, 823 and 753 cn ^Hrr (D».SO-0 6: 2.31 (3H, s, C-:,SQ,"), 2.62 (s, CH, or. pyridiniv^) , o 3 3 3 2.2-2; 9 (4H, SH, Ci on pyridini^) , 3.04 (2H, n, CH^S) , 4.53 (2K,t, J=5.4 Hz, CH2N+) , 8.01 (2H, d, J=6.6 Kr, Krs of pyridini'—} , 5.3= '.23., d, £=6.6 Hz, Ho 'of pyridiniun-.) , uv 255 (£4100), 221 (£7544) -V. . • —~ (2 —ri® r ca — toa} — 4—nsvlovridf.r' ■* • toluenes**; Lf~na -£ A + H(Q)^/?-sra pCsO To a suspension .cf p— ■toluenesulfcni.c acid (1.72 c, 0.01 —cl) in be.-.-ene ' (S. 5 =£.) was added 4-pi col ins (1.17 =iL, g. 012 nol) . The resulting fixture was stirred under a nitrogen atmosphere at. 22*C for 30 r-it, treated vith ethylenes-jlfids (0.63 :tL, C.C11 aol) and stirred at 75° for 24 h. More ethylenesulfice (0. =5 0.011 nol) vas added and the stirr ing was continued at 75°C for 24 i acre. The reaction mixture vas cooled to 23°C and diluted ---iti vater ' (5 il) ' and ether (3 mL) . The acueous layer vas separated and vashed vith ether (3x8 mL) . ' The traces cf organic solvents •-»:» rer^ved ■ under vacuum and the conpound vas chromatographed cr. y-bondapax C-lc vith water as eluting solvent to 'give 2.94 g (20^) cf the title * CC.T.DCwid as a colorless svru-s; ir (fiL*n) v : 251- (SH) 1S40 (?vri- r.ax ~ diniurn) , 1595, 1582, 1475, '.1200 (sulfonate), 1031, 1010. SIS c=_1. :>1- s I ?s 207346 - 83 - o (Dy.SO, d) o: 2.29 (3H, s, CF.^ cn pyridir.iv=t) , 2.51 (s, CH^ ?h), 2.4-2. S (4K, SH, CH3?h), 3.03 (2H, a [addition of -D,0 gave at:, £=6.4 Hz, at 3.04] , CH S) , 4. 68 (2H, t, £=6.4 Hz, CH^U')f 7.11, 7.49 0.3 Hr, C. 2d, 3-1.9 Hz, Phenyl), 8.00 (2H, d,' £= (2H, d, £=6.5 Hz, Ho of pyridir.itrO , (H20) 255 (£4315), 222 (C17045) aU Parar.itrcb-anzyl 3- T2- (1- (4-zvethylcv'ridir.iuz:) )etnylthio]-Sz- fl- (5)~. hydroxyethy 1 ] -7-oxg-l-azabicr-'clo (3.2. 0) bept-2-eze- 2-carbcxylate diohenvlchosbhate CH 1) K=t(i^>r)_ 9 2) d?(0?h)^, '"COO? S3 /©\- ilscf • CCC?>3 C~nO). ;'v> To a cold (0"C) sdl*jti.cn of p-r.itrobenzy I 6c- »i-(S)-hydrcxy-. ethyl] - 3,7-dicxo—1-azabicyclo (3.2.0) hettaze-2-carboxylase (0. 522g, 1.5 z=jo1) in acetonitrile. (6 si.) kept "uncer a ni.trocar. acrcsphere vas added diisopropylethylaz^Lne [0.314 :iL, 1.8-r^r.cl) folicked by" diphenyl chlcrophosphate (0.373 r_L, 1.9 zz^cl) . The reacticn =ixt^re vas stirred for 45 r-JLn and treated dropwise vith a solution cf 1-(2-mercaptoe tbyl)-4-hethylpyridiniu^ nethanesulfonate (C.533 a, 2.16 km!) in acetonitrile (1.8 rrZ,) followed trj diiscprcpylethylanir.e (0.314 nL, 1.3 nmol)'. The reacticn zixfrs vas stirred at C"C fcr 1 diluted wizh ccla (0"C) «a:er (24 nvl) anc oc-ired cn tea cf a col-jrin i • • - </ I " \ o'\ /P :5i 2 0 7 "x * ^ - w4o - 84 - (2.5 x 8.5 cn) of y-bondapak C-IS. Zlution of the colunn firsr with 25% acetonitrile -75% water mixture (100 aL) then vith 50* acetonitrile 50% water nixture (100 nL) afforded after lyophyligation of the appropriate fractions 0.91 g (83%) of the title compound as a yellowish powder, ir (K3r) : 3700-2800 (OH), 1770 (C=0 of 5-lacta.^), 1700 ' (C=0 of ?K3 ester) , 1640 (pyridiniua) , 1595 (phenyl) , 1520 (HO^) , 1340 (N02), 890 erf1 (N02), lKnr (DMSO, d&) 0: 1.16 (3H, d, J=5.2 Hi, C^EO 2.61 (s, CH^ on pyridiniua), 3.1-3.7 (3H, a,-.3-5, CS2S), 3.7-4.4 (23, a, 3-5, C3 CS0H), 4.79 (23, brt, £=6.3 Hz, CH N+), 5.17 (i, ' 5=4.9 Hi, OH), 5.37 (center of >3q, '2=14.1 Ez, C32 of ?K3), 5.7-7.4 (10 H, a, phenyl), 7.69 (23, d, J=8.8 Hz, So of ?K3) / 8.00' (23, d, £=6.5.Hz, 3=) of pyridiniua) , S.23 (23, d, J=S.8 Hz, Ha of ?K3), 8.52 .(23, d,'5=6.5'Hz, Ho of pyridiniua) , -.tv (3.0) X : 262 (£10335), 2 ZfiSLX • 311 .(CS570) ay. ' Anal. calcd. for C_.HJ,K,0, .S?-1.5 3„0: C 55.S4, —~36 3 10 2 3 5.17, N 5.52, S 4.21; found: C 56.39, 3 5.13, N 5.19, S 4.41. 3- [2- (1- (4-methyloyridiniim)) ethylthio'] -6c- fl- (5) -hycroxyethyl)-7-axo- l-azabicvclo (3.2.0)heot-2-ene-2-carboxylate " * - OH. - 85 - To-a solution of p-nitrobenzyl 3-[2-(1-(4-merhylpyridizium)) ethylthioj -6c- [1- (H) -hydroxyethyl)-7-oxo-l-azabicyclo (3.2.0)hept-2-ene-2-carboxylate diphenylphosphate (0.'537 g, 0.80 mmol) in vet tetrahydrofuraz (30 nL) vas added ether (30 mL)., potassium phosphate mono basic-sodiuzj hydroxide buffer ( 0.15 M, 14.7 nL, pH 7.22) and 10.% palladium cn charcoal (0.59 g) . The resulting mixture vas hydro-genated under 40 psi .at 23°C for 1.25 h. The organic layer vas separated and extracted with the buffer (2x6 mL) . The aqueous extracts were combined, filtered through a Celite pad, washed with ether (3.x 20 nL) , pimped to remove traces of organic solvents .and pcu on top of a column (2.5 x 10 cm) of y-bondapak C-13. Zlution.of the column with water and lyophylization'pf the.appropriate fractions gave 0.136 g (4 9%) of -the title conpound as a yellowish povder, ir (X3r) v_ : 3700-3000 (OH), *1770 (C=0 of 2-lactaz;) , 1S42 (pyridinium) 1592 cn 1 (carboxylate) , (D^C) 6: 1.19 (3H, t, J=S.3 Hz, CH O-OH) 2.59 (3H, s, CH on pyridiniua) , 2.64 (d, J=9.1 Hz,H-4), 2.90 (d, £= 9.1 Hz,H-4 ), 3.0-3.6 (3H, ra, C-^S, 2-6), 3.86 (dd, J=9.1 2 z, J=2.6 2: K-5) , 4.12 (ra, CI-^CHOH) , 4.5-4.9 (Cr^N* masked by HCD) , 7.80 <2H, d, £=*6.6 Hz, Ha of pyridiniua) , 8.58 - (2H,' d, J=5.6 Hz, Ho of pyridir.ium) , uv (HO) X : 256. (E5S10), 252 (C53S0), 296 (£7050) n , [c7 25 +20. d (c 0.48, H^O) , T^**12.8 h (measured at a .'concentration of 10 ^ Min a. phosphate, buffer pH 7.4 at 36.8°C). ~ "J/ 346 - 86 - 2xan"ol.e 7 Preparation of (5H) 3-t2-(4-methylthiopyridinio) ethylthio] - (6S) - [ (IR) -hydroxy ethyl] -7-oxo-l-azabicyclo[3.2.0]her> t-2-ene-2-carboxy late 'OH 4-Methvlthiooyridine* 1) Mel/EtOH "V 7/ JH V— / 2) NaOH 4-Mercaptopyridixse ■ (5.55 g, 50.0 isaol; Aldrich) was dissolved in boiling abs. EtOH(50 aL) . The insoluble aaterial vas " reaoved by filtration over Celite. ' The filtrate was heated to re-dissolve, and when it cooled to ca. 50°C, methyl iodide (3.17 aL, 51.0 rool; Aldrich) was added at once'. The nixt-ere 'was cooled to crystallize. Filtration of the solid cave 6.77 g (26.7 naol, y. 53.5%) of the title compound as the hydriodide: (D^O) "o: 2.70 (3H, s, -SCH3) and 7.65-7.77-8.35-8.48 ppai (4H, A232 type, arc^a Hs); ir (Nujol) : 1615, 15S5 (aromatic) and 730 cn -jv (H O) r.ax 2 % - 87 - * 227 (£2.02 x lO1*) and 293 ran (cl.64 x 10u). The hydriodide (6.33 g, 25.0 ssnol) vas dissolved in H O • 2 (40 mL) and the insoluble material vas. removed and washed with H^O (10 nL) . To the filtrate was added at 0-5° NaOS pellet (5 g) and extracted with 2^2® (3 ;< 25 rri) , saturating the acueous layer with NaCl. The corrbined organic, extracts were washed with brine (x 2), dried (MgS04) and evaporated, yielding 2.92 g (23.4 r=nol, overall yield 50%) of the title' co-pound as an oil: 1 Hrr- (CDC1 ) 6: 2.48 (3K, s, -SCH^) and 7.03-7.13-8.38-8.48 ppa (4K, ^^H^type, ar'c:r.atic-Ks) ; ir (fila) \> : 1580 and 800 ca"1. aax. . . . . - ♦preparation of this co-pound was reported iby Xing and Ware, J. Chera. Soc., 573 (1939). The procedure, described in this reference was followed. 4-Methvlthio-N- (2-inercapSoethyl) pyridiniua aethanesulfonate • 4— Hethylthiopyridine (2.75 g, 22.0 —.nol) was added slowly to methanesulfonic acid*- (0.65 aL, 10.5 -aol) by cooling in an ice-bath. To this solid was- added ethylene sulfide* (0.66 mL, 11.0 raol, Aldrich) and the nixture was heated at 50-60"C for 21.h. As reaction proceeds the solid went to solution. After coolinj, 2 073 * V. /- - 88 - the reaction mixture vas dissolved in H^O (5 mL) and washed vith Et^O (5x4 nL) . The cloudy aqueous layer was filtered over Celite and the filtrate was purified by reverse phase silica gel colunn chromatography (C micro boncapack 10 g) elutlng with H O. Each 18 " fraction of 10 nL was collected! Fractions 2 and 3 were ccnbined and repurified by the reverse phase column. Fraction 2 gave 1.258 g (4.48 laaol, v. 42.6%) of the title conpound as a viscous oil: 1Kmr (IMS'O-^ . CTT-20) 5: 2.32 (3H, s, MeSoQ , 2.72 (3H, s, -£>£e) , O w 2.68 (1Hr a, ;SH)', 2.9-3.2 (33, a, -CH S-), 4^59 (2H, t, J-5.4 Hz, -CH 7.97 (2H, "d", 3=1.2 Hz, arcmatic-Hs) and. 8.72 ppm (2H, "d", J=7.2 -Hz, aronatic-Hs) ; ir (neat) V : 1630, 1200 (br, -SO, — ei ax j 7 -85 and 770 cn-1. * These reagents vere distilled prior to use. C. (53) p-Ni trobenzyl 3-[2- (4-Methylthiooyridino)ethylthio]-(6g)-[(13)- hydroxvethyl)-7-cxo-l-azabicyclo[3.2.0)hept-2-ene-2-carboxylate chloride To a solution of (5.3) p-nitrobenzyl 3 ,7-dioxo- (6S) - [ (IS) -hydroxy ethyl) -1-azabicyclo [3 .2. 0)he?taner (2R)-carboxylate (475 ng", 1.36 mnol) and diisopropylethylanine (0.24'nL, 1.4 rc.ol) in CH^CN . (5 nL) was added at 0*-5cC under a nitrogen atmosphere diphenyl ch-lzro- - 89 - O r-'. 't ; phosphate (0.29 mL, 1.41 n.-nol) . The nixture was stirred at 0*-5®-, for 20 =in_ To this r.ixture was added an oily suspension of 4 -raethylthio-N- (2-:nercaptoethyl)pyridiniura methanesulfonate (678 mg, 1.45 csiol; 60* pure) in CH^CN (1.5 nL) followed by diisopropylethylanine (0.24 nL, 1.4 Etaol) . The mixture-was stirred at O'-S^C for 1 h. Immediately after addition of the base, yellowish precipitate foraed. "The precipitate was filtered and washed with cold CH^CN (3 »L) , yielding ^13 ng of yellowish solid. This was triturated from 10% MeOK in H^O ( 5 mL) to obtain 341 mg (0.618^rool, y. 45-. 4%) _ of the title compound as white crystals: np 118o-120°C; • JHnr (E£iSO-d , CFT-20) 6: 1.16 (3H, d, J=6.1 , l'-CH,), 2.72 (3H, "s, -SCH,), 3.1-3.7 (5H, m) , 3.7-4.3 — ^ J • © (2H, =0, 4.71. (2H, t, J=6.3 Hz, -CH2N) , 5.15 (1H, d, J=4.9 Hz, OH), 5.20-5.35-5.40-5:55 (2H, A3g, CX^CH -Ar) , 7.70 (2H, "d" 3=8.8Hz, nitrophenyl-Hs), 7.97 (2H, "d", 3=7.0 Hz, pyridinio-Hs), 8.25 (2H, "d", J=8.8 HZ/ nitrophenyl-Hs), and 8.76 ppra (2H, "d", 3=1.1 Hz, pyridinio-Hs); ~r (Nujol) v=iX: 3250 (OH), 1775 (B-lactaa), 1700 (ester) and -"I 1625 cn ' (pyridinio); uv (abs, StOH) A : 308 na (£4.47 x 104); + 24,8 HeOH); Anal, calcd. for C_.H.,N,0 S Cl-S O: => 24 26 3 6 2 2 C 50.56, H 4.95, N 7.37; found: 'C 50.63,- H 4.72, N 6.89. (55) 3- [2- (4-Methy Ithiopyridinio) ethylthioH65) - [ (IE) -hydroxyethyl) ~7-oxo-l-azabicyclo [3.2.0]he"Jt-2-ene-2-carboxvlate H2/?d-C OH A, pH 7. 4 buf - M * V 20^346 - 90 - i i (5SJ p-Nitrobenzyl 3- [2- (4-methyl thiopyridir.io) ethylthio] - ' (SS)- [ (IS) -hydroxyethyl]-7-oxo-l-azabicyclo [3.2. 0]hept-2-ene-2-carboxylate chloride (330 ng, 0.688 noaol) vas dissolved in TEF (31.5 nL)' and pH 7.40 phosphate buffer (31.5 mL; 0.05M Fisher) and diluted with Ft^O (31.5 nL). This solution was mixed with 10% Pd-C (380 sig, Engelhard) and hydrogenated at 35 psi on the Parr shaker at room temperature for 1 h. The aqueous layer was filtered over Celite to renove the catalyst, and the Celite pac was washed with H20 ( 2 x 5 mL). The filtrate and washing were ccnbined and washed with Ft^O (2 x 30 nL) . The acueous layer was pur.ped off to remove any organic solvents and purified by reverse phase col'jnn chrcr.a- tography (C _ nicrobondapak, 13 g, Waters Associates) eluting with K O. 2° 2 Fractions having a uv absorption at 307 nn were collected (ca. 1 L) and lyophilized to obtain 127 rag (0.334 smol, y.-48.5%) of the title compound as a yellowish powder: !Hnr (D^O, CFT-20)o: 1.20 (3H, d, J=6.4 Hz, I'-CH^), 2.64 (3H, S, "SCH ) , 2.81 (2H, n, -SC^-) , 3.19 (1H, dd, J. .,=6.1 Hz, J. ,=2.6 Hz, 6-H), 3.32 (2H, dd, J=ll Hz, 0=5.5 Kz, 4-Ks), 3.52 (IK, dt, J=9.2 Hz, J. =2.6 Hz, 5-K), 4.1 (IK, ra, .l'-K), o 4.61 (2H, t, J=5.9 Hz, -CH^) r 7.70 (2H, "ci", J=7.1 Hz, aronatic-Hs) , ■and 8.40 ppa • (2H, "d.", J=7.1 Hz, arorsatic-Hs); ir (K3r, disc) v : nw" 3400. (OH) , 1750 (5-lactam) , 1630 (pyridinius) and 1590 era"1 (carboxy-late); uv (J^O) X^^-. 231 (£9800) and '307 no (C25000) ; + 3.14 (c 0.5, H^O) . \ \ 207346 - 91 - Sxajr.pl e 8 Preparation of 3-[2-(3-msthoxy-l-~yrldinimn)ethylthio] 6e— [1' - (3) -hydroxy ethv 1 ] - 7-oxo-*l-azabicyclo(3.2.0)-heDt-2-ene-2-carboxvlate A. 1-(2-mercaotoethvl)- 3-methoxvpyridinium methanesulfonate **MeD\ • .V .-fx ' <Qf^d ■ To preceded (5*C) 3-s>ethoxypyridine (S9S ag, 6.4 =aol) vas . added drop-rise sietha.nesu.lfor.ic acid (0.216 =iL, 2.05 =ol) and f * * ethylene sulfide (0.19 mL, '3.2 rod). The rixture vas then heated at 60*C for 18 h, cooled to 20°C, diluted vith' vater (10 ru.) and . washed vith ether (3 x 10 nL) . The acueous phase vis p-i=ped under high vacuus for 15 min and poured on a C _ reverse phase 13 column. The title conpound vas eluted vith vater. The appropriate fractions were cor-hined and evaporated under high vacuus to give the desired thiol (61.6 sag, yield-76.3 V) ; ir (C-I CI J V :2550 (v, SS) 2 2' and 1620, 1600, 1585 ca"1 (ra, aromatic); JK.-^r (Df-SO d.) o: 8.90-7.90 © (4H, m, arcnatic C-K) , 4.72 (2K, t, 0_=6.6 V.z, CH^*) , 4.01 (SH, s, CC-^) , 3.5-3.0 (ra, hidden CH^S), 2.66 (IK,' dd, J=9.5 Hz, J=7.5 Hz, SH) and 2. 31 ppm ■ (3H, s, C-:3S03~) . 207346 . - 92 - 3. pa.ra-Nitrober.syl 3 [2- (3-nethoxy-l—pvridinixra chloride) ethylthio]-6c- [ 1' - (g) -hydroxyethyl] -7-cxo-l-asabicyclo (3.2.0) -hept-2-ane-2-carboxylate oh - A 1) 2) C1?0(0<£)„ ■N. XO) Mso" 0 CO ?N3 r - • A cold (0*C) solution of p-nitrobenzyl 6c- [11 - (3) -hydroxyethyl] -3,7-dioxo-l-azabicyclo-(3.2.0)-heptane-2-carbc:cylate (1.04 5, 3 'rr2oi) . • in acetonitrile (12 aL) • vas treated dropvise with diisopropylethyl- (0.63 mL, 3.6 nmol) and diphenylchlorophosphata (0.75 sL, 3.6 mrol) and stirred at 0°C for 30 nin. The resulting enol phosphate vas treated vith 1-( 2-niercaptoethyl)-3-rretho>ypyridiniim nethanesulfonate (1. 1U g, . 30 nnol) in CH^CNC? nL), diiso-propylethylanine (0.63 iL, U.30 nnol), stirred for 30 nin. and cooled at -10°C for 30 nin. The solid that precipi-atec out of the fixture vas filtered, vashed vith cold acetonitrile •w1 (2 aL) and dried to give the title compound- (1.32 g, yield 82%). i ir (nujol) V 3320 (ra, OH), 1780, 1765 (s, S-lactan ■ ,C=0) , 1700, 1625 (m, ester C=0) and 1520 cm"1 (s, NO,); ^Hmr (DMS0 d.) 0: 9.01'(1H, 2 o bs, H-3 aromatic), 8.75 (IK, bd, J=5.4 Hs, H-S aromatic), 8.35-7.95 (4K, m, H-aromatic), 7.70 -(2H, d,J=7.7 Hz, H-aromatic), 5.37 (2H, center of A3c, J=13 Hz, GJ2?N3) , 5.17 (IK, d, J=4.9 Hz, CH) , 4.87 (2H, t, 3=0.2 Hs, a2-N8), 4.35-3.75 (2H, m, H-5 ard H-l'), 4.00 (2H, s, 0CH3) ■ 3.56 (part cf a t, J=5. 3 Hz., CA S) , 3.5-3.20 (33, m, H-6, H-3) and 1.16 p?.t (3H, d, J=6.1 Kz, GLjOiO) . 207346 - 93 -i - i C- 3-f 2-(3-methoxy- 1-pyr idiniur:) ethylthio] - S-- [ 1' - (S) -hydroxyethyl] - " 7—cxc— 1-azabicyclo (3.2.0) -hept- 2-e.ne— 2-carbcxylate. /v 1 o A solution cf -P-ara-nitrobenzyl 3 [2- (2-:aethcxy-l-pyricir_i — chloride) ethylthio] -Sc- [1' - CZ) -hydroxyethyl] -7-cxo-l-azabicyclo (3.2.0) — hept-2-ene—2-carbcxyl'ate (=00 rig, 1.12 mol) i_n T^£? (25 rLL) , ether (25 rZ.) and. pH 7.4 phosphate buffer '(0.1M, 25 =lL) vas hydrocenated in a Parr shaker over 10* ?d/C (1-1 g) fcr.-l h at 40 psi . "The xr-^r; 'vas diluted vith ether and the ac-jeous phase vas filtered through. • a §52 hardened filter paper. The acieous layer was vashed vith ether (2 x 20 nL), p-.rr.ped under va^-r, and'poured or. a silica gel reverse phase colr~r..' The title co—pound was eluted vith '-"ater ■ containing 2 and St acetonitrile. The appropriate fractions were combined and lyophiliied to civs a yellow solid that vas rep\;rifie hplc to give the penen carbbxylate (150. rsg, 38%); ir (r.ujol) 1750 (s, D-lacta.- C=0) and 15S0 (s, carbcxylate); *H=r (D_£") c: 8.55-8.30 (2H, =1, H-2, 5-5 arc=atic) i 8.17-7.75 (25, =, H-3, 5-4 arcn-.atic) , 4.77 (2K, t, £=5.9 Hz, C= , 4.10 (1H, part oz 5 lines, £=6.3 'Hz, h— 11 ) , 3.97 (3K, s, OC5 ) , '3.85, 3. E2 (2 lines, part cf • dt, £=2.6 Hz, part cf H-5), 3.42 (2H, t, £=5.9'Er, C-: -S) , 3.25 (1H, cd, £-6.1 Hz, £=2.6 Kz, H-S) , 2.99- 2.60 (2H, S lines, pajrt cf ~ H-4) and 1.20 ppm (3H, d, £=5.4 H.Z, C-^); uv (J^O, c 0.05) 290 (C10517), 222 (C 6 S 4 2) ; T, (0.1 M ?H 7.4 phosphate buffer, 37 *C) n- 20/346 - 94 - r>, Example 9 ?reparat£on .of (53;6S)-3- [ 2- (3-aethylthiopyridinio) ethyl thio]-6- ^ —^-"hydroxyet-hyl] -7-oxo-l-a::abicryclo J3 .2.0] nept-2-er.e-2-carbcxyla.te S.Me 3-Methylthio-l— (2-as.rcaptoethyl) ovridir-i'-ra chlorlce 2 CI A -V HSG^CS N SMe CT- O o •To a solution of -3-aethylthiopyridine (2-00 g, 0.015 aol) in 10 aL of ether vas added 15 rr-.i of 1 5 KCl and the aixture vras veil shaken. .The aqueous phase vas separated, vashea vith 10 aL of ether and then evaporated. The residual hydrochloride vas then dried -£n vacuo (7 0)' to give a vhite solid. To this solid hydrochloride was added S-methylthiopyridine (1.33 g, 0.013 aol) and ethylene sulfide (0.S9 aL, 0.015 aol) and the resulting r-'xf~e vas heated (oil bath) at £5-65°C under N for 15 h. This ca.ve a slightly turbid oil vhich vas ta^en up in 125 nL cf- and vashed vith CH^Cl^- The aqueous solution vas concentrated to about 25 aL and then a few drops of acetonitrile were added to nia^e the aixrur-hcmoceneous. The resulting acueous solution was aoclied to a C IS reverse-phase column. El-jticn with "2® subsequent evaporation *1 /->• -t *■ 1 / : -> - 95 - of the relevant fractions afforded the product (2.66 e 3C^) as a pale yellow, viscous oil. ir- (fila) v 2410 (br, -SH) en"1; 1Zz=— td^-DXSO*-D^O) 0: 8.8S-7.83 (m, 4H, aromatic), 4.70 (t, _J-o.5 Hz, 2H, &-C32), 3.08 (skewed t, J=6.5 Hz, 23, S-CH }, 2.64 (s, 3H, S-He) . Prepared by the method of J.A. Zoltewiez and C. Nisi, J. Ore. Chen. 34, 765 (1969). B. p—• Nitrobenryl (5R,6_S)-3-[ 2- (3-aethyithiopyridinio) ethyl thio]-6- [1- (R)-hycroxyethyl) -7-oxo-l-azabicyclo [3.2. 0]hept-2-ene-2-carbcxylate chloride • x XQ- ■ -—> . CO„?N3 2 A solution of p-nitrobenzyl (5K, 6S_)-6-[ 1-(Rrhydroxyethyl]-. 3,7-dioxo-l-azabicyclo [3 .2. 0)heptane-2-carboxy late (0.522 g, 1.50.:mol) • in 7 al of dry acetonitrile was cooled at 0°C and then diisopropylethyl-anine (0.287 nL, 1.65 naol) vas acced drop vise.. 'To the resulting yellov-brow-n solution vas added drepvise diphenyl chlorophosphate (0.342 nL, 1.65 raol) and the reaction riixture vas kept at -0 °C for i - 30 nin. Diisopropylethylamine (0.313 TT.L, 1.80 ncnol) vas then added, L* followed by a solution of 3-.tiethylthio-1- (2-iaercaptoethy 1)pyridiniun chloride (0.393 g, 1.80 rmol) in 0.70 nL o'f dry DKT. About a minute after the addition was complete a precipitate separated from the_ reaction mixture and -further cooling at -ICC for 10 nin cave a solid orange-coloured mass. This solid vas subsequently triturated vith acetonitrile and the residue vas collected by filtration. The residue - 96 - 207346 was washed with acetonitrile, then acetone and finally dried -cn vazuo to give the product (0.455 g, 55%) as a ere an-coloured solid. The combined filtrate was evaporated to give a yellow oil which was taker.- up in a ninin\ra volume of acetonitrile and cooled at 0°C for 30 nin. ; filtration of this mixture afforded an additional 0.139 g of the product as a light yellow solid.. The ccnbined yield was 0.594 g (72%). - (J3r) Vrav: 3345 (br, -OH), 1770 (S-lactajn CO), 1680 (-CO^PKB) <=a~"; (d^-DSiSQ) d: -8.9S-7.S6 (m, 4K, pyridiniuni arcnatic) , 8.20- 7.65 (ABc, J=7.0 Hz, 42, PN3 arcmatic), 5.53-4.80_ (a, 4S) 4.3-3:7. (a,.2H), 3.6-3.25 (a, 6H) ^ 2.66"(s, 3H, S-.Me), 1-16 (d, J=6.0 Hz, 3H, CHMe) . C. (5R, 6^)-3.7 [ 2- (3-methylthio?yridinio) ethyl thiol-6-fl-(R^bydroxy-ethyl] -7-oxo-l-azabicyclo [3-2.0]hept-2-gn&-2-carboxylate SMe O To a nixture of p-nitrobenzyl (5£,6S)-3-[ 2-(3-aethylthio-pyridinio) ethyl thio]-5- [ ( ^;-hydrcxyethyl]-7-axo-l-azabicyclo [3.2.0]-hept-2-e.ne-2-carboxy late chloride (0.551 g, 1.0 nsiol) and 10% palladium on-charcoal (0.55 g) in 25 nL of.phosphate buffer (0.05 it, 'pH 7.4) was added 5 nL of TKF and 25 nL of ether. This nixture was hydrogenated (Parr) at 40 psi for 1 h. The reaction nixture was then filtered through Celite and the filter cake was washed_ with H^O and ether. The acueous phase was separated and washed with additional ether (3 x), r\ / /;■ - 97 - After removing residual'organic solvents vacuo the aqueous solution was cooled at C"C a_nd the pK was adjusted to 7.0 with saturated aqueous NaHCO. This solution was immediately applied to a C reverse- . 3 1® phase column. Elution with K^O and subsequent lyophilization of the relevant fractions afforded 0.25 g of a bright yellow solid'. This material was repurified by reverse-phase hplc to give the product (0.210 g, 55%) as a light yellow solid, ir (KBr) v : 3400 (br, -OH), max 1755 (g-lactaa CO), 1590 (-CO^") cs"1; 5Hnmr (DC) 0: S.60-7.76 (m, 4H, aromatic), 4.76 (t, 3=5.8 Hr, 2H, N-CH^), 4.13 (d of q, 3-3' = 6.3 Hz, 1H, K-1') , 3.95 (d of t, J=9.0 Hz, J'=2.8 Hz", IE, E-5) , 3.45-. .2.75 (n, 5H) , 2.59 (s, "3H, S-.Me), 1.20 (d/ J=6.4 Hz, 3H, CHMe); W (H20) \aix: 295 (£S509>' 273 (C13005), 231 (£11576) sn; (pH 7.4, 36.3"C) 20 h. Sxaspls 10 Preparation of 3- [ 2-(1- (2 , 6-cirasthyrpyridiniua) -ethylthio] -6c-ri~(B) "hydroxy ethyl] - 7-oxo-l -azaJbicycloQ. 2. 0) he? t-2 - en e-2- carboxy late - 98 - 207346 1-(2-mercaPtoethyl) -2,6-dimathylpyridinium methanesulfonate ^ + "O + Msca 100-C, 42 b • / _ Hs0 A nixture of 2,6-<iinethylpy-ridine ■ (19.2 nL, 0.155 sol) and E>eth'anesu.lfonic acid. (3.27 nL, 0.050 sol) vas stirred for 15 nin, treated vith ethylene sulfide (4.17 nL,. 0.070 aol). and stirred at 100 °C for 42 h under a. nitrogen atmosphere. After cooling to 25*C,. the reaction mixture vas diluted vith ether (45 nL) and vatsr (30 nL The two layers vere separated tnd the organic layer was extracted vith water (2x5 nL) . The aqueous layers were ccnbined, filtered ■ ' ■ through a Celite pad, washed with ether' (2 x 15 xaL) , pulped ta renove the traces of organic solvents and poured-on top of a coluna • (3.0 x 12 cn) of p-boncapaJc C-18. Llution vith 3% acetonitrile. 57i water nixture gave after lyophylization of'.the appropriate fractions 2.5 g of the impure title compound as a syrup. It vas repurified by hplc (y-bondapak C-18) to give 0.90 g (7%) of the title compound. ir (film) V : 2520 (SH), 1540 and 1625 (pyridiniura) , 1535, 1490, max 1200 cn 1 (sulfonate), 5Hnr (EMSO-cfg + D20) o: 2.36 (3H, s, 4.62 (2H, et, C-i^N ), 7.74 (2H, n, Ha of pyridiniua), 8.24 (IK, n, Kp of pyridiniua) , uv (H20) 272 ( 4080)ny - 99 - v :#? 3. ?aranitrobenzyl 3- [2- (1- (2, 6-dinethylpyridiniun)) ethylthio] -6S- [ 1- (H) -hydroxyethyl ] -7-oxo-l-azabicyclo (3.2.0) hept-2-ene-carboxylat dioheavlohosohate "* OH X 'COOPKB 4). NSXfiPr), e 9 _ (?h0)a?0 /-"N ...*s , '} To-a.cold (0°C) solution of p-nitrobenzyl 6c- [l-(3) -hydroxy ethyl] - 3, 7-dioxo-l-azabicyclo (3.2.0) ieptane-2-carboxylate (C.658 g, 1.85 =nol) •in acetonitrile (6 nL) kept uncer a nitrogen atnosphere was added . diisopropylethylanine (0.394 mL, 2.26 nrnol) and diphenyl chlorophosphate (0.463 nL, 2'. 2 6 mnol) . The reaction nixture was stirred 30 nin and. treated vith a solution of 1- (2-n»rcaptoethyl)-2,Grdinetbylpyridiniun ■ methane sulfonate (0.720 g, 2.73 rr.no 1)' in acetonitrile (3 nL) followed by diisoprcpylethylanine (0.394 nL, 2.25 mol) . The reaction nixture was stirred at 0°C for 2 h, diluted with cold (0°C). water (27 nL) and poured on top of a colunn (2.5 x 9.0 cn) of y-boncapa3< C-18. Zlution with acetonitrile-water' nixtures and lyophylization of-the appropriate fractions gave 0.92 g (55%) of the title conpound, ir (XBr) v : 3700-3000 (OH), 1765 (C=0 of S-lactan), 1690 (C=0 of PX3 ester), nax 1620 (pyridiniua), 1590 (phenyl), 1517 (N02), 1330 (NO^) , 880 cn 1 (NO.), JHnr (DMS0, d ) 5: 1.15 (3H, d, J=6.2 Hz, G.GOH), 2.7-3.7 2 o ""3 (11 H, Ci2S, 2-OJ3 on pyridiniun, H-4 , ■ H-6), 3.7-4.4 (2H, C^OJOH, K-5), 4,7 (2H, n, CH N+) , 5.14 (1H, d, J=4.5 Hz, OK), 5.37 (center of ABq, £=13.2 Hz, CH of PK3), 6.7-7.5 ' (10H, n, phenyl), 7.'5-8.7 (72, rsyridiniun, H's of ?N3) , uv (H„O) X : 274 (£14150), 319 (£9445) ny 2 nax - 100 - 207346 3 - [2- (I- (2 , 6-dinethyIpyridir.itrr,))ethylthio] -5c- [1- (3) -hvdroxyethvll -7-oxo-l-a::abicvclo (3. 2 . 0) heot-2-ene-2-ca.rboxvlate . COO?N3 10% ?d/C, K2 —b THT, ether, buffer Tt> a solution of p-r.itrobenryl 3- [2- (1- (2, S-diaethylpyridiniu=s)) ethylthio]-oc- [i-(P) - hydroxy ethyl] - 7-oxo-l-arabicyclo(3.2.0)hept-2-eae-2 carboxylate diphenylphosphate (0.80 g, 1.07 =aol) in wet -etrahydrofuran (42 nL) was added ether (42 nL), potassiim phosphate sonobasic— sodium . ' hydroxide buffer (0.1SW, pH 7.22, 21 nL) and 10% palladium on charcoa_1 (0.80 g) . The resulting fixture was hydrpgenated for 1 h under 40 psi at 23"C and filtered through a Celite pad. Ihe two layers vera separated aj>d the organic layer was extracted with the buffer (3 x 8' mL) The acueous phase were combined, washed with ether (50 rL), pumped to remove traces of ->rcanic solvent and poured on too of a column (3.0 x 10.2 cm) of p-bcndapak C-18. Elution of the colnnn with 5% acetonitrile - 95% water mixture ar.d lyophyligation of the appropriate fractions, cave the title compound 0.246 g (63%) as a yellowish powder, ir (KBr) V : 3700-2800 (OH), 1750 (C=0 of the S-lactaa) , 1620 max (pyridinium) , 1585 cm 1 (carboxylate), 1Hmr "(D20) o: 1.23 (3K, d, J=S. 4 Hz, CH CHOH), 2.5-3.5 (11H.H-4, K-6, C-'S, 2CH on pyridiniun), 3.8-4.4 (2H, Ch^CHOH, H-5), 4.5-4.9 (CH N+, HOD), 7.64 and 7.74 (2H, A part of A^3 system, Km of pyridinium),'8.07, 8.16, 8.18. and 8.27 (IK, B part cf A B system, K? of pyridinium), uv (H O) X : 2 * 277 (E9733), 300 .'(£8271) my, Tc] } 3 +50. 7' (C 0.48, HO), ? 2 • Anal, calcd. for C18H22N204S'5 H20: C 55-Sl, 3 6.<7, N 7.19; - 101 - 207346 Examole 11 o Preparation of (55, 65)-3-f2- (2-actbylthlo-3-methvliinidazolio) ethy1-thio] -6- [1- (S) -hvdroxv-ethy1]-7-oxo-l-azabicyclo[3.2.0]hept-2-ene-2-carboxvlate N-Me A. 2 -Me thy Ithio- 3-me thy 1-1- ( 2-mercaptoethy 1) imidazolium trifluoromethanesulfonate SMe f C?3SO3H + A KSCS2C£2 B SMe C?3S03 <7 . N-Me Trif luorome thanesulf onic acid (1.38 rrlL, 0.015 mol) was added dropwise to 2-methylthio-I-methyliiiiidazole^ (4.0 g) , 0.03 mol) at 0°C under Ethylene sulfide (0.9 ml, 0.015 mol) was then added and the nixture was heated at 55°C under N2 for 2 4 h. The reaction mixture was triturated with ether (3x) and the residue was taken up in acetone, filtered and evaporated. This gave the product (4.2 g, 82%) as a seni-crvstalline solid which -was used as such without further ourification, ir(film) v : 2550 (w; sh) cm max 1 Hnmr 2H, methylene), (d6-acetone) 6: 7.97 (s, 2H) , 4.66 (t, J=7 Hz .4.17 (s, 3H, N-Me), 3.20 (d of t, J=7 Kz, J'~9 Hz, 2H, methylene), 2.72 (s, 3H, S-Me), 2.20 (t, J=9 Hz, 1H, -SH) 1. Prepared as per A. Wohl and W. Marckwald, Chem. Eer. 2.2, 1353 (1889). - 10 2 - 207346 o B- o-Nltrobenzvl (55, 6S)-3-[ 2-(2-methvlthio-3-methvl-imicazolio)ethyl thio]-6-[1-(R) -hydroxyethyl]-7-oxo-l-azabxcvclo [3.2. 0]hept-2-ene-2-carboxvlate dioher.ylphosohate C02?N3 C02?K3 (4>O)2?O' 1 ..#> To a solution of p-nitrobenzyl (5R, 6S)-6-[ 1-(R)- hydroxyethyl] -3, 7-dioxo-l-asaJDicyclo [3. 2 . 0]heptane-2-carboxylate (1.40 g, 4.0 inmol) in 50 mL of dry acetonitrile, at 0°C under was added dropwise diisopropvlethylamine (0.76 mL, 4.4 mmol) followed by diphenyl chlorophosphate (0.91 mL, 4.1 nrnol) . After stirring the reaction mixture at room temperature for 1 h, diisopropylethvlamine (0.76 mL, 4.4 mmol) was added and then a solution cf 2-methylthio-3-methyl-l-(2-mercapto- ethyl) imidazolium trif luororne thanesulfonate (2.0 g, 5.9 mmol) in 5 mL of acetonitrile was added dropwise. The reaction mixture was kept at room temperature for 1.5 h and was then concentrated .in vacuo to give a gum. This gum was taken up in H20 and applied to a C,g reverse-phase column. Elution with E2O, then 20% acetonirrile-H^O and finally 30% acetonitrile-^O, followed by lyophilization of the appropriate fractions afforded the product (0.90 g, 30%) as a light yellow solid. ir(K3r) v : 3380 (br, OH), 1770 (s-lactam CO) . max cm ; 'Hnmr (dg-acetone) 0: 8.35 (br,s, 1H) , 8.24 , 7.78 (A3 q, J=8.8 Hz, 4H, aromatic), 7.89 (br s, 1H), 7.25-6.91 (m, 10K, ciphenylphosphate), 5.50, 5.25 (A3 q, J=12 Hz, 2H, benzvlic), 4. 75-4. 27 (m, 3*H) , 4.03 (s, 3H, N-Me), 4.15-2.75 (m, 8H),-2.59 (s, 3H, S-Me), 1.22 (d, J=6.2 Kz, 3H, -CHMe). 207346 - 103 - C. (55/ 6S)-3-[ 2- ( 2-Methy 1 thio-3-methy 11mldazolio) ethvl — thio]-6-[1-(R)-hydroxyethyl]-7-oxo-l-azabicyclo[3.2.0]heot-2-ene-2-carboxylate C02?K3 It-Mi To a solution of p-nitrobenzv1 (5R, £S)-3-[ 2- (2-methylthio-3-methylimidazolio) ethyl thio]-6-[1- (R) - hydroxyethyl]-7-oxo-l-azabicyclo [3.2.0]hept-2-ene-2- carboxylate diphenyIphosphate (1.20 g, 1.56 mmol) in a mixture of 70 mL of THF, 70 ml of ether and 31 mL of phosphate buffer (0.05 pH 7.4) was added 1.2 g of 10% palladium-on-charcoal. This mixture was hydrogenated (Parr) at 35 psi for 55 min. The reaction mixture was then filtered through Celite and the filter cake was washed with H20 and ether. The acueous phase was separated, cooled at 0°C and the pH was adjusted to 7.0 with saturated acueous NaHCO^. After removing residual organic solvents in vacuo the acueous solution was applied to a Cng reverse-phase column. Zlution with KjO and then 8% acetonitrile-H^O and subsequent lyophilization of the .relevant fractions gave 0.25 g of a solid. This material was repurified by reverse-phase hplc to give the product (0.114 g, 19%) as an off-white solid. ! ir (K3r) v : 3420 (OH), 1750 (S-lactam CO) 159 0 (-CO,8) cm""; m&x ^ Q 'Hnmr (D20) 6: 7.58 (s, 2H), 4.52 (t, J=6 Hz, 2K), 4.2S- | 3.82 (m, 2H) , 3.90 (s, 3H, N-Me) » 3 . 40-2 . 87 (m, 5H) , 2.40 j. (s, 3H, S-.Me), 1.20 (c, J = 6.4 Hz, 3K , -CH.Me); uv (K20) | 297 (t 7572), 262 (e 6259), 222 (e 7955) nai. - 104 - Example 12 Preparation of (53, 6^S) — 3 — [ 2-(3-aminopyridinio)sthy1 -thio]-6-[1-(R)-hydroxyethyl]-7-oxo-l-a2abicyclo[3.2.0]-hept-2-ene-2-carboxylate 3-Aminopyridine (1.50 g, 0.016 mol) was taken u? in 15 mL of 1 N methanolic HCl and the resulting solution was evaporated to give the hydrochloride as an oil-. To this oil was added 3-aminopyridine (1.32 g, 0.015 mol) and ethylene sulfide (0.89 ml, 0.015 mol) and the resulting mixture was heated (oil bath) at 60-65°C under ^ for 2 h. Another equivalent of ethylene sulfide (0.89 ml, 0.015 mol) was added and heating was continued at 55-65°C for 65 h. The reaction mixture was washed with and then taken up in ^0 (25 ml). The aqueous solution was applied to a C^g reverse-phase column which was eluted with H^O. Evaporation of the relevant fractions gave the product (1.26 44%) as a colorless, viscous oil. ir(film) v , : 3180 (NI-L, ) «. JHcLX £ cm ; 'Himr (d^-DMSO) 0: 8. 19-7. 59 (m, 4H, aromatic) , 4. 59 (t, J = S. 2 Hz, 2 K, N-CHj ) ; 1 5 (br s, 2H, -NH^ ) , 3.2 0-2.77 (m, 3H). \ 207346 - 105 - i> i " i O o B. p-Nitrobenzy1 f 53 - 6.S ) - 3-( . 2-( 3-aai n ooy r i di ni o ) ethy 1 - thio)-6-( 1- (B.) - hydroxy sthvl)-7-oxo-l-a.z abi eye lo( 3 • 2 . 0 )-h eat - 2-e a e - 2-c art oxy! at e diahe: c02?n3 C02?N3 ( (j)0) 'cP To a solution of p-nitrob ea zyl (53.» 6_S)-6-(1-(3R)-hydro xye'thyl)-3»7-di ox o-l-azabicyclo(3.2.0) heptaa e-2-carboxylate (O.696 g, 2.0 saol) in 10 aL of dry acetcaitrile, at 0°C under Ng, vas added dropvise diis o-oropylethylanine ( 0.382 nL, 2.2 aaol) folloved "by dipheayl chlorop'aosphate (0.1+57 nL, 2.2 aaol) . After stirring at 0°C for 30 nin. a solution of 3-aaino-l-( 2-aercaptoethy1)pyridiniua chloride (0.1+75 S, 2.5 saol) in 1 al of dry DM? vas added, folloved by additional dii sopropylethylaaine (0.^35 al», 2,5 aaol). The reaction aixture vas kept at 0°C for 1.5 h and vas then concentrated i_n vacuo. The resulting gua vas taken up in acetonitrile-H_0 (1:1) and applied to a C, 0 reverse-phase Elution vith HgO, folloved by 20% acetonitrile-H^O 2- -l8 coluan, and subsequent lyophilization of the relevant fractions afforded the product (0.730 g, 50£) as a beige-colored solid. ir(X3r) v : 3330 (br, OH), 3160 (br, NH-), 1770 (S-lactaa co), SI 2. X . & 1690 (-co ?N3)cn ; 'Hsar (dg-DMS0) 0: 8.29-7-63 (a, 3 aroaatic),: 7.2-6.7 (a, 10H, aipheaylphosphate), 5-^7, 5-18 (A3 q.,- J"= lk Hz, •. 2H , benzylic), li.73-ii.U5 (a, 3H), U. 2-3-8 (a, IS), 3.6-2.6 (a, 8h), 1.15 (d, J-6.2 Hz, 3H, chme). . ■ *^ * - 106 - C. (55< 6£)-3-( 2-(3-Aair. ooyridiaio^thyl -m-»o _ £ - ( (jj) -hydroxyethyl)-7-oxo-l-agabicyclo( 3 • 2 . 0)hett-2- ene-2-carboxvlate To a nixture of p-nitrobenzyl (53., 6s_)-3( 2- ( 3- aainopyridinio)ethyl thi o ) - 6-(1-(R) -hydroxyethyl)-7-oxo-l- azabicyclo(3.2.0)hept-2-ene-2-carboxylate dipheaylphosphate (0.730 g, 1.0 aaol) and 10? palladiua-on-charcoal (0.J s) in 25 aL of a phosphate buffer (0.05 pH 7 • *0 vas added 8 aL of TE? and 20 aL of ether. This aixture vas then hydrogenated (Parr) at 1*0 psi for 1 h. The resulting aixture vas filtered through a pad of Celite and the filter cake vas vashed vith E„0 and ether. The aaueous -abase vas 2 separated, vashed vith ether (2 x) and then residual volatiles vere reaoved i_n vacuo. The aqueous solution vas iaraediately applied to a Cng reverse-phase colnan vhich vas eluted vith E^O. Lyophilisat ion of the relevant fractions afforded 0.1*5 g of an off-vhite solid. This aaterial vas repurified by reverse-phase hplc to give the desired product (0.123 £ > 35?) as an ivory-colored solid. ir(KBr) v : 33^0 (br), 1750 (br, S-lactaa CO), 1580 (br, _ a ax -C0g ) ca"1 ; 'Enar (DgO) 6: 8.07-7.5 9 (a, 1*2, aronatic), . 61 (t, J= 5.8 Hz, 2E, N-CE2) , 1*. lU (d of q. , JrJ'^6.3 Hz, 1H, E-11 ) , 3-97 ( d of t, J=9.2 Hz, J'-2.6 Ez, IE, H-5), 3-38 (t, J= 5 • 8 Hz, 2 E, S-CH2), 3.21* (d of d, J=6.0 Hz, J'=2.6 Hz 1H, H-6), 3.17-2-57(n, 2H, K-M, 1.21 (d.J-6.3 Hz, 3E , CEMe); uv(E20) 2 99 (e 79^9), 256 (e 8822 ) 22; t1/2 (pH 7. 1* 36.8° C) 18.5 h. - 107 - 207346 Examole 13 PREPARATION 0? OH coo Q {5R, 6S_) 3- [1- (S_) -methyl-2- (1-pyridiniua) ethylthio] -6- II- (R) -hydroxyethyl]-7-oxo-l-azabicyclo (3 .2.0)hept--2-ene-2-carboxylate ind COO (5R, 6S).3r [1- (R) -nethyl-2- (l-pyridinium) ethylthio] -6- [1- (R) -hydroxyethyl]-7-oxo-l-azabicycio(3.2.0)hept-2-ens-2-carboxylate - 10 8 - 2073' A d.Z-1- [2-msrcaoto-2-net'nylethyl) pyridinium nethanesulfonate " dJL-1- (2-nsr capto-l-methylethyl) pyridinium methane sulfonate Ms°" sr® Ms0" Methanesuifonic acid (1.95.nL, 0.030 mol) vas added slowly to cold pyridine (7.'83, 0.097 aol) and the resulting mixture was stirred at 40°C for 15 min, treated with dZ-propylenesulfide (2.59 ml,. 0.033 mol) and stirred at 60"C under a nitrogen atmosphere for 90 h. Pyridine was removed uncer vacuum; the residue was mixed with water and purified by chromatography (hplc, ?rep. 3oncapa3< C-18) . The appropriate fractions were combined and lyophilized giving dZ-1-(2-mercapto-2-nethyl- ethyl)pyridiniua methanesulfonate 1.14 g (15%) as a colorless syrup; ir (film) 2520 (SH) , 1640 (pyridinium) , 11S0 (s, CH3SO ~) , 1040 (CH SO ") cm""1, 'l-imr (DMSO d.) 6: 1.25 ,'d, J=3.8 Hz, 3H, CH.CHS) , 2.30 [s, -» j 6 *"• 3H, CH3S0 "), 2.90 (d, J=8.5 Hz, IK, SH), 3.2-3.7 (n, CHSH) , 4.52 (dd, J =12.9 Hz, J=8.4 Hz, CHCr" N+) , 4.37 (dd, J =12.9 Hz, J=5.0 Hz, CHCH N+) , —gem — —2 —gem - —2 8.0-8.4 (m, 2H, Hm of pyridinium), 8.5-8.8 (m, 1H, Hp of pyridinium), 9.04 (dd, J=1.4 Hz, J=6.7 Hz, 2H, Ho of pyridinium), uv (HO) X : 208 (C5267), 259 (£3338), Anal, calcd. for C H NO S • 2H O; C 37.88, H 6.71, N 4.SI, • •? i ^ J * ^ S 22.47; found: C 37. 49, H 6.85, n'4.36, S 22.09 and <££-1- (2-nercapto-l- 207346 - 109 " methylethyl) pyridiniua aethanesulfonate 0.82 g. (lit) as a colourless syrup; ir (fila) vQax: 2500 (SH) , 1628 (pyridiniua)., 1180 (sulfonate, 1035 (sulfonate)' cn"1, JHnr (DMSO dc) 6: 1.69 (d, J=6.8 Hz, 3H, o — C^CHN ), 2.31 (s, 3H, c^3S03 ), 3.0-3.3. (a, 2H, C^S) , 4.2-5.2 (si, •• 1H, CHN ), 8.0-3.4 (m, 2K, Ha of pyridiniua), 3.5-8.3 (a, 1H, Ho of ' pyridinrua), S.0-9.2 (n, 2H, Ko of pyridiniua), uv (E 0) X : 209 2 max* (£4987), 258 (£3838). Anal, calcd. for C H NO S -1.53 0: C 39.11, 3 15 3 2. 2 H 6.56, N 5.07; found: C 39.13, H 5.92, H 5.20. (5R, 6S) paranitrobenzyl 3- [1- (R, S)aethyl-2- (1-pyridiniua) ethylthio!-6-II-(R)-hvdrcxyethyl}-7-oxo-l-asabicyclo(3.2.0)hept-2-ene-2—carboxylate diohenvlohosohate OH A. 1) NSt.(iPr) OH 2) Cl?(0?h) X COOPN3 3) HS HsO Nf + c0opn3 (PhO), 4) NBt(iPr) To a cold (0°C) solution cf (5_R, 6_S)paranitrobej^syl 6— [1-(R)~ hydroxyethyl]-3,7-dioxo-l-azabicyclo (3.2.0) he^tane-2-car'boxylate (0.523 g, 1.5 nrnol) in acetonitrile (6 :nX) kept under a nitrogen atmosphere was added diisopropylethylaaine (0.314 aL, 1.8 rnnxsl) followed by diphenyl chloropbospbate (0.373 mL, 1.8 aaol). The reacticn mixture was stirred for 30 ain and treated with a solution of dZ-1-(2-mercapto-2-methylethyl)pyridiniua methanesulf onate (0.539 g, 2.16 aaol) in acetonitrile (2 aL) and diisopropylethylajnine (0.314 mL, l.B aaol). The reaction mixture was stirred at 0°C for 1 h, diluted with cold (0°C) - 110 - water (24 mL) and chro.T.atographed- over prep bondapak C-18 column (2.5 x 3.5 cm) with 25-50% acetonitrile in water as eluting solvents to give 1.07 g (97%) of the title compound as a yellowish powder after lyophilization; ir (K3r) v : 3700-3100 (OH), 1770 (C=0 of S-lactaa), m3.x 1695 (C=0 of ?N3 ester),. 1630 (pyridiniuun), 1590 (phenyl), 1518 (NO), 1348 (N02), 885 (N02) cn'1, !Hmr (DMSO d ) 6: 1.14 (d, J=6.1 Hz, 3K, CS^CHO), 1.33 (d, J=6.3 Hz, 3H, C^CKS) , 4.6-5.0 (m, CH2N+), 5.14 (d, J=5.2 Hz, 1H, OH), 5.37 (center of A3q, £=12.4 Hz, 2H, Cn^ of PNB), 6.6-7.5 (m, 10H, phenyl of phosphate), 7.69 (d, J=8.7 Hz, 2H, Ho of ?N3), 8.0-8.4 (m, • 4H, Km of PN3,- of pyridinium), 8.4-3.8 (m, 1H, Hp of pyridinium), 9.08 (d, J=5.6 Kz, 2H, Ho of ovridinium), uv (HO) X : ~ 2 aax 263 (E13325) , 308 (E8915). Anal, calcd. for C.,K.J 0..SP-H.0: C57.52. 36 36 3 10 2 H 5.10, E 5.10, N 5.59, S 4.27; found: C 57.76, H 4.96, N 5.36, S 4-25. (5R, 6S)3-[1-(P and S)-nethyl-2-(1-pyridiniua)ethylthio)-6-fl-(R)-hydroxyethyl] -7-oxo-l-azabicyclo (3.2. 0) he?t-2-en&-2-carboWla te H rr> COOPN3 (PhO) 2d To a solution of (5H, 6 S_)parani trobenzyl 3-[1-(P., S_)nethyl -2- (1-pyridiniun) ethyl thio]-6.- [1- (P) -hydroxyethyl] -7-oxo-l-azabicyclc-(3.2.0)hept-2-ene-2-carboxylate diphenylphosphate (0.60 g, 0.82 r=r>ol) in wet tetrahydrofuran (33 nL) was added ether (33 mL) , potassiun phospha mono basic-sodium hydroxide buffer (17 nL, 0.15N, pH 7.22) and 10%., palladium on charcoal (0.60 g). The resulting mixture was hydrogenated for 1 h under 40 psi at 23'C. The two layers were separated and the. 2073/.^ - ill organic layer was extracted with water (3x7 nL). The aqueous layers were combined, filtered through a Celite pad, washed with ether (3 x 20 mL) and chromatographed on prep bondapak C-IS colur?.n (2.5 x 9.5 era) with water as eluting solvent to give 0.18 g (63%) of nixture of diastereoisomers. The two diastereoisomers were separated by hplc (prep bondapak C-18) with water as eluting solvent: isomer with lower retention tine, 0.063 g (23*.) compound "3", ir (J3r) V : 1770 (C=0 cf B-lactam), 1633 (pyridiniua), 1593 max (carboxylate) cn \ '.".nr (D^O) 6: 1.20 (d, £=6.3 Kz, 3H, CH^CHO) , 1.42 O (d, J=6.9 Hz, 3H, CH CHS), 2.3-3.2 (m, 3K, H-4, H-6) , 3.5-3.9 (la, 1H, SCH) , — —3 3.9-4.2 (ia, 2H. H-5, CH CHO) , 4.3-5.1 (in, <02N+) , 7.3-8.2 (m, 2H, Ss of py ridinium) , 8.4-8.7 (m, 1H, Hp of pyridinium), 3.7-9.0 <ra, 2H, Ho of pyridinium uv (H O) X : 260 (£6727) , 300 (E8245)',. [a) 23_.39.3* (c, HO), T, =12.6 h ^ mUoX D 2 i *"4 55 (measured at a concentration of 10 H in phosphate buffer pH 7.4 at 36.8°C); isomer with higher retention time, 0.081 g (28%), conpound "A", ir (X3r) "^rnaj£: 1755 (C=0 of S-lactam), 1630 (pyridinium), 1590' (carboxylate) era 1, ^.mr (D^) 5: 1.13 (d, J=6.3 Hz, 3H,"CH CHO), 1.40 (d, J=7.0 Hz, 3H, CHECKS), 2.84 (d, J=9.3 Hz, 2H, H-4), 3.'26 (cd, J=2.7 Hz, J=5.9 Kz," 1H, H-6), 3.4-4.2 (m, 3H, SCH, C^CHO, H-5), 4.2-5.1 (n, C32*0 , 7.7-8.1 (m, 2H, Hm of pyridinium), 8.3-8.65 (m, 1H7■ Hp of pyridinius), 3.65-8.9 ' (ra, 2H, Ho of pyridinium), uv (K,0) 1 : 259 (E5694), 296 (£6936), ? 2 max . 23 i [a]D . +96.9° (c 0.56, H20), =15.6 h (measured at a concentration 5 = -4 of 10 M in phosphate buffer pH 7.4 at 36.8°C). ) I ••v' ---• •«-— -- •- - - 112 - Example 14 PREPARATION OF (SR, 6S) 3- [2- I (S^) - (1-pyridiniun) ]-l- (£) -cyclohexylthio]-6-[1-(R)-hydroxyethyl]-7-oxo-l-azabicyclo(2.2.0)hept-2-ene-2-carboxvlate and (SH, 6S) 3- [2- [ (R) - (1-pyridiniua)]-l- (R) -cyclohexylthio]-6- [1- (R) hydroxyethyl]-7-oxo-l-azabicyclo(3.2.0)hept-2-ene-2-carboxy late - 113 - 207346 dt-l-[2-mercapto-l-cyclohexyl)pyridinium nethanesulfonate + N + KsOH HsO Me thane sulfonic acid (O.SS nL, 0.01 mol) was added dropwise to pyridine (2.42 nL, 0.03 mol) with cooling. The nixture was stirred under a nitrogen atmosphere for-10 nin, treated with dZ~cyclohexene-sulfide [1.377 g (85% pure), 0.0102 nol] and stirred at 72"C for 25 h. The excess of pyridine was removed under vacuum and the traces were ■ codistilled with water. The residue was mixed with water and chromatographed. through prepbondapak C-13 column (5 x 13 cm) with 0-2% acetonitril in water as eluting solvent giving after lyophilization a colourless syrup 1.57 g (53%), ir (film) V : 2500 (SK), 1625 (ovridinium), 1190 max (.S0 ~), :Hnr (DMSO 'd ) <5: 1.2-2.5 (n, 8H, cyclohexyl H) , 2.32 (s, 3H, J o CH3S03~), 2.82 (d,J=9.3Hz, SH), 3.0-3.5 (m, IK, CHSK), 4.2-4.9 (a, 1H, CHN ), 8.0-8.3 (m, 2H, Hm of pyridinium), 8.4-8.8 (m, IK, Hp of pyridiniua 8.9-9.3 (m, 2H, Ho of pyridinium), uv (H O) X : 214 (£5365) , 258 (£3500) 2 max Anal, calcd. for C12H19N°3S2" H2°'' C 46,83' H- 6-88' N 4-56; founc: C 46.61 H 6.46, N 4.65. rv - 114 - I B. (5R, 6S)paranitrobenzyl 3-[2-[(R or S) - (1-pyridinitm) ] -1- (R or S)-cyclohexylthio) -6-(1-(R)-hydroxyethyl]-7-oxo-l-azabicyclo (3.2.0)hept-2-ene-2-carbo-xylate dipheny1phosphate 1) NSt(i?r), C00PN3 9 •2) ClP(OPh), "30 -N 3) 4) NEt(iPr) MsO 9 - (PhO)2?0 -J To a cold (06C) solution of (5 R,SS_)parani trobenzyl 6-[l-(R)-hvdroxyethyl]-3, 7-dioxo-l-azabicyclo (3. 2. 0)heptase-2-carcoxylate '(!• 37 S > 3.93 maol) in acetonitrile (IS aL) kept under a nitrogen atmosphere was added diisopropylethylamine (0.S22 mL, 4.7 ircnol) and dip'nenyl chloro-phosphate (0.979 aL, 4.7 mmol). The resulting solution was stirred for 30 min and treated with a solution of dZ-1-(2-mercapto-1-cyclohexyl)pyridini', methanesulfonate (1.64 g, 5.66 mmol) in acetonitrile (4.7 mL) followed by diisopropylethylamine (0.S22 mL, 4.7 =ol). The reaction mixture was stirred at 0°C for 1 h, diluted with cold (0°C) water (75 nL) and chrcrr.a-tographed on prepbondapak C-18 with 25-50% acetonitrile in rater as eluting solvent giving after lyophilization of the appropriate fractions 1.9 c (53%) of the title comDound, ir (KBr) v : 3700-3000 (OH),'1770 (C=0 of max.: 6-lactam), 1700 (C=0 of PN3 ester), 1628 (pyridinium), 1590 (phenyl), 1515 (NO ), 1345 (N02) , 880 (N02) cm"1, 'Hmr (D20) 0: 1.13 (d, £=6.1 Kz, 3H, CH3CH0), 1.2-2.5 (m, 8H, cyclohexyl K) , 2.7-3.5 (m, 4H, H-4, H-6, CHS), 3.5-4.4 (m, 2H, CK CHO, K-5) , 4.4-5.0 (m, 1H, C-:N+) , 5.30 (center of"A3q, £=12.8 Hz, CK2 of PN2) , 6.7-7.4 (m, 10K, phenyl) , 7.65 (d, £=8-6 Kz, 2K, 2 0 73 ^ - 115 - Ho of Ph3) , 7.9-9.4 (m, 4H, Hm of ?N3, Kra of pyridinium) , 8.4-8.8 (m, 1H, Hp of pyridiniua), 9.0-9.4 (a, 2H, Ko of pyridiniua), uv (H^O) \ : 263 (E9038), 309 (£6394). Anal. calcd for C H „N O S?- H„0: max 39 40 3 10 2 C 59.16, H 5.35, N 5.31; found:' C 58.95, 5 5.15, N 5.57. (5R,6S) 3-[2-[(R or S)-(1-pyridiniun)]-1-(R or S)-cyclohexylthio)-6-[1- (R)-hydroxyethyl] -7-oxo-l-azabicyclo (3. 2 .0)hept-2-ene-2-carboxylate 10% ?d/C, H. OH X. THT, ether, buffer 0 If (PhO) ?0 r>: ^cco ^ To a solution of (5R,6_S) paranitrobenzyi 3-[2-[(R or S)-(l-pyridiniu=t) ] -1- (R or S^) -cyclohexylthio]-6- [1- (R)-hydroxyethyl]-7-oxo-1-azabicyclo(3. 2.0)hept-2-ene-2-carboxylate diphenylphosphate (1.85 g, 2.34 ssnol) in vet tetrahydrofuran (96 mL) was added ether (96 mL), potassium phosphate monobasic-sodium hydroxide buffer (0.15K, pH 7.22, 50 mL) and 10%-palladium on charcoal (1.9 g) . The resulting mixture was hydrogenated at 23° under 40 psi for 1.25 h.- The organic layer was separated and washed with water (3 x 20 mL) . The aqueous solutions were filtered through a Celite pad, washed with ether (2 x 60 pumped to remove the traces of organic solvents and chromatographed on prepbondapak C-18 column (4.5 x 9 cm) with 0-5% acetonitrile in water a eluting solvent giving after lyophilization 0.705 g (76%) of a Fixture of diastereoisomers. The separation of the diastereoisomers was done by hole (prepbondapak C-18) with 4% acetonitrile in water as eluting solvent; diastereoisomer with lower retention tine, coapouad a > (0.29 311), ir (K3r) v_ = 1750 [C=0 of S-lactam), 1620 (sh, pyridinium), 163S i7j2.x (carboxylate) cn \ 2Hmr (D^O) o: 1.21 (d, £=6.3 Hz, 3H, G^CHO) , 1.4-2.5 (m, SH, cyclohexyl H) , 2.5-3.05 (m, 2H, H-4), 3.05-3.25 (in, lH, H-6), 3.3- X 3.7 (m, 1H, CHS), 3.9-4.3 (m, 2H, H-5, CH^CHO), 4.3-4.8 (m, CHN'), 7.8- 8.2 (m, 2H, Hm of pyridinium), 8.3-8.7 (m, 1H, Hp of pyridiniua), 8.8-2.1 (m, 2H, Ho of pyridinium), uv (K.O) X : 260 (E7123), 300 (£8635), 2 max .. 23 +6.2" (£ 0.63, H^O) , Tj =16.6 h (measured at a concentration of -~4 10 M in phosphate buffer pH 7.4 at 3S.8°C); Anal, calcd. for C H N O S 20 24 2 4 •2H20: C 56.59, H 6.65, N 6.60, S 7.55; found: C 56.83, H 6.47, N 6.59, S 7.43; diastereoisomer wirh higher retention time, compound "3", (0.35 38%) ir (K3r) 1750 (C=0 of S-lactam), 1622 (sh,' pyridiniua) , 15S8 (carboxylate) cm 1, ■ ^-nr (D^) 0.- 1.19 (d, J=6.4 Hz, 3H, (3 CHO) , • 1.3-2.5 (m, 8H, cyclohexyl H) , 2.5-3.1 (m, 2H, H-4) ,' 3.1-3. 3 (m, 1H, H-6), 3.3-3.8 (m, 2H,K-5, CHS), 4.1 (center of m, 1h) CH^CHO) ,. 4.25-4.7 (m, 12, CHN ), 7.8-8.1 (m, 2H, Hm of pyridinium), 8.3-8.7 (m, IK, Hp of pyridinium) 8.75-9.0 (m, 2H, Ko of pyridinium), uv (H20) •' 259 (£5992), 296. (£7646) 23 0 +65.3° (£ 0.43, H^O), 7^ =20.2 h (measured at a concentration of 10 in phosphate buffer pH 7.4 at 36.8°C). "■"N " - 117 - Exanrole 15 2 07346 TV, 1 A o (5R) Allyl 3-((2-pyridinioethy1)thio]-(6S)-[(IK)-hydroxyethyl]-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate diphenylohosphate OH X 1) ClPO(O0)2 -= ^ 2) >ri© i ^ ~ HS — CO^ ®0?0(0$)2 To a solution of (SR) allyl 3,7-dioxo-(6£)-[(IR)-hydroxyethyl]-1-azabicyclo[3.2.0]heptane-(21*)-carboxylate (473 mg, 1.37 mmol) in CH^CN (6 mL) was added at ca. -106C under a nitrogen atmosphere diisopropylethyl-amine (0.42 mL, 2.4 mmol) followed by diphenyl chlorophosphate (0.50 mL, 2.4 mmol). The mixture was stirred at 0°C for 30 min, and then cooled to -15°C. To this was added an oily suspension of N-(2-mercaptoethyl)-pyridinium chloride (527 mg, 3.00 mmol) in CH^CN (1 mL) containing 5 drops of DMF, followed by diisopropylethylanine (0.42 mL, 2.4 mmol). The mixture was stirred at -15° for 30 min and then diluted with H20 (20 mL). This mixture was directly purified on a reverse phase silica gel (C PreoPAK, t lo 12. g, Waters Associates) eluting with H O (200 nL), 10% CH CN/H 0 b O 4m (100 mL) , 20% CH3CN/H20 (100 mL) , 30% (100 mL) and then 40% CH^CN/H^ (100 mL). Appropriate fractions were collected, the organic solvent removed by a vacuum pump and lyophilized to obtain 786 mg (1.26 mmol, y. 67.3%) of the title compound as brownish powder: 1Hmr (DMSO-d,, CFT-20) 6: 1.16 (311, d, J=6 Hz, l'-CH ), 2.6-3.7 (m) , 3.75-4.3 © — J (2H, m, 5-H and 1*-H) , 4.65 (2H, m, -C02CH2-) , 4.87 (2H, t, J=6 Hz, -CH2--N+) , \ i - 118 - 5-6.2 (3H, m, olefinic protons), 6.6-7.4 (m, aromatic protons), 8.15 (2H, "t", J=7.Hz, aromatic protons neta to the nitrogen), 8.63 (1H, "t", J=7 Hz, aromatic proton para to the nitrogen) and 9.07 pom (2H, "d", J=7 Hz, aromatic protons ortho to the nitrogen); ir (film) \J: 3400 (OH), 1770 (S-lactam)< 1690 (ester), 1625 (pyridinio). B . (5R) 3-[(2-pyridinioethyl)thio]-(6.S]- [ (lg)-hydroxyethyl]-7-oxo-l-azabicyclo[3.2.0]hept-2-ene-2-carboxylate OH ?d(?*3)4 ^ ' P*3 ®0?0(0(J))2 2 ^0 - x*> >-,K I r=) To a. solution of (5_R) allyl 3- [ (2-pyridi.*iioethyl) thio] ~ (6S) -[(IR)-hydroxyethyl]-7-oxo-1-azabicyclo [3.2.0]hept-2-ene-2-carboxylate diphenylphosphate (156 mg, 0.25 mmol) in CH^CN (2 mL) was successively added at ca_. 22 °C a solution of potassium 2-ethylhexanoate in EtOAc (0.5 M, 0.6 mL; 0.3 mmol), triphenylphosphine (15 mg, 0.057 mmol) and tetrakistriphenylphosphine palladium (15 mg, 0.013 mmol). The mixture was stirred at ca. 22°C under a nitrogen atmosphere for 2 h. After addition of anhydrous 2t^0 (7 mL), the precipitate was filtered, washed with anhydrous Et20 (7 mL) and dried -in vacuo to yield 101 mg of brownish solid.' This was purified by reverse phase column chromatography (C. Q ?rep?AX,'12 g, Waters Associates) eluting with H O. IS 2 Appropriate fractions (fr. 7-12, each 20 mL) were collected and lyophilized to obtain 53 mg (0.16 mmol/ y. 64%) of the title compound as__ yellowish powder. This material was contaminated with potassium diphenylphosphate and potassium 2-cthylhexanoate: 1Hmr (DO, CFT-20) 5: 0.80 207346 - 119 - (t, J=6.4 Hz, Me from ethylhexanoate) , 1.21 (3H, d', £=6.3 Hz, l'-Me), 2.93 (2H, dd, J, _=9 Hz, J =4 Hz, 1-Hs) , 3.28 (III, dd, Z, ..=6.2 Hz, —1- 5 —gem —o-l J, =2.5 Hz, 6-H), 3.42 (2H, t, J=6 Hz, -CH„S), 3.98 (1H, td, J. = —o-5 — 2 —d-1 9 Hz, J-_6=2.5 Hz, 5-H), 4.15 (1H, q, J=6.2 Hz, l'-H), 4.80 (2H, t, J=S.O Hz, -CH2N+), 7-7.5 (m, phenyl protons frora diphenyl phosphate),, n 8 .03 (2H, m, Hm of pyridinium), 8.56 (1H, m, Hp of pyridinium) and 8.81 ppra (2H, "d", J=6.5 Hz, Ho of pyridinium) . O o - 120 - Example ig Preparation of 3-[2-(N-Methvl-thiomorpholinium)ethylthio] 6a-[1'-(R)-hydroxyethyl]-7-oxo-l-azabicvclof3.2.0]-hept-2-ene-2-carboxvlate N-niethvl-N- (2-mercaotoethvl) thioiaoraholinium methanesulfonate Me o •w 1) Ms OH 2> A MsO To precooled (ice bath) N-methylthiomorpholine* (5.00 g, 42.7 mmol) was added nethanesulfonic acid (1.47 mL, 20.5 mmol) and ethylene sulfide (1.30 mL, 21.4 mmol) . The mixture was heated at 65°C for 24 h and diluted with water (25 mL). The aqueous solution was washed with diethyl ether (3 x 25 mL), pumped under vacuum and poured over a silica gel reverse phase column? the title compound being eluted with water. The appropriate fractions were combined and evaporated to afford the thiol as an oil (4.80 g, yield 861); ir (film) V v : 2550 cm-1 (w, SH) ; ^mr (DMSO d,)o: 3.25-2.95 (6H, m, CH2N ) , 3,32 (3H, s, CH3N ) , 3. 20-2. 65 (7H, m, CH2S, SH) and 2.32 pom (3H, s, CH^SO^) *J.M. Lehn and J. Wagner. Tetrahedron, 26, 4227 (1970) 207346 - 121 - 3. para-Nitrobenzyl 3- [2-(N-methy 1-thiomorpholinium diphenyl phosphate) ethylthio] -6c- [ 1' - ( R) -hvcroxvethv 1 ] -7-oxo-l-azabicvclo [3.2.0] -heat- 2-er.e- 2-carboxvlafce 1) 2). CIPO(C^) 2 3) H£ ©/ \ 4) Et_N(-<) ' CH A, • g© v_/ C02?NB A cold (ice bath) solution of oara-nitrobenzyl 6a- [11 - (R) -hydroxyethyl] - 3 , 7-dioxo-l-azabicyclo [3.2. 0jheptane-2-carboxvlate (557 mc, 1.60 naol] in CH^CN (3 mL) was treated dropwise with diisopropvlethvl amine (0 . 336 siL, 1.92 mol) and diphenylchlorophosphate (0.400 mL, 1.92 mmol) arid stirred for 30 min. The reaction mixture was treated with N-methvl-N-(2-mercaptoethvl)thiomorpholinium methanesulfonate (893 mg, 2.29 mmol) in CH^CN (4 mL) and ciisopropylethyl amine (0. 336 nL, 1.9 2 mmol) and stirred for 30 min. The solution was diluted with water (20 mL) and pourec over a silica eel reversed phase column. The desired compound was eluted with a 50% acetonitrile-water mixture. The appropriate fractions were combined, pumped under vacuum for 2 h. and lyophvlized to afford the title compound (1.01 g, yield 85%) : ir (nujol)v : 1760 (s, 3-Iactam -i lucLX C=0) and 1510 cm"1 (s, N02) ; Hmr (DMSO-dg) o: 8.25 (2H, d, J=8.8 Hz, H-aromatic), 7.70 (2K, d, J=8.8 Hz, K-aromatic ), 7.33-6.84 (10 K, m, H-aromatic), 5.37 (2K, center of ABq, J=14.2 Hz, CH2) , 5.14 (1H, d, J = 4. 5 Hz, OH), 4.35-3. 80, (2H, m, H-l' and H-5) , 3.75-3.45 (6H, m, CH2?0 , 3/31 (3K, s, CH3N~), 3.45-2.75 (9Hm, CH2S, H-6 and H-4) and l". 15 ppm (3H, d, J= 6.2 rv - 122 - 207346. C. 3-[2-(N-methy1-thiomorpholinium)ethylthio]-6a-[l'-(R)- hydroxvethv1]-7-oxo-l-azabicyclo[3.2.0j-hept-2-ene-2- carboxvlate jr ■ OH J H • 2 A solution of para-nitrobenzy1 3-[2-(N-methy1- thiomorpholinium dipheny Iphosphate) ethylthio] -6e-[l'-(R)- hydroxyethyl]-7-oxo-l-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (1.31 g, 1.76 mmol) in 0.1M pH 7.4 phosphate buffer (48.8 mL), tetrahydrofuran (20 mL) and diethyl ether (20 mL) was hvdrogenated over 10% pd/C (1.5 g) in a Parr shaker for 1 h at 40 psi. The reaction mixture was diluted with diethyl ether (40 mL) and the phases were separated. The organic phase was washed with water (2x5 mL). The aqueous phases were combined, filtered through a #52 hardened filter paper, washed with diethyl ether (2 x 20 mL) and pumped under vacuum. The aqueous solution was poured on a silica gel reverse phase column and the desired carbapenem was eluted with 5% acetonitrile-water. The appropriate fractions were combined, and lyophilized to give title compound as an amorphous solid (205 mg, 31%); ir (nujol)v : 1750 (s, S-lactam * -i max C=0) and 1590 cm"1 (s, C=0); Hmr (D20) 5:4.25-3.95 (2H, m, H-l', H-5) , 3. 70-3. 40 (6H, m, CH2N+) , 3.35 (1H, dd, J=6.1 Hz, J=2.6 Hz, H-6), 3.08 (3H, s, CH3N+) , 3. 25-2.75 ( 8H, CH2S, H-4), and 1.24 ppra <3H, d, J=6 . 4 Hz, CH3) ? uv (H20, c 0.062)Amax: 299 (elO ,962) Tl/2 17.7 h (0.1M pH 7 phosphate buffer., 37°C) . 2073 - 123 - Examole 17 Preparation of (5R,6S)-3- [2-(1-methylmorpholino)ethylthio]-6-[(R) -1-hydroxyethyl]-7-oxo-l-azabicyclo(3.2.0]hept-2-ene-2-carboxvlate A. 1-Methvl-l-(2-mercaotoethvl)moroholinium trifluoromethane- sulfonate Me V \ + C?3S03H + ^ HSOi2CH2N •N^ © ' CF,S0, Me 3 3 e To N-methylmorpholine (3.29 mL, 0.030 mol) was added dropwise trifluoromethanesulfonic acid (1.327 mL, 0.015 mol) at 10°C, followed by ethylene sulfide (0.89 mL, 0.015 mol). The resulting yellow-brown solution was heated (oil bath) at 50-60°C under N2 for 18 h. Volatile material was then removed in_ vacuo and the residual oil was taken up in 10 mL of H^O. The acueous solution was washed with diethyl ether (3x5 mL) and then residual organic solvent was removed in vacuo. The resulting aqueous solution was applied to a C^g reverse-phase column which was eluted with H20, then 5% acetonitrile-H20 and finally 10% acetonitrile-H20. Evaporation of the relevant fractions afforded a white solid which was dried in vacuo (P^O^) to give the product (1.92 g, 41%). ir (K3r)v : 2560 (-SH) cm ^"Hnmr (d^-acetane) in 3.x o 5: 4.25-3.6 (m, 8H), 3.49 (s, 3H, N-Me), 3.35-2.7 (m, 5H). - 124 - 207346 B. p-Nitrobenzv1 (5R,6S)-3- [2-(1-methylmorpholino)ethylthioJ-6-[(R)-1-hydroxyethyl]-7-oxo-1-azabicyclo [3.2.0]nept-2-ene-2-carboxvlate diohenvlohosohate " ~ " ~ hydroxyethyl]-3,7-dioxo-1-azabicyclo[3.2.0]heptane-2-carboxylate (0.34 8 g, 1.0 mmol) in 25 mL of dry acetonitrile was added drop-wise diisopropylethylamine (0.191 mL, 1.1 mmol) and then diphenyl chlorophosphate (0.228 mL, 1.1 mmol) at 0°C under Nj. After stirring at 0°C for 1 h diisopropylethylamine (0.226 mL, 1.3 mmol) was added to the resulting enol phosphate, followed by 1-methyl-1- (2-rr,ercaptoethy 1) morpholinium trif luoromethanesulf onate (0.373 g, 1.2 mmol). The reaction mixture was stirred at room temperature for 1.5 h and then concentrated in vacuo. The residual material was taken up in H^0 and applied to a C^g reverse-phase column. Elution with ^0, then 20% acetonitrile-I^O and finally 30% acetonitrile-H^O followed by lyophilization of the relevant fractions gave the product (0.360 g, 40%) as an amorphous solid, ir (film) 3300 (-OH), 1770 (0-lactam CO), 1700 (-C0-PN3) cm-1; 1 ^ Hnmr (dc-acetone) 6: 8.25, 7.80 (ABq, J=8.6 Hz, 4H, aromatic), b 7.4-6.8 (m, 10H, diphenyIphosphate), 5.56, 5.27 (A3q, J=14.2 Hz, 2H, benzylic) , 4.42 (d of t, J=9.2 Hz, J' = 2.7 Hz, 1H, H-5) , 4.1-2.7 (m, 17H), 3.40 (s, 3H, N-Me), 1.22 (d, J=6.2 H2, 3H, -CHMe) . (00) 2PO To a solution of p-nitrobenzyl ( 5R, 6£)-6-[(R)-1- - 125 - C. (5R,6S)-3-[2- (1-methylmorpholino)ethylthio]-6-[(R)-1-hydroxvethv1]-7-oxo-l-azabicyclo[3.2.0]hept-2-ene-2-carboxylate me thy lmorpholino) ethylthio] -6- [ (R) -1-hydroxy ethyl] - 7-oxo-l-azabicyclo[3.2.0]hept-2-ene-2-carboxylate diphenyIphosphate (0.360 g, 0.49 mmol) in 13 mL of phosphate buffer (0.05 M pH 7.4) was added 0.36 g of 10% palladium-on-charcoal, 20 mL of tetrahydrofuran and 20 mL of diethyl ether. This mixture was hydrogenated (Parr) at 32 psi for 1 h. The mixture was filtered through Celite and the filter pad was washed with ^0 and diethyl ether. The aqueous phase was separated and the pH was adjusted to 7.0 with additional pH 7.4 phosphate buffer. After removing residual organic solvents in vacuo the aqueous solution was applied to a C^g reverse-phase column. Elution with and lyophilization of the relevant fractions afforded 0.130 g of an amorphous solid. This material was repurified by reverse-phase•hplc to give the pure product (0.053 g, 34%) as an amorphous solid. ir (K3r)vm= : 3420 (br, OH) , 1750 -1 mfx (6-lactam CO), 1590 (-C02 ) cm ; Hnmr (D20) 6 : 4.35-2. 77 (m, 17H), 3.18 (s, 3H, N-Me), 1.23 (d, J=6.3 Hz, 3H, CHMe) ; uv (H-O)X : 300 (s 6344) nm; t, (pH 7.4, 36.8°C) 18.5 h. / max -L/ 4 OH To a solution of p-nitrobenzy1 (5R,6S)-3-[2-(1- - 126 - 207346 Example 18 Preparation of (5R, 6S ) 3- [ 2-(1, 4-dimethyl-l-piperazinium) -ethylthio]-6-(1-(R)-hydroxyethyl]-7-oxo-l-azabicyclo-[3.2.0]hept-2-ene-2-carboxvlate ~ A solution of 2-bromoethyl thiolacetate *(2.20 g, 0.012 mol) and 1,4-dimethylpiperazine (1.95 mL, 0.014 mol) in acetone (4 mL) was stirred at 50°C for 65 h. After cooling to 25®C, the liquid phase was decanted from the gum which was triturated twice in diethyl ether; a hygroscopic yellowish powder, 3.2 g (90%) was obtained; ir (Nujol) v ; 1685 (C=0 of 1 « iucLX thioester) cm ; Hmr (D^O) 6: 2.37, 2. 39 (2s, 6H, CH ^0, Sj-CH-J , 3.18 (s, 3H, ). J J / \ *B. Hansen, Acta Chem. Scand. 11, 537-40 (1957) * ^1 - 127 - B. 1,4-dimethvl-l-(2-mercaptoethyl)piperazinium bromide hvdro-chloride <0 CH3CS' -N' N— , 3r HCl A H< \/i \ 'N^ JH— , 3r , HCl A solution of 1-(2-acetylthioethyl)-1,4-dimethy1-piperazinium bromide (1.1 g, 3.7 mmol) in 6N hydrochloric acid (4 mL) was heated at 80°C under a nitrogen atmosphere for 1 h. The solution was concentrated under reduced pressure to give a white powder, 0.41 g (38%) , ^Hmr (DMSO, dfi) 6: 2.90 (s, —\ /H —\ /C-3 V >, 3.26 (s, y+ )m —/ ^ch3 —/ \— Anal, calcd. for CgH^NjSBrCl* H20: C 31.03, H 7.16, N 9.05, S 10.35; found: C 31.62, H 7.46, N 9.19, S 10.19. 207346 - 128 - C. (5R, 6S) Paranitrobenzvl 3- [2-(1, 4-dimethyl-l-piperaziniurn) - ethylthio]-6-[1-(R)-hydroxyethyl]-7-oxo-l-azabicyclo[3.2.0]-heot-2-ene-2-carboxvlate diphenyIphosphate OH o 1) N2t(i?r)2 o H 2) CI?(0?h)2 ' C00PN3 3) HS HCl \__y ,N-( (?hO)2 e COOPNH 4) NSt(iPr) o To a cold (0"C) solution of (5R,6S) paranitrobenzy1 6-[1 -[R)-hydroxyethyl]-3,7-dioxo-1-azabicyclo[3.2.0]heptane-2- (R)-carboxylate (0.465 g, 1.33 mmol) in acetonitrile (2 mL) kept under a nitrogen atmosphere was added diisopropylethvlamine (0.278 mL, 1.59 mmol) and diphenyl chlorophosphate (0.33 mL, 1.59 mmol). The reaction mixture was stirred for 30 min and treated with a suspension of 1,4-dimethy1-1-(2-mercaptoethyl) - piperazinium bromide hydrochloride (0.40 g, 1.37 mmol) in acetonitrile (3 mL)-water (1 mL) mixture and diisopropvlethylamine (0.278 mL, 1.59 mmol). After stirring for 18 h at 5°C, cold water (15 mL) was added to the mixture. The resulting solution was chromatographed over PrepPak-500/C^g (Waters Associates) column (2.5 x 7.5 cm) with 25-35% acetonitrile in water as eluting solvents to give a yellowish powder 0.SO g (50%) after lyophylization; ir (KBr) v : 1765 (C=0 of 0-lactam), 1690 max * (C=0 of PN3 ester), 1585 (phenyl), 1512 (NC^), 875 (NO^Jcm- , 1Hmr (DMSO, dg) 6: 1.16, 1.18 (2d, J=6.1 Hz, 3H, CH3CH0H), 2 44 39 <s, 3.14 ts, -^/qs3 5_31 (<5j i=6 H2; oh)j 5 (center of ABq, J=13 Hz, CH^ of PN3), 6.6-7.4 (m, 10H, phenyl of phosphate), 7.71 (d, J=8.8 Hz, 2H, Ho of PN3), 8.26 (d, J=8.8 Hz, Hm of PNB). I 207346 - 129 - D. (5R,6S)3- [2-(1,4-dimethy1-1-piperazinium)ethylthio]-6-[1-(R)-hydroxyethyl]- 7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxvlate To a solution of (5R,6S)paranitrobenzyl 3-[2-(1,4-dimethyl-l-piperaziniuiii) ethylthio] -6- [1- (R) -hydroxyethyl] -7-oxo-l-azabicyclo[3.2.0]hept-2-ene-2-carboxylate diphenyIphosphate (0.47 g, 0.623 mmol) in wet tetrahydrofuran (25 mL) was added diethyl ether (25 mL), potassium phosphate monobasic-sodium hydroxide buffer (13 mL, pH 7.22) and 10% palladium on charcoal (0.47 g) . The resulting mixture was hydrogenated at 23°C under 40 psi for 1 h. The two layers were separated and the organic layer was extracted with water (2x7 mL). The aqueous layers were combined, filtered through a Celite pad, washed with diethvl-ether (2 x 15 mL) and chromatographed on PrepPak-500/C^g (Waters Associates) column (2.5 x 9.5 cm) with water as eluting solvent to give, 0.097 g (43%) after lyophylization; ir (K3r) v : IHa-X ^ 3000-3700 (OH), 1750 (C=0 of 3-lactam), 1535 (carboxylate)cm , ^Hmr (D_0) <5: 1.24 (d, J=6.4Tiz, 3H, CH.CHOH) , 2.33 (s, 3H, ~\ —\ ^-3 ^ ^£*3) ' 3.15 (s, ), 4.0-4.5 (m, H-5, CH^CHOH), uv (H-0) X : 296 (e9476), [cxkT"3 61.1° (C 0.26, H_0) , tl/2=12.4 h ^ max D z ~ _ 4 (measured at a concentration of 10 "M in phosphate buffer pH 7.4 at 36.8°C) . 20^346 » - 130 - Example 19 Preparation of (5R,6S)-3-[2-(N-methy1-thiomorpholiniun-oxide)ethylthio]-6-(1-(R)-hydroxyethyl]-7-oxo-l-azabicy-clo(3.2.0)-hept-2-ene-2-carboxylate S~-0 rnCPBA ®r~~\ ^ \ / To a cold (-10°C) solution of (5R,6S)-3-[2-(N-methyl-thiomorpholinium)ethylthio]-6-[1-(R)-hydroxyethyl]-7-oxo-l-azabicvclo(3.2.0)-hept-2-ene-2-carboxylate (608 mg, 1.65 mmol) in a 1:1 mixture of acetonitrile-water (9 mL) was added m-chloroperbenzoic ac'id (334.8 mg, 1.65 mmol) in small portion over a 1 hour period. The mixture was then diluted - 131 - with water (15 mL) and washed with diethyl ether (3 x 15 mL). The aqueous phase was pumped under vacuum and passed through a reversed phase silica gel column (H20) to give a solid which consisted of a mixture of compounds. This mixture was separated by reversed phase HPLC and afforded fraction A 52.4 mg (yield 12%) and fraction 3 23.6 mg (yield 6%) as diastereomers of the title compound; Fraction A: ir (nujol) v : 1750 (s, S-lactarn C=0) i ^ max and 1580 cm (s, C=0); Hmr (D20) 6: 4.26-2.91 (20 H, m, H-4, H-5, H-6, H-l', CH2S, CH2S-0, CH3~N+ and CH2N+) and 1.2 4 ppm (3H, d, J=6. 4 Hz, CH,) ; uv (H-O, c 0.06) X _ : 302 (el0425); *■" J z max T 1/2:12 h (0. 065 iM, pH 7.4 phosphate buffer, 37°c). Fraction 3: ir (nujol) v : 1750 (s, S-lactam C=0) and 1585 cm 1 (s, C=0); ■, max Hmr (D20) 6: 3.86-2.90 (17 H, m, H-4, H-5, H-6, H-1',.CH2S, CH2S-0, CH2N+) , 3.25 (3H, s, CH3N+) and 1.24 ppm (3H., d, J=6.4 Hz, CH,) ; uv (H-O, c 0. 05) X : 2.99 (e6517); T 1/2:10.75 h (0.065 M, J 2 max pH 7.4 buffer solution, 37°C). t . I - 132 - 207346* Exanple 20 Preparation of (5R,6£)-3-[2-(1,4,4-Trimethy1-1-piperazinium)-ethylthio]-6-[IR-hydroxyethyl]-7-oxo-l-azabicyclo(3.2.0) hept-2-ene-2-carboxvlate chloride \ CH3 , ci® /^CH3 A. 1- ( 2-acetylthioethvl) -1,4, 4-trimsthvloiperazinium bromideiodide ' A suspension of 1-(2-acetylthioethyl)-1,4-cimethy1-piperazinium bromide (1.48 g, 5.0 mmol) in isopropyl alcohol (10 mL) was treated with methyliodide (0.373 mL, 6.0 mmol) and heated at 55-60°C for 30 h. The solvents were evaporated under reduced pressure; the residue was triturated in hexane and the solid was filtered, 1.85 g. The solid was dissolved in hot water (8 mL) and the solution was diluted with acetone until turbidity (70-80 mL). Two successive crystallizations gave 1.5 g, mp 220-5°C dec., 68% of the title compound; ir (K3r)vm=: 169 2 cm"1 (C=0) ; ^Hmr (D-,0) 6: 2.40 (s, 3H, JTiclX Z CH-COO) , 3.37 (s, N-CH,) , 3.39 (s , NrCH,), 3.99 (s) ; uv (H.O)X _ : J Z> ' J jL I713.X 226 (£13144). Anal, calcd for Ci;LH24N2OS3rI: C 30.08, H 5.51, N 6.38; found: C 30.48, H 5.53, N 6.86. J O 2 0/ - 133 - 3. 1- ( 2-rnercaptoethvl) -1,4, 4-trimethvlDioerazinium bischloride .HCl 6N Permutit ■> V- s-i cr 2C1 A mixture of 1-(2-acethylthioethvl)-1,4,4-trimethy1- piperazinium bromideiodide (1.84 g, 4.19 mmol) and 6N hydrochloric acid (15 mL) was heated at 57°C under a nitrogen atmosphere for 2.5 h. The solution was concentrated under reduced pressure to dryness. The solid was suspended in water (10 mL) and the well- stirred suspension was treated with permutit S-l Cl~ until a solution was obtained. The solution was poured on a column'(1.2 x 60 cm) of permutit S-l CI . The column was eluted with water (1.5 mL/min). The appropriate fractions were combined and lyophilized to give a white powder, 0.93 gf mD 190-191°C, 85%; ir (nujol)v : * max 2460 (SH) ; Hmr (D20) 5: 3.4 (s, N-CH.^) , 3.45 (s, N-CH-j) , 4.07 (s). • Anal, calcd for C9H22N2SC12* 0.75 KjO: C 39.34, H 8.62, N 10.20, S 11.67; found: C 39.48, H 8.39, N 10.55, S 11.15. C. (5R,6S) paranitrobenzyl 3-[ 2- ( i , 4 , 4-trirr.e thyl-1-piperazinium) -ethylthio]-6 - (IR-hycroxyethy1J- 7-oxo-1-azabicy clo(3.2.0)hspz-2-ene-2-carboxvlate bischloride 1) NEt(i?r, 9 To a cold (5°C) solution of (5R,6S) paranitrobenzyl 6-[IR-hydroxyethyl]- 3,7-dioxo-1-azabicyclo(3.2.0)heptane-2R-carboxylate (0.9 4 g, 2.7 ixnol) ir. acetonitrile (3 mL) kept under a nitrogen atmosphere was added diisopropylethv lamine (0.557 mL, 3.2 mmol) ar.c diphenyl chlorophosphate (0.663 mL, 3.2 mmol). The reaction mixture was stirred at 5°C for 30 min and treated with diisopropylethvlamine (0.599 mL, 3.44 mmol) and an aqueous solution (4 mL) of l-(2-mercaptoethyl)-1,4,4-trimethylpiperazinium bischloride (0.90 g, 3.44 mmol). After 1.25 h, ciisopropylethylamine (0.1 mL, 0.57 mmol) was added and the stirring was continued for 2 h. A part of the acetonitrile was eliminated under reduced pressure and the resulting red mixture was chromatographed on PrepPak -500/C1g (Water Associates) column with 25-7.5% acetonitrile in water as elutinc solvent to give a yellowish powder (1.4 g) after lycphvlization. The powder was solubilized in water anc the solution was passed on a column (1.2 x 58 cm) of permutit S-l Cl~ using water an eluting solvent. Lvophyli-zation of the appropriate fractions' gave 1.17 g of a powder that was repurified on a column of PrepPak -500/C^g. Lyophylization of the appropriate fractions gave a yellowish powder, 0.80 g (53%); ir (KBr)v . : 3400 (br, OH), 1770 (C=0 of the S-lactam), 1690 m&x _ ^ (C=0 of PNB ester) , 1605 (aromatic) , 1515 (N°2^ ' -^45 (NO^) cm. 1Hmr (D20) c: 1.26 (d, J = 6 . 3 Kz, 3H, -C^CHOH), 3.39 (s, NCH3), 4.00 (s), 5.37 (br, s, CH of PK3),'7.60 (d, J=8.6 Hz, 2H , Ho of-?N3) , 8.20 (d, J = 8. 7 Hz, 2H, Hm of P.M3) ; uv (H. 0) >. , : 276 (e 1209 4) , 30 6 — J. Jri&X (610752). Anal, calcd. for C25H3gN4OgSCl2"3H,0: C 46.51, H 6.56, ' N- c.cc, S 4.S7, CI 10.2 8; found: C 4 6.31, K 6.IS, N S.57, S 5.36,' CI 11.37. </div>

Claims

<div class="application article clearfix printTableText" id="claims"> - 135 - 20734$ D. (5R,6S)-3-[2-(l,4,4-trimethy1-1-piperazinium)ethylthio]-6-(IR-hydroxyethyl]-7-oxo-l-azabicvclo(3.2.0)hept-2-ene-2-carboxvlate chloride A mixture of (5R,6£) paranitrobenzyl 3-[2-(l,4,4-trimethyl-l-piperazinium) ethylthio] -6- [ IR-hydroxyethyl] -7-oxo-l-azabicvclo(3.2.0)hept-2-ene-2-carboxylate bischloride (0.40 g, 0.68 mmol), phosphate buffer (30 mL, 0.05M, pH 7.0) , tetrahvdrofurap. (10 mL), ether (30 mL) and 10% palladium on charcoal (0.40 g) was hvdrogenated at 23°C under 35 psi for 1 h. The two phases were separated. The organic phase was extracted with water (10 mL) . The aqueous phases were filtered on a Celite pad, washed with ether . (10 mL)/ concentrated under vacuum to 10 mL and chromatographed on PrePak-500/C^g column (2.2 x 11 cm) with water as eluting solvent to give 70 mg (25%) after ivonhylization; ir (X3r)v : 3400 (br, OH), •* - iilai 1755 (C=0 of the S-lactam) , 1585 (carboxylate) cm ; iHnir ® : 1.24 (3H, d, J = 6.3 Hz, CH^CHOH) , 3.35 (s, NCH3) , 3.93 (s) ; uv (HjO) Amax: 296 t£798'); [g] q3 35.9° (c, 0.30, H20) , ?1/2 = 9'8 h (measured at a concentration of 10 ^ M in phosphate buffer pH 7.4 at 36.8°C). o 207346 136 WHAT WE CLAIM IS: 1. A process for the preparation of a carbapenem derivative of the formula R' 1 S-A-R 14 fr— N 0 I 8 1 wherein R is hydrogen and R is selected from the group consisting of hydrogen; substituted and unsubstituted: alkyl, aikenyl and alkynyl, having from 1-10 carbon atoms; cycloalkyl and cycloalkyl-alkyl, having 3-6 carbon atoms in the cycloalkyl ring and 1-6 carbon atoms in the alkyl moieties; phenyl; aralkyl, aralkenyl and. aralkynyl wherein the aryl moiety is phenyl and the aliphatic portion has 1-6 carbon atoms; heteroaryl, heteroaralkyl, heterocyclyl and heterocyclylalkyl wherein the hetero atom or atoms in the above-named heterocyclic moieties are selected from the group consisting of 1-4 oxygen, nitrogen or sulfur atoms and the alkyl moieties associated with said heterocyclic moieties have 1-6 carbon atoms; wherein.the substituent or substituents . relative to the above-named radicals are selected from the group consisting of \\* n M 207346 - 137 - Q o Cj-Cg alkyl optionally substituted by amino, halo, hydroxy or carboxyl halo -or3 0 " 3 4 -ocnr r 0 -cnr3r4 -nr3r4 3 4 -s02nr r 0 " 3 4 -nhcnr r 0 3" 4 r cnr - -co2r3 =0 0 -ocr3 -sr3 0 H 9 -sr 0 II g -sr ' II 0 -cn -»3 3 -0S03R -oso,r3 3 4 -nr so_r 3 4 -nr c=nr A? 3 4 -nr c02r -no- v- 12 AUG 1985 E V > - 138 - 207346 wherein relative to the above-named substituents, the groups 3 4 R and R are independently selected from hydrogen; alkyl, .alkenyl and alkynyl, having from 1-10 carbon atoms; cycloalkyl, cycloalkylalkyl and alkvlcycloalky1, having 3-6 carbon atoms in the cycloalkyl ring and 1-6 carbon atoms in the alkyl moieties; phenyl; aralkyl, aralkenyl and aralkynyl wherein the aryl moiety is phenyl and the aliphatic portion has 1-6 carbon atoms; and heteroaryl, heteroaralkyl, heterocyclyl and heterocyclylalkyl wherein the hetero atom or atoms in the above-named heterocyclic moieties are selected from the group consisting of 1-4 oxygen, nitrogen or sulfur atoms and the alkyl moieties associated with said heterocyclic moieties have 3 4

1-6 carbon atoms, or R and R taken together with the nitrogen to which at least one is attached may form a 5- or 6- membered 9 3 nitrogen-containing heterocyclic ring; R is as defined for R 1 8 except that it may not be-hydrogen; or wherein R and R taken together represent c2~cio £lkyli<3ene or C2~C10 £lfcyli^ene substituted by hydroxy; A is cyclopentylene, cyclohexylene or C

2-C6 alkylene optionally substituted by one or more C1~C4 alkyl groups; R2 is hydrogen, an anionic charge or a conventional readily removable carboxyl protecting grouu, 2 providing that when R is hydrogen or a protecting group, there is also present a counter anion; and R1^ is a quaternized nitrogen-containing aromatic or non-aromatic heterocycle attached to A through a ring nitrogen, thereby forming a quaternary ammonium group, or a pharmaceutically acceptable salt, thereof, which process comprises reacting an intermediate of the formula 12 AUG V?81 c r - 139 - 207346 ,8 H IV COOR 18 2' wherein R and R are as defined above, R is a conventional; readily removable carboxyl protecting group and L is a conventional leaving group, with a thiol compound of the formula HS-A-R 14 VII 14 0 wherein A and R are as defined above and X is a counter anion, in an inert solvent and in the presence of base,to produce a carbapenem product of the formula ,8 COOR I' 1 8 21 14 © wherein R , R , R , A, R and X are as defined above and, 2' if desired, removing the carboxyl protecting group R to give the corresponding de-blocked compound of formula I, or a pharmaceutically acceptable salt thereof. u N ■— "S\-of "12 AUG 1986' /:• O 207346 - 140~- 2. The process according to Claim 1 for the preparation of a carbapenem derivative of the formula I wherein A is cyclopentylene, cyclohexylene or R10 R12 C R11 R13 in which R^, R^, R"^2 and R"*"3 are each independently hydrogen or C^-C^ alkyl.

3. A process according to Claim 1 or Claim 2 for the preparation of a carbapenem derivative of the formula 14 I wherein R represents a substituted or unsubstituted mono, bi- or poly-cyclic aromatic heterocyclic radical containing at least one nitrogen in the ring and attached to A through a ring nitrogen, thereby forming a quaternary ammonium group.

4. The process according to Claim 1, 2 or 3 wherein the base is a non-nucleophilic tertiary amine or a tri (C-^-C^ ) alkylamine.

5. The process according to Claim 4 wherein the base is a non-nucleophilic tertiary amine.

6. The process according to Claim 1, 2, 3, 4 or 5 wherein the reaction is carried out at a temperature of -15°C up to room temperature.

7. The process according to Claim 6 wherein the reaction is carried out at. a temperature in the range of from -15°C to substantially +15°C. f240CT1986»! - 141 - 207346

8. The process according to Claim 1, 2, 3, 4, 5, 6 or 7 wherein the inert solvent is selected from the group consisting of acetonitrile, a mixture of acetonitrile and dimethylformamide, tetrahydrofuran, a mixture of tetrahydrofuran and water, a mixture of acetonitrile and water, and acetone.

9. A process according to Claim 1 substantially as hereinbefore described with particular reference to any one of the foregoing Examples 1 to 20.

10. A carbapenem derivative of the formula I, as defined in Claim 1, when prepared by the process according to any one of the preceding claims. iC AftTI </div>