CA1284324C - Carbapenem process - Google Patents

Abstract

ABSTRACT

Disclosed is a new and more efficient process for producing carbapenem antibiotic derivatives having a 2-substituent of the general formula wherein A represents cyclopentylene, cyclohexylene or C2-C6 alkylene optionally substituted by one or more C1-C4 alkyl groups and R14 represents a quaternized nitrogen-containing aromatic or non-aromatic heterocycle attached to A through a quaternary nitrogen atom.

Description

~L2~

BACK('7ROUND OF THE INVEI`ITI01`1 The present invention is directed to a new process for the preparation of carbapenem derivatives having a 2-substituent of the formula -S-A-R
wherein A represents cyclopentylene, cyclohexylene or C2-C6 alkylene o~tionally substituted by one or more C1~C4 alkyl groups and R represents a quaternized nitrogen-containing aromatic or non-aromatic heterocycle attached to A through a quaternary nitrogen atom.

`~he carbapenem derivatives prepared by the process of the present invention are disclosed and claimed by my colleagues Choung U. Kim and Peter F. Misco, Jr., in U.S. Patent Applica-tion Se.rial No. 471,379 filed March 8, 1983, now U.S.
Patent No. 4,552,696. That patent, which corresponds to West Ge.rman Published Patent Applic~tion 3,312,533, discloses preparation of carbapenem antibiotics of the formula R
I ~ ~ S - A - N

RlF~ '' COOR
IA
wherein R8 is hydrogen and Rl is selected from the yroup consistin~
of hydrogen; substituted and unsubstituted: alkyl, alkenyl and alkynyl, having from l-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 th~ 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 qroup 3~

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 ,he above-named radicals are selected from the group consisting of Cl-C6 alkyl optionally substituted by amino, halo, hydroxy or carboxyl halo O
-OCNR R

-NR R
~R

~ ~3R4 -502~R3R4 Il 3 4 -~CN~ R
O
R3C~ ---Co2R3 O
-oCR3 ~3 --S R.
Il g -~~R

' -oSo3R3 -oSo2R3 -NR3So2R4 -NR3~=NR4 ~3 -NR3Co2R4 wherein, relative to the above-named substituents, the groups R3 and R4 are independently selected from hydrogen; alkyl, alkenyl and alkynyl, having from 1-10 carbon atoms; cycloalkyl, cycloalkylalkyl and alkylcycloalkyl, 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 1-6 carbon atoms, or R3 and R4 taken together with the nitrogen to which at least one is attached may form a 5-or 6-membered nitrogen-containing heterocyclic ring; R~
is as defined for R3 except that it may not be hydrogen;
or wherein Rl and ~8 taken together represent C2-C10 alkylidene or C2-C10 alkylidene substituted by hydroxy;
A is cyclopentylene, cyclohexylene or C2-C6 alkylene optionally substituted by one or more C1-C4 alkyl groups;
R2 is hydrogen, an anionic charge or a conventional X~ ; .
'~

.
-:
. ' ~ . ' : .

~x~

readily removable c~rboxyl protecting group, providing '.ha- when R is hydrogen or a protecting group, there is also present a counter anion; and -N

-represen,s a substituted or unsubstituted mono-, bi- or polycyclic aromatic heterocyclic radical con~aining at least one nitrogen ~n the ring and attached to A through a ring nitrogen, thereby ~orming a oua~ernary a~monium group; and pharmaceutically accept-able salts thereof by the pr~cess shown m the following reaction scheme:

R8 ~
Rl ~ diphenyl chlorophosphate N ~ COoR2 -~>

III -R H
Rl - ~ P(oc6H5)2 HS-A-OH

2' COOR A = aikylene or CS-C6 cycloallcylene ~:V

.R I ~ S-A-OH . methanesulfonyl .chloride ~ --jCOC~R2 .' ~ ~3,, V

R8 ~ .
R~ S-A-oso2cH3 ~) ~ ~--C

VI

Rl~ ~SOOR-2 ! AS~

II (~) = counter anion) R~

I 'A

Rl~ S-A-rC) IA

U.S. Pat~nt No. 4,536,335 of Choung U. Kim and Peter F. Micso, Jr., issued August 20, 1985, discloses preparation o carbapenem antibiotics of the formula l ~ - A -N

O ~oOR2 . IB

; wherein R8 is hydrogen and Rl is selected from the group consisting of hydrogen; substituted and unsubstituted: alkyl, alkenyl and t alkynyl, having from 1-10 carbon a~oms; 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 heterocyclylalXyl 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 substituent or substituents relative to the above-named radicals are independently selected from the group consisting of r~

~2~

Cl-C6 alkyl optionally substituted by amino, halo, hydroxy or carboxyl halo -oR3 O
oCNR3R4 ,~/
\NR3 R
-So2~R3R4 -NHC~NR3R4 O

-Co2R3 ~O

- ocR3 -S~3 Il 9 S R

-CN

-NR3So2R4 .

: ~ ' ~` ' .

', ~, -NR3Co2R4 wherein, relative to the above-named substituents, the groups R3 and R4 are independently selected from hydrogen; alkyl, alkenyl and alkynyl, having from 1-10 carbon atoms; cycloalkyl, cycloalkyalkyl and alkycycloalkyl, having 3-6 carbon atoms in the cycloalkyl ring and 1-6 carbon atoms in the alkyl moieties; phenyl;
arlakyl, a:ralkenyl and aralkynyl wherein the aryl moiety is phenyl and the aliphatic portion has 1-6 carbon atoms;
and heteroaryl, heteroaralkyl, heterocyclyl and heterocyclyalkyl 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, or R3 and R4 taken together with the nitrogen to which at least one is attached may form a 5- or 6-membered nitrogen-containing heterocyclic ring; R9 is as defined for R3 except that it may not be hydrogen; or wherein Rl and R8 taken together represent C2-C10 alkylidene or C2-C10 alkylidene substituted by hydroxy; R5 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 a~om or atoms in the above-named heterocyclic moieties are selected from the group consisting of 1-~ oxygen, nitrogen or sulfur atoms and the alkyl moieties --. ~
.

.

~l2~

9(a) associated with said heterocyclic moieties have 1-6 carbon atoms; wherein the above-named R5 radicals are optionally substituted by 1-3 substitu~nts independently selected from:

~2843~

Cl-C6 21kyl o~'ionzlly substi~u'ed by 2~no, fluoro, chloro, ca-boxyl, ~ydro~ or c~ aoyl;
fluoro, chloro or brom~;
_ 0~.3 ~ C2 ~3 - ocoR3 .
-oSo2R3;
--ox~
NR R ; .
R3 C o~R4 -~:R C02R ; . ..
-NR3 CoNR3 R
~NR3502R4;
-sR3 i ~ ,.
-S-R';
O O
9 :
-S-R
-S03:R3 ~
-C02~ ;
-CoNR3R4;
-CN; or phenyl optionzlly sl~'os~ituted ~y 1-3 'luoro, chloro, b:romo, Cl-C6 21kyl, -oR3 ~ 3:R4, -S03R, -C02"~ or -CoNR3R4, wherein R.3,. R 2~
R in such ~ subs ~ituen~s ~re 2S de ~ined æ~ove;

.
~ , . ' .

, 3~L

or RS may represent a di~alent phenylene or Cl-C4 alkylene group joined to the J

ring so as to form 2 bridged polycyclic group; A is cyclopentylene, or cyclohexylene or C2-C6 alkylene optionally substituted ~y one or more Cl-C4 alkyl gxoups; ~ is hydrogen,, an anionic charge o~
a.con~entional readily removable carboxyl protecting group, provid-ing that when R2 is hydrogen or a protecting group, there is also present 2 counter ion; and , N J

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 formlng' a quaternary ammonium group; and pharmaceutically accept-able salts thereof, by the process shown in the following reaction scheme:
R8 ~ ` ~

dipXenyl chlorophosphate 0~ --COOR2 ~ III

.

r R ~ ~ P(C6H5)2~
HS - A - OH

N COOR
A=alkylene or IV
C5-C6 cycloalkylene R
Rl ~ S -A - OH
methanesulfonyl chloride N ____I__ COOR

R H

Rl ~ S-A-OSO2CH3 I~
~ N 2' O COOR
VI

~ ~ S-A-I

O . COOR Ag X~

`
R8 HR ~
Rl 5-A ~ N J optional N ~ COOR2' de-blocking I'B

. .
-. , .

~ 13 -Rl ~ S-A-~ 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 ~I 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 a~ine in an inert organiC solvent and in the presence o silver ion to produce the quaternized product I'A or I'B which may then be de-blocked to give the corresponding de-blocked carbapenem of Formula IA or IB.
, The abo~e-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 quaternizatio~ 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 ~mine 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 PKb values. ~ ~

~ , .
.' ' 32~L

The present inven~ion provides a novel process for preparation of carbapenem deri~-atlves of the formula Rl~S_A_R14 O COOR

wherein R is hydrogen and Rl is selected from the group con~
sisting o~ 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, hetero-aralkyl, 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 sul~ur atoms and the alkyl moieties associated with said hetero-cyclic moieties have 1-6 carbon atomsi wherein the substituent:
or substituents relative to the above-named radicals are selected from the group consisting o~:

:

:
: : :
.. .
_ .

r ' ' ' ' ' . ' ' - : ' . , :, ', .
. " ' ~ , ' ' . ' ' I "

, ', ' ., ' ' , ' ~!L2~

Cl-C6 21~;yl c~p.io~21~y s~sti ,u~e~ by æ-nino, halo, hydroxy or car;)oxyl 3 ~ 4 R3 ~4 -NR R

~R3 R4 -S02~R ~

: ~I 3 4 -~nC~2 R

3 ll 4 Z
=~
ll 3 - OCR
~, ~
~ ~ 5~
o I g -SR

- ~ R
: : O ,, , :
3 ~ ~
: ~ :

-oSo3R3 -oso2-~3 -NR3-So2R4 -NR3~=NR4 -NR3Co2R4 wherein, relative to the above-name~ substituents, the groups R3 and R4 are independently selected from hydrogen; alkyl, alkenyl and alkynyl, having from 1-10 carbon atoms; cycloalkyl, cycloalkyalkyl and alkylcycloalkyl, 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 1-6 carbon atoms, or R3 and R4 taken together with the nitrogen to which at least one is attached may form a 5-or 6-membered nitrogen-containing heterocyclic ring; R9 is as defined for R3 except that it may not be hydrogen;
or wherein Rl and R8 taken together represent C2-C10 alkylidene or C2-C10 alkylidene substituted.by hydroxy; A
is cyclopentyl.ene, cyclohexylene or C2-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 group, providing that when R2 is hydrogen or a protecting group, there is also present a counter ' ~1 , i . - . . ,:
.~ ....
.

. . :

anion; and Rl4 is a quaternized nitrogen-con-aining 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 Rl ~--.

o N ~~ ~cooR2' IV
wherein Rl and R8 are as defined above, R2 is a conventional, readily removable carboxyl protecting group and L is a conventional leaving group)with a thiol compound of the formula ~S-A-Rl~2 X~3 VII

wherein A and Rl4 are as defined above and X~ is a counter anion in an inert solvent and in ~he presence of base~ to produce a carbapenem product of the formula R~ S_A_R14 ~ N ~ 2' xa o COOR

wherein Rl, R8, R2 , A, Rl4 and X3 are as defined above and, if desired, removing the carboxyl protecting group R to give the corresponding de-blocked compound of formula I, or a pharma-ceutically acceptable salt thereof. --~2~

~ 18 -Also provided by the present invention are intermediates of Formula VII and processes for preparing such intermediates.

The carbapenem compounds of Formula I are potent antibacterial agents or intermediates userul in the preparation of such agents.

The compounds of general Formula I above contain the carbapenem nucleus 6 ~ 2 ~ N 1 3 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 4 ` O ~ ~ 2 and named as 7-oxo-l-azabicyclot3.2.0)hept-2-ene-2-carboxylic acid derivatives. While the present invention includes compounds wherein the relative stereochemistry of the 5,6-protons is cls as well as transr 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, .
.

R8 may be hydrogen and R1 may be hydrogen or a non-hydrogen substituent disclosed, for example, in European Pa~ent Application 38,869 (see definition of R6). Alternatively, R8 and Rl taken together may be C2-C10 alkylidene or C2-C10 alkylidene substituted, for example, by hydroxy.

To elaborate on.the definitions for Rl and R8:

(a) The aliphatic "al~yl", "alkenyl" and "alkynyl" groups may be straight or branched chain having 1-10 carbon atoms;
preferred are 1-6, mos~ preferably 1-4, carbon groups;
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 O, N or S atoms; pre-ferred are 5- or 6- membered heterocyclic rings such as thienyl, furyl, thiadiazolyl, oxadiazolyl, triazolyl, isothiazolyl, thiazolyl, imidazolyl, isoxazolyl, tetrazolyl, oxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, pyrazolyl, etc.

(c) "heterocyclyl" includes mono-, bi- and polycyclic saturated or unsaturated non-aromatic heterocyclic groups con~aining 1-4 O, N or S atoms; preferred are 5- or 6-membered heterocyclic rings such as morpholinyl, piperazinyl, piperidyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, pyrrolinyl, pyrrolidinyl, etc.

(d) "halo" includes chloro, bromo, fluoro and iodo and i5 preferably chloro or bromo.

~4.~2~

The term "conventional readily removable carboxyl protecting group" refers to a known ester group which has been employed to block a carboxyl group dùring 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 ultraYiolet light or catalytic hydrogenation. Examples of such ester protecting groups include benzhydryl, p-nitrobenzyl, 2-naphthylmethyl, allyl, benzyl, trichloroethyl, silyl such as trimethylsilyl, phenacyl, p-methoxybenzyl, acetonyl~ o-nitrobenzyl, 4-pyridyl-methyl and Cl-C6 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 methoxymethyl.
Particularly advantageous carboxyl protecting groups are p-nitrobenzyl which may be readily remoYed by catalytic hydro-genolysis and allyl which can be removed by Pd(P~3)4-catalyzed reaction.

The pharmaceutically acceptable salts referred to 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 R~ ~ A R14 R - H or protecting group where X9 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 intermediate compounds of Formula I, X3 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 groups are present in the Rl group or on the quaternized Rl4 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, l-ephenamine, N-benzyl-~-phenethylamine, N,N'-dibenzylethylenediamine, etc.) in the case of an acidic group.

Compounds of Formula I wherein R2 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 conver~ed into ~he above-mentioned biologically active compounds.

A preferred embodiment of the present invention comprises compounds of Formula I wherein R8 is hydrogen and is hydrogen, CH3CH2-` CH CH OH OH
~ 1 ~ CH-, j C- or CH3CH-Among this subclass, the preferred compounds are those in which Rl is ---O~
CH3CH-, most preferably compounds having the ': ' , .

absolute conriguration 5R, 6S, 8R.

Another preerred embodiment comprises compounds of Formula I in which R~ and R8 taXen together form an alkylidene radical of the formula C- -. C~3 The alkylene or cycloalkylene radical A in the compounds of Formula I may be cyclopentylene \ 7 ~ ~2C CH2 .

cyclohexylene ( ~ ' ) f or C2-C6 alXylene optionally substituted by one or more Cl-C4 alkyl substituents. Preferred A substituents are cyclopentylene, cyclohexylene or alkylene of the formula ~,10 R12 --C~ ~
~ 13 i hich R10 Rll R12 and R13 are each independently hydrogen or Cl C4 alkyl. A preferred embodiment comprises those compounds of Formula I in which-substituent A is -CH2CH2-, -ICHCH2-, or -CH~CH-In the case of certain compounds of Formula I having acycloalkylene or branched alkylene A substituent, one or more --additional assymmetric carbon atoms may be created which resul~
in formation of diaster~éolsomers. The present invention includes mixtures of such diastereoisomers as well as the individual ourified diastereoisomers.

,.~, ~f ' .

The quaternized ~14 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.

One preferred class of R14 substituents may be represented by the general formula . ~I J

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 Cl-C4 alkyl, Cl-C4 alkyl substituted by hydroxy, amino, carboxy or halo, C3-C6 cycloalkyl, Cl-C4 alkoxy, Cl-C4 alkylthio, amino, Cl-C4 alkylamino, di(Cl-C4 alkyl)amino, halo, Cl-C4 alkanoylamino, Cl-C4 alkanoyloxy, carboxy, R
-C-OCl-C4 alkyl, hydroxy, amidino, guanidino, trifluoromethyl, phenyl, phenyl substituted by one, two or three amino, halo, hyd~roxyl, trifluoro-methyl, Cl-C4 alkyl or Cl-C4 alkoxy groups, heteroaryl and hetero-aralkyl in which the hetero a~om 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 hetexo-aralkyl has I-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 o' the alkylene or cycloalkylene radical) and, optionally, one or more additional hetero atoms selected .rom O, N or S. While, in general, any heteroaryl radical bonded to A

.
, via a quaternized nitrogen atom is found to produce biologically ac~ive carbapenem derivatives, a preferred embodiment comprises compounds in which - N J
/
represents a radical selected from the group consisting of (a) \ ~ ~ R6 R

wherein R5, R6 and R7 are independently selected from hydrogen;
Cl-C4 alkyl; Cl-C4 alkyl substituted by hydroxy, amino, carboxy or halo; C3-C6 cycloalkyl; Cl-C4 alkoxy; Cl-C4 alkylthio; amino;
Cl-C4 alkylamino; di(Cl-C4 alkyl)amino; halo; Cl-C4 alkanoylamino;
Cl-C4 alkanoyloxy; carboxy;

, -C-OCl-C4 alkyl; hydroxy, amidino;
guanidino; trifluoromethyl; phenyl; phenyl substituted by one, two or three amino, halo, hydroxyl, trifluoromethyl, Cl-C4 alkyl or Cl-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 or 1-4 oxygen, nitrogen or sulfur atoms and the alkyl moiety associated with said hetero-aralkyl moiety has 1-6 carbon atoms; or wherein two of R5, R6 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) ~ ~ or ~3 , , optionally su~stituted on a carbon atom by one or more sub-stituents independently selected from Cl-C4 alkyl; Cl-C4 alkyl substituted by hydroxy, amino, carboxy or halogen; C3-C6 cyclo-alXyl; Cl-C4 alkoxy; Cl-C4 alkylthio; amino; Cl-C4 alkylamino;
`di(Cl-C4 alkyl)amino; halo; Cl-C4 alkanoylamino; Cl-C4 alkanoyloxy;

carboxy; -C-OCl-C4 alkyl; hydroxy; amidinoi guanidino; trifluoro-methyl; phenyl; phenyl substituted by one, two or three amino, halo, hydroxyl, trifluoromethyl, Cl-C4 alkyl or Cl-C4 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~
N ~ N

~ 3 ~ 3 ~ 3 optionally substituted on a carbon atom by one or more substituents independently selected from C1-C4 alkyl; Cl-C4 alkyl substituted by hydroxy, amino, carboxy or halogen; C3-C6 cycloalkyl; C1-C~.
alkoxy; Cl-C4 alkylthio; amino; Cl-C4 alkylamino; di(Cl-C~ alkyl)-amino; halo; Cl-C4 alkanoylamino, Cl-C4 alkanoyloxy; carboxy;
q _.
-C-OCl-C4 alkyl; hydroxy; amidino; guanidino; trifluoromethyl phenyl; phenyl substitated by one, two or three amino, halo, .

.

' hydroxyl, trifluoromethyl, Cl-C4 alkyl or Cl-C4 alkoxy groups;
and heteroaryl or heteroaralkyl ln whlch 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 molety assoclated with said heteroaralkyl moiety has 1-6 carbon atoms, or optionally substituted so as to form a fused carbocyclic or heterocyclic ring;

(d) ~ ~ ~ ~ N

~ N ~ N N ~
~N 3 ~ ~N N N ~N

optionally substituted on a carbon atom by one or more substituents j independently selected from Cl-C4 alXyl; Cl-C4 alXyl substituted ; by hydroxy, amino, carboxy or halogen; C3-C6 cycloalkyl; Cl-C~
alkoxy; Cl-C4 alkylthio; amino; Cl-C4 alkylamino; di(Cl-C4 alXyl)-amino; halo; Cl-C4 alkanoylamino; Cl-C4 alkanoyloxy; carboxy;

-C-OCl-C4 alkyl; hydroxy; amidino; guanidino; trifluoromethyl;
l~ phenyl; phenyl substituted by one, two or three amino, halo, I hydroxyl, trifluoromethyl, Cl-C4 alkyl or Cl-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 ox~gen, 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, 3~

~ or ~ ~

wherein X is 0, S or NR in which R is Cl-C4 alkyl or phenyl, said radical being optionally substituted on a carbon atom by one or more substituents independently selected from Cl-C4 alkyl;
Cl-C4 alkyl substituted by hydroxy, amino, carboxy or halogen;
6 1 C4 alkoxy; Cl-C4 alkylthio; amino; C C
alkylamino; ~i(Cl-C4 alkyl)amino; halo; Cl-C4 alkanoylamino;
-Cl-C4 alkanoyloxy; carboxy;

-C-OCl-C4 alkyl; hydroxy; amidino; guanidino; trifluoro~ethyl;
phenyl; phenyl substituted by one, two or three amino, halo, hydroxyl, trifluoromethyl, Cl-C4 alkyl or Cl-C4 aIkoxy grou2s;
and he~eroaryl or heteroaralkyl in which the hetero atom or atoms in the abo~e-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 ~used carbocyclic or heterocyclic ring;

N- , \X ~

- or ~ -X >

_ .

:

.
- ~ . ::, ... . .

wherein X is 0, S or NR ln which R is Cl-C4 alkyl or phenyl, said radical being optionally substituted on a carbon atom by one or more substituents independently selected from Cl-C4 alkyl; Cl-C4 alkyl substituted by hydroxy, amino, carboxy or halogen; C3-C6 cycloalkyl; Cl-C4 alkoxy; Cl-C4 alXylthio; amino;
Cl-C4 alkylamino; di(Cl-C4 alkyl)amlno; halo; Cl-C4 alkanoylamino;
Cl-C4 alkanoyloxy; carboxy;
o -C-OCl-C4 alkyl; hydroxy; amidino;
guanidino; trifluoromethyl; phenyl; phenyl substituted by one, two or three amino, halo, hydroxyl, trifluoromethyl, Cl-C4 alkyl or Cl-C4 alkoxy groups; and heteroaryl or heteroaralkyl in which the hetero atom or atoms in the abo~e-named heterocyclic moieties are selected from ~he group consisting of 1-4 oxygen, nitrogen or sulfur atoms and the alkyl moiety associated with said hetero-aralXyl moiety has 1-6 carbon atoms; and N _ N\ j - ~
N ~ N-R N ~ N-R

N - N ~ N\-R
- ~ -R N ~ N-~~N ~-R or ~R

~8~

wherein R is Cl-C4 alkyl or phenyl, said radical being optionally substituted on ~he carbon atom by a substituent selected from Cl-C~
alXyl; Cl-C4 alkyl substituted by hydroxy; amino, carboxy or halogen C3-C6 cycloalkyl; Cl-C4 alkoxy; Cl-C4 alkylthio; amino; Cl-C4 alkylamino; di(Cl-C4 alkyl)amino; C1-C4 alkanoylaminoi carboxy;
11 .
-C-OCl-C4 alkylj hydroxy; amidino; guanidino; trifluoromethyl;
phenyl, phenyl substituted by one, two or three amino, halo, hydroxyl, trifluoromethyl, C1-C4 alkyl or C1-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 heteroaralXyl moiety has 1-6 carbon atoms.

Within the above subclass, the preferred compounds are those in which substituent A is -CH2CH2-, -~HCH2 , ~ or 1 8 CH3 ~
-CH~CH- and wherein either (a) R- and R taken together represent or (b) R is hydrogen and R represents hydrogen, CH3CH2-, C ~ ~ I OH
", CH- , ," C- or CH3CH-Particularly preferred are the compounds wherein R8 is hydrogen and Rl is OH
CH3CH-, preferably compounds having the absolute -.
configuration SR, 65, 8R.

:;
, - ~ ' A particularly preferred embodiment of the present invention comprises preparation of compounds wherein - ~ ) represents a radical of the formula I ~ R7 R ~
in which R5, R6 and R7 are independently selected from the group consisting bf hydrogen, C1-C4 alkyl, C1-C4 alkoxy, Cl-C4 alkyl substituted by a hydroxy group, Cl-C4 alkylthio, amino, carboxy and carbamoyl. Within this subclass, the preferred compounds are those wherein substitu~nt A is -CH CH -, -CHCH2- ' ~ or CH2CH

and wherein either (a) R1 and R8 taken together represent HOC ~

CH C=
or (b) R8 is hydrogen and Rl represents hydrogen, CH CH OH OH
~ ~1. .......... I
CH- , C- o~ CH3CH
` CH3 3 Particularly preferred are the compounds wherein R8 is .

~ .
,~

~, .
'. ' ' ' : . : '' . ' '.. :' . ' .

' ' ' ' ' ~'; '' ' .

- 30(a) -hydrogen and R1 is ~H
CH3CH-, preferably compounds having the absolute configuration 5R, 6S, 8R.

:
.

-" ~2~32~
~.

A most preerred embodi~ent of the present invention comprises preparation or compounds wherein represents a radical of the formula --~R7 in which R5, R6 and R7 are independently selected from the , group consisting of hydrogen, Cl-C4 alkyl, Cl-C4 alkoxy, Cl-C4 alkyl substituted by a hydroxy group, C1-C4 alkylthio and amino.
Within this subclass, the preferred compounds are those wherein substituent A is -CH2CH2-, -ICHCH2 ~ ~ or -CH2CH-and wherein either (a) Rl and R8 taken together represent HOC~
~ C=CH
or (b) R8 is hydrogen and Rl represents hydrogen, CH3CH~-, CH~ CH ~ OH OH
CX- , C- or CH3CH~

Particularly preferred are the compounds wherein R8 is hydrogen and Rl is ~H
CH3CH-, preferably compounds having the absolute configuration SR, 6S, 8R.

Another preferred,embodiment of the present inventlon comprises preparation of compounds wherein ~ ~0 ' ' ' , .
.
, ' ~ ' -.

~ 8 ~

represents a radical of the formula -N - \ SCH3 ~ -CH3 Within this subclass, the preferred compounds are those wherein substi~uent A is -CHCH2-, -CH2CH2- , ~ or -CH2CH-3 ~ CH3and wherein either (a) Rl and ~8 taXen together represent HOC~
C=
C~' or (b) R8 is hydrogen and Rl represents hydrogen, CH3CH2-, CX~ CH ~ OH OH
CH- C- or CH3CH-Particularly preferred are the compounds wherein R8 is hydrogen and Rl is OH
CH3CH-, preferably compounds having the absolute con~iguration 5R, 6S, 8R.

Another preferred em~odiment of the present invention comprises preparation of compounds wherein ~ ~ .
- N

represents a pyridinium radical. Within this subclass, the j preferred compounds are those wherein substituent A is -ICHCH2-, ' -CH2CH2- ~ ~ or -C~2C~- and wherein either (a) --¦1 Rl and R8 taken together represent .
.. . .

. .
. - . .

:. .. .
., . , , .
. - . . .

~8~3~

HOC\2 ~C=

or (b) R8 is hydrogen and Rl represents hydrogen, ~H3CH2-, CH~ CH ~ fH OH
CH- , C- or CH3CH-~articularly preferred are the compounds wherein R8 is hydrogen and R is ~H
CH3CH-, preferably compounds ha~ing 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) ~ ~ ~ 5 - A-N

~ .

wherein - A -N
represents ... ~

OE~ 3 ~
H2CH2 N~3 ( 2? -CH CH ~N~3 t 3 ) - CH 2 C~l 2 eN~ ( 4 ) 2 . 2 H
S) -Ch' CH ~N~ 3 ~ ~) SC~3 (7) -C1~2C~2 ~3C,a2oH ( 8 ) ~3 (9 ) C~2C~2 ~ I (iO ) CH2C~

( 11 ) - C~ CR2 e~N~3 , 3 . 3. ~ (12 ) -CR2C~2 N~
R or S di~stexeoisom~s ~N~3 N~ 2 ~ . . . ..... , .. _ , ( 13) ~ -CH2CH2~N~ o_ (14) 1~3J

R,R or S ,S
~J dizstereoisorners at two a~ylTmetric carbons of the cyclohexy l grou~
, .

~L~8~

and R2 is hydrogen, an anionic charge ox a conventional readily removable carboxyl protecting group, providlng that when R2 is hydrogen or a protecting group, there is also present a counter anion, and pharmaceutically acceptable acid addition salts thereof Another preferred class of quaternized R14 substituents may be represented by the general formula --N/~) in which R~6 is selected rom the group consisting of substitu.ed and unsubstituted: alkyl, alkenyl and alkynyl., having from 1-10 carbon atoms; cycloalkyl and cycloalkylalkyl, having 3-6 ca~bon atoms in the cycloalkyl ring and 1-6 carbon atoms in the alkyl moieties; phenyl; aralkyl, aralXenyl and aralkynyl wherein the aryl moiety is phenyl and the aliphatic portion has 1-6 carbon atoms; heteroaryl, he~eroaralXyl, heterocyclyl and heterocyclyl-alkyl wherein the hetero atom or atoms in the above-named hetero-cyclic 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 R15 radicals are optionally substituted by 1-3 substituents independently selected from:

~, ~ ' ' .

: - :' . ' Cl-C6 alkyl optionall~ subs~ilu~ed by ~ o, ^ uoro, chloro, c:a~boxyl, hydroxy or cæba~oyl, luoro, chloro or bromo ;
_ o~.3 -oco2~3 o23 .' ,, . C~S2R ; , ' --ox~
-NR :R ; .
_~. 3 ' ' -NR3CoNR3 ~ ; ' -S~.3;
O
S-R9;
,0 ~0 -S-R
` ~ -503R3;
.
--C2 R3 . ; , '.

- CON2 3~ ;
-CN; or phenyl option?lly substituted by 1-3 ~'luoro, chlo_o, bromo, Cl-C6 al~cyl, -oR3, -N~3R4~, ;
-~03R3 , ~ 2i?.3 or -coNx3~4 , wherei2l ~3, R4 .an R9 in such- ~16 sub5-t:i tuents are 2S de~ined 2~ove, ::.
:

; .

: , . .

, ~ .. . , . ' .

2~

or Rl6 may represent a divalent phenylene or C1-C4 alkylene group joined to the \~
-N

ring so as to form a bridged polycyclic group and \~
. - N J

represents a substituted or unsubstituted mono-, bi-or polycyclic non-aromatic (which may be fused to another aromatic o~ 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 O, S(O)m/ N, NR15 or NR17R18 wherein m is O, 1 or 2, R15 is hydrogen, optionally substituted C1-C6 alkyl or optionally substituted phenyl and R17 and R18 are each independently optionally substituted C1-C6 alkyl or optionally substituted phenyl.
In a preferred embodiment \~ ~

. . , 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-ato~s selected from O, S(O~m/ N, NR15 or NR17R18 wherein m is 0, 1 or 2, Rl5 is hydrogen, Cl-C6 alkyl optionally substituted by 1-.. ~, . .... ~ ...

_ 37(a) ~

2 substituents independently selected from -oR3, -NR3R4, -Co2R3, oxo, phenyl, fluoro, chloro, bromo, -S03R3 and -CoNR3R4 or phenyl optionally substituted by 1-3 substituents independently selected from C1-C6 alkyl, -oR3, -NR3R4, fluoro, chloro, bromo, -$03R3, -C02R2 and -CoNR3R4 and Rl7 and R18 are each independently C1-C6 alkyl optionally substituted by 1-2 substituents independently selected from -oR3, -NR3R4, -C02R3, oxo, phenyl, fluoro, chloro, bromo, -S03R3 and -CoNR3R4 or phenyl . . .

-o~tionally substituted by 1-3 substituents independ~ntly selec~d from Cl-C6 al~yl, -oR3~ -NR3R4, fluoro, chloro, bromo, SO3R , -CO R~ and -coNR3R4~ wherein ~3 and R4 in such heterocyclic N~15 or NRl~R18 gro-~ps are as defined above in connection with the R16 substituent. In such preferred embodiment the \1~
N

ring may be optionally substituted by 1-3 substituents independently selected from ~a) Cl-C6 alXyl optionally substituted by 1-2 substituents inde~endently selected from fluoro, chloro, bromo, -oR3, OCOR , -oCoNR3~4, oxo, -N~3R4 NR3CoR4 3 3 4 -NR3So2R4~ -SR , -S03R , -C02R and -CON~
(b) C2-C6 alkenyl optionally substituted by 1-2 substituents independently selected from fluoro, chloro, bromo, -oR3 -OCOR , -oCONR3R4, oxo, -NR3R4, -NR3CoR4, -NR3CoN~3R4 -NR3So2R4, -SR3 r -So3R3, -Co2R3 and -CoNR3R ;
tc) C2-C6 alXynyl optionally substituted by 1-2 substituents independently selected f~om fluoro, chloro, bromo, -OR, CoNR3~4 o~o -N~3R4r -NR3CoR , -NR CONR
-NR So2R4, -SR3, -So3R3, -C02R and -CONR R ;
(d) C3-C6 cycloalXyl optionally substituted by 1-2 substituents independently selected from fluoro, chloro, bromo, -oR3, -oCoR3, -oCoN~3R4, oxo, -NR3R4, -NR3CoR , -N~ CON~ R , -NR3So2R4, -SR3, -S03R3, -C02R and -CONR R ~
(e) cycloalkylalkyl ha~ing 3-6 carbon a,oms in the cycloalXyl ¦ ring and 1-6 carbon atoms in the alkyl moiety~ optionally ~ substituted by 1-2 substituents independently selected from ! fluoro, chloror bromo ~ -oR3 / -ocoR3 ~ -OCONR R , oxo, -N~3R4, -NR3CoR4, NR3CoNR R4, -NR3S02R , -SR , -SO3R , -CO2~ and ~C5NR R;

!

.

(r~ hete-oaryl wherein ~e hetero.atom or a~oms are selected from the group consisting of l-4 oxygen, n-t-ogen or sulfur atoms, optionally substituted by 1-2 substituents independently selected from fluoro, chloro, bromo, -oR3, -ocoR3~ -oCoNR3R4, oxo, -NR3R4, -N~3CoR4; -NR3coNR3R4 -N~3so2R , -SR3, -S03R3, -C02R3 and -coN~3~ ; pre'erred heteroaryl radicals are 5- or 6-~embered aromztic hetera-cyclic xings;
(g) heteroaralkyl wherein the hetero atom or atoms are selected from the sroup cons1sting of 1-4 oxygen, nitrogen or sulrur atoms and the alkyl moiety has 1-6 carbon ato~s, optionally substituted by 1-2 substituents independently selec,ed f om fluoro, chloro, bromo, -oR3r -OCOR, -OCONR R, oxo, -NR3R4, -NR3coR4r -N~3CoNR3R4, -N~3So2~4, -S~3, -S03R3, -C02R and -CoNR3R4; preferred heteroarai~yl are those in which t~e heteroa~yl _adical is a 5- or 6-membe~ed aromatic heterocyclic ring and the alkyl moiety has 1-2 carbon atoms;

(h) heterocyclyl wherein the hetero atom or atoms are selected from t~e group consisting of 1-4 oxygen, nitrogen or sulfur atoms, optionally substituted by 1-2 substituents independently selected from 'luoro, chloro, bromo, -oR3, -OCOR , -oCoNR3R4, oxo, -NR3R4, -NR3Co~4, -NR3CoNR3R4 -NR S02R , -SR , -S03R., -C02R and -CONR R ; preferred heterocyclyl are S- or 6-membered saturated or u~saturated rings; - -(i) heterocyclylalky.l wherein the hetero atom or atoms areselected from the group consisting o 1-4 oxygen, nitrogen or sulfur atoms and the al~yl moiety has 1-6 carbon atoms, optionally su~stituted by 1-2 substituents.independently selected from ~luoro, chloro, bromo, -oR3, -oCoR3, -oCoNR3R4, ox~, -NR3R4, -NR3CoR4, -NR3Co~R3R4, -N~3So2R4, -sR3, -S~3R3, -Co2R3 and -CoNR3R; prererred heterocyclylalkyl are those in which the heterocyclyl moiety is a S- or 6-mem~ered s aturated or unsaturated ring;

.

~j) fluoro, chloro or bromo;
(k) -oR3;
~ oCO~R3 ;
(m) -ocoR3;
(n) -oCoNR3R
( o) -oso2R3 (P) oxo;
(q) -NR3R
(r) R3CoN~4_;
(s) NR3Co2R4 ;
.(t) -NR3CoNR3R4 ; ~ - - -.... . _ (u) -N~3502R4 ;
(v) -SR3 ;
(w) o -S-R9 ;
(~) O O
~ 9 , (~) -503R3 ;
( Z) -Co2R3 (aa) -CONR R
(bb) -CN; or (cc) phenyl optionally substituted by 1-3 fluoro, chloro, bromo, .~ Cl-C6 alkyl, -OR , -NR R , -503R , C02R or CON~ R .
The R3, ~4 and R9 substituents mentioned a~o~e are as defined i~
connection wi',h substituent Rl.
The -N
. , ~

~8~3~

ring as defined above is a non-aromatic hetexocycle group.
This ring, however, may be fused to another ring which may be a saturated or unsaturated carbocyclic ring, preferably a C4-C7 carbocyclic ring, a phenyl ring, a 4-7 membered heterocyclic ~saturated or unsaturated) ring containing 1-3 hetero atoms selected from 0, N, S(O)~, NR15 or NX17R18 or a 5-6 membered heteroaromatic ring containing 1-3 hetero atoms selected 0, S(O) , N~ NR15 or NR17R18 in which m, R 5, Rl and R are as defined above.

The R16 substituent of the non-aromatic R14 radical may be either (a) an optionally substituted Cl-C6 alXyl, C2-C10 alkenyl, C2-C10 alkynyl, C3-C6 cycloalkyl, C3-C6 cycloalkyl~-Cl-C6 alkyl, phenyl, phenyl-Cl-C6 alkyl, phenyl-C2-C6 alkenyl, phenyl-C2-C6 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 Cl-C4 alkylene group joined to the :

~2~

ring so as to form a bridged rlng polycyclic sroup, e.g. a quinuclidine grou?. The heteroaryl (or he~eroaryl portion o' heteroaralkyl) substituent may be a mono-, bi- or polycyclic aroma,ic heterocyclic ~roup containing 1-4 O, N or S atoms;
preLerred are 5- or 6-membered heterocyclic rinss such as ~hienyl, furyl, thiadiazolyl, oxadiazolyl, triazolyl, isothiazolyl, ~hia~olyl, imidazolyl, isoxazolyl, tetrazolyl, oxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl znd pyrazolyl. The heterocyclyl ~or heterocyclyl portion of heterocyclylalkyl) sub-stituen. may be a mono-, bi- or polycyclic saturated or unsatu-ra~ed non-aromatic heterocyclic group containing 1-4 O, N or S
atoms; preferred are 5- or 6-membered heterocyclic rinss such 2S
mor~holinyl, piperazinyl, piperidyl, pyrazolinyi, pyrazolidinyl, imidazolinyl, imidazolidinyl, pyrrolinyl and pyrrolidinyl.
In the case where ~he R16 su~stituent is ~ alkyl, alkenyl, 21Xynyl, cycloalkyl, cycloaIXylalXyl; phenyl,_ phe~.yl-alXyl, phenylalkenyl, phenylalkynyl, hete_oaryl, hetero~ralXyl, he,erocyclyl or heterocyclylalkyl group, such s_oups may be optionally subs~ituted by 1-3 substituents independently selected rrom:
(a) Cl-C6 alXyl optionally substituted by, preferably 1-3, amino, 'l~oro, chloro, c2rboxy1, hydroxy or c~rb2moyl srou~s;
~b~ Lluoro, chloro or bromo;
( c) -oR3 (d~ -oC02R3 ;
:
~e) -oCoR3 ;
- ocoN~3 ~4 ;

( s) -oso2R3 (h) -oxo ;
(i) -NR3R4 ;
( j ~ R3CoNR4_ ;
(X~ -NR3C02R ;, ( 1~ -NR3CoNR3R4 ; - -(m) -~R3so2R4;
(n) -SR3;
(o) -SOR9;
(p) -S02R9;
( q) -S03R3;
(r) -C02R3;
( s ) -CoNR3R4;
(t) -CN; or (u) phenyl optionally substituted by 1-3 substituents independently selected from fluoro, chloro, bromo, Cl-C6 alkyl, -oR3~ -NR3R4, -S03R3, -Co2R3 or -CoNR3R4~ wherein relative to the above-named R16 substituents, the groups R3 and R4 are independently selected from hydrogen;
alkyl, alkenyl and alkynyl, having 1-10 carbon atoms;
cycloalkyl, cycloalkylalkyl and alkylcycloalkyl, 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 heteroaryl and heterocyclyl group or portion of a group is as defined above for R16 and the alkyl moieties associated with said heterocyclic moieties have 1-6 carbon atoms; or R3 and R4 taken together with the nitrogen to which at least one is attached may form a 5-or 6-membered nitrogen-containing heterocyclic (as defined above for R5) ring; and R9 is as defined above for R3 except that it may not be hydrogen. A most preferred R16 substituent is C1-C6 alkyl, especially methyl.
In the case where R16 is a divalent phenylene or C1-C6 alkylene group, ~uch group is bonded to another atom of the \1~

.;.

"

.

3~

_ 43(a) -ring so as to form a bridged polycyclic ring, e.g. a quaternized quinuclidine ring of the formula .. . ..
!i A particularly preferred embodiment of the present invention comprises preparation o~ compounds of Formula I wherein -\N

reDresents \ ~ y , _ ~ CH

\ ~,r~ \ ~ ~

y y Y

\ /f \ ~ ~N/f \N r -~N ~ _CH3 , -~N ~ ~ ' ~ ~ ~ CH3 CH~ ~ ~ C\ ~

wherein Y is hydrogen, Cl-C6 alkyl, hydroxy, -SCl-C6 alkyl, carboxyl, carbamoyl, chloro, bromo, iodo,~ fluoro or phenyl.
Within this subclass, the pre~erred compounds are those _.

.

~ fp ~

wherein A is - ~ CH2- , ~ \CH

-~CH2)n- in which n is 2, 3 or 4, more preferably those in which A is -CH2CH2-, -CH2CH2CH2-, -IHcH2 or c~2l~

and most preferably those in which A is -CH2CH2-, and wherein either (a) R1 and R8 taken together represent ~C~2 C
~ 3 or (b) ~8 is hydrogen and Rl represents hydrogen, CH3CH2- --O~E

C3 ~C C~3C3 3 C~3 Particularly preferred are the compounds wherein R8 is hydrogen and Rl is ~H
CH3~H-especially compounds having the absolute configuration 5R, 6S, 8R.
A still more preferred embodiment ~f the present invention comprises preparation of compounds of Formula I
wherein ~6 I -N~
represents /

.. ..... ....... _ .; .. . ....

' , CH3 ~ S-~0 , ~ ~ N-CH3 , ~ N

CH ~ ~ ~

Within ~his preferred subclass, the preferred compounds are those wherein A is -ICHCH2- , ~ , 21 or CH3 ~ CH3 -(CH2)n- in which A is 2, 3 or 4, more preferably those in which A is -CH2CH2-, -CH2CH2CH2-, -C~CH2- , ~ or -CH2CH- , and CH3 ~ C 3 most preferably those in which A is -CH2C~2-, and where~n eithe~
(a) Rl and R8 taken together represent ..
~OC~
C~
C~3 or (b) R8 is hydroge~ and Rl repres~n.s hydrogen, C~3C~2-, C~3~ ~ C~3 qH
~ C~ or C~3C~
CR3 . C~3 Pa~~icularly preferred a-e the com?ounds whe~ei~ ~8 is hydrogen and ~1 is C~3C~- , especially compounds having '~he absolute configuration 5R, 6S, 8R.
A still more preferred embodLment o~ the present inYen~;on comprises preparation of compound.s of Formula I wherein . ~ ~6 ~ _.
-N
\~ ~ .

rep_esen.s '~J ' . .

in which Y is hydrogen, Cl-C6 aIkyl, hyd,oxy, -S-Cl-C6 alkyl, carboxyl, carbamoyl, chloro, bromo, iodo, fluoro or phenyl.
Uithin this preferred subclas~, the p~eferred compounds ære those wherein ~ is -(C~2)~ in which n is 2, 3 or 4, ~ost pre-erably those in which A is -C~2~..2- and wherein ei,her (a) Rl æna R8 taXen togethe~ represent .
~C~2 ~ 3 or ~b) R8 is hydrogen 2nd Rl represents hydrogen, C~3C~2-, c~3 C~
C~_ , ~C- or C~3C~-C~3 C~3 Particula~ly preferred are the compounds wherein o8 is hyd~oge~
and ~1 is ' CH3C~ , i especially compounds having the 2b501ute configuhation 5~ 65 _ .

.

.~ :

,:

. .

~ *

A most preferred embodiment of ~.he present invention comprises preparation of compounds of Formula I wherein Rl 6~ ,.
-N

represen,s .. Wi~hin ~his pre erred subcl~ss, the preferred com~ounds ar2 ~,hose wherein A i5 ~(C~2)n i~ which n is 2, 3 or 4, most preferably those in which A is -C$2C~2- and whe ein either (à) Rl ~nd R8 t~ken together represent ~OCE~ z ~C
. C~3 or (~) R8 is hydrogen ænd Rl re~resents hydrogen, C~3C~2-, 3~ C~
C~- ~ C- o~ ~3C~-C~3 C~3 ~ ~ Particularly preferred are the compounds whexein R8 is hyd ogen ¦ and Rl is 0 C~3C~ ~
especially compounds having the absolute con,igura.ion 5R, 6S, . ., .` . , . ~ ;, . , ?~i~L
- 49 ~

A most preferred embodiment of the present invention comprises preparation of the compounds of the formula '- ~ ---S-C~.2CH2 O oOR2 wberein ~ ~ represents `C~ C~ ~' (both ~- and B~
di2stereoisomers) -~ N-C~3 or ~ N~/C~3 and R2 is hydrogen, an anionic charge or a conventional readily removable carboxyl protecting group, providing that when R2 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 or S configuration and the resulting isomers as well as epimeric mixtures thereof are encompassed by the present invention.

. ~, ~ . . , -,, :..... . ~ . ~ - . . . , , "

The process of the present invention utilizes the intermediate of the formula 1 ~ b ~

O COOR
IV
which has been disclosed, for example, in European Patent Application 38,~69 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, trifluoromethanesulronyloxy, diphenoxyphosphinyloxy or di(tri-chloroethoxy)phosphinyloxy. The preferred leaving group is diphenoxyphosphinyloxy Intermediates of Formula IV are generally ~ormed ln situ by reacting an intermediate of the formula R~ o O ~ N COOR

III
wherein Rl, R8 and R2 are as defined above with a suitable acylating agent R -L. The preferred intermediate IV where L
is diphenoxyphosphinyloxy may be prepared by reacting keto ester ~III in an inert organic $olvent such as methylene chloride, acetonitrile or dimethylformamide wlth about an equimolar amount of diphenyl chlorophosphate in the presence of a base such as diisopropylethylamine, triethylamine, 4-dimekhylaminopyridine or the like at a temperature of from about ~20C to +40C, most preferably at about 0C. ;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. : -.

, ~

. - :

In the prPsent process, carhapenem intermediate IV
is reacted with a quaternary amine thiol compound of the formula xe VII
wherein A is cyclopentylene, cyclohexylene or C2-C6 alkylene optionally substituted by one or more C1-C4 alkyl groups, most preferably cyclopentylene, cyclohexylene or :---C
` ~~ lll l13 in whieh R10, R11, R12 and R13 are each independently hydrogen or C1-C4 alkyl, X~ is a counter anion associated with a strong acid such as Cl-, Br~, CH3S03-, CF3S03- or C~3 ~ 3 . ~nd R14 is a quaternized nitrogen-containing aromatic or non-aromatic heterocycle as defined above. The reaction is earried out in an inert solvent sueh as acetonitrile, acetonitrile-dimethylformamide, tetrahydrofuran, tetrahydorfuran-H20, acetonitrile-H20 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 sueh as diisopropylethylamine, 1,8-diazabieyelo-[5.4.0]undec-7-ene, l,5-diazabicyclo[4.3.0]non-5-ene or tri~Cl-C4)alkylamine sueh as triethylamine, tributylamine or X~

~8~

tripropylamine is employed. Reaction of intermediate IV
with thiol VII may be carried out over a wide temperature range, e.g. -15C up to room temperature, but is preferably done at a temperature in the range of from about -15C to +15C, most preEerably at around 0C.
The carbapenem product produced by reaction of the quaternary amine thiol VII with intermediate IV will have a counter anion associated with it [e.g.(C6H5O)2PO2~, Cl or the anion associated with the quaternary thiol] which may at this stage be substituted by a different counter anion, e.g. one which 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 carboxyl protecting group R2 of Compound I may be optionally removed by conventional procedure such as solvolysis, chemical 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-aqueous dipotassium hydrogen phosphate-isopropanol 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 50C for from about 0.24 to 4 hours. When R2 is a group such as o-nitrobenzyl, photolysis may also be used for deblocking. Protecting groups such as 2,2,2-trichloroethyl may be removed by mild zinc reduction.

~r - 52(a) -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' where R2 is a physiologically hydrolyzable ester such as acetoxymethyl, phthalidyl, indanyl, pivaloyloxymethyl, methoxymethyl, etc. may be administered directly to the host without de-blocking since such esters are hydrolyzed n vivo under physiological conditions.
It will be understood that where the R1 andtor R8 substituent or the quaternized nucleophile R14 attached to substituent ~ contain a functional group which might interfere with the intended course of reaction, such group may be protected by a conventional blocking yroup and then subsequently de-blocked to regenerate the desired functional group. Suitable blocking groups and procedures for introducing and removing such groups are well know to those ; ~ ., i - . . - ~ , '. `: '. ~ : ~ . , ~ ~ , - : . ~ . :

` _ 53 _ sXilled in the art.

As in the case of o~her ~-lactam antibiotics, com?o7m ~s of general Formula I may be converted by known proceduIes to pharmaceutically acceptable salts which, for purposes of the present invention, are substantially equivalent ~o the non-salted compounds. Thus, for example, one may dissolve a compound o.
Formula I wherein R2 is an anionic charge in a suitable inert solvent and then add an equivalent of a pharmaceutically accep,aDle 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 sroups are present in the compound of ~ormula I, ~harmaceutically acceptable base addition salts and acid addition salts may be similarly oreoared by known methods.

A compound of Formula I where R2 is ~ydrogen or an anionic charge, or a pharmaceu~ically acceptable salt thereof may also be converted by conventional procedures to a corresponding compound where R2 is a physiologically hydrolyzable ester grouo, or a compound of Formula I wherein R2 is a conven~ional carboxyl pro~ecting group may be converted to the corresponding compound where R2 is hydrogen, an anionic charge or a physiologically hydrolyzable ester grou~, or a pharmaceutically acceptable salt thereof.

Certain of the thiol intermediates of ~orm~la VII may be prepared, for example, by reacting a sulfide of the formula ; ~ or VIIIa VIIIb Ri 7~ C--R 12 Rll R13 _.
VIIIc wherein R10, Rll, R12 and R13 are each lndependently hydrogen or Cl-C4 alkyl with a heteroaromatic amine (as defined above) of the formula ~ . ~ ' ~' ''' ' , ~ .

~ 54 --or a non-aroma~ic heterocyclic amine (as defi~ed above) of the formula \ f ~
N J

and a strong acid. The reaction may be carried out in ~he presence or absence of an inert organic solvent which is prerer-ably a non-polar organic solvent such as methylene chloride, ben2ene, xylene, toluene or the liXe. Where tne am~ne a~d sulfide reagen~s are liquids or where a solid amine is soluble in a liquid sulfide reagent, it is prefe_red to carry out ~he 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, methanesulfonic, p-toluenesulfonic, tri~luoromethanesulfonic, etc.

Formation of the quaternary amine thiol intermediate VII may be carried out at a temperature in ~he range of from about -20DC to about lOODC. Preferred te~peratures are ge~erally in the range of about 50-70DC.

The su~fide reagent, aromatic amine and acid are preferably employed so that the sulfide and acid are used ~n approxLmately equimolar amounts wi~th the amine being use~ ln excess, e.g. two to three moles of amine per mole of sulfide or acid.

The quaternary ~mine thiol intermediate will have a counter anion assoaiated with it which wiIl be determined by the particular acid employed. It is, of course,~ possible to --substitute at this point a different counter anion by conventi~nal procedures for use in the subsequent reaction with carbapenem intermediate IV.

' The carbapenem derivatives of general Formula I
where R2 is hydrogen, an anionic charge or a physiologically hydrolyzable carboxyl protecting group, or the pharmaceutically acceptable salts thereof, are potent antibiotics active against various gram-positive and gram-negative bacteria and they may be used, for example, as animal feed additives for promotion of growth, as preservatives in food, as bacteriocides 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 dental 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, topic 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 may 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.

~1 ' .
.

The dosage to be administered depends to a large ex,ent on the particular compound being used, the pax,icular composition rormulated, the route of ad~nistration, the nature and condition of the host and the particular situs and organism beins t-eated.
Selection of the particular pre~erred dosage and route of application, then, i5 left to the discretion of the therapist.
In general, however, the compounds may be administered parenterally ox orally to mamm21ian hosts in an amo~nt of from about 5 to 200 mg/kg/day. Ad~inistration is generally carried out in divided doses, e.g. three to four times 2 day.

The following examples illustrate but do not limit ~he scope OL =he present inventicn.

~ ~ :

:

:

.

.- .
: - ~ ~` ' ` '- ' ' `

:~

Exam~le 1 Preparation of 3-(2-~1-pyridinium~ethYlthio~-6 ~ ( R ) -hydroxyethyl)-7-oxo-1-azabicyclo(3.2.0)-hept-2-ene-2-carboxylate A. 1-(2-mercaptoethyl)pyridinium methanesulfonate t ~ ~. 55~C, 1- ~

To a suspension of pyridinium methanesulfonate in pyridine prepared by the dropwise addition of methanesulfonic acid (1.95 mL, 0.03 mol) to pyridine (8.0 mL, 0.099 mol) with cooling, was added ethylene sulfide (1.96 mL, 0.033 mol). The resulting mixture was stirred at 55C for 16 h and concentrated under reduced pressure to a thick syrup which was mixed with few mL of water.
The solution was poured on top of a column (40 x 16 cm) of ~-"Bondapak"* C-18 which was eluted with water.
Lyophilization of the appropriate fractions gave a colorless syrup 6.5 g (91%), ir (film.)~max: 2300-2600 (br, SH), 1635 (pyridinium), 1490, 1200 (sulfonate), 1068, 1060, 1045, 791, 780 cm 1, lHmr (DMSO-d6) ~:2.32 (3H, s, CH3SO3-); 2.61, 2.70, 2.73, 2.82 (lH, B part of A2B system, SH), 3.07 (2H, m [with D2O, 3008 (2H, t, J=6.5 Hz~], CH2S), 4.76 (2H, t, J=6.5 Hz, CH2N+), 8.19 (2H, m, Hm of pyridinium), 8.6 (lH, m, Ho of pyridinium), 9.08 (2H, dd, J=6.8 Hz, J=1.4 Hz Ho of pyridinium), uv (H2O) ~maX:206 (65230), 258 (~3760) m~.

* Trademark ,~1 .'' ..

.' . ' ' ~4;~

METHOD A
B. 1-(2-mercaptoethyl)~yridinium chloride e~uti "S-l Cl An aqueous solution of crude l-(Z-mercaptoethyl)pyridinium methanesulfo~ate (9.4 g, 0.04 mol) was ~oured on top of column (2.5 x 41 cm) of "Permutit S l"* Cl-. The column was eluted with water at a rate of 0.5 mL per min and the appropriate fractions were combined and lyophilized giving a yellowish syrup 7.0 g (100%) which was used as it was for the next step, Hmr (D2O) ~:3.22 (2H, m, CH2S), ~.88 (m, CH2N+), 8.18 (2H, m; Hm of pyridinium), 8.7 (lH, m, Hp of pyridinium), 9.0 ppm (2H, m. Ho of pyridinium).
METHOD B
~ ~ ~S ~> Cl . .

To a precooled (ice bath) pyridine (5.6 mL, 70 mmol) was added pyridine hydrochloride (4.05 g, 35 mmol) and ethylene sulfide (2.1 mL, 35 mmol). The mixture was heated at 65C and stirred 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 compound (90-100%) which was used as such for the next step *Trademark C. Paranitrobenzvl 3-~2-(1-pyridinium)ethYlthiol-6~- r 1-(~)-hydroxyethyl]-7-oxo-1-azabicyclo(3.2.0~hept-2-ene-2-carboxylate chloride 1- ~E~ r)2 ~"~C 2~ tOP~) 3-- XS~ N~
C00?~3 4- ~ (i~)2 ~ S~
` ~O?N3 Cl A solution of p-nitrobenzyl 6~-[1-(R)-hydroxyethyl]-3,7-dioxo-1-azabicyclo (3.2.0) heptane-2-carboxylate (6.09 g, 17.5 mmol) in acetonitrile (20 mL) was cooled to +5OC under a nitrogen atmosphere and treated successively with diisopropylethylamine (3.65 mL, 21.0 mmol) and diphenyl chlorophosphate (4.34 mL, 21.0 mmol). The resulting mixture was stirred for 30 min at 5C, cooled to -5C and treated successively with a solution of crude 1-(2-mercaptoethyl)pyridinium chloride (4.3 g, 24 mmol) in N,N-dimethylformamide (1.0 mL)~and dropwise with diisopropylethylamine (3.65 mL, 21.0 mmol). The reaction mixture was stirred at 0C for 1 h, cooled t~ -30C and stirred for 15 min. more. ~he solid was filtered off and washed with cold (-30C ) acetonitrile 5.77 g (65%), ir ("Nujol")* vmax:3300 (OH), 1775 (c=o of ~-lactam~ 90 (c=o of PNB ester), 1630 (pyridinium), 1605 (phenyl of PNB ester), 1515 (N02), 1335 cm~l (N02), 1Hmr (DMSO-d6) :

.

' '. ' , ~8~i~

- 59(a) -~:1.17 (3H, d, J=6.1 Hz, CH3 CHOH), 3.2-3.75 (5H, H-4, H-6, CH2S), 3.75-4.5 (2H, H-5, CH3CHOH), 4.92 (2H, brt, J=6.5 Hz, CH2N+), 5.18 (lH, d, J=4.9 Hz, OH), 5.37 (center of ABq, Ja b=l4.2 Hz, CH2 of PNB), 7.69 (2H, d, J=8.7 Hz, Ho of PNB), 8.24 (d, Jk=8.7 Hz, Hm of PNB), 8.0-8.4 (4H, Hm of PNB, Hm * Trademark :
:

:

~ ~ :
:
~ ~ :
:~ :

.
:
..
:, ~ i,'' ~ ' ', "

' ' `, ~; , ' ' ..

of pyridinium), 8.66 (lH, m, Hp of pyridinium), 9.17 ~2H, brd, J=5.5 Hz, Ho of pyridinium). The filtrate and washing were combined and diluted with ether (150 mL).
The supernatant was decanted and the gum was dissolved in water (40 mL) containing enough acetonitrile to have a solution which was poured on top of a column (3 x 10 cm) of ~-"Bondapak"* C-lg. The column was eluted with 10%
acetonitrile - 90% water (150 Ml) and 50% acetonitrile -50% water (100 mL) mixtures. The appropriate fractions were combined and lyophilized after the acetonitrile has been removed under vacuum giving a yellowish powder. An NMR of it showed the presence of the title compound mixed with some p-nitro-benzyl 3-[2-(1-pyridinium)ethylthio]-6~-[1-(_)-hydroxyethyl]-7-oxo-1-azabicyclo(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 cm) of "Permutit"* S-lC1- with water. Lyophilization of the appropriate fractions gave 1.8 g (20%) of the title compound.
* Trademark ' ' .
'~ ~' : ' ','' ' ' ' ' - - : ' , ' ' :
. ' ' ' 3~

D. Paranitrobenzyl 3-~2-(1-pyridinium)ethylthiol-6~-rl-)-hydroxyethyl]-7-oxo-1-azabicyclo(3.2 O)hept-2-ene-2-carhoxylate diphenYlphos~hate 1~ ~E~ )2.
J ~ 2) C~O~2 O ~ N ~ 3) ~S

0~
J~s ~ -~,~ ~
~hO)2?o A solution of p-nitrobenzyl 6~[1-(R)-hydroxyethyl]-3,7-dioxo-1-azabicyclo(3.2.0)heptane-2-carboxylate (0.174 g, 0.50 mmol) in acetonitrile (2 mL) was cooled to 0C
under a nitrogen atmosphere and treated successively with diisopropylethylamine (0.105 mL, 0.60 mmol) and diphenyl chlorophosphate (0.124 mL, 0.60 mmol). The resulting solution was stirred for 30 min at 0C and treated successively with a solution of 1-(2-mercaptoe~hyl)pyridinium methanesulfonate (0.170 g, 0.72 mmol) in acetonitrile (0.6 mL) and diisopropylethylamine (0.105 mL, 0.60 mmol). The reaction mixture was stirred at 0C for 15 min, diluted wlth cold (0C) water (7 mL) and poured on top of a column (1.5 x 6.4 cm) of ~-"Bondapak*" C-18. The column was eluted with a mixture of acetonitrile (25%-50%) in water (75%-50~). The appropriate fractions were combined and lyophilize.d after the acetonitrile has been removed under vacuum giving a yellowish powder 0.33 g (92%), ir (KBr) ~max: 3600-3000 .;, .

: . `
.

~;

- 61a -(OH), 1765 (C=O of B-lactam), 1690 (C=O of PNB ester), 1625 (pyridinium), 1585 (phenyl), 1510 (NO2), 1330 (NO2), 885 cm~1 (NO2), * Trademark ' ~ ' : , ;

;

: '~ , ' , .
.: ' ' ' . ' ' ' ' ' - '' ' , : ' ', . .

~8~

Hmr (DMSO-d6) : 1.16 (3H, d, J=6.2 Hz CH3CHOH), 4.87 (2H, brt, J=6.6 Hz, CH2S), 5.37 (center of Abq, Ja b=14 3 Hz, CH2 of PNB), 6.7-7.5 (phenyl), 7.68 (d, J~8.8 Hz, Ho of PNB), 8.23 (d, J=8.8 Hz, Hm of PNB), 8.0-~.3 (m, Hm of pyridinium), ~.4-8.8 (lH, Hp of pyridinium), 9.09 (2H, dd, J=6.7 Hz, J=1.3 Hz, Ho of pyridinium).

E. 3-~2-(1-pyridinium~ethylthio]-6~-~1-(R)-hydroxyethyl]-7-oxo-1-azabicyclot3.2.0)hept-2-ene-2-` carboxylate M

~ s N~
O N ~ ~-:~, E~-C00~3 t?~O~2~o O~;S ~ .., To a solution of p-nitrobenzyl 3-[2-(1-pyridinium)ethylthio]-6~-[1-(R)-hydroxyethyl]-7-oxo-1-azabicyclo (3.2.0) hept-2-3n3-2-carboxylate diphenylphosphate (0.16 g, 0.22 mmol) in wet tetrahydrofuran (10 mL) was added ether (10 mL), potassium phosphate monobasic-sodium hydroxide buffer pH
7.4 (16 mL, 0.05 M) and 10% palladium on charcoal (0.16 g). The resulting mixture was hydrogenated under 40 psi for 1 h at 25C. The two phases were separated and the organic phase was extracted with water (2 x 3 mL). The aqueous solutions were combined, washed with ether (2 x 10 mL) and poured on top of a column (1.5 x 6.2 cm) of ~-. : ' . ., , ~8~3;;~

- 62a -"Bondapak"* C-18 after the traces of organic solvents have been removed * Trademark .

.

:

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'.
:, ~2~

under vacuum. Elution of the column with water gave after lyophilization of the appropriate fractions a yellowish powder 0.062 g (84%), ir (KBr) vmaX : 3700-3000 (OH), 1755 (C=O of ~-lactam), 1630 (pyridinium), 1590 cm~l (carboxylate), lHmr (D2O): 1.22 (3H, d, J=6.4 Hz, C_3CHOH), 2.92 (d, J=9.1 Hæ, H-4), 2.97 (d, J=9.1 Hz, H-4), 3.20 (dd, J=2.5 Hz, J=6.1 Hz, H-6), 3.44 (t, J=6.0 Hz, CH2S), 3.93 (dd, J=9.1 Hz, J=2.5 Hz, H-5), 4.82 (t, J=6.0 Hz, CH2N+), 8.04 (m, Hm of pyridinium), 8.5 (m, Hp of pyridinium), 8.82 (dd, J=3.2 Hz, J=1.1 Hz, Ho of pyridinium), uv (H2O) max : 259 (~5800), 296 (~7030) m~
T~=13.5 h (measured at a concentration of 10 4 M in phosphate buffer pH 7.4 at 36.8C).

.
`:
' Method B

0~
~S~N~ ~
o ~u~_er 7 2 ~OO~

'J"~ ~ N~

COO ' .

-To a solution of-p-nitrobenzyl 3-[2-(1-pyridinium)ethylthio]-6~[1-(R)-hydroxyethyl]-7-oxo-1-azabicyclo (3.2.0) hept-2-ene-2-carboxylate chloride (5.77 g, 11.4 mmol) in potassium phosphate monobasic-~odium hydroxide buffer (170 mL, 0.2 M, pH 7.22) was added tetrahydrofuran (30 mL), ether (30 mL) and 10~
palladium on charcoal (5.7 g). The resulting mixture was hydrogenated at 22C under 40 psi for 1 h and filtered on a "Celite"* pad. The pad was washed with water t2 x 15 mL~. The filtrate and washings were combined and diluted with ether (100 mL). The aqueous phase was separated, washed with ether (3 x 100 mL) and poured on top of a column (4.5 x 20 cm) of ~-"Bondapak"* C-18 after the organic solvents have been removed under vacuum. Elution of the column with water followed by a mixture of 1%
acetonitrile in water gave after lyophilization of the appropriate fractions 2.48 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.

* Trademark X

~;.

Example 2 Preparation of 3-~2-(1-(3.5-dimethy~YridiniumL
ethylthiol]-6~ [1- (R) -hydroxyethyl]-7-ox~
azabicyclo(3.2.0!hept-2-ene-2-carboxylate ~-- C~3 ~ S ~ ~
COO C~3 A. 1-(2-merca~toethyl)-3 5-dimethylpyridinium ~ethanesulfonate 1 N~ I MsO~ S ~ ~

To a suspension of 3,5-lutidinium methanesulfonate in 3,6-lutidine prepared by the addition of methanesulfonic acid (0.65 mL, 0.010 mol) to cold 3,5-lutidine (2.51 mL, 0.022 mol) was added ethylene sulfide (0.655 mL, 0~011 mol). The resulting mixture was stirred under a nitrogen atmosphere at 55C for 24 h, cooled to 23C and diluted with water (5 mL) and ether t5 mL). The organic layer was separated and the aqueous solution was washed with ether (6 x 4.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 ~-"Bondapak"* C-18. The column was eluted with water and lyophilization of the * Tradem~rk ~7 , , appropriate fractions gave a colourless syrup 2.4 g (91~; ir (film) VmaX:2520 (SH), 1628 (pyridinium), 1600, 1495, 1325, 1305, 1283, 1200 (sulfonate), 1040, 938, 765, 680 cm~1, lHmr (DMSO d6) ~:2.31 (3H, s, CH3SO3-), 2.47 (6H, s, CH3 on pyridinium), 2.57, 2.66, 2.69, 2.78 (lH, B part of A2B system, SH), 3.06 (2H, m [with D2O added (2H, t, J-6.5 Hz)~, CH2S), 4.65 (2H, t, J=6.5 Hz, CH2N+), 8.34 (lH, s, Hp of pyridinium), 8.79 ~2H, s, Ho of pyridinium); uv (H2O) ~max 271 (~4 m~. Anal. calcd. for CloH17NO3S2 0.5H2O: C 44 6.66, N 5.14, S 23.54; found: C 44.26, H 6.49, N 5.17, S
24.18.

B. Paranitrobenzyl 3- r 2~ (3 5-dimethylpyridinium))ethylthio]-6~k-~1-(~)-hvdroxyethyll-7-oxo-1-azabicyclo(3.2.0)he~t-2-ene-2-carboxylate diphenylphosphate 0~ t(~?r)2 'o~y ~_~
~.~ 2J Cl~OPb)2 ~~S ~ ~>

O f ~_~ o~ N~
CO~?N3 3) ~ ~ N ~ M~0 \CCo~ R

4~ ~E~ r)2 (?~0)2~o To a cold (0C; solution of p-nitrobenzyl 6~-(1-(R)-hydroxyethyl)-3,7-dioxo-1-azabicyclo(3.2.0)heptane-2-carboxylate (0.523 g, 1.50 mmol) in acetonitrile (6.0 mL) kept under a nitrogen atmosphere was added diisopropylethylamine (0.314 mL, 1.8 mmol) followed by diphenyl chlorophosphate (0.373 mL, 1.8 mmol). The reaction mixture was stirred for 30 min and treated with ~2~3~

- 66a -a solution of 1-(2-mercaptoethyl)-3,5-dimethylpyridinium methanesulfonate (0.493 g, 1.87 mmol) in acetonitrile ~' , - ' : ' . ~ - ;
~. . .
: . . ,' ,.

(l.9 mL) followed by diisopropylethylamine (0.314 mL, 1.8 mmol). The reaction mixture was stirred at 0c for lh diluted with cold (0c) water (26 mL) and poured on top of a column (7.0 x 3.5 cm) of ~-I'Bondapak'' C-18. Elution of the column with 25-50% acetonitrile - 75-50% water mixture gave after lyophilization of the appropriake fractions 1.01 g (90%) of the title compound as yellowish powder, ir (KBr) ~max : 3700-3100 (OH), 1778 (C=O of ~-lactam), 1700 (C=O of PNB ester), 1635 (pyridinium), 1595 (phenyl), 1521 (NO2), 1335 (NO2), 895 cm~l (NO2), 1Hmr (DMSO d6) ~:1.16 (3H, d, J=6.1 Hz, CH3CHOH), 2.43 (s, CH3 on pyridinium), 4.75 (2H, m, CH2N+), 5.38 (center of ABq, Ja b=14-3 Hz, CH2 of PNB), 6.6-7.5 (lOH, m, phenyl), 7.70 (2H, d, J=8.7 Hz, Ho of PNB), 8.0-8.5 (3H, m, Hp of pyridinium, Hm of PNB), 8.82 (2H, s, Ho of pyridninium), uv (H2O) ~max : 270 (~11570), 306 (~7343) m~. anal.
calcd. for C37H38N310SP-H2O C 58 4.18; found: C 57.98, H 5.05, N 5.22, S 4.34 ;_ .
.

, 3'~

C. 3-[2-(1-(3,5-dimethylpyridinium))ethvlthiol-6~- r 1-(~)-hvdroxyethyll-7-oxo-1-azabicvclo(3.~.0) he~t-2-ene-2-carboxylate O;~
10~ ~d/C, ~2 S ~ ~ ~ ethe-, bu~-e-C~O NB (~h~) ~o~

To a solution of p-nitrobenzyl 3-[2-(1-(3,5-dimethylpyridinium))ethylthio]-~-[1-(R)-hydroxyethyl]-7-oxo-l-azabicyclo (3.2.0) hept-2-ene-2-carboxylate diphenylphosphate (0.600 g, 0.80 mmol) in wet tetrahydrofuran (36mL) was added ether (36 mL), potassium phosphate monobasicsodium hydroxide buffer (0.05M, pH
7.4, 44mL) and 10% palladium on charcoal (0.60 g). The resulting mixture was hydrogenated under 40 psi at 23C
for 1.25 h. The organic layer was separated and extracted with buffer (2 x 5 mL). Water layers were combined, filtered through a "Celite" pad, washed with ether (40 mL, pumpe~ to eliminate traces of organic solvents and poured on top of a column (2.5 x 10.0 cm) of ~-"Bondapak" C-18. Elution of the column with water and lyophilization of the appropriate fractions gave the title compound 0.186 g (64%) as a yellowish powder, ir (XBr).~max:3700-3100 (OH), 1760 (C=O of B-lactam), 1595 cm~l (carboxylate), 1Hmr (D2O) ~ 1.21 (3H, d, J=6.3 Hz, .

, CH3CHOH), 2.45 (6H, s, CH3 on pyridinium), 2.81 (d, J-9 2 Hz, H-4), 2.96 (d, J-9.2 Hz, H-4), 3.22 (dd, J-2.6 Hz, J=6.2 Hz, H-6), 3.40 (t, J-6.2 Hz, CH2S), 3.84 (dd, J-9.2 Hz, J-2.6 Hz, H-5), 4.15 (m, CH3CHOH), 4.71(t, J=6.2 Hz, CH2N+), 8.21 (lH, s, Hp of pyridinium), 8.46 (2H, s, Ho of pyridinium), uv (H2O) ~max 279 ( 8345), m, [~]~ + 40.7 (c 0.53, H2O), T~ ~ 16.9 h (measured at a concentration of 10-4 M in phosphate buffer pH 7.4 at 36.8C).
Example 3 Preparation of (5~,6~)-3-[[2-(3-hydroxymethylpyrid-inio)ethyl]thiol-6-[~ )-hydroxyethyl]-7-oxo-1-azabicyclo[3.2.01hept-2-ene-2-carboxylate 0~ .
J ~ ~ ~2~

A. 3-HYdroxymethyl-1-(2-merca~toethvl)pyridinium trifluoromethanesulfonate + OE3So ~2~
XS~32CH2N ~>

~ ~3S03~

Trifluromethanesulfonic acid (1.327 mL, 0.015 mol) was added dropwise to 3-pyridinemethanol (2.gl mL, 0.030 mol), followed by ethylene sulfide (0.89 mL, 0.015 mol).
The resulting homogeneous mixture was heated (oil bath) at 50-70C under N2 for 20 h. The reaction mixture was then taken up in H2O (15 mL) and extracted with CH2Cl2 (5 x 5 mL). The aqueous phase was concentrated in vacuo and then applied to C18 reverse-phase column. Elution with H2O followed by evaporation of the rele~ant fractions gave a pale yellow oil. This material was rechromatographed to give a nearly colourless oil. After drying in vacuo (P2O5) this afforded the product (4~50 g, 94%) as a viscous oil. ir (film) ~m~x:3450 (s, OH), 2560 (w, SH) cm~l; lHmr (d6-acetone) ~:9.10-8.05 (m, 4H, aromatic), 5.01 (t, J=5.5 Hz, 2H, N-CH2) 4.93 (s, 2H, -CH2OH), 4.43 (br S, lH, -OH), 3.43-3.18 (m, 2H, S-CH2), 2.34-2.l0 (m, lH, SH).

:: :
`,, ' ' ' ' ': . ` : ` ' .
: . . . ~ , .
- , ~ . . . . . . .
.
.
'~ ' ': ' ; ` . ',": ,, ', B. p-Nitrobenzyl (5B 6~)-3-~2-(3-hYdroxymeth~
pyridinio~ethyl thiol-6-[1-(B)-hydroxyethyll~7-oxo-azabicyclo[3.2.0]hept-2-ene-2-carpoxylate diphenylphosphate d~ ~-`<
CO ~3 ~2 '",cr~s~N~
,_ p To a solution of p-nitrobenzyl (5R, 6S)-6-[1-(R)-hydroxyethyl]-3,7-dioxo-1-azabicyclo[3.2.0]heptane-2-carboxylate (0.174 g, 0.50 mmol) in 2 mL of dry acetonitrile was added diisopropylethalimine (0.096 mL, 0.55 mmol) at 0C under N2. Diphenyl chlorophosphate (0.114 mL, 0.55 mmol was then added dropwise and the reaction mixture was stirred at 0C for 30 min. A
solution of 3-hydroxymethyl-1-(2-mercaptoethyl)pyridinium trifluoromethanesulfonate (0.223 g, 0.70 mmol) in 0.50 mL
of acetonitrile was then added, followed by diisopropylethylamine (0.122 mL, 0.70 mmol). After being kept at 0C for 30 min the reaction mixture was concentrated in vacuo and the residual yellow gum was taken up in H2O (enough acetonitrile was added to aid in dissolving the gum). This solution was applied to a C18 reverse-phase column which was eluted with 15%
acetonitrile-H2O. Lyophilization of the relevant fractions afforded the product (0~305 g, 81%) as a beige-coloured solid. ir (KRr) ~max:3420 (br, OH), 1775 (B-lactam CO), 1695 (-CO2PNB) cm~1; 1Hmr (d6-acetone) - 71(a) -~:9.44-7.72 ~m, 8H, aromatic), 7.22-6.91 (m, lOH, diphenylphosphate), 5~53, 5.27 (ABq, J=14Hz, 2H, benzylic), 5.04 (t, J=7.4 Hz, 2H, N-CH2), 4.75 (s, 2H, CH20H), 4.5-3.1 (m, 8H), 1.21 (d, J=6.3 Hz, 3H, CHMe).

~ -' C. (5B~6~)-3-~2-(3-hydrox~methylpYridinio)ethYll thiol-6-[1-(~-hydroxyethyll-7-oxo-1-azabicvclo-~3.2.0]hept-2-ene-2-carboxylate C~
o~ 4~ 2 ~ ~ ~ 2~ ~
0~ C32O~
;

To a solution of p-nitrobenzyl (5R,6S)-3-[2-(3-hydroxymethylpyridinio)ethyl thio]-6-[1-tR)-hydroxyethyl]-7-oxo-1-azabicyclo[3.2.0]-hept-2-ene-2-carboxylate diphenylphosphate (0.145 g, 0.194 mmol) in 10 mL of THF containing 5 drops of H2O, was added 6.0 mL of phosphate buffer (0.05 M, pH 7.4), 0.145 g of I0%
palladium-on-charcoal and 10 mL of ether. The mixture was hydrogenated (Parr) at 40 psi of 1 h and then filtered through a pad of "Celite". The filter cake was washed with a little H2O and ether and the aqueous phase was separated and extracted with ether (3x). The aqueous solution was then cooled at 0C and the pH was adjusted to 7.0 with pH 7.4 buffer. After removing residual volatiles in vac~o the aqueous solution was applied to a C18 reverse-phase column which was eluted with H2O.
Lyophilization:of the relevant fractions gave the product (36 mg, 51%) as a light yellow solid. Further purification by reverse-phase hplc gave the pure product (31 mg, 41~) as a solid- ir (KBr)~maX:330o (br~ OH)~
1755 (B-lactam CO)j 1590 (-CO2-) cm~1; lHnmr (D2O) :. :

' ~8~3~

- 72(a) -~:8.78-7.94 (m, 4H, aromatic), 4.83 ~t, J-6.0 Hz, 2H, N-CH2), 4.83 (s, 2H, CH2OH), ;

:~ .

: ~ ~
: ` ~' , '' ' .' ' - ;. ' ~

32~

4.16 (d of q, J=J'=6.2 Hz, lH, H-l'), 3.9B (d, of t, J=9.1 Hz, J'=2.6 Xz, lH, X-5), 3.75-3.20 (m, 3X), 3.20-2.65 (m, 2X), 1.22 (d, ~=6.4 Hz, 3X, CHMe); ~v (H20) ~ : 294 (~7614), 266 (~6936) n~; t~ (pH 7.4, 36.8C) 14.0 h.

Exzm~le 4 Preparation of (52,6S)-3-[2-(4-hydroxymethylpyridinio)ethyl-thio]-6-[1-(R)-hydroxye~hyl]-7-oxo-1-azabicyclo[3.2.0]hept-.
2-ene-2-carboxylate .

0~ . . ' ', .
'1~S~2C~ , , 3~

A. 4-Hydroxymethyl-1-(2-mercaptoethvl)pYridinium trifluoromethanesulfonate / ~ C3 0~ + -C~3SO3~

32Q ~ 3203 C~ , .

To a solution of 4-pyridinemethanol (1.635 g, 0.015 mol) in 10 mL of CH2Cl2, at 0C under N2, was added dropwise trifluoromethanesulfonic acid (1.327 mL, 0.015 mol~. A yellow-brown oil rapidly separated out. An additional equivalent of 4-pyridinemethanol (1.635 g, 0.015 mol) was added to this mixture and the solvent was removed under reduced pressure to give an oil. To this oil was added ethylene sulfide (0.891 mL, 0.015 mol) and the resulting homogeneous mixture was heated (oil bath) at about 60C for 3 h. The reaction mixture was then taken up in 15 mL of H2O and the aqueous solution was washed with CH2Cl2~(5 x 5 mL3. After removing residual organic solvent in vacuo the aqueous solution was applied to a Cl8 reverse-phase column. Elution with H2O and subsequent evaporation of the relevant fractions afforded an oil which was further dried in vacuo over P2O5 to give the product (4.64 g, 97%) as a colourless oil. ir (film) max:3455 (s, OH), 2565, (w, SH) cm~1; 1Hnmr~(d6~acetone) ~:9.07, 8.18 (ABq, 3=6.8 Hz, 4H, aromatic), 5.03~(s, 2H, CH2OH), 4.96 (t, J=6.5 Hz, 2H, N-CH2), 4.09 (br s, lH, -OH), 3.5-3.1 (m, 2H, S-CH2~, 2.25 (brs, lH, -SH).

~ .

~ ' ' .
,~
. . .

37~
- 75 ~

B. p-Nitrobenzyl (5~!6~)-3-r2-(4-hYdroxymethYlPyrid-inio)ethyl thiol-6- r~ -hvdroxyethyll-7 oxo-1-aza-bicyclo r 3.2.0lhept-2-ene-2-carboxylate diphenylE?hosphate 0:~

0~ . -~
CO2 ~3 20=

~ 3 (~~2 ~

To a solution of p-nitrobenzyl (5R,6S)-6-[1-(R)-hydroxvethyl~-3,7-dioxo-1-azabicyclo[3.2.0]heptane-2-carboxylate (0.34~ g, 1.0 mmol) in 5 mL of dry acetonitrile, at 0C under N2, was added dropwise diisopropylethylamine ~0.191 mL, 1.1 mmol) followed by diphenyl chlorophosphate (0.228 mL, 1.1 mmol). The resulting golden-yellow solution was stirred at 0C for 40 min. To this solution was added a solution of 4-hydroxymethyl-l-(2-mercaptoethyl)pyridinium trifluoro-methanesulfonate (0.447 g, 1.4 mmol) in 1 mL of acetonitrile, followed by diisopropylethalamine (0.191 mL, 1.1 mmol). A reddish-black gum separated from the reaction mixture. After 20 min at 0C the reaction mixture was filtered and concentrated in vacuo. The residue was taken up in a minimum volume of acetonitrile-H2O (1:1) and applied to a C18 reverse-phase column.
Elution with 25% acetonitrile-H2O and subsequent lyophilization of the relevant fractions gave the product X~ . :

`
.

z~

- 75(a) -~0.353 g, 47%) as a cream-coloured solid. ir (XBr) maX:3240 (br, OH~, 1775 (B-lactam CO), 1695 (-CO2PNB)cm 1; lHnmr (d6-acetone) ~: 9.24-7.84 (m, 8H, aromatic), 7.4-6.9 (m, 10H, diphenylphosphate), 5.52, 5.24 (ABq, J-14 Hz, 2H, benzylic), 5.15 4.80 (m, 4H), 4.45-3.05 (m, 7H), 1.35 (d, J=6.6 Hz, 3H, CHMe).

.

' . ,~: ' , ...

.: , : .. , . . .

.

C. (5~,6~)-3-[2-(4-hydroxymethvl~vridinio)ethvl thiol-6-[1-(B)-hydroxyethyll-7-oxo-1-azabicvclo[3.2.olhept-2-ene-2-carboxylate J'~ ~ ~ 2~

C02~3 (~)2 0'~
~ r~ ~ ~ C

A mixture of p-nitrobenzyl (5R,6S)-3-[2-(4hydroxy-methylpyridinio)ethyl thio]-6-[1-(R)-hydroxyethyl]-7-oxo-1-azabicyclo-[3.2.0]hept-2-ene-2-carboxylate diphenylphosphate (0.348 g, 0.465 mmol) and 10%
palladium-on-charcoal (0.35 g) in 11 mL of phosphate buffer (0.05 M p~ 7.~), 5 mL of THF and 10 mL of ether was hydrogenated at 4~ psi for 1.25 h. The mixture was then filtered through a "Celite" pad and the aqueous phase was washed with ether (3 x). The pH of the aqueous solution was then adjusted to 7.0 using additional pH
7.4 buffer. After removing residual volatiles in vacuo the aqueous solution was applied to a C18 reverse-phase column. Elution with 2% acetonitrile-H2O and subsequent lyophilization gave a yellow-brown solid. This material was rechromatographed (Cl~ reverse-phase/H2O) to give the desired product (0.060 g, 36%) as a light yellow solid.
ir (KBr) ~max:3400 (br, OH), 1755 (B-lactam CO), 1590 (-CO2-) cm~1; 1Hnmr (D2O) ~: 8.73, 7.96 (ABq, J=6.8 Hz, 4H, aromatic), 4.93 (s, 2H, CH2OH), 4.77 (t, J=6.0 Hz, 2H, N-- 76 (a) -CH2), 4.15 (d of q, J=JI=6.3 Hz, lH, H-l'), 3.96 (d of t, J=9.2 Hz, Jl=2.6 Hz, lH, h-5), 3.65-3.20 (m, 3H), 3.13-2.62 (m, 2H), 1.21 (d, J-6.3 Hz , 3H, CHMe); uv (H20) ~maX:295 (~6880), 256 (~5595), 224 (~8111) nm; ~ (pH
7.4, 36.8C) 14.5 h ; :
." :
- ` ' `

~2~3~3;~

Exam~le 5 Preparation of 3-[2-(1-(2-methylPYridinium)) ethylthio]-6~-[1-(RL=~ydroxyethyll- -oxo-1-azabicyclo(3.2.0)hept-2-ene-carboxy]ate A. 1-(2-mercaptoethyl)-2-methylpyridinium methanesulfonate N~ 55~C, 21 h N~ .~30 To a suspension of 2-methylpyridinium methanesulfonate in 2-methylpyridine prepared by the addition of methanesulfonic acid (0.65 mL, 0.010 mol) to cold 2-methylpyridine (2.17 mL, 0.022 mol) was added ethylene sulfide (0.655 mL, 0.011 mol). The reaction mixture was stirred under a nitrogen atmosphere at 55C
for 21 h, cooled to 23C and diluted with water (5 mL).
The aqueous solution was washed with ether (6 x 4 mL) pumped to remove traces of organic solvents and poured on top of a column (2.5 x 10.0 cm) -... . ~ . ... .. -- . -, . ~ --- ~ , ~, ` .

- 7~ -of ~-"Bondapak~" C-18. The column was eluted with water and lyophilization of the appropriate fractions gave 2.13 g (85%) of the title compound, ir (film max:2520 (SH)~
1623 (pyridinium) 1574, 1512, 1485, 1412, 1195 (sulfonates), 1038 cm~1, 1Hmr (DMSO-d6 ~ D2O) ~:2.37 (3H, s, CH3SO3-), 2.83 (3H, s, CH3 on pyridinium), 3.09 (2H, J=6.9 Hz, CH2S), 4.71 (2H, t, J-6.9 Hz, CH2N+), 7.93 (2H, m, Hm of pyridinium), 8.44 (lH, m, Hp of pyridinium), 8 89 (lH, m, Ho of pyridinium), uv (H2O) ~max 266 (~3550) m~.

B. Paranitrobenævl 3-[2-(1-(2-methylpyridinium))-ethylthio)-6~-[1-(R)-hydroxyethyl]-7-oxo-1-azabicyclo(3 2.0)hept-2-ene-2-carboxylate di~henylphosphate ~, ~ 2~ Cl~(O~'O 2 C00~3 3) ~ ~5 4) ~Et(i~r)2 i~
C00~
(PhOt2Po To a cold (0C) solution of p-nitrobenzyl 6~-[1-(R)-hydroxyethyl]-3,7-dioxo-1-azabicyclo(3.2.0)heptane-2-carboxylate (0.523 g, 1.50 mmol) in acetonitrile (6 mL) kept under a nitrogen atmosphere was added diisopropylethylamine t0.314 mL, 1.80 mmol) followed by diphenyl chlorophosphate (0.373 mL, 1.80 mmol). The ... .. _. . . . .. ~

- 78(a) -reaction mixture was stirred for 30 min at 0C and treated with solution of 1-(2-m~rcaptoethyl~-2-methylpyridinium methanesulfonate (0.530 g, 2.16 mmol) in acetonitrile (18 mL) followed by diisopropylethylamine ~0.314 mL, * Trademark . .

1.8 mmol). The reaction mixture was stirred at 0C for 1 h diluted with cold (0C) water (26 mL) and poured on top of a column (3.5 x 7.0 cm) of ~-"Bondapak"* C-18.
Elution of thee column with 25% acetonitrile ~ 75% water and with 50% acetonitrile - 50% water gave after lyophilization of the appropriate fractions 1.06 g, (96%) of the title compound as a yellowish powder, ir (KBr) ~max 3650-3100 (OH), 1770 (c=o of ~-lactam), 1695 and 1690 (C=O of PNB ester), 1630 (pyridinium), 1595 (phenyl), 1518 (NO2), 1335 (NO2), 890 cm~l (NO2), lHmr (DMSO, d6) ~: 1.15 (3H, d, J=6.1 Hz, CH3CHOH), 2.87 (s, CH3 on pyridinium), 3.6-4.4 (2H, m, H-5, CH3C_OH), 4.75 (2H, m, CH2N+), 5.37 (center of ABq, J=14 Hz, CH2 of PNB), 6.5-7.4 (10H~ m, phenyl), 7.70 (2H, d, J=8.8 Hz, Ho of PNB), 8.0 (2H, m, Hm of pyridinium), 8.24 (2H, d, J=8.8 Hz, Hm of PNB), 8.50 (lH, m, Hp of pyridinium), 8.95 (lH, brd, J=6.1 Hz, Ho of pyridinium), uv (H2O) :265 (E11990), 314 (~8020)m~

C. 3-~2-(1-(2-methylpyridinium)lethylthiol-6~ (B~-hvdroxyethyl~-7-oxo-1-azabicyclo(3.2.0)hept-2-ene-carboxylate OR

~,~ ~N~ --- ~ shér buSrer ~Coo?NB (P~O)~O

*Trademark , 3~

To a solution of p-nitrobenzyl 3-[2-(1-(2-methylpyridinium)) ethylthio]-6~-[1-(R)-hydroxyethyl]-7-oxo-1-azabicyclo(3.2.0)hept-2-ene-2-carboxylate diphenylphosphate (0.66 g, 0.90 mmol) in wet tetrahydrofuran (34 mL) was added ether (34 mL), potassium phosphate monobasic-sodium hydroxide buffer (0.15 M, 16.5 mL, pH 7.22) and 10% palladium on charcoal (0.66 g). The resulting mixture was hydrogenated under 40 psi at 23C for 1.25 h. The organic layer was separated and extracted with buffer (2 x 6 mL). Water layers were combined, filtered through a "Celite" pad, washed with ether (40 mL), pumped to eliminate traces of organic solvents and poured on top of a column (2.5 x 10 cm) of ~-"Bondapak"* C-18. Elution of the column with water and lyophilization of the appropriate fractions gave the title compound 0.098 g (31%) as a yellowish powder, ir (KBr) vmax: 3650-3100 (OH), 1755 (C=O of ~-lactam), 1630 (pyridinium), 1595 cm~1 (carboxylate), lHmr (D2O) S: 1.20 (3H, d, J=6.3 Hz, CH3CHOH), 2.83 (s, CH3 on pyridinium), 2.7-3.1 (5H, H-4, CH3 on pyridinium), 3.1-3.7 (3H, m, CH2S, H-6), 3.90 (dd, J=9.1 Hz, J-2.6 Hz, H-5), 3.1 (m, CH3CHOH), 4.78 (t, J=6.2 Hz, CH2N+), 7.8 (2H, m, Hm of pyridinium), 8.3 (lH, m, Hp of pyridinium), 8.65 (lH, m, Ho of pyridinium), uv (H2O) ~ max:268 (~9350), 296 (~8840) m~, [~]D23 +41 (C 0.5, H2O), ~ =
15.0 h (measured at a concentration of 10-4 M in phosphate buffer pH 7.4 at 36.8C)~

* Trademark .
- : ' ,. ... .
, . .
' ": ' ' ' Example 6 Preparation of ;~iL -(1-(4-methYlPYridinium~)-ethylthio]-6~-~1(~)-hydroxYethyl1-7-oxo-1-azabicy~lo(3.2.0)hept-2-ene-2-carboxYlate o~ ~ S~ N~3 -A. 1-t2-mercaptoethyl)-4-methylpyridinium methanesulfonate .sG~ ~ 55C, 24 h ~N~ M5 -To a suspension of 4-picolinium methanesulfonate in 4-picoline prepared by the addition of methanesulfonic acid (0.65 mL, 0.010 mol) to 4-picoline (2.14 mL, 0.022 mol) in cooling was added ethylene sulfide (0.655 mL, 0.011 mol). The reaction mixture was stirred under a nitrogen atmosphere at 55C for 24 h, cooled to 23C and diluted with water (5 mL) and ether (10 mL) . The organic layer was separated and the aqueous layer was washed with ether (5 x 5 mL) and applied on top of a column (2.5 x 10 cm) of ~-"Bondapak"* C-18 after traces of ether have been removed under reduced pressure. Elution of the column with 15~ acetonitrile 85% water mixture gave after lyophilization of the appropriate fractions a colorless * Trademark ' , ,, , ' : :

.

"' : ' syrup 2.66 g (100%), ir (film) vmax:2500 (SH~, 1540 (pyridinium), 1572, 1520, 1478, 1200 (sulfonate), 1040, 833 and 768 cm~l, 1Hmr (DMSO-d6) ~: 2.31 (3H, s, CH3S03-), 2.62 (s, CH3 on pyridinium), 2.2-2.9 (4H, SH, CH3 on pyridinium), 3.04 (2H, m, CH2S), 4.68 (2H, t, J=6.4 Hz, CH2N+), 8.01 (2H, d, J=6.6 Hz, Hm of pyridinium), 8.89 (2H, d, J=6.6 Hz, Ho of pyridinium), uv (H2O) ~max: 256 (~4100), 221 (~7544) m~.

B. 1-(2-mercaptoethyl)-4-methyl~yridinium p-toluenesulfonate N~ i 1~s~l ~_ 5' ,~N~ ~55 To a suspension of p-toluenesulfonic acid (1.72 g, 0.01 mol) in benzene (6.5 mL) was added 4-picoline (1.17 mL, 0.012 mol). The resulting mixture was stirred under a nitrogen atmosphere at 23C for 30 min, treated with ethylenesulfide (0.65 mL, 0.011 mol) and stirred at 75 for 24 h. More ethylenesulfide (0.65 mL, 0.011 mol) was added and the stirring was continued at 75~C for 24 h more. The reaction mixture was cooled to 23C and diluted with water (5 mL) and ether (8 mL). The aqueous layer was separated and washed with ether (3 x 8 mL).
The traces of organic solvents were removed under vacuum and the compound was chromatographed on ~-"Bondapak"* C-18 with water as eluting solvent to give 2.94 g (90%) of the title compound as a colorless syrup; ir (film) vmaX:
2510 (SH) 1640 (pyridinium), 1595, 1582, 1475, 1200 (sulfonate), 1031, 1010, 818 cm~1, lHmr * Trademark ' .

(DMSO, d6) ~: 2.29 (3H, s, CH3 on pyridinium), 2.61 15, CH3 Ph), 2.4-2.8 (4H, SH, CH3Ph), 3.03 (2H, m[addition of D2O gave a t, J=6.4 Hz, at 3.04], CH2S), 4.68 (2H, t, J=6.4 Hz, CH2N~), 7.11, 7.49 (4H, 2d, J=7.9 Hz, Phenyl), 8.00 (2H, d, J=6.5 Hz, Hm of pyridinium), 8.89 (2H, d, J=6.5 Hz, Ho of pyridinium), uv (H2O) ~max:256 (~4315), 222 (~17045) m~.

c. Paranitrobenzyl 3-[2-(1-4-methylpyridinium)~-ethylthiol-6~-~1-(~)-hydroxyethyl1-7 oxo-l-azabicyclo(3.2.0)hept-2-ene-2-carboxylate diphenylphosphate ~t (~3 2 2~ c1~(0~)2 I ~0 _ O ~XL~3 3) 0~
J"~ 2~

C~0~ ), Top a cold (0C) solution of p-nitrobenzyl 6~-[1-(R)-hydroxyethyl]-3 ! 7-dioxo-1-azabicyclo(3.2.0)heptane-2-carboxylate (0.522g, 1.5 mmol) in acetonitrile (6 mL) kept under a nitrogen atmosphere was added diisopropylethlamine (0.314 mL, 1.8 mmol) followed by diphenyl chlorophosphate (0.373 mL, 1.9 mmol). The reaction mixture was stirred for 45 min and treated dropwise with a solution of 1-(2-mercaptoethyl)-4-methylpyridinium methanesulfonate (0.539 g, 2.16 mmol) in ~_ , , -- 83(a~ -acetonitrile (1.8 mL) followed by diisopropylethylamine (0.314 mI" 1.8 mmol). The reaction mixture was stirred at 0C for 1 h, diluted with cold (0C) water (24 mL) and poured on top of a column .
, (2.5 x 8.5 cm) of ~-"Bondapak"*C-18. Elution of the column first with 25~ acetonitrile -75% water mixture (100 mL) then with 50% acetonitrile 50% water mixture (100 mL) afforded after lyophilization of the appropriate fractions 0.91 g (83%) of ~he title compound as a yellowish powder, ir (KBr~ vmaX:3700-2800 (OH), 1770 (c=o of ~-lactam), 1700 (c=o of PNB ester), 1640 (pyridinium), 1595 (phenyl), 1520 (NO2), 1340 (NO2), 890 cm~1 (NO2), Hmr (DMSO, d6) ~:1.16 (3H, d, J=6.2 Hz, CH3 CHOH), 2.61 (s, CH3 on pyridinium), 3.1-3.7 (3H, m, H-6, CH2S), 3.7-4.4 (2H, m, H-5, CH3CHOH), 4.79 (2H, brt, J=6.3 Hz, CH2N+), 5.17 (d, J=4.9 Hz, OH), 5.37 tcenter of ABq, J=14.1 Hz, CH2 of PNB), 6.7-7.4 (lOH, m, phenyl), 7.69 (2H, d, J=8.8 Hz, Ho of PNB), 8.00 (2H, d, J=6.5 Hz, Hm of pyridinium), 8.23 (2H, d, J=8.8 Hz, Hm of PNB), 8.92 (2H d J=6.5 Hz, Ho of pyridinium), uv (H2O ~maX:262 (~10835), 311 (~9670) m~. Anal. calcd. for -36H36-3QloSP 1 5 H2O: C 56-84, H 5.17, N 5. 52. S 4.21:
found: C 56.89 H 5.13, N 5.19. S 4.41.

D. 3- r 2-(l-(4-methylpyridinium))ethylthio]-6~- r 1- (R)-hydroxyethyl1-7-oxo-1-azabicvclo(3.2.0)hept-2-ene-2-carboxylate JD~ S~_~_~N ~ ~ 2 _~

~hO) ~O
CD5~3 2 0~
,J"~ ~
-C~

*Trademark ;_ 3~

To a solution of p-nitrobenzyl 3-[2-(1-(4-methylpyridinium))-ethylthio~-6~-[1-(R)-hydroxyethyl]-7-oxo-l-azabicyclo(3.2.0)hept-2-ene-2-carboxylate diphenylphosphate (0.587 g, 0.80 mmol) in wet tetrahydrofuran (30 mL) was added ether (30 mL) potassium phosphate mono basic-sodium hydroxide buffer (0O15 M, 14.7 mL, pH 7.22) and 10% palladium on charcoal (0.59 g).
The resulting mixture was hydrogenated under 40 psi at 23~C for 1.25 h. The organic layer was separated and extracted with the buffer (2 x 6 mL). The aqueous extracts were combined, filtered through a "Celite"* pad, washed with ether (3 x 20 mL), pumped to remove traces of organic solvents and poured on top of a column (2.5 x 10 cm) of ~-"Bondapak*" C-18. Elution of the column with water and lyophilization of the appropriate fractions gave 0.136 g (49%) of the title compound as a yellowish powd~r, ir (KBr) vmaX:37oo-3ooo (OH), 1770 (c=o of 8-lactam), 1642 (pyridinium) 1592 cm~1 (carboxylate), 1Hmr (D2O) ~:1.19 (3H, t, J=6.3 Hz, C_3CHOH), 2.59 (3H, s, CH3 on pyridinium), 2.84 (d, J=9.1 Hz,H-4~, 2,90 (d, J- 9.1 Hz, H-4), 3.0-3.6 (3H, m, CH2S, H-6), 3.86 (dd, J-9.1 Hz, J=2.6 Hz, H-5), 4.12 (m, CH3CHOH), 4.5-4.9 (CH2N+ masked by HOD), 7.80 (2H, d, J=6.6 Hz, Hm of pyridinium), 8.58 (2H, d, J=6.6 Hz, Ho of piperidinium), uv (H2O) ~maX:256 (~5510), 262 (~5360), 296 (~7050) m, [~]D23 +20.8 (C
0.48, H2O), ~=12.8 h (measured at a concentration of 10-4 M in a phosphate buffer pH 7.4 at 36.8C).

* Trademark ~ .
.

. .

Exam~le 7 Preparation of (5R) 3-[2~(4-methyl-thioPYridinio)-ethylthio~-(6~ ! - [ ( 1g~ oxyethyl~-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxYlate 0~ ' ' J ~ 5 ~ ~ C~3 ~2 A. 4-Methylthiopyridine*

N ~ H - ~ N ~ Me 4-Mercaptopyridine (5.55 g, 50.0 mmol; Aldrich*) was dissolved in boiling abs. EtOH(50 mL). The insoluble material was removed by filtration over "Celite"**. The filtrate was heated to xe-dissolve, and when it cooled to ca. 50C, methyl iodide (3.17 mL, 51.0 mmol; Aldrich) was added at once. The mixture was cooled to crystallize.
Filtration of the solid gave 6.77 g (26.7 mmol, y. 53.5%) of the title compound as the hydroiodide: 1Hmr (D2O) ~: 2.70 (3H, s, -SCH3) and 7.65 -7.77-8.35-8.48 ppm (4H, A2B2 type, aromatic Hs); ir ("Nujol")** ~max l6l5, 1585 (aromatic) and 780 cm~1; uv (H2O) * Trademark ** Trademark ~ ~ , . . :, ' . . ,~ ' :
; ' ' ': ' : ' ' . ' ' ~ ~ ' ~8~

~max 227 (~2.02 x 104) and 298 nm 1~1.64 x 104).
The hydroiodide (6.33 g, 25.0 mmol) was dissolved in H2O (40 mL) and the insoluble material was removed and washed with H2O (10 mL). To the filtrate was added at 0-5 NaOH pellet (5 g) and extracted with ~t2O (3 x 25 mL), saturating the aqueous layer with NaCl. The combined organic extracts were washed with brine (x 2), dried (MgSO4) and evaporated, yielding 2.92 g (23.4 mmol, overall yield 50%) of the title compound as an oil: lHmr (CDC13) ~: 2.4~ (3H, s, -SCH3) and 7.03-7.13-8.38-8.48 ppm (4H, A2B2type, aromatic-Hs); ir (film) ~max 1580 and 800 cm~l.
*Preparation of this compound was reported by Xing and Ware, J. Chem. Soc., 873(1939). The procedure, described in this ref~rence was followed.

B. 4-Methylthio-N-(2-mercaptoethyl)pyridinium methànesulfonate + N ~ 50-60 yS ~ ~ C335 ~

4-Methylthiopyridine (2.75 g, 22.0 mmol) was added slowly to methanesulfonic acid* (0.65 mL, 10.5 mmol) by cooling in an ice-bath. To this solid was added ethylene sulfide* (0.66 mL! 11.0 mmol, Aldrich) and the mixture was heated at 50-60C for 21 h. As reaction proceeds the solid went to solution. After cooling, ~;~

: ' '', ' ' '- . .

.
', , ' 1 ' ~ ' ' the reaction mixture was dissolved in H2O (5 mL) and washed with Et2O (5 x 4 mL). The cloudy aqueous layer was filtered over "Celite~' and the ~iltrate was purified by reverse phase silica gel column chromatography (C18 "Microbondapak"1 10 g) eluting with H2O. Each fraction of 10 mL was collected. Fractions 2 and 3 were combined and repurified by the reverse phase column. Fraction 2 gave 1.1258 g (4.48 mmol, y. 42.6%) of the title compound as a viscous oil: 1Hmr (DMSO-d6, CFT-20) ~: 2.32 (3H, s, MESO3~), 2.72 (3H, s, -SMe), 2.68 (lH, m, SH), 2.9-3.2 (3H, m, -CH2S-), 4.59 (2H, t, J=6.4 Hz, -CH2 ~, 7.97 (2H, "d", J=7.2 Hz, aromatic-Hs) and 8.72 ppm (2H, "d", J=7.2 Hz, aromatic-Hs); ir (neat) vmax:1630, 1200 (br, -SO3~), 7.85 and 770 cm~l.
* These reagents were distilled prior to use.

C. (5R) p-Nitrobenzyl 3-[2-(4-Methyl-thiopyridino~ethylthio]-(5S)-[(lB)-hydroxyethyl~-7 oxo-l-azabicyclo~3.2.0lhept-2-ene-2-carboxylate chloride OH - -J'~'~o ~

~O2P~3 Ms J ~ ~ ~,~_~ ~ ~.'e C~2~
To a solution of (SR3 p-nitrobenzyl 3,7-dioxo(6S)-[(lB)-hdyroxyethyl~ azabicyclo[3.2.0]heptane-(2B)-carboxylate (475 mg, 1.36 mmol) and diisopropylethlamine (0.24 mL, 1.4 mmol) in CH3CN (5 mL) was added at 0-5C
under a nitrogen atmosphere diphenyl chloro-1 Trademark , . .
.

phosphate (0.29 mL, 1.41 mmol). The mixture was stirred at 0-5, for 30 min. To this mixture was added an oily suspension of 4-methylthio-N-(2-mercaptoethyl)pyridiniUm methanesulfonate (678 mg, 1.45 mmol; 60% pure) in CH3CN
(1.5 mL) followed by diisopropylethylamine (0.24 mL, 1.4 mmol). The mixture was stirred at 0-5C for 1 h.
Immediately after addition of the base, yellowish precipitate formed. The precipitate was filtered and washed with cold CH3CN (3 mL), yielding 413 mg of yellowish solid. This was triturated from 10% MeOH in H2O (5 mL) to obtain 341 mg (0.618 mmol, y. 45.4%) of the title compound as white crystals: mp 118-120C; lHmr (DMSO-d6, CFT-20) ~: 1.16 (3H, d, J=601 Hz, 1'-CH3), 2.72 (3H, s, -SCH3), 3.1-3.7 (5H, m), 3.7-4.3 (2H, m), 4-71 (2H, t, J=6.3 Hz, -CH2 ~), 5.15 (lH, d, J=4.9 Hz, OH), 5.20-5.35-5.40-5.55 (2H, Abq, CO2CH2-Ar), 7.70 (2H, "d"
J=8.8 Hz, nitrophenyl-Hs), 7.97 (2H, "d", J=7.0 Hz, pyridinio-Hs), 8.25 (2H, "d", J=8.8 Hz, nitrophenyl-Hs), and 8.76 ppm (2H, "d", J=7.1 Hz, pyridinio-Hs); ir ("Nujol") vmaX: 3250 (OH), 1775 (~-lactam), 1700 (ester) and 1625 cm~1 (pyridinio); uv (abs, EtOH) ~maX:308 nm (~4.47 x 104); [~]n3 ~ 24.8 (c 0.5, MeOH); Anal. calcd.
for C24H26N3o6s2cl-H2o: C 50-56, H 4.95, N 7.37; found C 50.63, H 4.72, N 6.89.

D. (5R) 3-[2-(4-Methylthiopyridinio)ethylthio~-(6~)-[(l~)-hydroxyethyl)~7-oxo-l~azabicyclo[3.2.0~hept-2-ene-2-carboxylate ' .

.

.

- 89 (a) 0:~
J~ 2jPd C, :. 7. 4 buf'e~
Co2~1;3 OB
F~S ~5~
C02~' :

:

: : : ' ~ : :
X~

- :
, . ' ', ~ ' ' , ' ,, ~: : , `

- 9o -(5R) p Nitrobenzyl 3-[2-(4-methylthiopyridinio)ethylthio~-(6S)-[(1_)-hydroxyethyl]-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate chlorlde (380 mg, 0.68~ mmol) was dissolved in THF (31.5 mL) and pH 7.40 phosphate buffer (31.5 mL; 0.05M Fisher) and diluted with Et2O (31.5 mL). rrhis solution was mixed with 10% Pd-C (380 mg, Engelhard) and hydrogenated at 35 psi on the "Parr"* shaker at room temperature for 1 h.
The aqueous layer was filtered over "Celite"* to remove the catalyst and the "Celite"* pad was washed with H2O (2 x 5 mL). The filtrate and washing were combined and washed with Et2O (2 x 30 mL). The aqueous layer was pumped off to remove any organic solven~s and purified by reverse phase column chromatography (C18 'IMicrobondapak'l*
13 g, Waters Associates) eluting with H2O. Fractions having a uv absorption at 307 nm were collected (ca. 1 L) and lyophilized to obtain 127 mg (0.334) mmol, y. 48.5~) of the title compound as a yello~ish powder; 1Hmr (D2O, CFT-20) ~: 1.20 (3H, d, J=6.4 Hz, 1'-Ch3), 2.64 (3H, 5, -SCH3), 2.81 (2H, m, -SCH2-), 3.19 (lH, dd, J6-l' =6.1 Hz, J6_5=2.6 Hz, 6-H), 3.32 (2H, dd, J=11 Hz, J=5.5 Hz, 4-Hs), 3.92 (lH, dt, J=9.2 Hz, J5_6-2.6 Hz, 5-H), 4-1 (lH, m, 1'-H), 4.61 (2H, t, J=5.~ Hz, -CH2 ~), 7.70 (2H, "d", J=7.1 Hz, aromatic-Hs), and 8.40 ppm (2H, "d", J=7.1 Hz, aromatic-Hs); ir (KBr, disc) Vmax: 3400 (OH~ 1750 (~-lactam), 1630 (pyrindinium) and 1590 cm~1 (carboxylate);
uv (H2O) ~max 231 (~9800) and 307 nm (~25000); [~]n3 3.14 (c-0.5, H2O)-* Trademark . " .

,. . ~ .
.
. .
, ' ~

.

, ~2~2~

Example 8Pr~paration of 3-~-(3-methoxv-l-Pyridinium2 ethylthiol-6~-[.l'-(R~-hydroxyethyll-7-oxo-1-azabicyclo)3.2.0)-hept-2-ene-2-carboxylate 0~

A. 1-(2-mercaptoethyl)-3-methoxypyridinium methanesulfonate ~eO~> ~_ ~O~

To precooled (5C) 3-methoxypyridine (698 mg, 6.4 mmol) was added dropwise methanesulfonic acid (0.216 mL, 3.05 mmol) and ethylene sulfide (0.19 mL, 3.2 mmol). The mixture was then heated at 60C for 18 h, cooled to 20C, diluted with water (10 mL) and washed with ether (3 x 10 mL). The aqueous phase was pumped under high vacuum for 15 min and poured on a C18 reverse phase column. The title compound was eluted with water. The appropriate fractions were combined and evaporated under high vacuum to give the desired thiol (61.6 mg, yield 76.3%); ir (CH2C12) VmaX:2550 (w, SH) and I620, 1600, 1585 cm~1 (m, , .

- 91(a) -aromatic; lHmr (DMSO d6) ~: 8.90-7.90 (4H, m, aromatic C-H), 4.72 (2H, t, J-6.6 Hz, CH2N+), 4.01 (3H, s, OCH3), 3.5-3.0 (m, hidden CH2S), 2.66 (lH, dd, J=9.5 Hz, J=7.5 Hz, SH) and 2.31 ppm (3H, s, CH3SO3-).

X' :

.

'' ~g~

B. ~aL~-Nitrobenzyl 3~2-(3-methox~-1-pvridinium chloride?ethylthiol-6~ hydroxyethy~l-7-oxo l-azabicyclo(3.2.0)-hept-2-ene-2-carbox~late O~i ,1 ) ~ ~N ~--<) 2 J" ~ Z) ClPO(O~)2 O N ~ 3) ~ ~ ~` ~ ~sO
CO P~3 , -2 4~ N~2 ~ C~
0~
2~x3 A cold (0C) solution of p-nitrobenzyl 6a-[1'-(R)-hydroxyethyl]-3,7-dioxo-1-azabicyclo-(3.2.0)-heptane-2-carboxylate (1.04 g, 3 mmol), in acetonitrile (12 mL) was treated dropwise with diisopropylethylamine (0.63 mL, 3.6 mmol) and diphenylchlorophosphate (0.75 mL, 3.6 mmol) and stirred at 0C for 30 min. The resulting enol phosphate was treated with 1-(2-mercaptoethyl)-3-methoxypyridinium methanesulfonate (1.14 g, 4.30 mmol) in CH3CN(7mL), diisopropylethylamine (0.63 mL, 4.30 mmol), stirred for 30 min. and cooled at -10C for 30 min. The solid that precipitated out of the mixture was filtered, washed with cold acetonitrile (2 mL) and dried to give the title compound (1.32 g, yield 82%); ir (''Nujol'')Vmax 3320 (m, OH), 1780, 1765 (s, ~-lactam c=o), 1700, 1695 (m, ester c=o) and 1520 cm 1 (s, NO2); 1Hmr (DMSO d6) ~:
9.01 (lH, bs, H-3 aromatic), 8.75 (lH, bd, J-5.4 Hz, H-6 aromatic), 8.35-7.95 (4H, m, H-aromatic), 7.70 (2H, d, J-7.7 Hz, H-aromatic), 5.37 (2H, center of Abq, J=13 Hz, CH2PNB), 5.17 (lH, d, -4.9 Hz, OH), 4.87 (2H, t, J=6.3 Hz, CH2-N~), 4.35-3.75 (2H, m, H-5 and H-1'), 4.00 (3H, s, OCH3) 3.56 (part of a t, J=6.3 Hz, CH2S), 3.5-3.20 (3H, m, H-6, H-3) and 1.16 ppm (3H, d, J-6.1 Hz, C_3CHO).

~1 ~

.

C. 3-[2-(3-methoxy-1-pyridinium)ethylthiol-6~-[1'-(~)-hydroxyethyl~-7-oxo-1-azabicyclor3.2.0)-hept-2-ene-2-carboxylate 0~ Q~ ,0.~e ~2-N3 O~ Q~
. j~S

A solution of Para-nitrobenzyl 3[2-(3-methoxy-1-pyridinium chloride)ethylthio]-6~-[1'-(R)-hydroxyethyl]-7-oxo-1-azabicyclo(3.2.0)-hept-2-ene-2-carboxylate (600 mg, 1.12 mmol) in THF (25 mL), ether (25 mL) and pH 7.4 phosphate buffer (0.lM, 25 mL) was hydrogenated in a "Parr"* shaker over 10% PD/C (1.1 g) for 1 h at 40 psi.
The mixture was diluted with ether and the aqueous phase was filtered through a #52 hardened filter paper. The aqueous layer was washed with ether (2 x 20 mL), pumped under vacuum and poured on a silica gel reverse phase column. The title compound was eluted with water containing 2 and 5% acetonitrile. The appropriate fractions were combined and lyophilized to give a yellow solid that was repurified by hplc to give the penem carboxylate (150 mg, 38%); ir "(Nujol"*) vmaX 1750 (s, ~-lactam C=O) and 1580 cm~1 (s, carboxylate); 1Hmr (D2O) ~:
8.55-8.30 (2H, m, H-2, H-6 aromatic), 8.17-7.75 (2H, m, H-3~ H-4 aromatic), 4.77 (2H, t, J=5.9 Hz, CH2 ~), 4.10 (lH, part of 5 lines, J=6.3 Hz, H-l'), 3.97 (3H, s, OCH3), 3.85, 3.82 (2 lines, part of dt, J=2.6 Hz, part of ;_ .

_ 93(a) -H-5), 3.42 (2H, t, J=5.9 Hz, CH2-S), 3.25 (lH, dd, J=6.1 Hz, J=2.6 Hz, H-6), 2.g9-2.60 (2H, 6 lines, part of H-4) and 1.20 ppm (3H, d, J=6.4 Hz, CH3); uv (H20, c 0.05) ~ma~: 290 (~10517), 223 (~6643); ~% (0.1 M pH 7.4 phosphate buffer, 37C) 20 h.

* Trademark . ~ . . _ . ...

, Example 9 Preparation of (5 ,6~)-3-~2-(3-methylthiopyridinio~ethyl thiol-6- r 1_ (~) -hydroxyethyl]-7-oxo-1-azabicyclo[3 2.0]hept-2-ene-2-carboxylate 0~ S~

A. 3-Methylthio-1-(2-mercaptoethyl)pyridinium chloride 5~
N~ A

~2C,~
To a solution of 3-methylthiopyridine 1 (2.00 g, 0.016 mol) in 10 mL of ether was added 15 mL of 1 N HCl and the mixture was well shaken. The aqueous phase was separated, washed with 10 mL of ether and then evaporated. The residual hydrochloride was then dried in vacuo (P2O5) to give a white solid. To this solid hydrochloride was added 3-methylthiopyridine (1.88 g, 0.015 mol) and ethylene sulfide (0.89 mL, 0.015 mol) and the rustling mixture was heated (oil bath) at 55-65C
under N2 for 15 h. This gave a slightly turbid oil which was taken up in 125 mL of H2O and washed with CH2Cl2.
The aqueous solution was concentrated to about 25 mL and then a few drops of acetonitrile were added to make the mixture homogeneous. The resulting aqueous solution was applied to a C18 reverse phase column. Elution with H2O
and subsequent evaporation ,: ' , , ~8~

of the relevant fractions afforded the product (2.66 g 80%) as a pale yellow, viscous oil. ir (film) ~max 2410 (br, ~SH) cm~1; lHnmr (d6-DMSO+D2O) ~: 8.88-7.88 (m, 4H, aromatic), 4.70 (t, J=6.5 Hz, 2H, N-CH2), 3.08 (skewed t, J-6.5 Hz, 2H, S-CH2), 2.64 (s, 3H, S-Me).
1Prepared by the method of J.A. Zoltewiez and C. Nisi, J. Org. Chem. 34, 765 (1969).
B. p-Nitrobenzyl (5B,6~)-3- r 2-(3-methylthiopyridinio)ethyl thiol-6- r 1- (~) -hvdroxyethyl~-7-oxo-l-azabicyclo[3.2.o~hepto-2-ene 2-carboxylate chloride O N

o~ ~S4Ccl~

A solution of p-nitrobenzyl t5R,6S)-6-[1-(R)-hydroxyethyl]-3,7-dioxo-1-azabicyclo[3.2.0]heptane-2-carboxylate (0.522 g, 1.50 mmol) in 7 mL of dry acetonitrile was cooled at 0C and then diisopropylethylamine (0.287 mL, 1.65 mmol) was added dropwise. To the resulting yellow~brown solution was added dropwise diphenyl chlorophosphate (0.342 mL, 1.65 mmol) and the reaction mixture was kept at 0C for 30 min. Diisopropylethylamine (0.313 mL, 1.80 mmol) was then added, followed by a solution of 3-methylthio-1-(2-mercaptoethyl)pyridinium chloride (0.398 g, 1.80 mmol) in 0.70 mL of dry DMF. About a minute after the addition - 95(a) -was completP a precipitate separated from the reaction mixture and further cooling at -10C for 10 min gave a solid orange-coloured mass. This solid was subsequently triturated with acetonitrile and the residue was collected by filtration. The residue ' ' ' .
~, . .
' '. . :' d~L,~

was washed with acetonitrile, then acetone and finally dried in vacuo to give the product (0.455 g, 55%) as a cream-coloured solid. The combined filtrate was evaporated to give a yellow oil which was taken up in a minimum volume of acetonitrile and cooled at 0C for 30 min. Filtration of this mixture afforded an additional 0.139 g of the product as a light yellow solid. The combined yield was 0.594 g (72%). ir (KBr) ~max 3345 (br, -OH), 1770 (B-lactam CO), 1680 (-CO2PNB) cm~l; 1Hnmr (d6-DMSO) ~: 8.98-7.96 (m, 4H, pyridinium aromatic), 8.20-7.65 (ABq, J=7.0 Hz, 4H, PNB aromatic), 5.53-4.80 (m, 4H), 4.3-3.7 (m, 2H), 3.6-3.25 (M, 6H), 2.66 (s, 3H, S-Me), 1.16 (d, J-6.0 Hz, 3H, CHMe).

C. (5B.6S~-3-[2-(3-methylthiopyridiniolethyl thio~-6-r 1- (R) -hydroxyethyll-7-oxo-1-azabicYclo r 3.2.0]hept-2-ene-2-carboxylate ~ SMe . ~ ~
~ ~ \~Cl~ ' 22~
OH ~s ~'~S
C~;2 To a mixture of p-nitrobenzyl (5R,6S)-3-[2-(3-methylthiopyridinio)ethyl thio]-6-[1-(B)-hydroxyethyl]-7-oxo-l-azabicyclo[3.2.0]-hept-2-ene-2-carboxylate chloride (0.551 g, 1.0 mmol) and 10% palladium-on-charcoal (0.55 g) in 25 mL of phosphate buffer ~0.05 M, pH 7.4) was ~ L 'p~

- 9~(a) -added 5 mL of THF and 25 mL of ether. This mixture was hydrogenated (Parr) at 40 psi for 1 h, The reaction mixture was then filtered through "Celite" and the filter cake was washed with H20 and ether. The aqueous phase was separated and washed with additional ether (3 x).

.

'; . , : , .

After removing residual organic solvents in vacuo the aqueous solution was cooled at 0C and the pH was adjusted to 7.0 with saturated aqueous NaHCO3. This solution was immediately applied to a C18 reverse-phase column. Elution with H2O 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) vmaX:3400 (br, -OH), 1755 (~-lactam CO), 1590 (-CO2-) cm~1; lHnmr (D2O) ~:8.60-7.76 (m, 4H, aromatic), 4.76 (t, J=5.8 Hz, 2H, N-CH2), 4.13 (d of q, J=J'=6.3 Hz, lH, H-l'), 3.95 (d of t, J=9.0 Hz, J'-2.8 Hz, lH, H-5), 3.45-2.75 (M, 5H), 2.59 (s, 3H, S-Me), 1.20 (d, J=6.4 Hz, 3H, CHMe); uv (H2O) ~maX:296 (~8509), 273(~13005), 231(~11576) nm; ~ (ph 7.4, 36.8C) 20 h.

Example 10 Preparation of 3-[2-(1-(2,6-dimethylpyridinim)-ethylthiol-6~ -(~)hydroxyethyll-7-oxo-1-azabicyclo(3.2.0)hept-2-ene-2-carboxylate ~ C~3 J,,c;~
COo C~3 ._ .

A. 1-(2-mercaptoethyl-2!6-dimethylpyridinium methanesulfonate N~ ~ ~eO3 100~ ~ 42 ~ ~ y,5o A mixture of 2,6-diemthylpyridine tl9.2 mL, 0.165 mol) and methanesulfonic acid (3.27 mL, 0.050 mol) was stirred for 15 min, treated with ethylene sulfide (4.17 mL, 0.070 mol) and stirred at 100C for 42 h under a nitrogen atmosphere. After cooling to 25C, the reaction mixture was diluted with ether (45 mL) and water (30 mL). The two layers were separated and the organic layer was extracted with water (2 x 5 mL). The aqueous layers were combined, filtered through a "Celite" pad, washed with ether (2 x 15 mL), pumped to remove the traces of organic solvents and poured on top of a column (3.0 x 12 cm) of ~-"Bondapak"C-18. Elution with 3%
acetonitrile 97% water mixture gave after lyophilization of the appropriate fractions 2.5 g of the impure title compound as a syrup. It was repurified by hplc (~-"Bondapak" C-18) to give 0.90 g t7%) for the title compound. ir (film) VmaX:2520 (SH), 1640 and 1625 (pyridinium), 1585, 1490, 1200 cm~1 (sulfonate), lHmr (DMSO-d6 + D2O) ~: 2.36 (3H, s, CH3SO3-), 4.62 (2~, m, CH2N+), 7~74 (2H, m, Hm of pyridinium), 8.24 (lH, m, Hp of pyridinium), uv (H2O) ~maX:272 (4080)m~

r B . Paranitrobenzyl 3-[2-rl-(2 6-~ thvlro~ yn~ethylthiol-6~-~l-(~-hydroxyethyl]-7-oxo-1-azabicyclot3.2.0)he~t-2-ene-carboxYlate diphenylphosphate ~ 1) N:~, (i~._) J~= 2) cl~(a~?h).
3) ~(~
~sO
4) ~ ~i~)2 CC0~3 ~hO)2~o To a cold (0C) solution o~ p-nitrobenzyl 6~-[1-(~-hdyroxyethyl]-3,7-dioxo-1-azabicyclo(3.2.0)heptane-2-carboxylate (0.658 g, 1.89 mmol) in acetoni~rile (6 mL) kept under a nitrogen atmosphere was added diisopropylethylamine (0.394 mL, 2.26 mmol) and diphenyl chlorophosphate (0.~68 mL, 2.26 mmol). The reaction mixture was stirred 30 min and treated with a solution of 1-(2-mercaptoethyl)-2,6-dimethylpyridinium methanesulfonate (0.720 g, 2.73 mmol) in acetonitrile (3 mL) followed by diisopropylethylamine (0.394 mL, 2.26 mmol). The reaction mixture was stirred at 0C for 2 h, diluted with cold (0C) water (27 mL) and poured on top of a column (2.5 x 9.0 cm) of ~-"Bondapak" C-18. Elution with acetonitrile-water mixtures and lyophilization of the appropriate fractions gave 0.92 g (65~) of the title compound, ir (KBr) ~maX:3700-3000 (OH), 1765 (C=O of B-lactam), 1690 (c=o of PNB ester), 1620 (pyridinium), 1590 (phenyl), 1517 (NO2), 1330 ~NO2), 880 cm~l (NO2~, lHmr (DMSO, d6) ~: 1.15 (3H, d, J=6.2 Hz, CH3CHOH), 2.7-3.7 ~r ~L~89 - 99 (a (11 H, CH2S, 2-CH3 on pyridinium, H-4, H-6)j 3.7-4.4 (2H, CH3CHOH, H-5), 4.7 (2H, m, CH2N~), 5.14 (lH, d, J=4.5 Hz, OH), 5.37 (center of ABq, J=13.2 Hz, CH2 of PNB), 6.7-7.5 (lOH, m, phenyl), 7.5-8.7 (7H, pyridinium, H's of PNB), uv (H2O) ~maX 274 (~14150), 319 (~9445) m~

: : : :
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: .

8~

C. 3- r 2-(1-(2 6-dimethylpyridinium))ethylthio~-6~-rl-~ hYdroxyethyl]-7-oxo-1-azabicyclo(3.2.0!he~t-2-ene-2-carboxylate r~F, e~ er !
COO?~3 ~b~)2~O
OX
o5C~S~~

To a solution of p-nitrobenzyl 3-[2-(1-(2,6-dimethylpyridinium))-ethylthio]-6~-[1-(R)-hydroxyethyl]-7-oxo-1-azabicyclo(3.2.0)hept 2 ene-2 carboxylate diphenylphosphate (0.80 g, 1.07 mmol) in wet tetrahydrofuran (42 mL) was added ether (42 mL), potassium phosphate monobasic-sodium hydroxide buffer (0.15M, pH 7.22, 21 ML) and 10% palladium on charcoal (0.80 g). The result.ing mixture was hydrogenated for 1 h under 40 psi at 23C and filtered through a "Celite" pad.
The two layers were separated and the organic layer was extracted with the buffer (3 x 8 mL). The aqueous phase were combined, washed with ether (50 mL), pumped to remove traces of organic solvent and poured on top of a column (3.0 x 10.2 cm) of ~-"Bondapak" C-18. Elution of the column with 5% acetonitrile - 95% water mixture and lyophilization of the appropriate fractions gave the title compound 0.246 g (63%) as a yellowish powder, ir (KBr) ~maX:3700-2800 (OH), 1750 (c=o of the ~-lactam), 1620 (pyridinium), 1585 cm 1 (carboxylate~, lHmr (D2O) ~:1.23 (3H, d, J-6.4 Hz, CH3CHOH), 2.5-3.5 (llH, H 4, H-6, CH2S, 2CH3 on pyridinium), 3.8-4.4 (2H, CH3CHOH, H-5), ~i , ~.~84 - loo (a) 4.5-4.9 (CH2N+, HOD), 7.64 and 7.74 (2H, A part of A2B
system, Hm of pyridinium), 8.07, 8.16, 8.18 and 8.27 (lH, B part of A2B system, Hp of pyridinium), uv (H2O) ~max 277 (~9733), 300 (E8271) m~, [a] 23 +50.7'(C 0.48, H2O), Anal. calcd.for C18H22N~o4s-l-5 H2O: C 55-51 7.19; found: C 55.14, H 6.23, N 6.46 . ` . "' .

' Exa~n?le 11 Pre~ar2tio~ of (;~, 6~)-3-[2-(2-~ io-3-me.hylimidazolio)ethyl-thio~-6~ )-hydroxy-ethyl] -7-oxo-1-azabi~clo [ 3 . 2 . O ~ he~t-2-ene-2- caxboxy 12 .e 0~ ~Me ~ S ~ ~ N-Me o C023 . 2-Methyl~hio-3-methyl-1-(2-merc2ptoe'hyl)imud2zO1i~
tri'luoromethanesulfonate ' SMe ~3S3~ 5C~2c~2 ~ ~ ~-Me Me C~3S03 .
Trifluoromethanesul'onic acid (1.38 mL, 0.015 mol) was added dropwise to 2-methylthio-i-me.hylimidazolel (4.0 g~, 0.03 mol) at 0C under,N2. Ethylene su}.ide (0.9 mL, 0.015 mol) wzs then added and the mix~ure was heated at 5;C under N2 ror 24 h. The,reaction mixture was tri.urated with e~he~
~3x) and the residue was taXen up in aceto~e, filtered and evaporated. This gave the product (4.2 g, 8~2~) as a semi-c_Ysta~ e solid which was used.as such without rurther purification. ir( ilm),vmax:. 2550 (w, sh) c~ ; 'Hnmr (d6-acetone) ~: 7.97 (s, 2H)~ 4.66'(t, J=7 Hz , 2~, methylene), ~.17 ~s, 3~, N-Me), 3.20 (d of t, J=7 Hz, Jt~9 Hz, 2H, meehylene), 2.72 (s, 3H, S-Me), 2.20 (t,' J=9 Bz, l~ SH).

l. ?repared as ,Per A. Wohl and W. Mzrc~wzld, Chem. Ber. 2 , 1353 ~188 .-~\ '\

~ ~ :

3~

B. p-Nitrobenzyl (5~ 6 )-3_L_-(2-methylthio-3-methYl-imidazolio)ethyl thiol-6-[1-(~-hydroxyethyl~-?-oxo-1-azabicyclo~3.2.0lhept-2-ene-2-carboxyla~e diphenylphosphate 0~ 0~ ~e ~0 , r~ S~ h _ b ~ ~ ~ W
C02~N~ C02?~3 (~)2~~

To a solution of p-nitrobenzyl (5R,-6S)-6-[1-(R)-hydroxyethyl]-3,7-dioxo-1-azabicyclo[3.2.0]heptane-2-carboxylate (1.40 g, 4.0 mmol) in 50 mL of dry acetonitrile, at 0C under N2, was added dropwise diisopropylethylamine (0.76 mL, 4.4 mmol) followed by diphenyl chlorophosphate (0.91 mL, 4.1 mmol). After stirring the reaction mixture at room temperature for 1 h, diisopropylethylamine (0.76 mL, 4.4 mmol) was added and then a solution of 2-methylthio-3-methyl-1-(2-mercaptoethyl)imidazolium trifluoromethanesulfonate (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 ln vacuo to give a gum.
This gum was taken up in H2O and applied to a C18 reverse-phase column. Eution with H2O, then 20%
acetonitrile-H2O finally 30% acetonitrile-H2O, followed by lyophilization of the appropriate fractions afforded the product (0.90 g, 30%) as a light yellow solid. ir (KBr) VmaX:3380 (br, OH), 1770 (B-lactam CO) cm~1; ~Hnmr (d6-acetone) ~:8.35 (br,s, lH), 8.24, 7.78 (AB q, J=8.8 Hz, 4H, aromatic), 7.89 (br s, lH), 7.25-6.91 (m, 10H, diphenylphosphatej, 5.50, 5.25 (AB q, J=12 Hz, 2H, benzylic), 4.75-4.27 (m,3H), 4.03 (s, 3H, N-Me), 4.15-2.75 (m, 8H), 2.59 (s, 2H, S-MMe), 1.22 (d, J=6.2 Hz, 3H, ~CHMe).
r 3r~c C. (5R, 6S)-3-~2-(~-Methvlthio-3-methYlimidazolio)ethylthiol-6-Cl-iB)-hvdroxveth~ 7-oxo-l-azabicy~lor3.2.olhept-2-ene-2 carboxylate 0~ e -- - C 2 ~ 0 ) 2 ? 0 0~
Y SM.
r ~ -5 ~ ~ e co2~

To a solution of p-nitrobenzyl (5R, 6S)-3-[2-(2-methylthio-3-methylimidazolio)ethyl thio[-6-[1-(B)-hydroxyethyl]-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate diphenylphosphate (1.20 g, 1.56 mmol) in a mixture of 70 mL of THF, 70 mL of ether and 31 mL of phosphate buffer (0.05M, 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 H2O and ether. The aqueous phase was separated, cooled at 0C and the pH was adjusted to 7.0 with saturated aqueous NaHCO3. After removing residual organic solvents in vacuo the aqueous solution was applied to a C18 reverse-phase column. Elution with H2O and then 8%
acetonitrile-H2O 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(KBr),~max:3420 (OH), 1750 (B-lactam CO), 1590 (-CO2O) cm~l; 1Hnmr (D2O) ~: 7.58 (s, 2H), 4.52 (t, J=6 Hz, 2H), 4.28-3.82 (m, 2H), 3.90 (s, 3H, N-Me), 3.40-2.87 (m, 5H), 2.40 (s, 3H, S-Me), 1.20 (d, J=6.4 Hz, 3H, -CHMe); uv (H2O) ~maX:297 (E
7572), 262 (E6259), 222 (E 7955) nm.
.~

. ~

~xam~le 12 Pre~aration of (5~, 6S)-3-~ 2-(3-amino~r~dinio)e.hyl -thio]-6-[l~(R)-hydroxyethvl]-7-oxo-1-azabicyclo[3 2 0]-hept- 2-ene-2-c2rboxylate = 023 ~ 2 A 3-~mino-1-(2-merca?toethyl)pyridin~um chloride N2N~ 1~2N~Q 5 1~5C32C~I~C~ 2 3-Aminopyridine (1 50 g, 0 016 mol) W25 taXe~ U?
in 15 mL of 1 N meth~nolic HCl and the resulting solution w2s evapor2ted to give the hydrochloride 2s an oil To ~his oil was added 3-~mlnoDyridine (1.32 g, 0 015 mol) and ethylene sul'ide (0 89 ml, 0 01; mol) and the resulting mix!ure was heated (oil bzth) 2t 60-65C under N2 ~or 2 h Ano her equivalent of ethylene sul'ide (0 89 ml, O OlS mol) W25 added and heating was contlnued 2t 55-65C or 65 h.
The reaction mixture W2S washed wi'.h CH2C12 and hen tzken ` up in ~2 (25 ml). The zqueous solution was applied to a C18 reverse-phase column which was eluted with ~2 Evaporation of the relevant fractions gave the produc~ (1 26 g, ~4%) as a colorless, viscous oil ir(~ilm) ~max 3180 ~NP2) cm l; ~ mr (d6-D~5O) o: 8 19-7 59 (m, 4H, aromatic), 4 59 (t, J-6 2 Hz, 2H, N-C~ ); 3 5 (br s, 2H, -NH~), 3 20-2 7?
(m, 3~) _ j .~ :

B. p-Nitrobenzyl (5~ 65~-3-(2-(3-aminopyridinio)ethyl-thio~-6-( 1- ( R ) -hydroxyethyl)-7-oxo-1-azabicyclo(3.2 0)-hept-2-ene-2-carboxylate diphenylphosphate ~ ~2~3 (~ )2 To a solution of p-nitrobenzyl (5B, 6S)-6-(1-(B)-hydroxyethyl)-3,7-dioxo-1-azabicyclo(3.2.0)heptane-2-carboxylate (0.696 g, 2.0 mmol) in 10 mL of dry acetonitrile, at 0C under N2, was added dropwise diisopropylethylamine (0.382 mL, 2.2 mmol) followed by diphenyl chlorophosphate (0.457 m~, 2.2 mmol). After stirring at ooc for 30 min. a solution of 3-amino-1-(2-mercaptoethyl)pyridinium chloride (0.475 g, 2.5 mmol) in 1 ml of dry DMF was added, followed by additional diisopropylethylamine (0.435 mL, 2.5 mmol). The reaction mixture was kept at 0C for 1.5 h and was then concentrated in vacuo. The resulting gum was taken up in acetonitrile-H2O (1:1) and applied to a C18 reverse-phase column. Elution with H2O , followed by 20% acetonitrile-H2O and subsequent lyophilization of the relevant fractions afforded the product (0.730 g, 50%) as a beige-colored solid. ir(KBr) ~max:
3330 (br, OH), 3180 (br, NH2), 1770 (B-lactam CO), 1690 (-CO2PNB)cm~l; lHnmr (d6-DMSO) ~: 8.29-7.63 (m, 8 aromatic), 7.2-6.7 (m, 10H, diphenylphosphate), 5.47, 5.18 (AB q, J=14 H2, 2H, benzylic), 4.73-4.45 (m, 3H) 4.2-3.8 (m, lH), 3.6-2.6 (m, 8H), 1.15 (d, J-6.2 Hz, 3H, CHMe).

--10~--C. (5B. 6~)-3-(2-(3-Aminopyridinio)ethyl thio)-6~
hydroxyethyl)-7-oxo-l-azabicyclo(3.2.0)hept-2-ene-2-carboxylate ~ O
~ ~ 11 ~
C02 ( ~ 2PO

N
CO
To a mixture of p-nitrobenzyl (5R, 6S)-3(2-(3-aminopyridinio)ethyl thio)-6-(1-(R)-hydroxyethyl)-7-oxo-1-azabicyclo(3.2.0)hept-2-ene-2-carboxylate diphenylphosphate (0.730 g, l.0 mmol) and 10~ palladium-on-charcoal (0.7 g) in 25 mL of a phosphate buffer (0.05 M, pH 7.4) was added 8 mL of THF and 20 mL
of ether. This mixture was then hydrogenated (Parr) at 40 psi for l h. The resulting mixture was filtered through a pad of "Celite" and the filter cake was washed with H2O and ether. The aqueous phase was separated, washed with ether (2 x) and then residual volatiles were removed ln vacuo. The aqueous solution was immediately applied to a C18 reverse-phase column which was eluted with H2O. Lyophilization of the relevant fractions afforded 0.45 g of an off-white solid. This material was repurified by reverse-phase hplc to give the desired product (0.123 ~, 35%) as an ivory-colored solid.
ir(KBr)~max:3340 (br), 1750 (br, B-lactam CO), 1S80 (br, -CO20) cm l; 1Hnmr (D2O~ ~-8.0?-7.59 (m, 4H, aromatic), 4.61 (t, J= 5~8 Hz, 2H, N-CH2), 4.14 (d of q, J=J'=6.3 Hz, lH, H 1'), 3.97 (d of +, J-9.2 Hz, J'=2.6 Hz, lH, H-5), 3.38 (t, J=5.8 Hz, 2H, S-CH2), 3.24 (d of d, J=6.0 Hz, J'=2.6 Hz, lH, H-6), 3.17-2.57(m, 2H, H-4) 1.21 (d, J=6.3 Hz, 3H, CHMe); UV(H2) ~max:299 ( 7949), (8822) nm; ~1/2 (pH 7.4, 36.8C) 18.5 h.

. ~

Ex2m~le 13 PR~P~RATION O~

N

.
~5~,6S)3~ S)-methyl-2-(1-~y_idiniu~)e'hylthioj-6~ (R~-hy~roxyethyl~-7-oxo-1-zzabicyclo(3.2.0?hept-2- ene- 2-c2rboxylate a~

'"r;~
COO~ ' ' ' .. .

.
5~,6S)3~ tR)-methyl 2-(1-pyridinium)ethylthio]-6-[1~
hydroxyethyl~-7~ox=~ zablcyc~o~3.2.0~h-ot 2-ene-2-~- Osxyl~e , ' .`' " ~ ` .

'' ' , _ -t2-mercapto-2-methylethYl)pyridinium methanesulfonate d -1-(2-mercapto-1-methylethyl)pyridinium methanesulfonate ~ + ~ sO~ ~

~ N ~ MsO

~S~r ~ ~sO

Methanesulfonic acid (1.95 mL, 0.030 mol) was added slowly to cold pyridine (7.83, 0.097 mol) and the resulting mixturP was stirred at 40C for 15 min, treated with d -propylenesulfide (2.59 mL, 0.033 mol) and stirred at 60~C under a nitrogen atmosphere for 90 h. Pyridine was removed under vacuum; the residue was mixed with water and purified by chromatography (hplc, Prep. Bondapak C~
18). The appropriate fractions were combined and lyophilized giving d -1-(2-mercapto-2-methylethyl)pyridinium methanesulfonate 1.14 g (15~) as a colorless syrup, ir (film) Vmax:2520 (SH)~ 1640 (pyridinium), 1180 (s, CH3SO3-), 1040 (CH3SO3~)cm~l, 1Hmr (DMSO
d6) ~:1.35 (d, J=6.8 Hz, 3H, CH3CHS), 2.30 (s, 3H, CH3SO3-), 2.90 (d, J=8.5 Hz, lH, SH) 3.2-3.7 (m, CHSH), 4.52 (dd, Jgem=12~9 Hz, J=8.4 Hz, CHCH2N+), 4.87 (dd, Jgem=12~9 Hz, J=6.0 Hz, CHCH2N+), 8.0-8.4 (m, 2H, Hm of pyridinium), 8.5-8.8 (m, lH, Hp of pyridinium), 9.04 (dd, J=1.4 ~z, J=6.7 Hz, 2H, Ho of pyridinium) uv (H2O) ~max: 208 (~5267), 259 (3338), Anal. calcd. for CgH15NO3S2.2H2O; C 37.88, H 6.71, N 4.91, S 22.47; found: C 37.49, H 6.85, N 4.86, S 22.09 and d -1-(2-mercapto-1-' . , , ~, .: , . .
, methylethyl)pyridinium methanesulfonate 0.82 y (11%) as a colourless syrup; ir (film) ~max:2500 (S~), 1628 (pyridinium), 1180 (sulfonate), 1035 (sulfonate) cm~1, 1Hmr (DMSO d6) ~ :1.69 (d, J=6.8 Hz, 3H, CH3CHN+), 2.31 (s, 3H, CH3SO3 ), 3.0-3.3 (m, 2H, CH2S), 4.2-5.2 (m, lH, CHN+), 8.0-8.4 (m, 2H, Hm of pyridinium), 8.5-8.8 (m, lH, Hp of pyridinium), 9.0-9.2 (m, 2H, Ho of pyridinium)~ uv (H2O) ~max 209 ( 4987), 258 (E3838) . Anal.
calcd- for C9H15N3S2-1-5H2O: C 39.11, H 6.56, N 5.07; found C
39.13, H 5.92, N 5.20.
B. (5R!6S) paranitrobenzyl 3-[1-(R,S)methyl-2-(1-pyridinium)ethylthio~6-[1-(R)-hydroxyethyl]-7-oxo-1-azabicyclo(3.2.0)hept-2-ene-2-carboxylate diphenylphosphate 1) NE~ r)2 O _ 2 ~ _~

CCOPN~3 _ 3) RS~>
4) N2~(iPr)2 C)R

~S ~ ~

(?hO) 2gO

To a cold (0C) solution of (5R,6S)paranitrobenzyl 6-[1-(R)-hydroxyethyl]-3,7~dioxo-1-azabicyclo(3.2.0)heptane-2-carboxylate (0.523 g, 1.5 mmol) in acetonitrile (6 mL) kept under a nitrogen atmosphere was added diisopropylethylamine (0.314 mL, 1.8 mmol) followed by diphenyl chlorophosphate (0.373 mL, 1.8 mmol). The reaction mixture was stirred for 30 min and treated with a solution of d -1~(2-mercapto-2-methylethyl)pyridinium methanesulfonate (0.539 g, 2.16 mmol) in acetonitrile (2 mL) and diisopropylethylamine (0.314 mL, 1.8 mmol). The reaction mixture was stirred at 0C for 1 h, diluted with cold (0C) ~r '~ ~' " ' .

water (24 mL) and chromatographed over a "PrepBondapak" C-18 column (2.5 x 8.5 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 (KBr) v max 3700-3100 (OH), 1770 (C=O of ~-lactam), 1695 (C=O of PNB ester), 1630 (pyridinium), 1590 (phenyl), 1518 (NO2), 1348 (NO2), 885 (NO2) cm~1, lHmr (DMSO d6) ~
1.14 (d, J=6.1 Hz, 3H, CH3CHO), 1.33 (d, J=6.3 Hz, 3H, CH3CHS), 4.6-5.0 (m, CH2N+), 5.14 ~d, J=5.2 Hz, lH, OH), 5.37 (center of ABq, J=12.4 Hz, 2H, CH2 of PNB), 6.6-7.5 (m, 10H, phenyl of phosphate), 7.69 (d, J=8.7 Hz, 2H, Ho of PNB), 8.0-8.4 (m, 4H, Hm of PNB, Hm or pyridinium), 8.4-8.8 (m, lH, ~p of pyridinium) 9.08 (d, J=5.6 Hz, 2H, Ho of pyridinium), uv (H2O) ~max 263 (~13325), 308 (E8915).
Anal calcd. for C36H36N3olosp-H2o C57-52~ H 5 4.27; found: C 57.76, H 4.96, N 5.36, S 4.35.
C. (5B, 6S)3-[1-(B and S)-methYl-2-(1-pyridiniumlethylthio)-6-[1-(~)-hydroxyethyl]-7-oxo-1-azabicyclo(3.2.0)hept-2-ene~2-carboxylate 0~3 I ~5~ r,-~, et~ er CCOp~3 ~hO)2~O

,N~

-To a solution of (5B,6S)paranitrobenzyl 3-[1-(R,S)methyl-2-(1-pyridinium)ethylthio]-6-[1-(R)-hydroxyethyl]-7-oxo-1-azabicyclo-(3.2.0)hept-2-ene-2-carboxylate diphenylphosphate (0.60 g, 0.82 mmol) in wet tetrahydrofuran (33 mL) was added ether (33 mL), potassium phosphate mono basic-sodium hydroxide buffer (17 mL, 0.15N, pH 7.22) and 10% palladium on charcoal (0.60 g). The resulting mixture was hydrogenated for 1 h under ~0 psi at 23C.
The two layers were separated and the organic layer was extracted with water (3x7 mL).

`;~

': . ' . ' ' , ' , .

3~

The aqueous layers were combined, filtered through a "Celite" pad, washed with ether (3 x 20 mL) and chromatographed on a "PrepBondapak"* C-18 column (2.5 x 9.5 cm) with water as eluting solvent to give 0.18 g (63%) of mixture of diastereoisomers. The two diastereoisomers were separated by hplc ("PrepBondapak"* C-18) with water as eluting solvent: isomer with lower retention time, 0.068 g (23%) compound "B", ir (XBr) vmax: 1770 (C=O of ~-lactam), 1633 (pyridinium), 1593 (carboxylate)cm~l, lHmr(D2O) ~:1.20 (d, J=6.3 Hz, 3H, CH3C~0), 1.42 (d, J=6.9 Hz, 3H, CHCHS), 2.3-3.2 (m, 3H, H-4, H-6), 3.5-3.9 (m, lH, SCH), 3.9-4.2 (m, 2H, H-5, CH3CH0), 4.3-5.1 (m, CH2N~), 7.8-8.2 (m, 2H, Hm of pyridinium), 8.4-8.7 (m, lH, Hp of pyridinium), 8.7-9.0 (m, 2H, Ho of pyridinium uv (H2O) ~max 260 ( 6727), 300 ( 8245), [~]D23-39.3O (c, H2O), -~=12.6 h (measured at a concentration of 10-4M in phosphate ~uffer pH 7.4 at 36.8C);
isomer with higher retention time, 0.081 g (28%), compound "A", ir(KBr) max 1755 (C=O of ~ lactam), 1630 (pyridinium), 1590 ~carboxylate) cm~1, 1Hmr (D20) :1.18 (d, J=6.3 Hz, 3H, C_3CH0), 1.40 (d, J=7.0 Hz, 3H, CH3CHS), 2.84 (d, J=9.3 Hz, 2H, H-4), 3.26 (dd, J=2.7 Hz, J=5.9 Hz, lH, H-6), 3.4-4.2 (m, 3H, SCH, CH3CHO, H-5), 4.2-5.1 (m, CH2N+), 7.7-8.1 (m, 2H, Hm of pyridinium), 8.3-8.65 tm, lH, Hp of pyridinium~, 8.65-8.9 (m, 2H, Ho of pyridinium), uv (H20) maX:259 (~5694), 296 (~6936), [~]D23 ~96.9 (_ 0.56, HaO)~
~=15.6 h (measured at a concentration of 10-4 M in phosphate buffer pH 7.4 at 36.8C).

*Trademark ~43~a~

Example 14 PREPARATION OF
OH
S~
COO~ [~

(5-~6s)3-r2-[ts)-(l-pyridinium)]-l-(s) cyclohexylthio]-6-rl-(R)-hydroxyethyl]-7-oxo-1-azabicyclo(3.2.0)hept-2-ene-2-carboxylate and o}~

5""' ~ ~ ' (5_,6S)3-[2-[(R)-(l-pyridinium)]-1-(R)-cyclohexylthio]-6-[1-(R)-hydroxyethyl]-7-oxo-1-azabicyclo(3.2.0)hept-2-ene-2-carboxylate.

:' , .
,-.: . : .
.
: , , , A. d -1-~2-mercapto-1-cyclohexyl)pyridinium methanesulfonate C~ ~ N~> f ~5 03 SO-Methanesulfonic acid (0.65 mL, 0.01 mol) was added dropwise to pyridine (2.42 mL, 0.03 mol) with cooling. The mixture was stirred under a nitrogen atmosphere for 10 min, treated with d -cyclohexene-sulfide [1.377 g (85% pure), 0.0102 mol] and stirred at 72C 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 a "PrepBondapakl'*C-18 column (5 x 13 cm) with 0~2% acetonitrile in water as~eluting solvent giving after lyophilization a colourless syrup 1.57 g (53~), ir (film) vmax: 2500 (SH), 1625 (pyridinium), 1190 (SO3 ), 1Hmr (DMSO d6) ~:1.2-2.5 (m, 8H, cyclohexyl H), 2.32 (s, 3H, CH3S03-), 2.82 (d, J=9.8 Hz, SH), 3.0-3.5 (m, lH, CHSH), 4.2~4.9 (m, lH, CHN+), 8.0-8.3 (m, 2H, Hm of pyridinlum), 8.4-8.8 (m, lH,~Hp of pyridinium), 8.9-9.3 (m, 2H, Ho of pyridinium), uv (H20) ~max 214 (E5365)~ 258 (E
3500). Anal. calcd. for C12HlgNO3S2OH2O: C 46.88, H 6.88, N 4.56;
found: C 46.61, H~6.46, N 4.65.

*Trademark ' , .
'' v~

B. (5B 6~)~aranitrobenzyl 3-[2-L~ or S)-(1-pyridinium)l_l_(R or S)-cyclohexvlthiol-6-(1-(~)-hydroxyethyll-7-oxo-1-azabicyclo(3.2.0)hept-2-ene-2-carboxylate diphenvlPhosphate 1) NEt~i~r) .. ~ 2) Cl~(OPh)2 ,~c o ~
O~ N 3) ~ M~O
COOPNB N

4) N~:t(iPr)2 ~H
J~C~
COO~ hO)2~O

To a cold (0C) solution of (5R,6S)paranitrobenzyl 6-[1-tR)-hydroxyethyl]-3~7-dioxo-l azabicyclo- (3.2.0)heptane-2-carboxylate (1.37 g, 3.93 mmol) in acetonitrile (15 mL~ kept under a nitrogen atmosphere was added diisopropylethylamine (0.822 mL, 4.7 mmol) and diphenyl chlorophosphate (0.979 mL, 4.7 mmol). The resulting solution was stirred for 30 min and treated with a solution of d -1-(2-mercapto l cyclohexyl)- pyridinium methanesulfonate (1.64 g, 5.66 mmol) in acetonitrile (4.7 mL) followed by diisopropylethylamine (0.822 mL, 4.7 mmol). The reaction mixture was stirred at 0C for 1 h, diluted with cold (0C) water (75 mL) and chromatographed on "PrepBondapak"*C-18 with 25-50% acetonitrile in water as eluting solvent giving after lyophilization of the appropriate fractions 1.9 g (53%) of the title compound, ir (KBr) vmax: 3700-3000 (OH), 1770 (C=O of B-lactam), 1700 (C=O of PNB ester), 1628 (pyridinium), 1590 (phenyl), 1515 (NO2), 1345 (NO2) 880 (NO2) cm~1, 1Hmr (D2O) ~:1.13 (d, J=6.1 Hz, 3H, CH3CHO), 1.2-2.5 (m, 8H, cyclohexyl H), 2.7-3.5 (m, 4H, H-4, H-6, CHS), 3.5-4.4 (m, 2H, CH3CHO, H-5), 4.4-5.0 (m, lH, CHN+), 5.30 (center of ABq, J=12.8 Hz, CH2 of PNB), 6.7-7.4 (m, 10H, phenyl), 7.65 (d, J=8.6 Hz, 2H, *Trademark . .
' , ' '.

.

Ho of PNB), 7.9-8.4 (m, 4H, Hm of PNB, Hm of pyridinium), 8.4-8.8 (m, lH, Hp of pyridinium), 9.0-9.4 (m, 2H, Ho of pyridinium), uv (H20) ~max: 263 (~9038), 309 (~6394). Anal. calcd for C39H40N3010SP.H2o: C 59.16, H 5.35, N 5.31; found: C 58.95, H 5.15, N 5.57.
C; (5R. 6S) 3-~2-~(R or S)-(1-pyridinium)l-1-(R or S)-cyclohexylthio~-6-[1-(~)-hYdroxyethyl~-7-oxo-1-azabicyclo(3.2.0)hept-2-ene-2-carboxylate J` r l lO~ 2d~C, .~-, e ~e~, bUCr~
Oo~Ng ~ N~ O
~ (PhO)2~0 ~ -s.~

To a solution of (5R, 6S) paranitrobenzyl 3-[2-[(B or S)-(1-pyridinium)]-1-(R or S~-cyclohexylthio]-6-[1-(R)-hydroxyethyl]-7-oxo-l-azabicyclo(3.2.0)hept-2-ene-2-carboxylate diphenylphosphate (1.85 g, 2.34 mmol) in wet tetrahydrofuran (96 mL) was added ether (96 mL), potasslum phosphate monobasic-sodium hydroxide buffer (0.15M, 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 mL), pumped to remove the traces of organic solvents and chromatographed on a "PrepBondapak" C-18 column ~4.5 x 9 cm) with 0-5% acetonitrile in water as eluting solvent giving after lyophilization 0.705 g (76%) of a mixture of diastereoisomers.
The separation of the diastereoisomers was done by hplc ("PrepBondapak" C-18) with 4% acetonitrile in water as eluting .;.

solvent; diastereoisomer with lower retention time, compound "A", (0-29 g, 31~), ir (KBr) vmaX: 1750 (C=O of ~-lactam), 1620 (sh, pyridinium), 1685 (carboxylate) cm~l, lHmr (D2O) ~:1.21 (d, J=6.3 Hz, 3H, CH3CHO), 1.4-2.5 (m, 8H, cyclohexyl H), 2.5-3.05 (m, 2H, H-4), 3.05-3.25 (m, lH, H-6), 3.3-3.7 (m, lH, CHS), 3.9-4.3 (m, 2H, H-5, CH3CHO), 4.3-4.8 (m, CHN+), 7.8-8.2 (m, 2H, Hm of pyridinium), 8.3-8.7 (m, lH, Hp of pyridinium), 8.8-9.1 (m, 2H, Ho of pyridinium) uv (H2O) ~max 260 (~7123), 300 (~8685), [~]D23 ~6.2 (c 0.63, H2O), ~=16.6 h (measured at a concentration of 10-4M in phosphate buffer pH 7.4 at 36.8C); Anal. calcd- for C20H24N2O4S 2H2o 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 with higher retention time, compound "B", (0.35 g, 38%) ir (KBr) vmax: 1750 (C-O of ~-lactam), 1622 (sh, pyridinium), 1588 (carboxylate) cm~l, lHmr (D2O) ~:1.19 (d, J=6.4 Hz, 3H, CH3CHO), 1.3-2.5 (m, 8H, cyclohexyl H), 2.5-3.1 (m, 2H, H-4), 3.1-3.3 (m, lH, H-6), 3.3-3.8 (m, 2H, H-5, CHS), 4.1 (center of m, lH, CH3CHO), 4.25-4.7 (m, lH, CHN+), 7.8-8.1 (m, 2H, Hm of pyridinium), 8.3-8.7 (m, 1~, Hp of pyridinium), 8.75-9.0 (m, 2H, Ho of pyridinium), uv (H2O) ~max 259 (~5992), 296 (~7646), [~]D23 +65.3 (c 0.43, H2O) T~=20.2 h (measured at a concentration of 10 4 M in phosphate buffer pH 7.4 at 36.8C).

, Exam~le 15 A. (5E~ Allyl 3-[(2-pyridinioethyl~thiol-(6~-r(1~)-hydroxyethyl~-7-oxo-1-azabicyclo[3 2.0]hept-2-ene-2-carboxylate diphen~lphosphate O~H

O ~ 2) ~ ~ ~ Cl~
OH

o'l~S ~-C2 ~'~G~ ~30~0(0~)2 To a solution of (sR) allyi, 3,7-dioxo-~6S)-[(1g)-hydroxyethyl]-1-azabicyclo[3~2~o]heptane-(2~)-carboxylate (473 mg, 1.87 mmol) in CH3CN (6 mL) was added at ca. -10C under a nitrogen atmosphere diisopropylethylamine (0.42 mL, 2.4 mmol) followed by diphenyl chlorophosphate (0.50 mL, 2.4 mmol). The mixture was stirred at 0C
for 30 min. and then cooled to -15C. To this was added an oily suspension of N-(2-mercaptoethyl)-pyridinium chloride (527 mg, 3.00 mmol) in CH3CN (1 mL) containing 5 drops of DMF, followed by diisopropylethylamine (0.42 mL, 2.4 mmol). The mixture was stirred at -15 for 30 min and then diluted with H2O (20 mL). This mixture was directly purified on a reverse phase silica gel (C18 PrePAK*, 12. g, Waters Associates~ eluting with H2O (200 mL), 10% CH3CN/H2O
(100 mL), 20~ CH3CN/H2O (100 mL), 30% CH3CN/H2O (100 mL) and then 40% CH3CN/H2O (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-d6. CFT-20) ~ (3H, d J=6 Hz, l'-CH3), 2.6-3.7 (m), 3.75-4.3 (2H, m, 5-H and l'-H), 4.65 (2H, m, -CO2CH2-), 4.87 (2H, t, J=6 Hz, -CH2-N+), *Trademark ' ., , ~ .~ .

, ' , ~ ' ' '. ,' 5-6.2 (3H, m, olefinic protons), 6.6-7.4 (m, aromatic protons), 8.1;
(2H, "c`', J~7 ~z, aromatic protons meta tq the nitrogen), 8.63 (lr~, "t", J~7 Hz, aromatic proton para to the nitrogen) and 9.07 ppm (2:H, ~d", J~7 Hz, aromatic protons ortho to the nitrogen); ir (film) v: 3~00 ~Orl), 1770 (B-lactam), 1690 (ester), 1625 (pyridinio).

B. (5R) 3-[(~-~yridinioethyl)thio]-(65)-1(lR)-hydroxyethyl]-7-oxo-1-azabicyclo~3.2.0~hept-2-ene-2-car~oxylate 0~ ' ' 0 o ~C3 S ~ Pd ( P~ 3 ) 4 _ ~ J~L~_ ~ ~
` C02 ~ (~)OPO(0~)2 ~ C02~C co G

To a solution of (5~) allyl 3-[(2-pyridinioethyl)thio~ - (6S) -[(lR)-hyd~oxyethylj-7-oxo-1-a~abicyclo~3.2.0]hept-2-ene-2-carboxylate diphenylphosphate (156 mg, 0.25 mmol) in CH3CN (2 ~L) W25 successively ~dde~ ~t ca. 22C a solution of potassiu~ 2-ethylhexanoate in EtOAc (0.5 M, 0.6 m~; 0.3 mmol), triphenylphosphine (15 mg, 0 057 mmol) and tetrakistriphenylphosphine palladiu~ (15 mg, 0.013 ~mol). The mixture was stirred at-ca. 22C under a nitrogen atmosphere for 2 h.
A~ter addition of anhy2rous Et20 (7 mL), the precipitate was filtered, washed with anhydrous ~t20 (7 mL) and dried in vac~o to yield 101 mg of brownish solid.~ This was purified by reverse phase column c~roma-i tography (cla PrepPAK, 12 g, ~iaters Assoc{ates) eluting ~ith H~O.
Appropriate fractions (fr. 7-12, each 20 mL) were collected and lyo-philized to ~btain 53 mg (0.16 mmol, y. 6~) of the title compound as;

yellowish powder. This material was eontaminated with potassium diphe-ny phosphaee and potassium 2-cthylhs~noate: l8mr (D20, CFT-20~ ~: 0.80 " "~' ' .

.

3~4L

(t, J-6.4 Hz, Me fro~ ethylhexanoate), 1.21 ~3H, d, J=6.3 Hz, l'-Me), 2-93 (211, dd, Jl 5-9 ~z, ~ =4 Hz, l-Hs), 3.28 ~lH, dd, ~6 1,=6-2 Hz, J6 5=2.5 Hz, 6-H), 3.42 (2H, t, J=6 Hz, ~CH2S), 3.98 (lH, td, J5 1=
9 Hz, J5 6=2.5 Hz, S-H), 4.15 (lH, q, J=6.2 Hz, l'-H), 4.80 (2H, t, J=6.0 Hz, -CH2N ), 7-7.5 (m, phenyl protons from diphenyl phosphate),;
8.03 (2H, m, Hm of pyridinium), 8.56 (lH, m, Hp of pyridinium) and 8.81 ppm (2H, "d", ~-6.5 Hz, ~o of pyridinium) .

` : :

-~
- ~ : . .

. . ...

-. . . . .

32a~

Example 16 Preparation or 3-[2-(N-MethYl-thiomorpholinium)ethylthio]-6~-tl'-~R)-hydroxyethyl]-7-oxo-l--azabicyclo[3.2~o]-hept 2-ene-2-carboxylate OH o e Me A. N-methy~-N-(2-merca~toethyl)thiomorpholinium methanesulfonate ~ \ 1) MsOH HS Me Me ~ 2) ~ To precooled (ice bath) N-methylthiomorpholine*
(5.00 g, 42.7 mmol) was added methanesulfonic acid (1.47 mL, 20.5 mmol) and ethylene sulfide ~1.30 mL, 21.4 mmol). The mixture was heated at 65C 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 ~oured over a silica gel reverse phase column; the title compound being eluted with water. The appropriate fractions were combined and eYaporated to afford the thiol as an oil (4.~80 g, yield 86%); ir (film) ~max 2550 cm 1 (w, SH);~lHmr (DMSO d6)~: 3.25-2.95 (6~, m, CH~N~), 3j32 (3H, s, CH3N~)~; 3.20-2.65 (7H, m, CH2S, SH) and 2.32 ppm (3H, s~, CH3SO3).

*J.M. Lehn and J. Wagner. Tetrahedron, 26, 4227 (1970) :

.

, - ' ' , ' , .

~. : .

B. para-~itrobenzyl 3-[2-(N-methyl-thiomorpholiniUm diphenyl phosphate) ethylthio]-6~-[l'-tR)-hydroxyethyl]~7-o azabicyclo[3.2.0]-hept- 2-ene-2-carbo~ylate N ~) 2 J ~ 2 ) Cl~O ( ¢') 2 ~ 5 ~ \S
od~ N ~/ 3 ? ~5~ o N ~ ,~3 /
CO ~3 ~(e . C2P~ )2 2 4 ) E'~N (~ ) 2 A cold (ice bath) solution of para-nitrobenzyl 6~-[1'-(R)-hydroxyet~yi]-3,7-dioxo-1 azabicyclo[3.2.0]heptane-2-car~oxylate (557 mg, 1.60 mmol) in CH3CN (8 mL) was treated dropwise with diisopropylethyl amine (0.336 mL, 1.92 mmol) &nd diphenylchlorophosphate (0.400 mL, 1.92 mmol) and stirred for 30 min. The reaction mixture was treated with N-methyl-N-t2-mercaptoethyl)thiomorpholini~m methanesulfonate (~3 mg, 2,29mmol) in CH3CN (4 mL) and diisopropylethyl amine (0.336 mL, 1.92 mmoi) and stirred for 30 min. The solution W25 diluted with water (20 mL) a~d poured over a silica gel reversed phase column. The desired compound was eluted with a 50% acetonitrile-water mixture. The appropriate fractions were combined, pumped under ~acuum for 2 h. ~nd lyophilized to afford the title compound (1.01 g, yield 85%): ir t"Nujol~)vmax: 1760 (s, ~-lactam C=O) and 1510 cm 1 ~5, NO2); lHmr (DMSO-d6) ~: 8.25 (2H, d, i J=8.8 Hz, H-aromatic), 7.70 (2R, d, J=8.8 Hz, H-aromatic ), 7.33-6.84 (10 H, m, H-aromatic), 5.37 (2H, center of ABq, J=14.2 Hz, I CH2), 5.14 (lH, d, J=4.5 Hz, OH1, 4.35-3.80 (2H, m, H-l' and i H-5), 3.75-3.45 (6H, m, CH2N ), 3.31 (3H, s, CH3N ), 3.45-2.75 (9H, m, CH25, H-6 and H-4) and 1.15 ppo ~3H, d, J-6.2 Hz, CH3~.

`` ..~

~ :
.
.' 3~

C. 3-[2-(N-methyl-thiomor2holinium)ethylthio]-6~-~1'-(R)-hydroxvethyl]-7-oxo-1-azabicyclo[3.2.0]-he~t-2-ene-2-carboxylate . . .

. ~ , . 0~' U7 ~ Ue ~)2 , C)' .
A solution of ara-nitrobenzyl 3-[2-(N-methyl-thiomorpholinium diphenylphosphate)ethylthio]-6~-[1'-(R)-hydroxyethyl]-7-oxo-1-azabicyclo[3.2.03hept-2-ene-2-carboxylate (1.31 g, 1.76 mmol) ln O.lM pH 7.4 phosphate buffer (48.8 mL), tetrahydrofuran (20 mL) and diethyl ether ~20 ml) wzs hydrogenated over 10~ pd/C (1.5 g) in a"P ~ " ~ r for 1 h a~ 40 psi. The reaction mixture was diluted with diethyl ether ~40 mL) and the phases were separated~ The organic phase was washed with water ~2 x 5 mL). The aqueous phases were combined, riltered ~hrough a ~52 hardened filter paper, washed with diethyl ether (2 x 20 mL) and pumped under vacuum. The a~ueous 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, a~d lyophilized to gi~e title compound as an amorphous solid ~205 mg, 31%); ir ("~ujol'l)~max: 1750 (s, B-lactam C=O) and 1590 cm (s, C=O); lHmr (D2O) ô:4.25-3.95 (2H, m, H-l', H-5), 3.70-3.40 (6H, m, CH2N ), 3.35 (lH, dd, J-6.1 Hz, J=2.6 Hz, H-6)`, 3.08 (3H, s, CH3N ), 3.25-2.75 (8H, CH2S, H-4), znd 1.24 ppm (3H, d, J=6.4 Hz, C~3); uv (H2O, c 0.062)~max: 299 (~10,962) Tl/2 17.7 h (O.lM pH 7 phosphate buffer, 37C).

*Trademark ,~, Example 17 Preparation of (5R,6S)-3-[2-(1-me.hylmorpholino)ethylthio]-6-[(R)-l-hydroxyethyl]-7-oxo-1-azabicyclo~3.2.0]hept-2-ene-2-carboxylate JY Me ~
N

C02~

A. l-Methyl 1-(2-mercaptoethyl)morpholinium tri'].uoromethane-sulfonate C~3S03H ~ ~ HSC~2C~2N ~ O

Me C~3S039 To N-methylmorpholine (3.29 mL, 0.030 mol) was added dropwise trifluoromethanesulfonic acid (1.327 mL, 0.015 mol) at 10C, followed by ethylene sulfide (0.8g mL, 0.015 mol). The resulting yellow-brown solution was heated (oil bath) at 50-60C
under N2 for 18 h. Volatile material was then removed in vacuo and the~ residual oil was taXen up in 10 mL of H20. The aqueous solution was washed with diethyl ether ~3 x 5 mL) and then residual organic solvent was removed ln vacuo. The resulting aqueous solution was applied to a C18 reverse-phase column which was eluted with H2O, then 5~ acetonitrile-H2O and finally 10~
acetonitrile-H2O. Evaporation of the relevant fractions afforded i a white solid which was dried ln vacuo (P2O5) to give the produc~
(1.92 g, 41~). ir (KBr)~max: 25~0 ~SH) cm l; lHnmr (d6-acet~ne) ~: 4.25-3.6 (m, 8H), 3.49 (s, 3H, N-Me), 3.35-2.7 (m, 5H).

B. p-Nitrobenzyl (5R,6S)-3-[2-(1-methylmorpholino~ethylthio]-6-[(R)-l-hydroxyethyl]-7~oxo-1-azablcycloL3.2.0]hept-2-ene-2-carboxvlate diphenvl~hosphate OH OH M~
" ~ ~ " ~ N
L ~co ,> I />~ ~~ /
~ CO2PNB CO2PNB1l 3 (00) 2PO
To a solution of p-nitrobenzyl (5R,6S)-6-[(R)-l-hydroxyethyl]-3,7-dioxo-1-azabicyclo[3.2.0]heptane-2-carboxylate (0.348 g, 1.0 mmol) in 25 mL of dry acetonitrile was added drop-wise diisopropylethylamine t0.191 mL, l.l.mmol) and then diphenyl chlorophosphate (0.228 mL, 1.1 mmol) at 0C under ~2. A'ter stirring at 0C for 1 h diisopropylethylamine (0.226 mL, 1.3 mmol) was added to the resulting enol phosphate, followed by 1-me',hyl-1-(2-mercaptoethyl)morpholinium trifluoromethanesulfonate (O.373 g, 1.2 mmol). The reaction mixtuxe was stirred at room temperature for 1.5 h and then concentrated ln vacuo. The residual material was taken up in H2O and applied to a C18 reverse-phase column.
Elution with H2O, then 20~ acetonitrile-H2O and ~inally 30%
acetonitrile-H2O followed by lyophilization of the relevant fractions gave the product (0.360 g, 40%) as an amorphous solid.
ir (Lilm) 3300 (-OH), 1770 (~-lactam CO), 1700 (-CO2PNB) cm l;
Hnmr (d6-acetone) ~: 8.25, 7.80 (ABq, J=8.6 Hz, 4H, aromatic), 7.4-6.8 (m, 10H, diphenylphosphate), 5.56, 5.27 ~ABq, J=14.2 Hz, 2H, benzylic), 4.42 (d of t, J=9.2 Hz, J'=2.7 Hz, lH, H-5), 4.1-2.7 ~m, 17H), 3.4G (s, 3H, N-Me), 1.22 (d, J=6.2 Hz, 3H, -CHMe~.

'' .

C. (5R,6S)-3-~2-(1-methylmorpholino)ethylthio]-6-[(R)-1-hydroxvethyl~-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate ~3 2 ~) 2$~ C~2 -To a solution of p-nitrobenzyl (5R,6S)-3-[2~
methylmorpholino)ethylthio]-6-[(R)-l-hydroxye'~hyl]-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate diphenylphosphate ~0.360 g, 0.49 mmol) in 13 mL of phosphate buîfer (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 H20 and diethyl ether. The aqueous phase W2S separated and the pH
was adjusted to 7.0 with additional pH 7.4 phosphate buf~er.
A~ter removing residual organic solvents in vacuo the aqueous solution was applied to a C18 reverse-phase column. Elution with H2O and lyophilization of the relevant f-actions afforded 0.130 g of an amorphous solid. This ma~erial was repurified by reverse-phase hplc to give the pure product (0.058 g, 34%) as an amorphous solid. ir (KBr)vm~x: 3420 (br, OH), 1750 (~-lactam CO), 1590 (-CO2 ) cm 1; Hnmr (D2O) ~: 4.35-2.77 (m, 17~, 3.18 (s, 3H, N-Me), 1.23 (d, J=6.3 Hz, 3H, CHMe);
uv (H~O)~maX: 300 (~6344) nm; tl/2 (pH 7.4, 36.8C) 18.5 h.
.

- ~26~

ExamDle 18 Preparation of (5R,6S)3-[2-(1,4-dimethyl-1-piperazinium)-ethylthio]-6-[1-(R)-hydroxyethyl]-7-oxo-1-azabicyclo-[3.2.0]hept-2-ene-2-carboxylate OH
~ C~ ~

A. 1-(2-acetylthioethyl)-1,4-dimethylDi~erazinium bromide C~3CS r + _ ~ ~ _ Acetone C~ CS ~ N N-,8r 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 50C for 65 h. After cooling to 25C, 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) vmax: 1685 (C=O of thioester) cm l; lHmr (D2O) ~:
2.37, 2.39 (2s, 6H, CH3CO, ~ N-CH3), 3.18 (s, 3H, N ~ 3 ).

*B. Hansen, Acta Chem. Sca~d. 11, 537-40 (1957) 3~:~

B. 1 4-dimethyl-l-t2-mercaptoethyl)piperazinium bromide hydro-chloride CH3CS~'~_~'N ~ N - , Br ~ ~S ~ ~ - , Br , HCl A solution of 1-(2-acetylthioethyl)-1,4-dimethyl-piperazinium bromide (1.1 g, 3.7 mmol) in 6N hydrochloric acid (4 mL) was heated at 80C under a nitrogen atmosphere for 1 h.
The solution was concentrated under reduced pressure to give a white powder, 0.41 g (38~), lHmr (DMSO, d6) ~: 2.90 (s, ~H '~ CH3 ~ N~CH )~ ~ ~

Anal. calcd. for C8H20N2SBrCl~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.

'' ` ~

' ~. ' ' ' C. (5~,6O Paranitrobenzyl 3- r 2-(1 4-dimethvl-1-pi~erazinium)-ethylthio~-6-~l-(~)-hydrox~ethyl~-7-oxo-l-azabicyclo r 3.2.01-hept-2-ene-2-carboxylate di~henYlphosPhate O~ 1) NEt(iP~) 2 2) C~tOPh)2 ' COOPNB 3) ~S~~--, ~r , ~Cl 4) NEtli~r) 2 hO) 2 To a cold (0~C) solution of ~5R, 6S) paranitrobenzyl 6-~1-(R)-hydroxyethyl]-3,7-dioxo-l-azabicyclo[3.2.0]hePtane-2- (B) -carboxylate (0.465 g, 1.33 mmol) in acetonitrile (2 mL) kept under a nitrogen atmosphere was added diisopropylethylamine (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-dimethyl-1-(2-mercaptoethyl)-piperazinium bromide hydrochloride (0.40 g, 1.37 mmol) in acetonitrile (3 mL)-water (1 mL) mixture and diisopropylethylamine (0~278 mL, 1.59 mmol). After stirring for 18 h at 5C, cold water (15 mL) was added to the mixture. The resulting solution was chromatographed over a "PrepPak"-500/C-18 (Waters Associates) column (2.5 x 7.5 cm) with 25-35% acetonitrile in water as eluting solvents to give a yellowish powder 0.50 g (50%) after lyophilization; ir (KBr) ~max 1765 (C=O
of B-lactam), 1690 (C=O of PNB ester), 1585 (phenyl), 1512 (NO2), 875 (NO2)cm~l, lHmr (DMSO, d6) ~:1.16, 1.18 ~2d~, J=6.1 Hz, 3H, C_3CHOH), 2.44 (s, ~ N~-CH3), 3.14 (s, ~ ~ 3 ), 5.31 (d, J=6 Hz, OH), 5.39 (center of ABq, J=13 Hz, CH2 of PNB), 6.6-7.4 (m, 10H, phenyl of phosphate), 7.71 (d, J=8.8 Hz, 2H, Ho of PNB), 8.26 (d, J=8.8 Hz, Hm of PNB).

~ 2~2~

D. ~5R,6S)3-[2-(1,4-dimethyl-1-piperazinium) ethylthio] -6-[l-(R)-hydroxyethyl]-7-oxo-1-azabicyclo[3.2.0~hept-2-ene-2-carboxylate ~\N5~ PhO~ 2~ THI~ De~

COO

To a solution of (SR,6S)paranitrobenzyl 3-[2-~1,4-dimethyl-l-piperazinium)ethyl'hio]-6-[1-(R)-hydroxyethyl]-7-oxo-l-azabicyclo[3.2.0]hept-2-ene-2-carboxylate diphenylphosphate ~0.47 g, 0.623 mmol) in wet tetrahydrofuran (25 mL) was added diethyl ether (25 mL), potassium phosphate monobasic-sodi~m hydroxide buffer (13 mL, pH 7.22~ and 10% palladium on chzrcoal (0.47 g). The resulting mixture W2S hydrogenated at 23C under 40 psi for 1 h. The two layers were separated and the orsanic layer was extracted with water (2 x 7 mL). The a~ueous layers were combined, filtered through a Celite pad, washed with diethYl-ether (2 x 15 mL) and chromatographed on~PrepPak-500/C18 ~Waters Associates) column (2.5 x 9~5~cm~ ~lt fwater as eluting solvent to give, 0.097 g (43~) after ~y~ LLz~tiDn; ir (~Br) vmax:
3000-3700 (OH), 1750 (C-O of B-lactam), 1585 (carboxylate)cm 1, Hmr (D2O) ~: 1.24 (d, J=6.4 ~z, 3H, CH3CHOH), 2.33 (s, 3H, ~J N--CH3), 3.15 (s, ~ ~ ), 4.0-4.5 (m, H-5, CH3CHOH), uv (H2O) ~max 296 (~9476), ~a]D 61.1 (C 0.26, H2O), tl/2=12.4 h (measured at a concentration of 10 4 M in phosphate buffer pH 7.4 at 36.8C).

3%~

Example 19 Preparation o~ (SR,6S)-3-[2-(~-methyl-thiomorpholinium-oxide)ethylthio]-6-[1-(~)-hydroxyethyl]-7-oxo-l-azabicy clo(3.2.0)-hept-2-ene-2-carboxylate OH

~ ~ " /N ~ ~ -O

OH OH
\ mC~BA ~~\ (~

$S co ~ .

To~a cold (-10~C) solution of (SR,6S)-3-[2-(N-methyl-thiomorpholinium)ethylthio]-6-[1-(R)-hydroxyethyl]-7-oxo-1-azabicyclo(3.2.0)-hept-2-ene~2-carboxylate (608 mg, 1.65 mmol) in a 1:1 mixture of acetonitrile-water (9 mL) w2s added m-chloroper~enzoic acïd (334.8 mg, 1.65 mmol) in small portion over a l hour period. The mixture was then diluted ~~

.

, ~21~

with water (15 mL) and washed with diethyl ether (3 x 15 mL).
The aqueous phase W2S pumped under vacuum and passed through a reversed phase silica gel column (H2O) ~o give a solid which consisted of a mixture of compcunds. This mixture was separated by reversed phase HPLC and afforded fraction A 52.4 mg (yield 12%) and fraction B 23.6 mg (yield 6%) as diastereomers of the title com~ound; Fraction A: ir (nujol) vmax: 1750 (s, ~-lact~m C=O) and 1580 cm (s, C=O); Hmr (D2O) ~: 4.26-2.91 (20 H, m, H-4, H-5~ ~-6~ H-l', CH2S, CH2S-O, CH3-N and CH2N ) and 1.24 ppm (3H, d, J=6.4 Hz, CH3); uv (~2~ c 0-06) ~max 302 (~10425)i T 1/2`12 h ~0.065 M, pH 7.4 phosphate bufCer~ 37C). Fractio~ B:
ir (nujol) vmax: 1750 (s, 3-lactam C=O) and 1585 cm 1 (5, C=O);
Hmr (D2O) ~: 3.36-2.90 (17 H, m, H-4, H-5, H-6, H-l',.CH2S, CH2S-O, CH2N ), 3.25 (3H, s, CH3N ) and 1.24 ppm (3H., d, J=6.4 Hz, CH3); uv (H2O, c 0 05) ~max 2-99 (~6517); T 1/2:10.75 h tO.06; M, pH 7.4 buffer solution, 37C).

Exam~le 20 -Preparation of (5R,6S)-3-[2-(1,4,4-Trimethyl-1-piperazinium)-ethylthio~-6-[lR-hydroxyethyl]-7-oxo-l-azaDicyclo(3.2.o)hept 2-ene-2-carboxvlate chloride U~ \ ~ , C 1 o coo3 A~ 1-(2-acetylthioe hyl)-1,4,4-trimethYl~i~erazlniu~ bromideiodide 2'.eI ~ ~ ~ + N ', B~, I
/~ N + ~, Br ~ t CH3 S
CH3~S ~ \J ,~--OH, 55-60C

A suspension of 1-(2-acetylthioethyl)-~,4-dimethyl-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 5S-60C 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 dilu~ed with acetone until turbidity (70-80 mL). Two successive crystallizations gave 1.5 g, mp 220-5C dec., 68~ of the title com~ound; ir (XBr)~max: 1692 cm (C=O); Hmr (D2O) ~: 2.40 (s, 3H, CH3CO0), 3.37 (s, N-CH3), 3.39 (s, N-CH3), 3.99 (s); uv (H2O)~maX
226 (~13144). Anal. calcd for CllH24N20SBrI: C 30.08, H 5.51, N 6.38; round: C 30.48, H 5.53, N 6.86.

, 2~

B. l-t2-merca~toethyl)-1,4,4-trimethvlDiperaziniu~ b~schloride \ ~ / .Hcl 6N Per~utit ~ ~ /+
C~3CS ~ /N +, gr , I 5-1 Cl ~.S N~ , 2C:L

A mixture of 1-(2-acethylthioe~hyl)-1,4,4-trimethyl-piperazinium bromideiodide ~1.84~g, 4.19 mmol) and 6N hydrochloric acid ~15 mL) was heated at 57C under a nitrogen a~osphere for 2.5 h. The solution was concentrated under reduced pressure to dryness. The solid was"s~spended in water (10 mL) and the well-stirred suspension was treated with ~Permutit~ S-l Cl u~til a solution was obtained. The solution was pour.ed on a column (1.2 x 60 cm) ~ "Penmutit" S-l Cl . The column was eluted with water (1.5 mL/min). The appropriate fractions were combined and lyo?hilized to give a white powder, 0.93 g, mp l90-lglC, 85%; lr(''Nujol'')vmax:
2460 (SH); lHmr (D2C) ~: 3.4 (s, N-CH3), 3.45 (s, N-CH3), 4.07 (s).
Anal. calcd for CgH22N~SC12 0~75 H20: C 39.34, H 8.62, N 10.20, S 11.67; found: C 39.48, 3 8.39, N 10.55, S 11.15.

* trade mark.

. .
_ .
' ~9 :

.
':
.

, ~ .

C. (5~ 6~) paranitrobenzyl 3-[2-(1 4,4-trimethyl-1-piperazinium)-ethylthiol-6-(1~-hydroxyethyl1-7-oxo-1-azabicyclo(3.2.0)hept-2-ene-2-carboxylate bischloride 0~ Et(i?-)2 ~, ~ 2) Cl~(O?h)2 ~er~utit S-l Cl I ~--o6 ~ / , 3) N~ r?2~
.00?1~3 4~ XS~;~ N ~, 2Cl ~" ~ ~ N~
0 N ~ 2Ci To a cold (5~C) solution of (5R,6S) paranitrobenzyl 6-[lR-hydroxyethyl]-3,7-dioxo-1-azabicyclo(3.2.0)heptane-2R-carboxylate (0.94 g, 2.7 mmol) in acetonitrile (3mL) kept under a nitrogen atmosphere was added diisopropylethylamine (0.557 mL, 3.2 mmol) and diphenyl chlorophosphate (0.653 mL, 3.2 mmol). The reaction mixture was stirred at 5C for 30 min and treated with diisopropylethylamine (0.599 mL, 3.44 mmol) and an aqueous solution (4 mL) of 1-(2-mercaptoethyl)-1,4,4 trimethylpiperazinium bischloride (0.90 g, 3.44 mmol). After 1.25 h, diisopropylethylamine (0.1 mL, 0.57 mmol) was added and the stirring was continued for 2h.
A part of the acetonitrile was eliminated under reduced pressure and the resulting red mixture was chromatographed on a "PrepPak"*500/C-18 (Water Associates) column with 25-75%
acetonitrile in water as eluting solvent to give a yellowish powder (1.4 g) after lyophilization. The powder was solubilized in water and the solution was passed on a column (1.2 x 58 cm) of "Permutit"*
S-1 Cl- using water as an eluting solvent. Lyophilization of the appropriate fractions gave 1.17 g of powder that was repurified on a column of "PrepPak"*-500/C-18. Lyophilization of the appropriate fractions gave a yellowish powder, 0.80 g (53%); ir (KBr) vmax: 3400 ~8~

-134a-(br, OH), 1770 (C=O of the ~-lactam), 1690 (C=O of PNB ester), 1605 (aromatic), 1515 (NO2, 1345 (NO2) cm~1; lHmr (D2O) ~:1.26 (d, J=6.3 Hz, 3H, CH3CHOH), 3.39 (s, NCH3), 4.00 (s), 5.37 (br, s, CH2 of PNB), 7.60 (d, J=8.6 Hz, 2H, Ho of PNB), 8.20 (d, J=8.7 Hz, 2H, Hm of PNB); uv (H2O) ~max 276 (~12094), 306 (E10752). Anal. calcd.
for C25H36N4O6SCl2-3H2O: C 46.51, H 6.56, N 8.68, S 4.97, Cl 10.98;
found: C 46.31, H 6.18, N 8.57, S 5.36, Cl 11.37.

*Trademark . . . . . .

3~

D. (SR,6S)-3-[2-(1,4,4-trime~lyl-1-piperazinium)ethylthio~-6-~lR-hydroxyethyl]-7-oxo-l-az2bicyclo(3.2.0)hept-2-ene-2-carboxvlate chloride , 2Cl ~ ---+ ~ Cl CCOP~3 . COO
. ~

A mixture of (5~,6S) paranitrobenzyl 3-[2-(1,4,9-trimethyl-l-piperazini~)ethyl~hlo]-6-[lR-hydroxyethyl]-7-oxo-1-azabicyclo(3.2.0)hept-2-ene-2-carboxylate bischloride (0.40 g, 0.68 mmol), phosphate bu fer (30 mL, 0.0;M, pH 7.0), tetrahy~rofu~an (10 mL), ether (30 mL) and 10% palladium on charcoal (0.40 g) W25 hydrogenated at 23C under 35 psi for 1 h. The two phases were separated. The organic phase was extracted with wa.er (10 ~).
Th~ aqueous phases were filtered on a~Celite~ pad, washed wi~ e.he-(10 mL), concentrated under ~acuum to 10 mL and chromatogra?hed on a "PrePak" -SOO/C18 column (2.2 x 11 cm) with water 25 eluting solvent to give 70 mg (25~) after lyophilization n; ir (KBr)v~a~: 3400 (br, O~), 17;5 (C=O of the B-lactam), 1585 (carboxylate) cm 1; Hmr (D2O) ô:
1.24 ~3H, d, J=6.3 H2, CH3CHOH), 3.36 (s, NCH3), 3.98 ~s); uv (H20) ~max 296 (~7987); [~]D 35 9 (c, 0.30, H2O), Tl/2= 9.8 h (measured at a concentration of 10 M in phosphate buffer pH 7.4 at 36.8C).

~2~ ~ rk ', . ,, , . . . .. .

. . . ' , : ' ' ' ,, '

Claims (7)

1. A process for the preparation of a carbapenem derivative of the formula wherein R8 is hydrogen and R1 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, heteroaralkyl, 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 selected from the group consisting of C1-C6 alkyl optionally substituted by amino, halo, hydroxy or carboxyl halo -O?NR3R4 -?NR3R4 -NH?NR3R4 R3?NR4-=O
-O?R3 -?R9 -CN

-NR3?=NR4 wherein relative to the above-named substituents, the groups R3 and R4 are independently selected from hydrogen; alkyl, alkenyl and alkynyl, having from 1-10 carbon atoms; cycloalkyl, cycloalkylalkyl and alkylcycloalkyl, 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 1-6 carbon atoms, or R3 and R4 taken together with the nitrogen to which at least one is attached may form a 5- or 6- membered nitrogen-containing heterocyclic ring; R9 is as defined for R3 except that it may not be hydrogen; or wherein R1 and R8 taken together represent C2-C10 alkylidene or C2-C10 alkylidene substituted by hydroxy; A is cyclopentylene, cyclohexylene or C2-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 group, providing that when R2 is hydrogen or a protecting group, there is also present a counter anion; and R14 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 IV

wherein R1 and R8 are as defined above, R2' is a conventional readily removable carboxyl protecting group and L is a conventional leaving group, with a thiol compound of the formula VII
wherein A and R14 are as defined above and X.THETA. is a counter anion, in an inert solvent and in the presence of base, to produce a carbapenem product of the formula I' wherein R1, R8, R2', A, R14 and X.THETA. are as defined above and, if desired, removing the carboxyl protecting group R2' to give the corresponding de-blocked compound of formula I, or a pharmaceutically acceptable salt thereof.

2. The process according to Claim 1 wherein the base is a non-nucleophilic tertiary amine or a tri(C1-C4)alkyl-amine.

3. The process according to Claim 2 wherein the base is a non-nucleophilic tertiary amine.

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

5. The process according to Claim 1, 2 or 3 wherein the reaction is carried out at a temperature in the range of from about -15°C to +15°C.

6. The process according to Claim 1, 2 or 3, wherein the inert solvent is selected from the group con-sisting of acetonitrile, a mixture of acetonitrile, dimethyl-formamide, tetrahydrofuran, a mixture of tetrahydrofuran and water, a mixture of acetonitrile and water and acetone.

7. The process according to Claim 1, 2 or 3, wherein the inert solvent is selected from the group consisting of acetonitrile, a mixture of acetonitrile, dimethylforma-mide, tetrahydrofuran, a mixture of tetrahydrofuran and water, a mixture of acetonitrile and water and acetone, wherein the reaction is carried out at a temperature in the range of from about -15°C to +15°C.

(8) A process for the preparation of a carbapenem derivative of the formula I

wherein A is cyclopentylene, cyclohexylene or C2-C6 alkylene optionally substituted by one or more C1-C4 alkyl groups;
R2 is hydrogen, an anion charge or a conventional readily removable carboxyl protecting group, providing that when R2 is hydrogen or protecting group, there is also present a counter ion; and R14 is a quaternized nitrogen containing aromatic heterocycle attached to A
through a ring nitrogen, thereby forming a quaternary ammonium group, or a pharmceutically acceptable salt thereof, which process comprises reacting an intermediate of the formula wherein R2' is a conventional, readily removable carboxyl protecting group and L is a conventional leaving group, with a thiol compound of the formula wherein A and R14 are as defined above and X- is a counter ion, in an inert solvent and in the presence of base, to produce a carbapenem product of the formula X.THETA.

wherein R2' , A, R14 and X - are defined above and, if desired, removing the carboxyl protecting group R2' to give the corresponding de-blocked compound of formula I, a pharmaceutical salt thereof.