AU605491B2 - Quaternary amine thiol process - Google Patents

Description

r f"

AUSTRALIA

Patents Act 6 Vf'5 4 COMPLETE SPECIFICATION

(ORIGINAL)

Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Int. Class Related Art: This do:ument contains the amendments made under Section 49 and is correct for printing /PPLICANT'SREF.: Divisional of 25360/84 Name(s) of Applicant(s): f-R-S-T-OI--M-Y-E-R-S-GEOM-P-A-N-Y F ,gait'.L-- cv-( Sqo"sR coSoq- .'tFX, Address(es) of Applicant(s):. 345 Park Avenue New York, New York 10154 United States of America Actual Inventor(s): Pierre Dextraze Address for Service is: PHILLIPS, ORMONDE AND FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne, Australia, 3000 Complete Specification for the invention entitled: QUATERNARY AMINE THIOL PROCESS The following statement is a full description of this invention, including the best method of performing it known to applicant(s): A84 2 This invention relates to a process for the preparation of quaternary amine thiol compounds which may be used for the production of carbapenem derivatives claimed in Australian Patent Application 25360/84, from which the present invention is divided. The subject matter of Australian Patent Application 25360/84 is herein incorporated.

According to the present invention there is provided a process for the preparation of a quaternary amine thiol compound of the formula HS A R 14 VII XG wherein A is cyclopentylene, cyclohexylene or

R

1 0

R

1 2 I I -C C 111 113 R R 1 0 11 12 13 in which R 1 R R R are each independently hydrogen or C1-C 4 alkyl, X is a counter anion and

R

1 is represented by the general formula which defines a substituted or u26ubstituted mono-, bi- or polycyclic heteroaryl radical containing at least one N atom and optionally, one or more O, N or S atoms in the ring and 1 tattached to a carbon atom of A through a ring nitrogen, thereby forming a quaternary ammonium group which process comprises reacting a sulfide of the formula or R /R12

C-C

IR IR RIIRI3 3 wherein R R 1, R 12 and R 13 are each independently hydrogen or C 1

-C

4 alkyl with a strong acid and either a heteroaromatic amine of the formula 0 which represents a substituted or unsubstituted mono-, bior poly-cyclic aromatic heterocyclic radical containing at O1 least one N atom and optionally, one or more 0, N or S atoms in the ring and is capable of being quaternized and bonded to a carbon atom of A through a ring nitrogen or a heterocyclic amine of the formula

R

16 wherein represents a substituted or unsubstituted mono-, bi- or poly-cyclic non-aromatic heterocyclic radical containing at least one N atom and optionally one or more O, N or S atoms in the ring and is capable of being quaternized by R 6 and bonded to a carbon atom of A through a ring nitrogen and

R

16 represents either an optionally substitutedB aliphatic, cycloaliphatic, cycloaliphatic-aliphatic, aryl, araliphatic, heteroaryl, heteroaraliphatic., heterocyclyl or 4 heterocyclylaliphatic radical or a divalent phenylene or

C

1

-C

4 alkylene group joined to the

N

ring so as to form a bridged polycyclic group.

1 0 yurther, according to the present invention there is provided a quaternary amine thiol compound of the formula HS A R 1 4 VII X 9 wherein A is cyclopentylene, cyclohexylene or

R

1 0

R

1 2 R I C C-- R11 113 R R in which R 0, R R 12

R

13 are each independently hydrogen or C 1

-C

4 alkyl, X~ is a counter anion and 14 rr©0 R 4 is represented by the general formula t t-

N

r itt which defines a substituted or unsubstituted mono-, bi- or ,t polycyclic heteroaryl radical containing at least one N atom and optionally, one or more O, N or S atoms in the ring and attached to a carbon atom of A through a ring nitrogen, thereby forming a quaternary ammonium group.

The substituents encompassed by the term "substituted" as used herein the description and claims refers to C1-C 4 alkyl, C 1

-C

4 alkyl substituted by hydroxy, amino, carboxy or halogen, C 3

-C

6 cycloalkyl, C 1

-C

4 alkoxy, C 1

-C

4 alkylthio, amino, C 1

-C

4 alkylamino, di(C 1

-C

4 alkyl)amino; halo; C 1

-C

4 alkanoylamino;

C

1

-C

4 alkanoyloxy; carboxy; 0

II

A -C-OC 1

-C

4 alkyl; hydroxy; amidino, guanidino; trifluoromethyl; phenyl; phenyl

LS

A

/iVr O" J iht 5 substituted by one, two or three amino, halo, hydroxyl, trifluoromethyl, C 1

-C

4 alkyl or C 1

-C

4 alkoxy groups; heteroaryl and heteroaralkyl in which the hetero atom or atoms in the above-named heterocyclic moieties are selected from the group consisting of 1-4, O, N or S atoms and the alkyl moiety associated with heteroaralkyl has 1-6 carbon atoms.

The substituents encompassed by the term "substitutedl" as used herein the description and claims o1 refers to

C

1

-C

6 alkyl optionally substituted by amino, fluoro, chloro, carboxyl, hydroxy or carbamoyl; fluoro, chloro or bromo; -OR 2CO 2

R

3 3 -OCOR3 -OCONR R 4

-OSO

2 R3; -oxo; -NR R 3 4 R CONR t c -NR CO 2 R4 3 3 4 -NR3CONR3R -NR

SO

2 R4 -SR c 0 1 9 -S-R S 0 0 9

-S-R

-SO

3 R

-CO

2

R

3 2 3 4 -CONR R -CN; or phenyl optionally substituted by 1-3 fluoro, chloro, bromo, C.-C 6 alkyl, -OR -NR R 4 -S0 3

R

3 34 3 4 9 -CO2 R 3 or -CONR R wherein R R and R in such R 16 substituents are independently selected from hydrogen; alkyl, alkenyl and alkynyl, ha'ing from 1-10 carbona toms; cycloalkyl, cycloalkylalkyl and

LS

<cjrQ*(

J

5a 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 R and R 4 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 R 3 except that it may not be hydrogen.

R

14 is preferably a 5- or 6- membered aromatic heterocyclic radical containing a quaternized nitrogen atom (which is directly bonded to a carbon atom of the alkylene or cycloalkylene radical) and, optionally, one or more additional hetero atoms selected from O, N or S. While, in general, any heteroaryl radical bonded to A via a o 1 0 quaternized nitrogen atom is found to produce biologically active carbapenem derivatives, a preferred embodiment comprises compounds in which rse

I

represents a radical selected from the group consisting of ,y i iti' R 6

R

wherein R R and R are independently selected from hydrogen; C -C 4 alkyl; C1-C 4 alkyl substituted by hydroxy, amino, carboxy or halo; C 3

-C

6 cycloalkyl; C1-C4 alkoxy; C 1

-C

4 alkylthio; amino; C1-C 4 alkylamino; di(C 1

-C

4 alkyl)amino; halo; C 1

-C

4 alkanoylamino; C 1

-C

4 alkanoyloxy; carboxy;

O

-I

-C-OC

1

-C

4 alkyl; hydroxy, amidino; guanidino; trifluoromethyl; phenyl; phenyl substituted by one, two or three amino, halo, hydroxyl, trifluoromethyl,

C

1

-C

4 alkyl or C 1

-C

4 alkoxy groups; and heteroaryl and heteroaralkyl in which the hetero atom or atoms in the above-named heterocyclic moieties are selected from the group consisting of 1-4 oxygen, nitrogen or sulfur atoms and the alkyl moiety associated with said heteroaralkyl moiety has 1-6 carbon atoms; or wherein two of R 5

R

6 or

R

7 taken together may be a fused saturated carbocyclic ring, a fused aromatic carbocyclic ring, a fused saturated heterocyclic ring or a fused heteroaromatic ring, t t C t tiC

I

NN

N.

or optionally substituted on a carbon atom by one or more substituents independently selected from C 1

-C

4 alkyl;

C

1

-C

4 alkyl substituted by hydroxy, amino, carboxy or halogen; C 3

-C

6 cycloalkyl; C 1

-C

4 alkoxy; C,-C 4 alkylthio; amino; C 1

-C

4 alkylamino; di(C 1

-C

4 alkyl)amino; halo;

C

1

-C

4 alkanoylamino; C 1

-C

4 alkanoyloxy; 0.

i! carboxy; -C-OC 1

-C

4 alkyl; hydroxy; amidino, guanidino; trifluoromethyl; phenyl; phenyl substituted by one, two i b-

I

or three amino, halo, hydroxyl, trifluoromethyl,

C

1

-C

4 alkyl or C1-C 4 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 l [1 trI r o (N N optionally substituted on a carbon atom by one or more substituents independently selected from C 1

-C

4 alkyl;

C

1

-C

4 alkyl substituted by hydroxy, amino, carboxy or t halogen; C 3

-C

6 cycloalkyl; C 1

-C

4 alkoxy; C 1

-C

4 alkylthio; amino; C 1

-C

4 alkylamino; di(C 1

-C

4 alkyl)amino; halo;

S

t t t

C

1

-C

4 alkanoylamino; C 1

-C

4 alkanoyloxy; carboxy; 0

II

-C-OC

1

-C

4 alkyl; hydroxy; amidino; guanidino; trifluoro- V'fC methyl; phenyl; phenyl substituted by one, two or three "amino, halo, hydroxyl, trifluoromethyl, C 1

-C

4 alkyl or

C

1

-C

4 alkoxy groups; and heteroaryl or heteroaralkyl in which the hetero atom or atoms in the above-named hetercyclic 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; Cd) 4 7i r or optionally substituted on a carbon atom by one or more substituents independently selected from C-.C 4 alkyl; C1-C4 alkyl substituted by hydroxy, amino, carboxy or halogen;

C

3

-C

6 cycloalkyl; C1-C4 alkoxy; C1-C4 alkylthio; amino; C1-C4 alkylamino; di(C 1

-C

4 alkyl)-amino; halo; C 1

-C

4 alkanoylamino; C1-C 4 alkanoyloxv; carboxy; 0 -C-OC1-C4 alkyl; hydroxy; amidino; guanidino; trifluoromethyl; phenyl; phenyl substituted by one, two or three amino, halo, hydroxyl, trifluoromethyl, C1-C4 alkyl or C1-C4 alkoxy groups; r 4 and heteroaryl or heteroaralkyl in which the hetero atom or S. atoms in the above-named heterocyclic moieties are selected S t from the group consisting of 1-4 oxygen, nitrogen or sulfur 0* 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; 'is' or wherein X is O, S or NR in which R is C1-C4 alkyl or phenyl, ttsaid radical being optionally substituted on a carbon atom by t 4 one or more substituents independently selected from C1-C 4 alkyl; C1-C4 alkyl substituted by hydroxy, amino, carboxy or halogen; C3-C6 cycloalkyl; C 1

-C

4 alkoxy; C1-C4 alkylthio; amino; C1-C4 alkylamino; di(C1-C4 alkyl)amino; halo; C1-C4 alkanoylamino; C1-C4 alkanoyloxy; darboxy; 0

II

-C-OC

1

-C

4 alkyl; hydroxy; amidino; guanidino; trifluoromethyl; phenyl; phenyl substituted by one, two or three amino, halo, hydroxyl, trifluoromethyl, C 1

-C

4 alkyl or C -C 4 alkoxy groups; and heteroaryl or heteroaralkyl in which the hetero atom or atoms in the above-named heterocyclic moieties are selected 39 from the group JM I 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; f F N- X R N-N

XN

e or

X

wherein X is 0, S or NR in which R is C 1

-C

4 alkyl or phenyl, said radical being optionally substituted on a carbon atom by one or more substituents independently selected from C1-C 4 alkyl; C 1

-C

4 alkyl substituted by hydroxy, amino, carboxy or halogen; C 3

-C

6 cycloalkyl; Cl-C 4 alkoxy; C 1

-C

4 alkylthio; amino;

C

1

-C

4 alkylamino; di(C 1

-C

4 alkyl)amino; halo; C 1

-C

4 alkanoyl- S amino; C 1

-C

4 alkanoyloxy; carboxy; t IttII -C-OC -C 4 alkyl; hydroxy; amidino; guanidino; trifluoromethyl; phenyl; phenyl substituted C t C by one, two or three amino, halo, hydroxyl, trifluoromethyl,

C-C

4 alkyl or C 1

-C

4 alkoxy groups; and heteroaryl or heteroaralkyl in which the hetero atom or atoms in the above-named heterocyclic moieties are selected from the group consisting of 1-4 oxygen, nitrogen or sulfur atoms and the alkyl moiety associated with said heteroaralkyl moiety has 1-6 carbon atoms; tC C c and C t ==N ct C C

BR

L \e- N-N-R

N-R

or wherein R is C 1

-C

4 alkyl or phenyl, said radical being optionally substituted on the carbon atom by a substituent selected from C -C 4 alkyl; C 1

-C

4 alkyl substituted by hydroxy; amino, carboxy or halogen; C 3

-C

6 cycloalkyl; C 1

-C

4 alkoxy; C -C 4 alkylthio; amino; C -C 4 alkylamino; di(C 1

-C

4 alkyl)amino;

C

1

-C

4 alkanoylamino; carboxy; 0

-C

4 alkyl; hydroxy; amidino; guanidino; trifluoromethyl; phenyl, phenyl substituted by one, two or three amino, halo, hydroxyl, trifluoromethyl, C 1

-C

4 alkyl or

C

1

-C

4 alkoxy groups; and heteroaryl or heteroaralkyl in which the hetero atom or atoms in the above-named heterocyclic moieties are selected from the group consisting of 1-4 oxygen, nitrogen or sulfur atoms and the alkyl moiety associated with said heteroaralkyl moiety has 1-6 carbon atoms.

Within the above subclass, the preferred compounds are those in which substituent A is -CH2CH 2 -HCH2-,\ CH 3 or -CH CH-.

H

3 C r r 1- i A particularly preferred embodiment of the present invention comprises preparation of compounds wherein represents a radical of the formula 6

R

-N

N 7

R

in which R R and R are independently selected from the group consisting of hydrogen, C1-C alkyl, C1-C 4 alkoxy, S C 1

-C

4 alkyl substituted by a hydroxy group, C 1

-C

4 alkylthio, amino, carboxy and carbamoyl. Within this subclass, the preferred compounds are those wherein substituent A is -CH2CH2-," -CHCH or -CH 2

CH

SCH

3

CH

A most preferred embodiment of the present invention comprises preparation of compounds wherein I t S represents a radical of the formula R6 NI R 7

R

in which R, R 6 and R are independently selected from the group consisting of hydrogen, C 1

-C

4 alkyl, C -C 4 alkoxy, C1-C 4 alkyl substituted by a hydroxy group, Cl-C 4 alkylthio and amino. Within this subclass, the preferred compounds are those wherein substituent A is -CH2CH 2

-HCH

2 2 2y or -CH 2H-

CH

3 Another preferred embodiment of the present invention comprises preparation of compounds wherein -11-

-CH

2

CH

C )CH 3 H3C (10)

-CH

2 CH2 (11) -CHCH 2

N

CE 2 CH3 (12)

SCH

-CHCH2 2 2 3 R or S diastereoisomers (13) -CH2CH 2 or (14)

N

7 i, t f- .0 t "4 4 i

I

4 p n R,R or S,S diastereoisomers at two assymmetric carbons of the cyclohexyl group and R 2 is hydrogen, an anionic charge or a conventional readily removable carboxyl protecting group, providing that when R is hydrogen or a protecting group, there is also present a counter anion, and pharmaceutically acceptable acid addition salts thereof.

Another preferred class of quaternized R 1 substituents may be represented by the general formula 16

-N

in which R 16 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 cycloakyl ring and 1-6 carbon atoms in the alkyl moieties; phenyl; aralkyl, aralkenyl and aralkynyl wherein the aryl moiety is phenyl and the aliphatic portion has 1-6 carbon atoms; heteroaryl, heteroaralkyl, heterocyclyl and heterocyclylalkyl wherein the hetero atom or atoms in the above-named heterocyclic moieties are selected from the group consisting of 1-4

P,

A.-NO

represents _CH 2 CH2 -CH 2 CH 2 al CH OH C H 2 C H 2 aN -cH 2 CH 2 CH 3 Ct t Cr _CH 2 CH 2 N C3 (6

-CH

2

CH

2 0N CH c 2 OH (8) -CH CH 2 aN /F SCH 3 2CH Ct

C;

-CH 2

CH

2

OCH

3

H

3

C

-CH

2

CH

2 (10) -CH 2

CH

2

,N

CH

3 CH 3 (11) HCH2 N/ (12) -cH CH 2 3 R or S diastereoisomers

H

2 (13) -CH 2 CH 2 or (14)

U

R,R or S,S diastereoisomers at two assymmetric carbons of the cyclohexyl group and R 2 is hydrogen, an anionic charge or a conventional readily removable carboxyl protecting group, providing thatwhen R is hydrogen or a protecting group, there is also C present a.counter anion, and pharmaceutically acceptable acid addition salts thereof.

14 Another preferred class of quaternized R substituents may be represented by the general formula

R

1 6 C t C in which R 16 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 cycloakyl 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, Q L 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 DW I 14 oxygen, nitrogen or sulfur atoms and the alkyl moieties associated with said heterocyclic moieties have 1-6 carbon atoms; wherein the above-named R radicals are optionally substituted by 1-3 substituents'.

R may also preferably represent a divalent phenylene or C 1

-C

4 alkylene group joined to the ring so as to form a bridged polycyclic group wherein tCI- (r ii i r re e r C t ;i 1

-N

I

represents a substituted or unsubstituted mon-, bi- or polycyclic non-aromatic (which may be fused tc. another aromatic or non-aromatic ring) heterocyclic radical containing at least one nitrogen in the ring and attached to A through a ring nitrogen, thereby forming a quaternary ammonium group. The heterocyclic radical may be saturated or unsaturated (with 1-2 double bonds) and may contain up to two additional hetero atoms in addition to the quaternary nitrogen, such additional hetero atoms being selected from 0, S(O) m N, NR 1 5 or NR R 18 15 wherein m is 0, 1 or 2, R is hydrogen, optionally substituted C1-C 6 alkyl or optionally substituted phenyl and R 1 7 and R 1 8 are each independently optionally substituted C 1

-C

6 alkyl or optionally substituted phenyl.

In a preferred embodiment represents a non-aromatic 4-7 membered, preferably 5- or 6membered,.N-containing heterocyclic ring containing 0-2 double bonds and 0-2 additional hetero-atoms selected from 0, S(O) S NRi5 7 18 N, NR or NR R wherein m is 0, 1 or 2, R is hydrogen,

SC

1

-C

6 alkyloptionally substituted by 1-2 substituents independently selected from -OR 3

-NR

3

R

4

-CO

2 R 3 oxo, phenyl, 3 34 t fluoro, chloro, bromo, -SO 3 3R and -CONR R or phenyl optionally substituted by 1-3 substituents independently selected from

C

1

-C

6 alkyl, -OR 3 -NR3 R 4 fluoro, chloro, bromo, -SO 3 R3 C 2 3 4 17 is -CO2 R 2 and -CONR R and R and R are each independently C 1" C 1

-C

6 alkyl optionally substituted by 1-2 substituents independently selected from -OR 3 -NR3 R 4

-CO

2 R oxo, phenyl, fluoro, chloro, bromo, -SO 3

R

3 and -CONR3 R 4 or phenyl optionally substituted by 1-3 substituents independently selected from BR -iQ 3 3 4 3 C -C alkyl, -OR 3

-NR

3

R

4 fluoro, chloro, bromo, -SOR 3 3.62 34 3 4 -CO R and -CONR3R4, wherein R and R in such heterocyclic 33 3.7 18 NR or NR 17R groups are as defined above in connection with 16 the R16 substituent. In such preferred embodiment the ring may be optionally substituted by 1-3 substituents independently selected from

C

1 -C alkyl optionally substituted by 1-2 substituents 3 independently selected from fluoro, chloro, bromo, -OR 3 3 4 3 4 3 4 3 3 4 S, -R 3 -OCONR3R oxo, -NRR -NR 3 COR -NR CONR 3

R

4 3 4 3 3 3 3 4

-NR

3 so R -SR, -SO3R ,-CO 2 R and -CONR R 2 3 2

C

2

-C

6 alkenyl optionally substituted by 1-2 substituents 3 independently selected from fluoro, chloro, bromo, -OR

-OCOR.

3

-OCON

3

R

4 R oxo, -NR 3 4 3

C

4

-NR

3

CONR

3

R

4 3 4 3 34 2 R -SR -SO 3 R -COR and -CONR 3.R4 s2 R S so3 R n :t C 2

-C

6 alkynyl optionally substituted by 1-2 substituents iii. 3 independently selected from fluoro, chloro, bromo, -OR 3 3 4 3 4 3 4 3 34

-OCOR

3

-OCON

3

R

4 oxo, -NR R -NR3COR -NR CONR P I3 4 3 3C,~ 3 4 -NR. SO 2 -SR3 -SO 3 3 R CO2 3 and -CONR 3R4 It I

C

3 cycloalkyl optionally substituze. bv 1-2 substituents 3 6 independently selected frcm flucro, chizro, bromno, -OR 3 34 34 3 4 3 34

-OCOR

3 -OCONR oxo, -N -NI CONR P 2R SR s and -CON?.R -NR C 2

P

4

-SR

3 -SO 3-C23 an CN3R cycloalkylalkyl having 3-6 carbon atoms in the cycloalkyl ring and 1-6 carbon atoms in the alkyl moiety, optionally substituted by 1-2 substituents independently selected 3 3 3 4 from fluoro, chloro, bromo, -OR, -OCOR 3 -OCONR R Oxo, 3 43R 3 4 3 3 4 3 4 R3 -s

-NP.R

4 -NR COR -NR 3

CONR

4

-N

3

SO

2 R -SR -SO3 3 II 34

-CO

2

R

3 and -CONR R. heteroaryl wherein the hetero atom or atoms are selected from the group consisting of 1-4 oxygen, nitrogen or sulfur atoms, optionally substituted by 1-2 substituents L Iindependently selected from fluoro, chloro, bromo, -OR

-OCOR

3 -OCONR3 4 oxo, -NR 3

R

4

-NR

3

COR

4

-NR

3

CONR

3

R

4

-NR

3 SO R 4

-SR

3 -S0 3

R

3 -C0 2

R

3 and -CONR 3 R 4 preferred 2 heteroaryl radicals are 5- or 6-membered aromatic hetero-

BR

I

.0 cyclic rings; heteroaralkyl wherein the hetero atom or atoms are selected from the group consisting of 1-4 oxygen, nitrogen or sulfur atoms and the alkyl moiety has 1-6 carbon atoms, optionally substituted by 1-2 substituents independently selected from fluoro, chloro, bromo, -OR -OCOR 3

-OCONR

3

R

4 oxo, -NRR 4

-NRCOR

4

-NR

3

CONR

3

R

4

-NR

3

SO

2

R

4

-SR

3

-SO

3

R

3 -CO R3 and -CONR3R 4 preferred heteroaralyl are those in which the heteroaryl radical is a 5- or 6membered aromatic heterocyclic ring and the alkyl moiety has 1-2 carbon atoms; heterocyclyl wherein the hetero atom or atoms are selected from the group consisting of 1-4 oxygen, nitrogen or sulfur atoms, optionally substituted by 1-2 substituents independently selected from fluoro, chloro, bromo, -OR 3

-OCOR

3 -OCONR3R 4 oxo -NRR 4

-NRCOR

4 -NR3CONR 3

R

4 -NR3SO 2

R

4

-SR

3

-SO

3 R3, -CO 2 R3 and -CONR R 4 preferred heterocyclyl are 5- or 6-membered saturated or unsaturated rings; heterocyclylalkyl wherein the hetero atom or atoms are selected from the group consisting of 1-4 oxygen, nitrogen *t r« or sulfur atoms and the alkyl moiety has 1-6 carbon atoms, Soptionally substituted by 1-2 substituents independently 3 3 •1t t selected from fluoro, chloro, bromo, -OR -OCOR 34 34 3 4 3 34 3 4 -OCONR3R 4 oxo, -NRR 4 -NR COR 4 -NRCONRR -NR3SO R 3 3 3 34

-SR

3

-SO

3

R

3

-CO

2 R and -CONR R 4 preferred heterocvclylalkyl are those in which the heterocyclyl moiety is a or 6-membered saturated or unsaturated ring; S fluoro, chloro or bromo; S(k) -OR 3 S -OCO 2

R

3 3 -OCOR

-OCONR

3

R

4 -OSO R3 S oxo; 3 4 S\ -NR R

R

3

CONR

4 -NR3CO 2

R

4

BR

-7 ~1i:;

-NR

3

CONR

3

R

4 -NR3SO2R 4 32 -SR

-S-R

0 0 R 9

-S-R-

-S0 3 R -CO2R 3 (aa) -CONR 3

R

4 (bb) -CN; or (cc) phenyl optionally substituted by 1-3 fluoro, 3 34 3 3 chloro, bromo, C 1

-C

6 alkyl, -OR 3 -NR R 4

-SO

3 R3, -CO 2

R

3 4 or -CONR R 3 4 9 The R R and R substituents mentioned above are as defined in connection with substituent R The -0

N

t t t I J tI r S c 3 S f t If 't It t e 30 t gr t C c t (t

I

S P S1 ring as defined above is a non-aromatic heterocycle group.

This ring, however, may be fused to another ring which may be a saturated or unsaturated carbocyclic ring, preferably a C 4

-C

7 carbocyclic ring, a phenyl ring, a 4-7 mebered heterocyclic (saturated or unsaturated) ring containing 1-3 hetero atoms selected from 0, N, S(O) m 17 18

NR

1 or NR1 R or a 5-6 membered heteroaromatic ring containing 1-3 hetero atoms selected O, S(O) N, NR 1 17 18 15 17 18 or NR R in which m, R R and R are as defined above.

The R 16 substituent of the non-aromatic R 1 radical may be either an optionally substituted

C

1 -Cg alkyl, C 2

-C

10 alkenyl, C 2

-C

10 alkynyl, C 3

-C

6 cycloalkyl, C 3

-C

6 cycloalkyl-C 1

-C

6 alkyl, phenyl, phenyl-C 1

-C

6 alkyl, phenyl-C 2

-C

6 alkenyl, phenyl-C 2

-C

6 alkynyl, heteroaryl, heteroaralkyl in which the alkyl moiety has 1-6 carbon atoms, heterocyclyl or heterocyclylalkyl in which the alkyl moiety has 1-6 carbon atoms or a divalent phenylene or C -C 4 alkylene group I r joined to the ring so as to form a bridged ring polycyclic group, e.g. a quinuclidine group. The heteroaryl (or heteroaryl portion of heteroaralkyl) substituent may be a mono- bi- or polycyclic aromatic heterocyclic group containing 1-4 0, N or S atoms; preferred 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 and pyrazolyl. The heterocycly.

(or heterocyclyl portion of heterocyclylalkyl) substituent may be a mono-, bi- or polycyclic saturated or unsaturated non-aromatic heterocyclic group containing 1-4 0, N or S atoms; preferred are 5- or 6-membered heterocyclic rings such as morpholinyl, piperazinyl, piperidyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, pyrrolinyl and pyrrolidinyl.

In the case where the RP1 substituent is an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, t phenyl, phenylalkyl, phenylalkenyl, phenylalkynyl, heteroaryl, heteroaralkyl, heterocyclyl or heterocyclylt *t alkyl group, such groups may be optionally substituted by 1-3 substituents independently selected from: Crtr(a) C 1

-C

6 alkyl optionally substituted by, preferably ttkt t.1-3, amino, fluoro, chloro, carboxyl, hydroxy or carbainoyl groups; fluoro, chioro or bromo; -OR3 -0C0 2 R 3

-OCOR

3 -OCONR 3R4 -S0 2 R 3 -oxo; -R3 R4 R 3CONR 4_ Wk -NR 3Co 2 R4 -NR 3 CONR 3

R

4 11- -NR3SO 2

R

4

-SR

3 Co) -SOR -S02

R

9

-SO

3

R

3 -CO2R 3

-CONR

3

R

4 -CN or phenyl optionally substituted by 1-3 substituents independently selected from fluoro, chloro, bromo, 3 34 3 3 34 C1-C 6 alkyl, -OR 3 -NR R -SO 3 -CO R 3 or -CONR3R, wherein, relative to the above-named R substituents, the groups R 3 and R 4 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 S* 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 0 portion has 1-6 carbon atoms; and heteroaryl, heteroaralkyl, heterocyclyl and heterocyclylalkyl 8. wherein the heteroaryl and heterocyclyl group or 16 portion of a group is as defined above for R and the alkyl moieties associated with said heterocyclic t moieties have 1-6 carbon atoms; or R 3 and R 4 taken together with the nitrogen to which at least one is attached may form a 5- or 6-membered nitrogenr containing heterocyclic (as defined above for R S9 3 ring; and R is as defined above for R except that 16 it may not be hydrogen. A most preferred R 4* substituent is C 1

-C

6 alkyl, especially methyl.

16 In the case where R 1 is a divalent phenylene or C 1

-C

6 alkylene group, such group is bonded to another atom of the ring so as to form a bridged polycyclic ring, e.g. a quaternized quinuclidine ring of the formula 0 A particularly preferred embodiment of the present invention comprises preparation of compounds of Formula I wherein 16 e represents St .4 It CH3

Y

3

CHN-CH

.9 C 'H 3

CH

3

Y

CH

j; 3

CH_

t4 9?

Y

CH CH3 C -H

CH

or

I

I

S 3 4 Sf 4O 4 5'

I

0 4 iSA wherein Y is hydrogen, C -C 6 alkyl, hydroxy, -SC -C 6 alkyl, carboxyl, carbamoyl, chloro, bromo, iodo, fluoro or phenyl.

Within this subclass, the preferred compounds are those wherein A is -CHCH 2

-CH

2 CH- or CH3CH 3

CH

3

-(CH

2 n in which n is 2, 3 or 4, more preferably those in which A is -CH 2

CH

2

-CH

2

CH

2

CH

2

-CHCH

2

CH

3 or -CH 2

CH-

CH

3 and most preferably those in which A is -CH 2

CH

2

F-

VW

A still more preferred embodiment of the present invention comprises preparation of compounds of L'o,;.mula VII wherein R 1 6 represents r C t 9

C

CH

3 3\~

C""

3 j Li CH 3 CHi, J1 CH 3 or 1 Within this preferr~.d subclass, the preferred compounds are those wherein A is -CHCH 2

,,-CH

2

CII-

CH

3 CH 3 C t 2.1.

or -(CH 2 in which A is 2, 3 or 4, more preferably those in which A is -CH CH -,C-CH 2 -CHCH-,H2

H

or -CH CH-, and

CH

3 most preferably those in which A is -CH2CH2- A still more preferred embodiment of the present invention comprises preparation of compounds of Formula VII wherein 16

R

represents CH3 in which Y is hydrogen, C -C 6 alkyl, hydroxy, -S-C1-C 6 alkyl, carboxyl, carbamoyl, chloro, bromo, iodo, fluoro or phenyl.

S Within this preferred subclass, the preferred compounds are those wherein A is -(CH2) in which n is 2, 3 or 4, most preferably those in which A is -CH2CH2-.

A most preferred embodiment of the present invention comprises preparation of compounds of Formula VII wherein

R

1 6 represents ,2 n Within this preferred subclass, the preferred compounds are those wherein A is -(CH 2 in which n is 2, 3 or 4, most preferably those in which A is -CH 2 CH2-.

In the process described and claimed in Australian Patent Application 25360/84, carbapenem intermediate JW -23i IV is reacted with a quaternazy amine thiol compound of th2e formula HS-A R1

VII

C it z 9 t C C CC t C r t C t CC t C wherein A is cyclopentylene, cyclohexylene or C 2 -C 6 alkylene optionally substituted by one or more C 1

-_C

4 alkyl groups, most preferably cyclopentylene, cyclohexylene or R R01 Ill R 1 12 in which R 0, R 1, R 12and R 13 are each independently hydrogen or C 1

-C

4 alkyl, Xis a counter anion associated with a strong acid such as cl-, Br-, CH 3 so 3

CF

3 S0 3 -or CH 3 -o so3 and R 14is a quaternized nitrogen-containing aromatic or nonaromatic heterocycle as defined above. The reaction is carried out in an inert solvent such as acetonitrile, acetonitrile-dimethylformamide, tetrahydrofuran, tetrahydrofuran-H 2 0, acetonitrile-H 2 0 or acetone in the presence of base. The nature of the base is not critical.

Best results, however, have been obtained when a nonnuc-leophilic tertiary amine base such as diisopropylethylamine, l,8-diazabicyclo[5.4.Olundec-7-ene, f4.3.O1non-5-ene or a tri(C 1 -C 4 )alkylamine such as triethylamine, tributylamine or tripopylamine is employed.

Reaction of intermediate IV with thiol VII may be carried out over a wide temperature range, e.g. -15 0 C up to room temperature, but is preferably done at a temperature in the -range of from about -150C to +15 0 C, most preferably at around 0OC.

Uk.

Certain of the thiol intermediates of Formula VII may be prepared,-for example,.by reacting a sulfide of the formula o r VII~aVITIb Vrll t t t t it

C

It C I.

I

S

10 C/ C 12 R -C-C-R 11 \13 R R VIIIc wherein R 1 0

R

1 1 R and R 1 3 are each independently hydrogen or C 1

-C

4 alkyl with a heteroaromatic amine (as defined above) of the formula 100 C t t VC C fr C r rrr 39 or a non-aromatic heterocyclic amine (as defined above) of the formula 16 and a strong acid. The reaction may be carried out in the presence or absence of an inert organic solvent which is preferably a non-polar organic solvent such as methylene chloride, benzene, xylene, toluene or the like. Where the amine and sulfide reagents are liquids or where a solid amine is soluble in a liquid sulfide reagent, it is preferred to carry out the reaction without use of an additional solvent.

The particular strong acid used in the reaction is not critical and may be, for example, such strong inorganic or organic acids as hydrochloric, hydrobromic, methanesulfonic, p-toluenesulfonic, trifluoromethanesulfonic, etc.

Formation of the quaternary amine thiol intermediate VII may be carried out at a temperature in the range of from about -200C to about 100 0 C. Preferred temperatures are generally in the range of about 50-700C.

The sulfide reagent, aromatic amine and acid are preferably employed so that the sulfide and acid are used in approximately equimolar amounts with the amine being used in excess, e.g. two to three moles of amine per mole of sulfide or acid.

The quaternary amine thiol intermediate will have a counter anion associated with it which will be determined by

SI.

-2.6the particular acid employed. It is, of course, possible to substitute at this point a different counter anion by conventional procedures for use in the subsequent reaction with carbapenem intermediate IV.

C

CCZ

C r f tt

I

C C r r g It s, t t c c I t L I C C t I: tC' C C JW -2- U Ij i~I JWj -27 i: The following examples illustrate but do not limit the scope of the present invention.

Example 1 Preparation of 3-(2-(l-pyridinium)ethylthio)- 6a-(l-()-hydroxyethyl)-7-oxo-l-azabicycle hept-2-ene-2-carboxylate A. 1-(2-mercaptoethyl)pyridinium methanesulfonate MsOH 55°C, 16 550C, 16 h HS

MSO

'r.

Cr r i 9 14 4 To a suspension of pyridinium methanesulfonate 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 55 0 C 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 os a column (40 x 16 cm) of u-bondapak C-18 which was eluted with water.

Lyophylization of the appropriate fractions gave a colourless syrup 6.5 g ir (film) vmax: 2300-2600 (br, sH), 1635 (pyridinium), 1490, 1200 (sulfonate), 1068, 1060, 1045, 791, 780 cm-, 1 Hmr (DMSO-d 6 6: 2.32 (3H, s, CH 3

SO

3 2.61, 2.70, 2.73, 2.82 (1H, B part of

A

2 B system, SH), 3.07 (2H, m [with D 2 0, 3.08 (2H, t, J=6.

CH 4.76 (2H, t, J=6.5 Hz, CH 8.19 (2H, m, Hm of pyridinium), 8.6 (1H, m, Ho of pyridinium), 9.08 (2H, dd, J=6.8 Hz, J=1.4 Hz, Ho of pyridinium), uv (H 2 0) ma: 206 (e5230), 258 (c3760) mu.

mar 1 o -6 37 18 E CO 2 R and -CONR3R4 and R7 and R18 are each independently _C0 2 Ritent

C

1

-C

6 alkyl optionally substituted by 1-2 substituents C I 6 3 _N3 C oxo, phenyl, independently selected from -OR 3

-NR

3

R

4

-CO

2 R, oxo, phenyl, 11 3 and -CONRptonll fluoro, chloro, bromo, -SO 3

R

3 and -CONR 3

R

4 or phenyl optionally substituted by 1-3 substituents independently selected from

BR

METHOD A B. 1-(2-mercaptoethyl)pyridinium chloride H 0 N MsO Permutit S-I Cl, An aqueous solution of crude l-(2-mercaptoethyl)pyridiniumn methanesulfonate (9.4 g, 0.04 mol) was poured 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 lyophylized giving a yellowish syrup 7.0 g (100%) which was used as it was for the next step, 1 Hmr (D 2 0) 6:3.22 (2H, m, CH 2 4.88 CH 2 N 8.18 (2H, m, Hm of pyridinium), 8.7 (1H, m, Hp of pyridinium), ppm (2H, m, Ho of pyridinium).

METHOD B NH HS N Cl Cl up To a precooled (ice bath) pyridine (5.6 mL, 70 mmol) was S added pyridine hydrochloride (4.05 g, 35 mmol) and ethylene S sulfide (2.1 mL, 35 mmol). The mixture was heated at 65 0 C and S stirred for 75 min to give a two phases system. The lighter phase was removed. The remaining oil was washed with ether x 10 mL) and pumped under high vacuum to give the title compound (90-100%) which was used as such for the next step.

C. Paranitrobenzyl 3-[2-(1-pyridinium)ethylthio])-6a-l-(R)- 7 7-, hydroxyethyll-7-oxo-l-azabicyclo(3.2.0)hept-2-ene-2-carboxylate S chloride 1- NEt(iPr)

OH

0I *0 3- Hs C1 Cl COOPNB COOPNB 4- NEt(iPr) VM 4, sulfur atoms, optionally substituted by 1-2 substituents c3 independently selected from fluoro, chloro, bromo, -OR -oCOR 3 -OCONR3R 4, oxo, -NR3 R -NR COR4, -NR CONR 3

R

4

-NR

3 SO R 4

_SR

3 -SO R 3 -CO 2 3 and -CONR 3

R

4 preferred 2' 3' 2 heteroaryl radicals are 5- or 6-membered aromatic hetero-

BR

-Jb A solution of p-nitrobenzyl 6 -[1-(R)-hydroxyethyl]-3, 7-dioxo-l-azabicyclo heptane-2-carboxylate (6.09 g, 17.5 mmol) in acetonitrile (20 mL) was cooled to +50C 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 5 0 C, cooled to -S*C and treated successively with a solution of crude 1-(2-mercaptoethyl) pyridinium chloride (4.3 g, 24 mmol) in N,N-dimethylformamide 0 (1.0 and dropwise with diisopropylethylamine (3.65 mL, 21.0 mmol). The reaction mixture was stirred at OOC for 1 h cooled to -300C and stirred for 15 min. more. The solid was filtered off and washed with cold (-30 0 C) acetonitrile 5.77 g ir (nujol) vmax:3300 1775 (C=O ofB-lactam), 1690 (C=O of PNB ester), 1630 (pyridinium), 1605 (phenyl of PNB ester), 1315 (NO 2 1335 cm 1

(NO

2 1 Hmr (DMSO-6 6 6:1.17 (3H, d, J=6.1 Hz, CH 3 CHOH), 3.2-3.75 (5H,H-4, H-6, CH 2

S),

3.75-4.5 (2H, H-5, CH 3 CHOH), 4.92 (2H, brt, J=6.5 Hz, CH 2 N 3.18 (IH, d, J=4.9 Hz,0H), 5.37 (center of ABq, Ja ,b= 14 .2 Hz,

CH

2 of PNB), 7.69 (2H, d, J=8.7 Hz, Ho of PNB) 8.24 J=8.7 a ~Hz, Hm of PNB), 8.0-8.4 (4H, Hm of PNB, Hm of pyridinium), (itt c 8.66 m, Hp of pyridinium), 9.17 brd, J=5.5 Hz, Ho Sof 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 p-bondapak C-18. 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 lyophylized after the acetonitrile has been removed under vacuum giving a yellowish powder. An NMR of it t C C: C showed the presence of the title compound mixed with some pt"e nitrobenzyl 3-(2-(l-pyridinium)ethylthio]-6a-(l-(R)-hydroxyethy1(-7-oxo-l-azabicyclo(3.2.0)hept-2-ene-2-carboxylate diphenylphosphate The powder was dissolved in water C (Minimum amount) and passed through a column (1.5 x 21 cm) of permutit S-1C1 with water. Lyophylization of the appropriate fractions gave 1.8 g of the title compound.

D. Paranitrobenzyl 3-(2-(1-pyridinium)ethy1thiol-

-SO-

I

6a-[1-(R)-hydroxyethyl]- 7 -oxo-l-azabicyclo (3.2.0)hept-2-ene- 2-carboxylate diphenylphosphate.

1) NE (iPr)2 OH 0 OOP B 2) C1P(OPh)N N3 H 2 _M sS COOPN HSMSO 0 COOPNB 4) NEt(iPr)2 (Ph)2 A solution of p-nitrobenzyl 6a-[1-(R)-hydroxyethyl]- 3,7dioxo-l-azabicyclo(3.2.0)heptane-2-carboxylate (0.174 g, 0.50 mmol) in acetonitrile (2 mL) was cooled to 0 0 C 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 0 0 C and treated successively with a solution of l-(2-mercaptoethyl)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 0 0 C for 15 min, diluted with cold (0 0 C) water (7 mL) and poured on top of a column (1.5 x 6.4 cm) of p-bondapak C-18. The column was eluted with a mixture of acetonitrile (25%-50%) in water The appropriate fractions were combined and lyophylized after the acetonitrile has been removed under vacuum giving a yellowish powder 0.33 g ir (KBr) vmax: 3600-3000 1765 (C=O of 3-lactam), 1690 (C=O of PNB ester), 1625 (pyridinium), 1585 (phenyl), 1510 (NO 2 -1 1 1330 (NO 2 885 cm (NO 2 Hmr (DMSO-d 6 S:1.16 (3H, d, J=6.2 Hz, CH 3 CHOH), 4.87 (2H, brt, J=6.6 Hz, CH 2 5.37 (center of ABq, J =14.3 Hz, CH 2 of PNB), 6.7-7.5 (phenyl), -a,b 7.68 J=8.8 Hz, Ho of PNB), 8.23 J=8.8 Hz, Hm of PNB), 8.0-8.3 Hm of pyridinium), 8.4-8.8 (111, Hp of pyridinium), 9.09 (2H, dd, J=6.7 Hz, J=1.3 Hz, Ho of pyridinium).

E. 3-[2-(1-pyridinium)ethylthio]-6a-1-(R)-hydroxyethyl]-7oxo-1-azabicyclo 0)hept-2-ene-2-carboxylate c e C C t, fe -31- Method A OH Pd/C, H THF, Ether

COO

COOPNB (PhO)

PO

COOPNB 2 To a solution of p-nitrobenzyl 3-(2-(-pyridinium) ethylthio]-6c-(l-(R)-hydroxyethyll-7-oxo-l-azabicyclo(3.2.0) hept-2-ene-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 The resulting mixture was hydrogeneated under 40 psi for 1 h at 0 C. 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 W-bondapak C-18 after the traces of organic solvents have been removed under vacuum.

Elution of the column with water gave after lyophylization of f the appropriate fractions a yellowish powder 0.062 g 'I f it (KBr) v 3700-3000 1755 (C=O of 8-lactam), max S-1 1 1630 (pyridinium), 1590 cm (carboxylate), Hmr (D 2 1.22 (3H, d, J=6.4 Hz, CH 3 CHOH), 2.92 3=9.1 Hz, 2.97 J=9.1 Hz, 3.20 (dd, J=2.5 Hz, J=6.1 Hz, 3.44 S J=6.0 Hz, CH 2 3.93 (dd, J=9.1 Hz, J=2.5 Hz, 4.82 J=6.0 Hz, CH 2 8.04 Hm of pyridinium), 8.5 Hp of pyridinium), 8.82 (dd, 3=3.2 Hz, 3=1.1 Hz, Ho of pyridinium), uv (H 2 0) Xmax:259 (e5800), 296 (E7030) mu, t =13.5 h -4 .e (measured at a concentration of 10 M in phosphate buffer pH 7.4 at 36.8 0

C).

Method B OH

OH

r0 C- 10% Pd/C,H

A

THF, ether, O Cl T+0 0 buffer 7.2 coo

COOPNB

W0 e* 04

VM

I C -OS0 2 R i -oxo (i)-NR3 4 3 4 R CONR 3 4

-NR

3 CO R -NR CONR R

DW

To a solution of p-nitrobenzyl 3-[2-(-pyridinium) ethylthio1-6a-(-(R)-hydroxyethyll-7-oxo-l-azabicyclo (3.2.0) hept-2-ene-2-carboxylate cloride (5.77 g, 11.4 mmol) in potassium phosphate monobasic-sodium hydroxide buffer (170 mL, 0.2 M, pH 7.22) was added tetrahydrofuran (30 mL), ether mL) and 10% palladium on charcoal (5.7 The resulting mixture was hydrogenated at 22 0 C under 40 psi for 1 h and filtered on a Celite pad. The pad was washed with water (2 x 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 cm) of i-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 lyophylization of the appropriate fractions 2.48 g of the title compound as a yellowish powder. The analytical data were identical to those reported for the compound prepared in the method A.

Example 2 Preparation of 3-(2-(l-(3,5-dimethylpyridinium)ethylthio]-6art 1-(R)-hydroxyethyll-7-oxo-l-azabicyclo (3.2.0)hept-2-ene-2carboxylate H CH 3 O o+ co- CH 3 COO C3 3t A. l-(2-mercaptoethyl)-3,5-dimethylpyridinium methanesulfonate N, Mso N+ MsOHHS V14 0 VM ring so as to form a bridged polycyclic ring, e.g. a quaternized quinuclidine ring of the formula To a suspension of 3,5-lutidinium methanesulfonate in 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 550C for 24 h, cooled to 23*C and dilutedwith water (5 mL) and ether (5 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 i-bondapak C-18.

The column was eluted with water and lyophilization of the appropriate fractions gave a c .Aourless syrup 2.4 g ir (film) v max 252 0 1628 (pyridinium), 1600, 1495, 1325, 1305, 1283, 1200 (sulfonate), 1040, 938, 765, 680 cm- 1 1 Rnr (DMSOd 6 6:2.31 (3H, s, CH 3 so 3 2.47 (6H, s, CH 3 on pyridinium) 2. 57, 2. 66, 2. 69, 2. 78 (1H, B partof A 2 B system, SH) 3.06 (2H, m(with D 2 0 added (2H, t, J=6.5 Hz)]I CH 2 S) 4.65 (2H, t, J-6.5 Hz, CH 2 N 8.34 (1H, s, Hp of pyridinium), 8.79 (2H, s, Ho of pyridinium); uv MH 2 0) Xma :271 (c 4 8 6 0) mu 1 Anal. calcd. for C 1

OH

17 N0 3

S

2 *0.5H 2 0: C 44.09, H 6.66, N 5.14, S 23.54; found: C 44.26, H 6.49, N 5.17, S 24.18.

B. Paranitrobenzyl 3-(2-(l-(3,5-dinethylpyridiiiui) )ethylthio)-6a-(l-(R)-hydroxyethy11-7-oxo-1-azabicyclo (3.2.0)hept- 2-ene-2-carboxylate diphenylphosphate OH1) NEt(iPr) 2 H 2 d C1W(OPh) 2 w 03) e)MsO OON COOPNB

CON

4) NEt(iPr) 2 (PhO)2P .4 r To a cold (01C) solution of p-nitrobenzyl hydroxyethyl)-3,7-dioxo-l-azabicyclo(3.2.0)heptane-2carboxylate (0.523 g, 1.50 mmol) in acetonitrile (6.0 mL) kept under a nitrogen atmosphere was added diisopropylethylamine (0.314 mL, 1.8 nunol) followed by diphenyl chlorophos- AV phate (0.373 miL,. 1.8 muw1). The reaction mixture was stirred for 30 min anid treated with a solution of 1-(2-mercaptoethyl)- 39 3,5-dimethylpyridiniws mnethanesulfonate (0.493 g# 1.87 mmol) in Il acetonitrile (1.9 mL) followed by diisopropylethylamine (0.314 3 mm The reaction mixture was stiried at 0vc for I h,diluted with cold (OOW) water(26 m.L) and poured on too oo co1.'-L7fl (7.0 x 3.5 cm) of ;.-bondapak C-18. Slution of th.e colan with 23-50% acetonitrile 75-50% water mixture gave after lyophilization of the appropriate fractions 1.01 g (g0%) of the title compound as yellowish powder, ir (KBr) 1:3700- 31030 (oH) 1778 (C=O of 3-lactam), 1700 (C=O of PNB ester), 1635 (pyridiniui) 1595 (phenyl) 1521 (NO 2 1335 (NO 2 395 c 1

(NO

2 1Hmr (DMSO d6) 3:1.16 (3H, d, !16.1 Hz, CH 3 CHOM), 2.43 CH 3 on pyridinium) 4.75 (2H, m, CH 2 N,53 (center of ABq, Jab= 14 3 Hz, CH 2 of PNB), 6.6-7.5 (10H, m, p:henyl), 7.70 d, 3=8.-7 Hx, Ho of PNB) S. 0-8. 5 (3H, m, Ho o:4 Ovr-'oJ.nium, Hsi of PNB), 3.82 (2H, s, Ho of pyridiniumn), uv (H 0) ja: 270 306 (--7343) muj. Anal. calcd. for C 37 A 3 03 0 10 5P'H 2 O C 58.03, H 5.26, 4 5.48, S 4.18; found: C 5-7.93, H 5.35, N 5.22, S 4.34.

*se ViroxV/ethyI) -7-oxo- 1--azabicyclo hept-2-ene-2-carboxylate 2I 0 P C HI II I,+ 9 7oaslto fA-Lrbny -2('(,-inty etytho (1 RNhrxytyl7oo *31 a, G azb.. 3..0h t2-n lae -Lhnlhsh (0.60 q,08 I)i e erhdoua 3 L a de ete 3 ,L oasu hshaemnbsi-sdu yrxd -00M H74 4mL n 0 aldu ncaca (0o6 a) suTe n ofulin mixtu:roei 3a 2-rgeae un5dier 4ps 3 a 230bCycfor(3.25 h.)he 2ene2garbicYlayr wa separedhs a 011: exrce withbuffr( xH 7.54 m.L) and e 10 aladium onr carcoal 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 10.0 cm) of p-bondapak C-LB. El~ution

VX

I F of the column with water and lyophilization of the appropriate fractions gave the title compound 0.196 g (64%) as a yellowish powder, i: (KBr) v max: 3700-3100 1760 1 1 (C=O of S-actam) 1595 cm 1 (carboxylate), Hmr (D020) 6: 1.21 (3H, d, J=6.3 Hz, CH 3 CHOH), 2.45 (6H, s, CH 3 on pyridinium), 2.81 J=9.2 Hz, 2.96 J=9.2 Hz, 3.22 (dd, J=2.6 Hz, J=6.2 Hz, 3.40 J=6.2 Hz, CH 2 3,84 (dd, J=9.2 Hz, J=2.6 Hz, 4.15 CH 3 CHOH), 4.71 (t, J=6.2 Hz, CH 2 N 8.21 (1H, s, Hp of pyridinium), 8.46 (2H, s, Ho of pyridinium), uv (H20) max: 279 (E8345), 296 (7714) m, 2 3 40.7 (c 0.53, H 2 T 16.9 h -4 (measured at a concentration of 10 M in phosphate buffer pH 7.4 at 36.8 0

C).

Example 3 Preparation of (5R, 6S)-3-[(2-(3-hydroxymethylpyridino)ethyllthio]-6-[1-(R)-hydroxyethyll-7-oxo-1-azabicyclo(3.2.0]hept-2ene-2-carboxvlate Ctr OH CH OH 22 ,crA. 3 ydroxyme thy l-1 2-me rcaptoe thyl )py rid i nium tilco methanesulfonate 2C F H OH. v t

S

t t2 3 ce 3 HSCH2CH2N ft CO 9 t r fttt t2 CVrA. 3-H-ydroxyiethyl-l-_(2-mercaptoethyl)poyridiniumtru ne thanesul f on-ate CF so 3 H A MO HSCH CH N 9

CF

3

SO

3 Trifluoromethanesulfonic acid (1.327 mL, 0.015 mol) was added dropwise to 3-pyridinemethanol (2.91 mL, 0.030 nol), S' I followed by ethylene sulfide (0.89 mL, 0.015 mol). The resulting homogeneous mixture was heated (oil bath) at 50-700C under N 2 for 20 h. The reaction mixture was then taken up in H 2 0 (15 mL) and extracted with CH 2 Cl 2 (5 x 5 39 The aqueous phase was concentrated in vacuo and then applied JM

I

to a C18 reverse-phase column. Elution with H 2 0 followed by evaporation of the relevant fractions gave a pale yellow oil.

This material was rechromatographed to give a nearly colourless oil. After drying in vacuo (2205) this afforded the product (4.50 g, 94%) as a viscous oil. ir (film) vrnx: 3450 OH), 2560 SN) cm-; IHmr (d -acetone) 6: 9.10-8.05 4H, aromatic), 5.01 J=5.5 Hz, 2H, N-CH 2 4.93 2H, -CH 2 OH), 4.43 (br S, lH, 3.43-3.18 2H,

S-CH

2 2.34-2.10 SH).

B. p-Nitrobenzyl (5B,6j-3-( 2-(3-hydroxymethylpyridinio)ethyl thiol-6-tl-(.)-hydroxyethyll-7-oxo-l-azabicyclo- (3.2.01hept-2-ene-2-carboxylate diphenylphosphate OH OH H

CO

2 PNB CO 2 PNB (oW)2O To a solution of p-nitrobenzyl (5R, hydroxyethyl -3,7-dioxo-l-azabicyclo3.2.0heptane-2 -carboxyciI' late (0.174 g, 0.50 miol) in 2 mL of dry acetonitrile was added diisopropylethylaine (0.096 mL, 0.55 mmol) at 0 0

C

under N 2 Dichenyl chiorophosphate (0.114 mL, 0.55 mmol) uzas zhen added dropwise and the reaction stirred at o0C for 30 min. A solution of 3-hvdrox<vrethyi-l- (2-mercaptoethyl)pyridinium trifluoromethanesulfonate (0.223 g, 0.70 imol) in 0.50 mL of acetonitrile was then added, followed by diisopropylethylamine (0.122 mL, 0.70 mnol). After being kept at 0 0 C for 30 min the reaction mixture was concentrated 3 0 in vacuo and the residual yellow gum was taken up in H 0 (enough acetonitrile was added to aid in dissolving the gum).

This solution was applied to a C 18 reverse-phase column which was eluted with 15% acetonitrile-H20. Lyophilization of the relevant fractions afforded the product (0.305 g, 81%) as a beige-coloured solid. ir (Kr) V 3420 (br, OH),

TX

1 1775 (8-lactam CO), 1695 (-CO 2 tNB) cm ;Hmr (d 6 -acetone) 6: 9.44-7.72 8H, aromatic), 7.22-6.91 'OH, diphenylphosphate), 5.53, 5.27 (ABq, J=l4Hz,2H, benzylic), 5.04 (t, 39 J-7.4 Hz, 2H, N-CH 2 4.75 2H, CH 2 OH), 4.5-3.1 8H), JM -17 I F 1.21 J=6.3 Hz, 3H, CHMe).

C. 2-(3-hydroxymethylpyridinio)ethyl]thio]-6- (1-(R)-hydroxyethyll-7-oxo--azabicyclo(3.2.O]hept-2ene-2-carboxylate OH CH2H OH HO

(-D

0 0I 0 0 CO PNB (00)2PO CO2 2 2 To a solution of p-nitrobenzyl 2-(3hydroxymethylpyridinio)ethyl thio]-6-[l-(R)-hydroxyethyll-7oxo-l-azabicyclof3.2.0]hept-2-ene-2-carboxylate diphenylphosphate (0.145 g, 0.194 mmol) in 10 mL of THF containing drops of H 2 0, was added 6.0 mL of phosphate buffer (0.05 M, pH 0.l45 g of 10% palladium-on-charcoal and 10 mL of ether. The mixture was hydrogenated (Parr) at 40 psi for 1 h and then filtered through a pad of Celite. The filter cake was washed with a little H 2 0 and ether and the aqueous phase was separated and extracted with ether The aqueous 2Q 1 solution was then cooled at 0 0 C and the pH was adjusted to with pH 7.4 buffer. After removing residual volatiles in vacuo the aqueous solution was applied to a C 18 reverse- S phase column which was eluted with H20. Lyophilization of t St, 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) v max: 3300 (br, OH), 1755 (B-lactam CO), 1590 02 -1 1mx cm 1 Hnmr (D 2 0) 6: 8.78-7.94 4H, aromatic), 4.83 (t, e J=6.0 Hz, 2H, N-CH 2 4.83 2H, CH20H), 4.16 (d of q, J=J'=6.2 Hz, 1H, 3.98 of t, J=9.1 Hz, J'-2.6 Hz, 4r *r 1H, 3.75-3.20 3H), 3.20-2.65 2H), 1.22 (d, J=6.4 Hz, 3H, CHMe); uv (H 2 0) Xmax: 294 (s7614), 266 (E6936) nm; t (pH 7.4, 36.8 0 C) 14.0 h.

Example 4 SPreparation of (52,6S)-3-[2-(4-hydroxymethylpyridinio)ethylthiol -6-[1-(R)-hydroxyethyll-7-exo-1-azabicyclo 3.2.0] hept- 2-ene-2-carboxylate 39 JM -3

OH

OH 2OH CO 2 A. 4-Hydroxymethyl-l- (2-mercaptoethyl)pyridiniun trifluoromethanesulfonate 0 Ng CH O CFSO 3 H -L HSCH 2 CHN OH

CF

3 so 3 To a solution of 4-pyridinemethanol (1.635 g, 0.015 moL) in 10 mL of CH 2 0.

2 at 0 0 C under N 2 deddows 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 ~tt reduced pressure to give an oil. To this oil was added .0 4 ethylene sulfide (0.891 mL, 0.015 mol) and the resulting homogeneous mixture was heated (oil bath) at about 600C for 4[t 3 h. The reaction mixture was then taken up in 15 mL of H2 0 and the aqueous solution was washed with CH 2 012 After removing residual organic solvent in vacuo the aqueous solution was applied to a C018 reverse-phase column. Elution with H2 0 and subsequent evaporation of the relevant fractions afforded an oil which was further dried in vacuo over P20 2 to give the product (4.64 g, 97%) as a colourless oil. ir (film) V)max: 3455 OH) 2565, SH) cm ;Hnmr (d 6 acetone) 6: 9.07, 3.18 (ABq, J=6.8 Hz, 4H, aromatic), 5.03 2H, CH 2 OH), 4.96 J=6,5 Hz, 2H, N-OH 2 4.09 (br s, lH, 3.5-3.1 (in, 2H, S-OH 2 2.25 (brs, lH, -SH).

B. p-Nitrobenzyl 2- (4-hydroxymethylpyridino)ethyl thiol-6-[l-(R)-hydroxyethyl1-7-oxo-l-azabicyclo(3.- 2. 0]hept-2-ene--2-carboxylatediphenylphosphate 39 _'31-

OHOH

Co 2 PNB 0 2 2NB (00)2 o To a solution of p-nitrobenzyl hydroxyethyll-3,7-dioxo-l-azabicyclo[3.2.O]heptane-2carboxylate (0.348 g, 1.0 mmol) in 5 mL of dry acetonitrile, at OOC under N 2 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 OWC for 40 min. To this solution was added a so'ution of 4-hydrozymethyl-l-(2-mercaptoethyl)pyridinium trifluoromethanesulfonate (0.447 g, 1.4 mmol) in 1 mL of acetonitrile, followed by diisopropylethylamine (0.191 mL, 1.1 mmol). A reddish-black gum separated from the reaction mixture. After 20 min at O 0 C the reaction mixture was filtered and concentrated in vacuo. The residue was taken up in a minimum volume of acetonitrile-H 2 0 and applied to a C 18 reverse-ohase column. Elution with 25% acetonitrile- H 0 and. 3ubsequent lyophilization of the relevant fractions gave the oroduct (0.353 g, 47%) as a cream-coloured solid.

ir (Kr) v 3240 (br, OH), 1775 (2-lactam CO), 1695 max

(-C

2 PNB) cm Hnmr (d -acetone) 9.24-7.84 8H, aromatic), 7.4-6.9 lOH, diphenylchoshate) 5.52, 5.24 (ABq, J=14 Hz, 2H, benzylic), 5.15-4.80 4H), 4.45-3.05 7H), 1.35 J=6.6 Hz, 3H, CHMe) C. 2-(4-hydroxymethylpyridinio)ethyI thio] -6- 1- -hydroxyethyl) -7-oxo-l-azabicvclo 2.01 heot-2ene-2-carboxylate o 0 COH t Ie CO PNB (00)220 22

OH

HOH

39 0 Co 2

C

0 2 0 JMY -GO A irixture of p-nitrobenzyl 2-(4hydroxymethylpyri-dinio)ethyl thic] -hydroxyethyl3 7-oxo-l-azabicyclo[3.2.O]hept-2-ene-2-carboxylate diphenylphosphate (0.348 g, 0.465 mmol) and 10% palladium-oncharcoal (0.35 g) in 11 mL of phosphate buffer (0.05 M, pH 7.4) SrnL of THF and 10 mtL of ether was hydrogenated at 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 C 18 reversephase column. Elution with 2% acetonitrile-H 2 0 and subsequent lyophilization gave a yellow-brown solid. This material was rechromatographed (C 18 reverse-phase/H 2 O0) to give the desired product (0.060 g, 36%) as a light yellow solid. ir (KBr) v maf 3400 (br, OH), 1755 -lactam CO), 1590 (-CO 2 cm-. Hnmr (D 2 0) 6: 8.73, 7.96 (ABq, J=6.8 Hz, 4H, aromatic), 4.93 2H, CH 2 OH) 4.77 J=6.0 Hz, 2H,

N-CH

2 4.15 (d of q, J=J'=6.3 Hz, 1H, H-11), 3.96 (d of t, J=9.2 Hz, V=~216 Hz, 1H, H-5) 3.65-3.20 (in, 3H) 3.13-2.62 (in, 2H) 1.21 J=6.3 Hz, 3H, CHMe) uv (H 2 0) X max 295 (F-6880) 256 (F-5595) 224 (E:3111) nm; t (PH 7.4, 36.81C) t t' C 14.5.

Example Preparation of 3-12- (l-(2-methylpyridinium) )-ethylthio) (.E)-hydroxyethyl] -7-oxo-l-azabicyclo hept-2-enecarboxylate

OH

S 0 coo

CH

3 A. 1- (2-mercaptoethyl)-2-rnethylpyridinium methanesulfonate N\ H MsOH S N 55 0 C, 21 hQ 39

MJM

To a suspension of 2-methylpyridinium methanesulfonate in 2-methylpyridine prepared by the addition of rethanesulfonic acid (0.65 mL, 0.010 mol) to cold 2-methylpyridine (2.17 mt 1 0.022 mol) was added ethylene sulfide (0.655 mL, 0.011 mol). The reaction mixture was stirred under a nitrogen atmosphere at 550C for 21 h, cooled to 23 0 C 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) of vi-bondapak C-18. The column was eluted with water and lyophilization of the appropriate fractidns gave 2.13 g of the title compound, ir (film) vmax: 2520 1623 (pyridinium) 1574, 1512, 1485, 1412, 1195 (sulfonate), 1038 cm 1 m (DIISO-d 6

D

2 0) 2.37 (3H, s, CH SO 3 2.83 (3H, s, CH 3 on pyridinium), 3.09 (2H, J=6.9 Hz, CH 2 4.71 (2H, t, J=6.9 Hz, CH 2

N±,

7.93 (2H, m, Hm of pyridinium), 8.44 (1H, m, Hp of pyridinium), 8.89 (1H, r, Ho of pyridiniux) uv (H 2 0)6 max 266 (e3550) m~.

E 0 B. Paranitrobenzyl 3- (2-(l-(2-methylpyridinium )ethylthio]- 6- (-l-CR)-hydroxyethyl-7-oxo-l-azabicyclo(3.2.0)hept- 2 -ene-2-carboxylate diphenylphosphate OH 1) NEt (iPr) 2 cat c C ,lO 0 OPNB 0 2) CIF(OPh)-, 3) O MsO 4) NEt(iPr) 2 jij

B

/4!r r

C:.

I'

I $1 4e

I

(PhO) 2

ICOOPNB

To a cold (OOC) solution of p-nitrobenzyl hydroxyethyll-3,7-dioxo-1-azabicyclo(3.2.0)heptane-2carboxylate (0.523 g, 1.50 mmol) in acetonitrile (6 mL) kept under a nitrogen atmosphere was added diisopropylethylamine (0.314 mL, 1.80 mmol) followed by diphenyl chiorophosphate (0.373 m.L, 1.80 mmol). The reaction mixture was stirred for 30 min at OOC and treated with solution of 1- (2-mercaptoethyl) methylpyridinium DW -42.

I r 0 2- Clg(QPh) 2 S N O 3- Hs T 1) 3sQOPClCOPN Cl COOPNB COOPNB C 4- NEt(iPr)

VM

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 i-bondapak C-18. Elution of the column with water and lyophylization of the appropriate fractions give the title compound 0.098 g as a yellowish powder, ir (KBr) v 3650-3100 max 1755 (C=O of $-1actam), 1630 (pyridinium), 1595 cm (carboxylate), 1Hmr (D 20) 6: 1.20 (3H, d, J=6.3 Hz,

CH

3 CHOH), 2.83 CH 3 on pyridinium), 2.7-3.1 (5H, H-4,

CH

3 on pyridinium), 3.1-3.7 (3H, m, CH 2 S, 3.90 (dd, J=9.1 Hz, J=2.6 Hz, 3.1 CH 3 CHOH), 4.78 J=6.2 Hz, CH 2 N 7.8 (2H, m, Em of pyridinium), 8.3 (1H, m, Hp of pyridinium), 8.65 (1H, m, Ho of pyridinium), uv (H 2 0) max: 268 (e9350), 296 (E8840) mw, 23 [aID) +410 (c 0.5, H 2 15.0 h (measured at a -4 concentration of 10 M in phosphate buffer pH 7.4 at 36.8 0

C).

Example 6 Preparation of 3-[2-(1-(4-methylpyridinium))-ethylthio)- 0 6a-1(R)-hydroxyethyl]-7-oxo-l-azabicyclo(3.2.0)hept-2ene-2-carboxylate

OH

SI 3 XN H 3 0 00 A. 1-(2-mercaptoethyl)-4-methylpyridiniun -ethanesulfonate SIt Th 0 +MsOH HSXN\/ S +O 55-C, 24 h 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 55 0 C for 24 h, cooled to 230C and diluted with water mL) and ether (10 mL). The organic layer was separated and the aqueous layer was washed with ether (5 x 5 mL) and DW -44-

T:

sg~ 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 water mixture gave after lyophylization of the appropriate fractions a colorless syrup 2.66 g ir (film) v mnax 2500 1640 (pyridinium), 1572, 1520, 1478, 1200 (sulfon- -1 1 ate), 1040, 833 and 768 cm Hmr (DMSO-d 6 6: 2.31 (3H, s,

CH

3 S03 2.62 CH 3 on pyridinium), 2.2-2.9 (4H, SH, CH3 on pyridinium), 3.04 (2H, m, CH2S), 4.68 (2H, t, J=6.4 Hz, CH N 8.01 (2H, d, J=6.6 Hz, rm of pyridinium), 8.89 (2H, d, J=6.6 Hz, Ho of pyridinium), uv (H 2 0) Xm: 256 (E4100), 221 (0544) m.

B. 1-(2-mercaptoethyl)-4-methylpyridinium p-toluenesulfonate SN 4-pTsOH SHS pTSO o J te i C To a suspension of p-toluenesulfonic acid (172 g, 0.01 mol) in benzene (6.5 mL) was added 4-picoline (1.17 mL, 0.012 mot). The resulting mixture was stirred under a nitrogen atmosphere at 23 0 C for 30 min, treated with ethylenesulfide (0.65 mL, 0.011 mol) and stirred at 750 for 24 h. More ethylenesulfide (0.65 mL, 0.011 mol) was added and the stirring was continued at 75 0 C for 24 h more.

The reaction mixture was cooled to 23 0 C 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 p-bondapak C-18 with water as eluting solvent to give 2.94 g 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 Hmr (DMSO, ds 6 6: 2.29 (3H, s,

CH

3 on pyridinium),. 2.61 CH 3 Ph), 2.4-2.8 (4H, SH

CH

3 Ph), 3.03 (2H, m[addition of D 2 0 gave a t, J=6.4 Hz, at 3.041, CH 4.68 (2H, to J-6.4 Hz, CH 2 N 7.11, 7.49 (4H, 2d, J-7.9 Hz, Phenyl), 8.00 (2H, d, J-6.5 Hz, Mm of pyridinium), 8.89 (ZH, d, J-6.5 Hz, Ho of pyridinium), uv (H 2 0) A max: 256 (c4315), 222 (17045) my.

e: I r C. Paranitrobenzy 3-( 2 4 -rethvl vrid i )ethy1Lh o1 6 -al-(R)-hydroxYethYl)- 7 -oxo-l-azabicyc10(3.2.0)'et- 2-ene-2-carboxylate diphenylphosphate OH I NEt(ir) 2 0 0 2) CIP(OPh) 2 COOPNB 3) N_

OH

4) N;Et( lm r) CH 0 2 (PhO) 0 COOPNB To a cold (OOC) solution of p-nitrobenzyl 6a-[l- CR)-hydroxyethyll-3,7,-dioxo-l-azabicyclo(3.2.0)heptanecarboxylate (3,522,. 1.5 mol) in acetonitrile (6 mL) kept under a nitrogen atmosphere was added diisopropylethylamine (0.314 -mL, 1.3 mmol) followed by diphenyl chiorophosphate (0.373 1.9 -mmol). The reaction mixture was stirred -or 45 nin and treated dropwise with a solution of 1-(2-rercaptoethyl)-4-methylpyridinium methanesulfonzte (C.J33Z 2.16 in acetonitrile (1.8 followed by 4li i sosropylethylamine (0.314 L, 1.3 ocl) The reacticvx~ure was stirred at OOC for w ,l :z water (24 m.L) and o-red n of b-ondapak C-18. 1in ,on is acetonitrile -75% water mixture (130 mL) then wi;.

acetonitrile 50% water mixture (100 mL) afforded after lyophylization of the appropriate fractions 0.91 of the title compound as a yellowish powder, ir (Kr) )ax 3700-2800 1770 (C=O of 3-lactam), 1700 (C=0 of PNB ester), 1640 (pyridinium), 1595 (phenyl) 1523 (NO) 1340 (NO 2 890 cm 1

(NO

2 Hmr (DNMSO, d S: I. (3H, d, J=6.2 Hz, CH CHOH), 2.61 CH on pyridinium), 3.1-3.7 (3H, m, H-6, CH 2 3.7-4.4 (2H, m, H-5, CH 3

COH),

4.79 (2H, brt, -63 Hz, CH 2 N 5.17 J-4.9 Hz, OH), 5.37 (center of ABq, 1-4.1 CH 2 of PNB), 6.7-7.4 H, m, phenyl), 7.69 (2H, d, !18.8 Hz, Ho of PNB), 8.00 (2H, d, !16.5 Hz, Hm of pyridinium), 8.23 (2H, d, J-8.8 Hz, Kn of PUB), 8.92 (2H, d, J=6.5 Hz, Ho of pyridinium), uv W 4b- I iF 20Xmax 262 (1.e0835) 311 (E9670) Anal. calcd.

for C36H 3 6

N

3 0lSP.1.5 H 2 0:C 56.84, H 5.17, N 5.52, S 4.21; found: C 56.89, H 3.13, N 5.19, S 4.41.

P.3-[2-(I-(4-methylpyridiniurn))ethylhiol-a-I(.) hydroxyethyl]-7-oxo-1-azabicyclo(3.2.0)hept-2-ene- 2-carboxylate

OF

j 10% Pd/C, H2 0O THF, ether, buffer o (PhO) 102 COOPNB OH 0 00 To a solution of p-nitrobenzyl 3-[2-(1-(4-methylpyr idini=) -ethyl thio I 6 -hydroxyethyll -7 -oxo- Iazabicyclo(3.2.0)hept-2-ene-2-carboxylate diphenyiphosphate (0.587 g, 0.90 mnol) in wet tetrahydrofuran (30 mL) was added ether (30 mL), potassium phosphate mono basic-sodium hydroxide buffer (0.15 M, 14.7 mL, pH 7.22) and 10% palladium on charcoal (0.59 The resulting mixture was hydrogenated under 40 psi at 230C for 1.25 h. The organic layer was secarated and extracted with the buffer (2 x 6 mL).

7he acueous extracts were combined, filtered through a Celize cad, washed with ether :3 20 cpz 'zed to remove traces of organic solvents and cc-red on top of a column (2.5 x 10 cm) of u-bondapak 0-13. Elution of the column with water and lyophylization of the appropriate fractions gave 0.136 g of the title compound as a 3 0 yellowish powder, ir (KBr) ja: 3700-3000 1770 max -1 (C=O of 5-lactam), 1642 (pyridinium) 1592 cm (carboxylate) Hmr (D 2 0) 5: 1.19 (3H, t, J=6.3 Hz, CH 3 CHOH), 2.59 (3H, s, CH on pyridinium), 2.94 J=9.1 Hz, 2.90 J-9.1 Hz, 3.0-3.6 (3H, m, CH S, 3.86 VA C 2 (dd, J=9.1 Hz, J=2.6 Hz, 4.12 CH 3 CHOH), 4.5- .9 (CH2N matked by HOD), 7.80 (2H, d, !16.6 Hz, Hm of pyridinium), 8.58 (2H, d, !16.6 Hz, Ho of pyridinium), uv (H 2 0) X max: 256 (eSS10), 262 (e5360), 296 (c7050)m CcL 23 +20.80 (C 0.48, H 2 T,12.8 h (measured at a

DW

~I

concentration of 10-4 in a phosphate buffer pH 7.4 at 36.3 0

C)

Example 7 ?-.9aration of (5R) 3-f2-(4-methylthioyrid inio)-et-ytlthio]- (65)-(_1?R)-hydroxyethyl]- 7 -ox-l-azabicyclo.(3.2.01 hept-2are-2-carboxylate

OH

S I 0 a SCH 3 0 CO 9 2 A. 4-Methylthiopyridine* ~e/EtOX ,;SH Me/tHSMe 2) NaOHI 0 4-Mercaptopyridine (5.55 g, 50.0 mmol; Aldrich) was dissolved in boiling abs. EtOH(50 mL). The insoluble .aterial was removed by filtration over Celite. the -te was heated to re-dissolve, and when it cooled :0 3 C, methyl iodide (3.17 mL, 51.0 7mmol; Aldrich) as added at once. The mixt re was zcclad to crVstallize.

2 '::raion of the solid ;gave 6.77 6. 7 ol, j 53.3's) Of :itle compound as the ;',,dri.cdze 2.70 (3H, s, -SCH and 7.63-7.77-8.3-.43 Com (41,

A

2

B

2 type, aromatic Hs); ir (Nujol) Va: 1615, 1535 (aromatic) and 790 cm uv (H 2 0) max 227 (E2.02 x 10 4 4 and 298 nm (el.64 x 10 T the hydriodide (6.33 g, 25.0 mmol) was dissolved in H 2 0 (40 mL) and the insoluble material was removed and washed with H 0 (10 mL). To the filtrate was added at 2 .0-5 0 NaOH pellet (5 g) and extracted with Et O (3 x 25 mL), ,1 2 saturating the aqueous layer with NaCl. The combined organic extracts were washed with brine (x dried M4gSO 4 and evaporated, yielding 2.92 g (23.4 mmol, overall yield 50%) of the title compound as an oil: 1 Rmr (CDCl 3 6: 2.48 (3H, -SCH 3 and 7.03-7.13-8.38-8.48 PPM (4H, A 2 5a type, aromatic-HSab ir (film) v 1580 DW i 6- and 8Q0 cm-1, *Preparation of this compound was reported by King and Ware, J. Chem. Soc., 873(.1939). The procedure, described in this reference was followed.

B. 4-Methylthio-N-(2-mercaptoethyl)pyridinium methanesulfonate

CHJSQJH

Sme CH3 so3 5000 0 M HS CH3s CH3 3 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 50-60 0 C for 21 h. As reaction proceeds the solid went to solution. After cooling, the reaction mixture was dissolved in H 2 0 (5 mL) and washed with Et 2 0 x 4 mL). The cloudy aqueous layer was filtered over Celite and the filtrate was purified by reverse phase silica gel column chromatography (C 18 micro bondapack 10 g) eluting with H 2 0. Each fraction of 10 mL was collected.

Fractions 2 and 3 were combined and repurified by the reverse phase column. Fraction 2 gave 1.258 g (4.48 mmol, y. 42.6%) of the title compound as a viscous oil; IHmr (DMSO-d 6 CFT-20) 6: 2.32 (3H, s, MeS03 2.72 (3H, s, -SMe), 2.68 (1H, m, SH), 2.9-3.2 (3H, m, -CH 2 4.59 (2H, t, J=6.4 Hz, -CH 2 N 7.97 (2H, J=7.2 Hz, aromatic-Hs) and 8.72 ppm (2H, J=7.2 Hz, aromatic-Hs); e -1 S ;30 ir (neat) vmax: 1630, 1200 (br, -SO 785 and 770 cm.

max 3 *These reagents were distilled prior to use.

C. (5R) p-Nitrobenzyl 3-(2-(4-Methyl1thiopyridino)ethylthiol -((1)-hydroxyethyl]l-7-oxo-1-azabicyclo(3.2.0]hept- H4 2-ene-2-carboxylate chloride 1) ClPO(00) N 9 SMe CO PNB 9 Cl 2 MsO 0O 0 N 4CO PNB To a solution of (3R) p-nitrobenzyl 3,7-dioxo- (6)-((lR)-hydroxyethylV-l-azabicyclo(3.2,0]heptane- carboxylate (475 mg, 1.36 m. ol) and diisopropylethylairine (0.24 mL, 1.4 mmol) in CH 3 CN (5 mr) was added at 0 0 -5'C under a nitrogen atmosphere diphenyl chiorophosphate (0.29 mL, 1.41 mmol). The mixture was stirred at 00-5*, for 30 min.

:o this mixture was added an oily suspension of 4-methylthio- N-(2-ercaptoethyl)pyridinium methanesulfonate (678 mg, 1.45 mmol; 60% pure) in CH 3 CN (1.5 mL) followed by diisopropyl- .0 ethylamine (0.24 mL, 1.4 mmol). The mixture was stirred at 0 0 -5 0 C for 1 h. Immediately after addition of the base, yellowish precipitate formed. The precipitate was filtered and washed vith cold CH 3 CN (3 mL), yielding 413 mg of yellowish solid. This was triturated from 10% MeOH in H 2 0 mL) to obtain 341 mg (0.618 mmol, y. 45.4%) of the title compound as white crystals: mp 118 0 -120C; Lmr (DMSO-d 6 1.16 (3H, d, J=6.L Hz, l'-CH 3 2.72 (3H, s,

-SCH

3 3.1-3.7 (5H, 3.7-4.3 (2H, 4.71 (2H, t, J=6.3 Hz, -CH 2 5.15 (lH, d, J=4.9 Hz, OH), 5.20-5.35-5.40-5.55 (2H, A~a, CO 2

CH

2 Ar), 7.70 (2H, J=8.8 Hz, nitrophenyl-Hs), .97 J=7.0 Hz, nyridinio-Hs) 3.25 (211, >3.3 Hz, nitrohenyl-Hs) and 3.-6 zom (2H, H7.1 Hz, ir (nujol) 3250 L77 (3-lac::am) maxc and 1625 cm tbs 3332.3 L,

MH)

Anal. calcd. for C 2 4

H

26

N

3 0 6

S

2 C .H 2 3 C 50.36, H1 4.95, N 7.37; found: C 50.63, H 4.72, N 6.39.

0. 3-(2-(4-Methylthiopyridinio)ethylthio)-(6-(L) hvdroxyethyl)-7-oxo-1-azabicyclot3.2.0)hept-2-ene-2carboxylate

QH

H

2 /Pd-C pH 7.4 burfer CO 2 2NB s vM e Jo C00 CO 2 -v So 'ir p-Nitrobenzyl 3-12-(4-methylthiopyridinio)ethylthio] (Gs1 R)-hydroxyethyldro l-7-oxo-l-azabicycl (].2.01hept-2-ene-2-carboxylate chloride (280 mg, 0.638 mnvol) was dissolved in TUF (31.3 mL) and pH 7.40 phosphate buffer (21.5 mL; 0.05M Fisher) and diluted with Et 2 0 (31.5 mL). This solution was mixed with 10% Pd-C (380 mg, Engelhard) and hydrogenated at 35 psi on the Parr shaker at room temperature for I h. The aqueous layer was filtered over Celite to remove the catalyst and the Celite cad was washed with H 2 0 2 x 5 mL). The filtrate and washing were combined and washed with Et O (2 x 30 mL). The aqueous layer was pumped off to remove any organic solvents and purified by reverse phase column chromatography (C 18 microbondapak, 13 g, Waters Associates) eluting with H 2 0. Fractions having a uv absorption at 307 rnm were collected (ca. 1 L) and lyophilized to obtain 127 ng (0.334 mmol, y. 48.5%) of the title compound as a yellowish powder: IHmr (D20, CFT-20)i: 1.20 (3H, d, j=6.4 Hz, l'-CH 3 2.64 (3H, s, -SCH 3 2.81 (2H, m, -SCH2-), 3.19 (1H, dd, Hz, =2.6 Hz, 3.32 (2H, dd, J=11 Hz, J=5.3 Hz 4-Hs), 3.92 (lH, dt, j=9.2 Hz, j, =2.6 Hz, 3- 4.1 (1H, m, l t 4.61 (2H, t, J=5.9 Hz, 9 7.70 (2H, J.l Hz, aroatic-Hs) and 3.40 ppr (2H, J=7.1 Hz, arcnatic-Hs .r 1K3r, d-sc 3400 1730 (3-'acz) :630 az.r:~n- z 1 1390 (carboxylate) av: H.C fi 230H 308 nm (25000); 2a] 3 14 -*11 Example 8 Preparation of 3-(2-(3-methoxy-L-pyridin uzn)ethythiol- 6 i-(1'-(R)-hydroxyethylI-7-oxo-l-a zabicyc o(3.2.0)heot-2-ene-2-carboxylate ft.

OH

r* 4 1 S OMe C02 a~ A. L-(2-mercaptoethyl) -3-mthoxypyridinjum methanesulforate Or M4eo-<$Q MsOH Me YS09 \o H To precooled (50C) 3-methoxypyridine (698 mg, 6.4 mmol) was added dropwise methanesulfonic acid (0.216 mL, 3.05 rrmnol) and ethylene sulfide (0.19 raL, 3.2 mmol). The mixture was then heated at 60 0 C for 18 h, cooled to 20 0

C,

diluted with water (10 mL) and washed with ether (3 x 10 m.L).

The aqueous phase was pumped under high vacuum for 15 min and poured on a C 18 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 ir (CH 2 C1 2) max 2550 SH) and 1620, 1600, 1585 cm 1 aromatic); I a (DMSO d 6 5: 8.90-7.90 (4H, m, aromatic 4.72 (2H, t, J=6.6 Hz, CH 2 4.01 (3H, s, OCH 3 3.5-3.0 (m, hidden CH 2 2.66 (1H, dd, J=9.3 Hz, J=7.5 Hz, SH) and 2.3 ppm (3H, s, CH 3

SO

3 B. Zara-Nitrobenzyl 3[2-C3-methoxy-1-oyridinium chloride) ethyithiol-Ea- ('-(R)-hydroxyethyl-7-oxo-1-azabicyclo (3.2.0)-hept-2-ene-2-carboxylate OH 1) EtN(<)I 2) ClPO(00)2 3) 0

CO

2

PNB

Cl 2 120

I

*I

a I.

4) 2

CO

2

PNB

%~r3, A i t 0 A cold (OOC) solution of p-nitrobenzyl 6a-[1'- R)-hydroxyethyl]-3,7-dioxo-l-azabicyclo-C3.2.0)-heptane-2carboxylate (1.04 g, 3 mmol) in acetonitrile (12 mL) was treated dropwise with diisopropylethyla-mine (0.63 mL, 3.6 mmol) and diphenylchlorophosphate (0.75 mL, 3.5 mmol) and stirred at OOC for 30 min. The resulting enol phosphate was treated with l-(2-mercaptoethyl)-3-methoxypyridinium methanesulfonate (1.14 g, 4.30 mmol) in CH 3 CN(7 L), diisopropylethylamine (0.63 mL, 4.30 mmol), stirred for 0 rreparation or ,b-i -L I 4 -hyaroxyme tnyLpY.L.L".LL. r thio hydroxy yl ]-7-ol a c o 0 e 2-ene--carboxylate -3min. and cooled at for 30 mii. :he solid that precipitated out of the nixture was filtered, washed wih cold acetnitrile (2 mL) and dried to tz1e z:c;ncund (1.32 g, yield 82A) ir (nujol) 'Jnax 3320 :H) 1765 5-lactam 1700, 1695 ester C=O) and L320 c- I (s NO 2 I Hmr (DMSO d5) 5: 9.01 (IH, bS, H-3 aromatic), 8.75 (1H, bd, J=5.4 Hz, H-6 aromatic), 3.35-7.95 mn, H-aromat), 7.70 (2H, d, Hz, -arcmatic), 5.37 :enter of Aq, j=13 Hz, CH 2

?NB),

13 .7 (1M, d, 7=4.9 Hz, CH), 4.37 (2H, t, j=63 Hz,

CH

2 N 4.33-3.73 (2H, 1-5 and 4.00 (3H, s, OCH 3 35.6 i;art of a t, J=6.3 Hz, CH 2 3.5-3.20 H, H-3) and 1..6 ppm (3H, d, j=6.1 F, C.

3

CHO)

3<2- (3methoxy-l-1-zidinium3.ethyhthiO]-2-carboxylate.

ethl)~ -7-ox--e zabiCyjClo 2.0) -hept-2-ne- -carboxylate.

H3 C2 O1Me

O

2 ~NB COIe t It 4 o:Ci".on 0 'ara-n: bicclo Tt:1L L C 2 =0oL) in F (25 2 r 23-.4 :nos~hatb buffer (0.1M, 23 was -=eanated n a ?arr shaker over 10% Pd/C g1.. for L t 40 si. he -Jxture was diluted with ether and :he .cuecus -:h'ase was filtered through a ;52 hardened fU. :er :a-er. -he aqueous layer was washed with ether (2 x 0 :uL.:ed -nder and poured on a silica gel reverse phase colu,,. 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 hple to give the penem carboxylate (150 mg, ir (nujol) 1750 alactar. C-O) and 1580 cm Cs, carboxylate). I Hmr (D 2 0) aS5-3.30 (2H, m, H-2, H-6 arohatic), 8,17-7.75 (2H, m, H-3, H-4 aromatic), 4.77 (2ff t, j5.9 Hz, CH201) -M -2L 4.3.0 11.-a, part of 5 lines, J1=6.3 Hiz, e3.97 C3H, oCa.1, 3.85, 3.82 L2 lines, part of dt, Hz, part of H-5) 3.42 C2H, t, J=5.9 Hz CH- S1, 3. 25 (Li, dd, 1 iz 2.6 :Hz, H-61 2.99-2.60 (21f, 6 lines, part of H-4) an~d 1.20 con d, J= 6. 4 H1z uv (H 0 c 0 05) Y.k~ 290 C:-10517). 233 -66431 T(0.1 Y pH 7.4 phosph-ate buffer, 37 0C) 20 h.

.xarncle 9 Premaration of (5R;6S)-3-t 2-(3-methyLthiopyridionio) ethyl thiol-6-['C-(R)-hydroxyethyll-oxo-i-azabic,,clo(3.2.0] hept- -2-ene-2-carboxvlateSe

OH

CO 2 A. 3-Yethylthio-l-(2-m7ercaptoethyl)pyridiniun chloride 210 SMee 0 ECI -S SCH CH 2

Q

To a solution of 3-reth1iv-=r-,=ie .0 1 in 10 mL of ether was added 13 3f HC! and he mixture was well shaken. Teacaueous ochase was secarated, washed with 1.0 rnL of ether and then evaporated. The residual hydrochlorids was then dried in iacuo (P20 to 3 0 give a white solid. To this solid hydrochloride was added 3-methyithiopyridine (1,88 g, 0.015 mol) and ethylene sulfide (0.89, mL, 0.015 mol) aid the resulting mixture was heated (oil bath) at 55-65'ot under Nfor 15 h. This gave a slightly turbid oil which was taken up in 1.25 m.L and washed with CH 2 Cl 2 The aqueous solution was ',.oncentrated to about 25 rnL and then a few drops of acetonitrile wer:e added to make the mixture homogeneous. The resulting aqueous aolution was applied to a CIS reverse-phase column.

Elution with H 2 0 and subsequent evaporation of the relevant fractions afforded the product (2.66 g 80%) as a Pala

GB

I r Nob.

yellow, viscous oil. ir (film) 2410 Cbr, -SH) CM; 1-Einm (d 6 _DMSO+0 2 O) 5: 8.88-7.88 4H, aromatic), 4.70 Ct, J=6.5 Hz, 211, 2 3.08 (skewed t, Hz, 2H, S-C 2 2.64 3H, S-Me) prepared by the method of J.A. Zoltewiez and C. 'Nisi, j. Org. Chem. 34, 765 (]969).

B. p-Mitrobenzyl (5R, 2-(3-methylthiopyridilio) ethyl thia] -hydroxyethylj -7-oxo-] -azabicyclo 13. 2.0] h.ept-2-ene-2-carboxylate chloride OIM e OH E 0 N O O0 0 PNB 0 C02~ PNB2 A solution of p-nitrobenzyl hydroxyethyl)- 3,7-di-oxo-J -azabicycloV3.2.0)heptane-2carboxylate (0.522 g, 1.50 im.ol) in 7 mL of dry acetonitrile was cooled at 0 C and the diisopropylethylamine zc~t(0.287 .1.63 mrnol) was added dropwise. To the resulting C elo yelow- :rown solution was added dropwise d iphenyl chloro- P.-o sp (0.342 mL, L.65 -riol) and the reaction mixture was :keot at c 0 C for 30 mn. -isopropyvet:*ylamine (0.3'3 mL, 1.80 01no) was th.en added, f.-Lo,,ed by a sol-ition of3-ehtio -(2ecateh2 rdnun chloride (0.398 g, 1.80 mrnol) in 0.70 mL of dry D,1'F. About a minute after the addition was complete a precipitate separated from the reaction mixture and furth-er cooling at -10 0C 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 was Ct 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 0 0 C 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 ir v mx3345 1770 ($-lactam CO), GB gS II !630 (-CO 2 PNB) cn Hn j g6-.3-7.96 4H, zvridinix aromatic) 8,20-7.65 (ABa, >7.0 H, 4H P!;3 aromatic), 5.53-4.80 Cm, 4H), 4.3-3.7 2H), 3.6-3.:5 6 2.66 38, S-Me), 1.16 Cd, J =6.0 H z, 38, C'-Y e) C. 2-(3-,rethylthiopyridinio)ethly tnio3;-6- R) hydroxy ]tyl]-7-oxo-l-azabicyclo 3. 2. 01 ept-T_-ene- 2-carboxvlate S e /-J4, 3 \CO,,PNB 2~c 041 t 4 0000 00 0 0 0 04 *000 4 00 4 0 000 (0o 4o 0 4 0* 04 (0 0 0 To a mixture of p-nitrobenzyI (5R,6S)-3-C 2-(3- .ethylthio-pyridinio)ethyl thio]-6-(l-CR)-hydroxyethyll -7-oxo-1-azabicyclo[3.2.0]-hept-2-ene-2-carboxylate ch2.orlde (0.551 g, 1.0 -mol) and 10% palladium--on-charcoal (0.55 a) in 25 mL of phosahate buffer (0.05 p. pH 7.4) zo was added 5 mL of THF and 25 mL of ether. This mixture was .vYrzz.enated CParr) at 40 Psi for i h. -he reaction i was then filtered throuh Cel.: and the f :a-e .s ashed with H20 and e:1er. 7- aeous 3eara:ed and washed with adti:-:'.i e:er 3 x I residual organic solve':s _n 30:'1=:3n was cooled at 0.C ad z was -s wi.: saturated aqueous NaHCO This wolzizn was in.ediately applied- to a C 18 reverse-pase olz.

1lution with H2 0 and subsequent lyoph_,lzation of :he 30 relevant fractions afforded 0.25 g of a bricht yel1zw so> This material was repurified by reverse-phase hplc to c.

:ne product (0.210 g, 55%) as a light yellow solid. ir (KBr) v 3400 (br, 1755' (3-lactam GO), 1590 (-CO, cm IHn= (C 0) 5: 8.60-7.76 4H, aromatic), 4.76 Ct, J=5.8 Hz, 2H, N-C8 2 4. 13 of q, 6.3 .Hz, 1., 3.95 (d of t, J-9.0 Hz, J'=2.8 Hz, H, 3.45- 2.75 Cm, SH), 2.59 3H, S-Me), 1.20 J=6.4 Fz, 3H, CH1e); uv (H20) A 296 (8509), 273 (e13005), 231 (ellS7) nn; th CpH 7.4, 36.80C) 20 hi GB ,;Q I r 'I r

.D

I*

0 0 -A .04

I"_

Example Preparation of 3-j2-(1-( 2 ,6-dimethylpyridinium) ethylthio]-6a-[L1-(R- hydroxyethyll-7-oxo-1-azabicvclo (3.2.0)hept-2-ene-2-carboxylate OH

CH

3 93 S Q

N

COO CH3 A. l-(2-mercaptoethyl)-2,6-dimethylpyridinium methanesulfonate S+ O MsOH 100 0 C, 42 h H S 0 MsO A mixture of 2,6-dimethylpyridine (19.2 r1L, 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 100 C for 42 h under a nitrogen atmosphere. After cooling to 25 C, the reaction mixture was diluted with ether (45 rrL) and water (30 mL). The two layers were separated and the organic layer was extracted with water (2 x 5 irL.) The aqueous layers were combined, filtered through a Celite pad, washed with ether (2 x ]5 mL), pumped to remove ce,' the traces of organic solvents and poured on top of a column (3.0 x 12 cm) of 1-bondapak C-18. Elution with 3% acetonitrile 97% water mixture gave after lyophylization of the appropriate fractions 2.5 g of the E impure title compound as a syrup. It was repurified by hplc (p-bondapak C-18) to give 0.90 g of the title compound. ir (film) vmax: 2520 1640 and 1625 (pyridinium), 1585, 1490, 1200 cm (sulfonate), I-mr (DMSO-d 6

D

2 0) 6: 2.36 (3H, s, CH 3

SO

3 4.62 (2H, m, CH 2 N 7.74 (2H, m, Em of pyridinium, 8.24 GB -g7-

I

I Irr (1H. m, Hp of pyridini-un), uv (H 2 0) max: 272 4 080)mI B. Paranitrobenzyl 3-[2-Kl-(2,6-dimethypyridinium)) ethylthioj -6 1- (RY-hydroxyethyl] -7-oxo.-1-azabicyclo (3.2.0)h ept-2-ene-carboxylate diphenylphosphate 1) NEt(iPr) 2 0 0

II

2) CIP(OPh) 2 ICOOPNB /^0 3) HS II '0 (PO 2 MsO COOPNB 4) NEt(iPr) 2 To a cold (0 C) solution of p-nitrobenzyl 6a-[l- (R)-hydroxyethyl]-3,7-dioxo-l-azabicyclo (3.2.0)heptane-2 carboxylate (0.658 g, 1.8 mnmol) in acetonitrile (6 mL) kept under a nitrogen atmosphere was added diisbpropylethylamine (0.394 mL, 2.26 mmol) and diphenyl chiorophosphate (0.468 mL, 2.25 rnol). The reaction mixture was stirred 30 min and treated with a solution of 1-(2-mercaptoethyl)-2,6-diethylpyridinim methanesulfonate 29 (0.720 g, 2.73 mmol) in acetonitrile (3 rL) followed by diisopropylethylamine (0.394 mL, 2.26 mol). The reaction mixture was stirred at 0 0 for 2 h, diluted with cold (OC) water (27 mL) and poured on top of a column x 9.0 cm) of i-bondapak C-18. Elution with acetonitrile-water mixtures and lyophylization of the appropriate fractions gave 0.92 g of the title compound, ir (KBr) Vmax: 3700-3000 1765 (C=O of -lactam), 1690 (C=O of PNB ester), 1620 (pyridinium), -l 1590 (phenyl), 1517 (NO 2 1330 (NO 2 880 cm (NO 2 )1 1 Hmr (DMSO, d 6 6: 1.15 (3H, d J=6.2 Hz, CH CHOH), 2.7- 3.7 (11 H, CH 2 S, 2-CH 3 on pyridinium, H-4, 3.7-4.4 2 3 (2H, CH 3 CHOH, 4.7 (2H, m, CH 2 N 5.14 (1H, d, Hz, OH), 5.37 (center of ABq, J=13.2 Hz, CH 2 of PNB), 6.7-7.5 )10H, m, phenyl), 7.5-8.7 (7H, pyridinium, H's of PNB), uv (H 2 0 Xmax 274 (E14150), 319 (E9445) mp C. 3-12-(1-(2,6-dimethylpyridinium))ethylthio]-6c-[l- (R)-hydroxyethyll-7-oxo-l-azabicyclo(3.2.0)hept-2-ene-2carboxylate -CS S- Pd/C, H 2 THF, ether, butter (PhO) OH (PhO) 2 PO t I t i t t f i t 0 1 To a solution of p-nitrobenzyl 3-12-(1-(2,6dimethylpyridiniumn -ethylthio] -6a- -hydroxyethyl] -7-oxo-l-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 resulting mixture was hydrogenated for 1 h under psi at 23oC 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 p-bondapak C-18. Elution of the column with 5% acetonitrile 95% water mixture and lyophylization of the appropriate fractions gave the title compound 0.246 g as a yellowish powder, ir (KBr) v 3700-2800 1750 (C=O of the B-lactam), max -i 1620 (pyridinium), 1585 cm (carboxylate), Hmr (D 2 0) 6: 1.23 (3H, d, J=6.4 Hz, CH 3 CHOH), 2.5-3.5 (11H,H-4, H-6,

CH

2 S, 2CH 3 on pydridinium), 3.8-4.4 (2H, CH 3 CHOH, 4.5-4.9 (CH 2 N HOD), 7.64 and 7.74 (2H, A part of A 2

B

System, Hm of pyridinium), 8.07, 8.16, 8.18 and 8.27 (1H, B part of A 2 B system, Hp of pyridinium), uv (H 2 0) 23 Xmax: 277 (:9733), 300

E

8271) mp, 2 50.70 (C 0.48, H 20), Anal. calcd. for C 1 8

H

2 2 2 0 4 S-1.5 H 2 0: C 55.51, H 6.47, N 7.19; found: C 55.14, H 6.23, N 6.46.

Example 11 Preparation of (5R, 6S) 2-(2-methylthio-3-methylimidazo lo) etnyl-thio -6-11- -hydroxy-ethylj -7-oxo-1azabicyclol3.2.0]hept-2-ene-2-carboxylate '2 1 rl ~~1 OH SMe 6-Me A. 2-Methylthio-3-methyl-l- (2-mercaptoethyl) imidazolium trifluoromethanesulfonate SMe CF 3so3 H S\HSCH 2CH 2 N-NMe C SMe Me33 Trifluoromethanesulfonic acid (1.38 mL, 0.015 rnol) was added dropwise to 2-methylthio-l-methylimidazole 1 (4.0 g) 0.03 mol) at 0OC under NT 2 Ethylene .0 sulfide (0.9 mL, 0.015 rnol) was then added and the mixture was heated at 55C0C under N 2 for 24 h. The reaction mixture was triturated with ether (3x) and the residue was taken up in acetone, filtered and evaporated.

T11" gave the product (4.2 g, 82%) as a semi-crystalline solid which was used as such without further purification. ir(film)v max 2550 sh) cm- 1 Hninr (d 6 -acetone) 6: 7.97 2H) 4.66 J=7 Hz, 2H, methylene), 4.17 3H, N-Me), 3.20 (d of t, J=7 Hz, J'=9 Hz, 211, methylene) 2.72 3H, S-Me) 2.20 (t, J=9 Hz, 111, -SH).

1. Prepared as per A. Wohl and W. Marckwald, Chem. Ber. 22, 1353 (1889).

B. p-Nitrobenzyl (5R, 2-(2-methythio-3-methylimidazolio)ethyl thio]-6-[l-(R) -hydroxyethyl]-7-oxo- 1-azabicyclo[3 .2.Olhept-2-ene-2-carboxylate diphenylphosphate SMe

CO

2 PNB N -Me O 7H __j CO 2 PNB O 2 e To a solution of p-nitrobenzyl (5R, hydroxyethyll-3,7-dioxo-l-azabicyclol3.2.0]heptane-2carboxylate (1.40 g, 4.0 mmol) in 50 mL of dry acetonitrile, at 0 0 C under N 2 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-l-(2-mercapto-ethyl) 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 in vacuo to give a gum. This gum was taken up in H20 and applied to a C 1 8 reverse-phase column. Elution with H 2 0, then 20% acetonitrile-H 2 O and finally 30% acetonitrile- 2 O, followed by lyophilization of the appropriate fractions afforded the product (0.90 g, 30%) as a light yellow solid. ir(KBr) vmax 1 1 3380 (br, OH), 1770 (B-lactam CO) cm Hnmr (d 6 -acetone) 6: 8.35 (br,s, 1H), 8.24, 7.78 (AB q, J=8.8 Hz, 4H, aromatic), 7.89 (br s, 18), 7.25-6.91 O10H, diphenylphosphate), 5.50, 5.25 (AB q, J=12 Hz, 2H, benzylic), 4.75-4.27 3H), 4.03 3H, N-Me), 4.15-2.75 8H), 2.59 3H, S-Me), 1.22 J=6.2 Hz, 3H, -CHMe).

(5R, 6S)-3-1 2-(2-Methylthio-3-methylimidazolio) ethyl-thio]-6-11-(R)-hyroxyethyl]-7-oxo-l-azabicyclo3.2.01 nept-2-ene-2-carnoxylate OH e N-MeOH SMe S" l 0 2 PNB 20 O

GB

To a solution of p-nitrobenzyl (5R, 6S)-3-1 2-(2methylthio-3-methylimidazolio) ethyl thio]-6-11-CR)hydroxyethyl]-7-oxo-l-azabicyclc L3.2.0]hept-2-ene-2carboxylate diphenylphosphate (1.20 g, 1.56 mmol) in a mixture of 70 mL of THF, 70 ml of ether and 31 rnL of phosphate buffer (0.05 pH 7.4) was added 1.2 g of 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 H 2 0 and ether. The aqueous phase was separated, cooled at 0 C and the pH was adjusted to with saturated aqueous NaHco 3 After removing residual organic solvents in vacuo the aqueous solution was applied to a C 18 reverse-phase column. Elution with H 2 0 and then 8% acetonitrile-H 2 0 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) Sax: 3420 1750 (S-lactam CO), 1590 (-C02e) cm-l; 'Hn.r (D O0) 6: 7.58 2H), 4.52 J=6 Hz, 2H), 4.28- 3.82 2H), 3.90 3H, N-Me), 3.40-2.87 5H), 2.40 3H, S-Me), 1.20 J=6.4 Hz, 3H, -CHMe) uv (H 2 0) ,ax: 297 7572), 262 (c 6259), 222 7955) rm.

max Examole 12 Preparation of (5R, 2-(3-aminopyridinio)ethylthi ]-6-[-(R)-hydroxyethyll-7-oxo-1-azabicvclo[3.2.0:heDt-2-ene-2-carboxylate t Pt I 8 4 C I I Ab.

O* 8

I

9

CO

2 A. 3-Amino-l-(2-mercaptoethyl)pyridinium chloride +N Q L_\ HN H2N .HC1

HSCH

2

CH

2 Cl1 I r 3-Ainopyridine (1.50 a, 0.016 .7ol) was taken up in 1.5 m.L of .1 -1 methalic HCl and the resulting soluti~on was evaporated to give the hydrochloride as an oil. TO thi3 oil was added 3-aninopyridine (1.32 g, 0.015 moly and ethylene sulfide (0.039 ml, 0.015 mol) and zhe resulting mixture was heated (oil bath) at 60-65 0

C

.nder N2for 2 h. knother equivalent off ethylene sulfide (0.99 ml, 0.015 mol) was added and heating was continued at 3S-63 0 C for 65 h. The reaction mixture was washed with CH 2 )Cl 2 and then taken up in H 0 (25 ml) The aqueous solution was applied to a C 18 s reverse-phase column which was eluted with H 2 0. Evaporation of the relevant fractions gave the product (1.26 g, 44%) as a colorless, viscous oil. ir(film) j av 3190 (NH 2 cm 1

;I

'Hnmr (d 6 DMSO) 8.19-7.59 (in, 4H, aromatic), 4.59 j=6.2 Hz, 2H, 3.5 (br s, 2H, -NH 2 3.20-2.77 3H).

B. p-Nitrobenzyl (5R, 2-(3-aminopyridinio) ethyl hydroxyethyl) -7-oxo-l-azabicyclo (3.2.0)-hept-2-ene-2-carboxylate diphenylphosphate

OH

0 -oHl 2 CO 2NB 2 To a solution of p-nitrobenzyl CS5R, hydroxyethyl) -3,7-dioxo-l-azabicyclo(3.2.0)heptane-2carboxylate (0.696 g, 2.0 mjol) in 2.0 71L of dry acetonitrile, at 0 C under N, was added dropwise diisopropylethylamine (0.382 mL, 2.2 rmol) followed by diphenyl chlorophosphate (0.457 mL, 2.2 mol). After stirring at 0 aC for 30 min a solution of 3-amino-l-(2-mercaptoethyl)pvridinium chloride (0.475 g, 2.5 nmnol) in I MI of dry DMF was added, 4 Ottfollowed by additional diisopropylethylamine (0.435 mL, nmnol) The reaction mixture was kept at 0 C for h and was then concentrated in vacuo. The resulting gum was taken up in acetonitrile-H 2 0 and applied to a Cl, reverse-phase column. Elution with H 2 0, followed by GBLl I F j'=2.6 Hz. 1H, H-61, 3.17-2.57('M, 2H, H-41, 1.21 J=6.3 H-z, 3H, CHMej; UVCH 2 OY Xmax; 299 CE 7949) 256 (E bd22) ran; t12(pli 7.4, 36.8 0 C) 18.5 hL.

Example 13 PREPARATION OF

OH

O c (5R,6S)3-[l-(S)-methyl-2-(l-pyridinium)ethylthioll-6-[1- (R)-hydroxyethylll-7-oxo-l-azabicyclo(3.2.0)hept-2-ene-2 carboxylate and -hydroxyethyll -7 -oxo-dl-azabicyclo (3.2 hept-2-ene-2 carboxylate A. d9k-i-(2-rercapto-2-methylethyl)pyridiniun methaniesul.fonate dZ-1-(2-mercapto-1-methylethyl) pyridinium methanesulfonate

I

C

S ~0~

SH

N®o MsO- HS7 1 XN MsO MsOH GB3 Methanesulfonic acid C1.95 mL, 0.030 mol) was added slowly to cold pyridine 0.097 mol) and the resulting mixture was stirred at 40 0 C for 15 min, treated with dl-propylenesulfide (2.59 mL, 0.033 mol) and stirred at 0 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 dl-l- (2-mercapto-2-methyl-ethyl)pyridinium methanesulfonate 1.14 g as a colorless syrup; ir (film) v max: 2520 1640 (pyridinium), 1180 CH 3

SO

3 1040 CH So )cm 1 Hmr (DMSO d 6: 1.35 J=6.8 Hz, 3H, CH 3 CHS), 2.30 3H, CH 3 SO 2.90 J=8.5 Hz, 1H, SH), 3.2-3.7 CHSH), 4.52 (dd, J =12.9 Hz, J=8.4 Hz, CHCH N, -gem 4.87 (dd, J =12.9 Hz, J=6.0 Hz, CHCH N 2H, -ge 2 Hm of pyridinium), 8.5-8.8 1H, Hp of pyridinium), 9.04 (dd, J=1.4 Hz, J=6.7 Hz, 2H, Ho of pyridinium), uv max: 208 (E5267), 259 (E3338), Anal, calcd. for C 9

H

1 5 N0 3

S

2 .2H 2 0; 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 dZ-l-(2-mercapto-1-methylethyl) pyridinium methanesulfonate 0.82 g as a colorless syrup; ir (film) vmax: 2500 1628 (pyridinium), 1180 max (sulfonate, 1035 (sulfonate) cm Hmr (DMSO d 6 6: 1.69 J=6.8 Hz, 3H, CH 3 CHN 2.31 3H, CH 3 SO 3.0-3.3 tt 3 3 3 2H, CH 2 4.2-5.2 1H, CHN 8.0-8.4 2H, Hi of pyridinium), 8.5-8.8 1H, Hp of pyridinium), 9.0-9.2 2H, Ho of pyridinium), uv (H 2 0) max: 209 (E4987), 253 (E3838). Anal. calcd. for C 9

H

15 N0 3

S

2 .1.5H 2 0: C 39.11, H 6.56, N 5.07; found: C 39.13, H 5.92, N 5.20.

-4 B. (5R,6S) paranitrobenzyl 3-1[l-(R,S)methyl-2-(l-pyridinium)ethylthio]-6-[l-(R)-hydroxyethyl]-7-oxo-l-azabicyclo (3.2.0)hept-2-ene-2-carboxylate diphenylphosphate OH 1) NEt(iPr) 0 2 F~1\ I 0 2) CIP(OPh) 2

OH

00PNB 3) HS,.,N MsO N+ 4) NEt(iPr) 2

II

COOPNB (PhO) PO i bbcp~ia

I

j tt c .:rr 4 44r 04 4 *t 4 *r *G er 4 To a cold solution of (SR,6S) paranitrobenzy hydroxyethyll-3,7-dioxo-l-azabicy1o heptane-2-carboxylate (0-523 g, 1.5 mmcl) in acetoni -rile (6mL) kept under a nitrogen atmosphere was added diisopropylethylamine (0.14 tL, 1.8 rrol) followed by dipheny. chlorophosphate (0.373 mL, 1.8 mmol). The reaction mixture was stirred for 30 min and treated with a solution of dZ-l-C2-mercapto-2-methylethyl)pyridiniwm methanesulfonate (0.339 g, 2.16 mmcl) in acetonitrile (2 mL) and diisopropylethylamine (0.314 ,nL, 1.8 mmol). The reaction mixture was stirred at OOC for 1 h, diluted with cold (0 0 C) water (24mL) and chromatographed over prep bondapak C-I8 column (2.5 x 8.5 cm) with 25-50% acetonitrile in water as eluting solvents to give 1.07 g of the title compound as a yellowish powder after lyophilization; ir (KBr) m 3700-3100 1770 (C=O of 3-lactam), 1695 max of PNB ester), 1630 (pyridinium), 1590 (phenyl), 1518

(NO

2 1348 (NO 2 885 (NO 2 cm-1, 'Hmr (DMSO d 6 6: 1.14 j=6.1 Hz, 3H, CHCHO), 1.33 J=6.3 Hz, 3H, CH 3

CHS),

4.6-5.0 CH 2 N2 5.14 J=5.2 Hz, lH, OH), 5.37 (center of ABq, J12.4 Hz, 2H, CH 2 of PNB), 6.6-7.5 phenyl of phosphate), 7.69 Hz, 2H, Ho of ?NB), 3,3-3.4 4H, Hm of ?NB, Lm cf fvridiniu-,m) 3.4-8.3 1H, Hp of pyridinium), 9.03 z, 2H, Ho of nfium) uv (H 2 0) \max: 263 ri3325'i, 333 r3915) AnaL. calcd. for C 3 6

H

3 6 3 10 3 C7.5, H 5. H N 5.39, S 4.27; found: C 57.76, H 4.96, N 3.36, 9 4.35.

and S)-methy1-2-(L-pyridinium) ethylthiol- 6-(1-(R)-hydroxyethy1]-7-oxo-l-azabicycoC3.2.0)hept-2 ene-2-carboxylate i 10% Pd/C, H THFetherbuTer

PO

OH

coj I To a solution of (5R,6S) paranitrobenzyl methyl-2-(l-pyridinium)ethylthio]-6-[1-(R)-hydroxyethyl]- 7-oxo-l-azabicyclo-(3.2.0)hept-2-ene-2carboxylate 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 The resulting mixture was hydrogenated for 1 h under 40 psi at 23 0

C.

The two layers were separated and the organic layer was extracted with water (3 x 7 mL). The aqueous layers were combined, filtered through a Celite pad, washed with ether (3 x 20 mL) and chromatographed on prep bondapak C-18 column (2.5 x 9.5 cm) with water as eluting solvent to give 0.18 g of mixture of diastereoisomers. The two diastereoisomers were separated by hplc (prep bondapak C-18) with water as eluting solvent: isomer with lower retention time, 0.068 g compound f J ir (KBr) V 1770 (C=O of B-1actam), 1633 max -i 1 (pyridinium), 1593 (carboxylate)cm Hmr (D 2 0) 6: 1.20 J=6.3 Hz, 3H, CH CHO), 1.42 J=6.9 Hz, 3H,

CH

3 CHS), 2.3-3.2 3H, H-4, 3.5-3.9 1H, SCH), 3.9-4.2 2H, H-5, CH CHO), 4.3-5.1 CH 2 N 7.8-8.2 3 2H, Hm of pyridinium), 8.4-8.7 1H, Hp of pyridinium), 8.7-9.0 2H, Ho of pyridinium) uv (H 2 0) 23 Sm 260 (E6727), 300 (e8245), [a] 2 39.3° H 2 0), max -4 T 12.6 h (measured at a concentration of 10 M in phosphate buffer pH 7.4 at 36.8 0 isomer with higher retention time, 0.081 g compound ir (KBr) Ct t ~0'Vm v 1755 (C=O of B-lactam), 1630 (pyridinium), 1590 max -1 i (carboxylate) cm Hmr (D 2 0) 6: 1.18 J=6.3 Hz, 3H, CH CHO), 1.40 J=7.0 Hz, 2H, CH CHS), 2.84 (d, -3 3 J=9.3 Hz, 2H, 3.26 (dd, J=2.7 Hz, J=5.9 Hz, 1H, 3.4-4.2 3H, SCH, CH 3 CHO, 4.2-5.1 (m,

CH

2 7.7-8.1 2H, Hm of pyridinium), 8.3-8.65 (m, 1H, Hp of pyridinium), 8.65-8.9 2H, Ho of pyridinium), uv (H 2 max: 259 (E5694), 296 (E6936), [a]D 2 3 96.90 (c 0.56, H 2 T. =15.6 h (measured at a concentration of 10 4 M in phosphate buffer pH 7.4 at 36.8.

0

C).

Example 14 PREPARATION OF (5R,6S)3-C2-((S)-(1-pyridinium)-.- (S)-cyclohexyithiol-6fl-(R)-hydroxyethyll-7-oxo-l-azabicyclo(3.2.0)hept-2-ene-2carboxylate and

CC,,

C C CC r

C'

t (5R,6S)3-[2-i(R)-Cl-pyridinium))-(R)-cyclohexylthio]-6- Cl-(R)-hydroxyethyll-7-oxo-l-azabicyclo(3.2.0)hept-2-ene- 2-carboxylate.

A. d7-l-f2-mercapto-l-cclohexyl)yridiniulm methanesulfonate a s (D MsOH M 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 dt-cyclohexenesulfide [1.377 g (85% pure), 0.0102 moll and stirred at 72 0 C for 25 h. The excess of pyridine was removed under vacuum and the traces were codistilled with water.

The residue was mixed with water and chromatographed through prebondapak C-18 column 5 x 13 cm) with 0-2%

DV

I

V

Ai.-

A

acetonitrile in water as eluting solvent giving after lyophilization a colourless syrup 1.57 g ir (film) VJ 2500 1625 (pyridinium), 1190 max (S03 )I F= (DMSQ d 6 6: 1.2-2.5 88, cyclohexyl H), 2.32 3H, C 3 S0 3 2.82 J=9.8 Hz, SH), 3.0-3.5 1H, CHSH), 4.2-4.9 IH, CHN 8.0-8.3 2H, Fm of pyridinium), 8.4-8.8 18, Hp of pyridinium), 3.9-9.3 2H, Ho of pyridinium), uv (H 20) A 214 Smax (E5365), 258 (e3500). Anal. calcd. for C 12 H N0 3

S

2 1 2 40: C 46.88, H 6.38, N 4.36; found: C 46.61, H 6.46, N 4.65.

B. (5R,6S)paranitrobenzy 3-(2-r.C or j)-l-(pyridinium)1- 1-CR or s)-cyclohexylthio]--E-(-(R)-hydroxyethyl1-7oxo-l-azabicyclo C3.2.0)hept-2-ene-2-caboxylate diphenyiphosphate OH 1) NEt~iPr) OH 2) CIVCOPh) 2 _4 c Mso 0" COOPNB N (PhC 4) NEt(iPr) 2 To a cold CO 0 C) solution of CSR,GS)paranitrobenzyl '-2l-()-hdroyethyl-3,7-dioxo-l-azabicycloC3.2.0) 'e:tane-2-carboxylate (1.37 g, 3.93 =ol) .i acetonitrile kept under a nitrogen arosphere was added zi scpropylethylamine (0.822 7L, 4.7 mol) and diphenyl chlorophosphate (0.979 nL, 4.7 mmol). The resulting solution was stirred for 30 min and treated with a solution of dZ-l-(2-mercapto-l-cyclohexyl)pyridiniun methanesulfonate (1.64 g, 5.66 n'mol) in acetonitrile (4.7 mL) followed by diisopropylethylarine (0.822 mL, 4.7 mnol). The reaction mixture was stirred at OOW for I h, diluted with cold water (75 mL) and chroiatographed on prepbondapak C-18 with 25-50% acetonitrile in water as eluting solvent giving after lyophilization of the appropriate fractions 1.9 g of the title compound, ir v max: 3700-3000 1770 (C=O of 5-1actam), 1700 (C-O of PNB ester), 1628 (pyridinium), 1590 (phenyl), 1515 (NO 2 1345 (NO 2 )0 880 (N02) J '1{mr (D 2 0) 6: 1.13 J-6.1 Hz, 3H, CU 3 CHO), 1.2-2.5 (m,

DW

0

II

2 Yd c II 4C

'M-

8Hr CYclohexyl 2.7-3.5 (mn, 4H, H-4, H-6, CHS), 3.5-4.4 (Mn, 2H!, CH 3 CEoo H-5) 4.4-5.0 (mn, 1H, CHN) 5.30 (center of A~q, j=12.8 Hz, CH 2 Of PNB), 6.7-7.4 (mn, phenyl), 7.63 1j=8.6 Hz, 211 Ho of PNB), 7.9-8.4 (in, 4H, Hm of PNB, Him of pyridiniun), 8.4-8.8 (in, 18, Hp of pyridinium), 9.0-9.4 2H, Ho of 'pyridinium), uv (H2 0) X ax 263 (E9038) 309 (E:6394) Anal. calcd for C 39

H

40 N 3 0 10

SP.H

2 0: C 59.16, H 5.35, N 5.31; found: C 58.95, H 5.15, N 5.57.

C. (5R,6a) orS)-(l-pyridinium)-l-(R or cyclohexylthiol (&)-hydroxyethyll -7-oxo-lazabicyclo(C3.2. 0) hept-2-ene-2-carboxylate OH 10% Pd/c, H2 S ther, bufr O 01 /00PNB To a solution of (5R,6S) paranitrobenzyl or S)-(l-Pyridinium)1-l-CR or S)-cyclohexylthio1-6-11-(R)h.,droxyethyl]-7-oxo-l-azabicyclo(3.2.0).hept-2-ene-2carboxylate diphenylphosphate (1.35 g, 2.34 r'io1) in tetrahydrofuran (96 m.L) was added ether (96 rnL) potass:'-I phosphate ionobasic-sodium hydroxide bu-',er (0.15M, 2- 7.22, 50 inL) and 10% palladium on charcoal (1.9 7he resulting mixture was hydrogenated at 230 under 40 psi for 1.25 h. The organic layer was separated and washed with water (3 x 20 inL) 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 chroinatographed on prepbondapak C-l8 column (4.5 x 9 cm) with 0-5% acetonitrile in water as eluting solvent giving after lyophilization 0.705 g of a mixture of diastereoisomers. The separationi of the diastereoisomers was done by hplc (prepbondapak C-18) With 4% acetoflitril.e in water as eluting solvent; diastereoisomer with lower retention time, compound (0.29 g, i.r (KBr)

I

II

PP

Vm: 3,750 (C=O of -Iactam), 1620 pyridinium), 1685 (carboxylate) 1 Hmr (D 2 0) 5: 1.21 J=6.3 Hz, 3H,

CH

3 CHO), 1.4-2.5 8H, cyclohexyl 2.5-3.05 2H, 3.05-3.25 11, 3.3-3.7 1H, CHS), 3.9-4.3 2H, H-5, CH 3 CHO), 4.3-4.8 CHN+), 7.8-8.2 Cm, 2H, Hm of pyridinium), 8.3-8.7 JA, Hp of pyridinium), 8.8-9.1 2H, Ho of pyridinium), uv (H 2 0) X ma: 260 23ma (-7123), 300 (E8685), (a 3+6.20 (c 0.63, H =16.6 h (measured at a concentration of 10 M in phosphate buffer pH 7.4 at 36.8 0 Anal. calcd. for

C

20

H

24

N

2 0 4 S.2H 2 0: 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 (0.35 g, 38%) ir CKBr) v 1750 (C=O of -Iactam), 1622 Csh, pyridinium), 1588 (carboxylate) cm1, 1Hmr (D 2 O) 6: 1.19 J=6.4 Hz, 3H, CH 3 CHO), 1.3-2.5 Cm, 8H, cyclohexyl 2.5-3.1 2H, 3.1-3.3 Cm, 11, 3.3-3.8 2H, CHS), 4.1 Ccenter of m, 11, CH 3 CHO), 4.25-4.7 Cm, 1H, CHN 7.8-8.1 Cm, 2H, Hm of pyridinium), 8.3-8.7 Cm, t",2011, Hp of pyridinium), 8.75-9.0 Cm, 21, Ho of pyridiniu), 230 uv H 2 O) X: 259 CE5992), 296 (E7646), (al +65.30 (c 0.43, H 2 T 20.2 h (measured at a concentration of 104 M in phosphate buffer pH 7.4 at 36.8"C).

Example Allyl 3-j(2-pyridinioetbyl)t).io)-C63)-(CR)riv droxyethyl]-7-oxo-1-azabicyclo(3.2. 01ept-2-ene- 2-carboxylate diphenyiphosphate

OH

S1)C1POOO) 2 0OH 2 /N7L QO)

CO,

22 To a solution of C5R) allyl 3,7-dioxo-C6S)-E(lR)hydroxysthyll-l-azabicyclo[3.2.0)heptane-(2R)-carboxylate (473 mg, 1.87 mmol) in CH 3 CN (6 mL) was added at ca.

-100C under a nitrogen atmosphere diisopropylethylamine (0.42 mL, 2.4 mmol) followed by diphenyl chiorophosphate

DW

I7 I (0.50 mL, 2.4 ol). The mixture was stirred at 0 0 C for min, and then cooled to -1SC. TO this was added an oily suspension of N-(2-mercaptoethyl)yridifiufl chloride (527 mg, 3.00 rmol) in CH 3 CN (1 mL) containing 5 drops of DM.F, followed by diisopropylethylamine (0.42 mL, 2.4 mmol).

The mixture was stirred at -150 for 30 min and then diluted with H 2 0 (20 mL). This mixture was directly purified on a reverse phase silica gel (Cis PrepPAK, 12 g, Waters Associates) eluting with H 2 0 (200 mL),

CH

3 CN/H 2 0 (100 mL), 20% CH 3

CN/W

2 0 (100 mL),

CH

3

CN/H

2 0 (100 mL) and then 40% CW 3

CN/H

2 0 (100 ML).

Appropriate fractions were collected, the organic solvent removed by a vacuum pump and lyophilized to obtain 786 mg (1.26 rx'.ol, y. 67.3%) of the title compound as brownish powder: Iumr (DMSO-d 6 CFT-20) 6: 1.16 (3H, d, J=6 Hz, 1'-CW 3 2.6-3.7 3.75-Y;.3 (2H, m, 5-H and 4.65 (2H, m, -CO 2

CH

2 4.87 (2H, t, J=6 Hz, -CH 2 5-6.2 (3H, n, olefinic protons), 6.6-7.4 aromatic protons), 8.15 (2H, J3=7 Hz, aromatic protons meta t' 2 (1 to the nitrogen), 8.63 (1H, J=7 Hz, aromatic proton para to the nitrogen) and 9.07 ppm (2H, J7 Hz, aromatic protons ortho to the nitrogen); ir (film) v: 3400 etc '-7'70 (3-lactam), 1690 (ester), 1623 (pyridinio).

B. 15R) 3-((2-pyridinioethyl)thiiol-(6s)'-f(lR)-hydroxyethy2 J o-xo-1-azabicyclo(3.2.01hes t-2-ca

CH

2 S- r/ CO -3,3 7 CC O 3 e 2 *tTo a solution of (SR) allyl 3-[(2-pyridinioethyl)thio(6S)- (IR)-hydroxyethyl]-7-oxo-1-azabicyclo(3.2.0]hept- 2-ene-carboxylate diphenylphosphate (156 mg, 0.25 inmol) in CH 3 CN (2 mL) was successively added at ca. 22 0 C a solution of potassium 2-ethylhexanoate in EtOAc (0.5 M, 0.6 mL; 0.3 mmol), triphenylphosphine (15 mg, 0.057 mmol) and tetrakistriphenylphosphine palladium (15 mg 0.013 mmol).

DW

I

I r The mixture was stirred at ca. 220C under a nitrogen atmosphere for 2 h. After addition of anhydrous Et 2 O0 (7 mL), the precipitate was filtered, washed with anhydrous Et 2 O0 (7 mLs) and dried in vacuo to yield 101 mg of brownish solid. This was purified by reverse phase column chromatography (C 1 PrepPAK, 12 g, Waters Associates) eluting with H 2 0. Appropriate fractions 7-12, each mL) were collected and lyophilized to obtain 53 mg (0.16 inmol, y. 64%) of the title compound as yellowish powder. This material was contaminated with potassium diphenylphosphate and potassium 2-ethyihexanoate: 1Hmr

(D

2 0, CFT-20) 5: 0.80 J=6.4 Hz, Me from ethyihexanoate), 1.21 (3H, d, J=6.3 Hz, 1'Me), 2.93 (2H, dd, J 1 5 -29 Hz, gem 4 Hz, 1-Hs), 3.28 (1H, dd, Hz, Hz, 6-H), 3.42 (2H, t, J=6 Hz, -CH 2 3.98 (1H, td, J 5 1 9 Hz, J.5-6= Hz, 4.15 (1H, q, J=6.2 Hz, 11-H), 4.80 (2H, t, Hz, -CH 2 N 7-7.5 (mn, phenyl protons from diphenyl phosphate), 8.03 (2H, mn, Hxn of pyridinium), 8.56 (1H, mn, Hp of pyridiniun) and 8.81 ppm (2H, J=6.5 Hz, Ho' of pyridinium).

Example 16 Prearation of 3-[2-(N-Methyl.~thioiorpholinium)ethylthio] Gci -(11-(R)-hydroxyethylU-7-oxo-1-azabicycloII3.2.01-hept- 2-ene- 2-carboxylate

OH

:N/

0 Co2 A. N-methyl-N- (2-mercaptoethyl) thioinorpholinium methanesulfonate Me 1)MsOH

HS

MeN S N %N S 2) S s\2

M~V.J

reccoled (ice bath) meyico-.>.h 2. ~ojwas added etan e s u' f cnc -a J.2.c;L) -Ind ethylene sufd 1,0 ,21. 7-.C, :e ,as hneated -at 63*C r 24 h and w: w a er (25 :7.L Th-e ac'-:eous solution was washetl 4- et-e r (3 x 2 5 mL) Pumped under vacuum and ured cDer a silica zel reverse oh'ase column; the ti:Ie czmzcund elute'"~d with water. The approzoriate _fac:_4cns were :ccnnDned and va~orated to afford the thiol as an 2-Jio ~(4.30 g, yZe1l. 'z (film) v)ma 2550 cm- 1 w S I-Lnr (CMSC d; 3.25-2.95 (6H, M, CH 2 N ),33 3, cH.

3 N 3.20-2.65 (7H, m, CH 2 IS H8) 3 2 (3 H CH SC) Lehn and 7. Wagner. Tetrahedron, 26, 4227 (1970) diphenyl phosphate) ethylthio] -6a- [1'-(R)-hydrzx-yethvjlj-7-oxo-l-azabicyclo(3.2.01-hept-2-ene-2car'box;2.- a H 1) EtN (K2 2 C 2) CIPO(O:) 2 3) C H CO -PYB 4) EtN ol~d (ice bath) solution of =ara-nitr-cb.enzvl 6a-tl' (R)-h-y,:roxyethyl]-3,7-dioxo-azabicyclo(3.2.i^heptane-2-carboxylate (557 mg, 1.60 .7mo.) inCr 3

CN

(8 mL) was treated dropwise with diisc~ro cylethyl amine (0.336 1.92 mmol) and diphenylchl-oropohosphate (0.400 mL, 1.92 mmol) and stirred for 30 min. The reaction mixture was treated with N-methyl-N-(2t tvkmercaptoethyl)thiomorpholinium methanesulfonate (893 mg, t "tt 2.29 mmol) in CH CN (4 mL) and diisopropylethy. amine (0.336 m.L, 1.92 mmol) and stirred for 30 min. The solution was diluted with water (20 mL) and poured over a silica gel reversed phase column. The desired compound was eluted with a 50% acetonitrile-Water mixture.

I W TIhe appropriate fractions were combined, pumped under vac.um for 2 h. and lyophylized to afford the title compounld (1.01 go yield ir (nujol),vI 1760 $-lactam C=O) and 1510 cm 1 (so N0 2 2imr (DMSO-d 6 9.25 (2H, d, j=8.8 Hz, H-aromatic), 7.70 (2H, do 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, CH 2 5.14 (1H, d, Hz, OH), 4.35-3.80 (2H, m, H-l' and 3.75-3,45 (6H, m, CH 2 N 3.31 (3H, s, CH 3 N 3.45-2.75 (9H, m,

CH

2 S, H-6 and H-4) and 1.15 ppm (3H, d, J=6.2 Hz, CH3).

hydroxyethyl] -7-oxo-l-azabicyclo -hept-2-ene- 2-carboxy late

OH

J Me HOH

M

S Me C0 2 PNB op 2 S 'xNll C0 0 AI of CO 2

S

f Asolution of ara-nitrobenzy1 3-(2-(N-methylthi, ,or~holinium diphenylphosohate)ethylthio]-6ta-(l'-(R)d -,.ethvl-7-oxo--azabicyclo(3.2.0)heot-2-ene-2nu e r (48.8 %LL) tetrahvdro_ uian 123 0 and inv enr 7,r) was hydrogenated cv:er n a ~r shaker for 1 h at 40 osi. The reaction mlxzre -was ~:e with diethyl ether (40 mLr) and the phases were separated.

The organic phase was washed with water (2 x 5 mL) The 3,7 aqueous phases were combined, filtered through a t52 har~ened 4 ~filter paper, washed with diethyl ether (2 x 20 :nL) and pumped under vacuum. The aqueous solution was poured on a silica gel reverse phase column and the desired carbapenem was eluted with 5% acetonitrile-water. The appropriate fractions were combined, and lyophilized to give title 4 compound as an amorphous solid (205 mg, ir (nujol) limax: 1750 S-lactam C=O) and 1590 cm- 1 C0O); IHnir (0 2 0) 6:4.25-3.95 (2H, m, 3.70-3.40 (6H, m,

CH

2 N 3.35 (lH, dd, !1-6.1 Hz, !1-2.6 Hz, 3.08 (3H, So CH 3 N )P -r 3.25-2.75 (8H, CH 2 S, and 1.24 ppm (3H, d, J=6.4 Hz, CH ;uv (H 2 0, c 0.062))Lax 299 (el0,962) T1/2 17.7 h (0.lm pH 7 phosphate buffer, 371C).

Example 17 Preparation of (SR,6S) (1-methylmorpholino) ethyithiol 6- [(R)-l-hydroxyethyl]-7-oxo-l-azabicyclo[3.2.Olhept-2-ene- 2-carboxy late ICO 2 A. 1-Methyl-l- (2-mercaptoethyl) morpholiniun trifluoromethanesulfonate 0 CF 3 so 3 H

S

S HSCH CH 2 0 CF DSO

G

To N-methylmorpholine (3.29 mL, 0.030 mci) was added dropwise trifluoromethanesulfonic acid (1.327 ntL, 0.015 mol) at 10 0 C, followed by ethylene sulfide (0.89 mL, 0.015 mol) The resulting yelow-brown solution was heated (oil bath) at 50-600C under N 2 for 18 h. Volatile material was then removed in vacuo 'and the residual oil was taken up in m.L of H 2 0. The aqueous solution was washed with diethyl ether (3 x 5 mL) and then residual organic solvent was removed in vacuo. The resulting aqueous solution was applied to a Cl 8 reverse-phase column which was eluted with

H

2 0, then 5% acetonitrile-H 2 0 a,-d finally 10% acetonitrile- 77- H 0. Evaporation of the relevant fractions afforded a white solid whi.ch wps dried in vacuo (P20 to give the product (1.92 g, 41%) ir (KBr)vjmx 2560 cm 1 I; I Htm~r (d 6 acetone) 8: 4.25-3.6 (tn, 8H), 3.49 3H, N-Me), 3.35-27 B. p-Nitrobenzyl (5R,6S)-3-t2-(l-methylmorpholine) ethylthiol (tR)-l-hydroxyethyl]-7-oxo-l-azabicyclo[3.2.0]hept-2ene-2-carboxylate diphenylphosphate

OH

JOH Me 0 J" NL 0CO 2 PNB 0 CO 2 PNB (00)2 PO To a solution of p-nitrobenzyl hydroxyethylll-3,7-dioxo-l-azabicyclot3.2.0lheptane-2carboxyLate (0.348 g, 1.0 mmol) in 25 m.L of dry acetonitrile was added dropwise diisopropylethylamine (0.191 tnt, 1.1 mmcol) and then diphenyl chlorophosphate (0.228 mL, 1.1 rml) at 0 0 C under N 2' After stirring at 0 0 C for 1 h diisoprop~ylethylamine (0.226 m.L, 1.3 mmcl) was added to the resulting enol phosphate, followed by 1-methyl-i- (2-mercaptoethyL) morpholinium trifluoromethanesulfonate (0.373 g, 1.2 mmol).

The reaction mixture was stirred at room temperature for h and then concentrated in vacua. The residual material S 30 was taken up in H 2 0 and applied to a C 1 8 reverse-phase column.

Elution with H 2 0, then 20% acetonitrile-H 2 O0 and finally acetonitrile-H 20 followed by lyophilization of the relevant 22 PNB) cm 1 1 Hnr (d 6 acetone) 5: 8.25, 7.80 (ABq, J=8.6 Hz, 4H, aromatic), 7.4-6.8 (mn, IOH, diphenylphosphate), 5.56, (ABq, !1-14.2 Hz, 2H, benzylic), 4.42 (d ot t, J-9.2 Hz, J'-2.7 Hz, 1H, 4.1-2.7 (mn, 17H), 3.40 3H, N-Me), 1.22 (d,J=6.2 Hz, 3H, -CHZ4G).

1 'r C. (5R,6S)-3-[2-(1-methylmorpholino)ethylthiol-6-{(R)-lhydroxyethyl]-7-oxo-1-azabicyclo(3.2.01hapt-2-ene-2carboxylate OH M 0 Me

O

2 PNB ie OHMe 2 PO -J

:N

CO

2 To a solution of p-nitrobenzyl methylmorpholino)ethyithiol]-6-t(R)-l-hydroxyethyl]-7-oxo-lazabicyclo[3.2.0]hept-2-ene-2-carboxylate diphenylphosphate (0.360 g, 0.49 mmol) in 13 mL of phosphte buffer (0.05 M pH 7.4) was added 0.36 g of 10% palladium-on-charcaol, 20mL of ~tetrahydrofuran and 20 mL of diethyl ether. This mixture t2" was hydrogenated (Parr) at 32 psi for 1 h. The mixture was f.c filtered through Celite and the filter pad was washed with

H

2 0 and diethyl ether. The aqueous phase was separated and the pH was adjusted to 7.0 with additional pH 7.4 phosphate buffer. After removing residual organic solvents in vacuo the aqueous solution was applied to a C18 reverse-phase column. Elution with H 2 0 and lyophilization of the relevant fractions afforded 0.130 g of an amorphous solid. This V material was repurified by reverse-phase hplc to give the a pure product (0.058 g, 34%) as an amorphous solid. ir (KBr)v -1 Tax: 3420 (br, OH), 1750 (a-lactam CO), 1590 (-CO 2 cm Hnmr (D 2 0) 6: 4.35-2.77 17H), 3.18 3H, N-Me), 1.23 J=6.3 Hz, 3H, CHME); uv (H2O)Xmax: 300 (e6344) nm; tl/2 (pH 7.4, 36.80C) 18.5 h.

Example 18 Preparation of (5R,6S)3-[2-(1, 4 -dimethyl-1-Piperazinium)ethylthio]-6-[(1-(R)-hydroxyethyl]- 7 -oxo-l-azabicyclo- 13.2.01hept-2-ne-2-carboxylate i -CH 3 A. 1-(2-acetylthioethy.)-1,4-dimethylpiperazinium bromide 0 I I Br N CH 3CS Acetone 50 0

C

£0 0 CHcSZN NN -,Br A solution of 2-bromoethyl thiolacetate *(2.20 g, 0.012 mcl) and 1,4-dimethylpiperazine (1.95 nL, 0.014 mol) in acetone (4 mL was stirred at 50 0 C for 65 h. After cooling to 251C, the liquid phase was decanted from the gum which was triturated twice in diethyl ether; a hygroscopic yellowish powder, 3.2 g was obtained; ir (Nujol) v 1685 (C=0 of thioester) cm 1 Hmr (D 2 0) ma 2.37, 2.39 (2s, 6H, CH 3 CQ, N-CR 3 3.18 3H, Hansen, Acta Chem. Scand. 11, 537-40 (1957) B. 1,4-dimethyl--(2-mercaptoethyl) piperazinium bromide hydrochloride 3 NZ Br HC1 H s Br-, HC1 I I Br H S CH 3 jI -1 A solution of 1I(2-acetylthioethyl)-1,4direlyy.- ?iperazinium bromide (1.1 g, 3.7 mmo1) in 6N hydrochlorr: acid (4 nL) was heated at 80'C under a nitrogen atmosphere for Ih. 71e solution was concentrated under reduced pressure to give a white powdder, 0.41 g Hmr (-MSO, 4 d, :2.90 C 3 3.26 Aknal. calcd. for C 8

H

2 0

N

2 SBrCl.H 2 O: C 31.03, H 7.16, N 9.03, S 10.35; found: C31.62, H 7.46, N 9.19, S 10.19.

C. (5R,6S)Paranitrobenzyl 3-[2-(1,4-dimethyl-lpiperazinium)-ethylthil-6-(-(R)-hydroxyethyll-7-oxo- 1-azabicyclo(3.2.0]-hept-2-ene-2-carboxylate diphenylohosphate OH 1) NEt(iPr) 2 2) CI?(OPh), 3) 3 C

COOPNBHS

4) NEt(iPr) 2

OH

i2hO).

C _PN B To a cold (0 0 C) solution of (SR,53) ~aranrzenzv 6-'1 -hydroxyethyl)-3,7-d,.oxo-L.-azabiccLo 3.2.0he::e- 2-(R)-carboxylate (0.465 g, 1.33 :%mol) in acetonitrile 2 7.1) kept under a nitrogen atmosphere was added diisopropylethylamine (0.278 m.L, 1.59 mmol) and diphenyl chiorophosphate (0.33 mL, 1.59 mmol). The reaction mixture was stirred for 30 min and treated with a suspension of 1,4-dimethyl-l-(2mercaptoethyl)-piperazinium bromide hydrochloride (0.40 g, 1.37 mmol) inacetonitrile (3 mL)-water (1 mL) mixture and diisopropylethylamine (0.278 mL, 1.59 rmol). After stirring for 18 h at 50C, cold water (15mL) was added to the mixture.

The resulting solution was chromatographed over 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 (501) after lyophylization; ir (Kir) Y max: 1765 (C=O of S-lactam), 1690 (C*O of PN3 enter), 1585 (phenyl),

I-

I p I r y Li)l 1512 (NO 2 875 (NO )cm 1, lHmr (DI4SO, d 6 1.16, 1.18 (2d, J=6.1 Hz, 3H, CH 3 CHOH), 2.44 -3 -K OH), 5.39 (center of ABq, J=13 Hz, CH 2 of PNB), 6.6-7.4 (in, 10H, phenyl of phosphate), 7.71 J=8.8 Hz, 2H, Ho of PNB), 8.26 J=8.8 Hz, Hm of PNB).

D. (5R, 6 S) 3-12-(1,4-dimethyl-1-piperazinium)ethylthio]- 6-[1-(R)-hydroxyethyl]-7-oxo-1-azabicyclo[3.2.Olhept- 2-ene-2-carboxylate OH0 J N- (Pho) P0 10%, Pd/C 2 THF, Ether, COOPNBbuffer

OH

t N sL N 0CO To a solution of (5R,6S)paranitrobenzyl dirnethyl-1-piperaziniun) ethylthiol -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 diathyl ether (25 mL), potassium phosphate monobasic-sodiun hydroxide buffer (13 mL, pH 7.22) and J.0% palladium on charcoal (0.47 g).

The resulting mixture was hydrogenated at 23 0 C under psi for 1 h. The two layers were separated and the organic layer was extracted with water (2 x 7 ML). The aqueous 4, layers were combined, filtered through a Celite pad, washed with diethylether (2 x 15.mL) and chromatographed on PrepPak-500/C, 8 (Waters Associates) column (2.5 x 9.5 cm) with water as eluting solvent to give, 0.097 g after lyophilization; ir (KBr) vinx 3000-37 00 1750 max- 1; (C=0 of 5-lactam), 1585 (carboxylate)cm Hmr (D 2 0) 6: 1.24 J=6.4 Hz, 3H, CH 3 CHOH), 2.33 3H,

/CH

3 \-CH'3 3.15 DW 1

I

23 Sm, HC 3 COHI)., uv (H 20) max: 296 (9476), [aD2 61.10 CC 0.26, H 2 tl/2=12.4 h (measured at a concentration of 104 M in phosphate buffer pH 7.4 at 36.8 0

C).

Example 19 Preparation of (5R,6S)-3-[2-(N-methyl-thiomoflrpholiniumoxide)ethylthio]-6-[1-(R)-hydroxyethyl] -7-oxo-1-azabicyclo- (3.2.0)-hept-2-ene-2-carboxylate

OH

S

N\-

Me

CO

2 ~2 F~ t e mCA

OH

O Me

O

2

OH

:CO Me 02 To a cold (-10 0 C) solution of (5R,6S)-3-12-(Nmethyl-thiomorpholinium)ethylthio]-6-[1-(R)-hydroxyethyll-7oxo-l-azabicyclo(3.2.0)-hept-2-ene-2-carboxylate (608 mg, 1.65 mmol) in a 1:1 mixture of acetonitrile-water (9 mL) was added m-chloroperbenzoic acid (334.8 mg, 1.65 mmol) in small portion over a 1 hour period. The mixture was then diluted with water (15 mL) and washed with diethyl ether (3 c 15 mL).

The aqueous phase was pumped under vacuum and passed through a reversed phase silica gel column (H 2 0) to give a solid which consisted of a mixture of compounds. This mixture was separated by reversed phase HPLC and afforded fraction A 52.4 nq (yield 12%) and fraction B 23.6 mg (yield 61) as diasteremerS of the title compound; Fracton A: 4r L70 3-lactam C=O) and 1380 CM C=OC; L ~M max (02 3: 4.26-2.91 (20 H, M, H-4, H-5, H-6, CH 2 I C H S-0 C 3- N' and CH 2 )and 1.24 ppm (3H, d, J=6.4 Hz, CH 3 uV

(H

2 0, c 0.06) -max 302 (E10425); T 1/2:12 h (0.065 M, pH 7.4 ?hosphate buffer, 37 0 Fraction B: ir (nujol) 1750 3-lactm C=O) and 1385 cm 1 IHmr (D 0, 3.86-2.90 (L7 H, m, H-4, H-5, j-6, CH 2 S, CH 2

S-O,

CH

2 N 3.25 (3H, s, CH3N and 1.24 opm (3H, d, J6.4 Hz, CH; uv (H1O, c 0.05) 2.99 C26517); T 1/2:10.75 h 3 max (0.063 M, oh 7.4 buffer solution, 370C).

Example Preparation of (SR,6S)-3-(2-(l,4,4-Trimethyl-1-piperazinium)ethyl thio]-6-CIR-hydroxytehyl-7-xo-l-azabicyclo(3.2.0)hept- 2-ene-2-carboxylate chloride OH CH CH i 3 CH 3 xv t A 2 -aceyl thie thl) 4, 4 t r--methy Ip, az- 7 id od i d cV 'r 3 OH 00

COO

Br

B

CH

3

CS

A suspension of 1-(2-acetylthioethyl) -1,4-dimethylpiperazinium bromide (1.48 q, 5.0 mmol) in isopropyl alcohol mL) was treated with methyliodide (0.373 mL, 6.0 nmol) and heated at 55-60*C for 30 h. The solvents were evaporated under reduced pressure; the residue was triturated in hexane and the solid was filtered, 1.85 q. The solid was dissolved

DR

I r r

U

in hot water (8 mL) and the solution was diluted with acetone until turbidity (70-80 mL). Two successive crystallizations gave 1.5 g, mp 220-5 0 C dec., 68% of the title compound; ir (KBr) max: 1692 cm-1 Hmr (D 2 0) 6: 2.40 3H, CH3 COO), 3.37 N-CH 3 3.39 N-CH 3 3.99 uv (H 2 0) Xmax: 226 (13144). Anal. CalCd for C llH 24

N

2 0SBrl: C 30.08, H 5.51, N 6.38; found: C 30.48, H 5.53, N 6.86.

B. 1-(2-mercaptoethyl)-1,4,4-trimethylpiperazinium bischloride 01 v+ Br-, I-

CH

3 CS HC1 6N Permutit S-1 C1 2Cl A mixture of l-(2-acethylthioethyl)-1,4,4-trimethyl -piperazinium bromideiodide (1.84 g, 4.19 mmol) and 6N hydrochloric acid (15 mL) was heated at 57 0 C under a nitrogen i atmosphere for 2.5 h. The solution was concentrated under reduced pressure to dryness. The solid was suspended in water (10 m.L) and the well-stirred suspension was treated with permutit S-1 Cl- until a solution was obtained. The solution was poured on a column (1.2 x 60 cm) of permutit S-1 C1 The column was eluted with water (1.5 mL/min). The appropriate fractions were combined and lyophilized to give a white powder, 0.93 g, mp 190-1910C, 85%; ir (nujol) Vmax: 2460 1 Hmr (D 2 0) 6: 3.4 N-CH 3 3.45 N-CH3), 4.07 Anal. calcd for CH 22

N

2 SC1 2 .0.75 H20: C 39.34, I tr H 8.62, N 10.20, S 11.677 found: C 39.48, H 8.39, N 10.35, s 11.15.

C. (5R,6S) paranitrobenzyl 3-[2-(,4,4-trimethyl-Ipiperazinium)- ethyltLio]-6-(JR-hydroxyethyl(-7-oxo-lazabicyclo (3.2.0)hept-2-ene-2-carboxylate bischloride

OH

0 C

COOPNB

1) NEt(iPr) 2 0 I 2) CIP(OPh)2 Permutit S-1 11 ~.Ph 3) NEt(iPr) 2 4) 2C1

OH

t, t 1 4 N N t 0 2C1

COOPNB

To a cold (5 0 C) soluti.on of (5R,6S, paranitrobenzyl 6 -[!R-hydroxyethyll-3,7-dioxo-1-azabicyclo(3.2.0) ieptane-2Rfit carboxylate(0.94 g, 2.7 mol) in acetonitrile (3 mL) kept under a nitrogen atmosphere was added diisopropylethylamine (0.557 mL, 3.2 mmol) and diphenyl chlorophosphate (0.663 mL, 3 3.2 mmol). The reaction mixture was stirred at 50C for 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 mol) After 1.25 h, diisopropylethylamine mL, 0.57 mmoi) was added and the stirring was continued for 2 h. A part of the acetonitrile was eliminated under reduced pressure and the resulting red mixture was chromatographed on PrepPak -500/C 1 8 (Water Associates) column with 25-75% acetonitrile in water as eluting solvent to give a yellowish powder (1.4 g) after -a L- MMMIIIIIIII I lyophylization. The powder was solubilized in water and the solution was passed on a column (1.2 x 58 cm) of permutit S-1 C) using water an eluting solvent. Lyophylization of the appropriate fractions gave 1.17 g of a powder that was repurified on a column of PrepPak -500/C 18 Lyophylization of the appropriate fractions gave a yellowish powder, 0.80 g ir (KBr) Vmax: 3400 (br, OH), 1770 (C=O of the S-lactum), 1690 (C=O of PNB ester) 1605 (aromatic) 1515 (NO 2 1345

(NO

2 cm- 1 1 Hmr (D 2 0) 1.26 J=6.3 Hz, 3H, CH 3

CHOH),

3.39 NCH 3 4.00 5.37 Cbr, s, CH 2 of PNB), 7.60 J=8.6 Hz, 2H, Ho of PNB), 8.20 J=8.7 Hz, 2H, Hm of PNB); uv (H 2 0) max: 276 (E12094), 306 (E:10752). Anal. calcd.

for C 25

H

36

N

4 0 6 SC1,.3H 2 0: 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.

D. (5R,6S)-3-[2-(l,4,4-trimethyl-l-piperazinium)ethylthio]- 6-[lR-hydroxytehyl]-7-oxo-l-azabicyclo(3.2.0)hept-2-ene- 2-carboxylate chloride OH 2C1 0 COOP NB

OH

0) Cl coo A mixture of (5R,6S) paranitrobenzyl trimethyl-1-piperazinium) ethyithiol I1R-hydroxyethyl] -7oxo-l-azabicyclo(3.2.0) hept-2-ene-2-carboxylate bischloride (0.40 g, 0.68 mmol), phosphate buffer (30 mL, 0.05M, pH tetrahydrofuran (10 ether (30 niL) and 10% palladium on charcoal (0.40 g) was hydrogenated at 230C under 35 psi for -1- 1 h. The two phases were separated. The organic phase was extracted with water (10 mL). The aqueous phases were filtered on a Celite pad, washed with ether (10 mrL), concentrated under vacuum to 10 rnL and chromatographed on PrePak- 500/C 18 column (2.2 x 11 cm) with water as eluting solvent to give 70 mg after lyophylization; ir (KBr) V max: 3400 (br, OH) 1755 (C=O of the -Iactam), 1585 (carboxylate)cm1 1 Hmr (D 2 0) 6:1.24 (3H, d, J=6.3 Hz, CH 3 CHOH), 3.36 NCH 3 3.98 uv (H 0) Xmax: 296 (E7987); 2 35.90 0.30, 2 0) T 1 2 9.8 h (measured at a concentration ofl0 M in phosphate buffer pH 7.4 at 36.8 0

C).

t 4

Claims (8)

1. A process for the preparation of a quaternary amine thiol compound of the formula HS A R 14 VII wherein A is cyclopentylene, cyclohexylene or R 1 0 R 1 2 1 1 -C C-- I11 113 R R o0 in which R R R R 1 3 are each independently hydrogen or C 1 -C 4 alkyl, XG is a counter anion and 14 R is represented by the general formula -(N which defines a substituted or unsubstituted mono-, bi- or polycyclic heteroaryl radical containing at least one N atom and optionally, one or more 0, N or S atoms in the ring and attached to a carbon atom of A through a ring nitrogen, thereby forming a quaternary ammonium group which process comprises reacting a sulfide of the formula or S\ R 1 2 c-c RI R13 wherein R 1 0 R 1 1 R 12 and R 13 are each independently hydrogen or C 1 -C 4 alkyl with a strong acid and either VLS J 'vb_ WFLi i ltUI 90 a heteroaromatic amine of the formula 0 which represents a substituted or unsubstituted mono-, bi- or poly-cyclic aromatic heterocyclic radical containing at least one N atom and optionally, one or more O, N or S atoms in the ring and is capable of being quaternized and bonded 1o to a carbon atom of A through a ring nitrogen or a heterocyclic amine of the formula R16 wherein N represents a substituted or unsubstituted mono-, bi- or poly-cyclic non-aromatic heterocyclic radical containing at least one N atom and optionally one or more O, N or S atoms 16 in the ring and is capable of being quaternized by R and bonded to a carbon atom of A through a ring nitrogen and R 1 represents either an optionally substituted aliphatic, cycloaliphatic, cycloaliphatic-aliphatic, aryl, araliphatic, heteroaryl, heteroaraliphatic, heterocyclyl or heterocyclylaliphatic radical or a divalent phenylene or V Jc I 1-11.1.1 I 7 iII 91 C 1 -C 4 alkylene group joined to the ring so as to form a bridged polycyclic group. 1\

2. A process according to claim 1 wherein the reaction is carried out at a temperature in the range of from -20 0 C to 100°C.

3. A process according to claim 1 or claim 2 wherein the strong acid is hydrochloric, hydrobromic, methanesulfonic, p-toluenesulfonic or trifluoromethanesulfonic.

4. A process according to any one of the preceding claig- wherein the reaction is carried out in the presence of a non-polar organic solvent, preferably methylene chloride, benzene xylene or toluene. Qo

5. A process according to any one of the preceding claims wherein when the amine and sulfide reagents are liquids or when the amine reagent is a solid, the sulfide reagent is liquid and the solid amine is soluble in the liquid sulfide reagent, the reaction is carried out without additional solvent.

6. A quaternary amine thiol compound of the formula HS A- R 14 VII X O wherein A is cyclopentylene, cyclohexylene or 10 1 2 R R -C C- 111 113 R R in which R 1 0 R 1 1 R 12 R 13 are each independently hydrogen or C 1 -C 4 alkyl, X- is a counter anion and 92 R is represented by the general formula GN which defines a substituted or unsubstituted mono-, bi- or polycyclic heteroaryl radical containing at least one N atom and optionally, one or more O, N or S atoms in the ring and attached to a carbon atom of A through a ring nitrogen, I0 thereby forming a quaternary ammonium group.

7. A process according to claim 1 substantially as herein described with reference to any one of the examples.

8. A compound according to claim 6 substantially as herein described with reference to any one of the examples. DATED: 11 July 1990 PHILLIPS ORMONDE FITZPATRICK Attorneys For: i BRISTOL-MYERS COMPANY (7772h) 4