CA1175809A - 2.beta.-CHLOROMETHYL-2.alpha.-METHYLPENAM-3.alpha.-CARBOXYLIC ACID SULFONE AND SALTS AND ESTERS THEREOF - Google Patents

Abstract

Abstract:

2.beta.-Chloromethyl-2.alpha.-methylpenam-3.alpha.-carboxylic acid sulfone and salts and esters thereof were synthesized and found to be potent inhibitors of .beta.-lactamases.

Description

5~ DiV. II
I. Description:

The present inven~ion relates to 2~-chloromethyl-2~-methylpenam~3a-carboxylic acid sulfone, a pharmaceutically acceptable salt or an easily hydrolyzecl ester thereo~
which is useful as an inhibit~r of ~-lactamases.

The presumed association betw~en t:he resistance sh~wn by ~-}actam antibio~ics to certain bas:t:eria and the ability of those bacteria to produt:e ,B-lactamases has ~ed tu an intensive search for ~-lactamase inhibitors. Clavulanic .
acid is an example of such a compound presently under in~ensive study. A~o~her ~ lactamase inhibitor has in its acid form the structure B;!~HH3 SOOE~
and is disclosed in European Patent Application 2927 published July 11~ 1979.

- The compound having the ~tructur~
.

H2N ~c~Cl ~ . = .
Q COOH

is disclo~ed in U.S. Patents 4~036,.847, 4,009,159, .3,993,646, 3/989,685 and 3,954,732.

,~

, ~7~
U . S . Patent 4 ,155, 912 descxibes 2-pPn~m-3-car~oxylic acid compounds having the formula ' COOI~

and esters and salts, and see also Fa~mdoc Ab~tracts 82090A, 10336B and 44337B.

The cQmpound (under ~he number CP-45899) having the structure ~S~f ~

rf ~ 3 ~g--N
S:OOH

i~ an irrever~ibly acting ~-lactamase inhibitor with excellent ~olution stability. It has weak antibacterial a~tivity and potentiates the in vitro and in vivo activitieY
of amplcillin ~ersu~ ~lactamase~producing ~trains [A. R.
English e~ al., Antimicrobial ~gents and Che therapy, 14, 414-419 ~1918), ARwapokee et al., J. Antibiotlcs 31(12~, 1238-1244 (Dec. 1978) and Derwent's Fa~ndoc Abqtracts 89627A and 73866B~.

., j .
.
:
. . ' .

~5 It is disclosed by B. ~altzer et al., Mutual Pro Drugs of ~-Lactam Antibiotics and ~-Lactamase Inhibitors, J. Antibiotics~ 33(10), 1183-1192 (1980) that the principle of combining ~-lactam antibiotic with a ~-lactamase inhibitor in a single molecule functioning as pro-drug for the two acti~e components is illustrated by the linked esters 3 and 4 in which ampicillin and mecillinam, respectively, are combined with the ~-lactamase inhibitor penicillanic acid sulfone. It is shown that in man these esters are excel-lently absorbed from the gastro-intestina1 tract and after absorptivn hydrolyzed with simultaneous liberation of the active components. As a result high blood and tissue levels of antibiotic and ~-lactamase inhibitor in a balanced ratio are attained. The advantages of "mutual pro-dxugs" over simple combinations are discussed.

Esters 3 and 4 referred to therein have the structures ~NH ~3~ CN_CH=N~
N
N
o o co o o o //C~CO _O
F~CO-ICH2 ~7~
~4-It is stated in GB 2044255 published October 15, 1980 that the invention therein relates to hitherto unknown com-pounds of the general formula l:

H H
Rl- CH -CO -NH

~ H Cl- O - CH - A
O R~ 1 in which Rl stands for a phenyl, 4-hydroxyphenyl, 1,4- -cyclohexadienyl or a 3-thienyl group; R2 represents a primary amino or a carboxy group; R3 is a hydrogen atom, or a lower alkyl, aryl or aralkyl radical, and A stands for a radical of a ~-lactamase inhibitor containing a ~-lactam ring as well as a carboxy group, A being connected via the carboxy group.

The new compounds are use~ul in the treatment of bacterial infections and are in particular strongly active against ~-lactamase producing bacteria. See also Farmdoc Abstracts 60773C and 60776C.

Accordingly, the present invention provides ~he acid having the formula ~7~

O O
FN~CH2Cl OOH

or a pharmaceutically acceptable salt of said acid or an easily hydrolyzed ester of said acid.

The pharmaceutically acceptable salts referred to above include nontoxic metallic salts such as sodiwm, potassium, calcium and magnesium, the ammonium salt and ~ubstituted ammonium salts, e.g. sal~s of such nontoxic amines as trialkylamines (en g. triethylamine), procaine, dibenzylamine, N-benzyl-~-phene~hylamine, l-eph~namine, N,W'-dibenzyl-ethylenediamine, dehydroabietylamine, N,N'-bis(dehydroabietyl)ethylenediamine, N-(lower)alkyl-piperidine (e.g~ M-ethylpiperidine) and other amines which have been used to form pharmaceutically acceptable salts of penicillins and cephalosporins. The most preferred salts are the alkali metal salts, i.e. the sodium and potassium salts, and the ammonium salt.
, As used herein the term "physiologically hydrolyzed esters" refers to those pharmaceutically acceptable esters known in the art to hydrolyze to the free acid ~orm ln vivo.
Example~ of suitable physiologically hydrolyzed esters include phenacyl, acetoxymethyl, pivaloyloxymethyl, a-acetoxyethyl, a-acetoxybenzy}, a-pivaloyloxyethyl, ,:

, .1~ 7 5 ~ ~ ~

phthalidyl~3-phthalidyl), ind~n~l(5-indanyl)~ methoxymethyl~
benzoyloxymethyl, a-ethylbutyryloxymethyl~ propionylo.xy-me~hyl3 valeryloxymethyl, isobutyryloxymethyl, 6-[(R)-2-amino-2-phenylacetamido]-3~-dimethyl-7-oxo-4-thia-1-aza~icyclo[3.2 03heptane-2-carbonyloxymethyl and 6-~(R~2-amino-2-p-hydroxyphenylacetamido]-~ dimethyl-7-oxol4-thia-1-azabicyclo[3.2.0]heptane-2-carbonyloxymethyl.
The preferred esters are the acetox~methyl~ pi~alo~loxy-methyl, methoxymethyl, phthali~yl, 5-indanyl, 6-~(R)-2-amino-2-phenylacetamido~-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carbonyloxyme~hyl and 6-r(R)-2-~mino-2-p-h~droxyphenylacetamido]-3,~-dimethyl-7-ox~-4-thia-1-azabicyclo~3.2.0]heptane-2-carbonyloxymethyl.

There is further provlded by the present invention the process for the production as the desired product of the acid having the f`o~ula O o . ~s~ C~2C
I ~ 'CH
~ ~OOH

or a pharmaceutically acceptable salt of said acid wh:Lch compr~es the consecutive step5 of a) catalytically hydrogenatingg as with precious metal catalyst such as p~lladium,, an ester ha~ing the ~ormula .~7~

C~Cl ~ H3 A

o CO;2Rl where:~n Rl is benzyl or subst1tuted benzyl and then b) sub~ecting the hydrogenated product to oxidation to produce said desired acid or salt thereof and then, if desired, c ~ esterifying sai d ~cid or salt thereof to produce an ea~ily hydrol~rzed ester of said acid.

There is ~urther pro~ded by the present in~ention the process for the production as the desired product of' the acid having the formula : ' O 1) hC~CH2Cl N
~, ~
a or a pha~aceutically acceptable salt of s id acid ~hich comprises the con~ecutive steps of fl,) ox~d~zlng, as ~ th KMnO4, ~22 or like peroxide or peracid, a~ e~ter havlng the i~ox~ula Br ~C~12Cl ~N --h~
o - C- oCH2CC13 ~751~

to produce an ester sul:Eoxide having the formula ~ O
Br, \\ ~ ~CH2Cl CH~3 .
C-OC~I2CC13 and then b) reacting said ester sul~oxide with a metal :1~ acid, ~s with zinc in glaciaI acetic aeid, to produce said desired acid or salt and then, if desired, c ) esterifying ~aid acld or salt thereof to produce an ea~lly hydrol~ze~ ster ol' æaid acid.

A wide variety of oxidants known in the art for the oxidation o~ sulfides to sul~ones ean be used.
However, particularly convenient reagent~ for alkali metal permanganates~ e.g. potassium permanganate, and organic peracids, e.g. ~-chloroperbenzoic acid, Particularly useful protecting groups for Rl are the benzyl group and substituted benzyl groups, especially 4-nitr~benzyl. Benzyl and substituted benzyl groups can be removed conveniently by catalytic hydrogenatio~. In this ca~e, a solution in an inert solvent of the com~und of the ~ormula A7 wherein Rl is benzyl or substituted benzyl, is stirred or shaken under an at~osphere of ~ydroyen, or hydrogen mixed with an inert diluent such as nitrogen or argon, in ~he presence of a cat~lytic amount of a hydrogenation catalyst. ~onvenient solvents for this hydrogenation are lower-alkanols, such as methanol; ethers~
such as tetrahydrofuran and dioxan; low molecular weight esters, ~uch as ethyl acetate and butyl acetate; wa~er;
and mLxtures of these solvents. However, it is usual tn choose conditions under which the starting material is soluble. ~he hydrogenation is usually carried out at a temperature in the range from about 0 to about 60C. and at a pressure in ~he range from about 1 ~o about 100 kg./cm.2.
~e catalysts used i~ this hydrogenation reaction are the type o~ agents known in the art ~or this kind of trans-~ormation, and typical examples are the noble metals, such as nickel, palladium, platinum and rhodium. The cataly~t is u~ually present in an amount from about O . 01 to about

2.5 weight-percent, and preferably from about 0.1 to about loO weight-percent, based on the compound of formula A.
It is o~ten convenient to suspend the ~atalyst on an in~rt support; a particularly convenient catalyst is palladium suspended on a~ inert support such as carbon. Addition-`. ~ 5~

ally it is usu~l to bu~er the react~on mix~ure in orderto operate at a pH ~n the range ~rom about 4 to 9~ and preferably ~rom 6 to 8. Borate and phosphate buf~ers are commonly used. The reaction typ$cally takes about one hour.

There is ~urther provlded by the present ~n~ention the esters having the formuls C~
R - CH - CO -MH

O , ~ l-o O
~ S"` C ~ C1 ~ ~

F~CH~ H2 --O
: 8 ..
wherein R i8 or Rl ~ wherein Rl is h~drogen or hydroxy and R2 is hydrogeng hydroxy~ methyl, methoxy or chloro and : !

`'''`

.~17S~

pre~erably the ester having the formula ~ CH~5 H-CO~ ~S ~C~

NH2 ~', O 1=0 O O
O

~N 1, 3 7EE2 o . ~o o and th~ e~ter havi~ ~he rormula A CH~
HO ~ CH-CO-NH _~ ~

N~2 ~~"'C=O

.
O, O
' ' ~S"~ CH2Cl ! ~
~ ~ 7 TH2 o C , -.. o 8 and the proc2ss for producing such ~n ester which comprises th~
treatmen~ with acid o:E a solution o~ a compound having the fo~nula . ' ~ ` .

CH~
Rl C ~ ~ CO~
2 11 0 "' f=o R~ .
~ C~
O - ~0 O

~herein R ls ~5 or R4 ~ whereln R4 i~ hydrogen or hydroxy snd R5 is hydrogen, hydro~y, methyl, methoxy or chloro ~ld Rl i8 aIkyl~ aralkyl or aryl, R2 is hydrogen, alkyls aralkyl or aryl and R3 is alkyl, aralkyl, ar~l, aIkoxy, aralkoxy~
aryloxy or R4 `~R5 wherein R4 and R5 are eQch hydrogen, alkyl, aralkyl or ar~rl or, ~rhen taken together with the nitrogeIl atom, are piperidino or morpholino with said treatment wlth acid pre~erably being carrled out in an organic solvent such as acetone or chlorofo:~ or in aqueous or partly aqueous ~olution and pre~r~bly between pH 1 and pH 5 at room temperature.

~ 13- .
~l7;~
.

It is further preferred that, in the amino-protecting group, Rl is methyl, R~ is hydrogen and R~
is methoxy, ethoxy or methyl; this requires the use of methyl acetoacetate, ethyl acetoacetate or acetylacetone.

In the remo~al of the ~-~mino-protecting group it is preferred that use be made of a strong mineral acid such as hydrochloric acid or of ~ormic acid~

m ere is further provided by the present invention as a novel intermediate an ester having the formula ~r ~ H2cl -OR

wherein R i8 benzyl or substitute~ benzyl, and preferably ~p-nitrobenzyl~ and the process ~or its product$on which comprises heating~ pre~erably at reflux, a compound : having the ~o~mula o Br ~f~, ~S~1~
.' I I I .
O ~C-OR
o wherein R is benzyl or substituted benzyl ~nd pre~erably p-nitrobenzyl, in an ~nert~ anhydrous or~anic solvent~
pre~er~b~y dioxane, in the presence of large, and preferably .. -~ ~ -14- ..
~7S~

equimol~r, amounts of a weak tertiary amlne, preferably quinollne~ and an acid chloride, preferably benzoyl chloride, until the reaction is substRnti~lly complete.

There is provided by the present invention in addit~on as a novel lntermediate an ester having the formula O
r~H2Cl ~--OR

wherein R is benzyl or sub~tituted benzyl, and preferably p-nitrobenzyl3 and the process for its production which comprises oxidizing a solution in an inert solvent, preferably methylene chloride, Or a compound having ~he formula ~r~ c~
O C-OR

wherein R is benzyl or ~ubstituted b~nzyl, ~nd preferably p-nitrobenzyl~ at about room temperature by the use of a peracid, pre~erabl~ m-chloropero~ybenzoic acid.

s i ~7~

. .

There is further provided by the present inventlon a~ a novel intermediate the ester having the formula O O
Br ~ CH2Cl ""'r~S~'C~
O C-~C~2~
O

the pro~ess for itæ production which co~prises oxidizing a solution in an inert solvent5. preferably methylene chloride, of a compound having the formula ~C}~2Cl 0~ C- OC~2CC13 at about room temperature by the use of an oxidi~.ing agent ~uch as KMhO4, H202 or like peroxide or, preferably a peracid, preferably m-chloroperoxybenzoic acid.
"Skellysol~e B~ is a petroleum ether fraction of b.p. 60-68C. consisting e..sentlally o~' n-hex~ne ("Skellysolve" is a trade name o~' the Skelly Oil Co. ).
The following examples are provlded solely for the purpose of illustrating the preparakion o~ representa-tive compounds o~ the present invention ~nd are not ko be construeà as limiting the invent~ on.

* Trademark ~ --16--~.~

~ 3nam-3a-carboxvlate Sulfone (BI,-P2013) ,.
O
Br~ ~ 3x, r~S

O N "~CO ~ 6~a ~~ ~
: 2 ~o;2 - ~H2~No2 :~ _ ~, \ /
O
Elr,~H2cl 3r~CH3Cl C02-C~2~ N2 ~2-C~i2~tlo2 ~2 :~ 4 \/ 3 ~CU3 ~ H3 N N
. ~ -~O;~H ~ C02K

(BI.-P2 û13 ,~

~ 7~

~-Bro~openicillanic Acid S-Sulfoxide ~) 1. Di~solve 30 g (37.5 mmole~ 6a-bromopenic111anic acid N,~'-dibenzylethylenedi mine salt ~G. Cignarella et a J. Org. Che~. 27 2668 (1962) and E. Evrard~ Nature ?01, 1124 (1964)] in ~30 ml of methylene chloride. Agi.tate ~nd cool to 0C.

2. Slowly add 1~ ml (156 mmole) concentrated hydrochloric acid into the methylene chloride solution.
The precipitation of dibenzylethylenediamine HC1 salt (DBED~HCl) takes place within a minute. Sti-r the slurry at 0-5C for 10 minutes.

3. Filter to remove the DBED-HCl precipitate through a precoated dlatomaceous earth ("Dical te") filter.
Wash the cake with 150 ml of methylene chloride~ me filtration should be completed as quickly as possible.
~void holding the acidic methylene chloride solution for a~prolonged period. There m~y be some filtration problems becau~e of the fine nature of the precipitate. Addition of ~ilter aid to the slurry may be helpful.

4. Wa~h the combined methylene chloride ~lltrate and wash ~ith 60 ml of cold water. Agitate 5 minutes a~d discard the a~ueous phase. The pH of the wash is 2.0-2.~.

5. The methylene chloride solution containing

6~-bromope~icillanic acid 1s concentrated under reduced pressure to a volume of 65-80 ml. Cool and stir the solutio~ to ~C.
*Trademark ( -18- ) 6. With vigorous agitation cautiously add 13 ml (86.9 mmoles) of 40% peracetic acid over a period of 30 minutes. The reaction is exothermic. Maintaln temperature between 15-18~C with ice b~th cooling. m e sulfoxide begins to crystallize after 10 ml peracetic acicL is added. Cool and stir the slurry at 0-5C for two hoursD

7. Filter and wash the snow white cake with the following sequence: 10 ml 5C water, then 10 ml 0-5C
methylene chloride, ~d finally wash wlth 15 ml of heptane.

8. Dry the ca~s:e in 45C air oven to constant weight, about 6-10 hours should be su~ficient. Extended heatlng may generate a trace of pinkish color. The weight of 1 is about 16.26 gm, 73.24% yield.

9. The reaction mix and final product may be monitored by TIC uslng 15 toluene/4 acetone/l acetlc acid (~AC) or 8 acetone/8 methanol/3 toluene/l HAC solvent systems. me final product should be anal~zed by NMR and IR

p-~itrobenz~1 6a-bromopenicillanate S-Sulfoxide (2) To a 801ut~.0n 0~ 12 g ~0.0~ mole) o~ 6a-bromopenicillanic acid s su}foxide in 100 ml o~ acetone was added 7.5 g (0.041 mole) of potassium 2-ethylhexanoate.
~he salt was collected by filtration, washed with cold scetone and air dried to yield a total of 10 grams. The cr~stalline potassium salt waæ d~ssolved in 75 ml of dimethylacet~mide and 7.8 g (0.04 mole) of p-nitrobenzyl bromide was added. The solution was stirred at 23C for 24 hours. me mi~ture was diluted with 500 ml of water and extracted with ethyl acetate. The ethyl acetate layer ~,,, .
, ~ , ~s~

was washed four times with water and dr1ed over anhy~rous magnesium sul~ate. The ~olvent w~s e~aporated at 35C
(15 mm) to an oil which crystallized. m e light tan crystals of 2 were slurr~ed with ether and collected by filtration to yield 9 g (70~) mp 124-125~C dec.
. . .
Anal- calc'd for C15H15BrN206S: C, 41.98; ~, ~.05; N, 6,52 Found: C, 42.00; H, 3.48; N~ 6~98 IR(KBr): 1800(s), 1740(s), 1610(w), 1520(s), 1450(m~, 1350(s~, 1060(m), 740(m) cm~l. ~-NMR (60 mHz, DMS0): ~1.22 (s,3H), 1.6 (s,3H), 4.67 (s~lH), 5.2 ~d,~ 5 Hz,lE), 5.45 ts,2H), 5.68 (d,J~1-5 Hz,lX), 7.5-8.5 (mg4H).

p-Nitrobenzyl 2~-chloromethyl-2a-methyl-6-bromopenam-~a-carboxylate (~) A solution o~ 5 g (0.012 mole) of p-nitrobenzyl 6a-bromopenicillanate S-sulfoxide (2) in 120 ml anhydrous dioxane was heated at reflux under nitrogen for 4 hours with 1.5 g (0.012 mole) o~ ~uinoline and 1.6 g (0.012 mole) of benzoyl chloride. The solution was diluted with 600 ml o~ water and extracted wlth ethyl acetate. me ethyl acetate extract was washed w~th 5% sodium bicarbon~te olution~ 5% phosphoric acid solution and ~inally wlth water. The organic layer was dried over anhydrous magnesium sul~ate and evaporat~d to an oll at 35C (15 mm).
me oil crystallized ~nd wa~ collected, washed with ether, and ~lnall~ wlth cold toluene to yield ~, 3.5 g (65%) mp 130-1~5C dec.
Anal- calc'd for C15H15ClBrN2~S C, 40-06; H~ 3.14~ N~ 6-23 ~ound: C, 40.19; H, 3.12, N, 6.75 IR~KBr): 1792(s), 1740(~), 1610(w), 1520(s), 1353(s), 1280 tm~, 1025(w), 990(w), 750(w) cm NMR (60 mHz, DMS0): ~ 1.45 (s,3~), 3.5-4.3 (m,2H3, 5.05 (s,lH), 5.42 (s~2H)j 5.5(d,J~1-5 HZ~lH)~ 5~62 (d,J~1.5 Hz,l~)~ 7.5-8~5 (~

-20~

p-Nitrobenzyl ?,B-Chlor~methyl-2a-methylpenam-6~-carbox~late : Sul~oxide ~4) _~.., _ .

A solution of 1 g (0.0022 mole) of p-nitrobenzyl 3~-chloromethyl-2a-methyl-6a-bromopen~m-~a-carboxylate (3) dissolved in ~0 ml of methylene chloride was stirred with 473 mg of (0.0022 mole) of m-chloroperoxybenzoic acld.
me solu~ion was stirred at 23~C ~or 3 hour~. Ihe methylene chloride was evaporated to 20 ml at 15 mm and ~3C and the concentrated solution was diluted with ~0 ml of' heptane ( "Skellysolve B" ) . ~he solvent was decanted and the residue was slurried with ether and (4) soon crystallized yield 2~0 mg, 24% mp 136-1~7C dec.
alc'd for C15~143rClN206S ~ ~8-68; H~ 3-02; N, 6-02 Found: C, 39.14; H, ~ 5; N, 5.96 IR(KBr): 1800~s), 1760(s)~ 1520(s)~ 135~(~), 1200(s), 1050(m), 830(w), 740(w) cm 1, H-NMR (60 mHz~ DMS0):
2 (s,3H), ~.8~4.5 (m,2H), 4.97 ~s,lH), 5.25 (dzJ~1~5 Hz,~ . 5.45 (s,2H), 5.6 (d,J~1.5 Hz,lH), 7.8-8.5 (m,~H).

Potassium 2~-Chloromethy -2~-methyl~en~m-3a-carboxylate Sulfone (5) (BL-P201~) To a ~olution o~ 7 g ~0.015 mole) o~ p-nitrobenzyl 2~-chloromethyl-2a-methyl-6a-bromopenam-3a-c~rbox~late sulfoxide (4~ in 150 ml o~ ethyl acetate ~as added a suspension o~ 4 g o~ ~0% palladlum on diatom~ceous earth ("Celite") and 2.8 g of sodium bicarbonAte in 150 ml of water. The mixture was h~drogen~ted for 3 hours at 50 psi.

; .

( -21-~7S13~9 The catalyst was separated by filtration and the aqueous layer wa~ sep~rated and treated with 1.5 g of potassium permanganate in 50 ml of water. The ~lxture was stirred for 1 hour and 250 mg of ~odium bisulflte w~s added. m e mixture was ~iltered and the filtrate was ad~usted to pH 2 wi~h c.oncentrated hydrochloric acid. The solution w s lyophilized to give a white amorphous powder. The solid was extracted with ethyl acetate, evaporated to a volume of 20 ml ~nd diluted wlth 100 ml of heptanes ("Skellysolve B"). Whi~e3 hygroscopic, solid 2~-chloromethyl-2a-meth~lpenam-~a-carboxylic acid sul~one was collected. The ac~d was dissol~ed in acetone and treated with solid pot~ssium 2-ethylhexanoate. A
crystalline white salt precipitated to give, after ~iltratlon, 170 mg of 5 m2 ~140C dec.
Anal~ calc'd for C8 ~ ClKN05S'2H20: C, 28.27; H, ~.24; N, 4.12 Found: C, 28.27; H, 3.69; N, 3.84 IR(KBr): 1790(s), 1770(m), 1620(s), 1460(~, 1370(s)~
1~10~s), 1200(s), 1140(s), 955~m)~ 740(m~ cm 1, H-NM~
(100 mHz, D20): ~1.68(s,3H)3 3.2-3.9 tm,J~2~z~ æ~
J~6.Hz~2H)s 4.0-4.4 (m,2Hj, 4.~ (s,lH)9 5.02 (d d, J~ Hz), J~2 Hz,IH)~ ~

... . .

P~valoyl;ox~methx~ 2B-Chloromethyl-2-methyl,~enam-3~-carboxylate Sul~one 2~-Chloromethyl-2-methylpenam-3a-carboxylic acid sulfone in dimethylormamide is treated with one eguivalent of triethylamine and stirr~d to effect solution.
Bromomethyl pivalate (1 equivalent) ;n dimethylformamide 1 ~22-~L7~

is then added. The resulting mixture i5 stirred at room temperatura. m e mixture is then ~larified by filtration and the filtrate pour~d in~o ice w~ter~ $he separated solid is collected by iltration, washed with water and dried to give the title ester.

The respective acetoxymethyl, methoxymethyl, acetonyl and phenacyl esters of the ~ame acid are prepared by substituting in the method ahove for the bromomethyl pivalate used th~rein an e~uimolar weight of chloromethyl acetate, chloromethyl methyl ether, ~hloroace~one and phenacyl bromide, respectively~

~, Pivaloyloxy~ethvl 2~-Chloromethyl-2-methYlPen~n-3~-9b9L~ 9~'ea~ BL_P2024 :

O
~ ~y ~CH3 ~,S !~ O
0 ~ 2CI ~ NaI ~ N2 ~ ClCN2-0-C-C~CN3)3 c~

~BL--rP:!013 ) ~ P~
C0 C~ -0 C-C(CH3)3 tBL-P2024) To a ~tirred suspension of 14.fig (0.0487 mol) of BL P2013 (5) in 200 ml of acetone W?~ added 4 ml of a

10% aqueous ~olution of ~odium iodide and th~ mixture was broùgh~ to reflux on the steam bath. To this refluxing suspe~ion was added 14.8 ml ~0.1 mol) of xedistilled chloro~et~yl pival~te (~.P. 34C at 7 mm Hg) all at once.
The mixture was stirred at reflux for three hour~ and ~hen cooied to room t~mperature ~22~C~ The crystalline ~olids were collected by filtra~ion, washed with 3 x 30 ml of a~etone and the combined fîltrates were evaporated in a~
oil in vacuo at ~22C. The oil was then ~aken up in 500 ml o~ ethyl acetat~ and washed once with watex t200 mll and once with ~aturatea Na~SO4 while being stirred with 2 g.
o~ decolorizing carbcn with cooling tice bath). A~ter 20 minute~ the mixture was filtered through a diatomaceous tDicalitel pad with ~uction and the pad wa~ washed with 4 x 100 ml of ethyl acetate. The combined filtrate~ were concentrated in vacuo at 22C to an oil. The oil was then .,, , , . ~

S~

conoen~ated further at about 22C and cl mm ~Ig to r~nove most of the re~idual chloromethyl pivalate. Th~ remaining oil was th~n triturated t:wice with 50 ml portions of n-pent~ne and then l~f t over 'che weelcend in the cold room (about lObC) under n-pentane. The ~olid cry~talline mass wa~ therl broken up ~o a ~olid powder under 40 ml of 4:1 mixture of ether-n-pentane. The product was ~hen collected by filtration, washed with ether~p~ntane (1:1) the~ pentan~ and air dried . af te:c drying ir va uo f or four hours over P205 there wa~ obtained 13.37g. of pivaloyloxymethyl 2~-chloromethyl 2a-methylpenam-3u carboxylate sulfone (about 75% yld) M.P. 93-95C.
nal- Calc~- for ~14H20~lNo7s: ~:, 44.03; H, 5.27; ~, 3~;7n Found- C, 44.11; H, 5.0~; Nr ~,.85.

Example 4 Rec ~allization of Po~assium 2~-Chlorometh 1-2~-m~=~

To a mixture of 20 ~nl of n-butanol and one gram of ~L-P2013 (S) was add~d water, one ml at a time, wi.th shaking in ~ separatory fun~el until a pale yellow solution wa~ obtained. The clear solution was filtered throucih a 1uted filtOE paper and the flask and ~ilter paper washed ~lth about 10 ml of 9:1 n-butanol-H2O and the combined filtrates were diluted wi th a furth~r 20 ml o~ n-butanol.
The resulting ~olution was placed in a round-buttomed flask on the roto-vap and evaporated under reduced pressure to approxima~ely one hal the original vo:Luma~ The ~now white crystalline product was collected by ~iltration, wa~hed ~7~

with 6 x 10 ml of ace~one and air dried. Yield 810 mg.
After vacuum drying 6 ho~rs over P2O5 at ~1 mm H~o there wa~ obtai~ed 800 mg MoP~ 215~C ~dec.) (B0% yld)O
Anal. Calcd. for C8HgClN05SKolH~O C9 ~9.67; Hp 3.39; N, 4.63;
Cl~ :10.94; R~F.H2O~ 5.56.
Pound: ~, 29.23; }I, 3~38; N, 4.49;
Cl, 10.74; ~F.~2O, 5.74.
Thi~ recry~tallization procedure produces a cry~talli~e monohydra~e differing from ~he ~tar~lng material whi~h i9 esxen~ially anhydrou~.
.

Exam~le 5_ O O
Il C12 ClC ~ C-Cl CHL0-C-Cl ___________~
3 llght O
ClC ~ -0-S-Cl See Chemical Abstr~cts 27, 24271 and 22, 3828; a~d GB 299064~

-26- ~) 7S~

ClSO 5CH2Cl (6 o o o hCH3 ~ '` F

~OOK 8-OG~Cl . 7 NaI
ac etone O ~ ,0 ~C~2C

'C, OCH2I
O

8 ~ ~C~-CO-N~

rCOOK
H:3C N

~1 5 El C~ C~
I

OCH~ ' 9 (U.S. Patent 3,316,247) I

--27-- `I
~7.~g ~CH--CO--NH = ~

N O C=O
~C-17'~ l H C~C ~

`'S~ C~2Cl ~CH3, ~H2 O

~I

/9 C~
~CH CO~ ~CH3 o CGO
",_. I

o :Ll o O

~N2 ~ ) A solution of chloromethyl chlorosulfate (0.115 mol) in 40 ml. dichloromethane is added dropwise~ keep~ng the reaction temperature below 30C., to a s olution of compouna 5 (0,1 mol) ~d potassium bicarbonate (0.~ mol) and tetrabutylammonium hydrogen sulfate (0.01 mol) in 200 ml. dichlorometh~n~-water tl:l). At the end of the addition the mixture ls stirred at roo~ temperature ~or ~0 mi~ute~, the organic phase i~ separated and the a~ueous phase is extracted with dichloromethane (50 ml.).
me c~mbined organic phases are dried (Na~ 04) and ~vaporated in v~cuo to giYe a residue which is d~sol~ed ~n ether (150 ml.). Insoluble material is filtered off after add~ng diatomaceous earth and the filtrate is e~aporated in. acuo to provide compound 7.

b) To a ~uspen~ion o~ compound 5 (1.~ g.) in d~meth~l~ormamide (12 ml.) there is added 1.6 g. bi~-chlor~methyl sulfate and the mixture is ætirred at room t~mperature for 45 minutes. After dilution with ethyl acetate (50 ml. ) the mixture i8 washed with water and then aqueous ~odium bicarbonate, dried and evaporated in vacuo to leave compound 7 a~ ~n oil.

c) To a solut~on of compound 5 (0.005 mol) in dimethylformamlde (7.5 ml.~ there i8 ~dded triethylam~ne (0.007 mol) ~d chloroiodomethane (0.0~0 mol) and the mixture i~ stirred at room temperature for ~our hours.
~tsr dilution with ethyl acetate (30 ml.) the mixture i8 wa~hed with water (3 x 10 ml. ) ~ollowed by saturated aqueou~ sodlum chloride (5 ml.), dr~ed and evaporQted in vacuo to leave compound 7 a~ an oil.
____ i -29- ~

. d) To a mlxture of compound 5 ~0.15 mol) silver nitrate (0.15 mol) and ~ilver oxide (7.5 g.) in acetonitrate ~750 ml.) there is added chloroiodomethane (1.5 mol).
After stirring for ~8 hours at room temperature, the silver salts are flltered off and the filtrate is evaporated to dryness ln vacuo The resldue is dlssolved in ethyl acetate (200 ml. ) ~nd the solutlon is washed with saturated aqueous sodlum chloride, ~iltered, dried and evaporated in vacuo to gl~e compound 7.
.

Compound 7 and other intermediates and ~inal products of the present invention are pur~ied, if desired~
by column chromatography, as on "Sephadex" LH20 using chloroform-hexane~ 65:35 as the eluent for exemple or by silica gel chro~atography, e.g. using Mallinckrodt CC-7 and hexane-ethyl acetate, 3:2 or ethyl acetate-petroleum ether, 8:2 or 7:3 or 1:9 or 15:8~ or ethyl acetate-n-hexane, ~:6 or ~:1 hexane-ethyl acetate, ~:1 or 1:1 or 1:4 or cyclohexane-ethyl acetate, 1:1.

Thin layer chromatography is also uQeful.
"Sephadex" is cross-linked dextran 2-~diethylamino) ethyl 2-~r2-(diethylamino)ethyl~diethylammonlo}ethyl ether chloride hydrochloride epichlorhydrin (See Merck Index, Ninth Edition, item number 7337) .
A 801ution oi~ compound 7 (0.2 mol) and sodlum iodlde (0.~ mol) in ~cetone (150 ml.) i8 stirred at room temper~ture for 18 hours. The resulting suspenslon is coaled to about 0C. and ad~usted to about plI 7.2 by the addition o~ saturated aqueous ~odium bicarbonate with stirrinæ. After decolorlæing by titration with 0.5 M
aqueous sodium thiosulfate, water (150 ml.) is added ~ropwi~ to the stirred mlxture to precipitate solid compound 8 w~ich 1~ collected by filtration, washed with acetone-water 1:1 (2 x 20 ml.), lsopropanol (2 x 20 ml ) and ether (2 x 20 ml.) and dried.

.

--30-- j ~Q~

Ampicillin is converted to compound ~ by the use o~ methyl acetoacetate in the proc~dures Or U.S.
Patent 3,316,247. Then to a stirred solution of c~mpound 2 (~57 mol~ in d~methyl~ormamide (1 liter) there is added at 5C. 0.5 mol of campo~ld 8. ~fter stirring for 15 minutes at 5C. the reaction mixture is poured into an ice-cold mixture of ethyl acetate (4 liters) and saturated ~queous calcium chloride (2 liters) ~ith stirring. The organic layer is separatedg w~shed with saturated ~queous c~lcium chloride (2 x ~00 ml,), ~iltered a~d evaporated to abou~ one liter in ~acuo to provide ~ concentrated solution o~ compo~nd 10. To thls concentrate there is added water (500 ml.~ and n-butanol (500 ml.) and then, dropwise, 4 N hydrochloric acid with stirring until the ~mino-protecting group is remo~ed to provide a solution of compound 11. After the addition o~ the acid is ~ini~hed ether (1 liter) and water (500 ml.) are ~dded to the ~tirred mixture, the aqueouæ phase i~ separated and the organic phase is e:x:trac*ed with water ~800 ml. ). The c~mbined a~ueous extracts are wa~hed with ether (1 l~ter) and then sodium chloride (640 g. ) and di chloromethane ~2 liters ) are added and the m~ture i8 stirred ~or 15 minute~. Ihe organlc phase is separated and the aqueous phaæe is extracted with dichloromethane (1 liter) and the com~ned organic extracts are dried(MgS0~) ~nd evapora~ed to about 600 ml. under reduced pressure to give a concentrated ~olut~on o~ compound 11. Additlon to the conc~ntrate o~ 200 ml.2 butanona followed by coollng precipltate6 ~olld 6-~(R)-2-~min~-2~phenylacetamido1-3,3-d~methyl-7-~oxo-~-thia-l-azabicyclo~3.2.0Jheptane-2-carbonyloxymethyl 2~-chloromethyl-2-a-methylpenhm-3a-carboxylate sul~one

(11) which is collected by ~iltration.

ExELm21e 6 6-C~R)-?-Amino-2-p~hydr xyphenylacetamido]-3,3_ dimethyl-7-oxo-4-thia-1-azabicyclor3.2.0]heptane-2-carbonylox~methyl 2~-chloromethyl-2-a-methylpenam-3a-carboxylate sulfone having the formula ~o _~C~--CO--N~ ~CH3 NH2 0 C~O

O
~: .

is produced by substituting ~moxicillln for the ampiclllin used in the procedure o~ Example 5.
.

( -32- !
3~

ExRmple 7 . ~ N-Bromo~uccirlimide o a-~zo-isoblltyronitrile ~ ~' CH~Br O

~ AS taught by U.S~ Patent 3,860,579, recrystallized ; phthalidP (50 g.~ 0,~75 moI) and recry~tallized N-brom~-succinimlde (0.37~ mol) were refluxed by 4.5 hours ln the presence of about 100 mgm a-~zobutyronitrile in one liter CC14. The mixture was cooled to about 1~C. and ~iltered to remove succinlmide which was itsel~ washed with about 100 ml. CC14 and ~iltered. l'he c~mbined CC14 phases were concentrated in vacuo to about~ 150 ml. giving 501i~ ~-bromophthalide which was collected by ~iltration3 wa~hed with about 50 ml. CCll~ and ~ir-dried to yield 54 g.
which weigh~d 50 g. a~ter recrystallization froq~ boiling cyclohexane, m.p. 84-86C.
b) .. .. . _ _ .. . . ..
O O
CHBr ~~
C;zCl ~o C-OK

_ DMF
22C.

( -33~
~7~

o o ~S~ C~2~1 'CH
~ N ,~ *
u ~-0-CH ~ BL-P2036 O O l R ~ ' *dl O

To a stirred partial soluti~n and partial suspension of compound ~ (BL-P2013; 2,~ g.7 0.0075 mol~ in 20 ml, d~methylfo~mamide (DMF;. ~ried at least ~ weeks over 3A
molecular seives~ wa~ added 1.7 gO (o.oo8 mol) o~
3-br~mophthalide (12) and the mixture was stirred 4 hours ht 22G. The resulting mixture was poured into a mixture of 200 ml. ice-cold water and 200 ml. ice-cold ethyl acetate (rlnslng the fla~k with a little ethyl acetate) and the ~ixture was shaken. Then the organic solvent phase was separated ~nd washed with seven portions o~ ice-cold water (lOO.ml.). The ethyI ac~tate phase was ~shèd o~ce with saturated aqueou~ Na~S04~ dried ~n the cold o~er Na2S04, filtered and evaporated to dryness in vacuo to 12a~e as the residue ~n oil which was triturated twlce with methylcyclo-hexan~ (25 ml.)9 twice with "SkellysQlve B" (b.p. 60-68C., essentially n-hexane~ (25 ml.) and ~our times with 25 ml.
n-hexane to 2.5 g. compound 1~ as a nearly white solid after drying ~n a~r. Thi8 product was then dried over P20~ at less than } mm Hg to gi~e 2.5 g. c~mpound 13, m.p. 104C. (dec.). It~ estlmated purity was 85-95%.

~ 34-~7~ g Anal. Calcd. for C16Hl~ClN07S: C, ~1.61, H, ~.79; N, ~.77;
Cl, 9.5~.
Found: C, 52.59; H~ 4.67, N, 3.21;
Cl~ 7.r3; K.F.~20, 0.27.
.. . .. . .

P ~aloyl~ ethyl 2~-Chloromethyl-2a-methylpenam-~a-C ~lrb ~a ~9~Y~Df ~

A mixture of 1 g~ ~0.0031 mole) of potassium 2~-chloromethyl-2a-methylpenam-3a-carboxylate sulfone hydrate and 1 g. of ~A molecular sieves was stirred in 15 ml. o~ dimethylacetamide ~or 2 hours at 2~. To this mixture was added ~70 mg. (0.0031 mole) of pi~aloyloxymethyl chloride and the stirring was continued for 18 hours. me molecul~r sie~es were collected and the ~iltrate was diluted with 100 ml. of water ~nd ~xtracted with ethyl acetate. The ethyl acetate was wa~hed nlne times with water and dried:over anhydrous magnesium æulfate.
The solvent ~as removed at ~0 (15 mm) to le~ve an oil which was chromatographed on silic~ us~ng silicar CC-7 (methylene chlor~de 8, ethgl acet~te 2) showing 1 spot Rf .5. me residue obtained cr~stallized from heptane ("Skellysolve B") to yield 100 mg. (M.P. 94-95) of pi~al~yloxymeth~l 2~-chloromethyl-2a--methylpenam-~a-carboxylate sul~one.
Anal. Calcd.: C, 44.0~; H~ 5,27, N, 3.67.
Found : C, 44.20, H~ 5.24; N, 3.6~, ~he NMR and IR spectra were consi~tent ~or ~tructure.

, S~

Sodium ?~ Chloro~eth~1-2a-meth~ylpenam ~a-carboxylate Sul~one ~ CH2Cl I H~ NaEH~ ~ CH
CH
N _ ~ C2 2 C8HgClN05SNa (306) (2~9-673 __ _ _ .
To a stirred solution o~ 500 mg. of BL~P201~5 (potassium salt) in 5 ml. o~ H20 and 10 ml. o~ ethyl acetate was added 2N HCl until pH 1 was obtained (done in an ice-bath with vigorous st:lrring). The mi~cture was then saturated with Na2S04, the aqueous layer separated and the organic phase dried briefly in ice over Na2S04, ~lltered and treated dropwise with 50% NaEH (sodlum 2-eth~Flhexanoate ) in anhydrous n~butanol until neutral to moist pH paper. Product did not crystallize upon scratching and was then concentrated in vacuo to a~ oil which ~E~3 dissolved in acetone (5 ml.), scratched - no crystals, ether added to cloud point - no cyrstals. Concentrated in vacuo on rotovap. to ~n oil which was di~solved in ethyl acetate -added one drop H20 - scratched - no cry~tals. Concentrated ln vacuo and then resldue was triturated wlth 5 ml. of ~-butanol - 200 mg. o~ amorphous white powder obtained~ ether washed - air drled - vacuum dried over P205 for 24 hours.
180 mg. ~inal yield o~ sodlum 2~-chloromethyl-2a-methylpenam-3a-carboxylate sulfone; dec. pto ~100 inde~.
Anal. Calcd. for C8H~ClN0 SNa: C, 3~.10; ~, 3.1~; N, 4.89 Found: C, 33.~0; H, 3.69; N, 4.44 K.F~ ~ 0, 4.04 ~7~g Exampl~ 10 Potassium 2~-chlorome~ 2-meth~y~penam-~-carbox~late Sulfone (BL-P20133 To 10 L of water~ 1~0 g. (1.25 mole) of sodium hydrogen carbonate and 200 g. o~ 10 % Pcl on BaS0~ were added 272 g. (o.565 mole) o~ p-nitrobenzyl 6a-bromo-2~-chlorometh~l-2-methylpenam-7-carboxylate sul~on~ dissolved in 5 L o~ ethyl acetate. The mixture was hydrogenated at 40C and 1 Kg o~ pressure. A~ter 5 hours, the hydrogen uptake became very ~low and 200 g of 10% Pd on BaS0~
were ~dded and the mixture hydrogenat~d untll no ~urther signi~icant hydrogen ~bsorption was perceptible.
.
The slurry was ~iltered through a diatomaceous earth ( "Celite " 5 pad, the pad w~s washed with water and the aqueous ?hase washed with 3 L of ethyl acetate. To the aqueous solution, 3 L o~ ethyl acetate were added and the pH o~ the mixture adJusted to 1.5 with 150 ml o~ 12 N HCl at 10C. The org~nic ph~se was separated and the aqueous solution saturated with Na~S04 10 H20 and extracted with 2 x 1 L of ethyl acetate. The ombined extracts were dried with magnesium sulfate. me dry~ng agent was remo~ed and 260 ml of 2 N pota~sium 2-ethylhexanoic acid in butanol were addsd at 0C.
A~ter stirring 2 hour~ at 0C, the potasslum 2~
chloramethyl-Z-methylpenam-3-carbo~ylate sul~one (BL-P2013) was collected and dr~ed in a vacuum at room temperature~
~ ield : 134.~ g (about 70~).

*Trade~ark --37~
~ ~7~9 Example 11 p-Nitrobenzyl 6c~-Bromopenicillinate Sulfoxide __ . _ _ ......... .. . . .
o (101)~-~2 *02H ~216 ) ~296) ~ /
~: O
Br" ~ S ~

N ~ "~ ~===\
O C02CH2~ ~ N2 1.28) ... . . . . .. .
Procedure:
To 200 ml. of N~N-dimethylacetamide was added 44 g, (o.L48 mole) of 6a-bromopeniclllanic acid ~ulfoxide followed b~ 20.5 ml. (0.148 mole~ of trlethylamine and 38.2 ~. (0.177 mole) of p-nitrobenzyl bromide. It ~was ~tirred at 22 ~or 20 hours~
The reaction mixture was poured into l llter H20 and extracted into 3 x 300 ml. of methylene chloride. The comblned methylene chloride extracts were washed with 200 ml.
of 5% aqueous ~odlum bicarbonate solution and dried over ~odium ~ul~ate at 5 for half an hour. me solution was fllsered and ev~porated under vecuum to a resldue. The - .. . .

. ~ ~

~ 38-~7S~

. .
residue was diluted with ether and the soIid coliected by ~tltration to yield 54 g. p-nitrobenzyl 6a-bromop~nicillinate sulfoxide after drying.
85% y~eld.
nmr conslstent for structure, me yield ~or this ~tep was the same a~ for the K-salt esteri~icatlon. The advantage i~ there wa~ no need to make the K-salt. (A step which goes in 85~ to 90~ yield).

Pr~paration of~ itrobenzyl 6a-Bromopenicillanat~ Sulfoxide To 4.~75 L o~ N~N-dimethylacetamide wa~ added 873.o g (2.95 moles) of 6a bromopenicillan~c acid ~S) sulfo~ide and then with stirring and while keeping the internal temperature below 35C~ 29~ g (2.95 moles) of triethylamlne ~oIIowed by 764 g (3054 moles) o~ p-nitrobenzyl bromlde. The mixture was stirred then at room temperature for 5 hour~ and let stand overnight.
The reaction mlxture was poured lnto 20 L of wa~er and extracted with ~ x 7 L Or methylene chloride. The combined organic e~tracts we~e wa;hed 5 x 7 L of wat~r and then with 7 L of . 5% aqueous sodium bicarbonate solutlon and dried over anhydrou~ magnesium sulf~te.
The magneslum sulfate was filtered o~f and the solution evaporated to a crystalline residue; 4 L of diethyl ether were added ~nd the cryst~ls collected to y~eld a~ter drylng ~t room temperature 1171 g (92%) o~ p-nltrobenzyl 6a-bromopenicillan~te sul~oxide.
Br 18.~8% (calculated 18.53%), ~ (0.25% MeOH).+ 162.
. .

. .

~ 7~

Example 13 Preparation of p-Nit~obenz~:~
2 -me t?~ylpenam-3 -c~

To 16 L o~ acetic acid w~s added 364.6 g (0.812 mole) o~ p-nitrobenzyl 6a-bromo-2~-chloromethyl-2-methylpenam--~-carboxylate. To the solution so obtained and stirred at room temperature, a 601ution of 282 g (1.78 mole) o~ KMnO~
in 26 L o~ water was added drop~ise over ~ hours. me mixture was then stirred at room temperature ~or 1 hour ~nd H202 (~7%) was added dropwise until a colorless solution ~ wa obtained~ 30 L of water were th~n added, the mixture : stirred ~or 1 hour ~t ro~m ~em~erature~and the cr~stalline :~ precipitate was collected,.washed with 3 x ~ L o~ water ~nd :~ with 2 x 2 L of ethanol and dried o~er vacuum at room temper : ature.
Yield: 297 g (76j¢) ~: ~ : aD (~5% CH2C12) ~ 75.g : ~ :

:
Preparatlon o~ BL~ ~ee Acid ~C~12C1 ~3pO~ S~,CH2Cl r~ E~s ~ ~ I CH~
d~ "~C02~3K~ ~ '~C02H

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

, :

( -40-~'7~

To a mixture o~ 25 ml of ethyl acetate and lO
ml of water was added 800 mg (0.00261 mole) of BL~P201 potassium salt. After all of the solicl had dissolved, the mixture was treated dropwise with 50~ aqueous phosphoric acid with vigorous shalsing until no more material precipitated ~rom the aqueous layer. me ethyl acetate layer was separated, then washed with s~tuated sodium chloride ~olution and dried over ~ drou~ magneslum sul~ate. me drying agent was removed by filtra~ion and washed with lO ml of eth~l acetate. (The wash solvent was combined with the original filtrate). "Skellysolve B" was then added to the ethyl acetate to ~he cloud po~nt (approx. lO ml). The mlxture wa~ treated with 500 mg of acti~ated carbon ("Darko KB") and filtered. The ~lltrate was diluted w~th 15 ml of "Skellysolve ~", then seeded with crystals o~ BL-P2013 free ~cid. Af~er approx~ 3 hours at room temperature, the crystalline precipitate of ~ree acid was collected and dried: in vacuo (15 min) over P~05 to obta~ 323 mg (46~) mOp. slow decomp. over 100.
Anal. ~ Calcd, for C8~1oClN~ S: C~ ~5.89, H, ~.~7; ~"5~23, . 5 . .
C1, 13.2S
Found: C, 35.88, H, ~5.91~ Ng 5.41; Cl, 1~.52 This product was Pound to be unstable when stored at 2~C. ~or even days.

*Trademark 7~ g Example 15 6a-Bromopenicillanic Acid Sulfoxide _ O
Br"" ~ S Br" r S
(C6H5C~2NXCH2)2 ~
02E ' C0 H

MW 800.64 MW 296.14 To ~ 1 of methylene chloride w s added ~00 g (0.7~ mole) of 6a-bromopenicillanic acid N,N'-dibenzyl-ethylenediamine salt and this suspension was cooled to 5. Then over a 15 min. period, with good stirring, 130 ml of conc. HCl was added dropwise. The slurry was stirred at 5c for 2 hours~ It was then filtered through a ("Celite") pad of diatomaceous earth and the cake was washed with 3 x 250 ml of methylene chloride.
The c~mb~ned methylene chloride solutions were washed with 2 x 500 ml H20 and dried over sodium sulfate for 15 mln. The sodium sul~ate was removed by filtration and the ~iltrate evaporated under reduced pressure to approx.
750 ~1.
This æolution was cooled to 5 and, with vigorous stirring, 130 ml of 40~ peracetic acid was added dropwise such that the temperature was maintained at 5 to 12.
me addition was quite exothermic. At the end o~ the addition, the slurry wa~ st~rred at 5 ~or 2 hours and the product collected by filtration a~d washed with 100 ml of cold H20 (5) and 100 ml of cold methylene chlorlde (5). There was obtained 126 g (57%) o~ 6a-bromope~icillanic acid sulfoxide, m.p. 129. me ir and nmr spectra were consis~ent for the desired product.
Anal. Calcd. for C8HlQBr~04S: C, 32~44, H, 3.40; ~, 4.7~.
.. . . .
F~un~: C, 32~3~; H, 3u~5; ~, 4.71 H20, 2.18.

: , ( -42-Potassium 6a~Bromopenieillanate Sulfoxide O O
CO2H ~2K

.MW ~.24 To 3 1 of acetone was added 126 g (0.4~ mole) of 6a-bromopenicillanic acid sulfoxide and 162 ml of 50%
by weight potasslum 2-ethylhexanoir acid in n-butanol.
After stirring 1 hour at 22, the product was collected by filtration, washed with 2 ~ 250 ml of acetone and dried.
ere was obtained 127 g (90%) of potassium 6a-bromo-penicillanate sul~oxide, m.p. 185. m e ir and nmr spectra were consistent ~or the desired structure.
Anal. Calcd. for C8 ~ BrKN04S: C3 28.7~; X~ 2.71; N~ 4.19.
- Found: C, 29.03; H, 2.7~; N, 4.0~.

:,~

' 4 3 ~
a~8~9 p-Nitrobenzyl 6a-Bromo~en-icillanate Sulf oxide O O
Br ~ 1~

C2K ~ 2 C~I~No2 MW ~31.28 To 1 1 of N,N-dimethylacetamide w~ added 145 g (0.43 mole) pot~ssiuQn 6a-bromopenicillanate sulfoxide~
and, with ~tirring, there w~s then addPd 115 g (0.5~ mole) o~ p-nitrobenzyl bromide at 22~. The mixture was stirred at 22 for 20 hours.
The reaction mixture was poured into ~ 1 o~ E20 and e~ctracted with ~ x 1500 ml of ethyl acetate. The combined ethyl acetate extracts were washed with 2 x 500 ml of 5% aqueous sodium bicarbonate solut on and dried o~er sodium sulfate ~or 1/2 hour. The sodlum sulfate was ~iltered o~f and the filtrate e~aporated under reduced pressure to a residue to which 1 1 of diethyl ether was added causing the product to crystallize. The crystals were collected by ~iltration, washed with 2 x 100 ml o~
diethyl ether and dried to yield 162 g ~87~) o~ p nltro-benzyl 6a-bromopenicillanate sul~oxide, m.p. 111. The ir and nmr spectra were con~istent ~or the de~ired structure.
_ or C15H16Br~20~S: C, 41.78; H, 3-51; N 6 50 Foun~: C, 41.66; H, 3045; N, 6.85;
H20, o.69.

:~7~

~-carboxvlate v ~C02CII~ Br,~ N2 ~W 449,71 To 1 1 of p-dioxane was added to 70 g (O. 16 mole ) of p-nitrobenzyl . 6a-bromopenicillanate sulfoxide followed by 21.2 ml (0.10 mole) of' benzoyl chloride and 21.8 ml ~0.19 mole) o~ quinoline. The reaction mixture was reflwced for 4 ho-ars and then cooled to 22, poured into 2500 ml of ~I20 and extracted into 3 x 800 ml of ethyl acetate. The c~mbined ethyl acetate extracts were washed w~ 300 ml of ~% aqueous sodium bicarbonate solutior~, 300 ml o~ 5% aqueous pho~phoric ~c1a-an~ ~00 ml o~ ~0. The ethyl acet~te solution was dried over sodium sul~ate for l/2 hour and the sodium sul~ate was removed b~ filtration. The ~iltrate w~s evaporated under reduced pres~ure to a re~idue which was redissolved in 1 1. of eth~l acetate and again evapora~ed under reduced pressure to a resldue. m en 1 1. o~ diethyl ether was added and the product collected by ~iltration to yield 41 g (57%) o~ p nitrobenzyl 6a-bromo-2~-chloromethyl-2-methylpen~m-~-carboxylate, m.p. 1~2. The ir and nmr spectra were consis tent for the desired structure.
r C15H14BrClN205S: C, 40 . o6; H, 3-14; N 6 23 Found: C, 40.62; H, 3.11; N, 6.13.

( -~5-S~

p-Nitrobenz~l 6a-Bromo-2~-chloromet~ 2-methylpenam-3-carboxylate Sulfoxide .. _ . ~ . . O
13r~S~N32Cl r~S JC cl i N ~" N - ~, . N2 MW 465.71 ~ N02 To 1200 ml of methylPne chloride was addecl 51 g (0.11 mole) of p-nitrobenzyl 6a-bromo-2~-chloromethyl-2-methlypenam-~-carboxylate ~ollowed by 23 ~ (0.12 mole) of m-chIorop.eroxybenæoic ac~d. The solutlon was stirred at 22 ~or 2 hours and evaporated under reduced pressure to a wet residue. m e residue was stirred with 4 1 of diethyl ether ~or 1 hour and a~lowed to stand at 10 for 20 hours.
The product crystallized out and was collected by ~iltra~ion, washed wlth 2 ~ 200 ml of diethyl ether and dried, yielding ~9 g p-nitrobenzyl 6a-bro~o-2~-chloromethyl-2-methyl~enam-3-car~o~ylate sulfoxlde (75%)~ m.p. 1~2~. m e ir and nmr sp~ctra were consistent for the desired structure.
Anal Calcd. ~or C15~14BrClN206S: , ~
. Found: C, ~&.98; H, 3.04; N, 5.84;
0, -35~

( 46-Potassium ?~hlorometh~
. 5~1fone (BL-P201 ; . - . . _ . . _ . .
O O

C 2 ___~ c~3 N - ~" r-~ N
(~02CH~ N2 $`CQ2K

M ~05.77 _ _ .
To 600 ml of H?0 ~as added 8 g o~ 30% Pd on "Celite" and 16 g (0.19 mole) o~ sodium bicarbonate.
Then 32 g (o.69 mole) of p-nitrobenzyl 6a-bromo-2~-chloromethyl~2-methylpenam-~-carboxylate sul~ox~de was dissolved in ~00 ml ~f ethyl acetate and added to the aqueous slurry. me mlxture was hydrogenated on a Paar appaxatus at 50 p.s.i. at 22 ~or 4 hours. The slurry was filtered through a thin "Cellte" pad cn a sintered glass funnel, the p~d was washed ~ith 2 x 50 ml H 0 and the aqueous layer of the combi~ed filtrate and wash~lgs was separated~ me aqueous layer was washed with 200 ml of diethyl ether, then was cooled to 5~ and, with sti.rring, a solutlon of 12 g. (.076 mole) of KMnO4 in 200 ml o~` ~0 was added dropwise over a 1/2 hour period, keep~ng the pH
between 7..5 ~nd 8.o by the addition o~ 40%.~P04. When the pink color persisted ~or 5 minute~ no more KMnO4 solution was added. rhe reaction mixture was stirred wlth a small amcun~ (approx. 50 mg) of sodium b~sulflte ~or 1/2 hour, and then the slurry was ~lltered through ~ "Celite"
pad. The pad ~as washed with 2 x 50 ml of ~ 0. The .

~47- .

combined ~iltrate and washing~ wërP layered with 500 ml of ethyl acetate and, with stirring, the pH was adjusted to 1.5 by the addi.tion o~ 2 N HCl. The layers were ~eparated and the aqueous layer was saturated w~th sodium sulfate~
It was reextracted with 2 x 400 ml of ethyl acetate and the . .
combined ethyl acetate e~tractæ were drL~d over sodium sul~ate ~or 1/2 hour ~t 5. The sodium ~ulfate was removed b~ filtration and the ~iltrate evaporated under reduced pressure to a residue. That residue was dissolved in 1~0 ml o~ acetone and 160 ml of diethyl ether and 50%
by weight of potassium 2-RthyIhe~anoate in n-butanol ~as added until the solution ~as neutral to moist pH paper.
me potassium s lt o~ BL-P2013 crystalli~ed out, w~s collected b~ filtration, washed with diethyl ether and dried. Yield 16 g potassium 2~chloromethyl-2-methylpen~m-3-carboxylate ~ulfone (BL-P2013) ~76%), m.p. 202. ~he ir and nmr spectra were consistent ~or the desired structure.
Ar~ ~. Calcd. for C8H~ClKN05S: C, ~1.42; H, 2.97; N, ~.58.
Fo~n~: C, 31.18, H5 2.98; N3 4.51;
._ . .. - . - H203 0.93.

,~

' '.

.~ .

--Z~ 8-- !

Example 16 ~ =_ carbox~late Sul~one (Bl-P2024) - O O O O
~S~ C~ Cl ~ C1 ~,eH~ _ > ~'~"CE~3 C02K ~ C02CH~OCOc (cH~j )3 B~P2024 -_ _ MW ~81.8 _ . ~
To a stirred suspension of 14.6 g (o.0487 mole) of potasæium 2~-chlor~methyl-2-methylpen~m-~carboxylate sul~one (BL-P2013~ in 200 ml of acetone was add~d 4 ml of a 10% aqueous solution o~ sodium iodide and the mixture was brought to re~lux on the steam bath~ To this reflux ing suspension was added 1~.8 ml. (0.1 mole) of redistilled chloromethyl pi~alate (bp ~4CC ~t 7 mm Hg) all at o~ce.
The mixture was stirred at reflux for three hours and then cooled to room temperature (22 C). The cry~talline solids were collected by.filtration, washed with 3 x ~0 ml o~
a~etone a~d the combined :~lltrates were evaporated to an oil under reduced pressure at ~22~ C. The oll wa~ then taken up :ln 500 ml Qf ethyl acetate and washed once with water (200 ml) and once with 3aturated Na2S0~ ~olutlon (200 ml). The solution was then dried briePly over ~a2S04 whlle being stirred with Z g o~ de~oloriz~ng carbon with cooling (lce bath). A~ter 20 mln. the m~xture was ~iltered through a "Cel~te" pad Qnd the pad washed with 4 x 100 ml o~ ~thyl acetate~ The comb~ned filtrates were concentrated under reduced pre~sure at 22 C to an oil. The oil wa~
then further concentrated at about 22 C ~nd <1 mm ~g to remov~ most of the res~dual chloromethyl pivalate. The remaining oil was then trlturA~ed twice w~th 50 ml port~ons of n-pentane and then left over the weekend ~t about 10 C
:

under n~pentane. me resulting solid cr~stalline mass was then broken up to a powder under 40 ml of a 4:1 mixture o~ diethyl ether-n-pentane. The product was then collected by filtration, washed with diethyl ether-npentane (1:1) then n-pentane and air dried. A~ter drying under high vacuum ~or four hours over P205 thare was obtained 13.37 g pi~aloylox~methyl 2~-chlorometh~l-2-methylpen~m-3-carbo~ylate.sul~one (BL-P2024) about; (75%)~
m.p. ~3 - 9~ C
:
:
.

Puri~cation o~ ~L-P2OZ4 Approx~m~tely 3 g. o~ crude BL-P2024 ~obtalnad as described abo~e) w~s d~s~olved in 5 ml, of ethyl acetate, placed on a 4.5 x 40 cm, column of silica gel (Mallinckrodt CC-7), and eluted w~th ~ v C ~ Cl2-ethyl acetate. The ~ractions containing a s~ngle spot at RfO.8~ (TIC o~ æilica gel plates with 4:1 C~2Cl2-ethyl acetate, I2 detectlon) were combined and concentra-ted under reduced pressure to 1.38 g. of a ~rys~alline solid. A portion of this materlal (900 mg.) was dis-solved in 5 ml. of ethyl acetate; the resulting solution was ~iltered, diluted ~lmost to the cloud point with petrole~m ether ("Skellysol~e ~") and then store~ at room temperature ~or three days. The crystals which fo ~ ed were collected by filtr~tion, washed with petrole~m ether and dried ~o give 560 mg., m.p. 100-101., o~ purified BL-P2024, Anal.~Calcd. ~or C14 ~ oCl~O~S: C, 44.03; ~, 5.27; N, 3.67 - Found: C, 44.11; E, 5.o8; N, ~.85.
All t~mperatures in this application ar0 given ln degrèes Centlgrade.

...

Prepar~tion ofi BL-P2013 Ammo~ium Salt 1. The free acid of BL-P2013 (250 mg. ) dissolved in 20 ml. of acetolle-me~hanol (1:1 by volume) was filtered to get ~ clear solution.
2. Anhydrous ammoniu~h solution was prepared by adding 1 ml.
of ammorlium hydroxide (307~" reagent grade) to 10 ml~ of acetone-methanol (1:1 by volume3 solvent and then 1 gm.
of anhydrous magnesium sulfate was added to that solution with mild agitation and the mixture was filtered thrQugh a ~ilter paper; the ~iltrate was designated "anhydrous ammonillm solution. "
3. To the ~lltrate of Procedure 19 approximatel~ 2 ml. o~
"arihydrous aanmonium solutio~" was gradually added and mixed well.
4. A 100 ml, portion of diethyl ether was mixed with the mixture from Procedure 5 to precipitate the a;mmonium salt o~ 13L-P2013.
5. ~he white ~Lmmoni~am salt was iso}at0d from the sol~rent ànd washed with 2 portions of 50 ml. each o~ diethyl ether.
60 me isolated powder ~as dried at 55 C vacuum o-ren for overnight ., 7. Analytical data were as ~ollows:

Calculated ~ C 5~5.7; 4.6; N 9.8;
Found C ~5~i.66; H 4.63; ~ 10.12; dry by KF
Micro~coplc Examlnation: crgstalline ~ubstance --5 2-- ~ !

Ex~ple 18 Preparation of Non-hy~roscopic Sodium Salt o~ Bl-P201~

1. Dissolve 50 mg. of the free acid of ~L-P201~ i~ 4 ml.
of acetone-methanol (1:1 by rolume) mixture. Filter to get a clear solutlon.
2. Prep~-re sodium 2-ethylhexanoate solution b~ dissol~ing 40 mg. of sodium 2-ethyIhexanoate ln 10 ml. o~ acetone-methanol (1:1 by volume) mixture.
. To the ~ltrate of Yrocedure 15 ~dd the 10 ml. solution o~ Procedure 2 ~nd mix well.
. A 10 ml. portion o~ diethyl ether was mixed with the mixture from Pro~edure ~ to precipit~te ~he sodium salt of BL-P201~.
5. The white salt was ~mmersed in the diethyl ether ~or 1-2 hours and th~n was isolated from the ~olvent and washed with 3 portions of 5 ml. each of dlethyl ether.
6. The isol~ted powder was dried at 30 C vacuum oven rOr overnlght~ :

:

--53-- ~ ~
~s~

O O ~) O
~ S~ CH2Cl Recryst . \\~ C~I2G1 _~_ ~ f ~, ~
~' CE~; H20-acetone ~ ~CE3 "' C02E~I20 ~ C2 2 BI~P2013 (400 mg. ) w~s dissol~ed in a min~num amount of acetone~H20 (l:I) by volume and diluted with 10 ml. o~
acetone, :eiltered, then diluted with ac~tone to abalt 25 ml., ~cratched, and a~ter ~iO minutes t~e cryst~llirle hydrate was collected by filtration~ washed well with acetone, air dried and: then ~acuum dried at ~1 mm. H~
overnight.
Yield 280 mg.
Anal. Calcd~. ~or C8HgClNQSK~ H20: C, 29~67; H, ~.~9; 1~, 4.63;
... . : Cl, 10.94; ~I20, 5~55.
Found: C., 9. ~:2; H, ~,32, N, 4.44;
C1, 11.31, H~0, 5.90.

i -54-~e~
~ N'-Dibenzylethylenediamine S~lt.of BL-P201~
_...

BL-P2013 + 1/2 NgN~-Dibenzylethylenedi~m~nc diacetate ~. ~
Recryst.~ / 0~ ~/a C ~ Cl \ .
Aceto~e-ether ~ ~ C ~ ~

~ ' C 02H C6H5CH2N~-CH2t \ / 2 306 mg. (0.001 ml) of BL-P201~ was dissolved in 7 ml. H20 and added to solution o~ 180 mg. (0.000~ mol) o~ N~N'-dibenzylethylenediamlne diacetat~ in 7 ml. H20.
The mixture was stirred and the alt crystallized ~nd after stirring approximately 1~-15 minute~ the salt was collected by ~iltration and air dried to yield N,N'-dibenzylethylene-di~mine salt of BL-P201~ (~00 mg). The material was re-crystallized b~ dis~olving it in approximately 10 mln 0~
boiling acetone and dilutlng with ether to the cloud point.
260 m~. o~ air ~ried and vacuum dried material was obtained~
Anal. C lcd: C~ 51.69; H, 5.42~ N, 7.5~; Cl, 9.55.
Found: C, 49.39; H~ 5.49; NJ 7.05; C1~ 8.96;
~2' 1.23 (KF).

( --55-- ) ~7ASF~C99t Example 21 Chloromethyl E~ter of BL-P2013 .. ... . . .
- .. . .
O O
CH2Cl + ClCH2-0 S02Cl ~CF~ + (CH3cH2cx2cH2)~ ~fIso4 0~ ~COOK ~ 3 KXC03 (:~5 7) ~ CH2C12 -O O
~ r~Cl .
C/-ocN
(316 -17) ,.. .. . ... .. . . .... . .

?

To ~ vigorously stirred m~xtu:re of 15.25 g(0.0~ mol) of BL-P2013 (53, 15 ~. (0.15 mol) KHC0~ and 1.7 g (0.005 mol) of tetrabutyl~mmonium hydrogen sulfate (Aldrich Chem. Co.) ln a mixture o~ 50 ml. water ~nd 50 ml. CH2C12 there was added dropwise a solutlon o~ 9.5 g (0.0575 mol) of ClC ~ -0-S02Cl in 40 ml. CE2C12. me temperature rose to 26 C. and after the addition ~w~ich took about 15 minutes) the m~xture w~s stirred another ~0 inuteæ. Beca~se the product cr~stallized out more CH~C12 tabout 400 ml.) was addPd to obtain a solution. me separated CH2C12 layer and a 50 ml. C ~ C12 wash were com-bined, dried over MgS0~ with stirrlng and 2 g o~ decoloriz-ing carb~n ("Darco KB") was added. After about ~0 minutes the mixture was ~ltered, concentrated to a~out 50 ~1. and isopropyl alcohol (15~ ml.) was added. The rest of the CH2C12 was then removed under reduced pressure. Th~
resulting crystalline precipitate Wa8 col~ected by filtra-tio~, washed well with isopropyl alcohol and air-dried.
A~t~r vacuum drying at lesæ than 1 mm. Hg there was obtained 8.5 g o~ chloromethyl 2~-chloromethyl-2-methylpenam-3-carboxylate sulfone (1). M.p. 116 (decO~ darkens above 100C~.
Anal. Calcd. ~or CgHl~C12~0~S: Cg 34.18~ HJ 3.51; N~ 4.43;
Cl, ~2.43.
Found: C~ 34.16; H, ~.45; N, 4.47;
~ Cl, 2~ ~6, ~ 0~ 0-33 (KF)-Estimated purity i~ the 90-95~ range.

Iodomethyl Ester of 3L- 2013 \S ~ CH2Cl Na I ~"
r~ I CH~ CH3 0 ~ C-ocH2cl 8 C-0C~2I

To a stirred mixture of 5 g. (0.0159 mol) of ~the chlor~methyl ester of BL-P201~ (7) in 25 ml. acetone was added 3 g. (0.02 mol) of sod1um iodide. m e resulting ~lurry was stirred for 17 houræ and then cooled to about OC. Two drops of saturated a~ueous KHC03 were added and the mixture was slowly diluted dropwise with water over ten minutes until 50 ml. had been added. m e ; slurry underwent a sudden color change from yellow to grey to purple to black and therefore the crystals were immediately collected by filtration and w~shed with cold acetone-water (1:2) and then isopropyl alcohol (~ x 10 ml)~
then dieth~l ether and finally n-pentane and air-dried to yield 5.5~ g. (91~ yield) of the iodomethyl ester of BL-P201~ (8). M.p. 118 - 119C. with decomposition.
Purity estlmated at about 90%.
~ . , .
6-~ 5R?-2-~lno-2-phenylaceta~ido]-3,3-dimethyl-7-oxo-4-thia-l-azabicycloC ~ . 2 . 0~ heptane-Z-c arbonyloxymethyl 2~-chloromethyl-2-a-methylpenam-~a-carboxylate sulfone (11) _, .

--~8-- ~) 8~ C~CoN~ ~5~CR3 H~C ~ N

H C~ ,~0 ~:~C ~A Dane ~alt of ~mpic~llin; see U.S.
H~ Patent 3,~6,247) ~~
H~C--C H

I
OC~I3 0 lo J , ~_ . o ¦E

.
:~
.

. -59-~C~--CO~

O
~ 2 ~--O
: O
~:
To a stirred mixture cooIed ~n an ice-bath of ~.46 g. (0.01 mol) of the indicated Dane salt of ampicillin ; ~ (which was solvated with one molecule o~ propyl alcohol) in 60 ml. acetone there was added 4~o8 g. (0,01 ml) ~: : of th2 iodomethyl ester of BI-P201~ (8) and the resulting nearly clear ~olu^tion was stirred ~or fi~e hours with the ice-bath removed ~fter 30 minutes. m en most o~ the acetone was removed in vacuum on the roto-vap and the resulting concentrated solution was dis~olved in 2~0 ml. cold ethyl aceta~e which was then washed wit~ 2 x 50 ml. ice cold water and 2 x 100 ml. saturated a~ueous Na2SO~. The ethyl acetate solution was then dried over Na2S04, flltered and most of the ethyl acetate wa~ removed in vacuo on the roto-vap.
The residue wa~ tr~turated wtth 2 x 200 ml. dry diethyl ether and the resulting solids were collected by filtration to gi~e 5.5 e. o~ lo as a plnkish powder. This powder was , --6 0 ~

stirred ~n a m~xture of 50 ml. water, 50 ml. n-butanol and 20 ml. ethyl acetate while 6N HCl was added dropwise to pH 2 . 5 . Then occasionally a drop or two of HCl was added to keep the p~ at 2 . 2 - 2 . ~ over 45 minutes . When the pH no longer drifted upward there was added to this mixture 100 ml. diethyl ether with good stirring. The aqueous phase was separated and combined with a seeorld 25 ml. H20 extract of the organic layer The aqueous solution was extracted once with 50 ml. diethyl ether and the ether was discarded.

me ~queous layer was then stirred vigorously under a layer o~ 100 ml 2-butanone (methyl ethyl }~etone) while Na2S04 was added to saturate the aqueous layer. Ihe 2 butanone layer was separated, dried over ~a~,S04 for ~0 minutes in an ice-bath, :~iltered and concentrated in ~acuo to near dryness. The residual oil was triturated to a solid with n-butanol, wa~hed well with ether, then n-pentane, air-dried and then Yacuum-dried over P20~; at <1 mm Hg pressure to yield 1.6 g. 6-~ (R)-2-amlno-2-pbenylace-tamido]-~,3-d~methyl-7-oxo-4-thia-1-azabicyclor3.2.0}heptane-2-carbonylmethyl 2~-chloromethyl--2-a-methylpenam-3a-carboxylate sulfone (11) in crude fo~n. The IR and nmr spectra were consistent with structure 11 but not with high purity.
This solid product was estimated to contain at least 40% and perhaps as much as 80% 6-~ (R)-2-a:mino-2-phenylace-tamido] 3~3~dimethyl-7-oxo-4 -th~ a-l-azabicyc lo~ ~ . 2~0]
heptane-2-carbonyloxymethyl 2~-chloromethyl-2-a-methyl-penam-3a-carboxyla~e sulro~e.

~ -61-Improved Synthesis o~ BL-P2013 This procedure simplifies production of BL-P2013 by eliminating the previous use o~ cakalytic reduction.
St~p Br, 4 CH~CC~ + Dicyclohexylcarbodiimide 1 Pyrid~ne C~C12 O
Br~ ~ 2 O C-OCH2CC~
O
(See page 63~ of Cephalosporins and Penicillins, edlted by Edwin H. Flynn, Academlc Pres~, New York, 197 ) 6a-Bromopenicillanlc acid sulfoxide (1) (30 ~.
0.1 mol) ~a~ dissolved ln 1 1. dry C ~ Cl~ ~ollowed by the addition of 16.2 ml. (0.~ mol) p~ridine and 29.8 g.
(0.2 mol) trichloroethanol. Then 20 g. (0.1 mol) of dicyclohexylcarbodiimide wa~ ~dded and the mixture was stirred at 22 for 16 hours. Dlcyclohexylurea began , ( -62- .

to precipitate out, at the end it was removed by ~iltration.
The filtrate wa~ washed with 200 ml. o~ 5% ~queous ~odium bicarbonate3 200 ml. of 10~ phosphoric acid and 100 ml.
o~ saturated aqueous sodium sulfate. The organic phase was dried over sodium sulfate at 5C. ~or 30 minutes, ~iltered and evaporated to an oil. Diethyl ether was added and, with scratching, the product 2 cry~tallized out (27 g., 63% yield).
Step 2 2 1 ~ 0 - ~CCl Quinoline o ~ C- OCH2~C13 o C~mpound 2 (26.5 g.~ 0.062 mol~ wa8 dissolved in 500 ml. p-dioxane and there was added 8.5 ml. (o.o78 mole) ben~oyl chlorlde ~nd 8.75 ml. (o.o78 mole) quinoline. The solution was re~luxed for four hour~ and then poured ~nto 1100 ml. water and the product ~ w~s extracted into ~ x 400 ml. ethyl acetate. q~he ethyl acetate extracts w~re combined, succes~i~re~y washed with 200 ml. 5%
aqueous sodium b~carbonate, 200 ml. 5% phosphoric acid and 200 ml. saturated aqueous sodium. sul~ate, dried over sodlum sulrate at 5C. for th$rty m~utes and evaporated to an oil (~,) which was used l'a~ ls " for the next reactlon.

( -63- ~
L~7be~

; Step ~S ~0~ H202 in glaciPl acPtic acid ~ /
Q V
'F3~C 2C

O C-OCH2CCl~

;; :
Compound ~ obta~ned ~n the pre~iou~ step was : dissolv~d ln 1 1. glacial acetic acid and, with st~rring at 22Cog a saturated a~ueou~ solut~on o~ KMhO4 was added dropwise until a pink color persisted ~that is, a drop placed on a plece o~ filter paper ga~e a pink coloration). Then with cooling ~~ ~2 wa3 added dropw$se until a clear solution was obtained; some white precipitate was pre~ent. me solution was poured into 2.5 1. water and the product 4 was extracted into 3 x 500 ml. eth~yl acetate. The ethyl acetate was wa~hed with 5~t aqueous sod$um b~c~rbonate until neutral (that is, no more bubbling upon additlox~)g dried o~rer sod$um sul~ate a~d ev~porated to iea~e 4 as the residue.

~ ~7 ~

It was le~t ~t 10C. ~or one day and then triturated with "Skellysol~e B" to yield 9.1 g. solid 4. me yield was 28~ of theory for ~teps 2 and 3 comblned.

St~p ~

4 + ~ Zn in Acetic Acid KEH

~ CH Cl C~ 5 (BL-P201~) ~ N~
o COOK

(See U. SJ Patent 4,164,497) Zlnc dust (3.7~ g.) was slur~ied in 5 ml. glacial acetic acid and cooled to 5C. To thi~ ~xture th~r~ was added a solution of 4 (~ g.; 0.0057 mol~) in 15 ml.
dlmet~ylfo~mamide and the resulting slurry was stirred at 5 ~or 2.5 hours.

m e zinc wa~ then ~iltered o~ and the pale ~ellow solutlon was poured into 80 ml. o~ 5% aqueou~
hydrochloric acid. That mixture was extracted with ~ x 25 ml. ethyl acetate. The c~mbined ethyl acetate extracts were e~tracted wlth ~ x 20 ml. o~ 5~ aqueous sodium bicarbonate, savi~g the ethyl acetate phase a~ter separation.

7~

me bicarbon~te extracts were combined, placed under a layer o~ ethyl acetate~ ad~usted to pH 1.5 by the addition of 2N HCl and ~aturated with sodium sulfate.
The ethyl acetate was separated and the aqueous phase was extracted with 2 x ~0 ml. ethyl acet~te.

All of the ethyl acetate pha es above were combined, dried over sodium sulfate and evaporat~d to an oil (which was the ~ree acid fo~m of BL-P2013) which was dissolved in about 20 ml. acetone to which 20 ml. die~h~l ether was then added. Then 50% potassium 2-ethylhe~anoate (KEH) in d~y n-butanol was added to neu-tralit~. The product 5 ~BI-P2013~ crystalllzed out. After stirring 0.5 hour at 22 it was collected by flltration to yield 650 mgm. of 5 (~7% yi~ld).

A 50 mgm. sample of 5 was di~sol~ed in 0.5 ml.
water and 20 mgm. N~N'-dibenzylethylenediamine (D~ED) ~iacetate was added. The DBED salt of 5 crystallized out, was collected by filtration~ w~shed with water and dried over P205 under ~acuum to yield ~,NI-dibenzylethylenedi~mine 2~-chloro~ethyl-2-methylpenam-3-carboxylate sul~one (DBED
salt of ~ree acid 5).

Another sample o~ 5 (450 mgm.) was dis~ol~ed ln 3 ml. water to which wa~ added ~ solution o~ 270 m~m. DBED
d~acetate ~n 2 nLL. ~ 0. With scratching the DBED sa:Lt of ~ crystalllzed out (4~0 mgm.). Recrystallization from about 5 ml. boiling acetone yielded 270 mgm.

~cam~e 2 ~i 6_~ (R) -2-Amino- -p-hydroxyphenylacetamido~ dimethyl-7-oxo~4-thia-1 ~z~bicycloE3.2.0]hep ane-2-carb~nyl~ee~uq 2~chloromethyl-2-a-methyll?enam-3a-carboxylate sulfone _ _ _ __ _ ___ _ having the f o~mula N~2 o/~'C~13 ' .

.
1~ produced by substitutixlg the corresponding D~ne ~alt o~
amoxlciilin ~or the ampicillin used in the procedure of Example 21.

\~

BIOLOGICAL DATA

~ he product o~ aple 1~ Compound 5 , having the structure ~H;2Cl : :
r~ C~3 ,~ N g will ~e reerred t~ below as sL-p2ol3 ~;
Alt~iough at best a Yery ~eak a~ibacterial age~t i~self, BL-P2013 inhibits ~-lactamases and pro~et:ted ceforanide and amoxicillin from destruction by ~-lactama~e-: ~ producing ~acteria in vi~ro ~nd irl vi~o . when usedcombination with thoRe two age~ts, :; : :

.
~: :
.. .

, ~ , Table 1 ~tibacterial Activity of th~ _ 6~-lfone _~ ~

Organism BL--P2 013 ~npicill~
~_ ....... ~ :
. pneumoniae A-9585 16 0 . 004 ~- .
04 63 0. 00 15- aureusA-9537 5. Olre~ ' 5Q% sen~m A-9537 ~125 O. ~6 D. ~lr~
~es A-9606 ~125 ~125 S. aureus Meth- __ _ Res A15097 >125 125 S.. faecalis A20688 >125 0~13 E. coii ~A15119 ~125 ~ 1 Eo coli A20341-1 R. pne~mon~aeA15130~ ~125 ~ 125 K. pneumoniaeA2 04 68 >12 5 ~12 5 i~~ ~ A- 9 90 0 > 12 5 0 ~13 . ~ ._ . . . . ._ _ P. ~rulgari~;A21559 >125 125 P . Iw rganliI~l ~isi _ P. rettgeri~21203 ~12 5 . __ 4_ _ ¦
S~ marcescensA20019 >125 16:
. _ ~ . ~
E. cloacaeA-9659 ~125 63 Eo cloacaeA-9656 ~lZ 5 , ' P. aerug~nosaA-9843A ~125 >125 P,.aerllgirlosa A21213 > 125 ~1;25 _~9_ ) ~'7~

Tabl e 2 Bacteroides ~cti~rity of Ceforanide and Amoxicil lin Alone and in Combination with ~IL-P2 013 .

_ _ Eleta- ~
Organism lact- ~ Ce~o-CeforanideAmoxicilliI~ Amoaci-. amas~ ranide ~L-P2013;~oL13 +B~-2013cilli~
~ _, B a fragiIis A2191 l 63 2 ~125 Z 8 ~: ~22~53 ~ 32 ~q 63 2 8 AZ202 + 32 2 32 _2_ 4 A2187 ~ 32 4 63 2 8 A2253 ~ >125 32 125 16 >125 A2269 ~ 63 8 63 2 8 A2269 ~ 63 4 63 2 16 A22694 ~ 125 16 . 63 2 16 A2269 ~ ~125 _16_ 3Z 4 125 ::: ~2~69 + >125 _3~ 63 8 ~125 A2253 ~ ~125 _32 32 32 ~125 :: A2253 + ~125 32 125 32 ~125 A2279 ~ >125 ~ 32 4 125 A227 9 + 32 4 32 2 8 A2279 ~ 32 4 32 2 8 ~79 ~ ~3 ~ 32 ~ 16 A2279 + 63 4 63 2 16 ~2279 .~ 3~ ~ 63 2 8 3. theta-iotaomic- .
r~n A2227 + 125 4 63 2 16 A2227 + 125 8 63 q 16 . .~ ~ , ",~ :

~7~

Table 2 - cont ' d .

Anti-Bac:teroides Activity of Ceforanide and A~noxicillin ~ _ .
Alone a~d in Combination with BL-P2013 . . Beta-Organism lact- _ amas ranide I BL-P20132013 ~BL-P2013cillin _ ~ _ ~
acteroides pecies A2093 ~ 32 4 32 2 8 A21gS + 63 ~ 4 32 2 16 ~2092 ~ 63 4 32 2 16 A2195 ~ 6~ 16 63 2 16 A20~3 ~ 63 16 63 ~
~2093 ~ 125 ~ 125 4 32 : ~~2093 + 63 ~ 32 2 1~
' 1~20927- _ 0,5 1 63 ~.13 0.13 A2093' _ 2 2 125 0 ,131~ .13 ::
--good synergism --- marginal ~ynergi~m *Minimum inhibitory concentration ~MIIC~ dete~mined by the agar dilution method using SOX dilution~ o 2 4 hour cultures Z!~5 inocula dispensed by the 5teer ' ~ inoculatorO Assay ~editDn composed of Brucella Agar plu~ 5~ laked ~heep blood and 10 mcg/ml vitamin XO
. . .

: , , --71-- ) 7SI~

Table 3 Therapeutic Efficacy of Amoxicillin in Combination with Lac:tamase Strain of StaphYlococcus aureus ~ ' .
~: ~ Challenge ~
Orgarlism t:)rganisms) = IM PO
: . . . ~ .
~: S. aureus S x 108 >800 ~8Q0: ¦ ~50 ~200 6.3 44 ~; ~A 9506 5 x 108 >800 ~800 ¦ ~sn ~200 19 77 7 x 10~ ~ _ Treatment schedule: Drugs ad~i~inistered 0 and 2 hours post-ectic~nO . :' : ;

-72~ S~
. . __ ~ .
s~ ~n ~
~ o~ ~ ~ ~ ,~
ra_I ~ 1 0 O ~ O
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--80-- .

The compounds of the present invention are thus useful, given orally and parenterally, iEor enhanc:ing the effectiveness of ~-lactam ar~tibiotîcs against ~-lac~amase producing bacteria. On a weight basis, the dosage i~ from one-fifth to fi~re times, ana preferably equal to, that of th~ B-lactam a~tibiotics., As an exampl~, ~he compounds of the present invention as shown above when U9ed il'l a 1: 1 ratio markedly improved the activity of ceforanide and a~noxicill i~ against ~-lactamase producing strain~ o~
anaerobic Bacteroides such as B. fragilis ~ B. thetaiotaomicron and other species s~ ~at genus and also against resis~ant J l~ aureus. q~e compounds of the present inverltion are given eith~r in adm~ture with or concomitantly wit:h 'che ~-lactam antibi~tic with the dosage within the indicate~l ratio to the kno~ an~ cu~tomary dosage of the ~tibiotic .
:: ~
~ hus, the ability of ~e coMpounds o~ the present ~n~7ention to enhance the ePfectiveness of a ~-lactam an~-biotic again3t certain ~-lac~amase producing ~acteria makes them ~aluabl~ for co-adminis~xa~ion with certain ~ ctam antibiotics in the treatment of bactexial infections in mammals, particularly ~an. In the treatment o~ a bacterial infection, a compound o~ th~ present invention can be co-mingled with the ~-lactæm antibiotic, and the two agent~
; thereby administered simultaneou~ly. Alternatively, a compound o the present invention can be a~ministered as a ~eparate ag~nt during a cour~e of treatment with a B-lacta~ a~tibiotic.

:

Wherl using a compound of the prese~t invention or salt t~ereof, to enhance the antibactlerial activity of a ~-lactarn antibiotic, it can be adminis tered alone, or preferably, in ~o~mulation with sta~dard pharmaceutis::al carri~rs and diluents. A compound of the present invention which is irl t.he acid form or.as a pharmaceutically-acceptable sal~ thereof, can be administered orally or parenterally; a compound of the present invesltiv~ in the form of an est~r which i~ readily hydrolyzable ~n r vo, is best adminis1:ere~ orally. Parenteral administration includes intramuscular, subc:utaneous, intraperitoneal and in~a-venous administration.

When a compo~ind of the preserlt inventic~ is used in the presence of a ca~ r or diluent, said carrier or-diluent is chose~ on the basis of the intended mode of aaministration. ~or example, when consider ~ g the oral.
m ~ e o administration,.the compound can be used ~n the form of tablets,.capsules,.lozenges, troches, powdersl.
syrups~ elixir~, aqueous solutions and ~uspensio~s, aYla the like, in accordance with standard pharmac~utical practice.
~he proportional ~atio of acti~e ingredients to carrier will naturally depend on the chemical naturef solubility, stabili~y and potency of the active ingredients, as well as the dosage contempl~ted. However, these pharmaceutical co~positions will likely contain ~rom about 5~ to about 80% of caxrier. In the case o~ tablets ~or oral use, carriers which are commonly used include l~ctose, sodium citrate and salts of phosphoric acid. Various disintegrants such as starch, and lubricating agents, such as magnesium stearate, qodium l~uryl sul~te and talc, are commonly u~ed i~ tablets. For oral administration in cap$ule ~orm, useful diluents are lac~ose ~nd high molecul~r weight ;~

.

--8 2~
~:~Lt7S~

polyethylerle glyco:Ls~ When aqueous susp2nsions are re~uired for o~al use, the acti~re ingredies~ts are combined with emulsifying and suspend~g agents~. If desired, certain sweetening and/or f lavoring agents can ~e added. For parenteral administration, which inc:ludes intramuscular, intraperitoneal ~ subcutaneous and intravenous us~, steril~s solutions of the ac~ive ingredients are usually prepared, and the pH or the solutions are suitably adjusted and bu~fered. For intravenous use, the total concentration of solutes should be controlled to render ~he preparation isotonic.

Although the prescribing physician will ultimately decide the dosage to be used in a human subject, the ratio of ~he daily aosages of a compound o~ the present invention, or salt thereof~ and the ~-lactam antibiotic will normally be in the range from about 1:5 to 5:1, and preferably abou~
1:1. Additionally, the daily oral dosage of each component wîll normally be in the range rom about 10 to about 200 mg.
per kilogram o~ body weight and the daily parenteral dosage of ea~h component will normally be about 10 to about 100 mg.
per kil~gram of body weight~ These figures are illustrative ~ .. __ . _ . . .. . . . ... _ ., _ _ , _ ,,_, , ~_ only, however, and in some ca~es it may be necessary to u~e dosages outside these l~mit~.

This invention i~ capable of industrial appl ication .

:". '

Claims (12)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows.

1, A process for the preparation of an ester compound having the formula wherein R is benzyl, p-nitrobenzyl or .beta.,.beta.,.beta.-trichloroethyl and n is an integer of O to 2, which comprises:

(a) when n is O and R is benzyl or p-nitrobenzyl:
heating a compound having the formula wherein R is benzyl or p-nitrobenzyl in an inert, anhydrous organic solvent, in the presence of large equimolar amounts of a weak tertiary amine and an acid chloride, until the reaction is substantially complete;

(b) when n is 1 and R is benzyl, p-nitrobenzyl or .beta.,.beta.,.beta.- trichloroethyl:
oxidizing a solution in an inert solvent of a compound having the formula wherein R is benzyl, p-nitrobenzyl or .beta.,.beta.,.beta.-trichloxoethyl at about room temperature by use of a peracid; and (c) when n is 2 and R is .beta.,.beta.,.beta.-trichloroethyl:
oxidizing a solution in an inert solvent of a compound having the formula at about room temperature by the use of an oxidizing agent.

2. An ester compound having the formula wherein R is benzyl, p-nitrobenzyl or .beta.,.beta.,.beta.- trichloroethyl and n is an integer of 0 to 2 when prepared by the process of claim 1 or dn obvious chemical equivalent thereof.

3. A process for the preparation of an ester compound having the formula wherein R is benzyl or p-nitrobenzyl, which comprises heating a compound having the formula wherein R is as defined above in an inert, anhydrous organic solvent, in the presence of large equimolar amounts of a weak tertiary amine and an acid chloride, until the reaction is substantially complete.

4. An ester having the formula wherein R is benzyl or p-nitrobenzyl, when prepared by the process of claim 3 or an obvious chemical equivalent thereof.

5. A process for the preparation of an ester compound having the formula wherein R is benzyl, p-nitrobenzyl or .beta.,.beta.,.beta.-trichloroethyl, which comprises oxidizing a solution in an inert solvent, of a compound having the formula wherein R is as defined above, at about room temperature by the use of a peracid.

6. An ester having the formula wherein R is benzyl, p-nitrobenzyl or .beta.,.beta.,.beta.-trichloroethyl, when prepared by the process of claim 5 or an obvious chemical equivalent thereof.

7. A process as in claim 5 wherein R is .beta.,.beta.,.beta.-trichloroethyl.

8. The ester of Claim 6 wherein R is .beta.,.beta.,.beta.-trichloroethyl, when prepared by the process of claim 7 or an obvious chemical equivalent thereof.

9. A process for the preparation of an ester compound having the formula which comprises oxidizing a solution in an inert solvent, of a compound having the formula at about room temperature by the use of an oxidizing agent.

10. A process as in claim 9 wherein the oxidizing agent is a peroxide or a peracid.

11. A process as in claim 10 wherein the oxidizing agent is KMnO4 or H2O2.

12. An ester having the formula when prepared by the process of claim 9, 10 or 11 or an obvious chemical equivalent thereof.