CA1041481A - Process for preparing cephalosporin antibiotics - Google Patents

Abstract

ABSTRACT OF THE INVENTION:
An improved yield is obtained of the compound 7.beta.-(2-thienylacetamido)-7-methoxy-3-carbomoyloxymethyl-3-cephem-4-carboxylic acid or its esters, from the compound 7.beta.-(D-5-amino-5-carboxyvaleramido)-3-carbazoyloxymethyl-7-methyl-3-caphem-4-carboxylic acid, by conducting the acylation of the latter compound in the presence of com-merically available alumino-silicate zeolites, also known as "molecular sieves." The process can be employed more broadly to prepare a 7-acylamido cephalosporin from a ceph-alosporin having a different 7-acylamido group, without having to isolate and purify the 7-amino intermediate. The final products have utility as broad spectrum antibiotics.

Description

15393Y ~

.

:~

~04~8~l :~ ' RELATIONSHIP TO PRIOR APPLICATIONS~

his invention is an improvement over the acylation process for preparing 7-acylamido cephalosporins as disclosed and claimed in Canadian S.N. 115,151, filed June 8, 1971, now Can. Pat. 965,089.

BACKGROUND OF THE INVENTION:

One method of producing 7-acylamido cephalosporins used medicinally as antibiotics comprises preparing the analogous 7-aminocephalosporin and then acylating to produce ~ I 10 the~desired prod~uct. This method suffers from the disadvantage t ~ that it is necessary to first isolate and purify the inter-med~iate 7-aminocephalosporin. Accordingly, other methods j .

:

~ . - ' 15393IA
gl.09~
1 have been sought which would avoid the need of preparing the

2 7-aminocephalosporanic acid.

3 More recently it has also been found that cepha-

4 losporins having a methoxy substituent in place of the

5 hydrogen substituent at C-7 are produced by various micro- -

6 organisms. These cephalosporins contain an aminoadipoyl

7 sidechain which i5 preferably removed to provide new 7a-

8 methoxy cephalosporins of enhanced antibiotic activity.
g SUMMARY OF THE INVENTION: ~ -In accordance with the present invention, we have 11 found that cephalosporin compounds can be interchanged as 12 follows: -H ~1 S ______~ R'~ Rl S
B' N _ ~ ~ Q N ~ ~ O
N ~ CH2OcNH2 B' ~ N ~ _ CH2CNH2 COOR" COOR"
. `'.: :
, I II ~
. ;, R

T ~ ~ o .;~ ~ ~
_ N

, ~ COOR"

13~in~whlch B'~ and~R' represent different acyl groups, Rl ~ 14 ~repre~sents hydrogen or a substituent such as methoxy, and :~5,~ jlS~ ~Q'~represents hydrogen or~an easi1y removable blocking group.
6 ~ Thus, in the above flowsheet the cephalosporin 17~ compound I is interchanged wlth an acylatin~ agent~in the 18 ~presence of a molecular sieve catalyst to produce the 7-~' ~ :" .: . :
~l -2-~.:: . . . . .

1 diacylimido cephalosporin compound (II) which is then 2 cleaved to produce the new 7-acylamido cephalosporin 3 compound (III).
4 The step of producing the diacylated product is best effected by intimately contacting the cephalosporin 6 compound with an acylating agent in a suitable solvent 7 medium in the presence of the molecular sieve. The tem-8 perature at which the reaction is carried out is not

9 particularly critical and temperatures from about -20C.
to about 100C. are generally satisfactory, althou~h it is 11 preferred to carry out the reaction at temperatures from 12 about 50C. to 90C. Solvents which do not contain an 13 active hydrogen such as chloroform, acetonitrile, methyl- ;
-14 ene chloride, dioxane, benzene, haloben&ene, carbon tetrachloride, diethylether, and the like are suitable 16 mediums for carrying out this reaction.
17 The acylating agent can be an acyl halide, an 18 anhydri~e, or a mixed anhydride although generally it is 19 preferred to use an acyl halide, for example an acyl chloride, as the acylating agent.
~3~ 21 ~ ~he molecular sieves which are useful in this 22 invention are alumino-silicate zeolites. Generally speak-.~ . .
23 ing,naturally-occurring zeolites can be defined as a group 124 of crystalline solids, hydrated aluminosilicates of mono- ~

; 25 and divalent bases, which are capable of losing part or ;

26 ~all of their water wlthou~ change of crystal structure, ~-27 adsorbLng other compounds in plaoe of the water removed, ~28 and which are capable of undergoing base exchange. A

;t ~29 synthe~ic zeolite, on the other hand, is synthesized from a~oombination of basic oxides (A102, SiO2, Na20, K2O~

.. . .

41~
1 etc.) in an aqueous system to yield a hydrated or semi- ;
2 hydrated crystalline structure. Following heat treatment, 3 the zeoli-tes can be considered substantially anhydrous.
4 Synthetic zeolites are characterized and classified pri-marily by X-ray powder diffraction methods. Although there 6 is lack of a systematic chemical method for naming synthetic, 7 complex alumino-silicates, historically each new synthetic ~-8 zeolite is assigned an arbitrary letter or group of letters g and numbers. The meaning of these arbitrary sy~bols is well understood by those skilled in the art.
11 It has been found that synthetic zeolites o the 12 A and X classes are particularly advantageous to employ in 13 tha acylation process described above. The pore size of the 14 zeolites can be in the range of from about 3 to about 15 ~.
The zeolites can be substantially anhydrous or contain some 16 water of hydration. The amount of water by ~eight contained 17 in the zeolite can be from 0-30%.
18 The substltuent at the 3-position in I, II and III
H
19 above, is -CH2OCNH2 The transacylation reaction works 20 equally well when using compounds in which the 3-position 21 ca.n represented by -CH2A wherein ~ is hydrogen or other ;~
22 substituents known in the art. Thus, if A is hydroxy it 23 includes the lactone formed with the carboxy group at 4, 4; and~if A is amino it includes the lactam formed with the 25 carboxy group at 4. The substituent A can also represent 26 azido,~halo, cyano, alkoxy, aryloxy, aralkyloxy, hetero-J : . ~ . : .
!~ 27 cycleoxy, mercapto, alkylthio, arylthio, aralkylthio, 28~heterocyclethio, amino, alkylamino, alkanoylamino~ hydroxy-29 phenyl,~acylthio, acyloxy/ sulfamoyloxy, and the like. The ; 30 heterocycle.s can be a 5- or 6-membered hetero ring ~ `~

~Z ~ ~ ~
:
,~ , :

. . . .,, . . . . . - - . . . .. .

14~
1 containing one or more nitrogen, oxygen or sulEur 2 atoms, such as (1-methyl-1,2,3,4-tetrazolyl). The acyl 3 group can be a loweralkanoyl group of 2-6 carbon atoms,or ~ thiocarbamoyloxy and N-alkyl or N,N-dialkyl derivatives of carbamoyloxy or thiocarbamoyloxy. The alkyl group 6 of the foregoing substituents contains 1-6 carbon atoms 7 and may be further substituted radicals such as alkoxy, 8 halo, amino, cyano, carboxy, sulfo and the like. :
9 The acyl substituents represented by B' and R' in formulas I, II and III above are preferably carboxylic 11 acid radicals. B' is aminodipoyl when the compounds are 12 produced from certain microorganisms, such as S.
13 clavuligerus, S. lipmanii, or S. lactamdurans. However, 14 B' can also be any of the commonly employed acyl groups in the cephalosporin art. B' can be replaced by any R' 16 group aLso employed in the art. Both B' and R' can be 17 represented by the general formula ~llRloCHC0 wherein R
18 and Rll are as defined below, and represent a preferred 19 group of substituents because of their generally useful antibiotic activity. Rlo represents hydrogen, halo, amino, 21 guanidino, phosphono, hydroxy, tetrazolyl, carboxy, sulfo 22 or sulfamino. Rll represents phenyl, substituted phenyl, 23 a monocyclic heterocyclic 5- or 6-membered ring containing 24~ one or more oxygen, sulfur or nitrogen atoms in the ring, 25~ substituted heterocycles, phenylthio, heterocyclic or 26 substituted heterocyclic thio groups; or cyano. The sub-27 stituents can be halo, carboxymethyl, guanidino, guanidino-28 methyl,~carboxaminomethyl,~aminomethyl, nitro, methoxy or 29 methyl. Examples of the preferred acyl groups, either B' ~}~
~ 30~ or R',~that might be mentioned are phenacetyl, 3-bromo-., , S
. . .

15393IA ~
L4~3~

1 phenylacetyl, p-aminomethylphenylacetyl, 4~carboxylmethyl-2 phenylacetyl, 4-carboxamidomethylphenylacetyl~ 2-furyl-3 acetyl, 5-nitrofurylacetyl, 3-furylacetyl, 2-thienyl-4 acetyl, 5-chlorothienylacetyl, 5-methoxythienylacetyl, 5 a-guanidino-2-thienylacetyl, 3-thienylacetyl, 4-methylthi-6 enylacetyl, 3-isothiazolylacetyl, 4-methoxyisothiazolyl-7 acetyl, 4-isothiazolylacetyl, 3-methylisothiazolylacetyl, 8 5-isothiazolylacetyl, 3-chloroisothiazolylacetyl, 3-g methyl-1,2,5-oxadiazolylacetyl, 1,2,5-thiadiazolyl-4-

10 acetyl, 3-methyl 1,2,5-thiadiazolyl-4-acetyl, 3-chloro-

11 1,2,5-thiadiazolyl-4-acetyl, 3~methoxy-1,2,5-thiadiazo- ~ -

12 lyl-4-acetyl, phenylthioacetyl, 4-pyridylthioacetyl,

13 cyanoacetyl, tetrazolylacetyl, a-fluorophenylacety:L,

14 D-phenylglycyl, 3-hydroxy-D-phenylglycyl, 2-thieny:l~lycyl,

15 3-thienylgylcyl, phenylmalonyl, 3-chlorophenylmalonyl,

16 2-thienylmalonyl, 3-thienylmalonyl, a-phosphonophenyl-

17 acetyl! a-sulfaminophenylacetyl, a-hydroxyphenyla~etyl,

18 a-tetrazolylphenylacetyl and ~-sulfophenylac~tyl.

19 The reaction I~ II s~hematically represented ;~

20 above is an equilibrium reaction. An excess of the acyla-,': . - .:

21 ting agent ~containing the group R') is employed in order

22 to lncrease the yield of thedesired end product, III. The

23 intermediate diacyl product, II, must be cleared to remove

24 the group B' in order to prepare the end product III. This

25~cleavage is accomplished in several ways. Firstly, spon-

26 taneous cleavage takes place (in the presence of a mole-

27 cular excess of the acylating agent R'-hàlide~ merely by -;~

28 prolonging the reaction time. Secondly, when water is Z9 present in the molecular sieves, the water acts as a cleav-30 ing agent, and the final acylamido product is recovered in 31 high yiel~ Both of these methods can be characterized as .. 1 ; ~ ,:.
-6- ~
- : .. .':' ~0~ 8~

1 "passive" in the sense that there is no need to add a sepa-2 rate "cleaving agent" -to the reaction mixture~
3 A third method of cleavage results from the 4 addition of benzyl alcohol, an alkanol or a loweralkyl thiol, of 1-6 carbon atoms. Hydrochloric acid can also be 6 added as cleaving agent, as a fourth agent.

7 DESCRIPTION OF THE PREFERREO EMBODIMENTS:
_ 8 In accordance with a preferred embodiment of the 9 present invention, it is now found that cephalosporins such ~ .

10 as those obtained by fermentation by various Streptomyces .

11 species can be converted to derivatives having a different -.

,12 acyl group in place of the aminoadipoyl group without first '~13 cleaving this group and then reacylating the intermediate 14 7-amino compound. The general process is illustrated in the following flowsheet:

! I fOOH "H 1 S _ > CIOOH OR, 1 S
~ H ( CH2 ) 3CN_ ~ cH ( cH 2 ) 3c~
NH2 _ N ~ CH2A NH2 O~ N
: COOH COOH
IV ¦ VII

CIOOR" OH ¦l S R'- ~ ~
CH(CH2)3C ~ ~ O \ ~ _CH2A

NHR2 ~ N ~ CH2A COOR"
COOR"

~}~

,~ ~ ( 2)3 NHR2 ~ ~ ~ ~ H2A

Ri' ~ VI

.~ _7- :
; ~
,:~: :,.
. . . . ..
.. .

~0~4~ :
1 In tlle formulas of the above flowsheet, R1 2 represents hydrogen or methoxy; A represents hydrogen or a 3 substituent unaffected during the described reactions or 4 reconvertible thereto by the removal of any protecting or blocking groups, most desirably, acetoxy or carbamoyloxy; R' 6 represents an acyl group as defined; R" represents hydrogen 7 or a blocking or protecting substituent; and R2 represents 8 a blocking or protecting substituent, both of which are g easily removed using methods available in the art.
In accordance with the above flowsheet, the 11 cephalosporin compound IV or a derivative thereof wherein 12 the amino substituent and/or the carboxy groups are 13 optionally blocked or protected (V) is reacted with an 14 acylating agent in the presence of the molecular sieve to produce the intermediate diacylated product (VI or VII).
16 The aminoadipoyl moiety of the latter product is then 17 selectively cleaved to produce the new acylated cephalo-18 sporin compound (VIII), or a salt thereof when R" is 19 hydrogen. ;
Although this invention can be carried out without 21 blocking or protecting the amino and carboxy groups of the 22 starting cephalosporin compound (VII), it is generally 23 preferred to carry it ou-t by first blocking or protecting ,:
24; both the amino and carboxy groups since maximum yields of the desired new cephalosporin compound are obtained with 26 such protected compounds.

27 ~ An illustrative, more detailed description of 28~ thls preferred process of our invention is shown in the

29 following flowsheet:

:1 ~ - :
:
'~ ~ '' ':"
~:

~ -8-.' ~ ~, .
., . :
.. . . . .

4~
, 2 ,, H Rl El S
CH (CH2) 3C-N~/ ~
COOH 1 N ~CH 2A
O \~ ..
COOH
IX
:','~ ' R2 :`:
NH H S 1 _I S
CH ( CH 2 ) 3C-N
COOH ,L _N ~LCH2A ' ~ :
`r ,. ..
COOH .
X ` ' ' , R2 ;
': ~ NH H Rl H S
CEI (CE12) 3C~
COXR4 L_~f L C 2 i ~ ~ : OYR4 XI
~ , 1 ~"'''', H ~ R~ -` S ~ ~:
CH ~( CH 2 ) 3 -N ~ ~
coxR4; ~ 2A

' ~ : ~ . :
.: --9-- , :

15393IA : ~

4~4~
: .
R El ~S

N _~ CH2A' : :
O ~ "''.
COXR4 .`

XI I I ¦

. : , .
Rl H : :
H ~ S .-R ' N ~
N ~_ CH 2A ' o `r ~ ~
COOH : ;`~
''~'' XIV . . :

1 wherein Rl represents hydrogan or methoxy, A' is c~arbam-2 oyloxy or acetoxy, and R2 represents a blocking or pro-3: teoting group. The moiety COXR4 indicatas a blocked 4 ~carboxy group or:thiocarboxy group (X is O or S; R~ is a blocking group)~and R' represents an acyl group.
: 6 ~ In accordance with this process, the amino group 7: of the starting cephalosporin compound (IX) is the first ; 8:~blocked~R2) by~reaction with~ a suitable reagent to protect ~.
9~ the~5~ amlno-substltuent.~ Thus, the amino group i6 bloched . .
b~ amino~protecting groups such as acyl, aroyl, alkoxy~
c~arbonyl, alkylsulfonyl,~arylsulfonyl, and the like in l2~accordanae wlth methods well~known in this art. Speci~ic .-~: 13:~groups~suitablè;for blocking the amino group that might be :-J ~14~ mentioned~ are~richloroethoxycarbonyl, tertiary butoxy- . .
~5 ~carbonyl,~b nzoylmethoxycarbonyl, trimethylsilyl, p-methoxy- , ~.

:,::

8~L
1 benzyloxy, 2-nitrophenylsulfenyl/ 2l4-dinitrophenylsulfen 2 chloroacetyl, p-nitrophenylthio, p-nitrobenzenesulfonyl, 3 p-toluenesulfonyl, methanesulfonyl, benzoyl, p-chlorobenzoyl, 4 p-nitrobenzoyl, toluolyl, and the like, although we generally prefer to utilize the p-toluenesulfonylor benzoyl derivative 6 which is conveniently prepared by reacting the cephalosporin 7 compound with p-toluenesulfonyl chloride or benzoyl chloride 8 while keeping the pH of the mixture basic, i.e., between 9 9 and 10.
It is generally preferred to carry out the 11 above-described reactions with-a cephalosporin compound 12 wherein the carboxy groups on the aminoadipoyl side chain, 13 and at the 4-position are likewise blocked or protected (XI) 14 since maximum yields of the desired product are obtained 15 with such derivatives. Although the carboxy group on 16 the aminoadipoyl side chain is not necessarily deblocked, 17 since it is removed in the cleavage step, the blocking or 18 protecting group is preferably one which can be removed 19 easily at the 4-position to obtain the free acid without 20 disruption of the ~-lactam group since the cephalosporin 21 compounds are usually used in the form of salts such as 22 alkali metal salts or an amine salt. Protecting gxoups 3 suitakle for this purpose are well known in this art.
24 Examples of suitable derivatives that might be mentioned 25 are esters of alcohols, phenols, mercaptans, and thio~
,-., . ~ .
. ~! 26 phenols wherein the group -COXR4 represents the esters. In 27 that general formula, X is oxygen or sulfur, and R4 ~ 28 represents the radical of an alcohol or a thiol such as '' ~ '. .

~: ' ' .:
.j . .
. ~ . .

; ' '~ ' :

l methyl, ethyl, tertiary butyl, a substi-tuted alkyl such as phthalimidomethyl, succinimidomethyl, phenacyl r a 3 substituted phenacyl for example p-bromophenacyl, a ~-4 substituted ethyl group such as 2,2,2-trichloroethyl, .
2-methylthioethyl, 2-~p-methylphenyl)ethyl, 2-(p-~ methylphenyl)sulfonylethyl, 2-methylaminoethyl, 2-chloro-7 (or bromo)ethyl, benzyl, a substituted benzyl group such 8 as p-nitrobenzyl, p-methoxybenzyl, 3,5-dinitrobenzyl, 9 2,4,6-trimethylbenzyl, 3,5-dichloro-4-hydroxybenzyl, and the like, a benzhydryl or substituted benzhydryl group ll such as p-methoxybenzhydryl, an acyloxy alkyl group such ~ -12 as acetoxymethyl, pivaloyloxymethyl, an alkoxy group such `
13 as methoxymethyl, or a monocyclic aryl group for example 14 phenyl or substituted phenyl such as p-nitrophenyl or 3,5-dinitrophenyl. It has been found that the most 16 convenient group used for the purpose of blocking the 17 carboxy group is the methoxymethyl group, wherein X is ~ ~ 18 oxygen. These protecting or blocking groups for the ? ~ 19 carboxy substituents are readily prepared in accordance 20~with~processes well known in this art.
21 The protected cephalosporin compound is then 22 xeacted with an acylating agent in the presence of the 23 molecular sieve such as those described above to obtain ; 24 the imide or diacylated product (XII). The acylating ;~
25~ agent can be an~acid halide (chloride or bromide), or a 26~ functional equivalent thereof such as an acid anhydride, 27 ~a mèrcaptide, a mixed acid anhydride with other carboxylic 2~8~acids, an~activated~ester of the carboxylic acid such as ~i 29~ the~p-nLtropheny1 ester; and the like. ~ i , ~ -12-'.:~ .:
l .

1 The preferred acylating agents used in the process 2 of the present invention are those of carboxylic acids.
3 The preferred acyl groups representing R7 in the above ;.
4 flowsheet are those of the general formula:

3 ~ . .
X
wherein X is hydrogen, halogen, amino, azid~, guanidino, 6 phosphono, hydroxy, tetrazolyl, carboxy, sulfo, or 7 sulfamino; R3 is phenyl, substituted phenyl, a monocyclic 8 heterocyclic 5- or 6-membered ring containing one or more 9 oxygen, sulfur or nitrogen atoms in the ring, substituted heterocycles phenylthio, phenyloxy, heterocyclic or 11 substituted heterocyclic thio groups, loweralkyl (1-6 12 carbon atoms), or cyano; the substitutents on the R3 13 group being halo, carboxymethyl, guanidino, guanidino- : -, . . . . .
14 methyl, carboxamidomethyl, aminomethyl, nitro, methoxy : 15 or methyl. .
,! . . . ', ' .
16~ When the acylating agent contains groups such as ..

17 amino or carboxy, these groups can be blocked or protected ;

18~ daring the acylation reaction and later removed in accord~

19 ance with methods known in this art. Alternatively, the .. .

acylating agent can contain a substituent such as aziclo 21 which can be later reduced to an amino substituent 22~pursuant to~known:methods. :..... :.

23~ Éspecially preferred acylating agents that might ...

2~4~ bè~men~ioned~are thos~i~ having~an acetyl or substituted 2:5~ acetyl:group~such as phenylacetyl, thienylacetyl (2- and . ~

2~6~ 3-thienylacetyl), furylacetyl, (2- and 3-furylacetyl), ...

27~ a-hydroxyphenylacetyl~ phenoxyacetyl r ,~-formyloxyphenyl- -~

28~ acetyl~ c-tr ~ ol~.acetyl, a-amironh-~laFetyl, pheny1-~: , ~ 13- : ~
.. : :: ~ : : , ~ 15393IA
.

1 thioacetyl~ a-azidophenylacetyl, and others as the resulting 2 acylated cephalosporin compounds have enhanced antibiotic 3 activity.
4 The acylating agent is employed in amounts in molecular excess of that of the starting cephalosporin, 6 preferably from 2 to 6 times as much acylating agent as 7 cephalosporin.
8 The molecular sieve used is any of a number 9 readily and commer~!ially available. Preferably, a synthetic zeolite of regular crystal structure and uniform pore size 11 is used. The most commonly available sieve, Type 3A, 4A, ;;
1~ SA, and 13X, are all operable in the invention. These 13 sieves have the following properties-e Formula I Pore Diameter ~YP .
3A KqNa3[(Alo2? 12 (sio2)]27H2o 3A
4A Nal2[(AlO2)l25sio2)]27H2 4A
5~ Ca45Na3E(AlO2)12]3oH2o 5A ;, 13X Na86[(A1O2)86(sio2);o6-x~2O lOA
. . .
14 The sieves are available in substantially anhy-15 ~drous form; they can be used in this form ordehydra~edf~t~, 16 to 0% water ~ 2~ water, by heating to high temperatures .
17~ ~about 500C. or above) before use; or can be used when they 18 ~contain up to Gbout 30% water of hydration (weight ~ basis). ~ ;~
l9~ The hydrated sievas are prepared by allowing them to stand 20~ in~ a high humidity~chamber or environment or by slurrying in 21 water and then~adjusting to desired moisture content by v~uum 22 drying cr drying at room temper~ature or at elevated tempera- -, 5'~ Z 3 ~ture O ~ ~

, r~

1 Generally, this drying takes about 2-5 hours, 2 although it is not a critical time limit. Moisture level 3 can be measured using the Karl Fischevr method, a generally ~ accepted technique, or by other available methodology~
The step of converting the protected cephalo-6 sporin compound (XI) to the imide to diacylated product 7 (XII) is preferably effected by intimately contacting the 8 cephalosporin compound with the acylating agent in a g suitable solvent medium in the presence of the desired -molecular s~eve. The temperature at which this reaction ; 11 is carried out is not critical, and temperatures from about 12 -20C. to about 100C. are generally satisfactory. ~owever, 13 since the reaction appears to be temperature dependent, ~ 14 and proceeds faster at higher temperatures, it is preferred ! 15 to carry out the reaction at temperatuxes from about 50C.
16 to 90C. Various solvents which do not contain an active .~ ~ ' ',,.
~ 17 hydrogen such as chloroform, acetonitrile, methylene - : :
18 chloride, dioxane, benzene, halobenzene, carbon tetra-` ~ 19 chloride, 1,2-dichloroethane, and diethylether are most ;~
~j .
20 5uitable as mediums for the reaction mixture. It is impor-21 tant to keep the slurry ln motion by stirring or agitating 22 ~during the reaction.
`23 ~ ~ The amount of sieves necessary to the interchange j : . .
$~ 24~;~reaction can be varled to SUlt the chosen operating condi-25~tions. Generally, it is preferred to use approximately 26~ equal amounts by weight of the starting material and the ; 27 sie~es, although it is possible to use a weight ratio of 28 startlng compound to~sieves of 1 to 0.5~2.

.'.! , ' ' I ~ .
, -15-: ~ . ~',: :

1 As mention~d above, the original acyl group is 2 cleaved by a number of different routes. Simple "aging"
3 of the reaction mixture is enough in some cases, e.g., 4 when the sieves contain between 10 30~ water, for from 5 30 minutes to 30 hours. An alkanol,loweralkyl thiol, or 6 benzyl alcohol can be added following a briefer "aging"
7 period. The alkanol or loweralkyl thiol can have 1-6 8 carbon atoms, and preferably is methanol, ethanol, iso-9 propanol, or t-butanol. Hydrochloric acid can also be :
10 added to effect cleava~e. During the acylation reaction :: . .
11 some "spontaneous" cleavage of the aminoadipoyl group 12 occurs, due to the equilibrium nature of the xeaction, 13 depending upon the conditions under which tha acylation ~ ;
14 is e~fected. Prolonged heating o~ reaction mixture results ~;
15 in the cleavage of the aminoadipoyl group and the prepara- ~ ;
16 tion of the desired 7-acylated cephalosporin compound, 17 especially when the sieves contain above 10% water. -18 The removal of the protective or blocking group 19 on the carboxy function is accomplished in accordance with 20 procedures well known in this art. Thus, for example, the 21 methoxymethyl group is removed by the use of hydroch1oric 22 acid at 0-10C.; the trichloroethoxycarbonyl group is 24 removed by reaction with zinc and acetic acid; and the t-25 butoxycarbonyl and benzhydryl groups are removed by reaction 2~6~ with trifluoroacetic acid. Other removals are accomplished 27~ wLth similar ease.

.i~: , ~ 29 3-Carbamoyloxymethyl-7-methoxy-7~-thienylac2tamido-3-cephem-; ~ 30 4-carboxyIic Acid. ;
Step;A: 7~~(D-5-tosylamino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylic Acid _ ;

..
~ -16-. . 1~ , .

iL~43L9~
1 The mono~sodium salt of 7~-(D-5-amino-5-carboxy-2 valeramido)-3-carbamoyloxymeth~ 7-methoxy-3-cephem-4-3 carboxylic acid (45.0 ml. of 49O5 mg./ml. aqueous solution) 4 mixed with acetone (450 ml.) and water (450 ml.). The pH
of the mixture is adjusted to 9.5~9.6 with 50% NaOH solu-6 tion and tosyl chloride (19 g.) in acetone (100 ml.) is 7 added in~portion. The pH is maintained at 9.5-9.6 by 8 frequent addition of caustic solution. After 15-20 minutes 9 the pH becomes stable; sulfonylation continues for a total of one hour. The temperatures of the solution is 20-23C., 11 throughout the entire reaction period. --12 After this, the solution is cooled in an ice bath 13 and the pH is lowered to 7 by the addition of 1~1 HCl (ice 14 cold). The solution is extracted using ethyl acetate. The .
ethyl acetate layer is backwashed with 100 ml. 5% sodium 16 chloride solution. The organic layer is discarded and the ~ 17 aqueous layers together with 500 ml. of ethyl acetate are '~ 18 readjusted to 2.5 and the layers are separated. The aqueous i~ ~; 19~ layer is extracted further with 3 X 500 ml. EtOAc. The ; ~ .
20~ ethyl acetate layer is backwashed with 100 ml. sat. NaCl 21 solution. The extracts are dried with Na2SO4 and the solvent .~. ;~ . : .
, 22 is concentrated to a small volume. (Temperature 30C.) i~ 23 The concentrated solution is then dissolved in 24 200 ml~ of isopropanol, heated to 40-45C., and 5.8 ml.
! : : ., :.
25 ~acetic acid, and 21.6 ml. of dicyclohexylamine added.
26~ This slurry is allowed to cool slowly and is aged 27 overnight at room temperature. The product is filter~d, 28~washed with 100 ml. of isopropanol and dried overnight at ~;
29 room temperature under high vacuum.

30~ ~ The product, 7~-(D-5-kosylamino-5-carboxyvalera-

31~mido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylic ,.

32 acid, dicyclohexylamine salt, is obtained in a yield of 44.5g;

1 uv: (pH 7.0 buffer) 2 ~ ax. 2620 E% 94.7 3 Equivalent wt. (IIClO~ titration) 481.5 (theory 4 481 5) Anal. calcd- for C~7H74N6OllS2:
~:
6 C, 58.60, ~1, 7.74; N, 8.72;
. . .
7 Found:
8 C, 58.29; H, 7.23; N, 8.73.
g Step B: Dimethoxymethyl ester of 7~-(D-5~
tosylamino-5-carboxy valeramido)-3- ~ ~ -11 carbamoyloxymethyl-7--methoxy--3-cephem--12 4-carboxylic acid.
13 The tosyl salt from Step A, 20~., is charged to 14 a 3-necked flask. Methylene chloride (200 ml.) is added and the slurry is cooled to 0C. in an ice bath, under 16 nitrogen. Chloromethyl methylether (4.1 ml.) in 30 ml.
17 of methylene chloride is added to the reaction mixture over 18 a period of 90 minutesr with good agitation and ice cooling.
19 After one hour addition time, a solution o~ collide ¦~ ~ 20 (1~.58 ml~3 in 5 ml. of methylene chloride is introduced.
;21 ~ After additions, the mixture is agitated fox an 22 additional two hours, filtered, and the filter ca~e washed 23 with dry mèthylene chloride. ~fter extractlon with a~ueous 24 phosphorie aeid, sodium chloricle, sodium hicarbonate, and sodium chloride solutions, the filter cake is baek washed 26 with methylene chloride. The organic layer is dried, 27 ~filtered, coneentrated to a small volume, and crystallizecl.
2~8~ ~The product, the dimethoxymethyl ester of 7~-(D S--tosylamino- ;
29~ ~5~earboxy valeramido)-3-carbamoyloxymethyl-7--methoxy-3-~cephem-4-carboxylic acid, is obtained as 9.6 g. ~yielc1 83.5~ Both ultraviolet and thin layer chromatography 32 ~indieated only the single component in the product.

',~: : . :

1 Step C: 3-Carbamoyloxymethyl-7-~ethoxy-7~-2 thienylacetamido-3-cephem-4-carboxylic 3 acid 4 To a stirred slurry o~ 6.9 g. of tos~l methox~-5 methyl ester from Step B and 7.5 g. of type 4A powdered ~ 6 molecular sieves (600 mesh, hydrated to 17% + 2~ water) ; 7 in 85 ml. of 1,2-dichloroethane was added 5 ml. of distilled 8 2-thienylacetyl chloride. The stirred slurry was heated 9 at 65C. for 16 hrs. under a nitrogen atmosphere.
The reaction is monitored using thin layer 11 chromatography. After the time indicated, the major 12 component of the reaction mix~ure is the desired intermediate .
13 compound. Methanol (0.8 ml.) is then added, and the slurry ` 14 aged an additional 2 hours. At this point, the 4-methoxy-' 15 methylester of the desired product is the predominant il 16 compound in the slurry.
l 17 The ester is hydrolyzed by cooling the solution j 18 above to 25C., filtering, and washing with cold methanol.
~!~ 19 The filtrate and wash are combined and cooled to 0C. A
', 20 0C. solution of 20.8 ml. conc. HCl and 23.6 ml. methanol :
21 are added and the solution warmed to 15C. and stirred at ;l :
J 22 15C. for 2 hrs., 40 min. A thin layer chromatographic 23 analysis is run at this point. The pH of this mixture is 24 adjusted by first bubbling in ethylene oxide as necessary 25 to adjust the pH to between 2-2.5; then bringing up the pH ; `
26 to between 5-6 by addition of solid sodium hydroxide. Th~ ~-3 . : .
$~ 27 mixture is filtered, and the dichloroethane layer is sepa-~2 a rated. The cold aqueous layer contains the sodium salt of ~;
`~ 29 the product. This layer is purified using column chromato-;~ 30~ graphy, using IRA-68~resin on the nitrate cycle, the eluant ~ 31 being 0.02M phosphate buffer pH 7Ø The final yield of :~1~ , :, .

. j .
: ., :
;, . . .
-19- . :, 8~ ~ ~
1 product is 1.74 g., showing a single spot on thin layer 2 chromatography. The angle of rotation is 192C. Product 3 identi~y is confirmed using NMR as an analytic tool; the 4 product is the desired 3-carbamoyloxymethyl-7-methoxy-7 5 thienylacetamido-3-cephem-4-carboxylic acid, m.p. 165-167C. ~
6 The starting material, the monosodium salt of ~ -7 7~-(D-5-amino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-8 methoxy-3-cephem-4-carboxylic acid, used in the fore-9 going example is prepared as follows:
10 Preparation of Monosodium Salt of 7~-(D-5-amino-5-carboxy-11 valeramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-12 carboxylic Acid 13 Modified Fermentation Process:
14 Step 1: Slants A lyophilized tube of Streptomyces lactamdurans 16 culture (NRRL 3802) was opened asceptically and the 17 organism transferred to a medium of the following composi-18 tion:
`J ~ 19 Medium XI:
... . . .
i 20 1% Blackstrap Molasses : 21 1% National Brewerls Yeast i 22 2.5% Difco agar pH 7.0 ~ 23 ~ Water to volume ,ll 24 The slants are inoculated for 7 days at 28C. When stored 25 in the cold, the slants are stable for more than 13 weeks.
26 ~ Step 2. _Seed Stages: Two _tage System 27 ~ First Seed: The first seed is inoculated ; 2~B directly from ~he slant of Step 1 to 40 ml. of 1~ Primary 29~Dried;Yeast N.F.;~, pH 7.0 (obtained from the Yeast Product ~
30~Corporationj in a 250 ml. baffled Erlenmayer flask. The ~ -~ 31~1~asks were~then shaken on a 220 rpm. rotary shaker with ^3~ 3;2~;a~2~inch throw at 28C. for a period of from 2 to 3 days.
Second;Seed: A 2,5% inoculum from the firs^t ~ 34~seed stage was~added to a flask contain.ing a 2% Fleischmann J~

, ~ : .
:!:: `

g.041~
1 S-150 yeast autolysate, pEI 7Ø Tlle growth in this stage is 2 characteristically licsht and the incubation~ performed as 3 in the first sta~, was not extended beyond 48 hours.
Step 3: Production Medium llhe~procluction medium contains per liter of 6 distilled water 30 ~. distiller's solubles, 7.5 ~. of 7 primary dried yeast N.F. and 0.25% v/v of an emulsified 8 petroleum product (Mobilpar-S)defoamer. The medium is 9 adjusted to pH 7.0 with a small amount of concentrated sodium hydroxide solution dispensed into Erlenmeyer flas~s 11 and autoclaved for 15 or 20 minutes at 121C. After cool--12 in~ the medium received a 2.5% inoculum of the seed `' 13 obtained in Step 2. The time o~ incubation can vary from 14 about 50 to 100 hours but an incubation period o about 72 15 hours ls preferred. The volume of media in each flask can ~
16 vary from 30 to 50 ml. but 40 ml. was used routinely. I'he ;;
17 level of inoculum can vary from 1 to 5%; but, in practice, 18 a 2.5% level is generally employed.
19 Step 4: Assay 20 ~ When the fermentation was complete, the cells ; ~;
21 were removed by centrifugation and the broth was diluted 22 ~with phosphate buffer, pH 7.0, The concentration of 7~--23 (D-5-amino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-24~ me~thoxy-3-cephem-4-carboxylic acid in ~he fermentation 25 ~broth was determined by the standard biolo~ical-disc assay 26~ method. ~he~assay organism employed was Vibrio percolans 27~ (ATCC~8461~ Filter paper discs are immersed into the 28 diluted broths and placed on the surface of agar-containing ;~
29 Petri dishes whlah were inoculated with the assay organlsm . : .

1 Vibrio ~ercolans (ATCC 8461). Also placed on these Petri 2 dishes are discs that had been dipped previously in stan-3 dard solutions containing known concentrations of 7~-4 ~D-5-amino-5 carboxyvaleramido)-3-carbamoyloxymethyl--7-methoxy-3-cephem-4-carboxylic acid. The discs were 6 incubated overnight at 28C. and the diameters of the ~ones 7 of irhibition recordedO The concentration of product and 8 the fermented broth is calculated by interpolation from 9 the standard curve which relates zone diameter with the ~-nown concentrations of standard solutions of the product.
11 By this procedure it was calculated that Streptomyces lac-12 tamdurans NRRL-3802 produced 78.6 ~g.jml. of 7~-(D-5-amino-13 5-carboxyvaleramido)-3-carbamoyloxymethyl-7--methoxy-3-j 14 cephem 4-carboxylic acid in the modified ~ermentation ~rocess.
~; 16 Step 5: Isolation 17 The filtered broth is adjusted to pH 7.0 with ~i 18 dilute hydrochloric acid and 2900 ml. is passed through a 19 column containing a strongly basic anion exchange resin ~(100;g.) having ~a styrene-divinylbenzene matrix (Dowex 21 1 X 2 chloride cycle resin) at 10 ml./minute~ The spent l 22 solvent is aollected in 500 ml. fractions. The resin `1: .~ .
23 column is washed with water and eluted with 3~ ammonium ~ 24 chloride in 90% methanol. The eluate is collected in 100 ml.
Ji~ 25~ ractions.~ The actlvi~ty of the fraction ls monitored; the ; 2~ most~acti~:e~fractions are combined, the pH adjusted to pH
27 7.2 to 8.0 with~;dilute sodium hydroxide and adsorbed on a 28 ~strongly basic~anion exchange resin (100 g.) having a 29 styrene-divinylbenzene matrix (Dowex 1 X 2 chloride cycle resin) at 14 ml./minute, The column is washed . ~ : :' : .

3L4~3~
1 with water and eluted with 5~ aqueous sodium chloride. The 2 concentrate is diluted to 500 ml., adjusted from pH 8.8 to 3 pH 2.0 with dilute hydrochloric acid and adsorbe~ on 25 ml.
4 of a strongly acidic cation exchange resin of the sulfonate type having a styrene-divinylbenzene matrix IDowex 50 X 2 6 hydrogen cycle resin) at 2.5 ml./minute. The column is 7 washed with 25 ml. of water then eluted with 2~ pyridine 8 until the pH of the column effluent rose to pH 7.0 (54 ml.l.
9 The eluate thus obtained is adjusted to pH 8.0 with dilute 10 sodium hydroxide and concentrated under vacuum to remove ;
11 the pyridine and afford the monosodium salt of 7~-(D-5~
12 amino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy- -13 3-cephem-4-carboxylic acid. ;
14 Elemental analysis for C16H21N4SOgNa: ;
Calc.: C, 41.0%; H, 4.5%; N, 12.0%; S, 6.8%;
Found: C, 39.31%; H, 4.76%; N, 11.16%; S, 6.46%.
17 ~ EXAMPLE 2 ''~ 18 3-Carbamoyloxymethyl-7-methoxy-7~-phenylacetamido-3-cephem-~i~ 19 4-carboxylic Acld i ~ 20 ~ Step A: Dimethoxymethyl ester of 7~-[(D-5-tosylamino-21 5-carboxyvaleryl)phenylacetylamido]-3-carbam-22 oyloxymethyl-7-methoxy-3-cephem-4--carboxylic 23 Acid , 24~ A solution of the dimethoxymethyl ester of 7~- ~
,j . , .
(D-5-tosylamino-5-carboxyvaleramido)-3-carbamoyloxymethyl-26 ~;7-methoxy-3-cephem-4-carboxylic acid (9.3 gm., 10 mmoles), 27:~ type 12A powdered molecular sieves (hydrated to 20% ~ 2%
28 ~wàter)(10.~8 gm.,~) and phenylacetyl chloride (5.3 ml., 29~ 40 mmoles) in~5~0~ml. of acetonitrile is heated to 40C. for 3~0 ~20 hours. After this period the mixture is cooled to room 3~ temperature and filtered. ~he filtrate is evaporated to , . : .

~ 23-,: ' : ,.
~ :.

l dryness and triturated with hexane. The insoluble residue 2 containg dimethoxymethyl ester of 7~-[(~-5-tosylamino-5-3 carboxyvaleryl)phenylacetylamino]-3-carbamoyloxymethyl-7-4 methoxy-3-cephem-4-carboxylic acid, is used without purification in the next step.
6 Step B: 3-Carbamoyloxymethyl-7-methoxy-7~-phenyl--7 acetamido-3-ce~hem-4-carboxvlic Acid 8 The crude product from Step A is dissolved in 9 50 ml. 1,2-dichloroethane. l.0 Ml. of methanol is then added and the solution stirred for l hour. The~methoxy 11 methyl ester is hydrolyzed by adding a 0C. solution of 12 20 ml. concentrated HCl in 25 ml. methanol and stirring 13 at 15C. for 3 hours. The product is isolated and 14 purified using the same general procedure in Example 1.
The product, 3 earbamoyloxymethyl~7-methoxy-7~-phenyl-16 aeetamido-3-cephem-4-earboxylie aeid, is obtained having 17 a m.p. of 159-161~C., and having UV and NMR speetra 18 eonsistent with the assigned strueture.
, ' .
,, 19 EXAMP~E 3 i Z 20 3-Carbamoyloxymethyl-7-methoxy-7~-(2-furylacetamido)-3-;~ 21 eephem-4-earboxylic Acid 22 The dimethoxymethylester o~ 7~(D-5-tosylamino-23 S-earboxy~aleramido)-3-earbamoyloxymethyl-7-methoxy-3-24 eephem-4-ear~oxylie aeid is reaeted with 2-furylaeetyl ,:, ehloride is ~he~presence of 12 g~. of hydrated type 4A
26 moleeular sieves (hydrated to 15~ + 2~ water), following : 27 the procedures just described. The side chain, and ester 28 blocking group are removed also following the procedures ; 29 descrlbed. The produet obtained lS 3-carbamoyloxymethyl-7- ~ -j :
,~: ~ : :
~ -24-:~!: ::

.~ .

8~
1 methoxy-7~-(2--furylacetamido)--3-cephem-4-carboxylic acid, 2 m.p. 156-161C., UV ~pll 7.0 buffer)~ max. 265 Ilm. ~7200 3 and having IR and NMR consistent with the structure.
4 In the ~ame manner, the product 3-carbamoylox ,7- ~
5 methyl--7-methoxy-7~-thiophenoxyacetamido-3 cephem-4- ;
6 carboxylic acid is prepared, using phenylthioacetyl 7 chloride instead of 2--furyl acetyl chloride. The product 8 has a m.p. of 119-123C., UV (p~I 7.0 buffer)~ max 247 um.
.
9 c10400 and a consistent N~ spectrum. ~
:
EXAMæLE 4 ~;
; .
11 3-Acetoxymethyl-7~-(2-thienylacetamidO)-3-Cephem-4-12 carbox~vlic Acid 13 Step A: 7~-~D-5-Trichloroethoxycarbonylamino-5~ :,~ ,;',.':i:!".', ,'i 14 carboxyvaleramido)-3-acetylmethyl-3-cephem-4 ~'A.. ':',' ' carboxylic Acid 16 To a solution of 7~-(D~5-amino-5-carboxyvalerami-17 do)-3-acetoxymethyl-3-cephem-4-carhoxylic acid (2.5 g., 18 0.53 mole~ in acetone (13 ml.) and aqueous 10% dipotassium -:. . :
19 hydrogen phosphate (40 ml.) is added dropwise trichloro-1 20 ethoxycarbonyl chloride (3.35 g., 0.159 mole). During the .: , :
l ~ 21 addi~ion the pH of the solution is kept in the range of from ,. : .- .:
1~ 22 8~5 to 9.0 by the ~radual addition of a 17% aqueous solution ` 23 of sodium hydroxide. Ater 30 minutes the mixture is washed, :
24 with ethyl acetate and the aqueous layer is acidiied to ;~
pH 2.5 with concentrated hydrochloric acid. The precipi-26~;tated product is~extracted into ethyl acetate, the solution ~'2~ 27 is dried over sodium sulfate, filtered and the solvent 28 removed to afford 2.7 g. o 7~-~D-5-trichloroethoxycarbonyl- -29~ amino-5-carboxyvaleramido)~3-acetylmethyl-3-cephem-4-~carboxylic acid.
.t ~

~ ~ -2S-i "
3 ~

- ~ 15393IA
~L041~
1 Step ~: Dibenzhydryl ester of 7-(D-5--trichloroethoxy-2 carbonylamino-5-carboxyva]eramido)--3-acetoxy-3 _ethyl-3-cephem-4--carboxylic ~cid 4 To a solution of 7~--(D-5-trichloroethoxycarbonyl--amino-5-carboxyvaleramido)-3~acetylmethyl-3-cephem-4 6 carboxylic acid in ethyl ace-tate (30 ml.) is added diphenyl 7 diazomethane ~2.0 ~.) in ether (25 ml.). The mixture is 8 stirxed overnight and the solvent removed to afford 4.0 g.
9 of erude produet. The crude product is purified by chromatography on silica gel using chloroform as the eluant 11 to afford 2.3 g. of subst~Lntially pure dibsnzhydryl ester 12 of 7-(D-5-triehloroethoxyearbonylamino--5-carboxyvaleramido)-13 3 aeetoxymethyl-3-cephem-4-carboxylie acid. -14 NMR: (Solvent - CDC13) ~= 2.0 (methyl, s), 4.9 (10-H2, `
quartet), 3.2 (2-~H2, quartet), 4.95 (6~H, d), 5,92 16 (7-H), 7.0 (benzhydryl protons, 2 s).
17 Step C: Dibenzhydryl ester of 7--[(D-5-trichloro-18 ethoxyearbonylamino-5--earboxyvaleryl)-2-19 thienylaeetylamino]-3-acetoxymethyl-3-cephem-4-earboxylie ~eid 21 A mixture of the dibenzhydryl ester of 7~-(D-5 .
22~ triehloroethoxyearbonylamino-5-earboxyvaleramido)-3-23 aeetoxymethyl-3-eephem-4-earboxylie aeid (2.0 ~., 0.02 mole), 24 11.0 g. of type 5A moleeular sieves (hyclrated to 23% + 2%, 1: .,~,.. ,. ,;
2-thienylacetyl ehlorid~ (1.31 g., 0~0815 mole) a~.d 26 methylene ehloride (6 ml.) is warmed at 40-45C. in an oil 27 bath under a nitro~en atmosphere for 20 hours. The reaetion 28 mixture~is poured into hexane (lOO ml.) and filtered.
29~ Removal o f the solvent affords the dibenzhydxyl ester of -30 7-~(D-5-triehloroethoxyearbonylamino-5-earboxyvaleryl)-2-31 thienylaeetylamino]~-3-aeetoxymethyl-3-eephem-4-earboxylie 32 aeid.

9 ~

~ -26-"

'~: " ' , .

1 Step D: Benzhydryl ester of 3-acetoxymethyl 7--(2~
2 thi_nylacetamido)-3-cephem-4-carboxylic Acld 3 The dibenzhyclryl ester of 7-[(D-5-trichloroethoxy-4 carbonylamino-5-carboxyvaleryl)-~3-thienylacetylamino]-3-5 acetoxymethyl-3-cephem-4-carboxylic acid i'3 dissolved in ,~ ~i 6 ethyl acetate (10 ml.) and added to a mixture of 90%
7 aqueous acetic acid (10 ml.) and zinc dust (1.0 g.). The 8 mixture is stirred for two hours at room temperature. The g reaction mixture is filtered to remove the zinc. The reaction mixture is washed successively with 2 portions 11 of water, a cold sodium bicarbonate solution and then with ~ ;
12 a saturated sodium chloride solution (15.0 ml.). The ethyl 13 acetate solution is dried over sodium sulfate, filtered and 14 the solvent removed to afford 1.9 g. of crude product which is chromatographed on silica gel usin~ a mixture of chloro-,~` 16 form and ethyl acetate (50:1) as the eluant to afford 17 0.380 g. of praduct which, after recrystallization from 18 ethyl acetate, has a melting point of 141.5-143C~
19 UV: (CH30H) ~ max. 263 ~7580 ~ Elemental analysis for C23H26N206S2:
21 Calc.: C, 61.91; H, 4.66; N, 4.98; 1 1 22 Found: C, 62.14; H, ~.84; N, 4.91.
23 Step E: 3-(Acetoxymethyl)-7-(2~rthienylacetamido)~3-24 cephem-4-carboxylic Acid _ 25 ~ ~ A~cold solution of benzhydryl ester of 3-acetoxy-26 methyL-7-(2-thienylacetamido~-3-cephem-4-carboxylic acid 27 (103 mg,) in anisole (1.0 ml.) and trifluoroacetic acid 28 (0.5 ml.) is stirred at 0C. for 35 minutes. Carbon tetra~
29 chloride (50 ml. ) is added and the reaction mixture is . : : : , - :
30 ~concentrated to~dryness. The residue is triturate~ with 31~ ~hexane. The hexane is removed by decantation and this ~ 32 residUe is~dissolved in ethyl acetate (10 ml.), concentrated .;'.~:~ ~: '`' .
. :: .: .
r --2 ~
` ~ ~ ' ' .
.. , . :1.

~ 153931A

~O~IL4~
1 to 1 ml. ancl diethyl ether added to afford precipitate.
2 This precipitate is recrystalli~ed from a mixture of diethyl 3 ether and ethyl acetate to af~ord 0.025 ~. of 3-(acetoxy-4 methyl)--7-(2-tllienylacetamido)-3-cephem-4-carboxylic acid, 5 m.p. 164C. Mixed melting point with an authentic sample 6 was 163C. ; ,, 8 Sodium 7-(2-thienylacetamido)-7-methoxy-3-carbamoyloxy- ' 9 meth 1-3-ce hem-4-carbox late y p y .- .
Ste~ A: 7~-(D-5-benzoylamino-5-carboxyvaleramido)-3-1 11 carbamoyloxymethyl-7-methoxy-3-cephem-4-carbo-12 xylic acid, disodium salt 13 To 500 ml. of an a~ueous solution containing 14 48.5 mM of mono-sodium 7~-(D-5-amino 5-carhoxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylate is 16 added enough 50~ sodium hydroxide to bring the pH to 9.5. '~' ' 17 To this solution is added 15 ml. [128 mM] of benzovl 18 chloride with vi~orous stirring. The pH is maintained at 19 9.5 over 30 minutes by the addition of caustic soda upon demand~
1 , The pH of the solution is then adjusted to -~
~ 22 4.0 with concentrated hydrochloric acid, and washed twice ¦ 23 with ethyl acetate.
'I 24 The aqueous cut is cooled to 0C., and 2Q0 ml. , ', 25 ;~of isopropanol, and 300 ml. of ethyl aaetate added while ;
26 stirring.l The pH is a~justed to 2.0 with hydrochloric 27-~ac}d.~ The~organic cut is disaarded~and the aqueous 28~re-extracted three t1mes wi~th ethyl aoetate~ The combined '-9`~ ext,racts~are washed~with sodium chloride solution, dried 30~ with~sodium sulfate~and~concentrated~ln vacuo to yield }~h~ ~ ~31~43.o gms.~of~a dark~oil.~

"~32~ The oil is dissolved in 200 ml. of ethanol and

33~a~solution of~30 g. of 2~~ethyl hexanoic acid sodium salt '~' ' ' , ,, . 15393IA
~4~8~
1 added. The slurry is cooled to 0C., filtered, washed with 2 ethanol and dried in vacuo to yield 28.8 g. [102%] of 3 dlsodium 7~-(D-5-benzoylamino-5-carboxyvaleramido)-3-4 carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylate which 5 was 67% pure by chromatographic comparison with a pure 6 standard. ~-7 Step B: Dimethoxymethyl ester of 7~-(D-5-benzoylamino-5-8 carboxyvaleramZido)-3-carbamoyloxymethyl-7-methoxy-9 3-cephem--4-carboxylic Acid _ _ To a slurry of 20 g. of disodium 7~-(D-5-benzoyl-; 11 amino-5-carboxyvaleramido) 3-carbamoyloxymethyl-7-methoxy-12 3-cephem-4-carboxylate in 200 ml. of acetonitrile at 0C.
13 is added dropwise 16 ml. of 6M chloromethylmethyl ether 14 ovar 90 min. After one hour addition time 6 ml. of S-15 collidine is~ added, The slurry is stirred for an addi-16 tional 2 hours at 0C. The mixture is then diluted with 17 500 ml, of methylene chloride and washed twice with 18 dilute phosphoric acid once with dilute sodium bicarbonate 19 and once with 5~ sodium chloride. The aqueous cuts are ~ back washed with 50 ml. of methylene chloride. The organic , 21 phase is dried with sodium sulfate and concentrated ln ', 22 vacuo to about 100 ml.
` 23 The solution is passed through 200 ml. of Silica ~4 Gel G, washed with 200 ml. of methylene chloride, then 2S eluted with 800 ml. of ethyl acetate, The ethyl acetate eluates are concentrated ln vacuo to yield 18.5 gms. of ~27 yellow~oil.

The crude is recrystallized from 50 ml. of ethyl ; acetate to yleld 10.0 g. [67%]-of dimethoxymethyl ester of 7~-(D-5-benzoylamino-5-carboxyvaleramido)-3-carbamoyloxy-methyl-7-methoxy-3-cephem 4-carboxylic acid.

. ~ :
- 2 9 - ~::

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

L48~ ~

1 Step C: Sodium 7-(2-thienylacetamido)-7-methoxy-3-carbam 2 yloxymethyl-3-cephem-_-carboxylate A slurry containing 192 mg. (0.3 mM) of dimethoxy~
4 methyl ester of 7~-(D-5-benzoylamino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylic acid, 6 225 mg. pulverized Linde-type 4A molecular sieves contain-7 ing 10% + 2% water, 0.3 ml~ of 2-thienylacetyl chloride and ~-8 4 ml. of dichloroethane is refluxed with vigorous stirring 9 for 4 hours. The mixture is cooled to 65C., and 10 ml.
of 0.05M t-butanol in dichloroethane added over 2 hours, then 11 heated an additional 1 hr. at 65C. The reaction mixture is 12 cooled to 0C., filtexed and washed with 5 ml. of methanol.
13 The filtrate is cooled to 0C. with stirring, then 1.4 ml. ~ :
:1 .
14 of 1:1 hydrochloric acid : methanol is added, and the l 15 resultant mixture stirred at about 15C. for 3 hrs. The ;~
1 16 mixture is poured into 10 ml. of water containing 1.6 g. of " 17 sodium bicarbonate. The organic phase cuts are discarded.
18 The aqueous cuts are assayed for a 65% yield of the product, 19 sodium 7-(2-thienylacetamido)-7-methoxy-3-carbamoyloxy-~ ~ 20~ methyl-3-cephem-4-carboxylate.
ii~ 21 Step D:~ In analogous fashion, p-chlorobenzoyl chloride, p-~,' , . .
22 nitrobenzoyl chloride, or toluolyl chlor.ide are employed ~ 23 in Step A. The ultimate yield of the desired product S~ 24~sodium 7-(2-thienylacetamido~-7-methoxy-3-carbamoyloxy-methyl-3-cephem-4-carboxylate in each case, is, respectlvely:~
26~ 70% 68~; and 72%.
27 ~ E~AMPLE 6 ,--:: : : ~ ~ . .
28;~90dium 7-(2-thienylacetamido)-7-methoxy-3 carbamoyloxy-29 ~methyl-3-cephem-4-c~arboxylate ~
30~ A~mixture of 2.76 g. (4 Mmoles) of the dimethoxy-31~ methyl~e-ter Of 7~-tD-s-tcsylamino-5-carboxyvaleramido)- -32 3-carbamoyloxymethyl-7a-methoxy-3-cephem-4-carboxylate, ~30-1~' . ... ' :

1 3 g. of dry Linde-type 4A molecular sieves, having less than 2 2% water, 2 ml. of thienylacetyl chloride (16 Mmole) in 3 34 ml. of dichloroethane is stirred a~ reflux for 5 hrs.
4 t-Butanol, 0.38 ml. (4 Mmole) is then added and stirring continued for 2 hrs. At the end of this period, another 6 0.095 ml. (1 Mmole) of t buta~ol i9 introduced and the 7 reaction mixture was stirred at reflux for another 1/2 hr.
8 The reaction mixture is cooled to 0-5C. in an ice-water 9 bath. The molecular sieves are removed by suction-filtra-tion and then washed with 40 ml. of ice-cold methanol. The 11 filtrate and wash were combined and cooled to 0C. An 12 ice-cold solution of 8.3 ml. concentrated HCl and 9.5 ml.
13 MeOH is added and the solution warmed to 15C. and stirred ;
14 at 15C. for 2 hrs. 40 min. When the hydrolysis .i5 complete 15 the reaction is quenched by adding to a suspension of 22 g.
16 sodium bicarbonate in 120 ml. of water at 0-5C. The t~to-17 phase solution is stirred for l0 min. The heavy salt deposit , 18 that forms is removed by filtration and wt~.shed with a small . : . .
19 amount of 5% NaCl solution containing 0.5% sodium bicarbonate.

20 The dichloroethane layer is separated and extracted with 21 2 X 20 ml. of a solution of 0.5% Na~lCO3 -~ 5% NaCl. The 22 aqueous fractions are combined and washed with 20 ml. of 23 dichloroethane. The bicarbonate solution was assayed by , 24~liquid chromatography to contain 73go sodium 7-(2-thienyl-2~5 acetamldo)-7-methoxy-3-carbamoyloxymethyl-3-cephem-4-carbo~

26 xy~late~and 2.1% of the unchanged starting material.

27 ~ ~ EXAMEtLES 7-20 28 Sodium 7-(2-thienylacetamido)-7a-methoxy-3-carbam 2g methy~ E~n~4-carboxylate_ ~ ~ ~Following the general procedure outlined in 31 Example 6~, 4 Mmoles (2.8 g.) of the dimethoxymethyl ester of :~ .

32~7~-~(D-5-tosylamino-5-carboxyvaleramido)-3-carbclmoyloxymethyl-~
(` ~ , ~ -31-_ 15393IA
~4~4~ ~
l 7a-methoxy-3-cephem-4-carboxylate is reacted using the 2 following tabulated amounts of reagents and reaction condi-3 tions. The entire reaction time in each case is about 4 16 hours. The molecular sieves employed, in each case, are Linde-type 4A which are heated to 700~C. before the 6 reaction, exhibiting a weight loss of about 3% on drying.
7 The sieves are calculated to be substantia:Lly anhydrous, 8 having less than 2~i water content, by weightO The temp~era-9 ture of the reaction in each case is 67C.
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2 The same ~eneral procedure used in Examples 6-20 3 is employed, except that the reaction temperature is changed 4 as indicated in the tabulated data. The reaction mixture is 4Mmoles ~2.8 g.3 of the dimethoxymethy] ester of 7~-6 (D-5-tosylamino-5-carboxyvaleramido)-3-carbamoyloxymeth 7 7a-methoxy-3-cephem-4-carboxylate, 3 g. dried Linde-type .-............................... .
, 8 4A sieves (less than 2% water by weight) and 16 Mmole 9 thienylacetyl chloride in 34 ml. of dichloroethane.

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~L04~3i 1 The final product in all cases is sodium 7-(2-2 thienylacetamido)-7Q-~ethoxy-3 carbamoyloxymethyl-3-cephem-3 4-carboxylate. .

7-(2-Thienylacetamido)-7-methoxy-3-carbamoyloxymethyl-3-6 cephem-4-carboxylic acid 7 One gram of sodium 7~-(D-5-amino-5-carboxyval-8 eramido)-3-carbamoyloxymethyl-7-methoxy~3-cephem-4-carbo-g xylate to a reaction vessel containing 20 ml. dichloro- ~-athane. To this is added 1 g. of Linde molecular sieves 11 type 4A powder having 15~ water and 8 Mmoles (1 ml.) 12 thienylacetyl chloride. The slurry is heated at reflux for 13 8 hrs. After filtering the molecular sieves are washed 14 with methanol. The methanol wash of the sieves is analyzed 15 and contains a 25~ yield of the desired product, 7- i 16 thi¢nylacetamido-7a-methoxy-3-carbamoyloxymethyl-3-cephem-17 4-carboxylic acid, by tlc and liquid chromatography assay.
18 The dichloroethane layer of the reaction mixture is extrac-19 ted with 5% Na~CO3 and shown by `tlc and li~uid chromato- .
graphy to contain another 5% of the desired product. Thin 21 layer chromatography of the oryanic layer at this point 22 shows the ~resence of the mixed anhydride; after removal of 23 the solvent by evapoxation, the mixed anhydride is hydro-~24 lyzed i.n 50% acetone-water (50 ml.) in the presence of 10 mole ~ pyridine. Tlc and li~uid chromatography show the 2~6~ product~ion of another 13~ of the final product free acid.
27:~The total yield is l5S mg. of the acid7 23% yield.
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~ -36- ~

Claims (31)

WHAT IS CLAIMED IS:

1. The process of preparing a compound of the formula:

wherein R1 is hydrogen or methoxy;
A' is carbamoyloxy or loweralkanoyloxy; and R' is an acyl group having the formula wherein X is hydrogen, hydroxy, amino, protected amino, or protected hydroxy, or carboxy; R3 is phenyl, 2-thienyl, 3-thienyl, 2-furyl, 3-furyl, 1-tetrazyl, C1-C4 lower-alkylphenyl, halophenyl, hydroxyphenyl, a C1-C4 lower-alkyloxyphenyl;
and R" is an easily removable ester blocking group;
which comprises interchanging a compound of the formula wherein B is blocked amino adipoyl, or an acyl group R' which is different than the final desired substituent with an acylating agent having the formula wherein R3 and X are the same as before defined, in the presence of a molecular sieve catalyst, and then cleaving the B substituent to yield the final product.

2. The process of Claim 1 wherein the molecular sieve catalyst is a synthetic zeolite of the type 3A, 4A, 5A, or 13X.

3. The process of Claim 2 wherein the molecular sieve catalyst has from between about 0% to about 30% by weight water of hydration.

4. The process of Claim 3 wherein the molecular sieve catalyst is employed in approximately 0.5 to 2 times the amount by weight as the starting material.

5. The process of Claim 4 wherein the molecular sieve catalyst is employed in approximately equal, or slightly greater amounts (weight basis) as the starting material.

6. The process of Claim 5 wherein the interchange reaction takes place in a solvent inert to the reactants.

7. The process of Claim 6 wherein the reaction mixture is continually agitated during the interchange reaction.

8. The process of Claim 7 wherein the cleavage of the B substituent is effected by prolonging the reaction time to between about 30 minutes to about 30 hours.

9. The process of Claim 8 wherein the cleavage is effected by addition of a lower alkanol or lower alkyl thiol following the prolonged reaction time.

10. The process of Claim 1 wherein R1 is methoxy.

11. The process of Claim 10 wherein B is amino-adipoyl having blocking groups on the amino and carboxy functions.

12. The process of Claim 11 wherein R' is 2-thienylacetyl.

13. The process of Claim 10 wherein R' is 1-tetrazolylacetyl.

14. The process of Claim 10 wherein R' is 2-amino-2-phenylacetyl, the amino being blocked.

15. The process of Claim 10 wherein R' is 2-amino-2-phenylacetyl, the amino being blocked.

16. The process of Claim 10 wherein R' is 2-hydroxy-2-phenylacetyl, the hydroxy being blocked.

17. The process of Claim 12 wherein A' is carbamoyloxy.

18. The process of Claim 17 wherein R" is methoxymethyl, benzyl, benzhydryl, methylthiomethyl, methylthioethyl, or 2-butenyl.

19. The process of Claim 18 wherein R" is methoxymethyl.

20. The process of Claim 12 wherein A' is loweralkanoyloxy of 1-6 carbon atoms.

21. The process of Claim 20 wherein A' is acetoxy.

22. The process of Claim 21 wherein R" is methoxymethyl.

23. The process of preparing the compound wherein R" represents hydrogen, or an easily removable blocking group; and A is carbamoyloxy or acetoxy;
which comprises mixing the starting compound of the formula:
wherein A is carbamoyloxy or acetoxy; and each R" is hydrogen or an easily removable blocking group, provided that at least one R" group is not hydrogen, with from about 0.5 to about 2 times (weight basis) of a synthetic zeolite, type 3A, 4A, 5A, or 13X, containing from about 0 to 30% by weight water of hydration;
at a temperature of between about 50°-90°C.;
in an inert solvent;
for a period of from about 30 minutes to 30 hours;
then optionally adding a cleaving agent; and recovering:the desired product.

24. The process of Claim 23 wherein the cleavage agent is employed in amounts ranging from about 0.5 molar equivalent to about 3 molar equivalents, based on the amount of the starting compound.

25. The process of Claim 24 wherein the cleavage agent is permitted to react after addition, for a reaction period of from 1 to 15 hours.

26. The process of Claim 25 wherein the cleavage agent is added in aliquots over the reaction period time.

27. The process of Claim 24 wherein the cleavage agent is a lower alcohol or a loweralkyl thiol of 1-6 carbon atoms, benzyl alcohol, or hydrochloric acid.

28. The process of Claim 27 wherein the cleavage agent is isoproponal or t-butanol.

29. The process of Claim 28 wherein the cleavage agent is employed in amounts ranging from approximately equivalent, to about 3 molar equivalents, based on the amount of the starting compound.

30. The process of Claim 27 wherein the cleavage agent is hydrochloric acid.

31. The process of Claim 30 wherein the cleavage agent is employed in amounts ranging from 0. 5 molar equi-valent, to approximately equivalent, based on the amount of the starting compound.