Note: Descriptions are shown in the official language in which they were submitted.
108357~
,.` ' :: .
:~ :
:~ .... :,
...
17 BACRGROUND OF THB INVENTION
18 This invention relates to certain substituted N- ~ .
19 methylene derivatives of the new antibiotic thienamycin.
20 Such compounds and their pharmaceutically acceptable salt,
21 ether, ester, and amide derivatives are useful as antibiotics. ... .:
22 This invention also relates to processes for the preparation
23 of such compounds; pharmaceutical compositions comprising - .
24 such compounds; and methods of treatment comprising
~ ' .
.
- 1 - , ~
: .. , . .. . .. ~. . . .. . . .
15834IB
iL~8;~S~7
``
administering such compounds and compositions when an
antibiotic effect is indicated.
Thienamycin is disclosed and claimed in U.S. Patent
3,950,357 issued April 13, 1976, and may be employed as a
starting material in the preparation of the compounds of the
present invention.
Thienamycin is known to have the followiny
structure:
OH
~ ScH2cH2NH
O N COOH
: I
The substituted N-methylene thienamycin derivatives
of the present invention may be depicted by the following
structural formula:
OH
2CH2N=f X
O N COOH y
II
which, depending upon the basicity of the amino nitrogen
,
(a function of the identity of the methylene substituents
X and Y), may equivalently be represented as an inner salt:
OH
. Y
II
.. ... . ..
.. . . ...
1583~IB
~(~8357~7
.~
1 which is one canonical form of a single resonant structure,
2 which, for example, when Y is -NRlR2 and X is R is:
OH
~SCH2CH2N ' C '--NRlR
N 1I COO~
II
3 For convenience, the compounds of the present invention
4 may be represented by the symbol:
-OH
Th. -N=C-X
COOH
II
wherein "Th~ indicates ~he bicyclic nucleus of thienamycin
6 and its hydroxyl, amino, and carboxyl functional groups are
7 shown wherein X and Y are independently selected from the
8 group consisting of hydrogen, -R, -OR, -SR, and -NRlR2;
g R1 and R are independently selected from R, hydrogen, nitro,
hydroxyl, alkoxyl having 1-6 carbon atoms, amino, mono- di-
11 and trialkylamino wherein the alkyl moieties each comprise
12 1-6 carbon atoms; Rl and R2 may be joined together to form
13 a substituted or unsubstituted mono- or bicyclic heteroaryl
14 or heterocyclyl comprising (together with the nitrogen atom
to which they are attached) 4-10 atoms one or more of which
16 may be an additional hetero atom selected from oxygen,
17 sulphur or nitrogen: R, ~1 and R~ are subs~ituted or un~ub-
18 stituted: cyano; carbamoyl; carboxyl; alkoxycarbonyl and alkyl
19 having from 1 to about 10 carbon atoms; alkenyl having
from 2 to about 10 carbon atoms; alkynyl having Erom 2 to
21 about 10 carbon a~oms; c~cloalkyl having from 3 to 10 ~ :
22 carbon atoms; cycloalkylalkyl and cyaloalkylalkenyl having
- 3 -
. .
. ~: ... . . .
( 1583~r~
, _
~)83S7~
1 from 4 to 12 carbon atoms; cycloalkenyl, cycloalkenyl-
2 alkenyl, and cycloalkenylalkyl having 3-10,4-12 and
3 4-12 carbon atoms, respectively; aryl having from 6 to
4 10 carbon atoms, aralkyl, aralkenyl, and aralkynyl having
5 from 7 to 16 carbon atoms; mono- and bicyclic heteroaryl
6 and heteroaralkyl which typically comprise 4 to 10 ring
7 atoms one or more of which is a hetero atom selected from
8 oxygen, sulphur, or nitrogen and wherein the al~yl moiety
g of the heteroaralkyl radical comprises 1 to about 6 carbon
atoms; mono- and bicyclic heterocyclyl and heterocyclylalkyl
11 which typically comprises 4 to 10 ring atoms one or more of
12 which is a hetero atom selected from oxygen, sulphur or nitro-
13 gen and wherein the alkyl moiety of the heterocyclylalkyl
14 radical comprises from 1 to about 6 carbon atoms; ana wherein
15 the above-mentioned substituent or substituents on R, Rl, R ~ -
16 or on the ring formed by the joinder of Rl and R2, are
17 selected from tne group consisting of: halo, such as chloro,
18 bromo, iodo and fluoro; azido; alkyl having 1-4 carbon atoms;
19 thio; sulpho; phosphono; cyanothio (-SCN); nitro; cyano;
amino; hydrazino; mono-, di- and trialkyl substituted amino,
21 and hydrazino wherein the alkyl has 1-6 carbon atoms; hydrox- -
22 yl; alkoxyl having 1-6 carbon
23 atoms; alXylthio having 1-6 carbon atoms; carboxyl; oxo;
24 alkoxylcarbonyl having 1-6 carbon atoms in the alkoxyl
moiety; acyloxy comprising 2-10 carbon atoms; carbamoyl and
26 mono- and dialkylcarbamoyl wherein the alkyl grou~s have
27 1-4 carbon atoms.
28 TAe compounds of the present invention also
29 e,~brace embodiments of the following structure:
oR3
; ~ ~ ScH2cH2N-lc-x
O~ ~ co
IIa
--4--
~ ' ~'.' '; : ,' - , . . . .
~ 1583~B
~83S7q
1 which may also exist as a salt:
oR3
CoX3R3 Y
IIa
2 or, more conveniently by the previously introduced symbol:
.. --oR3
. Th _ ~ =C-X A~ :
.~,.,,, ~
.; ': coX~R3 .:
. IIa _
3 wherein the non-critical counter anion, A, i~ representa-
4 tively selected ~o provide pharmaceutically acceptable
salts ~uch as halides (chloro, bromo and the like), sulfate,
6 phosphate, citrate, acetate, benzoate and ~he like; and
7 R3, X' and R3 are independently selected from the groups
8 hereinafter defined:
9 X' is oxygen, sulphur or NR' (R' i6 hydrogen or R3 );
10 R3 is hydrogen, or, inter alia, i6 representatively
-. 11 selected to pxovide the pharmaceutically
: 12 acceptable salt, ester, anhydride (R3 is acyl~, and amide ~.
13 moieties known in the bicyclic ~-lactam antibiotic art--
14 such moieties are enumerated in greater detail below; and
15 A3 i~ acyl (generically the group oR3 is classifiable
~ 16 as an este~); or 2.) R3 i5 selected from alkyl, aryl, alkenyl,
;~- 17 aralkyl and the like (such that the group oR3 i~ generically
18 clas~i~iable as an ether). R3 may also be hydrogen. The
19 term ~acyl" i~ by de~inition inclusive of the alkanoyls
20 incIuding derivative~ and analogues thereof such as thio
21 analogues wherein the caxbonyl oxygen is replaced by
. 5
~ ~ .
.
, - .
158'1,4IB
~01~3S7t~
1 sulphur; as well as sulphur and phosphorous acyl analogues
2 such as substituted sulfonyl-, sulfinyl-, and sulfenyl
3 radicals, and substituted P(III and ~) radicals such as
4 substituted phosphorous-, phosphoric-, phosphonous- and
phosphonic radicals. Such radicals, R3, of the present
6 invention are enumerated in greater detail below. -~
7 There is a continuing need for new antibiotics.
8 For unfortunately, there is no static effectiveness of a
9 given antibiotic because continued wide scale usage of any
10 such antibiotic selectively gives rise to resistant strains
11 of pathogens. In addi~ion, the known antibiotics suffer from
12 the disadvantage of being effective only against certain
13 types of microorganisms. ~ccordingly, the search for new
14 antibiotics continues.
Unexpectedly, it has been found that the compounds
16 of the present invention are broad spectrum antibiotics,
17 whibh are useful in animal and human therapy and in inani-
18 mate systems.
19 Thus, it is an object of the present invention
20 to provide a novel class of antibiotics which possess the
21 basic nuclear structure of thienamycin (1), but which are
22 characterized as the substituted N-methylene derivatives
23 thereof. These antibiotics are active against a broad range
24 of pathogens which representatively include both gram po3itive
25 bacteria such as S. aureus, Str~ pyogenes, and B. ~ubtilis
26 and gram negati~e bacteria such as E. coli, Proteus mor~anii,
27 Klebsiella, Serratia, and Pseudomonas. Further objects
28 of this invention are to provid~ chemical processes for
29 the preparation of such antibiotic~ and their non-toxic
30 pharmaceutically acceptable salt, ether, ester and amide
- 6 -
15834Ih
~3357~
1 derivatives; pharmaceutical compositions comrpising such
2 antibiotIcs; and to provide methods of treatment comprising
3 administering such antibiotics and compositions when an
4 antibiotic effect is indicated.
DETAILED DESCRIPTION OF THE INVENTION
6 The compounds of the present invention (Structures
7 II and IIa, a~ove) may be divided into four classes:
8 1.) ~midines; wherein: Y= -NRlR and
9 X = hydrogen or R:
OH
2CH2N=C-NR R
~ COOH R
10 which may be represented by the resonant structure~
OH
~SC 2C~2NEI~ NRlR2
11 wherein R, Rl and R2 are as previously defined. Species
12 IIa may similarly be depicted:
oR3
~/~ SCH2CH2N=C-NRlR2
O,~ COX R
13 wherein R3, X' and R3 are fully defined below.
14 Representative examples of such amidine embodiments
15 ~the substituent plu~ the amino group of thienamycin form
16the amidine ~tructure) are:
17 The benzamidine: Y = -NH2, X = phenyl~
18 The formamidine: Y = -NH2, X - H;
19 The acetamidine: Y ~ -NH2, X ~ CH3;
The 4-pyridylcarboxamidine: Y--NH ,
21 X = 4-pyridyl; 2
22 The N-isopropyl formamidine: Y Y -NHCH~CH3)~,
, '., - . , :
$, '
15B34IB
1()1~3S f'7
2 The N-methyl formamidine: Y = -NHCH3,
X - H;
4 The N,~-dimethylformamidine: Y = -N(CH3)2,
The piperidinyl methylenimine: Y = l-piperidyl
6 X = H.
7 Other preferred amidines are those wherein Y = NRlR2 and
8 Rl and ~2 are independently selected from the group
9 consisting of:.hydrogen; substituted and unsubstituted:
10 alkyl having 1-6 carbon atoms, such as methyl, ethylt i80-
ll propyl, butyl, t-butyl, N,N-dimethylaminoethyl, 2,2,2-
12 trifluoroethyl, 2-methylthioethyl, and the like; alkenyl
13 having ~-6 carbon atoms, such as allyl, methallyl, 2-butenyl,
14 l-buten-3-yl, and ~he like; cycloalkyl, cycloalkylalkyl,
15 cycloalkenyl, and cycloalkenylalkyl having 3-6, 4-7, 4-6,
16 and 4-7 carbon atoms, respectively, such as cyclopropyl,
17 cyclopentyl, cyclohexyl, cyclopropylmethyl, cyclohexylmethyl,
18 2 cyclopropenyl, 1,4-cyclohexadienylmethyl, and the like;
19 aralkyl and aralkenyl having 7-10 carbon atoms, such as
20 benzyl, p-methoxybenzyl, p-dimethylaminobenzyl, cinnamyl,
21 and the like; and monocyclic heteroaralkyl having 5-6 ring
22 atoms, one or more of which is selected from oxygen, sulphur,
23 and nitrogen and 1-3 carbon atoms in the alkyl moiety, such
24 as 2-thienylmethyl~ 3-thienyl~ethyl, 2-furylmethyl,
25 l~methyl-5-tetrazolylmethyl, and the like; wherein the ring
26 or chain substituent relative to the definition of ~l and
~7 R may be ohloro, fluoro, hydroxyl, alkoxyl having 1-3
; 2a car~on atoms, aialkylamino having 1-3 carbon atoms in each
2g alkyl moiety, and alkylthio having 1-3 carbon atom~;
1 5~33~ I B
'`: `
`` ~L)83S7
X is selected From the yroup defined above for R and in par-
. ticular is selected from the group consisting of: hydrogen,
alkyl having from 1 to about 6 carbon atoms; aminoalkyl and
:: aminoalkenyl having 1-6 carbon atoms; alkenyl having 2-6 carbon
atoms; alkoxylalkyl llaving from 2 to about 6 carbon atoms such
as methoxymethyl, ethoxyethyl and the like, mono, di- and tri-
loweralkylaminoalkyl having from 2 to 12 carbon atoms such as
dimethylaminomethyl, methylaminomethyl, trimethylammoniummethyl
and the li~.e; perhaloalkyl having from 1 to 6 carbon atoms such
as trifluoromethyl; alkylthioalkyl having from 2 to about 6
carbon atoms such as methylthiomethyl, ethylthioethyl and the
like, substituted and unsubstituted: aryl and aralkyl such as
phenyl and benzyl; monocyclic heteroaryl, heteroaralkyl,
heterocycly1 and heterocyclylalkyl such as 2-, 3- and 4-pyridyl
and 2-thiazolyl wherein the substituent or substituents are as
. defined above. The amidine embodiments of the present
invention represent a preferred class. Further, amidine
embodiments wherein X' is oxygen and R3 and R3 are hydrogen
are particularly preferred.
Especially preferred amidines of the present
invention are those wherein Y =-NRlRZ and X-R; wherein: Rl, R
and R are selected from hydrogen and the above-listed preferred
substituted and unsubstituted: a.lkyl an.d alkenyl radicals.
2.) Guanidines; wherein: Y = -NRlR2, and
X - -NRlR2 ..
OR
Th - -N-C-NRlR2
NRlR2
C o x ~ R 3
wherein all symbolism is as deFined above.
1583~IB
lU183SJ^~r~'
Represent~tive examples of such guanidine
2 embodiments (the ~ubstituent plus the amino group of
3 thienamycin form the guanidine structure) are:
4 The guani~ine: Y a NH2 ~ X ~ -NH2;
The N-met~ylguanidine: Y - -NHCH3, X = -NH2;
6 The N,N dimethylguanidine: Y = -N(CH3)2,
7 X = -NH2;
8 The N,N,N-trlmethylguanidine: Y = -N(CH3)2
X = -NHC~;
The N-phenylguanidine: Y = ~NH(C6H5), X = -NH2;
11 The nitroguanidine: Y = -NHNO2, X = -NH2;
12 The aminoguanidine: Y = -NHNH2, X = -NH2.
13 Other preferred guanidine6 are those wherein
14 Rl and R2 are independently selected ~rom the group
15 consisting of:..hydrogen; substituted and unsubstituted:
16 alkyl having 1-6 carbon atoms, such as methyl, ethyl, i80-
17 propyl, butyl, t-butyl, N~N-dimethylaminoethyl~ 2,2,2-
18 trifluoroethyl, 2-methylthioethyl, and the like; alkenyl
19 having ~-6 carbon atoms, such as allyl; methallyl, 2-butenyl,
20 1-buten-3-yl, and the like cycloalkyl, cycloalkylalkyl,
21 cycloalkenyl, and cycloalkenylalkyl having 3-6, 4-7, 4-6,
22 and 4-7 carbon atoms, respectively, ~uch as c~clopropyl,
23 cyclopen~yl, cyclohexyl, cyclopropylmethyl, cyclohexylmethyl,
24 2-~yclopropenyl, 1~4-cyclohexadien~lmethyl, and the l~kes
25 aralkyl and aralkenyl having 7-10 carbon atoms, such as
26 benzyl, p-methoxybenzyl, p-dimethylaminobenzyl, cinnamyl,
27 a~d the l~ke; and ~onocyclic heteroaralkyl having 5-6 r~ng
28 a~oms, one or more of which is ~elected ~rom oxygen, sulphur,
29 and nitrogen and 1-3 carbon atoms in the alkyl moiety, such
30 ~8 2-thienylmethyl, 3-thienylmethyl, 2-furylmethyl,
31 1-me~hyl-5-tetrazolylmethyl, and ~he lik~t wherein the rlng
3~ or chain substituent relstive to the definition of ~1 and ..
-- 10 --
. .
15~3~IB
R may be chloro, fluoro, hydroxyl, alkoxyl having 1-3 carbon
atoms, dialkylamino haviny 1-3 carbon atoms in each alkyl
moiety, and alkylthio having 1-3 carbon atoms; X' is oxygen
and R3 and R3 are hydrogen.
3.) Substituted Pseudoureas; wherein:
Y = -NRlR2 and X = -OR or -SR:
OR
Th~N=C-llR R2
COX'R
wherein R3, R3 , X', Rl, and R2 are as defined, and X = -OR
or -SR.
Representative examples of such substituted pseudo-
urea embodiments (the substituent plus the amino group of ~ ~ -
thienamycin form the substituted pseudourea structure) are: ~ ;
The N,N-dimethyl-O-methyl pseudourea:
y = -N(CH3)2~ X = OcH3;
The N,N-dimethyl-S-ethyl pseudothiourea:
y = -N(CH3)2~ X = SCH2CH3;
The N-phenyl-S-ethyl pseudothiourea:
Y = -NHC6H5, X = -SCH2CH3;
The N-methyl-S-methyl pseudothiourea: - -
y = -NHCH3, X = -SCH3 .
Particularly preferred compounds are those wherein R3 and R3
; are hydrogen and X' is oxygen.
. .
-- 11 --
;
.- - . ~
~L083~
1 4.) ImidD Ethers, and ImidD ~hioethers
-OR
~h- -N'C-X
_ Y
~oX'~3
3 wherein R3, X', ~ and ~ are as defined and X is -OR or
4 -SR and Y is hydroge~, R, -OR and - SRo
Representative example6 of such imido ester and
6 imido thioester embodime~ts (the substituent plus the amino
7 group of thienamycin form the imido ester or imiao thioester
8 structure) are:
9 ~he methyl formimidate:
Y = -OC~3, ~= -~;
11 ~he S-methyl thiobenzimidate:
12 Y = -SCH3, X ~ phenyl;
13 The methyl benzyloxycarbimidate: ;
14 3~ X ~CH2C6H5;
15 ~he Diethyl dithiocarbimidate:
16 y = -S-C2H5- X ~ S C2~5
,,
- 12 -
1583~IB
.
gL083~7r,'
IDENTIFICATION OF STARTING MATERIALS
The compounds of the present invention are con-
veniently prepared from thienamycin (I, above). Embodiments
of the present invention such as IIa, above, wherein the
secondary alcoholic group and/or the carboxyl group are
derivatized are conveniently prepared either from the corre-
sponding O-, carboxyl, or O- and carboxyl derivative of
thienamycin or from II or thienamycin followed by subsequent
reaction to establish the radicals R3 and R3 (or -X'R3) and
combinations thereof.
As starting material there may be used O- deriva-
tiyes of thienamyc;n (ester or ether derivatives of the
~econdary alcoholic group of thienamycin) having the following
structural formula:
oR3
J~-~SCH2CH2NH2 ~; :
O N COOH.
Ia
or the N-acyl derivatives of thienamycin having the following
structural for~ula:
OH ~ :
--~SCH2CH2NRl R2 , ~ ~
o~N COOH ~ -
Ib
:'
1 3
ls~34Is
10~3S'-~)7
wherein R and R are select0d from the group consisting of
hydrogen and acyl; the term "acyl" is deined, as it is in the
incorporated by reference application, below. Such N-acyl
thienamycins are useful starting materials for the preparation
of the substituted pseudourea (3.) and the imido ether and
imido thioether (4.) embodiments of the present invention.
As starting materials there may also be used
carboxyl derivatives of thienamycin having the following
structural formula:
OH
-J` ~ SCH2CH2NH2
O N COX'R
Ic
and N-acyl and carboxyl derivatives of thienamycin having the
following structural formula:
:
LSCH2CH2NRl R2 " : -
o~N cox ~ R3
Id
or N-acyl, O- and carboxyl derivatives of thienamycin having
the following structural formula:
.~. ........
'
. .
- 14 -
; . .,
.. ... , , . . ~ . : -.. :. ; . . ,
1583~
-
~(~83~7~
OR
~ SCH2CH2NRl R2
o~N COX'R
Ie
Thus embodiments of the present invention depicted
as IIa, above, may be prepared by starting with the corre-
sponding derivative Ia, Ib, Ic, Id, Ie; or embodiments IIa may
be prepared directly starting with Thienamycin, I, (I~
followed by the desired derivatization procedure to establish
R and/or X'R (II-~IIa).
Relative to structures Ia, Ib, Ic, Id, and Ie, the
radicals R3, R3 , X' and acyl (Rl and R2 ) are defined as
follows:
.
. ,~ .
i . . .
- 1583~1
~ ~ 3S~7
1 ~dentification of the Radical -COX'R
2 In the generic representation of the compounds
3 of the present invention t~Ia,above), the radical ~epre-
4 sented by-COX~R3 is, inter alia, -COOH (X'is oxygen and
is hydrogen) and all radicals known to be effective as
6 pharmaceutically acceptable ester, anhydride ~3 is acyl)
7 and amide radicals in the bicyclic ~-lactam antibiotic
8 art, such as the cephalosporins and penicillins and the
9 nuclear analogues thereof.
Suitable radicals '(R ) include conventional
11 protecting or carboxyl bl~cking groups. The term
12 ~blocking groupa as ~tilized herein is empl~yed i~ the
13 same manner and ~n accordance with the *eaching o~ ~.S.
1~ Patent 3,697,~15 which is incorporated herein by ~eference.
15 Pharmaceutically acceptable thienamycin derivatives of
16 the present invention falling in this class are given
17 below. Suitable ~locking esters thus include tho~e
18 ~elected from the ~ollowing list which is represe~tative
19 and not intended to be an exhaustive list of possible
20 ester gr~ups, wherein X'=O and R3 i8 given:
21 (i) R' -CRaRbRF wherein at least one oi Ra,Rb and
22 Rc i an electron-donor, e.g., ~-methoxyphenyl, 2,4,6-tri-
23 methylphenyl,9-anthryl,methoxy, CH2SCH3, tetrahydro~ur-2-yl,
':
~ 16 -
, . ,.., ,... . ,
. . .
15~3a,~[B
~L~)133S~
tetrahydropyran-2-yl or fur-2-yl. The remaininy Ra, Rb and R
groups may be hydrogen or organic substituting groups. Suita-
ble ester groups of this type include p-methoxybenzyloxy-
carbonyl and 2,4,6-trimethylbenzyloxycarbonyl.
(ii) R =CRaRbRC wherein at least one of Ra, Rb and
Rc is an electron-attracting group, e.g., benzoyl, _-ni-tro-
phenyl, 4-pyridyl, trichlorome-thyl, tribromomethyl, iodo-
methyl, cyanomethyl, ethoxycarbonylmethyl, arylsulphonyl-
methyl, 2-dimethylsulphoniummethyl, o-nitrophenyl or cyano.
Suitable esters of this type include benzoylmethoxycarbonyl,
p-nitrobenzyloxycarbonyl, 4-pyridylmethoxycarbonyl, 2,2,2-
trichloroethoxycarbonyl and 2,2,2-tribromoethoxycarbonyl.
(iii) R3 =CRaRbRC wherein at least two of R , R and
R are hydrocarbon such as alkyl, e.g., methyl or ethyl, or
aryl, e.g, phenyl and the remaining Ra, Rb and Rc group, if
there is one, is hydrogen. Suitable esters of this type
include t-butyloxycarbonyl, t-amyloxycarbonyl, diphenyl-
methoxycarbonyl and triphenylmethoxycarbonyl.
(iv) R3 =Rd, wherein Rd is adamantyl, 2-benzyloxy-
phenyl, 4-methylthiophenyl or tetrahydropyran-2-yl.
Silyl esters, under this category of blocking
groups, may conveniently be prepared from a halosilane or a
silazane of the formula:
R 3SiX'; R 2SiX'2; R 3Si.NR 2i R 3Si-NH-COR ;
R43Si.NH.Co.NH.SiR43; R NH.Co.NH.SiR43; or R4C(oSiR43);
HN(SiR43)2 wherein X' is a halogen such as chloro or bromo and
the various groups R4, which can be the same or different,
represent hydrogen atoms or alkyl, e.g., methyl, ethyl, n-
propyl, iso-propyl; aryl, e.g., phenyl; or aralkyl, e.g.,
benzyl groups.
: , -
:- ~ , .
.
.- 15~3a,I~
. .
1()1~35~
More generally stated, pharmaceutically acceptable
carboxyl derivatives of the present invenkion are those
derived by reacting thienamycin or an N-protected thienamycin,
such as N-acylated thienamycin, wikh alcohols, phenols,
mercaptans, thiophenols, acylating reagents and the like. For
: example, esters and amides of interest are the above-listed
starting materials and final products having the following
group at the 2-position of the thienamycin nucleus: -CoX'R3
wherein X' is oxygen, sulfur, or NR' (R' is H or R3 ), and R3
is alkyl hav~ng 1-10 carbon àtoms, straight or branched, such
as methyl, ethyl, t-butyl, pentyl, decyl and the like;
:` carbonylmethyl, including phenacyl, p-bromophenacyl, p-t-
butylphenacyl, acetoxyacetylmethyl, pivaloxyacetylmethyl,
carboxymethyl, and its alkyl and aryl esters, a-carboxy-a-
isopropyl; aminoalkyl including 2-methylaminoethyl, 2-diethyl-
~ aminoethyl, 2-acetamidoethyl, phthalimidomethyl, succinimido-
methyl; alkoxyalkyl wherein the alkoxy portion has 1-10 and
preferably 1-6 carbon atoms, but can be branched, straight or
. cyclic, and the alkyl portion has 1-6 carbon atoms, such as
methoxymethyl, ethoxymethyl, isopropoxymethyl, decyloxymethyl,
ethoxypropyl, decyloxypentyl, cyclohexyloxymethyl and the
like; alkanoyloxyalkyl wherein the alkanoyloxy portion is
straight or branched and has 1-6 carbon atoms and the alkyl : :
portion has 1-6 carbon atoms, such as acetoxymethyl, pivaloyl-
oxymethyl, acetoxyethyl, propionyloxyethyl, acetoxypropyl, and
the like; haloalkyl wherein halo is chloro, bromo, fluoro, or
iodo, and the alkyl portion is straight or branched having 1-6
carbon atoms, e.g., 2,2,2-trichloroethyl, triEluoroethyl, 2-
bromopropyl, diiodomethyl, 2-chloroethyl, 2-bromoethyl, and
the like; alkenyl having 1-10 carbon atoms, either straight or
- 18 -
.` :,
':~ : ~' . `' . . - ... .
. .
.. . . . .
15~34~s
~(~83S~
branched, e.g., allyl, 2-propenyl, 3-bu-tenyl, 4-butenyl, 4-
pentenyl, 2-butenyl, 3-pentenyl, 3-methyl-3-butenyl, meth-
allyl, l,4-cyclohexadien-1-yl-methyl, and the like; alkynyl
having 1-10 carbon atoms, either straight or branched e.g.,
3-pentenyl, propargyl, ethynyl, 3-butyn-1-yl, and the like;
alkanoyl, either straight or branched, having 1-10 carbon
atoms, such as pivaloyl, acetyl, propionyl, and the like;
aralkyl or heteroaralkyl wherein alkyl has 1-3 carbon atoms,
and hetero means 1-4 hetero atoms being selected from the
group consisting of 0, S, or N, such as benzyl, benzhydryl,
and substituted benzyl, benzhydryl, or e.g., benzyl or benz-
hydryl substituted with 1-3 substituents such as benzyl,
phenoxy, halo, loweralkyl, loweralkanoyloxy of 1-5 carbon
atoms, lower alkoxy, hydroxy, nitro, blocked carboxy, or
combinations thereof, e.g., p~chlorobenzyl, o-nikrobenzyl,
3,5-dinitrobenzyl, p-methoxybenzyl, m-benzoylbenzyl, p-t-
butylbenzyl, m-phenoxybenzyl, p-benzoylbe~zyl, p-nitrobenzyl,
3,5-dichloro-4-hydroxybenzyl, p-methoxycarbonylbenzyl,
p-methoxybenzhydryl, p-carboxybenzyl, the latter being either
the free acid, ester or the sodium salt, 2,4,6-trimethyl-
benzyl, p-pivaloyloxybenzyl, p-t-butoxycarbonyl benzyl, p- ~; -
methylbenzyl, p-benzoyloxybenzyl, p-acetoxybenzyl, p-2-ethyl-
hexanoylbenzyl, p-ethoxycarbonylbenzyl, p-benzoylthiobenzyl,
p-benæamidobenzyl, o-pivaloyloxybenzyl, m-pivaloyloxybenzyl,
p-isopropoxybenzyl, p-t-butoxybenzyl, as well as the cyclic
analogues thereof, 2,2 dimethyl-5-coumaranmethyl, 5-indanyl-
methyl, p-trimethylsilylbenzyl, 3,5-bis-t-butoxy-4-hydroxy-
benzyl; 2-thienylmethyl, 2-furylmethyl, 3-t-butyl-5-isothi-
azolmethyl, 6-pivaloyloxy-3-pyridazinylethyl, 5-phenylthio-1-
- 19 - :
,. . . .. . . . . .
- ~ .: . ~ .
- ,: ` . . :.: . ,. . ' ~' ,, ' :
15834~B
~L~)83S7~7
1 tetra201ylmethyl, or the like (the use of the terms lower
2 alkyl or lower alkoxy in this context means 1-4 carbon atoms
3 chain); or phthalidyl; or phenylethyl, 2-(p-methylphenyl)-
4 ethyl, and the arylthioalkyl analogues, aryloxyalkyl wherein
5 aryl is preferably a phenyl ring having 0-3 substituents
6 preferably 0 or 1 substit~ents in the ortho or para
7 positions and alkyl is 1-6 carbon atoms, e.g., (4-methoxy)-
8 phenoxymethyl, phenoxymethyl, (4-chloro)phenoxymethyl,
9 (4-nitro)phenoxymethyl, (4-benzyloxy)phenoxymethyl,
(4-methyl)phenoxymethyl, (4-benzyloxy)phenoxymethyl, ~4-
11 methyl)phenoxymethyl, t~-methoxy)phenoxymethyl, (l-phenoxy)-
12 ethyl, (4-amino)phenoxymethyl, (4-methoxy)phenylthiomethyl,
13 (4-chloro)phenylthiomethyl, phenylthioethyl; aryl wherein
14 aryl is phenyl, ~-indanyl, or substituted phenyl having
0-3 substituents, preferably 0 or 1 substituent in the
16 ortho or para position, e.g., (4-methyl)phenyl, 14-hydroxy)-
17 phenyl,(4-t-butyl)pheny]., p-nitrophenyl, 3,5-dinitro- :
18 phenyl, or p-carboxyphenyl, the latter having either *he :
19 free acid or the sodium salt form; araikenyl wherein aryl .
is phenyl and alkenyl has 1-6 carbon atoms, such as 3-
21 phenyl-2-propenyl; aralkoxya-kyl wherein aralkoxy is -
22 benzyloxy, and alkyl has 1-3 carbon atoms, ~uch as benzyloxy-
23 methyl, t4-nitro)benzyloxymethyl, (4-chloro)benzyloxymethyl;
24 alkylthioalkyl wherein the alkylthio portion has 1-10
~5 and preferably-1-6 rarbon atoms, but can be branched,
26 straight or cyclic, and the alkyl porticn has 1-6 carbon
27 atoms, s~ch as methylthioethyl, ethylthioethyl, cyclo- :
28 hexylthiomethyl, decylthiobutyl, methylthiopropyl, iso-
29 propylthioethyl, methylthiobutyl and the like.
~0
.. . -,
15834~B
~(~83~
1 . In addition to the esters (and thio ester~) listed
2 above, amides are also embraced by the present invention,
3 i.e., wherein X'is the -N- group. ~epresentative of ~uch
4 amides are those wherein R' is selected from the group
consisting of hydrogen, methyl, ethyl, phenyl, p-methoxy-
6 phenyl, ~enzyl, carboxymethyl, methylthioethyl, and hetero-
7 aryl; also embraced by -CoX'R3 are anhydrides wherein
8 R3 is acyl, for example, benzyloxycarbonyl, ethoxycarbonyl~
9 benzoyl, and pivaloyl.
The most preferred -CoX'R3 radicals of the present
11 invention are those wherein (relative to Structure IIa abov ~ :
12 ~ is oxygen, sulphur or NR' ~R' is selected from the
13 group consisting of hydrogen and lower alkyl); and R3 is
14 selected from the group consisting of: loweralkyl, lower
alkenyl, such a5 methallyl, 3-methylbutenyl, 3-butenyl,
16 and the like; methylthioethyl; benzyl and substituted benzyl
17 such as p-t-butylbenzyl, m-phenoxybenzyl, ~-pivaloyloxy-
18 benzyl, -nitrobenzyl and the like; pivaloyloxymethyl,
ig 3-phthalidyl and acetoxymethyl, propionyloxymethyl,
acetylthiomethyI, pivaloylthiomethyl, allyl, 4-butenyl, -~
~1 2-butenyl, 3-methyl-2-butenyl, phenacyl, acetoxyacetylmethyl,
22 methoxymethyl, p-acetoxybenzyl, p-pivaloyloxybenzyl,
23 p-isopr~poxybenzyl, S-indanylmethyl, 5-indanyl, benzyloxy- :
methyl, ethylthioethyl, methylthiopropyl, methoxycarbonyloxy-
25 methyl, ethoxycarbonyloxymethyl, dimethylaminoacetoxymethyl,
26 crotonolacton-3-yl, and acetamidomethyl.
~ 2~ ~
.7 5 a 3 4 IB
3S~7
1 Identifi~ation ~f R3 (Rl and R2 )
2 In the generic representati~n of the present
3 invention, structure IIa(above), the radical R3 is, in
4 addition t3 hydrogen, 1.) acyl (generically the group
-oR3 is classifiable as an ester); or 2.~ R3 is seleet~d
6 from alkyl, aryl, aralkyl, and the like such that the
7 group -oR3 is classifiable as an ether. For the ~ster
8 embodiments tl) R3 i~ selected from the following definition
9 of acyl radicals (p=l~. In the so-called ether ~mbodiments ~2.)
of the present invention, R3 is selected from the same acyl
11 radicals wherein the carbonyl moiety, -C-, or more generally ~ .
X
12 -C-, is deleted (p=0); thus R3 is selected from the follow- -~
13 ing radicals wherein all symbolism is defined below: .
~ ~ -R' p (CH2)nzR ~C~p -CHR"
~Rnl .
~C~p-C~R R ~C~p (CH2)m ~ ~CH2)~ Y
16 Rl and R2 ~are selected fro~ ~he above radicals
17 wherein p=l. Thus, relative to the definition of R3, Rl and
18 R2 , the acyl radical can, înter alia, be substituted or
19 unsubstituted aliphatic, aromatic or heterocyclic, araliphatic
20 or heterocyclylaliphatic carboxylic acid radical, a subs~i-
21 tuted or unsubstituted carbamyl radical or a carbothioic
22 acid radical. One group of acyl radicals can be represented
23 by the general formula:
24 1l
--C-Rn
26 wherein X is O or S and Rr represents hydrogen; amino;
27 substituted amino such as alkyl- and dialkylamino wherein the
28 alkyl radical comprises 1 to about 6 carbon atoms; substituted
29 or unsubstituted: ~traight or branched chain alkyl wherein the
30 ~lkyl radical comprises 1 t~ about 6 carbon atoms; ~ercapto;
- 22 -
..
15834IB
3~
1 aryloxy, typically comprising 6 to 10 carbon atoms;
2 alkenyl, or alkynyl groups typically comprising 2 to 6
3 car~on atoms; aryl such as phenyl; aralkyl such as benzyl;
4 cycloalkyl, typically comprising 3 to 6 carbon a~oms; ~r
a heteroaryl ~r heteroaralkyl group ~mono- and bicyclic)
6 wherein the alkyl moiety typically comprises 1 to 3 carbon
7 atoms and the heterocyclic ring comprises typically 4 ~o 10 -
8 atoms and the hetero atom or atoms are selected from 0, N
g and S; such above-listed groups can be unsubs~ituted or can
be substituted by radicals s~ch as OH, SH, SR (R is lower alkyl
11 or aryl such as phenyl), alkyl or alkoxy groups having 1 to
12 about 6 carbon atoms, halo, such as Cl, Br, F and I,
13 cyano, carboxy, sulfamino, carbamoyl, sul~onyl, azido, amino,
14 substituted amino such as alkylamino including quaternary
ammonium wherein the alkyl group comprises 1 to 6 carbon
16 atoms, haloalkyl such as trifluoromethyl, car~oxyalkyl, ~:
17 carbamoylalkyl, N-substituted ca bamoylalkyl, wherein the
18 alkyl moiety of the foregoing four radicals comprises 1 to
19 about 6 carbon atoms, amidino, guanidino, N-substituted
guanidino, yuanidino lower alkyl and the like. ~epresentative
21 examples of s~ch acyl groups that might ~e mentioned are
22 those wherein R" is ~enzyl, p-hydroxybenzyl, 4-amino-4-
carboxybutyl, methyl, cyanomethyl, 2-pentenyl, n-amyl, n-
heptyl, ethyl 3- ~r 4-nitrobenzyl, phenethyl, ~
~iphenylethyl, methyldiphenylmethyl, triphenylmethyl, 2-
26 methoxyphenyl, 2,6-dimethoxyphenyl, 2,4,6-trimethoxyphenyl,
27 3,5-dimethyl-4-isoxazolyl, 3-butyl-5-methyl-4-isoxa7.olyl,
28 5-methyl-3-phenyl~4-isoxazolyl, 3-(2-chlorophenyl)-5- ..
_ 23 _
, ' ~, , , . ~ , .
1583~I~
~)193S7~
methyl~4-isoxazolyl, 3-(2,6-dichlorophenyl)-5-me-thyl-4-
isoxazolyl, D-4-amino-4-carboxybutyl, D-4N-benzoylamino-4-
carboxy-n-butyl, p-aminobenzyl, o-aminobenzyl, m-aminobenzyl,
p-dimethylaminobenzyl, (3-pyridyl)methyl, 2-ethoxy-1-naphthyl,
3-carboxy-2-quinoxalinyl, 3-(2,6-dichlorophenyl)-5-(2-furyl)-
4-isoxazolyl, 3-phenyl-4-isoxazolyl, 5-methyl-3-(4-guanidino-
phenyl)-4-isoxazolyl, 4-guanidinomethylphenyl, 4-guanidino- ~-
methylbenzyl, 4-guanidinobenzyl, 4-guanidinophenyl, 2,6-
dimethoxy-4-guanidino, o-sulfobenzyl, p-carboxymethylbenzyl,
p-carbamoylmethylbenzyl, m-fluorobenzyl, m-bromobenzyl,
p-chlorobenzyl, p-methoxybenzyl, l-naphthylmethyl, 3-isothi-
azolylmethyl, 4-isothiazolylmethyl, 5-isothiazolylmethyl,
guanylthiomethyl, 4-pyridylmethyl, 5-isoxazolylmethyl,
4-methoxy-5-isoxazolylmethyl, 4-methyl-5-isoxazolylmethyl,
l-imidazolylmethyl, 2-benzofuranylmethyl, 2-indolylmethyl,
2-phenylvinyl, 2-phenylethynyl, l-aminocyclohexyl, 2- and
3-thienylaminomethyl, 2-(5-nitrofuranyl)vinyl, phenyl,
o-methoxyphenyl, o-chlorophenyl, o-phenylphenyl, p-amino-
methylbenzyl, 1-(5-cyanotriazolyl)methyl, difluoromethyl,
dichloromethyl, dibromomethyl, 1-(3-methylimidazolyl)methyl,
2- or 3-(5-carboxyme~hylthienyl)methyl, 2- or 3-(4-carbamoyl-
thienyl)methyl, 2- or 3-(5-methylthienyl)methyl, 2- or 3-
(methoxythienyl)methyl, 2- or 3-(4-chlorothienyl)methyl,
2- or 3-(5-sulfothienyl)methyl, 2- or 3-(5-carboxythienyl)-
~, ,.
methyl, 3-(1,2,5-thiadiazolyl)methyl, 3~(4-methoxy-1,2,5-
thiadiazolyl3methyl, 2-furylmethyl, 2-(5-nitrofuryl)methyl,
3-furylmethyl, 2-thienylmethyl, 3-thienylmethyl, tetrazolyl-
methyl, benzamidinomethyl and cyclohexylamidinomethyl.
The acyl group can also be a radical of the formula:
- 24 -
- , .
..
15834I~
: ~083S~7 :~
X
Il
--C (CH2) nZR . ~
2 wherein X is O or S and n is 0-4, Z represents oxygen,
3 sulfur, carbonyl or nitrogen and R" is defined as above.
4 Representative members of the substi~uent
5 ~(CH2)n~R , :~
6 that might be mentioned are allylthiomethyl, phenylthio-
7 methyl, butylmercaptomethyl, ~-chlorocrotylmercaptomethyl,
8 phenoxymethyl, phenoxyethyl, pheroxybutyl, phenoxybenzyl,
9 diphenoxymethyl, dimethylmethoxyethyl, dimethylbutoxymethyl,
dimethylphenoxymethyl, 4-guanidinophenoxymethyl, 4-pyridyl-
11 thiomethyl, p-(carboxymethyl)phenoxymethyl, p-(carboxymethyl)~
12 phenylthiomethyl, 2-thiazolylthiomethyl, p-(sulfo)phenoxy-
13 methyl, p-(carboxymethyl)phenylthiomethyl, 2-pyrimidinyl- -
14 thiomethyl, phenethylthiomethyl, 1-(5,6,7,8-tetrahydro-
lS naphthyl)oxomethyl, N-methyl~4-pyridylthio, benzyloxy,
lS methoxy, ethoxy, phenoxy, phenylthio, amino, methylamino,
17 dimethylamino, pyridinium methyl, trimethylammonium-methyl,
18 cyanometh~ylthiomethyl, trifluoromethylthiomethyl,
19 4-pyridylethyl, 4-pyridylpropyl, 4-pyridylbutyl,
Zo 3-imidazolylethyl, 3-imidazolylpropyl, 3-imidazolylbutyl,
pyrroloethyl, l-pyrrolopropyl, and l-pyrrolobutyl.
22 Alternatively, the acyl group can be a radical
23 of the formula:
24
--C--ÇHRn
;
- 25 -
::
: .: ~:,: , . ...
15~34I~
3S ~ ~
wherein R" is defined as above and R"' is a radical such :
as amino, hydroxy, azido, carbamoyl, yuanidino, amidino,
acyloxy, halo, such as Cl, F, Br, I, sulfamino, tetrazolyl,
sulfo, carboxy, carbalkoxy, phosphono and the like.
Representative members of the substituent
f HR"
R"'
that might be mentioned are a aminobenzyl, a-aminO-(2-
thienyl)methyl, a-(methylamino)benzyl, a-amino-methylmercapto-
propyl, a-amino-3- or 4-chlorobenzyl, a-amino-3- or 4-hydroxy-
benzyl, a-amino-2,4-dichlorobenzyl, a-amino-3,4-dichloro-
benzyl, D(-)-a-hydroxybenzyl, a-carboxybenzyl, a-amino-
(3-thienyl)methyl, D(-)-~i-amino-3-chloro-4-hydroxybenzyl,
a-amino(cyclohexyl)methyl, a-(5-tetrazolyl)benzyl, 2-thienyl-
carboxymethyl, 3-thienyl-carboxymethyl, 2-furyl-carboxymethyl,
3-furyl-carboxymethyl, a-sulfaminobenzyl, 3-thienyl-sulfamino-
methyl, a-(N-methylsulfamino)benzyl, D(-)-2-thienyl-guanidino-
methyl, D(-)-a-guanidinobenzyl, a-guanylureidobenzyl, ~-
a-hydroxybenzyl, a-azidobenzyl, a-fluorobenzyl, 4-(5-methoxy-
1,3-oxadiazolyl)aminomethyl, 4-(5-methoxy-1,3-oxadiazolyl)-
hydroxymethyl, 4-(5-methoxy-1,3-sulfadiazolyl)hydroxymethyl,
4-(5-chlorothienyl)aminomethyl, 2-(5-chlorothienyl)hydroxy-
methyl, 2-(5-chlorothienyl)carboxy-methyl, 3-(1,2-thiazolyl)-
aminomethyl, 3-(1,2-thiazolyl)hydroxymethyl, 3-(1,2-thiazolyl)-
carboxymethyl, 2-(1,4-thiazolyl)aminomethyl, 2-(1,4-thiazolyl)-
hydroxymethyl, 2-(1,4-thiazolyl)carboxymethyl, 2-benzothienyl-
aminomethyl, 2-benzothienylhydroxymethyl, 2-benzothienyl-
carboxymethyl, a-sulfobenzyl, a-phosphonobenzyl, ~-diethyl-
phosphono, and a-monoethylphosphono. Further acyl radicals of
interest in this class when ~ - oxygen are:
- 26 -
: ... . .. , . : .
15~341B
~35r~r4~
0
-CcHR4R
2 wherein R4 and R5 are as defined below. ~ ~epresents
3 hydrogen, halo, such as chloro, fluoro, bromo,iodo, ~nino,
4 guanidino, phosphono, hydroxy, tetrazolyl, carboxy, sulfo,
or sulfamino and ~ represents phenyl, substituted phenyl,
6 a mono- or bicyclic heter~cyclyl containing one or more
7 oxygen, sulfur or nitrogen atoms in the ring, such as furyl,
8 ~uinoxalyl, thienyl, quinolyl, quin~zolyl, ~hiazolyl,
9 isothiazolyl, tetrazolyl, oxadiazolyl, thiadiazolyl and
10 and the like substitued heterocycles, phenylthio, phenyloxy,
11 lower alkyl of 1-6 carbon atoms, heterocyclic or
12 substituted heterocyclic thio gxoups; or cyano. ~he
13 substituents on the moieties, R4 and ~5, can be halo,
14 carboxymethyl, guanidino, guanidinomethyl, carboxamido-
15 methyl, aminomethyl, nitro, methoxy or methyl. When
16 R is selected from the group consisting of hydrogen,
17 hydroxy, amino or caxboxy and R is selected from the group
18 consisting of phenyl, or a 5- or 6-membered heterocyclic
19 Iing having one or two ~ulfur, oxygen or nitrogen hetero atom -
20 such as tetrazolyl, thienyl, furyl ~nd phenyl, the
21 following acyl radicals are representative: phenylacetyl
22 3-bromophenylacetyl, p-aminomethylphenylacetyl, 4-
23 carboxymethylphenylacetyl, 4-carboxyamidomethylphenylacetyl,
24 2-furylacetyl, 5-nitro-2-furylacetyl, 3-furylacetyl, 2-
25 thienylacetyl, 5-chloro-2-thienylacetyl, ~-rnethoxy-2-
26 thienylacetyl, a-guanidino-2-thienylacetyl, 3-thienylacetyl,
27 2-(4-methylthienyl)acetyl, 3 isothiazolylacetyl, 4-methoxy-
'~;
.`
- 27 -
:,. , . .. ~ ..
1-583~IB
1()~i~5~7
1 3~isothiazolylacetyl, 4-isothiazolylacetyl, 3-methyl-4-
2 isothiazolylacetyl, ~-isothiazolylacetyl, 3-chloro-~-
3 isothiazolylacetyl, 3-methyl-1,2,5-oxadiazolyla~etyl,
4 1,2,5-thiadiazolyl-4-acetyl, 3-methyl-1,2,5-thiadiazolyl-
acetyl, 3-chloro-1,2,5-thiadiazolylacetyl, 3-methoxy-1,2,~-
6 thiadiazolylacetyl, phenylthioacetyl, 4-pyridylthioacetyl,
7 cyanoacetyl, l-tetrazolylacetyl, Q-fluorophenylacetyl,
8 D-phenylglycyl, 4-hydroxy-D-phenylglycyl, 2-thienylglycyl,
9 3-theinylglycyl, phenylmalonyl, 3-chlorophenylmalonyl,
2-thienylmalonyl, 3-thienylmalonyl, a-phosphonophenylacetyl,
11 ~-amino cyclohexadienylacetyl, Q-sulfaminophenylacetyl,
12 Q-hydroxyphenylacetyl, ~-tetrazolylphenylacetyl a~d
13 Q~ sulfophenylacetyl.
14 ~he acyl radical may also be selected ~rom
sulphur (1) and phosphorous (2) radicals:
_,
16 ~O)m (X)n
11
--S--Y _~_y ~
11 1
(O)n
17 1 2
18 wherein with respect to 1 , m and n are integer~ selected
19 from 0 or 1 and Y~= 0~ M ~ -N(Rn)2, and Rn; wherein M :
is selected from hydrogen, alkali metal cations and
21 organic bases; and Rw is as defined above, e.g., alkyl,
22 alkenyl, aryl and heteroaryl. With respect to 2 ~ z
3 O or S; nzO or l; and Y' and ~ are selected from the
e:, kl~
24 group consisting of o M , -N(Rn)2, R" and ZR" wherein
all symbolism is as defined above, e.g., R" and ZR" are
26 representatively: al~yl, alkenyl, aryl, heteroaryloxy,
,
~ 28 ~
: . ,
.
15834IB
lUiY3~
1 Y' and Y", including R" moieties, can be joined together
to form cyclic ester, ester- amide and amide functions.
3 Illustrative examples of ~ are O-(methylsulphonyl)-
4 theinamycin, O-(p-nitrophenylsulphonyl)thienamycin,
5 ~-(p-chlorophenylsulphinyl)thienamycin, ~-(o-nitrophenyl-
6 sulphenyl)thienamycin, O-sulfamoylthienamycin, O-
7 dimethylsulphamoylthienamycin and thienamycin OLsulphonic
8 acid sodium salt. Illustrative examples of 2 are
9 O~dimethoxyphos~hino)thienamycin, -tdibenzyloxyphosphino)-
10 theinamycin, O- (dihydroxyphos~hino)thienamycin disodium
11 salt, O-(dimethoxyphosphinyl)thienamycin, O-(dimethoxy-
12 phosphino~hioyl)thienamycin,O-[dibenzyloxyphosphinyl)-
13 thienamycin, and O-(dihydroxyphosphinyl)thienamycin disodium
14 salt. ~he definitio~ of Rl and R2 does not embrace radicals
15 1 and ~
16 An acyl class ~f particular interest is those -
i7 acyl radicals which are selected from the group c~nsisting
i8 f conventionally known N-acyl blocking or protective groups
ag such as carbobenzyloxy, ring-~ubstituted carbobenzyloxy ~uch
as o- and p-nitrocarbobenzyloxy, p-methoxycarbobenzyloxy,
~1 chloroacetyl, bromoacetyl, phenylacetyl, t-butoxycarbonyl,
2 trifluoroacetyl, bromoethoxycarbonyl, 9-fluorenylmethoxy-
carbonyl, dichloroacetyl, o-nitrophenylsulfenyl, 2,2,2-
trichloroethoxycarbonyl, bromo-t-butoxycarbonyl, phenoxy-
~5 acetyl, non-acyl protective groups such as trilower alk-~l
2~ silyl, for example, trimethylsilyl and t-butyldimethyl
2~ are also of interest.
~:
.
.
15~34IB
J.01~35'~
1 The ~ollowing radicals, according to the foregoing
2 definition o~ acyl, are especially preferred for R3 of struc-
3 ture IIa: formyl, acetylj propionyl, ~utyryl, chloroacetyl,m~-
4 oxyacetyl, aminoacetyl, methoxycarbonyl, ethoxycarbonyl,
methylcarbamoyl, ethylcarbamoyl, phenylthiocarbonyl, 3-amino-
6 propio~yl, 4-aminobutyryl, N-methylaminoacetyl, N,N-dimethyl-
7 amino-acetyl, N,N,N-trimethylaminoacetyl, 3-(N,N-dimethyl)-
8 aminopropionyl, 3-(N,N,~-trimethyl)aminopropiOnyl,
9 N,N,N-triethylaminoacetyl, pyridiniumacetyl, guanidino-
acetyl, 3-guanidinopropionyl, N3-methylguanidinopionyl,
ll hydroxyacetyl, 3-hycroxypropionyl, acryloy~, propynoyl,
12 malonyl, phenoxycarbonyl, amidinoa~etyl, acetamidinoacetyl,
13 amidinopropionyl, acetamidinopropionyl, guanylureidoacetyl,
14 guanylcarbamoyl, carboxymethylaminoacetyl, sulfoacetyl-
- 15 aminoacetyl, phosphonoacetylaminoacetyl, N3-dimethylamino-
16 acetamidinopropionyl, ureidocarbonyl, dimethylamiDoguanyl-
17 thioacetyl, 3-(l-methyl-4-pyridinium)propionyl, 3-(5-
18 aminoimidazol l-yl)propionyl, 3-methyl-l-imidazoliumacetyl,
l9 3-sydnonylacetyl, o-aminomethylbenzoyl, o-aminobenzoyl, sulfo,
phosphono,
O S S
-P(OCH3)2 , -P(OCH3)2 , Il~~H3
ONa
S O O
--1[N(CH3)2]2~ P~NNCEI3)2 , --P[N~CH3)2]2 ~
~' , .
~ -P-N(CH3)2
; ONa
15834IB
IIUB3S7'~7
l Another class of acyl radicals
2 are terminally substituted acyls wherein the substituent
3 is a basic group such as substituted and unsubstituted:
4 amino, amidino, guanidino, guanyl and nitrogen-containing
mono- an~ bicyclic heterocyles ~aromatic and non-aromatic)
6 wherein the hetero atom or atoms, in addition to nitrogen,
7 are selected from oxygen and sulphur. Such -- -
8 substituted acyls may be represented by the following formula:
g q
- 11CH2)m - A- (CH2)n
lO wherein m, and n are integers se~ected from 0 to 5;
11 A is O, NR' ~R' is hydrogen or loweralkyl having 1-6 carbon
12 atoms) r S or A represents a single ~ond; and
13 ~is selected from the following group:
14 l.) amino or substituted am no:
-N(R~2 and -N(R)3
16 wherein the values for ~ are independently selected from:
17 hydrogen; N(R')2 (R' is hydrogen or loweralkyl having
18 1-6 carbon atoms); loweralkyl and loweralkoxyl having from
l9 1 to 6 oarbon atoms; loweralkoxyloweralkyl wherein the
20 alkoxyl moiety comprises l to 6 carbon atoms and the
21 alkyl moiety comprises 2-6 carbon atoms; cycloalkyl and
22 cycloalkylalkyl wherein the cycloalkyl moiety comprises
23 3-6 carbon atoms and the alkyl moiety comprises 1-3
24 carbon atoms, two ~ ~roups may be joined together with the
25 N atom to which they are attache d to form a ring having
26 3-6 atoms.
. ~
.
1583~IB
~(1 8~S~7
1 2.) amidin~ and substituted amidino:
-N=C-N(R)2
~, 0
2 wherein the value of Ris independently selected from the
3 group consisting of: hydrogen; N(R')2 (R' is hydrogen ~r
4 loweralkyl having 1-6 carbon atoms); loweralkyl and
loweralkoxyl having from 1 to 6 carbon atoms, :Loweralkoxy-
b loweralkyl wherei.n the alkoxyl moiety comprises 1 to 6
7 carbon atoms and the alkyl moiety comprises 2 to 6 carbon
8 atoms (when the loweralkoxyloweralkyl radical is attached
9 to carbon the alkyl moiety comprises 1 to 6 carbon atoms);
cycloalkyl and cycloalkylalkyl wherein the alkyl moiety
11 comprises 1 to 3 carbon atoms; two Rgroups may be joined -
12 together with the atoms to which they are attached to form .
13 a ring having 3 to 6 atoms;
14 3.) guanidino and substituted guanidino:
.:-
-NH-C-N(RD)~
NR
16 wherein F~is as defined in 2.(above).
17 4-) guanyl and substituted guanyl:
18 -C=N~
-2
19 wherein ~ is as defined in 2. ~abvve).
20 5-~ . nitrogen-containing mono- and bicyclic heterocyclyls
21 (aromatic and non-aromatic~ having 4 to 10 nuclear atoms
22 wherein the hetero atom or atoms, in addition to nitrogen,
23 are selected from oxygen and sulphur. 5uch heterocyclyls
24 are representatively illustrated by the Pollowing list of
l 25 radicals (R' is H or loweralkyl having 1 6 carbon atoms):
'
~2
.. . . . . .. ..
1-583~IB
13S~
~(R )2
$~
O
O ~ --R
'
~ 3 ~R
,~
.; ' .
_ ~3 --
-
15834~B
1()8;3~57~7
1 The following specific acyl radicals falling
2 within this class are additi~nally representatiYe:
O NH
Il I
C~H2CH2NHC CH3
p NH
--CCH2CE12NHC--H
O NH
Il I
CCH2CH2NHC NH2
O '
-CCH2CH2N~CH3)2
Il ~) '.
CCH2CH2N(CH3)3
Q NH
Il I . .
CCH2CH2C NH2
..
O NH
Il I . .
: 2 2 2 3
O
--ccH2cH2cH2NtcH3)3
fj `:
--CCH2~H2~H2N(C~3~2
O
~,NH
--CCH2S-
~
~NH~
O
-CCH2S-CH~- ~
NH2
o
-ICC~2-O-C~2C~
N~2 . :
- 34 -
- : '.: .' . . ' .. , ' . ': .
15834I~
35~f~
1 Preparation of Starting M~terials la, Ib, Ic, Id and Ie
2 The above-described starting materials are
3 conveniently prepared from an N-protected thienamycin
4 (1), such as an N-acylated thienamycin (1).
,-- .
~ ~ OH
Th - _ NR~R2
. ~ COOH
wherein ~1 and ~2 are selected from hydrogen and the
6 above-defined acyl radicals. Preferably Rl is hydrogen
7 and R2 is an easily r~movable blocking group such as: ;
8 carbobenzyloxy, ring-substituted carbobenzyloxy such as
; g o- and p-nitrocarbobenzyloxy, p-methoxycarbobenzyloxy,
chloroacetyl, bromoacetyl, phenylacetyl, t-butoxycarbonyl
11 trifluoroacetyl, bromoethoxycarbonyl, 9-fluorenylmethoxy-
: 12 ~carbonyl, dichloroacetyl, o-nitrophenylsulfenyl,
13 2,2,2-trichloroethoxycarbonyl~ bromo-t~butoxycarbonyl,
14 phenoxyacetyl;non-acyl protective groups 6uch as trilower-
alkylsilyl, for example, trimethylsilyl, and t-butyldimethyl-
16 silyl are also of interest. The most preferred N-blocking
17 groups are the substituted and unsubstituted carbobenzyloxy
18. radical:
~2 = _ C - OCH2 ~ ~ R ' ) n
19 wherein n is 0-2 tnY0, R'=hydrogen) and
~0 R' is lower alkoxy or nitr~ and ~xomo-t-butoxycarbonyl,
~:~. O ~CH3
21 -C-O-C~CH2Br
~ 35 ~ ::
lSg3D,:[B
`:
3S~
The ultimate N-deblocking procedure ~or the prepa-
ration of Ia, Ic or Ie .is accomplished by any of a variety of
well-known procedures which include hydrolysis or hydrogen-
ation; when hydrogenation is employed suitable conditions
involve a solvent such as a loweralkanol in the presence of a
hydrogenation catalyst such as palladium, platinum or oxides
thereof.
The N-acylated intermediate ~, (or Ia) abov ~ is
prepared by treating thienamycin (~) with an acylating agent,
lQ for example, an acyl halide or acyl anhydride such as an
aliphatic, aromatic, heterocyclic, araliphatic or heterocyclic
aliphatic carboxylic acid halide or anhydride. Other acyl-
ating agents may also be employed, for example, mixed carbox-
ylic acid anhydrides and particularly lower alkyl esters of
mixed carboxylic-carbonic anhydrides; also, carboxylic acids
in the presence of a carbodiimide such as 1,3-dicyclohexyl-
carbodiimide, and an activated ester of a carboxylic acid such
as the _-nitrophenyl ester.
The acylation reaction may be conducted at a
20 temperature in the range of from about -20 to about 100C.,
but is preferably conducted at a temperature in the range of
from -9C. to 25C. Any solvent in which the reactants are
soluble and substantially inert may be employed, for example
.``' ' ~'.
,:
- 36 -
. .
:, . .... . . .
15834IB
1 polar solvents such as water, alcohols and polar organic
2 solvents in general such as dimethylformamide tDMF),
3 hexamethyl, phsophoramide (HMPA), acetone, dioxane
4 tetrahydrofuran (THF), acetonitrile, heterocyclic amines
such as pyridine, ethylacetate, aqueous mixtures o~ ~he
6 above, as well as halogenated solvents such as methylene
7 chloride and chloroform~ ~he reaction is conducte~
8 ~or a period o~ time of from about five minutes to a maxinum
9 of three hours, ~ut in general, a reaction time of about
0.5 to about one hour is sufficient. The following
11 eguation illustrates this process employing a carboxylic
12 acid halide; however, it is to be understood that by substi-
13 tuting a carboxylic acid anhydride or other functionally
14 e~uivalent acylating agent similar products may be obtained.
OH OH
SCH2CH2NH2 ~ CH~CH2NR1 R
COOH acyl halide~ N COOH
0~ ' 0~ ~
16~ Generally when the above-described ~acylating
17 reaction employs an acid halide (suitable halides are chloro,
18 i~do, or bromo) or anhydride the resction is conducted in
19 water or an aqueous mixture of a polar organic solvent such
as aceto~e, dioxane, THF, ~MF, acetonitrile or the like
21 in the presence of a suitable acceptor base ~uch as NaHCO3,
22 MgO, NaOH, R2~PO4 and the like.
23 In carrying out the reactions described herein,
24 it is generally not necessary to protect the 2-car~oxy
group or the 1'-hydroxy group; however, in cases where the
26 acylating reagent is exceedingly water sensitive it is
27 sometimes advantageous to perform the acylation in a non
28 agueous ~olvent gystem. ~rioryanosilyl ~or tin) derivatives
~ 37 -
',, .' ' '
~35~q
1 of thienamycin proceeds rapidly to give the tri~-triorgaDo-
2 silyl derivative, for example tris-trimethylsilyl thienamycin
3 Th(TMS~3:
~ OTMS
Th_ _ NXTMS
--COOTMS
Such derivatives, which are readily soluble in organic
solvents, are conveniently prepared by treating thienamycin
6 with an excess of hexamethyldisilazane and a stoi~hiome~ric
7 amount of trimethylchlorosilane at 25C., with vigorous
8 ~tirring under a N2 atmosphere. The resulting NH4~1 is
9 removed by centrifugation and the solvent is ~emoved by
evaporation to provide the desired silyl derivative.
11 ~hé intermediate starting materials Ic are
12 prepared according to the following scheme; however, it should
13 be noted that direct esterification, without protection of
14 the amino group, is also possible.
rOH ! -OH -0
Th~ NRlR2 -~ ~h- -NR~R2 deblock ~ Th-
COOH ~OX'~ COX'R
1 Id ~c
16 wherein all symbolism i8 as previously defined.
17 In general, the transformation (l--~IC)
18 is accomplished by conventional procedures ~nown in the
19 art. Such procedures include:
~0 1.) Reacti~n of 1 (or I~ with a diazoal~ane ~uch
21 as diazomethane, phenyldiazomethane, diphenyldiazomethane,
22 and the like, in a solvent such as dioxane, ethylacetate,
;
23 acetonitrile and the like at a temperature of from 0C
24 to reflux for from a few minutes to 2 hour~.
2.) Reaction of an alkali metal salt of 1 with an
26 activated al~yl halide such as methyliod~de~ benzyl bromide,
.~ .
38
. ,.: . . ,
3~i7~ 5 g 3 4 I B
1 or m-phenoxybenzyl bromide, ~ butylbenzyl bromide,
2 pivaloyloxymethyl chloride, and the like. Suitable reaction
3 conditions include solvents such as hexamethylphosph~ramide
4 and the like at a temperature of from nDC. to 60C. for
from a few minutes to 4 hours.
~ 3.) Reaction of 1 with an alcohol such as
7 methanol, ethanol, benzyl alcohol, and the like. ~This
8 reaction may be conducted in the presence of a carbodi-
9 imide condensing agent such as dicyclohexylcarbodiimide
or the like. Suitable solvent, at a temperature of from
11 0C to reflux ~or from 15 minu~es to 18 hours, include
12 CHC13, CH3Cl, CH2C12 and the like.
13 4.) Reaction of an N-acylated acid anhydride ~f 1
1~ prepared by reacting the ~ree acid 1 with an acid ~hloride
such as ethylchloroformate, benzylchloroformate and the like,
16 with an alcohol such as those listed in 3.1 under the ~ame
17 conditions of leaction as given above for 3.). The anhydride
18 is prepared by reacting 1 and the acid chloride in a solvent
19 ~uch as tetrahydrofuran (THF), ~H2C12 and the like at a -
temperature of from 25C., to reflux for from 1~ minutes
21 to 10 hours.
~2 5.) Reaction of labile esters of 1
23 such as the trimethylsilyl ester, dimethyl-t-butylsilyl
24 ester or the like with R3 X wherein X is halogen ~uch as
bromo and chloro and ~3 is as defined, in a solv~nt ~uch
2Ç as THF, CH2C12 and the like at a temperature of from 0DC
27 to reflux for from 15 minutes to 16 hours. For example
~` 28 according to the following scheme:
-OTMS -OTMS esteri- -OTMS
Th -NHTMs N-acylatiOn ~ NRlTM5 fication~ Th- -NRlTMS
COOTMS COO~MS CoOR3
_ - 3~ -
-15834IB
~83~q
. ~OH
1 mild hydrolysis~ Th- -N~Rl'
-COOR
2 wherein TMS is triorganosilyl such as trLmethylsilyl and all
3 other symbolism is as previously defined.
4 The amides of the present in~entio~ are most
conveniently prepared by reacting the acid anhydridetI
6 XLo~R =acyl) with ammonia or with the amine of choice,
7 e.g., the alkyl-, aialkyl-, aralkyl- or heterocyclic amines
8 listed above.
9 The above-recited schemes of esterification are
well known in the related bicyclic ~-lactam antibiotic art
11 and indeed in all of general ~rganic synthesis and it is
12 to be noted that there is no undue criticality of Ieac~ion
13 parameters in the preparation of the N-acylated and carboxyl -:
14 derivatiYes Ic useful as starting materials in the practice
of the present invention.
16 Starting materials la and ~e are conveniently
17 prepared by any of a variety of well-known e~terification .
18 or etherification reactions upon the secondary alcoholic
19 group of Id. Such procedures include:
~ -O~ -~R3
~h- _NRlR2 ) Th_ NRlR2 -~:
_COXR COXR
Id Ie
21 1.) For the preparation of ether embodLments
22 of the present invention, the.acid catalized reaction Of ~d
'`
_ ~9 _ :
.
.
1583~IB--
~VB3S7~7
1 with a diazoalkane such as diaz~methane, phenyldiazomethane,
2 diphenyldiazomethane and the like in an inert solvent such
3 as dioxane, tetrahydrofuran ~THF), halohydrocarbons such
4 as CH2C12, ethylacetate and ~he like in the presence of a
catalytic amount of a strong acid or ~ewis acid such ~s
6 toluenesulfonic acid, trifluoroacetic acid, ~luoboric acid,
7 boron trifluoride and the like at a temperature of from
8 -78C to ~5~C for from a few minutes to 2 hours.
9 2.) For the ~reparation of ether embodiments of
the present invention, the reaction of Id with an alkylating
11 a~ent such as ~ctive halides, for example methyliodide,
12 benzylbromide, m-phenoxybenzylbromide and the like; alkyl- :
13 sulphonates such as dimethylsulphate, diethylsulphate,
14 methylfluorosulphonate and the like in the presence of ~
1~ strong base capable of forming the alcoholate anion of Ib.
16 Suitable bases include alkali and alkaline earth metal oxides
17 and hydroxi de, alkali metal alkoxides such as potassium,
1~ tertiarybutoxide, tertiary amines such as triethylamine,
19 alkali metal alkyls and aryls such as phenyllithium, and
20 alkali metal amides such as sodium amide. Suitable solvents
21 include any inert anhydrous solvent such as t-butanol,
22 dimethylformamide (DMF), THF, hexamethylphosphoramide ~HMPA)
23 dioxane and the like at a temperature of from -78C to
24 25C., for from a few minutes to 4 hours.
3.) For the preparation of ester embodiments,
26 of the present invention, the reaction of Id with any of
27 the above-listed acyl radicals in their acid form. This
28 reaction may be conducted in the presence of a carbodiimide
29 conGensing agent such a.s dicyclohexylcarbodiimide or the
- 30 like. Suitable solvents include any inert solvent such as
.
~ 41 ~
.
1~83~IB
~83S~
CHC13, CH2Cl~, DM~`, HMPA, acetone, dioY.ane and the like
2 at a temperature o~ fro~ 0C to 6~C. for from 15 minutes
3 to 12 hours.
4 4 . ) ~r the preparation of ester emb~diments of
the present invention, the reac~ion of Ia with an acyl
6 halide or an acid anhydride, wherein the acyl moiety is :.
7 described above. Generally, when the above-described
8 acylating reaction employs an acid halide (suitable halides
9 are chloro, i~do, or bromo or acid anhydride) the reaction is
conducted in an anhydrous organic solvent such as acetone,
11 dioxane, methylenechloride chloroform, DMF, or the like in
12 the presence of a suitable acceptor base such as NaHC03, MgO,
13 triethylamine, pyridine, and the like at a temperature ; :
14 of ~rom 0C. to 40~C for from 1 to 4 hours.
15 Suitable acyl halides and anhydrides include: ;
16 acetic anhydride, bromoacetic anhydride, propionic anhy- ~ :
17 dride, benzoylchloride, phenylacetyl, chloride azidoace- :~
18 tyl chloride, 2-thienylace~yl chloride, 2-, 3- and 4~ -
19 nicotinyl chloride, p-nitrobenzoyl ~chloride, 2,6-
dime~hoxybenzoyl chloride, 4-guanidinophenylacetyl chlDride,
~1 methanesulfonyl chloride, dibenzylphosphoro-
22 chloridate, dimethylthiophosphorochloridate, 2-furoyl
23 ethyl carbonic anhydride, methylchloroformate,
24 bis(p-ni~robenzy~)phosphorochloridate and the like.
5.) For the preparation of ester embodiments
26 of the present invention, ~he reaction of Id with a
27 suitably substituted ketene or isocyanate such as ketene,
28 dimethyl ~etene, methylisocyanate, methylisothiocyanatet
29 chlorosulfonyl isocyanate and the like. Suitable solvents
include dioxane, tetrahydrofuran, chloroform and the like
31 at a temperature of from -70C. to 60C. for from 15
32 ~ ute~ ~o lB hours. ~ 42 ~
.
.... , ~: . . ,
15~3~IB
~.o83s7~7
1 The intermediateIe is then N-~eblocked as descsibed
2 above to provide starting materials,Ie tR1 and R2 =H) and la.
3 From le, la is prepared by debl~cking the carboxyl group:
l oR3 r oR3 -OR
~h- -NRlR2 t NH2
COX R Cox!R COOH
Ie Ie la
Starting material Ia is conveniently and
6 preferably obtained when X'is oxygen and R3 i5 a readily
7 removable carboxyl protecting or blocking group (see
8 above). Starting material la is prepared by deblocking
g according to any of a variety of well known procedures
which include hydrolysis and hydrogenation. When the
11 preferred carboxyl-blocking groups are employed (below),
12 the preferred aeblocking procedure is hydrogenation,
13 w~erein the intermediate species tIe) in a solvent
14 such as a lower alkanol, is hydrogenated in the presence
of a hydrogenation catalyst ~uch as palladi~m, platinum
16 or oxides thereo~.
17 In this connection, it is noted that suitable
18 'blocking groupsr~3 incl~de the sub-generic groups de~ined
19 above as aralkyl, haloalkyl, alkanoyloxyalkyl, alkoxy-
alkyl, alkenyl, substituted alkyl, or aralkoxyalkyl, and
21 a1so lncluding alkylsilyl, wherein alkyl has 1-10 carbon
22 atoms. ~or example, suitable ~blocking groups"
23 include benzyl, phenacyl, p-nitrobenzyl~ methoxymethyl,
2~ trichloroethyl, trimethylsilyl, tributyltin, p-methoxybenzyl,
benzhydryl. These blocking group~ are preferred since they
26 are generally recognized easily-removable blocking groups
27 in cephalosp~rin and penicillin art.
_ 43 ~
:
l5834Is
~0~35~
l The preferred carboxyl blocking groups, are
2 benzyl and substituted benzyl:
R = - CH2 ~
R')n
3 wherein n is 0-2 (n= O, R'=H) and R' is loweralkoxyl or
4 nitro.
In the alternative it should be noted that the
6 compounds of the present invention, IIa, may be arrived
7 at by operating upon the substituted N-methylene thien-
8 amycin derivatives, II, to achieve derivatization by es-
g tablishment of R3 and/or -CoX'R3 . Such procedure is ex-
actly as described above except that species II Ieplaces
ll the above-described starting materials, such as Ia, Ic ;~
12 and Ie, and, of course, there is no need to ~-deblock.
- 49 -
.. . . .. . . . ... . . .. . . . .
` 158~4IB
3~
Preparation
2 The preparation of the compounds of the
3 present invention is conveniently described according to
4 the above-defined four classes, or embodiments, namely:
1.) Amidines; 2.) Guanidines; 3.) Substituted Pseudoureas;
6 and ~) Imido Esters, and Imido Thioesters.
7 1.) Amidines. In general, the compounds of
8 Class 1.) may conveniently be prepared by reacting thiena-
g mycin (I) or a derivative th~reof (Ia,Ic or Ie, when Rl =
R2 =H) or a suitably protected form of thienamycin such as
11 its silylated derivative (1) with an imido ester (a.) or a
12 substituted imido halide (b.):
13 OTMS
~ SC~I2CH2NEITMS
COOTMS
1 TMS = trimethylsilyl
NR [lR N=C-X' ~ X'~
a.) b.)
1~ wherein Rl, R , and R are as defined above; X' is halo
such as chloro; and -OR" is a leaving group wherein R" is
16 loweralkyl such as methyl, ethyl and the like. Alternatively,
17 the compounds of Class 1 may be prepared by reacting a com-
18 pound of Class 4. with NH3 oraprimary or secondary amino
19 compounds (c.) calcula~ed to provide the desired species of
Class 1. Reagent3 a., b., and c., are representafi~ely
21 enumerated below.
. .. ,: . , , - .
15834IB
35~q
1 Suitable solvents for the pxeparation of the
2 compounds of Class 1 according to the above reaction
3 schemes, depending upon the identity of the thienamycin
4 substIate a~d reagent, include water, dioxane, tetrahydro-
furan (THF), dimethylformamide (DMF), chloroform,
6 acetone, acetonitrile or mixtures thereof. The reaction
7 is conducted at a temperature of from 0~ to about 25C
8 for from 1 to about 6 hours. There is no criticality as
9 to the precise identity of the reaction solvent nor the
10 variables of reaction within the limits described above,
11 provided only that the reaction solvent is inert or
12 substantially inert to the intended course of reaction.
13 Suitable reagents representatively include:
14 a.) Imido Esters: R~2
N ~ N
R-C-~Rn , R-C-XR"
X = 0 or S
15 Methyl formimidate, ethyl formimidate, methyl acetimidate,
16 ethyl acetimidate, methyl benzimidate, ethyl 4-pyridyl
17 carboximidate, methyl phenylacetimidate, methyl 3-thienyl-
18 carboximidate, methyl azidoacetimidate, methyl chloro-
19 acetimidate, methyl cyclohexylcarboximidate, methyl 2-
20 urylcarboximidate, methyl p-nitrobenzimidate, methyl 2,4-
21 dimethoxybenzimidate, ethyl N=methyl formimidate, methyl N-
22 methyl formimidate, methyl N-isopropyl formimidate, and the
23 like.
24 Such imido ester reagent~ ~a.) are conveniently
25 prepared by any of a variety of known procedures, such as:
,
.
~ 46 ~
.~'' ' ' ' . , ,. ' . ' "' ' ' '
15B3~Is
~L0~3S~
1 1.) The reaction of a nitrile, RCN, wikh a
2 lower alkanol in the presence of HCl according to the
3 well-known Pinner synthesis.
2.) The reaction of a nitrile, RCN, with a
lower alkanol in the presence of a base. Typically, the
6 reaction is conducted at 0-40C in the presence of an
7 excess of the alcohol with a catalytic amount of an alkali
8 metal alkoxide for from 15 minutes to 4 hours.
9 3.) The reaction of an amide, RCNHRl,
with an alkylcnioroformate, sucn as metnylchloroformate
11 at 25G-45C for 1-4 hours.
12 O 4.) The reaction of an N-substituted amide,
~. . .. ...
13 RCNHR~ or RGNRlR~, with an eguivalen~ of an alkylating
14 agent such as triethyloxonium fluoroborate in an inert
601vent such as ether, chloroform or the like at 0-23C
16 for from 10 minutes to 2 hours.
17 5) The conversion of a readily available
18 imido ester, RÇNR' (R' may be hydrogen), to
OR"
~9 a desired imido ester, RCNRl , by reaction of the first-
OR"
20 mentioned with an alkylamine, R'NH2, in a mixture of water
21 and an immiscible solvent such as ether or chloroform at
22 0-23C for from 5 minutes to 1 hour.
, ~ .
- 47 ~
.
.' ', . :" :' , .
15~3~IB
~3B357~
1 b.) Substituted Imido Halides:
2 Chloropiperidino methyli~n chloride, chloro-
3 dimethylforminium chloride, chlorodiethyl forminium
chloride, and the like.
Such imido halide reagents (b.) are
6 conveniently prepared by any of a variety of known procedures,
7 such as:
B l.) The reaction of an N,N-disubstituted amide,
g R~NRlR2 , with a halogenating agent such as thionyl chloride,
phosgene, phosphorous pentachloride or the like in an inert
ll solvent such as chloroform, methylene chloride and the like
12 at 0-40C for from 1-5 hours.
I3 c.) Primary and Secondarv Amino Compoundst
14 Methylamine, ethylamine, 2-aminopyrimidine, dimethylamine,
methyl benzylamine, 3-aminomethyl pyridine, 2-aminomethyl
16 thiophene, ethanolamine, dimethylaminoethylamine, N-
17 2-~aminoethyl)pyrrolidine, cyclohexylamine, n-heptylamine,
18 isopropylamine, 2-methylallylamine, 3-phenyl-l-propylamine,
19 2-amino 4-picoline, 2-amino pyridine, 3-amino-4-carbethoxy-
pyrazolej 2-aminothiazole, 5-amino-3-methyl isothiazole,
21 and 3-amino-1,2,4-triazole.
22 The reaction involving the reagents (a.),
23 may be representatively shown bv the following diagram:
OR
~3~ 5CN2CN2NN2
~8
1 5 8 3 4 I B
35~
R'
~ .
I R-C-OR"
OR
2CH2N=C-NHR'
,~N~L COX ' R3
1 wherein OR~ is the leaving group of the imido ester
2 reagent and R, Rl, R3 , R3 and X' are as defined above.
This reaction is particularly suitable for embodiments
4 wherein R3 and R3 are hydrogen and X' is oxygen.
The reaction involving the reagents, (b.),
6 may representatively ~e shown by the follo~-ing diagram;
~=Cx, ~ Xl~ - oR3 H 1 2 ¦ -
SC 2C 2N=C-NR R ~ X~ ~ ~
- '":'~ ' .'
OH
mild hydrolysis~ ~ /~ 1 2
pH 3-6 ' ~ ~ ~ SCH~CH2N=C-NR R
COOH
2 -
7 wherein all sy~bolism is as previously defined. When
product 2 is d~esired, suitable values for ~3 and R3 are
9 erimethyls~lyl and .' is oxyyen.
:
:; , '
: .
' . '.
,~ g
15~34I~
~ 3S7~7
The reaction involving the reagents, ~c.), may
representatively be shown by the following diagram:
OR
-SCR2CR2N~I=C-X ~ 1 2
Compound of Class 4.
OR
,~ ~ SCR2CR2N::=CI NR R AO
O .
wherein all symbolism is as previously defined and X is -OR
or -SR wherein R is preferably lower alkyl such as methyl or
ethyl. When R3 and R3 are readily removable blocking or
protecting groups they may independently be removed by well :
known procedures to provide species ~, ~ and 5.
OH
2CH2N=~-NR R
O ~ COOH
', '.
,, oR3 :
SCH2CH2N=CI-NRlR
N COOH
OH
1 2 A~
N COXIR
: - 50 -
,
15~34IB
108357~7
2.) _anidines:
In general, the compounds of Class 2. may convenient-
ly be prepared by reacting thienamycin or an o- or carboxyl
derivative thereof (Ia, Ic or Ie, when Rl =R2 =H) with (a.) an
-OR" (e.g., O-alkyl, O-aryl) pseudourea or an S-alkyl or
S-aryl pseudothiourea; or (b.) by reacting a compound of
Class 3. (above) with ammonia or an amino compound such as an
alkyl, aralkyl or heteroaralkyl amine.
Suitable solvents for such reactions include water
and buffered aqueous polar organic solvent mixtures at pH 7-9
or anhydrous polar organic solvents such as dimethylformamide
or hexamethylphosphoramide at a temperature of from 0C. to
40C. for from 1 to 24 hours. -
Suitable reagents, (a.) and (b.), include: -
(a.) -OR pseudoureas and -SR pseudothioureas:
O-Methyl pseudourea, S-methylpseudothiourea,
S-methylpseudothionitrourea, 0-2,4-dichlorophenyl pseudourea,
S-p-nitrophenyl pseudothiourea, O-N,N-trimethylpseudourea, and
the like.
(b.) Amino reagents:
These reagents are the same as those given for the
preparation of Class 1, (c.), above.
The reaction involving the reagents, (a.), may
~ representatively be shown by the following diagram:
; IOR
~SCH2CH2NH2 1 " _
~, ~
/~8C~12C~12N~2
O
- 51 -
.. . .
'~~ 1583~IB
~83S7t7
1 wherein R3, X', R3 , Rl and R2 are as defined above; X"
2 is O or S and R" is as defined and preferably is lower alkyl
3 or aryl.
4 The reaction involving the reagents, (b.),
may representatively be shown by the following diagram:
Compound of Class 3.
OH
~\~ C3 NRlRZ
6 wherein all symbolism is as previously defined.
7 3.) Substi~uted Pseudoureas:
8 In general, the compounds of Class 3. may
g conveniently be prepared by reacting a carbamyl or thio-
carb~myl N-substi-uted Thienamycin species (a.), for
11 example. OIH
2 2 3
COOH
or
OIH S
2CH2NHCNHCH3
~ ~ COOH
O
12 with an alkylating agent (b.) such as an active alkyl
13 or aralkyl nalide or sulfate ester.
~2
.
..
15834IB
~01~57~7
1 Suitable solvents ~or the above reaction
2 include lower alkanols, dioxane and acetonitrile at a
3 temperature of from 20C. to 60C. for from 1 to 4 hours.
4 Suitable reagents (a.) for above reaction
scheme incl~ae N-acyl Thienamycins:
oR3
SCH2CH2NHR
~ ~ N ~ CoX'R3'
6 wherein R3, X' and R3 are as defined above and Rl is
7 acyl~as defined above and preferably is selected from
8 the group consisting of: -C-N~lR2 and - ~ RlR2 (Rl and R2
g are as defined above), such as: carbamyl, methylcarbamyl~
10 eth~lcarbamyl, phenylcarbamyl, p-bromophenylcarbamyl,
11 phenylthiocarbamyl, methylthiocarbamyl, dimethylcarbamyl,
12 and the like.
13 S~itable reagents (b.), alkylating agents,
14 include: methyl iodide, benzyl bromide, dimethylsulfate,
diethylsulfate, allyl bromide, 2-thienyl bromide, methallyl
16 bromide, p-nitrobenzyl bromide, methyl chloromethyl ether,
17 and the like.
18 The reaction involving the above reagents (a.)
19 and (b.) may representatively be shown by the following
20 diagram: ~R3
H2cH2NH-c-NR R
/
~ pR3 ~
/~SCH2CH2NH-~-NRlR2
X~R3 X R
~ 53 -
lS83~IB
357q
1 wherein X" is o or S; X is halogen such as bromo, iodo
2 or alkyl sulphate; R~ is the alkylating agent; and R1,
3 R , R3, X', R3 and R are as previously defined.
4 4.) Imido Esters and Im~ o Thio Esters:
In general, the compounds of Class 4. may
6 conveniently be prepared by reacting a suitable protected
7 N-acyl, N-thioacyl or N-alkoxy carbonyl derivative of
8 tilienamycin (a.) with an alkylating agent (b.).
9 Suitable solvents for tbe above reaction include
Io metnylene chloride, tetrahydrofuran, dioxane, chloroform,
11 and the like at a temperature of from -78C. ~o 25C.
12 for from 5 minutes to 3 hours.
.
13 Suitable N-acyl thienamycin starting materials
14 (a.) include: oR3
~f~LSCH2CH2NHRl
N CoX'R3
wherein Rl is acyl such as formyl, benzoyl, thiobenzoyl,
16 thioacetyl, and the like; R3, ~3 and X' are as defined.
17 Suitable alkylating agénts, (b.) include:
18 triethyl oxonium fluoroborate, methyl fluorosulphonate,
19 and trimethyloxonium hexafluorophosphate.
~0 ~he reaction involving the above reagents
21 (a. and b.) may representativeIy~be shown by the following
22 diagram:
` oR3 Xn
CH2CH2N~IC R
RX
15~3~Is
33S~
oR3
/~-- f ~ ScH2cH2NH=c-R
N cox~R3 X"R
1 4~7 deblocking
OH
F~ 0011
1 wherein X~ = O or S; and R3, X', R3 and R are as
2 previou91y defined. When the deblocked species is desired
3 suitable values for X' and R3, R3 are oxygen and trimethyl-
4 silyl; in which case, deblocking ls conveniently achieved
by mild aqueous hydrolysis of pH 3-6. It is to be
6 noted that the above eraction mixture may be used directly
7 in reaction with the amine (c.) as described in the
~ preparation of the amidines of Class L above.
/
-~ .
; ~ ... .
- 55
:., , .,. , . - . , :
. . . . . . .
15834~B
1~83S7~
~ e ~roducts of -tnis invention (II and IIa)
2 form a wide variety of phannacologically acceptable salts
3 such as acid addition salts, e.g., with hydrochloric,
4 hydro~romic, sulfuric, nitric, tol-~e~e-p-sulphonic and
methane sulpnonic acids. The salts of this invention are
6 ~harmacologically acceptable non-toxic derivatives which
7 can be used as the active ingredient in suitable unit-
8 dosage pharmaceutical forms. Also, they may ~e combined
9 with other drugs to provide compositions having a broad
spectrum of activity.
11 The novel compounds are valuable antibiotics
12 active against various gram-positive and gram-negative
13 bacteria and, accordingly, find utility i~ human and
14 veterinary medicine. Tile compounds of this invention can
therefore be used as antibacterial drugs for treating
16 infections caused by gram positive or gram-negative
17 bacteria, for example, against taphylococcus aureus,
18 Escherichia coli, Klebsiella pneumoniae, Serratia, Salmonella
19 typhosa, Pseudomonas and Bacterium proteus. The anti-
bacterials of the invention may further be utilized as
21 additives to animal feeding stuffs, for preserving food-
22 stuf~s and as disinfectants. For example, they may be
23 employed in a~ueous cor~lpositions in concentrations ranging
24 from 0.1 to 100 parts of antibiotic per million parts of
solution in order to destroy and inhibit the growth of
26 harmful bacteria on medical and dental e~uipment and as
27 bactericides in industrial applications, ~or example, in
28 waterbased paints and in tne white water of paper mills
29 to inhibit tne growth of harm~ul bacteria.
56
, . . .
. .
., .~ . , ,. ' ~
1583~Is
357f7
The products o~ this invention may be used alone or
in combination as an active ingredient in any one of a variety
of pharmaceutical preparations. These antibiotics and their
corresponding salts may be employed in capsule form or as
tablets, powders or liquid solutions or as suspensions or
elixirs. They may be administered orally, intravenously or
intramuscularly.
The compositions are preferably presented in a form
- suitable for absorption by the gastro-intestinal tract.
Tablets and capsules for oral administration may be in unit
dose presentation ~orm, and may contain conventional excipi-
ents such as binding agents, for example, syrup, acacia,
gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone;
fillers for example, lactose, sugar, maize-starch, calcium
phosphate, sorbitol or glycine; lubricants, for example,
magnesium stearate, talc, polyethylene glycol, silica; dis-
integrants, for example, potato starch or acceptable wetting
agents such as sodium lauryl sulphate. The tablets may be ~ `
coated according to methods well known in the art. Oral `
liquid preparations may be in the form of aqueous or oily
suspension, solution, emulsions, syrups, elixirs, etc. or may
be presented as a dry product, for reconstitution with water
or other suitable vehicles before use. Such liquid prepa-
rations may contain conventional additives such as suspending
agents, for example, sorbitol syrup, methyl cellulose,
glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxy- ;
~`~ methyl cellulose, aluminum stearate gel or hydrogenated edible
oils, ~or example almond oil, fractionated coconut oil, oily
.
:
- 57 -
. . - . . - . . - ,
~ . : - - .. , .. ,, :
15~3~s
35i7'7
esters, propylene glycol, or ethyl alcohol; preservatives, for
example methyl or propyl p-hydroxybenzoates or sorbic acid.
Suppositories will contain conventional suppository bases,
e.g. cocoa butter or other glyceride.
Compositions for injection may be presented in unit
dose form in ampules, or in multidose containers with an added
preservative. The compositions may take such forms as sus-
pensions, solutions, or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabi-
lizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for reconstitution with a
suitable vehicle, e.g. sterile, pyrogen-free water, before
use.
The compositions may also be prepared in suitable
forms for absorption through the mucous membranes of the nose
and throat or bronchial tissues and may conveniently take the
form of powder or liquid sprays or inhalants, lozenges, throat
paints, etc. For medication of the eyes or ears, the prepa~
rations may be presented as individual capsules, in liquid or
semi-solid form, or may be used as drops etc. Topical appli-
cations may be formulated in hydrophobic or hydrophilic bases
as ointments, creams, lotions, paints, powders, etc.
Alsa, in addition to a carrier, the instant
compositions may include other ingredients such as
stabilizers, binders, antioxidants, preservatives, lubri-
cators, suspending agents, viscosity agents or flavoring
agents and the like. In addition, there may also be
included in the composition other active ingredients to
,
- 58 -
.. . . . .
,
158341:B
~01 33S7~
1 provide a broader spectrum of antibiotic sctivity.
2 For veterinary medicine the composition may,
3 for example, be formulated as an intramammary preparation
4 in either long or quick-release bases.
The dosage to be administered depends to a
6 large extent upon the condition of the subject being
7 treated and the weight of the host, the route and frequency
8 of administration, the parenteral route being preferred for
9 generalized infections and the oral route for intestinal
infections. In general, a daily oral dosage consists of
ll from about 2 to about 600 mg. of active ingredient per kg.
12 of body weight of the subject in one or more applications
13 per day. A preferred daily dosage for adult humans lies
14 in the range of from about 15 to 150 mg. of active ingre-
dient per kg. of body weight.
16 The instant compositions may be administered in
17 several unit dosage forms as, for example, in solid or
18 liquid orally ingestible do~age form. The compositions per
l9 unit dosage, whether liquid or solid may contain from 0.1%
to 99% of active material, the preferred range being from
21 about 10-60~. The composition will generally contain
22 from about 15 mg. to about 1500 mg~ of the active ingredient;
23 however, in general, it i5 preferable to employ a dosage
24 amount in the range of from about lO0 mg. to lO00 mg. In
:- . .
~ 25 parenteral admini~tration the unit dosage is usually the
.
26 pure compound in a ~lightly acidified ~terile water solution
27 or in the form of a soluble powder intended for ~lution.
:
- S9 -
.. . : . .
1583~IB
~(~8~S7~7
1 The following Examples, illustrate but do not
2 limit the product, process, compositional or method of
3 trea.ment aspects of the present invention~ In the
4 Examples, the tnien~mycin nucleus (I, above) is represented
by the following symbol:
-OH
Th - -NH2
COOH
6 wllerein the secondary alcoholic group, the amino group
7 and tne carboxyl group are illustrated. Thus, compounds
8 of tne present invention can conveniently be represented
9 as follows: . _
Th ~-C-X and ~Th-- H=C-X
COOH OX ' R
II IIa
wherein X, Y, R , X', R an~ A are as previously defined.
: '
-6~ -
.. ,, . :
15834IB
~()83S~
1 EX~LE 1
~TMS
Th- -NHTr~ or TH(TMS)3
_COOTMS
TMS=trimethylsilyl
2 Preparation of Silylated-~hienamycin
3 Thienamycin (80.0 mg.) is suspended in 40 ml~
4 tetrahydrofuran (THF) ~nder a N2 atmospher~ ~n~ i~
concentrated to 10 ml.; hexamethyldisilazane (l.Oml.) and
6 trimethylchlorosilane (300 ~1) is added. The mixture
7 is reacted for 20 mins. at 25~C. with vigorous stirring.
~ 8 The suspension is then centrifuged to remove ammonium
- 9 chloride. The supernatant is evaporated to an oil under a ;--
nitrogen stream for future reaction.
.':
11 EXAMPLE la
'
Cl~
~ ~ Th ~ zC
Thienamycin _
12 Preparation of Thienamycin N-Piperidin-l-yl Methylene -
13 Derivative
14 Thienamycin (57 mg., 162 ~mol) is silylated
accordi~g to the procedure previously described. The
16 silylated antibiotic Th(TMS)3, 18 dissolved in methylene
17 chloride (6 cc) in a septum stoppered flask under positive
18 nitrogen pressure and cooled in a dry ice-acetone bath.
l9 To the magnetically stirred solution is added a solution
(180 ~1) of triethylamine (644 ~mol) in methylene chloride.
'.
~1
.
15834 IB
~L()8357~7
1 This is followed by the addition of a solution of
2 chloropiperidinomethylium chloride (67 mg., 405 ~mol)
` 3 in methylene chloride (465 ~1). After 1 hour in the dry
4 ice bath, the reaction solution is rapidly added to a
tetrahydrofuran - pH 7,0.1N phosphate buffer (1:1)
6 solution (50 ml.). The mixture is then concentrated under
7 vacuum to 10 ml. to give a homogeneous solution. The
8 solution is washed twice with ethyl acetate (2 x 5 ml.)
9 and ether (2 x 5 ml.) and briefly pumped under vacuum.
This aqueous solution is then chromatographed on an XAD-2
;11 resin column (60 ml. bed). The product is eluted i~ 10%
12 aqueous tetrahydrofuran (following water elution) to give
13 12.9 mg. (22%) product (as measured in solution assumlng
14 ~ 8,030 same as ~ïenamycin. Pape~ chrom~tography Rf 0.42
(4:1:5, n-BuOH:EtOH:water).
16 EXP~LE 2
. ~
~H H NH
H3C-O-C ~~ Th-
Thienamycin ) - O~
C 2
17 Preparation of N-~en~imi~yl ~hienamyci~
18 Thienamycin (59 mg., 212 ~mol) is dissolved in a
19 33% -~,N-dimethylformamide pH 7 phosphate buffer (~.05N)
solution (4.5 ml.) and adjusted to pH 9.5 using 2.5N
21 NaOH with an automatic dispensing burette. The solution is
22 magnetically stirred at 25C. and methylbenzimidate.HCl
23 (340 mg., 1981 ~mol) is added at once. After 30 min. the
24 solution is extracted twice with an equal volume of
chloroform and adjus~ed with dilute aqueous phosphoric acid
6~
- ~
,: ,
15 ~3~
~!L08357~
1 to pH 7Ø The buffered solution is chromatographed on
2 XAD-2 resin (65 ml.). The column is first eluted with
3 water followed by 10~ aqueous tetrahydrofuran which elutes
4 the product. This fraction is concentrated to one-half
volume and freeze-dried to give 50 mg. of the product.
6 Electrophoretic mobility (50 V/cm., 20 min., p~ 7
7 O.lN phosphate buffer) is 1.5 cm. towards the anode.
8 W ~ max = 300nm(~ 6,960) pH 7 O.lN phsophate buffer.
9 EXAMPLE 3
ThJ~NHC~ BrCH2~C (CH3) 3 ~ rh~N~C~
C2H l ~ (CH3~ Hl!3
Preparation of N-Benzimidoyl Thienamycin, p-tert-Butyl-
11 benzyl Ester
12 Benzimidoyl Thienamycin (3.2 mg.) is suspended
13 in hexamethylphosphoram~ae (75 ~ containing ~-tert-butyl-
14 benzyl bromide (3.8 ~L) and magnetically stirred at 22C.
After 45 minutes a solution results which is stirred an
16 additional hour. The product is then precipitated out of
17 solution with ether and the crude product chromatographed
18 on a 250 ~ thick silica gel plate developed in 7:3, chloro-
19 form ethanol.- The band at Rf 0.6 is removed and eluted
with ethanol to give N-benzimidoyl thienamycin, ~-tert-
21 butylbenzyl ester hydrobromide. Mass Spec. ~/~ 521
22 (M ), 487, 444, 418, 341, 323, 297, 226, 147.
.
~ 63 ~
:, . ... . ~ ,. ~ ,.. : .. . . . : .
15834 Is
57~7
1 EXAMæLE 4
H 0
Th ~ HC ~
02CH2~CH3
/= C~
H CH3 Br
_ l
2 Preparation of N-Benzimidoyl Thienamycin, 3-Methyl-2-
3 Buten~-l-yl Ester
4 Benzimidoyl thienamycin (5.9 mg.) is dissolved
in hexamethylphosphoramide (100 ~1.) containing l-bromo-
6 3-methyl-2-butene (4.8 ~l.) and triethylamine (0.5 ~1.)
7 and magnetically stirred at 22C. After l hour the crude
8 reaction is chromatographed on a 250 ~ thick silica gel
9 plate developed in 8:2, chloroform, ethanol. The band
~ lO of Rf 0.1 - Rf 0.3 is removed and eluted with ethanol.
; ll Benzimidoyl thienamycin, 3-methyl-2-buten-1-yl ester hydro-
12 bromide is isolated as a solid after precipitation from an
l3 ethanol-chloroforn ~olution with hexane.
;`
: 64
.' - ' . ' '. .' . - , ' ' ' ' ' , .
~5B34 IB
3S7~7
EXAMPLE 5
2 Preparation of N-Formimidoyl Thienamycin
OH
~ SCH2CH2N=C NH2
.; O
3 Thienarnycin (517 mg) is dissolved in pE'. 7
4 0.lN phcsphate buffer (25 ml) and cooled in an ice bath
with magnetic stirring. The solution is adjusted to pH
6 - 8.5 using 2.5N sodium hydroxide solution dispensed from an
7 automatic burette. While maintaining a pH of 8.5, methyl
8 formimidate hydrochloride (711 mg) is added portionwise
9 over a 2-3 minutes. After an additional 10 minutes, the
pEI of the solution i5 brought to 7.0 using 2.5N hydrochloric
- 11 acid. 'rhe solution is chromatographed on a column of
12 XAD-2 resin (150 cc) which is eluted tvith water. The
13 N-formimidoyl Thienamycin derivative elutes in 1.5-2.0
14 column volumes (200-300 cc) and is lyophilized to a white
solid (217 mg).
16 W (pH 7 0.1N phosphate buffer) )~max 297 nm ( 8,590)-
17 IR (Nujol mull) 1767 cm 1 (~-lactam)
18 nmr (D20)Jl.37 (d, J=6Hz, CH3-CH), 3.D - 3.75
19 (m, -CH2-), 4.2 - 4-8 (m~ CsH~ C6H' C7H)'
(s, -C-H)-
.
~,
15834:t~
-
~8357~
EXAMPLE 6
OH
Th- -N=C/ 2
Preparation of N-Guanyl Thienamycin
Thienamycin (8.9 mg.) is dissolved in pH 7 0.lN
phosphate buffer (0.7 ml.) and N,N-dimethylformamide (0.3 ml.)
and the solution brought to pH 9.5 by the addition of 2.5N
sodium hydroxide solution. To the magnetically solution is
; added O-methylisourea.hydrogen sulfate (43 mg.) causing a
slight drop in pH. Additional sodium hydroxide solution is
added to bring the pH back to 9.5 and the solution is stirred
30 minutes at 23C. ~he solution is then acidified to pH 7Ø
; A sample of the solution containing a mixture of thienamycin
and N-guanyl thienamycin shows two bioactive zones after
electrophoresis (50 V/cm., 20 minutes, 0.05N pH 7 phosphate
buffer) and bioautography on S. aureus plates.
- 66 -
J
,,,, ,, , . , . ~ .
.
` 15834Is
~.~83~7~7
EX~MPLE 7
C1
Thienamycin Cl~ ~NH2 r NH2
Cl ~Th- -N=C
~NH2
C02H
Preparation of N-Guanyl Thienamycin
, .
Thienamycin (11 mg.) is dissolved in pH 7 O.lN
phosphate buffer (1 ml.) and adjusted to pH 8.3 with O.lN
sodium hydroxide by means of an automatic dispensing burette.
To the magnetically stirred solution is added 0-2,4,5-tri-
chlorophenylisourea.hydrochloride (76 mg.) portionwise to
allow the auto burette to maintain a nearly constant pH. The
reaction is run 4 hours at 22C. and is then readjusted to
pH 7.0 by the addition of dilute acid. A sample of this
solution containing thienamycin and N-guanyl thienamycin is
electrophoresed (50 V/cm., 25 minutes, pH 7 O.lN phosphate
buffer) and shows a positive Sakaguchi spray zone at 2.0 cm.
towards the anode and a positive ninhydrin spray zone at 1.5
cm. in the same direction.
- 67 -
.i 1
'~?- `-`'
15834 IB
~V~;~S7~7
1 EXAMPLE 8
C~
Cl ~ ~ ~NC~ Cl ~ O-C ~
2 Pre~aration of 0-2,4,5-Trichlorophenylisourea Hydrochloride
3 A solution ofcyanami~ (0.28 mg.) in ether
4 (0.50 ml.) is mixed with 2,4,5-trichlorophenol (12.5 g.);
the mixture is heated to 70C. and the melt magnetically
6 stirred while the reaction flask is flushed wi~h nitrogen.
7 Dry hydrogen chloride gas is then slowly bubbled into the
8 melt and the reaction is allowed to cool to 22C. The
9 resulting solid is washed thoroughly with ether and filtered
to give 0-2,4,5-trichlorophenylisourea-hydrochloride as a
11 white solid, m.p. 205-206C.
12 EXAMPLE 9
Cl~
(CEI ) 2N=CHCl roH
Th(TM$)3 3 ) Th- - N= ~ 3
~ H
-CO~ '~
13 Preparation of N-Dimethylaminomethylene Thienamycin
14 Thienamycin (16.5 mg.) is silylated with
hexamethyldisilazane (200 ~1.) and trimethylchlorosilane
16 (60 ~1.) in the usual manner. The silylated thienamycin
17 is suspended in (ethanol free) chloroform (1 ml.) with
18 magnetic stirring under a nitrogen atmosphere. The
19 mixture is cooled to -45C. and a solution of triethylamine
(21 ~1.) in chloroform (21 ~1.) is added followed by a
_l solution of (chloromethylene)-dimethylammonium chloride
22 (11.5 mgOI in chlor9form (50 ~1.). The mixture is warmed to
68
.
15834 IB
S7~7
1 -25C. over l hour and O.lN pH 7 phosphate buffer (5 ml.)
2 is added. The mixture is vigorously stirred 15 minutes.
3 The aqueous phase is separated and contains N-dimethylamino-
4 methylene thienamycin which has an electrophoretic mobil~ty
(50 V/cm., 1 hour, pH 7 buffer) of 3.6 cm. towards the
6 cathode-
7 EXAMPLE 10
Th ~ NHC ~rcH2a~c(c~3)3 Th ~ NHC~
C2 ~-O-CH2O~C(CH3)3
8 Preparation of N-Formimidoyl Thienamycin Pivaloxymethyl
9 Ester Hydrobromide
N-aminomethylene thienamycin (10 mg.) is
11 dissolved in hexamethylphosphoramide (200 ~l.) containing
12 bromomethyl pivalate (10 ~1.) and triethyla~ine (1 ~l.)
13 ana magnetically stirred at 22C. After 2 hours the
14 hexamethylphosphoramide solution is dissolved in 2 ml.
lS methylene chloride and the product precipitated with a 50%
16 hexane-ether ~olution. The precipitate is dissolved in
17 an a~ueous 10% tetrahydrofuran solution and chromatographed
18 on an XAD-2 resin packed column. N-Formimidoyl
19 thienamycin pivaloxyme~hyl ester is isolated as a solid
after tetrahydro~uran elution o the column and
21 lyophilization.
.~ ~
.
- 69 ~
15834IB
357~7
EXAMPLE 11
Preparation of N-Trifluoroacetimidoyl Thienamycin
OH
~ SCH2CH2N=C NH2
3 Thienamycin (199 mg) is di~solved in pH 7
4 0.lN phosphate buffer (7 ml) and adjusted to pH 8.5 with
lN sodium hydroxide solution. While maintaining this pH
6 with an automatic burette, a solution of methyl trifluoro-
7 acetimidate (355 ~1) in dioxane (2.5 ml) is added at once.
8 After 30 minutes the pH is readjusted to 7.0 by the
g addition of lN hydrochloric acid. The solution is then
chromatographed on Dowex 50-X4 resin (200 cc, Na+ cycle,
11 200-400 mesh) and is eluted with water. The N-trifluoro-
12 acetimidoyl Thienamycin derivative elutes in the first half -
13 column volume. This eluate is rechromatographed in a
14 similar manner on Dowex 50-X4 (100cc~ Na+ cycle, 200-400
lS mesh) and the first column volume concentrated and
16 chromatographed on XAD-2 resin (30 cc). The N-trifluoro-
17 acetimidoyl Thienamycin derivative elutes in 2.5 - 5.0
18 column volumes which is lyophilized to a white solid ~15 mg).
19 W (pH 7 0.1N phosphate buffer) ~ max 302 nm (~ 4,450).
ir tNujol mull? 1750 cm~ -lactam).
21 Elec~rophoresis: (50 v/cm,20 min, p~ 7, 0.1N phosphate
22 bu~fer) mobility
23 2.0 cm (toward cathode)
,~
1583~ IB
3Sq~
1 XAMPLE 12
2 Preparation of N-Acetimidoyl Thienamycin
OH
/~ SCH2CH2N=C NH2
N COOH
3 Thienamycin (190 mg) is dissolved in pH 7
4 0.1N phosphate buffer (13 ml) and cooled in an ice bath
with magnetic stirring. The solution is adjusted to pH 8.5
6 using 2.5N sodium hydroxide solution dispensed from an
7 automatic burette. While maintaining a pH of 8.5,
8 ethyl acetimidate hydrochloride (400 mg) is added portionwise
g over a few minutes. After an additional 40 minutes the
solution is adjusted to pH 7.0 with 2.5N hydrochloric acid.
11 The solution is then chromatographed on Dowex 50-X8 resin
12 (250 cc, Na+ cycle, 100-200 mesh) and is eluted with water. -~
13 The N-acetimidoyl derivative elutes in 1-2 column volumes
14 (240-520 cc) and is lyophilized to a white ~olid ~88 mg).
W (pH 7 0.1N phosphat~ buffer)~ max 297 nm (~ 7,6
16 ir (NujoI mull) 1774 cm 1, ~-lactam.
NH
17 nmr (D2O) Jl . 27 (d, J=6 Hz, CH3-CH) 2.24 (S, -C-CH
18 3.2 - 3.5 (m; -CH2), 3,S - 3.9 (m, -CH2-)
19 4.2 -- 4.6 (m; C5N~ C6~1, C7~
:`
:
~ ,
` 71 _
~ . ', , ; :
1583~IB
~0~35i7~
1 ~XAMPLE 13
2 Preparation of N-[(4-~ridyl)(imino)methyl]thienamycin
OH
~ SCH2CH2N= I NH2
~ COOH ~ -
'
3 Thien2my_in (80 mg., 0.2~ mmole) is dissolved in
4 a~ueou~ sodium bicarbonate (24.7 mg., 0.294 mmole in 2.0 ml.)
at 25C. Methyl isonico inimidate t83 mg., 0.588 ~ole) is
6 dissolved in the solution and progress of the reaction is
7 rollowed by t~led aliquots using high performance liquid
8 chromatography (HPLC): Waters instrument; ~.2 x 61 cm.
g Cl~ Bondapak reverse phase column; l.S ml/m-n aqu~ous 10
T~F; W (254 nm.) and R.I~ monitors. The reaction is
ll essentially complete in 40 minutes, and the reactic~
2 801utio~ iS chromatographed directly over an 1~.4 x 270 Imm,
13 X~D resin column, first eluting with deionised, dist-lle~ :
14 watex, then changing to aqueous 10~ THF. The eluate is
monitored by W and HPLC is used to loc~te the pure product.
16 Correct fractions are combined and lyophylized to yield a
17 colorless, fluffy pow~er (80 mg. 73~)~ W ~ max ~98 nm
18 (~,800); IR (Nujol mull) 1762 cm~l (~-lactam); NMR
19 ~60 MHz, ~,,O),~ 1.27, 3H (d, J-7Hz, CH3-CH(OH)); ~7.75 and
8.30, 4~, (m, m, 4-pyri~yl3; EPLC, 1~5~ x re enti4n of
21 thienamycin r conditions aY akove.
-`; ' .
'
7 2
15~34 1~
3S~7
E XAl~ LE 14
2 Following the procedure of ~xample 13, but - -
3 replacing the reagent with methyl picolinimidate, ~here is
4 obtained: N-[(2-~y___yl)(imino)methyl]thienamycin (85 mg
77~) W 1 max 267, 300 nm (~, 8,150, 7,600); IR (Nujol
6 mull) 1764 cm 1 (~-lactam); NMR (60 MHz, D2O),~ 1.24,3H
7 (d, J=7Hz, CH3-CH(OH));~ 7.80, 8.07, 8.80, 4H, (m,m,m,
8 2-pyridyl); HPLC, 1.8 x retention of thi~namycin.
''
g - ExAMæLE 15
Following the procedure of Example 13, but
11 replacing the reagent with methyl nicotinimidate, there is
12 obtained: N-[(3-pyridyl(imino)methyl]thienamycin (77 mg.,
~ H20 ,.
13 70%): W ~max ~64, 299 nm, (~ 5570, 6120); IR, (Nujol
14 mull), 1766 cm~l (~-lactam); NMR, (60 MHz, D2O), ~ 1.24,
15 3H, (d, J=7Hz, CH3 CH(OH));J 7.6, 8.2, 8.9, 4H,
16 (m,m,m, 3-pyridyl); HPLC, 1.57 x retention of thienamycin.
17 EXAMPLE 16
18 Following the procedure of Example 13, but
19 replacing the reagent with methyl 4-thiazolecarboximidate,
there is obtained: N-[(4-thiazolyl imino)methyl]thienamycin
21 (99 mg, 89%): W~max 300 nm, (~ 7530); IR (Nujol Mull)
22 1764 cm 1 (~-lactam~; NMR (60 MHz~ D2O), J 1.23, 3H, (d,
23 J=7Hz, CH3-CH(OH)); ~ 8.60, 9.17, 2H (d,d, J=2Hz, 4-thiazolyl);
24 HPLC, 1.8 x retention time of thienamycin.
, , .
., ,~ ..
' .
~ . . . . .
15834IB
~0~33S7~7
1 EXAMPLE 17
2 Preparation of N-Allylformamide
3 A mixture of allylamine (5.00 g., 87.6 mmole) and
4 methylformate (5.26 g., 87.6 mmole) is stirred at
25C., for 2 hours. At the end of this time, the reaction
6 flask is fitted with a short path distillation head ~nd the
7 desired N-allylformamide is collected at 89-90C./0.7 mm as
8 a colorless oil~ Yield 7.0 g. (94%). IR(CHC13) 3380, 1680 cm
9 nmr (CHC13) ~ 8.1 (lH, br s), ~6.4-7.9 (lH, very ~road),
lo J 5-5-6-3 tlH, m), ~4.9-5.5 (2~,m), ~3.85 (2H, m).
11 EXAMæLE 18
12 Preparation of Ethyl Allylimidate ~ydrochloride
13 Ethyl chloroformate (2.66 g., 24.47 mmole~ is
14 added by syringe to N-allylformamide (2.08 g., 24.47 mmole)
15 in a dry flask under N2. The resulting mixture is then
16 stirred at 25C., for 2 hours during which time
17 CO2 is rapidly evolved, The reaction mixture is then heated
18 to 45C. until no further evolution of gas is evident (2 hours).
19 The viscous product is then cooled and held at a vacuum of
20 0.2 mm for 2 hours to remove all volatiles.
i4 ; ~. .
.
15834~B
~13357t7
EX~MPLE 1 q
2 Preparation of N' (2-Methylthioethyl) N-Formimidoyl ThienamYcin
~-~2CH2SCH3
Th - JN~C~
OOH
3 Thienamycin (105 mg.) is dissolved in pH 7 0.1N
4 phosphate buffer (5 ml.) and to this is added a solution of
S ethyl N-2-methylthioethyl formimidate (300 ~1) in tetra-
6 hydrofuran (2 ml.). The pH of the solution is adjusted to
7 and maintained at 8.5 using an autoburette dispensing lN
8 NaOH. After 30 minutes the pH is adjusted to 7.0 with 2.5N
9 ~Cl. The solution is chromatographed on an ice water jacketed
10 column of Dowex~50-X4 resin (53cc, Na~ cycle, 200-400 mesh)
11 eluted with deionized water. The N' [2-methylthioethyl~N-
12 formimido~l derivative elutes in 2-4 column volumes and is
13 lyophilized to give a white solid.
14 U.V. (pX 7 0.1N phosphate buffer) 7~max 298 nm (~ 7,760)
lS i.r. (Nujoi mull) 1760 cm 1 (~-lact _).
.
.~ .
'
15834IB
57~7
1 EXAMPLE 20
2 Preparation of N'-Tert-Butyl-N-Formimidoyl Thienamycin
. .
OH
N-C(C~3)3
Th - - N~ ~ :
COOEl
.
3 Thienamycin (105 mg.) is dissolved in pH 7 0.1N
4 pnosphate buffer (5 ml.) and to this is added a solution of
ethyl N-tert-butyl formimidate (290 mg.) in tetrahydrofuran
6 (1 ml.). The pH of the solution is adjusted to and main-
7 tained at 8.5 using an autoburette dispensing lN NaO~. ,, :--
~ After 30 minutes, the pH is adjusted to 7.0 with 2.5N HCl.
9 The solution is chromatographed on an ice water jacketed
10 column of Dowex 50-X4 resin (53 cc, Na cycle, 20~-400 mesh)
11 eluted with deionized water. The fractions containing the
12 title product are co~oined and lyophilized.
~ 76 ~
.. . .. . . .
.. . .
15834I~
~83~7'7
EXAMPLE 21
Preparation of N' ~-Methyl-2-Propeny ~N-Formimidoyl
Thienamycin
- OH
/ NH-CH(CH3)CH=CH2
Th- -N=C
\H
-COOH
Thienamycin (126 mg.) is dissolved in pH 7 0.lN
phosphate buffer (6 ml.) and the pH of the solution is
adjusted to 8.5 using an automatic burette dispensing lN NaOH.
To this stirred solution is added ethyl N-l-methyl 2-propenyl-
formimidate hydrochloride (300 ~1) while the pH is maintained
at 8.5. After 30 minutes, the pH of the solution is adjusted
to 7.0 with 2.5N HCl and the solution is chromatographed on an
ice water jacketed column of Dowex~50-X4 resin (49 cc, Na+
cycle 200-400 mesh) eluted with deionized water. The N'
~-methyl-2-propeny ~N-formimidoyl derivative elutes in 2-4
column volumes and is lyophilized to a white solid (59 mg.).
.V. (pH 7 0.lN phosphate buffer) wave length of maximum
absorption = 299 nm (absorptivity = 7,820) i.r. (Nujol mull)
1760 cm 1 (~-lactam).
.
.
( 15834Ig
3S~7
EXAMPLE 22
2 Preparation of N~ Buten-3-yl)Formalnide
.
3 A solution of 3.5 g. (0.05 mole) 3-amino-1-
4 butene in 12 ml. of methyl~ormate is kept at 25C. for
hours; the solution is then concentrated under reduced
6 pressure to remove excess methylformate~ The residual N-
7 (1-buten-3-yl)formamide is distilled under reduced pressure.
8 A fraction amounting to 3 g. (b.p. 58-60C./0.5 mm) of
9 N-(1-buten-3-yl)formamide is obtained.
EXAMæLE 23
11 Preparation of Ethyl-N-(l-suten-3-yl)Formimidate
12 A mixture of 1.O g. of N-(l-buten-3-yl)formamide
13 and one equivalent of ethylchloroformate is stirred under
14 N2 for 4 hours during which time C02 is evolved. The
solution is stirred under reduced pressure for 3 hours
16 to remove any unreacted ethylchloroformate, and a residue
17 of ethyl-N-(l-buten-3-yl)formimidate is obtained.
", . .
18 EXAMPLE 24
:
19 Preparation of Methyl N-Dimethylaminoformimidate
To a stirred solution of N,N-dimethylformhydrazide
21 ~0.22 g) in 2.0 ml of chloroform, under nitrogen, is
22 added methylchloroformate (0.5 ml). The mixture is heated
23 at 40Co for three hours then evaporated under nitrogen.
24 The mixutre is triturated with anhydrous ether. ~he
supernatant solution is decanted and the residue dried
26 in a stream of nitrogen.
27 Yield: 284 mg. nmr CDC13 J, 9.13(C~); 3.80(0CH3),
28 3.01(N(C~3)2).
, _ jg~ _
, . ;. , . , ., . :, ,
15834IB
3357~
1 EXAMPJ.E 25
2 Preparation of Cyclopropyl Formamide
3 A mixture of cyclopropylamine (5.00 g, 87.6 mmole~
4 and methylformate (5.26 g, 87.6 mmole) is stirred at
25C., for 2 hours. (an initial exotherm is noted).
6 The mixture is then placed on the rotary evaporator to
7 remove the MeOH formed in the reaction. The remaining
8 material is distilled through a short path head to yield
9 6.92 g (93~) of the desired N-cyclopropyl formamide as
- 10 a colorless oil, n.m.r. (CDC13) Lf8.1 (1~, br S),
11 6.8-8.5 (lH, br), ~ 2.4-3.0 (lH, m~, ~ 0.4-1.0 (4H,m).
13 EXAMp~E 26
14 Preparation of Ethyl N-Cyclopropyl-Formimidate
Ethylchloroformate (4.078 g, 37.58 mmole) is added
16 by syringe to N-cyclopropylformamide (3.194 g, 37.58
17 mmole) in a dry flask under N2. A~ter an induction period
18 o~ 30 sec.,a rapid evolution of gas begins. The resulting
19 reaction mixture is stirred at 25C until no further
evolution of gas ~an be detected (~4 hr), then the
21 viscous product is subjected to a vacuum of 0.5 mm for 1 hr
2~ to remove any unreacted ethyl chloroformate. NMR analysis
23 Of the product shows the formyl proton at ~ 9.37 as a broad
24 singlet- (CDC13 solution).
~-
_ j9 _
: . .. ... .... .
: . ,: " . .
1583~IB
~ 83S7~7
EXAMPLE 27
Preparation of Ethyl N(~ethylthioethyl)formimida~e
To a 60 ml separatory funnel are added ethyl
formimidate hydrochloride (0.97 g, 8.8 mmole) a solution of
~-methylthioethylamine (0.80 g, 8.8 mmole) in CH2C12 (35 ml),
and H2O (35 ml.). The mixture is shaken vigorously for 5
minutes. The CH2C12 layer is separated, washed with brine,
- dried with MgS~4, filtered, and evaporated under reduced
; pressure to give the crude imidate (0.59 g) as a hazy, pale,
- 10 yellow liquid.
i.r. 1660, 1230 cm 1.
EXAMPLE 28
Preparation of N'-Dimethylamino-N-Formimidoyl Thienamycin
OHN N / 3
ll H3
Th - - NHC~
COOH - - -
- Thienamycin (115 mg.) is dissolved in pH 7 0.1 N
phosphate buffer (7 ml.) and the pH of the solution is adjust-
ed to 8.5 using an automatic burette dispensing 1 N NaOH. To
this stirred solution is added methyl N-dimethylamino form-
imidate hydrochloride (284 mg.) while the pH is maintained at
2Q 8.5. After 20 minutes the pH of the solution is adjusted to
7.0 using 2.5 N HCl and the solution is chromatographed on
Dowex 50-X4 resin (53 cc, Na cycle, 200-400 mesh) eluted with
deionized water. The chromatography is carried out in a water
jacketed column at 3. The N'-dimethylamino-N-formimidoyl
derivative elutes in 2 column volumes and is lyophilized -to a
white solid (40 mg.) UV (pEI 7, 0.1 N phosphate buf~cr ~max
298 nm (~6, 910) ir (Nujol mull) 1760 cm 1 (~-lactam) nmr ~ -~
(D2O~ ~ 1.29 (d, J=6 Hz, CH3-CH), 2.59 (s, N (CH3)2), 7.76
(s, NCH).
- 80 -
:-- -. -- ~ -. ~ . . . ................. .
. .
15834IB
~3S7q
1 EXAMPLE 29
2 Pre~aration of Metnyl Oxalimidoyl Thienamycin
N~ NH
ll ll
Tn -NHC-C-OCH3
-COOH
3 Thienamycin (105 mg.) is dissolved in pH 7
4 0.1 N phosphate buffer (5 ml.) and the pH of the solution
is adjusted to 8.5 usin~ an automatic burette dispensing 1 N
6 NaOH. To this solution is added metnyl oxalimidate (200
7 ~1) while the pH is maintained at 8.5. After 30 minutes
8 the pH is adjusted to 7.0 using 2.5 N HCl and the solution
9 is chromatographed on Dowex~50-X4 resin (53 cc, Na~ cycle,
10 200-400 mesh) eluted with deionized water. The chromato~
11 grapny is carried out in water jacKeted column at 3.
12 The methyl oxalimidoyl derivative elutes in 2 column
13 volumes and is lyophilized to a white solid (44 mg.) uv
14 (pH 7 0.1 N phosphate buffer) ~ max 298 nm (~ 6, 230) ir
15 (Nujol mull) 1760 cm 1 (~-lactam); nmr (D2O) ~1.27 (d,
16 J=6 Hz, CH ~C~), 3.87 (s,~OCH3).
.' ' .
.
'~ . ' '
.' '
.~
81
1583~ IB
3~i7~7
.
1 EXAMPLE 30
2 Preparation of N-Propionimidoyl Thiena~ycin
-OH
N~
Th -NHC~
. ~H2CH3 -
COO~I
3 Thienamycin (114 mg.) is dissolved in pH 7 0.1 N
4 phosphate buffer (10 ml.) and the pH Or the solution is
5 adjusted to 8.5 using an automatic burette dispensing
6 l N NaOH. Solid ethyl propionimidate hydrochloride (231 mg.
7 is added portionwise as rapidly as possible allowing the
8 pH to be maintained near 8.5. After 30 minutes the pH is
g adjusted to 7.0 using 2.5 N HCl and the solution is
10 chromatographed on Dowex~50-X4 resin (72 cc, Na+ cycle,
ll 200-400 mesh) eluted with deionized water. The N-propionimidoyl
12 derivative elutes in 2 column volumes and is lyophilized to a
white solid (76 mg.). uv (pH 7 0.1 N phosphate buffer)
14~MaX 298 nm (E7,830) nmr (D20) ~ 1.28 (d, J= 6 Hz, CH3 C~(OH)),
15 1.23 (t, J= 8 Hz, -C82-C~3), 2.50 ~q, J= 8 ~z, C~2CH3).
:` :
,
.
- 82
~ - 15~3~ IB
~LV~357q
1 EX~PLE 31
2 Preparation of N'-Methyl-N-Formimidoyl Thienamycin
-OH
~-CH3
Th - -NH~
OOH
3 Thienamycin (140 mg.) is dissolved in pH 7 0.1 N
4 phosphate buf'er (10 ml.) and the p~ of the solution is
adjusted to 8.5 using an automatic burette dispensing 1 N NaOH.
6 To this solution is added methyl N-methyl formimidate
7 hydrochloride (200 ~1) while the pH is maintained at 8.5.
8 After 40 minutes the pH is adjusted to 7.0 using 2.5 N HCl
g and the solution is chro~atographed on Dowex 50-X4 resin
10 (72 cc, Na cycle, 200-400 mesh) eluted with deionized water.
11 The N'-methyl-N-formimidoyl derivative elutes in 2 column
12 volumes and is lyophilized to a white solid (43 mg.3.
13 uv (pH 7 0.1 N phosphate buffer) ~max 298 nm (~7,250) ir
14 (Nujol mull) 1765 cm 1 (~-lactam). nmr (D20) ~ 1.29 (d, J= -~
15 6 Hz, CH3-CH), 2.92 (8, N-CH3) 7.80 ~s, N-CH).
83
,
15834 IB
~35~7
B.~P LE~ 3 2
~ 2 Preparation of N'-Benzyl-N-Formimidoyl Thienamycin
~ . ~ . _
~ .
Tfi ~ ~C 2
OOH
3 Thien~mycin (110 mg.) is dissolved in pH 7 0.1 N
4 phosphate buffer (7 ml.) and the pH of the solution i9 adju~ted
to 8~5 using an automatic burette dispensing 1 N NaOH.
6 A solution of ethyl N-benzyl formimidate fluroborate (572 m~.)
7 in p-dioxane (2 ml.) is added to the buffered solution while
8 the pH is maintained at 8.5. After 20 minutes the pH of the
9 solution is adjusted to 7.0 using 2.5 N HCl and chromatogra2hed
10 on Dowex 50-X4 resin (53 cc, Na+ cycle, 200-400 mesh) ell~ted
11 with deionized water. The chromatography is carried out in
O : , .
12 a water jacketed column at 3 . The N'-benzyl-N-formimidoyl
13 deriva~ive elutes in 2 column volumes and is lyophilized to
14 a white solid ~5 mg.). uv (pH 7 0.1 N phosphate buffer)
15 ~max 295 nm (~3,980) ir (NUjol mull) 1765 cm 1 (~-lactam)
16 nmr (D20) ~ 1~29 (d, J= 6 Hz, C~3CH), 4.44 (s, CH2-Ar),
17 7.37 (s, Aryl), 8.14 (s, NCH). ~ -
.' .,:
.
~`
. ~
~ 84
~. ,
15834Is
~OB357~
EXAMPLE 33
Preparation of N'-Isopropyl-N-Formimidoyl Thienamycin
. _
- OH
Th- - N=C~ NH CH(cH3)2
COOH
Thienamycin (110 mg.) is dissolved in pH 7 0.1 N
phosphate buffer (7 ml.) and the pH of the solution is ad-
justed to 8.5 using an automatic burette dispensing 1 N NaOH.
A solution of methyl N-isopropyl formimida-te hydrochloride
(300 mg.) in p-dioxane (1 ml.) is added to the magnetically
; stirred buffered solution while the pH is maintained at 8.5.
After 25 minutes the pH of the solution is adjusted to 7.0
using 2.5 N NaOH and chromatographed on Dowex~50-X4 resin
(53 cc, Na~ cycle, 200-400 mesh) eluted with deionized water.
The chromatography is carried out in a water jacketed column
at 3 C. The N'-isopropyl-N-formimidoyl derivative elutes in
2 column volumes and is lyophilized to a white solid (12 mg.).
UV (pH 7 0.1N phosphate buffer) ~max 299 nm (~8,130) ir
(Nujol mull) 1760 cm 1 (~-lactam) nmr (D2O) ~1.26 (d, J=6Hz,
CH3CH(OH)), 1.29 (d, J=6Hz, CH(CH3)3), 7.89 (s, NHCH), 7.96
(s, NHCH).
- 85 -
~ .
. . ~ . . :
15~341B
~83S7~7
_XAMPLE 34
Preparat;on of N(N'-Allyl-Formimidoyl)T_ienamycin
r N~cH2cH-cH2
Th ~ NH-C
COOH
To a prechilled sample of Thienamycin (123 mg., 0.452
mmols) is added 13 ml. of cold 0.1 N phosphate buffer. The
solution is adjusted to pH 9 with lN sodium hydroxide. To this
basic solution at 2C. is added all at once ethyl N-allyl-
formimidate hydrochloride (0.3 9.). The pH dropped to 7.3 and : .
is brought back to 8.5 with additional sodium hydroxide. The
reaction mixture is stirred at 2C. for an ad.ditional 30 min.
and the pH is adjusted to 7 with cold 0.1 N sulfuric acid. The
reaction mixture is assayed using high pressure liquid chroma~
tography on a C18-Porosil column, developed with 10% aqueous
tetrahydrofuran and is found to show only trace a~ounts of
Thienamycin (retention time, 5 min.) and substantially pure
product (retention time, 10.5 min.). The reaction mixture is
chromatographed on a Dowex-50x4 column (60 ml., Na cycle 200-
4QO mesh) eluting with water at a flow rate of 0.5 ml/min/cm2
. of resin bed. After discarding the first 400 ml. of eluate,
: 20 the next 150 ml. is lyophilized to give the product. Yield 96
mg. (63%). U.V. ~max 301 nm, 24.6 optical density units/mg.
(NH20H extinguished) 90% purity. IR Nujol exhibits C=O at
. 5.67 ~ and 5.90 ~. NMR 100 MHz D20 shows its a 1:1 m;xture of
syn- and anti-N(N'-allyl-formimidoyl)Thienamycin.
.
- 86 -
... . . . .
:, . , ... . , :
... , .- ,
15834lB
335~
_XAMPIE 35
Preparation of N(N'-Trifluoroethyl-Formimidoyl)Thienamyc~n
- - OH
Nl-CH2CF3
Th - - NH-C~
COOH
To a prechilled sample of Thienamycin (123 mg., 0.452
mmoles) is added 15 ml. of cold O.lN phosphate buffer. The
solution is adjusted to pH 9 with lN sodium hydroxide. To this
basic solution at 0-2C. is added ethyl N-trifluoroethyl-
formimidate (0.3 ml.) in dioxane (2 ml.) portionwise over 30
` min. The pH of the reaction is maintained at 8.5-9 during the
addition. The reaction mixture is stirred for a few minutes.
After the addition of imidate is completed and the pH is
brought to 7 with cold O.lN H2S04 HPLC, C18 Porosil reverse
phase, using 10% aqueous-tetrahydrofuran exhibits a new peak at
12.2 min assayed to the desired product. The mixture is
chromatographed on a Dowex~50x4 column (60 ml 200-400 mesh).
The column is eluted with water at a flow rate of 0.5 ml/min/
cm of resin bed. The forerun is discarded and fractions con-
taining the product are combined and lyophilized to give a
hydroscopic solid, 10.2 mg. ~max 302 nm.
- 87 -
~ ~ .
.
... . . - .
\
15834IB
~33S7~
EXA~PLB 36
2 PsredPiaratsiln c~f N (N~ rboxymethyl-~Formimidoyl) T;~ienamycin
--OH
N--CEI2COOH
Th _ ~ HC ~H
OONa
3 Thienamycin (130 mg.) is dissolved in pH 7
4 0.lN phosphate buffer (4 ml.) and solid sodium ethyl N-
carboxymethyl formimidate (500 mg.) is added at once.
6 The pH of the solution is adjusted to 8~5 using an auto-
7 matic burette dispensing lN NaOH. After 25 min. at pH
8 8.5 the solution is adjusted to 7.0 with 2.5 N HCl. The
0 solution is then chromatographed on an ice-water jacketed
10 column of Dowex 50-x4 resin (51cc, Na~ cycle, 200-400 mesh)
11 eluted with deionized water. The eluate of the first col-
lZ umn volume was combined and concentrated to 7 ml. This
13 solution was then cnromatographed on an ice-water jacketed
14 column of XAD-2 re~in (53 cc) eluted with deionized water.
The second through fourth column volumes were collected
16 and combined and lyophilized to give sodium N,.~ carboxy-
17 methylformimidoyl)Thienamycin (25 mg). uv (pH 7 0.1N phos-
18 phate buffer)~ max 300 nm (~ 6, 390) ir (Nujol mull)
1755 cm (~-lactnm) nmr (D2O)d~1.29 (d, J=6Hz, CH3C~),
20 7.85 (s, NCH~.
.,
:;
. .
`8B-
~a34~s
~V83S7~
1 EXAMPLE 37
2 Preparation of N(3-azidopropionimidoyl)~hienamycin
{)HNH
Th -NHccH2cH2N3
-COOH
3 To a solution of Thienamycin (133 mg.) in 10 ml.
4 0.1~ pH 7.0 phosphate buffer is added 1.2 g. of O-ethyl-3-
azid~propionimidate HCl while the solution is maintained
6 at pH 8.5 with 2.5 N NaOH. The mixture is stirred at 0C.
7 for 0.5 hr., then is neutralized with 2.5N HCl to pH 7.0,
8 concentrated to 5 ml. and chromatographed on a Dowex 50W x 8
q (Na form) column (1.5" x 12") which is eluted with water to
give 30 mg. of the desired product. The product shows
11 w 2 300 nm; high pressure li~uid chromatography
12 (HPLC) retention time of 10 min. with comparison to that of
13 4.8 min. of the starting material under the same conditions
14 (1~8" x 2', Bondapak C18 reverse phase column eluted with
10% THF in water at flow rate of 1.5 ml./min.); Electro-
16 phoretic mobllity 5 mm toward cathode at 50V/CM for 20 min.
17 in 0.05M pH 7.0 phosphate buffer.
-~
~ 8~ ~
,, . , ~ . : , . . . :
: . ~ . : .: - :
~ 1583~IB
.
: ~o~3357~7
.
EXAMP E_38
Preyaratjon of N(3-aminoprop~onimidoyl)thienamycin
_ _ _ _
--OH - OH
NH NH
Th _ NHCCH2CH2N3 H2/Pd > Th----NHCCH2CH2NH
. --C 0 2(~N a~) C 2
` (I) (II)
N-(3-azidopropionimidozyl)thienamycin (I) (~3 mg in 40 ml
water) is hydrogenated under 1 atm of H2 in the presence of
0.19 Pd catalyst (10 percent palladium on charcoal) for 30 min.
Electrophoresis of the resulting mixture shows a new bio-active
product which moves 30 mm toward cathode (50V/CM for 20 min in
0.05 M pH 7.0 phosphate buffer) in addition to the starting
material (I) which moves 5 mm toward cathode. The electro-
` phoretic mobility of the product is consistent with that of the
expected product (II). The resulting reaction mixture from the
hydrogenation reaction is neutralized with 2.5 N HCl and
filtered from the catalyst. The filtrate is concentrated to 10
ml and chromatographed on XAD-2 resin (2.3 x 16 cm column).
; The column is eluted with water to provide the desired product
II as the hydrochloride after lyophilization (23 mg, N-(3-
aminopropionimidoyl)thienamycin hydrochloride).
U.V. Absorbance maximum in water equals 301 nm (absorptivity
equals 7080)i
I.R.: Nujol mull 1765 cm~l;
" NMR: 60 MHz, D20 ~ 1.30 ppm, (doublet, 3), ~ 2.60-3.72 ppm,
(multiplet, 11) and ~ 4.18 ppm, (multiplet, 2).
- 90 -
.
~ 15834IB
la~3s7~
1 BXAMPLE 39
2 Preparation of N-Nitroguanyl Thienamycin
3 Thienamycin (131 mg) is dissolved in a
4 solution of dimethyl sulfoxide ( 10 ml), tri-n-kutylamine
(0.30 ml), and 2-methyl-l-nitro-2-thiopseudourea(0~3g)- The
6 solution is heated in a water bath at 45C.while a stream
7 of nitrogen is vigorously bubbled into the solution7
8 After 50 min. the solution is concentrated under high
g vacuum to 1.0ml. and dissolved in 0.05N pH7 phosphate
10buffer (7 ml). The unreacted thiopseudourea is
llprecipitated and removed by filtration. The solution is
12then chromatographed on Dowex 50-X4 resin (53 cm3, 200 -
13400 mesh, Na cycle) and eluted with water. The N-nitroguanyl
14derivative elutes in the first column volume and is -
15lyophilized to a solid (23%).
16 W (pH7 0.1N phosphate buffer)~ max 269 nm (~ 000)
17Electrophoresis (40 v/cm, pH7 0.1N phosphate buffer,
18 20 min) 3.0 cm toward cathode.
19 EXAMPLE 40
Preparation of the N-Isobut~rimidoyl Thienamycin
21 Following the procedure of Example 12
22bUt replacing ethyl acetimidate hydrochloride with
23isobutyrimidate hydrochloride and allowing the reaction
24to proceed ~t 20GCand pH 8.2 there is obtained N-
251sobutyrimidoyl ~hienamycin (14%).
26 W (pH7 0.1~ phosphate buffer ) ~ max 298 nm (~ 8~290)
27NMR (D2O S 1.27 (d, J=7Hz, CH(CH3)2, 1.29
27 (d, J=6Hz, CH3CH(OH) , 2.79 (heptet, J=7Hz, CH(CH3)2).
..:
.
-- 91 --
.. . .. . . .
15834ïB
it3S7~7
:
1 EXAMPLE 41
2 Preparation of N'-Methyl-N-Acetimidoy__Thienamycin
3 Following the procedure of Example 12, but
4 replacing ethyl acetimidate hydrochloride with Methyl-
5 N-methyl acetimidate, there is obtained N-methyl-N'-
6 acetimidoyl thienamycin (10%).
7 W ~pH7 0.1N phosphate buffer)~ max 298nm (~ 6,700)
8 IR (Nujol mull) 1750 cm 1 (~-lactam), 1660 cm 1
9 ( C=NCH3)
10 NMR (D2O) ~1.27 (d, J=6Hz, CH3CH(OH), 2.22 and 2.25
11 (S, N-CCH3), 2.97 (S, NCH3)
. '.
12 EXAMPLE 42
~` 13 Preparation of N'-Methyl-N-Formimidoyl Thienamycin
i 14 Following the procedure of Example 12,
15 but replacing ethyl acetimidate hydrochloride with ~-
16 ethyl N-methyl formimidate hydrochloride there is
17 obtained N'-methyl-N-formimidoyl thienamycin (10~).
18 W (pH7 0.lN phosphate buffer) ~ max 298 nm
19 NMR(D2O) ~ 1.30 (d, J-6Hz, CH3 CH(OH), 2.92 (S, N-CH3),
7.78 (S, -CH)
.- :
21 EXAMPLE 43
: 22 Following the procedure of Example 12, but
23 replacing the reagent with an e~uivalent arnount of metnyl
24 methoxyacetimidate, there is obtained: N(methoxyacet-
25 imidoyl)thienamycin ~34~);
- 92
. . .
15834Ig
33S~7
H20
UV l max 198, 301 nm ~ ,180, 8,700);
IR (Nujol mull) 1760 cm 1 (~-lactam);
NMR (60 MHz, D2O) ~ 1.28, 3H, ld, J=6Hz, CH3 CH(OH);
3.50, 3H, (S, CH3-O-CH2) ~ 4.35, 2H, (S, CH3-O CH2);
HPLC, 1.50 x retention time of thienamycin.
EXAMPLE 44
Preparation of Ethyl N-Methoxyformimidate
A mixture of methoxyamine hydrochloride
(0.020 mole, 1.6700 g) and anhydrous potassium carbonate
(0.010 mole, 1.3821 g) is dissolved in 7.0 ml water.
Ether 80 ml is added and the reaction mixture is treated
with ethyl formimidate hydrochloride (0.02 mole, 2.1900
-~` g). The mixture is shaken for 15 minutes. The ether
layer is separated and the aqueous layer is extracted with
two portions of ether (30 ml). The combined and dried
ether solution is evaporated to give 0.8433 g of ethyl
N-methoxyformimidate.
nmr ~ 1.36 (triplet)
3.83 (singlet)
4.13 (quartet)
6.56 (~inglet)
'
. ~ .
.
~ 93 -
~083S7t7
15834IB
1 EXAMPLE 45
2 Ethyl N-(2,2,2-trifluoroethyl)formimidate
3 Ethyl formimidate hydrochloride (0.555 g,
4 5 mmole), 2,2,2 ~rifluoroe~hylamine hydrochloride
(0.677 g, 5 mmole) and potassium carbonate (0.345 g,
6 2,5 mmole) are suspended in 20 ml CH2C12 and treated
7 with 2 ml H2O. The mixture is shaken vigorously for
8 3 minutes. The organic phase is separated and the
9 aqueous extracted twice with 10 ml portions of CH2C12
; 10 The combined organic phase is dried and the CH2C12
11 distilled through a Vigreauxe column to give the Ethyl
12 N-(2,2,2-~trifluoroethyl)formimidate. n.m.r. ~ 1.33 t -~
13 (CH3CH2); 3.8 ~ (j = 10 c.p.s., CF3 CH2) 4.23 q (j = 7.5,
14 CH3CH2O); 7.6 S (H-C=N).
-
15 ~ EXAMPLE 46
. . . _
~ 16 Preparation of Ethyl N-ethoxycarbonylethyl-formimidate
.
17 Ethyl formimidate hydrochloride (0.55 g, 5 mmole)
18 ethyl glycinate hydrochloride (0.697 g, 5 mmole~ and
19 potassium carbonate (0.345 g, 2.5 mmole) are suspended in
20 20 ml CH2C12 and txeated with 2 ml H2O. The mixture is
21 shaken vigorously for 4 minutes. The organic phase is
22 separated, the aqueous phase is extracted twice with
23 CH2C12 (10 ml) and the combined organic phase is dried
24 and evaporated to give Ethyl N-ethoxycarbonylmethyl-formi- ;
25 midate. n.m.r. ~ : 1.26 t (CH2-CH2); 4.06 S (N-CH2-Cl;
26 4-23 g (CH3CH2-O); 7.5 S (N=CH).
.~ ,
,,
~ 94
, ~ ~, -, .... .
15834IB
357t7
1 EXAMPLE 47
2 Preparation of Potassium N-ethoxycarbonylmethyl-formi-
3 midate
5 ~ KOEt, -/=N-CH2-C~OK
6 C=N-CH2-COOEt Et2O, Et ~ EtO
7 EtO
8 Potassium t0.18 g) is dissolved in a mixture
9 of 0.6 g EtOH and 4 ml B~2O under N2. The solution is
diluted with 50 ml of Et2O and ethyl N-ethoxycarbonyl-
11 methyl-formimidate (0.79 g) in 2 ml Et20 is added, follow-
12 ed by 0.1 ml ~2 Rapid crystallization of the sal~
13 takes place. The solid is filtered, washed with ether
14 and dried under vacuum to give Potassium N-ethoxycarbonyl-
lS ~methylformimidate. n.m.rO (D2O) 1.13 t (C~3CH2);
16 3.63 g tC~3_CH2O); 3.8 S (N-CH2-C); 8.06 S N=CH. ~-
17 EXAMPLE
18 Preparation of Ethyl N-Benzylformimidate
19 A solution of 690 mg (5.1 mmoles) of N-benzyl-
formamide in 5 ml of methylene chloride is cooled in an
21 ice-water bath and put under an argon bla~ket. The
22 solution is stirred while 4.9 m~ t4.9 mmoles) of lM
23 triethyloxonium fluoroborate in methylene chloride is
24 added dropwise. After a 45 minute reaction time, the
mixture is concentrated to dryness under reduced pre~sure
; 26 at room temperature, and the residue i5 dried under
27 reduced pressure over P2O5. The nuclear magnetic re-
28 sonance spectrum of the product in deuterochloroform is
29 fully in acaord with the product being a fluoroborate
etherate complex of ethyl ~-benzylformimidate.
. .
- . .
~8357~ 15834IB
~ 1 EXAM~'LE 4g
1 2 Preparat N-isopropyl formamide
Formamide (1.13 g, 0.98 ml) is dissolved in
`~ 10 ml of toluene, containing toluenesulfonic acid
(4.7 g). To the above mixture is added isopropylamine
6 (2.95 g,,4.25 ml). The mixtuxe is refluxed overnight
7 under a gentle stream of N2. The solution is filtered
8 and the toluene is evaporated under reduced pressure.
9 The residual oil is distilled at 59-62C/0.07 mm to
give 1.0 g of the desired product.
11 EXA~LE 50 -.
12 Pre~aration o~ Methy~ isoproEyl formimidate
Isopropyl formamide (535 mg) is treated with ''~
14 an e~uivalent amount of ethyl chloroformate !440 ~
for 2-3 hours under N2 at 40-45C. The mixture is washed
16 successively with petroleum ether anhydrous ether and
17 benzene leaving the product as an oil.
, ,
,
.
. ~
96 _
.~ -: . ~,' ', '- -
sa3s.
~0~3S7t7
1 EXAMPI,E 51
2 Preparation of N-[N'-Ethy~ormimldoyl~thienamyCin
3 ____OH
4 Th - NHC-H
C02H
7 Thienamycin (100 mg) in 10 ml O.lM pH 7.0 ,
8 phosphate bu~fer is adjusted and maintained at pH 8.5-
9 9.0 with 2.5 N sodium hydroxide. To the solution is
added 300 mg. of ethyl N-ethylformimidate hyarochloride.
11 The mixture is stirred at 23C for 20 minutes, then is
12 neutralized to-pH 7.0 with 2.5 N HCl and chromatographed
13 on a Dowex 50-X8 (Na form) ion-exchange column (1.5"
14 X 10"). The column is eluted with water taking 6.7
ml fractions. Fractions 40-90 are combined, concentrated
16 and freeze-dried ~o give 15 mg of the solid product. ;
17 Electrophoresis of the product at 50V/cM for 20 minutes
18 in 0.1 M, pH 7.0 phosphate buffer shows a single bioactive
H O
19 zone which moves 2 mm toward the cathode.uv,~ max 301 nm;
nmr (100 MH2, D2O);~7.77 (S) and 7.82 (S) (formimidoyl CH).
-.
'
' ` :
-:
.
357~ 15834Ig
' ,
1 EXAMPLE 52
Preparation of N-[N'-cyclopropylformimidoYl]thienamYcin
4 r N ~
I~ tNHC
6 COoH
7 Thienamycin (100 mg) in 10 ml 0.1 M, pH 7.0
8 phosphate buffer is adjusted and maintained at pH 8.5 - 9.0
9 while 300 mg of ethyl N-cyclopropylformimidate hydrochloride
is added dropwise to the solution. The mixture is stirred
11 at 23 for 40 minutes, then is neutralized, and chroma-
12 tographed on a Dowex - 50 X 8 (Na form) ion-exchange
13 column (1.5"X 10"). The column is eluted with water,
14 collecting 6.5 ml fractions. Fractions 43-95 are combinedr
concentrated and freeze-dried to give 54 mg of the solid
16 product. Electrophoresis of the product shows a single
17 bio-active zone which moves 10 mm toward the cathode
18 (50 V/CM, 1 hour in 0.05 M pH 7.0 phosphate buffer).
19 uv ~ 301 nm; Nmr (100 MHz, D2O): 0.60 - 1.30 ppm
(m, cyclopropyl) a~d 7.80 ppm (~ormimidoyl CH).
:. :
: .
` ~ 98
. . : .:: , . -
15834I~
~083S7~7
1 EXAMPLE 53
2 Following the procedure set forth in the
3 foregoing text and examples, the following compounds
4 of the present invention are obtained. The reagents,
S imido ethers and imido halides, utilized in the reaction
6 with thienamycin, or a derivative thereof, to provide ~ :~
7 the following compounds are either known, or may be
8 prepared as described above.
~CR2 CE~ 21:=C~
~` '
Com- ~ ~
9 pound R Rl R2
lO l.) H -CH2CH2CH2CH
ll 2.) ~ ~ 3 H
H3
; 12 3.) H -CHCH2CH3
CH3
13 4~) H -CH2CH2CH2CH2CH3 H
.
14 5.) H -~H-CH2CH2CH3 H
'
''
99
... . . .
.. , , . ; .
.
1583~B
~83S~q
com-
1 pound R Rl 2
_ R
2 6.) CH3 2 3 H
3 7.) H -CH ---ÇH - CH H
CH3 CH3
4 8.) H ÇH3 H
CH3
5 9~) H -cH2-c(~H3)3
6 10.) H ~cH2cH2cH2cH2cHcH3 H
7 11.) H - $ -cH2cH(cH3)2 H
12 . ) 2 , 2 H
CH3
9 13.) H --CH2-CH=CH-CH3
10 14.) H -cH-cH=cH2 H
11 15. ) H --CH2CH2CH=CH2 H
12 16.) H -CH2~H2-CH CH-CH3 H
13 17 . ~ H --CH2-CH2--C~H--CH2 H
CH3
14 18.) ~ -CH2-CH-CH=cH
: CH3
15 19. ) , 2 H
CH3 CH3
16 20.~ ~I -CH-CH:=CH2 H
C2H5
-- 100
15834IB
~35i7
com~
2 pound R Rl R _ .
3 21.) H -cH2-cH=cH-cH2cH2cH3 H
4 22. ) H -cH2-cH2-c~=cH-cH2cH3 H
5 23.) H - CH - CH=CH - CH2CH3
CH3
7 24.) , 2 z C~ C~2-C33 ~3
H -CHCH2-CH CH =CH H
11 26.) H ~ H
12 27.) H ~ H
13 28.) H ~ H
14 29. ) H ~ H
15 30.) H -CH2- ~ H
16 31. ) H --CH2~ H
17 32. ) H --CH24~ H
18 33 . ) H --CH2~3 H
19 34 . ) H --CH2~ H
20 35.) H --CH2~ H
21 36.) H -CH2 CH~O H
101 --
:' ' . -, ', ' " ' , ., `. ': ` .
1583~IB
~ 83S7~
1 Com-
2 pound R Rl R2
3 37.) H O H
4 38.) ~ ~ H
39-) H O H
6 40. ) H --CH2~ H
7 41-) H ~ H
8 42.) H --CH2~ H
9 43 . ) H --CH2{~) B
44 ) H --CH2CH2--O H
11 45. ) H l-adamantyl
12 4 6 . ) H --CH2--O H
13 4 7 . ) H ~ H
14 ~8.) H ~ H
CH
4 9 . ) H ~OCH3 H
16 50. ~ H ~NH2 H
17 51. ) H ~ Cl H
18 52. ) H --CH2CH2N~D H
: ~,
.~ -- lo~ --
.
. ... . ... : -, .
1583~
~ 35~
1 Com-
2 pound R . Rl R2
3 53 . ) H CH2CH2~3 H
54.) 1~ H
CH3
55. ) H --CH2CH=CH~) H
6 56. ) H --CH ~) H
\~ ' :
7 57 . ) H --CH~OH H
8 58 . ) H --CH~SCH3 H
9 59. ) H -CH~3N- (CH3~ 2 H
.
10 60D) H -C~:l X , - -
CE~3
11 61. ) H --C2~5 2 5
12 62.) H --CH~CH3)2 CH(CH3)2
13 63 . ) H --CH2CH2CEI3 --CH2CH2CH
14 64 . ) . H --CH3 C2~5
15 65. ) }1 --~13 --CH (CE~3) 2
16 66.) H --CH3 --CH2CH=CH2
17 67 . ) H --CH2CH'CH2 --CH2CH=CH2
18 68 . ) H --C'~CH3) 3 C~3
.
.
-- 1 0 3
~. . ....
15B3~IB
S7~
.
Com- 1
1 Pound R R R2
2 69.) H -CH3 -CH-C2H5
CH3
3 70.) H -C2H5 -CH(CH3)2
4 71.) H -CH3 -CHCH=CH2
. CH3 .,
72.) H -CH3 -CH2-CH(CH3)2
6 73.) H -CH3 -CH2CH2CH3
7 74.) H -CH3 -CH20
8 75-) 3 0
9 76.) H -CH3 H2
10 77.) H -CH3 CH3
11 78.) C~3
12 79.) H -CH3 2
13 80.) H -CH3 -CH
14 81.) -CH3 C2H5 H
15 82.) -CH3 -CH2CH=CH2
16 83,) -CH3 -CH(CH3)2 H
17 8~,~ CH=CH2 -CH3 H
.
- 104 ~
15834IB
357~7
Com 1 2
1 pound R R _ R
2 85.) -CH=CH2 C2H5
3 86.) -C~=CH2 CH(CH3)2
4 87.) -CH3 - C(CH3)3 H
5 88.) -CH3 -CH2 ~
6 89.) -CH3 ~ H
7 90.) -CH3 ~ H
,;, ,
8 91.) -CH3 -CH20 H
9 92.) -CH3 -CH2 ~ H
10 93 ) -CH3 -CHCH=CH2 H .
11 94 ) -CH3 -CH2-C-CH2 H
CH3
12 95 ) -CH3 -CH2-CH2CH3 H
GH3
13 96.) -CN=CH2 H H
1497 ) CH3 CH2
1598O) CH3 ~ H
1699.) -CH3 ~ H
'
17100.) -CH3 H~ I H
-CH ~ )
- 105
. , , ~ , ..
.
1583~IB
~08357~7
- 1 com- -
2 pound R _ . R _ R
3 101.) CH3 CH3 CH3
4 102-) CH3 CH3 C2H5
5 103.) CH3 CH3 ~CH3)2
6 104.) CH3 CH3 CH2CH=CH2
7 105.) CH3 C2H5 C2H5
8 106.) CH3 CH3 -CH2~CH=CH2
CH3
107.) CH3 CH3 CH2
11 108.) CH3 -CH2CH=CH3 ~
. : , . '
12 109.) CH3 C2H$ -
13 110,) CH3 CH3 CH2
S~/N
14 111.) CDOH ~ H
112.) ~ ~ ~ H -
16 113.) H ~ H
17 114.) H ~ H
18 115.) H H
~: '
.
_ 106 -
.~ . -- ' . ... :
:- ~ .,
.. ~, ,
15834IB
~o~3~7~7
Com- 1 2
2 pound R R _ R -
3 116-) ~TOCH3 ~ H
~ ,, .
4 117. ) H _~3 H
5 118.) ~ H H
6 119.) H ~ H : .
H
7 1 0 . ) H ~ CH3 H
H
~ 1 ~.
3 121. ) ~
9 122.~ ;3 H
:~ CH3
.- . .. .
123. ) ~ H
Q
11 C~3
12 124 . ) N ~ H H
13 CH3
~ -:
-- 107 --
;, . ... .. ~ ',
15834IB
10~357~7
Com- 1 2
Pound R R R
2 125.) H --~S> H
3 126.) H ~
4 127.) H ~3 H
/~ '
128.) H ~ H
6 129. ) ~ / H H
H
7 130 . ) H ~ H
8 131. )H ~ H
' , N'C~3
i`: 9 132.) ~ H H
10 1~3,) H --CH2 ~j7 H
11 134.) H ~N~ H
-
H3
12 135 . ) H N- CE13 H
--CH2 ~ ~
13136. j H N H
-C~
- 14 137.) El N H
: -CEI21~3
-- 10~ --
. ~ . , .
lSB3~1IB
~0~357~
Com- , 2
pound R Rl R
2 138 . ) H 7 =~ H
CH 2 ~,/
3 139. ) H H
CH2 ~3
:
4 140.~ N --CN
141.) H--CH2~ H :
O ''' ' "
6 142. ) H t H
`' ~5~ ,
7 143,) H --CH~3 H
.
8 144.) H--CH2--~N H
: 9 145.) H--CH2~ H
14 6 . ) ~ --CEI2--~ H
11 147. ) H--CH2~)
12 148. ) H-C~2-CEl2~ H
13 149.~ N--CN2_~3 N
~14 1500 ) ~CH 2 H H
- 109 -
.. , . -. ' ' . . . ' , , j~,,: ', , . '
,,
. . ~ . . ... . . .
1583'aIB
~33~7~
Com- l 2
pound R R R
2 151.)i~CH2_ H H
3 152. ) H --CH2-O-CH3 H
4 153 . ) C 2C 2 CH3 H
154 . ) CH3 CH2CH2 S CH3 H
155. ) H --CH2CH2--OH H
7 156. ) H --CH2CH--CH3 H
OC~I3
8 157 . ) H --CH2CH2--CH2--C--N H
9 158.) H CH2CH2~H--(OcH3)2 H
lO 159. ) 2 0 H
ll 160. ) H --CH--CH2CH3 H
C:H20H
12161. ) H -CH2CH2CONH2 H
13 162. ) H -CH2COOC2H5 H
14 163 . ) H --CCH2CH2CH2N (c2H5) 2
CEI3
15 164 . ) H --CH2CH2-SH H
16 165~) H CC,H ~3OH H
17 166.) H -CH2CH2 N(CH3)2 H
18 167 . ) K -CK2CH2CK2-8r K
-- 110 --
1583'~IB
357
com-
2 Pound R Rl R2
3 168 . ~ H --CH2CH2CH-N (CH3) 2 H
4 169. ) H -CH2CH2 N (CH3) 3 H
!i 17 0 . ) NEI2 CH3 H
6 171. ) NH2 CH3 CH3
7 17 2 . ) NHCH3 CH3 CH
8173 ~ ) N (CE~3 ) 2 CH3 CH3
9174 ' ) NH2 C2H5 H
10175. ) NH2 --CH ~CH3) 2 H
11176 . ) NH2 --CHCH=CH2 H
12177. ~ NHCH3 --CH (CH3) 2 H
1317 8 . ) NHCH3 CH3 ~ .
14179.) NH2 N(CH3)2 H
15180.) NH2 NEINH2 H :
16181. ) OC~13 H H
17}82 . ) OCH3 CH3 H
18183 . ) OC~3 CH3 ~3
19184 . ) OC~3 C2E~5 H
20185.) OCH3 CH(CH3)2 H
2118 6 . ) OCH3 H H
22187. ) SCH3 CH3 H
~ .
- 111 -
', ' '
15834IB
~1335i7~
Com- 1 2
Pound R R R
_
2 188.) SC~13 CH3 CH3
3 189.) SCH3 CH(CH3)2
4 190.) S-CH2--CH=CH2 CH3 H
191.) S-CH2-0 CH3 H
6 192.) --SCH2CH=CH2 H H
7 193.) SCH20 H H
8 194.) H --OH H
9 195.) H --OCH3 H
196.) CH3 --OH H
11 197.) H --C=N H
12 198.) H --NHCH3 H
13 199.) H NH2 H
14 200.) CH3 CH3 2
201.) H CH3 ( 3)2
16 202.) CH2Br H H
17 203.) --CH2N(CH3)2 H H : :
18 204.) --CH2~CH3 H H
i9 205.) --~-NH2 H H
206.) --CH2N(CH3)3 H H
21 207.) --C(CH3)3 H -C(CH3)3
22 208.) --ICHCH3 CH3 -~HCH3
CH3 H3
23 209-) -~HCH3 -ICHCH3 -CIHCH3
H3 H3 H3
24 210.) -C(CH3)3 -C(CH3)3 -C(CH3)3
.~ .
-- 112 --
,: , .
1~83~1B
F3357q
; ~R ~
S-CH2CH2-N=C-X A~ .
, ~ ~ 1I COOR~ Y
Com-
pound R R _ X Y A __
211.) H -CH2CHeC(CH3)2 NH2 H Cl
212.) H -CH ~ CH3 NH2 H Cl
213.) H -CH2--nc-c~c~3)3 NHCH3 H HS04
214.) 2 n ( 3)3 NH2 CH3 CH3COO
215.) H 2CH2 CH CH2 NHCH(CH3)2 H Cl
216.) H CH2CH2 S CH3 NHCH3 CH3 ~ .:
217.) H -CH2-0-C-CH3 NHCH3 CH3 Cl
O
218.) H CH2 nC 0 NH2 H Cl
O , ~, '
219.) H -5 indanyl N(CH3)2 H C1
220.) H -phthalidyl CH2CH=CH2 H Cl
-~ 221.) S03 Na N~2 H ~ : ~ :
~ 222.) P4H2-CH20CCH(CH3) NH2 CH3
O
223.) S03--CH2-CH=C~CH3)2 NHCH3 H
; 224.) P4~2Na NHCH(CH3)2 H
.` .
113 -
15~34I:E~
~B35~r7
EXAMPLE 54
Preparation of Pharmaceutical Compositions
One such unit dosage form consists in mlxing 120 mg.
of N-acetimidoyl Thienamycin (product of Example 12) with 20
mg. of lactose and 5 mg. of magnesium stearate and placing the
145 mg. mixture into a No. 3 gelatin capsule. Similarly, by
employing more of the active ingredient and less lactose,
other dosage forms can be put up in No. 3 gelatin capsules and
should it be necessary to mix more than 145 mg. of ingredients
together, larger capsules such as compressed tablets and pills
can also be prepared. The following examples are illustrative
of the preparation of pharmaceutical formulations:
TABLET PER TABLET
N-acetimidoyl
Thienamycin 125 mg.
Cornstarch, U.S.P. 6 mg.
Dicalcium Phosphate 192 mg.
Lactose, U.S.P. 190 mg.
The active ingredient is blended with the dicalcium -
phosphate, lactose and about half of the cornstarch. The
mixture is then granulated with a 15~ cornstarch paste (6 mg.)
and rough-screened. It is dried at 45C. and screened again
through No. 1~ screens. The balance of the cornstarch and the
magnesium stearate is added and the mixture is compressed into
tablets, approximately 0.5 inch in diameter each weighing
800 mg.
PARENTERAL SOLUTION
Ampou`le:
N-acetimidoyl
30Thienamycin 500 mg.
Sterile Water 2 ml.
114 -
,~ ~ ,;
,. .
,
1583~IB
~L~83S7t7
OPHTHALMIC SOLUTION
N-acetimidoyl Thienamycin 100 mg.
Hydroxypropylmethylcellulose 5 mg.
Sterile Water to1 ml.
OTIC SOLUTION
N-acetimidoyl
Thienamycin 100 mg.
Benzalkonium Chloride 0.1 mg.
Sterile Water to1 ml.
10 TOPICAL OINT~ENT
N-acetimidoyl
Thienamycin 100 mg.
Polyethylene Glycol 4000 U.S.P. 400 mg. ~ - -
Polyethylene Glycol 400 U.S.P. 1.0 gram
The active ingredient in the above formulations
; may be administered alone or in combination with other
biologically active ingredients as, for example, with other
- antibacterial agents such as lincomycin, a penicillin,
streptomycin, novobiocin, gentamicin, neomycin, colistin
and kanamycin, or with other therapeutic agents such as
probenecid
. . .
` - 115 -
: .. : . . . :
: '. - : . ,
lV83S7~
1 PREPARATION OF ~LTERNATE ST~RTING MATE~IALS
2 In addition to the thienamycin, one ~killed in
3 the art will recognize th~t it~ variou6 isomer~, alon~ or
4 as mixtures, may serve as ~targing materials in the prep~ra-
tion of the compounds of the present invention. Some of
6 these isomers are obtainable from natural pr~ducts of
7 fermentation (~ee below). However, ~y ~otal 6ynthesis
8 all isomers are made availAble tbelow) a6 a mixture of
9 4 diastereomers which possess antibacterial acti~ity and
which are amenable to resolution by conventional techni~ues.
11 The 4 diastereoi~omers (2 cis, 2 trans) are separable by
12 chromatography. Resolution of any given d/l pair with
13 optically active acids or bases proceeds according to
14 conventional techniques. It should be noted that the
absolute configuration of the first-identified starting
16 material (I) is 5R 6S 8~.
- 116 -
I l583~IB
~ ~357~
1 Preparation of Thiena~ycin by Total ~ynthesis
OH
~ COOH
2 Step A:
3 Preparation of 4-(2-acetoxyvinyl)azetidine-~ one
4 H2C=C~-CH=CHOC-CH3 ~ 0=C=N-S02Cl
O '~ .
O O ' .
CH=CHOCC~3 CH=CHOCCH3
C~' ' > ~ " ~
~/ S02Cl 0~
':
A solution of 1.0 ml distilled chlorosulfonyl~
6 isocyanate (1.65 g; 11.7 mmoles) in 2.5 ml anhydrous
7 diethyl ether is cooled under N2 in a -20C bath.
8 A ~olution of 2.5 g l-acetoxybutadiene (22
g mmoles) in 2.5 ml anhydrous ether is fiimilarly cooled
10 under N2 in a -20C bath.
11 The chlorosulfonylisocyanate solution is added
12 dropwise to the acetoxybutadiene solution by means of a
13 Teflon tube immersed in the CSI solution and pressurized
14 with N~. The addition takes 10 minutes. Little or no
15 color is seen and the reaction is stirred at -20C for
16 0.~ hour. The solution i5 clèar and has a light yellow
17 color,
,
. - ' ~.
.' : .
; - 117 -
~ - , ~ ~. . . '' .
~o~s~
1 A solut~on of 2 g sodium sulfite and 5 g R2E~04
2 in 20 ml H20 is prepared during the above 0.~ hour
3 reaction time ~nd is cooled in an ice bath; 20 ml of
4 ether is added and ~he mixture i6 vigorously stirred in
an ice bath. At the end of the 30 minute reaction time,
~ the reaction mixture i8 transferred, again using N2
7 pressure and the Teflon tube, from the reaction flask
8 which is ~aintained in the -20~C bath, to the vigorously
9 stirred hydrolysis mixture. Rap~d dropwise addition is
completed in 5 minutes . ~he hydrolysis i5 allowed to
11 continue for 5 additional minutes. ~he hydrolysis mix
12 has a pH of 6-8, preferably pH 8.
13 The phases are eeparated, leaving a yellowish-
14 orange gum with the aqueous phase. Ihe e~her phase is
dried directly with MgS04. The aqueous/gum phase is
16 extracted three more times with 50 ml portions of ether,
17 each being added to the initial ether/MgS04.
18 The dried extracts are filtered and concentrated
19 under a N2 stream to 5 ml; a portion of the product is
crystalline at this stage.
21 A column of 10 g Baker silica gel, packed in
22 ether is prepared, and the ether concentrate is applied
23 to the top and run in. Ihe flask/solids are rinsed three
24 times with 2 ml ether, each being pipetted off and run into
the column. Elution is then begun with ether. The first
26 25 ~1 is primarily void volume. The next five 10 ml
27 fractions are collected followed by three ~0 ml fractions,
28 and all are reduced in Yolume under a N2 ~tream. The pro-
29 duct crystallizes from fractions 4-6, with traces in 3 and 7.
Fractions 1-3 contain a yellowish sharp-8melling material
31 which resinifies on 8tanding. Yield: 100 mg as a mixture
32 of the cis and trans isomers.
- 118 -
-.
:
- .
1 Step B:
2 Preparation of 4- (?-Acetoxyethyl)-2-Azetidinone
O O
~ ~ CC 3 ~ OCC~3
3 A solution of 4-(2-acetoxyvinyl)-2-azetidinone
4 (10.0 g, 0.065 mole)in 200 ml ethyl acetate containing
5 100 mg of 10% Pd/C is hydrogenated on a Parr ~haker at
6 25C under 40 psi hydrogen for 15 minutes. Ihe mixture
7 is filtered through a bed of Supercel and washed with
8 additional ethyl acetate. The combined filtrate is
g evaporated in vacuo to give 4-(2-acetoxyethyl)-2-
10 azetidinone (10.0 8) as a crystalline ~olid. Recrystal-
11 lization from ether affords white crystals: M.P.
12 44-7 ; ir (CHC13)~ ~.66, 5.74; nmr (CDC13)1~ 3.44
13 (broad ~, 1, NH), ~.82 (m, 2, CH20COCH3), 6.29 (m,
14 1, C-4H), 6.87 ( 1/2 AB pattern further split in four ~y
C-4H and NH, 1, Jgem ~ 12.~Hz, J~4.5 N JNH ~ 1.9 ~z,
16 7.38 ( 1/2 AB pattern ~urther 8pl~ in four ~y C-4H and
17 MH, 1, Jgem ~ 12.8Hz, J ~ 2.3Hz, JNH ~ l.OHz), 7.93 and 8.02
18 (s on m, total ~, OCOCH3 and CH2CH20COC~3, respectively).
19 Step C
PreParation of 4-t2 _Hydroxyethyl)-2-Azet~dinone
OCCH3 \ . ~
21 Under nitrogen at 0, a ~olution of 4-(~-
22 acetoxyethyl)-2-azetid~none ~2.24 8. .014 mole) in 25 ml
-- ~19 --
. -. . . .
. ~ .
~ ~ 3 S7~
1 anhydrous methanol is treated with a solution of sotium
2 methoxide (77 mg, 1.4 mmoles) in 5 ml anhydrou~ methanol.
3 After stirring for 1 hour, the 601ution is neutralized
4 with glacial acetic acid. Removal of the me~hanol
_ vacuo gives crude ~-~2-hydroxyet~yl)-2^az~tidinone
6 as an oil. The product i6 purified by chromatography
7 on silica gel eluting wlth 10% MeOH/CHC13 to give 1.~5 g
8 o~ ~he alcohol: m.p. 5~; ir (CHCl3) ~ ~.67; nmr
9 (CDC13)~3.20 (broad s, 1, NH), 6.24 and 6.28 (m on t,
10 total 3, C-4H and CH20H respectively), 6.90 (broad s on
11 1/2 AB pattern further split in four by C-4H and NH, total
12 2, OH and C-3H respectively, Jgem ~ 13.0Hz, Jvic ~ 4.2Hz,
13 JNH = 1.6Hz), 7.42 (1/2 AB pattern further spl~ in four
14 by C-4H and NH, 1, C-3H, Jgem = 13.0Hz, Jvi~ = 2.2Hz,
15 JNH = l.lHz), 8.16 (m, 2, C~ CH20H).
16 Step D:
17 PreParation of 8~0xo-2,2-dimethyl-3-oxa-1-azabicyclo
18r4.~.o~octane
~H
l9 A solution of 4-l2-hydroxyethyl)-2-azetidinone
20 (1.87 g, .016 mole) and 2,2-dimethoxypropane tl.69 g,
21 .016 mole) in 25 ml anhydrous methylene chloride is treated
22 with boron trifluoride etherate (.201 ml, .002 mole) at
23 25C The resulting solution i8 6tirred for ten minutes.
24 Removal of the solvent under reduced pressure gives
25 an oLl (2.5 g). Chromatography of the crude product
';
- 120 -
.. , ,, . -
.. . ..
~t~83S7t7
1 on silic~ gel using 2:1 ethyl acet~e/benzene a6 elutlng
2 ~olvent gives ~-o~o-2,2~dimethyl-3-oxa-1-~zabicyclo/~.2.07-
3 octane tl-59 g) as a crys~alline solid. Recrystallization
4 from ether/hexane gives product of m.p. 60-1.
ir (CHC13)~ : ~.73 (~-lactam)
6 nmr (CDC13)~: 6.02 - 6.28, m, 2H, C-4 methylene
7 6.22 - 6.62, m, lH, C-6 methine
8 6.90. dd, lH, J7,7 14Hz. J6.7
9 C-7 proton cis to C-6H
7.47, dd, lH, J7 7 ~ 14HZ- J6.7
11 C-7 proton trans to C-6H
12 7.82 - 8.68, m, 2H, C-5 methylene
13 8.23, 8, 3H~ -
C-2 methyls
14 8.57, 8, 3H
- 121 -
- , . .
'
. . .
~0~3~7
1 ~
2 Preparationbof B-oxo-2,2-dimethyl_7a and~-(l hydroxyethyl)-
3 -oxa-l-aza lCyc o . . octane
pH
o//~
4 To a 601ution of 1.1 equi~alents of freshly
prepared lithium diisopropylamite in anhydrous tetrahydro-
6 furan under a nitrogen atmosphere at --78 is added a
7 solution of 8-oxo-2,2-dimethyl-3-oxa-1-azabicyclo/~.Z.07-
8 octane in anhydrous tetrahydrofuran which has been cooled ~-~
9 to -78C. After two minutes, the resulting lithium enolate
is treated with excess acetaldehyde. Ihe ~olution is
11 stirred for 30 minutes a~ -78 and then poured into water.
12 The ~queous phase is ~aturated with sodium chloride and
13 extracted with ethyl acetate. ~he combined ethyl acetate ,;
14 solutions are dried over m~gnesium sulfate and filtered.
15 Ihe filtrate is evaporated under reduced pressure to
16 give the crude product. Purification by chromatography on
17 silica gel using ethyl acetate/benzene gives 8-oxo-Z,2-dimeth-
18 yl-7a and B-(l-hydroxyethyl~-3-oxa-1-azabioyclol4 2 O]octane.
:`
.
- 122 -
1~8357~ :LS~3~
1 Data ~or 8-ox~-2,2-dimethyl-7~-~1-hydroxyethyl)-3-
2 oxa-1-azabicyclo[4 2 0]~ctane:
3 ir (CH2C12)~ : ~.72~ (B-lactam)
4 nmr (CDC13) ~ : ~.53 - 6.43, m, 4H, C-4 methylene
S C-6 methine ~ C-9 methine
6 6.90, dd on broad ~, 2H, J7 9 = 9Hz
7 J6 7 = ~.5Hz, C-7 methine I OH
8 7~70 - 8.83, m, 2H, C-~ methylene
9 8.27, s, 3H~
~ C-2 methyl
8.60j s, 3H~
Jg,lo = 6.5Hz, C-10 methyl
12 ~ata for 8-oxo-2,2-dimethyl-7 Q- ~ l-hydroxyethyl)-3-oxa-1-
13 azabicycl~[4 2 0]octane:
14 ir (CHC13)~ :2.9 ~road O-H
5.73 ~-lactam
16 nmr (acetone - d6)~: 4.23 - 3.33, m, C-9 methine I C-4
17 methylene ~ C-6 methine
18 3.33,broad s, OH :
19 2.83, dd, J=2Hz, 6Hz~
~ C-7 methine
2.67, dd, J=2Hz, 8HzJ
21 1.93 - 1.63, m, C-5 methylene
22 1.63, s~
C-2 methyls
23 1.40, s
24 1.23, d, J=6.5Hz, C-10 methyl
~123_
~ ~,, . ` '! .
3S7~7
l Step F
2 ~reparation of 8-Oxo-2,2-dimethyl-7atl -~-nitro~enzyl-
3 carbonyldioxyethyl)-~-oxa-l-azabicyclo~4.2.Q7Octane
~H OR
R ~ ~OCH2~ N02
o
4 Under anhydrous conditions at OC. a solution
of 8-oxo-2,2-dimethyl-7a~ hydroxyethyl)-3-oxa-1-
6 azabicyclo/Z 2 0~octane (60 mg, .302 mmole~ inO,6 ml
7 ether is treated with powdered potassium hydroxide (19 mg,
8 .332 ~mole). After a period of 15 minutes, ~-nitrobenzyl
9 chloroformate (65 mg, .30~ mmole) is added ~o ~he leaction
~ixture. Stirring is continued at 25C for an adtitional
11 15 hours. The mixture i8 partitioned between lM pH 7
12 phospha~e buffer and more ether. The ether phase is washed
13 with water and brine, dried over magnesium sulfate and
14 filtered. Evaporatlon of the filtrate under reduced
pressure gi~es 67 mg of a colorless oil. Purification
16 by preparative thick-l~yer chromatography on silica gel
17 developing with 1:9 ethyl acetate/benzene gives 8-oxo-
18 2,2-dimethyl-7a~ -nitrobenzylcarbonyldioxyethyl)-3-
l9 oxa-1-azabicyclo/Z.2.0~octane (40 mg) as a mixture of
dias~ereomers.
21 ir (CH2Cl2) ~ : 5.68 (~-lactam and carbonate), 6.19 and 6.54
(nitro)
: . .
~ - 124 _
-, . . . .
~8;~
1nmr(CDC13) : 1.67, d, 2H, ArH
2 2.37, d, 2H, ArH
3 4.67, 8, 2H, ArC-~2
4 4.67 - 5.22, m, CH3CH
5.98 - 6.25, m, 2H, C-4 methylene
6 6.25 - 6.62, m, lH, C-6 methine
7 6.75 - 7.12, m, lH, C-7 methine
8 7.75 - 8.83, m, 2H, C-5 methylene
9 8.22, s, 3H, C-2 methyl
8.~0 - 8.58~ m, ~H, C-2 methyl ~ CH3CH
The 7~-diastereoisomers or the 7a and ~-mixture are obtained
12 in an anal~gous manner.
13 Step G:
14 Preparation of Cis and Trans-3-(1-p-nitrobenzylcarbonyl-
15 dioxyethyl)-4~2-hvdroxyethyl)-2-azetidinone
.. ..
: OR ~R
~>~0 > ~~ ~
`~ O
R = -C-O-CH2 ~ NO2
16 8^Qxo-3-oxa-2,2-dimethyl-7~ p-nitrobenzyl-
17 carbonyldioxyethyl)-1-azabicyclo/~.2.07~ctane (1.0 g)
iS dissolved in 8 ml acetic ac~d and 2 ml water and
9heated at 65C for 1.25 hours. The acetic acid and water
20are removed under reduced pressure and the residue i~
21taken up in benzene and evaporated to give trans-3-
22(1-R-ni~robenzylcarbonyldioxyethyl)-4-(2-hydroxyethyl)-2-
23azetidinone as a mixture of dia~tereoisomers.
- 12~ _
` 1083S~
1 ir (CH2C12)~ : 5.67 (~-lact~m), 5.72 fihoulder, 6.20 ~nd 6.~7
(nitro)
nmr (~DC13) : 1.73, d, 2H, J=8.5 Hz, ArH
2.43, d, 2H, Jz8.~ Hz, ~rH
3.63, broad s, lH, NH
4.37 - 5.13, m, lH, ~H3CH
6 4.72, s~ 2H, ~rCH2
7 6.07 - 6.53, m, lH, C-4 methine
8 6.23, t, 2H, J=5.5 ~z, CH2OH
9 6.73 - 6.93, ~, lH, C-3 methine
7.63 - 8.97, m, 3H, CH2CH20H
11 8.53, d~ J=6.~ Hz, CH3CH
12 The cis diastereoisomers or the cis-trans mixture are
~_ ,
13 obtained in an analogous manner.
:' .
14 Steps D', E', F' and G' as alternative to Steps D, E, F, -:
15 and G for the preparation of 3~ nitrobenzylcarbonyl- :
16 dioxyethyl)-4-(2-hydroxyethyl)azetldinone
'" '
OR
OH
,l ~H
R = -COCH2 ~ 2
;
' '
:
. - 126 -
. , :. . ., , : -; .
~083S7'7
1 Steps D', E', F' _nd ~'
2 Preparation of 1-(2-TetrahYdr~PYranYl~-4~ (2-tetr~hydr
3 pyranyl)oxyet~y~7~ azetidinone
~ ~ F~
4 Under nitrogen and at 25C, a ~olution
of 4-(2-hydroxyethyl)-~-azetidinone (62 mg, .539 mmole) in
6 .5 ml of anhydrous P-dioxane is treated with 2,3-dihydro-
7 pyran (.98 ml, 1.08 mmoles) and p-toluenesulfonic acid
8 monohydrate (19 mg, .10 mmole). ~he resulting solution is
g stirred for a period of 60 minutes and then partitioned
between 10 ~1 of .SM pH7 phosphate buffer and 10 ml of
11 ethyl acetate. The aqueous phase i5 extracted a second
12 time with ethyl acetate. The eombined ethyl acetate
13 solutions are washed with brine, dri~d over magnesium
~4 sulfste and filtered. The filtrate is evaporated under
reduced pressure to give 216 mg of crude product. Purifi-
16 cation by preparative thick-layer chromatography developing
17 with ethyl acetate gives 80 mg of 1-(2-tetrahydropyranyl)-4-L~-
I8 ~2-tetrahydropyrany~oxyethy ~ 2-azetidinone as an oil.
19 nmr (CDC13)1r: 5.13-5.60, ~, OCH
5.83-6.85, ~, C-4H ~ OCH2
21 6.95, dd, J ~ 5Hz and 15 Hz~
t C-3 methylene
22 7.35, dd, J ~ 3Hz and 15 HzJ
23 7-62-8-95, m, CHCH2CH2CH2CH2 ~ CHCH2CH~0
-127_
~ ,. .. . ..... . .
- - . . .
~(~83~
1 Preparation ~f Ci6 and TrAns-1-(2-t~trahydropyranyl)-3-
3 azetldlnone
~,~ l~ Q
o
4 Following the procedure described for the preparation of
8-oxo-2,2-dimethyl-7a and ~-tl-hydroxyethyl)-3-oxa-1-
6 azabicyclo L~.2.07 octane from 8-oxo-2,2-dimethyl-3-oxa- .~ -
7 l-azabicyclo /Z.2.0~ octane and using 1-(2-tetrahydropyranyl)-
8 4-L~- (2-tetrahydropyranyl)oxyethyl~-2-azetidinone one obtains
9 a diastereomeric mixture of both cis and trans -1-(2-tetra-
hydropyranyl)-3-(1-hydroxyethyl) -4-L~- (2-tetrahydropyranyl)-
ll oxyethyl7-2-azetidinone.
- -128- ,
~83S~
1 Preparation ~ Cis and Tran~-1-(2-tetrahydropyr~nyl~-3-
2 (l-~-nitrobenzy~ nyldioxyethyl~-~-LZ-~Z-tetr~hydro-
3 pyranyl) oxyethyV-~-azetidinonë ~~
/t~o
l~J ~1
R - ~O~H2- ~ ~ No2
4 Following the prDcedure descri~ed for the preparation of
5 8-oxo-2,~-dimethyl-7Q~ -nitrobenzylcarbonyldioxyethyl)-
6 3-oxa-1-azabicyclo/~.2.Q~octane from 8-oxo-2,2-dime~hyl-
7 7Q-tl-hydroxyethyl)-3-oxa-1-azabicyclo/~.2.0~octane and
8 using trans-1-(2-tetrahydropyranyl)-3-(1-hydroxyethyl)-
: 9 4-L~- ~2-tetrahydropyranyl)oxyethyl~-2-azetidinone thPre is
lO obtained trans-l-(2-tetrahydropyranyl)-3-(l-~-nitroben
11 carbonyldioxyethyl)-4-/~-(2-tetrahydropyranyl)oxyethyl702-
12 azetidinone. The cis diastereoisomers are obtained in an
13 analo~ous manner.
. .
-129-
,
.. : . . .
: . . . . ..
: . :. .. -.
L0~357~
1 Preparatlon of Cis and Trans-3-(1-~-nitrobenzylcarbon~l-
2 dioxyethy~ Z-hydroxyethyl)-Z-szetidinone -~~
~ H
~lOCllz ~3 N2
3 A solution o~ trans~l-(2-tetrahydropyranyl) 3-tl-~-nitrobenzyl-
4 carbonyldioxyethyl)-4-/~-(2-tetrahydropyranyl)oxyethyl7-2-
5 azetidinone in methanol at 25C. is treated ~ith
6 .1 molar equivalent of ~-toluenesulfonic acid ~onohydrate.
7 The solution i6 stirred for a period of 2 hours and then
8 neutralized with lM pH7 phosphate buffer. The product is
~ n
extracted into ethyl acetate. The ethyl acetate solution
10 is washed with brine, dried over magnesium sulf~te and
11 filtered. The filtrate iB evaporated under reduced pressure
12 to give trans-3-(1-~-nitrobenzylcarbonyldioxyethyl)-4-
13 (2-hydroxyethyl)-2-azetidinone. The cis diastereoisomers
14 are obtained in an analogous manner.
. .
;
, :
-130-
.. .. .. - . ~... . .
~0835~
1 Step H:
2 Preparation ~f Cis and ~rans-3~ nitrobenzylcarbonyl-
3 ~ioxyethyl)-4-L2~2-~is~-hydroxyethyI)thioethyI7
4 azeti inone
L~ :
/ ~ - OH
0~ ~ ~ OH
R ~ COCH2- ~ 2
Under nitrogen at 25C, a mixture
; 6 of anhydrous pyridine (.146 ml, 1.81 mmoles) and anhydrous,
7 powdered chromium trioxide (92 mg, .916 mmole) in 8 ml
8 anhydrous acetonitrile is stirred for a period of 30 minutes.
9 ~o the resulting dark brown 801ution iS added 250 mg of
dry SuperCel followed by a solution of trans-3~ -nitro-
11 benzylcarbonyldioxyethyl)-4-(2-hydroxyethyl)-2-azotidinone
12 (186 mg, .550 mmole) in 1 ml anhydrous acetonitrile. After
13 stirring for a period sf 1 hour, the reaction mixture is
14 filtered through a mixed, packed bed of 2 g each of silica
gel and magnesium sulfate. The bed is washed repeatedly
16 with a total of 30 ml of additional acetonitrile. The
-131-
3357~
1 filtra~e is concentrated under reduced pre85ure at
2 25~C. ~o a volume of ~ ml. By thin-layer chroma-
3 tography (silica gel; ethyl acetate/benzene 2:1) this
4 solution contains a product ~Rf~.38) less pslar ~han the~
startin~ material (~ .21).
6 Ihe ~cetonitrile solution of trans-3-(1-~-
7 ni~robenzylcarbonyldioxyethyl~-4-(2-oxoethyl)-2-azetidinone
8 prepared above is, under nitrogen and at 0, treated with
9 2-mercaptoethanol (.386 ml, 5.5 mmoles) followed i~mediately
by boron triflu~ride etherate (.176 ml, 1.43 mmoles).
ll After stirring for a period of 15 minutes, this solutio~ :
12 is partitioned between ~queous dipotassium hydrogen phos-
13 phate (1.5 g. in 4 ~1 of water) and 1~ ~1 of ethyl acetate.
14 ~he aqueous phase is extracted a second time with ethyl
acetate. The combined ethyl acetate solutions are washed
16 with brine, dried o~er magnesium sulfate and filtered. The
17 filtrate is evaporated under reduced pressure to give 229 mg
18 of an oil. The product is purified by preparative thick-
l9 layer chromat~graphy on silica gel developing with ethyl
acetate to give 118 mg of trans-3-(1-~-nitrobenzylcarbonyl-
21 dioxyethyl)-4-L~,2-bis(2-hydroxyethyl)thioethyl~-2-azetidinone
22 as a colorless oil.
23 ir (CH2Cl,~ 5.75 (5.79 shoulder) ~-lactam and carbonate
24 6.~0, 6.~5 nitro
nmr (acetone-d6)^r: 1.70, d, J ~ 8.5 ~z, 2H, ArH
26 2.28, d, .l ~ B.5H2, 2H, ArH
27 2.48-2.~8, ~, lH, NH
28 4.63, ~, ArCH2
29 3H
4.63-5.12, m, CH3CH
-132-
.; . .
~r
~L08357~
1 5.80, t, J ~ 7H2, CH2 ~ S
13H
2 ~.B0-7.45, ~, C-4H + C-3H + SCH2CH20H,
3 7.63-8.33, m, 2H, CH2CH
4 8.53, d, J ~ 6.5 Hz 3H, H3CH
5 ~e cis diastereoisomers are obtained $n an analogous
6 manner. Alternatively, the mixed diastereoisomers are
7 obtained when the starting materials comprise a ~ixture
8 of the diastereoisomers.
-133-
.- . . ,., - . :
,. - . , ~. .. . . .
1~8357~
1 ~p~
2 Pr ~ a2a~ion(2of Tdans-31(1-p-nitrl7enzylcarbonyldioxyeth~
3 4~ s -azl oet y t loet y - -azet none
OCOOPNB 0~
~ S~ ~ ~ 2CH3SO2Cl 2Et N) NaN
0//
COOPNB S/ \ ~ 3
"` ~ ~ S ~ 3
~ NH
4 To a s~lution of 211 mg (mw - 474; O ~ 445 mmole)
trans-3-(1-p-nitrobenzylcarbonyldioxyethyl) -4-L~. 2-bis(2-
6 hydroxyethyl~thioethyl7-2-azetidinone in 5 ml tetrahydro-
7 furan (THF)~distilled from lithium aluminum hydride)
8 at OC is added 103 mg mesylchloride cmw = 114; 0.904
g mmole) in 1 ml THF followed immediately by 134 ~1 triethyl-
amine (mw = 101; e 0~29; 0~967 ~mole). The reac~ion
11 mixture is stirred for 1 hour under N2. The triethylamine
12 hydrochloride is filtered under N2 washing with a few
13 milliliters additional THF. The clear colorless filtrate
14 is concentrated under a stream of N2 followed by pumping
under high vacuum for 10 minutes. Ihe dimesylate is
16 immediately dissolvet in 5 ml DMSO (distilled from CaH2
17 at 8 mm and stored over 4A Linde Molecular sieves) in
18 the presence of 347 m~ NaN3 (mw ~ 65; 5.34 mmole).
l9 After stirring overnight under N2, 10 ml H20 and 20 ml
ethyl acetate tEA) are added. The lsyer6 are sepa-
21 rated,and th~ aqueous one is washed three times with 10 ml EA,
22 each organic layer then being backwashed with 10 ml H20 and
23 10 ml brine. The com~ined EA layers are dried o~er
24 anhydrous MgS0~, filtered and c~ncentrated under a N2
-13~-
... . . .. . . . . . .
~8~7~
l stream to give the crude diazide. Preparative t~in layer
2 chrom~tography on ~ilica gel yields ~rans-3-~l-p-~itro-
3 benzylcarbonyldioxyethyl) -4-L~, 2-bis(2-az~doethyl)thio-
4 ethyl~-2-azetidinone. ~he cis dias~ereoisomer~ o~ the
cis-trans mixture are obtained in an analogou~ manner.
Step J:
Cl 02H C02PNB
C(OH)2 ~ 2NO2 ~ CHN2 EA/Et20 ~ C(OH)2
C02H C02PNB
OCOOPNB OCOOPNB
. CH CH,N ~ SCH2CH2N
" . ~ ~ 2 ~ 3 Tol. / ~
r~ SCH2CH,N3 ~~~~~~ L I SCH2CH2N3
o~ o~ ~ ~ H20 .,
(C02PNB)2
PNB = CR,~_~/ \ ~ NO.)
; -135-
. ., . : .
. . . . .. . .
3 ~
l A freshly prepared (H. Davies and M. Sch~arz,
2 J.O.C., 30, 1242 (1965)) ~olution of p-nitrophenyldiazo-
3 methane (29 mmole) in 150 ml of ether is added with stirring
4 to a solution of l.0 g oxomalonic acid, monohydrste
(mw = 136; 7.35 mmole) in 50 ml ethylacetate (EA) at 0C.
6 After 2 l/2 hours the yellow solution is concentrated on
7 a rotary evaporator with mild heating to approximately half
8 the volume, dried over anhydrous sodium sulfate, filtered and
: g concentrated as above to an oil. To the crude p-nitro-
benzyl ester in 50 ml toluene (Tol.) is added 3.54 g of
ll trans-3~ p-nitrobenzylcarbonyldioxyethyl)-4-L~t2-bis(~-
12 azidoethyl)th~oethyl7 ~-azetidinone (mw ~ 524; 6.75 mmole).
13 With stirring the reaction mixture is heated in an oil
14 bath allowing approximately 1/3 of the toluene to boil
off. Toluene (dried over 3A 1/16" Linde Molecular sieves)
16 is added to again bring the volume to 50 ml, and the azeo-
17 drying process is repeated three additional times. Ihe
18 solution is then refluxed under N2 for one hour, the
19 azeodrying process repeated a ~inal time, and refluxing
continued for an additional hour. Concentration of ~he
21 resulting solution under a stream o~ N2 yields crude 1.
22 The crude material is chromatographed on silica gel to
23 give 1- Th~ cis diastereoisomers or ~he _ -trans
24 mixture is obtained io sn analogous manner.
:
~: :
,
-136-
.
' .' ' ' " ' ' '
~VI!33~7t7
Step K:
OCOOPNB
D~ SCH,~CH2N3
C~ ~<SCH2CH2N3
OH ~ SOC1
02PNB) 2
'~
OCOOPNB ~ ~
/ ~/~SCH2CH2N3
O~ ~/Cl ~ 03P
( C02PNB ) 2
OCOOPNB
SCH2CH2N3
SCH2CH2N3
~I N~/H
02PNB ) 2
~
,.....
:`;
.
--137 -
,: . , - ., . , , , :
10~3357~7
1 ~o a ~olution of Z.80 g 1 (mw - 912; 3.07 mmole)
2 in 35 ml THF (distilled from lithium aluminum hydride) at
3 -20C is added 0.3 ml pyridine (mw G 79; ~ - .982; 3.73
4 mmole) (distilled from NaH and stored over 4A Linde Molecu-
lar sieves). With ~tirrln~ under N2, 0.438 g thionyl
6 chloride (mw = 119; 3.68 mmole) in 1 ~1 TXF is added drop-
7 wise. The reaction mixture is ~tirred under N2 ~or 5 minutes
8 at -20C, then 1/2 hour at 0C, and finally 1 hour at 25C.
9 The pyridine hydrochloride is filtered under N~ and washed
twice with benzene (dr~d over 3A 1/16" LiDde Molecular
11 sieves~. The combined filtrate and washings are concen-
12 trated under a N2 stream, clurried in a ~mall volume of
13 benzene with anhydrous MgS04, filtered under N2 and then
14 concentrated under a N2 stream. Pumping on high vacuum
15 for 1/2 hour yields an oil. To this freshly prepared
16 chloro compound is added with stirring-0.885g triphenyl
phosphine (mw = 262; 3.38 mmole) in 66 ml 9:1 dimethyl-
formamide ~DMF~/H20 followed by 550 mg K2~PD4 (mw ~ 174;
19 3.16 mmole). The reaction mixture i8 stirred at 25C for
20 35 minutes. After dilution with EA and brine,the layers
21 are separated and the aqueous one extracted three times
22 with EA. The cQmbined EA layers are wàshed with brine,
23 dried over anhydrous MgS04, filtered, and concentrated
24 under a stream of N2 to give crude 2. The material is
25 chromatographed on silica gel to give 2. The cis diastereo-
26 isomers or the cis-trans mixture is obtained in an analogous
27 manner.
138-
, . .
1~33S7~7
Step_L:
OCOOP~B
~S-CH2CH2N
S-CH2CH2N3 ~ ~3r2 Et2/C5H12 ,_~
02PNB) 2
. _
OCOOPNB Br ~rl C3
2CH2N3 ( 1 )
¦ I SCH CH N . ~ -
N~H 2 2 3 (2) NaH,DMF
(CO2PN~) 2
_ ~ ~,-
OCOOPNB . .
2CH2N3 ~ 3
N~ \.~ Br
(C02PNB)2
;'. :
3 ~ .. , .: -
'
':`
::
`: _
l~)B357~7
1 To 7.8 ml pentaneCdried over 4A L~nde molecular
2 sieves) is added 0.2 ml Br2 ~mw - 160;P -3-12; 3-9
3 mmole). To a Rolution of 950 mg 2 (mw - 896; 1.06 mmole)
4 in 15 ml Et20 (dried over 3A 1/16" Linde molecular ~ie~e )
at 0C. under N2 with stirring is ~dded dropwise 2.3 ml
6 of the above 0.49 M Br2 solution (1.13 mmole). After stir-
7 ring for 10 minutes at 0C., 114,ul cyclohexene
8 (mw ~ 82, ~ = .81; 1.13 mmole) is added. After 5 minutes
g at 0C,53 mg 57% NaH (57% of 53 mg = 30.2 mg, ~w - 24,
lO 1.26 mmole) in mineral oil is added to the stirred reaction
11 mixture. This is followed immediately by the addition
12 of 14 ml ice cold DMF (distilled from anhydrous CaS04 at
13 40 mm and stored over 4A Linde molecular ieves). Stirring
14 at 0C under N2 is con~inued for ~ hours. The reaction
. ~ .
15 mixture is poured into a stirred ice-cold mixture of
16 2.5 ml lM KH2P04-40 ml H~0-75 ml EA. After separation
17 of the layers, the aqueous one is saturated with NaCl
18 and re-extracted with EA. The combined organic layers .~:
19 are extracted once with brine, dried over anhydrous ~gS04,
20 filtered and concentrated under a N2 stream followed by
21 pumping on a high vacuum pump to provide crude 3
22 Preparative thin layer chromatography on silica gel
23 yields ~. ~he cis diastereoisomers or the cis-trans
24 mixeure 1~ obtained in an ~nalogou~ manner.
~` ~
: .
.~
-1~ O-
~0~3S~7
Step M:
OCOOPNB
, ~ ~ ~ SCH2CH2N3
I Br2 Et~/C51!12
02PNB)2
~v ~
, _ ::
OCOOPNB :
" CHCH
I r ~2CH2N3
0~ ~ Na~ DMF _
tC02PNB) 2
: OCOOPNB Br
~SCH2~H2N3 :
(Co2pNB)2
~.
--141--
- , . .
. ~ :
~L08357~7
1 To 9.16 ml pentane ~dried over 4A Linde Molecular
2 sieves) i9 added 0.2 ml Br2 ~mw = 160, 3.9 mmole).
3 To 474 mg 3 (mw e 793; 0.598 mmole) in 13 ml Et20 (dried
4 over 3A 1/16" Linde Molecular sieves) at 0C under ~
with stirring is added dropwise 1.52 ml of the above 0.42 M
6 Br2 ~olution (0.63 mmole). After 15 minutes at 0C, 33 mg
7 57% NaH (57% of 33 mg = 18.8 mg; mw = 24; 0.78 mmole) i8
~` 8 added followed immediately by the addition of 6.35 ml ice-
g cold DMF (distilled from anhydrous CaS0~ at 40 mm and
stored over 4A Linde Molecular sieves). The reaction mixture
11 is stirred for 1 1/~ hours at 0C., then poured into a
12 stirred ice-cold mixture of 1.6 ml lM KH2P04 - 20 ml H20, ~-
13 and 20 ml EA. The layers are separated and the aqueous one
14 saturated with NaCl and re-extracted with additional EA.
The combined organic layers are washed once with brine,
16 dried over anhydrous MgS04 and filtered. The filtrate
17 is concentrated under a N~ stream and then pumped on high
18 vacuum to give crude 4. Preparative thin layer chroma-
19 tography on silica gel gives 4 The cis diastereoisomers
or the cis-trans mixture is obtained in an analogous
-- - ,
21 manner.
.
( ~
. .
-142- -~
~083S~7
5teP N:
OCOOPNB ~r
'F~SC~2CH2~3
(~02PNB)2
~OCOOPNB
`1 ~--SC~2C~2N3
(Co2pNg) 2
~
1 To 210 mg 4 (mw 5 871; 0.241 mmole) dissolved in
2 0.5 ml DM~O (distilled from CaH2 at 8mm and stored over
4A Linde molecular sieve~) is added at 25C. with stirring
~ 4~ mg 1,5-diazobicyclo/~.4.0~undec-5-ene (distilled at
5 80C./2 mm) (mw ~ 152; 0.263 mmole) in 0.7 ml dimethyl- ~
6 sulfoxide (DM$0). The solution is stirred undec N2 for ~-
7 4 hours, and then added to a ~tirred ice-cold mixture of
8 0.48 ml lM KH2P04, 7 ml H2~, and 10 ml EA. After separation
9 of the layers, the aqueous layer is again extracted with EA.
10 The combined organic layers are washed ~ne time with brine,
11 dried over anhydrou~ MgS04, iiltered, and concentrated under
12 a N2 stream followed by pumping on high vacuum, to provide
13 crud~ ~. Preparative thin layer chromatography on 8 ilica
14 gel yields 5. The ci~ dia~tereoisomers or the cis-trans
5 mixture i~ obtained in an analogou~ manner.
-143-
. ' ' , ,, . . ' ' , ,'. ',
~357~7
Step O:
OCOOPNB
~2CN2~3+LiI s-collidine >
(CO2PNB)2
~COOPNB
a~C~12Ct~2N3
H ~O2PNB
1 A solution of 187 mg ~ (mw ~ 791; 0.~36 mmo~e)
2 in 2.5 ml S-collidine (distilled from powdered KOH at
3 30 mm pressure) is added to 45 mg anhydrous LiI (dr~ed
4 for a few hours at 100C o~er P2O5 under vacuum)
(mw - 134; 0.336 mmole). With stirring under N2, the
6 reaction mixture is heated ~n an oil bath at 120C. After
7 a total of 25 minutes, the reaction mixture is cooled to :
8 25C., diluted with CH2C12 and transferred to a round
9 bottom flask for concentration under a N2 stream and
~hen on high vacuu:m. Partitioning of the residue between
lL 10 mL EA and 1.8 ml lM KH2P04 in 10 ml H20 is followed
12 by extraction of the aqueous layer two additional times
13 with EA. Ihe combined organic layers are extracted with
14 brine, dried over anhydrou~ MgSO4, ~iltered and concentratsd
under a stream of N2 to give crude 6 Preparative thin
16 layer chromatography on silica gel yields 6. The Ci8
17 dias~ereoisomers or the cis-trans mixture is obt~ined in
18 an analogous manner.
-144-
, ~ '.~ . ., ,,, ' : ' ' '.
.. , -., . .. . .. . :
357~7
SteP P:
o
2C~2N3
o~ 6 C02PNB +
OCOPNB
`~ DMS0 ~ ~ ~ SCH2CH2N
N C02PNB
1 To a solution of the mixed diastereoisomers,
2 6, (34 ~g; mM ~ 612; 0.055 mmole) in 0.2 ml DMS0 ~dis~illed
3 from CaH2 a~ 8 mm and stored over 4A Linde Molecular sieves)
4 with stirring is added 9.5 ~1 1,5-diazobicyclo/~.4.0~undec-
5 5-ene (distilled at- 80C/2mm), (mw ~ 152; ~ - 1 0.0625 ...
6 mmoLe). The solution is stirred under N2 for 15 minutes
t diluted to 1 ml total ~olume with CHC13 and
8 applied immediately to 2-1000~ silica gel plates. The
g product band yields 7.as a mixture of cis and trans
10 diastereoisomers .
.~ ~
;
-145-
. . . ,:
. .. . . . . .
- . . . ~ ,
~o~3357t7
steP Q
~COoPNB
/~SCH2CH2N3 /~ SCH2CH;~NH2
2/40 p.~-i- l 11
N J CO2PNB ~ ~ - ~ - I CO~H
1 In the presence of 61 mg PtO2, 61 mg of 7 r.
2 (mw = 612; O.l mmole) in 6 ml dioxane, 6 ml IHF, 3 ml H20
3 is hydrogenated at 40 p.8.i. - ~ for 4 hours. Ihe reac~on
4 mixture is then filtered through Celite washing with 2 ml
5 0.1N pH7 phosphate buffer. After concentration in v cuo
6 to the~cloud point, the aqueous mixture is extracted
7 with ethyl acetate. The water layer i8 concentrated to
, 8 a small volume and applied to a column of 100 g XAD-2
9 resin. Upon elution with H20 and discarding the ini~ial
10 fractions, those fractions containing product are lyophilized
11 to give 8 as a mixture of cis and trans diastereoisomers.
,
.1 .
.~
. :
6-
- .
~ .... . ...... .... .
.. . . . . .... .. . . .
. .. .. .
~(~83S~
1 The following procedure for the enzymatic
2 N-deacylation of thienamycin is applicable for all
3 isomers of thienamycin -- particularly the aistinct
4 N-acetyl isomers 890A, and 890~3, which are described
5 below.
6 Deacetylation of N-Acetyl Thienam~cin
7 A 1% (w/v) suspension of fertile lawn soil is
8 prepared by suspending 1 gm. of lawn soil in 100 ml.
~ sterile phosphate buffer-saline solution wherein the
10 phosphate buffer-saline solution has the following
11 Compositon
12 Phosphate Buffer-Saline Solution
13 NaCl 8.8 g.
14 lM Phosphate Buffer, pH 7.5~ 10 ml~ `~
Distilled ~2 1000 ml.
16 ~lM Phosphate Buffer, pH ?.5
17 16 ml. lM KH2PO are mixed with 84 ml. lM
18 K~HPOA. The pH~f the phosphate buffer is
aajus~ed to 7.5 by adding small quantities
of either lM RH2P04 or lM R2HP04.
21 Aliquots of this 1~ stock soil suspension are
22 used to prepare 10x, 100x and 1,000x dil~tions.
23 One ml. portions of the stock suspension or 1
24 ml. portions of the 10x, 100x and 1,000x dilutions are
25 added to 2 ml. portions of sterile, 1.0% agar solutions
26 at 48C. The mixtures are quickly poured over the surface
27 Of sterile petri dishes of 85 mm. diameter containing
28 20 ml~ of ~edium A. Medium A has the following composition:
29 Medium A
KH2PO4 3.0 g.
31 K2HPO4 7.0 g.
32 MgSO4 0.1 g.
33 Distllled H2O 1000 ml.
N-Acetylethanol-
amine 301utionn 8.5 ml.
. -
., . , ., ~ . . . . .
15834IB
~LOi!~3S7~7
1 ~ -acetylethanolamine So~ution
2 N-acetylethanol~mine is diluted 10x in H O
3 and membrane sterilized. This solution 25
4 added after autoclaving
For solid media: Add 20 g. agar
6 The petri dishes are incubated for 18 days at 28~C. A
7 well-isolated colony is picked and streaked on a petri
8 dish containing Medium 8. Medium B has the following
9 composition:
Medium 8
11 Tomato paste 40 g.
12 Ground oatmeal 15 g.
13 Distilled ~21000 ml.
14 pH: adjust to 6 using NaO~
For solid media: Add 20 g. agar
16 An individual clone is selected and grown for 2 days at
17 28C. on a slant of Medium B. A portion of the
18 growth on this slant is streaked on the surface of six
19 slants pxepared from Medium B. These slants are incubated
20 for 2 days at 28C. This culture was identified as
21 Protaminobacter ruber and has been designated M~-3528 in the
22 culture collection of Merck & Co., Inc., Rahway, New Jersey,
23 ~.S.A. and a sample deposited with the Agrlcultural Research
24 Service, U,S. Department of Agriculture, Accession No. NRRL B-8143
A portlon of the growth on the slant of
26 Protaminobacter ruber MB-3528 is used to inoculate a 250
27 ml~ Erlenmeyer flask containing 50 ml of Medium C. Medium
~8 C has the following composition:
29 Medium C
Dextrose 20 g.
31 Pharmamedia 8 g.
32 Corn Steep Liquor
33 (wet basis) 5 g.
34 Distilled ~zO1000 ml.
pH: adjust to 7 with NaOH or ~Cl
36 N-acetylethanolamine
37 solution 8.5 ml.
-148-
.
~3S~q
1 ~ -acetylethanolamine Solution
2 N-acetylethanolamine is diluted with lOx in
3 H20 and membrane sterilized. This solution
4 is added after autoclaving.
The flask is skaken at 28C o~ a 220 rpm
6 (2" throw) shaker for 4 days. A 25 ml. portion from the
7 flask is centrifuged for 15 minutes at 8,000 rpm. The
8 supernatant is removed and the cells on the surface of
9 the media solids scraped oEf into 0.5 ml. 0.05M potassium
lO phosphate buffer, p~ 7.4. T~e resulting suspension is
ll subjected to ultrasonic disruption using a Branson
12 Instrument Model LS-75 Sonifier with a l/2 inch probe at
13 setting 4 for 4, 15 second intervals, while chilling the
14 suspension in ice water during and between disruption.
15 A lO~l portio~ of the sonicate is mixed with a 25 ~1
16 solution containing 840 ~g/ml. of N~acetylthienamycin in
17 lOmM potassium phosphate buffer, p~ 7 and incubated
18 overnight at 28C. Controls containing antibiotic
19 and ~uffer alone; and sonicated cells and buff~r without
20 antibiotic are also run~ After incubation overnight at 28C.,
21 2 ~l guantities are applied on cellulose coated TLC plates,
22 which are developed in EtOH:H2O, 70:30. After ~ir drying,
23 the TLC plate is placed on a Staphylococcus aureus ATCC
24 6538P assay plate for 5 minutes.
The ass y plates are prepared as follows:
26 An overnight growth of the assay organism, Staphylococcus
27 aureus ATCC 6538P, in nutrient broth plus 0.2% yeast
28 extract is diluted with nutrient broth, plus 0.2% yeast
29 extract to a suspension havlng 60~ transmittance at a
,
-149_
.. _ . .. ..
, ~ .~ ,f ' ...
~08357~
1 wavelength of 660 nm. This suspension is added to Di~co
2 nutrient agar supplemented with 2.0 g./l. Difco yeast
3 extract at 37C. to 48C., to make a composition containing
4 33.2 ml. of the suspension per liter of agar. Forty ml.
of this suspension is poured into 22.5 cm. x 22.5 cm. petri
6 plates, and these plates are chilled and held at 4C.
7 until used (5 day maximum).
8 The TLC plate is removed and the assay
9 plste incubated overnight at 37~C. In addition to the
10 unreacted bioactive ~-acetyl thienamycin spot at Rf 0.7-
11 0.89, a bioactive spot is observed at Rf 0.44-0~47 due
12 to thienamycin. Control incubation mixtures of antibiotic
13 plus buffer, cell sonica~e plus ~uffer, and antibiotic plus
14 buffer to which cell sonicate is added just prior to TLC
15 application produce no bioactive material at Rf 0.44-0.47
16 Production of B9OAl, A Distinct Isomer
OH
2CH2NHCCE~3
~L__N COOH
17 A tube of lyophilized culture of Streptomyces
18 flavoqriseus MA-4434 (NRRL 8139) is opened aseptically
19 and contents suspended in a tube containing 0.8 ml. of
sterile Davis salts having the following composition:
-150-
3577
1 Da~is Salts
2 Sodium citrate0~, 5 g.
3 K2HPo4 7. 0 g.
4 KH2P04 3. 0 g.
(NH4) 2S04 1. 0 g.
6 MgS04 7H~O 0.1 g.
7 Distllle~ H201000 ml.
8 This suspension is used to inoculate four slants
g of medium A ha~ing the followin~ composition:
Medium A
11
12 Glycerol 20~0 g.
3 Primary Yeast 5. 0 g.
Fish Meal 15. 0 g.
14 Distilled H2O -1000 ml.
Agar 2 0 . 0 g .
16 pH: ad just to 7 . 2 using ~aO~ .
The inoculated slants ~re incubated for one
18 week at 27-28Co and then stored at 4-6C. until used.
.~
; 1~ A one-th~-d portion of the growth from three
20 slantsis used to inoculate 9 baffled 250 ml. Erlenmeyer ~ ;
21 flask containing 50 ml. medium B having the following :
22 composition:
' ~'
, .. `'
'~
: ` :
.
.
~ -151-
.
357t7
1 Medium B
2 Yeast Autolysate (+Ardamine) 10.0 g.
3 Glucose ~ 10.0 g.
~hosphate buffer 2.0 ml.
~gS04~7H20 50 ~g.
6 Distilled H20 1000 ml.
7 ~H: adjust ~o 6.~ using KCl or NaOH
8 Ardamine: Yeast Products Corporation
3 ~Phosphate Buffer solution
i~H2P4 91. 0 ~.
11 ~a2HPO4 95.0 g.
12 Distilled ~2 1000 ml.
13 The seed flask is shaken for one day at
1427-28C. on a 220 r~n shaker (2" throw). The flask and
lScontents are stored stationary for one day at 4C.
16 Thirty three 2 liter Erlenmeyer production ~lasks,
17each containing 250 ml. of ~edium C, are inoculated with
188 ml per flask of the growth from the seed flask. The
l9medium C has the following composition:
Medium C
21 Tomato Paste 20.0 g
22 PrLmary Yeast 10.0 g
23 Dextrin ~Amidex) 20.0 g.
24 CoC12 6H~O 5.0 mg.
Distille~ H2O 1000 ml.
26 pH: adjus~ to 7.2-7.4 using NaOH
27 ~fter inoculation, the pro~uction flasks are
28incubated at 24C., with agitation on a 21Z rpm shaker
29(2'9 throw), for four days. The flasks are harvested and
30assayed for activity by using standard Salmonella
31gallinarum MB1287 and Vibrio percolans ATCC 8461 as8ay
32plates using 1/2 inch assay di~cs dipped into centri~uged
broth samples. Sample5 are diluted with 0.02M phosphate
34buffex, pH 7.0 when necessary. The results are tabulated
-152-
_ ... . .
~ . . ~ . .
: - - . . :
33S~
1 below:
~arvest Age hours 96
pH 7.2
Salmonella gallinarum
(mm. zone) 29.5
Vibrio percolans 1/10 dil
(mm. zone) 31
890 Assay Units 40
2 Seven liters of whole fermentation broth is
3 chilled to 3C. and centrifuged in 200 ml. portions at
4 9000 rpm for 15 minutes each.
To the combined supernatants is added 7 ml.
6 o~ O.lM neutral EDTA, and the entire sample is adsorbed
7 on a Dowex 1 x 2 (Cl ), 50-100 mesh column, bed dLmensions
8 ~.1 x 25 cm., at a flow rate of 40 ml. per minute. The
9 column is washed with 500 ml. of deionized water containing
5 ml. of lM Tris-HCl buffer, p~ 7.0, and 25~M neutral
11 EDTA. The antibiotic is eluted with 1 liter of deionized
12 water containing 50 g. of sodium chloride, and the column
13 is then washed with 30~ ml. of deionized water. Fractions
14 of 100 ml. are collected, starting ~rom the first appearance
of salt at the column outlet. Bioactivity appears in
16 fractions 1 through 10 with a peak at fraction 2.
17 Fractions 2 through 5 t:ontaining l7% of the applied
18 ac~ivity, are pooled.
19
~he pooled fractions are soncentrated to 110 ml.
21 by rotary evaporation under reduced pressure, and the pH
22 is adjusted to 5.8 by addition of 4.2 ml. of lM HCl.
23 The adjusted concentrate is applied on a column o~ XAD-2,
2~ bed aimensions 3 . 8 by 50 cm., which had been previously
~5 washed with 3 liters of 60% aqueous acetone (v/v), followed
26 by 3 liters of deionized water, 3 liters o~ 5% (w/v)
:
; . . ~
~ -153-
. .~ .
l~B~577
1 sodium chloride, and 1 liter of 25% (w/~) sodium chloride
- 2 in deionized water. The applied concentrate i~ allowed
3 to drain to bed level . The antibiotic is eluted with
4 deionized water at a flow rate of 15 ml./minute. Twenty-
two fractions of 100 ml. each are collected, co~nting
6 from the first application of sample. Bioactivity appears
7 in fractions 6 through 22, with a peak at fractions
8 9 and ~0. Frac~ions 9 through 20 are comblne~ for
9 further processing. Similarly prepared fractions are
10 combined and the pool is concentrated to 70 ml. by rotary
11 evaporation un~er reduced pressure.
12 The concentrate is adsorbed on a Dowex-l x 4
13 (Cl ) minus 400 mesh column with bed dimensions 2.2 x 27
14 cm. The column is washed with 50 ml. of deionized water,
and the antibiotic eluted with 2 liters of 0.070M
16 NaCl + 0.005M NH4Cl I O.OOOLM NH3 in deionized water
17 at a flow rate of 2 ml. per minute. Fractions of
18 9,5 ml. are collected.
19 The main peak of antibiotic is eluted in
fractions 142 to 163. Fractions 146 through 157 contained
21 were combined for further processing.
22 The combined fractions 146-157 are concentrated
23 to 3 ml. by rotary evaporation under reduced pressure,
24 and the concentrate is brought to pH 6.5 by addition of
25 5 ~1~ of lM NH3. The concentrate is applied on a column
26 (2-2 x 70 cm.) o~ Bio-Gel P-2 (200-400 mesh), which had
27 been washed with 20 ml. o~ SM NaCl and 500 ml. of
.~ :
~ -154-
~V8357~7
..
1 deionize~ water. After the concentrate has drained to bed
2 level, two rin~es of 1 ml. each of deionized water are
3 applied and allowed to drain to bed lèvel. The column is
4 then eluted with deionized water at a flow rate of 0.6 ml.
per minute. Fractions of 2.9 ml. are collected.
6 The main peak of antibiotic is eluted in fractions
7 63 to 70. Fractions 64 and 65 are pooled for further
8 workup.
3 Ihe combined fractions 64 and 65 are rotary
evaporated under reduced pressure to 2 ml., and then shell
ll frozen and lyophilized in a 14 ml. screw-cap ~ial for 8
12 hours ~o give 2.25 mg. substantially pure antibiotic 890Al.
13 The N-acetyl group is cleaved as above described to provide
14 the free base: ~H
/~M/~SCH2cH2NH,~ ,
~ COOH
'~ 0~
15 Production of ~ 30A3, A Distinct Isomer
`:
OH O
\I rSCH2CH2~1CCH3
~ N COOH
16 A tube of lyophilized culture of Streptomyces
flavo~riseus MA-4434 (NRRL 8139) is opened aseptically and
18 the contents suspended in a tube containing 0.8 ml. of
sterile Davis salts having the following composition:
Davis Salts
21 Sodium citrate 0.5 g.
~- 22 K2~PO4 7.0 g-
23 KH2P4 3~0 g-
24 (NH )2S04 1.0 g.
25 MgS~ .7H O 0.1 g.
26 Dist~lle~ H2O 1000 ml
27 This suspension i8 used to inoculate four slants
28 of medi~m A having the following composition:
-155-
. ' ' .: ' .
~83S7~
1 Medium A
-
2 Glycerol 20.0 g.
3 Primary Yeast 5.0 g.
4 Fish Meal 15.0 g.
Di~tilled H2O 1000 ml.
6 Agar 20.0 g~
7 pH~ adjus~ to 7.2 using NaOH
8 The inoculated slants are incubated for one
9 week at 27-28C. and then stored at 4-6C. until used
~not longer than 21 days).
11 ~ one-third portion of the growth from four
12 slants is used to inoculate 12 baffled 250 ml. Erlenmeyer
13 flask containing 50 ml. medium B having the following
14 cOmposi~ion:
Medium B
-
16 Yeast Autolysate (+Ardamine) 10.0 g.
17 Glucose 10.0 g.
18 Phosphate Buffer ~ 2.0 ml. :
19 MgSO~-7H2O 50 mg.
Distllled H2O 1000 ml.
21 pH: a~just to 6.5 using HCl or NaOH
2~ +Ardamine: Yeast Products Corporation
23 ~Phosphate Buffer solution
24 KH2P04 91.0 ~. :
: 25 Na2HPO4 95-0 g~
27 Distilled H2O 1000 ml. ~:
27 The seed flask is shaken for one day at .
2~ 27-28C. on a 220 rpm shaker (2" throw). The flask
29 ~and contents are stored stationary for one day at 4C.
3Q Forty-four 2 liter Erlenmeyer production
31 flasks, each containing 20G ml. of Medium C, are inoculated
32 with 8 ml. pex flask of the growth from the seed flask.
33 The medium C has the following composition:
. ~.. .
-156-
:... . .
. . - : . . .
~8;3 S7t7
1 Medium C
2 Tomato Paste 20.0 g.
3 Primary Yeast 10.0 g.
4 Dextrin (Amidex~ 20.0 g.
CoCl~ 6H2O 5.0 ~g.
6 Distllled H2O 1000 ml.
7 pH: adjust to 7.2-7.4 usi~g NaOH
8 After inoculation, the production flasks are
g incubated at 24C., with agitation on a 212 rpm shaker
(2" throw), for ~our days and five hours. The flasks are
11 harvested and assayed for activity by using standard
12 Salmonella gallinarum MB1287 and Vibro percolans ATCC 8461
13 assay plates using 1/2 inch as~ay discs dipped into
14 centrifuged broth samples. Samples are diluted with 0.02
phosphate bu~fer, pH 7.0 when nece~sary. ~he results are
16 tabulated below:
17 Harvest Age hours 101
18 pH 7.2
19 Salmonella gallinaru~
(mm. zone) 35
21 Vibrio percolans 1/10 dil
22 (mm. zone) 34
23 890 Assay Units 121
24 The total of 7.0 liters of whole broth obtained
from this fermentation is chilled to 3C. and centrifuged
26 in 200 ml. portions at 9000 rpm for 15 minutes each. ~o the
27 combined supernatant is added 1~7 ml. of 0.lM neutral EDTA ~
28 and the batch is held at 3~C. ;:
29 The abovs fermentation is repeated under identical
condi~ions with the exception that the 44 two liter
31 Erlenmeyer produation flasks are inoculated with 7 ml.
32 per flask with growth from the seed flask. The pH and
33 assay results are tabulated below:
-157-
_. . _ .. __ . _ _ ... . . _ ... . . .
~83S7q
1 Harvest Age hour6 101
2 pH 7.3
3 Salmonella gallinarum
4 (mm. zone) 38
Vibrio percolans 1/10 dil
6 (mm. zone) ~-- 39
7 B90 Assay Units 92.8
8 The total of 7.4 liters of whole broth obtained
9 from this fermentation is chilled to 3C. and centrifugea
in 200 ml. portions at 9000 rpm for 15 minutes each. To
11 the combined supernatant i8 added 1.8 ml. of 0.lM
12 neutral EDTA.
13 The supernatant from the centrifuged broth
14 resulting from the two abo~e fermentations in this Example
are combined to give a ~ota~ Yolume of 13 liter~.
16 The combined supernatant is passed through a
17 column of Dowe~-l x 2 (Cl ), 50-100 ~esh, with bed
18 dimensions 4.7 cm. x 50 cm., at a flow rate of 60 ml.
19 per minute. The column is washed with 1 liter of
20 deionized water, and the antibiotic is eluted with 5 liters ~. :
21 o~ 5% (w/v) NaCl solution containing O.OlM Tris-HCl buffer,
22 pH 7.0, and 25~M neutral EDTA. Fractions of 220 ~1. are
23 collected at a ~low rate of 50 ml. per minute, and the
24 fractions are assayed on Salmonella gallinarum MB1287
._ ~
25 plateS.
26 Antibiotic activity appears in fractions 3 through
27 26, with a peak at fractions 5 and 6. Fractions 5 through 9
~re com~ined for further processing. The pH of the
29 pooled fractions i5 7~8, and the pooled fractions contain
,
'
-158-
.
~L083S7~
1 24% of the initial bioactivity, as measured on Salmonella
2 gallinarum MB1287 plates.
3 The combined fractions S through 9 are concen-
4 trated to 150 ml. by rotary evaporation under reduced
S pressure, and applied to a column (4.9 cm. x 47 cm. bed
6 dimensions) of XAD-2 which had been washed with 5 liters
7 of 60% (v/v) aqueous acetone followed by ~ liter~ of
8 deionized water, and ~ liters of 50 g./liter NaCl in
9 deionized water. The sample is applied in 20 ml. portions,
draining the column to bed level each tL~e. When appli-
11 cation is complete, three 20 ml. portions of deionized
12 water are applied and drained to bed level each time.
13 The sample is eluted with deionized water at a flow rate
14 of 20 ml~ per minute. All operations involving the XAD
column are carried out at room temperature (24C.), and
16 the fractions which are eluted are chilled rapidly in
17 an ice bath immediately after collection. Fractions of
18 95 to 230 ml. are collected.
19 Antibiotic activity appears in fractions 2
through 21, as measured by assay on Salmonella gallinarum
21 MB1287 plates~ with a peak at ~ractions 5 through 7 (being
22 510 to 895 ml. of eluted ~olume, counting from the first
23 application of dei~nlzed water). Fractions 6 through 21
24 are pooled~
The pooled fractions 6 through 21 are concentrated
to 68 ml. by rotary evaporation under reduced pre~sure, and
then diluted to 112 ml. by addition o~ deionized water~
~he concentrate is applied on a column (Z.2 cm. x 21 cm.
--159--
~. , ;
lV8357~7
1 bed dLmensions) of ~owex-l x 4 (Cl ) minu6 400 me~h.
2 The column is washed with 20 ml. of deionized water,
3 and the antibiotics are eluted with 2 liters of 0.07M
4 NaCl ~ 0.005M NH4Cl I 0.0001~ NH3 in deionized water,
at a flow rate o~ 1.6 ~l. per minuts. Fractions of
6 8 ml. each are collected. ~ractions 157-179 have the
7 greatest acti~ity and are pooled.
8 These pooled fractions are concentrated by
rotary evaporation under reduced pressure to 7 ml.,
and the pH adjusted to 7.5 by addition of 20 ~l. of
ll lM NaOH. The solution is concentrated further to 5 ml.,
12 and applied on a column (2.2 x 75 cm.) of Bio-~el P-2,
13 200-400 mesh. The sample is washed into the column bed -~-
14 with two rinses of 1 ml. each of deionized water, ana
eluted with deionized water at a flow rate of 0.6 ml. per
16 minute. Ten fractions of 3.3 ml. followed by sixty
17 fractions of Z.65 ml. and ten fractions of 2.0 ml. are
18 collected-
.
l9 The fractions are adjusted in the range pH 7
to 8.0 by addition of fxom 1~5 to 2.5 ~1. of 0.1M NaOH
21 Fractions 62, 63, 64 and 65 are frozen and lyophilized
`22 individually in 14 ml. glass vials, and stored at -20C.
23 under vacuum.
.
.
:
~... . ..
-160- -
. _
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., . :
15834IB
In order to isolate antibiotic 890A3 free from 890Al,
advantage is taken of the relatively higher resistance of
antibiotic 890A3 to degradation by penicillinase, as follows:
Bio-Gel fraction 63 is combined with fractions 61,
66 and 67 from the Bio-Gel column. To these four ccmbined
fractions is added 0.2 ml of lM Tris-HCl buffer, pH 7.5, and
0.2 ml. of penicillinase ~ifco "Bacto-Penase ~. After 113
minutes at 23C., an additional 0.2 ml of penicillinase is
added. After an additional 7 hours at room temperature, an-
other 0.2 ml of penicillinase is added, and after an additionaltwo hours at room temperature, the reaction mixture is chilled
in an ice bath. The terminated reaction mixture is diluted to
15 ml. by addition of 5 ml. of deionized ~ater.
The reaction mixture, is adsorbed on a Dowex 1 x 4
; (Cl ) minus 400 mesh column, bed dimensions 2.15 x 40 cm. The
antibiotic 890A3 is eluted with 0.07M NaCl ~ 0.005M ~IH4Cl +
O.OOOlM NH3 in deionized water, at a flow rate of 3 ml. per
minute. Fractiors of 13.8 ml. each are pooled.
~'
- 161 -
, ,;, .
S~
1 The combined fractions are concentrated to
2 4 ml. by rotary evaporation under reduced pres~ure, and
3 the concentrate is applied on a column (2.2 x 70 cm.) of
4 Bio-Gel P-~, 200-400 me~h. The antibiotic 890A3 is
elu~et with deionized water at a flow rate of 0.6 ml.
6 per minute. Thirty fractions of 3.3 ml. followed by
7 fifty fractions of 2.65 ml. are collected.
8 Fractions 66 through 70 are combined and the pooled
9 samples are concentrated to 1.5 ~l. by rotary evaporation
u~der reduced pressure, and the concentrate frozen and
11 lyophilized to give 5.4 mg. of solids containing the
12 antibiotic 890A3 plus residual salts. The N-acetyl group
13 is cleaved as above described to provide the free base
14 OH
FcH2cH2NH2
N ~ COOH
'.~ '
... .
.
-162-
.. ~
.
..
