Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
- 2 - ~34os97
Field of the Invention
The present invention relates to a selection of
cephalosporin compounds having the 3-((Z)-1-propenyl) and
7-phenylglycyla.mido groups, the latter may be substituted,
(Class 544, Subclass 16) and to methods of treating
bacterial infections employing these compounds (Class 424,
Subclass 246).
Description of the Prior Act
The 3-formylceph-3-em compounds used as
intermediates in one method of preparation of the
3-substituted vinyl cephalosporins of the present
invention may be prepared by oxidation of the
corresponding 3~-hydroxymethylceph-3-ems obtained by
enzymatic hydrolysis of the corresponding cephalosporins.
This process is~ represented in the prior art by
Chamberlin, U.S. Patent No. 3, 351, 596 (November 7, 1967)
who disclosed inter alia Compounds II, and III.
S
RCONH
~~J CHO I
O
C02CH3
R -
a~d
S --
NH2
II III
13469 7
- 3 -
Chamberlin (loc. cit.) disclosed derivatives at the 3-CHO
group with carbonyl reagents such as semicarbazide and
hydroxylamine. but there was no disclosure of any carbon
alkylation of the 3-CHO.
The correspanding sulfoxides are more stable and
can be prepared. in better yield (Webber, U.K. Patent
Specification 1,341,712, published December 23, 1973).
The first disclosure of 3-alkenyl substituted
cephalosporins was by Clark et al. in U.K. Patent
Specification 1,342,241, published January 3, 1974
(corresponding U.S. Patent Nos. 3, 769, 277, and 3, 994, 884,
granted October 30, 1973, and November 30, 1976. The
Compounds IV anal V are disclosed on pp.25 and 29 of the
U.K. Specification.
S j~H2CONH - S
~ \ CHCON ~ S
CH=CHCH
NH2 ~- H=CH2 / N ~ 3
O 0
C02H C02H
LV . V
These compounds were prepared by reacting the
corresponding 3~-triphenylphosphoniummethyl cephalosporin
with formaldehyde or acetaldehyde. The inverse process of
reacting a phos~phoranylidine derivative of the formula
R3P=CR3R4 with a 3-CHO cephalosporin is also
disclosed in the specification on page 5. Compound IV is
~3~os97
- 4 -
stated to be absorbed when given by the oral route in U.S.
Patent No. 4, 107, 431.
Another early disclosure of compounds of this
type was by Webber et: al. J. Med. Chem. 18(10) 986-992,
(1975), and in U.S. Patent No. 4,065,620 patented December
27, 1977 which discloses at columns 3, 4, and 5 the genus
to which the present compounds belong. Specific compounds
disclosed are represented by Formula VI.
j l C'ON ~ S
/ N ~ CH=CHC02H
_ 0 ~ (C02Et)
~ COZH (CN)
VI
Other variations of this type are disclosed in
U . S . Patent Nos . 4, 094, 978 ( June 13, 1978 ) , and 4, 112, 087
(September 5, 1978) where Compounds VII and VIII are
disclosed.
CH=CHCH OH
2
0
C02H VII
U.S. 4,094,978 Col. 44
HC~ ~ ~~CHCONH S
~1
NH2 N /
1340697
- 5 -
HO-- ~ ~ HCON
I . ~
NH2 N ~ OH=CHCH- NCH
/ ~ 2 3 VIII
O CO H (trans)
2
U.S. 4,112,087 Col. 31
Other substituted 3-alkenyl cephalosporins are
disclosed in the following patent publications.
U.S. 3, 830, 700, O' Callaghan et al. (April 20,
1974)
3-(nitrostyryl)cephalexin analogs
U.S. 3, 983, 113, Beeby (September 28, 1976),
U.S. 4, 049, 806, Beeby (September 20, 1977), and
U.S. 4,, 139, 618, Beeby (February 13, 1979),
3~-(heterocyclothio)propenyl cephalosporins
U.S. 4,, 147, 863, Miyadera et al. (April 3, 1979),
3~-(1-methyl-5-tetrazolyl)vinyl cephalosporins
Ger. Offen DE 3019445 (December, 1980)
3~-(sulfonyloxy)vinyl cephalosporins
Fr. 2400302 (January 23, 1981)
3~-(dimethylamino)vinyl cephalexin analogs
Eu 30630 ( June 24, 1981 )
7~-[(3-methanesulfonamidophenyl)- a
-~3minoacetamido]-3-
v:inylceph-3-em-4-carboxylic acid
U.S. 4,, 255, 423, Beattie et al. (March 10, 1981)
U.S. 4,, 390, 693, Beattie et al. (June 28, 1983)
7~-(2-thienyl)acetamido-3-(3-acetoxy-1-
p:ropenyl) and -3-(heterocyclovinyl)
ceph-3-em-4-carboxylic acids, and 7a-
methoxy analogs.
1340697
- 6 -
The principal commercially available orally
active cephalosporins, the use for which the present
compounds are intended, are cephalexin. cefadroxil,
cephradine, an~i cefaclor. These substances have Formulas
IX, X, XI, and XII.
S ~ S
HCONH HC
ONH
NH2 ~ ~ CH3 I
NH2. ~ 1
0 O
C02H C02H
R=H cephalexin iX Cefaclor XI
R=OH cefadroxil X
S
~HCON
_. NH2 ~ ~ CH3
C02H
cephradine XII
These compounds are the subjects of the following
patents.
cephalexin - U.S. 3,507,861 (April 21, 1970)
cefadroxil - U.S. 3,489,752 (January 13, 1970)
( Re 2 9, 164 )
cefaclor - U.S. 3, 925, 372 (December 9, 1975)
cephradine - U.S. 3,485,819 (December 23, 1969)
Relatead structures which have been disclosed are
3-chlorocefadroxil and 3-hydroxycefadroxil respectively in
U.S. 3,489,751 (January 13, 1970) and U.K. Specification
1,472,174 (published May 4, 1977).
- ' - ~13~069'~
Summary of the Invention
Thus the present invention provides a process for
the preparation of a cephalosporin of the formula
~~~ n
R2 / ~ CHCONH .
NF3P1 ' CH=CHR3
R1 O
C02p2
and the Z configuration about the exocyclic double bond
wherein
n is they integer 0,
R1 is hydrogen, OP3, lower alkoxy or halogen,
. Pl, P2, and P3 are hydrogen atoms or
conventianal protecting groups used in
. cep~halosporin chemistry respectively with
amino, carboxy, and hydroxy groups,
R2 is hydrogen, OP3 or lower alkoxy and
R3 is selected from the group consisting of
Cl_.4 alkyl and C~-14 aralkyl,
which comprises reacting in a reaction inert organic
liquid vehicle at 20 to~ 150°C a halide reactant of the
formula QCH2X and R3CH2X wherein X is C1, Br, or I
with a triarylpho;sphine to yield a phosphonium salt and
conversion of the latter in a water immiscible liauid
organic solvent with aqueous base to a phosphoranyl
intermediate of the formula QCH=PAr3 or R3CH=PAr3
followed by reaction of the latter under dry conditions at
-40° to +50° in said water immiscible liquid organic
solvent with a carbonyl reactant of the formula QCHO and
R3CH0 wherein one and only one of said halide reactant
and said cazbonyl reactant contains the group Q and Q is
selected from the group consisting of the following
formulas:
134os97
_ _8_
P (O) n
HCONx
I
1 NxP N /
C02P' R
O C02P2
(~)n
AcNH
8=N
N
O
COZP2 O
COZp2
wherein n is the integer 0 and Rl, Pl, P2, P3 and
R3 have the same meaning as previously and
Ac refers to an acyl group of the sort ordinarily
found in a cephalosporin, and
B is an alkylidene or aralkylidene protecting
group and thereafter converting the reaction product to
the desired product having the formula first given above
by a combination as necessary of one or more of removing
said blocking groups of the formulas P1, p2, P3 , Ac
and B and introducing the 7-acyl group of the formula
R2 ~ ~ CFiCO-
f
R1 NH2
into the resulting 3-substituted-7-aminoceph-3-em compound
wherein R1 and R2 are as previously defined and, if
desired, converting the resultant substance to a
pharmaceutically acceptable acid addition salt, wherein
~34os97
7-acyl group of the formula
2
R ~ ~ CHCO
R i NHZ
into the resulting 3-substituted-7-aminoceph-3-em compound
wherein R1 and RZ are as previously defined.
In certain a:>pects the present invention provides
compounds of Formulas ?~;III, and XIV
~D~n
2
R - ~HCO
NHP -~ N ~ CH=CHCH3
R O/
OzPz
Formula XIII
~O~n
2
R ~ ~ ~ HCONH ,
NHl?1 ~ CH=CHAIkX
R i
O
02P2
Formula XIV
In these formulas
N is th~~ integer 0, or 1
R1 is h~~drogen, OP3, lower alkoxy, or halogen
in~~luding chlorine, bromine, fluorine, and
iodine
-9-
X
.~ ~34os~7
- 10 -
P1, P'') and P3 are hvdroaen atoms or
conventional protecting groups used in
cephalosporin chemistry respectively with
avmino, carboxy, and hydroxy groups.
R2 is hydrogen, OP3, or lower alkoxy
Alk is an alkylidene or alkylene group having
from 1 to 4 carbon atoms, and
X is chlorine) bromine, or iodine.
Those compounds wherein n is 1, and P1, P2~
and P3 are conventional protecting groups, are
intermediates for making the biologically active end
products of the present invention which are represented by
Formula XIII when n is 0, and Pl, P2, and P3 are
hydrogen. These products are of interest as orally
effective cephalosporin antibiotics having strong activity
against Gram-positive bacteria and an improved spectrum of
activity against Gram-negative bacteria, various
fastidious bacteria, and anaerobes relative to cephalexin,
cefadroxil, cefaclor, and cephradine. They provide
prolonged antibiotic concentrations in the blood stream
following oral administration and are suitable for
administration to humans on a once or twice a day basis.
As such they are administered in doses ranging from 100
mg. to 5,000 mg, per day depending upon the size of the
patient and the disease condition. They may be
administered parenterally in similar dosage amounts.
The products of Formula XIV are of interest
chiefly as intermediates. Those, however, wherein n is 0,
and P1, P2, and P3 are hydrogen possess
antibacterial activity and are also useful as antibiotics.
In view of these properties. the compounds of
Formula XIII and Formula XIV wherein n is 0, and P1,
P2 and P3 are hydrogen are useful for the treatment of
bacterial infections caused by sensitive organisms in
mammals. For this purpose they are administered orally or
parenterally in antibacterially effective non-toxic doses
- 11 -
134069
as such or in t'he form of one of their pharmaceutically
acceptable acid addition salts, pharmaceutically
acceptable metal or amine salts, or as a pharmaceutically
acceptable ester.
The pharmaceutically acceptable acid addition
salts are those in which the anion does not contribute
significantly to the toxicity of the salt and which salts
are compatible with the customary pharmaceutical vehicles
and adapted for oral or parenteral administration. They
include the salts of Formulas XIII and XIV wherein n = 0,
and Pl is hydrogen with mineral acids such as
hydrochloric acid, hydrobromic acid, phosphoric acid, and
sulfuric acid, with organic carboxylic acids or organic
sulfonic acids such as acetic acid, citric acid, malefic
acid, succinic acid, benzoic acid, tartaric acid, fumaric
acid, mandelic acid, ascorbic acid, malic acid,
methanesulfonic acid, p-toluenesulfonic acid, and other
acids known and used in the penicillin and cephalosporin
arts. Preparation of these salts is carried out by
conventional techniques involving reaction of one of the
substances of Formulas XIII or XIV wherein n is 0 and P1
is hydrogen with the acid in a substantially equivalent
amount.
Pharmaceutically acceptable metal and amine salts
similarly are those salts of the compounds of Formulas
XIII and XIV wherein n is 0 and P2 is hydrogen which are
stable under ambient conditions, and in which the cation
does not contribute significantly to the toxicity or
biological activity of the salt. Suitable metal salts
include the sodium, potassium, barium, zinc, and aluminum
salts. The sodium or potassium salts are preferred.
Amine salts prepared from amines used for instance with
benzyl penicillin which are capable of forming stable
salts with the .acidic carboxyl group include
trialkylamines such as triethylamine, procaine,
dibenzylamine, 1~T-benzyl- ~ -phenethylamine, 1-ephenamine,
- 4340697
- 12 -
N,N'-dibenzylet:hylenediamine, dehydroabietylamine,
N-ethylpiperidi.ne, benzylamine, and dicyclohexylamine.
Pharmaceutically acceptable esters include those
esters which are active ~ se, or which serve as
pro-drugs by being hydrolyzed in the body to yield the
antibiotic ger se. Suitable esters of the latter type are
the phenacyl, a~cetoxymethyl, pivaloyloxmethyl,
a -acetoxyethyl., a-acetoxybenzyl, a -pivaloyloxyethyl,
3-phthalidyl, ~~-indanyl, methoxymethyl, benzoyloxymethyl)
a -ethylbutyryl.oxymethyl, propionyloxymethyl,
valeryloxymethyl) isobutyryloxymethyl, glycyloxymethyl,
and others known in the penicillin and cephalosporin arts.
The compounds of Formulas XIII and XIV wherein n
is 0, and P1, F'2, and P3 are hydrogen atoms and
their salts as defined above may be formulated for oral or
parenteral use in conventional manner using known
pharmaceutical carriers and excipients, and they may be
presented in unit dose form or in multiple dose
containers. The compositions may be in the form of
tablets, capsules, solutions, suspensions, or emulsions.
These compounds. may also be formulated as suppositories
utilizing conventional suppository bases such as cocoa
butter or other fatty materials. The compounds may, if
desired, be administered in combination with other
antibiotics including cephalosporins, penicillins, and
aminoglycosides;.
Detailed Description of the Invention
Table 1 contains a summary of the structures of
the products disclosed in Procedures 1-43. Most of these
compounds are TS -(D-phenylglycylamido)cephalosporins
having the 1-propen-1-yl group in the 3-position. The
terminal carbon atom of the propenyl substituent of some
of them bears a. subst_ituent such as an alkyl group
(methyl), halogen (chlorine or iodine), an aryl group
(phenyl), a het.erocyclothio group (1,2,3-triazol
5-ylthio), or a.n alkoxy group (methoxy). The
~3~069~
- 13 -
phenylglycylami.do group may be unsubstituted, or mono, or
disubstituted by hydroxy, alkoxy, or halogen.
Table 1
Products Disclosed in Procedures 1-43
R 2-
~~ CHCONH
1
R.1 NHZ N / CH=CHR3
O
02~
Com pound No.- Rf R2 R3 (configuration)
9 (BMY-28100) H OH -CH3 (Z)
13 (BBS-1058) H OH -C2H5 (Z)
11 (BBS-1064) H OH -H -
24 (BBS-1065) H H -CH3 (Z)
26 (BBS-1066) H H -CH2C1 (Z)
8 (BBS-1067) H OH -CH3 (E)
15 (BBS-1076) H OH -CH2C6H5 (Z)
r
21 (BBS-1091) H OH -C~' -5-~~~
2 r
H
17 (BBS-1092) H OH -CH20CH3 (Z
)
32 (BMY-28060) Cl OH -CH3 (Z
)
37 (BMY-28068) HO OH -CH3 (Z)
42 (BMY-28097) CH30 OH -CH3 (Z)
Table 2 provid es a summary of in vitro
the
antibacterial activity of the substances isclosed in
d the
present specif ication. Minimum inhibitory concentrations
determined by the agar dilution technique for threegroups
or organisms d esignatedGp-Ia, Gp-Ib, and Gn-Ia
are
.~340~97
- 14 -
provided. Each. of these groups of organisms is
constituted of five individual strains of microorganism
which are identified in a footnote to the table. The
Gp-Ia organisms are Gram+ staphylococci which are
sensitive to penicillin. The Gp-Ib organisms are Gram+
staphylococci which are resistant to penicillin and
produce penicillinase. The Gn-Ia organisms are Gram-
bacteria which are sensitive to ampicillin and
cephalothin. The present substances have generally low
activity against ampicillin and cephalothin resistant
Gram- bacteria. The following conclusions can be drawn
from Table 2 concerning the in vitro antibacterial
activity of these compounds.
All-of the compounds have good activity against
penicillin sensitive staphylococci (Gp-Ia). They are
generally less active against the penicillin resistant
staphylococci (Gp-Ib) by a factor of three or more. In
each instance, however, the compounds are several fold
more active than cephalexin and cefadroxil.
Only those compounds having the unsubstituted
cis(Z)- propenyl group in the 3-position have good
activity against the Gram- bacteria (Gn-Ia). Refer to
Compound Nos. 9, 24, 32, and 42. The trans(E)-propenyl
compound, Compound No. 8, is less active against the Gram-
bacteria by a factor of 8 relative to the corresponding
cis-propenyl compound., Compound No. 9. Similarly,
substitution on the terminal methyl group of the propenyl
subsituent in the 3-position appears to result in a
reduction of Gram- activity. Refer to Compound Nos. 13,
15, 21, and 17. This is true of the vinyl compound also,
No. 11. These compounds are nevertheless potent
antibacterial agents being substantially equivalent to
cephalexin and cefadroxil. Ring substitution is in no way
detrimental to antibacterial activity. Compare Compound
Nos. 9, 24, 32, and 42. Compound No. 37 appears to be an
exception to each of the foregoing conclusions. but in
15 - 134Q 69?'
fac t, it is ghly active substance against
a hi both the
Gra m+ and Gram-bacteria as will be shown in
Table 3.
Table 2
Agar I>ilution Agar)
Technique
(Mueller-Hinton
Minimum Inhibitory Concentration (mcg/ml)
Gp Ia3 Gp Ib3 Gn Ia3
Com pound No. 1 2 1 2 1 2
9 (BMY-28100) 0.23 0.35 0.92 0.8 0.8 0.7
13 (BBS-1058) 0.40 1.4 4.1
11 (BBS-1064) 0.40 1.2 3.6
24 (BBS-1065) 0.23 0.3 0.92 0.92 0.8 0.8
8 (BBS-1067) 0.26 1.4 6.3
(BBS-1076) 0.20 0.7 >50
21 (BBS-1091) 0.61 2.7 2.7
15 17 (BBS-1092) 0.53 2.1 2.7
32 (BMY-28060) 0.13 0.53 1.1
37 (BMY-28068) 6.30 7.2 6.3
42 (BMY-28097) 0.354 1.24 0.534
cephalexin 1.2 0.70 4.1 3.6 6.2 4.1
Cef adroxil 1.2 1.10 3.6 4.1 8.3 8.3
1. Columns 1 and 2 report separate test
runs.
2
3. Average of five organisms each group - GP IA
Gra;m+ s taphylococci: penicillin sensitive:
no
penicil linase produced.
S. aureus Smith A9537
S. aureus A9497
S. aureus Terajima
S. aureus A9534
S. aureus A9601
Gp IB G ram+ staphylococci; penicillin resistant;
penicil linase producers.
S. aureus 193
S. aureus BX-1633.-2 A9606
S, aureus A15092
1340697
- 16 -
S. aureus Russell
S.. aureus A9602
Gn Ia Gram- bacteria: ampicillin and cephalothin
sensitive.
E'., calf Juhl A15119
E ( calf A9660
K.. pneumoniae D11
P'. mirabilis A9554
P'. mi.rabilis A9900
4. No part of run 1: tested separately.
Table 3 contains comparative data for in vitro
antibacterial activity against the same organisms as in
Table 2 employing two different bacteriological culture
media. Mueller-Hinton agar is the standard medium
employed in the tests referred to in Table 2. Table 3
contains a comparison of the minimum inhibitory
concentrations of three of the test compounds determined
first in Mueller-Hint.on medium and then in nutrient agar.
Compound No._9 which contains 4-hydroxy substitution in
the phenyl ring and Compound No. 42 which contains the
3-methoxy-4-hydroxy substitution in the phenyl ring
reflect only a moderate medium effect. By this it is
meant that the differences in MIC are less than three
fold. Compound No. 37, the 3,4-dihydroxyphenyl
substituted compound reflects differences in activity
between the two media. of from 6 to 12 fold, the minimum
inhibitory concentrations in nutrient agar being much
lower than those determined Mueller-Hinton agar.
Accordingly, Compound No. 37 was concluded to be
comparable in antibacterial effect to the other
cephalosporins having the cis-propenyl group in the
3-position which are referred to in Table 2. This
phenomenon whereby an antibiotic shows greater activity in
one type of nutrient medium than in another has been
reported and studied previously. Refer to T.A. Pursiano
et al. Antimicrobial. Agents and Chemotherapy, Vol. 3, No.
1, pp.33-39 (1973).
- 17 - 1340697
Table 3
Test Medium Comparison
Agar Dilution Technique
Minimuia inhibitory Concentration (mc g/ml)
Compound No. Gp Ia2 Gp Ib2 Gn Ia2
9 (BMY-28100)A.l C1,23 0.92 0.70
B C1.17 0.35 0.70
37 (BMY-28068)A. 4,8 6.3 5.5
B 0.40 0.61 0.92
42 (BMY-28097)A. 0.35 1.2 0.53
B G.23 0.40 0.40
lA Mueller-Hint:on agar
B Nutrient agar
2 Average values for the same groups of organisms
as in Table 2
The structure activity correlations drawn from
the foregoing in vitro studies are born out
by the results
of in vivo studies in mice. Table 4 is a tabulation
of
the protective doses for mice infected with lethal
a
inoculum of a bacteria. Two different bacter ia were
employed in the studies, one a Gram+ organism
and the
other a Gram- organism. The protective dose (PD50) is
that dose which when administered to a group of infected
mice results in 50$ survival after five days. Normally
untreated infected mice die within three days following
injection of the lethal inoculum.
Table 4
Protective Dose for Mice Infected with Lethal
Inoculuml
Oral Treatment
Compound No. S. aureus Smith E , coli Juhl
9 (BMY-28100) 0.14 (0.31)2 1.2 (8.4)2
13 (BBS-1058) 0.32 (0.31) 3.0 (8.4)
11 (BBS-1064) 0,18 (0.31) 3.8 (8.4)
24 (BBS-1065) 0.18 (0.27) 1.5 (8.2)
8 (BBS-1067) 0.20 (0.31) 7.5 (8.2)
32 (BMY-28060) 0.17 (0.22) 3.04(8.4)
1340697
- 18 -
37 (BMY-28068) 0.13 (0.27) 0.44(8.2)
42 (BMY-28097) 0.093
1 Dose in mg/kg. preventing death for 5 days in 50%
of the animals in groups of 5 mice treated with
various doses of the test compound on the day of
infection determined by interpolation from the
dose/response curve: untreated animals die within
3 days.
2 Values in parentheses are for cephalexin in the
same run.
3 In this run a value of 0.16 mg/kg. was obtained
for BM.Y-28100: control values for cephalexin or
cefadroxil not available.
The_.data in Table 4 are drawn from several
different experiments. In these experiments cephalexin
was used as a control. treatment. The PD50 value
determined for cephal.exin in the same experiment is given
in parentheses next t.o the PD50 values of the test
compounds. It is evident that each of the cephalosporins
possesses good activity against the Gram+ Staphylococcus
aureus infection, and that the compounds bearing the cis
propenyl group in they 3-position are more active against
the Gram- infection, Compounds 9, 24, and 37.
Table 5 contains comparative blood-level data for
mice treated orally and intramuscularly with the test
compounds listed in Z'able 1. Uniformly good oral
absorption is reflected except for Compound No. 21 which
bears a heterocyclothio substituent on the 3-propenyl
group. Compound No. 37 exhibits exceptionally high blood
levels in the mouse following oral administration. This
compound has been shown to be metabolized in the rat to
Compound No. 42. Refer to Procedure 43. Compound No. 37
is the 3,4-dihydroxyphenyl compound and Compound No. 42 is
the 3-methoxy-4-hydraxyphenyl compound. The latter has
been shown to have high in vitro and in vivo activity.
. .- ~ ~34os97
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C ~ W tn Cl) tl) fn V) U! tn N O N i~ ~ O
O ~ O ft~ fn OO GI ft1 CL1 W ..~ a m ~ ~ a G
ri pp r1 tp fa ~C GC _In m _LC N 'O N <t) fa OD (r1 'O rt
t Ip b d
O ' 01 " M r-1 V' O 1!1 r-1 n Q~ N ~ 01 V~ N n d
V Q', .-1 ri N r1 N .-1 V V ~" N M M V
u1 O ~f1 O
r~1 r~1 N
- 20 - 1340697
Table 6 contains additional in vivo data for
Compound No. 9 against four other organisms compared to
cephalexin, cefachlor, and cefadroxil. Tables 7 and 8
contain comparative in vitro data for Compound No. 9
versus cephalexin, cefadroxil, and cephachlor with respect
to a nummer of Streptococci, Neisseria, Haemophilia, and
various anaerobes.
In rat urinary recovery experiments, the 24 hour
recovery of Compound No. 9 from the urine of rats treated
orally is comparable to that of cephalexin and cefadroxil,
and greater than that. of cefachlor. Stability studies
comparing Compound No. 9 with cephalexin and cefachlor in
solution using phosphate buffer at pH 6.5 and pH 7.0,
human serum (pH 6.8), horse serum (pH 7.6), and calf serum
(pH 8.2) as vehicles has revealed that Compound No. 9 is
remarkably more stable than cefachlor and comparable to
cephalexin.
Table 6
_, 1?rotective Dose PD50 for Mice
:infected with Lethal Inoculum
Oral Treatment
Organism 9 (BMY-28100) Cephalexin Cefachlor Cefadroxil
S. aureus
BX-1633 2.2 17 2.2 7.2
S. pyogenes
A20201 0.11 0.74 0.14 0.25
H. influenza
A9729 1.8 18 1.6 25
P. mirabilis
A9554 1..8 12.5 1.8 14
1340697
- 21 -
a
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01
pp py r ~p r t~M M M N 1~ .1 ~ir-1 C1
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O O d'd' ar N '-1.1 111M d'~ .1 d' ehN 1~
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d
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ro ro
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- 23 -
134~s97
TABLE 8
In Vitro Activity Against Anaerobes
MIC (mcg/ml)
S-~c 9(BMY-
Organi.sm tamase 28100) CephalexinCefachlor
Gn, rods B. Fragi.lis A20928-1 (-) 0.8 12.5 3.1
" A21900 (-)* 50 12.5 6.3
A20935 (-) 0.8 6.3 3.1
Geometric Mean 3.2 9.9 3.9
Gn, rodsB. Fragi.lis A22053 (+) 50 25 100
A22021 (+) > 100 100 >100
A22693 (+) > 100 >100 >100
Geometric Mean > 100 >75 > 100
A22695 (+) >100 100 100
A22533 (+) >100 >100 >100
Geometric Mean >100 >100 >100
Gp, rods C. difficile A21675 * 6.3 100 25
C. perfringens A9645 0.4 12.5 1.6
Gp, cocci P. acnes~ A21933 0.4 1.6 0.8
P. anaerobius A21905 0.8 6.3 0.4
Geometric Mean 0.95 11
* clindamycin resistant
1340697
The compounds of the present invention are
prepared by application of the synthetic routes disclosed in
U.K. Specification No. 1,342,241, U.S. patent No. 3,994,884,
and U.S. Patent No. 4,107,431, which are cited above, to the
appropriately selected starting materials. In essence,
formation of the substituted vinyl group in the 3-position
of the cephalosporins of the present invention involves
reaction of a halide rE:actant with a triarylphosphine to
yield a phosphonium salt which on treatment with base yields
a phosphoranyl intermediate. The latter is then treated
with a carbonyl reactant to produce the compound of the
present invention. Either the halide reactant or the
carbonyl reactant contains the cephalosporin nucleus. This
is illustrated in the following reaction schemes.
15Halide Ph.osphoranyl Carbonyl
Reactant Intermediate Reactant '~
U
b
QCHZX (C6Hs ) 3P H
x
~
--~ C QCH=P ( CsHs ) 3 ] 0
C=0
base
R3
m
Reaction Scheme 1
20Reaction Scheme ? QCH=CHR3"~Formulas
XIII or
H XIV
'CHX
( C6Hs ) 3P H QCH=0
C=P
C
H
(
3 6
s) s]
R base
R
.
25 In the foregoing reaction schemes, R3
is H, C1_9
alkyl, C1_4alkoxy--C1_9-alkyl, C~_14 aralkyl, or group alkX
the
wherein k and X are as previously defined. The symbol Q
Al
refers to the 7-amino-3-cephem-3-yl-4-carboxylic acid
nucleus erein the amino and carboxylic acid ups may
wh gro
30bear prote cting groups such as the silyl group other
or
groups whi ch are well-known to
-24-
134069?'
- 25 -
those skilled :in the chemistry of the beta-lactam
antibiotics, o:r Q may be a 7-acylamino-3-
cephem-3-yl-4-carboxylic acid nucleus where the
7-acylamino group may be one which conventionally appears
in cephalospor:in antibiotics including the a-amino-a
substituted phenylacetamido group of the present invention
as defined with respect to Formulas XIII and IX. The
sulfoxides of the foregoing have advantages.
Specifically, Q has one of the following formulas:
( ) (q)
S
P1N H ~ S
1 I
N I R NHP1
O ~ ' ~ i
_. C02P2 O C02P2
(i ) n (0) n
~S S
. Ac:NH'~I B=N
DT ~ ( ~ N
O ( O
C02P2 C02P2
35
1340697
- 26 -
wherein:
R1 has the same meaning as previously
n is the integer 0, or 1 referring to the number
of oxygen atoms attached to sulfur,
Ac refers to an acyl group of the sort ordinarily
found in the 7-acylaminocephalosporins such as
phenylacetyl. phenoxyacetyl and
B is an alkylidene or aralkylidene protecting
group derived from an aldehyde or ketone such as the
benzylidene group which is easily removed at a subsequent
stage for instance by hydrolysis using Girard's Reagent T.
P1, P2, and P3 are hydrogen atoms or
protecting groups of the sort conventionally used in
cephalosporin clhemistry with amino groups, hydroxy groups,
and the carboxyl group)
Suitable carbonyl protecting groups (P2)
include aralkyl groups such as benzyl, p-methoxybenzyl,
p-nitrobenzyl, and diphenylmethyl (benzhydryl) alkyl
groups such as 't-butyl; haloalkyl groups such as
2,2,2-trichloroethyl, and other carboxyl protecting groups
described in the literature, for instance, in British
Specification 1"399,086. We prefer to utilize carboxyl
protecting groups which are readily removed by treatment
with acid, particularly benzyhydryl or t-butyl.
Amino and hydroxy protecting groups (P1 and
P3) are well-known in the art and include the trityl and
acyl groups such as c'hloroacetyl, formyl, trichloro-
ethoxycarbonyl, tert.-butoxycarbonyl, carbobenzyloxy,
etc. Again amino protecting groups which are readily
removed by treatment with acid are preferred, particularly
the tert.-butox~tcarbonyl group.
In Reaction Schemes 1 and 2 when cephalosporin
nucleus Q is utilized in the form of the 1-oxide (n=1) the
oxides are prepared by known procedures such as by
oxidation of then corresponding cephalosporin (n=0) with
m-chloroperbenzoic acid or peracetic acid. At some later
1340697
- 27 -
stage in the synthesis the 1-oxide is reduced by known
procedures, for example by reduction with iodide ion in an
aqueous medium.
Conversion of the halide reactant of the formula
QCH2X according to Scheme 1 to the phosphoranyl
intermediate is preferably carried out employing a halide
reactant wherein X is iodide. If a chloride or bromide
halide reactant is used it may be first transformed into
the iodide by treatment with sodium iodide in
dimethylformamide or acetone solution. The iodide
reactant readihcr reacts with a triarylphosphine such as
triphenylphosph:ine in an organic liquid vehicle which is
inert to the reactants under the reaction conditions.
Room temperature for a brief period of up to several hours
constitute suitable conditions. Suitable triaryl-
phosphines in addition to triphenylphosphine include the
readily availab:Le compounds having reaction compatible
aryl groups such as substituted phenyl e.g. tolyl,
naphthyl, substituted naphthyl, and heteroaromatic or
substituted heteroaromatic groups. The first stage of the
reaction involves formation of the triarylphosphonium salt
which ordinaril:~ precipitates from solution and is
collected on a :filter. The triarylphosphonium salt is
then dissolved :in a suitable liquid organic solvent which
is water immisc:ible and inert under the reaction
conditions such as chloroform, trichloroethylene, or other
polychlorinated or brominated methane or ethane. The
phosphoranyl intermediate is then produced in situ by
treatment of the solution with aqueous alkali metal
carbonate, bicarbonate, or hydroxide at room temperature.
The organic layer containing the phosphoranyl intermediate
is separated, washed with water, and dried in the usual
fashion. The carbonyl reactant shown in the reaction
scheme is them :added to the dry solution of the
phosphoranyl intermediate and the final step of the
reaction then t~3kes places at room temperature again
1340697
- 28 -
within a relatively brief reaction time of from about 2 to
20 hours. The desired product represented by the formula
QCH=CAR3 is recovered by techniques known to those
skilled in organic chemical laboratory procedures such as
chromatography on a silica gel column.
The halide reactants of the formula QCH2X of
Scheme 1 are produced from the corresponding 7-amino or
7-acylamino-3-h.ydroxymethyl-ceph-3-em-4-carboxylic acid
derivatives by methods which are known in principle.
Conversion of the halide reactant of the formula
R3CH2X according to Scheme 2 to the phosphoranyl
intermediate may be carried out with either the chloride,
bromide or iodide (X=C1, Br, or I). If desired the
chloride or bromide may be transformed to the iodide as
before, but this is not essential. Reaction with the
triarylphosphin.e such as triphenylphosphine is carried out
either without a solvent or in an organic liquid vehicle
which is inert under the reaction conditions. Room
temperature or elevated temperatures for a period of from
1 to 24 hours at 20°C: to 150°C may be employed. The
triarylphosphon.ium salt ordinarily precipitates and is
collected on a filter. It is then dissolved in a suitable
liquid organic solvent such as dimethylsulfoxide or one
which is immiscible with water such as ether, or
tetrahydrofuran., and treated with a base such as butyl
lithium, phenyl lithium, sodium methoxide, or sodium
hydride for a ~~eriod of from several minutes to several
hours at a temperature in the range of -40°C to +50°C.
The carbonyl reactant: is then added to the dry reaction
solution and th.e reaction is allowed to take place at
-40°C to +50°C for from one to several hours. The desired
product represented by the formula
B
Q~C ~
.1340697
- 29 -
is recovered as. before.
Scheme: 1 has been found convenient for
preparation of those substances of the formula QCA=CHR3
in which R3 is lower alkyl, phenylalkyl, naphthalkyl,
haloalkyl, or alkoxyalkyl in the cis-(Z) configuration.
According to one variation of Scheme 1, which we refer to
as Method A, 7~~ -[a(N-t-butoxy-carbonylamino)-2-
(p-hydroxyphenyl)acetamido]-3-chloromethyl-3-cephem-4-
carboxylic acidi benzhydryl ester is used as halide
reactant. This is illustrated in Procedures 4, 5, and 6
hereof.
A further variation of Scheme 1 which we have
found convenient is similar to Method A in that 7S-
[a(N-t-butoxyca.rbonylamino)-2-(p-hydroxyphenyl)
acetamido]-3-chloromethyl-3-cephem-4-carboxylic acid
benzhydryl ester is employed as starting material, but in
method B chloroacetal.dehyde is employed to produce the
blocked 7-aminocephalosporanic acid having the
3-chloro-1-propen-1-yl group in the 3-position. The
latter material. possesses antibacterial activity, but not
to an outstanding extent. In Method B the
3-chloro-1-proF~en-1-yl compound is employed as an
intermediate ar.~d concerted first to the corresponding
3-iodo-1-propen~-1-yl compound which is then converted with
heteroaromatic thiols to produce 3-heteroarylthioprop-1-
en-1-yl-cephalosporin derivatives.
A further variation of Scheme 1 we refer to as
Method C. In this variation 7-amino-3-chloromethyl-
3-cephem-4-carboxylic: acid benzhydryl ester is prepared as
before and the 7-amino group is protected by reaction with
benzaldehyde to produce the benzylidene protecting group.
The latter is then treated with triphenyl-
phosphine to provide the phosphonium salt which is then
converted with base to the phosphoranyl intermediate and
the latter is treated with an aldehyde to give the
3-substituted vinyl-7-aminocephalosporanic acid which then
1340697
- 30 -
may be acylated to introduce the desired acyl group into
the 7-position.
Two variations of Scheme 2 are proposed. In the
first, Method D,
3-hydroxymethyl-7-phenylacetamido-3-cephem-4-carboxylic
acid prepared as described above in which the carboxylic
acid is protected as the benzhydryl ester is converted to
the corresponding 3-formyl compound. The latter is then
allowed to react with the phosphoranyl intermediate
derived from a 'halide of the formula R3CH2X as shown
in Scheme 2, and the desired 7-acylamino group is
introduced by acyl exchange.
Method E is a further variation of Reaction
Scheme 2 in which blocked 7-p-hydroxyphenylglycyl-
amido-3-formyl-3-cephem-4-carboxylic acid is used as
carbonyl reactant.
7-Phen,ylacetamidocephalosporanic acid is a
convenient starting material in view of its ready
availability. 'The acetoxy group thereof may be readily
hydrolyzed enzymatically employing wheat bran as the
enzyme source to yield 7-phenylacetamido-3-hydroxymethyl-
ceph-3-em-4-carboxylic acid. The carboxylic acid group
may be protected by conversion to the benzhydryl ester by
treatment of the acid with diphenyldiazomethane. The
ester is then treated with phosphorus pentachloride under
known conditions which result in cleavage of the 7-
phenylacetyl group and conversion of the 3-hydroxymethyl
group to a 3-chloromethyl group. The production of
7-amino-3-chloromethyl-3-cephem-4-carboxylic acid
benzhydryl ester by these methods is illustrated in
Procedures 1 and 2.
Alternatively the 7-phenylacetamido-3-
hydroxymethylce;ph-3-em-4-carboxylic acid may be converted
to the 3-halomethyl compound and thence to the
phosphoranyl intermediate followed by reaction with an
aldehyde to produce the substituted 3-vinyl-cephalosporin
according to one of the variants of Reaction Scheme 1.
-- 1340697
- 31 -
The cephalosporin-3-carboxaldehyde represented by
the formula QCH==0 in the above reaction scheme which
serves as carbonyl reactant in Reaction Scheme 2 is
produced by oxidation of a 7-acylamino-3-hydroxymethyl-
ceph-3-em-4-carboxylic acid ester as is described in U.S.
Patent No. 3, 35:L, 596 cited above. Reaction Scheme 2 is
the less preferred of the two routes shown, and does not
seem to be suitable for the propenyl products of Formula
XIII.
The compounds having the formula QCH=CHR3
exists in the _c:i_s(Z)- and trans(E)-configurations. Those
compounds which have the cis(or Z)-configuration are
preferred. The;~r have greater antibacterial activity than
the corresponding substances having the trans(or
E)-configuration. The compounds of Formula XIV are useful
as intermediates for the preparation of other
cephalosporins 'having the formula QCH~CHR3 wherein R3
is the methylen~e group substituted with the residue of a
nucleophilic group such as the mercapto, alkylmercapto,
arylmercapto, or heteroarylmercapto groups such as 1,2,3-
triazol-5-ylmercapto and 2-methyl-6-pyridinylmercapto.
This is illustrated below in Procedure 20. The iodomethyl
compounds are preferred as intermediates for nucleophilic
displacement processes.
Scheme 1 is adapted to preparation of a product
of Formula XIV 'by substitution of the appropriate carbonyl
reactant of the formula XAIkCHO for the R3CH0 reactant
shown.
35
1340697
- 32 -
Preparative Procedures
Procedure 1
Benzhydryl 3-Hy~droxymethyl-7S-phenylacetamido-3-cephem-4-
carboxylate (Compound 1)
To a stirred suspension of phosphate buffer (pH
7, 162.5 ml) and wheat bran (20 g, dry) at room
temperature was added 7-phenylacetamidocephalosporanic
acid sodium salt (5 gm, 12.1 mmole) in one portion. The
progress of the reaction was monitored by HPLC until the
hydrolysis was complete (5 hours). The suspension was
filtered to remove the wheat bran and the filtrate was
cooled to 5-10°C for extractive esterification. To the
cooled solution was added methylene chloride (32 ml)
followed by a 0.5 M solution of diphenyldiazomethane in
methylene chloride (24 ml). The pH was then adjusted to
3.0 with 28$ phosphoric acid. After 1 hour the reaction
mixture was allowed to rise to 20°C. Heptane (56 ml) was
slowly added and the resulting crystalline title product
was recovered by filtration. Yield of product Was 3.0 gm
(50$).
Procedure 2
Benzhydryl 7S-Amino-3-chloromethyl-3-cephem-4-carboxy-
late (2)
S
13 N
2
N
~g2C1
C~
COOCH(Ph)2
434069?'
- 33 -
To a slurry of PC15 (8.3 g, 40 mmoles) in
CH2C12 (1000 ml) was added pyridine (3.2 g, 40 mmoles)
and the mixture was stirred for 20 minutes at 20°C. To
the mixture was added benzhydryl 3-hydroxymethyl-7-
phenylacetamido-3-cephem-4-carboxylate (1), 5.1 g, 10
mmoles, with stirring at -40°C, in one portion. The
mixture was stirred at -10°C for 15 minutes and allowed to
stand at -10°C to -15°C for 7 hours. To the cooled
solution (1-20°C) was added propane-1,3-diol (10 ml) and
the mixture was allowed to stand at -20°C for 16 hours and
then at room temperature for 20 minutes with stirring.
The resulting solution was washed with ice-water (2 x 20
ml) and saturated aqueous NaCl (10 ml), dried over MgS04
and concentrated _in vacuo The gummy residue (12 g) was
dissolved in a mixture of CHC13 and n-hexane (2:1), and
subjected to chromatography using a silica gel column
(200 g) and the same solvent as eluant. Fractions
containing the 'title compound were evaporated in vacuo and
the residue triturated with n-hexane to give 2 (2.1 g,
51% ), melting a~t 110°C (dec. ) .
IR . ~~r 3400, 2800, 1785, 1725 cm 1.
UV: ~ Etc~H 265 nm (E1$ 160).
ma:K 1 cm
NMR: d DMSO--d6 + CDC13 3. 69 ( 2H, s ) 4.43
ppm ( 2H, s ) , 5 . 09 ( 1H, d, J = 4. 5
Hz), 5.24 (1H, d, J = 4.5
Hz ) , 6 . 87 ( 1H, s ) , 7 . 3 ( lOH,
m))
Procedure 3
Benzhydryl 7S-L1~-2-(t-butoxycarbonylamino)-2-(p-hydroxy-
phenyl)-acetamido]-3-chloromethyl-3-cephem-4-carboxylate
(Compound 3)
1340697
- 34 -
HO-
~ H2C1
NH
O
C02 C(CH 3 )'3 CO CH lC H )
2 6 5 2
To a mixture of 20.7 g (0.05 mol) of benzyhydryl
7-amino-3-chloromethyl-3-cephem-4-carboxylate (2) and 20 g
(0.075 mol) of D-2-(t.-butoxycarbonylamino)-
2-(p-hydroxyphenyl)-acetic acid in 500 ml of dry
tetrahydrofuran (THF) was added 15.45 g (0.075 mol) of
N,N'-dicyclohexylcarbodiimide (DCC) and the mixture was
stirred at room temperature for 2 hours and evaporated to
dryness. The residue was dissolved in 1 1. of ethyl
acetate (AcOEt) and the insoluble dicyclohexylurea was
removed by filtration. The filtrate was washed with an
aqueous sodium bicarbonate solution, water and saturated
aqueous NaCl~solutiora, dried on anhydrous sodium sulfate
and evaporated to dryness. The oily residue was
chromatographed on a column of, silica gel (Wako* gel
C-100, 500 g) (Trade Mark) by eluting with 4 liters of
chloroform and 6 liters of 1% chloroform-methanol. The
desired fractions were combined and evaporated to
dryness. The oily residue was triturated with
ether-isopropyl ether to give 30.6 g (92%) of 3.
IR : ~KBr cm 1 1790, 1710, 1670, 1500,
max 1360, 1230, 1150.
NMR , 6 CDC1 ;~ ppm l . 45 ( 9H, s, C-CH3 ) , 3 ( 4
( 2H, br-s, 2-H ) , 4 . 28 ( 2H, s, CH 2C1 ) , 4. 86 ( 1H, d, 4. 5
Hz, 6-H) 5.12 (1H, d, 6 Hz, CH-CO), 5.68 (1H, d-d, 8 & 4.5
Hz, 7-H ) , 6. 63 ( 2H, d, 9 Hz, phenyl-H ) , 6. 93 ( 1H, s,
CH-Ph2 ) , 7 . 08 ( 2H, d, 9Hz, phenyl-H ) , 7 . 0-7. 5 ( lOH, m,
Phenyl-H).
x.340697
- 35 -
The oily residue may be used without chromatographic
purification in. Procedure 4.
Procedure 4
Benzhydryl 7R-[D-2-(t-butoxycarbonylamino)-2-(p-hydroxy
phenyl)- acetam,ido]-3-iodomethyl-3-cephem-4-carboxylate
(Compound 4)
A mixture of 26.6 g (0.04 mol) of 3 and 18 g
(0.12 mol) of sodium iodide in 400 ml of acetone was
stirred at roomy temperature for 2 hours and evaporated to
dryness. The residue was extracted with 400 ml of ethyl
acetate and the: extract was washed with an aqueous
Na2S203 solution, wager, and a saturated aqueous
NaCl solution. After evaporation of the solvent, the
residue was triturated with ether-isopropyl ether to give
27 g (89%) of the title compound. The ethyl acetate
solution may be used directly in the next step (Compound
5) without isolation of Compound 4 if desired.
IR . ~K3r
-- max cm 1 1790, 1710, 1670, 1500,
1360, 1220, 1150.
CDC1
NMR: 8 3 ppm 1.47 (9H, s, C-CH3), 3.3-3.6
( 2H, m, 2-H ) , 4 . 20 ( 2H, s, CH2 ) , 4. 89 ( 1H, d, 4. 5 Hz,
6-H ) , 5. 12 ( 1H, d, 6 Hz, CH-CO ) , 5. 68 ( 1H, d-d, 8 & 4. 5
Hz, 7-H ) , 6. 62 ( 2H, d, 9 Hz, phenyl-H ) ( 6 . 92 ( 1H, s,
CHPh2), 7.08 (2H, d, 9 Hz, phenyl-H), 7-7.5 (lOH, m,
phenyl-H).
35
- 36 -
Procedure 5
Benzhydryl 7S-[;D-2-(t-butoxycarbonylamino)-2-
(p-hydroxyphenyl)-acetamido]-3-(triphenylphosphonio)methyl-
3-cephem-4-carboxylate iodide (Compound 5)
HO ~ ~ - CHCONFi
NH N / CH~~(c6~~) 3 I~
COZC (CFi3) 3 0
C02CH(C6H5)2
A mixture of 15.1 g. (0.02 mol.) of 4 and 15.7 g.
(0.06 mol.) of triphenylphosphine in 200 ml. of ethyl
acetate was stirred at room temperature for one hour. The
resulting precipitate was collected by filtration to give
17.4 g. (85.5%) of 5, melting at 170-180°C. The filtrate
was concentrated to :L00 ml. and the concentrate was
diluted with 500 ml. of ether to give the second crop 1.1
g.) of 5. The total yield was 18.5 g. (91%). The overall
yield of 5 from 2 is 74.5%. This can be increased to
87.5% by omission of the purification and isolation steps
as indicated at~ove.
IR . ~ ICar
max cm 1 1780, 1610, 1490, 1420, 1350,
1240, 1150, 1090.
NMR: dI)MSO ppm 1. 42 ( 9H, s, C-CH3 ) , 3 . 45 ( 2H,
br-s, 2-H ) , 5- °.i . 4 ( 3H, m, 3-H & 6-H ) , 5 . 7 ( 1H, m, 7-H ) ,
6 . 63 ( 2H, d, 9 Hz, phenyl-H ) , 7 .1-7 . 45 ( 12H, m, phenyl-H ) ,
7 . 5-7 . 9 ( 15H, n~, phenyl-H ) .
Anal. Calcd for C52H49N307SPI: C, 61.36;
H, 4.85; N, 4.7.3; S, 3.15
Found.. C, 61.26; H, 4.82; N, 4.11; S, 3.92.
X340697
- 37 -
Procedure 6
Benzhydryl 7B-[D-2-(t-butoxycarbonylamino)-2-
(p-hvdroxyphenyl)-acetamido~-3-[(Z)-1-propen-1-yl]ceph-3-em-
4-carboxylate
(Compound 6)
\ S
HO ~ ~~CHC:ONH
!~H N / CH=CHCH3
CO.,C (CH3) 3 0
..
C02CH(C6H5)2
To a solution of 1.8 g. (1.77 m mol) of 5 in 100
ml. of chloroform was added 100 ml. of water containing
2 ml. (2 m mol.) of N sodium hydroxide and the mixture was
shaken for 5 minutes. The organic layer was separated,
washed with water and dried on anhydrous sodium sulfate.
The chloroform solution being filtered, the filtrate was
concentrated..to 50 ml. under reduced pressure. To the
concentrate wa.s added 1 g. of acetaldehyde and the mixture
was stirred at room temperature for 2 hours and evaporated
to dryness. T'he oily residue was chromatographed on a
silica gel column (Wako-gel C-200) 50 g.) by eluting with
chloroform anf~ chloroform-methanol (99:1). The desired
fractions were collected and evaporated to give 318 mg.
(28%) of the product _6, m.p. 120-130'C (dec.).
IR:vn ax cm 1 1780, 1670, 1710, 1490, 1360,
1210, 1150.
NMR: dCDCl3 ppm 1.3-1.5 (12H, m, C-CH3), 3.22
( 2H) br-s, 2H ) , 4. 90 ( 1H, d. 4. 5 Hz, 6-H ) , 5.15 ( 1H, br-d,
~-CO)) 5. 5-6.1 ( 3H) m, CH=CH & 7-H), 2 6.63 ( 2H, d, 9
Hz, phenyl-H), 6.91 (1H, s, CH-Ph). 7.09 (ZH, d, 9Hz,
phenyl-H ) , 7. 's'-7 . 5 (; lOH, m. phenyl-H ) .
* Trade-mark
-- 1340697
- 38 -
Procedure 7
Sodium 7~-[D-2-amino-2-(p-hydroxyphenyl)acetamido]-
3-[(Z)-1-propen-1-yl]-3-ceph-em-4-carboxylate (Compound 7,
BMY-28100 Sodium Salt.)
HO ~ '~HCONH .
NH2 N / CH=CHGH3 (cis)
0~
COOH
A mixture of 318 mg. (0.48 m mol) of 6 and 2.5
ml. of trifluoroaceti.c acid (TFA) was stirred at room
temperature for one r~our and then diluted with 50 ml. of
ether and 50 ml. of isopropyl ether. The precipitate
separated was collected by filtration and washed with
ether to give 188 mg. (77%) of the trifluoroacetate of 7,
which was dissolved i.n 2 ml. of methanol (MeOH). To the
solution was added 2 ml. (2 m mol) of a solution of sodium
2-ethylhexanoate (SEH) in ethyl acetate and the mixture
was diluted with 30 ml. of ethyl acetate to separate the
precipitate, which was collected by filtration, washed
with ether and dried in vacuo over P205 to give 144
mg. (73% from 6.) of crude 7. The crude product (135 mg.)
was dissolved in 10 ml. of water and the solution was
chromatographed on a column (25 mm x 100 mm) using about
20 ml. of the packing in the PrepPak-500/C18 (Trade
Mark) (Waters). The column was eluted with water and the
eluate containing the desired product were concentrated to
5 ml. and lyophilized to give 93 mg. (69%) of 7. M.p.
200°C (grad, de~c.). Estimated purity 60% (by HPLC).
I R . ~ KB r cm 1 1760, 1660, 1590,
max
1400, 1360, 1250
~34~697
- 39 -
UV . ~ maxsphate Buffer pH 7 nm(c ) 227
(11300), 280 (8200).
NMR : d D2~ ppm 1. 65 ( 3H, d 6 Hz, -C-CH3 ) ,
3 . 21 ( 1H, d, 18 Hz, 2-H ) , 3 . 52 ( 1H, d, 18 Hz, 2-H ) , 5. 12
( 1H, d, 4. 5 Hz, 6-H ) , 5 . 68 ( 1H, d, 4 . 5 Hz, 7-H ) , 5 . 5-5 . 9
( 1H, m, vinyl-H ), 5) 95 ( 1H, d, 11. 5 Hz, vinyl-H ), 6. 94
( 2H, d, 8 Hz, phenyl-H ) , 7 . 36 ( 2H, d, 8 Hz, phenyl-H ) .
Procedure 8
7S-[D-2-Amino-2-(p-hydroxyphenyl) acetamido]-3-[(Z)-1
-propen-1-yl~-3-cephem-4-carboxylic Acid (Compound 8,
BB-S1067)
The crude product produced in Procedure 7, crude
_7 prior to chromatographic purification, 11.9 g., was
dissolved in 50 ml. of 0.01 M phosphate buffer (pH
7.2)-methanol (85 . 1.5) and the solution was adjusted to
pH 6 with 6 N hydrochloric acid. This solution was
subjected to preparative high performance liquid
chromatography (PHLC) (prepPAK-500/C18, System 500,
Waters) (Trade Mark) by eluting with 0.01 M phosphate
buffer (pH 7.2) containing 15% methanol. The eluate was
monitored by analytical HPLC and the first 4 1. fraction
was found to contain cis isomer(BMY-281001. The second 1
1. fraction containing the trans isomer was collected and
concentrated to 500 ml. The concentrate was adjusted to
pH 3 with dilute hydrochloric acid and chromatographed on
an HP-20 column (100 ml.) by eluting with 1 1. each of
water and 30% methanol. The latter eluate, volume about
300 ml., was concentrated to 10 ml. and lyophilized to
give 290 mg. of the crude trans isomer (55% pure). This
material was dissolved in 100 ml. of 50% methanol and
treated with activated carbon. The filtrate was
concentrated to a volume of 20 ml. and allowed to stand
overnight at 5°C. The product crystallized as colourless
prisms which were collected by filtration and dried in
vacuo, 129 mg.. m.p. 230°C (dec.).
1340697
- 40 -
IR . ~ KBr cm 1 1760, 1680, 1590, 1550,
max 1520, 1450, 1390, 1350, 1240.
UV : phosphate buffer (pH 7) nm(e) 228
max (13000), 292 (16900).
NMR: sD20+Na2C03 ppm 1. 89 (3H, d, 6Hz,
C=C-CH3 ) , 3. 60 ( 2H, s, 2-H ) , 5. 13 ( 1H, d, 4. 5 Hz, 6-H ) ,
5. 20 ( 1H, s, CH--CO) ( 5. 68 ( 1H, d, 4. 5 Hz, 7-H), 5. 99 ( 1H,
d-q, 16 & 6 Hz ) ,, 6. 54 ( 1H, d, l6Hz ) , 6. 98 ( 2H, d, 9 Hz,
phenyl-H ) , 7 . 41 ( 2H, ~d, 9 Hz, phenyl-H ) .
Procedure 9
Crystalline 7S - [D-2-amino-(p-hydroxyphenyl) acetamido]-3
-[(Z)-1-propen-:L-yl]-3-cephem-4-carboxylic Acid
( Compound 9, BM's'-28100 )
The first 4 1. fraction obtained in the
preparative HPL~~ in Procedure 8 containing the cis isomer
(BMY-28100) was concentrated to a volume of 2 1. and the
concentrate adjnssted to pH 3 with dilute hydrochloric
acid. The solution was charged to a column containing
HP-20 (1 1.) and the column was washed with 6 1, of water
until the pH of the effluent was pH 7. The column was
then eluted with 4 1. of 30% aqueous methanol. The eluate
solution was monitored by HPLC and the appropriate
fractions were combined (about 2.5 1.) and concetrated to
50 ml. at a temperature less than 40°C at reduced
pressure. A crystalline precipitate formed. The
concentrate was cooled at 0°C for two hours and the
crystalline precipitate collected by filtration, washed
with 80$ aqueous acetone, then with 100% acetone and then
dried _in vacuo over P205 yielding 4.09 g, of the pure
crystalline desired product, melting point 218-220°C
(dec.), colorless prisms 95% pure as determined by HPLC
assay.
IR . ~ KBr cm 1 1750, 1680, 1560, 1520,
'max.
1460, 1390, 1350, 1270,
1235
UV : ~ phosphate buffer (pH 7) nm(e ) 228
max.
(12300), 279 (9800).
j340697
- 41 -
NMR: SD20+NaHC03 ppm 1.71 (3H, d, 6 Hz,
CH3 ) , 3 ( 27 ( 1H) d, 18 Hz, 2-H ) , 3. 59 ( 1H, d, 18 Hz, 2-H ) ,
5.18 ( 1H, d, 4., 5 Hz, 6-H), 5. 22 ( 1H, s, CHCO), 5. 73 ( 1H,
d, 4.5 Hz, 7-H), 5.5-6.0 (1H, m, CH=C), 6.02 (1H, d, 11
Hz, CH=C) , 6. 98 ( 2H, d, 9 Hz, phenyl-H ) ( 7 . 41 ( 2H, d, 9Hz,
phenyl-H).
Anal. Calcd for C18H19N305S.1/2H20: C,
54.26; H, 5.06 N, 10.55; S, 8.05 Found: C, 54.15, 54.19;
H, 5.13, 5.08: N, 10.30, 10.42; S, 8.38, 8.04.
The mother :liquor from the foregoing
crystallization was concentrated to a volume of 10 ml. and
treated with 20 ml. of acetone. After keeping the
solution overnight in the refrigerator a crystalline
precipitate ha~i formed which was collected by filtration
and dried _in vacuo over P205, weight 670 mg. (90% pure
by HPLC). A portion of this material, 560 mg.. was
dissolved in 200 ml. of 50% aqueous methanol and the
solution was treated with 0.5 g, of activated carbon and
filtered. The filtrate was concentrated at reduced
pressure and 40°C to a volume of 20 ml. and then kept for
five hours at °i°C. The product crystallized and was
collected by filtration, washed with acetone, and dried in
vacuo over P20,~ to yield 227 mg. of crystalline
BMY-28100 (98$'pure by HPLC). Lyophilization of the
mother liquor )yielded 181 mg. of SMY-28100 which was 95$
pure (HPL C).
Procedure 10
Diphenylmethyl 7 g-[D-2-(t-butoxycarbonylamino)-2-(p-
hydroxy-phenyl;lacetamido]-3-vinyl-3-cephem-4-carboxylate
(Compound 10)
A solution of 3 g. (2.95 m. mol.) of benzhydryl
7-[2-(N-t-butoaycarbonylamino)-2-(p-hydroxyphenyl)
acetamido]-3-(r.riphenylphosphonio)methyl-3-cephem-4-
carboxylate iodide (5) in 50 ml, of chloroform was shaken
with a mixture of 3 :ml. (3m. mol.) of 1 N NaOH and 50 ml.
of water at room temperature for 1 minute. The organic
layer was separated after the addition of a saturated NaCl
....
- 42 -
~~4~697
solution (20 ml) and washed with water (3 x 30 ml.). To
the organic solution was added 2.5 ml. of 35% aqueous
formaldehyde with vigorous stirring under water-cooling. '
The stirring was corit:inued for 20 minutes. The organic
layer was separated, dried over anhydrous Na2S04 and
concentrated in vacuo. The concentrate was placed on a
column of silica gel, which was eluted with CHC13
(600 ml.) and 2% MeOH in CHC13 (800 ml.) to give 850 mg.
(45%) of the title compound. TLC: Rf 0.48 [silica gel,
MeOH-CHC13 (1 . 10)].
Procedure 11
7~S-[D-2-Amino-2--(p-hydroxyphenyl)acetamido]-3-vinyl-3-
cephem-4-carboxylic Acid (Compound 11, BB-S1064)
A mixture of 850 mg. (1.32 m. mol.) of 10, and 5
ml. of 90% aqueous trifluoroacetic acid (TFA) was allowed
to stand at rooTn temperature for one hour and concentrated
to ca. 1 ml. _in vacuo. The concentrate was triturated
with 20 ml. of diisopropyl ether to give 679 mg. of yellow
powder, which was dissolved in 3 ml. of methanol and
subsequently di:Luted with 30 ml. of water. The solution
was passed through a column of HP-20 (50 ml.), which was
washed with 200 ml. of water and eluted with 250 ml. of
30% methanol. '.Phe eluate containing the desired compound
was concentrated and lyophilized to give 197 mg. (31%) of
the title compound, estimated purity, 60% by HPLC, m.p.
190°C (dec.).
IR , v~XCm 1 1760, 1680, 1615-1570, 1520.
UV : Phosphate buffer (pH 7) nm( a ) 228
max
(13500), 283 (14400).
NMR: dD20 ppm 3. 6 ( 2H, s, SCH2 ) , 5. 51
( 1H, d, SHz, 6-:H ) , 5. 73 ( 1H, d, 5 Hz, 7-H ) , 7 . 03 ( 2H, d, 8
Hz, phenyl-H ) ( 7 . 45 ( 2H, d, 9 Hz, phenyl-H ) .
Procedure 12
Di henylmethyl 7~-[D-2-(t-butoxycarbonylamino)-2-(p-
hydroxyphenyl)-acetamido]-3-[(Z)-1-buten-1-yl]-3-cephem-4-
carboxylate (Compound 12)
~34os97
- 43 -
A solution of 3 g. (2.95 m. mol.) of 5 in 50 ml.
of CHC13 was mixed with a mixture of 3.2 ml. (3.2 m.
mol.) of 1 _N NaOH and 50 ml. of water and the mixture was
shaken at room temperature for 3 minutes. The organic
layer was separated, washed with water (3 x 30 ml.) and a
saturated NaCl solution, and dried over anhydrous
Na2S04. To they solution was added 1.71 g. (29.5 m.
mol.) of propic>naldehyde. The mixture was stirred
overnight at room temperature and concentrated under
reduced pressure. The concentrate was charged on a column
of silica gel, which was eluted with 1-2% methanol in
CHC13. The fractions showing a spot at Rf 0.30 (TLC,
MeOH-CHC13=1 . 10) were combined and evaporated to give
1.08 g. (55%) of the title compound.
IR : ~r cm 1 1780, 1680, 1500.
Procedure 13
Sodium 7S-[D-2-amino-2-(p-hydroxyphenyl)acetamido]-3-
[ (Z ) (-1-buten-l.-yl ]-:3-cephem-4-carboxylate ( Compound 13,
BB-51058 Sodium Salt;l
A solution of 1.08 g. (1.61 m. mol.) of 12 in 11
ml. of TFA cont:airing 1% of water was allowed to stand for
one hour at room temperature. The mixture was
concentrated to about 2 ml. in vacuo and the resulting
syrup was triturated with about 20 ml. of diisopropyl
ether to give .'96 mg.. of yellow powder. The powder was
dissolved in 3 ml. of methanol and the solution was
treated with 3 ml. of 0.8 M SEH in ethyl acetate (AcOEt)
to afford a prescipitate, which was filtered, washed with
diisopropyl ether an<i dissolved in 5 ml. of water. The
solution was passed through a column, packed with the
packing (80 ml.,) of a prepPAK-500/C18 cartridge
(Waters), which was washed with water and eluted
successively with 10% methanol, 20% methanol and 30%
methanol. The desired fractions (monitored by HPLC) were
combined, concentrated and lyophilized to give 118 mg.
(9.4%) of the title compound, estimated purity 55% (by
HPLC) , darkene~t when heated in a glass capi llary tube
> 180'C.
X340697
- 44 - _
IR : ~ ~X cm 1 1755, 1660, 1580.
UV : ~Phos,phate buffer (pH 7) nm(E) 228
max
(10900), 278 (7200).
NMR: a D20 ;ppm 0.81 ( 3H, t, 7.5 Hz), 1. 7-2.2
(2H, m), 3.25 (2H, ABq), 5.01 (1H, d, 5 Hz). 5.50 (1H,
d-t, 7. 5 & 12 H;a ) , 5 . 58 ( 1H, d, 5Hz ) ( 5 . 78 ( 1H, d, 12 Hz ) ,
6. 86 ( 2H, d, 8 Hz ) , 7 . 26 ( 2H, d, 8 Hz ) .
Procedure 14
Diphenylmethyl '7B-[D-2-(t-butoxycarbonylamino)-2-
(p-hydroxyl-phenyl)acetamido]-3-[(Z)-3-phenyl-1-propen-1-yl]
-3-cephem-4-carbox late (Compound 14)
A solution of 3 g. (2.95 m. mol.) of 5 in 50 ml
of CHC13 was shaken with a mixture of 3.2 ml. (3.2 m.
mol.) of 1 _N NaOH and 50 ml. of water for one minute. The
organic layer was separated after the addition of a
saturated NaCl aolution (20 ml.), washed with water (3 x
30 ml.) and a saturated NaCl solution and dried with
anhydrous Na2S0,4. To the solution was added 7.2 g.
(30 m. mol.) of 50% phenylacetaldehyde and the mixture was
stirred overnight at room temperature. The reaction
mixture was con~~entrated in vacuo and the concentrate was
purified on a column of silica gel (75 g.) using 1%
MeOH/CHC13 to give 800 mg. (37$) of the title compound.
Thin layer chromatography (TLC): Rf 0.33 (silica gel,
MeOH-CHC13 1 . 10). IR (KBr) : 1780, 1710 -1680
cm 1. This compound was used for Procedure 15 without
further purification.
Procedure 15
7~-[D-2-Amino-2-(p-hydroxyphenyl)acetamido]-3-[(Z)-3-
phenyl-1-propen-1-yl]-3-cephem-4-carboxylic acid
(Compound 15, BB-S10T6)
A solution of 800 mg. (1.09 m. mol.) of 14 in 4
ml of 90% TFA was allowed to stand for two hours. The
reaction mixture was concentrated and the concentrate was
triturated with diisopropyl ether to give 490 mg. of
yellow powder. A solution of the powder in 2 ml. of
4340697
- 45 -
methanol was mixed with 20 ml. of water and charged on a
column of HP-20 (50 ml.), which was washed with water
(250 ml.) and eluted with 30% methanol (250 ml.) and 75%
methanol (300 ml.) successively. The 75$ methanol eluate
was concentrated and lyophilized to give 302 mg, of the
crude product, which was dissolved in 10 ml. of 75%
methanol and chromatographed on a column using the packing
(80 ml.) of a PrepPAK.-500/C18 cartridge (Waters). The
column was eluted with 75% methanol to afford 158 mg.
(31%) of the desired product. Estimated purity, 65% (by
HPLC). It darkened when heated in a capillary tube over
175°C.
IR : ~maXCm 1 1760, 1680, 1600-1580, 1520.
UV : amaXSphate buffer (pH 7) nm(E ) 280
(8900).
NMR : a~s0-D6 /D20 ( 5/1 ) ppm 4. 45 ( 2H, d, 4 Hz,
CH2Ph), 4.87 (1H, s, CHND2), 6.7 (2H, d, 9 Hz, Ph),
6 . 9- 7 . 5 ( 7H, m Ph ) .
Procedure 16
Diphenylmethyl 7S-[D-2-(t-butoxycarbonylamino)-2-(p-
hydroxyphenyl)-acetamido]-3-[(Z)-3-methoxy-1-propen-1-yl]-3-
cephem-4-carbox ly ate (Compound 16)
A solution of 3.0 g. (2.95 m. mol.) of 5 in
CHC13 (100 ml.) was treated with a mixture of 2 N NaOH
(1.8 ml.) and water (100 ml.) at room temperature for 5
minutes. The organic' phase was separated, washed with
water (50 ml.) and agueous NaCl (50 ml.) dried and
evaporated to ca. 10 ml. The resulting red yield solution
was treated with methoxyacetaldehyde (1.3 ml., 15 m. mol.)
at room temperature for 15 minutes. After evaporation of
the solvent, the residue was chromatographed on a column
of silica gel (100 g.), eluting with toluene-AcOEt (3 . 1
and 1 . If to afford the title compound (750 mg., 38%).
NMR: g~Cl.3+D20ppm 1. 45 ( 9H, s, t-Bu) ,
3~ 15 ( 3H, s, OC'H3 ) , 3 . 27 ( 2H, s, 2-CH2 ) , ca. 3. 5 ( 2H,
1340697
- 46 -
m, -CH2-OMe ) , 4" 90 ( 1H, d, 5. 0 Hz, 6-H ) , 5. 12 ( 1H, s,
-CH-ND-), ca. 5" 5 ( 1H, m, =CH-CH2-), 5.72 ( 1H, d, 7-H),
6.18 ( 1H, d. 12 Hz, -CH=CH-CH2- ) , 6. 65 & 7.10 ( each 2H,
each d, HO-Ph- ) , 6 . 90 ( 1H, s, -CHPh 2 ) , 7 . 3 ( lOH, s, Ph ) .
Procedure 17
7S-[D-2-Amino-2--(p-hydroxphenyl)acetamido]-3-[(Z)-3-
methoxy-1-propen-1yl]-3-cephem-4-carboxylic Acid
( Compound 17, BF3-S 109 2 )
Compound 16 was deblocked with TFA (3 ml.) at
room temperature for one hour. Evaporation of the solvent
followed by precipitation from isopropyl ether gave the
trifluoroacetate~ of t'he product, which was purified by
HP-20 column chromatography. The column was washed with
H20 (500 ml.) and eluted with 30$ MeOH (500 ml.) to
afford 350 mg. (75%) .of desired product. Estimated
purity, 90% (by HPLC). M.p. 160°C (dec.).
IR : v~X cm 1 3400, 3180, 1760, 1680.
UV : ~maxsphate buffer pH 7 nm(e } 228
(11500), 279 (9400).
NMR: dD20p;pm 3.40 ( 3H) s, OCH3), 3.40
( 2H, ABq, 2-CH2 ) , 4. 0 ( 2H, m, -CH20Me ) , 5. 19 ( 1H, d,
4) 5 Hz, 6-H ) , 5 . 25 ( 1H, s, -CH-ND 2 ) , 5. 77 ( 1H, d, 7-H ) ,
ca. 5.8 (1H, m, =CH-GH2-}, 6.20 (1H, d, 11 Hz,
-CH=CH-CH2), 7.05 & 7.45 (each 2H, each d, HO-Ph-) .
Procedure 18
Diphenylmethyl '7~-[D-2-(t-butoxycarbonylamino)-2-
(p-hydroxypheny:L)-acetamido]-3-[(Z)-3-chloro-1-propen-1-yl]-
3-cephem-4- carbox late (Compound 18)
A solution of 5 ( 5.0 g., 4.9 m, mol. ) in CHC13
(100 ml.) Was treated with a mixture of 2 N NaOH (2.9 ml.,
5.8 m. mol.) an~~ water (100 ml.) at room temperature for 5
minutes. The organic phase was separated and washed with
water (50 ml.) .and a saturated NaCl solution (50 ml.). and
dried over anhydrous Na2S04. The filtrate was
evaporated to ca. 20 ml. and chloroacetaldehyde (2.0 ml.,
25 m, mol.) was added. The mixture was stirred at room
..-. - 47 -
~34069~
temperature for 30 minutes and evaporated in vacuo, The
residual syrup was chromatographed on a column of silica
gel (100 g.), eluting with toluene-AcOEt (3/1) to afford
the title compound 18 (900 mg.. 27$).
NuR: d~Cl.3+D20ppm 1.45 (9H, s, t-Bu),
ca . 3 . 3 ( 2H, m, 2-CHI, ) , 3 . 5-4. 0 ( 2H, m, -CH2-C1 ) , 4. 92
( 1H, d, 5. 0 Hz, 6-H ) , 5) 12 ( 1H, s, -CH-ND- ) , ca . 5 . 7
( 2H, m, -7-H&=CH-CH2 ) ( 6.15 ( 1H, d, 11 Hz, 3-CH=CH-CH2- )
6. 63 & 7.10 ( each 2H, each d, HO-Ph-) , 6. 89 ( 1H, s,
CHPh2). 7.3 (lOH, s, Ph).
Deblocking of this substance with TFA as
described in the preceding examples (e.g. Proc. 7, 11,
etc.) yielded 7 - [D-2-amino-2-(p-hydroxyphenyl)acetamido]
-3-[(Z)-3-chloro-1-propen-1-yl]-3-cephem-4-carboxylic acid.
Procedure 19
Diphenylmethyl 7~-[D-2-(t-butoxycarbonylamino)-2-
(p-hydroxy-phenyl)acetamido]-3-[(E)-3-iodo-1-propen-1-yl]
-3-cephem-4-carbo_ xylate (Compound 19)
A mixture of: 18 (900 mg., 1.3 m. mol.) and NaI
(590 mg.. 3.9 m. mol.) in acetone (18 ml) was stirred at
room temperature for one hour. After evaporation of the
solvent, the residue was dissolved in AcOEt (100 ml.),
washed successively with water, aqueous Na2S203 and
aqueous NaCl, dried and evaporated to give the title
compound (1.02 g.).
~R: CDC1.3+D20 ppm 1,45 (9H, s, t-Bu),
ca, 3. 4 ( 2H, m, 2-CH2 ) , ca. 3. 8 ( 2H, m, -CH2-I ) ( 4. 90
( 1H, d, 5. 0 Hz, 6-H ) ( 5 .14 ( 1H, s, -CH-ND- ) , 5 . 73 ( 1H, d,
7-H ) , ca, 5 . 5-6 . 0 ( lei. m, =CH-CH2- ) , 6. 68 & 7. 10 ( each
2H, each d, HO-Ph- ) , 6. 78 ( 1H, d, 15 Hz, 3-CH=CH-CH2- ) ,
6. 99 ( 1H, s, CH.Ph2 ) , 7. 35 ( lOH, s, Ph) .
Procedure 20
Diphenylmethyl 7S-[D-2-(t-butoxycarbonylamino)-2-(p-
hydrox~-phenyl)acetamido,]-3-[3-(1H-1,2,3-triazol-5-yl)
thio-1-propen-1-yl]-:3-cephem-4-carboxylate (Compound 20)
4340697
- 48 -
To a solution of 19 (1.0 g., 1.3 m. mol.) in
ethyl acetate (:ZO ml.) were added propylene oxice (.027
ml., 3.8 m. mol.) and 0.1 M (1H-1,2,3-triazol-4-yl)thiol
in ethyl acetate (19. ml.). The mixture was stirred at
room temperature for 30 minutes and evaporated under
diminished pressure. The residual syrup was
chromatographed on a column of silica gel C-200 (50 g.).
The desired product was eluted with CHC13-MeOH (10 . 1)
to afford 800 mc3. (83%) of the title compound.
NMR : a~Cl 3+H20 ppm 1 , 45 ( 9H, s, t-3 a ) ,
ca. 3. 3 ( 4H, m, 2-CH2- & -CH2-S-) 4.80 ( 1H, d, 5. 0 Hz,
6-H), 5. 20 ( 1H, s, -CH-ND-), 5.70 ( 1H, d, 7-H), ca. 5.95
( 1H, m, =CH-CH2~- ) , 6. 68 ( 2H, d, HO-Ph- ) , 6. 90 ( 1H, s,
-CHPh2), 7.25 (:LOH, s, Ph), 7.52 (1H, s, triazole-4-H).
Procedure 21
7S-[D-2-Amino-2--(p-hydroxyphenyl)acetamido]-3-[3-
(1H-1,2,3,-triazol-5-Y1)thio-1-propen-1-yl]-3-cephem-4-
carboxylic Acid (Compound 21, BB-S1091)
A mixture of 20 (800 mg.) and TFA (2 ml.) was
kept at room temperature for one hour and then evaporated
to dryness. To the residue was added isopropyl ether to
give yellow precipitate (600 mg.), which was dissolved in
water (1 ml.) and charged onto an HP-20 column (100 ml.).
The column was washed with water (500 ml.) and eluted with
30% MeOH and subsequently with 50% MeOH. The fraction
containing the desired compound was collected, evaporated
and lyophilized to afford 170 mg. (33%) of desired
product, estimated purity, 50$ (by HPLC), m.p. 180°C
(dec.).
IR : ~maXCm-1 3360, 3280, 1755, 1670
UV : amaxsphate buffernm( E) 235 (14100),
252 (12300)
NMR: aD20+DC1 ppm ca. 3.4 ( 4H, m, 2-CH2-,
-CH2-S- ) , 5 . 43 ( 1H, d ( 4. 5 Hz, 6-H ) , 5. 15 ( 1H, s,
' -~-ND2 ) ~ ca. 6. 0 l 2H, m, 7-H and =CH-CH2- ) , 6 . 70 &
7.15 (each 2H, each d., HO-Ph-), 8.05 (1H, s, triazol-4-H).
j34~697
- 49 -
Procedure 22
Benzhydryl 7~-[D-2-(t-Butoxycarbonylamino)-2-
phenylacetamido]-3- (triphenylphosphonio)methyl-3-cephem-
4-carboxylate Iodide (Compound 22)
A mixture of 14.5 g. (0.0196 m mol) of benzhydryl
7-[D(-)- -(t-butoxycarbonylamino)- -phenylacetamido]-3-
iodomethyl-3-cephem-4-carboxylate and 5.24 g. (0.02 mol)
of triphenylphosphine in 300 ml of ethyl acetate was
stirred at room temperature for 2 hours. To the reaction
mixture was added 200 ml of ether to form precipitate.
which was collected by filtration and washed with ether to
give 14,3 g. (73%) of the title compound. The filtrate
was concentrated to 50 ml and the concentrate was diluted
with ether to give 2.4 g of the second crop of the
product. Total yield 16.7 g. (85%).
IR . ~~rcm-1 1780, 1690, 1480, 1420, 1350,
max
1240, 1150
Procedure 23
Benzhydryl 7S-[D-2-(t-Butoxycarbonylamino)-2-
phenylacetamido]-3-[(Z)-1-propen-1-yl]-3-cephem-4-
carboxylate (Compound. 23)
To a solution of 5 g. (5 m mol) of 22 in 200 ml
of chloroform was added a mixture of 100 ml of water and 5
ml 5 (m mol) of N sodium hydroxide and the mixture was
shaken for 3 minutes. The organic layer separated was
washed with water and a saturated NaCl solution, and dried
on anhydrous magnesium sulfate. The chloroform solution
being filtered, the filtrate was concentrated to 100 ml
under reduced pressure. To the concentrate was added 3 ml
of acetaldehyde and the mixture was stirred at room
temperature for 1.5 hours and evaporated to dryness. The
oily residue was chromatographed on a column of silica gel
(Kiesel gel 60, 50 g) (trade mark) by eluting with
chloroform. The desired fractions were collected and
.340697
- 50 -
evaporated to dryness and the residue was triturated with
n-hexane to give 990 mg (31%) of the title compound (23).
IR . v~XCm 1 1780, 1710, 1660, 1510, 1490,
1360, 1240, 1210, 1150.
NMR: dCDCl3 ppm 1.3-1.5 (12H, m, -C-CH3),
3. 22 ( 2H, s, 2-H ) , 4. 93 ( 1H, d, 4. 5 Hz, 6-H ) , 5. 23 ( 1H, d,
8 Hz, C_H-CO), 5..5-6.2 (3H, m, 7-H & vinyl-H), 6.94 (1H, s,
c:EiPh ) , 7 . 2-7. 5 15H, m, phenyl-H ) .
Procedure 24
Sodium 7~-[D-2-amino-2-phenylacetamido]-3-[(Z)-1-
propenyl]-3-cephem-4-carboxylate (Compound 24, BB-51065)
A mixture of 0.94 g. (1.47 m mol) of 23 and 3 ml
of TFA was stirred at room temperature for 30 minutes then
diluted with 50 ml of a 1 . 1 mixture of ether-isopropyl
ether to separate ca. 800 mg of precipitate, which was
collected by filtration and dissolved in 3 ml of
methanol. To the solution was added 4.5 ml (4.5 m mol) of
1 M_ sodium 2-ethylhex.anoate (SEH) in ethyl acetate and the
mixture was diluted with 50 ml of ether and 50 ml of
isopropyl ether successively. The precipitate was
collected by filtration to give 710 mg of the crude
product 24, which was dissolved in 20 ml of water and
chromatographed on a column using 50 ml of the packing in
a PrepPAK/C18 cartridge (Waters). The column was eluted
with water and 10% methanol. The fractions containing the
desired product were collected monitoring by HPLC and
concentrated to 5 ml and lyophilized to give 182 mg (31%)
of desired product, melting at 200°C. Estimated purity,
50$ by HPL C.
IR: v~XCm 1 1760, 1660, 1600, 1400,
1180, 1100.
UV: ~maxsphate buffer (pH 7)nm( E ) 282 (5500).
NMR: gD20ppm 1.60 (3H, d, 6 Hz -C-CH3),
3.12 ( 1H, d, 18 Hz, 2-H ) , 3 . 48 ( 1H, d, 18 Hz, 2-H ) , 5. 03
( 1H, d, 4. 5 Hz, 6-H), 5.62 ( 1H, d, 4. 5 Hz, 7-H), 5.93 ( 1H,
d, 10 Hz, vinyl-H ) , 5. 2-5. 8 ( 1H, m, vinyl-H ) , 7. 41 ( 5H, s,
phenyl-H).
.~
j34469~'
- 51 -
Procedure 25
Benzhydryl 7 S-[;D-2-( t-Butoxycarbonylamino)-2-
phenylacetamido]-3-[(Z)-3-chloro-1-propen-1-yl]-3-cephem-4-
carboxylate
(Compound 25)
To a solution of 2 g. (2 m mol) of 22 in 50 ml of
chloroform was added 50 ml of water containing~2 ml (2 m
mol) of N sodium hydroxide and the mixture was shaken for
3 minutes. They organic layer was separated and washed
with water and a saturated NaCl solution successively.
The dried chloroform solution was concentrated to 30 ml
under reduced pressure. To the concentrate was added 2 ml
of chloroacetal.dehyde and the mixture was stirred at room
temperature -for one haur, washed with water, and
subsequently with a saturated NaCl solution. The organic
solution was d:vied and evaporated to dryness. The oily
residue was chromatographed on a column of silica gel
(Wako-gel C-200, 50 g) by eluting with chloroform. The
desired fractions were collected and evaporated to dryness
to give 534 mg of the crude product.
IR: v~ aXCm '1780, 1710, 1660, 1500, 1490,
1360, 1240, 1210, 1150.
The structure of this sample was not confirmed
because of its poor nmr spectrum.
30
~3~~697
- 52 -
Procedure 26
Sodium 7S -(D-2-amino-2-phenylacetamido)-3-[(Z)-3-chloro-1
propen-1-yl]-3-cephem-4-carboxylate (Compound 26, BB-51066)
A mixture of 472 mg (0.7 m mol) of 25 and 1.5 ml
of TFA was stirred at 10-15°C for 15 minutes and diluted
with 30 ml of a mixture of ether and isopropyl ether
(1 . 1) to afford 330 mg of pale yellow precipitate, which
was collected by filtration. To a solution of the
precipitate in 3 ml of methanol was added 2 ml (2 m mol)
of SEH in ethyl acetate and the mixture was diluted with
50 ml of ethyl acetate. The resulting precipitate was
collected by filtration and washed with ether to give 244
mg of a crude product. A solution of the crude product in
10 ml of water was chromatographed on a column using 50 ml
of the packing in a PrepPAK-500/C18 cartridge (Waters).
The column was eluted. with water and 10% methanol. The
desired fractions of 10% methanol were combined and
concentrated to 5 ml and lyophilized to give 60 mg of the
solid product melting at 200°C (grad. dec.).
IR: vm~XCm 11760, 1660, 1630, 1360,
1120, 1070.
UV: phosphate buffernm(e ) 243 (12700), 200sh
max
(4200).
Procedure 27
7 S -(D(-)-2-Amino-2-phenylacetamido)-3-[(Z)-1-propen-1-yl]-
-3 -cephem-4-carboxylic acid (Compound 24, BS-S 1065
zwitterion form)
S
._CHCONH
I
NH2 N / H=CHCH3 (Z)
O
C02 H
1340697
- 53 -
Diphenylmethyl 7- ~ -[D-2-(t-butoxycarbonylamino)-2
phenylacetamido]-3-(1-propenyl)-3-cephem-4-carboxylate
(compound 23) 1.5 g)(2.34 m moles), was treated with 3 ml
of trifluoroacetic acid and the mixture was stirred at
room temperature for 20 min, and diluted with 100 ml of
ether to give 1.15 g (96%) of the crude trifluoroacetate
of BB-S 1065.
it : vmax ( KB r ) in cm 1 1760, 1670, 1200, 1130
uv: ~;max(pH 7 phosphate buffer) 283 nm
( E : 8300 )
The trifluoroacetate (1.1 g, 2.25 m moles) was
dissolved in 20 ml of water and the solution was
chromatographed on a column using 100 ml of the packing
obtained fro~i prepPAK/C18 cartridge (Waters). The
column was eluted with water, 10% methanol and 30%
methanol. The eluate with 30% methanol was concentrated
to 10 ml. The crystalline product was separated. The
product was collected and washed with acetone and dried in
vacuo over P205 to give 505 mg (46%) of pure BB-S 1065
(zwitterion form) melting at 180-183°C(dec.). Est'd
purity 95%.
ir: vmax(~r) in cm 1 1750, 1690, 1590,
1400, 1350
uv: ~ (pH 7 phosphate buffer) 282 nm
max
( a :8800).
nmr: d (D20 + NaHC03) in ppm 1.58 (3H, d,
J=6 Hz, C-CH3 ) , 3 . 3 ( 2H, d, 2-H ) , 5. 03 ( 1H, d, J=4. 5 Hz,
6-H), 5.20 (1H, S, CH.-CO), 5.1-5.8 (1H, m, CH=C), 5.63
( 1H, d, J=4. 5 Hz, 7-H: ), 5. 92 ( 1H, d, J=12 Hz, CH=C) ( 7. 4
(5H, s, phenyl-H).
Procecure 28
D(-)-2-(t-Butoxyca,rbonylamino)-2-(3-chloro-4-hydroxyphenyl)
acetic acid (Compound. 28)
A mixture of 6 g (0.03 mole) of 3-chloro-4-
hydroxyphenylglycine and 9.8 g (0.045 mole) of di-t-butyl
dicarbonate in 120 ml of a 50% aqueous tetrahydrofuran
434069 ~
- 54 -
(THF) solution containing 10 ml (0.071 mole) of
triethylamine wras stirred at room temperature for 3
hours. The mica ure was concentrated to 60 ml and the
concentrate was: washed with ether. The aqueous layer was
acidified with 6 N hydrochloric acid and extracted with
200 ml of ether. The extract was washed with water and a
saturated NaCl solution, dried on MgS04, and evaporated
to dryness to give 10 g of an oily residue, which did not
solidify by attempted trituration with ether-n-hexane.
Procedure 29
Benzhydryl 7 S -[D-2--(t-butoxycarbonylamino)-2-(3-chloro-4-
hydroxyphenyl)acetamido]-3-chloromethyl-3-cephem-4-
carboxylate (Compound 29)
S
HO ~ ' CONH
_ NH ~ CH2C1
C1 ~ 0
C02C (CH3) 3 C02CH (C6H5) 2
To a solution of 6.2 g (0.015 mole) of Compound 2
and 5.4 g (0.07.8 mole) of Compound 28 in 150 ml. of dry
THF was added 3.7 g (0.018 mole) of DCC and the mixture
was stirred at room temperature for one hour.
Dicyclohexylure~a, which separated during stirring, was
removed by filtration and the filtrate was evaporated to
dryness. The residue being extracted with 200 ml of ethyl
acetate, the e~aract was washed with an aqueous NaHC03
solution, water and a saturated NaCl solution, and dried
with MgS04. The filtrate was evaporated to dryness and
the oily residue was chromatographed on a silica gel
column (Wako gel C-200, 140 g) by eluting with
toluene-ethyl acetate: (10 . 1). The desired fractions
X340697
- 55 -
were collected and evaporated to dryness to give 10 g of
the product 29.
i r : v max ( ~ r ) in cm 1 1790, 1720, 1680,
1500, 1370, 1240, 1160.
Procedure 30
Benzhydryl 7 ~ [D-2-(t-Butoxycarbonylamino-2-(3-chloro-4-
hydroxyphenyl)acetamido]-3-(triphenylphosphonio)methyl-3-
cephem-4-carbox ly ate iodide (Compound 30)
S
HO ~ ,\ ~:HCONH~ O O
C1~ ;H ~ CH2P (C6H5) 3 I
C'~ZC (CH3) 3
C02CH(C6H5)2
To a solution of 10 g (0.0143 mole) of Compound
29 in 100 ml of acetone was added 11.2 g (0.075 mole) of
sodium iodide and the mixture was stirred at room
temperature for 30 min. The mixture was concentrated to
ml. To the concentrate was added 200 ml of ethyl
acetate and the mixture was washed with an aqueous
25 Na2S2p3 solution, water and a saturated NaCl
solution, and dried with MgS04. The ethyl acetate
solution was filtered and the filtrate was concentrated to
a half the volume. Z'o the concentrate was added 3.9 g
(0.015 mole) of triphenylphosphine and the mixture was
30 stirred at room temperature for 2 hours. To the solution
was added 300 m.l of ether to separate a precipitate, which
was collected by filtration and dried to give 9.2 g of the
phosphonium iodide 30.
i r : Amax ( KB r ) in cm 1 1780, 1680, 1490,
1350, 1240, 1150.
130697
- 56 -
Procedure 31
Benzhydryl 7 R [D-2-(t-butoxycarbonylamino)-2-(3-chloro-4-
hydroxyphenyl) acetamido]-3-[(Z)-1-propen-1-yl)-3-cephem-4-
carboxylate (Compound 31)
H O ~ ~ ~S
CHCONH
~H / CH=CHCH.3 ( Z )
C1 O
C02C (CH3 ) 3 _
C02CH(C6H5)2
A solution of 9.5 g (9 m moles) of Compound 30 in
200 ml of chloroform was layered with a mixture of water
(100 ml) and N NaOH (.10 ml) and the mixture was shaken for
3 min. The organic layer was washed with water and a
saturated NaCl solution, dried with MgS04 and
concentrated tc~ about a half the volume. To the
concentrate was added 20 ml of 90$ acetaldehyde and the
mixture was stirred at room temperature for 3 hours,
treated with anhydrous MgS04, and filtered. The
filtrate was evaporated to dryness and the residue was
chromatographed on Kiesel gel 60-(Merck, 120 g) by eluting
with toluene-ethyl acetate (4 . 1). The desired fractions
were collected and e~~aporated to dryness and the residue
was triturated with a mixture of ether, isopropyl ether
and n-hexane to~ give 1.33 g of the blocked product _31.
ir: (KBr) in cm 1 1770, 1700 1660, 1480,
i'ma x
1350, 1210, 1150.
Procedure 32
7 S -[D-2-Amino-2-(3-~chloro-4-hydroxyphenyl)acetamido]-3-
[(Z)-1-propen-1-yl]-3-cephem-4-carboxylic acid (Compound
32, BMY28060)
1340697
- 57 -
S
HO " CHCONH~
-- NH 2 N ~ CH=CHCH 3 ( Z )
CT p
C02H
A mixture of 1.33 g (1.93 m moles) of Compound 31
and 3 ml of trifluoraacetic acid was stirred at room
temperature for 30 min. and the mixture was diluted with
50 ml of ether-isopropyl ether (1 : 1) to give 1.072 g of
the crude trifluoroacetate of 32, which was
chromatographed on a column packed with the packing of a
prepPAK-C18 cartridge (Waters) (80 ml). The column was
eluted with water and 10$ methanol. The eluate with 10$
methanol was..concentrated to 10 ml of the volume to
separate a crystalline precipitate, which was collected by
filtration and washed with acetone and dried in vacuo over
P205 to give 238 mg of 32 (95$ pure) melting at
180-185°C (grad. dec.). The filtrate was concentrated to
5 ml and lyophilized to afford 154 mg of a second crop
which was 80$ pure by HPLC.
ir: vmax(~'r) in cm 1 1760, 1680, 1570,
1410, 1390, 1350, 1290,
1270.
uv: amax(pH7 phosphate buffer) in nm (E ) 232
(10000), 280 (10500).
nmr: (D20 + NaHC03) in ppm 1, 68 ( 3H, d, J=6
Hz, C=C-CH3 ) , 3. 25 ( 1.H, d, J=18 Hz, 2-H) 3. 57 ( 1H, d,
J=18 Hz, 2-H ) , 4 . 90 ( 1H, s, CH-CO ) , 5 .18 ( 1H, d, J=4 . 5 Hz,
6-H) , 5.72 ( 1H, d) J= 4. 5 Hz, 7-H), 5. 5-5. 9 ( 1H, m, CH=C),
5. 97 ( 1H, d, J=12 Hz, CH=C) , 7. 02 ( 1H, d, J=8 Hz,
phenyl-H), 7.30 (1H, d-d, J=8 & 1.5 Hz, phenyl-H), 7.50
( 1H, d, J=1. 5 Hz, phenyl-H) .
4340697
- 58 -
Procedure 33
D(-)-2-(t-Butoxycarbo~lamino)-2-(3,4-dihydroxyphenyl)acetic
acid (33a) Mixture with Its 3-(and 4)-Mono-O-butoxycarbonyl-
Derivatives(33b).
A mixture of 3.66 g (0.02 mole) of
3,4-dihydroxyph~enyl-glycine and 9.24 g (0.04 mole) of
di-t-butyl dicarbonate in 120 ml of a 50% aqueous THF
solution containing 10 ml (0.071 mole) of triethylamine
was stirred at room temperature for 16 hours and the
mixture was concentrated to 60 ml. The concentrate was
washed with 100 ml of ether, acidified with N hydrochloric
acid and extracted with ether (100 x 2 ml). The combined
extracts were washed with water and a saturated NaCl
solution, dried with MgS04 and evaporated in dryness to
give 8 g of an oily residue which was a mixture of the
desired 3,4-dihydroxyphenyl derivative and the 3- and
4-mono-O-BOC-protected derivatives (30C refers to t-butoxy
carbonyl).
Procedure 34-
Benzhydryl 7 S -CD(-)-2-(t-Butoxycarbonylamino)-2-(3,4-
dihydroxy-phenyl) acetamido]-3-chloromethyl-3-cephem-4-
carboxylate (34a) and. Mixture of its 3-(and 4-)
Mono-0-butoxycarbon 1 Derivatives (34b).
HO ~ ~ S
ONH
NH ~ CH2C1
HO ~ 0
C02C (CH3) 3 C02CH (C5H5) 2
A mixture of 8 g (0.0193 mole) of Compound 2, 8 g
of the mixed product of Procedure 33, and 4.12 g (0.02
mole) of DCC im 200 ml of dry THF was stirred at room
- 59 _ .340697
temperature for one hour. The reaction mixture was
evaporated to dryness. The residue was dissolved in 200
ml of ethyl acetate and insoluble material
(dicyclohexylur~ea) was removed by filtration. The
filtrate was washed with an aqueous NaHC03 solution,
water and a saturated NaCl solution, dried with MgS04
and evaporated to dryness under reduced pressure. The
oily residue wars chromatographed on a silica gel column
(Kiesel gel 60, 130 g) by eluting with toluene-ethyl
acetate (5 . 1) and toluene-ethyl acetate (2 . 1). The
eluate with toluene-ethyl acetate (5 : 1) was collected
and evaporated to dryness to give 9.5 g of a mixture of
the mono-O-BOC-1~1-BOC diprotected derivatives (34b). The
eluate with tolvuene-ethyl acetate (2 : 1) was collected
and evaporated to dryness to give 3 g of the
3,4-dihydroxyphenyl derivative (34a).
Compound 34a
ir: v;~ax(~r) in cm 1 1770, 1720, 1690,
1500, 1370, 1240, 1150.
nmr : s ( CDCl 3 ) in ppm 1, 42 ( 9H, s, CH-CH3 ) ,
3 . 4 ( 2H, br-s, 2-H ) , 4. 30 ( 2H, br-s, CH2-Cl ) , 4. 85 ( 1H,
d, J=4. 5 Hz, 6-:H ) , 5 . 07 ( 1H, d, J=6 Hz, CH-NH ) , 5 . 74 ( 1H,
d-d, J=9 & 4. 5 :Hz, 7-H ) ( 6. 6-6. 9 ( 3H, m, phenyl-H ) , 6 . 93
( 1H, s, CHPh ) , 7 . 3 ( lOH, s, phenyl-H ) .
Mixture 34b
ir: v,~ax(KBr) in cm 1 1770, 1720, 1690,
1500, 1370, 1240, 1150.
nmr: s (CDC13) in ppm 1, 42 (9H, s, C-CH3),
1. 55 ( 9H, s, C-CH3 ) , 3. 4 ( 2H, br-s, 2-H) , 4. 35 ( 2H,
br-s, CH2-Cl), 6.9-7.1 (4H, m, CHPh & phenyl-H), 7.3.
( lOH, s, phenyl-H ) .
Procedure 35
Benzhydryl 7 g [D(-)-2-(t-Butoxycarbonylamino)-2-(3,4-
dihydroxy-phenyl)acetamido]-3-triphenylphosphoniomethyl-3-
cephem-4-carbox ly ate Iodide (35a).
1340697
- 60 -
HO ~ ~ S
IHCONH
~. NH r~ ~ CH2P (C6H5) 3 I-
HO ~ O
C02C (CH3) 3 C02CH (C6H5) 2
A mixture of 3 g (4.4 m moles) of 34a and 3.3 g
(22 m moles) oi: sodium iodide in 50 ml of acetone was
stirred at room temperature for 30 min. and the mixture
was concentrated to dryness. The residue was extracted
with 100 ml of ethyl acetate and the extract was washed
with an aqueou~~ Na2S203 solution, water and a
saturated NaCl solution. After drying with MgS04 the
extract was concentrated to 60 ml. To the concentrate was
added 1.4 g (5.,3 m males) of triphenylphosphine and the
mixture was stirred at room temperature for one hour. To
the mixture was added 100 ml of ether to separate a
precipitate, wriich was collected by filtration and washed
with ether to dive 3.2 g (70%) of the phosphonium iodide
(35a).
lr ' ~~max ( ~3 r ) in cm 1 1780, 1680, 1480,
1430, 1360, 1240, 1150.
By a similar procedure, 9.5 g (12 m moles) of the
mixture of mono-O-BOC-protected derivatives (34b) was
allowed to react with sodium iodide and subsequently with
triphenylphosphine to give 10.7 g (77%) of a mixture of
the corresponding mono-O-BOC-N-BOC
triphenylphosphoniomethyl derivatives (35b).
ir: omax(Kj3r) in cm 1 1770, 1720, 1680,
1480, 1430, 1360,
1240, 1140.
X340697
- 61 -
Procedure 36
Benzhydryl 7 S -[D(-)-2-(t-Butoxycarbonylamino)-2-(3,4-
dihydroxyphenyl)acetamido]-3-[(Z)-1-propen-1-yl]-3-cephem-
4-carboxylate (Compound 36a).
S
HO ~ \ HCONH
NH / CH=CHCH (Z)
HO ~ O 3
C02C (CH3) 3 C02CH (C6H5) 2
To a stirred solution of 3.15 g (3 m moles) of
Compound 35a and 10 nul of acetaldehyde in 50 ml of
chloroform was added dropwise 8 ml (4 m moles) of 0.5 N
sodium hydroxide over a period of 10 min. and the mixture
was stirred at room temperature for one hour. The
reaction mixture was washed with water and a saturated
NaCl solution, dried with MgS04 and evaporated under
reduced pressure. The oily residue was chromatographed on
a silica gel column (Wako gel C-200, 60 g), which was
eluted with chloroform (2 L) and 2% methanol in chloroform
under monitoring by TLC (chloroform . methanol = 10 . 1).
The desired fractions from the 2% methanol eluate were
collected and evaporated to dryness to give 0.8 g (40%) of
the propenyl derivative 36a.
nmr : d ( CDC:13 ) in ppm 1. 28 ( 3H, d, J=6 Hz,
C-CH3 ) , 1. 42 ( 9H, s, C-CH3 ) , 3 . 25 ( 2H, s, 2-H ) ( 4. 92
( 1H, d, J=4. 5 Hz, 6-H ) , 5 . 08 ( 1H, d, J=6 Hz, CH-NH ) ,
5. 3-5. 8 ( 1H, m, CH=C) , 5. 80 ( 1H, d, J=4. 5 Hz, 7-H ), 6. 04
( 1H, d, J=11 Hz, CH=C:), 6. 70 ( 2H, s, phenyl-H) ( 6.82 ( 1H,
s, phenyl-H ) , 6 . 92 ( l.H, s, CHPh ) , 7 . 3 ( lOH, s, phenyl-H ) .
By a similar procedure to that described above,
10.5 g (9.3 m moles) of the mixture of the 3- and
X340697
- 62 -
4-O-BOC-N-BOC d.iprotected derivatives 35b was allowed to
react with acetaldehyde to give 3.3 g (46%) of the
corresponding ~~-propenyl derivative 36b.
ir: vmax(~r) in cm 1 1770, 1700, 1500,
1370, 1240, 1150.
nmr: ~i (CDC13) in ppm 1.4 (9H, s, C-CH3),
1.55 (9H, s, C-~CH3), 3.25 (2H, s, 2-H), 6.07 (1H, d,
J-11 Hz, CH=C ) , 6 . 9-7 .1 ( 4H, m, CH-Ph & phenyl-H ) , 7 . 3-7 . 5
( l OH, m, phenyl.-H ) .
Procedure 37
7 S -[D(-)-2-Amino-2--(3,4-dihydroxyphenyl)acetamido]-3-[(Z)
-1-propen-1-yl~-3-cephem-4-carboxylic Acid (Compound 37,
BMY-28068).
.S
CHCONH
1
NH2 N / CH~CHCH3 (Z)
HO O
CO H
2
A mixture of 0.8 g (1.2 m moles) of compound 36a,
0,8 ml of anisole and 3 ml of trifluoroacetic acid was
stirred at room temperature for 5 min. and diluted with 25
ml of ether and 25 m:l of isopropyl ether. The resulting
precipitate was collected by filtration and washed with
isopropyl ether to give 557 mg of the crude
trifluoroacetat:e salt of Compound 37. A solution of the
crude product ~Ln 10 ml of water was purified by column
chromatography using 100 ml of the packing of a
prepPAK-C18 cartridge (Waters) and the column was eluted
with water and 5% methanol successively. The 5% methanol
eluate containing the desired product was concentrated to
5 ml and lyophilized to give 231 mg (47%) of Compound 37
(zwitterion form, 90% pure). M, p. 200°C (grad, dec.).
1340697
- 63 -
ir: Amax In cm 1 1760, 1690, 1580, 1530,
1400, 1360, 1290, 1270.
uv: Amax (pH7 phosphate buffer) in nm (~ ) 233
(9200), 281 (11000)
nmr: d (D20) in ppm 1.68 (3H, d, J=6 Hz,
C-CH3 ) , 3 . 26 ( 1H, d, J=18 Hz, 2-H ) , 3 . 58 ( 1H, d, J=18
Hz, 2-H ) , 5 .18 ( 1H, s, CHNH ) , 5 . 22 ( 1H, d, J=4. 5 Hz, 6-H ) ,
5. 5-5. 9 ( 2H, m, CH=C & 7-H ) , 5. 97 ( 1H, d, J=11 Hz, CH=C) ,
7. 05 ( 3H, m, phenyl-H:) .
According to a similar procedure, 3.3 g (4.3 m
moles) of the N,O-di-t-BOC-protected derivative mixture
36b gave 1.3 g (75$) of Compound 37 as the zwitterion form
(90$ pure), which gave the spectral data identical with
those given above.
Procedure 38
D(-)-2-(t-Butox~rcarbonylamino)-2-(4-hydroxy-3-methyoxy-
phenyl) acetic Acid (Compound 38)
Hp - ~ ~ CHC0;2H
NH
t
CH30 C02C (CH3) 3
A mixture of: 2.96 g (0.015 mole) of
D(-)-2-amino-2-(4-hydroxy-3-methoxyphenyl) acetic acid and
3,6 g (0.0165 mole) c>f di-t-butyl dicarbonate in 100 ml of
50$ aqueous THF containing 4.2 ml (0.03 mole) of
triethylamine was stirred at room temperature for 16 hours
and the reaction mixture was concentrated to 50 ml. The
concentrate was washed with 50 ml of ether, acidified with
N hydrochloric acid and extracted twice with ether (100 x
2 ml). The combined extracts were washed with water and a
X340697
- 64 -
saturated NaCl solution. The dried extracts were
evaporated to dryness to give 4.38 g of Compound 38 as
foamy solid.
nmr : d ( CD~13 ) in ppm 1. 4 ( 9H, s, -C-CH 3 )
3 . 8 ( 3H, s, OCH.3 ) , 5 .15 ( 1H, d, J=6 Hz CH-NH ) , 6 . 85 ( 3H,
s, phenyl-H ) .
Procedure 39
Benzhydryl 7 S -[D(-)-2-(t-Butoxycarbonylamino)-2-(4-
hvdroxv-3-methoxyphenyl)-acetamido]-3-chloromethyl-3-cephem-
4-carboxylate (Compound 39)
S
H O -y--a ~ ~ H°C ONH
~ NH / CH2C1
CFf30 C02C (CH3) 3
C02CH (C6H5) 2
A mixture of 4.3 g of Compound 38, 5 g (0.012
mole) of Compound 2, and 3 g (0.015 mole) of DCC in 150 ml
of dry THF was stirred at room temperature for 2 hours.
The precipitated urea was removed by filtration and the
filtrate was avaporated to dryness. A solution of the
residue in 200 ml of ethyl acetate was washed with an
aqueous NaHC03 solution, water, and a saturated NaCl
solution, dried with MgS04 and evaporated to dryness.
The oily residue was chromatographed on a silica gel
column (Kiesel gel 60, 100 g) which was eluted with
toluene-ethyl e~cetata_ (4 . 1) under monitoring by TLC
[toluene-ethyl acetate (1 . 1) or chloroform-methanol
(50 . 1)]. ThES desired fractions were collected and
evaporated to dryness to give 7 g of the desired
3-chloromethyl cephem, Compound 39, as a foamy solid.
nmr : 8 in ppm 1. 4 ( 9H, s, C-CH3 ) , 3 . 45 ( 2H,
br-s, 2-H ) , 3 , f33 ( 3H,, s, OCH3 ) , 4. 32 ( 2H, s, -CH2C1 ) ,
- 65 - 1340697
4. 92 ( 1H, d, J=4. 5 Hz, 6-H ) , 5. 13 ( 1H, d, J=6 Hz CH-NH ) ,
. 65 ( 1H, d, J=6 Hz, NH ) , 5 . 80 ( 1H, d-d, J=8 & 4. 5 Hz,
7-H ) , 6. 85 ( 3H, s, phenyl-H ) , 6. 95 ( 1H, s CH-Ph ) , 7 . 2-7 . 5
( 10-H, m . pheny:L-H ) .
5 Procedure 40
Benzhydryl 7 S -[D(-)-2-(t-Butoxycarbonylamino)-2-(4-
hydroxy-3-metho:Kyphen~l)-acetamido]-3-triphenylphosphonio-
methyl-3-cephem~-4-carboxylate Iodide (Compound 40)
S
HO ~ ' CHCONH
NH / H2p (C6H5) 3
CH30 C02C (CH3) 3
C02CH (C6H5) 7
A mixture of 7 g (0.01 mole) of Compound 39, and
7,5 g (0.05 mole) of sodium iodide in 100 ml of acetone
was stirred at room temperature for 30 min. and evaporated
to dryness. A solution of the residue in 200 ml of ethyl
acetate was washed with an aqueous Na2S203 solution,
water and a saturated NaCl solution, dried with NgS04
and concentrated to 100 ml. To the concentrate was added
3.1 g (0.012 mole) of triphenylphosphine and the mixture
was stirred at room temperature for one hour. To the
reaction mixture was added 100 ml of ether and the
separated solid was collected by filtration, washed with
ether and dried to give 5.8 g the triphenylphosphonium
derivative Compound 40. The ethereal filtrate was
concentrated to 10 ml. and to the concentrate was added 300
ml of ether to give 0.9 g of the product as a second
crop. The total yield was 6.7 g.
-- 1340fi97
- 66 -
Procedure 41
Benzhydryl 7 R -[D-(-)-2-(t-Butoxycarbonylamino)-2-(4-
hydroxy-3-methoxyphenyl-acetamido]-3-[(Z)-1-propen-1-yl]
-3-cephem-4-ca:rboxylate (Compound 41)
S
HO ~ \ CHCONH~
CH=CHCH_3 ( Z )
NH
r
' CH30 C02C (CH3 ) 3
C02CH(C6H5)2
To a stirred mixture of 5.8 g (5.5 m moles) of
Compound 40 and 10 ml of 90% acetaldehyde in 100 ml of
chloroform was added dropwise 11 ml (5.5 m moles) of 0.5 N
sodium hydroxide over a period of 25 min. and the mixture
was stirred at room temperature for 2 hours. The reaction
mixture was washed with water, then with a saturated NaCl
solution, dried with MgS04, and evaporated to dryness.
The oily residue was chromatographed on a silica gel
column (Kiesel gel 60, 130 g) by eluting with a mixture of
toluene and ethyl acetate [the ratio was changed stepwise;
4 . 1 (1.3 L), 3 . 1 (1.1 L), 2 . 1 (1.0 L) and the eluate
was collected in 20-ml fraction. Fractions No. 26 through
fraction No. 5'9 were combined and evaporated to dryness to
give 830 mg of the desired 3-propenyl derivative Compound
41 as a foamy .solid.
nmr of 41: d (CDC13) in ppm, 1.35 (3H, d,
CH-CH3), 1.4 (9H, s, C-CH3) 3.85 (3H, s, O-CH3),
6. 07 ( 1H, d, J-11 Hz, -CH=C) .
~34~69~
- 67 -
Procedure 42
7 S -[D(-)-2-Amino-2-(4 hydroxy-3-methoxyphenyl)acetamido]
-3-[(Z)-1-propen[-~~pen] 1-yl]-3-cephem-4-carboxylic
Acid (Compound 42, BMY 28097)
/ y s
HO ?.---CHCONH
I
NH;Z N ~ CH=CHCH3 (2)
C~H 3 0
C02H
A mixture of 830 mg (1.2 m moles) of Compound 41,
0.5 ml of anisole and 2 ml of trifluoroacetic acid was
stirred at room temperature for 5 min. and the mixture was
diluted with 30 ml of ether and 30 ml of isopropyl ether.
The resulting precipitate was collected by filtration,
washed with isopropyl ether and dried to give 437 mg of
the crude trifluoroacetate of Compound 42. The crude
product was chromatographed on a column packed with 100 ml
of the packing of a prepPAK-C18 cartridge column
(Waters), which was eluted with water and 5$ methanol.
The eluate with 5$ methanol was concentrated to 5 ml and
lyophilized to give 225 mg of Compound 42 (zwitterion, 90$
pure). M. p. 1.76-180°C (dec.).
ir: ~'maxln cm 1 1760, 1690, 1590, 1530,
1400, 1360, 1280.
uv: h (pH 7 phosphate buffer) in nm (e ) 235
max
(10000), 280 (11000).
nmr : i$ (D20) in ppm 1. 68 ( 3H, d, J=6 Hz,
C-CH3 ) , 3. 25 ( :LH, d. J=18 Hz, 2-H) , 3 . 57 ( 1H, d. J=18
Hz, 2-H ) , 4. O1 ( 3H, s, OCH3 ) , 5 .10 ( 1H, s, CH-CO ) , 5 .19
( 1H, d, J=4. 5 13z, 6-H ) , 5 . 78 ( 1H, d, J=4. 5 Hz, 7-H ) ,
5. 5-5. 9 ( 1H, m, CH=C) . 5. 98 ( 1H, d, J=11 Hz, CH=C) ( 7 . 07
( 2H, s, phenyl-H), 7. 17 ( 1H, br-s, phenyl-H) .
1340697
- 68 -
HPLC: retention time 9.3 min. (0.02 M acetate
buffer (pH 4) containing 15$ acetonitrile).
Procedure 43
Isolation of Compound 42 from the Urine of Rats fed
Compound 37.
Six male blister rats (400-600g) were placed in
steel metabolic cages after the oral administration of
Compound 37 at the dase of 100 mg/kg and urine was
collected over a period of 24 hours. The rats were fed
their regular diet and given water during the experiment.
The following table shows the volume of urine collected
from time to time.
0-2 hr 2-4 hr 4-6 hr 6-24 hr Total
Urine volume-(mil) 18 19.5 13 42 92.5
The urine (ca. 90 ml) was adjusted to pH 3 with N
hydrochloric acid and filtered to remove a precipitate.
The filtrate wa.s chromatographed on a column packed with
300 ml of HP-2C~ by eluting with 2 L of water and 2 L of
30% methanol-un.der monitoring with HPLC. The fractions
containing the bioact:ive components of the 30$ methanol
eluate were collected, concentrated to 10 ml and
lyophilized to give :390 mg of brown solid. A solution of
the solid in 2C~ ml o!: water was chromatographed on a
column packed with 200 ml of the packing of a
prepPAK-C18 cartridge (Waters) by eluting with water, 5$
methanol, and 10$ methanol, successively. The first half
of the 5$ methanol eluate was concentrated to 5 ml and
lyophilized to give 44 mg of Compound 37 (70$ pure)
containing impurities derived from urine. The second half
of the 5% methanol eluate was concentrated to 5 ml and
lyophilized to give 36 mg of product, which was a mixture
of Compound 37, Compound 42, and impurities derived from
urine. The elu.ate with 10% methanol (ca. 600 ml) was
concentrated tc~ 5 ml and lyophilized to give 38 mg of
Compound 42 (7C~% pure by HPLC), which was
re-chromatograp~hed on a column of the same packing as
above (40 ml) by eluting with water,
1340697
- 69 -
5% methanol and 10% methanol. The desired fractions
eluted with 10% methanol were combined and concentrated to
ml and lyophilized to give 16 mg of Compound 42 which
was 90% pure by HPLC (0.02 M acetate buffer (pH
5 4)-acetonitrile (85 . 15). M, p. 180°C (grad. dec.).
ir: vinax(~r) in cm 1 1760, 1690, 1590,
1530, 1400, 1360, 1280.
uv: ~~nax(pH 7 phosphate buffer) in nm ( a ) 233
(8200), 280 (8800).
nmr: d (D20) in ppm 1.68 ( 3H, d, J=6 Hz,
-C-CH3 ) , 326 ( 1 H, d, J=18 Hz, 2-H ) , 3 . 58 ( 1H, d, J=18
Hz, 2-H ) , 4. Ol ( 3H, s, OCH3 ) , 5 .12 ( 1H, s, CH-CO ) , 5. 21
( 1H, d, J=4. 5 H:Z, 6-H ) , 5 . 78 ( 1H, d, J=4. 5 Hz, 7-H ) ,
5.5-5.9 ( 1H, m, CH=C-), 5.98 ( 1H, d, J=11 Hz, CH=C-), 7.07
( 2H, s, phenyl-IH), 7. 17 ( 1H, br-s, phenyl-H) .
The structure of the metabolite was established
as 7 S-[D(-)-2-;amino-2-(4-hydroxy-3-methoxyphenyl)
acetamide]-3-[(;~)-1-propen-1-yl]-3-cephem-4-carboxylic
acid by comparison (nmr, ir, uv, HPLC) with the Compound
42 prepared by Procedure 38-42.
30
