Anti-MRSA cephems. Part 2: C-7 cinnamic acid derivatives

Article abstract of DOI:10.1016/S0968-0896(02)00336-X

Forty-five novel cephalosporin derivatives with activity against methicillin-resistant Staphylococcus aureus (MRSA) are described. The compounds contain novel cinnamic acid moieties at C-7 that were synthesized using a key Heck reaction followed by nucleophilic aromatic substitution reactions. The most active compound (41) displayed an MIC₉₀ against MRSA of 1.0 μg/mL, and a PD₅₀ of 0.8 mg/kg. Compound 14 was found to be very safe in a mouse model of acute toxicity.

Full text of DOI:10.1016/S0968-0896(02)00336-X

Bioorganic & Medicinal Chemistry 11 (2003) 265–279
Anti-MRSA Cephems. Part 2:
C-7 Cinnamic Acid Derivatives
Dane M. Springer,* Bing-Yu Luh, Jason Goodrich and Joanne J. Bronson
Anti-infective Chemistry, Bristol-Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway, POBox 5100,
Wallingford, CT 06492, USA
Received 6 November 2001; accepted 17 June 2002
Abstract—Forty-five novel cephalosporin derivatives with activity against methicillin-resistant Staphylococcus aureus (MRSA) are
described. The compounds contain novel cinnamic acid moieties at C-7 that were synthesized using a key Heck reaction followed by
nucleophilic aromatic substitution reactions. The most active compound (41) displayed an MIC90 against MRSA of 1.0 mg/mL, and
a PD50 of 0.8 mg/kg. Compound 14 was found to be very safe in a mouse model of acute toxicity.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
moieties provided compounds with exceptional anti-
MRSA activity.6 An early lead, aminopropyl derivative
1, satisfied our goals for in vitro and in vivo activity.
Unfortunately, compound 1 was found to be acutely
toxic to mice upon iv bolus delivery to the tail vein. It
was subsequently found that the C-3 amino acid deri-
vative 2 had good activity against MRSA, and was less
acutely toxic than 1 in mice.7 However, bis-zwitterion 2
was not very soluble in a variety of aqueous media (<3
mg/mL).8 As a result of this low solubility, a large
number of derivatives were synthesized that had either a
carboxylic or sulfonic acid group on the side-chain of
the thiopyridinium moiety at C-3, or an acid substituted
off the pyridinium ring itself.4i The goal was to move the
acid group around the C-3 moiety in the hope of
improving upon the solubility of our derivatives while
maintaining sufficient anti-MRSA activity. As a result
of these efforts, some general trends were observed
regarding the inclusion of acid groups into our cephems.
First, the extra acid functions improved the acute toxi-
city profiles of our compounds (vide supra regarding the
toxicity of compound 1 vs 2). In general, it was observed
that positively charged molecules (such as 1) were
usually quite toxic in our acute toxicity assay. Zwitter-
ions or bis-zwitterions (such as 2) usually fared better in
the acute toxicity screen, while compounds carrying a
net negative charge were usually non-toxic in our assay.
While the inclusion of an extra acid improved the solu-
bility of our compounds (except in the case of zwitter-
ions), and helped improve their toxicity profile, the
addition of each extra acid group at the C-3 position
also resulted in a concomitant loss of potency in vitro
(generally 4–8 fold less active by MIC). We therefore
embarked upon a program to introduce an acid at C-7
Post-surgical morbidity from bacterial infections of
methicillin-resistant Staphylococcus aureus (MRSA) is
increasing as most strains have become multi-drug
resistant.1 The only effective treatment of MRSA cur-
rently in the clinic is vancomycin. The advent of vanco-
mycin resistance in enterococci has raised concern that
this resistance might eventually be transferred to MRSA,
creating a lethal pathogen of epidemic proportions.2
Strains of MRSA with reduced susceptibility to vanco-
mycin have begun to appear in the clinic, heralding the
beginning of the end of vancomycin’s utility in the
management of these infections.3 Therefore, the search
for new antibiotics with anti-MRSA activity is critical
to the future maintenance of public health.4
We wished to develop an injectable anti-MRSA cephalo-
sporin that would manage these life-threatening infections.
Our biological and solubility criteria for a suitably
active clinical candidate were as follows: MIC90s against
MRSA ꢀ4 mg/mL; PD50 in a mouse systemic MRSA
infection ꢀ5 mg/kg; safety in a mouse model of acute
toxicity; and, aqueous solubility ꢁ20 mg/mL.
Our program began with the observation that C-7 2,5-
dichlorothiophenyl acetamides imparted excellent anti-
staphylococcal activity to cephems.5 The combination
of this lipophilic C-7 group with C-3 thiopyridinium
*Corresponding author at current address: Rib-X-Pharmaceuticals,
300 George Street, New Haven, CT 06511, USA; e-mail: springer@
rib-x.com
0968-0896/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00336-X

266
D. M. Springer et al. / Bioorg. Med. Chem. 11 (2003) 265–279
of our cephems in the hopes of improving solubility,
maintaining safety in the acute toxicity assay, and
retaining excellent in vitro potency against MRSA. The
results of some of our efforts in this area are hereby
presented.
series, it was a bit long (six steps to key diester 6) and
suffered a low yield in the Heck reaction step. We
desired a shorter route to diester 6 and discovered the
chemistry shown in Scheme 2. We found that 2,4,5-tri-
chloro iodobenzene underwent clean conversion to
acrylate 8 via the Heck reaction. Subsequent nucleo-
philic aromatic substitution of ester 8 could be achieved
in good yield using the sodium salt of methyl mercapto-
acetate in DMF to produce the desired diester 6 in just
two steps. The efficient acquisition of 6 by this process
facilitated the synthesis of our analogues as exemplified
by the remaining chemistry shown in Scheme 2. Hydro-
lysis of 6 to acid 9 followed by DCC coupling to cephem
amine 10 led to the chloromethyl diester 11.10 TFA
deprotection of 11 gave intermediate diacid 12, which
could be treated with thiopyridones such as 13 to provide
the final antibiotic targets (such as 14).
Chemistry
The C-7 amide-linked thiophenyl group is undoubtedly
a major contributor to the lipophilicity of our deriva-
tives. We had long maintained an interest in introducing
functional groups onto the C-7 dichlorothiophenyl ring
of our compounds that would improve their water
solubility. One of our early routes to C-7 acid compounds
is illustrated by Scheme 1. Iodination of 2,5-dichloro-
phenol in the para position, followed by conversion
to the O-aryl dimethyl thiocarbamate, provided
intermediate 3. Newman–Kwart rearrangement of 3
gave S-aryl thiocarbamate 4, which could be hydrolyzed
to the thiol.9 Alkylation of the thiol derived from 4 with
methyl bromoacetate yielded iodo ester 5. The Heck
reaction of 5 with t-butyl acrylate afforded diester 6 in a
40% yield. Hydrolysis of the methyl ester of 6 gave the
corresponding acid which could be coupled with an
appropriate C-7 amino cephalosporin using DCC, and
subsequently utilized to produce final cephem targets of
type 7.
We wished to explore the use of other substrates for the
Heck reaction in an attempt to determine structure–
activity relationships in this series of C-7 cinnamic acid
derivatives. Unfortunately, aryl iodide 5 did not react
successfully with either t-butyl methacrylate or t-butyl
propiolate. Similarly disappointing was the unreactivity
of aryl chlorides 15–19 towards nucleophilic aromatic
substitution with the sodium thiolate derived from
methyl mercaptoacetate. Aryl chloride 15 lacks the
easily replaceable chlorine atom para to the acrylate
group, leaving only the more hindered ortho chlorine
available for displacement. Compounds 16 and 17 bear
two ortho chlorine substituents that may force the acrylate
While the chemistry of Scheme 1 was successful in pro-
ducing the first cephalosporin targets in the C-7 acid
Scheme 1. Reaction conditions: (a) I2, AgSO4, CH2Cl2; (b) DABCO, Me2NC(S)Cl, DMF; (c) heat, 220 ꢂC, 33% from 2,5-dichlorophenol; (d) 3 N
ꢂ
KOH, EtOH, reflux, 81%; (e) Et3N, BrCH2CO2CH3, CH2Cl2, 90%; (f) CH2¼CHCO2t-Bu, PPh3, Bu3N, DMF, Pd(OAc)2, 12 0 C, 67%.

D. M. Springer et al. / Bioorg. Med. Chem. 11 (2003) 265–279
267
Scheme 2. Reaction conditions: (a) CH2¼CHCO2t-Bu, PPh3, Bu3N, DMF, Pd(OAc)2, 80 ꢂC, 75%; (b) NaSCH2CO2CH3, DMF, 75%; (c) NaOH,
92%; (d) CDD, THF; (e) TFA, CH2Cl2, 71% from 9; (f) CH3OH, 61%.
residue more out of plane with the aryl ring than is the
case for compound 8. This would reduce the ability of
the acrylate p-system to stabilize the anion derived from
addition of the thiolate to the aryl ring. Compound 18
has an additional methyl substituent at the b-carbon of
the acrylate group that could similarly force the aryl
ring slightly more out of conjugation with the acrylate
group, reducing its reactivity relative to 8. The failure of
thiophene ester 19 to undergo the desired nucleophilic
aromatic substitution of one of the chlorine atoms is not
too surprising since thiophenes usually require the
presence of a nitro group on the ring for nucleo-
philic addition to proceed efficiently. We had hoped
that the aldehyde group of the bis-ortho substituted
derivative 20 might maintain good planarity with
the aryl ring, allowing the displacement of the para
chlorine atom. Interestingly, the reaction afforded
instead the benzothiophene 21 via ortho substitution
of the thiolate, followed by intramolecular aldol
condensation.
Since it appeared that we could not easily synthesize
alternate chloroaryl derivatives using the aromatic sub-
stitution reaction, or easily introduce other groups on
the olefinic moiety of our derivatives, we decided to
make amide derivatives of the C-7 acid. Towards these
ends, diester 6 was treated with TFA to afford acid 22
(Scheme 3). Acid 22 could be directly coupled with
amines using DCC, however, the yields were not opti-
mal, and purification of the products was occasionally
problematic. Better results were obtained by first cou-
pling acid 22 with N-hydroxy succinimide to give the
activated ester 23, and then treating 23 with amines such
as t-butyl glycine (to provide glycinamide 24). The cor-
responding diesters were selectively hydrolyzed at the
methyl ester position, similarly to diester 6, and the
derived acids were then coupled to cephem amine 10 as
previously described. Using the aromatic substitution
chemistry described above, and a variety of different
thiopyridones related to 13, the compounds shown in
Tables 1–4 were synthesized for biological evaluation.

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D. M. Springer et al. / Bioorg. Med. Chem. 11 (2003) 265–279
Scheme 3. Reaction conditions: (a) TFA, CH2Cl2, 79%; (b) N-hydroxysuccinimide, DCC, THF; (c) t-butyl glycine, THF, 89% from 22; (d) NaOH,
80%.
Results and Discussion
5.8 mg/mL for compound 45. The remaining members of
The first aspect of SAR that was investigated was the
effect of chlorine substitution on activity. As Table 1
demonstrates, the C-7 dichloro aryl derivative 25 is
more active than the corresponding monochloro com-
pounds 27 and 28. Similarly, the dichloro cephem 38 is
more active than its related monochloro derivatives 39
and 40. Amongst the monochloro compounds, the
ortho-substituted (relative to sulfur) 28 and 40 were less
active than their meta-substituted counterparts 27 and
39. The bis meta-chlorinated cephem 29 appears to be
slightly less active than compound 25, and trichloro
cephem 30 was much less potent than 25.
Table 2, the N-methyl thiopyridinium derivatives 14, 48
and 49, are quite potent in vitro. The only one of these
tested in vivo was 14, and it was found to have good
activity (PD50=5.4, 9.5 mg/kg).11 Compound 14 dis-
played an excellent MIC90 against MRSA (3.4 mg/mL).
Cephem 14 was also found to be quite safe in our mouse
acute toxicity assay. The compound was found to be
safe when given iv to mice at a bolus dose of at least 850
mg/kg (the highest dose tested). By comparison, cephem
2 was safe at a dose of 240 mg/kg, but higher doses
began to result in toxic effects in mice.
Table 3 contains the biological data for a series of C-3
salicylic acid pyridinium derivatives. Compound 50 was
found to be the most interesting of the group displaying
an excellent MIC90 (3.6 mg/mL) as well as very good
activity in vivo (PD50=3.6, 4.1 mg/kg). Unfortunately,
cephem 50 was found to have a short half-life in mice (7
min). The data for cephems 51–53 indicates that methyl
substitution on the pyridinium ring is tolerated in vitro,
but has deleterious effects on the in vivo activity of these
compounds relative to cephem 50.12 Replacement of the
salicylic hydroxyl group with a chlorine atom resulted in
a loss of in vitro potency (cf. 56 with 50). While the C-7
amide-linked acids 57 and 59 were quite active in vitro,
the C-3 dimethyl pyridinium compound 58 was mark-
edly less active than its unsubstituted counterpart
cephem 57.
Dimethyl substitution on the C-3 pyridinium ring is
often well tolerated (i.e., MIC of 31 and 33), but on
occasion results in loss of potency; thus, no general
trend can be predicted. The glycine acids 32–34, the
alanines 35–36, and serine 37 were fairly active deriva-
tives. An interesting feature of these C-7 amide-linked
acids is that they often have an improved pharmaco-
kinetic (PK) profile relative to the corresponding cinnamic
acid derivative. For example, both 25 and 32 were eval-
uated in a mouse PK study (20 mg/kg bolus dose given
iv). The following data was obtained for cinnamic acid
25: C5min=21 mg/mL; AUC=11 mg h/mL; and, t1/2=6
min. The data obtained from testing of 32 was:
C5min=53 mg/mL; AUC=38 mgh/mL; and, t1/2=43 min.
The slightly improved in vivo activity of 32 (PD50=1.8,
4.1 mg/kg) relative to 25 (PD50=4.1, 5.4 mg/kg) might
be a consequence of the compound’s improved PK
characteristics.
Table 4 shows the biological activity for some addi-
tional C-3 derivatives in the C-7 cinnamic acid, and
amide-linked acid, series of cephems. As the data in
Table 4 indicate, good in vitro activity can be attained
with a variety of substitution patterns. Unfortunately,
good in vivo activity is lacking in most of these com-
pounds, except for cephems 60 and 66.12 Cephem 66 did
not meet our criteria for a clinical candidate due to its
relatively high MIC90. Compound 60, while meeting all
our biological goals, was found to be insoluble in a
variety of aqueous media at pH 7.
Table 2illustrates the activity of the most potent C-7
acid derivative (in this series of cephems), the C-3
hydroxy aminopropyl thiopyridinium cephem 41. This
compound had excellent in vitro and in vivo activity,
and the following mouse PK parameters: C5min=51 mg/
mL; AUC=22 mg h/mL; and, t1/2=25 min. Unfortu-
nately, cephem 41 did not meet our solubility criteria,
being rather insoluble at pH 7 (<3 mg/mL).8 The poor
solubility of 41 is likely due to its bis-zwitterionic nature
at physiological pH.
Table 5 illustrates the in vitro antibacterial activity
against some Gram-positive organisms for the more
interesting compounds generated in the present work.
These compounds are quite active against Gram-posi-
tive bacteria such as streptococci, staphylococci, and
enterococci. The cephems in this class have little activity
against the representative Gram-negative organisms
The imidazolium compounds 45–47 had good activity in
vivo, and in vitro against our marker strain of MRSA
(A27223). However, the imidazolium class of C-3 deri-
vatives (paired with a C-7 acid group) suffered from
high MIC90s, as illustrated in this work by the value of

D. M. Springer et al. / Bioorg. Med. Chem. 11 (2003) 265–279
269
Table 1.
b
c
No.
R1
R2
H
R3
MICa
1 (4)
MIC90
3.3
PD50
25
4.1, 5.4
26
27
H
H
4 (4)
2(4)
16.5, >25
3.6
9.5, 16.5
28
29
H
16 (16)
2(2)
—
—
5.4, 7.2
30
31
32
H
—
H
8 (32)
2(4)
—
>25, >25
1.8, 4.1
4 (4)
33
34
35
36
—
—
—
—
1 (2)
2(4)
2(4)
2(4)
12.5, 12.5
9.5, 12.5
9.5, 12.5
21.8, >25
37
—
H
H
H
4 (4)
2(4)
5.4
16.5, 16.5
12.5, >25
—
3813
3913
4013
8 (8)
32(32)
aMIC (in mg/mL) versus MRSA A27223, value in parentheses is MIC in presence of 50% calf serum. The MIC of vancomycin (vs MRSA A27223) in
our assay was 0.25 mg/mL, and that of imipenem was 32 mg/mL.
bMIC90 (in mg/mL) for 58 strains of methicillin-resistant S. aureus.
cPD50 (in mg/kg) for activity against MRSA A27223 in a mouse model of systemic infection (vancomycin PD50 ꢃ0.3–0.8 mg/kg). Multiple values
denote separate experiments.

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D. M. Springer et al. / Bioorg. Med. Chem. 11 (2003) 265–279
Table 2.
R1
R2
H
R3
MICa
MIC90
1.0
PD50
b
c
No.
41
0.25 (0.5)
0.8, 0.8
42
H
4 (4)
12.5, 16.5
43
44
H
H
1 (2)
1 (2)
3.6
5.4, 12.5
9.5, 9.5
45
46
47
14
48
—
—
—
H
2(4)
2 (4)
1 (2)
2(4)
5.8
4.1, 9.5
1.4, 2.8, 4.2, 12.5
7.2
3.4
1.4, 5.4, 9.5
—
1 (2 )
—
49
—
1 (2 )
—
aMIC (in mg/mL) versus MRSA A27223, value in parentheses is MIC in presence of 50% calf serum. The MIC of vancomycin (vs MRSA A27223) in
our assay was 0.25 mg/mL, and that of imipenem was 32 mg/mL.
bMIC90 (in mg/mL) for 58 strains of methicillin-resistant S. aureus.
cPD50 (in mg/kg) for activity against MRSA A27223 in a mouse model of systemic infection (vancomycin PD50 ꢃ0.3–0.8 mg/kg). Multiple values
denote separate experiments.
included in our screening panel (Escherichia coli, Klebsiella
pneumoniae, Enterobacter cloacae, Proteus mirabilis,
Pseudomonas aeruginosa; data not shown in Table 5).
anti-MRSA cephalosporin into the clinic. The chemistry
developed to synthesize these C-7 cinnamic acid deriva-
tives will be of use to those interested in developing anti-
MRSA cephems. The activity of compounds derived
from further combinations of these of C-7 acid deriva-
tives with a variety of additional C-3 substituents
remains to be disclosed in future communications from
the laboratories of Bristol-Myers Squibb.
Conclusions
We have discovered a series C-7 cinnamic acid deriva-
tives that often display excellent in vitro and in vivo
activity against MRSA. The introduction of an extra
acidic group at C-7 onto leads such as cephem 1 was
found to be less deleterious to antibacterial potency
than placing the acid group at C-3. Many of these
compounds had good PK characteristics, and cephem
14 was shown to be a very safe compound in an acute
toxicity assay in mice. Cephems such as 14, 25, 41, 50
and 60 are exciting leads in the effort to bring a viable
Experimental
Biology
Antibacterial MICs were determined in broth according
to the standard conditions recommended by the
National Committee for Clinical Laboratory Standards

D. M. Springer et al. / Bioorg. Med. Chem. 11 (2003) 265–279
271
Table 3.
R1
R2
H
R3
MICa
2(8)
MIC90
3.6
PD50
b
c
No.
50
3.6, 4.1
51
52
53
—
H
4 (8)
4 (8)
4 (8)
>25, >25
12.5, 16.5
9.5, 12.5
—
54
55
56
—
H
H
4 (4)
2(8)
9.5, 21.8
21.8, >25
—
8 (16)
57
58
—
—
1 (2)
8 (8)
7.2, 21.8
—
59
—
1 (1)
9.5, 21.8
aMIC (in mg/mL) versus MRSA A27223, value in parentheses is MIC in presence of 50% calf serum. The MIC of vancomycin (vs MRSA A27223) in
our assay was 0.25 mg/mL, and that of imipenem was 32 mg/mL.
bMIC90 (in mg/mL) for 58 strains of methicillin-resistant S. aureus.
cPD50 (in mg/kg) for activity against MRSA A27223 in a mouse model of systemic infection (vancomycin PD50 ꢃ0.3–0.8 mg/kg). Multiple values
denote separate experiments.
(NCCLS). MIC assays against MRSA utilized Mueller-
Hinton broth+2% NaCl, a bacterial inoculum size
ꢃ5Â105 CFU/mL, and were incubated at 35 ꢂC for 24
h. MIC was defined as the lowest drug concentration
inhibiting all visible growth. MIC90s were determined
against a panel of 58 strains of MRSA.
Animals were monitored until the end of day five for
survival.
Compounds were assayed for acute toxicity in mice as
follows: the test compound was dissolved in 5% aqu-
eous dextrose at a concentration between 5 and 25 mg/
mL. The solution was filtered through a 0.2 m filter, and
between 0.1 and 1 mL of the solution was injected (in
usually less than a min) into the tail vein of three mice.
The usual toxic reaction observed after injection of these
types of compounds was red coloration of the feet, ears, tail
and muzzle of the mice, followed by respiratory distress
and death. Death, when observed, usually occurred within
min of injection. Mice surviving 1 h post injection usually
recovered with no obvious adverse effects.
The PD50 values reported represent the concentration of
the compound that protects 50% of the infected animals
from death in a mouse model of systemic infection.
Mice were given (ip) a lethal dose of MRSA A27233
(homogeneous strain, 2.4Â108 CFU) on day 1, and the
test compound was administered (im) twice on day 1 at
0.2h and 2h post infection [five animals per dose level,
five animals for infected (but untreated) control animals].

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D. M. Springer et al. / Bioorg. Med. Chem. 11 (2003) 265–279
Table 4.
b
c
No.
R1
R2
H
R3
MICa
1 (2)
MIC90
1.8
PD50
60
>25, >25
61
62
63
H
H
2 (4)
4 (8)
2(4)
21.8, 21.8
21.8, >25
21.8, >25
—
64
—
—
H
2(2)
>25, >25
>25, >25
5.4, 9.5
65
4 (4)
2(16)
6614
4.9
6714
H
H
1 (32)
1 (64)
>25, >25
6814
9.5, 12.5
aMIC (in mg/mL) versus MRSA A27223, value in parentheses is MIC in presence of 50% calf serum. The MIC of vancomycin (versus MRSA
A27223) in our assay was 0.25 mg/mL, and that of imipenem was 32 mg/mL.
bMIC90 (in mg/mL) for 58 strains of methicillin-resistant S. aureus.
cPD50 (in mg/kg) for activity against MRSA A27223 in a mouse model of systemic infection (vancomycin PD50 ꢃ0.3–0.8 mg/kg). Multiple values
denote separate experiments.
Chemistry
are reported in Hertz (Hz). Mass spectra were recorded on
a Kratos MS-50 or a Finnegan 4500 instrument utilizing
direct chemical ionization (DCI, isobutene) or fast atom
bombardment (FAB), or on a Shimadzu/Micromass
LCMS array (for ESI).
Unless otherwise indicated all reactions were performed
under a nitrogen atmosphere and all solvents were of
reagent grade (anhydrous if available). Analytical thin-
layer chromatography (TLC) was carried out on silica
gel plates (60F-254) and visualized using UV light,
iodine vapors, and/or staining by heating with ethanolic
phosphomolybdic acid. ‘Chromatography’ or ‘Chromato-
graphy on silica gel’ refers to flash column chromato-
graphy using E-Merck silica gel 60 (230–400 mesh)
unless otherwise noted. C-18 silica gel was purchased
from Whatman.
Synthesis of thiocarbamate 3. A. 2,5-Dichlorophenol
(20.4 g, 0.125 mol) is placed in a 1-L round-bottom flask
equipped with a large egg-shaped stir bar and is dis-
solved in 307 mL CH2Cl2. With rapid stirring, iodine
(46.6 g, 0.183 mol) is added, followed by silver sulfate
(42.3 g, 0.136 mol). The purple solution is stirred 1 day,
at which point NMR analysis of an aliquot indicates the
reaction is complete. The reaction is diluted with
CH2Cl2 (ꢃ200 mL) and filtered through a fritted
Buchner funnel to remove silver salts. The salts are
washed with additional CH2Cl2 (ꢃ100 mL). The
organic filtrate is transferred to a separatory funnel and
is washed first with a solution of sodium thiosulfate
Proton and carbon-13 nuclear magnetic resonance (1H
and 13C NMR) spectra were recorded on a Bruker AM-
300 or a Varian Gemini 300 spectrometer. Chemical
shifts are reported in d (ppm) units relative to tetra-
methylsilane (TMS) and interproton coupling constants

D. M. Springer et al. / Bioorg. Med. Chem. 11 (2003) 265–279
273
Table 5.
MIC of compounds
41
Organism
A No.
14
25
32
0.03
46
0.25
1
0.015
50
60
1
2
3
4
5
6
7
8
S. pneumoniae/Pen. I
S. pneumoniae/Pen. Resist.
S. pyogenes/Todd Hewitt
E. faecalis
E. faecalis/+ 50% calf serum
E. faecium
A27881
A28272
A9604
A20688
A20688
A24885
A28156
A9497
0.125
0.5
0.015
1
8
8
0.125
0.25
0.007
1
4
8
128
0.015
0.5
0.5
0.5
0.5
2
0.5
1
4
1
2
0.5
1
0.06
0.5
0.5
0.125
4
0.125
0.5
0.0005
0.25
0.5
0.06
0.25
0.007
1
2
4
64
0.015
0.5
0.5
0.5
0.5
1
0.5
2
8
0.03
0.25
0.003
0.25
1
0.25
0.001
0.5
1
2
2
8
>128
2
32
16
>128
0.06
4
128
E. faecium/Amp. R, Vanco. S
S. aureus/Pen.À
128
0.03
0.25
0.5
0.25
0.5
1
0.25
2
4
1
4
1
2
0.015
0.25
0.25
0.25
1
0.003
0.125
0.25
0.125
0.25
0.5
0.015
0.125
0.25
0.25
0.5
0.5
0.25
1
2
1
1
0.5
0.5
9
S. aureus/Pen.+
A9606
A9606
1
1
1
2
2
1
2
4
2
4
2
2
0.06
1
1
0.25
4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
S. aureus/+ 50% calf serum
S. aureus/MS/Pen. +
S. aureus/Hetero MR
S. aureus/+ 50% calf serum
S. aureus/Hetero MR
S. aureus/Homo MR
S. aureus/+ 50% calf serum
S. aureus/Homo MR
S. aureus/Homo MR
S. aureus/Homo MR
S. aureus/MR, PÀ
A20241
A27218
A27218
A27217
A27223
A27223
A27621
A27295
A27226
A27225
A24548
A25783
A27368
A21638
A27229
1
1
4
4
4
2
2
1
0.25
0.25
0.5
0.25
0.5
2
1
2
1
0.25
0.125
0.015
0.125
0.125
0.06
0.5
S. epidermidis
S. epidermidis/MR
S. epidermidis/Imipenem R
S. haemolyticus
S. haemolyticus/Homo MR
0.06
—
0.5
0.125
2
0.03
0.125
0.125
0.06
8
0.06
1
1
0.125
8
0.03
0.125
0.125
0.06
1
(ꢃ40 g in ꢃ200 mL water; this removes excess iodine),
and then brine. The organic phase is dried (MgSO4),
and evaporated to give 2,5-dichloro-4-iodophenol
(34.59 g, 0.108 mol; 86% yield) as a pale pink/yellow
solid. 1H NMR (300 MHz, CDCl3): d 7.75 (s, 1H, ArH),
7.14 (s, 1H, ArH), 5.62(br, 1H, OH).
minimum amount of CH2Cl2 for column loading), and
then ꢃ70% CH2Cl2/hexane. Compound 4 is obtained
as a yellow crystalline solid (13.0 g, 36.0 mmol; 33%
yield from 2,5-dichlorophenol). 1H NMR (300 MHz,
CDCl3): d 7.95 (s, 1H, ArH), 7.62(s, 1H, ArH), 3.10 (br
s, 3H, CH3), 3.00 (br s, 3H, CH3).
Synthesis of ester 5. A. Thiocarbamate 4 (9.80 g, 0.026
mol) is dissolved in 40 mL EtOH and treated with 30
mL 3 N aqueous KOH. The mixture is heated to reflux
with stirring under nitrogen for 3 h. The solution is
allowed to cool and is then acidified with 3 N HCl until
pH ꢃ3. The mixture is extracted with CH2Cl2 (three
times), and the combined organic phase washed with
water and then brine. The extracts are dried (MgSO4)
and evaporated. The crude material is chromatographed
on silica using 1:1 CH2Cl2/hexane. 2,5-Dichloro-4-
iodothiophenol (6.43 g, 0.021 mol; 81% yield) is
B. 2,5-Dichloro-4-iodophenol (34.59 g, 0.108 mol) is
placed into a 500-mL round-bottom flask equipped with
septa, N2 inlet, and a stir bar. The phenol is then dis-
solved in 130 mL DMF. DABCO (24.2 g, 0.216 mol) is
added followed by dimethylthiocarbamyl chloride (21.6
g, 0.175 mol). The mixture is stirred at room tempera-
ture for ꢃ1 h, then diluted with EtOAc (ꢃ400 mL) and
poured into a separatory funnel containing ꢃ300 mL of
ice-water. The phases are separated, and the aqueous
extracted twice with ꢃ200 mL of EtOAc. The combined
organic extracts are washed twice with water (ꢃ100
mL), and then brine. The organic phase is dried
(MgSO4), and evaporated to afford 3 as a dark oil. This
material is dissolved in CH2Cl2 and dried again
(MgSO4). After evaporation a yellow solid (ꢃ35 g) is
obtained. The compound was of sufficient purity to be
1
obtained as a white solid. H NMR (300 MHz, CDCl3):
d 7.83 (s, 1H, ArH), 7.56 (s, 1H, ArH).
B. 2,5-Dichloro-4-iodothiophenol (6.43 g, 0.021 mol)
is dissolved in 50 mL CH2Cl2 and triethylamine (2.52 g,
0.025 mol) is added. Methyl bromoacetate (3.82 g, 0.025
mol) is then added over 5 min. The resultant mixture is
1
used in the next reaction. H NMR (300 MHz, CDCl3):
d 7.90 (s, 1H, ArH), 7.24 (s, 1H, ArH), 3.46 (s, 3H,
CH3), 3.37 (s, 3H, CH3).
1
stirred at room temperature for 1.5 h, at which time H
NMR analysis indicated the reaction was complete. The
mixture was diluted with CH2Cl2 (ꢃ200 mL) and was
washed with water, 1 N HCl, water, and then brine. The
organic layer was dried (MgSO4) and evaporated. The
crude material is chromatographed on silica using 70%
CH2Cl2/hexane. Ester 5 is obtained as a white solid
(7.20 g, 0.019 mol; 90% yield). 1H NMR (300 MHz,
CDCl3): d 7.82(s, 1H, ArH), 7.42(s, 1H, ArH), 3.75 (s,
3H, OCH3), 3.68 (s, 2H, SCH2).
Synthesis of thiocarbamate 4. The crude material
obtained above (ꢃ35 g) was heated neat under N2 at
220 ꢂC for 2h. After cooling, the material was dissolved
in CH2Cl2 and filtered through a plug of silica gel. The
fractions containing the product are evaporated to afford
30.2g of a brown solid. This material was chromato-
graphed on silica (in portions) using a gradient elution
starting with 1:1 CH2Cl2/hexane (material dissolved in a

274
D. M. Springer et al. / Bioorg. Med. Chem. 11 (2003) 265–279
Synthesis of diester 6 from ester 5. Ester 5 (9.00 g, 0.024
mol) is dissolved in 20 mL DMF. Triphenylphosphine
(1.20 g, 0.005 mol), tributylamine (13.3 g, 0.071 mol),
and t-butyl acrylate (17.3 g, 0.140 mol) are then added
to the flask. Palladium acetate (0.95 g, 0.004 mol) is
added, and the mixture heated under nitrogen to 80 ꢂC
for 4 h. The solvents are evaporated, and the crude
material is partitioned between EtOAc and water. The
organic phase is washed thrice with 1 N HCl, once with
water, and then brine. The organic solution was dried
(MgSO4) and evaporated. Chromatography on silica gel
using 1:1 CH2Cl2/hexane, followed by CH2Cl2 as eluant
affords diester 6 (4.40 g, 0.012mol; 50% yield) as a yel-
2equivalents of this reagent by weight for the reaction
to go to completion. Presumably the solid is a solvate/
hydrate which is only about 50% pure anion by weight.
This solid is usually stored in the freezer and may
remain stable anywhere from a few days to a few weeks
(months?). If the color changes from white to faint beige
it has usually decomposed. However, white batches that
have been stored in the freezer for extended periods
must also be used with caution!
1
The progress of this reaction is best monitored by H
NMR of aliquots. TLC is misleading at times. Use of an
excess of thiolate anion results in further reaction to
substitute another chlorine with a thioacetate chain.
1
low solid. H NMR (300 MHz, CDCl3): d 7.86 (d, 1H,
J=16 Hz, ArCH¼C), 7.60 (s, 1H, ArH), 7.36 (s, 1H,
ArH), 6.35 (d, 1H, J=16 Hz, C¼CHCO2t-Bu), 3.77 (s,
3H, OCH3), 3.72(s, 2H, SCH 2), 1.51 (s, 9H, C(CH3)3).
Synthesis of acid 9. Diester 6 (4.40 g, 0.012mol) is dis-
solved in 30 mL THF. To this solution is added 13 mL
of 1 N NaOH, and the mixture is allowed to stir at room
temperature for 1.5 h. At this time 1H NMR analysis of
an aliquot indicates that the reaction is complete. The
THF is removed under vacuum, and the concentrate is
diluted with water and extracted with EtOAc. The aqueous
layer is then acidified with 1 N HCl to pH 4, and then
extracted with CH2Cl2. The organic phase is washed
with brine, dried (MgSO4), and then evaporated. Acid 9
is obtained as a tan solid (3.80 g, 0.011 mol; 92% yield).
1H NMR (300 MHz, CDCl3): d 7.82(d, 1H, J=16 Hz,
ArCH¼C), 7.56 (s, 1H, ArH), 7.33 (s, 1H, ArH), 6.29
(d, 1H, J=16 Hz, C¼CHCO2t-Bu), 3.72(s, 2H, SCH 2),
1.51 (s, 9H, C(CH3)3).
Synthesis of ester 8. 2,4,5-Trichloroiodobenzene (25 g,
81.3 mmol) is dissolved in 80 mL DMF. tert-Butyl
acrylate (48 mL, 328 mmol), tributylamine (58 mL, 243
mmol), triphenylphosphine (4.08 g, 15.5 mmol), and
palladium acetate (3.23 g, 14.4 mmol) are added, and
the mixture heated to 80 ꢂC for 3 h. The solvents are
evaporated and the residue is partitioned with EtOAc
and water. The aqueous phase is extracted with EtOAc,
and the combined organic phase is washed with brine,
dried (MgSO4), and evaporated. The dark red-brown oil
is chromatographed on silica in a fritted Buchner funnel
using vacuum filtration, with 30% CH2Cl2/hexane fol-
lowed by 50% CH2Cl2/hexane as eluant. Acrylate 8 is
obtained (18.7 g, 60.8 mmol; 75% yield) as a mauve
solid. 1H NMR (300 MHz, CDCl3): d 7.82(d, 1H, J=16
Hz, ArCH¼C), 7.67 (s, 1H, ArH), 7.51 (s, 1H, ArH),
6.34 (d, 1H, J=16 Hz, C¼CHCO2t-Bu), 1.51 (s, 9H,
C(CH3)3).
Note: This reaction was later found to be complete in
less than 30 min.
Synthesis of chloromethyl cephem diester 11. Cephem
amine 10 (15.04 g, 0.035 mol) is suspended under a
nitrogen atmosphere in 65 mL THF. A solution of DCC
in methylene chloride (1 M, 36.2mL, 0.036 mol) is
added, and the mixture allowed to stir for 5 min. Acid 9
(13.15 g, 0.035 mol) is added and the mixture is stirred
for 1.5 h. Ether (ꢃ30 mL) is added, and the solids
(mostly DCU) are filtered off. The red-colored filtrate is
evaporated to ꢃ25–30 mL and ether and pentane are
added to precipitate the cephem product. The solid
cephem is collected, washed with ether, and dried under
vacuum to afford diester 11 (14.2g, 0.019 mol; 54%
yield). 1H NMR (300 MHz, CDCl3): d 7.53 (d, 1H,
J=16 Hz, ArCH¼C), 7.45–7.20 (m, 12H, ArH), 6.98 (s,
1H, Ph2CH), 6.35 (d, 1H, J=16 Hz, C¼CHCO2t-Bu),
5.82(dd, 1H, J=5, 8 Hz, R1R2CHNR3), 4.97 (d, 1H,
J=5 Hz, CH(CNR)(SR)), 4.37 (m, 2H, CH2Cl), 3.76 (d,
1H, J=16 Hz, ArSCH2), 3.55 (d, 1H, J=16 Hz,
ArSCH2), 3.40 (d, 1H, J=16 Hz, RSCH2R), 1.54 (s,
9H, C(CH3)3).
Synthesis of diester 6 from acrylate 8. Acrylate 8 (21.23
g, 69 mmol) is dissolved in 131 mL DMF. The sodium
salt of methyl mercaptoacetate (17.7 g, crude: see note
below) is added and the mixture stirred at room tem-
perature for 1 h. The mixture is partitioned with EtOAc
and water. The aqueous phase is extracted with EtOAc,
and the combined organic phase is washed with brine,
dried (MgSO4), and evaporated. Chromatography on
silica in a fritted Buchner funnel (vacuum filtration)
using 50% CH2Cl2/hexane followed by 90% CH2Cl2/
hexane as eluant affords diester 6 (19.6 g, 52.0 mmol;
75% yield). The spectral properties of 6 generated from
8 are identical to those determined for 6 obtained from
5, and are reported above.
Note: The sodium salt of methyl mercaptoacetate is best
made fresh before use. Approximately 30 mL of methyl
mercaptoacetate is dissolved in ꢃ250 mL THF. One
equivalent of 5 N NaOH is added slowly in pipetfulls,
and the mixture allowed to stir for 5 min. The solvents
are removed in vacuo (including water) and the sticky
solid is co-evaporated with ether (ꢃ200 mLs) and then
dry THF (2Â200 mLs). The solid is pumped dry for
about 12h under high vacuum until the flask is no
longer cool due to evaporation. The freely mobile white
solid is used as obtained. Generally it takes from 1.5 to
Synthesis of chloromethyl diacid cephem 12. Diester 11
(0.760 g, 1.00 mmol) is dissolved in 4 mL CH2Cl2 and
0.8 mL anisole. Trifluoroacetic acid (2mL) is added,
and the mixture is stirred for 4 h. The solvents are eva-
porated, and the residue triturated with CH2Cl2/ether.
The solid is collected, washed with EtOAc and then
dried under vacuum. Diacid 12 is obtained (0.420 g,

D. M. Springer et al. / Bioorg. Med. Chem. 11 (2003) 265–279
275
0.780 mmol; 78% yield) as a light yellow solid. 1H
NMR (300 MHz, DMSO): d 9.30 (d, 1H, J=8 Hz,
RC(O)NH), 8.07 (s, 1H, ArH), 7.72(d, 1H, J=16 Hz,
ArCH¼C), 7.54 (s, 1H, ArH), 6.68 (d, 1H, J=16 Hz,
C¼CHCO2H), 5.71 (dd, 1H, J=5, 8 Hz, R1R2CHNR3),
5.13 (d, 1H, J=5 Hz, CH(CNR)(SR)), 4.55 (m, 2H,
CH2Cl), 3.97 (m, 2H, ArSCH2), 3.70 (d, 1H, J=16 Hz,
RSCH2R), 3.53 (d, 1H, J=16 Hz, RSCH2R).
7.50 (s, 1H, ArH), 7.17 (d, 1H, J=16 Hz, C¼CHCO2),
4.23 (s, 2H, SCH2), 3.68 (s, 3H, OCH3), 2.84 (m, 4H,
CH2CH2).
Synthesis of diester 24. tert-Butylglycine hydrochloride
(5.19 g, 0.031 mol) is suspended under a nitrogen
atmosphere in 60 mL dry DMF. N-Methylmorpholine
(3.90 mL, 0.036 mol) is added, and then the mixture is
cooled to 0 ꢂC. Crude hydroxysuccinimide ester 23 (12.3
g) is added, and the mixture allowed to stir for 10 min at
0 ꢂC. The cooling bath is removed and the reaction is
stirred for 1 h. The mixture is concentrated, and the
residue dissolved in ethyl acetate and placed in a
separatory funnel. The solution is washed with 0.4 N
aqueous HCl, 0.1 N aqueous NaHCO3, water and then
brine. The organic phase is dried (MgSO4) and evapo-
rated to afford clean diester 24 as a light yellow solid
Synthesis of thiopyridone 13. Pyran-4-thione (3.63 g,
32.4 mmol) is dissolved in a 2 M solution of methylamine
in methanol (19.4 mL, 38.9 mmol), and the mixture
stirred for 3 h at room temperature. The mixture is
concentrated, and then chromatographed on silica using
methanol/CH2Cl2 as eluants. 1H NMR (300 MHz,
DMSO): d 7.55 (d, 2H, J=8 Hz, C¼CH), 7.16 (d, 2H,
J=8 Hz, C¼CH), 3.71 (s, 3H, CH3).
1
(10.4 g, 0.024 mol; 89% yield from acid 22). H NMR
Synthesis of cephem 14. Cephem diacid 12 (5.50 g, 10.2
mmol) is dissolved in ꢃ75 mL methanol. Thiopyridone
13 (1.28 g, 10.2 mmol) is added, and the mixture
allowed to stir for ꢃ1 h. The mixture is concentrated,
and the residue is carefully treated with dilute (ꢃ0.5 N)
NaOH until the pH reaches ꢃ8.5. The solution is then
chromatographed on reverse-phase C-18 silica gel, and
the fractions containing the product are lyophillized.
Cephem 14 (4.08 g, 6.15 mmol) is obtained as the mono
sodium salt, zwitterion (a light tan lyophillate). 1H
NMR (300 MHz, 1:1 CD3OD/DMSO-d6): d 8.44 (d, 2H,
J=7 Hz, C¼CH), 8.00 (d, 2H, J=7 Hz, C¼CH), 7.73
(s, 1H, ArH), 7.43 (s, 1H, ArH), 7.20 (d, 1H, J=16 Hz,
ArCH¼C), 6.39 (d, 1H, J=16 Hz, C=CHCO2), 5.39 (d,
1H, J=5 Hz, R1R2CHNR3), 4.83 (d, 1H, J=5 Hz, CH(-
CNR)(SR)), 4.40 (s, 2H, SCH2), 4.10 (s, 3H, CH3), 3.83 (m,
2H, SCH2), 3.45 (d, 1H, J=16 Hz, SCH), 3.2 4 (d, 1H,
(300 MHz, DMSO-d6): d 8.42(t, 1H, J=8 Hz, NH),
7.81 (s, 1H, ArH), 7.59 (d, 1H, J=16 Hz, ArCH¼C),
7.47 (s, 1H, ArH), 6.80 (d, 1H, J=16 Hz,
C¼CHCONH), 4.20 (s, 2H, SCH2), 3.85 (d, 2H, J=8
Hz, NCH2CO2), 3.67 (s, 3H, OCH3), 1.41 (s, 9H,
C(CH3)3).
Synthesis of acid 24b. Diester 24 (0.600 g, 1.40 mmol) is
dissolved in 5 mL THF. Aqueous 1 N NaOH (1.40 mL,
1.40 mmol) is added and the mixture is allowed to stir at
room temperature for 1 h. The THF is evaporated, and
the residue taken up in 20 mL water. The solution is
acidified to pH=3 with 1 N HCl, and the mixture is
partitioned with EtOAc and water. The organic phase is
washed with brine, dried (MgSO4), and evaporated.
Pure acid 24b is obtained (0.470 g, 1.12mmol; 80%
yield). 1H NMR (300 MHz, DMSO): d 8.45 (t, 1H, J=7
Hz, NH), 7.83 (s, 1H, ArH), 7.59 (d, 1H, J=17 Hz,
C¼CH), 7.43 (s, 1H, ArH), 6.80 (d, 1H, J=17 Hz,
C¼CH), 4.08 (s, 2H, SCH2), 3.85 (d, 2H, J=7 Hz,
NCH2), 1.43 (s, 9H, C(CH3)3).
.
J=16 Hz, SCH). Anal. (C25H20Cl2N3Na1O6S3 2.6H2O) C,
H.
Synthesis of acid 22. Diester 6 (7.00 g, 0.019 mol) is
suspended in 40 mL CH2Cl2. Anisole (1 mL) is added,
followed by 15 mL of trifluoroacetic acid. The mixture
is stirred for 1 h at room temperature. The solvents are
concentrated to ꢃ15 mL, and excess ether is added to
precipitate a white solid. The solid is collected, washed
with ether, and dried under vacuum to yield acid 22
(4.85 g, 0.015 mol; 79% yield). 1H NMR (300 MHz,
DMSO-d6): d 8.08 (s, 1H, ArH), 7.71 (d, 1H, J=16 Hz,
ArCH¼C), 7.43 (s, 1H, ArH), 6.69 (d, 1H, J=16 Hz,
C¼CHCO2), 4.19 (s, 2H, SCH2), 3.66 (s, 3H, OCH3).
Synthesis of cephem 25. Cephem diester 11 (0.609 g,
0.802mmol) is dissolved in 8 mL DMF. Sodium iodide
(0.361 g, 2.40 mmol) is added, followed by 1-(2-
hydroxy-ethyl)-1H-pyridine-4-thione (0.155 g, 1.00
mmol), and the mixture is allowed to stir for 3 h at
room temperature. The DMF is evaporated, and the
residue is triturated with CH2Cl2 and ether to afford the
C-3 alkylated cephem as a solid (0.503 g, 0.557 mmol).
The protected cephem is dissolved in 2mL CH Cl2 and
2
0.5 mL anisole, and 1.5 mL of TFA is added. The mix-
ture is stirred 4 h at room temperature, and is then
evaporated to dryness. The residue is redissolved in
methanol and triturated with ether. The solid obtained
is then stirred in ethyl acetate for 1 h, and then collected
by filtration and dried over P2O5. Cephem 25 is
Synthesis of hydroxysuccinimide ester 23. Acid 22 (8.75
g, 0.027 mol) is suspended in 55 mL THF under an
atmosphere of nitrogen. Dicyclohexylcarbodiimide (1 M
in CH2Cl2, 28.7 mL, 0.029 mol) is added, followed by
N-hydroxysuccinimide (3.14 g, 0.027 mol). The reaction
is allowed to stir for 3 h at room temperature. The
mixture is diluted with ꢃ30 mL acetone, filtered to
remove dicyclohexylurea, and concentrated to ꢃ2 5 mL.
A solid forms which is filtered off, and the filtrate is
evaporated to dryness. Crude 23 is obtained as a white
solid (12.3 g) which is of sufficient purity for use in
1
obtained as a light tan solid (0.206 g, 0.268 mmol). H
NMR (300 MHz, DMSO; partial): d 9.24 (d, 1H, J=8
Hz, C(O)NH), 8.69 (d, 2H, J=8 Hz, C¼CH), 8.09 (s,
1H, ArH), 8.01 (d, 2H, J=8 Hz, C¼CH), 7.73 (d, 1H,
J=16 Hz, ArCH¼C), 7.56 (s, 1H, ArH), 6.71 (d, 1H,
J=16 Hz, C¼CHCO2H), 5.71 (dd, 1H, J=5, 8 Hz,
R1R2CHNR3), 5.16 (d, 1H, J=5 Hz, CH(CNR)(SR)),
4.55–4.44 (m, 2H), 4.43–4.36 (m, 2H), 3.99 (s, 2H,
1
subsequent reactions. H NMR (300 MHz, DMSO-d6):
d 8.27 (s, 1H, ArH), 8.01 (d, 1H, J=16 Hz, ArCH¼C),

276
D. M. Springer et al. / Bioorg. Med. Chem. 11 (2003) 265–279
ArSCH2). MS (ESI) m/e 656 (M+H)+. Anal.
mixture is allowed to stir for 35 min. The solvents were
evaporated, and the residue triturated with CH2Cl2 and
ether. The yellow solid is collected (0.805 g), and the pH
is adjusted to neutral with aqueous NaHCO3. The aqu-
eous mixture is chromatographed on C-18 silica gel
using water, then 5% CH3CN/water and finally 15%
CH3CN/water as eluants. Cephem 32 is obtained as
yellow crystals of the mono sodium salt, zwitterion
(0.305 g, 0.421; 31% yield). 1H NMR (300 MHz,
DMSO-d6/D2O, 2:1; partial): d 8.52(d, 2H, J=8 Hz,
C¼CH), 8.10 (d, 2H, J=8 Hz, C¼CH), 7.82(s, 1H,
ArH), 7.53 (d, 1H, J=16 Hz, ArCH¼C), 7.51 (s, 1H,
ArH), 6.87 (d, 1H, J=16 Hz, C¼CHC(O)NH), 5.42(d,
1H, J=6 Hz, R1R2CHNR3), 4.89 (d, 1H, J=6 Hz,
CH(CNR)(SR)), 4.50–4.42(m, 5H), 3.51 (s, 2H, SCH 2),
3.48 (d, 2H, J=17 Hz, RSCH2R), 3.28 (d, 2H, J=17
Hz, RSCH2R). MS (ESI) m/e 711 (M-H)À. Anal.
.
(C26H24Cl2N3O7S3 C2F3O2–) C, H, N.
Synthesis of cephem 32. Cephem amine 10 (6.22 g, 15.0
mmol) is suspended in 30 mL THF. DCC (1M in
CH2Cl2; 15 mL, 15.0 mmol) is added, followed by acid
24b (6.30 g, 15.0 mmol), and the mixture is allowed to
stir for 1 h at room temperature. THF (ꢃ1 L) is added,
and stirring is continued for another hour. The solids
remaining are filtered off (dicyclohexylurea) and the fil-
trate is treated with ether to precipitate 7-(2-{4-[2-(tert-
butoxycarbonylmethyl-carbamoyl)-vinyl]-2,5-dichloro-
phenylsulfanyl}-acetylamino)-3-chloromethyl-8-oxo-5-
thia-1-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylic acid benz-
hydryl ester as a white solid (10.0 g, 12.2 mmol; 81%
yield). 1H NMR (300 MHz, DMSO): d 9.31 (d, 1H, J=8
Hz, C(O)NH), 8.42(t, 1H, J=7 Hz, C(O)NH), 7.81 (s,
1H, ArH), 7.60 (d, 1H, J=16 Hz, ArCH¼C), 7.57 (s,
1H, ArH), 7.52–7.24 (m, 10H, ArH), 6.96 (s, 1H,
CHPh2), 6.70 (d, 1H, J=16 Hz, C¼CHCO2H), 5.80
(dd, 1H, J=5, 8 Hz, R1R2CHNR3), 5.20 (d, 1H, J=5
Hz, CH(CNR)(SR)), 4.41 (m, 2H, CH2Cl), 3.97 (s, 2H,
ArSCH2), 3.84 (d, 2H, J=7 Hz, NCH2C(O)), 3.73 (d,
1H, J=16 Hz, RSCH2R), 3.55 (d, 1H, J=16 Hz,
RSCH2R), 1.39 (s, 9H, C(CH3)3). MS (ESI) m/e 814
(M-H)À. Anal. (C38H36Cl3N3O7S2) C, H.
.
(C28H25Cl2N4Na1O8S3 2.7H2O) C, H.
Synthesis of cephem 41. A. Synthesis of [2-hydroxy-3-(4-
thioxo-4H-pyridin-1-yl)-propyl]-carbamic acid tert-butyl
ester. A mixture of pyran-4-thione (1.10 g, 10.0 mmol)
and 1-(N-boc)amino-3-amino-2-propanol (1.90 g, 10.0
mmol) in 25 mL absolute ethanol is stirred for 18 h at
room temperature. The solution is concentrated, and
the residue is triturated with a small amount of diethyl
ether and filtered to provide the title thiopyridone (0.850
g, 3.01 mmol; 30%) as a tan solid (more material is
7-(2-{4-[2-(tert-Butoxycarbonylmethyl-carbamoyl)-vinyl]-
2,5-dichloro-phenylsulfanyl}-acetylamino)-3-chloromethyl-
8-oxo-5-thia-1-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylic acid
benzhydryl ester as obtained above (7.20 g, 8.81 mmol)
is suspended in 40 mL CH2Cl2, and anisole (2mL) fol-
lowed by TFA (15 mL) are added, and the mixture is
allowed to stir 4 h at room temperature. The solvents
are evaporated, and the residue triturated with ether and
CH2Cl2 to afford an orange solid. The solid is collected
and pumped dry to afford 7-(2-{4-[2-(carboxymethyl-
carbamoyl)-vinyl]-2,5-dichlorophenylsulfanyl}-acetyl-
amino)-3-chloromethyl-8-oxo-5-thia-1-aza-bicyclo[4.2.0]
oct-2-ene-2-carboxylic acid (4.40 g, 7.40 mmol; 84%
yield). [Note: An impurity, (3-{4-[(carboxymethyl-carb-
amoyl)-methylsulfanyl]-2,5-dichloro-phenyl}-acryloyl-
amino)-acetic acid, was often present in an amount of
ꢃ3–10% after this TFA deprotection. The cephem diacid
material obtained is suitable for further use since the
impurity is removed by trituration of the products from
the subsequent C-3 alkylation reactions with thiopyr-
idones. For a discussion on how this impurity arises in
1
present in the filtrate). H NMR (300 MHz, DMSO): d
7.54 (d, 2H, J=7 Hz, C¼CH), 7.14 (d, 2H, J=7 Hz,
C¼CH), 6.88 (br t, 1H, J=6 Hz, NH), 5.35 (d, 1H, J=6
Hz, OH), 4.06 (d, 1H, J=13 Hz, NCH), 3.82–3.65 (m,
2H), 2.96 (br dd, 2H, J1=J2=6 Hz, NCH2), 1.39 (s, 9H,
C(CH3)3).
B. Synthesis of 1-[2-hydroxy-3-amino-prop-1-yl]-4-[(6R)-
trans-2-carboxy-8-oxo-7-[(2,5-dichloro-4-(2-carboxy-
ethenyl)phenylthio)acetamido]-5-thia-1-azabicyclo[4.2.0]-
oct-2-en-3-yl]methylthio]pyridinium bis-trifluoroacetate.
Diacid 12 (0.780 g, 1.45 mmol) is dissolved in 3 mL
methanol and 8 mL of CH2Cl2. The above thiopyridone
(0.395 g, 1.45 mmol) is added and the reaction mixture
is stirred at room temperature for 4 h. The reaction
mixture is then stored at 4 ꢂC overnight. The reaction
mixture is then concentrated in vacuo and the crude
product is precipitated with diethyl ether. The crude
product is collected by filtration and is then stirred in
EtOAc for 30 min. The product is collected by filtration
and dried under vacuum to provide the intermediate
BOC-protected cephem as a tan solid (0.690 g, 0.850
mmol; 59% yield of a mixture of two diastereomers).
1H NMR (300 MHz, DMSO, partial): d 9.31 (d, 1H,
J=8 Hz), 8.67 (d, 2H, J=7 Hz), 8.07 (s, 1H), 7.98 (d,
2H, J=7 Hz), 7.72(d, 1H, J=16 Hz), 7.54 (s, 1H), 5.70
(dd, 1H, J=5, 8 Hz), 5.14 (d, 1H, J=5 Hz), 4.59–4.45
(m, 2H), 4.40–4.32 (m, 2H), 4.05–3.95 (m, 2H), 1.38
(s, 9H).
1
the TFA deprotection see ref. 4k.] H NMR (300 MHz,
DMSO; partial): d 9.30 (d, 1H, J=8 Hz, C(O)NH), 8.40
(t, 1H, J=7 Hz, C(O)NH), 7.81 (s, 1H, ArH), 7.58 (d,
1H, J=16 Hz, ArCH¼C), 7.55 (s, 1H, ArH), 6.81 (d,
1H, J=16 Hz, C¼CHCO2H), 5.73 (dd, 1H, J=5, 8 Hz,
R1R2CHNR3), 5.14 (d, 1H, J=5 Hz, CH(CNR)(SR)),
4.55 (m, 2H, CH2Cl), 3.97 (s, 2H, ArSCH2), 3.89 (d, 2H,
J=7 Hz, NCH2C(O)). MS (ESI) m/e 588 (MÀH)À.
7-(2-{4-[2-(Carboxymethyl-carbamoyl)-vinyl]-2,5-dichloro-
phenylsulfanyl}-acetylamino)-3-chloromethyl-8-oxo-5-
thia-1-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylic acid as
obtained above (0.800 g, 1.34 mmol) is dissolved in 4
mL methanol and 1 mL DMF. 1-(2-Hydroxy-ethyl)-1H-
pyridine-4-thione (0.208 g, 1.34 mmol) is added, and the
The above BOC-protected cephem (0.605 g, 0.747
mmol) is suspended in 3 mL of CH2Cl2 then 1 mL of
trifluoroacetic acid is added. The reaction mixture is
stirred at room temperature for 2h. The reaction mixture
is concentrated in vacuo then the residue is dissolved in

D. M. Springer et al. / Bioorg. Med. Chem. 11 (2003) 265–279
277
CH2Cl2 and precipitated with diethyl ether. The solids
are collected by filtration, stirred in EtOAc for 30 min
then again collected by filtration and dried under
vacuum in the presence of P2O5. Cephem 41 is obtained
(0.410 g, 0.609 mol; 82% yield) as the bis-trifluoro-
acetate salt. 1H NMR (300 MHz, DMSO, partial): d
9.40–9.35 (m, 1H, RC(O)NH), 8.77–8.72(m, H2,
SpyrH), 8.23–8.18 (m, 2H, SpyrH), 8.12 (s, 1H, ArH),
7.77 (d, 1H, J=16 Hz, ArCH¼C), 7.62(s, 1H, ArH),
6.75 (d, 1H, J=16 Hz, C¼CHCO2H), 5.68–5.61 (m,
1H, R1R2CHNR3), 5.11 (d, 1H, J=5 Hz,
CH(CNR)(SR)), 4.07–3.97 (m, 2H, ArSCH2). MS
(ESI) m/e 685 (M+).
extracts are washed with water and then brine. The
organic layer is dried (MgSO4), and concentrated in
vacuo. The residue is filtered through a short plug of
silica gel by eluting with 5% methanol/CH2Cl2 as the
solvent. The title thiopyridone was obtained as the
chloride salt (1.70 g, 5.46 mmol; 54%). 1H NMR
(300 MHz, DMSO): d 7.72(s, 1H, imidazolium), 7.61 (s,
1H, imidazolium), 7.03 (s, 2H, 2ÂC¼CH), 4.23 (t, 2H,
J=8 Hz, NCH2), 4.00–3.92(m, 2H, NCH 2), 3.75 (s, 3H,
NCH3), 2.69 (s, 3H, CH3), 2.37 (s, 6H, 2ÂCH3), 2.18–
2.02 (m, 2H, CH2).
Synthesis of cephem 46. Cephem diacid 12 (0.550 g, 1.02
mmol) is dissolved in 3 mL DMF, and 1-[3-(2,6-dimethyl-
4-thioxo-4H-pyridin-1-yl)-propyl]-2,3-dimethyl-3H-imid-
azol-1-ium; chloride salt (0.270 g, 0.877 mmol) is added.
The mixture is allowed to stir for 20 min at room tem-
perature, and then the solvents are evaporated. The
residue is triturated with ether, and the solids that are
collected are washed with ether, ethyl acetate and acetone.
The material is then treated with 0.4 N aqueous NaOH
until the pH is ꢃ7.5, and the solution is chromato-
graphed on reverse-phase C-18 silica. A gradient elution
beginning with water, and ending with 20% CH3CN/
water, delivers bis-zwitterion cephem 46 (0.310 g, 0.399
mmol) as a light yellow lyophillate. 1H NMR (300 MHz,
DMSO; partial): d 9.12(d, 1H, J=8 Hz, RC(O)NH),
8.13 (s, 2H, SpyrH), 7.79 (s, 1H, imidazolium), 7.71 (s,
1H, ArH), 7.64 (s, 1H, imidazolium), 7.45 (s, 1H, ArH),
7.16 (d, 1H, J=16 Hz, ArCH¼C), 6.39 (d, 1H, J=16
Hz, C¼CHCO2H), 5.36 (dd, J=5, 8 Hz, R1R2CHNR3),
4.86 (d, 1H, J=5 Hz, CH(CNR)(SR)), 4.42–4.21 (m,
6H), 3.99–3.80 (m, 2H), 3.75 (s, 3H, CH3), 2..70 (s,
6H, 2ÂCH3), 2.63 (s, 3H, CH3). MS (ESI) m/e 777
(M+).
Synthesis of 1-[3-(2,6-dimethyl-4-thioxo-4H-pyridin-1-
yl)-propyl]-2,3-dimethyl-3H-imidazol-1-ium; chloride salt.
A. 3-Bromopropylamine hydrobromide (20.5 g, 93.6
mmol) and triphenylmethyl chloride (25.0 g, 103 mmol)
are dissolved in 200 mL CH2Cl2. A solution of 2 5 g of
triethylamine in 10 mL of CH2Cl2 is added dropwise, and
the mixture is stirred for 2h. The mixture is washed
with water, 10% phosphoric acid, water, and then brine.
The organic phase is dried (MgSO4), and concentrated
in vacuo. Upon concentration, a solid forms that is
collected by filtration and washed with diethyl ether and
hexane to provide 1-triphenylmethyl-3-bromopropyl-
amine (30.0 g, 87.2mmol; 93% yield).
B. 1-Triphenylmethyl-3-bromopropylamine as obtained
above (6.87 g, 20.0 mmol) and 1,2-dimethylimidazole (2.2
g, 22.9 mmol) are dissolved in 80 mL DMF and heated
at 80 ꢂC for 3 h. The reaction mixture is concentrated in
vacuo, and the residue is diluted with diethyl ether. A
precipitate forms that is collected by filtration and
washed with diethyl ether to provide 4.5 g of product.
The filtrate is concentrated in vacuo, then treated with a
small amount of diethyl ether to afford an additional 2.2
grams of product. The solids are combined to yield 1-(3-
triphenylmethylamino)propyl-2,3-dimethylimidazolium
bromide (6.70 g, 15.3 mmol; 77%).
Synthesis of cephem 50. A. Synthesis of 2-hydroxy-4-(4-
thioxo-4H-pyridin-1-yl)-benzoic acid, sodium salt. 3-
Aminosalicylic acid (1.50 g, 9.87 mmol), pyran-4-thione
(0.640 g, 5.71 mmol) and sodium bicarbonate (0.840 g,
10.0 mmol) are suspended in 20 mL absolute ethanol,
and the mixture is refluxed for 6 h. The mixture is con-
centrated, and placed on a reverse-phase C-18 silica gel
column. Elution with water and then acetonitrile/water
gives the title thiopyridone as a red sodium salt. 1H
NMR (300 MHz, DMSO): d 7.87 (d, 2H, J=8 Hz,
C¼CH), 7.80 (d, 1H, J=8 Hz, ArH), 7.21 (d, 2H, J=8
Hz, C¼CH), 6.81 (d, 1H, J=2Hz, ArH), 6.72(dd, 1H,
J=2, 8 Hz, ArH).
C. 1-(3-Triphenylmethylamino)propyl-2,3-dimethylimid-
azolium bromide as obtained above (6.50 g, 14.8 mmol)
is dissolved in 70 mL methanol and cooled to 0 ꢂC. 3 N
HCl (15 mL) is added, and the cooling bath was
removed. The reaction mixture is stirred 30 min at
ambient temperature, and then the organic solvents are
evaporated. The aqueous phase is washed with diethyl
ether (two times), and with CH2Cl2 (three times), and
then concentrated. The 1-(3-amino)propyl-2,3-dimethyl-
imidazolium dichloride obtained (2.30 g, 10.2 mmol;
69%) is used in subsequent reactions without further
purification.
B. Cephem diacid 12 (0.430 g, 0.799 mmol) is dis-
solved in 2mL DMF, and 2-hydroxy-4-(4-thioxo-4H-
pyridin-1-yl)-benzoic acid, sodium salt (0.210 g, 0.781
mmol) is added and the mixture stirred at room tem-
perature for 20 min. The crude product is precipitated
from the solution with ether. The material obtained is
treated with 0.5 N NaOH to ꢃpH 8, and chromato-
graphed on a reverse-phase C-18 silica gel column. Elu-
tion with water and then 10% acetonitrile/water affords
cephem 50 (0.131 g, 0.170 mmol) as a light yellow lyo-
D. 1-(3-Amino)propyl-2,3-dimethylimidazolium dichloride
as obtained above (2.30 g, 10.2 mmol) and 2,6-dimethyl-
pyran-4-thione (1.6 g, 11.4 mmol) are dissolved in 30
mL absolute ethanol. 2N NaOH (6.3 mL, 12.6 mmol) is
added, and the reaction mixture is stirred at room tem-
perature for 18 h. The reaction mixture is heated at
60 ꢂC for 6 h. The mixture is concentrated, and acidified
with 1 N HCl to pH ꢃ2. The mixture is extracted with
CH2Cl2 (three times), and the combined organic
1
phillate. H NMR (300 MHz, DMSO; partial): d 9.05
(d, 1H, J=8 Hz, RC(O)NH), 8.87 (d, 2H, J=7 Hz,
SpyrH), 8.34 (d, 2H, J=7 Hz, SpyrH), 7.82(d, 1H, J=7

278
D. M. Springer et al. / Bioorg. Med. Chem. 11 (2003) 265–279
Hz, ArH), 7.46 (s, 1H, ArH), 7.09 (d, 1H, J=16 Hz,
ArCH¼C), 7.01 (d, 1H, J=2Hz, ArH), 6.89 (dd, 1H,
J=2, 7 Hz, ArH), 6.30 (d, 1H, J=16 Hz, C¼CHCO2H),
5.40 (dd, J=5, 8 Hz, R1R2CHNR3), 4.92(d, 1H, J=5
Hz, CH(CNR)(SR)), 4.66 (d, 1H, J=13 Hz, SCH2),
4.45 (d, 1H, J=13 Hz, SCH2), 3.96–3.75 (m, 2H), 3.50
(d, 1H, J=17 Hz, SCH2), 3.36 (d, 1H, J=17 Hz, SCH2).
MS (ESI) m/e 748 (M+H)+. Anal. (C31H21Cl2N3Na2-
Brunswick, NJ for solubility determinations in support
of this work.
References and Notes
1. (a) Ayliffe, G. A. J. Clinical Infectious Diseases 1997, 24,
S74. (b) Ehlert, K. Curr. Pharm. Des. 1999, 5, 45.
2. Rotschafer, J. C.; Petersen, M.; Hoang, A. T. D.; Wright,
D. J. Infect. Dis. Pharm. 1998, 3, 2 9.
.
O9S3 4.7H2O) C, H, N.
3. (a) Hiramatsu, K.; Hanaki, H.; Ino, T.; Yabuta, K.; Oguri,
T.; Tenover, F. C. J. Antimicrob. Chemother. 1997, 40, 135. (b)
Smith, T. L.; Pearson, M. L.; Wilcox, K. R.; Cruz, C.; Lan-
caster, M. V.; Robinson-Dunn, B.; Tenover, F. C.; Zervos,
M. J.; Band, J. D.; White, E.; Jarvis, W. R. New Eng. J. Med.
1999, 340, 493.
Synthesis of cephem 60. A. Synthesis of N,N0-di(tert-
butoxycarbamyl)-N00-[2-(4-thioxo-4H-pyridin-1-yl)-ethyl]-
guanidine. Pyran-4-thione (0.110 g, 0.982mmol) and
N,N0-di(tert-butoxycarbamyl)-N00-[2-aminoethyl]-guanidine
(0.385 g, 1.28 mmol) are dissolved in 10 mL absolute
ethanol and allowed to stir 18 h at room temperature.
1H NMR analysis indicates the reaction is only ꢃ50%
complete. The mixture is heated to 80ꢂC for 4 h, at which
4. (a) For some recent publications concerning the develop-
ment of anti-MRSA cephems see: Hecker, S. J.; Glinka, T. W.;
Cho, A.; Zhang, Z. J.; Price, M. E.; Chamberland, S.; Griffith,
D.; Lee, V. J. J. Antibiot. 2000, 53, 1272. (b) Glinka, T. W.;
Cho, A.; Zhang, Z. J.; Ludwikow, M.; Griffith, D.; Huie, K.;
Hecker, S. J.; Dudley, M. N.; Lee, V. J.; Chamberland, S. J.
Antibiot. 2000, 53, 1045. (c) Ishikawa, T.; Iizawa, Y.; Okonogi,
K.; Miyake, A. J. Antibiot. 2000, 53, 1053. (d) Ishikawa, T.;
Kamiyama, K.; Matsunaga, N.; Tawada, H.; Iizawa, Y.;
Okonogi, K.; Miyake, A. J. Antibiot. 2000, 53, 1071. (e)
Tsushima, M.; Iwamatsu, K.; Umemura, E.; Kudo, T.; Sato,
Y.; Shiokawa, S.; Takizawa, H.; Kano, Y.; Kobayashi, K.;
Ida, T.; Tamura, A.; Atsumi, K. Bioorg. Med. Chem. 2000, 8,
2781. (f) Pae, A. N.; Lee, J. E.; Kim, B. H.; Cha, J. H.; Kim,
H. Y.; Cho, Y. S.; Choi, K. I.; Koh, H. Y.; Lee, E.; Kim, J. H.
Tetrahedron 2000, 56, 5657. (g) Yamazaki, H.; Tsuchida, Y.;
Satoh, H.; Kawashima, S.; Hanaki, H.; Hiramatsu, K. J.
Antibiot. 2000, 53, 546. (h) Yamazaki, H.; Tsuchida, Y.;
Satoh, H.; Kawashima, S.; Hanaki, H.; Hiramatsu, K. J.
Antibiot. 2000, 53, 551. (i) For prior publications from the
laboratories of Bristol-Myers Squibb see: Springer, D. M.;
Luh, B.-Y.; Bronson, J. J. Bioorg. Med. Chem. Lett. 2001, 11,
797. (j) D’Andrea, S. V.; Bonner, D.; Bronson, J. J.; Clark, J.;
Denbleyker, K.; Fung-Tomc, J.; Hoeft, S. E.; Hudyma, T. W.;
Matiskella, J. D.; Miller, R. F.; Misco, P.F.; Pucci, M.; Ster-
zycki, R.; Tsai, Y.; Ueda, Y.; Wichtowski, J. A.; Singh, J.;
Kissick, T. P.; North, J. T.; Pullockaran, A.; Humora, M.;
Boyhan, B.; Vu, T.; Fritz, A.; Heikes, J.; Fox, R.; Godfrey,
J. D; Perrone, R.; Kaplan, M.; Kronenthal, D.; Mueller, R. H.
Tetrahedron 2000, 56, 5687. (k) Singh, J.; Kim, O. K.; Kissick,
T. P.; Natalie, K. J.; Zhang, B.; Crispino, G. A.; Springer,
D. M.; Wichtowski, J. A.; Zhang, Y.; Goodrich, J.; Ueda, Y.;
Luh, B. Y.; Burke, B. D.; Brown, M.; Dutka, A. P.; Zheng, B.;
Hsieh, D.-M.; Humora, M. J.; North, J. T.; Pullockaran, A. J.;
Livshits, J.; Swaminathan, S.; Gao, Z.; Schierling, P.; Ermann,
P.; Perrone, R. K.; Lai, M. C.; Gougoutas, J. Z.; DiMarco,
J. D.; Bronson, J. J.; Heikes, J. E.; Grosso, J. A.; Kronenthal,
D. R.; Denzel, T. W.; Mueller, R. H. Org. Process Res. Dev.
2000, 4, 488. (l) Kim, O. K.; Hudyma, T. W.; Matiskella, J. D.;
Ueda, Y.; Bronson, J. J.; Mansuri, M. M. Bioorg. Med. Chem.
Lett. 1997, 7, 2753. (m) Kim, O. K.; Ueda, Y.; Mansuri,
M. M.; Russell, J. W.; Bidwell, V. W. Bioorg. Med. Chem.
Lett. 1997, 7, 1945.
1
time H NMR analysis indicates no more product is
forming, but the presence of decomposition products is
increasing. The reaction is concentrated, and then
chromatographed on silica using CH3OH/CH2Cl2 as
eluants to afford the title thiopyridone (0.110 g, 0.285
mmol). 1H NMR (300 MHz, CDCl3): d 8.48 (t, 1H, J=7
Hz, CNH), 7.41 (d, 2H, J=8 Hz, C¼CH), ), 7.15 (d,
2H, J=8 Hz, C¼CH), 4.06 (t, 2H, J=7 Hz, CH2), 3.63
(dt, J=7, 7 Hz, CH2), 1.47 (s, 9H, C(CH3)3), 1.46 (s,
9H, C(CH3)3).
B. Cephem diacid 12 (0.153 g, 0.285 mmol) is dissolved
in 5 mL of 1:1 CH3OH/CH2Cl2, and N,N0-di(tert-butoxy-
carbamyl)-N00-[2-(4-thioxo-4H-pyridin-1-yl)-ethyl]-guani-
dine (0.110 g, 0.285 mmol) is added. The mixture is
stirred at room temperature for 1 h, and then con-
centrated. The residue is triturated with ether and ethyl
acetate, and the solids collected are then stirred in ethyl
acetate for ꢃ30 min and then filtered. The material is
suspended in 3 mL CH2Cl2, and 1 mL TFA is added.
The mixture is stirred at rt for 3 h and then evaporated.
The residue is suspended in some CCl4, which is then
evaporated. Ether is added to triturate the cephem, and
this material is collected by filtration, redissolved in
methanol, and then triturated again using ether. The
solids are collected and washed with ethyl acetate to
afford cephem 60 bis-trifluoroacetate (0.063 g, 0.068
1
mmol) as a yellow solid. H NMR (300 MHz, DMSO):
d 8.69 (d, 2H, J=8 Hz, C¼CH), 8.07 (s, 1H, ArH), 8.02
(d, 2H, J=8 Hz, C¼CH), 7.72(d, 1H, J=16 Hz,
ArCH¼C), 7.55 (s, 1H, ArH), 6.69 (d, 1H, J=16 Hz,
C¼CHCO2H), 5.69 (dd, J=5, 8 Hz, R1R2CHNR3),
5.14 (d, 1H, J=5 Hz, CH(CNR)(SR)), 4.56–4.46 (m,
2H), 4.40–4.31 (m, 2H), 3.97 (s, 2H, SCH2). MS (ESI)
m/e 697 (M+).
5. A group from Eli Lilly found that in a series of C-3 acetate
cephems, a derivative bearing a 2,5-dichlorothiophenyl acetamide
group at C-7 was extremely potent against staphylococcus species.
The optimal halogen substitution to maximize anti-staphylococcus
activity on the thiophenyl ring was the 2,5-dichloro pattern. In
largely unpublished observations, our group at Bristol-Myers
Squibb has found this SAR trend at C-7 to be quite general for
anti-MRSA activity irrespective of the nature of the C-3
group. For the original work by Lilly, see: Huffman, G. W. US
Patent 3,907,784, 1975. Chem. Abstr. 1975, 84, 17395.
Acknowledgements
We thank our colleagues in the Department of Micro-
biology in Wallingford, CT for the biological data
reported in this work. We also thank the Department of
Analytical Research in Wallingford, CT, for analytical
data, and Mr. Robert Perrone of the Department of
Pharmaceutics Research and Development in New

D. M. Springer et al. / Bioorg. Med. Chem. 11 (2003) 265–279
279
6. Kim, C. U.; Misco, P. F.; Wichtowski, J. A.; Ueda, Y.;
Hudyma, T. W.; Matiskella, J. D.; D’Andrea, S. V.; Hoeft,
S. E.; Miller, R. F. US Patent 5,567,698, 1996. Chem. Abstr.
1996, 126, 7903.
7. Compound 1, given at a dose of ꢃ200 mg/kg, resulted in
death to all three mice tested. Compound 2, given at a dose of
ꢃ300 mg/kg, was safe to all three mice tested. See the
Experimental details of the acute toxicity assay.
8. For solubility determinations, a known quantity of test
compound is dissolved in water, 0.9% saline, or any other
buffer and the pH adjusted to the desired point by the addition
of min amounts of 1 N NaOH. The solutions are stirred for 2
h, and then filtered through a 0.2-m filter. The filtrate is then
assayed by HPLC and the concentration of dissolved com-
pound determined by comparison to a standard curve.
9. (a) Kwart, H.; Evans, E. R. J. Org. Chem. 1966, 31, 410. (b)
Newman, M. S.; Karnes, H. A. J. Org. Chem. 1966, 31, 3980.
(c) Sebok, P.; Timar, T.; Eszenyi, T.; Patonay, T. J. Org.
Chem. 1994, 59, 6318. (d) Robertson, D. W.; Beedle, E. E.;
Krushinski, J. H.; Pollock, G. D.; Wilson, H.; Wyss, V. L.;
Hayes, J. S. J. Med. Chem. 1985, 28, 717.
macodynamic studies. Thus, there remain many possible (but
unevaluated) explanations for the lack of in vivo activity of
many of our cephems. Depending on the nature of the pendant
groups on the C-3 thiopyridinium ring, the compounds may
have differential metabolic and distribution profiles. One pos-
sible common explanation could be that inactive compounds
remain localized at the site of intramuscular injection limiting
the exposure of the animal to the test drug. This could either
be a consequence of low solubility, or poor absorption into the
vascular system from the muscle tissue, or both.
13. (a) These compounds were synthesized from the known t-
butyl ester of 7-aminocephalosporanic acid by acylation and
then TFA deprotection, as described for other derivatives in
the experimental section. For syntheses of t-butyl 7-amino-
cephalosporanate see: Blacklock, T. J.; Butcher, J. W.; Sohar,
P.; Lamanec, T. R.; Grabowski, E. J. J. J. Org. Chem. 1989, 54,
3907. (b) Mangia, A.; Scandroglio, A. Org. Prep. Proced. Int.
1986, 18, 13. For reference purposes, the C-3 acetate derivative
with a 2,5-dichlorothiophenyl group at C-7 (no acrylic acid
substituent) had an MIC of 2 mg/mL versus MRSA A27223.
Thus, with this C-3 group, the inclusion of the acid substituent
at C-7 (compare with compound 38) caused no loss of in vitro
potency. Usually, a 2-fold loss of in vitro potency is observed
by incuding an acid at C-7, while in general an acid placed at
C-3 results in a 4-fold loss of activity.
10. Chloromethyl cephem amine 10 was purchased from
Otsuka Chemical Co., Ltd.
11. Unfortunately, compounds 48 and 49, synthesized
towards the end of our MRSA cephem program, remained
unevaluated in vivo at the close of our work.
14. These compounds were generally prepared in methanol
using the requisite chloromethyl cephem diacid, and the
sodium thiolate of the C-3 side-chain group.
12. Compounds that were found to be inactive in vivo were
not generally evaluated in mouse pharmacokinetic or phar-