Relationships between structure, antibacterial activity, serum stability, pharmacokinetics and efficacy in 3-(heteroarylthio)cephems. Discovery of RWJ-333441 (MC-04,546)

Article abstract of DOI:10.1016/S0968-0896(02)00431-5

SAR studies in a series of related 3-(heteroarylthio)cephems determined that a relatively high chemical reactivity of the β-lactam ring, modulated by electronic effects of substituents at C-3 and C-7, is necessary to achieve high in vitro activity against methicillin-resistant Staphylococcus aureus (MRSA). Such high reactivity results in lowered hydrolytic stability and concomitantly increases susceptibility to β-lactam ring opening mediated by serum enzymes. Therefore, optimization of anti-MRSA activity versus stability toward serum-mediated degradation required a fine balance of substituent effects. Serum stability studies (measured as percentage of parent drug degraded after 60 min incubation) revealed up to 80-fold difference in degradation rate in a series of closely related (3-heteroarylthio)cephems. Of the compounds evaluated, RWJ-333441 (MC-04,546) possessed the best balance of serum stability (6% degradation after 60 min incubation) and in vitro activity versus MRSA (S. aureus COL MIC=1 μg/mL). Accordingly, RWJ-333441 displayed excellent in vivo efficacy versus methicillin-susceptible Staphylococcus aureus (MSSA, ED₅₀=0.39 mg/kg in mouse sepsis model with S. aureus Smith) and good pharmacokinetic properties in the rat (Cl_total=0.39 L/h/kg).

Full text of DOI:10.1016/S0968-0896(02)00431-5

Bioorganic & Medicinal Chemistry 11 (2003) 591–600
Relationships Between Structure, Antibacterial Activity, Serum
Stability, Pharmacokinetics and Efficacy in
3-(Heteroarylthio)cephems. Discovery of
RWJ-333441 (MC-04,546)
Tomasz Glinka,* Keith Huie, Aesop Cho, Maria Ludwikow, Johanne Blais,
David Griffith, Scott Hecker and Michael Dudley
Essential Therapeutics, Inc.,y 850 Maude Ave., Mountain View, CA 94043, USA
Received 9 May 2002; accepted 7 August 2002
Abstract—SAR studies in a series of related 3-(heteroarylthio)cephems determined that a relatively high chemical reactivity of the
b-lactam ring, modulated by electronic effects of substituents at C-3 and C-7, is necessary to achieve high in vitro activity against
methicillin-resistant Staphylococcus aureus (MRSA). Such high reactivity results in lowered hydrolytic stability and concomitantly
increases susceptibility to b-lactam ring opening mediated by serum enzymes. Therefore, optimization of anti-MRSA activity versus
stability toward serum-mediated degradation required a fine balance of substituent effects. Serum stability studies (measured as
percentage of parent drug degraded after 60 min incubation) revealed up to 80-fold difference in degradation rate in a series of
closely related (3-heteroarylthio)cephems. Of the compounds evaluated, RWJ-333441 (MC-04,546) possessed the best balance of
serum stability (6% degradation after 60 min incubation) and in vitro activity versus MRSA (S. aureus COL MIC=1 mg/mL).
Accordingly, RWJ-333441 displayed excellent in vivo efficacy versus methicillin-susceptible Staphylococcus aureus (MSSA,
ED50=0.39 mg/kg in mouse sepsis model with S. aureus Smith) and good pharmacokinetic properties in the rat (Cltotal=0.39 L/h/
kg).
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
b-lactams in which the affinity toward PBP protein 2a of
S. aureus is restored.5
The incidence of life-threatening nosocomial MRSA
infections has been on the rise over the last three dec-
ades.1 Since PBP2a-mediated MRSA resistance has
rendered all clinically used b-lactams ineffective against
such infections, the glycopeptide vancomycin, and to a
lesser extent, new agents quinupristin/dalfopristin and
linezolid,2 represent the only available therapies. How-
ever, the recent occurrences of GISA (glycopeptide
intermediate-resistant Staphylococcus aureus) infections3
have prompted recommendation that vancomycin use be
limited in order to control the emergence of vancomycin-
resistant gram-positive pathogens.4 The MRSA-active
cephalosporin RWJ-54428 (MC-02,479), presently
under development by R. W. Johnson Pharmaceutical
Research Institute and Essential Therapeutics (formerly
Microcide), represents one of the recent examples of
We have reported previously on discovery of a wide
variety of 3-heteroarylthio analogues with antimicrobial
activities comparable to RWJ-54428.6,7 The present
publication describes our efforts aimed toward better
understanding of the structural factors that govern phar-
macokinetic properties within this class of compounds. In
particular, the effects of structural modifications at posi-
tions C-3 and C-7 of 3-(heteroarylthio)cephalosporins on
stability in serum, drug clearance and efficacy were
studied in detail.
*Corresponding author. Tel.: +1-650-428-3512; fax: +1-650-428-
3550; e-mail: tglinka@etrx.com
yFormerly Microcide Pharmaceuticals, Inc.
0968-0896/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00431-5

592
T. Glinka et al. / Bioorg. Med. Chem. 11 (2003) 591–600
Chemistry
obtained by standard deprotection of 10 followed by
desalting on an HP20 column and lyophilization. In
order to synthesize the sidechain of cephalosporin 18, 2-
mercaptoisonicotinic acid was reduced to the corre-
sponding alcohol and S-tritylated to produce inter-
mediate 11. Treatment with the Vilsmeyer reagent
followed by reaction with N-Boc cysteamine gave the
intermediate 12, which upon further manipulation
furnished the sidechain thiol 13 in suitably protected
form. Coupling with the cephem 3-mesylate 14 fol-
lowed by deprotection of acid-labile groups resulted in the
target cephem. Preparations of other cephem analogues,
which often required multi-step syntheses of C-3 sidechain
thiols, generally followed similar synthetic schemes.
The syntheses of cephalosporin 29 (RWJ-333441/MC-
04,546) and cephalosporin 18 exemplify the routes
employed for the preparation of other cephalosporin
analogues (Schemes 1 and 2). The 7-amino-3-chloro-
cephem carboxylic acid 1 (Otsuka Chemical Co.) was
converted into the corresponding t-Bu ester 2 using a
procedure described for other b-lactam intermediates.8
Protected
(5-amino-[1,2,4]thiadiazol-3-yl)-trityloxy-
imino-acetic acid (4) was synthesized by a modified
Katayama route9 starting from 3-aminoisoxazole (3).
Acylation of 2 with the acid 4 provided the key
3-chlorocephem intermediate 5. In the synthesis of the
C-3 sidechain, 3-tert-butylthiopyridine-2-carboxylic
acid (6), prepared from 3-bromopyridine,10 was con-
verted into the key intermediate 7, which was then
deprotected and oxidized to disulfide 8. An aqueous
solution of this hydrophilic intermediate was reacted in
a biphasic system with Boc-anhydride in ethyl acetate
solution to produce protected disulfide 9 in high yield.
In situ reduction of 9 with triphenylphosphine provided
the corresponding thiol, which was coupled with
chlorocephem 5 to produce protected cephem 10.
Cephalosporin 29 (RWJ-333441/MC-04,546) was
Results and Discussion
We have found that in order to obtain the desired level
of anti-MRSA activity in the 3-(heteroarylthio)cephem
series, it is necessary to append electron-withdrawing
substituents at positions C-7 and especially C-3 of the
cephem core, which increase the reactivity of the b-lac-
tam ring. We observed that certain of these compounds
displayed reduced stability in rat serum, and hypothesized
ꢀ
.
Scheme 1. (a) t-BuOAc, BF3 Et2O; (b) (i) KSCN, MeOC(O)Cl; (ii) MeOH, 60 ; (iii) peracetic acid; (iv) MeOH, SOCl2; (c) (i) Br2, MeOH; (ii) pyr-
idine N-oxide, acetonitrile; (iii) NH2OH, EtOH; (iv) trityl chloride, Et3N, DMF; (v) NaOH, EtOH, water; (d) (PhO)2P(O)Cl, 2,6-lutidine, THF; (e)
(i) ClC(O)OEt, Et3N; (ii) NaBH4, THF; (iii) SOCl2, DMF; (iv) N-Boc-cysteamine; (f) (i) 6M HCl; reflux; (ii) air, water; (g) Boc-anhydride, Et3N,
ethyl acetate, MeOH; (h) Ph3P, NaHCO3, DMF, water; (j) (i) trifluoroacetic acid, triethylsilane, CH2Cl2; (ii) HP20 resin, acetonitrile/water.

T. Glinka et al. / Bioorg. Med. Chem. 11 (2003) 591–600
593
Scheme 2. (a) (i) B2H6, THF; (ii) TrCl, Et3N, DMF; (b) (i) SOCl2, DMF; (ii) HSCH2CH2NHC(O)O-t-Bu, DMF, K2CO3; (c) (i) trifluoroacetic acid,
triethylsilane, CH2Cl2; (ii) di-t-butyldicarbonate, (iPr)2NEt; (iii) sodium methoxide, MeOH; (d) (i) 13, ethyl acetate; (ii) trifluoroacetic acid, tri-
ethylsilane, CH2Cl2.
Table 1. Structures of RWJ-333441 (MC-04,546; 29) and related compounds
R2
R2
R2
15: Y¼H; R¼H
26: R¼H
32:
16: Y¼Cl; R¼H
27: R¼C(NH)NH2
17: Y¼Cl; R¼C(NH)NH2
18: Y¼Cl
28:
33:
34:
19: Y¼Cl: R¼H
29: R¼H
20: Y¼Cl; R¼C(NH)NH2
30: R¼C(NH)NH2
21: Y¼Cl; R¼H
31:
35:
36:
37:
22: Y¼Cl; R¼C(NH)NH2
23: X¼S; Y¼Cl; R¼H
24: X¼NH; Y¼Cl; R¼H
25: X¼NH; Y¼Cl; R¼C(NH)NH2

594
T. Glinka et al. / Bioorg. Med. Chem. 11 (2003) 591–600
that this feature might influence their pharmacokinetic
profiles (Table 1).
the anti-MRSA activity of cephalosporins. Therefore
while optimizing the pharmacokinetic properties of the
C-3 heteroarylthio cephalosporins we maintained the
presence of either amino or guanidino functionality in
the structure. Several guanidine-substituted cephalos-
porins (17, 20, 22, 25) displayed improved gram-positive
In our earlier SAR studies leading to the discovery of
MC-02,331, basic functionality attached to the C-3 het-
eroarylthio substituent was found to be beneficial for
Table 2. Antimicrobial activity of cephalosporins 15–37 against gram-positive bacteria
S.a.1
S.a.2
S.a.3
S.a.4
S.a.5
S.h.
E.fs.
E.fm.1
E.fm.2
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
IMI
0.25
0.25
0.13
0.13
0.25
0.13
0.25
0.06
ꢁ0.06
0.13
ꢁ0.06
0.13
0.25
0.5
0.13
0.13
1
0.5
0.5
1
0.5
0.5
1
0.25
0.5
1
0.5
1
1
8
1
1
1
1
8
2
1
1
0.5
1
1
0.5
1
0.5
0.5
1
0.5
1
1
8
1
1
1
2
8
2
1
1
0.5
1
1
0.5
2
0.5
0.5
1
0.5
1
1
8
1
1
1
2
4
1
2
2
1
2
1
0.13
ꢁ0.06
ꢁ0.06
0.13
0.06
0.06
0.13
0.06
ꢁ0.06
ꢁ0.06
ꢁ0.06
0.13
0.13
1
1
1
0.5
0.25
0.5
0.25
0.13
0.25
0.25
ꢁ0.25
1
2
0.5
0.25
1
0.5
0.25
1
0.5
0.5
0.5
0.25
0.5
0.5
4
1
0.5
2
1
4
1
1
1
1
0.25
0.5
2
1
0.5
8
1
4
4
1
1
1
8
2
1
8
4
4
ꢁ0.06
0.13
0.13
0.06
0.13
0.06
0.13
1
1
0.5
0.5
1
0.5
2
2
16
2
2
2
4
8
2
2
32
1
64
0.13
ꢁ0.06
0.13
0.13
0.13
0.25
0.13
0.13
0.25
0.13
0.5
0.5
0.25
0.13
0.13
0.13
0.25
0.13
ꢁ0.008
ꢁ0.06
ꢁ0.06
ꢁ0.06
ꢁ0.06
ꢁ0.06
ꢁ0.008
2
1
2
2
1
8
1
32
1
16
1
1
8
1
32
32
4
4
S.a.1, S. aureus ATCC 29213 (methicillin-susceptible); S.a. 2, S. aureus ATCC 13709 Smith (methicillin-susceptible); S.a.3, S. aureus Col (methicillin-
resistant, beta-lactamase negative); S.a. 4, S. aureus 76 (methicillin-resistant, beta-lactamase positive); S.a. 5, S. aureus ATCC 33593 (methicillin-
resistant); S.h., S. haemolyticus 05 (methicillin-resistant); E.fs., E. faecalis ATCC 29212; E.fm.1, E. faecium ATCC 35667; E.fm.2, E. faecium (van-
comycin resistant).
Table 3. Pharmacokinetic properties and serum stability of cephalosporins 15–37
ED50 (mg/kg)
(95% CL)a
Total drug
clearance in rat
(L/h/kg) (cassette dosing)a
Human serum
binding (%)
(HSMHB/MHB
MIC ratio)a
Aqueous
solution
decomposition
(%/ha)
Rat serum
decomposition
(%/h)
(human serum
decomposition)a
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
IMI
1.3 (0.8–1.8)
1.0 (0.7–1.6)
—
0.8 (0.5–1.0)
<0.31
—
0.2 (0.17–0.5)
—
1.25
5.26 (2.41)
1.35 (1.67)
0.73
0.79
—
0.95 (0.72)
—
(14.7)
—
—
2.17 (1.59)
1.12 (0.50)
—
0.51 (0.39)
68 (–)
84 (3)
70 (–)
90 (2)
90 (2)
83 (–)
88 (2)
– (2)
83 (–)
—
4.7
2.4
2.6
1.4
2
1.2
1.7
—
5.1
—
—
—
—
—
2
—
—
2.9
—
3.2
—
—
4.8
—
26
55 (22)
82
3
9
38 (4)
3
40 (1)
47 (15)
16 (6)
63 (8)
19
45
1
6 (1)
10 (<1)
31 (13)
66
1
15
70
63
42 (11)
—
—
—
—
0.46 (0.3–0.6)
<0.31
—
66 (2)
47 (1)
– (4)
66 (2)
60 (2)
—
87 (–)
– (4)
94 (4)
– (4)
—
—
0.39 (0.3–0.5)
<0.31
—
0.8 (0.6–1.0)
—
0.4 (0.3–0.5)
2.0(1.3–2.7)
—
—
—
2.85
—
0.81 (0.95)
—
—
—
0.07
5.39
—
91 (–)
– (2)
aFor details see Experimental.

T. Glinka et al. / Bioorg. Med. Chem. 11 (2003) 591–600
595
activity (including MRSA) in comparison to their
Conclusions
amino analogues. The chlorine substituent present in
the C-7 aminothiazole also proved to be beneficial to
the anti-MRSA activity as evidenced by comparison
with other C-7 substituents, of which the aminopyridine
contributed the least to the anti-MRSA activity (e.g., 16
vs 15, 26 and 32). Interestingly, the 4-(2-amino-
ethylsulfanylmethyl)pyridin-3-yl substituent at C-3
position (e.g., 33) consistently provided less anti-MRSA
potency than the other two isomeric pyridines. In gen-
eral, with the exception of a few analogues, the whole
series of C-3 heteroarylthio cephalosporins displayed
excellent gram-positive activity including b-lactam
resistant strains of staphylococci and enterococci (Table
2).
Considerable differences in stability of 3-(hetero-
arylthio)cephems toward enzyme-mediated decomposi-
tion in rat serum were observed. These differences
appear to be related to the electron-withdrawing effects
of both C-7 and C-3 substituents on the cephalosporin
core, which influence the reactivity of the b-lactam ring.
Accordingly, analogues with increased stability in rat
serum displayed low clearance and improved efficacy in
an animal model of infection. Based on these attributes,
cephalosporin RWJ-333441 (MC-04,546), which dis-
played an excellent antimicrobial profile (MRSA
MIC90=2 mg/mL), high stability in rat serum (6%
decomposition over 60 min) and improved pharmaco-
kinetics in rat (total clearance 0.39 L/h/kg) was selected
for further evaluation. Evaluation of pharmacokinetic
properties and antimicrobial in vivo efficacy of RWJ-
333441 (MC-04,546) will be described elsewhere.11
Assessment of cephalosporin stability in rat serum was
performed in fresh rat serum, and the results are repor-
ted in Table 3 as the percentage of compound decom-
position over a period of 60 min (human serum stability
was tested for only a limited number of analogues).
With the exception of compound 16 (RWJ-54428/MC-
02,479), where essentially all drug loss was accounted
for in the form of b-lactam ring-opened product (data
not shown), no attempt was made to rigorously identify
the decomposition products. In several other cases
however, the LC/MS/MS profiling of degradation mix-
tures suggested that the hydrolysis of the b-lactam ring
represented the major decomposition pathway. The
study revealed an 80-fold range in rat serum stability of
(3-heteroarylthio)cephems (1% decomposition for pyr-
idyl-3-thio compounds 28 and 33 versus 81% decom-
position for pyridyl-4-thio compound 17). A plausible
explanation of this observation is that the effect of the
more electronegative C-3 substituent extends over the
cephem framework to the b-lactam ring making it more
susceptible to the ring opening by serum enzymes. A
considerable change in stability resulting from minor
structural change in the 23/24 pair of analogues (2-(2-ami-
noethyl)thiothiazol-5-yl-thio versus 2-(2-aminoethyl)-
aminothiazol-5-yl-thio substituent) points to a delicate
balance between structure and serum stability. The
lower stability of guanidine analogues revealed by com-
parison of pairs of analogues which differ only in a
guanidine group replacing primary amine at the C-3
sidechain (e.g., 27 vs 26) shows that the character of the
C-3 heterocycle is not the only factor determining rat
serum stability. Human serum stability data obtained
for selected compounds showed less hydrolysis by
human serum as compared to rat serum. Analogues
tested in pH 7.9 buffer alone showed low levels of base-
line aqueous hydrolysis, which supports the hypothesis
that the decomposition observed in serum is enzyme-
assisted (Table 3).
Experimental
Chemistry
Syntheses of cephalosporin analogues 29 and 18 repre-
sent the general routes employed in the preparation of
the 3-(heteroarylthio)cephems.
(7R,6R)-7-Amino-3-chloro-8-oxo-5-thia-1-aza-bicyclo[4.2.0]-
oct-2-ene-2-carboxylic acid tert-butyl ester (2). To a sus-
pension of (7R,6R)-7-amino-3-chloro-8-oxo-5-thia-1-
aza-bicyclo[4.2.0]oct-2-ene-2-carboxylic acid (1) (23.7 g,
101.2 mmol) in t-butyl acetate (510 mL), under nitro-
gen, was added boron trifluoride–ethyl ether complex
(80 mL) and the mixture was stirred vigorously at room
temperature until complete dissolution. The reaction
mixture was poured into stirred ice water (1000 mL) and
the organic layer was discarded. Aqueous layer was
washed with 1:1 ethyl acetate/hexane (200 mL) and
separated. To this aqueous solution with ice cooling and
stirring was added ethyl acetate (500 mL) followed by
portion-wise addition of sodium carbonate (216 g) until
pH of 8–8.5 is reached. Organic layer was separated and
the aqueous layer was extracted with ethyl acetate (100
mL). Combined aqueous extracts were dried over
anhydrous sodium sulfate and concentrated under
reduced pressure (not exceeding 30 ꢀC) to oily residue
(28 g). To this residue was added toluene (100 mL) fol-
lowed by hexane (100 mL). After 10 min stirring crys-
talline title product (2) was filtered, washed with hexane
and dried under reduced pressure (15.4 g, 52%). 1H
NMR (CDCl3/CD3OD) d 1.40 (s, 9H), 3.30 (d, J=17
Hz, 1H), 3.63 (d, J=17 Hz, 1H), 4.59 (d, J=6 Hz, 1H),
4.86 (d, J=6, 1H).
Among the compounds showing high serum stability,
compound 29, the potent anti-MRSA cephem RWJ-
333441 (MC-04,546) with low serum binding and good
aqueous solubility (4.4 mg/mL at pH 7.2) was selected
for further evaluation. RWJ-333441 (MC-04,546) also
possessed the lowest rat clearance and a low ED50 in a
mouse septicemia model against MSSA infection
(MSSA Smith strain).
N-(Isoxazol-3-yl)-N 0-(carbomethoxy)thiourea. A suspen-
sion of potassium thiocyanate (225 g, 2.3 mol) in fresh
acetonitrile (1.65 L) was mechanically stirred at room
temperature for 15 min and was then treated, dropwise,
with methyl chloroformate (202 g, 2.1 mol). The sus-
pension was warmed to 60 ꢀC where it was stirred for 30
min becoming a thick, yellow mixture. The mixture was

596
T. Glinka et al. / Bioorg. Med. Chem. 11 (2003) 591–600
cooled to 0 ꢀC and treated dropwise with isoxazol-3-y-
lamine (3) (150 g, 1.8 mol). After additional 30 min at
0 ꢀC the mixture was allowed to reach room tempera-
ture. The mixture was poured into rapidly stirred ice
water (5.0 L) and was then stirred for 15 min. The pre-
cipitate was allowed to settle. After decanting the aqu-
eous phase the yellow solid was washed with ice water
(4.0 L). The aqueous phase was again decanted from the
yellow solid. This was repeated twice more, the solid
becoming more powdery with each wash. The solid was
dried on the vacuum filter overnight yielding title pro-
duct as an orange powder, 185 g (52%). 1H NMR
(DMSO-d6) d 3.95 (s, 3H), 7.52 (s, 1H), 9.14 (s, 1H).
sodium bicarbonate (2ꢂ0.5 L), then brine. The organic
layer was dried with sodium sulfate and concentrated to
produce a white solid (66.5 g). NMR revealed presence
of 80% (molar) of title product contaminated with
about 10% of both non-brominated starting material
and bis-brominated by-product. 1H NMR (CDCl3) d
3.80 (s, 3H), 3.95 (s, 3H), 5.73 (s, 1H).
(5-Methoxycarbonylamino-[1,2,4]thiadiazol-3-yl)-oxo-
acetic acid methyl ester. A stirred solution of bromo-(5-
methoxycarbonylamino-[1,2,4]thiadiazol-3-yl)-acetic acid
methyl ester (50 g, 0.13 mol, about 80% pure) in aceto-
nitrile (0.3 L) was treated with pyridine-N-oxide (38.3 g,
0.40 mol). After stirring at reflux for 1 h the reaction
was concentrated. The residue was partitioned between
dichloromethane (0.5 L) and brine (0.15 L). The brine
was washed with dichloromethane. The organic layers
were combined, dried on sodium sulfate, and concentrated
to produce crude title product as a brown oil (45.5 g). This
material was taken into next step without purification.
1H NMR (CDCl3) d 3.92 (s, 3H), 3.96 (s, 3H).
[3-(2-Hydroxy-2-methoxy-ethyl)-[1,2,4]thiadiazol-5-yl]-
carbamic acid methyl ester. A mechanically stirred solu-
tion of N-(isoxazol-3-yl)-N0-(carbomethoxy)thiourea
(185 g, 0.9 mol) in methanol (2 L) was warmed to 60 ꢀC
and was stirred for 1 h. Concentration under reduced
pressure gave a pale, yellow, moist solid. Residual water
was removed azeotropically with toluene and the pre-
cipitate was filtered off producing title product as a
cream colored solid (180 g, 86%). 1H NMR (DMSO-d6)
d 2.85–3.00 (m, 2H), 3.18 (s, 3H), 3.80 (s, 3H), 4.92
(br.s, 1H), 6.20 (br.s, 1H).
(Z)-Hydroxyimino-(5-methoxycarbonylamino-[1,2,4]thia-
diazol-3-yl)-acetic acid methyl ester. A solution of (5-
methoxycarbonylamino-[1,2,4]thiadiazol-3-yl)-oxo-acetic
acid methyl ester (45.5 g, 0.18 mol), pyridine (19.6 g,
0.25 mol), and hydroxylamine hydrochloride (16.8 g,
0.24 mol) in absolute ethanol (0.4 L) was stirred at room
temperature for 16 h. Concentration gave a yellow
syrup which was partitioned between ethyl acetate (0.5
L) and water (0.15 L). The organic was again washed
with water (0.15 L) then one molar hydrochloric acid
(0.2 L) then brine. The organic was dried on sodium
sulfate and concentrated to 0.1 L. The solution was
allowed to crystallize. The product was collected,
washed with ether and dried yielding pure title product as
white crystals (12.3 g, 26%). Concentration of mother
liquors gave an orange glass (15.1 g), which by NMR was
(5-Methoxycarbonylamino-[1,2,4]thiadiazol-3-yl)-acetic
acid. A solution of [3-(2-hydroxy-2-methoxy-ethyl)-
[1,2,4]thiadiazol-5-yl]-carbamic acid methyl ester (178 g,
0.76 mol) in glacial acetic acid (1500 mL) was stirred for
2 h then filtered, removing small amount of insoluble
material. The filtrate was treated dropwise over 20 min
with peracetic acid (32 wt.%, 195 mL). The reaction was
stirred at room temperature for 16 h. The reaction mix-
ture was then filtered and the resulting solid was washed
repeatedly with ether, air-dried, then dried in vacuum
yielding title product (110 g) as a white powder. 1H
NMR (DMSO-d6) d 3.75 (s, 2H), 3.80 (s, 3H).
1
estimated to contain 60–70% of the desired material. H
NMR (CDCl3) d 3.85 (s, 3H), 3.93 (s, 3H).
(5-Methoxycarbonylamino-[1,2,4]thiadiazol-3-yl)-acetic
acid methyl ester. A mechanically stirred suspension of
(5-methoxycarbonylamino-[1,2,4]thiadiazol-3-yl)-acetic
acid (108 g, 0.5 mol) in methanol, cooled to 0 ꢀC (3 L),
was treated dropwise with thionyl chloride (297 g, 2.5
mol). After stirring for 16 h at room temperature the
mixture was filtered. The resulting white crystals were
washed with ether, air-dried, then dried in vacuum
yielding title product (60 g). Concentration of the
mother liquors produced second crop of the product
(53.3 g). Combined yield, 113 g (98%). 1H NMR
(CDCl3) d 3.75 (s, 3H), 3.92 (s, 3H), 4.00 (s, 2H).
(5-Methoxycarbonylamino-[1,2,4]thiadiazol-3-yl)-(Z)-tri-
tyloxyimino-acetic acid methyl ester. A solution of (Z)-
hydroxyimino-(5-methoxycarbonylamino-[1,2,4]thiadia-
zol-3-yl)-acetic acid methyl ester (10.0 g, 38.5 mmol) in
dichloromethane (0.15 L) was cooled to 0 ꢀC. The cold
mixture was treated with triethylamine (4.0 g, 39.5
mmol) then portion-wise with triphenylmethyl chloride
(10.7 g, 38.5 mmol). The resulting yellow solution was
stirred at 0 ꢀC until TLC showed the reaction complete
(approximately 2 h). The reaction solution was washed
with brine, dried on sodium sulfate then concentrated to
100 mL. The slightly opaque mixture was placed in the
freezer. The resulting thick mixture was filtered. The white
crystals were washed with ether and dried under reduced
pressure yielding title product as colorless crystals (12.0
g, 62%, pure syn isomer as confirmed by NMR). 1H NMR
(CDCl3) d 3.53 (s, 3H), 4.05 (s, 3H), 7.20–7.35 (m, 15H).
Bromo-(5-methoxycarbonylamino-[1,2,4]thiadiazol-3-yl)-
acetic acid methyl ester. A stirred solution of (5-meth-
oxycarbonylamino-[1,2,4]thiadiazol-3-yl)-acetic
acid
methyl ester (58 g, 0.25 mol) in 1:1 methanol/dichloro-
methane mixture (0.6 L) was cooled to 0 ꢀC and treated
dropwise with bromine (40.1 g, 0.25 mol). The red-
brown solution was allowed to rise slowly to room
temperature and stirred for 16 h. The resulting pale
yellow solution was partitioned between ethyl acetate
(0.75 L) and water (0.5 L). The organic layer was
washed further with water (0.5 L) then saturated
(5-Amino-[1,2,4]thiadiazol-3-yl)-(Z)-trityloxyimino-acetic
acid sodium salt (4). A suspension of (5-methoxy-
carbonylamino-[1,2,4]thiadiazol-3-yl)-(Z)-trityloxyimino-
acetic acid methyl ester (9.5 g, 18.9 mmol) in 2:1 mixture

T. Glinka et al. / Bioorg. Med. Chem. 11 (2003) 591–600
597
of 2.5 M aqueous NaOH and ethanol (75 mL) was
heated to gentle reflux for 8 h and stirring was con-
tinued for 16 h at room temperature (initial suspension
becomes clear solution on heating). Crystalline product
was collected by filtration, washed with water, air dried
and then dried in high vacuum for 72 h producing white
crystalline title product (6.0 g, 70%).
(1.09 g, 9.17 mmol) to dry dimethylformamide (10 mL)
at room temperature. After 30 min the above solution
was transferred to a solution of (3-tert-butylsulfanyl-
pyridin-2-yl)-methanol (1.20 g, 6.09 mmol) in dry dime-
thylformamide (5 mL). After stirring for 30 min at room
temperature, powdered potassium carbonate (4.15 g, 30
mmol) was added followed by addition of (2-mercapto-
ethyl)-carbamic acid tert-butyl ester (5.73 g, 30.0 mmol)
and sodium iodide (0.15 g, 1.05 mmol) and vigorous
stirring was continued for 16 h. Reaction mixture was
partitioned between ethyl acetate and water. Organic
extract was thoroughly washed with water, then dried
over sodium sulfate and evaporated to produce oily
residue, which was purified by flash chromatography on
silica gel (ethyl acetate/ hexane 1:2) to afford oily title
(6R,7R)-7-[2-(5-Amino-[1,2,4]thiadiazol-3-yl)-2-(Z)-
trityloxyimino-acetylamino]-3-chloro-8-oxo-5-thia-1-aza-
bicyclo[4.2.0]oct-2-ene-2-carboxylic acid tert-butyl ester
(5). To a stirred suspension of (5-amino-[1,2,4]thiadia-
zol-3-yl)-(Z)-trityloxyimino-acetic acid sodium salt (4)
(14.38 g, 31.7 mmol, containing 0.33 equiv of water
according to Karl Fisher method determination) in dry
THF (170 g) was added diphenylchlorophosphonate
(9.9 mL, 47.5 mmol). After a few minutes of stirring
most of starting material dissolved. Stirring was con-
tinued for 1 h at ambient temperature and solid
(6R,7R)-7-amino-3-chloro - 8 - oxo - 5 - thia - 1 - aza - bicy-
clo[4.2.0]oct-2-ene-2-carboxylic acid tert-butyl ester (2)
(9.19 g, 31.7 mmol) was added followed by addition of
2,6-lutidine (3.7 mL, 31.7 mmol). After 3 h at room
temperature reaction mixture was partitioned between
ethyl acetate (200 mL) and 0.5 M hydrochloric acid (100
mL). Organic layer was washed with 0.5 M hydrochloric
acid (100 mL) and then washed twice with 0.5 M aqu-
eous sodium bicarbonate (100 mL). Organic solution
was dried over anhydrous sodium sulfate and con-
centrated to about 60 mL volume. To this solution 1:2
mixture of ethyl acetate/hexane (80 mL) was added and
the product was allowed to crystallize overnight. Filtra-
tion and drying under reduced pressure yielded 14.1 g of
1
product (1.10 g). H NMR (CDCl3) d 1.40 (s, 9H), 1.57
(s, 9H), 2.80 (t, J=6, 2H), 3.43 (t, J=6, 2H), 4.35 (s,
2H), 5.40 (br.s, 1H), 7.28 (dd, J=6, J=4, 1H), 7.95 (d,
J=6, 1H), 8.63 (d, J=4, 1H).
2-(2-Amino-ethylsulfanylmethyl)-pyridine-3-thiol dihydro-
chloride. A solution of [2-(3-tert-butylsulfanyl-pyridin-
2-ylmethylsulfanyl)-ethyl]-carbamic acid tert-butyl ester
(7) (0.60 g) in hydrochloric acid (5 mL, 6.0 M) was
refluxed for 72 h (until NMR of a sample in D2O does
not show any t-butyl signal) and the reaction mixture
was evaporated to dryness to produce solid material of
dihydrochloride of title product (0.40 g), which was
used for next step without further purification. 1H NMR
(D2O) d 2.85 (t, J=6, 2H), 3.15 (t, J=6, 2H), 4.20 (s,
2H), 7.62 (m, 1H), 7.30 (d, J=4, 1H), 7.43 (d, J=6, 1H).
2-{3-[2-(2-Amino-ethylsulfanylmethyl)-pyridin-3-yldisul-
fanyl]-pyridin-2-ylmethylsulfanyl}-ethylamine (8).
1
crystalline product. H NMR (CDCl3/CD3OD) d 1.40
(s, 9H), 3.20 (d, J=17, 1H), 3.60 (d, J=17, 1H), 4.97 (d,
J=6, 1H), 5.80 (d, J=6, 1H), 7.10–7.30 (m, 15H).
A
solution of 2-(2-amino-ethylsulfanylmethyl)-pyridine-3-
thiol dihydrochloride (0.40 g) in water (4 mL) basified
with addition of concentrated ammonium hydroxide
and a stream of air was bubbled through it for 16 h with
occasional addition of more concentrated ammonium
hydroxide. Reaction mixture was evaporated to dryness
to produce solid residue of title product and ammonium
chloride, which was used for next step without further
(3-tert-Butylsulfanyl-pyridin-2-yl)-methanol. To a sus-
pension of 3-tert-butylsulfanyl-pyridine-2-carboxylic
acid (6) (10.0 g, 47.4 mmol) in tetrahydrofuran (200
mL) cooled to ꢃ5 ꢀC was added triethylamine (8.25 mL,
47.4 mmol) followed by addition of ethyl chloroformate
(4.38 g, 47.4 mmol) and reaction was stirred for 30 min
at 0 ꢀC. Lithium borohydride (2.58 g, 118 mmol) was
added in portions, maintaining the temperature below
ꢃ5 ꢀC. After the addition was complete the reaction was
allowed to warm to room temperature and stirred for 1
h. Temperature was lowered to ꢃ5 ꢀC and methanol (10
mL) was added followed by addition of aqueous sodium
hydroxide (10 mL, 10%). After the addition of ethyl
acetate (50 mL) and water (40 mL) dilute hydrochloric
acid was added to obtain pH=5.0. Precipitated inor-
ganic salt was filtered off and organic layer of the filtrate
was separated. After washing aqueous layer thoroughly
with ethyl acetate the combined organic extracts were
dried over sodium sulfate and concentrated to produce
1
purification. H NMR (D2O) d 2.75 (t, J=6, 2H), 3.15
(t, J=6, 2H), 4.00 (s, 2H), 7.38 (dd, J=4, J=6, 1H),
8.21 (d, J=6, 1H), 8.38 (d, J=4, 1H).
(2-{3-[2-(2-tert-Butoxycarbonylamino-ethylsulfanylme-
thyl)-pyridin-3-yldisulfanyl]-pyridin-2-ylmethylsulfanyl}-
ethyl)-carbamic acid tert-butyl ester (9). To a solution
of crude 2-{3-[2-(2-amino-ethylsulfanylmethyl)-pyridin-
3-yldisulfanyl]-pyridin-2-ylmethylsulfanyl}-ethylamine
(8) in methanol (50 mL) were added di-t-butyldicarbo-
nate (1.19 g, 5.46 mmol) and triethylamine (0.73 g, 7.20
mmol). After 45 min at room temperature reaction
mixture was evaporated to dryness and re-dissolved in
dichloromethane. The insoluble residue was filtered off
and the filtrate was concentrated under reduced pres-
sure to produce oily residue. Flash chromatography on
silica gel (5% methanol in dichloromethane) yielded oily
1
yellow oil (7.21 g) of title product. H NMR (CDCl3) d
1.40 (s, 9H), 4.50 (br.s, 1H), 4.90 (s, 2H), 7.20 (m, 1H),
7.80 (d, J=7, 1H), 8.55 (d, J=5, 1H).
1
title product (0.28 g). H NMR (CDCl3) d 1.32 (s, 9H),
[2-(3-tert-Butylsulfanyl-pyridin-2-ylmethylsulfanyl)-ethyl]-
carbamic acid tert-butyl ester (7). A solution of Vilsme-
ier reagent was prepared by addition of thionyl chloride
2.60–2.70 (m, 2H), 3.20–3.30 (m, 2H), 4.00 (s, 2H), 5.10
(br.s, 2H), 4.00 (s, 2H), 7.17 (dd, J=5, J=8, 1H), 7.90
(d, J=8, 1H), 8.38 (d, J=5, 1H).

598
T. Glinka et al. / Bioorg. Med. Chem. 11 (2003) 591–600
(7R,6R)-7-[2-(5-Amino-[1,2,4]thiadiazol-3-yl)-2-(Z)-
trityloxyimino-acetylamino]-3-[2-(2-tert-butoxycarbony-
lamino-ethylsulfanylmethyl)-pyridin-3-ylsulfanyl]-8-oxo-5
-thia-1-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylic acid tert-
butyl ester (10). To a solution of (6R,7R)-7-[2-(5-
amino-[1,2,4]thiadiazol-3-yl)-2-(Z)-trityloxyimino-acet-
ylamino]-3-chloro-8-oxo-5-thia-1-aza-bicyclo[4.2.0]oct-2-
ene-2-carboxylic acid tert-butyl ester (5) (2.00 g, 2.84
mmol) in DMF (30 mL), under nitrogen, was added (2-
{3-[2-(2-tert-butoxycarbonylamino-ethylsulfanylmethyl)-
pyridin-3-yldisulfanyl]-pyridin-2-ylmethylsulfanyl}-ethyl)
-carbamic acid tert-butyl ester (9) (852 mg, 1.42 mmol)
followed by addition of triphenylphosphine (1.12 g, 4.27
mmol), water (0.41 mL) and 0.5 M aqueous sodium
bicarbonate (5.68 mL, 2.84 mmol). After stirring at
ambient temperature overnight the reaction mixture was
diluted with ice cold water and thoroughly extracted
with ethyl acetate/hexane mixture (3:1). The combined
extracts were washed with water. The title product was
isolated by chromatography on silica gel column to
produce quantitative yield of final product (2.91 g) as
1.0 M, 52 mmol) and the reaction mixture was stirred
for 30 min. Solvent was evaporated under reduced
pressure, methanol (40 mL) was added and after gas
evolution stopped concentrated hydrochloric acid (3.6
mL) was added. The solution was filtered and the fil-
trate was evaporated to dryness. The residue was re-
dissolved in small volume of water, concentrated aqu-
eous ammonia (3.6 mL) was added and the reaction
mixture was evaporated to dryness. After overnight
drying under vacuum quantitative yield of the title pro-
duct was obtained, which was used for next step without
1
purification. H NMR (CD3OD) d 4.786 (s, 2H), 8.13
(d, J=6, 2H), 8.60 (d, J=6, 2H), 8.70 (s, 1H).
(3-Tritylsulfanyl-pyridin-4-yl)-methanol (11). (3-Mer-
capto-pyridin-4-yl)-methanol (100 mg, 0.71 mmol) was
dissolved in DMF (5 mL) and diisipropylethylamine
(0.12 mL, 0.71 mmol) was added followed by trityl
chloride (197 mg, 0.71 mmol). After 30 min the reaction
mixture was partitioned between water and ethyl ace-
tate, the organic layer was thoroughly washed with
water and dried with anhydrous sodium sulfate. Pur-
ification by radial chromatography on silica gel pro-
1
yellow-orange foam. H NMR (CDCl3/CD3OD) d 1.35
(s, 9H), 1.49 (s, 9H), 2.62 (t, J=7, 9H), 3.02 (s, J=17,
9H), 3.10–3.20 (m, 3H), 3.96 (s, 2H), 5.07 (d, J=6, 1H),
6.00 (d, J=6, 1H), 7.14 (dd, J=5, J=8, 1H), 7.20–7.35
(m, 15H), 8.40 (br.s, 1H).
1
duced pure title material (67 mg, 25% yield). H NMR
(CDCl3) d 4.17 (s, 2H), 7.20–7.40 (m, 15H), 7.42 (d,
J=6, 1H), 8.22 (s, 1H), 8.40 (d, J=6, 1H).
(7R,6R)-3-[2-(2-Amino-ethylsulfanylmethyl)-pyridin-3-yl-
sulfanyl]-7-[2-(5-amino-[1,2,4]thiadiazol-3-yl)-2-(Z)-hydro-
xyimino-acetylamino]-8-oxo-5-thia-1-aza-bicyclo[4.2.0]oct-
2-ene-2-carboxylic acid (29). To a solution of (7R,6R)-7-
[2-(5-amino-[1,2,4]thiadiazol-3-yl)-2-(Z)-trityloxyimino-
acetylamino]-3-[2-(2-tert-butoxycarbonylamino-ethylsul-
fanylmethyl)-pyridin-3-ylsulfanyl]-8-oxo-5-thia-1-aza-
bicyclo[4.2.0]oct-2-ene-2-carboxylic acid tert-butyl ester
(10) (2.91 g, 3.0 mmol) in dichloromethane (15 mL) was
added triethylsilane (15 mL). To this mixture tri-
fluoroacetic acid (58 mL) was added slowly with ice
cooling. The reaction was stirred for 2 h at 10 ꢀC and then
for 3 h at ambient temperature. The bis-trifluoroacetic
acid salt of the title product (2.13 g, 89%) was isolated
by precipitation with slow addition of diisopropyl ether
(120 mL) with ice cooling, filtration and thorough
[2-(3-Tritylsulfanyl-pyridin-4-ylmethylsulfanyl)-ethyl]-car-
bamic acid tert-butyl ester (12). A solution of thionyl
chloride (126 mg, 1.05 mmol) in dry DMF (2 mL) was
stirred for 30 min at room temperature. Such solution
was then cannulated into a solution of (3-tritylsulfanyl-
pyridin-4-yl)-methanol (11) (270 mg, 0.70 mmol) in
DMF (2 mL) at room temperature and the reaction was
stirred for 30 min. (2-Mercapto-ethyl)-carbamic acid
tert-butyl ester (187 mg, 1.05 mmol) and powdered
potassium carbonate (486 mg, 3.52 mmol) were added
to the reaction mixture and stirring was continued for
additional 30 min. The reaction was partitioned
between water and ethyl acetate and the organic layer
was thoroughly washed with water and dried. After
removing the solvent under reduced pressure the residue
was purified by radial chromatography on silica gel
(hexane/ethyl acetate 4:1) to yield title material as off-
1
washing with diisopropyl ether. H NMR (D2O) d 2.91
1
(t, J=6, 2H), 3.31 (t, J=6, 2H), 3.36 (d, J=16, 1H),
3.76 (d, J=16, 1H), 4.28 (s, 2H), 5.37 (d, J=6, 2H), 5.91
(d, J=6, 1H), 7.92 (dd, J=7, J=6, 1H), 8.43 (d, J=7,
1H), 8.60 (d, J=6, 1H).
white solid (220 mg, 58%). H NMR (CDCl3) d 1.46 (s,
9H), 2.42 (t, J=6, 2H), 3.18 (s, 2H), 3.23 (m, 2H), 4.80
(br.s, 1H), 7.10 (d, J=6, 1H), 7.20–7.45 (m, 15H), 8.28
(m, 2H).
A 1 inch diameter column was packed with 30 g of
HP20 resin and the solution of the bis-trifluoroacetic
acid salt of the title product (1.20 g) in water (20 mL)
was loaded. Distilled water was run through the column
until the pH of the effluent reached 5.5. The solvent was
changed immediately to 4:1 water/acetonitrile and the
zwitterionic product was eluted in approximately 100
mL of effluent. The acetonitrile was removed in vacuum
at <40 ꢀC and the remaining water was lyophilized to
give the title compound (0.79 g, 92%).
Thiocarbonic acid S-[4-(2-tert-butoxycarbonylamino-
ethylsulfanylmethyl)-pyridin-3-yl] ester O-tert-butyl es-
ter. To [2-(3-tritylsulfanyl-pyridin-4-ylmethylsulfanyl)-
ethyl]-carbamic acid tert-butyl ester (12) (790 mg, 1.45
mmol) and triethylsilane (2 mL, 12.5 mmol) dissolved in
dichloromethane (15 mL) was added trifluoroacetic acid
(15 mL). After 1 h stirring at room temperature reaction
mixture was evaporated to dryness under reduced pres-
sure and the residue was dissolved in dry tetra-
hydrofuran
(10
mL).
To
this
solution
diisopropylethylamine (1.40 mL, 7.84 mmol) was added,
followed by di-t-butyldicarbonate (1.28 g, 5.88 mmol)
and after 3 h reaction at room temperature solvent was
removed at reduced pressure. Purification on silica gel
(3-Mercapto-pyridin-4-yl)-methanol. To a suspension of
3-mercaptoisonicotinic acid (1.80 g, 11.6 mmol) in dry
THF (70 mL) was added slowly borane in THF (52 mL,

T. Glinka et al. / Bioorg. Med. Chem. 11 (2003) 591–600
599
by radial chromatography (dichloromethane/methanol
Microbiology and pharmacology
50:1) produced pure title product as yellow foam (460 mg,
79%). 1H NMR (CDCl3) d 1.42 (s, 9H), 1.50 (s, 9H), 2.58
(t, J=6, 2H), 3.23 (t, J=6, 2H), 3.84 (s, 2H), 4.95 (br.s,
1H), 7.42 (d, J=6, 1H), 8.58 (d, J=6, 1H), 8.70 (s, 1H).
Susceptibility testing. Compounds were evaluated for
antimicrobial activity against a panel of bacterial strains
using a broth microdilution assay performed using
NCCLS reference methods.12 The minimum inhibitory
concentration (MIC) is defined as the lowest concentra-
tion of a compound that prevents the growth of the
bacteria.
[2-(3-Mercapto-pyridin-4-ylmethylsulfanyl)-ethyl]-car-
bamic acid tert-butyl ester (13). To a solution of thio-
carbonic
acid
S-[4-(2-tert-butoxycarbonylamino-
ethylsulfanylmethyl)-pyridin-3-yl] ester O-tert-butyl
ester (415 mg, 1.04 mmol) in methanol (2 mL) was
added under nitrogen methanolic solution of sodium
methoxide (1.04 mL, 1.0 M) and the reaction mixture
was heated to 50 ꢀC for 30 min. After evaporating the
solvent in vacuum the residue was partitioned between
water and ethyl acetate with addition of acetic acid (125
mg, 2.08 mmol). Evaporation of the solvent under
reduced pressure yielded title material (298 mg, 96%
yield), which was used in the next step without further
purification. 1H NMR (CD3OD) d 1.42 (s, 9H), 2.60 (m,
2H), 3.20 (m, 2H), 4.0 (s, 2H), 4.95 (br.s, 1H), 7.52 (d,
J=6, 1H), 8.00 (d, J=6, 1H), 8.40 (s, 1H).
Effect of human serum on MIC; serum binding determi-
nation. The effect of human serum on antimicrobial
activity against S. aureus ATCC 29213 was determined
using a 1:1 mixture of human serum and growth med-
ium (HS–MHB). The magnitude of change in MIC is
expressed as a ratio of the MIC in serum-supplemented
to unsupplemented media (Table 3 — ‘MIC ratio
HSMHB/MHB’). For selected compounds, the binding
in pooled human serum was determined using ultra-
filtration. Compounds were incubated in serum for 10
min at 37 ꢀC in a shaking water bath. Serum ultrafiltrate
was obtained by centrifugation of ultrafiltration units
(Amicon Centrifree) for 20 min at 25 ꢀC. Drug content
in ultrafiltrate was quantified by HPLC using standards
prepared in blank ultrafiltrate undergoing similar
processing.
(7R,6R)-7-[2-(2-Amino-5-chloro-thiazol-4-yl)-2-(Z)-trityl-
oxyimino-acetylamino]-3-[4-(2-tert-butoxycarbonylamino-
ethylsulfanylmethyl)-pyridin-3-ylsulfanyl]-8-oxo-5-thia-1-
aza-bicyclo[4.2.0]oct-2-ene-2-carboxylic acid benzhydryl
ester. To a solution of [2-(3-mercapto-pyridin-4-ylme-
thylsulfanyl)-ethyl]-carbamic acid tert-butyl ester (13)
(298 mg, 0.99 mmol) in ethyl acetate (5 mL) was added
(7R,6R)-7-[2-(2-amino-5-chloro-thiazol-4-yl)-2-(Z)-trityl-
oxyimino-acetylamino]-3-methanesulfonyloxy-8-oxo-5-
thia-1-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylic acid benz-
hydryl ester. After 1 h stirring at room temperature the
reaction was partitioned between ethyl acetate and
dilute sodium bicarbonate solution (30 mL, 1%). The
organic layer was dried over anhydrous sodium sulfate
and evaporated under reduced pressure. The residue
was purified by radial chromatography on silica gel to
yield pure title product (366 mg, 33%). 1H NMR
(CD3OD) d 1.40 (s, 9H), 2.50 (m, 2H), 3.20 (m, 2H), 3.12
(d, J=16, 1H), 3.80 (s, 2H), 3.38 (d, J=16, 1H), 5.25 (d,
J=6, 1H), 6.00 (d, J=6, 1H), 7.00 (s, 1H), 7.20–7.40 (m,
25H), 7.42 (d, J=6, 1H), 8.40 (d, J=6, 1H), 8.50 (s, 1H).
Mouse model of sepsis. Inoculum preparation: Staphyl-
ococcus aureus (strain Smith; ATCC 13709, penicillin-
susceptible) was grown overnight at 37 ꢀC in brain–
heart infusion broth (BHIB). The following morning, it
was subcultured to fresh BHIB and incubated for 4–5 h
at 37 ꢀC. The cells were harvested by centrifugation,
washed twice with PBS, and adjusted to the desired
inoculum. The cell suspension was mixed with an equal
volume of sterile 14% hog-gastric mucin.13 The inocu-
lum was kept in an ice bath until used (<1 h).
Experimental infection. Male Swiss–Webster mice were
challenged intraperitoneally with 0.5 mL of bacterial
suspension (ca 10ꢂLD50). Test compounds were admi-
nistered subcutaneously in 0.1 mL volumes immediately
after inoculation and 2 h later. The total dose associated
with 50% survival (ED50) at 72 h was determined using
the probit method.14
(7R,6R)-3-[4-(2-Amino-ethylsulfanylmethyl)-pyridin-3-yl-
sulfanyl]-7-[2-(2-amino-5-chloro-thiazol-4-yl)-2-(Z)-
hydroxyimino - acetylamino] - 8 - oxo - 5 - thia - 1 - aza - bicy-
clo[4.2.0]oct-2-ene-2-carboxylic acid bis-trifluoroacetic acid
salt (18). To a suspension of protected cephem (342 mg,
0.31 mmol) precursor in dichloromethane (3.5 mL) was
added triethylsilane (1.7 mL, 4.06 mmol) followed by
addition of trifluoroacetic acid (4.5 mL). After stirring
at room temperature for 1 h, reaction was cooled to
0 ꢀC and isopropyl ether (30 mL) was added. Stirring
was continued at 0 ꢀC for 10 min and the resulting pre-
cipitate was filtered off and washed thoroughly with
diisopropyl ether and dried in vacuum, yielding title
deprotected cephem bis-trifluoroacetate (222 mg, 85%).
1H NMR (D2O) d 2.90 (t, J=6, 2H), 3.31 (t, J=6, 2H),
3.43 (d, J=17, 1H), 3.85 (d, J=17, 1H), 4.18 (s, 2H),
5.43 (d, J=4.5, 1H), 5.97 (d, J=4.5, 1H), 8.10 (d, J=6,
1H), 8.66–8.68 (m, 2H).
Pharmacokinetic studies in rats. Single dose pharmaco-
kinetic (PK) studies were conducted in male Sprague–
Dawley (CD) rats (250–300 g). Drugs were studied as
single compounds (20 mg/kg) or in a cassette up to 4
compounds (1 mg/kg of each compound) with a control
agent RWJ-54428. Doses were administered through a
jugular venous catheter as a bolus injection (cassette) or
over 20 min (single compounds). Blood (serum) samples
were collected from tail vein or the venous catheter and
assayed for drug content using HPLC. For selected
compounds, PK parameters were determined in cassette
experiments. Cassette dosing of a cocktail of up to 4
compounds (1 mg/kg of each compound) and a control
of RWJ-54428 was conducted in single catheterized
(jugular vein) male CD rats. Serum samples were
assayed using LC/MS/MS and the data were analyzed
using WinNonlin program (Pharsight).

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T. Glinka et al. / Bioorg. Med. Chem. 11 (2003) 591–600
Serum and buffer stability. Rat serum was obtained
from a euthanized rat immediately prior to the experi-
ment. Solutions of b-lactam (50 mL, 1 mg/mL) were
added to fresh rat serum (950 mL, preincubated at 37 ꢀC
for 10 min). Aliquots were removed at varying time
points. The pH prior to and after the addition of com-
pound was in the 7.5–8.0 range and at the end of the 1 h
incubation was in the 8.5–9.0 range. Samples for HPLC
were prepared by addition of 100 mL aliquots to tri-
chloroacetic acid solution (200 mL, 4%), vortexing and
centrifugation at 14,000 rpm in an Eppendorf micro-
centrifuge. A 25 mL sample of each supernatant was
injected onto the HPLC. Human serum stabilities were
determined in analogous fashion.
References and Notes
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Henderson, T. S.; Tolson, J. S.; Martone, W. J. Infect Control
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2000, 53, 1272.
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C.; Cho, A.; Crawford, J.; Ludwikow, M.; Calkins, T.; Cham-
berland, S.; Price, M.; Dudley, M.; Hecker, S.; Lee. V. Abstracts
of papers, 39th Interscience Conference on Antimicrobial Agents
and Chemotherapy, San Francisco, CA, 1999; Abstract F-0391.
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V. J; Hecker S. J. Abstracts of papers, 39th Interscience Con-
ference on Antimicrobial Agents and Chemotherapy, San Fran-
cisco, CA, 1999; Abstract F-0392.
For buffer stability determinations, each compound was
dissolved at a concentration of 500 mg/mL (or 250 mg/
mL for less soluble compounds) in a 0.1 M MOPS buf-
fer, pH 7.9. Solutions were placed in 37 ꢀC bath. At 0, 1,
2, 4 and 6 h time an aliquot of solution was removed
and diluted to concentration that fit into predetermined
calibration curve. Quantification was done by reverse
phase HPLC using a five-point curve.
8. Grigan, N.; Musel, D.; Veinberg, G. A.; Lukevics, E.
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Glinka, T. W.; Dudley M. N.; manuscript in preparation.
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Wayne, PA, 4th Ed.; Methods for Dilution Antimicrobial Sus-
ceptibility Tests for Bacteria that Grow Aerobically, vol 17, no.
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microb. Agents Chemother. 1975, 7, 179.
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Acknowledgements
The authors gratefully acknowledge Doug Clark and
Vrushali Tembe for measurement of serum protein
binding and Monica Hoang for MIC measurements.
The work described herein was conducted as part of a
research collaboration with the R. W. Johnson Phar-
maceutical Research Institute.