S-Aminosulfeniminocephalosporins
J . Org. Chem., Vol. 64, No. 9, 1999 3137
target,25-27 the use of a prodrug to corelease two distinct
structures could provide a route to establishing useful
synergistic effects.
mixture was stirred for 15 min. Water (2 mL) was added,
followed by the slow addition of light petroleum (30 mL); the
reaction mixture was then stirred for a further 10 min.
Precipitated sulfonamide was filtered off, and the filtrate was
washed successively with water (3 × 500 mL) and saturated
brine solution (100 mL), separated, dried, and concentrated
under reduced pressure to yield crude 3. This was purified by
flash chromatography using 90/10 dichloromethane/ethyl ac-
etate. Removal of solvent under reduced pressure gave 3 as a
yellow solid: yield 1.05 g (1.65 mmol, 72%): mp 96-99 °C; 1H
NMR (CDCl3, 90 MHz) δ 2.03 (s, 3H), 2.42 (s, 3H), 3.32 and
3.62 (AB q, J ) 18 Hz, 2H), 4.67 and 5.03 (AB q, J ) 13 Hz,
2H), 5.36 (s, 1H), 7.00 (s, 1H), 7.20-7.85 (m, 14 H); IR (film)
1779, 1735 cm-1; IR (film) 3214, 1780, 1736, 1674 cm-1; UV
log ꢀ ) 4.06, λ290 (MeOH). Anal. Calcd for C30H27N3O7S3: C,
56.50.; H, 4.27; N, 6.59. Found: C, 56.64; H, 4.29; N, 6.54.
Dip h en ylm eth yl N-Meth yl-N-tosyl-S-a m in osu lfen im i-
n ocep h a losp or a n a te (3m ). A solution of diazomethane was
prepared from Diazald.29 Using a stream of nitrogen the
diazomethane was entrained through a cold trap into a
solution of 3 (1 g, 1.57 mmol) in dichloromethane (25 mL);
reaction progress was monitored using TLC. Removal of
solvent under reduced pressure yielded a sticky solid. This was
purified by flash chromatography using dichloromethane as
eluant. Pure 3m was obtained as a light yellow solid (650 mg,
Con clu sion s
We have demonstrated that S-aminosulfeniminocepha-
losporins are prototypic examples of a novel class of
â-lactamase-dependent prodrug. In these the cepha-
losporin acts as a nucleus from which the corelease of
two distinct structural components can be effected through
enzyme-catalyzed hydrolysis of the â-lactam ring. The
potential to elaborate and exploit this structure type in
the ADEPT approach to cancer chemotherapy and in the
further development of antibiotic agents is clear.
Exp er im en ta l Section
Column chromatography (flash technique)28 was carried out
using Kieselgel S (32-63 µm). â-Lactamase type I from
Bacillus cereus (EC 3.5.2.6) was obtained from Sigma; the
specific activity for the hydrolysis of benzylpenicillin at pH 7.2
and 25 °C was 2.70 mmol min-1 (mg of protein)-1. Elemental
analyses were performed by the Microanalytical Laboratory,
UCD, Dublin, Ireland.
1
1.00 mol, 63%): mp 87-92 °C; H NMR (90 MHz, CDCl3) δ
2.02 (s, 3H), 2.46 (s, 3H), 3.31 (s, 3H), 3.35 and 3.67 (AB q, J
) 18 Hz, 2H), 4.76 and 5.02 (AB q, J ) 13 Hz, 2H), 5.50 (s,
1H), 7.00 (s, 1H), 7.22-7.90 (m, 14 H); IR (film) 1781, 1738,
1675 cm-1; UV log ꢀ ) 4.13, λ298 (MeOH). Anal. Calcd for
Dip h en ylm eth yl 7-Am in ocep h a losp or a n a te (1). To a
suspension of 7-ACA (28.9 g, 0.106 mol) in methanol (100 mL)
was added a solution of diphenyldiazomethane (0.1 mol) in
dichloromethane (40 mL), and the mixture was stirred for 2
days at room temperature until the purple color disappeared.
The unreacted solid was removed by filtration, and the organic
layer was concentrated under reduced pressure to yield a
sticky solid. This was purified by flash column chromatography
using 50/50 ethyl acetate/dichloromethane as eluant. Removal
of solvent under reduced pressure yielded 1 as a pale yellow
powder (23.3 g, 53.2 mmol, 50%): mp 120-121 C; 1H NMR
(CDCl3, 90 MHz) δ 1.85 (br s, NH2), 2.00 (s, 3H), 3.32 and 3.60
(AB q, J ) 18 Hz, 2H), 4.73 and 5.01 (AB q, J ) 13.5 Hz, 2H),
4.77 (d, J ) 5 Hz, 1H), 4.91 (d, J ) 5 Hz, 1H), 6.98 (s, 1H),
C
31H29N3O7S3: C, 57.13; H, 4.48; N, 6.44. Found: C, 56.86; H,
4.38; N, 6.18.
Dep r otection a n d Isola tion of th e Sod iu m Sa lts. The
cephem esters (typically 0.5 mmol) were deprotected as previ-
ously described for the S-aminosulfeniminopenicillanates.5 The
sodium salts were obtained by extracting a dichloromethane
solution (20 mL) of an accurately weighed amount of the free
acid (typically 0.25 mmol) with aqueous sodium bicarbonate
(0.20 mmol). The aqueous layer was separated and freeze-dried
to yield the sodium salts as yellow solids.
Sod iu m N-tosyl-S-a m in osu lfen im in oceph a losp or a n a te
(4) was obtained as a bright yellow foam (137 mg, 0.28 mmol,
45%): 1H NMR (90 MHz, D2O) δ 2.12 (s, 3H), 2.40 (s, 3H),
3.35 and 3.67 (AB q, J ) 18 Hz, 2H), 4.70 and 4.92 (AB q J )
12.5 Hz, 2H), 5.30 (s, 1H), 7.3-7.8 (m, 4H); UV, log ꢀ ) 4.16,
7.20-7.92 (m, 10 H); IR (film) 1779, 1735 cm-1
.
Dip h en ylm eth yl N-Tosyl-S-a m in osu lfen im in ocep h a -
losp or a n a te (3). 1 (1 g, 2.28 mmol) was dissolved in dichlo-
romethane (35 mL). 25 (1.0 g, 2.7 mmol) was added, and the
λmax 361 nm (H2O buffer pH 7.2). Anal. Calcd for C17H16N3O7S3-
Na‚2H2O: C, 38.56; H, 3.81; N, 7.94. Found: C, 38.16; H, 3.47;
N, 7.66. Corresponding free acid: 1H NMR (90 MHz, CDCl3)
δ 2.12 (s, 3H), 2.42 (s, 3H), 3.40 and 3.61 (AB q, J ) 18 Hz,
2H), 4.96 and 5.18 (AB q, J ) 13.5 Hz, 2H), 5.38 (s, 1H), 7.23-
7.90 (m, 4H).
(23) Appropriately substituted phenols, and related structures, are
very effective inhibitors of oxidative phosphorylation, which is a key
metabolic process: Terada, H.; Goto, S.; Yamamoto, K.; Takeuchi, I.;
Hamada, Y.; Miyake, K. Biochim. Biophys. Acta 1988, 936, 504 and
references therein. It is relevant to note that some phenolic structures
(salicylanilides) which have been used as topical antibiotics have been
found to act as inhibitors of autophosphorylation of KinA kinase (which
is part of a TCS pathway26a). The phenolic group provides a useful
linkage for attachment to a prodrug nucleus, and this linkage should
also mask the inherent toxicity of the phenolic moiety within the
prodrug, as the key acid-dissociable site would be blocked.
Sod iu m N-m eth yl-N-tosyl-S-a m in osu lfen im in ocep h a -
losp or in a te (4m ) was obtained as a bright yellow solid (73
mg, 0.14 mmol, 32%): 1H NMR (90 MHz, D2O) δ 2.11 (s, 3H),
2.42 (s, 3H), 3.37 (s, 3H), 3.40 and 3.70 (AB q, J ) 18 Hz, 2H),
4.71 and 4.92 (AB q, J ) 12.5 Hz, 2H) 5.47 (s, 1H), 7.39-7.90
(m, 4H);. UV, log ꢀ ) 4.11, λmax 298 nm (H2O buffer pH 7.2).
Anal. Calcd for C18H18N3O7S3Na‚1.75H2O: C, 40.11; H, 4.02;
N, 7.79. Found: C, 39.77; H, 3.69; N, 7.59. Corresponding free
acid: 1H NMR (90 MHz, CDCl3) δ 2.12 (s, 3H), 2.43 (s, 3H),
3.32 (s, 3H), 3.41 and 3.72 (AB q, J ) 18 Hz, 2H), 4.97 and
5.20 (AB q, J ) 13 Hz, 2H), 5.51 (s, 1H), 7.27-7.88 (m, 4H).
Deter m in a tion of kcat/Km for 4 a n d 4m . The measurement
of kcat/Km was carried out as described previously for the
corresponding penicillins.5 For 4 and 4m the decrease in
absorbance at 361 and 298 nm, respectively, was monitored
as a function of time; the initial rate was determined from the
linear absorbance change within the first 10 s. The enzyme
concentration in each case was 28.2 nM, while the concentra-
tion of 4 was 112 µM and for 4m it was 120 µM. ∆ꢀ values for
4 and 4m were 10 000 and 6500 M-1 cm-1, respectively.
En zym e In h ibition Stu d ies. Benzylpenicillin was used as
the assay substrate, and measurement of the rate of its
(24) Examples of synergy in the field of antibiotics include (i)
combination of pristinamycin IA and IIA in streptogramins (Barrie`re,
J . C.; Paris, J . M. Drugs of the Future 1993, 18, 833), (ii) combination
of trimethoprim with
â-lactamase inhibitor (e.g. clavulanic acid) with
inhibitor (e.g. amoxycillin).
a sulfonamide, and (iii) combination of a
a transpeptidase
(25) For leading references see: (a) Two-Component Signal Trans-
duction; Hoch, J . A., Silhavy, T. J ., Eds.; ASM Press: Washington,
DC, 1995. (b) Barrett, J . F.; Isaacson, R. E. In Annual Reports in
Medicinal Chemistry; Bristol, J . A., Ed.; Academic Press: San Diego,
CA, 1995; Chapter 12. (c) Goldschmidt, R. M.; Macielag, M. J .; Hlasta,
D. J .; Barrett, J . F. Curr. Pharm. Des. 1997, 3, 125.
(26) (a) Macielag, M. J .; Demers, J . P.; Fraga-Spano, S. A.; Hlasta,
D. J .; J ohnson, S. G.; Kanojia, R. M.; Russell, R. K.; Sui, Z.; Weidner-
Wells, M. A.; Werblood, H.; Foleno, B. D.; Goldschmidt, R. M.; Loeloff,
M. J .; Webb, G. C.; Barrett, J . F. J . Med. Chem. 1998, 41, 2939. (b)
Lam, C.; Turnowsky, F.; Ho¨genauer, G.; Schutze, E. J . Antimicrob.
Chemther. 1987, 20, 37.
(27) Volz, K. In Two-Component Signal Transduction; Hoch, J . A.,
Silhavy, T. J ., Eds.; ASM Press: Washington, DC, 1995; Chapter 4,
and references therein.
(28) Casey, M.; Leonard, J .; Lygo, B.; Procter, G. Advanced Practical
Organic Chemistry; Blackie: Glasgow, Scotland, 1990; Chapter 9.
(29) See Chapter 5 of ref 28.