Brief Articles
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 1 131
Trifluoroacetic acid (10 mL) was added to a solution of 6 (0.98
g, 2.58 mmol) in CH2Cl2 (10 mL) at room temperature, followed
by 50 min of stirring at that temperature. The solvent was
removed in vacuo to give the product as a solid (0.54 g,
100%): mp 215-218 °C; IR (KBr) 3311, 3065-2513 (br), 1614,
The bacterial strains used were K. pneumoniae ATCC 10031,
E. faecalis ATCC 29212, S. aureus ATCC 25923, and B. subtilis
ATCC 33608. The MIC was defined as the lowest concentration
of antibiotic that prevented growth, which was determined by
the appearance of turbidity after 24 h of incubation at 37 °C.
P h otod egr a d a tion of Com p ou n d 1. Compound 1 (10 mg)
dissolved in 1 mL of phosphate buffer (100 mM, pH 7.0) in a
Pyrex vessel was irradiated at 350 nm (64 W, Hg lamp) in a
Rayonet RPR-100 photoreactor for 10 h at ∼35 °C (normal
operating temperature in the reactor). The resultant yellow
reaction mixture was concentrated to one-half volume in vacuo,
treated with active charcoal, and filtered, and the filtrate was
evaporated to dryness to obtain the crude product. The TLC
and HPLC showed no trace of starting material, but it was
constituted of several different products. The NMR gave a
complex pattern for which no specific structure could be
assigned.
NMR Exp er im en t of th e P h otor ea ction . A CD3OD
solution of compound 1 (10 mg in 0.8 mL) in an NMR tube
(Pyrex) was irradiated at 350 nm (64 W) in a Rayonet RPR-
100 photoreactor (Hg lamp) at ∼35 °C. The light-induced
degradation of the compound was monitored by taking its
NMR spectra every 30 min until the spectrum showed the
absence of 1. Whenever this compound was handled outside
the photoreactor, it was protected from light.
1
1594, 1428, 1355 cm-1; H NMR (CD3OD) δ 2.98 (dd, J ) 14,
8 Hz, 1H), 3.17 (dd, J ) 14, 5 Hz, 1H), 3.91 (dd, J ) 8, 5 Hz,
1H), 7.22-7.29 (m, 5H); 13C NMR (CDCl3) δ 37.5, 63.3, 128.1,
129.6, 130.5, 137.6, 174.4; EI HRMS 180.0895 (M+, calcd for
C9H12N2O2 180.0899).
(R)-2-Nb-o-Nitr oben zyloxyca r bon ylh yd r a zin o-3-p h en -
ylp r op a n oic Acid (8). A solution of 7 (90 mg, 0.5 mmol) in
1:1 dioxane/water (10 mL) and 1 N NaOH (0.5 mL, 0.5 mmol)
was stirred at ice-water temperature. Additional 1 N NaOH
(0.55 mL, 0.55 mmol) and a solution of 2-nitrobenzyl chloro-
formate9 (0.12 g, 0.55 mmol in 7 mL of dioxane) were added
to the mixture gradually. After the addition of the reagents,
the solution was stirred at that temperature for an additional
30 min. Dioxane was removed in vacuo, the aqueous solution
was acidified to pH 2 with 2 N HCl, and the product was
extracted into CH2Cl2. The combined organic portion was
washed with saturated NaCl and was dried over MgSO4. The
solvent was removed in vacuo to give the crude product (90
mg, 50%), which was used in the next step without further
purification: 1H NMR (CD3OD) δ 3.03 (br m, 2H), 3.87 (br s,
1H), 5.49 (s, 2H), 7.22-7.29 (m, 5H), 7.55-8.12 (m, 4H); 13C
NMR (CDCl3) δ 37.12, 63.81, 64.59, 125.07, 125.16, 126.98,
128.71, 129.04, 129.15, 129.68, 134.29, 137.78, 148.02.
Ben zh yd r yl 7â-((R)-2-Nb-o-Nitr oben zyloxyca r bon ylh y-
d r a zin o-3-p h en ylp r op a n a m id o)cep h a losp or a n a te (9). A
solution of 7 (0.15 g, 0.42 mmol) in THF (8 mL) was added to
a solution of benzhydryl 7-aminocephalosporate14 (0.20 g, 0.45
mmol), DCC (0.08 g, 0.41 mmol), and HOBt (0.056 g, 0.42
mmol) in CH2Cl2 (12 mL), and the mixture was stirred at room
temperature for 24 h. The precipitate was filtered and the
filtrate was concentrated under reduced pressure. A 50-mL
portion of EtOAc was added to precipitate another crop of
DCU, which was then filtered out. The product was passed
through a short column of silica gel to give the crude product
as a white solid (95 mg, 29%), which was used in the next step
without further purification: 1H NMR (CDCl3) δ 2.00 (s, 3H),
2.75 (dd, J ) 14, 10.5 Hz, 1H), 3.25 (dd, J ) 14, 4 Hz, 1H),
3.37 (d, J ) 18 Hz, 1H), 3.53 (d, J ) 18 Hz, 1H), 3.91 (m, 1H),
4.79 (d, J ) 13.5 Hz, 1H), 5.01 (m, 2H), 5.46 (s, 2H), 5.79 (dd,
J ) 9, 5 Hz, 1H), 6.53 (br s, 1H), 6.95 (s, 1H), 7.28-8.06 (m,
19H); 13C NMR (CDCl3) δ 20.6, 26.6, 37.3, 57.6, 59.2, 62.9, 64.0,
65.4, 79.8, 124.9, 126.8, 127.0, 127.4, 127.6, 128.1, 128.2, 128.4,
128.5, 128.8, 129.0, 133.8, 135.9, 139.0, 139.2, 156.3, 160.6,
164.2, 170.5, 172.6.
Sodiu m 7â-((R)-2-Nb-o-Nitr oben zyloxycar bon ylh ydr azi-
n o-3-p h en ylp r op a n a m id o)cep h a losp or a n a te (1). Anisole
(1 mL) and TFA (2 mL) were added to a solution of 9 (40 mg)
in CH2Cl2 (10 mL) at 0 °C, and the mixture was stirred for 3
h at that temperature. After removal of solvent in vacuo,
NaHCO3 (5 mg) in water (5 mL) was added to the residue and
the mixture was stirred at room temperature for 10 min. Water
was removed in vacuo to give the product as a white solid (40
mg, 99%): mp 140 °C dec; IR (KBr) 3158-3680 (br), 1760,
1724, 1701, 1604, 1520 cm-1; 1H NMR (CD3OD) δ 2.03 (s, 3H,
CH3CO-), 2.87 (dd, J ) 14, 7 Hz, 1H, benzyl CHH), 3.05
(dd, J ) 14, 7 Hz, 1H, benzyl CHH), 3.22 (d, J ) 17.5 Hz, 1H,
C2-H), 3.53 (d, J ) 17.5 Hz, 1H, C2-H′), 3.88 (br t, J ) 7 Hz,
1H, CH on C7-side chain), 4.82 (d, J ) 12.5 Hz, 1H, C10-H),
4.98 (m, 2H, C10-H′ and C6-H), 5.46 (br s, 2H, o-nitrobenzyl
CH2), 5.61 (d, J ) 4.5 Hz, 1H, C7-H), 7.21-8.11 (m, 9H,
aromatic); 13C NMR (CD3OD) δ 19.99, 26.03, 37.75, 57.96,
59.70, 63.74, 64.73, 115.79, 125.10, 127.19, 128.96, 129.10,
129.59, 133.37, 134.46, 164.06, 172.03, 174.90; HRFAB 612.1374
(M- + H, calcd for C27H27N5O10S 612.1400). Anal. (C27H34N5-
NaO14S-compound plus 4 waters of hydration) C, N; H: calcd,
4.84; found, 4.09.
Ack n ow led gm en t. This research was supported by
the National Institutes of Allergy and Infectious Dis-
eases.
Refer en ces
(1) Levy, S. B. Antibiotic resistance: An Ecological Imbalance. In
Antibiotic Resistance: Origins, Evolution, Selection and Spread;
Chadwick, D. J ., Goode, J ., Eds.; Ciba Foundation Symposium
207; Wiley: Chichester, 1997; pp 1-14.
(2) Levy, S. B. The Antibiotic Paradox. How Miracle Drugs Are
Destroying the Miracle; Plenum Press: New York, 1992; pp 143-
150.
(3) Amit, B.; Zehavi, U.; Patchornik, A. Photosensitive Protecting
Groups of Amino Sugars and Their Use in Glycoside Synthesis.
2-Nitrobenzyloxycarbonylamino and 6-Nitroveratryloxycarbo-
nylamino Derivatives. J . Org. Chem. 1974, 39, 192-196.
Patchornik, A.; Amit, B.; Woodward, R. B. Photosensitive
Protecting Groups. J . Am. Chem. Soc. 1970, 6333-6335. Barl-
trop, J . A.; Plant, P. J .; Schofield, P. Photosensitive Protective
Groups. Chem. Commun. 1966, 822-823.
(4) Hoz, S. The
R Effect: On the Origin of Transition-State
Stabilization. J . Org. Chem. 1982, 47, 3545-3547. DePuy, C.
H.; Della E. W.; Filley, J .; Grabowski, J . J .; Bierbaum, V. M.
Absence of an R-Effect in the Gas-Phase Nucleophilic Reactions
of HOO-. J . Am. Chem. Soc. 1983, 105, 2481-2482. Klopman,
G.; Fierson, M. R. The Alpha-Effect. A Theoretical Study
Incorporating Solvent Effects. Croatica Chem. Acta 1984, 57,
1411-1415.
(5) Page, M. I. The Mechanisms of Reactions of â-Lactam Antibiot-
ics. Acc. Chem. Res. 1984, 17, 144-151. Page, M. I.; Laws, A. P.
The Mechanism of Catalysis and The Inhibition of â-Lactamases.
Chem. Commun. 1998, 1609-1617.
(6) Reaction of a penicillin with hydrozine has been investigated
before: Morris, J . J .; Page, M. I. Buffer Catalysis in the
Hydrazinolysis of Benzylpenicillin. J . Chem. Soc., Perkin Trans.
II 1980, 220-224.
(7) The concept of caged functionalities finds precedent in the
literature: Curley, K.; Lawrence, D. S. Light-Activated Proteins.
Curr. Opin. Chem. Biol. 1999, 3, 84-88. Furuta, T.; Wang, S.
S. H.; Dantzker, J . L.; Dore, T. M.; Bybee, W. J .; Callaway, E.
M.; Denk, W.; Tsien, R. Y. Brominated 7-Hydroxycoumarin-4-
ylmethyls: Photolabile Protecting Groups With Biologically
Useful Cross-sections for Two Photon Photolysis. Proc. Natl.
Acad. Sci. U.S.A. 1999, 96, 1193-1200. Wood, J . S.; Koszelak,
M.; Liu, J .; Lawrence, D. S. A Caged Protein Kinase Inhibitor.
J . Am. Chem. Soc. 1998, 120, 7145-7146. Salerno, C. P.; Resat,
M.; Magde, D.; Kraut, J . Synthesis of Caged NAD(P)+ Coen-
zymes: Photorelease of NADP+. J . Am. Chem. Soc. 1997, 119,
3403-3404. Gee, K. R.; Kueper, L. W.; Barnes, J .; Dudley, G.;
Givens, R. S. Desyl Esters of Amino Acid Neurotransmitters.
Phototriggers for Biologically Active Neurotransmitters. J . Org.
Chem. 1996, 61, 1228-1233. Holmes, C. P. Model Studies for
New o-Nitrobenzyl Photolabile Linkers: Substituent Effects on
the Rates of Photochemical Cleavage. J . Org. Chem. 1997, 62,
2370-2380.
Deter m in a tion of MICs. The MICs for 1 were determined
by the broth microdilution method with inocula of 5 × 105
colony-forming units per milliliter in Mueller-Hinton broth.15