Synthesis of beta-lactamase activated nitric oxide donors

Article abstract of DOI:10.1016/S0960-894X(03)00242-7

In order to achieve site specific delivery of NO, we designed conjugates of cephalosporin with NO donors. NO donors such as cupferron and SIN-1 were evaluated as potential choices for conjugates. Cephalosporin conjugated with SIN-1 demonstrated promising beta-lactamase dependent NO releasing ability.

Full text of DOI:10.1016/S0960-894X(03)00242-7

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1687–1690
Synthesis of Beta-Lactamase Activated Nitric Oxide Donors
Xiaoping Tang, Tingwei Cai and Peng George Wang*
Department of Chemistry, Wayne State University, Detroit, MI 48202, USA
Received 16 December 2002; revised 26 February 2003; accepted 7 March 2003
Abstract—In order to achieve site specific delivery of NO, we designed conjugates of cephalosporin with NO donors. NO donors
such as cupferron and SIN-1 were evaluated as potential choices for conjugates. Cephalosporin conjugated with SIN-1 demon-
strated promising beta-lactamase dependent NO releasing ability.
# 2003 Elsevier Science Ltd. All rights reserved.
Nitric oxide (NO) plays important roles in a number of
physiological processes including vasodilatation, anti-
platelet effects, macrophage-induced cytotoxicity, and
neurotransmission.1,2 To deliver NO to specific targeted
cells or sites, one can convert the spontaneous NO
releasing agents to stable prodrugs by attaching them to
carriers which can be selectively removed in certain tar-
get sites. As a result, NO dosage could be concentrated
at a specific site even if the prodrugs were systemically
distributed. Recently, we reported several classes of
enzyme activated NO donors based on this concept. For
example, peptide–diazeniumdiolate conjugates were
synthesized by attaching diazeniumdiolate to prostate
specific antigen (PSA) substrates, these conjugates
demonstrated NO release upon activation by PSA.3 In
addition, glycosylated diazeniumdiolates could readily
release NO in the presence of certain glycosidase.4
in the expulsion of NO donors at the C-30 position. The
released cephalosporin is nontoxic to mammalian cells
and the NO donors would generate active NO to kill
sensitive bacteria. In fact, the development of b-lacta-
mase dependent prodrugs with the applications in anti-
body-directed enzyme prodrug therapy (ADEPT) has
been an area of particular interest.6,7 b-Lactamases are
small, soluble monomeric enzymes without a mamma-
lian counterpart. They are abundant, available in mul-
tiple forms, inexpensive, and retain their activities in
vivo. Several studies have demonstrated that b-lacta-
mase can release nitrogen mustard,8ꢀ11 doxorubicin,12,13
vinca alkaloid,14 mitomycin C,15 platinum com-
pounds,16 and paclitaxel17 from cephalosporin pro-
drugs. Our designed b-lactamase activated NO donors
may be also useful for ADEPT. If the bacteria antibody
and b-lactamase conjugates were applied in vivo, and
then our designed NO donors were used, NO would be
released around the bacteria and demonstrate bacter-
icidal activities.
Antibacterial effects of NO have been well documented.5
To address microbial resistance to current antibiotics, we
present here a new type of NO prodrugs, conjugates of
cephalosporin with NO donors. As shown in Scheme 1,
this design is based on the knowledge that hydrolysis of
cephalosporin’s b-lactam bond by b-lactamase can result
Cupferron releases NO upon enzymatic,18 electro-
chemical,19 as well as chemical oxidation.20 O-alkyl
cupferron derivatives exhibited significantly improved
*Corresponding author. Tel.:+1-313-993-6759; fax:+1-313-577-2554; e-mail: pwang@chem.wayne.edu
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00242-7

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X. Tanget al. / Bioorg. Med. Chem. Lett. 13 (2003) 1687–1690
stability compared to their corresponding parent com-
pounds.21 We first synthesized a conjugate of cephalo-
sporin with cupferron as a model compound. As
illustrated in Scheme 2, esterification of cephalothin
with allyl bromide gave the ester 1 in good yield.14
oxidation to the final product 3b. Similar observation
has been reported in the O-alkylation chemistry of neo-
cupferron.23 Unfortunately, attempts to remove the
allylic protecting group of the conjugate 3a by palla-
dium (0) catalysed hydrolysis24 did not work, only
starting material was recovered. Instead of using allylic
protecting group for the acid, a variety of silyate pro-
tecting groups such as TBDMS, TMS or TPS were tes-
ted, but all of these approaches failed to provide the
desired product.
Treatment of
1 with two equivalents of iodo-
trimethylsilane cleaved the acetoxyl moiety to provide
the iodide 2, which was immediately used in the follow-
ing step without further purification. Subsequent addi-
tion of cupferron gave a mixture of compounds 3a and
3b in a molar ratio of 1:2. Separation of the two pro-
ducts was readily achieved by silica gel flash column
chromatography. Both compounds 3a and 3b have been
3-Morphorlinosydnonimine (SIN-1) belongs to another
important class of NO donors known as sydnonimines.
In addition to its potent vasorelaxant and antiplatelet
effects,25 it also displays bactericidal activity.26 We
decided to synthesize a cephalosporin carbamate deri-
vative of the SIN-1, because it has been known that a
carbamate moiety at the C-30 position of the cephem
nucleus serves as a good leaving group upon enzyme-
catalysed hydrolysis of the b-lactam.
1
characterized by H, 13C NMR, as well as HRMS.22
The presence of NO2 group in 3b was confirmed by its
very strong IR stretches at 1530 and 1341 cmꢀ1. The
major product was compound 3b instead of the expec-
ted 3a. A possible reaction mechanism is proposed in
Scheme 3. The O-alkylation of cupferron can occur via
pathway A and B. When the terminal oxygen attacks
the electrophile, the O-alkylation reaction occurs via
path A and product 3a is obtained as the major product.
In the case of path B, the interior oxygen reacts with the
electrophile, which leads to the formation of an inter-
mediate A. The N–N bond can be cleaved homolytically
to form a NO radical. The addition of the NO radical to
the para position of the benzene ring affords another
intermediate B. This unstable intermediate undergoes
Scheme 4 illustrates the synthetic route undertaken.
Selective hydrolysis of the C-30 acetate of the commer-
cially available cephalothin gave the (hydroxy-
methyl)cephem
4
in high yield.27 Subsequent
esterification of 4 with diphenyldiazomethane provided
the benzhydryl ester 5.14 Chloroformate formation in
situ(C 6F5OH/NaH/COCl2) followed by addition of 5 in
Scheme 2.
Scheme 3.

X. Tanget al. / Bioorg. Med. Chem. Lett. 13 (2003) 1687–1690
1689
Scheme 4.
the presence of pyridine gave the pentafluorophenyl car-
bonate 6. After purification, the carbonate 6 was reacted
with one equivalent of SIN-1.HCl in anhydrous pyridine
at 0 ꢁC for 2 h to afford an inseparable 4:1 mixture of
cephem-30-carbamate 7 and, possibly, its corresponding
20-isomer. Acid-catalyzed hydrolysis of the benzhydryl
ester in the presence of triethylsilane, as a carbonium ion
trap, provided the desired conjugate 8.28
donor prodrugs. The cephalosporin carbamate of SIN-1
was successfully synthesized and its b-lactamase depen-
dent NO releasing ability was demonstrated. Using this
established synthetic strategy, we plan to generate a
series of cephalosporin carbamate derivatives. The
synthesis of cephalosprin conjugates using other NO
donors is also in progress. We hypothesize that this class
of NO donors may be a potential drug in the field of
ADEPT, as well as a new class of antibiotics.
The NO releasing ability of the conjugate 8 was mea-
sured through the formation of nitrite (NOꢀ2 ) in solu-
tion,29 following incubation of the compound in the
presence or the absence of b-lactamase. SIN-1 was used
as control. A stock solution of 8 was prepared in 1.0 mL
of methanol. Then 100 mL of the solution was diluted
with 500 mL of Tris–HCl buffer (pH=7.0) to a final
concentration of 1.2 mM. Then 100 mL of the solution
of penicillinase (0.5 units, SIGMA E.C. 3.5.2.6, Type
III: from Enterobacter cloacae) in the Tris–HCl buffer
(pH=7.0) was added. The mixture incubated at room
temperature for about 1 h before the addition of Griese
reagent (150 mL of 1% sulfaniamide in 1 N HCl and 150
mL of 0.1% N-(1-naphthyl)-ethylenediamine in 1 N
HCl). The absorbance of the solution was then mea-
sured at 548 nm. The calibration curves were made from
known concentrations of NaNO2 in the same buffer
solution to determine the amount of NOꢀ2 formed in the
reactions. The results are summarized in Table 1. As
expected, the SIN-1 decomposes easily in the aqueous
solution. The conjugate 8 is more stable than SIN-1
under the test conditions, and it releases NO in the pre-
sence of b-lactamase.
Acknowledgements
Financial support from the National Institutes of
Health (GM 54074) is greatly appreciated.
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[NOꢀ2 ] mM
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8+penicillinase
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2.2
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(d, J=9.0 Hz, 1H), 5.98–5.87 (m, 1H), 5.85 (dd, J=5.0, 9.0
Hz, 1H), 5.50 (d, J=13.5 Hz, 1H), 5.38 (d, J=16 Hz, 1H),
5.30 (d, J=12 Hz, 1H), 5.18 (d, J=14 Hz, 1H), 4.98 (d, J=5.0
Hz, 1H), 4.76 (m, 2H), 3.85 (s, 2H), 3.62 (d, J=18.5 Hz, 1H),
3.48 (d, J=18 Hz, 1H); 13C NMR (CDCl3, 125 MHz) d
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127.90, 127.56, 126.11, 125.89, 125.70, 121.13, 119.70, 72.70,
67.03, 59.20, 57.43, 37.08, 26.24; MS (ESI): m/z 537 (M+Na),
553 (M+K); HRMS (FAB) calcd for C23H22N4O6S2Na
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NMR (acetone-d6, 500 MHz) d 9.34 (s, 1H), 8.10 (dd, J=9.0
Hz, 2H), 7.32 (d, J=5.0 Hz, 1H), 7.07–6.95 (m, 4H), 5.96–5.86
(m, 2H), 5.36 (d, J=17.5 Hz, 1H), 5.18 (m, 2H), 4.88 (d,
J=12.5 Hz, 1H), 4.71 (m, 3H), 3.85 (m, 3H), 3.71 (d, J=18
Hz, 1H); 13C NMR (acetone-d6, 125 MHz) d 170.58, 165.70,
162.20, 155.54, 137.52, 132.77, 127.42, 127.35, 127.22, 126.18,
126.02, 125.66, 118.82, 112.88, 112.78, 74.21, 66.94, 60.48,
58.65, 37.02, 27.27. MS (ESI) 553 (M+Na), 569 (M+K);
HRMS (FAB) calcd for C23H22N4O7S2Na [M+Na]:
553.0828, found: 553.0828.
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(d, J=13.0 Hz, 1H), 5.07 (d, J=5.0 Hz, 1H), 5.00 (d, J=14.0
Hz, 1H), 3.97 (m, 4H), 3.80 (d, J=5.0 Hz, 2H), 3.69 (m, 4H),
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