Design and synthesis of a β-lactamase activated 5-fluorouracil prodrug

Article abstract of DOI:10.1016/j.bmcl.2008.12.057

An efficient synthesis of a 5-fluorouracil-cephalosporin prodrug is described for use against colorectal and other cancers in antibody and gene-directed therapies. The compound shows stability in aqueous media until specifically activated by β-lactamase (

Full text of DOI:10.1016/j.bmcl.2008.12.057

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1261–1263
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of a b-lactamase activated 5-fluorouracil prodrug
Ryan M. Phelan a, Marc Ostermeier b, Craig A. Townsend a,
*
a Department of Chemistry, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21212, USA
b Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21212, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient synthesis of a 5-fluorouracil-cephalosporin prodrug is described for use against colorectal
and other cancers in antibody and gene-directed therapies. The compound shows stability in aqueous
media until specifically activated by b-lactamase (bL). The kinetic parameters of the 5-fluorouracil-ceph-
alosporin conjugate were determined in the presence of Enterobacter cloacae P99 bL (ECl bL) revealing a
Received 10 November 2008
Revised 9 December 2008
Accepted 11 December 2008
Available online 24 December 2008
Km = 95.4 lM and Vmax = 3.21 l
Mol minꢀ1 mgꢀ1. The data compare favorably to related systems that have
been reported and enable testing of this prodrug against cancer cell lines in vitro and in vivo.
Keywords:
Beta-lactam
Ó 2009 Elsevier Ltd. All rights reserved.
Cephalosporin
5-Fluorouracil
Cephalosporin derivatization
Colorectal cancer is the third most common cancer in both men
and women in the United States today. American Cancer Society
projections for 2008 estimate that 150,000 cases of colorectal can-
cer will occur, claiming the lives of approximately one-third of
those diagnosed. Strategies to better combat colorectal cancer
would be of great benefit in lowering the mortality rate of this dis-
ease. Current methodology using targeted drug delivery through
antibody (ADEPT) or gene-directed prodrug therapy (GDEPT) is
able to specifically activate prodrugs at precise locations.1,2 The
benefit of an ADEPT or GDEPT strategy is that heightened concen-
trations of the desired chemotherapeutic agent can be delivered
directly to infected cells, while avoiding their release in benign re-
gions. This strategy is represented in the literature through the use
of a b-lactamase (bL) to activate b-lactam based prodrugs, specifi-
cally targeting infection or disease.3–5 One particular b-lactam
class, the cephalosporins, have proved to be very useful reporter,
signaling and antibacterial compounds that are able to lie dormant
until activation by a bL (Fig. 1).6–9 Many chemotherapeutic com-
pounds using the cephem scaffold have been reported including;
mitomycin C,4 nitrogen mustards,3,10–13 doxarubacin,14,15 platinum
compounds,16 taxol5 and a derivative of the vinca alkaloid vinblas-
tine.17 These compounds have been tested in vivo and show prom-
ising activity against various cancer lines when used in conjunction
with an ADEPT strategy. Although many of the aforementioned
prodrug conjugates have been evaluated in vivo, a common FDA
approved drug for colorectal cancer, 5-fluorouracil (5-FU), has
not been evaluated in an analogous fashion. 5-Fluorouracil is a
main component of the currently used colorectal cancer therapy
regimen FOLFOX, along with oxaliplatin and folinic acid.18 Ability
to specifically deliver 5-fluorouracil to cancerous cells provides
protection to benign regions while targeting drug concentrations
to areas of concern. It would be a benefit to create a simple,
straight-forward synthesis of a cephalosporin-5-fluorouracil pro-
drug that can then be evaluated in vivo. We describe here a facile
synthesis of a cephalosporin-5-fluorouracil prodrug that shows
stability in aqueous environments until specifically activated by a
b-lactamase.
In order to create a 5-FU-cephalosporin conjugate, it was of pri-
mary importance to determine the manner in which 5-FU could be
attached to the 30 position of the cephalosporin. More often than
not the drug of choice is attached to the 30 carbon by the use of a
carbonate or carbamate linker that decomposes upon expulsion
from the prodrug to yield the desired free compound. In some in-
stances the compound may be attached directly to the 30 position
through a displacement reaction between a 30 leaving group and
a suitable nucleophile. With both means of linkage being equal,
the deciding factor was the ready availability of a commercial
starting material that led to the most direct synthetic route. 7-Ami-
no-3-chlormethyl-3-cephem-4-carboxylic acid p-methoxybenzyl
ester (3) was selected as a suitable starting material (Otsuka, Osaka
Japan).
Cephem 3 provided a nucleus that could be modified to the
desired prodrug (Scheme 1). This starting material was amenable
to direct attachment of 5-FU via displacement of the 30 chloride
with 5-FU. Treatment of 3 with 2-thiophene acetyl chloride and
2,6-lutidine afforded the intermediate 4 in excellent yield.8,19 With
compound 4 in hand it was desired to then attach 5-FU. This
* Corresponding author. Tel.: +1 410 516 7444; fax: +1 410 261 1233.
E-mail address: ctownsend@jhu.edu (C.A. Townsend).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.057

1262
R. M. Phelan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1261–1263
Figure 1. Activation of a cephalosporin prodrug by a b-lactamase. In general, lactam cleavage releases the chemical moiety at the 30-position, in this case 5-fluorouracil.
Scheme 1. Reagents and conditions: (a) Boc2O, DMAP, 78%; (b) 2-thiopheneacetyl chloride, TEA, 2,6-lutidine, 92%; (c) NaI, KOSiMe3, 3, 60%; (d) CF3COOH, Et3SiH, 90%.
proved more difficult than originally thought. A primary difficulty
with using 5-FU in organic reactions is its relative insolubility in all
common organic solvents. In addition to its poor solubility, it was
of concern that both the amide and imide positions could act as the
nucleophile, giving rise to regioisomers. To overcome both of these
constraints, protection of the amide nitrogen with t-butyl carba-
mate (Boc) was performed.20,21 The Boc protection increased 5-
FU solubility while also limiting the imide to function as the sole
nucleophile. With compounds 2 and 4 in hand, direct coupling to
the 30 position was attempted. It was observed that when using
carbonate and tertiary amine bases, as well as pyridine, partial
isomerization of the cephem nucleus occurred yielding a mixture
of the D2 and D3 isomers. Formation of the D2 isomer inactivates
the compound by taking the nitrogen of the b-lactam out of conju-
gation with the 30 position, thus blocking release of the attached
compound upon bL activation. A common solution to this problem
is oxidation of the cephem sulfur to restore the active D3 isomer,
followed by reduction back to the thioether. Oxidation of com-
pound 5 containing a mixture of the D2 and D3 isomers was pre-
sumed to restore conjugation.22 It was observed, however, that
oxidation of the bridgehead sulfur was not possible as with other
related systems, providing no trace of the sulfoxide as a product.
One may rationalize this by noting that the cephem contains a
highly puckered ring in which the 5-FU moiety could block ap-
proach of the oxidant, resulting in no reaction. At this point any
attachment of the 5-FU moiety needed to provide a single isomer
due to the inability of the oxidation/reduction sequence to restore
the required conjugation. A previous report had shown that potas-
sium trimethyl silanoate (KOSiMe3) could be used to perform the
acylation reaction producing 4 without formation of the D2 iso-
mer.23 Indeed, use of KOSiMe3 as the stoichiometric base in the for-
mation of compound 5 provided the D3 isomer in good yield.24 It
was then possible for acidic deprotection of both the Boc and p-
methoxybenzyl moieties of 5 providing the final compound in high
yield.25 The use of KOSiMe3 in the direct displacement reaction
afforded a facile route to the desired 6 in 3 linear steps. This route
to cephem derivatives avoids the oxidation/reduction sequence
that has traditionally dominated in syntheses of similar
compounds.
ADEPT and GDEPT strategies to combat disease have the distinct
advantage that the prodrug is specifically activated in areas of
infection while benign regions are left relatively untouched. For
any ADEPT or GDPET strategy to be successful, the prodrug must
first be stable under non-activating conditions, yet quickly acti-
vated under the desired conditions. In the case of 6 this would
amount to the compound lying dormant until acted upon by a
bL. In addition to the specific activation by bL, it is important to
verify that 6 is a good substrate for bL by evaluation of its funda-
mental kinetic parameters. Compound
6 was prepared in a
10 mg/mL stock solution and subsequently diluted for all in vitro
experiments. To first verify that the compound can specifically be
activated only in the presence of a bL, a HPLC assay was pre-
formed.26 The prodrug substrate 6 was incubated in phosphate
buffered saline (50 mM, pH 7.0) for 12 h at 28 °C under activating
and non-activating conditions. The results show that 6, in the pres-
ence of bL, was completely cleaved to 5-FU and the hydrolyzed
Figure 2. HPLC trace of compound 6 and products released by bL activation. Front
to back: 6 incubated 20 h in 50 mM PBS (pH 7.0) without bL, 6 incubated 20 h in
50 mM PBS (pH 7.0) with 1 mg/mL EClbL, Compound 6 standard, 5-FU standard. (ꢁ)
5-FU. (ꢂ) Compound 6.

R. M. Phelan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1261–1263
1263
cephalosporin while 6 was otherwise stable in the absence of bL
References and notes
(Fig. 2). To better evaluate how efficient a substrate 6 is for the
aforementioned bL, kinetic parameters were measured using an as-
say previously described.13 It was determined that compound 6
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had a molar absorbtivity (e) of 9640 l
molꢀ1 cmꢀ1, which enabled
monitoring of b-lactam hydrolysis at 267 nm. Compound 6 was
evaluated against the commercially available Enterobacter cloacae
b-lactamase (EClbL) (Sigma, St. Louis, MO). Initial velocities were
measured at varying concentrations of 6 (25–250
the Michaelis–Menton equation to establish Km and Vmax. Com-
pound 6 was found to have a Km of 95.4 M and a Vmax of
3.21
mol minꢀ1 mgꢀ1, which compare favorably to previously re-
lM) and fit to
l
l
ported cephalosporin conjugates.3,4,13 To determine the 5-FU yield
when 6 is activated, a reaction of 6 with the EClbL was run under
conditions identical to the previous stability assay. The products
of this reaction were compared to a standard curve of known con-
centrations of 5-FU to determine the percentage of 5-FU released
using the previously established HPLC method.26 It was deter-
mined that compound 6 is activated to 5-FU in a 78 2% yield.
The combined data show that compound 6 is a good substrate
for a bL, activation occurs in an efficient manner and compound
6 is stable in a neutral aqueous environment until specifically acti-
vated by the bL (Fig. 2).
13. Svensson, H. P.; Kadow, J. F.; Vrudhula, V. M.; Wallace, P. M.; Senter, P. D.
Bioconjugate Chem. 1992, 3, 176.
14. Vrudhula, V. M.; Svensson, H. P.; Senter, P. D. J. Med. Chem. 1995, 38, 1380.
15. Jungheim, L. N.; Shepherd, T. A.; Kling, J. K. Heterocycles 1993, 35, 339.
16. Hanessian, S.; Wang, J. G. Can. J. Chem. 1993, 71, 896.
17. Jungheim, L. N.; Shepherd, T. A.; Meyer, D. L. J. Org. Chem. 1992, 57, 2334.
18. Rothenberg, M.; Navarro, M.; Butts, C.; Bang, Y.; Cox, J.; Goel, R.; Gollins, S.; Siu,
L.; Cunningham, D. Ann. Oncol. 2007, 18, 17.
19. Compound 4: 1H NMR (300 MHz, CDCl3) d 3.53 (2H, qab, J = 18.6, 30.6 Hz), 3.81
(3H, s), 3.85 (2H, s), 4.45 (2H, qab, J = 11.7, 17.7 Hz), 4.94 (1H, d, J = 7.8 Hz), 5.12
(2H, s), 5.82 (1H, dd, J = 3.0 Hz), 6.23 (1H, d, J = 9.3 Hz), 6.89 (2H, d, J = 11.7 Hz),
6.96 (2H, m), 7.30 (1H, m), 7.34 (2H, d, J = 11.7 Hz); 13C NMR (100 MHz, CDCl3)
d 27.9, 37.0, 43.2, 55.2, 57.5, 59.1, 68.2, 114.0, 125.5, 126.0, 126.1, 126.5, 127.6,
127.9, 130.7, 134.6, 159.9, 161.0, 164.5, 169.9; HRMS (FAB, MH+) calcd for
C22H21ClN2O5S2: 493.1. Found: 493.1.
We have designed a straight-forward synthesis of a bL-activat-
able 5-FU-cephalosporin conjugate to release the cytotoxic agent
5-FU. Preparation of this compound was readily achieved through
careful attention to two key steps in the synthesis. The protection
of the 5-FU amide nitrogen with a Boc group improved solubility in
organic solvents and avoided the formation of regioisomers. Sec-
ond, the use of KOSiMe3 in the cephem 30-chloride displacement
reaction proceeded without formation of the D2 isomer, circum-
venting the customary oxidation and reduction sequence at the
cephem sulfur to restore the D3 double bond. The use of KOSiMe3
offers a general approach for attachment of nucleophilic chemical
moieties at the 30 carbon while keeping the D3 cephem intact.
The stability of compound 6 in the absence of a bL and its favorable
kinetic parameters for hydrolysis in its presence demonstrate the
potential for compound 6 to be used in ADEPT or GDEPT strategies
against a range of human carcinoma cell lines, and open the way
for its evaluation it in vivo.
20. Compound 2: 1H NMR (300 MHz, CDCl3) d 1.52 (9H, s), 8.18 (1H, d, J = 7.2 MHz),
11.9 (1H, s); 13C NMR (75 MHz, CDCl3) d 27.7, 88.1, 123.5, 139.0, 146.1, 147.5;
HRMS (FAB, MH+) calcd for C9H11FN2O4: 231. Found: 231.
21. Jacobsen, M. F.; Knudsen, M. M.; Gothelf, K. V. J. Org. Chem. 2006, 71,
9183.
22. Kaiser, G. V.; Cooper, R. D. G.; Koehler, R. E.; Murphy, C. F.; Webber, J. A.;
Wright, I. G.; Vanheyni, E. J. Org. Chem. 1970, 35, 2430.
23. Lee, M.; Hesek, D.; Mobashery, S. J. Org. Chem. 2005, 70, 367.
24. Compound 5: 1H NMR (400 MHz, CDCl3) d 1.60 (9H, s) 3.36 (2H, d, J = 7.6 Hz),
3.81 (3H, s), 3.84 (2H, s), 4.89 (1H, d, J = 4.8 Hz), 4.95 (2H, qab, Jab = 15.2,
116.0 Hz), 5.24 (2H, s), 5.78 (1H, dd, J = 4.8 Hz), 6.41 (1H, d, J = 9.2 Hz), 6.98
(2H, d, J = 6.8 Hz), 7.02 (3H, m), 7.36 (2H, d, J = 6.4 Hz), 7.93 (1H, d, J = 5.0 Hz);
13C NMR (100 MHz, CDCl3) d 26.1, 27.7, 29.6, 37.0, 43.1, 55.2, 57.1, 59.1, 68.0,
88.1, 113.9, 122.3, 122.7, 123.3, 126.0, 126.5, 126.8, 127.5, 127.8, 130.5, 134.5,
138.3, 140.7, 161.4, 164.2, 169.9; HRMS (FAB, MH+) calcd for C31H31FN4O9S2:
687.1. Found: 687.1.
25. Compound 6: 1H (300 MHz, DMSO-d6) d 3.51 (2H, qab, J = 17.4, 29.4 Hz), 3.75
(2H, s), 4.79 (2H, qab, J = 14.7, 67.2 Hz), 5.00 (1H, d, J = 4.8 Hz), 5.64 (1H, q,
J = 4.2 Hz), 6.95 (2H, m), 7.35 (1H, d, J = 3.6 Hz), 9.08 (1H, d, J = 8.4 Hz); 13C
(75 MHz, DMSO-d6) d 24.7, 35.5, 57.0, 58.6, 124.9, 126.0, 126.4, 129.2, 136.6,
137.7, 140.7, 149.9, 157.3, 157.6, 162.8, 164.3, 169.7; HRMS (FAB, MH+) calcd
for C18H15FN4O6S2: 467. Found: 467.
26. The bL cleavage assay was preformed on an Agilent (Santa Clara, CA) 1100
HPLC using a Phenomenex (Torrance, CA) Luna C-18 reverse phase column. All
compounds were run using an isocratic mobile phase consisting of 30%
acetonitrile in 0.1% trifluoroacetic acid. All compounds were monitored at
267 nm.
Acknowledgments
We gratefully acknowledge a seed grant from the Institute for
NanoBioTechnology at The Johns Hopkins University for financial
support. Otsuka Chemical Corporation (Osaka, Japan) is thanked
for donation of a sample of 7-amino-3-chloromethyl-3-cephem-
4-carboxylic acid p-methoxy-benzyl ester. We thank T. Soka for
helpful discussions in preparation for the in vivo applications of
the conjugate.