Cephalosporin prodrugs of paclitaxel for immunologically specific activation by L-49-sFv-β-lactamase fusion protein

Article abstract of DOI:10.1016/S0960-894X(02)00935-6

Paclitaxel conjugates of 7-phenylacetamidocephalosporanic acid were prepared as prodrugs for site specific activation by targeted β-lactamase. Immunologically specific activation of the prodrug 5 containing 3,3-dimethyl-4-amino-butyric acid as linker in combination with the fusion protein L-49-sFv-β-lactamase was demonstrated in vitro on 3677 melanoma cells.

Full text of DOI:10.1016/S0960-894X(02)00935-6

Bioorganic & Medicinal Chemistry Letters 13 (2003) 539–542
Cephalosporin Prodrugs of Paclitaxel for Immunologically
Specific Activation by L-49-sFv-ꢀ-Lactamase Fusion Protein
Vivekananda M. Vrudhula,* David E. Kerr,y Nathan O. Siemers,{
Gene M. Dubowchik and Peter D. Senterx
Bristol-Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway, Wallingford, CT 06492, USA
Received 6 September 2002; accepted 1 October 2002
Abstract—Paclitaxel conjugates of 7-phenylacetamidocephalosporanic acid were prepared as prodrugs for site specific activation by
targeted b-lactamase. Immunologically specific activation of the prodrug 5 containing 3,3-dimethyl-4-amino-butyric acid as linker
in combination with the fusion protein L-49-sFv-b-lactamase was demonstrated in vitro on 3677 melanoma cells.
# 2002 Elsevier Science Ltd. All rights reserved.
The aim of Antibody Directed Enzyme Prodrug
Therapy (ADEPT) is to achieve selective toxicity to
tumor cells while sparing normal cells from chemo-
therapeutic damage. In this approach a monoclonal
antibody (mAb) enzyme conjugate is administered
first and localizes at the tumor. After allowing suffi-
cient time for systemic clearance of the mAb con-
jugate, a prodrug of a cytotoxic agent capable of
being activated by the targeted enzyme is adminis-
tered, leading to site selective generation of the cyto-
toxic substance at the tumor site. A plethora of
prodrugs of potent cytotoxic agents have been
employed in combination with a variety of mAb
enzyme conjugates, and this methodology has been
thoroughly reviewed.1
KB cells, thus illustrating the importance of this side
chain for activity.2b Esters derived from the 20-OH
group of 1 generally exhibit diminished cytotoxicity.2b
The 20-OH therefore serves as a good handle for the
design of prodrugs. Because paclitaxel is a bulky mole-
cule, it was anticipated that successful ADEPT pro-
drugs would require the intermediacy of a less sterically-
demanding spacer that would release free drug quickly
following enzymatic hydrolysis. During the synthesis of
water soluble esters of paclitaxel, Kingston and co-
workers found that the 20-g-aminobutyrate ester of 1
underwent cyclization slowly in methanol to release
The diterpenoid natural product paclitaxel2a (1) is an
important anticancer drug discovered in 1971. Paclitaxel
exerts its antitumor activity by stabilizing tubulin poly-
mers. The N-benzoyl-b-phenyl isoserine side chain in 1
is crucial for its activity. Baccatin III, a taxoid devoid of
this side chain is 50-fold less effective in tubulin poly-
merization assay in vitro and 1000-fold less cytotoxic to
paclitaxel in 15 h.3 Utilizing this observation,
a
cephalosporin paclitaxel prodrug (2) incorporating
g-aminobutyric acid (GABA) as a linker was prepared
for ADEPT.4 When exposed to b-lactamase, 2 released
1 slowly in phosphate buffered saline (PBS). The half-
life for this rate limiting cyclization was found to be
approximately 6 h at 37ꢀ.4 While this compound was
the first published example of a cephalosporin prodrug
of paclitaxel, we sought to further improve upon the
kinetics of the rate limiting cyclization reaction. A fur-
ther limitation with sterically unhindered 20-esters of
*Corresponding author. Fax +1-203-677-7702; e-mail: vivekananda.
vrudhula@bms.com
yPresent address: Biocontrol Systems, Inc., Bellevue, WA 98005, USA.
{Present address: Bristol-Myers Squibb Pharmaceutical Research
Institute, Princeton, NJ 08543, USA.
xPresent address: Seattle Genetics, 21823-30th Dr. S.E., Bothell, WA
98021, USA.
0960-894X/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00935-6

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V. M. Vrudhula et al. / Bioorg. Med. Chem. Lett. 13 (2003) 539–542
paclitaxel is the potential for direct hydrolysis by
endogenous esterases.5 The important issues to be
addressed therefore were: (a) modifications of the lin-
ker to ensure more rapid release of 1, (b) stabilization
of the 20-ester bond to prevent premature hydrolysis by
endogenous esterases and finally (c) demonstration of
immunologically specific prodrug activation. Efforts in
this direction and the results obtained are described
here.
The 7-phenylacetamido side chain-containing cephalo-
sporin prodrugs of doxorubicin, phenylenediamine mus-
tard and melphalan displayed favorable properties and
pronounced antitumor effects in our earlier studies.6,7
Hence, it was decided to retain this side chain at
position 7 on the cephem nucleus. The sulfoxide oxi-
dation state of the cephem sulfur prevents undesirable
Á3 to Á2 isomerization of the double bond, a com-
mon occurrence with many esters of 3-cephem-4-car-
boxylic acid.8 In designing the linkers, a geminal
dimethyl group in the vicinity of the carbonyl group
was expected to increase the rate of cyclization to
form both five and six-membered rings, due to the
Thorpe–Ingold effect.9 In addition, such a steric con-
straint was also expected to improve the stability
toward hydrolysis by endogenous esterases. For a
different linker strategy, we tried to take advantage of
the cyclization of the aminoacid linkers which leads to
the formation of diketopiperazine. This is a facile
process employed in the synthesis of Ara C pro-
drugs.10 Since an l-aminoacid derived peptide may be
a substrate for endogenous amidases, the d-alanyl-
amide of a-aminoisobutyric acid was chosen as the
linker for the diketopiperazine strategy. Compounds
4–6 (Scheme 1) thus became the preferred targets. In
addition, 3 was prepared as an analogue of 2 in the
present series.
Scheme 2. (a) Methoxy trityl chloride, Et3N, rt, 14 h, 96%; (b) LDA
(2.2 equiv), À78 ꢀC; (c) MeI (À78 ꢀC to rt); (d) repeat (b) and (c); (e)
KOH, EtOH reflux 24 h, then acidification (pH 4); (f) TFA, CH2Cl2
over all 70% yield; (g) DSC, Et3N, DMF 0 ꢀC, 1 h/H2N–
C(CH3)2COOH 43%; (h) H2–Pd, MeOH.
lysis to give the zwitterionic N-p-MeO-trityl-GABA.
Detritylation with TFA provided the necessary linker
with the reported properties.11 Michael addition of
nitromethane to ethyl 3,3-dimethylacrylate followed by
reduction of the nitro group and hydrolysis provided
the necessary 3,3-dimethyl-4-aminobutyric acid.12 The
CBZ protected peptide linker necessary for the diketo-
piperazine strategy was prepared by coupling a-amino-
isobutyric
acid
with
in
situ
prepared
N-hydroxysuccinimidate ester of CBZ-d-alanine using
disuccinimidyl carbonate (DSC).13 After reductive
removal of the CBZ group the crude product was used
to condense with 7.
Coupling of 7 with these linkers (Scheme 3) in acetone
containing aqueous NaHCO3 proceeded in ꢁ60% yield
to give 8–11. While 8, 10 and 11 underwent DCC cou-
pling with paclitaxel smoothly, compound 9 gave only
N-acyl-dicyclohexyl urea under the same conditions of
coupling. Removal of the DPM ester group was carried
out using TFA anisole to give 3, 5 and 6.15
The linkers for attachment to the activated sulfoxide 77
were prepared as shown in Scheme 2: The necessary
2,2-dimethyl-4-aminobutyric acid was prepared by
alkylation of the lithium enolate of N-p-MeO-trityl-
GABA methyl ester in THF followed by basic hydro-
Reversed phase analytical HPLC analyses were carried
out to study the stability of 3, 5, and 6 in human serum
at 37 ꢀC. The rate of cyclization of the intermediate
20-esters generated by b-lactamase in DMSO:phosphate
buffered saline (PBS pH 7.4, 1:9) were also evaluated. A
high concentration of Enterobacter cloacae P99 b-lacta-
mase (0.07 mg/mL) was used in these studies. Under
these conditions the enzymatic hydrolysis is almost
instantaneous to form the intermediate 20-esters. The
half-life values for cyclization and the half life values in
Scheme 1.
Scheme 3.

V. M. Vrudhula et al. / Bioorg. Med. Chem. Lett. 13 (2003) 539–542
541
Table 1. Half lives of cephalosporin paclitaxel derivatives in the
human serum and in presence of b-lactamase at 37 ꢀC
immolative linkers. The rate limiting step for release of
paclitaxel from these derivatives is the linker cyclization
reaction, the kinetics of which is influenced by alkyl
group substitution. Compound 5, with the fastest rate
of release of 1 in the presence of b-lactamase, was
shown to be a prodrug which can be activated in an
immunologically specific manner.
Compd
t1/2 in human
serum (min)
t1/2 for cyclization after
b-lactamase hydrolysis (min)
3
5
6
275
350
410
230
50
60
human serum are listed in Table 1. Geminal dimethyl-
ation not only improved serum stability, but also resulted
in approximately 4- to 5-fold increase in the rate of
cyclization irrespective of whether the cyclization led
to the formation of five-membered lactam from com-
pound 5 or the diketopiperazine from compound 6.
References and Notes
1. Senter, P. D.; Springer, C. J. Adv. Drug Deliv. Rev. 2001,
53, 247.
2. (a) Wani, M. C.; Taylor, H. L.; Wall, M. E.; Coggin, P.;
McPhail, A. T. J. Am. Chem. Soc. 1971, 93, 2325. (b) Taxane
Anticancer Agents Basic Science and Current Status; Georg, G.
I., Chen, T. T., Ojima, I., Vyas, D. M., Eds. ACS Symposium
Series 583; American Chemical Society: Washington, DC,
1995.
3. Zhao, Z.; Kingston, D. G. I. J. Nat. Prod. 1991, 54, 1607.
4. Rodrigues, M. L.; Carter, P.; Wirth, C.; Mullins, S.; Lee,
A.; Blackburn, B. K. Chem. Biol. 1995, 2, 223.
5. Senter, P. D.; Marquardt, H.; Thomas, B. A.; Hammock,
B. D.; Frank, I. S.; Svensson, H. P. Cancer Res. 1996, 56,
1471.
6. Kerr, D. E.; Screiber, G. J.; Vrudhula, V. M.; Svensson,
H. P.; Hellstrom, I.; Hellstrom, K. E.; Senter, P. D. Cancer
Res. 1995, 55, 3558.
7. Kerr, D. E.; Li, Z.; Siemers, N. O.; Senter, P. D.; Vrudhula,
V. M. Bioconj. Chem. 1998, 9, 255.
In vitro cytotoxicity assays were performed with human
3677 melanoma cells expressing the melanotransferrin
(p97) antigen. L-49-sFv-bL is a recombinant fusion
protein comprised of the sFv fragment of the anti-
melanoma mAb L49 fused to a mutated form of
E. cloacae b-lactamase. The protein was expressed in
Escherichia coli as described earlier14 and purified by
affinity chromatography. Cells were treated with the
fusion protein and after washing off the unbound
material various concentrations of 5 were added and
incubated at 37 ꢀC for 4 h. Incorporation of [3H] thymi-
dine was used as a measure of cytotoxic activity.
8. Murphy, C. F.; Webber, A. J. In Cephalosporins and Peni-
cillins: Chemistry and Biology; Flynn, E. H. Ed.; Academic:
New York and London, 1972; p 147.
9. Eliel, E. L. In Stereochemistry of Carbon Compounds;
McGraw Hill: New York, 1962; p 197.
10. Wipf, P.; Li, W.; Sekhar, V. Bioorg. Med. Chem. Lett.
1991, 1, 745.
11. Royer, F.; Felpin, F.; Doris, E. J. Org. Chem. 2001, 66,
6487.
12. Andruszkiewitz, R.; Silverman, R. B. J. Biol. Chem. 1990,
265, 22288.
13. l-Isomer reported in: Nagaoka, Y.; Iida, A.; Fujita, T.
Chem. Pharm. Bull. 1994, 42, 1258.
14. Siemers, N. O.; Kerr, D. E.; Yarnold, S.; Stebbins, M. R.;
Vrudhula, V. M.; Hellstrom, I.; Hellstrom, K. E.; Senter, P. D.
Bioconj. Chem. 1997, 8, 510.
15. Spectral characteristics of key intermediates and final
compounds: In the assignment of NMRspectra of cephalos-
porin derivatives of 1, the notation ceph for cephem nucleus, L
for linker and T- for paclitaxel portion with appropriate
numbering system was used. Compd 8: MS: [MÀH]=658; IR
(KBr) 3294 cmÀ1 (carboxyl), 1793 cmÀ1 (b-lactam), 1032 cmÀ1
(S¼O); 1H NMR(DMSO- d6+D2O) d 7.54–7.20 (m, 15H,
ArH), 6.94 (s, 1H, Ph2CH–), 5.91 (d, 1H, H-7, J6,7=4.8 Hz),
5.00 (d, 1H, CH2A–O–CO–N, JAB=13.4 Hz), 4.93 (d, 1H, H-
6), 4.55 (d, 1H, CH2B–O–CO–N), 3.69 (d, 1H, H-2A,
JAB=14.0 Hz), 3.56 (d, 1H, H-2B), 3.42 (s, 2H, PhCH2–), 2.96
(t, 2H, –CO–N–CH2–, Jvic,=7.1 Hz), 2.20 (t, 2H, –N–C–C–
CH2–CO–), 1.60 (m, 2H, N–C–CH2–C–CO–).
The prodrug 5 was found to be 12-fold less toxic than 1
on 3677 melanoma cells (Fig. 1). The fusion protein
activated the prodrug to a level comparable to that of
the free drug. When 3677 cells were first saturated with
unconjugated L49 mAb prior to the addition of the
fusion protein (blocking the antigenic sites), no acti-
vation of prodrug 5 took place, suggesting that the
activation is immunologically specific. In this assay, 6
was 30 fold less toxic than 1 (IC50 for 6=388 nM, IC50
for 1=13 nM). However, the activation of 6 in combi-
nation with L-49-sFv-bL was poor (IC50 for L-49-sFv-
bL+6=189 nM) when compared with compound 5.
In conclusion, we have prepared a series of cephalo-
sporin derivatives of paclitaxel containing different self-
À1
Compd 9: MS: [MÀH]=686; IR(KBr) 3288 cm
(car-
boxyl), 1790 cmÀ1 (b-lactam), 1391 cmÀ1 (gem di Me),
1065 cmÀ1 (S¼O); 1H NMR(DMSO- d6+D2O) d 7.49–7.27
(m, 15H, ArH), 6.84 (s, 1H, Ph2CH–), 5.85 (d, 1H, H-7,
J6,7=4.8 Hz), 4.94 (d, 1H, CH2A–O–CO–N, JAB=13.5 Hz),
4.84 (d, 1H, H-6), 4.53 (d, 1H, CH2B–O–CO–N), 3.58 (AB q,
2H, H-2A, JAB=14.3 Hz), 2.90 (t, 2H, –CO–N–CH2–C–,
Figure 1. Cytotoxicity assay on 3677 melanoma cells demonstrating
immunologically specific activation of 5.

542
V. M. Vrudhula et al. / Bioorg. Med. Chem. Lett. 13 (2003) 539–542
Jvic=8.2 Hz), 1.56 (t, 2H, –C–CH2–C(Me)2–CO–), 1.05 (s, 6H,
2 ꢂ gem-CH3). Compd 10: MS: [MÀH]=686; IR(KBr)
3288 cmÀ1 carboxyl), 1794 cmÀ1, 1384 cmÀ1 (gem di Me),
1040 cmÀ1 (S¼O); 1H NMR(DMSO- d6+D2O) d 7.54–7.29
(m, 15H, ArH), 6.91 (s, 1H, Ph2CH–), 5.91 (d, 1H, H-7,
J6,7=4.8 Hz), 5.00 (d, 1H, CH2A–O–CO–N, JAB=13.2 Hz),
4.92 (d, 1H, H-6), 4.60 (d, 1H, CH2B–O–CO–N), 3.81 (s, 2H,
PhCH2–), 3.68 (d, 1H, H-2A, JAB=14.0 Hz), 3.57 (d, 1H, H-
2B), 2.92 (s, 2H, –CO–N–CH2–C(Me)2–), 2.08 (s, 2H, –C–
C(Me)2–CH2–CO–), 0.89 (s, 6H, 2 ꢂ gem-CH3).
6A, 2 ꢂ COCH3), 1.78 (s, 3H, T-18), 1.49 (s, 3H, T-angular
CH3), 1.01 and 0.99 (2 ꢂ s, 6H, T-16 and T-17).
Compd. 5: HRMS: [M+Na]+=137À91.4570 (found),
1
[M]+=1356.4672 (calcd); IR(KBr) 1778 cm
(b-lactam); H
NMR(DMSO- d6) d 8.27–7.17 (m, 21H, ArH and amide), 6.29
(s, 1H, T-10), 5.87 (t, 1H, T-13, J13,14=9.0 Hz), 5.57–5.53 (2 ꢂ
dd, 2H, ceph-7and T-30, JNH,7=8.4 Hz, J6,7=4.4 Hz,
0
0
0
J2 ,3 =5.1 Hz), 5.42–5.35 (m, 2H, T-2 and T-2), 5.09 (d, 1H,
ceph-CH2A–OCO–, JAB=12.2 Hz), 4.94 (m, 2H, T-5 and NH
or OH), 4.67–4.30 (m, 3H, ceph-CH2B–OCO–, ceph-6, NH or
OH), 4.12 (m, 1H, T-7), 4.01 (m, 1H, T-20), 3.68 (d, 1H, ceph-
2A, JAB=14.1 Hz), 3.58 (m, 1H, T-3), 3.53 (d, 1H, ceph-2B),
2.89 (s, 2H, L-NH–CH2–), 2.40–2.20 (m and s, 6H, T-6A, T-
COCH3, L-CH2CO–), 2.10 (s, 3H, T-COCH3), 1.99–1.77 (m
and s, 4H, T-14 and T-18), 1.66–1.49 (m and s, 4H, T-6B, T-
angular CH3), 1.07 (s, 1H, OH), 1.01 and 0.99 (2 ꢂ s, 6H, T-16
and T-17), 0.88 and 0.84 (2 ꢂ s, 6H, L-gem-CH3).
À1
Compd. 11: MS: [MÀH]=729; IR(KBr) 3289 cm
(car-
boxyl), 1793 cmÀ1, 1388 cmÀ1 (gem di Me), 1074 cmÀ1 (S¼O);
1H NMR(DMSO- d6+D2O) d 7.54–7.18 (m, 15H, ArH), 6.92
(s, 1H, Ph2CH–), 5.90 (d, 1H, H-7, J6,7=4.8 Hz), 5.04 (d, 1H,
CH2A–O–CO–N, JAB=13.6 Hz), 4.94 (d, 1H, H-6), 4.61 (d,
1H, CH2B–O–CO–N), 3.99 (q, 1H, d-alanyl–CH(Me)–CO–,
Jvic=7.1 HZ), 3.70 (d, 1H, H-2A, JAB=14.0 Hz), 3.56 (d, 1H,
H-2B), 3.41 (s, 2H, PhCH2–) 1.35 and 1.33 (2 ꢂ s, 6H, 2 ꢂ
gem-CH3), 1.16 (d, 3H, d-alanyl–CH3).
Compd. 6: HRMS: [M+Na]+=142À21.4628 (found),
1
[M]+=1399.4730 (calcd); IR(KBr) 1780 cm
(b-lactam); H
Compd. 3: HRMS: [M+Na]+=1351.4257 (found),
NMR(DMSO- d6) d 8.37–7.15 (m, 23H, ArH and amide), 6.28
(s, 1H, T-10), 5.81 (t, 1H, T-13, J13,14=9.3 Hz), 5.57–5.53 (m,
2H, ceph-7 and T-30), 5.42–5.36 (m, 2H, T-20 and T-2), 5.06 (d,
1H, ceph-CH2A–OCO–, JAB=11.9 Hz), 4.91 (m, 2H, T-5),
4.67–4.30 (m, 2H, ceph-CH2B–OCO–, ceph-6), 4.10 (m, 1H, T-
7), 4.00 (m, 1H, T-20), 3.94 (m, 1H, L-CH–Me), 3.68 (d, 1H,
ceph-2A, JAB=14.0 Hz), 3.62–3.50 (m and d, 3H, T-3 and
ceph-2B), 2.30–2.20 (m and s, 4H, T-6A, T-COCH3), 2.10 (s,
3H, T-COCH3), 1.79–1.72 (m and s, 4H, T-14 and T-18), 1.59
(m, 1H, T-6B), 1.49 (s, 3H, T-angular CH3), 1.40 and 1.35 (2
ꢂ s, 6H, L-gem-CH3), 1.13 (d, 3H, L-CH–CH3), 1.01 and 0.99
(2 ꢂ s, 6H, T-16 and T-17).
À1
[M]+=1328.44 (calcd); IR(KBr) 1781 cm
(b-lactam); 1H
NMR(DMSO- d6) d 8.31–7.22 (m, 23H, ArH and amide), 6.29
(s, 1H, T-10), 5.87 (t, 1H, T-13, J13,14=9.0 Hz), 5.58 (dd, 1H,
ceph-7, JNH,7=8.6 Hz, J6,7=4.4 Hz), 5.53 (dd, 1H, T-30,
0
0
0
JNH,3 =J2 ,3 =9.0 Hz), 5.40 (d, 1H, T-2, J2,3=7.1 Hz), 5.34 (d,
1H, T-20), 5.04 (d, 1H, ceph-CH2A–OCO–, JAB=12.3 Hz),
4.93 (m, 1H, T-5), 4.69–4.60 (m, 3H, ceph-CH2B–OCO–, ceph-
6, NH or OH), 4.10 (m, 1H, T-7), 4.00 (m, 1H, T-20), 3.68 (d,
1H, ceph-2A, JAB=14.3 Hz), 3.60–3.50 (d and m, ceph-
PhCH2–CO–, ceph-2B and T-3), 2.98 (m, 2H, L-NH–CH2–),
2.42 (t, 2H, L-CH2–CO, Jvic=7.1 Hz), 2.25 (m and 2 ꢂ s, T-