Reductively activated disulfide prodrugs of paclitaxel.

Article abstract of DOI:10.1016/S0960-894X(02)00784-9

A series of unsymmetrical polar disulfide prodrugs 2-5 of paclitaxel were designed and synthesized as reductively activated prodrugs. These compounds behaved as prodrugs in vitro on L2987 lung carcinoma cells. In vivo evaluation in mice demonstrated that the mutual prodrug 5 with captopril exhibited significant regressions and cures.

Full text of DOI:10.1016/S0960-894X(02)00784-9

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3591–3594
Reductively Activated Disulfide Prodrugs of Paclitaxel
Vivekananda M. Vrudhula,*,y John F. MacMaster,{ Zhengong Li,x David E. Kerrk
and Peter D. Senter{
Bristol-Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway, Wallingford, CT 06492, USA
Received 21 August 2002; accepted 11 September 2002
Abstract—A series of unsymmetrical polar disulfide prodrugs 2–5 of paclitaxel were designed and synthesized as reductively acti-
vated prodrugs. These compounds behaved as prodrugs in vitro on L2987 lung carcinoma cells. In vivo evaluation in mice
demonstrated that the mutual prodrug 5 with captopril exhibited significant regressions and cures.
# 2002 Elsevier Science Ltd. All rights reserved.
The microenvironment in which solid tumors thrive is
characterized by hypoxia due to abnormal vasculature.
Tumor hypoxia leads to diminished supplies of oxygen
and nutrients. The hypoxic environment of tumors has
been delineated in cervical cancer, squamous cell carci-
noma of the head and neck, melanoma, and mammary
tumors. Hypoxia has also been implicated in resistance
to chemotherapy and radiation and is an excellent
target for development of cancer therapeutics.1 The
hypoxic environment is also congenial for reductive
transformations. It is known that the anticancer drug
mitomycin C2a,2b and agents such as tirapazamine elicit
their cytotoxicity through reductive activation.3 Nitro-
reductase mediated reductive activation of a prodrug of
the minor groove alkylating agent amino-seco-CBI-
TMI,4a and a number of other cytotoxic agents has
recently been reported.4b The use of aromatic nitro and
azido groups as bioreductive triggers to release pacli-
taxel (1) from its 20-ester prodrugs via 1,6- or 1,8-elim-
ination reactions has also been described.5 Cleavage of a
disulfide bond is a facile biochemical transformation in
a reductive environment. In an attempt to make more
potent mitosane derivatives, several disulfide containing
mitosanes were prepared and tested. Compounds such
as BMY-25067 and RR-150 are examples of such
agents.6a,6b
*Corresponding author. Fax:+1-203-677-7702; e-mail: vivekananda.
vrudhula@bms.com
Paclitaxel (1) is one of the most widely used chemo-
therapeutics and has activities in many types of cancer.7
Due to its aqueous insolubility, paclitaxel is adminis-
tered in a vehicle containing cremophor EL1, which
can produce hypersensitivity reactions in patients.7
Water soluble prodrugs of paclitaxel, which would be
activated in the reductive hypoxic environment of
tumors may comprise useful new drugs in cancer
chemotherapy.
yThis research was conducted at the Seattle site of the Pharmaceutical
Research Institute.
{Present Address: Bristol-Myers Squibb Pharamaceutical Research
Institute, Princeton, NJ 08543, USA.
xPresent address: Central Research Division, Pfizer Inc., Eastern Point
Rd., Groton, CT 06340, USA.
kPresent address: Biocontrol Systems Inc., Bellevue, WA 98005, USA.
{Present address: Seattle Genetics, 21823-30th Dr. S.E., Bothell, WA
98021, USA.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00784-9

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V. M. Vrudhula et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3591–3594
The synthesis of pyridyldisulfide carboxylic acid (7)
necessary for coupling to paclitaxel was carried out
as shown in Scheme 1. a,a-Dimethylbutyrolactone13
was subjected to nucleophilic ring opening with
benzylmercaptan. The resulting S-benzyl derivative
(6) was debenzylated with sodium in liquid ammo-
nia to give the disodium salt which upon treatment
in situ with 2,20-dipyridyl disulfide in methanol gave (7)
in 62% yield. DCC mediated esterification of paclitaxel
with the hindered carboxylic acid was slow and resul-
ted in the formation of 2 in a 43% isolated yield. In
order to prepare disulfides 3–5, the activated disulfide 2
was treated with excess thiol under basic conditons and
the excess thiolate was quenched with N-ethylmalei-
mide. In this manner disulfides 3–5 were prepared in 45–
53% yields.14
Esterification of the 20-OH group of paclitaxel generally
results in paclitaxel esters of diminished cytotoxicity.
Paclitaxel esters derived from 20-OH that release pacli-
taxel either by endogenous esterase activity8 or by a
cyclization reaction resulting in expulsion of paclitaxel
have been prepared previously.9 In this report we
describe the synthesis of unsymmetrical disulfide pro-
drugs of 1 derived from 2,2-dimethyl-4-mercaptobutyric
acid, which contain polar moieties as part of the disulfide
residue for increased hydrophilicity. Geminal dimethyl-
ation of carbon a to the carbonyl group in the linker
may offer stability toward serum esterases in addition to
facilitating cyclization of the intermediate thiol due to
the Thorpe–Ingold effect.10
Reversed-phase analytical HPLC studies were carried
out to investigate the release of 1 from 2–5 under redu-
cing conditions. When treated with dithiothreitol
(DTT), disulfides 2–5 underwent reduction and released
paclitaxel. Since the steric and electronic environment
around the disulfide bond is different in each of these
disulfides, they were expected to undergo reductive
cleavage at differing rates. The half-lives for the release
of paclitaxel after cyclization (Table 1) ranged from 4
min for the activated pyridyl disulfide (2) to >60 min
for the glutathione derived disulfide (4).
Human serum stability studies were done at 37 ꢀC
with compounds 2 and 3 using reversed-phase HPLC
methods. Both these compounds released <10%
paclitaxel after 125 min.
Cytotoxicity assays using L2987 lung carcinoma cells
were carried out using incorporation of [3H]-thymidine
as a measure of cellular replication. Cells were treated
with various concentrations of disulfides or a combina-
tion of disulfides and DTT for reductive activation. The
percentage of control of [3H]-thymidine incorporation
against concentration was plotted. Prior treatment of
disulfide derivatives with DTT (10 mM) for an hour was
used to measure reductive activation in this assay. IC50
values shown in Table 2 indicated that compounds 2–5
were all prodrugs. The diminishment in cytotoxicity in
comparison with 1 ranged from 30-fold for the activated
pyridyl disulfide 2 to 650-fold with captopril prodrug 5.
Compounds 2–5 underwent reductive activation in pre-
sence of DTT. The cytotoxicity of 2 and 3 in reductive
It was expected that the enhanced hydrophilicity of
compounds 2–5 would impair their abilities to traverse
cell membranes and would therefore diminish their cyto-
toxic activities. The key intermediate for the syntheses of
these disulfides is the activated 2-thiopyridyldisulfide (2).
To enhance hydrophilicity, compounds in which the
variable component of the unsymmetrical disulfide con-
tained highly polar moieties such as thioglucose (3) and
glutathione (4) were prepared. The ACE inhibitor capto-
pril was recently reported to have antiangiogenic
effects.11 Compound 5 utilizing captopril as one of the
disulfide components is thus a mutual prodrug.12
Scheme 1. (a) MeI, NaH, dioxane; (b) NaH, PhCH2SH, toluene,
reflux; (c) Na, liqNH3; (d) PyS-SPy, MeOH; (e) DCC, 1, CH2Cl2;
(f) RSH, MeOH, Et3N.

V. M. Vrudhula et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3591–3594
Table 1. Half lives values for release of 1 from 2–5
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mg/kg dose, there was a 60% tumor cure rate and the
remaining animals experienced marked regressions.
Prodrug 5 was clearly superior to paclitaxel in this
model. Paclitaxel did not regress tumors at the max-
imum tolerated dose of 30 mg/kg (Fig. 1). It is possible
that some of the superiority of 5 can be attributed to the
possible anti-angiogenic effects of captopril combined
with the cytotoxic effects of paclitaxel. Further studies
are required to establish if this is the case.
Compd
Half-life for release of paclitaxel (min)
2
3
4
5
4
12
>60
25
Table 2. IC50 valuesa from cytotoxicity assay on L2987 lung carci-
noma cells
In conclusion, we have demonstrated that reductively
activated polar disulfide derivatives of 1, incorporating
a range of thiol-containing agents, can be prepared. The
disulfide derivatives have reduced cytotoxic activities,
and are reductively activated leading to the formation of
paclitaxel. The mutual prodrug 5 served as a prodrug of
both paclitaxel and captopril, and led to regressions and
cures of established tumor xenografts. The activities
were superior to the clinically approved anticancer drug
paclitaxel.
Compd
IC50, mM of compd
IC50, mM of compd+DTT
1
2
3
4
5
0.2
6.20.2
5.7
10.1
130.0
0.2
0.4
2.3
aValues are averages from experiments run in triplicate.
environment matched that of 1 while 4 and 5 in the
presence of DTT were slightly less toxic.
References and Notes
Prodrugs 2–5 were evaluated for antitumor activity in
vivo in athymic nude mice that had subcutaneous L2987
human lung adenocarcinoma xenografts. Treatment
was initiated on day 20-post implant and was repeated
every two days for a total of three treatments. Paclitaxel
(1) and the prodrugs were administered in a 1:1 solution
of cremaphor ethanol intravenously. Control animals
received only the vehicle diluted in saline. The data were
plotted until any of the animals within the group had a
tumor that reached 1000 mm3.
1. Wouters, B. G.; Weppler, S. A.; Koritzinsky, M.; Landuyt,
W.; Nuyts, S.; Theys, J.; Chiu, R. K.; Lambin, P. Eur. J.
Cancer 2002, 38, 240.
2. (a) Iyer, V. N.; Szybalski, W. Science 1964, 145, 55. (b)
Tomasz, M. Curr. Biol. 1995, 9, 575.
3. Brown, J. M. Br. J. Cancer 1993, 67, 1163.
4. (a) Hay, M. P.; Sykes, B. M.; Denny, W. A.; Wilson, W. R.
Bioorg. Med. Chem. Lett. 1999, 9, 2237. (b) Denny, W. A.
Curr. Pharm. Des. 2002, 8, 1349.
5. Damen, E. W. P.; Nevalainen, T. J.; van den Bergh, T. J. M.;
de Groot, F. M. H.; Scheeren, H. W. Bioorg. Med. Chem.
2002, 10, 71.
6. (a) Vyas, D. M.; Chang, Y.; Benigni, D.; Rose, W. C.;
Bradner, W. T.; Doyle, T. W. In Recent Advances in Chemo-
therapy, Anticancer Section 1; Ishigami, J. Ed.; University of
Tokyo: Tokyo, 1985; pp 485–486. (b) Senter, P. D.; Langley,
D. R.; Manger, W. E.; Vyas, D. M. J. Antibiot. 1988, 41, 199.
7. 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 D.C., 1995.
While 2–4 at doses of 125 mg/kg for 2, 85 mg/kg for 3
and 150 mg/kg for 4, proved to be no more effective
than those with paclitaxel (data not shown), the results
obtained with the mutual (i.e., captopril containing)
prodrug 5 were very pronounced (Fig. 1). Prodrug 5
was well tolerated with a 10–20% loss of body weight
even at a dose of 125 mg/kg. In both groups no toxi-
cities were observed. In animals treated with 5 at 125
8. Senter, P. D.; Marquardt, H.; Thomas, B. A.; Hammock,
B. D.; Frank, I. S.; Svensson, H. P. Cancer Res. 1996, 56,
1471.
9. Zhao, Z.; Kingston, D. G. I. J. Nat. Prod. 1991, 54, 1607.
10. Eliel, E. L. In Stereochemistry of Carbon Compounds:
McGraw Hill: New York, 1962: pp 197–202.
11. Volpert, O. V.; Ward, W. F.; Lingen, M. W.; Chesler, L.;
Solt, D. B.; Johnson, M. D.; Molteni, A.; Polverini, P. J.;
Bouck, N. P. J: Clin. Invest. 1996, 98, 671.
12. Singh, G.; Sharma, P. D. Indian J. Pharm. Sci. 1994, 56,
69.
13. Baas, J. L.; Davies-Fidder, A.; Huisman, H. O. Tetra-
hedron 1966, 22, 285.
14. Spectral characteristics of 2: Letter T is used to denote
paclitaxel. MS: [M+H]=1093, [M+NH4+MeCN]=1151.
1
IR (KBr) 532.8 cmꢁ1 (–S–S– stretching). H NMR (CDCl3) d
8.37 (m, Py-H), 8.16–7.31 (5ꢂm, 16H, ArH, PyH,), 7.06–7.03
(m, 2H, NHCO, PyH), 6.28 (s, 1H, T-10), 6.23 (t, 1H, T-13,
J12,13=9.0 Hz), 5.98 (dd, 1H, T-30, JNH,3=9.2Hz, J2,3=3.2
Hz), 5.67 (d, 1H, T-2, J2,3=7.1 Hz), 5.40 (d, 1H, T-20), 4.97
(m, 1H, T-5) 4.97 (m, 1H, T-5), 4.44 (dd, H-7, T-7, J=6.7 Hz,
Figure 1. In vivo evaluation of 5 in athymic nude mice implanted with
L2987 lung carcinoma.

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V. M. Vrudhula et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3591–3594
and 10.9 Hz), 4.32(d, 1H, T-20A, JAB=8.4 Hz), 4.20 (d, 1H,
T-20B), 3.81 (d, 1H, T-3), 2.79 (dt, 1H, S–CH2A-, JAB=4.1,
Jvic=12.3 Hz), 2.58–2.34 (m and s, 7H, T-6A,–S–CH2B–, T-14,
COCH3), 2.22–1.60 (m and 3ꢂs, 13H,–S–C–CH2–, T-6B,
COCH3, OH, T-18 and angular CH3), 1.20 and 1.04 (2ꢂs, 6H,
T-gem di Me), 1.22 (s, 6H, linker gem di Me). Representa-
tive procedure for the synthesis of disulfides. Preparation of
3: A solution of 2 (0.116 g, 0.106 mmol) and b-d-thioglu-
cose sodium salt (76% free SH content by estimation with
Ellman’s reagent, 36 mg) in argon bubbled MeOH (10 mL)
at 0 ꢀC was stirred for 30 min. At the end N-ethylmalei-
mide (14 mg, 0.106 mmol) in MeOH (1 mL) was added.
After 10 min purified by C18 Column chromatography with
a step gradient of MeCN/H2O (2:3) to MeCN/H2O (3:2).
Fractions containing required compound were analyzed on
5 m Brownlee reversed-phase column with MeCN: Sod.phos-
phate buffer (50 mM, pH 3.5, 2:3) at l 230 nm. Fractions
containing pure compound were combined and lyophilized to
give 3 (63 mg, 53% yield).
stretching), 1H NMR (DMSO+D2O) d 7.96–7.17 (6ꢂm,
ArH), 6.27 (s, 1H, T-10), 5.79 (t, 1H, T-13, J12,13=9.0
Hz), 5.55 (d, 1H, T-30, J2,3=9.6 Hz), 5.39 (d, 1H, T-2,
J2,3=7.2Hz), 5.30 (d, 1H, T-2 0), 4.90 (m, 1H, T-5), 4.48
(dd, 1H, T-7, J6,7=4.3 Hz and 9.9 Hz), 4.51–3.95 (m, 3H,
Cys–CH- and T-20), 3.28 (t, 1H, Glu–CH–, Jvic=6.6 Hz),
3.2–2.5 (3ꢂm, Cys–CH2–, Linker–S–CH2–, T-6A), 2.45–1.47
(2ꢂm and 4ꢂs, 16H, 16H, T-14,–CH2C(Me)2CO–, T-18,
2ꢂCOCH3 and angular Me), 1.13–0.98 (4ꢂs, 2ꢂgem di
Me).
Spectral characteristics for 5: MS: [M+H]=1199, [M
+NH4+MeCN]=1257, [M–H]=1198, IR (KBr) 535.1 cmꢁ1
(–S–S– stretching), 1H NMR (CDCl3+D2O) d 8.17–7.31
(5ꢂm, 15H, ArH), 6.28 (s, 1H, T-10), 6.26 (t, 1H, J12,13=9.0
Hz), 6.02(d, 1H, T-3 0, J2,3=3.1 Hz), 5.68 (d, 1H, T-2,
0
J2,3=7.2Hz), 5.43 (d, 1H, T-2 , 4.97 (m, 1H, T-5), 4.98–4.41
(m, 2H, T-5, prolyl–CH–), 4.32(d, 1H, T-20 A, JAB=8.4 Hz),
4.20 (d, 1H, T-20B), 3.81 (d, 1H, T-3), 3.60–3.40 (m, 2H, Pro-
lyl–CH2–NCO), 3.00–2.80 (m, 2H, Prolyl–CH—S–, Prolyl–
CH2A), 2.8–1.6 (5ꢂm and 4ꢂs, 24H, T-6, T-14,–S–S–CH2–
CH2–,Prolyl–CH2B, Prolyl–C–CH2–C, 2ꢂCOCH3, T-18 and
angular CH3), 1.77, 1.66, 1.21 (3ꢂs, 12H, 2ꢂgem diMe), 1.08
(d, 3H, Cys–CH3).
Spectral characteristics of 3: MS=[M
ꢁ1
– H]=1176,
(–S–S– stretching),
[M+NH4]=1195, IR (KBr) 536.2cm
1H NMR (CDCl3) d 8.14–7.29 (5ꢂm, 15H, ArH,), 7.00 (m,
1H, NHCO), 6.31 (s, 1H, T-10), 6.20 (t, 1H, T-13, J12,13=9.0
Spectral characteristics for 6: MS: [M–H]=237. 1H NMR
(DMSO-d6) d 12.20 (s, 1H, COOH), 7.38–7.20 (m, 5H, ArH),
3.71 (s, 2H. PhCH2), 2.33–2.26 (m, 2H, SCH2), 1.74–1.67(m,
2H, CH2COOH), 1.05 (s, 6H, CMe2); 13C NMR (DMSO–d6) d
178.22, 138.64, 128.82, 128.29, 126.70, 41.32, 34.90, 26.15,
24.78.
Spectral characteristics for 7: 1H NMR (CDCl3) d 8.47–8.41
(m, 1H, ArH), 7.74–7.56 (m, 2H, ArH), 7.10–7.02(m, 1H,
ArH), 2.84–2.70 (m, 2H, SCH2), 2.05–1.86 (m, 2H,
CH2COOH), 1.17 (s, 6H, CMe2).
0
Hz), 5.94 (dd, 1H, T-3, JNH, 3 =9.0 Hz, J2,3=3.2Hz), 5.67 (d,
1H, T-2, J2,3=7.2Hz), 5.46 (d, 1H, T-2 ), 4.96 (m, 1H, T-5),
0
4.42(m, 1H, T-7), 4.30 (d, 1H, T-20A, JAB=8.4 Hz), 4.20–
4.16 (m, 2H, T-20B and anomeric H), 3.85–3.0 (4ꢂm, 10H, T-
3, 4ꢂthioglucose-CH, thioglucose–CH2–, and 3ꢂOH), 3.10
(br.s, 1H, OH), 2.71–2.52 (m, 4H, S-CH2–, T-6A, 2.45– 1.80
(2ꢂm, and 3ꢂs, 13H, T-14, 3ꢂMe,–CH2–C(Me)2–CO–, 1.67
(s, 3H, T-18), 1.24–1.12 (4ꢂs, 12H, 2 sets of gem di Me).
Spectral characteristics for 4: MS: [M+H]=1289,
[M+MeCN+H]=1330, IR (KBr) 536.8 cmꢁ1 (–S–S–