G. B. Jones et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1987±1989
1989
Scheme 4. Enzymatic activation of Tyr-linker-mechlorethamine conjugates.
a
Table 1. Cytotoxicity of conjugates [IC50
]
PBS (180 mL) and substrate (20 mL) solutions were incu-
bated under identical conditions. p-Aminobenzyl alcohol
was quanti®ed against authentic standards by HPLC
(C18 mBondpak, 1 mL/min, 100% iPrOH, tR=8.2 min).
Entry
Substrate
LNCaP
MCF-7
HEK-293
1
2
3
4
6a
>500 mM Æ 18 >900 mM Æ 15 >600 mM Æ 48
33 mM Æ 3 >900 mM Æ 17 176 mM Æ 14
7a
Mechlorethamineb
7ac
64 mM Æ 5
34 mM Æ 3
70 mM Æ 6
173 mM Æ 15
Acknowledgements
aCells treated in sextuplicate with candidate compounds [72 h] and
growth assessed by monitoring uptake of 3H thymidine. IC50 expres-
sed as concentration [mM] required for 50% reduction in cell growth.
bCH3N(CH2CH2Cl)2.
We thank the American Cancer Society [MA Division],
Schering-Plough Inc., and the Hershey Family Founda-
tion for ®nancial support, and Dr. Walter Samaniego
and Dr. Duckhee Lee for additional experimentation.
cDenotes PSA [1 mg] added to assay medium.
Cytotoxicity studies performed using a PSA secreting
cell line (LNCaP) and non-PSA secreting lines (MCF-7,
HEK-293) clearly demonstrated selective cytotoxicity
for 7a in the PSA rich cells, which may imply that
enzymatic activation is plausible under cellular condi-
tions (Table 1). However, when non-PSA secreting cells
(HEK-293) are `doped' with PSA, the cytotoxicity of 7a
remains unchanged (within standard error), pointing
either to some alternate mode of activation unique to
LNCaP cells, or PSA inactivation on binding to serum
(entry 4). Opportunity to improve the therapeutic index
of these agents exists, as a number of polypeptide
sequences with high anity for PSA have been repor-
ted,5 which could be expected to reduce competing non-
speci®c hydrolysis and improve prodrug solubility under
aqueous conditions. Encouraged by the present ®ndings,
we are currently engaged in preparation of conjugates of
other antitumoral agents including anthracyclines,11
where this activation method, or related ADEPT directed
strategies may prove promising,12 and also enzyme acti-
vated imaging systems, where controlled release may be
useful for mapping of the prostatic microenvironment.13
References and Notes
1. Parker, S. L.; Tong, T.; Bolden, S. Can. Cancer J. Clin.
1996, 46, 5.
2. Bubley, G. J.; Balk, S. P. Hem/Onc Clin. NA. 1996, 10, 713.
3. (a) Kelly, W. K.; Scher, H. I.; Mazumdar, M.; Vlamis, V.;
Schwartz, M.; Fossa, S. D. J. Clin. Oncol. 1993 11, 607. (b)
Bates, S. E. Ann. Intern. Med. 1991 115, 623.
4. Lilja, H.; Abrahamsson, P.; Lundwall, P.-A. J. Biol. Chem.
1989, 264, 1894.
5. Denmeade, S. R.; Nagy, A.; Gao, J.; Lilja, H.; Schally, A.
V.; Isaacs, J. T. Cancer Res. 1998, 58, 2537.
6. Christensson, A.; Laurell, C.; Lilja, H. Eur. J. Biochem.
1990, 194, 755.
7. Carl, P. L.; Chakravarty, P. K.; Katzenellenbogen, J. A. J.
Med. Chem. 1981, 24, 479.
8. Denny, W. A. In Advances in DNA Sequence-Speci®c
Agents; Jones, G. B., Ed.; JAI: Greenwich, 1998; Vol. 3, pp
157±178.
9. (a) Seidman, A. D.; Scher, H. I.; Petrylak, D.; Dershaw, D.
D.; Curley, T. J. Urol. 1992, 147, 931. (b) Teicher, B. A.;
Herman, T.; Tanaka, J.; Eder, J. P.; Holden, S.; Bubley, G.;
Coleman, C. N.; Frei, E. Cancer Res. 1991, 51, 1086.
10. a-Chymotrypsin was also able to cleave substrate 6a but
not 6b, wheras PSA failed to cleave either.
11. Sweatman, T. W.; Israel, M. In Anthracyclines in Cancer
Therapeutics: Experimental and Clinical Agents; Teicher, B.,
Ed.; Humana: Totowa, 1997; pp 113±136.
12. (a) Jungheim, L. N.; Shepherd, T. A. Chem. Rev. 1994, 94,
1553. (b) Alexander, R. P.; Beeley, N. R. A.; O'Driscoll, M.;
O'Neill, F. P.; Millican, T. A.; Pratt, A. J.; Willenbrock, F. W.
Tetrahedron Lett. 1991, 32, 3269.
Representative Enzymatic Conditions
In duplicate, PSA (Cortex Biochem), 20 mL, 1.0 mg/mL,
pH 7.4 0.01 M phosphate buer saline [PBS]) or a-chy-
motrypsin (Sigma, TLCK-treated, 20 mL, 5.0 mg/mL,
pH 7.4 0.01 M PBS), 160 mL PBS, and 20 mL substrate
(1.0 mg/mL; 1:1, EtOH:H2O) were incubated for 12±18 h
at 37 ꢁC. As a control, duplicate reactions containing
13. D'Amico, A. V.; Debruyne, F.; Huland, H.; Richie, J. P.
The Prostate 1999, 41, 208.