260 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1
Wei et al.
6′), 7.57 (dd, J ) 8.2, J′1.6, 2H, Ar-o), 7.45 (d, J ) 8.7, 1H,
O2NArH-5′), 7.41-7.35 (m, 3H, Ar-m, p), 7.26 (broad s,1H, H-9,
exchanges with D2O), 5.59 (s, 2H, O2NArCH2), 5.23 (d, J )
4.6, 1H, OH-2′′, exchanges with D2O), 5.18 (s, 2H, CH2Ar), 5.08
(d, J ) 7.3, 1H, H-1′′), 5.01 (d, J ) 4.5, 1H, OH-3′′), 3.47 (d, J
) 10.0, 1H, H-5′′), 3.26-3.11 (m, 4H, OH-4′′ and H-2′′,3′′,4′′);
MS [LC (0.1% HCOOH)] m/z 613.2 [M(acid form) + H]+, 635.2
[M(acid form) + Na]+; Anal. (C26H23N6NaO12‚2H2O) C, H, N.
O6-Benzyl-N2-[[[[ 4′′-[(â-D-glucopyranuronosyl)oxy]-3′′-
nitrophenyl]methyl]oxy]carbonyl]-2′-deoxyguanosine,
Monosodium Salt (2). Using the above procedure for 1,
compound 2, which eluted from the Sephadex LH-20 column
in fractions 35-48, was obtained in 64% yield. UV [0.05 M
phosphate buffer (pH 7.0)] λmax 267 (ꢀ ) 1.97 × 104 M-1‚cm-1);
1H NMR δH(DMSO-d6) 10.49 (s, 1H, N2H, exchanges with D2O),
8.40 (s, 1H, H-8), 7.97 (d, J ) 2.1, 1H, O2NArH-2′′), 7.73 (dd,
J ) 8.8, J′2.2, 1H, O2NArH-6′′), 7.56 (dd, J ) 8.2, J′1.7, 2H,
Ar-o), 7.48 (d, J ) 8.8, 1H, O2NArH-5′′), 7.41-7.35 (m, 3H,
Ar-m, p), 7.19 (broad, 1H, OH-2′′′, exchanges with D2O), 6.31
(t, J ) 7.1, 1H, H-1′), 5.60 (s, 2H, O2NArCH2), 5.43 (d, J )
3.9, 1H, OH-3′, exchanges in D2O), 5.23 (d, J ) 4.7, 1H, OH-
3′′′, exchanges with D2O), 5.20 (s, 2H, CH2Ar), 5.08 (d, J )
7.3, 1H, H-1′′′), 5.01 (d, J ) 4.8, 1H, OH-4′′′, exchanges with
D2O), 4.89 (t, J ) 5.4, 1H, OH-5′), 4.41-4.39 (m, 1H, H-3′),
3.87-3.84 (m, 1H, H-4′), 3.61-3.50 (m, 2H, H-5′), 3.47 (d, J )
9.9, 1H, H-5′′′), 3.29-3.11 (m, 3H, H-2′′′,3′′′,4′′′), 2.75-2.68 (m,
1H, H-2′R), 2.29-2.23 (m, 1H, H-2′â); MS [LC (H2O/acetoni-
trile)] m/z 727.1[M - Na]-; Anal. (C31H31N6NaO15‚1.5H2O) C,
H, N.
Prodrug Stability and Enzyme Kinetic Analyses. Pro-
drug stability and purity was determined by HPLC on a
Hewlett-Packard LC 1090 Series II system equipped with a
Phenomenex 250 × 4 mm column (5 µm particle size) eluted
isocratically at 1 mL/min with acetonitrile/0.1 M triethylam-
monium acetate (TEAA), pH 7.0, (3:7). Aliquots (100 µL) from
prodrug solutions were withdrawn and diluted with 100 µL of
a solution of p-nitrobenzyl alcohol (an internal standard) in
acetonitrile/0.1 M TEAA (6:4). UV detection was at 254 and
280 nm. Retention times for 1, 2, O6-benzylguanine, O6-benzyl-
2′-deoxyguanosine, and p-nitrobenzyl alcohol were 5.20, 4.97,
7.13, 7.18, and 8.64 min, respectively. All determinations were
carried out in duplicate or triplicate.
ferent concentrations of prodrugs in 0.5 mL of reaction buffer
(50 mM Tris-HCl, pH 7.6, 0.1 mM EDTA, 5.0 mM dithiothrei-
tol) containing 50 µg of hemocyanin for 30 min at 37 °C. For
experiments involving â-glucuronidase, the bovine liver pro-
tein, prodrugs, and alkyltransferase were incubated together
in the above hemocyanin-containing buffer for 30 min at 37
°C. The remaining alkyltransferase activity was determined
after incubation with a [3H]-methylated calf thymus DNA
substrate for 30 min at 37 °C by measuring the [3H]-
methylated protein formed, which was collected on nitrocel-
lulose filters. The results were expressed as the percentage of
the alkyltransferase activity remaining. The concentration of
inhibitor that led to a 50% loss of alkyltransferase activity
(ED50) was calculated from graphs of the percentage of
remaining alkyltransferase activity against inactivator con-
centration.
Cell Culture Cytotoxicity Assay. HT29 cells were grown
in RPMI 1640 medium in the presence of 10% fetal bovine
serum. The effect of alkyltransferase inactivators on the
sensitivity of cells to BCNU was determined using a colony-
forming assay. Cells were plated at a density of 106 in 25 cm2
flasks and 24 h later were incubated with different concentra-
tions of prodrugs for the time indicated before exposure to 40
mM BCNU for 2 h. For experiments involving â-glucuronidase,
the bovine liver protein was added to the cell cultures at 20
units/mL of medium and incubated along with the prodrug.
BCNU was dissolved in absolute ethanol at a concentration
of 8 mM. It was diluted with the same volume of ice-cold
phosphate-buffered saline and was immediately administered
to cells. After 2 h, the medium was replaced with fresh medium
and the cells were left to grow for an additional 16-18 h. The
cells were then replated at densities of 250 cells per 25 cm2
flask and grown for 8 days until discrete colonies had formed.
The colonies were washed with 0.9% saline solution, stained
with 0.5% crystal violet in ethanol, and counted. The plating
efficiency of cells not treated with drugs was about 50%.
References
(1) Sinkula, A. A.; Yalkowsky, S. H. Rationale for design of biologi-
cally reversible drug derivatives: prodrugs. J. Pharm. Sci. 1975,
64, 181-210.
(2) Stella, V. J.; Himmelstein, K. J. Prodrugs and site-specific drug
delivery. J. Med. Chem. 1980, 23, 1276-1282.
(3) Bagshawe, K. D. Antibody directed enzymes revive anticancer
prodrugs concept. Br. J. Cancer 1987, 56, 531-532.
(4) Bagshawe, K. D.; Springer, C. J.; Searle, F.; Antoniw, P.;
Sharma, S. K.; Melton, R. G.; Sherwood, R. F. A cytotoxic agent
can be generated selectively at cancer sites. Br. J. Cancer 1988,
58, 700-703.
(5) Bosslet, K.; Czech, J.; Hoffmann, D. A novel one-step tumor-
selective prodrug activation system. Tumor Targeting 1995, 1,
45-50.
The stability of prodrugs was determined in phosphate-
buffered saline (pH 7.2) (Life Technologie, Inc), a Tris buffer
containing 50 mM Tris-HCl (pH 7.5) (Life Technologies, Inc),
5 mM dithiothreitol, and 0.1 mM EDTA, a MOPS buffer (pH
7.0) containing 50 mM morpholinopropane sulfonic acid, 0.01%
bovine serum albumin, and 0.01% NaCl, and a modified
Dulbecco’s medium prepared by combining 400 mL of Dulbec-
co’s medium with 7 mL of 7.5% NaHCO3, 4 mL of 15 mM
glutamine, 2 mL of gentamicin (10 mg/mL), and 40 mL of fetal
calf serum. Solutions were incubated at 37 °C, and prodrug
concentrations were analyzed by HPLC at various times as
indicated above.
(6) de Groot, F. M. H.; Damen, E. W. P.; Scheeren, H. W. Anticancer
prodrugs for application in monotherapy: Targeting hypoxia,
tumor-associated enzymes, and receptors. Curr. Med. Chem.
2001, 8, 1093-1122.
Enzymatic Cleavage by â-Glucuronidase. Prodrugs and
p-nitrobenzyl alcohol (HPLC internal standard) were dissolved
in the MOPS buffer (pH 7.0) at 37 °C. Enzymatic cleavage was
initiated by adding 0.2 Fishman units of E. coli â-glucu-
ronidase (Sigma type IX-A) (42.8 units/mg of protein) or 10
Fishman units of bovine liver â-glucuronidase (Sigma type
B-10) (10.2 units/mg of protein) to the incubation buffer.
Aliquots (100 µL) of the reaction mixture were withdrawn at
various times and were mixed with 100 µL of acetonitrile/0.1
M TEAA (6:4) to quench the enzymatic reaction. Prodrug and
product concentrations were determined by HPLC. For deter-
mination of enzyme kinetic parameters, prodrug solutions at
concentrations between 5 and 300 µM were incubated with a
fixed amount of enzyme. Aliquots were withdrawn at time
intervals varying from 1 to 20 min and the reactions were
quenched as described above. Initial reaction velocities were
determined at each substrate concentration. Nonlinear regres-
sion methods were used to determine KM and Vmax values. Data
were processed with Prism 3.0 software.
(7) Bosslet, K.; Straub, R.; Blumrich, M.; Czech, J.; Gerken, M.;
Sperker, B.; Kroemer, H. K.; Gesson, J.-P.; Koch, M.; Monneret,
C. Elucidation of the mechanism enabling tumor selective
prodrug monotherapy. Cancer Res. 1998, 58, 1195-1201.
(8) de Graaf, M.; Boven, E.; Scheeren, H. W.; Haisma, H. J.; Pinedo,
H. M. Beta-glucuronidase-mediated drug release. Curr. Pharm.
Des. 2002, 8, 1391-1403.
(9) Leenders, R. G. G.; Damen, E. W. P.; Bijsterveld, E. J. A.;
Scheeren, H. W.; Houba, P. H. J.; van der Meulen-Muileman, I.
H.; Boven, E.; Hidde, J. H. Novel anthracycline-spacer-beta-
glucuronide, -beta-glucoside, and -beta-galactoside prodrugs for
application in selective chemotherapy. Bioorg. Med. Chem. 1999,
7, 1597-1610.
(10) Houba, P. H. J.; Boven, E.; van der Meulen-Muileman, I. H.;
Leenders, R. G. G.; Scheeren, J. W.; Pinedo, H. M.; Haisma, H.
J. A novel doxorubicin-glucuronide prodrug DOX-GA3 for tu-
mour-selective chemotherapy: distribution and efficacy in ex-
perimental human ovarian cancer. Br. J. Cancer 2001, 84, 550-
557.
(11) Schmidt, F.; Florent, J. C.; Monneret, C.; Straub, R.; Czech, J.;
Gerken, M.; Bosslet, K. Glucuronide prodrugs of hydroxy
compounds for antibody directed enzyme prodrug therapy
(ADEPT): A phenol nitrogen mustard carbamate. Bioorg. Med.
Chem. Lett. 1997, 7, 1071-1076.
In Vitro Alkyltransferase Activity Assay. Purified re-
combinant human alkyltransferase was incubated with dif-