Antitumor activity of S-(p-bromobenzyl)glutathione diesters in vitro: A structure-activity study

Article abstract of DOI:10.1021/jm960129c

S-(p-Bromobenzyl)glutathione is a competitive inhibitor of human glyoxalase I which is part of the cytosolic glyoxalase system. It may be delivered into the cytosol of cells by diesterification wherein it is deesterified by cytosolic nonspecific esterases

Full text of DOI:10.1021/jm960129c

J . Med. Chem. 1996, 39, 3409-3411
3409
Notes
An titu m or Activity of S-(p-Br om oben zyl)glu ta th ion e Diester s in Vitr o: A
Str u ctu r e-Activity Stu d y
Paul J . Thornalley,* Muhati J . Ladan, Simon J . S. Ridgway, and Yubin Kang
Department of Biological and Chemical Sciences, University of Essex, Central Campus, Wivenhoe Park,
Colchester, Essex CO4 3SQ, U.K.
Received February 9, 1996X
S-(p-Bromobenzyl)glutathione is a competitive inhibitor of human glyoxalase I which is part
of the cytosolic glyoxalase system. It may be delivered into the cytosol of cells by diesterification
wherein it is deesterified by cytosolic nonspecific esterases. S-(p-Bromobenzyl)glutathione
diesters had antitumor activity in vitro and in vivo. The inhibition of human leukemia 60 cell
growth in vitro by a series of alkyl and cycloalkyl diesters of S-(p-bromobenzyl)glutathione
was investigated. For n-alkyl diesters, the n-propyl diester was the most potent derivative
with a median growth inhibitory concentration GC50 value of 7.77 ( 0.01 µM (N ) 18). The
most potent derivative was S-(p-bromobenzyl)glutathione cyclopentyl diester which had a GC50
value of 4.23 ( 0.01 µM (N ) 21) and also had antitumor activity in vivo.
Ch a r t 1. Ester Derivatives of S-(p-Bromobenzyl)-
In tr od u ction
glutathionea
Inhibition of glyoxalase I (EC 3.2.1.6) was proposed
as a novel strategy for the development of antitumor
agents by Vince and Wadd in 1969.1 The consequent
accumulation of the physiological R-oxoaldehyde me-
tabolite methylglyoxal was expected to lead to antipro-
liferative activity which had previously been found for
high concentrations of exogenous methylglyoxal.2 The
poor efficacy of methylglyoxal was attributed to rapid
detoxification by the glyoxalase system. Glutathione
S-conjugates were evaluated as competitive inhibitors
of glyoxalase I;3 S-(p-bromobenzyl)glutathione (Chart 1)
was the most potent analogue studied3 and had an
inhibition constant Ki value of 0.08 µM4. S-(p-Bro-
mobenzyl)glutathione was not a potent antitumor agent,
however, which has now been attributed to the poor
delivery of this glutathione S-conjugate into cells to
reach the glyoxalase I enzyme target in the cytosol of
cells and extracellular degradation by γ-glutamyl trans-
ferase.5
a
S-(p-Bromobenzyl)glutathione R1 ) R2 ) H; S-(p-bromoben-
zyl)glutathione monoester R1 ) H, R2 ) alkyl, cycloalkyl; S-(p-
bromobenzyl)glutathione diester R1 ) R2 ) alkyl, cycloalkyl.
diester derivatives of the glyoxalase I inhibitor S-(p-
bromobenzyl)glutathione in vitro.
Ch em istr y
S-(p-Bromobenzyl)glutathione diesters were synthe-
sized by acid-catalyzed esterification of S-(p-bromoben-
zyl)glutathione in the appropriate alcohol.7 The ester-
ified product was contaminated with the corresponding
monoester derivative. S-(p-Bromobenzyl)glutathione
diesters were purified by hydrophilic interaction column
chromatography using Dowex-1 anion exchange resin
in the formate form with methanol as eluent: the
diester was eluted shortly after the void volume, well-
resolved from the monoester derivative. A series of
n-alkyl diester derivatives, ethyl, n-propyl, n-butyl, and
n-pentyl diesters; isopropyl diester; and cyclopentyl and
cyclohexyl diesters were prepared; corresponding mo-
noesters were also isolated and characterized. This
series of diesters was chosen following findings of the
delivery of cysteine into tissues in vivo by ester deriva-
tives where cyclopentyl and cyclohexyl esters were found
to be the most effective prodrugs.8
S-(p-Bromobenzyl)glutathione ethyl diester (1) was
developed as a prodrug vehicle for the delivery of S-(p-
bromobenzyl)glutathione into cells. Unlike the ethyl
monoester derivative, it inhibited the growth of human
leukemia 60 (HL-60) cells in vitro.5 Since then, dies-
terification has also been found to be a much more
potent strategy than monoesterification for the delivery
of reduced glutathione into cells.6 Incubation of HL-60
cells with S-(p-bromobenzyl)glutathione cyclopentyl di-
ester lead to the delivery of diester into the cell cytosol
wherein it was hydrolyzed sequentially to S-(p-bro-
mobenzyl)glutathione cyclopentyl monoester and S-(p-
bromobenzyl)glutathione. There was a subsequent
increase in the concentration of methylglyoxal and
induction of apoptosis.7 In this report, we describe a
structure-activity study of the antileukemia activity of
Resu lts a n d Discu ssion
S-(p-Bromobenzyl)glutathione diesters were evalu-
ated for antileukemia activity by determining the
median growth inhibitory concentration (GC50) and
median toxic concentration (TC50) values with HL-60
cells in vitro. For the n-alkyl series of S-(p-bromoben-
* Address correspondence to this author.
X Abstract published in Advance ACS Abstracts, August 1, 1996.
S0022-2623(96)00129-X CCC: $12.00 © 1996 American Chemical Society

3410 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17
Notes
Ta ble 1. Physicochemical and Biological Properties of
S-(p-Bromobenzyl)glutathione Diesters
zyl)glutathione diesters, the propyl diester had the
lowest GC50 and TC50 values. The cyclopentyl diester,
however, was the most potent of the S-(p-bromobenzyl)-
glutathione diesters evaluated with a GC50 value of 4.23
( 0.01 µM (N ) 21) and a TC50 value of 8.86 ( 0.01 µM
(N ) 21). The antitumor activity of S-(p-bromobenzyl)-
glutathione diesters was not limited to activity against
HL-60 cells. For the National Cancer Institute panel
of leukemia, non-small lung cancer, colon cancer, CNS
cancer, melanoma, ovarian cancer, renal cancer, pros-
tate cancer, and breast cancer cell lines, S-(p-bromoben-
zyl)glutathione ethyl diester gave GC50 values in the
range 7-20 µM,11 and S-(p-bromobenzyl)glutathione
cyclopentyl diester had antitumor activity in vivo against
murine adenocarcinoma 15A.7 Incubation of HL-60 cells
with S-(p-bromobenzyl)glutathione cyclopentyl diester
lead to the delivery of diester into the cell cytosol
wherein it was hydrolyzed to S-(p-bromobenzyl)glu-
tathione cyclopentyl monoester and S-(p-bromobenzyl)-
glutathione.7
S-(p-Bromobenzyl)glutathione and S-(p-bromobenzyl)-
glutathione monoesters were inactive (GC50 and TC50
values >300 µM) although some monoester derivatives
were potent inhibitors of human glyoxalase I: the
inhibitor constant Ki value of S-(p-bromobenzyl)glu-
tathione ethyl monoester was 2.36 µM.9 In contrast,
S-(p-bromobenzyl)glutathione ethyl diester was a poor
inhibitor of human glyoxalase I and had a Ki value of
235 µM.9 This is consistent with the diesterification of
S-(p-bromobenzyl)glutathione being important for de-
livery into the cell cytosol, cf. the delivery of GSH into
the cytosol of cells by GSH diester, and also important
to confer resistance to degradation by γ-glutamyl
transpeptidase on the external surface of the plasma
membrane of cells.6
com-
pound R1,R2
yield
mp (°C) (%) GC50 (µM) N TC50 (µM) N
M+1
1
2
3
4
5
6
7
Et
532 & 534 107-109 35
8.3 ( 0.1 12 16.5 ( 0.1 27
n-Pr 560 & 562 118-120 70 7.8 ( 0.1 18 10.3 ( 0.3 18
n-Bu 588 & 590 128-130 75 18.5 ( 1.5 21 26.3 ( 1.0 21
n-Pt 616 & 618 133-135 66 22.2 ( 2.5 12 24.9 ( 1.3 12
i-Pr 560 & 562 120-122 13 19.6 ( 0.1 12 20.9 ( 1.1 12
cPt 612 & 614 136-138 71
cHx 640 & 642 121-123 60 29.2 ( 1.2 21 33.1 ( 4.7 21
4.2 ( 0.1 21 8.9 ( 0.1 21
1640 and fetal calf serum were purchased from Gibco Europe
Ltd. (Paisley, Scotland). Methanol (HPLC grade) was pur-
chased from Rathburn Chemicals Ltd. (Walkerburn, Scotland).
Silica gel 60 F234 TLC plates were purchased from BDH
Chemicals (Poole, Dorset, U.K.). S-(p-Bromobenzyl)glu-
tathione was synthesized by modification of the the method
of Vince et al.3,7 NMR spectra were recorded on a J eol ex270
MHz NMR spectrometer. FAB mass spectra were recorded
on a Kratos MS50 FAB mass spectrometer with compounds
dissolved in a glycerol matrix. S-(p-Bromobenzyl)glutathione
ethyl diester was synthesized as previously described.7
S-(p-Br om oben zyl)glu ta th ion e cyclop en tyl d iester (6).
S-(p-Bromobenzyl)glutathione cyclopentyl diester was pre-
pared and purified by methods similar to those described for
the corresponding ethyl diester except the acid-catalyzed
esterification of S-(p-bromobenzyl)glutathione was performed
in cyclopentanol for 7 days at room temperature. It was
characterized by melting point, FAB mass spectrometry (Table
1
1), H and 13C NMR spectroscopy, TLC, and elemental CHN
analysis: 1H NMR (DMSO-d6) δ 8.54 (t, 1H, J ) 5.86 Hz, glycyl
NH), 8.23 (d, 1H, J ) 7.81 Hz, cysteinyl NH), 7.49 (d, 2H, J )
8.43 Hz, benzyl m-H), 7.28 (d, 2H, J ) 8.43 Hz, benzyl ï-H),
5.06 (m, 2H, cyclopentyl 1-H), 4.54 (m, 1H, cysteinyl 2-H), 3.76
(s, 2H, benzyl 1-H and d, 2H, J ) 5.86 Hz, glycyl 2-H), 3.41 (t,
1H, J ) 5.35 Hz, γ-glutamyl 1-H), 2.74 (q, 1H, J ) 4.94 Hz
and -12.03 Hz, cysteinyl 3A-H), 2.52 (q, 1H, J ) 4.94 Hz and
-12.03 Hz, cysteinyl 3B-H), 2.25 (t, 2H, J ) 8.12 Hz,
γ-glutamyl 4-H), 1.6-1.9 (m, 10H, γ-glutamyl 3-H and cyclo-
pentyl-2,3,4,5-H); 13C NMR (DMSO-d6) δ 174.5 (γ-glutamyl
C-5), 172.3 (γ-glutamyl C-1), 171.1 (cysteinyl C-1), 169.4 (glycyl
C-1), 138.1 (phenyl C-4), 131.4 (phenyl C-3,5), 131.3 (phenyl
C-2,6), 120.1 (phenyl C-1), 77.4 (cyclopentyl(γ-glutamyl) C-1),
77.2 (cyclopentyl(glycyl) C-1), 53.5 (cysteinyl C-2), 51.8 (γ-
glutamyl C-2), 41.3 (glycyl C-2), 34.5 (cysteinyl C-3), 33.2
(benzyl C-1), 32.4 (cyclopentyl(γ-glutamyl) C-2,5), 32.2 (cyclo-
pentyl(glycyl) C-2,5), 31.7 (γ-glutamyl C-4), 29.8 (γ-glutamyl
C-3), 23.4 (cyclopentyl(γ-glutamyl) C-3,4 & cyclopentyl(glycyl)
C-3,4). The TLC Rf value on silica gel was 0.90 (mobile
phase: chloroform, methanol, acetic acid, 8:1:1). Anal.
(C27H40N3O7SBr) C, H, N, for S-(p-bromobenzyl)glutathione
cyclopentyl diester‚H2O.
Precedents for maximal pharmacological response of
the n-propyl and cyclopentyl diesters were in a series
of n-alkyl esters of ibuprofen,10 and the delivery of
cysteine into rat lung tissue by cysteine esters.8 This
ester structure-activity effect is probably due to a
balance of factors influential on the ability of S-(p-
bromobenzyl)glutathione diesters to deliver S-(p-bro-
mobenzyl)glutathione into cells: (i) the ability of the
ester groups to be resistant to cleavage by plasma and
extracellular plasma membrane-bound nonspecific es-
terases, and (ii) the susceptibility of the ester groups to
hydrolysis by nonspecific esterase in the cytosol of the
target cells.
Other S-(p-bromobenzyl)glutathione diesters were prepared,
purified, and characterized similarly.
Evalu ation of An tileu kem ia Activity of S-(p-Br om oben -
zyl)glu ta th ion e Diester s in Vitr o. HL-60 cells were incu-
bated at 37 °C in RPMI 1640 media containing 10% fetal calf
serum under an atmosphere of 5% CO2 in air, 100% humidity.7
Cells were seeded at an initial density of 5 × 104/mL and
incubated with 0.5-300 µM S-(p-bromobenzyl)glutathione
diester for 2 days.7 A stock solution of S-(p-bromobenzyl)-
glutathione diester was prepared in dimethyl sulfoxide and
diluted into the growth medium such that the final concentra-
tion of dimethyl sulfoxide did not exceed 5 mM, a concentration
which did not induce differentiation or toxicity in HL-60 cells.
Cell viability was judged by the ability of cells to exclude
trypan blue. GC50 and TC50 values were determined by logistic
regression of viable cell number and cytotoxicity data on
diester concentration, respectively. Nonlinear regression was
performed using the ENZFITTER program (Biosoft, Cam-
bridge, U.K.).
Con clu sion
S-(p-Bromobenzyl)glutathione diesters, glyoxalase I
inhibitor prodrugs, have antitumor activity in vitro
where a limited ester structure-activity study sug-
gested that n-propyl and cyclopentyl diesters have
potent antitumor activity. Their prospective mechanism
of action is different from current clinical antitumor
agents,7,11 and they are good candidates for further
pharmacological evaluation.
Exp er im en ta l Section
Trypan blue, reduced glutathione, Dowex 1 (chloride form),
and dimethylsulfoxide were purchased from Sigma Chemical
Co. Ltd. (Poole, Dorset, U.K.). p-Bromobenzyl bromide, hy-
drogen chloride gas, trifluoroacetic acid, dimethyl sulfoxide-
d6, and alcohols used were purchased from Aldrich Chemical
Co. Ltd. (Poole, Dorset, U.K.). Tissue culture medium RPMI
Ack n ow led gm en t. M.J .L. was on study leave from
the Department of Biochemistry, Usamanu Danfodiyo

Notes
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17 3411
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(7) Thornalley, P. J .; Edwards, L. G.; Kang, Y.; Wyatt, C.; Davies,
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(8) Hobbs, M. J .; Butterworth, M.; Cohen, G. M.; Upshall, D. G.
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University, Sokoto, Nigeria. This is a contribution from
the Glyoxalase Research Group at the University of
Essex, U.K.
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J M960129C