Design, synthesis, and evaluation of latent alkylating agents activated by glutathione S-transferase

Article abstract of DOI:10.1021/jm950005k

In search of compounds with improved specificity for targeting the important cancer-associated P1-1 glutathione S-transferase (GST) isozyme, new analogs 4 and 5 of the previously reported glutathione S-transferase (GST)- activated latent alkylating agent γ-glutamyl-α-amino-β-[[[2[[bis[bis(2- chloroethyl)amino]phosphoryl]oxy]ethyl]sulfonyl]propionyl]-(R)-(-)- phenylglycine (3) have been designed, synthesized, and evaluated. One of the diastereomers of 4 exhibited good selectivity for GST P1-1. The tetrabromo analog 5 of the tetrachloro compound 3 maintained its specificity and was found to be more readily activated by GSTs than 3. The GST activation concept was further broadened through design, synthesis, and evaluation of a novel latent urethane mustard 8 and its diethyl ester 9. Interestingly, 8 showed very good specificity for P1-1 GST. Cell culture studies were carried out on 4, 5, 8, and 9 using cell lines engineered to have varying levels of GST P1- 1 isozyme. New analogs 4 and 5 exhibited increased toxicity to cell lines with overexpressed GST P1-1 isozyme. The urethane mustard 8 and its diethyl ester 9 were found to be not as toxic. However, they too exhibited more toxicity to a cell line engineered to have elevated P1-1 levels, which was in agreement with the observed in vitro specificity of 8 for P1-1 GST isozyme. Mechanistic studies on alkaline as well as enzyme-catalyzed decomposition of latent mustard 3 provided experimental proof for the hypothesis that 3 breaks down into an active phosphoramidate mustard and a reactive vinyl sulfone. The alkylating nature of the decomposition products was further demonstrated by trapping those transient species as relatively stable diethyldithiocarbamic acid adducts. These results substantially extend previous efforts to develop drugs targeting GST and provide a paradigm for development of other latent drugs.

Full text of DOI:10.1021/jm950005k

1736
J . Med. Chem. 1996, 39, 1736-1747
Design , Syn th esis, a n d Eva lu a tion of La ten t Alk yla tin g Agen ts Activa ted by
Glu ta th ion e S-Tr a n sfer a se
Apparao Satyam,* Michael D. Hocker, Kim A. Kane-Maguire, Amy S. Morgan, Hugo O. Villar, and
Matthew H. Lyttle
Terrapin Technologies, Inc., 750-H Gateway Boulevard, South San Francisco, California 94080
Received J anuary 6, 1996X
In search of compounds with improved specificity for targeting the important cancer-associated
P1-1 glutathione S-transferase (GST) isozyme, new analogs 4 and 5 of the previously reported
glutathione S-transferase (GST)-activated latent alkylating agent γ-glutamyl-R-amino-â-[[[2-
[[bis[bis(2-chloroethyl)amino]phosphoryl]oxy]ethyl]sulfonyl]propionyl]-(R)-(-)-phenylglycine (3)
have been designed, synthesized, and evaluated. One of the diastereomers of 4 exhibited good
selectivity for GST P1-1. The tetrabromo analog 5 of the tetrachloro compound 3 maintained
its specificity and was found to be more readily activated by GSTs than 3. The GST activation
concept was further broadened through design, synthesis, and evaluation of a novel latent
urethane mustard 8 and its diethyl ester 9. Interestingly, 8 showed very good specificity for
P1-1 GST. Cell culture studies were carried out on 4, 5, 8, and 9 using cell lines engineered
to have varying levels of GST P1-1 isozyme. New analogs 4 and 5 exhibited increased toxicity
to cell lines with overexpressed GST P1-1 isozyme. The urethane mustard 8 and its diethyl
ester 9 were found to be not as toxic. However, they too exhibited more toxicity to a cell line
engineered to have elevated P1-1 levels, which was in agreement with the observed in vitro
specificity of 8 for P1-1 GST isozyme. Mechanistic studies on alkaline as well as enzyme-
catalyzed decomposition of latent mustard 3 provided experimental proof for the hypothesis
that 3 breaks down into an active phosphoramidate mustard and a reactive vinyl sulfone. The
alkylating nature of the decomposition products was further demonstrated by trapping those
transient species as relatively stable diethyldithiocarbamic acid adducts. These results
substantially extend previous efforts to develop drugs targeting GST and provide a paradigm
for development of other latent drugs.
In tr od u ction
isozymes. The second way, on which this article is
based, involves the use of an appropriately functional-
ized GST analog that would serve both as a modified
GSH substrate and a latent alkylating agent. In the
past, on the basis of this latter concept, we designed and
successfully demonstrated11 that the glutathione-linked
nitrogen mustards γ-glutamyl-R-amino-â-[[[2-[[bis[bis-
(2-chloroethyl)amino]phosphoryl]oxy]ethyl]sulfonyl]-
propionyl]glycine (2) and γ-glutamyl-R-amino-â-[[[2-
[[bis[bis(2-chloroethyl)amino]phosphoryl]oxy]ethyl]-
sulfonyl]propionyl]-(R)-(-)-phenylglycine (3) are cleaved
by physiological concentrations of GSTs at physiological
temperature and pH to release a toxic phosphorodi-
amidate mustard 7b (Scheme 1). As an extension of
this work, we designed, synthesized, and evaluated two
new compounds 4 and 5 (Scheme 1) which provide more
GST P1-1 isozyme selectivity. More importantly, be-
cause of its potential therapeutic importance, we suc-
ceeded in broadening the concept further by the design,
synthesis, and evaluation of novel GST P1-1 selective
urethane mustards 8 and 9 (Scheme 2), enabling the
concept to be applied to a much wider range of toxic
moieties than the phosphorodiamidate mustards previ-
ously reported.
A major cellular defense mechanism utilizes a trip-
eptide, glutathione (γ-glutamylcysteinylglycine, GSH),
which acts as a scavenger molecule able to couple to and
neutralize many toxic electrophiles, including chemo-
therapeutic agents. Such coupling is mediated by a
family of enzymes named glutathione S-transferases
(GSTs) (EC 2.5.1.18). At least 10 different types of
human GSTs have been identified from four different
isozyme families named A, M, P, and T. It has been
observed that many types of cancer tissue often have
elevated levels of GSTs compared to corresponding
healthy tissue.1 There are a few reports demonstrating
the association of GSTs with resistance to alkylating
agents, a major class of anticancer drugs.2-4 A chemo-
therapeutic agent that takes advantage of this intrinsic
property of many types of cancer cells may prove to be
a valuable anticancer drug.
Interestingly, different cancers show different GST
isozyme distribution, but it is most often the P1-1
isozyme that is elevated to the greatest extent in many
types of cancer tissue.5,6 As a result, an isozyme
selective drug can be targeted to a specific cancer tissue
on the basis of its GST isozyme distribution profile. In
principle, targeting of GSTs can be accomplished in two
ways. The first way is to develop isozyme specific/
selective GST inhibitors. Several laboratories,7-9 in-
cluding ours,10 have reported the synthesis and evalu-
ation of a variety of GSH analogs as GST inhibitors,
which exhibited varying affinities toward different GST
Design
(a ) P h osp h or od ia m id a te Mu sta r d s. In all the
structures generated to date for the different GST
families,12-15 a Tyr residue is present proximal to the
S atom of the Cys in glutathione. It has been speculated
that the Tyr is in the phenoxide form and that its
function is to facilitate the deprotonation of the sulfhy-
X Abstract published in Advance ACS Abstracts, March 15, 1996.
0022-2623/96/1839-1736$12.00/0 © 1996 American Chemical Society

Latent Alkylating Agents
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8 1737
Sch em e 1. Proposed Mechanism of GST-Mediated Cleavage of 1-5
Sch em e 2. Proposed Mechanism of GST-Mediated
Cleavage of 8 and 9
dryl group in GSH, making it able to react with
electrophilic species.16 By taking advantage of this
active-site geometry, we previously designed compounds
1-3 (Scheme 1) where an active methylene group was
placed adjacent to the glutathione sulfur in such a way
that the Tyr phenoxide (Tyr7 in the case of GST P class
and Tyr6 in case of GST M class) would be able to
abstract one of those acidic methylene protons. Such a
deprotonation should trigger a â-elimination, resulting
in the cleavage of the phosphorodiamidate moiety 7.11
To lend additional support to this hypothesis, a
computer model of 3 (TER-286) at the active site of GST
P1-1 isozyme was generated (Figure 1). The model
shows that it is possible to accommodate 3 in the active
site of this enzyme in such a way that the Tyr7 is close
to the active methylene protons adjacent to the sulfone
group in 3.
Design of 3 was based on earlier work from several
laboratories,7-9 but primarily from our laboratory,10
where the C-terminal (R)-(-)-phenylglycine GSH ana-
logs were discovered to exhibit a high degree of potency
and specificity in inhibition of P1-1 GST isozyme.
Interestingly, these GSH variants exhibited different
isozyme selectivity in the context of the latent alkylating
agent: 1 and 2 were M1a-1a selective and 3 was equally
selective to both P1-1 and A1-1 GST isozymes. As
expected, only 2 and 3 were cytotoxic. The placebo
compound γ-glutamyl-R-amino-â-[[[2-[[bis(diethylami-
no)phosphoryl]oxy]ethyl]sulfonyl]propionyl]glycine (1),
which contains the diethylamine group instead of bis-
(2-chloroethyl)amine, was found to be noncytotoxic
although as readily cleaved by GST as its toxic analogs,
thereby suggesting that the cytotoxicity is due to the
liberation of chlorinated species 7b. The human breast

1738 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8
Satyam et al.
glutamyl-S-benzylcysteinyl-(R)-(-)-phenylglycine dieth-
yl ester)10 has been shown in preclinical (in vivo) studies
not only to potentiate cytotoxicity of common therapau-
tic drug19 but also to accelerate the recovery of bone
marrow cells, as well as circulating neutrophils and
platelets, following their suppression by cancer chemo-
therapy.18 Interestingly, compound 3 as well as one of
its decomposition products (see Scheme 1) bear struc-
tural similarities (same peptide backbone) to TER-199
and may therefore have beneficial effects with regard
to myelostimulation.
Since GST P1-1 is elevated in many types of cancer
cells, we continued our efforts to design more P1-1
selective analogs and synthesized compounds 4 and 5
(Schemes 1, 3, and 4). Compound 4 differs from
M1a-1a specific 2 by having a phenyl group on the
methylene group adjacent to the sulfone. The design
of 4 was based on our speculation that introduction of
a bulky phenyl group at the sulfone R-carbon would
change its selectivity from M1a-1a to either A1-1 or P1-1
GST isozymes. With a desire to enhance the toxicity of
3, we also synthesized and evaluated its tetrabromo
analog 5 (Scheme 1 and 4).
(b) Ur eth a n e Mu sta r d . Introduction of a urethane
protecting group on the toxic bis(2-chloroethyl)amine
should deactivate its alkylating ability at physiological
pH and temperatures. By combining this attractive
feature with our GST activation concept, we designed
a novel urethane mustard 8 and its diethyl ester 9
containing a latent alkylating group (Scheme 2). It was
anticipated that 8 or 9, upon GST-assisted proton
abstraction, would trigger a â-elimination with concomi-
tant decarboxylation to release a toxic nornitrogen
mustard, bis(2-chloroethyl)amine (10)20 (Scheme 2). A
literature survey revealed that there are at least two
examples that utilized a similar concept. The first
F igu r e 1. Schematic two-dimensional representation of 3
(TER-286) at the active site of the GST P1-1 isozyme. Indicated
are the potential hydrogen bond sites as well as the distance
between the the phenoxide oxygen of Tyr7 of the enzyme and
the methylene group adjacent to the sulfone group in 3.
tumor cell line MCF-7, transfected with GST P1-1 gene,
showed 2-fold increased sensitivity to 3 as compared to
2, which correlated well with the increased cleavage
rates observed with P1-1 GST in vitro. 3 not only
appears to be effective against cancer in a variety of
animal models but also seems to be minimally toxic to
bone marrow GM progenitor cells.17,18 It is of interest
to note that a GST P1-1 selective inhibitor TER-199 (γ-
Sch em e 3. Synthesis of Compound 4, the R-Phenyl Analog of 3a
a
(a) BH3‚THF, THF; (b) bromotrimethylsilane, CH3CN; (c) POCl3, bis(2-chloroethyl)amine hydrochloride (14), Et3N, CH2Cl2; (d) 14,
Et3N, CH2Cl2 or EtOAc; (e) glutathione (17), NaOH, H2O, EtOH, CH3CN, pH 9-10; (f) AcOH, aqueous H2O2, CH3CO3H.

Latent Alkylating Agents
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8 1739
Sch em e 4. Synthesis of Compound 5, the Tetrabromo
Analog of 3a
may be activated in a very specific, disease related,
fashion in sharp contrast to prodrugs which are acti-
vated by mechanisms that are unrelated to the disease
they are intended to treat.
Ch em istr y23
The compound 4 was synthesized as outlined in
Scheme 3. 2-Bromophenethyl alcohol (13) was synthe-
sized either by reduction of R-bromophenylacetic acid
(11) or by treatment of styrene oxide (12) with bromo-
trimethylsilane. In the latter method, the major prod-
uct was a polymer that was formed by the acid-catalyzed
self-condensation of the initially formed 13. As a result,
13 was subsequently prepared only by the first condi-
tion. Compound 16 was synthesized by treating 13 with
phosphorus oxychloride and bis(2-chloroethyl)amine
hydrochloride (14) in the presence of triethylamine. The
major product obtained from this preparation was
identified as 15, but this could be converted to 16 by
treatment with excess 14 and triethylamine. The
sulfide 18 was synthesized by treating the bromide 16
with glutathione (17) at pH 9-10. Oxidation of sulfide
18 by treating with hydrogen peroxide and peracetic
acid yielded the sulfone 4. As expected, the sulfide 18
as well as the sulfone 4 were obtained as mixtures of
diastereomers.24 Our attempts to separate these dia-
stereomers at the sulfide stage were unsuccessful.
However, with some difficulty, we separated the dia-
stereomeric sulfones 4A24 and 4B24 by HPLC.
a
(a) LiBr, acetone/acetonitrile.
The tetrabromo derivative 5 was synthesized by
treating the corresponding tetrachloro derivative 3 with
lithium bromide as shown in Scheme 4, and the reaction
progress was monitored by analytical HPLC. The
reaction did not go to completion even after 46 h reaction
time, and the mixture at that stage indicated the
presence of varying amounts of monobromo, dibromo,
and tribromo derivatives, along with a major amount
(>50%) of tetrabromo derivative 5. The reaction was
stopped at 46 h reaction time because of the progressive
appearance of other side products, and the compound 5
was isolated from the mixture by HPLC.
Synthesis of urethane mustards 8 and 9 was achieved
as shown in Scheme 5. Treatment of 2-bromoethyl
chloroformate (19) with 14 in the presence of triethyl-
amine yielded the bromide 20. Reaction of glutathione
(17) with 20 at pH 9-10 gave glutathione conjugate 21.
Oxidation of the sulfide 21 with hydrogen peroxide and
peracetic acid yielded the sulfone 8. Esterification of
the diacid 8 with thionyl chloride in the presence of
ethanol yielded the corresponding diethyl ester hydro-
chloride 9.
F igu r e 2. Structures of mitomycin C prodrug I, and dyne-
mycin A analog prodrug II; note that prodrug activation
mechanism by 1,6-elimination/â-elimination is illustrated.
example was the prodrug I21 (Figure 2), where an
anticancer drug mitomycin C was masked by an ap-
propriately substituted benzyl carbamate disulfide.
Upon treatment with a thiol, a thiophenoxide was
generated that triggered 1,6-elimination, followed by
decarboxylation to release the cytotoxic free mytomycin
C. Surprisingly, in vitro studies showed that the
prodrug I was 40-70-fold more toxic than mitomycin
C. The authors speculated that the enhanced cytotox-
icity of the prodrug I could be due to its lipophilic
character and the ease with which it can penetrate into
the cell and undergo subsequent fragmentation. The
second example was the compound II22 (Figure 2), where
an analog of the enediyne anticancer drug dynemycin
A was masked by a phenyl sulfone ethylene carbamate.
Upon treatment with a base, a â-elimination with
concomitant decarboxylation released the free dynemy-
cin A analog. These examples clearly demonstrate the
potential utility of a urethane mustard bridge and could
be conveniently extended to the targeted and controlled
delivery of other amine-containing anticancer drugs
when it is beneficial to mask the reactive amino function
of the drug during its transportation. Such latent drugs
Resu lts a n d Discu ssion
In Vitr o Resu lts. Compounds 4, 4A, 4B, 5, and 8
were individually treated with physiological concentra-
tions (usually 0.003-0.006 mM) of recombinant human
A1-1, M1a-1a, and P1-1 GST isozymes at physiologically
relevant pH (7.1-7.3) and temperature (37 °C). Control
experiments without GSTs were run simultaneously.
Aliquots of the reaction mixture were taken out at
specific intervals and quenched with acetic acid to stop
further decomposition, and the amount of cleavage was
assayed by reversed-phase HPLC. The rates of decom-
position were calculated by measuring the ratio of peak
heights of test compound to a nonreactive internal

1740 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8
Sch em e 5. Synthesis of Urethane Mustards 8 and 9a
Satyam et al.
a
(a) Bis(2-chloroethyl)amine hydrochloride (14), Et3N, CH2Cl2; (b) glutathione (17), NaOH, H2O, EtOH, CH3CN, pH 9-10; (c) AcOH,
aqueous H2O2, CH3CO3H; (d) SOCl2, EtOH.
F igu r e 4. Differential cleavage of 5 by a panel of GST A1-1,
M1a-1a, and P1-1 isozymes; note that complete cleavage of 5
had occurred in <50 min by both GST A1-1 and P1-1 isozymes.
accelerated decomposition of 4A about 100 times faster
than background, 80 times faster than A1-1, and about
11 times faster than M1a-1a (Figure 3). Contrarily,
when the diastereomer 4B was subjected to activation
F igu r e 3. GST P1-1 catalyzed decomposition of 4 and
by GST P1-1, only about 20% decomposition was ob-
served in 1 h (data not shown). Compound 5, which is
a tetrabromo analog of P1-1 and A1-1 specific 3, was
similarly subjected to the panel of three GST isozymes.
5 was found to be very reactive and was completely
cleaved by P1-1 and A1-1 in about 45 and 30 min,
respectively (Figure 4). In contrast, only 35-40% of 3
was cleaved by P1-1 and A1-1 in 45 min.11 The most
isozyme selective result was obtained from the in vitro
GST assay of latent urethane mustard 8. As shown in
Figure 5, 8 exhibited very good activation by GST P1-1
isozyme and almost negligible activation by A1-1 and
M1a-1a isozymes.
differential cleavage of 4A by a panel of GST A1-1, M1a-1a,
and P1-1 isozymes; note that >50% of decomposition of 4 had
occurred in first 20 min, but it took nearly 7 h to achieve an
additional 40% decomposition. Also note that complete decom-
position of 4A had occurred in 20 min with GST P1-1 isozyme.
standard (an oligopeptide DQQNAFYEILHOPN-NH2)
at specific time intervals, and the graphs were drawn
accordingly. Interestingly, as shown in Figure 3, when
a diastereomeric mixture of 4 was treated with GST
P1-1 isozyme, it followed a decomposition pattern that
is suggestive of a condition where only one of the
diastereomers of 4 is preferentially and selectively
cleaved faster by the isozyme. To confirm this interest-
ing feature, we separated the diastereomer 4A by a
preparative HPLC and subjected it to the panel of GSTs.
As anticipated, the rate of decomposition of 4A by GST
P1-1 isozyme was dramatic with 100% decomposition
in just 20 min (see Figure 3). Interestingly, P1-1
Cell Cu ltu r e Resu lts. The toxicity of compounds 3
(TER-286)11 (included for comparison), 4A (TER-296),
5 (TER-338), 8 (TER-322), and 9 (TER-324) was deter-
mined in MCF-7 cell lines transfected with either a P1-
1-containing vector (pi) or a control (neo) vector. Trans-

Latent Alkylating Agents
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8 1741
rigorous statistical analysis such as nonparametric
determinations show such results to be highly signifi-
cant. Among the test compounds, 3, 4A, and 5 were
the most toxic while 8 and 9 were the least toxic to
either cell line. One explanation for the disparity
between in vitro enzymatic and cell culture results may
be the relative ability of the different compounds enter-
ing the cell. Because of the presence of a hydrophobic
phenyl ring in compounds 3, 4, and 5, they may be
entering cells more readily than 8 or 9. It has been
reported that glutathione diethyl esters can easily
penetrate cellular membranes and be hydrolyzed enzy-
matically to glutathione once inside the cell.19,30 Com-
pound 8 was significantly less toxic in the cell culture
model than could be anticipated from its in vitro
specificity for GST P1-1 isozyme. This led us to examine
its diethyl ester form (compound 9) to see if it can
exhibit improved toxicity. Compound 9 exhibited a 30%
increase in toxicity in the cell culture model compared
to compound 8 (Table 1), which was encouraging but
not statistically significant. Since the differential toxic-
ity between pi and neo transfects was maintained for 8
and 9, the increased toxicity to pi transfects is unlikely
to be a function of the cell’s ability to internalize the
compounds.
F igu r e 5. Differential cleavage of 8 by a panel of GST A1-1,
M1a-1a, and P1-1 isozymes; note that only GST P1-1 isozyme
could catalyze/accelerate the decomposition of 8.
Ta ble 1. Cytotoxicity Data for 3, 4, 5, 8, and 9
IC50, µMa
5c
treatment
time, min
cell line
3b
4Ac
8c,d
9b,d
MCF-7 neo
120
39.9
21.3
16.1
132.8
102.1
Mech a n istic Stu d ies. Some experimental proof of
the GST activation concept was presented in our
preliminary communication11 where the compounds 1
and 2 were individually subjected to both base-(chemi-
cal) and enzyme-catalyzed decomposition and the de-
composition products identified by mass spectrometry.
1 and 2 gave mass peaks attributable to vinyl sulfone
6a (m/z 366 amu) and their respective phosphorodia-
midate 7a (m/z 231 amu, MNa+), and the monoaziri-
dinium (m/z 275 amu) and diaziridinium (m/z 333 amu,
MNa+) derivatives of 7b (Scheme 1). Additionally, in a
separate experiment, 1 was subjected to a base-
catalyzed (chemical) decomposition and the decomposi-
tion product 7a was isolated and characterized (1H-
NMR and mass spectrometry). The vinyl sulfone 6a
could not be isolated in pure form probably because of
its inherent reactivity/instability. Control experiments
in which other proteins, such as ovalbumin and human
serum albumin (HSA), showed no activity combined
with the GST isozyme selectivity for decomposition of
test compounds not only supported the proposed mech-
anism but also ruled out the possibility of a nonspecific
protein-dependent degradation.
(4.4) (7.8) (2.7) (29.5) (10.8)
MCF-7 pi
120
14.4
13.5
11.4
85.8
(8.5)
68.8
(6.7)
(3.0) (5.1) (1.8)
a
Value is the mean of four or five experiments. Value in
b
parentheses is the sem. Difference between MCF-7 neo and
MCF-7 pi is statistically significant, p < 0.05. c Difference between
d
MCF-7 neo and MCF-7 pi is not statistically significant. Differ-
ence between 8 and 9 is not statistically significant.
fection with P1-1 resulted in approximately 4-fold
increase in P1-1 protein levels (2.5 µg/mg cytosolic
protein) compared to the neo transfect (0.6 µg/mg/
cytosolic protein).25 While it was too low for accurate
determination of the GST activity in MCF-7 neo cells,
previously published levels were approximately 7 nmol/
min per mg of protein; MCF-7 pi total GST π activity
was reported to be approximately 90 nmol/min per mg
protein.26 Determination of GST pi activities in MCF-7
pi and MCF-7 neo cell lines was carried out again in
our laboratory using the CDNB method.27 Here again,
the GST pi activity in MCF-7 neo cells was too low to
be accurately measured and seemed to be less than 20
nmol/min per mg of protein. The GST pi activity in
MCF-7 pi cells was about 176 nmol/min per mg of
protein.25 Glutathione (GSH) levels were not signifi-
cantly different between MCF-7 neo and MCF-7 pi28 and
were similar to other MCF-7 cell lines transfected with
µ or R GSTs (approximately 120 nmol/mg protein).29
This rules out the possibility that the different sensitivi-
ties exhibited by neo and pi transfects to the test
compounds is based on their GSH content. As shown
in Table 1, all of the test compounds were found to be
more toxic in the pi-containing MCF-7 cells than in the
neo-containing MCF-7s, which was in good agreement
with their in vitro GST P1-1 selectivities. The dif-
ferential toxicity between MCF-7 neo and MCF-7 pi was
statistically significant for 3 and 9, but not for 4A, 5,
and 8. Lack of statistically significant differential
toxicity for compounds 4A, 5, and 8 was due to inter-
experimental variation since these compounds were
more toxic to MCF-7 pi cell lines in all experiments. Less
We present here additional experimental proof of the
concept. Our main aim during the present study was
to probe whether the anticipated decomposition prod-
ucts can be trapped as relatively stable diethyl dithio-
carbamate adducts.31 Experiments were designed to
provide proof of not only the decomposition pattern but
also the alkylating ability of the decomposition products.
We have selected compound 3 (TER-286) for this
important study because it has been the most exten-
sively studied member of the family.17,18 In a separate
experiment, as shown in Scheme 6, 3 was first subjected
to a base-catalyzed chemical decomposition in the
presence of diethyldithiocarbamic acid (DTC) at pH 11-
12 while monitoring the reaction by HPLC. 3 decom-
posed completely within 1 h. Purification of the mixture
by HPLC led to the isolation of the vinyl sulfone-DTC
adduct 22 (tR ) 17.23 min, m/z 591 amu, MH+) and an

1742 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8
Satyam et al.
Sch em e 6. Base- (Chemical) and GST P1-1 Isozyme-Catalyzed Decomposition of 3 (TER 286) in the Presence of
Diethyldithiocarbamic Acid (DTC), Trapping Unstable Decomposition Products 6b and 7b as Stable DTC Adducts 22,
24, and 25
a
(A) Acetonitrile/water (3:1), diethyldithiocarbamic acid sodium salt (DTC), 1 N Na2CO3 to pH 11-12; (B) acetonitrile/water (3:1),
DTC, 1 N HCl to pH 7.2; (C) DTC, 0.0096 mM GST P1-1, phosphate buffer, pH 7.2.
impure fraction containing some amount each of phos-
phoramidate 24 and diethylamine-DTC adduct 25
(based on mass spectrometry; see the Experimental
Section). Obviously, 24 and 25 must have formed from
the probably unstable phosphorodiamidate-DTC ad-
duct 23.32 Further structural proof of 22 was estab-
without DTC and without GST P1-1; c, with both DTC
and GST P1-1 (Scheme 6); and d, without DTC and with
GST P1-1.11 As anticipated, only experiments c and d
containing GST P1-1 isozyme exhibited decomposition
of 3 roughly 3-4 times faster than control (experiments
a and b) (see Figure 6). Similarly, as shown in Figure
6, generation of vinyl sulfone-DTC adduct 22 in experi-
ment c was roughly 3 times faster than that in control
(experiment a) and that the formation of DTC-adduct
22 was at a rate proportional to the decomposition of
test compound 3, thereby indicating that the major
pathway of decomposition of the test compound is likely
to be as proposed.
1
lished from its H-NMR, HRMS, and elemental analysis
(see the Experimental Section). As anticipated, 31P-
NMR of 22 did not show any phosphorus-31 peak,
thereby confirming the fact that the phosphate portion
of the molecule had been cleaved off. Convincingly, the
presence of the phosphate fragment in the product
mixture of 24 and 25 was evidenced by two phosphorus-
31 peaks in its 31P-NMR spectrum (data not shown).
The above mechanistic studies clearly provided ex-
perimental proof to our hypothesis that GSTs catalyze
the decomposition of the latent mustard 3 and its
reported analogs. Isolation and characterization of DTC
adducts 22, 24, and 25 demonstrated that these com-
pounds are cleaved in the prescribed manner and the
decomposition products 6b and 7b are indeed alkylating
agents. With limited data in hand, it is premature to
conclude firmly that the vinyl sulfone 6b, which is a very
good Michael acceptor, is nontoxic. However, previous
In a separate experiment at pH 7.2, chemical decom-
position of 3 in the presence of DTC resulted in only
about 10-15% decompostion in 1 h and about 30%
decomposition in 24 h periods (Scheme 6, condition B,
data not shown).
To probe further, as shown in Scheme 6 and Figure
6, 3 was simultaneously subjected to decomposition in
phosphate buffer at pH 7.2 under the following four
conditions: a, with DTC and without GST P1-1; b,

Latent Alkylating Agents
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8 1743
mental proof of the GST activation concept and the
resulting fragmentation pattern. Trapping of transient
decomposition products as relatively stable diethyldithio-
carbamic acid adducts established the alkylating ability
of those reactive species. Novel GST-activated urethane
mustard compounds demonstrated the potential for
targeted and controlled delivery of a wide variety of
other amine-functionalized pharmaceutical agents into
specific cancer and other tissues with elevated levels of
a particular GST isozyme. This highly flexible latent
drug activation paradigm may, in principle, be extended
to other disease-associated enzymes, providing a sub-
stantial advantage over prodrugs that are activated in
a manner unrelated to the disease tissue’s properties.
As an alternative to enzyme inhibition, therefore, this
kind of latent drug shows considerable promise.
Exp er im en ta l Section
General information, including procedures for in vitro
enzymatic and cell culture studies, are as previously re-
ported.11 The preparative HPLC column used was a YMC 18
ODS-AQ column (YMC Corp., Wilmington, NC; Catalog No.
AQ-323-5). Unless otherwise noted, the buffers used for
preparative HPLC were as follows: buffer A, 0.1% TFA in
water; buffer B, 0.1% TFA in 9:1 acetonitrile/water. After
equilibration of the column with buffer A, elution of compounds
was done by running a gradient of 0-100% buffer B at 12 mL/
min over 120 min and the UV detector was set at 220 nm.
The analytical HPLC column used was a Bakerbond C-18
column (VWR, Catalog No. J T7098-0); the buffers used for
analytical HPLC were as follows: buffer A, 0.05 M NH4H2-
PO4 in 95:5 water/acetonitrile; buffer B, 0.015 M NH4H2PO4
in 3:7 water/acetonitrile. After equilibration of the column
with buffer A, a gradient of 0-100% buffer B was run over 30
min and the UV detector was set at 214 nm. Retention times
(tR) reported were usually for >90% pure products by analytical
HPLC. 1H, 13C, and 31P NMR spectra were recorded on a
Bruker instrument at 400, 100, and 162 MHz, respectively (UC
F igu r e 6. Differential cleavage of 3 (TER-286) with and
without GST P1-1 isozyme in presence or absence of dieth-
yldithiocarbamic acid (DTC); note that 3 had decomposed
faster only in the experiments containing GST P1-1 isozyme.
Also shown is the differential generation of vinyl sulfone-DTC
adduct 22 from the decomposition reaction of 3 (TER 286) with
or without GST P1-1 isozyme in presence of DTC; note that
formation of DTC-adduct 22 was about 3 times faster in the
experiment containing GST P1-1 isozyme and the formation
of 22 was at a rate proportional to the decomposition of test
compound 3.
in vitro and cell culture studies on placebo compound
1,11 which was found to be noncytotoxic although readily
cleaved by GST M1a-1a isozyme, suggest that the
reactive vinyl sulfone 6b may be quickly neutralized by
a mild nucleophilic species (including water), or by any
of more than 20 intracellular detoxification enzymes,
to a nontoxic byproduct as soon as it is formed. The
observed cytotoxicity of 3, therefore, is apparently
contributed solely by the phosphorodiamidate mustard
fragment 7b.
1
Berkeley, California); some H NMR spectra were recorded on
Nicolett instrument at 360 MHz (Acorn NMR Inc., Fremont,
California); HRMS and elemental analysis were obtained from
UC Berkeley. Mass spectral analyses of compounds were
performed on a Finnigan matrix-assisted laser desorption
ionization time of flight (MALDI TOF) mass spectrometer
using a 50 mM solution of R-cyano-4-hydroxycinnamic acid
(Aldrich) and 0.01% TFA in 7:3 acetonitrile/water as the
matrix. Bis(2-chloroethyl)amine hydrochloride, Borane-tet-
rahydrofuran complex, 2-bromoethanol, 2-bromethyl chloro-
formate, R-bromophenylacetic acid, bromotrimethylsilane, di-
ethyldithiocarbamic acid sodium salt, diisopropylethylamine,
hydrogen peroxide, lithium bromide, peracetic acid, styrene
oxide, and thionyl chloride were obtained from Aldrich. Glu-
tathione (reduced) was obtained from Sigma.
2-Br om op h en eth yl Alcoh ol (13): Con d ition a . A 100
mL (100 mmol) sample of a 1.0 M solution of borane-
tetrahydrofuran complex was added via cannula to a solution
of R-bromophenylacetic acid (11) (10.75 g, 50 mmol) in 50 mL
of dry tetrahydrofuran at 0-5 °C under argon over 15 min.
After the mixture was stirred at 5-10 °C for 3 h, it was
rechilled and quenched with 100 mL of 10% acetic acid in
methanol over 20 min. After 15 min of additional stirring,
the mixture was concentrated in vacuo. The oily residue was
redissolved in 300 mL of ethyl acetate, washed with water (2
× 250 mL) and brine (1 × 250 mL), and dried over anhydrous
Na2SO4. Evaporation and purification by flash column chro-
matography (35- × 3.7-cm silica gel bed and eluted isocratically
with methylene chloride) gave 13 (7.25 g, 72%) as a colorless
viscous oil: 1H NMR (300 MHz, CDCl3) δ 2.53 (br s, 1H, OH,
exchanged with D2O), 3.90-4.11 (m, 2H, CHCH2), 5.06 (m, 1H,
CHCH2), 7.25-7.45 (m, 5H, C6H5). This compound was found
to be unstable and was used immediately after preparation.
Con clu sion
Two new analogs, 4 and 5, of the previously reported
GST-activated latent alkylating agent 3 have been
designed, synthesized, and evaluated yielding a more
selective compound for the key target GST P1-1 isozyme.
The GST activation concept was further broadened
through design, synthesis and evaluation of novel latent
urethane mustards 8 and 9. From in vitro enzymatic
studies, 4A and 8 were identified as selective for GST
P1-1 isozyme which is known to be elevated in several
types of cancer. Cell culture studies with MCF-7 cell
lines transfected with high and low levels of GST P1-1
isozyme revealed that all the test compounds were more
toxic to MCF-7 pi than to MCF-7 neo cell lines. How-
ever, a statistically significant differential toxicity be-
tween pi and neo transfects was achieved only by 3 and
9. The tetrabromo derivative 5 was found not only to
be more reactive and cytotoxic than the corresponding
tetrachloro derivative 3 but also to have lost its statisti-
cally significant differential toxicity between pi and neo
transfects. Mechanistic studies on base- (chemical) and
enzyme-catalyzed decomposition of 3 provided experi-

1744 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8
Satyam et al.
Con d ition b. To a stirred solution of styrene oxide (12) (6
g, 50 mmol) in 50 mL of acetonitrile placed in a 2-neck reaction
flask fitted with a reflux condenser and a dropping funnel
under argon at room temperature was added bromotrimeth-
ylsilane (7.26 mL, 55 mmol) over 20 min. After an exothermic
reaction which subsided within a few minutes, the mixture
was stirred at room temperature under argon for 22 h. The
mixture was concentrated in vacuo, and the residue was
redissolved in 200 mL of ethyl acetate, washed successively
with water (2 × 100 mL), 5% sodium bicarbonate (1 × 100
mL), water (1 × 100 mL), and brine (1 × 250 mL), and dried
over anhydrous Na2SO4. Evaporation and purification by flash
column chromatography (30- × 3.7-cm silica gel bed and eluted
isocratically with 1:1 petroleum ether/methylene chloride) gave
a colorless viscous oil (2.2 g, 22%) which was identical, by TLC
remaining aqueous mixture was lyophilized and purified by
HPLC: after equilibration of the column with buffer A (0.1%
TFA in water), an aqueous solution of the crude product was
loaded onto the column. The column was eluted first with
buffer A followed by a gradient of 0-50% buffer B (0.1% TFA
in 9:1 acetonitrile/water) over 120 min at 12 mL/min. Frac-
tions which appeared pure by TLC were combined and
lyophilized to give 3.2 g (65%) of 18 as a white fluffy powder
which was found by HPLC to be a 1:1 mixture of diastereomers
18A and 18B: mp 63-67 °C, tR ) 19.33 min (A), and 19.63
min (B); 1H NMR (400 MHz, D2O) δ 1.60-1.80 (m, 2H, CHCH2-
CH2CON), 2.08 (t, 1H, J ) 7.8 Hz, CHCH2CH2CON), 2.13 (q,
1H, J ) 6.6, 8.8 Hz, CHCH2CH2CON), 2.24-2.55 (m, 1H,
CHCH2S), 2.70 (dd, 0.5H, J ) 5, 14 Hz, CHCH2S), 2.83 (dd,
0.5H, J ) 5, 14 Hz, CHCH2S), 2.90-3.10 (m, 8H, N(CH2CH2-
Cl)2), 3.20-3.35 (m, 9H, N(CH2CH2Cl)2 + R-H), 3.40 (dq, 2H,
J ) 11, 17 Hz, HNCH2CO), 3.95-4.05 (m, 1H, R-H), 4.05-
4.10 (m, 2.5H, SCHCH2), 4.17 (q, 0.5H, J ) 5, 9 Hz, SCHCH2),
7.05-7.15 (m, 5H, C6H5); 31P NMR (162 MHz, D2O) δ 28.73
and 28.77 (two s, 1:1.05); MS m/z 777.85 (MNa+). Anal.
(C26H40Cl4N5O8PS‚TFA‚H2O) C, H, N.
γ-Glu ta m yl-r-a m in o-â-[[[2-[[bis[bis(2-ch lor oeth yl)a m i-
n o]p h osp h or yl]oxy]-1-p h en yleth yl]su lfon yl]p r op ion yl]-
glycin e (4). To a stirred solution of 18 (755.5 mg, 1 mmol) in
10 mL of glacial acetic acid at room temperature was added
0.39 mL (2 mmol) of 30% H2O2. The reaction flask was covered
with aluminum foil to exclude light, and the mixture was
stirred at room temperature for 2 h when the mass spectrum
of the crude indicated complete conversion to the sulfoxide.
Next, 0.26 mL (1.25 mmol) of 32% peracetic acid in acetic acid
was added to the mixture, and it was stirred at room temper-
ature overnight, whereupon the mass spectrum of the crude
indicated formation of the sulfone 4. The mixture was
lyophilized and purified by HPLC using the same column and
procedure as for 18, except that the column was eluted
isocratically with 0.1% TFA in 3:7 acetonitrile/water to give
220 mg (28%) of 4 as a white fluffy powder which was found
by HPLC to be a 1:1 mixture of diastereomers 4A and 4B: tR
) 22.17 min (A) and 22.48 min (B); 1H NMR (400 MHz, DMSO-
d6) δ 1.90-2.00 (m, 2H, CHCH2CH2CON), 2.15-2.35 (m, 2H,
CHCH2CH2CON), 3.05-3.21 (m, 8H, (N(CH2CH2Cl)2)2), 3.25-
3.60 (m, 11H, (N(CH2CH2Cl)2)2 + HNCH2CO + R-H), 3.67 (d,
1H, CH CH2SO2, J ) 6.4 Hz), 3.82 (t, 1H, J ) 6.8 Hz, CH
CH2SO2), 4.40-4.45 (m, 1H, SO2CHCH2O), 4.50-4.61 (m, 1H,
SO2CHCH2O), 4.70-4.85 (m, 1H, SO2CHCH2O), 5.02 (q, 1H,
J ) 5.2, 8.4 Hz, R-H), 7.30-7.50 (m, 5H, C6H5), 8.20 (br s, 2H,
NH2), 8.31 (t, 1H, J ) 6 Hz, NH)), 8.45 (d, 1H, J ) 8 Hz, NH);
13C NMR (100 MHz, DMSO-d6) δ 25.72 (â-C, Glu), 30.80 (γ-C,
Glu), 41.07 (â-C, Cys), 42.18 (Cl-C), 46.71 (R-C, Glu), 48.20
(q, P-N-C, J ) 3.8, 9.1 Hz), 51.71 (R-C, Gly), 52.01 (R-C, Cys),
61.76 (Ph-C-SO2), 66.77 (d, P-O-C, J ) 8.3 Hz), 128.82 (m-
C, phenyl), 129.19 (p-C, phenyl), 129.93 (o-C, phenyl), 130.60
(1-C, phenyl); 169.19 (CdO), 170.76 (CdO), 170.83 (CdO),
170.94 (CdO); MS m/z 788.32 (MH+), 811.30 (MNa+). Anal.
(C26H40Cl4N5O10PS‚TFA‚H2O) C, H, N.
1
and H NMR, to that obtained from above. The major product
obtained from this reaction was found to be, by 1H NMR, a
polymer probably formed by the acid-catalyzed self-condensa-
tion of the initially formed unstable 13.
2-Br om op h en eth yl N,N,N′,N′-Tetr a k is(2-ch lor oeth yl)-
p h osp h or od ia m id a te (16). To a stirred solution of freshly
distilled phosphorus oxychloride (6.15 mL, 66 mmol) in 150
mL of dry methylene chloride at 0-5 °C in a reaction flask
fitted with a pressure-equalizing dropping funnel under argon
was added triethylamine (4.9 mL, 35 mmol) over 5 min. Then
a solution of 13 (6.65 g, 33 mmol) in 33 mL of dry methylene
chloride was added over a period of 4.5 h. After the mixture
was stirred at room temperature for 17 h, it was rechilled to
0 °C and bis(2-chloroethyl)amine hydrochloride (14) (17.8 g,
100 mmol) was added as a solid in one lot. To this stirred
suspension was added dropwise a solution of triethylamine (28
mL, 200 mmol) in 50 mL of methylene chloride over 2 h. The
mixture was allowed to warm to room temperature and stirred
for 3 days. Then, the mixture was suction filtered through a
thin layer of Celite, and the filtrate was concentrated in vacuo.
The brown oily residue was redissolved in 200 mL of ethyl
acetate and suction filtered again to remove the insoluble
triethylamine hydrochloride. The filtrate was concentrated in
vacuo, and the oily residue, which still contains some triethyl-
amine hydrochloride, was purified by flash column chroma-
tography (48- × 4.3-cm silica gel bed and eluted isocratically
with methylene chloride) to give 8 g (57%) of 2-bromophenethyl
N,N-bis(2-chloroethyl)chlorophosphoramidite (15) as colorless
viscous oil [1H NMR (300 MHz, CDCl3) δ 3.30-3.75 (m, 8H,
N(CH2CH2Cl)2), 4.50-4.75 (m, 2H, CHCH2O), 5.10-5.20 (m,
1H, CHCH2O), 7.30-7.50 (m, 5H, C6H5); MS m/z 446.66
(MNa+)] and 5.5 g (32%) of 16 as colorless viscous oil: 1H NMR
(400 MHz, CDCl3) δ 3.31-3.42 (m, 8H, (N(CH2CH2Cl)2)2),
3.52-3.63 (m, 8H, (N(CH2CH2Cl)2)2), 4.36 (t, 2H, J ) 6.8 Hz,
CHCH2O), 5.09 (t, 1H, J ) 6.8 Hz, CHCH2O), 7.36-7.42 (m,
5H, C6H5); 31P NMR (162 MHz, CDCl3) δ 13.55 (s); MS m/z
552.58 (MNa+).
Con ver sion of 15 to 16. A mixture of 15 (5.17 g, 12.2
mmol), 14 (4.46 g, 25 mmol), and triethylamine (7.0 mL, 50
mmol) in 120 mL of ethyl acetate was stirred at reflux
temperature for 3 days, whereupon TLC indicated >95%
conversion. The mixture was cooled to room temperature and
suction filtered through a thin layer of celite. The solids were
washed with 50 mL of ethyl acetate. The filtrate was washed
successively with 100 mL each of 1 N HCl, 5% NaHCO3, water,
and brine. The organic phase was dried over anhydrous Na2-
SO4, filtered, and concentrated in vacuo. Flash column
chromatography of the crude product as described above
yielded 3.8 g (56.4%) of pure 16, which was identical, by TLC,
mass spectrometry, and 1H NMR, to that obtained above.
γ-Glu ta m yl-r-a m in o-â-[[[2-[[bis[bis(2-ch lor oeth yl)a m i-
n o]p h osp h or yl]oxy]-1-p h en ylet h yl]t h io]p r op ion yl]gly-
cin e (18). Glutathione (17) (3.07 g, 10.00 mmol) was dissolved
in 40 mL of deionized water, and the pH was adjusted to
between 9 and 10 by adding 1 N NaOH. To this stirred
solution at room temperature was added a solution of 16 (3.46
g, 6.54 mmol) in 40 mL of ethanol. The resulting turbid
mixture was made clear by adding 20 mL of acetonitrile, and
the mixture was stirred at room temperature for 3 days. The
mixture was neutralized to pH 5-6 with 10% acetic acid, and
most of the organic solvent was removed in vacuo. The
The diastereomers were carefully separated by HPLC
(eluted isocratically with 0.1% acetic acid in 3:7 acetonitrile/
water). The diastereomer 4A was obtained (27 mg) as white
pluffy powder: mp 114-118 °C; tR ) 22.17 min; 1H NMR (360
MHz, CD3CN/D2O) (assignment of protons was done with the
help of 2D (COSY) spectrum) δ 2.02 (q, 2H, J ) 7.3, 14.0 Hz,
CHCH2CH2CON), 2.37 (dt, 2H, J ) 2.0, 7.3 Hz, CHCH2CH2-
CON), 3.16-3.33 (m, 9H, (N(CH2CH2Cl)2)2 + CHCH2SO2),
3.44-3.64 (m, 8H, (N(CH2CH2Cl)2)2), 3.66-3.72 (m, 2H, CHCH2-
CH2CON + CHCH2SO2), 3.83 (d, 2H, J ) 3.7 Hz, NCH2CO2H),
4.56-4.61 (m, 1H, SO2CHCH2O), 4.70-4.79 (m, 1H, SO2-
CHCH2O), 4.85 (dd, 1H, J ) 5.6, 8.6 Hz, SO2CHCH2O), 4.90
(dd, 1H, J ) 3.9, 9.4 Hz, CHCH2SO2), 7.46 (s, 5H, C6H5); 31P
NMR (162 MHz, CD3CN/D2O) δ 15.73 ppm; MS m/z 788.13
(MH+), 833.2 (M2Na+); HRMS calcd for C26H40Cl4N5O10PS +
H+ 786.1066, found 786.1072.
The diastereomer 4B was obtained (25 mg) as a white
powder: mp 116-120 °C; tR ) 22.48 min; 1H NMR (360 MHz,
CD3CN/D2O) (assignment of protons was done with the help
of 2D (COSY) spectrum) δ 2.02 (q, 2H, J ) 7.0, 14.4 Hz,
CHCH2CH2CON), 2.38 (t, 2H, J ) 8.2 Hz, CHCH2CH2CON),

Latent Alkylating Agents
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8 1745
3.17-3.29 (m, 8H, (N(CH2CH2Cl)2)2), 3.45-3.53 (m, 9H, (N(CH2-
CH2Cl)2)2 + CHCH2SO2), 3.63-3.73 (m, 2H, CHCH2CH2CON
+ CHCH2SO2), 3.80 (d, 2H, J ) 3.7 Hz, NCH2CO2H), 4.56-
4.63 (m, 1H, SO2CHCH2O), 4.69-4.75 (m, 1H, SO2CHCH2O),
4.81-4.88 (m, 2H, SO2CHCH2O + CHCH2SO2), 7.45 (s, 5H,
C6H5); 31P NMR (162 MHz, CD3CN/D2O) δ 16.02 ppm; MS m/z
790 (M3H+), 833 (M2Na+); HRMS calcd for C26H40Cl4N5O10PS
+ H+ 786.1066, found 786.1072.
min) to give 1.7 g (33%) of 21 as a white fluffy hygroscopic
powder: mp 85-113 °C; tR ) 15 min; 1H NMR (400 MHz, D2O)
δ 1.80 (q, 2H, J ) 7.6 Hz, CHCH2CH2CON), 2.19 (dt, 2H, J )
7.3, 7.9 Hz, CHCH2CH2CON), 2.50-2.60 (m, 3H, CHCH2-
SCH2), 2.77 (dd, 1H, J ) 5, 14 Hz, SCH2), 3.34-3.50 (m, 11H,
N(CH2CH2Cl)2 + NHCH2CO2H + R-H), 3.95 (t, 2H, J ) 6 Hz,
SCH2CH2O), 4.24 (q, 1H, J ) 5 Hz, R-H); MS m/z 542.69
(MNa+). Anal. (C17H28Cl2N4O8S‚TFA‚2.5H2O) C, H, N.
γ-Glu ta m yl-r-a m in o-â-[[[2-[[[bis(2-ch lor oeth yl)a m in o]-
ca r bon yl]oxy]eth yl]su lfon yl]p r op ion yl]glycin e (8). To a
stirred solution of 21 (0.519 g, 1 mmol) in 10 mL of glacial
acetic acid at room temperature was added 30% H2O2 (0.39
mL, 2 mmol). The reaction flask was covered with aluminum
foil to exclude light, and the mixture was stirred at room
temperature for 4 h, when mass spectrum of the mixture
indicated complete conversion to sulfoxide. Then, 0.26 mL
(1.25 mmol) of 32% peracetic acid in acetic acid was added to
the mixture, and it was stirred at room temperature for an
additional 4 h, whereupon the mass spectral analysis of the
mixture indicated formation of the sulfone 8. The mixture was
lyophilized and purified by HPLC as for 21 (except that the
elution rate was 20 mL/min) to give 0.44 g (80%) of 8 as a
hygroscopic fluffy white powder: mp 82-93 °C; tR ) 15.2 min;
1H NMR (400 MHz, CD3CN/D2O) δ 1.90-2.00 (m, 2H, CHCH2-
CH2CON), 2.31 (dt, 2H, J ) 2.0, 7.5 Hz, CHCH2CH2CON),
3.38-3.66 (m, 14H, N(CH2CH2Cl)2 + NHCH2CO2H + CH2-
SO2CH2), 3.71 (dd, 1H, J ) 3.5, 14.7 Hz, R-H), 4.37 (t, 2H, J )
5 Hz, SO2CH2CH2O), 4.88 (dd, 1H, J ) 3.4, 9.7 Hz, R-H); MS
m/z 552.46 (MH+). Anal. (C17H28Cl2N4O10S‚TFA‚2H2O) C, H,
N.
γ-Glu ta m yl-r-a m in o-â-[[[2-[[[bis(2-ch lor oeth yl)a m in o]-
ca r bon yl]oxy]eth yl]su lfon yl]p r op ion yl]glycin e Dieth yl
Diester (9). To a stirred suspension of 8 (0.39 g, 0.7 mmol)
in 28 mL of dry ethanol in a 100 mL reaction flask fitted with
a reflux condenser under argon at room temperature was
added thionyl chloride (1.1 mL, 15 mmol) from the top of the
condenser. The resulting clear colorless solution was stirred
at gentle reflux temperature for 2.5 h, whereupon mass
spectral analysis of the mixture indicated formation of the
diethyl diester. The mixture was concentrated in vacuo, and
the gummy residue was purified by HPLC as for 21 to give
0.17 g (40%) of 9 as a hygroscopic fluffy white powder: mp
54-60 °C; tR ) 20.38 min: 1H NMR (Varian 300 MHz, CD3-
CN/D2O) δ 1.25 (t, 3H, J ) 7.1 Hz, CO2CH2CH3), 1.30 (t, 3H,
J ) 6.2 Hz, CO2CH2CH3), 2.05-2.30 (m, 2H, CHCH2CH2-
CONH), 2.53 (t, 2H, J ) 7.1 Hz, CHCH2CH2CONH), 3.52-
3.83 (m, 12H, N(CH2CH2Cl)2 + CH2SO2CH2), 3.92 (d, 2H, J )
2 Hz, NHCH2CO2H), 4.00 (dd, 1H, J ) 4.5, 7.5 Hz, R-H), 4.17
(q, 2H, J ) 7.2 Hz, CO2CH2CH3), 4.26 (q, 2H, J ) 7 Hz,
CO2CH2CH3), 4.50 (t, 2H, J ) 5.3 Hz, SO2CH2CH2O), 4.99 (dd,
1H, J ) 3, 9 Hz, R-H); MS m/z 608.31 (MH+), 631.26 (MNa+).
Anal. (C21H36Cl2N4O10S‚HCl) C, H, N.
γ-Glu t a m yl-r-a m in o-â-[[2-et h yl-N,N,N′,N′-t et r a k is(2-
br om oeth yl)p h op h or od ia m id a te]su lfon yl]p r op ion yl-(R)-
(-)-p h en ylglycin e (5). A solution of 3 (2.17 g, 2.76 mmol)
and lithium bromide (13 g, 150 mmol) in 180 mL of 5:1 acetone/
acetonitrile was stirred at gentle reflux temperature while the
reaction progress was monitored by HPLC. After 46 h reaction
time, there was >50% conversion to tetrabromo derivative 5
(51%, tR ) 15.95 min), along with varying amounts of unre-
acted starting material 3 (<1%, tR ) 13.46 min), monobromo
(4%, tR ) 14.05 min), dibromo (13%, tR ) 14.63 min), and
tribromo (24%, tR ) 15.20 min) derivatives. The mixture was
concentrated in vacuo, and the residue was purified twice by
HPLC: buffer A, 0.1% TFA in 1:4 acetonitrile/water; buffer
B, 0.1% TFA in 9:1 acetonitrile/water; for the first purification,
a gradient of 0-100% buffer B was run at 20 mL/min over
120 min. Fractions containing 5, which were also contami-
nated with varying amounts of dibromo and tribromo deriva-
tives, were pooled and repurified by running a gradient of
0-50% buffer B at 20 mL/min over 120 min. Here again a
majority of fractions were obtained as mixtures of 5, dibromo,
and tribromo derivatives. These were mixed and kept for
further purification. Fractions containing pure 5 were pooled
and lyophilized to give 135 mg (5%) of a fluffy white powder:
mp 75-93 °C; tR ) 25.24 min (94% pure); 1H NMR (400 MHz,
CD3CN/D2O) δ 2.19-2.36 (m, 2H, CHCH2CH2CON), 2.57-2.66
(m, 2H, CHCH2CH2CON), 3.44-3.65 (two m, 19H, CH2SO2CH2
+ (NCH2CH2Cl)2)2), 3.81 (dd, 1H, J ) 5, 14.7 Hz, SO2CH2),
4.08 (t, 1H, J ) 6.5 Hz, R-H), 4,45-4.50 (m, 2H, SO2CH2CH2O),
5.12 (dd, 1H, J ) 5, 8 Hz, R-H), 5.52 (s, 1H, C6H5CH), 7.47-
7.55 (m, 5H, C6H5); 31P NMR (162 MHz, D2O/CD3CN) δ 14.93
(s) and 15.10 (s). Anal. (C26H40Br4N5O10PS‚TFA‚H2O) C, H,
N.
[(2-B r o m o e t h o x y )c a r b o n y l][b i s (2-c h lo r o e t h y l)]-
a m in e (20). 2-Bromoethyl chloroformate (19) (5.6 mL, 50
mmol) was added to a stirred suspension of bis(2-chloroethyl)-
amine hydrochloride (14) (9.8 g, 55 mmol) in 250 mL of dry
dichloromethane at 0-5 °C under argon over 2 min followed
by 28 mL (200 mmol) of triethylamine over 20 min. The
mixture was stirred at 5-10 °C for 3 h and at room temper-
ature for 18 h. The mixture was suction filtered, and the
filtrate was concentrated in vacuo. The residue was dissolved
in 200 mL of ethyl acetate and suction filtered to remove the
triethylamine hydrochloride. The filtrate was successively
washed with 100 mL each of 2 N HCl, 5% NaHCO3, water,
and brine. It was dried over anhydrous Na2SO4, filtered, and
concentrated in vacuo to give 13 g of crude product as a
colorless oil, which was further purified by flash column
chromatography (31- × 3.7-cm of silica gel bed and eluted
isocratically with dichloromethane) to give 12.5 g (85%) of 20
as a colorless oil: 1H NMR (400 MHz, CDCl3) δ 3.55 (t, 2H, J
) 5.7 Hz, BrCH2CH2O), 3.69 (s, 8H, N(CH2CH2Cl)2), 4.43 (t,
2H, J ) 5.7 Hz, BrCH2CH2O); MS m/z 316.31 (MNa+). Anal.
(C7H12BrCl2NO2) C, H, N.
Ch em ica l Decom p osition of 3: Con d ition A (Sch em e
6). A mixture of 3 (1.24 g, 1.5 mmol) and diethyldithiocar-
bamic acid sodium salt (3.38 g, 15 mmol) was dissolved in 40
mL of 3:1 acetonitrile/water, and the solution pH was adjusted
to 11 by addition of 1 N sodium carbonate (10 mL). The
mixture was stirred at room temperature for 1 h whereupon
HPLC analysis of the mixture indicated disappearance of 3
(tR ) ∼20 min) and appearance of a prominent product (tR
)
∼17 min) along with two or three other decomposition products
whose retention times changed variously with time. The
mixture was purified three times by HPLC (buffer A, 0.1% TFA
in water; buffer B, 0.1% TFA in 9:1 acetonitrile/water; eluted
by running a gradient from 0-100% buffer B at an elution
rate of 12 mL/min at 214 nm) to give a fraction (∼150 mg)
containing some amount each of the decomposition products
24 (HRMS calcd for C9H21N2O4P1S2 + H+ 317.0759, found
317.0753) (24 also existed as a dimer with m/z 633 amu which
was not a true molecular ion) and diethylamine-DTC adduct
25 (HRMS calcd for C14H30N3S4 + H+ 368.1323, found 368.1323)
(24 and 25 also existed as a dimer with m/z 684 amu which
was not a true molecular ion). The main product (tR ) ∼17
min) was isolated (650 mg, 66%) and characterized as the
expected vinyl sulfone-DTC adduct 22: a white powder; mp
156-158 °C; 1H-NMR (400 MHz, DMSO-d6/D2O) δ 1.11-1.21
γ-Glu ta m yl-r-a m in o-â-[[[2-[ca r bon yl[bis(2-ch lor oeth -
yl)a m in o]oxy]et h yl]t h io]p r op ion yl]glycin e (21). Glu-
tathione (17) (6.14 g, 20 mmol) was dissolved in 100 mL of
water, and the pH was adjusted to between 9 and 10 by adding
1 N NaOH. To this stirred solution at room temperature was
added a solution of 20 (2.93 g 10 mmol) in 100 mL of 1:1
ethanol/acetonitrile, and the resulting clear colorless solution
was stirred at room temperature under argon for 3 days,
whereupon TLC of the mixture indicated completion of the
reaction. The mixture was acidified to pH 5-6 with 10% acetic
acid, and most of the organic solvent portion was removed in
vacuo. The aqueous portion was lyophilized and purified by
HPLC (buffer A, 0.1% TFA in 1:9 acetonitrile/water; buffer B,
0.1% TFA in 9:1 acetonitrile/water; eluted by running a
gradient from 0-100% buffer B at an elution rate of 12 mL/

1746 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8
Satyam et al.
(m, 6H, N(CH2CH3)2), 1.90-2.04 (m, 2H, CH2CH2CH), 2.25-
2.36 (m, 2H, CH2CH2CH), 3.14-3.91 (m, 11H, CH2CH2CH +
CH2SO2CH2CH2S + N(CH2CH3)2), 4.93 (dd, 1H, J ) 4.0 and
9.0 Hz, SO2CH2CH), 5.24 (s, 1H, C6H5CH), 7.28-7.43 (m, 5H,
C6H5CH); HRMS calcd for C23H34N4O8S3 + H+ 591.1617, found
591.1619. Anal. (C23H34N4O8S3‚0.5TFA‚H2O) C, H, N, S.
Ch em ica l Decom p osition of 3: Con d ition B/Con tr ol
Exp er im en t (Sch em e 6). Diethyldithiocarbamic acid sodium
salt (225 mg, 1 mmol) was dissolved in 4 mL of 3:1 acetonitrile/
water, and the solution pH was adjusted to 7.3 with 1 N HCl.
An 82 mg (0.1 mmol) sample of 3 was added to the above
solution, and the pH of the resulting mixture was adjusted
back to 7.3 with 1 N sodium carbonate, and the mixture was
stirred at room temperature for 1 h when HPLC analysis of
the mixture indicated only 10-15% decomposition of 3. When
the mixture was stirred further for 24 h, only 25-30%
decomposition of 3 was observed with the formation of a small
amount of vinyl sulfone-DTC adduct 22 which was confirmed
by coelution with a pure sample of 22 (data not shown).
Higgins, Polly Sanderson, Don Schmidt, and Marvin
Seigel for many helpful discussions and suggestions.
Refer en ces
(1) Howie, A. F.; Farrester, L. M.; Glancey, M. J .; Schlager, J . J .;
Powis, G. G.; Beckett, G. J .; Hayes, J . D.; Wolf, C. R. Glutathione-
S-transferase and glutathione peroxidase expression in normal
and tumor human tissues. Carcinogenesis 1990, 11, 451-458.
(2) Tew, K. D.; Clapper, M. L. Glutathione-S-transferases and
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(3) Kuzmich, S.; Vanderveer, L.; Walsh, E.; La Creta, F.; Tew, K.
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GST-Ca ta lyzed Decom p osition of 3: Con d ition C in
Sch em e 6/Exp er im en t c in F igu r e 6 (w ith Both DTC a n d
GST P 1-1 Isozym e). Standards of 0.003 M solution of 3 and
0.0015 M solution of an internal standard peptide DQQN-
AFYEILHOPN-NH2 were prepared. Also prepared were buffer
1, 200 mM sodium phosphate at pH 7.2, and buffer 2, 200 mM
sodium phosphate and 0.009 M diethyldithiocarbamic acid at
pH 7.2. The reaction was initiated by mixing 0.2 mL of 0.003
M solution of 3, 0.04 mL of 0.0015 M solution of internal
standard, and 0.05 mL of 9.6 × 10-6 M solution of GST P1-1
in 1.71 mL of buffer 2, and the reaction progress was monitored
by HPLC at 1 h intervals for 6 h. Progressive decomposition
of 3 (tR ) ∼20 min) and formation of vinyl sulfone-DTC adduct
22 at tR ∼17 min was identified by coelution with a pure
sample of 22. Also shown in the Figure 6 is the relative
formation of DTC adduct 22 in the experiment with GST P1-1
isozyme.
GST-Ca ta lyzed Decom p osition of 3: Exp er im en t d
(F igu r e 6) (w ith ou t DTC a n d w ith GST P 1-1 Isozym e).11
The reaction was initiated by mixing 0.2 mL of 0.003 M
solution of 3, 0.04 mL of 0.0015 M solution of internal
standard, and 0.05 mL of 9.6 × 10-6 M solution of GST P1-1
in 1.71 mL of buffer 1 (see the above experiment), and the
reaction progress was monitored by HPLC at 1 h intervals for
6 h. The rate of decomposition of 3 was almost close to that
observed in the above experiment.
(5) (a) Montali, J . A.; Wheatley, J . B.; Schmidt, D. E., J r. Comparison
of glutathione S-transferase levels in predicting the efficacy of
a novel alkylating agent. Cell. Pharmacol. 1995, 2, 241-247.
(6) Kauvar, L. M. Pharmaceutcal targeting of GST isozymes. In
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(7) Armstrong, R. N. Glutathione S-transferases: Structure and
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references cited therein.
(8) Armstrong, R. N. Glutathione S-transferases: Reaction Mech-
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131-140 and related references cited therein.
(9) Adang, A. E. P.; Meyer, D. J .; Brussee, J .; Van der Gen, A.;
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S-transferases 7-7 and 8-8 with γ-glutamyl or glycyl modified
glutathione analogs. Biochem. J . 1989, 264, 759-764 and related
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(10) Lyttle, M. H.; Hocker, M. D.; Hui, H. C.; Caldwell, C. G.; Aaron,
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Glutathione-S-transferase Activates Novel Alkylating Agents.
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(13) Reinemer, P.; Dirr, H. W.; Ladenstein, R.; Huber, R.; Lo Bello,
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Con tr ol Exp er im en ts: Exp er im en t a in F igu r e 6 (w ith
DTC a n d w ith ou t GST P 1-1). The same amounts of
reactants and conditions as described in experiment c, but
without GST P1-1 isozyme, were used. The reaction progress
was monitored by HPLC at 1 h intervals for 6 h. Also shown
in the Figure 6 is the relative formation of DTC adduct 22 in
the experiment without GST P1-1 isozyme.
Con tr ol Exp er im en ts: Exp er im en t
b in F igu r e 6
(w ith ou t DTC a n d w ith ou t GST P 1-1). The same amounts
of reactants and conditions as described for experiment d, but
without GST P1-1 isozyme, were used. The reaction progress
was monitored by HPLC at 1 h intervals for 6 h.
Molecu la r Mod elin g. A computer model of 3 at the active
site of the GST P1-1 isozyme (Figure 1) was generated using
the Insight II software (Biosym Technologies, Inc., San Diego,
CA). The crystal structure of GST P1-1 complexed with
hexylglutathione was used as the starting point.13 The Builder
facility of the Insight software was utilized to modify the
structure of the substrate into that of 3. Manual modification
of the torsion angles were carried out on the modified hexyl
portion, continuously checking in order to avoid steric clashes.
Schematic two-dimensional representations of the site were
then generated, respecting all the information contained in
the model.
(16) Liu, S.; J i, X.; Gilliland, G. L.; Stevens, W. J .; Armstrong, R. N.
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Ack n ow led gm en t. We thank Larry Kauvar for his
suggestions on the manuscript and for the overall
project guidance. We also thank Rey Gomez, Deb

Latent Alkylating Agents
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8 1747
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