Hypoxia-selective nitrobenzyloxycarbonyl derivatives of 1,2- bis(methylsulfonyl)-1-(2-chloroethyl)hydrazines

Article abstract of DOI:10.1021/jm9805891

Some 4- and 2-(nitrobenzyloxycarbonyl)-1,2-bis(methylsulfonyl)-1-(2- chloroethyl)hydrazines (4, 6, and 7) were synthesized and evaluated for their ability to exert preferential toxicity to hypoxic EMT6 mammary carcinoma cells using a colony-forming assay. Of these, the 4,5-dimethoxy-2-nitro analogue 6 (50 μM, 1-h exposure) caused greater than 3 logs of kill of hypoxic cells, with relatively minor toxicity to corresponding aerobic cells. The ability of 4-nitro (4) and 4,5-dimethoxy-2-nitro (6) analogues to reach and kill hypoxic cells of solid tumors was also demonstrated using intradermally implanted EMT6 solid tumors in mice. In addition, a possible source of toxicity to normal tissue, i.e., the activation of the 4- nitrobenzyl derivative 4 by glutathione S-transferase-catalyzed thiolysis, was essentially eliminated by replacing one of the benzylic methylene protons by a methyl group. The 4-nitro (4) and 4,5-dimethoxy-2-nitro (6) analogues also appear to be reduced more easily under acidic conditions (pH 6.0) than under neutral conditions, as measured by differential pulse polarography. Since the pH in hypoxic regions is often lower than that in adjacent aerobic regions, this property should aid in the cytotoxic action of these agents against hypoxic cells of solid tumors.

Full text of DOI:10.1021/jm9805891

J . Med. Chem. 1999, 42, 941-946
941
Hyp oxia -Selective Nitr oben zyloxyca r bon yl Der iva tives of
1,2-Bis(m eth ylsu lfon yl)-1-(2-ch lor oeth yl)h yd r a zin es
Krishnamurthy Shyam,† Philip G. Penketh,† Maxim Shapiro,† Michael F. Belcourt,† Regina H. Loomis,†
Sara Rockwell,‡ and Alan C. Sartorelli*,†
Departments of Pharmacology and of Therapeutic Radiology and Developmental Therapeutics Program, Cancer Center,
Yale University School of Medicine, New Haven, Connecticut 06520
Received October 15, 1998
Some 4- and 2-(nitrobenzyloxycarbonyl)-1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazines (4,
6, and 7) were synthesized and evaluated for their ability to exert preferential toxicity to hypoxic
EMT6 mammary carcinoma cells using a colony-forming assay. Of these, the 4,5-dimethoxy-
2-nitro analogue 6 (50 µM, 1-h exposure) caused greater than 3 logs of kill of hypoxic cells,
with relatively minor toxicity to corresponding aerobic cells. The ability of 4-nitro (4) and 4,5-
dimethoxy-2-nitro (6) analogues to reach and kill hypoxic cells of solid tumors was also
demonstrated using intradermally implanted EMT6 solid tumors in mice. In addition, a possible
source of toxicity to normal tissue, i.e., the activation of the 4-nitrobenzyl derivative 4 by
glutathione S-transferase-catalyzed thiolysis, was essentially eliminated by replacing one of
the benzylic methylene protons by a methyl group. The 4-nitro (4) and 4,5-dimethoxy-2-nitro
(6) analogues also appear to be reduced more easily under acidic conditions (pH 6.0) than under
neutral conditions, as measured by differential pulse polarography. Since the pH in hypoxic
regions is often lower than that in adjacent aerobic regions, this property should aid in the
cytotoxic action of these agents against hypoxic cells of solid tumors.
Solid tumors contain regions of hypoxia which fre-
quently constitute 5-30% of the total viable tumor cell
population. These oxygen-deficient tumor cells limit the
curability of many solid tumors by radiation and most
chemotherapeutic agents. Thus, hypoxic cancer cells
may be capable of proliferating and causing tumor
regrowth after treatments that produce tumor regres-
sion. However, hypoxia also creates an environment
conducive to reductive processes, which results in an
important exploitable difference between normal and
neoplastic tissues.1 This observation has led to extensive
efforts to design and develop several classes of synthetic
nitro-containing hypoxia-selective agents, including ana-
logues of nitroimidazoles (for a review, see ref 2),
nitroacridines,3 benzotriazine N-oxides,4 nitrobenzyl
halides and carbamates,5,6 nitrobenzyl mustard quater-
nary salts,7,8 and nitrobenzyl phosphorodiamidates.9
This report describes our efforts to employ the exceed-
ingly efficacious and broad-spectrum activity of the 1,2-
bis(sulfonyl)-1-(2-chloroethyl)hydrazine family of cyto-
toxic agents10-14 to develop prodrugs of this class with
preferential toxicity to hypoxic cells of solid tumors
relative to their aerobic counterparts.
cyclophosphamide, and the nitrosoureas, evaluated
concomitantly.12,13 More recently, we have reported the
synthesis of (alkoxycarbonyl)- and (aryloxycarbonyl)-1,2-
bis(methylsulfonyl)-1-(2-chloroethyl)hydrazines, which
are preferentially activated by glutathione (GSH) and
glutathione S-transferase (GST), making these agents
potentially useful in the treatment of multidrug-
resistant tumors with increased intracellular levels of
GSH and/or GST.14 We have also measured the rates
of activation of the synthesized agents (0.2 mM drug
concentration) in phosphate buffer (pH 7.4, 37 °C) alone,
in 1 mM phosphate buffer containing 5 mM GSH, and
in 1 mM phosphate buffer containing 5 mM GSH and
17.5 U/mL GST.14 The most interesting compound to
emerge from this study was the nitrobenzyl analogue
4, which was found to undergo little or no hydrolysis in
phosphate buffer and an extremely slow rate of thiolysis
by GSH, but a relatively high rate of activation when
thiolysis by GSH was catalyzed by GST. Although this
preferential activation occurs to some degree with
almost all of the alkoxycarbonyl and aryloxycarbonyl
derivatives examined, compound 4 was quite unique in
the magnitude of this activation. For example, the GST-
catalyzed thiolysis rate for compound 4 was found to
be approximately 4 times faster than the second most
labile prodrug under these conditions.
Several classes of prodrugs of 1,2-bis(methylsulfonyl)-
1-(2-chloroethyl)hydrazine (1) have been synthesized by
our laboratory and evaluated for antineoplastic activity
against a number of transplanted tumors in mice.11-14
Of these, compounds 2 and 3 produced 80-100% cures
of mice bearing the L1210 leukemia and were active
against a spectrum of transplanted murine and human
tumors, with activities that compared favorably with
those of the clinically useful agents, mitomycin C,
* To whom correspondence should be addressed.
† Department of Pharmacology.
‡ Department of Therapeutic Radiology.
10.1021/jm9805891 CCC: $18.00 © 1999 American Chemical Society
Published on Web 02/17/1999

942 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 5
Brief Articles
Sch em e 1
of tumors which have become resistant to reactive
compounds through elevation of GST. However, GST-
catalyzed activation may also result in toxicity to normal
tissues because GSTs are not confined to tumor cells.
Two approaches may be used to minimize normal
tissue toxicity created by GST activation. One involves
the introduction of different functional groups on the
aromatic ring (e.g., compound 6). A second approach
toward minimizing the potential toxicity created by GST
activation is to eliminate the structural feature in
compound 4 which makes it a good SN2 substrate. The
reactivity of substrates in SN2 reactions follows the rank
order CH3W (where W is a leaving group) > primary
(e.g., CH3CH2W) > secondary [e.g., (CH3)2CHW] >
tertiary [e.g., (CH3)3CW].15 Since steric factors play an
important role in SN2 reactions, branching at the
R-carbon to give compound 7, which would transform
compound 4 into a secondary substrate, should decrease
the rate of GST-catalyzed activation appreciably. How-
ever, one would still expect compound 7 to be activated
in hypoxic cells by bioreduction in a manner analogous
to that of compound 4 (Scheme 3). Compound 7 was
synthesized and its capacity to undergo GSH/GST-
catalyzed activation was evaluated. In addition, the
ability of compound 7 to cause preferential cytotoxicity
to EMT6 cells was also determined.
We reasoned that compound 4 would also have the
potential to be activated under hypoxic conditions by a
reductive mechanism similar to the one described for
nitrobenzyl phosphorodiamidates,9 whereby lability of
the carbamate moiety could be induced by the enzymatic
reduction of the electron-withdrawing nitro group to an
electron-releasing hydroxylamino or amino group
(Scheme 1). Lone pair-assisted expulsion of carbon
dioxide and 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-
hydrazine (1) as the anion would then be expected to
occur, followed by the fragmentation of the latter to
generate the putative alkylating species CH3SO2NdN-
CH2CH2Cl.
This paper reports the synthesis and biological evalu-
ation of some structural analogues of compound 4
(5-7). It also reports the successful resolution of the
two activation mechanisms to which compound 4 is
prone, i.e., thiol-catalyzed activation and bioreductive
activation. As observed earlier, activation studies with
compound 4 demonstrated that this agent is relatively
refractory not only to hydrolysis in phosphate buffer but
also to thiolysis by GSH.14 In contrast, a relatively high
rate of activation of compound 4 occurs when thiolysis
is catalyzed by GST. Activation by thiols could occur by
two possible mechanisms: (a) nucleophilic attack at the
carbonyl carbon and/or (b) nucleophilic attack at the
benzylic methylene. An SN1 mechanism involving a
carbocation intermediate can be ruled out because of
the instability of the 4-nitrobenzyl carbocation. In an
earlier study we found that with 2-(alkoxycarbonyl)- and
2-(aryloxycarbonyl)-1,2-bis(methylsulfonyl)-1-(2-chloro-
ethyl)hydrazines lacking the benzylic methylene group,
the rate of GST-catalyzed activation ranged from 0 to
only 6 times the rate of activation produced by GSH
alone.14 In contrast, for compound 4, the rate of GST-
catalyzed activation is about 340 times greater than that
of uncatalyzed thiolysis. This enormous difference in
relative rates of activation between compounds lacking
the benzylic methylene and compound 4 implicates the
benzylic methylene moiety in compound 4 as the pre-
dominant site at which the nucleophilic attack takes
place. Since activation by an SN1 mechanism is unlikely,
this reaction is probably an SN2 process in which
nucleophilic attack at the benzylic methylene portion
of the molecule results in activation (Scheme 2). Com-
pound 4 could be particularly useful in the treatment
Ch em istr y
All of the new compounds reported in this paper
(5-7) were prepared by reacting 1,2-bis(methylsulfonyl)-
1-(2-chloroethyl)hydrazine (1) with the appropriate
benzyl chloroformate (Scheme 4) using a procedure
similar to the one we have described for the preparation
of compound 4. Where necessary, the chloroformate was
synthesized by reacting the appropriate alcohol with a
solution of phosgene in toluene.16 Compound 7 was
prepared using 1-(4-nitrobenzyl)ethanol as the starting
material. The latter was synthesized by adapting a
literature procedure.17
Resu lts a n d Discu ssion
Compound 4 was evaluated against EMT6 mouse
carcinoma cells in vitro under aerobic and hypoxic
conditions using a colony-forming assay described by our
laboratory.18,19 At a concentration of 40 µM, a 1-h
exposure to compound 4 caused 2 logs of kill of hypoxic
EMT6 cells, with relatively minor toxicity to corre-
sponding aerobic cells (Figure 1).
To ensure that the nitro group in compound 4 is
important to its preferential cytotoxicity to hypoxic cells,
the unsubstituted benzyl derivative 5 was synthesized
and evaluated against EMT6 cells. As shown in Figure
1, compound 5 not only was considerably less potent
than compound 4 but also was essentially equitoxic to
EMT6 cells under both conditions of oxygenation. This
finding confirmed the importance of the nitro group for
the preferential toxicity of compound 4 to hypoxic cells.

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 5 943
Sch em e 2
Sch em e 3
Sch em e 4
As described earlier, the cytotoxicity of compound 4
is postulated to result from the release of a chloroethy-
lating species following preferential 4- or 6-electron
reduction of the nitro group to a hydroxylamino or
amino function, respectively, under hypoxic conditions.
A similar reductive activation mechanism may also be
invoked for the 4,5-dimethoxy-2-nitro analogue, com-
pound 6, a representative of the 2-nitro class. Thus,
reduction of the 2-nitro group in compound 6 to the
corresponding amino or hydroxylamino substituent
would also be expected to result in the generation of a
chloroethylating species. In addition, the close proximity

944 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 5
Brief Articles
logue 6 with compound 4 have shown that the functional
groups present on the aromatic ring can markedly affect
the rate of GST activation. Thus, the rate of activation
of compound 6 by GSH/GST was 8.7 compared to 20.5
nmol/mL/min for compound 4. Both steric and electronic
factors may be responsible for the observed differences
in the rate of activation.
As anticipated, the rate of activation of compound 7,
which differs from compound 4 in possessing a methyl
group in place of one of the benzylic methylene hydro-
gens, in the presence of 5 mM GSH and 17.5 U/mL GST
was considerably slower than that of compound 4. Thus,
the rate of activation of compound 7 (0.1 mM) under
these conditions was at least 150 times slower than that
of compound 4 (0.1 mM). In addition, compound 7
appeared to be less toxic to aerobic EMT6 cells than
compound 4, while its hypoxic cell toxicity was es-
sentially the same as that of compound 4 (compare
graphs for compounds 4 and 7 in Figure 1). In addition,
we have found that it is possible to eliminate hypoxia-
selective cytotoxicity while retaining the approximate
magnitude of the GSH/GST-catalyzed rate of activation
of compound 4 by merely removing the nitro group from
4 (data not shown).
F igu r e 1. Survival of EMT6 cells exposed to various concen-
trations of compound 4, 5, 6, or 7 for 1 h under hypoxic or
aerobic conditions in vitro. Points are geometric means of two
or more independent determinations of surviving fractions.
The SEMs are shown where n ) 3 and where the error bars
are larger than the point sizes. Note the differences in scales.
Key: aerobic (4), hypoxic (2).
The capacity of agents to reach and kill hypoxic cells
of solid tumors was tested using the in vivo-in vitro
test system we described previously.18,20,21 The radiation
dose used in these studies (15 Gy) was large enough that
the tumor cell population surviving after irradiation is
composed almost entirely of radioresistant, hypoxic
cells. The fact that concomitant treatment of X irradia-
tion with compound 4 or 6 killed cells which survived
the radiation treatment showed that these compounds
were capable of reaching hypoxic areas of the solid
tumor and killing oxygen-deficient cells in these regions
(Table 1).
of the nitro group makes possible other activation
mechanisms. Thus, once the nitro group is converted
to the nucleophilic amino or hydroxylamino function,
activation can be envisioned to occur via a six-membered
cyclic transition state (Scheme 5). Activation via a five-
membered cyclic transition state may also be envisioned
to occur for the hydroxylamino metabolite, whereby
nucleophilic attack of the benzylic methylene carbon by
the hydroxylamino oxygen results in the generation of
compound 1 (not shown). The ability of compound 6 to
exert preferential toxicity to hypoxic EMT6 cells was
evaluated. As shown in Figure 1, at a concentration of
50 µM, a 1-h exposure to compound 6 caused greater
than 3 logs of kill of hypoxic EMT6 cells, with relatively
minor toxicity to corresponding aerobic cells.
Hypoxic regions have a greater reliance on anaerobic
metabolism; as a consequence, the pH in these regions
is lower than that generally found in adjacent well-
oxygenated tissues.22 The H+ concentration greatly
influences the chemistry in these regions.23-25 Because
of the low oxygen tension, the rate of oxidation of NADH
is decreased, resulting in an increase in the ratio of
NADH/NAD+, as well as an increase in the redox
potential of this couple.26 However, this effect is par-
One of the approaches that may be used to minimize
normal tissue toxicity created by GST activation is to
introduce different functional groups on the aromatic
ring. Studies comparing the 4,5-dimethoxy-2-nitro ana-
Sch em e 5

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 5 945
Ta ble 1. Effects of Compounds 4 and 6 on the Survival of
and 6 (Table 2) will increase the action of these agents
in acidic regions of solid tumors and thereby comple-
ment the hypoxic/oxic differential activity of these
compounds.
Cells from EMT6 Solid Tumorsa
surviving fraction
treatment
none
compd 4
compd 6
1.00
1.00
Con clu sion
drug alone
X-ray alone
drug + X-ray
0.841
0.043
0.013
0.856
0.043
0.024
The nitrobenzyloxycarbonyl derivatives of 1,2-bis-
(methylsulfonyl)-1-(2-chloroethyl)hydrazines described
in this paper exhibit exceptional preferential toxicity to
hypoxic EMT6 cells relative to their aerobic counter-
parts. It also appears that their preferential toxicity to
hypoxic cells can be improved further by replacing one
of the benzylic methylene protons with a methyl group.
Two representative agents of this class possess signifi-
cantly higher (more positive) redox potentials at pH 6.0
than at pH 7.0, a property that should enhance the
preferential toxicity of these agents to hypoxic tumor
cells, which tend to be in more acidic environments than
their well-oxygenated counterparts. Furthermore, the
observation that compounds 4 and 6 are capable of
reaching and killing hypoxic cells in solid tumors should
make these lead agents, and possibly other members of
this class, promising new candidates for potential clini-
cal use as adjuvants to radiation therapy.
a
BALB/c mice bearing well-established (ca. 100 mm3) intrad-
ermal EMT6 solid tumors were treated with a single 60 mg/kg
intraperitoneal injection of compound 4 or 6, followed 100 min later
by 15 Gy of total body radiation to the tumor-bearing animals.
Mice were then killed, solid tumors excised, single cell suspensions
prepared, and their cloning efficiencies ascertained. Results shown
are averages of two experiments.
Ta ble 2. Half-Wave Potentials of Nitrobenzyloxycarbonyl
Derivatives of 1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-
hydrazinesa
E1/2 (mV)
compd
pH 6.0
pH 7.0
∆V (mV)
4
6
-360
-467
-473
-545
-113
-78
a
Half-wave reduction potentials (E1/2) were calculated from
values of the potentials at the differential pulse polarographic
current peak in 100 mM potassium chloride, 50 mM potassium
phosphate buffer/electrolyte at pH values of 6.0 and 7.0 as
described in the Experimental Section.
Exp er im en ta l Section
Syn th esis. Melting points were determined with a Thomas-
Hoover melting point apparatus and are uncorrected. Proton
magnetic resonance spectra were recorded on a Varian EM-
390 spectrometer with tetramethylsilane as an internal stan-
dard. Elemental analyses were performed by the Baron
Consulting Co., Orange, CT, and were within (0.4% of the
calculated values for all compounds reported.
tially countered by the increase in H+ concentration due
to the impact of the H+ ion concentration term in the
equation below, which describes the redox potential of
the NADH/NAD+ couple:
NAD+ + H+ + 2e- ) NADH
Compounds 5 and 6 were synthesized using procedures
analogous to the one reported for compound 4.14
E ) E° - (RT/2F) ln [NADH]/[NAD+][H+]
2-(Ben zyloxyca r b on yl)-1,2-b is(m et h ylsu lfon yl)-1-(2-
ch lor oeth yl)h yd r a zin e (5). This compound was prepared by
reacting compound 1 (1.0 g, 4.0 mmol) with benzyl chlorofor-
mate (1.8 g, 10.5 mmol). The title compound (0.64 g, 41.3%)
The redox potential of the NADH/NAD+ couple is 30
mV greater (more positive) at pH 6.0 than at pH 7.0.
Therefore, a drop in pH of 1 unit would require a 10-
fold increase in the NADH/NAD+ ratio to maintain the
same redox potential. Despite these effects, the redox
potential of the NADH/NAD+ couple probably still
increases significantly under hypoxic conditions, as
indicated by a shift in the pyruvate/lactate equilibri-
um.27 Many oxidative and reductive processes (or sub-
sequent steps) involve [H+]; therefore, how readily these
steps occur will be influenced by pH. Ideally, a hypoxia-
selective agent should be reduced more readily under
more acidic conditions to exploit this difference. We have
measured the E1/2 of compounds 4 and 6 at both pH 6.0
and pH 7.0 (Table 2). Redox potential shifts of 113 and
78 mV were measured for compounds 4 and 6, respec-
tively. The shifts in redox potential probably occur as a
consequence of the interaction of H+ with the initial
1-electron reduction products (and the involvement of
H+ in subsequent steps), resulting in inhibition of the
reverse reaction.28 Attainment of a maximum dif-
ferential in the reduction rate between two pH values
requires the synthesis of compounds with large shifts
in redox potential in the pH range of interest. Studies
correlating the rate of reduction of various nitroimida-
zoles by xanthine oxidase with the redox potential have
demonstrated a 10-fold change in the reduction rate for
only a 70-80 mV change in redox potential.29 We believe
that the large redox shifts observed with compounds 4
1
was isolated as an oil. H NMR (acetone-d6): δ 7.2-7.6 (5H,
m, aromatic H), 5.4 (2H, s, ArCH2), 3.5-4.0 (4H, m, CH2CH2-
Cl), 3.4 and 3.1 (6H, 2s, 2 CH3SO2). Anal. (C12H17ClN2O6S2)
C, H, N.
1,2-B is (m e t h y ls u lfo n y l)-1-(2-c h lo r o e t h y l)-2-[(4,5-
dim eth oxy-2-n itr oben zyloxy)car bon yl]h ydr azin e (6). Com-
pound 1 (1.8 g, 7.2 mmol) was reacted with 4,5-dimethoxy-2-
nitrobenzyl chloroformate (2.0 g, 7.3 mmol) to give 0.556 g of
the title compound. This compound was recrystallized from
1
ethanol. Mp: 155-155.5 °C. Yield: 15.8%. H NMR (acetone-
d6): δ 7.7 and 7.4 (2H, 2s, aromatic H), 5.8 (2H, d, ArCH2),
3.7-4.1 (4H, m, CH2CH2Cl), 3.9-4.0 (6H, 2s, 2 OCH3), 3.5 and
3.2 (6H, 2s, 2 CH3SO2). Anal. (C14H20ClN3O10S2) C, H, N.
1,2-Bis(m et h ylsu lfon yl)-1-(2-ch lor oet h yl)-2-[[1-(4-n i-
t r op h en yl)et h oxy]ca r b on yl]h yd r a zin e (7). 1-(4-Nitro-
phenyl)ethanol (2.4 g, 14 mmol) in tetrahydrofuran (5 mL) was
added dropwise to a stirred, ice-cold solution of phosgene in
toluene (20% w/v, 30 mL). The flask was then wrapped in
aluminum foil and allowed to equilibrate to room temperature,
and the reaction mixture was stirred for an additional 24 h. A
dark oil was obtained upon evaporation of the reaction mixture
in vacuo at a temperature not exceeding 30 °C. To this oil were
added anhydrous acetonitrile (20 mL) and compound 1 (1.5 g,
6 mmol). After this mixture had cooled in an ice bath,
triethylamine (1.7 mL, 12 mmol) was added dropwise and the
reaction mixture stirred for 17 h at 4 °C. The reaction mixture
was evaporated to dryness on
a rotary evaporator at a
temperature not exceeding 30 °C. The residue was taken up
in ethyl acetate (150 mL) and washed with 2 × 50 and 1 ×
100 mL of hydrochloric acid (5%, w/v). The combined aqueous
layers were extracted with ethyl acetate (50 mL), and the

946 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 5
Brief Articles
(6) Kirkpatrick, D. L; J ohnson, K. E.; Sartorelli, A. C. Nitrobenzyl
derivatives as bioreductive alkylating agents: evidence for the
reductive formation of a reactive intermediate. J . Med. Chem.
1986, 29, 2048-2052.
(7) Tercel, M.; Wilson, W. R.; Denny, W. A. Nitrobenzyl mustard
quaternary salts: a new class of hypoxia-selective cytotoxin
showing very high in vitro selectivity. J . Med. Chem. 1993, 36,
2578-2579.
(8) Tercel, M.; Wilson, W. R.; Anderson, R. F.; Denny, W. A.
Hypoxia-selective antitumor agents. 12. Nitrobenzyl quaternary
salts as bioreductive prodrugs of the alkylating agent me-
chlorethamine. J . Med. Chem. 1996, 39, 1084-1094.
(9) Mulcahy, R. T.; Gipp, J . J .; Schmidt, J . P.; J oswig, C.; Borch, R.
F. Nitrobenzyl phosphorodiamidates as potential hypoxia-selec-
tive alkylating agents. J . Med. Chem. 1994, 37, 1610-1615.
(10) Shyam, K.; Hrubiec, R. T.; Furubayashi, R.; Cosby, L. A.;
Sartorelli, A. C. 1,2-Bis(sulfonyl)hydrazines. 3. Effects of struc-
tural modification on antineoplastic activity. J . Med. Chem.
1987, 30, 2157-2161.
(11) Shyam, K.; Penketh, P. G.; Divo, A. A.; Loomis, R. H.; Patton,
C. L.; Sartorelli, A. C. Synthesis and evaluation of 1,2,2-tris-
(sulfonyl)hydrazines as antineoplastic and trypanocidal agents.
J . Med. Chem. 1990, 33, 2259-2264.
(12) Shyam, K.; Penketh, P. G.; Divo, A. A.; Loomis, R. H.; Rose, W.
C.; Sartorelli, A. C. Synthesis and evaluation of 1-acyl-1,2-bis-
(methylsulfonyl)-2-(2-chloroethyl)hydrazines as antineoplastic
agents. J . Med. Chem. 1993, 36, 3496-3502.
(13) Shyam, K.; Penketh, P. G.; Loomis, R. H.; Rose, W. C.; Sartorelli,
A. C. Antitumor 2-(aminocarbonyl)-1,2-bis(methylsulfonyl)-1-(2-
chloroethyl)hydrazines. J . Med. Chem. 1996, 39, 796-801.
(14) Shyam, K.; Penketh, P. G.; Loomis, R. H.; Sartorelli, A. C.
Thiolysable prodrugs of 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-
hydrazine with antineoplastic activity. Eur. J . Med. Chem. 1998,
33, 609-615.
(15) Sykes, P. Guidebook to Mechanism in Organic Chemistry, 6th
ed.; J ohn Wiley: New York, 1986; pp 82-87.
(16) Carpenter, F. H.; Gish, D. T. The application of p-nitrobenzyl
chloroformate to peptide synthesis. J . Am. Chem. Soc. 1952, 74,
3818-3821.
(17) Furniss, B. S.; Hannaford, A. J .; Rogers, V.; Smith, P. W. G.;
Tatchell, A. R. Cognate preparation: m-nitrobenzyl alcohol.
Vogel’s Textbook of Practical Organic Chemistry, 4th ed.; Long-
man: London, 1978; p 357.
(18) Rockwell, S.; Keyes, S. R.; Sartorelli, A. C. Preclinical studies
of porfiromycin as an adjuvant to radiotherapy. Radiat. Res.
1988, 116, 100-113.
(19) Keyes, S. R.; Rockwell, S.; Sartorelli, A. C. Enhancement of
mitomycin C cytotoxicity to hypoxic tumor cells by dicumarol in
vivo and in vitro. Cancer Res. 1985, 45, 213-216.
(20) Rockwell, S.; Sartorelli, A. C. Mitomycin C and radiation. In
Antitumor Drug-Radiation Interactions; Hill, B. T., Bellamy, A.
S., Eds.; CRC Press: Boca Raton, FL, 1990; pp 125-139.
(21) Rockwell, S. In vivo-in vitro tumor systems: new models for
studying the response of tumors to therapy. Lab. Animal Sci.
1977, 27, 831-851.
(22) Wike-Hooley, J . L.; Haveman, J .; Reinhold, H. S. The relevance
of tumor pH to the treatment of malignant disease. Radiother.
Oncol. 1984, 2, 343-366.
(23) Vaupel, P. W.; Frinak, S.; Bicher, H. I. Heterogeneous oxygen
partial pressure and pH distribution in C3H mouse mammary
adenocarcinoma. Cancer Res. 1981, 41, 2008-2013.
(24) Gullino, P. M.; Grantham, F. H.; Smith, S. H.; Haggerty, A. C.
Modifications of the acid-base status of the internal milieu of
tumors. J . Natl. Cancer Inst. 1965, 34, 857-869.
extract was combined with the organic (ethyl acetate) layer
from the previous step. The combined ethyl acetate extracts
were washed with brine (100 mL), dried over anhydrous
magnesium sulfate, and filtered. The filtrate was evaporated
to dryness to give a viscous oil. The desired product was
obtained by column chromatography on silica gel (70-270
mesh, 60 A, chloroform-methylene chloride), followed by re-
crystallization from ethanol. Yield: 35%. Mp: 94-95 °C. 1H
NMR (acetone-d6): δ 8.3 and 7.8 (4H, 2d, aromatic H), 6.2 (1H,
m, ArCH), 3.6-4.1 (4H, m, CH2CH2Cl), 3.5 and 3.2 (6H, 2s, 2
CH3SO2), 1.7 (3H, dd, CH3). Anal. (C13H18ClN3O8S2) C, H, N.
Cytotoxicity Stu d ies. The ability of compounds 4-7 to
exert preferential toxicity to hypoxic cells was evaluated using
EMT6 mouse mammary carcinoma cells, by methodology
described previously.18,19 Briefly, exponentially growing mono-
layers of EMT6 cells were exposed to a continuously flowing
95% N2/5% CO2 humidified atmosphere for 2 h to produce
radiobiologic hypoxia. Parallel flasks were maintained simi-
larly in humidified 95% air/5% CO2. Without breaking the
hypoxia, cells were exposed to various concentrations of the
test agent for 1 h. Cell survival was then measured by colony
formation.
The capacity of the synthesized agents to reach and kill
hypoxic cells of solid tumors was evaluated using the in vivo-
in vitro test system described previously.20,21 BALB/c mice
bearing well-established (ca. 100 mm3) intradermal EMT6
solid tumors were treated with a single 60 mg/kg intraperi-
toneal injection of compound 4 or 6, followed 100 min later by
15 Gy of total body X-irradiation to the tumor-bearing animals;
this irradiation eliminated >99% of the oxygenated tumor
cells. Mice were then killed, the solid tumors excised, single-
cell suspensions prepared, and cloning efficiencies ascertained.
Decom p osition Stu d ies. The rates of decomposition of
compounds 4, 6, and 7 in the presence and absence of GSH
and/or GST were studied by following the acidification of
weakly buffered (1 mM potassium phosphate, pH 7.4, at 37
°C) solutions of phenol red (20 mg/L) spectrophotometrically
at 560 nm, as descibed previously.14
Deter m in a tion of Ha lf-Wa ve P oten tia ls. The polaro-
graphic half-wave potentials (E1/2) for compounds 4 and 6 were
calculated from potentials at half-peak current (Ep) measured
by differential pulse polarography, using the equation E1/2
)
Ep - Pa/2, where Pa is the pulse amplitude. Polarograms were
obtained using a PAR model 174A polarographic analyzer with
a PAR model 9346 dropping mercury electrode. A silver/silver
chloride half-cell was used as the reference electrode. Solutions
of compounds 4 and 6 (2.5 mM) in dimethyl sulfoxide were
added to the buffer/electrolyte (100 mM KCl, 50 mM KH2PO4/
K2HPO4) at various pH values to give a final concentration of
25 µM. The E1/2 values were recorded at room temperature
and represent the mean values of 3-5 determinations.
Ack n ow led gm en t. This research was supported in
part by U.S. Public Health Service Grant CA-74970
from the National Cancer Institute.
Refer en ces
(1) Kennedy, K. A.; Teicher, B. A.; Rockwell, S.; Sartorelli, A. C.
The hypoxic tumor cell: a target for selective cancer chemo-
therapy. Biochem. Pharmacol. 1980, 29, 1-8.
(25) Tannock, I. F.; Rotin, D. Acid pH in tumors and its potential for
therapeutic exploitation. Cancer Res. 1989, 49, 4373-4384.
(26) J ones, D. P.; Mason, H. S. Gradients of O2 concentration in
hepatocytes. J . Biol. Chem. 1978, 253, 4874-4880.
(2) J enkins, T. C. Hypoxia-selective agents: radiosensitizers and
cytotoxins. In The Chemistry of Antitumor Agents; Wilman, D.
E. V., Ed.; Blackie: Glasgow, 1990; pp 342-369.
(27) Newsholme, E. A.; Start, C. The [NAD+]/[NADH] ratios in the
cytoplasm and mitochondrial compartments of the liver cell.
Regulation in Metabolism; J ohn Wiley: London, 1977; pp 324-
328.
(3) Wilson, W. R.; Denny, W. A.; Twigden, S. J .; Baguely, B. C.;
Probert, J . C. Selective toxicity of nitracrine to hypoxic mam-
malian cells. Br. J . Cancer 1984, 49, 215-223.
(28) Edwards, D. I. Reduction of nitroimidazoles in vitro and DNA
damage. Biochem. Pharmacol. 1986, 35, 53-58.
(29) Clarke, E. D.; Wardman, P.; Goulding, K. H. Anaerobic reduction
of nitroimidazoles by reduced flavin mononucleotides and by
xanthine oxidase. Biochem. Pharmacol. 1980, 29, 2684-2687.
(4) Zeman, E. M.; Brown, J . M.; Lemmon, M. J .; Hirst, V. K.; Lee,
W. W. SR-4233: a new bioreductive agent with high selective
toxicity for hypoxic cells. Int. J . Radiat. Oncol. Biol. Phys. 1986,
12, 1239-1242.
(5) Teicher, B. A.; Sartorelli, A. C. Nitrobenzyl halides and carbam-
ates as prototype bioreductive alkylating agents. J . Med. Chem.
1980, 23, 955-960.
J M9805891