Synthesis and Evaluation of Nitroheterocyclic Carbamate Prodrugs for Use with Nitroreductase-Mediated Gene-Directed Enzyme Prodrug Therapy

Article abstract of DOI:10.1021/jm030308b

A variety of nitroheterocyclic carbamate prodrugs of phenylenediamine mustard and 5-amino-1-(chloromethyl)-3-[(5,6,7-trimethoxyindol-2-yl)carbonyl]-1, 2-dihydro-3H-benz[e]indoline (aminoseco-CBI-TMI), covering a wide range of reduction potential, were pre

Full text of DOI:10.1021/jm030308b

J . Med. Chem. 2003, 46, 5533-5545
5533
Syn th esis a n d Eva lu a tion of Nitr oh eter ocyclic Ca r ba m a te P r od r u gs for Use
w ith Nitr or ed u cta se-Med ia ted Gen e-Dir ected En zym e P r od r u g Th er a p y
Michael P. Hay,* Robert F. Anderson, Dianne M. Ferry, William R. Wilson, and William A. Denny
Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University of Auckland,
Private Bag 92019, Auckland, New Zealand
Received J une 24, 2003
A variety of nitroheterocyclic carbamate prodrugs of phenylenediamine mustard and 5-amino-
1-(chloromethyl)-3-[(5,6,7-trimethoxyindol-2-yl)carbonyl]-1,2-dihydro-3H-benz[e]indoline (amino-
seco-CBI-TMI), covering a wide range of reduction potential, were prepared and evaluated for
use in gene-directed enzyme prodrug therapy (GDEPT) using a two-electron nitroreductase
(NTR) from Escherichia coli B. The carbamate prodrugs and corresponding amine effectors
were tested in a cell line panel comprising parental and NTR-transfected human (SKOV3/
SKOV3-NTRneo, WiDr/WiDr-NTRneo), Chinese hamster (V79puro/V79-NTRpuro), and murine
(EMT6/EMT6-NTRpuro) cell line pairs and were compared with the established NTR substrates
CB1954 (an aziridinyl dinitrobenzamide) and the analogous dibromomustard. The 1-methyl-
2-nitroimidazol-5-ylmethyl carbamate of phenylenediamine mustard was metabolized rapidly
by EMT6-NTRneo but not EMT6 cells, demonstrating that it is an efficient substrates for NTR.
Despite this, the carbamates of phenylenediamine mustards show relatively low differential
cytotoxicity for NTR+ve cells in IC50 assays, apparently because they retain sufficient alkylating
reactivity that most of the prodrug reacts with nucleophiles during the drug exposure period.
In contrast, the corresponding amino-seco-CBI-TMI prodrugs were less efficient NTR substrates
but had greater chemical stability, were more potent, and showed substantial NTR-ve/NTR+ve
ratios in the cell line panel, with ratios of 15-100-fold for the 1-methyl-2-nitro-1H-imidazol-
5-ylmethyl and 1-methyl-5-nitro-1H-imidazol-2-ylmethyl carbamates of amino-seco-CBI-TMI.
The activity of these two prodrugs was evaluated against NTR-expressing EMT6 tumors
comprising ca. 10% NTR+ve cells. Small but not statistically significant killing of NTR+ve
cells was observed, with no effect against NTR-ve target cells. The lack of activity against
NTR+ve cells in tumors, despite potent and selective activity in culture, indicates that
pharmacokinetic optimization will be required if in vivo efficacy against solid tumors is to be
achieved with this new class of NTR prodrugs.
In tr od u ction
Gene-directed enzyme prodrug therapy (GDEPT) is
a novel approach to the treatment of cancer.1-3 This
approach uses various vector systems to selectively
deliver a gene encoding a nonhuman prodrug-activating
enzyme to tumor tissue. Administration of a suitable
prodrug for the enzyme results in generation of the drug
selectively in transfected tumor cells. Given the expec-
tation that only a small proportion of tumor cells will
express the activating enzyme, the released drug must
diffuse to kill neighboring tumor cells (the bystander
effect).
The minor aerobic nitroreductase (NTR) from Es-
cherichia coli B4-7 has been widely used in GDEPT,
activating nitroaromatic prodrugs by reducing the nitro
group (Hammet σp ) 0.78) to the corresponding hy-
droxylamine (σp ) -0.34). This electronic release acti-
vates a prepositioned functional group within the mol-
ecule to provide an active form of the drug.
Two main classes of prodrugs for NTR have been
evaluated. The prototypical example of the dinitrobenz-
amide class of prodrug, CB1954 (1),4,8,9 has advanced
to clinical trial,10,11 and the structure-activity relation-
ships (SAR) of related nitrogen mustards have been
determined empirically.12-14 The bromomustard 2 has
shown increased in vivo activity and a larger bystander
effect compared to CB1954 in preclinical models.15
The second class comprises 4-nitrobenzylcarbamates
of cytotoxic amines, exemplified by 3. Stabilization by
the carbamate occurs either because of the steric influ-
ence of the attached carbamate and/or because of the
* Corresponding author: Phone: 64-9-3737599, ext. 86598. Fax: 64-
9-3737502. E-mail: m.hay@auckland.ac.nz.
10.1021/jm030308b CCC: $25.00 © 2003 American Chemical Society
Published on Web 10/31/2003

5534 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Ch a r t 1. Compounds of Tables 1 and 2
Hay et al.
Sch em e 1
Sch em e 2a
electronic difference between the amine (σp ) -0.66)
and the carbamate (σp ) -0.17). Reduction of the 4-NO2
group to the hydroxylamine results in the fragmentation
of the carbamate linkage, releasing the active amine.
A wide range of amine-bearing DNA-reactive agents,
including aniline mustard,16 mitomycin,16 enediynes,17,18
seco-cyclopropylindoline derivatives,19 pyrrolobenzodi-
azepines,20 and DNA-binding agents including anthra-
cyclines16 and tallimustine analogues21 have been pro-
tected as 4-nitrobenzyl carbamates. However, efficacy
studies have been either rudimentary or failed to
identify a lead compound with in vivo activity in a
GDEPT protocol. We have recently shown that 2-alkoxy-
4-nitrobenzylcarbamates of 5-aminobenz[e]indolines are
good substrates for NTR in human cell lines in vitro,
being more selective than the unsubstituted 4-nitroben-
zyl carbamate.22 However, these analogues were spar-
ingly soluble, had reduction potentials considerably
lower than those of the established substrates 1 and 2,
and placed considerable steric demand on the NTR
binding site.
a
Reagents: (a) BH3‚DMS; (b) NaOMe, PhCHO, DMSO; (c)
TBDMSTf, pyridine, DCM; (d) O3, DCM, MeOH, then NaBH4,
EtOH.
In an effort to improve all of these factors, we
considered using a range of nitroheterocyclic carbamates
6c,d -18c,d (Chart 1) as potential prodrugs for NTR.
Fragmentation of 5-nitroimidazol-2-ylmethyl carbam-
ates after nitro group reduction has been documented
for ronidazole23 and for aniline mustard derivatives.24,25
Reactions of these 5-nitroimidazol-2-ylmethyl carbam-
ates have been suggested23-25 to proceed via reduction
to the hydroxylamine and nucleophilic attack by thiols
or water at the 4-position, with subsequent release of
the amine (Scheme 1). The chemical reduction of 5-ni-
trofuran-2-ylmethyl carbamates (7; R ) phenethyl) to
the corresponding amines has been explored as a model
for prodrug activation.26 We chose two amine cytotoxin
effectors for this study: the moderately potent phen-
ylenediamine mustard27 (4) and the very potent 5-amino-
1-(chloromethyl)-3-[(5,6,7-trimethoxyindol-2-yl)carbonyl]-
1,2-dihydro-3H-benz[e]indoline (amino-seco-CBI-TMI)
(5).28,29 These are both DNA alkylators30,31 capable of
killing both cycling and noncycling cells in a variety of
hypoxic and acidic microenvironments.32
In a preliminary study33 we showed that chemical,
radiolytic, or enzymic reduction of the 2-nitroimidazol-
5-ylmethyl carbamate model compound 9b induced
fragmentation of the carbamate and release of anisidine.
Furthermore, the 2-nitroimidazol-5-ylmethyl carbamate
9d of amino-seco-CBI-TMI (5) was 21-fold more toxic
in cell culture against a cell line (SKOV3-NTRneo
)
transfected with the NTR gene than the parent human
ovarian carcinoma line SKOV3.
With these precedents in mind, we sought to expand
the range of nitroheterocyclic carbamate-based prodrugs
for use with NTR. We report here the preparation and
in vitro and in vivo studies on a small set of 2- and
5-nitroimidazole, nitrothiophene, and nitrofuran pro-

Nitroheterocyclic Carbamate Prodrugs
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5535
Sch em e 3a
a
Reagents: (a) NO2PhOCOCl, pyridine, THF; (b) 35, pyridine; (c) TBAF, THF; (d) MsCl, pyridine; then LiCl, DMF.
Sch em e 4a
drugs (7c-10c) of phenylenediamine mustard 4 and a
larger range of prodrugs (spanning a wider range of
reduction potential and substitution pattern) of amino-
seco-CBI-TMI (5) (6d -18d ).
Ch em istr y
The alcohols 7a ,26 8a ,34 9a ,35 10a ,35 12a ,36 13a ,37 and
15a -18a 36 required for carbamate formation were
synthesized as previously described. (1-Methyl-5-nitro-
1H-pyrazol-4-yl)methanol (6a ) was prepared by BH3‚
DMS reduction of the corresponding acid38 19 (Scheme
2). Introduction of the hydroxymethyl substituent in the
a
Reagents: (a) TBDMSCl, imidazole, DMF; (b) Pd/C, H2, EtOH;
2-position of the imidazole derivatives 23 was conve-
niently achieved by condensation of the corresponding
methyl analogue 20 with benzaldehyde to give the
styrene 21.39 This approach allowed selective protection
of the side chain alcohol to give 22, thus allowing
regiospecific carbamate formation with alcohol 23. Ozo-
nolysis of the styrenes 22 and 24, followed by a reductive
workup, gave alcohols 23 and 14a in good yields (88%
and 78%, respectively).
(c) MeOPhNCO, nBu2Sn(OAc)2, DCM.
Sch em e 5a
Two general methods were used for carbamate forma-
tion. For the more nucleophilic anilines, reaction with
activated 4-nitrophenyl carbonates was used. Thus,
alcohols 7a and 8a were activated as 4-nitrophenyl
carbonates 25 and 26, and displacement with 35 gave
diols 29 and 30, respectively, in moderate yields for the
two steps (Scheme 3). Formation of mustards 7c and
8c was achieved in good yield via mesylation and
displacement with LiCl in DMF. Similarly, activation
of the nitroimidazoles 9a and 10a as carbonates 27 and
28 ensured facile reaction with aniline 38, prepared
from diol 34 (Scheme 4), to give the protected carbam-
ates 31 and 33 in good yields (82 and 84%, respectively).
The silyl protecting groups were readily removed using
TBAF, and the resulting diols 32 and 34 elaborated to
the mustards 9c and 10c using standard conditions. The
use of the silyl protecting groups gave an improved yield
in the coupling step compared to 25 and 26, primarily
due to improved isolation of the products. However,
when combined with only good yields (88 and 82%) in
the deprotection steps, this approach did not provide a
significant benefit over the direct route. An alternate,
more direct approach with activation of the alcohol 10a
using diphosgene and reaction of the resulting chloro-
formate with phenylenediamine mustard 4 gave a poor
yield of carbamate 10c. Although giving 10c directly,
a
Reagents: (a) triphosgene, pyridine, DCM, then 5; (b) 5,
triphosgene, Et3N, DCM, then 6a -18a , nBu2Sn(OAc)2, DCM; (c)
5, DIEA, HOBT, 4 Å molecular sieves, DMF; (d) HCl, aq MeOH.
this approach is less effective than the multistep ap-
proach used above.
For the more weakly nucleophilic amino-seco-CBI-
TMI (5), attempts to form carbamates by in situ forma-
tion of the chloroformate of alcohol 9a and reaction with
5 were plagued by low and variable yields (0-33%)
(Scheme 5). Reaction of the carbonate 27 with amine 5
was extremely slow, and while addition of HOBT40
provided an increase in rate, the reaction was still low-
yielding (Scheme 5). Formation of the isocyanate of 5
in situ,22 and subsequent reaction with alcohol 9a in
the presence of catalytic dibutyltin diacetate,41 was
found to be a superior method. The reaction did not
proceed in the absence of dibutyltin diacetate. Thus,
reaction of amine 5 with triphosgene and Et3N in DCM
followed by addition of alcohols 6a -18a and dibutyltin
diacetate gave variable yields (28-89%) of carbamates
6d -18d .

5536 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Hay et al.
Ta ble 1. Physicochemical Data, IC50 Values for Parental Cells Lines, and IC50 Ratios (NTR-ve/NTR+ve) for Phenylenediamine
Mustard (4) and Its Nitroheterocyclic Carbamate Prodrugs 8c-10c; Comparison with the Reference Dinitrobenzamide Prodrugs CB
1954 (1) and 2
SKOV3/
SKOV3-
NTRneo d,e
WiDr/
WiDr-
V79/
V79-
EMT6/
EMT6-
E(1) pH 7.0
SKOV3
WiDr
V79
EMT6
no. sol.a
(mV)b
refc
(µM)d
(µM)d
NTRneo d,e
(µM)d
NTRpuro d,e
(µM)d
NTRpuro d,e
1
2
4
8c
9c
10c
160 0
40
nd
10
nd
393 ( 7
ndf
nd
-394 ( 7 MV 1.7 ( 0.2
-421 ( 8 MV 2.3 ( 0.2
-483 ( 8 MV 2.1 ( 0.2
BV
nd
174 ( 10
58 ( 10
317 ( 21
211 ( 16
54 ( 3
40 ( 3
3.9 ( 1.0
1.39 ( 0.07
1.7 ( 0.2
2.0 ( 0.1
51 ( 2
174 ( 31
1.3 ( 0.2
2.8 ( 0.3
6.7 ( 1.8
2.01 ( 0.02
374 ( 14
43 ( 5
1.9 ( 0.2
0.92 ( 0.02
1.6 ( 0.2
1.7 ( 0.2
2090 ( 210
302 ( 93
71 ( 7
930 ( 140
54
1380
nd 3.74 ( 0.06 0.97 ( 0.02
0.57 ( 0.05 1.06 ( 0.05 0.51 ( 0.04
2.9 ( 0.5
28 ( 3
3.7 ( 0.8
2.3
5.3 ( 0.2
1.3 ( 0.2
1.16 ( 0.07
1.3 ( 0.3
1.63 ( 0.03
6.1 ( 1.3
22 ( 6
6.1 ( 1.1
11
a
b
Solubility (µM) in R-MEM culture medium, determined by HPLC. Measured on 8a -10a ; one-electron potential in mV determined
by pulse radiolysis. c BV, benzyl viologen (1,1′-dibenzyl-4,4′-bipyridinium) ) -375 ( 10 mV;55 MV, methyl viologen (1,1′-dimethyl-4,4′-
bipyridinium) ) -447 ( 7 mV.56 Values are mean ( SEM for up to five independent experiments. e Intraexperiment ratios. f Not
d
determined.
Ta ble 2. Physicochemical Data, IC50 Values for Parental Cells Lines, and IC50 Ratios (NTR-ve/NTR+ve) for Amino-seco-CBI-TMI (5)
and Its Nitroheterocyclic Carbamate Prodrugs 6d -18d
E(1)b
pH 7.0
(mV)
SKOV3/
SKOV3-
NTRneo d,e
WiDR/
WiDr-
V79/
V79-
EMT6/
EMT6-
SKOV3
(nM)d
WiDr
(nM)d
V79
EMT6
(nM)d
no. sol.a
refc
nd
NTRneo d,e
(nM)d
NTRpuro d,e
NTRpuro d,e
5
6d
197
8
ndf
1.1 ( 0.08 0.55 ( 0.04 1.6 ( 0.1
0.9 ( 0.3
0.33 ( 0.07 0.9 ( 0.1 0.37 ( 0.06 2.0 ( 0.7
-222 ( 6 MEN
32 ( 2
76 ( 7
MV 10.6 ( 0.8
30.3 ( 0.7 11.3 ( 0.6 13.5 ( 1.8
41 ( 5
20 ( 2
3.5 ( 0.3
24 ( 2
68 ( 10
94 ( 11
24 ( 2
44 ( 9
55 ( 4
53 ( 23
52 ( 5
62 ( 5
6 ( 1
37 ( 4
1.1 ( 0.2
3.1 ( 0.9
39 ( 8
18 ( 1
4.3 ( 1.0
87 ( 21
1.2 ( 0.2
16 ( 3
62( 4
71 ( 5
11 ( 3
12 ( 3
6.5 ( 0.3
2.1
7d
8d
11
4
-381 ( 10
-394 ( 7
-421 ( 8
-483 ( 8
-482 ( 8
-496 ( 9
-525 ( 8
-577 ( 8
-605 ( 11
-611 ( 7
-627 ( 7
-671 ( 7
BV
1.2 ( 0.2
7 ( 0.8
21 ( 2
50 ( 1
26 ( 2
9.6 ( 1.6
5.6 ( 0.2
4.8
89 ( 8
18 ( 4
75 ( 11
230 ( 1.0
1.9 ( 0.2
13.5 ( 1.9
40 ( 6
9d
17
8
MV
MV
TQ
TQ
MV
MV
TQ
TQ
TQ
TQ
75 ( 7
146 ( 18
378 ( 79
97 ( 8
14.7 ( 1.7 19.6 ( 0.4
10d
11d
12d
13d
14d
15d
16d
17d
18d
100 ( 19
427 ( 41 12.4 ( 0.02
10 ( 4
1.2 ( 0.1
2.0 ( 0.0
1.4 ( 0.2
1.2 ( 0.0
1.0 ( 0.1
1.8
78 ( 5
129 ( 32
28 ( 3
63 ( 5
73 ( 18
49 ( 5
nd
20
nd
5.5
13
9
21
18
8.4
94 ( 13
24 ( 1
201 ( 9
182 ( 31
223 ( 17
219 ( 27
28 ( 4
13 ( 3
3.4 ( 0.7
2.8 ( 0.8
2.5 ( 0.1
1.3
1.8 ( 0.4
1.4 ( 0.2
61 ( 3
185 ( 13
265 ( 60
98 ( 22
116 ( 4
25 ( 6
1.3 ( 0.5
1.2 ( 0.6
0.8
nd
1.3
1.5
0.5 ( 0.0
0.7 ( 0.1
1.5 ( 0.3
0.9 ( 0.2
64 ( 3
15 ( 6
a
b
Solubility (µM) in R-MEM culture medium, determined by HPLC. Measured on 6a -18a ; one-electron potential in mV determined
by pulse radiolysis. c MEN, menadione ) -203 ( 5 mV;42 BV, benzyl viologen (1,1′-dibenzyl-4,4′-bipyridinium) ) -375 ( 10 mV;55 MV,
methyl viologen (1,1′-dimethyl-4,4′-bipyridinium) ) -447 ( 7 mV;56 TQ, triquat (7,8-dihydrodipyrido-[1,2-a:2′,1′-c][1,4]diazepinediium)
) -548 ( 7 mV.56 Values are mean ( SEM for up to five independent experiments. e Intraexperiment ratios. f Not determined.
d
Model carbamate 9b (Scheme 4) was prepared as
described previously35 by reaction with 4-methoxyphen-
ylisocyanate and di-n-butyltin diacetate in DCM, and
10b was prepared similarly from 10a .
The prodrugs 8c-10c were unstable in R-MEM
culture medium + 5% FCS over a 24 h period, whereas
the prodrugs of 5 (with the exception of the 5-nitroim-
idazole 11d and the pyrrole esters 12d and 16d ) were
stable under these conditions (as were the mustards 1
and 2). 5-Nitroimidazole 11d gave 30% of a more polar
metabolite after 24 h, while the pyrrole esters 12d and
16d both underwent ca. 50% conversion to more polar
metabolites, consistent with hydrolysis of the ester
groups. Importantly, formation of the parent drug 5 was
not observed for any of the amino-seco-CBI-TMI pro-
drugs 6d -18d .
One-electron reduction potentials [E(1)], determined
by pulse radiolysis, have been used as a surrogate
measurement of the potential for hydride transfer from
FMN to the substrate in reductase enzymes.12-14 For
most two-electron reduction systems, the potential for
addition of the second electron [E(2)] is more positive
than for the first [E(1)]43 and the two-electron reduction
potential is half the sum of E(1) and E(2).44 Studies with
a related oxygen-insensitive nitroreductase (NR; EC
1.6.99.7) from Enterobacter cloacae found a relationship
between the initial rates of reduction and E(1) for a
series of para-substituted nitrobenzenes.45 Further stud-
ies with E. cloacae suggested a more complex parabolic
relationship between E(1) and reduction rate.46 None-
theless, E(1) is expected to provide a guide to the rate
of nitro group reduction for a substrate by NTR. The
Pulse radiolysis experiments were performed on a
Dynaray 4 (4 MeV) linear accelerator (200 ns pulse
length with a custom-built optical radical detection
system). One-electron reduction potentials [E(1)] for the
compounds were determined in anaerobic aqueous solu-
tions containing 2-propanol (0.1 M) buffered at pH 7.0
(10 mM phosphate) by measuring the equilibrium
constant42 for the electron transfer between the radical
anions of the compounds and the appropriate viologen
or quinone reference standard. Data were obtained at
three concentration ratios.
Resu lts a n d Discu ssion
The solubility of compounds in R-MEM culture me-
dium with 5% added fetal calf serum (FCS) was deter-
mined by HPLC analysis of the supernatant of a
saturated solution. All the prodrugs (8c, 10c, and 6d -
18d ) were considerably less soluble than CB1954 (1) (4-
21 µM, compared with 1600 µM) and slightly less soluble
than the bromomustard 2 (Table 1). The prodrugs 6d -
18d were also less soluble than the corresponding amine
5 (197 µM) (Table 2). However, they were more soluble
(5-40-fold) than the corresponding 4-nitrobenzyl car-
bamate of 5 (0.8 µM).22

Nitroheterocyclic Carbamate Prodrugs
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5537
F igu r e 1. (A) Stability of 9c (10 µM) in RMEM culture medium, O; with EMT6 cells (106 mL-1), b; with EMT6-NTRpuro
(106 mL-1), 2; under aerobic conditions. (B). Stability of 9d (10 µM)) in RMEM culture medium with EMT6 cells (106 mL-1), b;
with EMT6-NTRpuro (106 mL-1), 2; under aerobic conditions.
low solubility of the prodrugs 6d -18d precluded E(1)
measurements on these compounds, but these were
determined for the corresponding methanols 6a -18a
(Table 2) and two model carbamates 9b and 10b. The
values for the latter (-419 ( 8 mV and -477 ( 9 mV,
respectively) were indistinguishable from those of the
parent alcohols, suggesting that the E(1) values for 6a -
18a would reasonably approximate E(1) values for 6d -
18d . The reduction potentials of these alcohols spanned
a wide range [-222 ( 6 mV (6a ) to -671 ( 7 mV (18a )]
(Table 2), much larger than that previously covered.
4-Nitrobenzyl alcohol, which has been widely used
previously to prepare carbamate substrates (3) for
NTR,16-22 has a reduction potential of -494 mV, while
CB1954 (1) has a reduction potential of -385 mV47 and
the chloromustard analogue of 2 is intermediate at
-421 ( 8 mV.30
The effectors and prodrugs were evaluated for cyto-
toxicity in four pairs of cell lines, each comprising a non-
NTR-expressing cell line [SKOV3 (human ovarian car-
cinoma), WiDr (human colon carcinoma), V79puro (Chinese
hamster fibroblast), and EMT6 (mouse mammary car-
cinoma)], and the corresponding transfectants stably
expressing NTR. Cytotoxicity was measured as IC50
values (following an 18 h drug exposure) in the NTR-
ve lines, together with the ratios of the IC50 values
between the NTR-ve and NTR+ve lines (Tables 1 and
2) as a measure of selectivity for NTR-expressing cells.
The compounds were compared with CB1954 (1) and
the related dibromomustard 2 in order to evaluate their
potential as GDEPT candidates.
expressing cells, with the largest NTR-ve/+ve ratios
(5-28-fold) for the 2-nitroimidazole 9c.
In an effort to explain the increase in toxicity dis-
played by 8c-10c relative to 4, the stability of 9c and
9d in culture medium was examined by LC/MS, follow-
ing incubation in RMEM culture medium at 37 °C, with
and without EMT6 or the transfected EMT6-NTRpuro
cells at 106/mL, under aerobic conditions. Rapid loss of
9c was observed in culture medium with a T1/2 of ca.
1.0 h (Figure 1A), with no appreciable change in T1/2 in
the presence of EMT6 cells (T1/2 ) 1.1 h). The major
products observed by mass spectrometry were consistent
with the monohydroxy and dihydroxy hydrolysis prod-
ucts of 9c (MH+ ) 398 and 380, respectively). In the
presence of EMT6-NTRpuro cells the rate of loss of 9c
from the extracellular medium was increased 18-fold
(T1/2 ) 0.06 h) and only very low levels of the mustard
hydrolysis products were detected. In contrast, 9d was
stable in culture medium over 24 h and in the presence
of EMT6 cells but was consumed (T1/2 ) 0.7 h) in the
presence of EMT6-NTRpuro cells (Figure 1B). The rela-
tive rates of consumption indicate that 9c is a better
substrate than 9d , but 9c is hydrolytically unstable,
despite the presumed deactivating effect of the carbam-
ate linkage on mustard reactivity. Thus, the cytotoxicity
of 4 (IC50 ca. 1-4 µM) observed in the cell line panel
with an 18 h drug exposure appears underestimated due
to rapid hydrolysis of the mustard group.48 The mustard
group in 9c may be sufficiently stabilized by the increase
in σ [σp(OCONH) ) -0.17; σp(NH2) ) -0.66] to provide
improved delivery of the mustard to the nuclear com-
partment and consequently increased cytotoxicity, but
it is not sufficiently stabilized for it to be a useful
prodrug.
Phenylenediamine mustard (4) is an alkylating agent
that has been used in a number of bioreductive prodrug
approaches.16,24,25 It has only moderate potency, with
IC50 values of ca. 1-4 µM in the cell line panel (Table
1). The carbamate derivatives 8c-10c were slightly
more toxic (ca. 2-fold) than 4 in the wild-type human
lines and equitoxic in the rodent cell lines. Given this
lack of deactivation, the carbamates would not be
expected to act as NTR prodrugs. However, surprisingly,
all three showed significant increases in toxicity in NTR-
This unfavorable balance between chemical reactivity
and biological cytotoxicity for carbamates 7c-10c
prompted us to consider the more potent alkylating
cytotoxin amino-seco-CBI-TMI (5), in which the chemical
reactivity and cytotoxicity are less strongly linked. The
effector 5 is ca. 1000-fold more potent than 4,28,29 with
IC50 values of 0.3-1.6 nM across the panel of parental

5538 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Hay et al.
(non-NTR-expressing) cell lines. Masking the amino
group as a nitroheterocyclic carbamate (6d -18d ) pro-
vided a variable (7-348-fold) decrease in cytotoxic
potency in the NTR-negative cell lines, with the 5-ni-
troimidazole 11d providing the greatest deactivation
(66-348-fold).
For these compounds, the reduction potential signifi-
cantly influenced selectivity. The nitropyrrole analogues
(15d -18d ), with E(1) values below -600 mV, were not
substrates for NTR, as shown by cell line ratios (NTR-
ve/NTR+ve) of ca. 1. Analogues 12d -14d , with E(1)
values between -500 and -600 mV, were poor sub-
strates, inferred by cell line ratios of ca. 10 [conjugation
of the ethyl carboxylate with the nitro group in 12d
raises the E(1) sufficiently for reduction to occur, despite
the nitro group being in a sterically hindered environ-
ment]. For compounds with E(1) above ca. -500 mV,
the data are more complex. As the E(1) increases to ca.
-394 mV (10d -8d ) there is a corresponding increase
in cytotoxic potency in the NTR-ve cell lines, yet the
most electron-deficient compounds (the nitrofuran 7d
and the nitropyrazole 6d ) are relatively nontoxic. There
is also a decrease in selectivity for NTR with increasing
E(1) for compounds 10d -7d with the nitropyrazole 6d
displaying moderate selectivity. The 2-nitroimidazole 9d
had a selectivity of 15-62-fold while the 5-nitroimida-
zole 10d was less potent (IC50 ) 68-146 nM) but more
selective (50-99-fold) for cells expressing NTR.
The addition of a larger N-1 side chain in compound
11d resulted in lower cytotoxic potency (266-348 nM)
and selectivity (1.2-26-fold). This suggests that steric
bulk adjacent to the nitro group is not tolerated by the
NTR enzyme in this series of prodrugs. Overall, the IC50
ratios (NTR-ve/NTR+ve) of prodrugs 6d , 9d , and 10d
were similar to that of the dinitrobenzamides 1 and 2
for the human cell lines (SKOV3 and WiDr), although
their ratios in the two rodent cell line pairs were much
lower. The reduction potentials, potencies, and cell line
ratios for 9d and 10d are broadly similar to those
previously seen for the related 2-alkoxy-4-nitrobenzyl
carbamates of amino-seco-CBI-TMI.22
F igu r e 2. Comparison of in vivo activity and bystander effects
for CB1954 (1), bromomustard 2, 2-nitroimidazole carbamate
9d , and 5-nitroimidazole carbamate 10d . Prodrugs given at
200, 1330, 1000, and 1000 µmol kg-1 ip. In this experiment,
tumors at excision comprised 9.2% EMT6-NTRpuro as assessed
by the proportion of puromycin-resistant cells. Filled bars,
EMT6-NTRpuro; shaded bars, EMT6. * EMT6-NTRpuro < 10-4
clonogens/g.
respectively, show significant killing of activator (EMT6-
NTRpuro) cells and target (EMT6) cells, indicating the
operation of a bystander effect. The lack of activity of
9d and 10d against solid tumors expressing only a
fraction of NTR+ve cells may have several origins. The
pharmacokinetics of these compounds have yet to be
investigated and may not be optimal. Recent studies
have also highlighted the requirement for high ex-
travascular diffusion for bioreductive drugs targeted to
the hypoxic subfraction of tumors.50 Analogously, it is
possible that the diffusion of these NTR prodrugs
through the extravascular compartment to a limited
number of NTR-transfected cells within the tumor may
also be critical for in vivo activity. These possibilities
require further investigation.
Prodrugs 9d and 10d were selected for in vivo
evaluation because of their good selectivity in vitro. The
maximum tolerated dose (MTD) in C3H mice of effector
5 given ip in DMSO was 2.37 µmol kg-1, whereas the
MTD of 9d and 10d was >1000 µmol kg-1, demonstrat-
ing substantial masking of toxicity of the CBI effector
in the prodrug form in vivo. In comparison, the MTD of
dinitrobenzamide 1 was 240 µmol kg-1 given ip in 10%
DMA/40% PEG400/50% water, whereas 2 (DMSO) was
considerably less toxic (>1330 µmol kg-1). The activities
of 9d and 10d were evaluated against NTR-expressing
EMT6 tumors containing mixtures of NTR-ve and
NTR+ve cells. In this model nude mice are inoculated
with 2:1 mixtures of EMT6-NTRpuro and EMT6 (NTR-
ve) cells respectively, providing ca. 10% NTR+ve cells
at the time of tumor treatment.49 This tumor model
represents the likely situation in a GDEPT protocol,
where low rates of transfection of tumor tissue are
expected. Although a small decrease in tumor cells was
observed after treatment with 9d or 10d , these effects
were not statistically significant (Figure 2). In contrast,
as we have previously demonstrated,22 dinitrobenz-
Con clu sion s
A range of nitroheterocyclic carbamates of the phen-
ylenediamine mustard (4) showed low selectivity for
NTR and were unsuitable as prodrugs because of their
chemical reactivity and consequent instability in culture
medium. A study of similar analogues of the amino-seco-
CBI-TMI alkylating agent 5, spanning a wider range
of reduction potentials and structural variation, showed
the important influence of reduction potential on both
potency and selectivity for NTR-transduced cells. The
selectivity of carbamates 6d -18d for NTR across the
cell line panel was greatest for those with E(1) values
between ca. -400 mV and ca. -500 mV, with the
exception of the nitropyrazole carbamate 6d . These
prodrugs had solubilities broadly comparable to those
of the “solubilized” nitrobenzyl carbamate prodrugs
previously described.22 This study identified analogues
(6d , 9d , and 10d ) with good selectivity for NTR in the
human (SKOV3 and WiDr) and murine (EMT6) cell line
pairs (although not as selective as the established
dinitrobenzamide NTR prodrugs 1 and 2). In contrast
amides 1 and 2 given at 200 and 1330 µmol kg-1
,

Nitroheterocyclic Carbamate Prodrugs
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5539
a yellow powder: mp (EtOAc/pet. ether) 155 °C (lit.39 mp 156-
157 °C); 1H NMR δ 8.06 (s, 1 H, H-4′), 7.83 (d, J ) 15.8 Hz, 1
H, CHd), 7.52-7.58 (m, 2 H, H-2′′, H-6′′), 7.33-7.38 (m, 3 H,
H-3′′, H-4′′, H-5′′), 7.05 (d, J ) 15.8 Hz, 1 H, CHd), 4.64 (dd,
J ) 5.1, 5.0 Hz, 2 H, H-1), 4.07 (dd, J ) 5.1, 5.0 Hz, 2 H, H-2),
2.42 (br s, 1 H, OH); 13C NMR δ 150.9, 140.0, 138.5, 135.3,
134.6, 129.7, 128.9 (2), 127.6 (2), 112.1, 61.8, 47.7.
to 1 and 2, carbamates 9d and 10d did not show
significant antitumor activity in the EMT6/EMT6-
NTRpuro tumor model. The lack of activity against
NTR+ve cells in tumors, despite potent and selective
activity in culture, indicates that pharmacokinetic
optimization will be required if in vivo efficacy against
solid tumors is to be achieved with this new class of NTR
prodrugs.
TBDMS triflate (2.7 mL, 11.8 mmol) was added dropwise
to a stirred solution of alcohol 21 (2.77 g, 10.7 mmol) and
pyridine (1.3 mL, 16.0 mmol) in DCM (100 mL) at -5 °C and
the solution stirred at 20 °C for 16 h. The reaction was
quenched with MeOH (5 mL) and poured into saturated
aqueous KHCO3 (100 mL). The mixture was extracted with
DCM (3 × 50 mL), the combined organic fraction dried, and
the solvent evaporated. The residue was purified by chroma-
tography, eluting with 20% EtOAc/pet. ether, to give tert-butyl-
(dimethyl)silyl 2-{5-nitro-2-[(E)-2-phenylethenyl]-1H-imidazol-
1-yl}ethyl ether (22) (4.00 g, 100%) as a yellow solid: mp
(EtOAc/pet. ether) 99-100.5 °C; 1H NMR δ 8.13 (s, 1 H, H-4′),
7.87 (d, J ) 15.8 Hz, 1 H, CHd), 7.57 (d, J ) 6.8 Hz, 2 H,
H-2′′, H-6′′), 7.34-7.41 (m, 3 H, H-3′′, H-4′′, H-5′′), 7.06 (d,
J ) 15.8 Hz, 1 H, CHd), 4.62 (dd, J ) 5.0, 4.8 Hz, 2 H, H-1),
4.00 (dd, J ) 5.0, 4.8 Hz, 2 H, H-2), 0.77 [s, 9 H, SiC(CH3)3],
0.10 [s, 6 H, Si(CH3)2]; 13C NMR δ 151.2, 139.3, 138.4, 135.5,
134.8, 129.6, 128.9 (2), 127.5 (2), 112.9, 62.3, 47.8, 25.7 (3),
18.1, -5.8 (2). Anal. (C19H27N3O3Si) C, H, N.
Ozone was bubbled into a solution of imidazole 22 (1.3 g,
3.5 mmol) in DCM/MeOH (1:1, 120 mL) at -78 °C until a blue
color persisted. The solution was warmed to -40 °C with a N2
purge to remove excess ozone. A solution of NaBH4 (132 mg,
3.5 mmol) in EtOH (10 mL) was added dropwise over 15 min
and the mixture stirred for 30 min. The mixture was treated
with acetic acid (0.5 mL) and stirred for 10 min, and the
solvent was evaporated. The residue was partitioned between
EtOAc (100 mL) and water (100 mL). The organic fraction was
washed with water (50 mL) and brine (25 mL) and dried, and
the solvent was evaporated. The residue was purified by
chromatography, eluting with 50% EtOAc/pet. ether, to give
23 (0.92 g, 88%) as a white solid: mp 104-105 °C; 1H NMR δ
7.97 (s, 1 H, H-4), 4.79 (s, 2 H, CH2O), 4.62 (t, J ) 4.8 Hz, 2
H, CH2O), 3.97 (t, J ) 4.8 Hz, 2 H, CH2N), 3.80 (br s, 1 H,
OH), 0.81 [s, 9 H, SiC(CH3)3], 0.10 [s, 6 H, Si(CH3)2]; 13C NMR
δ 157.2, 138.8, 132.3, 62.0, 57.2, 48.3, 25.7 (3), 18.2, -5.8 (2).
Anal. (C12H23N3O4Si) C, H, N.
(1-Met h yl-4-n it r o-1H -im id a zol-5-yl)m et h a n ol (14a ).
Ozone was bubbled through a solution of 1-methyl-4-nitro-5-
[(E)-2-phenylethenyl]-1H-imidazole37 (24) (1.0 g, 4.4 mmol) in
DCM/MeOH (1:1, 120 mL) at -78 °C until a blue color
persisted. The solution was warmed to -40 °C with a nitrogen
purge to remove excess ozone. A solution of NaBH4 (165 mg,
4.4 mmol) in EtOH (10 mL) was added dropwise over 15 min
and the mixture stirred for 30 min. Acetic acid (0.5 mL) was
added and the solvent evaporated. The residue was partitioned
between water (50 mL) and pet. ether (50 mL). The aqueous
fraction was evaporated and the residue triturated with hot
acetone (60 mL). The mixture was filtered and the solution
concentrated to give 14a (523 mg, 78%) as a tan powder: mp
(acetone) 135-137 °C [lit.51 mp (CHCl3) 239-240 °C]; 1H NMR
δ 7.78 (s, 1 H, H-2), 5.48 (t, J ) 5.6 Hz, 1 H, OH), 4.85 (d, J )
5.6 Hz, 2 H, CH2O), 3.75 (s, 3 H, NCH3); 13C NMR δ 143.5,
136.8, 133.2, 51.4, 32.5. Anal. (C5H7N3O3) C, H, N.
Exp er im en ta l Section
Analyses were carried out in the Microchemical Laboratory,
University of Otago, Dunedin, NZ. Melting points were
determined on an Electrothermal 2300 melting point ap-
paratus. IR spectra were recorded on a Midac FT-IR as KBr
disks, unless otherwise stated. NMR spectra were obtained
on a Bruker Avance 400 spectrometer at 400 MHz for 1H and
100 MHz for 13C spectra. Spectra were obtained in CDCl3
unless otherwise specified, and are referenced to Me4Si.
Chemical shifts and coupling constants were recorded in units
of ppm and Hz, respectively. Assignments were determined
using COSY, HSQC, and HMBC two-dimensional experiments.
Mass spectra were determined on a VG-70SE mass spectrom-
eter using an ionizing potential of 70 eV at
a nominal
resolution of 1000. High-resolution spectra were obtained at
nominal resolutions of 3000, 5000, or 10 000 as appropriate.
All spectra were obtained as electron impact (EI) using PFK
as the reference unless otherwise stated. Solutions in organic
solvents were dried with anhydrous Na2SO4 and solvents were
evaporated under reduced pressure on a rotary evaporator.
Thin-layer chromatography was carried out on aluminum-
backed silica gel plates (Merck 60 F254) with visualization of
components by UV light (254 nm) or exposure to I2. Column
chromatography was carried out on silica gel, (Merck 230-
400 mesh). All compounds designated for testing were analyzed
at >99% purity by reverse phase HPLC using an Agilent 1100
liquid chromatograph, an Altima C18 (5 µM) stainless steel
column (150 mm × 3.2 mm i.d.) and an Agilent 1100 diode
array detector. Chromatograms were run using various gra-
dients of aqueous (0.045 M ammonium formate and formic acid
at pH 3.5) and organic (80% MeCN/MilliQ water) phases. DCM
refers to dichloromethane; DMF refers to dry dimethyl form-
amide; ether refers to diethyl ether; EtOAc refers to ethyl
acetate; EtOH refers to ethanol; MeOH refers to methanol;
pet. ether refers to petroleum ether, boiling range 40-60 °C;
THF refers to tetrahydrofuran dried over sodium benzophe-
none ketyl. All solvents were freshly distilled. Alcohols 7a ,26
8a ,34 9a ,35 10a ,35 12a ,36 13a ,37 and 15a -18a 36 were synthesized
as previously described.
(1-Meth yl-5-n itr o-1H-p yr a zol-4-yl)m eth a n ol (6a ). Bo-
rane dimethyl sulfide (2 M solution in THF, 4.2 mL, 8.4 mmol)
was added to a solution of 1-methyl-5-nitro-1H-pyrazole-4-
carboxylic acid38 (19) (1.11 g, 6.5 mmol) in THF (50 mL) under
N2, and the mixture stirred at reflux temperature for 80 min
and then cooled. MeOH (5 mL) and then water (5 mL) and
then 2 M HCl (5 mL) were added, the THF was evaporated,
and the residue was diluted with water and extracted with
EtOAc (3 × 50 mL). The combined organic extract was dried,
the solvent evaporated, and the residue purified by chroma-
tography, eluting with 50% EtOAc/pet. ether, to give 6a (0.52
1
g, 51%) as a white solid: mp (benzene) 78-80 °C; H NMR δ
7.58 (s, 1 H, H-3), 4.82 (d, J ) 3.4 Hz, 2 H, CH2O), 4.25 (s, 3
H, NCH3), 2.39 (br s, 1 H, OH). Anal. (C5H7N3O3) C, H, N.
[1-(2-{[ter t-Bu tyl(d im eth yl)silyl]oxy}eth yl)-5-n itr o-1H-
im id a zol-2-yl]m eth a n ol (23). A solution of Na (2.0 g, 87.6
mmol) in dry MeOH (30 mL) was added in one portion to a
stirred solution of metronidazole (20) (10.0 g, 58.4 mmol) and
benzaldehyde (7.1 mL, 70 mmol) in DMSO (30 mL) at 20 °C.
The mixture stood at 20 °C for 24 h and then water (80 mL)
was added and the resulting solid filtered. The solid was
dissolved in EtOAc (100 mL) and dried, and the solvent was
evaporated. The residue was purified by chromatography,
eluting with 50% EtOAc/pet. ether, to give 2-{5-nitro-2-[(E)-
2-phenylethenyl]-1H-imidazol-1-yl}ethanol (21) (4.0 g, 26%) as
(5-Nitr o-2-fu r yl)m eth yl 4-[Bis(2-ch lor oeth yl)a m in o]-
p h en ylca r ba m a te (7c). 4-Nitrophenyl chloroformate (4.17 g,
20.7 mmol) in dry THF (20 mL) was added slowly to a stirred
solution of (5-nitro-2-furyl)methanol26 (7a ) (2.69 g, 18.7 mmol)
and pyridine (1.67 mL, 20.7 mmol) in dry THF (100 mL) at
20 °C under N2. The mixture was stirred at 20 °C for 4 h and
then partitioned between EtOAc/water (200 mL). The organic
layer was washed with water (100 mL) and brine (80 mL) and
dried, and the solvent was evaporated to give (5-nitro-2-furyl)-
methyl 4-nitrophenyl carbonate (25) (4.79 g, 83%) as a white
solid: mp (EtOAc/pet. ether) 93-94 °C; IR ν 1775, 1501, 1352,
1
1215 cm-1; H NMR [(CD3)2SO] δ 8.34 (ddd, J ) 9.2, 3.2, 2.1
Hz, 2 H, H-3, H-5), 7.72 (d, J ) 3.8 Hz, 1 H, H-4′), 7.61 (ddd,

5540 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Hay et al.
J ) 9.2, 3.2, 2.1 Hz, 2 H, H-2, H-6), 7.07 (d, J ) 3.8 Hz, 1 H,
H-3′), 5.43 (s, 2 H, CH2O); 13C NMR [(CD3)2SO] δ 155.0, 151.8,
151.5, 146.0, 145.2, 125.4 (2), 122.5 (2), 115.1, 113.2, 61.5. Anal.
(C12H8N2O8) C, H, N.
2 × CH2O), 3.36 (t, J ) 6.2 Hz, 4 H, 2 × CH2N); 13C NMR
[(CD3)2SO] δ 153.0, 150.8, 148.1, 144.3, 129.6, 127.5, 126.9,
120.5 (2), 111.4 (2), 60.1, 58.2 (2), 53.4 (2). Anal. (C16H19N3O6S)
C, H; N, calcd 11.0, found 10.5%.
A solution of carbonate 25 (1.0 g, 3.2 mmol), N,N-bis(2-
hydroxyethyl)-1,4-benzenediamine (35) (3.6 mmol), and pyri-
dine (0.26 mL, 3.2 mmol) in THF (80 mL) was stirred at 20 °C
for 16 h. The solvent was evaporated and the residue purified
by chromatography, eluting with EtOAc to give (5-nitro-2-
furyl)methyl 4-[bis(2-hydroxyethyl)amino]phenylcarbamate (29)
(0.56 g, 64%) as an colorless oil: 1H NMR [(CD3)2SO] δ 9.44
(s, 1 H, OCONH), 7.68 (d, J ) 3.7 Hz, 1 H, H-4′), 7.20 (br d,
J ) 9.1 Hz, 2 H, H-2, H-6), 6.93 (d, J ) 3.7 Hz, 1 H, H-3′),
6.62 (d, J ) 9.1 Hz, 2 H, H-3, H-5), 5.19 (s, 2 H, CH2O), 4.71
(t, J ) 5.4 Hz, 2 H, 2 × OH), 3.48-3.54 (m, 4 H, 2 × CH2O),
3.33-3.38 (m, 4 H, 2 × CH2N); 13C NMR [(CD3)2SO] δ 154.0,
152.7, 151.5, 144.2, 127.0, 120.4 (2), 113.8, 113.6, 111.4 (2),
58.1 (2), 57.1, 53.4 (2); MS (DEI) m/z 365 (M+, 15%), 334 (70),
222 (20), 196 (40), 191 (100); HRMS (DEI) calcd for C16H19N3O7
(M+) m/z 365.1223, found 365.1218.
Methanesulfonyl chloride (260 µL, 3.4 mmol) was added
dropwise to a stirred solution of diol 30 (0.43 g, 1.1 mmol) in
pyridine (10 mL) at 5 °C and the solution stirred at 20 °C for
2 h. The solvent was evaporated and the residue partitioned
between DCM (50 mL) and water (50 mL). The aqueous
fraction was washed with DCM (2 × 50 mL), the combined
organic extracts were dried, and the solvent was evaporated.
The residue was dissolved in DMF (10 mL), LiCl (0.29 g, 6.8
mmol) added, and the mixture stirred at 80 °C for 3 h. The
solvent was evaporated and the residue partitioned between
EtOAc (100 mL) and water (100 mL). The aqueous fraction
was extracted with EtOAc (2 × 50 mL), the combined extracts
were dried, and the solvent was evaporated. The residue was
purified by chromatography, eluting with 50% EtOAc/pet.
ether, to give 8c (0.35 g, 69%) as pale green needles: mp
(EtOAc/pet. ether) 99-100 °C; IR ν 3353, 1723, 1547, 1530,
1339, 1219 cm-1 1H NMR [(CD3)2SO] δ 9.58 (br s, 1 H,
;
Methanesulfonyl chloride (170 µL, 2.1 mmol) was added
dropwise to a stirred solution of diol 29 (260 mg, 0.7 mmol) in
pyridine (5 mL) at 5 °C and the solution stirred at 20 °C for 1
h. The solvent was evaporated and the residue partitioned
between DCM (80 mL) and water (80 mL). The aqueous
fraction was washed with DCM (2 × 50 mL), the combined
organic extracts were dried, and the solvent was evaporated.
The residue was dissolved in DMF (10 mL), LiCl (180 mg, 4.3
mmol) added, and the mixture stirred at 80 °C for 3 h. The
solvent was evaporated and the residue partitioned between
EtOAc (100 mL) and water (100 mL). The aqueous fraction
was extracted with EtOAc (2 × 50 mL), the combined extracts
were dried, and the solvent was evaporated. The residue was
purified by chromatography, eluting with 50% EtOAc/pet.
ether, to give 7c (237 mg, 83%) as an oil: 1H NMR δ 9.57 (s,
1 H, OCONH), 7.69 (d, J ) 3.7 Hz, 1 H, H-4′), 7.28 (br d, J )
9.1 Hz, 2 H, H-2, H-6), 6.95 (d, J ) 3.7 Hz, 1 H, H-3′), 6.70 (d,
J ) 9.1 Hz, 2 H, H-3, H-5), 5.21 (s, 2 H, CH2O), 3.63-3.72 (m,
8 H, 2 × CH2N, 2 × CH2Cl); 13C NMR [(CD3)2SO] δ 153.9,
152.7, 151.5, 142.3, 128.7, 120.3 (2), 113.8, 113.6, 112.3 (2),
57.2, 52.3 (2), 41.1 (2); MS m/z 405 (M+, 10%), 403 (M+, 30),
401 (M+, 50), 354 (40), 352 (100); HRMS (DEI) calcd for
OCONH), 8.04 (d, J ) 4.2 Hz, 1 H, H-4′), 7.28-7.30 (m, 3 H,
H-3′, H-2, H-6), 6.71 (d, J ) 9.1 Hz, 2 H, H-3, H-5), 5.34 (s, 2
H, CH2O), 3.65-3.72 (m, 8 H, 2 × CH2N, 2 × CH2Cl); 13C NMR
[(CD3)2SO] δ 153.0, 150.8, 148.2, 142.4, 129.5, 128.6, 127.5,
120.4 (2), 112.3 (2), 60.1, 52.2 (2), 41.1 (2). Anal. (C16H17
Cl2N3O5S) C, H, N, Cl.
-
N,N-Bis(2-{[ter t-b u t yl(d im et h yl)silyl]oxy}et h yl)-4-n i-
tr oa n ilin e (37). A solution of TBDMSCl (4.20 g, 27.9 mmol)
in DMF (15 mL) was added to a stirred solution of N,N-bis-
(2-hydroxyethyl)-4-nitroaniline (36) (3.0 g, 13.3 mmol) and
imidazole (3.79 g, 55.7 mmol) in DMF (50 mL), and the solution
stirred at 20 °C for 48 h. The solvent was evaporated and the
residue partitioned between EtOAc (150 mL) and water (150
mL). The organic fraction was washed with water (2 × 200
mL) and dried, and the solvent was evaporated. The residue
was purified by chromatography, eluting with 10% EtOAc/pet.
ether, to give 37 (5.72 g, 95%) as a white solid: mp (pet. ether)
48-49 °C; IR ν 1597, 1520, 1300, 1202, 1107 cm-1; 1H NMR δ
8.07 (ddd, J ) 9.5, 3.5, 2.1 Hz, 2 H, H-3, H-5), 6.67 (ddd, J )
9.5, 3.5, 2.1 Hz, 2 H, H-2, H-6), 3.80 (dd, J ) 6.0, 5.7 Hz, 4 H,
2 × CH2N), 3.63 (dd, J ) 5.9, 5.7 Hz, 4 H, 2 × CH2O), 0.86 [s,
18 H, 2 × SiC(CH3)3], 0.01 [s, 12 H, 2 × Si(CH3)2]; 13C NMR δ
153.0, 138.6, 126.2 (2), 110.4 (2), 60.2 (2), 53.6 (2), 25.8 (6),
18.2 (2), -5.5 (4); MS (DEI) m/z 454 (M+, 10%), 439 (5), 397
(10), 309 (100); HRMS (DEI) calcd for C22H42N2O4Si2 (M+) m/z
454.2683, found 454.2668. Anal. (C22H42N2O4Si2) C, H, N.
C
16H1735Cl2N3O5 (M+) m/z 401.0545, found 401.0546; calcd for
C16H1735Cl37ClN3O5 (M+) m/z 403.0516, found 403.0521; calcd
for C16H1737Cl2N3O5 (M+) m/z 405.0486, found 405.0498.
(5-Nitr o-2-th ien yl)m eth yl 4-[Bis(2-ch lor oeth yl)a m in o]-
p h en ylca r ba m a te (8c). 4-Nitrophenyl chloroformate (2.58 g,
12.8 mmol) in dry THF (20 mL) was added slowly to a stirred
solution of (5-nitrothien-2-yl)methanol34 (8a ) (1.85 g, 11.6
mmol) and pyridine (1.03 mL, 12.8 mmol) in dry THF (50 mL)
at 20 °C under N2. The mixture was stirred at 20 °C for 16 h
and then partitioned between EtOAc and water. The organic
layer was washed with saturated aqueous NaHCO3 (50 mL)
and water (50 mL) and dried and the solvent evaporated to
give 4-nitrophenyl (5-nitro-2-thienyl)methyl carbonate (26)
(1.86 g, 49%) as white solid: mp (EtOAc/pet. ether) 121-122
N,N-Bis(2-{[ter t-b u t yl(d im et h yl)silyl]oxy}et h yl)-1,4-
ben zen ed ia m in e (38). A mixture of 37 (1.54 g, 3.4 mmol)
and Pd/C (50 mg) in EtOAc/EtOH (1:1, 50 mL) was stirred
under hydrogen (60 psi) for 30 min, filtered through Celite,
and washed with EtOH (2 × 10 mL), and the solvent was
evaporated to give crude benzenediamine 38 as an oil that was
used directly without further purification or characterization.
(1-Met h yl-2-n it r o-1H -im id a zol-5-yl)m et h yl 4-[Bis(2-
ch lor oeth yl)a m in o]p h en ylca r ba m a te (9c). (1-Methyl-2-
nitro-1H-imidazol-5-yl)methyl 4-nitrophenyl carbonate35 (27)
was prepared as previously described from alcohol 9a . A
solution of carbonate 27 (0.87 g, 2.7 mmol), aniline 38 (3.4
mmol), and pyridine (217 µL, 2.7 mmol) in THF (50 mL) was
stirred at 20 °C for 48 h. The solvent was evaporated and the
residue partitioned between EtOAc (100 mL) and water (100
mL). The organic fraction was washed with water (2 × 50 mL)
and brine (50 mL) and dried, and the solvent was evaporated.
The residue was purified by chromatography, eluting with a
gradient (20-50%) of EtOAc/pet. ether, to give (1-methyl-2-
nitro-1H-imidazol-5-yl)methyl 4-[bis(2-{[tert-butyl(dimethyl)-
silyl]oxy}ethyl)amino]phenylcarbamate (31) (1.37 g, 84%) as
a white solid: mp 143-144 °C; IR ν 3258, 1721, 1539, 1257,
1
°C; IR ν 1763, 1522, 1345, 1231 cm-1; H NMR [(CD3)2SO] δ
8.33 (ddd, J ) 9.2, 3.4, 2.2 Hz, 2 H, H-3, H-5), 8.08 (d, J ) 4.2
Hz, 1 H, H-4′), 7.60 (ddd, J ) 9.2, 3.4, 2.2 Hz, 2 H, H-2, H-6),
7.40 (d, J ) 4.2 Hz, 1 H, H-3′), 5.56 (s, 2 H, CH2O); 13C NMR
[(CD3)2SO] δ 155.0, 151.6, 151.5, 145.2, 144.8, 129.5, 129.0,
125.4 (2), 122.6 (2), 64.4. Anal. (C12H8N2O7S) C, H, N.
A solution of 26 (0.75 g, 2.3 mmol), N,N-bis(2-hydroxyethyl)-
1,4-benzenediamine (35) (2.5 mmol), and pyridine (206 µL, 2.5
mmol) in THF (50 mL) was stirred at 20 °C for 16 h. The
solvent was evaporated and the residue purified by chroma-
tography, eluting with EtOAc to give (5-nitro-2-thienyl)methyl
4-[bis(2-hydroxyethyl)amino]phenylcarbamate (30) (0.56 g,
64%) as a cream solid: mp (EtOAc/pet. ether) 139-140.5 °C;
IR ν 3360, 3208, 1730, 1530, 1337, 1215 cm-1; 1H NMR [(CD3)2-
SO] δ 9.46 (s, 1 H, OCONH), 8.02 (d, J ) 4.2 Hz, 1 H, H-4′),
7.29 (d, J ) 4.2 Hz, 1 H, H-3′), 7.21 (br d, J ) 9.1 Hz, 2 H,
H-2, H-6), 6.62 (d, J ) 9.1 Hz, 2 H, H-3, H-5), 5.33 (s, 2 H,
CH2O), 4.72 (t, J ) 5.5 Hz, 2 H, 2 × OH), 3.49-3.56 (m, 4 H,
1
1103 cm-1; H NMR [(CD3)2SO] δ 7.23 (s, 1 H, H-4′), 7.15 (br
d, J ) 8.9 Hz, 2 H, H-2, H-6), 6.63 (d, J ) 8.9 Hz, 2 H, H-3,
H-5), 6.52 (br s, 1 H, OCONH), 5.20 (s, 2 H, CH2O), 4.06 (s, 3
H, NCH3), 3.73 (dd, J ) 6.5, 6.3 Hz, 4 H, 2 × CH2O), 3.47 (dd,
J ) 6.5, 6.3 Hz, 4 H, 2 × CH2N), 0.88 [s, 18 H, 2 × SiC(CH3)3],

Nitroheterocyclic Carbamate Prodrugs
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5541
0.02 [s, 12 H, 2 × Si(CH3)2]; 13C NMR [(CD3)2SO] δ 152.9, 146.1,
145.4, 132.5, 129.6, 125.4, 121.7 (2), 111.8 (2), 60.3 (2), 55.2,
53.6 (2), 34.3, 26.0 (6), 18.2 (2), -5.4 (4). Anal. (C28H49N5O6-
Si2) C, H, N.
amino]phenylcarbamate (34) (0.54 g, 82%) as a tan solid: mp
(EtOAc/pet. ether) 157-158 °C; IR ν 3446, 3371, 1728, 1547,
1471, 1375, 1267 cm-1; 1H NMR [(CD3)2SO] δ 10.50 (br s, 1 H,
OCONH), 8.02 (s, 1 H, H-4′), 7.20 (br d, J ) 9.0 Hz, 2 H, H-2,
H-6), 6.61 (br d, J ) 9.0 Hz, 2 H, H-3, H-5), 5.25 (s, 2 H, CH2O),
4.71 (br s, 2 H, 2 × OH), 3.95 (s, 3 H, NCH3), 3.51 (dd, J )
6.1, 6.0 Hz, 4 H, 2 × CH2O), 3.36 (dd, J ) 6.2, 6.1 Hz, 4 H,
2 × CH2N); 13C NMR [(CD3)2SO] δ 152.7, 148.1, 144.2, 139.3,
131.9, 127.0, 120.3 (2), 111.4 (2), 58.2 (2), 57.3, 53.4 (2), 33.4;
MS (DEI) m/z 379 (M+, 35%), 348 (100), 318 (60); HRMS (DEI)
calcd for C16H21N5O6 (M+) m/z 379.1492, found 379.1482. Anal.
(C16H21N5O6) C, H, N.
TBAF (1 M in THF, 3.9 mL, 3.9 mmol) was added to
dropwise to a stirred solution of 31 (1.07 g, 1.8 mmol) in THF
(30 mL) at 5 °C. The solution was stirred for 30 min and the
solvent evaporated. The residue was purified by chromatog-
raphy, eluting with a gradient (0-10%) of MeOH/EtOAc, to
give (1-methyl-2-nitro-1H-imidazol-5-yl)methyl 4-[bis(2-hy-
droxyethyl)amino]phenylcarbamate (32) (0.59 g, 88%) as a
white solid: mp (MeOH) 171-174 °C; IR ν 3445, 3329, 3266,
1717, 1609, 1549, 1491, 1375, 1248 cm-1; 1H NMR [(CD3)2SO]
δ 9.37 (s, 1 H, OCONH), 7.28 (s, 1 H, H-4′), 7.19 (br d, J ) 9.1
Hz, 2 H, H-2, H-6), 6.61 (d, J ) 9.1 Hz, 2 H, H-3, H-5), 5.23 (s,
2 H, CH2O), 4.71 (t, J ) 5.4 Hz, 2 H, 2 × OH), 3.96 (s, 3 H,
NCH3), 3.51 (dd, J ) 6.4, 5.9 Hz, 4 H, 2 × CH2O), 3.34 (dd,
J ) 6.2, 5.9 Hz, 4 H, 2 × CH2N); 13C NMR [(CD3)2SO] δ 152.8,
146.0, 144.2, 133.6, 128.6, 127.1, 120.3 (2), 111.4 (2), 59.7, 58.2
(2), 53.4 (2), 34.2; MS (FAB+) m/z 380 (MH+, 10%), 348 (5);
HRMS (FAB+) m/z calcd for C16H22N5O6 (MH+) 380.1570, found
380.1579. Anal. (C16H21N5O6) C, H, N.
Methanesulfonyl chloride (190 µL, 2.5 mmol) was added
dropwise to a stirred solution of diol 32 (312 mg, 0.8 mmol) in
pyridine (10 mL) at 5 °C and the solution stirred at 20 °C for
1 h. The solvent was evaporated and the residue partitioned
between DCM (100 mL) and water (100 mL). The aqueous
fraction was washed with DCM (2 × 50 mL), the combined
organic extracts were dried, and the solvent was evaporated.
The residue was dissolved in DMF (10 mL), LiCl (210 mg, 4.9
mmol) added, and the mixture stirred at 80 °C for 3 h. The
solvent was evaporated and the residue partitioned between
EtOAc (100 mL) and water (100 mL). The aqueous fraction
was extracted with EtOAc (2 × 50 mL), the combined extracts
were dried, and the solvent was evaporated. The residue was
purified by chromatography, eluting with 50% EtOAc/pet.
ether, to give 9c (227 mg, 66%) as a white solid: mp (MeOH)
Methanesulfonyl chloride (240 µL, 3.1 mmol) was added
dropwise to a stirred solution of diol 34 (390 mg, 1.0 mmol) in
pyridine (10 mL) at 5 °C and the solution stirred at 20 °C for
1 h. The solvent was evaporated and the residue partitioned
between DCM (100 mL) and water (100 mL). The aqueous
fraction was washed with DCM (2 × 50 mL), the combined
organic extracts were dried and the solvent evaporated. The
residue was dissolved in DMF (10 mL), LiCl (260 mg, 6.2
mmol) added, and the mixture stirred at 80 °C for 3 h. The
solvent was evaporated and the residue partitioned between
EtOAc (100 mL) and water (100 mL). The aqueous fraction
was extracted with EtOAc (2 × 50 mL), the combined extracts
were dried, and the solvent was evaporated. The residue was
purified by chromatography, eluting with 50% EtOAc/pet.
ether, to give 10c (0.31 g, 72%) as a cream solid: mp (CHCl3/
pet. ether) 164-164.5 °C; IR ν 3250, 3185, 3127, 1723, 1603,
1
1547, 1516, 1381 cm-1; H NMR [(CD3)2SO] δ 9.57 (br s, 1 H,
OCONH), 8.08 (s, 1 H, H-4′), 7.27 (br d, J ) 8.0 Hz, 2 H, H-2,
H-6), 6.70 (d, J ) 9.1 Hz, 2 H, H-3, H-5), 5.26 (s, 2 H, CH2O),
3.95 (s, 3 H, NCH3), 3.65-3.72 (m, 8 H, 2 × CH2N, 2 × CH2-
Cl); 13C NMR [(CD3)2SO] δ 152.7, 148.0, 142.3, 139.3, 131.7,
128.7, 120.3 (2), 112.3 (2), 57.4, 52.2 (2), 41.1 (2), 33.4; MS
(DEI) m/z 415 (M+, 1%), 366 (2), 316 (2), 258 (20), 211 (30),
209 (100); HRMS (DEI) calcd for C16H1935Cl2N5O4 (M+) m/z
415.0814, found 415.0808. Anal. (C16H19Cl2N5O4) C, H, N.
156-157 °C; IR ν 3408, 3246, 1725, 1531, 1354, 1221 cm-1
;
Alter n a tive P r ep a r a tion of 10c. Diphosgene (85 µL, 0.7
mmol) was added dropwise to a stirred solution of alcohol 10a
(0.2 g, 1.3 mmol) and Et3N (98 µL, 0.7 mmol) in THF (10 mL)
at 5 °C. The suspension was stirred at 5 °C for 30 min and a
mixture of N,N-bis(2-chloroethyl)-1,4-benzenediamine hydro-
chloride27 (4) (0.38 g, 1.4 mmol) and Et3N (195 µL, 1.4 mmol)
in THF (4 mL) was added dropwise to the above suspension.
The mixture was stirred at 20 °C for 4 h, the solvent
evaporated, and the residue purified by chromatography,
eluting with 50% EtOAc/pet. ether, to give carbamate 10c (0.19
g, 36%); spectroscopically identical to the sample prepared
above.
(1-Met h yl-5-n it r o-1H -p yr a zol-4-yl)m et h yl 1-(Ch lor o-
m eth yl)-3-[(5,6,7-tr im eth oxy-1H-in d ol-1-yl)ca r bon yl]-2,3-
d ih yd r o-1H-ben zo[e]in d ol-5-ylca r ba m a te (6d ). A solution
of triphosgene (14.3 mg, 48 µmol) in DCM (2 mL) was added
dropwise to a stirred solution of amine 5 (57 mg, 122 µmol)
and Et3N (38 µL, 275 µmol) in DCM (10 mL) and stirred at 20
°C for 2 h. A solution of alcohol 6a (26 mg, 165 µmol) in DCM
(2 mL) was added, followed by nBu2Sn(OAc)2 (2 drops), and
the solution stirred at 20 °C for 24 h. The solvent was
evaporated and the residue purified by chromatography,
eluting with 20% EtOAc/DCM, to give 6d (41 mg, 52%) as a
white solid: mp (EtOAc/pet. ether) 201-202 °C; 1H NMR
[(CD3)2SO] δ 11.47 (br s, 1 H, indole-NH), 9.80 (br s, 1 H,
OCONH), 8.56 (br s, 1 H, H-4), 8.06 (d, J ) 8.5 Hz, 1 H, H-6),
7.98 (d, J ) 8.3 Hz, 1 H, H-9), 7.74 (br s, 1 H, H-3′′), 7.58 (dd,
J ) 8.3, 7.4 Hz, 1 H, H-8), 7.47 (dd, J ) 8.5, 7.4 Hz, 1 H, H-7),
7.45 (d, J ) 1.6 Hz, 1 H, H-3′), 6.98 (s, 1 H, H-4′), 5.33 (s, 2 H,
CH2O), 4.80 (dd, J ) 11.0, 9.4 Hz, 1 H, H-2), 4.53 (dd, J )
11.0, 1.8 Hz, 1 H, H-2), 4.32-4.38 (m, 1 H, H-1), 4.17 (s, 3 H,
NCH3), 4.07 (dd, J ) 11.0, 3.1 Hz, 1 H, CH2Cl), 3.91-3.96 (m,
4 H, OCH3, CH2Cl), 3.82 (s, 3 H, OCH3), 3.80 (s, 3 H, OCH3);
13C NMR [(CD3)2SO] δ 160.2, 154.3, 149.1, 142.5, 141.5, 139.9,
139.0, 137.5, 134.2, 130.7, 129.4, 127.1, 125.4 (2), 124.3, 123.7,
123.3, 123.1, 122.0, 117.4, 113.0, 106.2, 98.0, 61.0, 60.9, 57.2,
55.9, 54.8, 47.5, 41.1, 40.8; MS (FAB+) m/z 650 (MH+, 2%),
1H NMR [(CD3)2SO] δ 9.49 (s, 1 H, OCONH), 7.26-7.29 (m, 3
H, H 4′, H-2, H-6), 6.70 (d, J ) 9.1 Hz, 2 H, H-3, H-5), 5.24 (s,
2 H, CH2O), 3.96 (s, 3 H, NCH3), 3.64-3.70 (m, 8 H, 2 × CH2N,
2 × CH2Cl); 13C NMR [(CD3)2SO] δ 152.8, 146.0, 142.3, 133.5,
128.7, 128.6, 120.3 (2), 112.3 (2), 54.9, 52.2 (2), 41.1 (2), 34.1.
Anal. (C16H19Cl2N5O4) C, H, N, Cl.
(1-Met h yl-5-n it r o-1H -im id a zol-2-yl)m et h yl 4-[Bis(2-
ch lor oeth yl)a m in o]p h en ylca r ba m a te (10c). (1-Methyl-5-
nitro-1H-imidazol-2-yl)methyl 4-nitrophenyl carbonate35 (28)
was prepared as previously described from alcohol 10a . A
solution of carbonate 28 (1.00 g, 3.1 mmol), aniline 38 (3.7
mmol), and pyridine (270 µL, 3.4 mmol) in THF (100 mL) was
stirred at 20 °C for 16 h. The solvent was evaporated and the
residue partitioned between EtOAc (100 mL) and water (100
mL). The organic fraction was washed with water (2 × 50 mL)
and brine (50 mL) and dried, and the solvent was evaporated.
The residue was purified by chromatography, eluting with a
gradient (20-50%) of EtOAc/pet. ether, to give (1-methyl-5-
nitro-1H-imidazol-2-yl)methyl 4-[bis(2-{[tert-butyl(dimethyl)-
silyl]oxy}ethyl)amino]phenylcarbamate (33) (1.54 g, 82%) as
a white solid: mp 117-118 °C; IR ν 3277, 1730, 1535, 1471,
1215, 1194 cm-1; 1H NMR [(CD3)2SO] δ 7.99 (s, 1 H, H-4′), 7.15
(br d, J ) 8.9 Hz, 2 H, H-2, H-6), 6.63 (d, J ) 8.9 Hz, 2 H, H-3,
H-5), 6.59 (br s, 1 H, OCONH), 5.28 (s, 2 H, CH2O), 4.05 (s, 3
H, NCH3), 3.73 (dd, J ) 6.5, 6.4 Hz, 4 H, 2 × CH2O), 3.47 (dd,
J ) 6.5, 6.4 Hz, 4 H, 2 × CH2N), 0.88 [s, 18 H, 2 × SiC(CH3)3],
0.02 [s, 12 H, 2 × Si(CH3)2]; 13C NMR [(CD3)2SO] δ 152.9, 147.2,
145.4, 139.5, 132.1, 125.4, 121.7 (2), 111.8 (2), 60.3 (2), 57.8,
53.6 (2), 33.7, 25.9 (6), 18.3 (2), -5.4 (4). Anal. (C28H49N5O6-
Si2) C, H, N.
TBAF (3.8 mL, 3.8 mmol) was added to dropwise to a stirred
solution of carbamate 33 (1.06 g, 1.7 mmol) in THF (50 mL)
at 5 °C. The solution was stirred for 30 min and the solvent
evaporated. The residue was purified by chromatography,
eluting with a gradient (0-20%) of MeOH/EtOAc, to give (1-
methyl-5-nitro-1H-imidazol-2-yl)methyl 4-[bis(2-hydroxyethyl)-

5542 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Hay et al.
648 (MH+, 5); HRMS (FAB+) calcd for C31H3035ClN6O8 (MH+)
m/z 649.1814, found 649.1803; calcd for C31H3037ClN6O8 (MH+)
m/z 651.1784, found 651.1796. Anal. (C31H29ClN6O8) C, H, N.
(br dd, J ) 8.3, 7.4 Hz, 1 H, H-8), 7.44 (ddd, J ) 8.5, 7.4, 0.7
Hz, 1 H, H-7), 7.37 (br s, 1 H, H-4), 6.99 (d, J ) 2.3 Hz, 1 H,
H-3′), 6.87 (s, 1 H, H-4′), 5.38 (d, J ) 13.4 Hz, 1 H, CH2O),
5.34 (d, J ) 13.4 Hz, 1 H, CH2O), 4.79 (dd, J ) 10.7, 1.6 Hz,
1 H, H-2), 4.65 (dd, J ) 10.7, 8.7 Hz, 1 H, H-2), 4.15-4.19 (m,
1 H, H-1), 4.09 (s, 3 H, OCH3), 4.02 (br s, 3 H, NCH3), 3.95 (s,
3 H, OCH3), 3.92-3.94 (m, 4 H, OCH3, CH2Cl), 3.45 (dd, J )
10.9, 10.7 Hz, 1 H, CH2Cl); 13C NMR δ 160.4, 153.4, 150.2,
146.9, 141.6, 140.7, 139.6, 138.9, 133.3, 132.1, 129.7, 129.6,
127.6, 125.7, 125.2 (2), 123.6, 123.2, 122.3, 122.2, 112.8, 106.6,
97.7, 61.5, 61.2, 58.4, 56.3, 54.9, 45.8, 43.4, 33.8; MS (FAB+)
(5-Nitr o-2-fu r yl)m eth yl 1-(Ch lor om eth yl)-3-[(5,6,7-tr i-
m eth oxy-1H-in d ol-1-yl)ca r bon yl]-2,3-d ih yd r o-1H-ben zo-
[e]in d ol-5-ylca r ba m a te (7d ). Similarly, reaction of amine 5
and alcohol 7a gave 7d (84%) as a white solid: mp (EtOAc/
1
pet. ether) 185-187 °C; H NMR δ 11.46 (s, 1 H, indole-NH),
9.92 (s, 1 H, OCONH), 8.54 (s, 1 H, H-4), 8.04 (d, J ) 8.5 Hz,
1 H, H-6), 7.98 (d, J ) 8.3 Hz, 1 H, H-9), 7.72 (d, J ) 3.8 Hz,
1 H, H-4′′), 7.58 (ddd, J ) 8.3, 7.2, 0.7 Hz, 1 H, H-8), 7.45
(ddd, J ) 8.5, 7.2, 0.7 Hz, 1 H, H-7), 7.09 (d, J ) 2.1 Hz, 1 H,
H-3′), 6.98-7.00 (m, 2 H, H-4′, H-3′′), 5.30 (s, 2 H, CH2O), 4.80
(dd, J ) 10.8, 9.5 Hz, 1 H, H-2), 4.53 (dd, J ) 10.8, 1.9 Hz, 1
H, H-2), 4.32-4.37 (m, 1 H, H-1), 4.07 (dd, J ) 11.1, 3.0 Hz,
1 H, CH2Cl), 3.91-3.96 (m, 4 H, OCH3, CH2Cl), 3.83 (s, 3 H,
OCH3), 3.80 (s, 3 H, OCH3); 13C NMR δ 160.1, 153.9, 153.8,
151.1, 149.2, 141.4, 139.8, 139.0, 134.0, 130.7, 129.4, 127.1,
125.4, 125.3, 124.3, 123.7, 123.3, 123.1, 122.2, 113.9, 113.6,
113.1, 106.2, 98.0, 61.0, 60.9, 57.7, 55.9, 54.8, 47.5, 41.1; MS
(FAB+) m/z 635 (MH+, 6%), 637 (MH+, 3); HRMS (FAB+) calcd
for C31H2835ClN4O9 (MH+) m/z 635.1545, found 635.1552; calcd
for C31H2837ClN4O9 (MH+) m/z 637.1515, found 637.1514. Anal.
(C31H27ClN4O9) C, H, N.
m/z 649 (MH+, 3%), 651 (1.5); HRMS (FAB+) calcd for C31H30
-
35ClN6O8 (MH+) m/z 649.1814, found 649.1797; calcd for
C31H3037ClN6O8 (MH+) m/z 651.1784, found 651.1802. Anal.
(C31H29ClN6O8) C, H, N.
1-(2-H yd r oxyet h yl)-5-n it r o-1H -im id a zol-2-yl]m et h yl
1-(Ch lor om eth yl)-3-[(5,6,7-tr im eth oxy-1H-in d ol-2-yl)ca r -
bon yl]-2,3-dih ydr o-1H-ben zo[e]in dol-5-ylcar bam ate (11d).
Similarly, reaction of amine 5 and alcohol 23 gave [1-(2-{[tert-
butyl(dimethyl)silyl]oxy}ethyl)-5-nitro-1H-imidazol-2-yl]meth-
yl 1-(chloromethyl)-3-[(5,6,7-trimethoxy-1H-indol-2-yl)carbonyl]-
2,3-dihydro-1H-benzo[e]indol-5-ylcarbamate (39) (49%) as a
colorless oil: 1H NMR δ 9.41 (s, 1 H, indole-NH), 8.88 (s, 1 H,
OCONH), 8.06 (s, 1 H, H-4′′), 7.87 (d, J ) 8.5 Hz, 1 H, H-6),
7.79 (d, J ) 8.3 Hz, 1 H, H-9), 7.57 (dd, J ) 8.3, 7.5 Hz, 1 H,
H-8), 7.46 (dd, J ) 8.5, 7.5 Hz, 1 H, H-7), 7.17 (br s, 1 H, H-4),
7.00 (d, J ) 2.2 Hz, 1 H, H-3′), 6.88 (s, 1 H, H-4′), 5.44 (d, J )
13.5 Hz, 1 H, CH2O), 5.39 (d, J ) 13.5 Hz, 1 H, CH2O), 4.81
(dd, J ) 10.7, 1.5 Hz, 1 H, H-2), 4.65-4.74 (m, 3 H, H-2, CH2N),
4.16-4.22 (m, 1 H, H-1), 4.10 (s, 3 H, OCH3), 3.95-3.99 (m, 4
H, CH2Cl, OCH3), 3.89-3.93 (m, 5 H, OCH3, CH2N), 3.48 (t,
J ) 10.9 Hz, 1 H, CH2Cl), 0.81 [s, 9 H, SiC(CH3)3], -0.08 [s, 6
H, Si(CH3)2]; MS (FAB+) m/z 795 (MH+, 12%), 793 (25); HRMS
(FAB+) calcd for C38H4635ClN6O9Si (MH+) m/z 793.2784, found
793.2762; calcd for C38H4637ClN6O9Si (MH+) m/z 795.2755,
found 795.2751.
HCl (1 M, 0.23 mL, 230 µmol) was added to a stirred solution
of silyl ether 39 (91 mg, 115 µmol) in MeOH (5 mL) and the
solution stirred at 20 °C for 4 h. The solvent was evaporated
and the residue partitioned between EtOAc (40 mL) and water
(40 mL). The organic fraction was washed with water (25 mL)
and brine (20 mL) and dried, and the solvent was evaporated.
The residue was purified by chromatography, eluting with a
gradient (0-10%) of MeOH/40% EtOAc/DCM, to give 11d as
a white solid: mp (EtOAc/pet. ether) 148-150 °C (dec); 1H
NMR δ [(CD3)2SO] 11.45 (s, 1 H, indole-NH), 9.92 (s, 1 H,
OCONH), 8.45 (s, 1 H, H-4), 8.16 (s, 1 H, H-4′′), 8.06 (d, J )
8.5 Hz, 1 H, H-6), 7.97 (d, J ) 8.3 Hz, 1 H, H-9), 7.58 (ddd,
J ) 8.3, 7.2, 0.8 Hz, 1 H, H-8), 7.46 (ddd, J ) 8.5, 7.2, 0.8 Hz,
1 H, H-7), 7.09 (d, J ) 2.0 Hz, 1 H, H-3′), 6.97 (s, 1 H, H-4′),
5.37 (s, 2 H, CH2O), 5.12 (t, J ) 5.4 Hz, 1 H, OH), 4.80 (dd,
J ) 10.7, 9.4 Hz, 1 H, H-2), 4.56-4.60 (m, 3 H, H-2, CH2O),
4.32-4.38 (m, 1 H, H-1), 4.06 (dd, J ) 11.1, 3.2 Hz, 1 H, CH2-
Cl), 3.91-3.95 (m, 4 H, OCH3, CH2Cl), 3.83 (s, 3 H, OCH3),
3.81 (s, 3 H, OCH3), 3.70-3.75 (m, 2 H, CH2N); 13C NMR δ
[(CD3)2SO] 160.1, 154.0, 149.1, 148.7, 141.4, 139.9, 139.0,
138.9, 134.1, 132.5, 130.7, 129.4, 127.1, 125.4, 125.3, 124.3,
123.8, 123.3, 123.1, 122.2, 113.2, 106.2, 98.0, 61.0, 60.9, 59.7,
58.1, 55.9, 54.9, 48.2, 47.8, 41.1; MS (FAB+) m/z 681 (MH+,
5%), 679 (MH+, 12%); HRMS (FAB+) calcd for C32H3235ClN6O9
(MH+) m/z 679.1919, found 679.1797; calcd for C32H3237ClN6O9
(MH+) m/z 681.1890, found 681.1892. Anal. (C32H31ClN6O9) C,
H, N.
(5-Nitr o-2-th ien yl)m eth yl 1-(Ch lor om eth yl)-3-[(5,6,7-
tr im eth oxy-1H-in d ol-1-yl)ca r bon yl]-2,3-d ih yd r o-1H-ben -
zo[e]in d ol-5-ylca r ba m a te (8d ). Similarly, reaction of amine
5 and alcohol 8a gave 8d (89%) as a white solid: mp (EtOAc/
1
pet. ether) 218-219 °C; H NMR [(CD3)2SO] δ 11.48 (br s, 1
H, indole-NH), 9.34 (br s, 1 H, OCONH), 8.55 (br s, 1 H, H-4),
8.08 (d, J ) 4.1 Hz, 1 H, H-4′′), 8.04 (d, J ) 8.5 Hz, 1 H, H-6),
7.99 (d, J ) 8.3 Hz, 1 H, H-9), 7.58 (dd, J ) 8.3, 7.4 Hz, 1 H,
H-8), 7.47 (dd, J ) 8.5, 7.4 Hz, 1 H, H-7), 7.33 (d, J ) 4.1 Hz,
1 H, H-3′′), 7.10 (d, J ) 1.9 Hz, 1 H, H-3′), 6.97 (s, 1 H, H-4′),
5.43 (s, 2 H, CH2O), 4.80 (dd, J ) 11.0, 9.5 Hz, 1 H, H-2), 4.54
(dd, J ) 11.0, 1.8 Hz, 1 H, H-2), 4.32-4.36 (m, 1 H, H-1), 4.07
(dd, J ) 11.0, 3.0 Hz, 1 H, CH2Cl), 3.92-3.96 (m, 4 H, CH2Cl,
OCH3), 3.92 (m, 3 H, OCH3), 3.81 (s, 3 H, OCH3); 13C NMR
[(CD3)2SO] δ 160.3, 154.4, 151.1, 149.3, 148.2, 141.6, 140.0,
139.1, 134.1, 130.8, 129.7, 129.6, 127.8, 127.4, 125.6, 125.5,
124.6, 123.8, 123.3, 123.2, 122.5, 113.5, 106.4, 98.1, 61.2, 61.0,
60.8, 56.0, 55.0, 47.7, 41.2; MS (FAB+) m/z 653 (MH+, 4%),
651 (MH+, 8); HRMS (FAB+) calcd for C31H2835ClN4O8S (MH+)
m/z 651.1316, found 651.1311; calcd for C31H2837ClN4O8S
(MH+) m/z 653.1287, found 653.1307. Anal. (C31H27ClN4O8S)
C, H, N.
(1-Meth yl-2-n itr o-1H-im id a zol-5-yl)m eth yl 1-(Ch lor o-
m eth yl)-3-[(5,6,7-tr im eth oxy-1H-in d ol-1-yl)ca r bon yl]-2,3-
d ih yd r o-1H-ben zo[e]in d ol-5-ylca r ba m a te (9d ). Similarly,
reaction of amine 5 and alcohol 9a gave 9d (68%) as a tan
1
powder: mp (EtOAc) 202-204 °C; H NMR δ 9.49 (br s, 1 H,
indole-NH), 8.81 (br s, 1 H, H-4), 7.86 (d, J ) 8.5 Hz, 1 H,
H-6), 7.78 (d, J ) 8.3 Hz, 1 H, H-9), 7.57 (m, 1 H, H-8), 7.43
(m, 1 H, H-7), 7.25 (s, 1 H, H-4′′), 7.21 (br s, 1 H, OCONH),
7.00 (d, J ) 1.6 Hz, 1 H, H-3′), 6.87 (s, 1 H, H-4′), 5.31 (d, J )
13.6 Hz, 1 H, CH2O), 5.25 (d, J ) 13.6 Hz, 1 H, CH2O), 4.80
(dd, J ) 10.5, 1.6 Hz, 1 H, H-2), 4.65 (dd, J ) 10.5, 8.7 Hz, 1
H, H-2), 4.13-4.20 (m, 1 H, H-1), 4.11 (s, 3 H, OCH3), 4.01 (br
s, 3 H, NCH3), 3.94-3.98 (m, 4 H, CH2Cl, OCH3), 3.92 (s, 3 H,
OCH3), 3.47 (dd, J ) 10.8, 10.8 Hz, 1 H, CH2Cl); 13C NMR δ
160.4, 153.5, 150.2, 146.4, 141.6, 140.7, 138.9, 133.3, 132.1,
129.8, 129.7, 129.5, 127.6, 125.7 (2), 125.1 (2), 123.6, 123.3,
122.3 (2), 106.6, 97.6, 61.5, 61.2, 56.3, 55.8, 54.9, 45.8, 43.4,
34.3; MS (FAB+) m/z 649 (MH+, 2%); HRMS (FAB+) calcd for
C31H3035ClN6O8 (MH+) m/z 649.1814, found 649.1767; calcd for
C31H3037ClN6O8 (MH+) m/z 651.1784, found 651.1819. Anal.
(C31H29ClN6O8‚1/2H2O) C, H, N.
Eth yl 4-({[({1-(Ch lor om eth yl)-3-[(5,6,7-tr im eth oxy-1H-
in d ol-1-yl)ca r bon yl]-2,3-d ih yd r o-1H-ben zo[e]in d ol-5-yl}-
am in o)car bon yl]oxy}m eth yl)-1-m eth yl-5-n itr o-1H-pyr r ole-
2-ca r boxyla te (12d ). Similarly, reaction of amine 5 and
alcohol 12a gave 12d (31%) as a white solid: mp (EtOAc/pet.
ether) 248-250 °C; 1H NMR [(CD3)2SO] δ 11.45 (s, 1 H, indole-
NH), 9.86 (s, 1 H, OCONH), 8.56 (br s, 1 H, H-4′′), 8.09 (d,
J ) 8.5 Hz, 1 H, H-6′′), 7.99 (d, J ) 8.3 Hz, 1 H, H-9′′), 7.59
(dd, J ) 8.3, 7.7 Hz, 1 H, H-8′′), 7.48 (dd, J ) 8.5, 7.7 Hz, 1 H,
H-7′′), 7.10 (d, J ) 2.0 Hz, 1 H, H-3′′), 7.06 (br s, 1 H, H-3),
6.97 (s, 1 H, H-4′′), 5.38 (s, 2 H, CH2O), 4.80 (dd, J ) 11.0, 9.6
(1-Meth yl-5-n itr o-1H-im id a zol-2-yl)m eth yl 1-(Ch lor o-
m eth yl)-3-[(5,6,7-tr im eth oxy-1H-in d ol-2-yl)ca r bon yl]-2,3-
d ih yd r o-1H-ben zo[e]in d ol-5-ylca r ba m a te (10d ). Similarly,
reaction of amine 5 and alcohol 10a gave 10d (34%) as a tan
powder: mp (EtOAc) 169-170 °C; 1H NMR δ 9.47 (s, 1 H,
indole-NH), 8.85 (s, 1 H, OCONH), 7.98 (s, 1 H, H-4′′), 7.86
(d, J ) 8.5 Hz, 1 H, H-6), 7.77 (d, J ) 8.3 Hz, 1 H, H-9), 7.57

Nitroheterocyclic Carbamate Prodrugs
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5543
Hz, 1 H, H-2′′), 4.53 (dd, J ) 11.0, 2.0 Hz, 1 H, H-2′′), 4.35-
4.40 (m, 1 H, H-1′′), 4.29 (q, J ) 7.1 Hz, 2 H, H-1′), 4.19 (s, 3
H, NCH3), 4.07 (dd, J ) 11.0, 3.0 Hz, 1 H, CH2Cl), 3.92-3.96
(m, 4 H, CH2Cl, OCH3), 3.83 (m, 3 H, OCH3), 3.81 (s, 3 H,
OCH3), 1.31 (t, J ) 7.1 Hz, 3 H, H-2′); 13C NMR [(CD3)2SO] δ
160.2, 159.4, 154.3, 149.1, 141.5, 139.9, 139.0, 137.1, 134.2,
133.9, 130.7, 129.5, 127.1, 125.6, 125.5, 125.4, 124.3, 123.7,
123.3, 123.1, 122.1, 114.6, 113.0, 106.2, 98.0, 61.2, 61.0, 60.9,
59.6, 55.9, 54.9, 47.5, 41.1, 35.2, 14.0; MS (FAB+) m/z 722
C32H3135ClN5O8 (MH+) m/z 648.1861, found 648.1852; calcd for
C32H3137ClN5O8 (MH+) m/z 650.1832, found 650.1836. Anal.
(C32H30ClN5O8) C, H, N.
Eth yl 5-({[({1-(Ch lor om eth yl)-3-[(5,6,7-tr im eth oxy-1H-
in d ol-1-yl)ca r bon yl]-2,3-d ih yd r o-1H-ben zo[e]in d ol-5-yl}-
am in o)car bon yl]oxy}m eth yl)-1-m eth yl-4-n itr o-1H-pyr r ole-
2-ca r boxyla te (16d ). Similarly, reaction of amine 5 and
alcohol 16a gave 16d (62%) as a white solid: mp (EtOAc/pet.
ether) 227-229 °C; 1H NMR [(CD3)2SO] δ 11.46 (s, 1 H, indole-
NH), 9.83 (s, 1 H, OCONH), 8.56 (br s, 1 H, H-4′′), 8.02 (d,
J ) 8.5 Hz, 1 H, H-6′′), 7.97 (d, J ) 8.3 Hz, 1 H, H-9′′), 7.57
(dd, J ) 8.3, 7.4 Hz, 1 H, H-8′′), 7.46 (dd, J ) 8.5, 7.4 Hz, 1 H,
H-7′′), 7.43 (s, 1 H, H-3), 7.10 (d, J ) 2.0 Hz, 1 H, H-3′′), 6.98
(s, 1 H, H-4′′), 5.63 (s, 2 H, CH2O), 4.80 (dd, J ) 11.0, 9.4 Hz,
1 H, H-2′′), 4.53 (dd, J ) 11.0, 1.9 Hz, 1 H, H-2′′), 4.33-4.37
(m, 1 H, H-1′′), 4.29 (q, J ) 7.1 Hz, 2 H, H-1′), 4.00-4.08 (m,
4 H, CH2Cl, NCH3), 3.91-3.95 (m, 4 H, CH2Cl, OCH3), 3.83
(m, 3 H, OCH3), 3.80 (s, 3 H, OCH3), 1.31 (t, J ) 7.1 Hz, 3 H,
H-2′); 13C NMR [(CD3)2SO] δ 160.1, 159.3, 154.0, 149.1, 141.4,
139.9, 139.0, 134.1, 133.6, 133.4, 130.7, 129.4, 127.1, 125.4,
125.3, 124.3, 123.7, 123.3, 123.1, 122.4, 122.1, 113.0, 111.2,
106.2, 98.0, 61.0, 60.9, 60.8, 55.9, 54.8, 54.6, 47.5, 41.1, 33.6,
13.9; MS (FAB+) m/z 722 (MH+, 2.5%), 720 (MH+, 6); HRMS
(FAB+) calcd for C35H3535ClN5O10 (MH+) m/z 720.2073, found
720.2045; calcd for C35H3537ClN5O10 (MH+) m/z 722.2043, found
722.2039. Anal. (C35H34ClN5O10) C, H, N.
(MH+, 0.3%), 720 (MH+, 0.6); HRMS (FAB+) calcd for C35H35
35ClN5O10 (MH+) m/z 720.2073, found 720.2059; calcd for
35H3537ClN5O10 (MH+) m/z 722.2043, found 722.2031. Anal.
-
C
(C35H34ClN5O10) C, H, N.
(1-Meth yl-5-n itr o-1H-im id a zol-4-yl)m eth yl 1-(Ch lor o-
m eth yl)-3-[(5,6,7-tr im eth oxy-1H-in d ol-1-yl)ca r bon yl]-2,3-
d ih yd r o-1H-ben zo[e]in d ol-5-ylca r ba m a te (13d ). Similarly,
reaction of amine 5 and alcohol 13a gave 13d (79%) as a pale
yellow powder: mp (EtOAc) 219-220 °C; 1H NMR [(CD3)2SO]
δ 11.47 (s, 1 H, indole-NH), 9.80 (s, 1 H, OCONH), 8.54 (br s,
1 H, H-4), 8.08-8.11 (m, 2 H, H-6, H-2′′), 7.97 (d, J ) 8.4 Hz,
1 H, H-9), 7.57 (ddd, J ) 8.4, 7.2, 0.7 Hz, 1 H, H-8), 7.45 (ddd,
J ) 8.5, 7.2, 0.7 Hz, 1 H, H-7), 7.09 (d, J ) 2.0 Hz, 1 H, H-3′),
6.97 (s, 1 H, H-4′), 5.40 (s, 2 H, CH2O), 4.79 (dd, J ) 10.8, 1.4
Hz, 1 H, H-2), 4.52 (dd, J ) 11.0, 1.9 Hz, 1 H, H-2), 4.31-4.36
(m, 1 H, H-1), 4.07 (dd, J ) 11.1, 3.0 Hz, 1 H, CH2Cl), 3.89-
3.95 (m, 7 H, OCH3, CH2Cl, NCH3), 3.83 (s, 3 H, OCH3), 3.81
(s, 3 H, OCH3); 13C NMR [(CD3)2SO] δ 160.1, 154.3, 154.2,
149.1, 141.4, 141.2, 139.9, 139.0, 135.1, 134.4, 130.8, 129.4,
127.0, 125.5, 125.4, 124.2, 123.9, 123.2, 123.1, 122.0, 113.1,
106.2, 98.0, 61.0, 60.9, 59.6, 55.9, 54.8, 47.5, 41.1, 35.1; MS
(FAB+) m/z 651 (MH+, 1%), 649 (MH+, 2); HRMS (FAB+) calcd
for C31H3035ClN6O8 (MH+) m/z 649.1814, found 649.1802; calcd
for C31H3037ClN6O8 (MH+) m/z 651.1784, found 651.1761. Anal.
(C31H29ClN6O8) C, H, N.
(1-Meth yl-2-n itr o-1H-pyr r ol-3-yl)m eth yl 1-(Ch lor om eth -
yl)-3-[(5,6,7-tr im eth oxy-1H-in d ol-1-yl)ca r bon yl]-2,3-d ih y-
d r o-1H-ben zo[e]in d ol-5-ylca r ba m a te (17d ). Similarly, re-
action of amine 5 and alcohol 17a gave 17d (28%) as a white
1
solid: mp (EtOAc) 218-220 °C; H NMR [(CD3)2SO] δ 11.46
(br s, 1 H, indole-NH), 9.80 (br s, 1 H, OCONH), 8.55 (br s, 1
H, H-4), 8.09 (d, J ) 8.5 Hz, 1 H, H-6), 7.98 (d, J ) 8.3 Hz, 1
H, H-9), 7.58 (dd, J ) 8.3, 7.3 Hz, 1 H, H-8), 7.48 (dd, J ) 8.5,
7.3 Hz, 1 H, H-7), 7.33 (d, J ) 2.6 Hz, 1 H, H-4′′), 7.09 (d, J )
1.9 Hz, 1 H, H-3′), 6.98 (s, 1 H, H-4′), 6.37 (br s, 1 H, H-3′′),
5.37 (s, 2 H, CH2O), 4.80 (dd, J ) 11.0, 9.3 Hz, 1 H, H-2), 4.52
(dd, J ) 11.0, 1.9 Hz, 1 H, H-2), 4.31-4.37 (m, 1 H, H-1), 4.07
(dd, J ) 11.1, 3.0 Hz, 1 H, CH2Cl), 3.96 (s, 3 H, NCH3), 3.91-
3.94 (m, 4 H, CH2Cl, OCH3), 3.82 (s, 3 H, OCH3), 3.80 (s, 3 H,
OCH3); 13C NMR [(CD3)2SO] δ 160.2, 154.4, 149.1, 141.5, 139.9,
139.0, 134.3, 133.2, 130.7, 130.4, 129.4, 127.1, 125.4, 125.3,
124.3, 123.8, 123.3, 123.1, 122.6, 122.0, 113.0, 108.1, 106.1,
98.0, 61.0, 60.9, 60.2, 55.9, 54.8, 47.5, 41.1, 37.9; MS (FAB+)
m/z 650 (MH+, 1.5%), 648 (MH+, 3.5); HRMS (FAB+) calcd for
C32H3135ClN5O8 (MH+) m/z 648.1861, found 648.1844; calcd for
C32H3137ClN5O8 (MH+) m/z 650.1832, found 650.1826. Anal.
(C32H30ClN5O8) C, H, N.
(1-Meth yl-4-n itr o-1H-im id a zol-5-yl)m eth yl 1-(Ch lor o-
m eth yl)-3-[(5,6,7-tr im eth oxy-1H-in d ol-1-yl)ca r bon yl]-2,3-
d ih yd r o-1H-ben zo[e]in d ol-5-ylca r ba m a te (14d ). Similarly,
reaction of amine 5 and alcohol 14a gave 14d (56%) as a white
1
powder: mp (EtOAc/pet. ether) 229-231 °C; H NMR δ 9.48
(s, 1 H, indole-NH), 8.87 (s, 1 H, OCONH), 7.87 (d, J ) 8.5
Hz, 1 H, H-6), 7.76 (d, J ) 8.3 Hz, 1 H, H-9), 7.55 (ddd, J )
8.3, 7.4, 0.7 Hz, 1 H, H-8), 7.44 (ddd, J ) 8.5, 7.4, 0.7 Hz, 1 H,
H-7), 7.40 (s, 1 H, H-2′′), 7.33 (br s, 1 H, H-4), 7.00 (d, J ) 2.3
Hz, 1 H, H-3′), 6.87 (s, 1 H, H-4′), 5.61 (s, 2 H, CH2O), 4.78
(dd, J ) 10.7, 1.7 Hz, 1 H, H-2), 4.65 (dd, J ) 10.7, 8.7 Hz, 1
H, H-2), 4.15-4.20 (m, 1 H, H-1), 4.05-4.10 (m, 4 H, OCH3,
CH2Cl), 3.95 (s, 3 H, OCH3), 3.92 (s, 3 H, OCH3), 3.83 (br s, 3
H, NCH3), 3.44 (dd, J ) 10.9, 10.7 Hz, 1 H, CH2Cl); 13C NMR
δ 160.4, 153.8, 150.2, 146.3, 141.5, 140.6, 138.8, 136.3, 133.4,
129.7, 129.5, 127.6, 126.9, 125.7, 125.2, 125.2, 123.6, 123.1,
122.3, 121.9, 112.5, 106.6, 97.7, 61.5, 61.1, 56.3, 54.9, 54.4, 45.8,
43.4, 33.2; MS (FAB+) m/z 651 (MH+, 1%), 649 (MH+, 2);
HRMS (FAB+) calcd for C31H3035ClN6O8 (MH+) m/z 649.1814,
found 649.1818; calcd for C31H3037ClN6O8 (MH+) m/z 651.1784,
found 651.1805. Anal. (C31H29ClN6O8) C, H, N.
(1-Meth yl-3-n itr o-1H-pyr r ol-2-yl)m eth yl 1-(Ch lor om eth -
yl)-3-[(5,6,7-tr im eth oxy-1H-in d ol-1-yl)ca r bon yl]-2,3-d ih y-
d r o-1H-ben zo[e]in d ol-5-ylca r ba m a te (18d ). Similarly, re-
action of amine 5 and alcohol 18a gave 18d (28%) as a white
solid: mp (EtOAc) 218-220 °C; 1H NMR δ 9.42 (s, 1 H, indole-
NH), 8.94 (s, 1 H, OCONH), 7.87 (d, J ) 8.4 Hz, 1 H, H-6),
7.79 (d, J ) 8.2 Hz, 1 H, H-9), 7.57 (ddd, J ) 8.2, 7.4, 0.9 Hz,
1 H, H-8), 7.47 (ddd, J ) 8.4, 7.4, 0.9 Hz, 1 H, H-7), 7.08 (br
s, 1 H, H-4), 7.01 (d, J ) 2.2 Hz, 1 H, H-3′), 6.89 (s, 1 H, H-4′),
6.80 (d, J ) 3.3 Hz, 1 H, H-5′′), 6.57 (d, J ) 3.3 Hz, 1 H, H-4′′),
5.65 (s, 2 H, CH2O), 4.81 (dd, J ) 10.7, 9.7 Hz, 1 H, H-2), 4.67
(dd, J ) 10.7, 8.6 Hz, 1 H, H-2), 4.15-4.20 (m, 1 H, H-1), 4.10
(s, 3 H, OCH3), 3.95-3.99 (m, 4 H, OCH3, CH2Cl), 3.92 (s, 3
H, OCH3), 3.80 (br s, 3 H, NCH3), 3.48 (dd, J ) 11.0, 10.7 Hz,
1 H, CH2Cl); 13C NMR δ 160.4, 153.9, 150.2, 141.6, 140.6,
138.9, 133.7, 130.9, 129.7, 129.6, 128.8, 127.5, 125.6, 125.1 (2),
123.6, 123.1, 122.6, 122.2, 121.6, 113.0, 106.5, 106.1, 97.7, 61.5,
61.1, 56.3, 55.3, 54.9, 45.8, 43.4, 35.3; MS (FAB+) m/z 650
(1-Meth yl-5-n itr o-1H-pyr r ol-2-yl)m eth yl 1-(Ch lor om eth -
yl)-3-[(5,6,7-tr im eth oxy-1H-in d ol-1-yl)ca r bon yl]-2,3-d ih y-
d r o-1H-ben zo[e]in d ol-5-ylca r ba m a te (15d ). Similarly, re-
action of amine 5 and alcohol 15a gave 15d (63%) as a white
1
solid: mp (EtOAc) 212-214 °C; H NMR [(CD3)2SO] δ 11.45
(br s, 1 H, indole-NH), 9.82 (br s, 1 H, OCONH), 8.55 (br s, 1
H, H-4), 8.04 (d, J ) 8.5 Hz, 1 H, H-6), 7.89 (d, J ) 8.3 Hz, 1
H, H-9), 7.57 (dd, J ) 8.3, 7.2 Hz, 1 H, H-8), 7.45 (dd, J ) 8.5,
7.2 Hz, 1 H, H-7), 7.24 (d, J ) 4.4 Hz, 1 H, H-4′′), 7.09 (d, J )
2.0 Hz, 1 H, H-3′), 6.98 (s, 1 H, H-4′), 6.45 (d, J ) 4.4 Hz, 1 H,
H-3′′), 5.30 (s, 2 H, CH2O), 4.80 (dd, J ) 11.0, 9.4 Hz, 1 H,
H-2), 4.53 (dd, J ) 11.0, 1.8 Hz, 1 H, H-2), 4.32-4.37 (m, 1 H,
H-1), 4.07 (dd, J ) 11.1, 3.1 Hz, 1 H, CH2Cl), 3.91-3.96 (m, 7
H, CH2Cl, NCH3, OCH3), 3.83 (s, 3 H, OCH3), 3.81 (s, 3 H,
OCH3); 13C NMR [(CD3)2SO] δ 160.2, 154.0, 149.1, 142.1, 141.4,
139.9, 138.2, 136.1, 134.2, 130.7, 129.4, 127.1, 125.3, 125.2,
124.3, 123.7, 123.3, 123.1, 122.1, 113.1, 113.0, 110.6, 106.2,
98.0, 61.0, 60.9, 57.6, 55.9, 54.8, 47.5, 41.1, 33.9; MS (FAB+)
m/z 650 (MH+, 1%), 648 (MH+, 2); HRMS (FAB+) calcd for
(MH+, 0.6%), 648 (MH+, 1.5); HRMS (FAB+) calcd for C32H31
35ClN5O8 (MH+) m/z 648.1861, found 648.1850; calcd for C32H31
37ClN5O8 (MH+) m/z 650.1832, found 650.1841. Anal. (C32H30
ClN5O8) C, H, N.
-
-
-
Alter n a tive P r ep a r a tion s of 9d . Meth od 1. A solution
of 9a (17 mg, 110 µmol) in DCM (2 mL) was added dropwise
to a stirred solution of triphosgene (12 mg, 40 µmol) and
pyridine (9 µL, 110 µmol) in DCM (2 mL) at 20 °C. The mixture

5544 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Hay et al.
was stirred at 20 °C for 2 h, the solvent removed under reduced
pressure, and the residue dissolved in anhydrous THF (5 mL).
A solution of amine 5 (50 mg, 110 µmol) in THF (5 mL) was
added and the solution stirred at 20 °C for 16 h. The mixture
was partitioned between EtOAc (50 mL) and saturated aque-
ous KHCO3 solution (50 mL), the organic fraction dried, and
the solvent evaporated. The residue was purified by chroma-
tography, eluting with a gradient (50-100%) of EtOAc/pet.
ether, to give 9d (23 mg, 33%) as a tan solid: mp 200-205 °C
(dec); spectroscopically identical with an authentic sample
prepared above.
Meth od 2. Diethylisopropylamine (25 µL, 140 µmol) was
added to a stirred mixture of carbonate 27 (42 mg, 130 µmol),
amine 5 (57 mg, 140 µmol), HOBT (19 mg, 140 µmol), and 4 Å
powdered molecular sieves (200 mg) in dry DMF (5 mL) under
N2. The mixture was stirred for 6 days, filtered, diluted with
EtOAc (50 mL), washed with water (4 × 30 mL) and brine (25
mL), and dried, and the solvent was evaporated. The residue
was purified by chromatography, eluting with 50% EtOAc/pet.
ether, to give (i) starting amine 5 (40 mg, 70%) and (ii)
carbamate 9d (19 mg, 19%), spectroscopically identical with
an authentic sample prepared above.
EMT6 cells was estimated from the difference between plating
efficiency in puromycin and nonselective medium, and the
number of clonogens of both types was calculated per gram of
tumor tissue for control and treated tumors. Statistical
significance of drug effects was determined by ANOVA using
Dunnett’s test to compare groups.
Ack n ow led gm en t. The authors thank Dr. Maruta
Boyd, Alison Hogg, Li Fong Leong, and Susan Pullen,
for technical assistance; Dr. Moana Tercel, for providing
compounds 6a , 12a , and 16a ; Graham Atwell, for
providing amino-seco-CBI-TMI (5); Frank Friedlos, for
providing the V79puro and V79-NTRpuro cell lines; and
Dr. Martin Ford, Glaxo-Wellcome, Stevenage, U.K., for
providing the SKOV-NTRneo and WiDr-NTRneo cell lines.
This work was supported supported by the Marsden
Fund of New Zealand (M.P.H.), the Australian Institute
of Nuclear Sciences and Engineering (M.P.H.), the
Health Research Council of New Zealand (R.F.A.,
D.M.F., W.R.W.), and the Auckland Division of the
Cancer Society of New Zealand (W.A.D.).
Cell Lin es. Four pairs of cell lines, each comprising a tumor
cell line and corresponding transfectant stably expressing
NTR, were grown as monolayers in RMEM containing 5% fetal
bovine serum. V79-NTRpuro, also known as T79-A3, is a
Chinese hamster fibroblast which expresses NTR from an
CMV promoter; the corresponding NTR-ve line, here referred
to as V79puro, has been transfected with the empty shuttle
vector and is also known as T78-1.14 SKOV3-NTRneo and WiDr-
NTRneo, also known as SC3.2 and WC14, respectively, are
human ovarian and colon carcinoma lines derived from SKOV3
and WiDr, which also express NTR from a CMV promoter.52
EMT6-NTRpuro, also known as EN2A, is a murine breast
carcinoma line derived from EMT6 and expresses NTR from
a bicistronic cassette with an EF-1R promoter.49 Selection for
NTR expression was maintained during passage, but not
during experiments, using 15 µM puromycin (V79-NTRpuro), 5
µM puromycin (EMT6-NTRpuro), or 300 µg/mL G418 (WiDr-
NTRneo; SKOV3-NTRneo).
Gr ow th In h ibition Assa ys. Growth inhibitory potencies
were determined under aerobic conditions using log-phase
cultures in 96-well plates, as described previously.53,54 Cultures
were initiated 24 h before an 18 h drug exposure, with cell
densities determined 4-5 days later by staining with sulfo-
rhodamine B. IC50 values were calculated as the drug concen-
tration providing 50% inhibition of growth relative to controls
on the same plate.
Mou se Toxicity. Compound 1 was formulated in 10%
DMA/40% PEG400/50% water, and compounds 2, 9d , and 10d
were formulated in DMSO immediately before use. Groups of
six male C3H mice (ca. 25 g) were treated ip with single doses
of compounds at 10 µL/g body weight for 1 and 1 µL/g for 2,
9d , and 10d using 101/8-fold dose increments and were
observed daily for 60 days. Any animals losing >15% body
weight or becoming moribund during the study were termi-
nated.
In Vivo Excision Assa y. Activity was assessed by treating
mice with tumors containing mixtures of EMT6-NTRpuro cells
and EMT6 cells. CD-1 nude mice were inoculated subcutane-
ously with 3 × 106 cells using a 2:1 mixture of EMT6-NTRpuro
and EMT6 cells. When the tumors reached a mean diameter
(length × width) of 9 ( 1 mm, the animals were randomized
to treatment groups (five animals/group). Mice were treated
ip with single doses of prodrugs, at the MTD as determined
in C3H mice, and tumors were removed 18 h later to determine
cell killing by clonogenic assay as reported elsewhere.49 Briefly,
tumors were dissected, weighed, and dissociated in a Pronase/
collagenase/DNAase cocktail. Cell numbers were determined
with a particle counter (Coulter Electronics) and up to 105 cells
were plated in medium containing 3 µM puromycin or nonse-
lective medium to quantify survival of EMT6-NTRpuro and total
tumor cells, respectively. Plates were incubated for 8 days, and
colonies of >50 cells were counted. The plating efficiency of
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