Structure-activity relationships for 4-nitrobenzyl carbamates of 5-aminobenz[e]indoline minor groove alkylating agents as prodrugs for GDEPT in conjunction with E. coli nitroreductase

DOI: 10.1021/jm0205191

Source and publish data:

Journal of Medicinal Chemistry p. 2456 - 2466 (2003)

Update date:2022-09-26

Topics:

Authors:

Hay, Michael P.

Atwell, Graham J.

Wilson, William R.

Pullen, Susan M.

Denny, William A.

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Article abstract of DOI:10.1021/jm0205191

Twelve substituted 4-nitrobenzyl carbamate prodrugs of the 5-aminobenz[e]indoline class of DNA minor groove alkylating agents were prepared and tested as prodrugs for gene-directed enzyme prodrug therapy (GDEPT) using a two-electron nitroreductase (NTR) f

Full text of DOI:10.1021/jm0205191

2456

J . Med. Chem. 2003, 46, 2456-2466

Str u ctu r e-Activity Rela tion sh ip s for 4-Nitr oben zyl Ca r ba m a tes of

5-Am in oben z[e]in d olin e Min or Gr oove Alk yla tin g Agen ts a s P r od r u gs for

GDEP T in Con ju n ction w ith E. coli Nitr or ed u cta se

Michael P. Hay,* Graham J . Atwell, William R. Wilson, Susan M. Pullen, and William A. Denny

Auckland Cancer Society Research Laboratory, Faculty of Medical and Health Science, The University of Auckland,

Private Bag 92019, Auckland, New Zealand

Received November 14, 2002

Twelve substituted 4-nitrobenzyl carbamate prodrugs of the 5-aminobenz[e]indoline class of

DNA minor groove alkylating agents were prepared and tested as prodrugs for gene-directed

enzyme prodrug therapy (GDEPT) using a two-electron nitroreductase (NTR) from E. coli B.

The prodrugs and effectors were tested in a cell line panel comprising parental and transfected

human (SKOV/Skov-NTR^neo, WiDr/WiDr-NTR^neo), Chinese hamster (V79^puro/V79-NTR^puro), and

murine (EMT6/EMT6-NTR^puro) cell line pairs. In the human cell line pairs, several analogues

bearing neutral methoxyethoxy-, 2-hydroxyethoxy-, or 3-hydroxypropoxy-substituted side chains

were good substrates for NTR as measured by cytotoxicity ratios, with NTR-ve/NTR+ve ratios

similar to the established NTR substrates CB1954 (an aziridinyl dinitrobenzamide) and the

analogous bromomustard. Selectivity for NTR decreased with increasing side-chain size or the

presence of a basic amine group. Low to modest selectivity was observed in the Chinese hamster-

derived cell line pair; however, in the murine EMT6/EMT6-NTR^purocell line pair, the above

hydroxyalkoxy analogues again showed significant selectivity for NTR. The activity of the

2-hydroxyethoxy analogue was evaluated against NTR-expressing EMT6 tumors comprising

ca. 10% NTR+ve cells at the time of tumor treatment. A small decrease in NTR+ve cells was

observed after treatment, with a lesser effect against NTR-ve target cells, but these effects

were not statistically significant and were much less than for the dinitrobenzamides. These

results suggest that useful GDEPT prodrugs based on the 4-nitrobenzyl carbamate and

5-aminobenz[e]indoline motifs may be developed if optimization of pharmacokinetics can be

addressed.

In tr od u ction

nitrogen mustards have been developed as prodrugs for

NTR^14-16and the bromomustard SN 24927 (2) has

shown superior in vivo activity and a larger bystander

effect compared to CB1954 in preclinical models.¹⁷The

second class of substrates for NTR features a 4-ni-

trobenzyl carbamate motif (4-NO₂PhCH₂OCONHR).

The reduction of such 4-nitrobenzyl carbamates to the

corresponding hydroxylamines has been shown to in-

duce spontaneous fragmentation, releasing amines

RNH₂.^18-20Examples of 4-nitrobenzyl carbamate pro-

drugs of DNA-reactive agents include aniline mus-

tards,²¹mitomycin C,²¹enediynes,²²and pyrrolobenzo-

diazepines.²³Examples involving DNA-binding species

include prodrugs of tallimustine derivatives,²⁴doxoru-

bicin, and actinomycin D.²¹Notably, all of these ex-

amples have used the unsubstituted 4-nitrobenzyl car-

bamate as a trigger.

We recently studied^19,20the factors affecting the

kinetics of spontaneous fragmentation of 4-hydroxy-

laminobenzyl carbamates (HOHNPhCH₂OCONR₁R₂).

We showed that the unsubstituted 4-hydroxylami-

nobenzyl carbamate fragmented relatively slowly (t¹/₂:

16 min at 20 °C). Consistent with the proposed mech-

anism of fragmentation, this process could be acceler-

ated by electron-donating substituents on the benzyl

ring (stabilizing the developing positive charge on the

benzylic carbon), according to the equation: log(t_1/2) )

0.57σ + 1.30. This study suggested that appropriately

Gene-directed enzyme prodrug therapy (GDEPT) for

cancer uses various vector systems to deliver genes for

prodrug-activating enzymes (usually nonhuman) selec-

tively to tumor tissue.^1,2Subsequent systemic admin-

istration of a nontoxic prodrug that is a good substrate

for the introduced enzyme then results in selective

generation of active drug (“effector”)³in the tumor cells.

Provided this compound is able to diffuse to kill neigh-

boring tumor cells (bystander effect),⁴the approach can

compensate for the low efficiencies of gene transduction

for most gene vector delivery systems. Nitroreductases

are suitable enzymes for prodrug activation, since the

very large electronic change generated in a prodrug by

reduction of an aromatic nitro group (Hammett σ_p)

0.78) to the corresponding hydroxylamine (σ_p) -0.34)

provides an efficient “switch” mechanism.⁵The most

widely studied nitroreductase for GDEPT is the oxygen-

insensitive enzyme from E. coli (the nfsB gene product

NTR).^6,7

The substrates reported for prodrug approaches with

NTR fall into two classes. The first, dinitrobenzamide

alkylating agents, is exemplified by the aziridine CB

1954 (1)^8-11which is now in clinical trial as a prodrug

for GDEPT using NTR.^12,13More recently, related

* Corresponding author. Phone: 64-9-3737599 ext. 86598. Fax: 64-

9-3737502. E-mail: m.hay@auckland.ac.nz.

Published on Web 05/02/2003

Minor Groove Alkylating Agents as Prodrugs

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 12 2457

Sch em e 1^a

substituted 4-nitrobenzyl carbamates, particularly 2′-

alkoxy analogues, could be superior triggers because of

faster fragmentaion kinetics.

Reagents: (i) K₂CO₃, DMF, then Br(CH₂)₂OMe (17), Br-

In this paper we build on these kinetic studies by

linking modified 4-nitrobenzyl triggers (including 2′-

alkoxy derivatives) to very potent 5-aminobenz[e]indo-

line cytotoxins. We have reported recently^25,26on 5-amino-

benz[e]indolines (e.g., 3a -c), which are compounds

related to the duocarmycins²⁷that alkylate DNA in the

minor groove at the N3 of adenines, preferentially in

5′-polyA sequences.²⁸This process is deactivated by

acylation of the amine, which makes these compounds

of interest as effectors for 4-nitrobenzyl carbamate

prodrugs. We report our in vitro and in vivo studies on

the effectiveness of modified 4-nitrobenzylcarbamates

4-15 as prodrugs for a NTR-mediated GDEPT ap-

proach.

(CH₂)₂OTBDMS (18), Br(CH₂)₃OTBDMS (19), Cl(CH₂)₃NMe₂(20)

or Cl(CH₂)₃Nmorpholide (21); (ii) DIBALH, THF; (iii) 3a , tri-

phosgene, Et₃N, DCM, then 22-26, ⁿBu₂Sn(OAc)₂; (iv) for 27 and

28 HCl/MeOH.

Sch em e 2^a

Ch em istr y

The benzyl alcohols 22-26, required for carbamate

formation, were prepared by alkylation of methyl 2-hy-

droxy-4-nitrobenzoate¹⁹(16), with the appropriate ha-

lides to give ethers 17-21, followed by reduction with

DIBAL-H (Scheme 1). The preparation of alcohol 32

involved alkylation of 16 with epichlorohydrin under

basic conditions to give epoxide 29, which was hydro-

lyzed with perchloric acid to the diol 30. This was

protected as the acetonide 31 and then reduced with

DIBAL-H to the required benzyl alcohol 32 (Scheme 2).

Alcohol 37 was prepared by activation of alcohol 34 as

the 4-nitrophenyl carbonate 35 and subsequent reaction

with 4-aminobenzyl-TBDMS ether to give 36 which was

deprotected to provide 37 (Scheme 3).

Two methods were used to couple amines 3a ²⁵and

3b²⁵with the various alcohols. Reaction of the chloro-

formates of 34 and 38 wth 3a , formed in situ, gave

carbamates 5 and 12 in moderate yields (Scheme 4).

Reaction of 4-nitrobenzyl chloroformate with 3a and 3b

gave carbamates 4 and 14, in modest yield (Scheme 5).

Similarly, reaction of 3-NBoc derivative 39 with 4-nitro-

benzyl chloroformate gave carbamate 40, which was

deprotected and coupled with 5-[2-(dimethylamino)-

ethoxy]-1H-indole-2-carboxylic acid²⁶to give carbamate

15. Significantly higher yields (47-96%) were obtained

by in situ formation of the isocyanate of 3a , followed by

reaction with alcohols and catalytic dibutyltindiacetate,

Reagents: (i) epichlorohydrin, K₂CO₃, DMF; (ii) HClO₄, THF;

(iii) dimethoxypropane, PPTS, DMF; (iv) DIBAL-H, THF; (v) 3a ,

triphosgene, Et₃N, DCM, then 32, ⁿBu₂Sn(OAc)₂; (vi) 1 M HCl,

THF.

to give carbamates 6-11 and 13 (Schemes 1-3). This

method is the preferred option for carbamate formation

with deactivated amines such as 3.

Resu lts a n d Discu ssion

The prodrugs studied, together with the three amine

effectors (3a for compounds 4-13; 3b for compound 14,

and 3c for 15), are listed in Table 1 along with CB1954

(1) and the dibromomustard 2. The solubility of com-

pounds 1-15 in R-MEM culture medium with 5% added

fetal calf serum was determined by HPLC analysis of

the supernatant of a saturated solution. The prodrugs

4, 14, and 15 were considerably less soluble than their

corresponding amines 3a -c. The most dramatic loss in

solubility was seen for the unsubstituted nitrobenzyl

prodrug 4 (ca. 246-fold). The addition of a 2-substituent

to the nitrobenzyl group provided significant increases

of solubility: 6-fold for the 2-OMe group 5, up to 36-

fold for the dihydroxypropyl side chain 9. Addition of

an R-methyl substituent 12 gave a 28-fold increase in

solubility over 4. The addition of a basic amino side

chain positioned either on the nitrobenzyl unit, 10 and

11, or on the indole unit 15 provided no increase in

2458 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 12

Hay et al.

Sch em e 3^a

Sch em e 5^a

Reagents: (i) 4-nitrobenzyl chloroformate, THF; (ii) HCl,

dioxan; then 5-[2-(dimethylamino)ethoxy]-1H-indole-2-carboxylic

acid, EDCI‚HCl, DMA.

Reagents: (i) 4-nitrophenyl chloroformate, pyr; (ii) HOBT,

Et₃N, 4A sieves, THF; (iii) 1 M HCl, MeOH; (iv) 3a , triphosgene,

Et₃N, then 37, ⁿBu₂Sn(OAc)₂.

Ta ble 1. Structures and Physicochemical Properties of

4-Nitrobenzyl Carbamate Prodrugs

Sch em e 4^a

Reagents: (i) COCl₂, THF, then 3a , THF; (ii) triphosgene, pyr,

DCM; then 3a , THF.

solubility at pH 7 over neutral side chains despite their

lower logD values. Remarkably, the addition of a second

benzyl group in 13 reduced solubility by only a small

degree. The low solubilities were not unexpected, as

their calculated logP values [determined using ACDlogP

software (Advanced Chemistry Development Inc., Tor-

onto, Canada, v. 4.5)], were high, ranging from 3.55 for

9 to 6.04 for 13, considerably greater than those of the

amines themselves (1.87 to 2.87). The prodrugs 4-15

were considerably less soluble than 1, but were similar

to 2 despite having higher calculated logP values.

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

carcinoma), WiDr (human colon carcinoma), V79^puro

(Chinese hamster fibroblast) and EMT6 (mouse mam-

mary carcinoma), and the corresponding transfectants

stably expressing NTR. Cytotoxicity was measured as

IC₅₀values (nM following an 18 h drug exposure) in the

NTR-ve lines, together with the ratios of the IC₅₀values

between the NTR-ve and NTR+ve lines (Table 2) as a

measure of selectivity for NTR-expressing cells. The

compounds were compared with CB1954 (1) and SN

24927 (2) in order to evaluate their potential value as

GDEPT candidates.

no. formula

solubility^a

LogP^b

1600

197

310

0.8

1.28^c

2.68^c

1.87

2.87

2.26 (0.53)^d

4.55

2-OMe

4.77

4.47

3.79

4.57

2-O(CH₂)₂OMe

2-O(CH₂)₂OH

2-O(CH₂)₃OH

2-OCH₂CH(OH)CH₂OH

2-O(CH₂)₃NMe₂

2O(CH₂)₃Nmorpholide

3.38

4.95 (2.74)^d

4.55 (4.06)^d

4.90

6.09

3.98

4.94 (3.24)^d

Solubility (µM) in R-MEM culture medium, determined by

HPLC. ^blogP calculated using the ACDLogP prediction software(v

4.5). ^cMeasured values [1, 0.26; 2, 1.97 (ref 16)]. ^dlogD at pH 7 for

compounds with ionizable groups (in brackets).

The effector 3a had an average cytotoxicity across the

four parental cell lines of 0.85 nM while the methy-

lamino analogue 3b was on average ca. 6-fold more

Minor Groove Alkylating Agents as Prodrugs

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 12 2459

Ta ble 2. IC₅₀Values and IC₅₀Ratios (NTR-ve/NTR+ve) for the Amino-CBI Cytotoxins 3a -c and 4-Nitrobenzylcarbamate Prodrugs

4-15

SKOV/Skov-

NTR^{neo a,b}

WiDr/WiDr-

NTR^{neo a,b}

V79^puro/V79-

NTR^{puro a,b}

EMT6/EMT6-

NTR^{puro a,b}

no.

SKOV (nM)^a

WiDr (nM)^a

V79 (nM)^a

EMT6 (nM)^a

174 000 ( 10 000

58 000 ( 10 000

1.1 ( 0.1

0.2 ( 0.0

13 ( 1

317 ( 21

211 ( 16

0.6 ( 0.0

0.7 ( 0.2

1.6 ( 0.2

12 (1

54 000 ( 3000 51.2 ( 3

374 000 ( 14 000 2090 ( 210 710 000 ( 7 000

930 ( 140

1380

40 000 ( 3000

1.6 ( 0.1

0.2 ( 0.0

6 ( 2

174 ( 31

0.9 ( 0.3

1.1 ( 0.1

1.9 ( 0.8

33 ( 6

43 000 ( 5 000

0.33 ( 0.07

0.08 ( 0.02

2.6 ( 0.3

89 ( 23

39 ( 11

31 ( 1

302 ( 93

0.9 ( 0.1

1.2 ( 0.2

10 ( 3.3

6 ( 3

54 000

0.37 ( 0.06

2.0 ( 0.7

0.74 ( 0.18

0.6 ( 0.3

112 ( 44

108 ( 29

78 ( 2

0.11 ( 0.05

1.9 ( 0.8

193 ( 46

174 ( 28

33 ( 6

62 ( 8

391 ( 75

141 ( 17

110 ( 15

295 ( 127

191 ( 15

129 ( 41

269 ( 60

98 ( 13

59 ( 13

17 ( 4

47 ( 13

45 (4

106 ( 23

224 ( 37

145 ( 12

114 ( 26

189 ( 10

88 ( 10

67 ( 8

17 ( 4

84 ( 4

148 ( 48

91 ( 2

25 (1

3.8 ( 1.1

9.0 ( 0.1

6.60

63 ( 22

48 ( 5

11 ( 2

107 ( 22

47 ( 9

104 ( 11

91 ( 25

13 ( 3

58 ( 7

11 ( 1

40 ( 1

45 ( 7

20 ( 5

62 ( 10

54 ( 8

1.75

44 ( 18

74 ( 14

24 ( 7

79 ( 12

63 ( 1

3.6 ( 0.4

6.5 ( 1.0

5.65

1.45 ( 0.03

2.6 ( 1.1

1.67

3.4 ( 0.3

6 ( 3

158 ( 5

154 ( 3

11 ( 5

223 ( 48

152 ( 27

770 ( 107

0.7 ( 0.0

295 ( 61

101 ( 24

403 ( 24

1.8 ( 0.8

15 ( 1

1.03 ( 0.01

8.4 ( 2.1

4.3 ( 1.0

1.8 ( 0.6

27 ( 7

10 ( 4

48 ( 15

157 ( 20

48 ( 13

208 ( 54

41 ( 4

20 ( 2

31 ( 13

Values are mean ( SEM for up to five independent experiments. ^bIntraexperiment ratios. Cell lines: SKOV3 (human ovarian

carcinoma), WiDr (human colon carcinoma), V79^puro(Chinese hamster fibroblast), and EMT6 (mouse mammary carcinoma).

potent and the solubilized amine 3c was ca. 7-fold less

potent than 3a . Prodrugs 4-13, which share the same

effector 3a , were overall less cytotoxic in the human

SKOV and WiDr wild-type lines (average IC₅₀s 127 and

207 nM, respectively), compared with the rodent lines

T-78 and EMT6 (average IC₅₀s 49 and 83 nM, respec-

tively). Across all cell lines, prodrugs 4-13 were, on

average, deactivated relative to the effector 3a by factors

ranging from 66 to 273-fold. Prodrug 14 showed an even

greater degree of deactivation (average of 575-fold) than

4-13, primarily due to the enhanced cytotoxicity of 3b.

Prodrug 15 displayed modest deactivation (average 74-

fold). However, the prodrugs 4-15 were still ca. 1000-

fold more potent than the dinitrobenzamide prodrugs

1 and 2.

Prodrugs 4 and 5 generally showed a similar degree

of activation by the NTR-expressing cell lines (mean IC₅₀

ratio 42 and 45, respectively) but this was substantially

less than the differential between effector 3a and

prodrugs 4 and 5, suggesting partial metabolic conver-

sion to 3a . The methoxyethoxy prodrug 6 and alcohols

7 and 8 showed the largest degree of activation by NTR

across the four cell lines (mean IC₅₀ratios 52, 87, and

63, respectively), while the larger diol 9 showed lesser

activation (19-fold). Compounds 6-8 were clearly su-

perior to 4 in the human cell lines, but showed only

equivalent activation in the rodent cell lines. The

amines 10 and 11 showed little activation, suggesting

an unfavorable interaction between the side chain and

the binding pocket of NTR. Compound 12 was examined

to determine the effect of an R-methyl substituent on

prodrug selectivity, since we had previously showed¹⁹

that this accelerated the fragmentation of the corre-

sponding hydroxylamines. This prodrug was somewhat

less selective than the nonmethylated derivative 4

(mean activation 6-fold), with lower levels of activation

in the NTR+ve lines. There have been several reports

that placing large effector units close to the trigger

domain of prodrugs results in a loss of enzyme binding,

presumably due to steric hindrance. In those cases (e.g.,

prodrugs for carboxypeptidase²⁹and â-glucuronidase³⁰),

interposition of a “spacer” group can restore activity.

This was evaluated in the present case with compound

13, where a 4-aminobenzyl carbamate unit was inter-

posed. However, this showed no selectivity, with no

increase in potency in the NR+ve lines, suggesting

limited fragmentation of the more complex spacer or

unfavorable binding interactions in the active site. To

determine the effect of substitution on the carbamate

amine, the NMe analogue 14 was evaluated. This

prodrug achieved the highest degree of deactivation

compared to its effector 3b (430-760-fold), but showed

only moderate differentials between NTR-expressing

and parental lines (average of 15-fold) indicating rela-

tively poor levels of activation by NTR. Limited studies¹⁹

have suggested that the fragmentation of a similar NMe

carbamate is a less efficient process than for the

corresponding NH carbamate.

Overall, the IC₅₀ratios (NTR-ve/NTR+ve) of prodrugs

6-8 were similar to that of the dinitrobenzamides 1 and

2 for the human cell lines (SKOV3 and WiDr) but the

ratios for the two rodent cell line pairs were much lower

for the nitrobenzylcarbamate derivatives. Maximum in

vitro selectivity for NTR was seen with compound 7

which has a 2-hydroxyethoxy side chain. Increasing the

length of this by one carbon (6 or 8) was accompanied

by a loss of selectivity which continued with diol 9. Low

selectivity was seen with 10 and 11 which bear basic

side chains. The lack of tolerance for charged side chains

has been observed for analogues of 2.¹⁴Other modifica-

tions, e.g., 12-15 did not result in significant selectivity

for NTR. Assuming comparable uptake and formation

of a common cytotoxic metabolite, the ratios of the IC₅₀s

in the NTR-ve and NTR+ve cells are a measure of the

effectiveness of the prodrugs as substrates for the NTR

enzyme. This will depend on a number of factors, the

most important likely to be the reduction potential of

the nitro group and the ability to fit the enzyme binding

site.

Generally, 4-nitrobenzyl carbamates are likely to have

considerably lower reduction potentials than the estab-

lished NTR substrates such as CB1954 (E(1) -385

mV).³¹In contrast, the reduction potential of 4-nitroben-

zyl alcohol is considerably lower (-494 ( 8 mV), which

is likely to make the 4-nitrobenzyl carbamates harder

to reduce. In agreement with this, various nitrobenzyl

carbamates showed little activity when evaluated as

bioreductive prodrugs (activation under hypoxic condi-

2460 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 12

Hay et al.

tions by endogenous enzymes).^32-34Addition of a 2′-

methoxy group (electron-withdrawing when meta to the

NO₂) is likely to raise the E(1) of compounds such as

6-8 slightly (2-methoxy-4-nitrobenzyl alcohol has an

E(1) of -470 ( 12 mV; R. F. Anderson, personal

communication).

The ability of prodrugs to access the enzyme binding

site is more difficult to evaluate, but X-ray studies of

NTR reveal a V-shaped pocket narrowing toward the

FMN cofactor which is only accessible from the re

face.^7,35The prodrugs were docked into the active site

of the enzyme generated from the coordinates³⁵for the

crystal structure of NTR complexed with nicotinic acid

as a mimic for the cofactor NADPH. Nicotinic acid was

removed from the active site and energy-minimized

prodrugs docked using GOLD (v1.2, Cambridge Crystal-

lographic Centre) and Sybyl (v6.8, Tripos Inc., St Louis,

MO). Docking of prodrug 4 into the active site showed

a variety of binding modes, indicating that the cleft is

large enough to accommodate it readily. Constraining

the 4-nitro group to within 3.5 Å of N5 of FMN located

the nitrobenzyl group above the FMN with the CBI-TMI

unit adopting a range of configurations extending out

into the binding cleft. Smaller side chains at the

2-position, e.g., methoxy 5 and 2-hydroxyethyl 7, were

also aligned into the cleft whereas longer side chains,

e.g., 9-11 were aligned along the channel formed by

Phe124 and Tyr68. The placement of the side chain

along the channel resulted in displacement of the nitro

group from above the N5 of FMN. This provides a

possible explanation for the waning in selectivity for

NTR seen from 7 down to 11.

F igu r e 1. Comparison of in vivo activity and bystander effects

for CB1954 (1), SN 24927 (2), and the nitrobenzylcarbamate

7. Prodrugs given at 240, 1330, and 421 µmol kg^-1ip. In this

experiment, tumors at excision comprised 9.9% EMT6-NTR^puro

as assessed by the proportion of puromycin-resistant cells. *

EMT6-NTR^puro< 10^-4clonogens/g.

lines in vitro and were significantly more selective for

NTR in these cell lines than the unsubstituted nitroben-

zyl carbamate 4. These compounds, bearing neutral

methoxyethoxy- (6), 2-hydroxyethoxy- (7), or 3-hydrox-

ypropoxy-substituted (8) side chains, displayed NTR+ve/

NTR-ve ratios similar to the dinitrobenzamides 1 and

2, which are well established as useful substrates for

NTR. Compounds 6-8 were active in vitro despite

having lower reduction potentials and a large steric

demand on the binding site relative to 1 and 2. Com-

pounds 6-8 showed modest selectivity (11-17-fold) for

NTR in the V79^puro/T79-NTR^purolines and good selectiv-

ity (78-104-fold) for NTR in the EMT6/EMT6-NTR^puro

murine lines, matching the selectivity seen for 4 in these

cell lines. Compound 7 did not display significant

antitumor activity in the mixed EMT6/EMT6-NTR^puro

tumor model. However, the dinitrobenzamides 1 and 2

which were considerably more selective for NTR in the

rodent cell lines, displayed in vivo activity in the EMT6

model. These results suggest potentially useful GDEPT

prodrugs of 5-aminobenz[e]indolines using the 4-ni-

trobenzyl carbamate motif may be developed if a num-

ber of factors can be addressed. In particular the

optimization of pharmacokinetics, especially extravas-

cular diffusion, may be required for the translation of

the observed in vitro activity to in vivo efficacy. Im-

proved activity might also be achieved through increas-

ing the reduction potential of the nitro group and

reducing the steric demand in the active site of the

enzyme.

The maximum tolerated dose (MTD) of effector 3a

given ip in DMSO to C3H mice was 2.37 µmol kg^-1

whereas the MTD of 7 was 421 µmol kg^-1demonstrating

considerable deactivation of the toxicity of the effector

by the prodrug in vivo. The MTD of 1 was 240 µmol kg^-1

given ip in DMSO whereas 2 was considerably less toxic

(>1330 µmol kg^-1). The activity of 7 was evaluated

against NTR-expressing EMT6 tumors comprising mix-

tures of NTR-ve and NTR+ve cells and compared with

1 and 2. In this model nude mice are inoculated with

2:1 mixtures of EMT6-NTR^puroand EMT6 (NTR-ve)

cells, respectively, providing ca. 5-10% NTR+ve cells

at the time of tumor treatment.³⁶This tumor model

represents the likely situation in a GDEPT protocol

where low rates of transfection of tumor tissue are

expected. A small decrease in NTR+ve cells was ob-

served after treatment with 7, with a lesser effect

against NTR-ve target cells, but neither of these effects

were statistically significant (Figure 1). In contrast,

dinitrobenzamides 1 and 2 given at 200 and 1330 µmol

kg^-1, respectively, showed significant killing of activator

(EMT6-NTR^puro) cells and target (EMT6) cells, indicat-

ing the operation of a bystander effect. Given that killing

of NTR+ve cells in the tumors was at least as great for

1 as for 2, but killing of NTR-ve cells was greater for 2,

the latter appears to have a more efficient bystander

effect in this system as previously reported with WiDr

tumor xenografts¹⁷

Exp er im en ta l Section

Ch em istr y. Analyses were carried out in the Microchemical

Laboratory, University of Otago, Dunedin, NZ. Melting points

were determined on an Electrothermal 2300 Melting Point

Apparatus. NMR spectra were obtained on a Bruker AM-400

spectrometer at 400 MHz for H and 100 MHz for ¹³C spectra.

Spectra were obtained in CDCl₃unless otherwise specified and

are referenced to Me₄Si. Chemical shifts and coupling con-

stants were recorded in units of ppm and Hz, respectively.

Assignments were determined using COSY, HSQC, and HMBC

two-dimensional experiments. Mass spectra were determined

Con clu sion s

Several nitrobenzyl carbamate prodrugs, e.g., 6-8,

proved to be good substrates for NTR in the human cell

Minor Groove Alkylating Agents as Prodrugs

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 12 2461

on a VG-70SE mass spectrometer 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 10000 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 anhy-

drous Na₂SO₄. Solvents were evaporated under reduced pres-

sure on a rotary evaporator. Thin-layer chromatography was

carried out on aluminum-backed silica gel plates (Merck 60

Meth yl 2-[3-(4-Mor p h olin yl)p r op oxy]-4-n itr oben zoa te

(21). Similarly, reaction of 16 and 4-(3-chloropropyl)morpho-

line gave ether 21 (80%) as an oil, H NMR δ 7.88 (d, J ) 9.1

Hz, 1 H, H-6), 7.81 (m, 2 H, H-3, H-5), 4.21 (t, J ) 6.3 Hz, 2

H, CH₂O), 3.93 (s, 3 H, OCH₃), 3.69-3.74 (m, 4 H, 2 × CH₂O),

2.57 (t, J ) 7.0 Hz, 2 H, CH₂N), 2.47-2.51 (m, 4 H, 2 × CH₂N),

2.02-2.07 (m, 2 H, CH₂); ¹³C NMR δ 165.3, 158.6, 150.6, 132.0,

126.2, 114.8, 107.8, 67.6, 66.9 (2), 55.0, 53.7 (2), 52.5, 25.9.

The hydrochloride salt had mp (EtOAc) 160-163 °C. Anal.

(C₁₅H₂₀N₂O₆.HCl) C, H, Cl.

₂₅₄) 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 biological testing were analyzed at >99% purity by reverse

phase HPLC using a Philips PU4100 liquid chromatograph, a

Phenomenex BondClone 10-C18 stainless steel column (300

mm × 3.9 mm i.d.) and a Philips PU4120 diode array detector.

Chromatograms were run using various gradients of aqueous

(1 M NaH₂PO₄, 0.75 M heptanesulfonic acid, 0.5 M dibuty-

lammonium phosphate, and MilliQ water in a 1:1:1:97 ratio)

and organic (80% MeOH/MilliQ water) phases. DCM refers to

dichloromethane, DMF refers to dry dimethylformamide, ether

refers to diethyl ether, EtOAc refers to ethyl acetate, EtOH

Met h yl 4-Nit r o-2-(2-oxir a n ylm et h oxy)b en zoa t e (29).

Similarly, reaction of 16 and epichlorohydrin followed by

chromatography successively gave starting material (18%),

followed by ether 29 (59%) as a white solid, mp (EtOAc/pet.

ether) 62-63 °C; ¹H NMR δ 7.91 (dd, J ) 7.7, 1.0 Hz, 1 H,

H-5), 7.84-7.86 (m, 2 H, H-3, H-6), 4.49 (dd, J ) 11.2, 2.4 Hz,

1 H, H-3′), 4.14 (dd, J ) 11.2, 5.2 Hz, 1 H, H-3′), 3.94 (s, 3 H,

OCH₃), 3.40-3.44 (m, 1 H, H-2′), 2.91-2.97 (m, 2 H, H-1′);

¹³C NMR δ 165.0, 158.1, 150.6, 132.3, 126.1, 115.6, 108.4, 69.6,

52.6, 49.7, 44.3; MS (CI, NH₃) m/z 295 (M + CH₃CN⁺, 70%),

259 (MH⁺, 100%). Anal. (C₁₁H₁₁NO₆) C, H, N.

Meth yl 2-(2,3-Dih ydr oxypr opoxy)-4-n itr oben zoate (30).

Perchloric acid (1 mL) and water (3 mL) were added to a

stirred solution of epoxide 29 (205 mg, 0.81 mmol) in THF (20

mL), and the solution was stirred at 20 °C for 16 h. The solvent

was evaporated and the residue partitioned between EtOAc

(50 mL) and water (50 mL). The organic fraction was washed

with water (50 mL) and brine (25 mL) and dried and the

solvent evaporated. The residue was purified by chromatog-

raphy, eluting with 70% EtOAc/pet. ether, to give diol 30 (172

mg, 78%) as an oil which solidified on standing, mp 60-65

°C; ¹H NMR δ 8.02 (d, J ) 8.5 Hz, 1 H, H-6), 7.87 (dd, J ) 8.5,

2.0 Hz, 1 H, H-5), 7.84 (d, J ) 2.0 Hz, 1 H, H-3), 4.38 (dd, J )

9.3, 3.1 Hz, 1 H, H-3′), 4.23 (dd, J ) 9.3, 5.4 Hz, 1 H, H-3′),

4.10-4.14 (m, 1 H, H-2′), 3.95 (s, 3 H, OCH₃), 3.88 (br d, J )

4.1 Hz, 2 H, H-1′), 3.05 (br s, 1 H, OH), 1.95 (br s, 1 H, OH);

¹³C NMR δ 164.8, 159.1, 151.0, 132.8, 124.9, 115.6, 108.8, 73.0,

69.2, 63.2, 52.8; MS (CI, NH₃) m/z 272 (MH⁺, 1%), 240 (50%),

165 (100); HRMS (CI, NH₃) calcd for C₁₁H₁₄NO₇(MH⁺) m/z

272.0770, found 272.0766. Anal. (C₁₁H₁₃NO₇) C, H, N.

refers to ethanol, Pr₂O refers to diisopropyl ether, MeOH

refers to methanol, pet. ether refers to petroleum ether, boiling

range 40-60 °C, and THF refers to tetrahydrofuran dried over

sodium benzophenone ketyl. All solvents were freshly distilled.

Alk yla tion of P h en ols. Meth yl 2-(2-Meth oxyeth oxy)-

4-n itr oben zoa te (17). A mixture of methyl 2-hydroxy-4-

nitrobenzoate (16)¹⁹(1.0 g, 5.07 mmol) and K₂CO₃(1.05 g, 7.61

mmol) in DMF (25 mL) was stirred at 20 °C for 30 min. A

solution of 2-bromoethyl methyl ether (0.72 mL, 7.61 mmol)

in DMF (3 mL) was added and the mixture was stirred at 100

°C for 4 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), dried, and the solvent evaporated. The residue was

purified by chromatography, eluting with 30% EtOAc/pet.

ether, to give ether 17 (1.27 g, 98%) as a white solid, mp

(EtOAc/pet. ether) 45-46 °C; H NMR δ 7.90 (d, J ) 8.3 Hz,

1 H, H-6), 7.82-7.86 (m, 2 H, H-3, H-5), 4.28-4.30 (m, 2 H,

CH₂O), 3.93 (s, 3 H, OCH₃), 3.82-3.87 (m, 2 H, CH₂O), 3.48

(s, 3 H, OCH₃); ¹³C NMR δ 165.1, 158.6, 150.6, 132.1, 126.4,

115.2, 108.4, 70.5, 69.4, 59.4, 52.5. Anal. (C₁₁H₁₃NO₆) C, H,

Meth yl 2-[(2,2-Dim eth yl-1,3-d ioxola n -4-yl)m eth oxy]-4-

n itr oben zoa te (31). 2,2-Dimethoxypropane (0.91 mL, 7.37

mmol) was added dropwise to a stirred solution of diol 30 (400

mg, 1.47 mmol) and PPTS (37 mg, 0.15 mmol) in DMF (20

mL) under N₂and stirred at 20 °C for 24 h. The solvent was

evaporated and the residue 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 evaporated. The residue was purified by chromatog-

raphy, eluting with 30% EtOAc/pet. ether, to give acetonide

Meth yl 2-(2-{[ter t-Bu tyl(d im eth yl)silyl]oxy}eth oxy)-4-

n itr oben zoa te (18). Similarly, reaction of 16 and 2-bromo-

ethyl tert-butyl(dimethyl)silyl ether gave ether 18 (77%) as a

white solid, mp (EtOAc/pet. ether) 47-48 °C; ¹H NMR δ 7.89

(d, J ) 1.3 Hz, 1 H, H-3), 7.81-7.85 (m, 2 H, H-5, H-6), 4.24

(t, J ) 5.0 Hz, 2 H, CH₂O), 4.03 (t, J ) 5.0 Hz, 2 H, CH₂O),

3.92 (s, 3 H, OCH₃), 0.88 (s, 9 H, SiC(CH₃)₃), 0.08 (s, 6 H, Si-

(CH₃)₂); ¹³C NMR δ 165.5, 158.6, 150.4, 131.8, 126.7, 115.0,

31 (458 mg, 100%) as a yellow oil, H NMR δ 7.90 (d, J ) 8.2

Hz, 1 H, H-6), 7.84-7.88 (m, 2 H, H-3, H-5), 4.49-4.54 (m, 1

H, H-4′′), 4.25 (dd, J ) 9.6, 4.6 Hz, 1 H, H-5′′), 4.19 (dd, J )

8.5, 6.4 Hz, 1 H, H-2′), 4.15 (dd, J ) 9.6, 4.6 Hz, 1 H, H-5′′),

4.03 (dd, J ) 8.5, 5.8 Hz, 1 H, H-2′), 3.94 (s, 3 H, OCH₃), 1.46

(s, 3 H, CH₃), 1.41 (s, 3 H, CH₃); ¹³C NMR δ 165.1, 158.2, 150.6,

132.2, 126.4, 115.5, 109.9, 108.4, 73.6, 69.8, 66.5, 52.6, 26.6,

25.3; MS (CI, NH₃) m/z 312 (MH⁺, 15%), 296 (95), 101 (95), 71

(100); HRMS (CI, NH₃) calcd for C₁₄H₁₈NO₇(MH⁺) m/z

312.1083, found 312.1092.

108.3, 71.1, 61.7, 52.5, 25.7 (3), 18.3, -5.5 (2). Anal. (C₁₆H₂₅

NO₆Si) C, H, N.

Meth yl 2-(3-{[ter t-Bu tyl(d im eth yl)silyl]oxy}p r op oxy)-

4-n itr oben zoa te (19). Similarly, reaction of 16 and 3-bro-

mopropyl tert-butyl(dimethyl)silyl ether gave ether 19 (93%)

as a waxy solid, mp (EtOAc) 36.5-37 °C; ¹H NMR δ 7.88 (d, J

) 8.9 Hz, 1 H, H-6), 7.80-7.84 (m, 2 H, H-3, H-5), 4.24 (t, J )

6.0 Hz, 2 H, CH₂O), 3.92 (s, 3 H, OCH₃), 3.85 (t, J ) 5.9 Hz,

2 H, CH₂O), 2.04-2.09 (m, 2 H, CH₂), 0.88 (s, 9 H, OSi(CH₃)₃),

0.04 (s, 6 H, OSi(CH₃)₂); ¹³C NMR δ 165.4, 158.7, 150.7, 132.0,

126.1, 114.8, 107.7, 64.0, 59.0, 52.5, 32.0, 25.9 (3), 18.3, -5.5

(2). Anal. (C₁₇H₂₇NO₆Si) C, H, N.

Meth yl 2-[3-(Dim eth yla m in o)p r op yloxy]-4-n itr oben -

zoa te (20). Similarly, reaction of 16 and N-(3-chloropropyl)-

N,N-dimethylamine gave ether 20 (71%) as a pale yellow oil

which converted to the HCl salt, mp (EtOAc) 175-177 °C; ¹H

NMR [(CD₃)₂SO] δ 10.90 (br s, 1 H, NH⁺Cl^-), 7.87-7.93 (m, 3

H, H-3, H-5, H-6), 4.32 (t, J ) 6.0 Hz, 2 H, CH₂O), 3.89 (s, 3

H, OCH₃), 3.18-3.23 (m, 2 H, CH₂N), 2.77 (d, J ) 4.8 Hz, 6

H, N(CH₃)₂), 2.18-2.24 (m, 2 H, CH₂); ¹³C NMR [(CD₃)₂SO] δ

165.0, 157.3, 150.2, 131.6, 126.0, 115.3, 108.4, 66.5, 53.7, 52.6,

42.0 (2), 23.3. Anal. (C₁₃H₁₈N₂O₅.HCl) C, H, N, Cl.

DIBAL-H Red u ction of Ester s. 2-[2-(Meth oxy)eth oxy]-

4-n itr oben zyl Alcoh ol (22). DIBAL-H (1 M in DCM, 16.4

mL, 16.4 mmol) was added dropwise to a stirred solution of

ester 17 (1.27 g, 4.97 mmol) in THF (100 mL) at 5 °C and the

solution stirred at 5 °C for 1 h. The solution was poured into

a solution of potassium sodium tartrate (1 M, 100 mL) and

stirred vigorously for 30 min. The mixture was extracted with

EtOAc (2 × 100 mL), the combined organic fraction washed

with water (50 mL) and brine (50 mL) and dried, and the

solvent was evaporated. The residue was purified by chroma-

tography, eluting with a gradient (30-50%) of EtOAc/pet.

ether, to give alcohol 22 (1.03 g, 91%) as a white solid, mp

(EtOAc/pet. ether) 89-90.5 °C; H NMR δ 7.86 (dd, J ) 8.2,

2.1 Hz, 1 H, H-5), 7.72 (d, J ) 2.1 Hz, 1 H, H-3), 7.47 (d, J )

2462 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 12

Hay et al.

8.2 Hz, 1 H, H-6), 4.74 (br s, 2 H, CH₂O), 4.26-4.29 (m, 2 H,

CH₂O), 3.78-3.80 (m, 2 H, CH₂O), 3.45 (s, 3 H, OCH₃), 3.10

(br s, 1 H, OH); ¹³C NMR δ 156.8, 148.2, 137.5, 128.6, 116.5,

107.0, 70.5, 68.4, 61.1, 59.1. Anal. (C₁₀H₁₃NO₅) C, H, N.

2-Meth oxy-4-n itr oben zyl 4-({[ter t-Bu tyl(dim eth yl)silyl]-

oxy}m eth yl)p h en ylca r ba m a te (36). Et₃N (0.40 mL, 2.84

mmol) was added to a stirred suspension of carbonate 35 (0.90

g, 2.58 mmol), 4-({[tert-butyl(dimethyl)silyl]oxy}methyl)aniline

(0.64 g, 2.71 mmol), HOBT (0.35 g, 2.58 mmol), and 4 Å

molecular sieves (900 mg) in THF (80 mL) and the mixture

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 1 M HCl (2 × 40

mL), water (100 mL), and brine (50 mL) and dried and the

solvent evaporated. The residue was purified by chromatog-

raphy, eluting with 20% EtOAc/pet. ether, to give silyl ether

36 (0.89 g, 77%), mp (EtOAc/pet. ether) 120-122 °C; ¹H NMR

δ 7.84 (dd, J ) 8.3, 2.1 Hz, 1 H, H-5′), 7.72 (d, J ) 2.1 Hz, 1

H, H-3′), 7.51 (d, J ) 8.3 Hz, 1 H, H-6′), 7.35 (d, J ) 8.3 Hz,

2 H, H-2, H-6), 7.26 (d, J ) 8.3 Hz, 2 H, H-3, H-5), 6.76 (br s,

1 H, OCONH), 5.30 (s, 2 H, CH₂O), 4.69 (s, 2 H, CH₂OSi), 3.93

(s, 3 H, OCH₃), 0.92 (s, 9 H, SiC(CH₃)₃), 0.09 (s, 6 H, Si(CH₃)₂);

¹³C NMR δ 157.3, 153.0, 148.7, 137.0, 136.4, 132.1, 128.7, 126.9

(2), 118.6 (2), 115.7, 105.2, 64.6, 61.4, 56.0, 26.9 (3), 18.4, -5.2

(2). Anal. (C₂₂H₃₀N₂O₆Si) C, H, N.

[2-(2-{[ter t-Bu t yl(d im et h yl)silyl]oxy}et h oxy)-4-n it r o-

p h en yl]m eth a n ol (23). Similarly, reduction of ester 18 gave

alcohol 23 (97%) as a white solid, mp (EtOAc/pet. ether) 89-

90 °C; ¹H NMR δ 7.85 (dd, J ) 8.2, 2.1 Hz, 1 H, H-5), 7.74 (d,

J ) 2.1 Hz, 1 H, H-3), 7.46 (d, J ) 8.2 Hz, 1 H, H-6), 4.75 (s,

2 H, CH₂O), 4.21 (dd, J ) 4.9, 4.4 Hz, 2 H, CH₂O), 4.01 dd, J

) 4.9, 4.4 Hz, 2 H, CH₂O), 2.84 (br s, 1 H, OH), 0.90 (s, 9 H,

SiC(CH₃)₃), 0.06 (s, 6 H, Si(CH₃)₂); ¹³C NMR δ 157.0, 148.3,

137.1, 128.4, 116.3, 106.8, 70.5, 61.6, 61.3, 25.8 (3), 18.3, -5.4

(2); Anal. (C₁₅H₂₅NO₅Si) C, H, N.

[2-(3-{[ter t-Bu tyl(d im eth yl)silyl]oxy}p r op oxy)-4-n itr o-

p h en yl]m eth a n ol (24). Similarly, reduction of ester 19 gave

alcohol 24 (94%) as a white solid, mp (EtOAc/pet. ether) 48-

49 °C; ¹H NMR δ 7.84 (dd, J ) 8.3, 2.1 Hz, 1 H, H-5), 7.71 (d,

J ) 2.1 Hz, 1 H, H-3), 7.51 (d, J ) 8.3 Hz, 1 H, H-6), 4.76 (d,

J ) 6.3 Hz, 2 H, CH₂O), 4.21 (t, J ) 6.1 Hz, 2 H, CH₂O), 3.82

(t, J ) 5.9 Hz, 2 H, CH₂OSi), 2.40 (t, J ) 6.3 Hz, 1 H, OH),

2.02-2.08 (m, 2 H, CH₂), 0.89 (s, 9 H, OSiC(CH₃)₃), 0.06 (s, 6

H, OSi(CH₃)₂); ¹³C NMR δ 156.5, 148.2, 136.7, 127.8, 115.9,

105.8, 65.5, 60.8, 59.3, 32.0, 25.9 (3), 18.3, -5.4 (2). Anal.

(C₁₆H₂₇NO₅Si) C, H, N.

{2-[3-(Dim eth yla m in o)p r op oxy]-4-n itr op h en yl}m eth a -

n ol (25). Similarly, reduction of ester 20 gave alcohol 25 (86%)

as a tan solid, mp (EtOAc) 104-105 °C; ¹H NMR δ 7.87 (dd, J

) 8.3, 2.1 Hz, 1 H, H-5), 7.69 (d, J ) 2.1 Hz, 1 H, H-3), 7.64

(d, J ) 8.3 Hz, 1 H, H-6), 5.43 (br s, 1 H, OH), 4.58 (s, 2 H,

CH₂O), 4.14 (t, J ) 6.5 Hz, 2 H, CH₂O), 2.36 (t, J ) 7.0 Hz, 2

H, CH₂N), 2.15 (s, 6 H, N(CH₃)₂), 1.85-1.91 (m, 2 H, CH₂);

¹³C NMR δ 155.2, 147.0, 138.9, 126.7, 115.3, 105.2, 66.5, 57.6,

55.5, 45.1 (2), 26.5. Anal. (C₁₂H₁₈N₂O₄) C, H, N.

{2-[3-(4-Mor p h olin yl)p r op oxy]-4-n it r op h en yl}m et h a -

n ol (26). Similarly, reduction of ester 21 gave alcohol 26 (88%)

as a tan solid, mp (EtOAc) 105-106 °C; ¹H NMR δ 7.83 (dd, J

) 8.2, 2.1 Hz, 1 H, H-5), 7.70 (d, J ) 2.1 Hz, 1 H, H-3), 7.46

(d, J ) 8.2 Hz, 1 H, H-6), 4.71 (s, 2 H, CH₂O), 4.18 (t, J ) 6.0

Hz, 2 H, CH₂O), 3.74-3.77 (m, 4 H, 2 × CH₂O), 3.60 (br s, 1

H, OH), 2.56 (dd, J ) 6.7, 6.5 Hz, 2 H, CH₂N), 2.45-2.49 (m,

4 H, 2 × CH₂N), 2.01-2.06 (m, 2 H, CH₂); ¹³C NMR δ 156.7,

148.2, 137.2, 128.2, 116.1, 106.2, 67.7, 66.5 (2), 60.7, 56.3, 53.9

(2), 25.5. Anal. (C₁₄H₂₀N₂O₅) C, H, N.

{2-[(2,2-Dim et h yl-1,3-d ioxola n -4-yl)m et h oxy]-4-n it r o-

p h en yl}m eth a n ol (32). Similarly, reduction of ester 31 gave

alcohol 32 (92%) as a white solid, mp (EtOAc/pet. ether) 90-

92 °C; ¹H NMR δ 7.87 (dd, J ) 8.2, 2.1 Hz, 1 H, H-5), 7.72 (d,

J ) 2.1 Hz, 1 H, H-3), 7.50 (d, J ) 8.2 Hz, 1 H, H-6), 4.82 (d,

J ) 14.1 Hz, 1 H, CH₂O), 4.70 (d, J ) 14.1 Hz, 1 H, CH₂O),

4.51-4.57 (m, 1 H, H-4′′), 4.23 (dd, J ) 9.8, 4.0 Hz, 1 H, H-5′′),

4.19 (dd, J ) 9.8, 5.4 Hz, 1 H, H-2′), 4.11 (dd, J ) 9.8, 5.4 Hz,

1 H, H-5′′), 3.95 (dd, J ) 8.7, 5.4 Hz, 1 H, H-2′), 3.25 (br s, 1

H, OH), 1.48 (s, 3 H, CH₃), 1.41 (s, 3 H, CH₃); ¹³C NMR δ 156.5,

148.2, 137.2, 128.6, 116.6, 110.0, 106.4, 73.7, 69.7, 66.0, 61.0,

26.6, 25.0; MS m/z 283 (M⁺, 3%), 268 (20), 225 (30), 101 (100);

HRMS calcd for C₁₃H₁₇NO₆(M⁺) m/z 283.1056, found 283.1055.

Anal. (C₁₃H₁₇NO₆) C, H, N.

Syn th esis of Alcoh ol 37. 2-Meth oxy-4-n itr oben zyl 4-Ni-

tr op h en yl Ca r bon a te (35). A solution of 4-nitrophenyl

chloroformate (1.00 g, 4.97 mmol) in pyridine (4 mL) was added

dropwise to a stirred solution of 2-methoxy-4-nitrobenzyl

alcohol (34) (617 mg, 3.31 mmol) in pyridine (15 mL) at 20 °C,

and the solution was stirred for 16 h. The solvent was

evaporated and the residue purified by chromatography,

eluting with a gradient (20-50%) EtOAc/light petroleum, to

give carbonate 35 (928 mg, 80%) as a white solid, mp (EtOAc/

pet. ether) 105-106 °C; ¹H NMR δ 8.28 (ddd, J ) 9.2, 3.1, 2.1

Hz, 2 H, H-3′), 7.89 (dd, J ) 8.3, 2.1 Hz, 1 H, H-5), 7.77 (d, J

) 2.1 Hz, 1 H, H-3), 7.58 (d, J ) 8.3 Hz, 1 H, H-6), 7.40 (ddd,

J ) 8.3, 3.1, 2.1 Hz, 2 H, H-2′), 5.41 (s, 2 H, CH₂O), 4.00 (s, 3

H, OCH₃); ¹³C NMR δ 157.6, 155.4, 152.3, 149.2, 145.5, 129.8,

129.3, 125.3 (2), 121.7 (2), 115.8, 105.5, 65.3, 56.2. Anal.

(C₁₅H₁₂N₂O₈) C, H, N.

2-Met h oxy-4-n it r ob en zyl 4-(Hyd r oxym et h yl)p h en yl-

ca r ba m a te (37). A stirred solution of silyl ether 36 (0.89 g,

0.2 mmol) in MeOH (10 mL) was treated with 1 N HCl (4 mL,

4 mmol) and stirred at 20 °C for 1 h. The solution was poured

into brine (50 mL) and extracted with EtOAc (3 × 50 mL).

The combined organic fraction was washed with water (50 mL)

and dried and the solvent evaporated. The residue was purified

by chromatography, eluting with a gradient (20-50%) of

EtOAc/pet. ether, to give alcohol 37 (628 mg, 95%) as a white

solid, mp (EtOAc/pet. ether) 164-165 °C; ¹H NMR [(CD₃)₂SO]

δ 9.83 (br s, 1 H, OCONH), 7.90 (dd, J ) 8.3, 2.1 Hz, 1 H,

H-5′), 7.80 (d, J ) 2.1 Hz, 1 H, H-3′), 7.63 (d, J ) 8.3 Hz, 1 H,

H-6′), 7.41 (d, J ) 8.4 Hz, 2 H, H-2, H-6), 7.22 (d, J ) 8.4 Hz,

2 H, H-3, H-5), 5.21 (s, 2 H, CH₂O), 5.07 (t, J ) 5.6 Hz, 1 H,

OH), 4.41 (t, J ) 5.6 Hz, 2 H, CH₂O), 3.97 (s, 3 H, OCH₃); ¹³

NMR [(CD₃)₂SO] δ 157.0, 153.0, 148.2, 137.4, 136.7, 132.3,

128.8, 127.0 (2), 117.9 (2), 115.5, 105.4, 62.5, 60.4, 56.0. Anal.

(C₁₆H₁₆N₂O₆) C, H, N.

Cou p lin g of Alcoh ols to 3a -c. 4-Nitr oben zyl 1-(Ch lo-

r om 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 (4). A solu-

tion of 1-(chloromethyl)-3-[(5,6,7-trimethoxy-1H-indol-2-yl)-

carbonyl]-2,3-dihydro-1H-benzo[e]indol-5-ylamine (3a )²⁵(430

mg, 0.92 mmol) in THF (50 mL) was treated at -5 °C with

4-nitrobenzyl chloroformate (298 mg, 1.38 mmol). The mixture

was stirred at 10 °C for 30 min and then at 20 °C for 2 h,

before being diluted with Pr₂O/pet. ether. The precipitate was

collected and extracted with DCM. The combined extracts were

evaporated, and the residue was purified by chromatography,

eluting with 10% EtOAc/DCM, to give 4 (290 mg, 49%) as a

tan solid, mp (ⁱPr₂O/DCM) 191-192 °C; H NMR [(CD₃)₂SO]

δ 11.48 (d, J ) 1.7 Hz, 1 H, indole-NH), 9.91 (s, 1 H, NHCO₂),

8.57 (br s, 1 H, H-4), 8.29 (d, J ) 8.7 Hz, 2 H, H-3′′, H-5′′),

8.09 (d, J ) 8.5 Hz, 1 H, H-6), 7.99 (d, J ) 8.3 Hz, 1 H, H-9),

7.73 (d, J ) 8.7 Hz, 2 H, H-2′′, H-6′′), 7.58 (t, J ) 7.6 Hz, 1 H,

H-8), 7.48 (t, J ) 7.6 Hz, 1 H, H-7), 7.10 (d, J ) 2.2 Hz, 1 H,

H-3′), 6.98 (s, 1 H, H-4′), 5.36 (s, 2 H, CH₂O), 4.80 (dd, J )

10.8, 9.4 Hz, 1 H, H 2), 4.53 (dd, J ) 11.1, 1.9 Hz, 1 H, H-2),

4.31-4.39 (m, 1 H, H-1), 4.07 (dd, J ) 11.1, 3.1 Hz, 1 H, CH₂-

Cl), 3.91-3.97 (m, 4 H, OCH₃, CH₂Cl), 3.82 (s, 3 H, OCH₃),

3.80 (s, 3 H, OCH₃). Anal. (C₃₃H₂₉ClN₄O₈) C, H, N.

2-Met h oxy-4-n it r ob en zyl 1-(Ch lor om et h 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 (5). Phosgene (300 µL, 0.3 mmol,

1 M in toluene) was added to a stirred solution of 2-methoxy-

4-nitrobenzyl alcohol (34)¹⁹(20 mg, 0.11 mmol) in THF (10 mL)

and stirred at 20 °C for 16 h. The solvent was evaporated, the

residue dissolved in THF (10 mL), a solution of 3a (50 mg,

0.11 mmol) in THF (10 mL) added, and the solution stirred at

20 °C for 4 days. The solvent was evaporated and the residue

purified by chromatography, eluting with 50% EtOAc/pet.

ether, to give 5 (31 mg, 43%) as a tan solid, mp (EtOAc/pet.

ether) 162-165 °C; H NMR δ 9.52 (s, 1 H, indole-NH), 8.90

Minor Groove Alkylating Agents as Prodrugs

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 12 2463

(s, 1 H, OCONH), 7.90 (d, J ) 8.7 Hz, 1 H, H-6), 7.80 (d, J )

8.7 Hz, 1 H, H-5′′), 7.77 (d, J ) 8.4 Hz, 1 H, H-9), 7.70 (br s,

1 H, H-3′′), 7.50-7.57 (m, 2 H, H 8, H-6′′), 7.42-7.47 (m, 1 H,

H-7), 7.25 (br s, 1 H, H-4), 6.99 (d, J ) 2.2 Hz, 1 H, H-3′), 6.87

(s, 1 H, H-4′), 5.34 (d, J ) 1.9 Hz, 2 H, CH₂O), 4.78 (dd, J )

10.7, 1.6 Hz, 1 H, H-2), 4.64 (dd, J ) 10.7, 8.8 Hz, 1 H, H-2),

4.07-4.17 (m, 5 H, H-1, CH₂Cl, OCH₃), 3.95 (s, 3 H, OCH₃),

3.94 (s, 3 H, OCH₃), 3.91 (s, 3 H, OCH₃), 3.45 (t, J ) 10.9 Hz,

1 H, CH₂Cl); ¹³C NMR δ 160.3, 157.2, 154.0, 150.2, 148.6,

141.6, 140.6, 138.9, 133.9, 132.0, 129.7, 129.6, 128.8, 127.4,

127.2, 125.6, 125.0, 123.6, 123.1, 123.0, 122.4, 121.8, 115.7,

106.5, 105.1, 97.6, 61.8, 61.5, 61.1, 56.2, 56.0, 54.9, 45.8, 43.4;

MS (FAB⁺) m/z 675 (MH⁺, 10%), 677 (4), 659 (1), 639 (1), 517

(5), 234 (25); HRMS (FAB⁺) calcd for C₃₄H₃₂³⁵ClN₄O₉(MH⁺)

m/z 675.1858, found 675.1832; calcd for C₃₄H₃₂³⁷ClN₄O₉(MH⁺)

m/z 677.1828, found 677.1834; Anal. (C₃₄H₃₁ClN₄O₉‚H₂O) C,

H, N.

HCl (1 M) (0.4 mL, 0.40 mmol) was added to a stirred

solution of silyl ether 27 (157 mg, 0.19 mmol) in MeOH (5 mL)

and the solution stirred at 20 °C for 1 h. The solvent was

evaporated and the residue partitoned between EtOAc (50 mL)

and water (50 mL). The organic fraction was washed with

water (50 mL) and brine (25 mL) and dried and the solvent

evaporated. The residue was purified by chromatography,

eluting with a gradient (20-50%) of EtOAc/pet. ether, to give

carbamate 7 (119 mg, 88%) as a hygroscopic white solid, ¹H

NMR δ 9.72 (s, 1 H, indole-NH), 8.80 (s, 1 H, OCONH), 7.86

(d, J ) 8.5 Hz, 1 H, H-6), 7.79 (br d, J ) 8.1 Hz, 1 H, H-5′′),

7.67-7.73 (m, 2 H, H 9, H-3′′), 7.47-7.53 (m, 3 H, H-4, H-8,

H-6′′), 7.37 (ddd, J ) 8.5, 7.1, 0.8 Hz, 1 H, H-7), 6.97 (d, J )

2.2 Hz, 1 H, H-3′), 6.87 (s, 1 H, H-4′), 5.40 (s, 2 H, CH₂O), 4.73

(dd, J ) 10.7, 1.6 Hz, 1 H, H-2), 4.59 (dd, J ) 10.7, 8.7 Hz, 1

H, H-2), 4.21 (br dd, J ) 4.6, 4.0 Hz, 2 H, CH₂O), 4.07-4.11

(m, 4 H, H-1, OCH₃), 4.00-4.04 (m, 2 H, CH₂O), 3.95 (s, 3 H,

OCH₃), 3.92 (s, 3 H, OCH₃), 3.85 (d, J ) 11.3, 3.0 Hz, 1 H,

CH₂Cl), 3.39 (br s, 1 H, OH), 3.28 (dd, J ) 11.3, 10.9 Hz, 1 H,

CH₂Cl); ¹³C NMR δ 160.5, 157.2, 154.4, 150.2, 148.9, 141.4,

140.6, 138.9, 133.8, 131.9, 130.3, 129.6 (2), 127.4, 125.8, 125.0,

123.6, 123.0, 122.5 (2), 121.9, 115.9, 112.8, 106.7, 106.6, 97.7,

70.7, 62.0, 61.5, 61.1, 60.9, 56.3, 55.1, 45.6, 43.3; MS (FAB⁺)

m/z 707 (MH⁺, 5%), 705 (MH⁺, 14); HRMS (FAB⁺) calcd for

2-(2-Meth oxyeth oxy)-4-n itr oben zyl 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-d ih yd r o-

1H-ben zo[e]in d ol-5-ylca r ba m a te (6). A solution of triphos-

gene (12 mg, 40 µmol) in DCM (2 mL) was added dropwise to

a stirred solution of amine 3a (53 mg, 0.114 mmol) and Et₃N

(32 µL, 0.228 mmol) in DCM (10 mL) and stirred at 20 °C for

2 h. A solution of alcohol 22 (28 mg, 0.125 mmol) in DCM (2

mL) was added, followed by ⁿBu₂Sn(OAc)₂(2 drops), and the

solution was stirred at 20 °C for 24 h. The solvent was

evaporated and the residue purified by chromatography,

eluting with 20% EtOAc/DCM, to give 6 (75 mg, 91%) as a

tan gum, ¹H NMR δ 9.49 (s, 1 H, indole-NH), 8.19 (s, 1 H,

OCONH), 7.92 (d, J ) 8.5 Hz, 1 H, H-6), 7.80-7.82 (m, 1 H,

H-5′′), 7.78 (d, J ) 8.3 Hz, 1 H, H-9), 7.71 (d, J ) 1.8 Hz, 1 H,

H-3′′), 7.56 (ddd, J ) 8.3, 7.1, 0.8 Hz, 1 H, H-8), 7.49-7.54 (m,

1 H, H-6′′), 7.45 (ddd, J ) 8.5, 7.1, 0.8 Hz, 1 H, H-7), 7.27 (br

s, 1 H, H-4), 7.00 (d, J ) 2.3 Hz, 1 H, H-3′), 6.87 (s, 1 H, H-4′),

5.39 (s, 2 H, CH₂O), 4.79 (dd, J ) 10.7, 1.7 Hz, 1 H, H-2), 4.66

(dd, J ) 10.7, 8.7 Hz, 1 H, H-2), 4.23 (dd, J ) 4.6, 4.4 Hz, 2 H,

CH₂O), 4.15-4.20 (m, 1 H, H-1), 4.08 (s, 3 H, OCH₃), 3.94-

3.98 (m, 4 H, OCH₃, CH₂Cl), 3.91 (s, 3 H, OCH₃), 3.80 (dd, J

) 4.6, 4.4 Hz, 2 H, CH₂O), 3.47 (d, J ) 10.9 Hz, 1 H, CH₂Cl),

3.44 (s, 3 H, OCH₃); ¹³C NMR δ 160.3, 156.5, 154.0, 150.2,

148.5, 141.7, 140.6, 138.9, 133.9, 132.4, 129.7, 129.6, 128.7,

127.5, 125.6, 125.0, 123.6, 123.1, 122.5 (2), 121.8, 116.0, 112.7,

106.5, 106.3, 97.7, 70.7, 68.5, 61.9, 61.5, 61.1, 59.3, 56.3, 54.9,

45.9, 43.1; MS (FAB⁺) m/z 721 (MH⁺, 1.5%), 719 (MH⁺, 3.5);

HRMS (FAB⁺) calcd for C₃₆H₃₆³⁷ClN₄O₁₀(MH⁺) m/z 721.2091,

found 721.2131; calcd for C₃₆H₃₆³⁵ClN₄O₁₀(MH⁺) m/z 719.2120,

found 719.2133. Anal. (C₃₆H₃₅ClN₄O₁₀) C, H, N.

₃₅H₃₄³⁵ClN₄O₁₀(MH⁺) m/z 705.1964, found 705.1919; calcd

for C₃₅H₃₄³⁷ClN₄O₁₀(MH⁺) m/z 707.1934, found 707.1931. Anal.

(C₃₅H₃₃ClN₄O₁₀) C, H, N.

2-(3-Hydr oxypr opoxy)-4-n itr oben zyl 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-d ih yd r o-

1H-ben zo[e]in d ol-5-ylca r ba m a te (8). Similarly, reaction of

amine 3a and alcohol 24 gave 2-(3-{[tert-butyl(dimethyl)silyl]-

oxy}propoxy)-4-nitrobenzyl 1-(chloromethyl)-3-[(5,6,7-trimethoxy-

1H-indol-2-yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5-ylcar-

bamate (28) (67%) as a white solid, mp (MeOH) 149-150 °C;

¹H NMR δ 9.42 (s, 1 H, indole-NH), 8.96 (s, 1 H, OCONH),

7.91 (d, J ) 8.4 Hz, 1 H, H-6), 7.78-7.85 (m, 2 H, H 9, H-5′′),

7.75 (d, J ) 1.7 Hz, 1 H, H-3′′), 7.53-7.59 (m, 2 H, H-8, H-6′′),

7.47 (ddd, J ) 8.4, 7.4, 0.8 Hz, 1 H, H-7), 7.08 (br s, 1 H, H-4),

7.02 (d, J ) 2.2 Hz, 1 H, H-3′), 6.89 (s, 1 H, H-4′), 5.38 (s, 2 H,

CH₂O), 4.82 (dd, J ) 10.7, 1.7 Hz, 1 H, H-2), 4.69 (dd, J )

10.7, 8.7 Hz, 1 H, H-2), 4.21 (t, J ) 6.0 Hz, 2 H, CH₂O), 4.17-

4.20 (m, 1 H, CH₂Cl), 4.09 (s, 3 H, OCH₃), 3.99 (dd, J ) 11.3,

2.9 Hz, 1 H, H-1), 3.95 (s, 3 H, OCH₃), 3.92 (s, 3 H, OCH₃),

3.83 (t, J ) 5.9 Hz, 2 H, CH₂O), 3.49 (t, J ) 11.0 Hz, 1 H,

CH₂Cl), 2.02-2.08 (m, 2 H, CH₂), 0.88 (s, 9 H, OSiC(CH₃)₃),

0.04 (s, 6 H, OSi(CH₃)₂; MS (FAB⁺) m/z 833 (MH⁺, 25%), 835

(MH⁺, 12), 775 (5), 599 (5); HRMS (FAB⁺) calcd for C₄₂H₅₀

ClN₄O₁₀Si (MH⁺) m/z 833.2985, found 833.3008; calcd for

₄₂H₅₀³⁷ClN₄O₁₀Si (MH⁺) m/z 835.2955, found 835.2982. Anal.

2-(2-Hyd r oxyeth oxy)-4-n itr oben zyl 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-d ih yd r o-

1H-ben zo[e]in d ol-5-ylca r ba m a te (7). Similarly, reaction of

amine 3a with alcohol 23 gave 2-(2-{[tert-butyl(dimethyl)silyl]-

oxy}ethoxy)-4-nitrobenzyl 1-(chloromethyl)-3-[(5,6,7-trimethoxy-

1H-indol-2-yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5-ylcar-

bamate (27) (94%) as a white solid, mp (EtOAc/pet. ether) 182-

185 °C; ¹H NMR δ 9.45 (s, 1 H, indole-NH), 8.93 (s, 1 H,

OCONH), 7.92 (d, J ) 8.5 Hz, 1 H, H-6), 7.76-7.83 (m, 3 H,

H-9, H-3′′, H-5′′), 7.53-7.60 (m, 2 H, H-8, H-6′′), 7.47 (ddd, J

) 8.5, 7.1, 0.8 Hz, 1 H, H-7), 7.13 (br s, 1 H, H-4), 7.01 (d, J )

2.2 Hz, 1 H, H-3′), 6.88 (s, 1 H, H-4′), 5.39 (s, 2 H, CH₂O), 4.81

(dd, J ) 10.7, 1.8 Hz, 1 H, H-2), 4.67 (dd, J ) 10.7, 8.7 Hz, 1

H, H-2), 4.21 (br dd, J ) 5.0, 4.8 Hz, 2 H, CH₂O), 4.15-4.18

(m, 1 H, H-1), 4.09 (s, 3 H, OCH₃), 4.02 (br d, J ) 5.0 Hz, 2 H,

CH₂O), 3.97 (dd, J ) 11.5, 3.1 Hz, 1 H, CH₂Cl), 3.95 (s, 3 H,

OCH₃), 3.92 (s, 3 H, OCH₃), 3.48 (dd, J ) 11.5, 10.9 Hz, 1 H,

CH₂Cl), 0.90 (s, 9 H, SiC(CH₃)₃), 0.10 (s, 6 H, Si(CH₃)₂); ¹³C

NMR δ 160.3, 156.7, 154.0, 150.2, 148.5, 141.7, 140.6, 138.9,

133.8, 132.3, 129.7, 129.6, 128.7, 127.5, 125.6, 125.0, 123.6,

123.1, 122.4 (2), 121.8, 115.8, 112.8, 106.5 (2), 97.7, 70.7, 61.8,

61.7, 61.5, 61.1, 56.3, 54.9, 45.8, 43.5, 25.8 (3), 18.3, -5.4 (2);

MS (FAB⁺) m/z 819 (MH⁺, 25%), 821 (MH⁺, 12); HRMS (FAB⁺)

calcd for C₄₁H₄₈³⁵ClN₄O₁₀Si (MH⁺) m/z 819.2828, found 819.2804;

calcd for C₄₁H₄₈³⁷ClN₄O₁₀Si (MH⁺) m/z 821.2799, found 821.2803;

Anal. (C₄₁H₄₇ClN₄O₁₀Si) C, H, N.

(C₄₂H₄₉ClN₄O₁₀Si) C, H, N.

HCl (1 M) (0.2 mL, 0.20 mmol) was added to a stirred

solution of silyl ether 28 (64 mg, 0.08 mmol) in MeOH (5 mL)

and the solution stirred at 20 °C for 30 min. The solvent was

evaporated, the residue dissolved in EtOAc (50 mL), washed

with water (2 × 50 mL) and brine (25 mL), and dried, and the

solvent evaporated. The residue was purified by chromatog-

raphy, eluting with a gradient (50-100%) of EtOAc/pet. ether,

to give carbamate 8 (52 mg, 94%) as a white solid, mp (EtOAc)

122-126 °C; ¹H NMR δ 9.51 (s, 1 H, indole-NH), 8.90 (s, 1 H,

OCONH), 7.92 (d, J ) 8.5 Hz, 1 H, H-6), 7.80 (d, J ) 8.2 Hz,

1 H, H-5′′), 7.77 (d, J ) 8.3 Hz, 1 H, H-9), 7.73 (d, J ) 1.8 Hz,

1 H, H-3′′), 7.50-7.57 (m, 2 H, H-8, H-6′′), 7.40-7.46 (m, 2 H,

H-4, H-7), 6.99 (d, J ) 2.2 Hz, 1 H, H-3′), 6.87 (s, 1 H, H-4′),

5.37 (d, J ) 13.1 Hz, 1 H, CH₂O), 5.32 (d, J ) 13.1 Hz, 1 H,

CH₂O), 4.77 (dd, J ) 10.8, 1.6 Hz, 1 H, H-2), 4.64 (dd, J )

10.8, 8.6 Hz, 1 H, H-2), 4.27 (t, J ) 5.7 Hz, 2 H, CH₂O), 4.11-

4.18 (m, 1 H, CH₂Cl), 4.09 (s, 3 H, OCH₃), 3.96 (s, 3 H, OCH₃),

3.91-3.95 (m, 3 H, H 1, CH₂O), 3.90 (s, 3 H, OCH₃), 3.44 (t, J

) 10.9 Hz, 1 H, CH₂Cl), 2.75 (br s, 1 H, OH), 2.12-2.18 (m, 2

H, CH₂); ¹³C NMR δ 160.4, 157.2, 153.8, 150.2, 148.9, 141.6,

140.6, 138.9, 134.0, 131.6, 130.1, 129.7, 129.6, 127.5, 125.7,

125.0, 123.6, 123.1, 122.4 (2), 121.6, 115.7, 112.2, 106.6, 106.1,

97.7, 66.8, 62.2, 61.5, 61.1, 60.1, 56.3, 55.0, 45.8, 43.4, 31.6;

MS (FAB⁺) m/z 721 (MH⁺, 2%), 719 (MH⁺, 4); HRMS (FAB⁺)

2464 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 12

Hay et al.

calcd for C₃₆H₃₅³⁵ClN₄O₁₀(MH⁺) m/z 719.2120, found 719.2107;

calcd for C₃₆H₃₅³⁷ClN₄O₁₀(MH⁺) m/z 721.2091, found 721.2093.

Anal. (C₃₆H₃₅ClN₄O₁₀) C, H, N.

123.7, 123.3, 123.1, 122.1, 121.2, 115.7, 113.0, 106.3 (2), 98.0,

66.8, 61.0, 60.9, 60.7, 55.9, 54.9, 53.7, 47.5, 42.0 (2), 41.1, 23.6;

MS (FAB⁺) m/z 746 (MH⁺, 16%), 748 (MH⁺, 7); HRMS (FAB⁺)

calcd for C₃₈H₄₁³⁵ClN₅O₉(MH⁺) m/z 746.2593, found 746.2582;

calcd for C₃₈H₄₁³⁷ClN₅O₉(MH⁺) m/z 748.2563, found 748.2577.

Anal. (C₃₈H₄₀ClN₅O₉‚2HCl) C, H, N.

2-(2,3-Dih yd r oxyp r op oxy)-4-n it r ob en zyl 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 (9). Similarly,

reaction of amine 3a and alcohol 32 gave 2-[(2,2-dimethyl-1,3-

dioxolan-4-yl)methoxy]-4-nitrobenzyl 1-(chloromethyl)-3-[(5,6,7-

trimethoxy-1H-indol-2-yl)carbonyl]-2,3-dihydro-1H-benzo[e]-

indol-5-ylcarbamate (33) (96%) as a gum, ¹H NMR δ 9.44 (s, 1

H, indole-NH), 8.94 (s, 1 H, OCONH), 7.92 (d, J ) 8.5 Hz, 1

H, H-6), 7.87 (dd, J ) 8.2, 2.1 Hz, 1 H, H-5′′), 7.81 (d, J ) 8.2

Hz, 1 H, H-9), 7.73 (d, J ) 2.1 Hz, 1 H, H-3′′), 7.58 (ddd, J )

8.2, 7.3, 0.7 Hz, 1 H, H-8), 7.45-7.51 (m, 2 H, H-7, H-6′′), 7.13

(br s, 1 H, H-4), 7.02 (d, J ) 2.2 Hz, 1 H, H-3′), 6.89 (s, 1 H,

H-4′), 5.38 (s, 2 H, CH₂O), 4.83 (dd, J ) 10.8, 1.7 Hz, 1 H,

H-2), 4.69 (dd, J ) 10.8, 8.7 Hz, 1 H, H-2), 4.50-4.56 (m, 1 H,

H-4′′′′), 4.23 (dd, J ) 9.8, 4.0 Hz, 1 H, H-5′′′′), 4.15-4.20 (m, 2

H, H-1, H-2′′′), 4.09-4.14 (m, 4 H, OCH₃, H-5′′′′), 3.95-4.00

(m, 5 H, OCH_3,CH₂Cl, H-2′′′), 3.92 (s, 3 H, OCH₃), 3.50 (dd, J

) 10.9, 10.8 Hz, 1 H, CH₂Cl), 1.45 (s, 3 H, CH₃), 1.39 (s, 3 H,

CH₃); MS (FAB⁺) m/z 777 (MH⁺, 10%), 775 (MH⁺, 35); HRMS

(FAB⁺) calcd for C₃₉H₄₀³⁵ClN₄O₁₁(MH⁺) m/z 775.2381, found

777.2379; calcd for C₃₉H₄₀³⁷ClN₄O₁₁(MH⁺) m/z 777.2535, found

777.2354.

2-[3-(4-Mor p h olin yl)p r op oxy]-4-n itr oben zyl 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 (11). Similarly,

reaction of amine 3a and alcohol 26 gave carbamate 11 (92%)

as a tan solid, mp (EtOAc) 102-107 °C; H NMR δ 9.48 (s, 1

H, indole-NH), 8.93 (s, 1 H, OCONH), 7.91 (d, J ) 8.5 Hz, 1

H, H-6), 7.78-7.84 (m, 2 H, H 9, H-5′′), 7.73 (d, J ) 1.8 Hz, 1

H, H-3′′), 7.57 (ddd, J ) 8.2, 7.0, 1.0 Hz, 1 H, H-8), 7.50-7.53

(m, 1 H, H-6′′), 7.45 (ddd, J ) 8.5, 7.0, 1.0 Hz, 1 H, H-7), 7.28

(br s, 1 H, H-4), 7.00 (d, J ) 2.2 Hz, 1 H, H-3′), 6.88 (s, 1 H,

H-4′), 5.36 (s, 2 H, CH₂O), 4.81 (dd, J ) 10.8, 1.8 Hz, 1 H,

H-2), 4.68 (dd, J ) 10.8, 8.7 Hz, 1 H, H-2), 4.16-4.22 (m, 3 H,

CH₂O, CH₂Cl), 4.08 (s, 3 H, OCH₃), 3.93-3.99 (m, 4 H, H-1,

OCH₃), 3.92 (s, 3 H, OCH₃), 3.67-3.69 (m, 4 H, 2 × CH₂O),

3.49 (t, J ) 10.9 Hz, 1 H, CH₂Cl), 2.55 (t, J ) 7.1 Hz, 2 H,

CH₂N), 2.43-2.49 (m, 4 H, 2 × CH₂N), 2.00-2.08 (m, 2 H,

CH₂); ¹³C NMR δ 160.4, 156.7, 154.0, 150.2, 148.6, 141.7, 140.6,

138.9, 133.9, 132.2, 129.8, 129.6, 128.8, 127.5, 125.7, 125.0,

123.6, 123.2, 122.4 (2), 121.6, 115.7, 112.2, 106.5, 106.1, 97.7,

67.1, 66.8 (2), 61.9, 61.5, 61.1, 56.3, 55.2, 54.9, 53.6 (2), 45.8,

43.4, 26.1; MS (FAB⁺) m/z 788 (MH⁺, 6%), 790 (MH⁺, 3);

HRMS (FAB⁺) calcd for C₄₀H₄₃³⁵ClN₅O₁₀(MH⁺) m/z 788.2699,

found 788.2721; calcd for C₄₀H₄₃³⁷ClN₅O₁₀(MH⁺) m/z 790.2699,

found 790.2728. Anal. (C₄₀H₄₂ClN₅O₁₀‚¹/₂H₂O) C, H, N.

HCl (1 M) (1 mL) was added to a stirred suspension of

acetonide 33 (160 mg, 0.06 mmol) in THF (20 mL) and the

mixture stirred at 20 °C for 16 h. The mixture was evaporated

and the residue partitioned between DCM (50 mL) and water

(50 mL). The organic fraction was washed with water (30 mL)

and brine (30 mL) and dried and the solvent evaporated. The

residue was purified by chromatography, eluting with 50%

EtOAc/DCM, to give carbamate 9 (87 mg, 56%), as a tan solid,

mp (MeOH/ⁱPr₂O) 147-149 °C; ¹H NMR [(CD₃)₂SO] δ 11.46

(s, 1 H, indole-NH), 9.90 (s, 1 H, OCONH), 8.56 (s, 1 H, H-4),

8.12 (d, J ) 8.5 Hz, 1 H, H-6), 7.98 (d, J ) 8.3 Hz, 1 H, H-9),

7.92 (dd, J ) 8.3, 1.9 Hz, 1 H, H-5′′), 7.83 (d, J ) 1.9 Hz, 1 H,

H-3′′), 7.69 (d, J ) 8.3 Hz, 1 H, H-6′′), 7.59 (dd, J ) 8.2, 7.6

Hz, 1 H, H-8), 7.49 (dd, J ) 8.5, 7.6 Hz, 1 H, H-7), 7.09 (d, J

) 2.1 Hz, 1 H, H-3′), 6.98 (s, 1 H, H-4′), 5.33 (s, 2 H, CH₂O),

5.07 (d, J ) 5.2 Hz, 1 H, OH), 4.81 (dd, J ) 11.0, 9.7 Hz, 1 H,

H-2), 4.73 (t, J ) 5.7 Hz, 1 H, H-3′′′), 4.53 (dd, J ) 11.0, 3.5

Hz, 1 H, H-2), 4.32-4.37 (m, 1 H, H-1), 4.24 (dd, J ) 10.0, 3.9

Hz, 1 H, CH₂Cl), 4.09-4.13 (m, 1 H, H-2′′′′), 4.02-4.06 (m, 1

H, H-3′), 3.93-3.96 (m, 4 H, OCH_3,CH₂Cl), 3.84-3.89 (m, 1

H, OH), 3.83 (s, 3 H, OCH₃), 3.81 (s, 3 H, OCH₃), 3.51 (t, J )

5.7 Hz, 2 H, H-1′′′′); ¹³C NMR [(CD₃)₂SO] δ 160.2, 156.2, 154.3,

149.2, 148.0, 141.5, 139.9, 139.0, 134.3, 133.0, 130.7, 129.4,

128.0, 127.1, 125.4 (2), 124.3, 123.9, 123.3, 123.1, 122.0, 115.4,

113.0, 106.3, 106.2, 98.0, 70.7, 69.7, 62.4, 61.0, 60.9, 60.7, 55.9,

54.9, 47.5, 41.4; MS (FAB⁺) m/z 737 (MH⁺, 3%), 735 (MH⁺, 8);

HRMS (FAB⁺) calcd for C₃₆H₃₆³⁵ClN₄O₁₁(MH⁺) m/z 735.2069,

found 735.2050; calcd for C₃₆H₃₆³⁷ClN₄O₁₁(MH⁺) m/z 737.2040,

found 737.2000. Anal. (C₃₆H₃₅ClN₄O₁₁‚CH₃OH) C, H, N.

4-({[(2-Met h oxy-4-n it r ob en zyl)oxy]ca r b on yl}a m in o)-

ben zyl 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-d ih yd r o-1H-ben zo[e]in d ol-5-ylca r ba m -

a te (13). Similarly, reaction of amine 3a and alcohol 37 gave

carbamate 13 (47%) as a white solid, mp 135-141 °C; ¹H NMR

δ 11.47 (s, 1 H, indole-NH), 9.97 (s, 1 H, NHCO₂), 9.71 (s, 1 H,

NHCO₂), 8.56 (s, 1 H, H-4), 8.05 (d, J ) 8.6 Hz, 1 H, H-6),

7.97 (d, J ) 8.3 Hz, 1 H, H-9), 7.91 (dd, J ) 8.3, 2.1 Hz, 1 H,

H-5′′′), 7.81 (d, J ) 2.1 Hz, 1 H, H-3′′′), 7.64 (d, J ) 8.3 Hz, 1

H, H-6′′′), 7.57 (t, J ) 7.6 Hz, 1 H, H-8), 7.51 (d, J ) 8.4 Hz,

2 H, H-3′′, H-5′′), 7.44 (t, J ) 7.7 Hz, 1 H, H-7), 7.40 (d, J )

8.5 Hz, 2 H, H-2′′, H-6′′), 7.09 (s, 1 H, H-3′), 6.98 (s, 1 H, H-4′),

5.23 (s, 2 H, CH₂O), 5.12 (s, 2 H, CH₂O), 4.80 (t, J ) 10.1 Hz,

1 H, 1 H, H-2), 4.52 (dd, J ) 11.1, 1.6 Hz, 1 H, H-2), 4.28-

4.38 (m, 1 H, H-1), 4.06 (dd, J ) 11.2, 3.1 Hz, 1 H, CH₂Cl),

3.97 (s, 3 H, OCH₃), 3.89-3.96 (m, 4 H, CH₂Cl, OCH₃), 3.82

(s, 3 H, OCH₃), 3.80 (s, 3 H, OCH₃). Anal. (C₄₂H₃₈ClN₅O₁₁) C,

H, N, Cl.

1-(4-Nitr op h en yl)eth yl 1-(Ch lor om eth yl)-3-[(5,6,7-tr i-

m 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 (12). A solution of 1-(4-nitrophenyl)-

ethanol³⁷(38) (18 mg, 0.11 mmol) in DCM (2 mL) was added

dropwise to a stirred solution of triphosgene (16 mg, 0.054

mmol) and pyridine (9 µL, 0.11 mmol) in DCM (2 mL) at 20

°C. The mixture was stirred at 20 °C for 2 h, the solvent

evaporated, and the residue dissolved in THF (5 mL). A

solution of 3a (50 mg, 0.11 mmol) 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 aqueous

KHCO₃solution (50 mL), the organic fraction dried, and the

solvent evaporated. The residue was purified by chromatog-

raphy, eluting with 25% EtOAc/pet. ether, to give 12 (23 mg,

32%) as a tan solid, mp (EtOAc/pet. ether) 175-178 °C; ¹H

NMR [(CD₃)₂SO] δ 9.49 (s, 1 H, indole-NH), 8.88 (s, 1 H,

OCONH), 8.18 (d, J ) 7.6 Hz, 2 H, H-3′′, H-5′′), 7.88 (d, J )

8.3 Hz, 1 H, H-6), 7.78 (d, J ) 8.3 Hz, 1 H, H-9), 7.52-7.58

(m, 3 H, H-8, H-2′′, H-6′′), 7.45 (dd, J ) 7.8, 7.5 Hz, 1 H, H-7),

7.16 (br s, 1 H, H-4), 7.00 (d, J ) 1.9 Hz, 1 H, H-3′), 6.87 (s, 1

H, H-4′), 6.00 (q, J ) 6.6 Hz, 1 H, CHO), 4.80 (dd, J ) 10.7,

1.2 Hz, 1 H, H-2), 4.65 (dd, J ) 10.7, 8.8 Hz, 1 H, H-2), 4.11-

4.17 (m, 1 H, CH₂Cl), 4.08 (s, 3 H, OCH₃), 3.93-3.97 (m, 4 H,

OCH₃, CH₂Cl), 3.91 (s, 3 H, OCH₃), 3.45 (dt, J ) 10.7, 3.3 Hz,

1 H, H-1), 1.65 (br d, J ) 6.6 Hz, 3 H, CH₃); ¹³C NMR δ 160.3,

2-[3-(Dim et h yla m in o)p r op oxy]-4-n it r ob en zyl

(Ch lor om eth yl)-3-[(5,6,7-tr im eth oxy-1H-in d ol-2-yl)ca r bo-

n yl]-2,3-d ih yd r o-1H -b en zo[e]in d ol-5-ylca r b a m a t e (10).

Similarly, reaction of amine 3a and alcohol 25 gave carbamate

10 (55%) as a yellow solid which was converted to the

hydrochloride salt, mp (MeOH) 176-180 °C; ¹H NMR [(CD₃)₂-

SO] δ 11.43 (s, 1 H, indole-NH), 10.47 (br s, 1 H, NH⁺Cl^-),

9.92 (s, 1 H, OCONH), 8.58 (s, 1 H, H 4), 8.10 (d, J ) 8.5 Hz,

1 H, H-6), 7.97 (d, J ) 8.3 Hz, 1 H, H-9), 7.93 (dd, J ) 8.4, 2.0

Hz, 1 H, H-5′′), 7.82 (d, J ) 2.0 Hz, 1 H, H-3′′), 7.71 (br d, J )

8.4 Hz, 1 H, H-6′′), 7.56-7.61 (m, 1 H, H-8), 7.46-7.51 (m, 1

H, H-7), 7.10 (d, J ) 2.1 Hz, 1 H, H-3′), 6.97 (s, 1 H, H-4′),

5.34 (s, 2 H, CH₂O), 4.81 (dd, J ) 10.8, 9.5 Hz, 1 H, H-2), 4.53

(dd, J ) 10.8, 1.7 Hz, 1 H, H-2), 4.33-4.38 (m, 1 H, H-1), 4.30

(t, J ) 5.9 Hz, 2 H, CH₂O), 4.07 (dd, J ) 11.1, 2.9 Hz, 1 H,

CH₂Cl), 3.94-3.97 (m, 4 H, CH₂Cl, OCH₃), 3.83 (s, 3 H, OCH₃),

3.81 (s, 3 H, OCH₃), 3.23-3.27 (t, J ) 7.5 Hz, 2 H, CH₂N),

2.75 (s, 6 H, N(CH₃)₂), 2.17-2.23 (m, 2 H, CH₂); ¹³C NMR

[(CD₃)₂SO] δ 160.2, 155.8, 154.3, 149.2, 148.0, 141.5, 139.9,

138.9, 134.3, 132.9, 130.7, 129.5, 128.5, 127.2, 125.4, 124.4,

Minor Groove Alkylating Agents as Prodrugs

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 12 2465

153.4, 150.2, 149.0, 147.5, 141.6, 140.6, 138.9, 133.8, 130.4,

129.7, 129.6, 127.4, 126.8 (2), 125.6, 125.0, 123.9, 123.8 (2),

123.6, 123.1, 122.3, 121.7, 106.5, 97.6, 72.6, 61.5, 61.1, 56.3,

54.9, 45.8, 43.4, 22.6; MS (FAB⁺) m/z 659 (MH⁺, 6%), 658 (6),

510 (1), 234 (10); HRMS (FAB⁺) calcd for C₃₄H₃₂³⁵ClN₄O₈

(MH⁺) m/z 659.1909, found 659.1881; calcd for C₃₄H₃₂³⁷ClN₄O₈

(MH⁺) m/z 661.1879, found 661.1882. Anal. (C₃₄H₃₁ClN₄O₈) C,

H, N. Starting material 3a (30 mg, 60%) was also recovered.

11.1, 6.7 Hz, 1 H, CH₂Cl), 2.65 (t, J ) 5.8 Hz, 2 H, CH₂N),

2.24 (s, 6 H, N(CH₃)₂). Anal. (C₃₄H₃₂ClN₅O₆) C, H, N, Cl.

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-NTR^puro, 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 V79^purohas been transfected with the empty shuttle

vector and is also known as T78-1.¹⁶Skov-NTR^neoand WiDr-

NTR^neo, 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.¹¹

EMT6-NTR^puro, 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.³⁶Selection for

NTR expression was maintained during passage, but not

during experiments, using 15 µM puromycin (V79-NTR^puro), 5

µM puromycin (EMT6-NTR^puro), or 300 µg/mL G418 (WiDr-

NTR^neo; SKOV-NTR^neo).

4-Nitr oben zyl 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-d ih yd r o-1H-ben zo[e]in d ol-5-

yl(m eth yl)ca r ba m a te (14). A solution of 1-(chloromethyl)-

3-[(5,6,7-trimethoxy-1H-indol-2-yl)carbonyl]-2,3-dihydro-1H-

benzo[e]indol-5-yl(methyl)amine (3b)²⁵(77 mg, 0.16 mmol) in

dry DCM containing pyridine (32 µL, 0.40 mmol) was treated

portionwise at 0 °C with 4-nitrobenzyl chloroformate (69 mg,

0.32 mmol). The mixture was stirred at 10 °C for a further 45

min, then diluted with DCM (20 mL) and washed with water

(2 × 10 mL) and dried. Solvent was evaporated and the residue

was purified on alumina, eluting with 15% EtOAc/DCM, to

give 14 (84 mg, 79%) as a tan solid, mp (EtOAc/ⁱPr₂O) 125-

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.^38,39Cultures

were initiated 24 h before an 18 h drug exposure, with cell

densities determined 4-5 days later by staining with sulfor-

hodamine B. IC₅₀values were calculated as the drug concen-

tration providing 50% inhibition of growth relative to controls

on the same plate.

128 °C; H NMR δ (2 rotamers) 9.38 (br s, 1 H, indole-NH),

8.55 (br s, 1 H, H-4), 8.02 and 8.07 (2 × d, J ) 8.5 Hz, 2 H,

H-3′′, H-5′′), 7.86 (br d, J ) 8.3 Hz, 1 H, H-6), 7.78-7.82 (m 1

H, H-9), 7.60-7.64 (m, 1 H, H-8), 7.47 (ddd, J ) 8.3, 7.0, 0.9

Hz, 1 H, H-7), 7.11 and 7.19 (2 × d, J ) 8.5 Hz, 2 H, H-2′′,

H-6′′), 7.04 (d, J ) 2.2 Hz, 1 H, H-3′), 6.89 (s, 1 H, H-4′), 5.12-

5.20 (m, 2 H, CH₂O), 4.86 (br d, J ) 10.7 Hz, 1 H, H-2), 4.73

(br dd, J ) 10.7, 8.7 Hz, 1 H, H-2), 4.24-4.28 (m, 1 H, H-1),

4.11 (s, 3 H, OCH₃), 4.00-4.07 (m, 1 H, CH₂Cl), 3.96 (s, 3 H,

OCH₃), 3.92 (s, 3 H, OCH₃), 3.50-3.57 (m, 1 H, CH₂Cl), 3.40

and 3.46 (2 × s, 3 H, NCH₃). Anal. (C₃₄H₃₁ClN₄O₈) C, H, N.

Mou se Toxicity. Compounds 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 1 µL/g

body weight, using 10^1/8-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.

4-Nitr oben zyl 1-(Ch lor om eth yl)-3-({5-[2-(d im eth yla m i-

n o)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 (15). A stirred solution of tert-

butyl 5-amino-1-(chloromethyl)-1,2-dihydro-3H-benzo[e]indole-

3-carboxylate (39)²⁶[(400 mg, 1.20 mmol) in THF (25 mL) was

treated at 0 °C with 4-nitrobenzyl chloroformate (388 mg, 1.80

mmol). The mixture was stirred at 0 °C for 45 min and stirred

for a further 1 h at 20 °C. The mixture was concentrated to

10 mL, DCM (40 mL) was added, the solution was washed with

dilute KHCO₃(50 mL) and water (50 mL) and then dried, and

the solvent was evaporated. The residue was purified by

chromatography, eluting with 5% EtOAc/DCM, to give tert-

butyl 1-(chloromethyl)-5-({[(4-nitrobenzyl)oxy]carbonyl}amino)-

1,2-dihydro-3H-benzo[e]indole-3-carboxylate (40) (344 mg, 56%)

In Vivo Excision Assa y. Activity against NTR-expressing

and parental (NTR-ve) tumor cells was assessed by treating

mice with tumors containing mixtures of EMT6-NTR^purocells

and EMT6 cells. CD-1 nude mice were inoculated subcutane-

ously with 3 × 10⁶cells using a 2:1 mixture of EMT6-NTR^puro

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.³⁶Briefly,

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 10⁵cells

were plated in medium containing 3 µM puromycin or nonse-

lective medium to quantify survival of EMT6-NTR^puroand total

tumor cells, respectively. Plates were incubated for 8 days, and

colonies of >50 cells counted. The plating efficiency of 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.

as a white solid, mp (EtOAc/ⁱPr₂O) 152 °C; H NMR [(CD₃)₂-

SO] δ 9.86 (s, 1 H, NH), 8.28 (d, J ) 8.7 Hz, 2 H, H-3′, H-5′),

8.10 (br s, 1 H, H-4), 8.06 (d, J ) 8.5 Hz, 1 H, H-6), 7.88 (d, J

) 8.3 Hz, 1 H, H-9), 7.73 (d, J ) 8.7 Hz, 2 H, H-2′, H-6′), 7.53

(t, J ) 7.6 Hz, 1 H, H-8), 7.40 (t, J ) 7.6 Hz, 1 H, H-7), 5.35

(s, 2 H, CH₂O), 4.27-4.11 (m, 2 H, H-2), 4.11-3.97 (m, 2 H,

H-1, CH₂Cl), 3.88 (dd, J ) 10.8, 6.7 Hz, 1 H, CH₂Cl), 1.52 (s,

9 H, C(CH₃)₃). Anal. (C₂₆H₂₆ClN₃O₆) C, H, N, Cl.

A solution of 40 (296 mg, 0.58 mmol) in HCl-saturated

dioxane (10 mL) was stirred at 20 °C for 45 min, then the

solvent evaporated below 30 °C. 5-[2-(Dimethylamino)ethoxy]-

1H-indole-2-carboxylic acid²⁶(174 mg, 0.61 mmol), EDCI‚HCl

(278 mg, 1.45 mmol), and DMA (2 mL) were then added, and

the mixture was stirred at 20 °C for 6 h. The mixture was

basified with dilute NH₃, and the precipitated solid was

collected and purified by chromatography on alumina-90.

Elution with 5% MeOH/EtOAc gave 15 (219 mg, 59%), mp (ⁱ-

Pr₂O/EtOAc) 115-118 °C; ¹H NMR [(CD₃)₂SO] δ 11.62 (s, 1

H, indole NH), 9.91 (s, 1 H, NHCO₂), 8.61 (s, 1 H, H-4), 8.29

(d, J ) 8.6 Hz, 2 H, H-3′′, H-5′′), 8.08 (d, J ) 8.5 Hz, 1 H,

H-6), 8.00 (d, J ) 8.3 Hz, 1 H, H-9), 7.74 (d, J ) 8.6 Hz, 2 H,

H-2′′, H 6′′), 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), 7.40 (d, J ) 8.9 Hz, 1 H, H-7′), 7.18 (d,

J ) 2.2 Hz, 1 H, H-4′), 7.12 (s, 1 H, H-3′), 6.92 (dd, J ) 8.9,

2.3 Hz, 1 H, H-6′), 5.36 (s, 2 H, CH₂O), 4.85 (t, J ) 10.1 Hz, 1

H, H-2), 4.61 (dd, J ) 10.1, 1.9 Hz, 1 H, H-2), 4.35-4.43 (m,

1 H, H-1), 4.04-4.12 (m, 3 H, CH₂O, CH₂Cl), 3.98 (dd, J )

Ack n ow led gm en t. The authors thank Dr Maruta

Boyd, Dianne Ferry, Alison Hogg, and Li Fong Leong

for technical assistance, Dr Brian Palmer for helpful

discussions, Frank Friedlos for providing the V79^puro

and V79-NTR^purocell lines, and Dr. Martin Ford, Glaxo-

Wellcome, Stevenage, U.K., for providing the SKOV-

NTR^neoand WiDr-NTR^neocell lines. This work was

supported by the Marsden Fund of New Zealand

(M.P.H.), the Health Research Council of New Zealand

(W.R.W., G.J .A.), and the Auckland Division of the

Cancer Society of New Zealand (W.A.D.).

2466 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 12

Hay et al.

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J M0205191

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