Synthesis and antitumor activity of duocarmycin derivatives: A-ring pyrrole compounds bearing β-(5',6',7'-trimethoxy-2'-indolyl)acryloyl group

Article abstract of DOI:10.1016/S0968-0896(00)00086-9

A series of A-ring pyrrole derivatives of duocarmycin bearing β-(5',6',7'-trimethoxy-2'-indolyl)acryloyl group were synthesized, and evaluated for in vitro anticellular activity against HeLa S₃ cells and in vivo antitumor activity against murine sarcoma 180 in mice. New Seg-B analogues bearing β-(5',6',7'-trimethoxy-2'-indolyl)acryloyl group containing double bond as spacer had lower peripheral blood toxicity than the derivatives bearing 5',6',7'-trimethoxyindole-2'-carboxyl group in Seg-B of the natural type. Moreover, most of them exhibited potent antitumor activity against in vivo murine tumor models. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00086-9

Bioorganic & Medicinal Chemistry 8 (2000) 1637±1643
Synthesis and Antitumor Activity of Duocarmycin Derivatives:
A-Ring Pyrrole Compounds Bearing
ꢀ-(50,60,70-Trimethoxy-20-indolyl)acryloyl Group
Nobuyoshi Amishiro,* Satoru Nagamura, Eiji Kobayashi, Akihiko Okamoto,
Katsushige Gomi, Masami Okabe and Hiromitsu Saito
Pharmaceutical Research Institute, Kyowa Hakko Kogyo Company, Ltd., 1188 Shimotogari, Nagaizumi, Sunto, Shizuoka,
411-8731, Japan
Received 8 February 2000; accepted 7 March 2000
AbstractÐA series of A-ring pyrrole derivatives of duocarmycin bearing b-(50,60,70-trimethoxy-20-indolyl)acryloyl group were syn-
thesized, and evaluated for in vitro anticellular activity against HeLa S3 cells and in vivo antitumor activity against murine sarcoma
180 in mice. New Seg-B analogues bearing b-(50,60,70-trimethoxy-20-indolyl)acryloyl group containing double bond as spacer had
lower peripheral blood toxicity than the derivatives bearing 50,60,70-trimethoxyindole-20-carboxyl group in Seg-B of the natural type.
Moreover, most of them exhibited potent antitumor activity against in vivo murine tumor models. # 2000 Elsevier Science Ltd. All
rights reserved.
Introduction
segment-B (Seg-B) has been considered to play an
important role for noncovalent binding to the minor
groove of DNA.8 We have previously synthesized a
series of DUM analogues bearing the simpli®ed DNA-
binding moieties.9 Among them, A-ring pyrrole DUMs
bearing cinnamoyl (3b)9b or heteroarylacryoyl groups10
showed remarkably potent in vivo antitumor activity
and low peripheral blood toxicity, compared with the
A-ring pyrrole derivatives having 50,60,70-trimethoxy-
indole-20-carboxyl group in Seg-B. The previous
structure±activity relationships (SAR) of the Seg-B
analogues suggest that our approach to synthesize the
new Seg-B analogues bearing b-(50,60,70-trimethoxy-20-
indolyl)acryloyl group containing double bond as
spacer may enhance the potency and decrease toxicity.
This paper describes the synthesis, in vitro anticellular
and in vivo antitumor activity, and hematotoxicity of
new Seg-B duocarmycin analogues.
A new class of antitumor antibiotics produced by
Streptomyces sp., including duocarmycins (DUMs) A
(1a), B1, B2 (1b), C1, C2 (1c) and SA possess excep-
tionally potent cytotoxicity (Fig. 1).1,2 DUMA (1a),
which is considered to be an active form among these
DUMs, possesses a unique cyclopropane ring with the
alkylating ability of DNA. DUMA (1a) and DUMSA
have been reported to show this cytotoxicity through a
sequence-selective alkylation of double-stranded DNA
mediating N3 adenine covalent adduct formation.3
KW-2189 (2),4 selected as the best compound in ana-
logues of A-ring pyrrole derivatives of DUMB2 (1b),
showed good stability in the culture medium and aqu-
eous solubility greater than 10 mg/mL.5,6 It showed
strong activities against murine ascitic and human solid
tumors.4b KW-2189 (2) was designed as a prodrug
which requires enzymatic hydrolysis followed by regen-
eration of DU-86 (3a) as an active metabolite.7
Chemistry
The segment-A (Seg-A) containing a spirocyclopropyl-
hexadienone moiety is necessary for the formation of
covalent bonding with DNA.4c On the other hand, the
Initially, the 2-methyl-3-methoxycarbonyl A-ring pyr-
role compound (3a) was treated with NaOMe in MeOH
to a€ord compound 4a (Seg-A) and methyl 5,6,7-tri-
methoxyindole-2-carboxylate (5), quantitatively (Scheme
1).9b,11 The obtained compound 5 was allowed to reduce
using DIBAL-H and then to oxidize using CrO3±Py to
*Corresponding author. Tel.: +81-559-89-2024; fax: +81-559-86-
7430; e-mail: cmurakata@kyowa.co.jp
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00086-9

1638
N. Amishiro et al. / Bioorg. Med. Chem. 8 (2000) 1637±1643
Figure 1. Structure of duocarmycins and duocarmycin derivatives.
Scheme 1. (a) NaOMe, MeOH, rt (76%); (b) (1) DIBAL-H, THF, 0 ꢀC; (2) CrO3±Py, CH2Cl2, 0 ꢀC±rt (36%); (c) (1) NaH, (EtO)2P(ˆO)CH2CO2Et;
(2) 4 N KOH, MeOH, 50 ꢀC (92%); (d) p-nitrophenol, Et3N, 2-chloro-1-methylpyridinium iodide, CH2Cl2, re¯ux (94%).
yield 2-formyl-5,6,7-trimethoxyindole (6). Ethyl 3-
(5,6,7-trimethoxy-2-indolyl)acrylate was synthesized
from compound 6 and triethyl phosphonoacetate by
Horner±Emmons reaction.12 The ethyl 3-(5,6,7-tri-
methoxy-2-indolyl)acrylate was treated with 4 N KOH
to a€ord compound 7, which was converted to p-nitro-
phenyl esters (8) by the reaction with p-nitrophenol
using 1-methy-2-chloropyridinium iodide (Mukaiyama's
method).13
In order to enhance in vivo antitumor activities of
b-(50,60,70-trimethoxy-20-indoyl)acrylate (9), the 8-O-
substituted derivatives were prepared (as described in
preceding papers).4a,15 The preparation of the 8-O-sub-
stituted analogues is outlined in Scheme 3. The 8-O-
acetate (15) was prepared by the reaction of 9 with HBr
in CH3CN followed by the addition of acetic anhydride
in the presence of 4-dimethylaminopyridine (DMAP).
Compound 9 was treated with HBr, followed by con-
version to the 4-nitrophenylcarbonate by the reaction
with 4-nitrophenyl chloroformate in the presence of
triethylamine. The carbonate was allowed to react with
N-methylpiperazine or methylhydrazine to produce the
8-O-[(N-methylpiperazinyl)carbonyl] (the free base of
14) and 8-O-(2-methylcarbazoyl) (16) compounds. The
obtained 8-O-[(N-methylpiperazinyl)carbonyl] deriva-
tive was converted to the HBr salt (14) upon treatment
with HBr in AcOEt. The aqueous solubility of this salt
(14) was found to be 1.2 mg/mL.
The preparation of compounds 9±13 has been investi-
gated by two approaches.14 Compounds 4a, 4c, 4d were
allowed to react with (E)-p-nitrophenyl 3-(5,6,7-tri-
methoxy-2-indolyl)acrylate (8) in the presence of NaH
to yield the corresponding cyclopropane compounds (9,
11, 12) in reasonable yield (see Scheme 2, method A).
On the other hand, the intermediate of 10 and the com-
pound 13 were prepared by the reaction of 4b, 4e with 4
N HCl in AcOEt followed by the addition of compound
7 in the presence of 1-(3-dimethylaminopropyl)-3-ethyl-
carbodiimide hydrochloride (EDCI), respectively (see
Scheme 2, method B). The obtained intermediate of
10 was converted to 10 upon treatment with DBU in
CH3CN. However, compound 13 caused decomposition
under the same conditions.
Results and Discussion
The antitumor activity of some representative deriva-
tives was evaluated primarily by assays of the inhibition

N. Amishiro et al. / Bioorg. Med. Chem. 8 (2000) 1637±1643
1639
Scheme 2. (a) NaH, 8, DMF, 20 ꢀC; (b) (1) 4 N HCl, AcOEt; (2) 7, EDCl, DMF, rt; (c) DBU, CH3CN, rt.
Scheme 3. (a) (1) HBr, CH3CN, rt; (2) 4-nitrophenyl chloroformate, Et3N, CH2Cl2, 78 ꢀC; (3) N-methylpiperazine, 0 ꢀC; (4) HBr; (b) (1) HBr,
CH3CN, rt; (2) Ac2O, DMAP, CH2Cl2, 0 ꢀC; (c) (1) HBr, CH3CN, rt; (2) 4-nitrophenyl chloroformate, Et3N, CH2Cl2, 78 ꢀC; (3) MeHNNH2.
of HeLa S3 cell growth (in vitro), and antitumor activity
against murine sarcoma 180 (in vivo). As shown in
Table 1, the ecacy in vivo is expressed as T/C, where T
and C represent means of tumor volume in treated and
control mice, respectively. The 3-formyl (10) and 3-
bromo (12) compounds exhibited strong anticellular
activity with IC50 values below 0.1 nM at 1 h exposure,
which was almost equivalent to compound 3a (DU-86).
This tendency was identical with SAR of 3-substituted
A-ring pyrrole DUM analogues in a previous paper.16
Table 1. Anticellular activity, antitumor activity and hematotoxicity
of duocarmycin derivatives
No.
HeLa S3
IC50 (nM)a
Sarcoma
180 (sc-iv)b
Hematotoxicity
1 h
72 h
Dose (mg/kg) T/Cc WBCd (%) PLe (%)
9
0.36
0.19
0.25
0.13
0.25
0.13
8.0
4.0
0.5
2.0
0.25
0.20
0.24
0.20
0.42
0.19
0.22
0.07
0.21
0.15
20
32
21
26
50
22
29
26
22
24
76
74
58
82
91
94
87
91
38
10
10
11
12
13
14
15
16
3a
2
0.044
0.49
0.058
71
59
0.64
4.2
0.045
53
0.091
0.14
0.022
56
3.2
0.082
0.27
0.0052
1.6
Peripheral blood toxicity (reduction of the number of
peripheral blood platelets) of b-(50,60,70-trimethoxy-20-
indolyl)acrylates (9±16) was lower than that of the deri-
vative bearing Seg-B of natural type. This tendency was
similar to that of cinnamates (3b)9b and hetero-
arylacrylates.10 These results suggest that new Seg-B
analogues bearing b-(50,60,70-trimethoxy-20-indolyl)acry-
loyl group containing double bond as spacer decrease
peripheral blood toxicity by noncovalent binding site
change attributable to double bond as spacer.
0.25
0.63
aDrug concentration required to inhibit the growth of HeLa S3 cells by
50%.
bMice (®ve mice/group) were implanted subcutaneously (sc) with
tumor cells, and the drug was dosed (mg/kg) intravenously (iv).
cT and C are the values of mean tumor volume of treated and control
mice, respectively.
dNumber of white blood cells of tumor-bearing mice on day 4 (% of
control).
eNumber of peripheral platelets of normal mice on day 7 (% of control).
The cyclopropane compounds (9±12) showed strong
antitumor activity against murine solid tumor, which
was almost equivalent to that of compound 3a. In con-
trast, 7-formyl compound (13) showed inferior anti-
cellular and antitumor activity to that of 7-hydro
compounds (9±12). All of the 8-O-substituted deriva-
tives (14±16) of 9 exhibited sucient ecacy with low
peripheral blood toxicity. Among them, 8-O-(2-methyl-
carbazoyl) compound (16) showed excellent activity in
vivo with T/C values of 0.07. Compound 16 was eval-
uated in vivo for ecacy in nude mice bearing human
xenograft St-4 (poor di€erentiated stomach adenocarci-
noma). Compound 16 showed e€ective activity T/C
values of 0.34 (3 mg/kg dose). Its potency against St-4
human stomach tumor xenograft was nearly comparable

1640
N. Amishiro et al. / Bioorg. Med. Chem. 8 (2000) 1637±1643
to the potency of our clinical candidate KW-2189 (2)
(T/C=0.12 (0.63 mg/kg dose)).4
20 min, a solution of 6 (98 mg, 0.42 mmol) in THF
(1.5 mL) was added, and the mixture was stirred for 1 h
20 min. The reaction was quenced by addition of water,
and the resulting mixture was poured into 0.5 N HCl.
The whole was extracted with AcOEt and worked up as
usual. Thereafter, the residue was puri®ed by column
chromatography (hexane:AcOEt, 7:1±5:1) to give
117 mg (93%) of ethyl 3-(5,6,7-trimethoxy-2-indolyl)-
acrylate. To a solution of ethyl 3-(5,6,7-trimethoxy-2-
indolyl)-acrylate (135 mg, 0.445 mmol) in MeOH (3 mL)
was added 4 N KOH (0.33 mL, 1.34 mmol). The mixture
was stirred at 50 ꢀC for 1 h 30 min. The reaction mixture
was poured into 0.5 N HCl, and the combine was
extracted AcOEt. The usual work up a€orded 121 mg
Conclusions
A series of A-ring pyrrole compounds of duocarmycin
bearing b-(50,60,70-trimethoxy-20-indolyl)acryloyl group
were prepared and evaluated for their anticellular
activity against Hela S3 cells and for antitumor activity
against sarcoma 180 murine solid tumor and St-4
human stomach tumor xenograft. The A-ring pyrrole
derivatives bearing b-(50,60,70-trimethoxy-20-indolyl)-
acryloyl group containing double bond as spacer to
Seg-B of the natural type showed weaker peripheral
blood toxicity than derivatives having the Seg-B of the
natural type. Moreover, most of them exhibited potent
antitumor activity against in vivo murine tumor
models.
1
(99%) of 7: H NMR (90 MHz, CDCl3): d 8.44 (1H,
brs), 7.72 (1H, d, J=16.0 Hz), 6.79 (1H, s), 6.75 (1H, d,
J=2.0 Hz), 6.20 (1H, d, J=15.8 Hz), 4.09 (3H, s), 3.93
(3H, s), 3.89 (3H, s).
To a solution of 7 (500 mg, 3.24 mmol) in CH2Cl2
(20 mL) were added p-nitrophenol (766 mg, 5.51 mmol),
triethylamine (1.54 mL, 11.0 mmol), 2-chloro-1-methyl-
pyridinium iodide (1.41 g, 5.51 mmol) and the mixture
was heated under re¯ux (bath temperature, 50 ꢀC) for
2 h 30 min. The reaction mixture was poured into aqu-
eous NaHCO3, and the combine was extracted with
CHCl3 and worked up as usual. Thereafter, the residue
was puri®ed by column chromatography (CHCl3) to
give 842 mg (94%) of 8: 1H NMR (270 MHz, CDCl3): d
8.44 (1H, brs), 8.30 (2H, d, J=8.9 Hz), 7.84 (1H, d,
J=15.8 Hz), 7.37 (2H, d, J=9.2 Hz), 6.82 (1H, d,
J=2.0 Hz), 6.80 (1H, s), 6.34 (1H, d, J=15.8 Hz), 4.10
(3H, s), 3.94 (3H, s), 3.90 (3H, s); FAB±MS m/z 399
(M+H)+.
Experimental
Infra-red spectra (IR) were recorded on a JASCO IR-810
spectrometer. 1H NMR spectra were measured on
JEOL JNM-EX270 and Hitachi R-90H spectrometers
and are reported in d units. Mass spectra were measured
with JEOL JMS-DX303 and Shimazu QP-1000 spec-
trometers. Elemental analyses were performed with a
Perkin±Elmer 2400 C, H, N analyzer. For column
chromatography, silica gel (SiO2, Merck Kieselgel 60
F254) was used. Preparative TLC (PTLC) was carried
out on glass plates coated with Merck Kieselgel 60
F254s. The usual work up refers to washing of organic
layers with brine, drying over anhydrous Na2SO4, and
evaporating o€ the solvents under reduced pressure.
ꢀ-(50,60,70-Trimethoxy-20-indolyl)acryloyl 2-methyl-3-
methoxycarbonyl-A-ring pyrrole duocarmycin (9). To a
solution of NaH (60%, 39 mg, 0.93 mmol) in DMF
(1 mL) was added a DMF solution (3.5 mL) of 4a (Seg-
A) (200 mg, 0.775 mmol), and the mixture was stirred
under Ar atomosphere at 20 ꢀC for 2 h 40 min. Then, a
solution of 8 (340 mg, 0.853 mmol) in DMF (3.5 mL)
was added and stirred for 3 h 10 min. 0.01 M phosphate
bu€er (pH 7) was added to the mixture. The whole was
extracted with AcOEt and then worked up as usual.
Thereafter, the residue was puri®ed by column chroma-
tography (CHCl3:MeOH, 100:1±80:1) to give 347 mg
(E)-p-Nitrophenyl 3-(5,6,7-trimethoxy-2-indolyl)acrylate
(8). To a solution of 5 (265 mg, 1.00 mmol) in dry THF
(15 mL) was added DIBAL-H in toluene (1 M, 3 mL,
3 mmol), and the mixture was stirred at 0 ꢀC for 3 h. The
reaction was quenched by addition of water, and then
the whole was extracted with AcOEt. The usual work
up a€orded 237 mg (100%) of 2-hydroxymethyl-5,6,7-
trimethoxyindole. CrO3 (348 mg, 3.48 mmol) was added
to pyridine (1.2 mL), and the mixture was stirred at
room temperature for 20 min. After adding CH2Cl2
(2 mL) to this mixture, a solution of 2-hydroxymethyl-
5,6,7-trimethoxyindole (276 mg, 1.16 mmol) in CH2Cl2
(8mL) was added to the mixture and stirred from 0 ꢀC to
the room temperature for 17 h. 0.5 N HCl was added to
the mixture, and then the whole was ®ltered. The ®ltrate
was extracted with AcOEt and then worked up as usual.
Thereafter, the residue was puri®ed by column chroma-
tography (hexane:AcOEt, 5:1) to give 98 mg (36%) of 6:
1H NMR (90 MHz, CDCl3): d 9.75 (1H, s), 9.14 (1H,
brs), 7.13 (1H, d, J=2.2 Hz), 6.85 (1H, s), 4.06 (3H, s),
3.94 (3H, s), 3.90 (3H, s).
1
(87%) of 9: H NMR (270 MHz, CDCl3): d 11.88 (1H,
br), 10.64 (1H, brs), 7.65 (1H, d, J=15.5 Hz), 7.23 (1H,
d, J=17.2 Hz), 6.73 (1H, s), 6.58 (1H, s), 6.43 (1H, brs),
4.23±4.27 (2H, br), 3.86 (3H, s), 3.84 (3H, s), 3.80 (3H,
s), 3.64 (3H, s), 3.37 (1H, br), 2.35 (3H, s), 2.22 (1H, br),
1.53 (1H, br). IR (KBr): 1705, 1660, 1608, 1464, 1390,
1360, 1294, 1219, 1109 cm 1. FAB±MS: m/z 518
(M+H)+. FAB±HRMS calcd for C28H28N3O7
(M+H)+ m/z 518.1927, found 518.1939. Anal. calcd
.
for C28H27N3O7 1.7H2O: C, 61.35; H, 5.59; N, 7.67;
found: C, 61.30; H, 5.21; N, 7.02.
To a solution of NaH (60%, 30 mg, 0.75 mmol) in THF
(1 mL) was added a solution of triethyl phosphono-
acetate (168 mg, 0.751 mmol) in THF (0.5 mL). The
mixture was stirred under Ar atmosphere at 0 ꢀC. After
ꢀ-(50,60,70-Trimethoxy-20-indolyl)acryloyl 2-methyl-3-for-
myl-A-ring pyrrole duocarmycin (10). To a solution of
4b (Seg-A) (15 mg, 0.066 mmol) in AcOEt (1 mL) was
added 4 N HCl in AcOEt (0.082 mL, 0.33 mmol), and

N. Amishiro et al. / Bioorg. Med. Chem. 8 (2000) 1637±1643
1641
the mixture was stirred at room temperature for 40 min.
After concentrating in vacuo, 7 (54 mg, 0.20 mmol) and
EDCI (38 mg, 0.20 mmol) were added to a solution of
the residue in DMF (1 mL), and the mixture was stirred
at room temperature for 22 h. 0.01 M phosphate bu€er
(pH 7) was added to the mixture. The whole was
extracted with AcOEt and then worked up as usual.
Thereafter, the residue was puri®ed by PTLC (CHCl3:
MeOH, 10:1) to give 7.5 mg (22%) of b-(50,60,70-tri-
methoxy-20-indolyl)acryloyl 8-hydroxy 2-methyl-3-for-
myl-A-ring pyrrole duocarmycin C2: 1H NMR
(270 MHz, CDCl3+CD3OD): d 9.79 (1H, s), 7.73 (1H,
s), 7.59 (1H, d, J=15.2 Hz), 6.88 (1H, d, J=15.5 Hz),
6.69 (1H, s), 6.62 (1H, s), 4.41 (1H, d, J=10.2 Hz),
4.31±4.33 (1H, m), 4.23 (1H, dd, J=9.6, 8.9 Hz), 4.00
(3H, s), 3.84 (3H, s), 3.80 (3H, s), 3.47 (1H, br), 3.28
(1H, dd, J=8.6, 7.6 Hz), 2.59 (3H, s). FAB±MS: m/z
524 (M+H)+.
57.05; H, 4.60; N, 7.68; found: C, 57.09; H, 4.61; N,
7.49.
ꢀ-(50,60,70-Trimethoxy-20-indolyl)acryloyl 7-formyl 8-
hydroxy 2-methyl-3-methoxycarbonyl-A-ring pyrrole
1
duocarmycin C2 (13). Method B: Yield 11%; H NMR
(270 MHz, CDCl3): d 12.32 (1H, brs), 9.90 (1H, s), 9.55
(1H, brs), 8.71 (1H, brs), 7.81 (1H, d, J=15.2 Hz),
6.79 (1H, s), 6.75 (1H, s), 6.69 (1H, d, J=15.8 Hz),
4.59 (1H, d, J=9.9 Hz), 4.30 (1H, dd, J=9.6, 9.6 Hz),
4.20±4.30 (1H, m), 4.10 (3H, s), 3.93 (6H, s), 3.90 (3H,
s), 3.85 (1H, br), 3.29 (1H, dd, J=10.2, 9.9 Hz),
2.71 (3H, s). IR (KBr): 1606, 1576, 1464, 1392, 1277,
1221, 1097, 1051 cm 1. FAB±MS: m/z 582 (M+H)+.
35
FAB±HRMS calcd for C29H29 ClN3O8 (M+H)+ m/z
582.1644, found 582.1643. Anal. calcd for C29H28
.
ClN3O8 2.0H2O: C, 56.36; H, 5.22; N, 6.80; found: C,
56.53; H, 5.12; N, 6.36.
To a solution of b-(50,60,70-trimethoxy-20-indolyl)acryl-
oyl 8-hydroxy 2-methyl-3-formyl-A-ring pyrrole duo-
carmycin C2 (7.5 mg, 0.014 mmol) in CH3CN (0.8 mL)
was added DBU (0.0064 mL, 0.043 mmol), and the
mixture was stirred at room temperature for 40 min.
0.01 M phosphate bu€er (pH 7) was added to the mix-
ture. The whole was extracted with CHCl3 and then
worked up as usual. Thereafter, the residue was puri®ed
by PTLC (CHCl3:acetone, 10:1) to give 3.3 mg (47%) of
ꢀ-(50,60,70-Trimethoxy-20-indolyl)acryloyl 8-O-[(N-me-
thylpiperazinyl)carbonyl] 2-methyl-3-methoxycarbonyl-
A-ring pyrrole duocarmycin B2 hydrobromide (14).
Hydrobromic acid in methanol (5%, 250 mg, 0.154
mmol) was added to a solution of 9 (20 mg, 0.039 mmol)
in CH2Cl2 (1.3 mL), and the mixture was stirred at room
temperature for 1 h 10 min. After the mixture was
cooled at 78 ꢀC, p-nitrophenyl chloroformate (23 mg,
0.12 mmol) and triethylamine (0.022 mL, 0.15 mmol)
were added to the mixture, and the mixture was stirred
1
10: H NMR (270 MHz, CDCl3+CD3OD): d 9.74 (1H,
s), 7.69 (1H, d, J=15.5 Hz), 6.76 (1H, d, J=15.5 Hz),
6.73 (1H, s), 6.70 (1H, s), 4.23 (1H, d, J=10.9 Hz), 4.13
(1H, dd, J=11.2, 5.0 Hz), 4.00 (3H, s), 3.86 (3H, s), 3.83
(3H, s), 3.46±3.50 (1H, m), 2.57 (3H, s), 2.31 (1H, dd,
J=7.3, 3.3 Hz), 1.29 (1H, dd, J=5.0, 3.6 Hz). IR (KBr):
1666, 1606, 1466, 1390, 1340, 1279, 1240, 1221, 1169,
1124 cm 1. FAB±MS: m/z 488 (M+H)+. Anal. calcd
at
78 ꢀC for 30 min. Then, N-methypiperazine
(0.015 mL, 0.14 mmol) was added, and the mixture was
stirred continually at 0 ꢀC from 78 ꢀC for 20 min. The
mixture was extracted with CHCl3 and washed with
aqueous NaHCO3 and worked up as usual. Thereafter,
the residue was puri®ed by PTLC (CHCl3:MeOH, 15:1)
to give 21 mg (76%) of the free base of 14. A solution of
the free base of 14 (16 mg, 0.022 mmol) in AcOEt
(1.6 mL) was treated with anhydrous hydrobromic acid
in methanol (5%, 108 mg) at room temperature for
30 min. The mixture was concentrated under reduced
pressure to give 17 mg of 14: 1H NMR (270 MHz,
DMSO-d6): d 12.00 (1H, s), 11.49 (1H, s), 9.87 (1H, br),
8.13 (1H, s), 7.56 (1H, d, J=15.2 Hz), 7.30 (1H, d,
J=15.2 Hz), 6.86 (1H, s), 6.79 (1H, d, J=1.7 Hz), 4.37±
4.59 (3H, m), 4.00 (3H, s), 3.86 (3H, s), 3.80 (6H, s),
3.71 (1H, br), 3.53±3.58 (5H, br), 3.15±3.34 (4H, br),
2.90 (3H, d, J=4.0 Hz), 2.69 (3H, s). IR (KBr): 1699,
.
.
for C27H25N3O6 0.5H2O 0.5CHCl3: C, 59.38; H, 4.80;
N, 7.55; found: C, 59.34; H, 5.10; N, 7.24.
ꢀ-(50,60,70-Trimethoxy-20-indolyl)acryloyl
2-methyl-A-
ring pyrrole duocarmycin (11). Method A: Yield 71%;
1H NMR (270 MHz, CDCl3): d 11.22 (1H, brs), 10.85
(1H, brs), 7.63 (1H, d, J=15.5 Hz), 7.30 (1H, d,
J=17.2 Hz), 6.73 (1H, s), 6.56 (1H, s), 6.34 (1H, brs),
5.34 (1H, s), 4.24 (1H, d, J=11.6 Hz), 4.17 (1H, dd,
J=11.7, 4.5 Hz), 3.86 (3H, s), 3.83 (3H, s), 3.81 (3H, s),
2.49 (1H, m), 2.12 (3H, s), 1.43 (1H, dd, J=7.1, 3.8 Hz),
1.25 (1H, m). IR (KBr): 1659, 1608, 1535, 1479, 1392,
1244, 1221, 1103 cm 1. FAB±MS: m/z 460 (M+H)+.
1695, 1645, 1470, 1435, 1423, 1416, 1254, 1219,
1
1095 cm
.
FAB±MS (the free base): m/z 726,
Anal. calcd for C26H25N3O5 1.2H2O: C, 64.91; H, 5.74;
N, 8.73; found: C, 64.90; H, 5.68; N, 8.48.
724 (M+H)+. FAB±HRMS (the free base) calcd
.
for C34H3979BrN5O8 (M+H)+ m/z 724.1982, found
724.1957. Anal. calcd for C34H38BrN5O8 HBr 5.0H2O:
C, 45.60; H, 5.51; N, 7.82; found: C, 45.76; H, 5.38; N,
7.75.
.
.
ꢀ-(50,60,70-Trimethoxy-20-indolyl)acryloyl
bromo-A-ring pyrrole duocarmycin (12). Method A:
2-methyl-3-
1
Yield 74%; H NMR (270 MHz, CDCl3+CD3OD): d
7.56 (1H, d, J=15.2 Hz), 6.74 (1H, d, J=15.2 Hz), 6.64
(1H, s), 6.59 (1H, s), 6.55 (1H, br), 4.14 (1H, d,
J=10.9 Hz), 4.03 (1H, dd, J=11.1, 4.8 Hz), 3.89 (3H, s),
3.76 (3H, s), 3.73 (3H, s), 3.05±3.11 (1H, m), 2.23
(1H, dd, J=7.6, 4.0 Hz), 2.15 (3H, s), 1.16 (1H, dd,
J=4.6, 4.6 Hz). IR (KBr): 1666, 1606, 1464, 1392, 1356,
ꢀ-(50,60,70-Trimethoxy-20-indolyl)acryloyl 8-O-acetyl 2-
methyl-3-methoxycarbonyl-A-ring pyrrole duocarmycin
B2 (15). Hydrobromic acid in methanol (5%, 244 mg,
0.151 mmol) was added to a solution of 9 (20 mg,
0.038 mmol) in CH2Cl2 (1.3 mL), and the mixture was
stirred at room temperature for 60 min. After the mix-
ture was cooled at 0 ꢀC, Ac2O (0.011 mL, 0.011 mmol)
and DMAP (14 mg, 0.011 mmol) were added to the
1277, 1221, 1174, 1051 cm 1. FAB±MS: m/z 540, 538
+
.
(M+H) . Anal. calcd for C26H24BrN3O5 0.5H2O: C,

1642
N. Amishiro et al. / Bioorg. Med. Chem. 8 (2000) 1637±1643
stirred solution, and the mixture was stirred at 0 ꢀC for
1 h. 0.01 M phosphate bu€er (pH 7) was added to the
mixture. The whole was extracted with CHCl3, and the
organic layer was worked up as usual. Thereafter, the
reside was puri®ed by HPLC (HPLC conditions: col-
umn: YMC-Pack ODS, S-5 mm, 120A, 250 mmÂ20 mm
(YMC, Co, Ltd, Japan). Mobile Phase: CH3CN:H2O=
70:30. Detection: 330 nm. Flow rate: 30 mL/min) to give
14 mg (57%) of 15: 1H NMR (270 MHz, CDCl3): d 8.68
(1H, brs), 8.55 (1H, brs), 7.56 (1H, d, J=15.2 Hz), 6.79
(1H, s), 6.60 (1H, s), 6.46 (1H, d, J=15.2 Hz), 4.42±4.53
(1H, m), 4.33 (1H, d, J=10.2 Hz), 4.23 (1H, dd, J=9.9,
8.6 Hz), 4.11 (3H, s), 3.96 (3H, s), 3.95 (3H, s), 3.90 (3H,
s), 3.78 (1H, dd, J=9.7, 2.5 Hz), 3.31 (1H, dd, J=10.2,
10.2 Hz), 2.44 (3H, s), 2.34 (3H, s). IR (KBr): 1678,
1643, 1468, 1416, 1306, 1217, 1201, 1122, 1107,
1088 cm 1. FAB±MS: m/z 642, 640 (M+H)+. FAB±
HRMS calcd for C30H31 79BrN3O8 (M+H)+ m/z
640.1295, found 640.1318.
medium for 71 h. For the continuous exposure experi-
ment, cells were treated with each compound for 72 h.
Then, cells were treated with PBS( ) containing 0.05%
trypsin (Difco Laboratories, Detroit, MI) and 0.02%
EDTA (Wako Pure Chemical Industries Co., Ltd.,
Osaka, Japan) and counted using a Microcell Counter
(CC-180A, Toa Medical Electronics Co., Ltd., Kobe,
Japan). The IC50 values (drug concentration required
for 50% inhibition of the cell growth) were determined.
Sarcoma 180, St-4 (poorly di€erentiated stomach ade-
nocarcinoma) cells were kindly supplied by the National
Cancer Center (Tokyo, Japan). Sarcoma 180 cells were
passaged and used for the experiment in adult male ddY
mice. Human xenografts were passaged and used in
adult male BALB/c-nu/nu mice. Murine solid tumor was
inoculated subcutaneously (sc) at the axillary region of
mice. Human xenografts were inoculated sc in the ¯ank
of nude mice. Drugs were administered intravenously
(iv) beginning 1 day after tumor inoculation. Antitumor
ecacy is expressed as T/C, where T and C are the
values of mean tumor volume of treated and control
mice. The length and width of the tumors were mea-
sured, and tumor volume was calculated as tumor
volume (mm3)=length (mm)Â[width (mm)]2/2 accord-
ing to the method of the National Cancer Institute.17
ꢀ-(50,60,70-Trimethoxy-20-indolyl)acryloyl 8-O-(2-methyl-
carbazoyl) 2-methyl-3-methoxycarbonyl-A-ring pyrrole
duocarmycin B2 (16). Hydrobromic acid in methanol
(5%, 376 mg, 0.232 mmol) was added to a solution of 9
(30 mg, 0.058 mmol) in CH2Cl2 (1.4 mL), and the mix-
ture was stirred at room temperature for 50 min. After
the mixture was cooled at 78 ꢀC p-nitrophenyl chloro-
formate (35 mg, 0.17 mmol) and triethylamine
(0.040 mL, 0.29 mmol) were added to a stirred solution
at 78 ꢀC for 25 min. Methylhydrazine (0.015 mL,
0.29 mmol) was added to the mixture, and the mixture
was attained at 0 ꢀC for 30 min. The mixture was diluted
with CHCl3, and washed with aqueous NaHCO3. The
usual work up and puri®cation by PTLC (CHCl3:
MeOH, 25:1) gave 28 mg (72%) of 16: 1H NMR
(270 MHz, CDCl3): d 10.52 (1H, brs), 9.98 (1H, brs),
8.11 (1H, s), 7.46 (1H, d, J=15.2 Hz), 6.72 (1H, s), 6.66
(1H, d, J=15.5 Hz), 6.53 (1H, s), 4.27±4.39 (1H, br),
4.30 (1H, d, J=10.2 Hz), 4.10 (1H, dd, J=9.9, 9.2 Hz),
4.02 (3H, s), 3.89 (3H, s), 3.86 (3H, s), 3.83 (3H, s), 3.70
(1H, dd, J=9.6, 2.3 Hz), 3.28 (3H, brs), 3.20 (1H, dd,
J=10.2, 9.9 Hz), 2.30 (3H, brs). IR (KBr): 1701, 1697,
1645, 1466, 1414, 1350, 1306, 1219, 1159, 1109,
1090 cm 1. FAB±MS: m/z 672, 670 (M+H)+. FAB±
HRMS calcd for C30H3379BrN5O8 (M+H)+ m/z
670.1513, found 670.1492. Anal. calcd for C30H32
The criteria for e€ectiveness against murine solid
tumors were the percentage T/C values with 42% and
less, and statistical signi®cance was determined by the
Mann±Whitney U test (p<0.05). Drug ecacy against
human xenografts was expressed as the percentage of
mean V/V0 value against that of the control group,
where V is the tumor volume on the day of evaluation
and V0 is the tumor volume on the day of initial drug
treatment. The criteria for e€ectiveness were T/C values
with 50% and less, and statistical signi®cance was
determined by the Mann±Whitney U test (p<0.01,
one-sided).18
Hematotoxicity (e€ect of compounds on peripheral blood
(PB) platelet counts and white blood cell counts). E€ect
on PB platelet counts. Each drug was dissolved with
saline and was administered into the tail vein of normal
male ddY mice (mean weight 20Æ1 g). After 7 days,
peripheral blood was obtained from the orbital vein to
measure the platelet counts using a microcell counter
(CC-180A, Toa Medical Electronics Co., Ltd., Kobe,
Japan). Results are presented as percentage of the
absolute value of the treated group versus that of con-
trol (percent of control).
.
BrN5O8 2.5H2O: C, 50.36; H, 5.21; N, 9.79; found: C,
50.42; H, 4.83; N, 9.45.
Biological studies
Human uterine cervix carcinoma HeLa S3 cells were
obtained from American Type Culture Collection
through Dainippon Pharmaceutical Co. (Osaka, Japan).
The cells (2Â104/well) were precultured in the culture
medium in 24-well multidishes (Nunc, Roskilde, Den-
mark) for 24 h at 37 ꢀC in a humidi®ed atmosphere of
5% CO2. For the pulse exposure experiment, cells
were treated with each compound for 1 h, washed with
E€ect on PB white blood cell counts. Drugs were
administered intravenously (iv) beginning 1 day after
tumor inoculation. After 4 days, peripheral blood was
obtained from the orbital vein of tumor-bearing mice to
measure the white blood cell counts using a microcell
counter (CC-180A, Toa Medical Electronics Co., Ltd.,
Kobe, Japan). Results are presented as percentage of
the absolute value of the treated group versus that of
control (percent of control).
Dulbecco's phosphate-bu€ered saline ((Ca2+
Mg2+-free, PBS( )), and further incubated in fresh
- and

N. Amishiro et al. / Bioorg. Med. Chem. 8 (2000) 1637±1643
1643
Acknowledgements
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Bioorg. Med. Chem. Lett. 1996, 6, 1955. (e) Boger, D. L.;
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We express our gratitude to Mikiko Saito, Etsuko
Koshimura and Kumiko Masunaga for their excellent
technical assistance. We also thank Mariko Yoshida,
Atsuko Kobayashi and Hideko Yokoyama for measur-
ing elemental analyses and MS spectra.
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