Synthesis and antitumor activity of duocarmycin derivatives: Modification at the C-7 position of segment-A of A-ring pyrrole compounds

Article abstract of DOI:10.1016/S0968-0896(00)00046-8

A series of the C7-substituted A-ring pyrrole derivatives of duocarmycin were synthesized, and evaluated for in vitro anticellular activity against HeLa S₃ cells and in vivo antitumor activity against murine sarcoma 180 in mice. All of the C7-substituted A-ring pyrrole compounds decreased potency in vitro and in vivo. However, some showed strong antitumor activity with T/C values less than 0.3. Among them, the 7-formyl compound 5d showed remarkable potent in vivo antitumor activity and low peripheral blood toxicity, which were equal to 2c. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00046-8

Bioorganic & Medicinal Chemistry 8 (2000) 1195±1201
Synthesis and Antitumor Activity of Duocarmycin Derivatives:
Modi®cation at the C-7 Position of Segment-A of A-Ring Pyrrole
Compounds
Nobuyoshi Amishiro,* Akihiko Okamoto, Masami Okabe and Hiromitsu Saito
Pharmaceutical Research Institute, Kyowa Hakko Kogyo Company, Ltd., 1188 Shimotogari, Nagaizumi, Sunto, Shizuoka,
411-8731, Japan
Received 11 January 2000; accepted 9 February 2000
AbstractÐA series of the C7-substituted A-ring pyrrole derivatives of duocarmycin were synthesized, and evaluated for in vitro
anticellular activity against HeLa S3 cells and in vivo antitumor activity against murine sarcoma 180 in mice. All of the C7-sub-
stituted A-ring pyrrole compounds decreased potency in vitro and in vivo. However, some showed strong antitumor activity with
T/C values less than 0.3. Among them, the 7-formyl compound 5d showed remarkable potent in vivo antitumor activity and low
peripheral blood toxicity, which were equal to 2c. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
segment-B (Seg-B) of DUM has been considered to play
an important role for noncovalent binding to the minor
groove of DNA.9 We have previously synthesized a
series of DUM analogues bearing the simpli®ed DNA-
binding moieties.10 Among them, A-ring pyrrole DUM
bearing cinnamoyl10b or heteroarylacryloyl10c groups
showed remarkable potent in vivo antitumor activity
and low peripheral blood toxicity.
A new class of antitumor antibiotics produced by
Streptomyces sp., including duocarmycin(DUM)s A
(1a), B1, B2 (1b), C1, C2 (1c) and SA possess excep-
tionally potent cytotoxicity (Fig. 1).1,2 Since DUMB1,
B2 (1b), C1, and C2 (1c) readily yield DUMA (1a) in
an aqueous solution, DUMA is thought to be an
active form among these antibiotics. DUMA (1a) and
DUMSA have a unique cyclopropane ring responsible
for the sequence-selective alkylation of double-stran-
ded DNA mediating N3 adenine covalent adduct for-
mation.3 This mechanism is similar to that of CC-1065
which has been reported to show high cytotoxicity.4,5
KW-2189 (2a),6 selected as the best compound in
analogues of A-ring pyrrole derivatives of DUMB2
(1b), showed good stability in the culture medium and
aqueous solubility greater than 10 mg/mL.7,8 It showed
strong activities against murine ascitic and human solid
tumors.6b KW-2189 (2a) is currently under phase II
clinical evaluation.
In the previous papers,11 we have reported our investi-
gation into the synthesis, anticellular and antitumor
activity, and toxicity of C3-substituted A-ring pyrrole
DUM derivatives containing various modi®cations. Our
study showed a new approach in the modi®cation of
C7-substituted A-ring pyrrole derivatives of DUM. The
modi®cation at the C-7 position of Seg-A containing
cyclopropane ring has been little reported as DUM or
CC-1065 analogues, and would a€ect electrophilicity of
the cyclopropane ring. Interestingly, the substituents at
the C-7 position of Seg-A would in¯uence the reversible
DNA-alkylating activity of DUM analogues.12
The segment-A (Seg-A) containing a spirocyclopropyl-
hexadienone moiety is necessary for the formation of
covalent bonding with DNA. Our previous results indi-
cate that the Seg-A structure in¯uences the electro-
philicity of cyclopropane.6c On the other hand, the
Chemistry
First, we have investigated various reaction conditions
to compound 2b or the 8-O-protected DUMB2 of A-ring
pyrrole, however, the modi®cation at the C-7 position
of Seg-A was unsuccessful. Next, Seg-A moiety (3a)
was treated with NBS or NCS to yield the 7-bromo (4a)
or 7-chloro (4b) compound (Scheme 1). The Seg-A
*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)00046-8

1196
N. Amishiro et al. / Bioorg. Med. Chem. 8 (2000) 1195±1201
Figure 1. Structure of duocarmycins and duocarmycin derivatives.
.
Scheme 1. (a) NBS or NCS, CCl4, rt; (b) Ac2O, BF3 Et2O, rt; (c) Cl2CHOMe, TiCl4, CH2Cl2, rt; (d) MeOCH2COCl, AlCl3, CH2Cl2, rt; (e) NaNO2,
AcOH, dioxane, 20 ꢀC; (f) 1) NaH, 2) p-nitrophenyl 4-methoxycinnamate, DMF, 20 ꢀC; (g) 1) HCl, 2) 4-methoxycinnamic acid, EDCI, rt; (h)
DBU, CH3CN, rt.
derivatives 3a±c were treated with electrophilic reaction,
such as Friedel±Crafts reaction using Lewis acids, to
a€ord the 7-acetyl (4c), 7-formyl (4d, g, h), 7-methoxy-
methylcarbonyl (4e) compound. Moreover, the treat-
ment of 3a with NaNO2 in acetic acid and 1,4-dioxane
a€orded the 7-nitroso (4f) compound in 70% yield.
Then, the preparation of N-40-methoxycinnamoyl deri-
vatives have been investigated by two approaches.
g, h decomposed under the same condition. Compound
4f was allowed to react with p-nitrophenyl 4-methoxy-
cinnamate in the presence of NaH to yield the corre-
sponding 40-methoxycinnamate (6f) in reasonable yield
(method B).
Since the formation of the 7-formyl compounds 4d, g, h
had low yields (15±29%), we have investigated other
methods. Treatment of 3a with POCl3 in DMF (Vils-
meier reaction) didn't a€ord the desired 7-formyl (4d)
but N-formyl compound of 3a. Furthermore, com-
pound 3a was treated with hexamethylenetetramine
(HMT) in TFA, and the four rings compound 7 was
produced (Scheme 2). It was assumed that the produce
of compound 7 was caused by the intermediate attack-
ing the amine moiety of C-ring part as an electrophilic
reaction. The obtained compound 7 was allowed to
Compounds 5a±e, 5g, 5h were prepared by the reaction
of 4a±e, 4g, 4h with 4 N HCl in CH3CN followed by the
addition of 4-methoxycinnamic acid in the presence of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro-
chloride (EDCI), respectively. The obtained compounds
5a±c were converted to 6a±c upon treatment with DBU
in CH3CN (method A). The cyclopropane compounds
6d, e, g, h were not obtained, because compounds 5d, e,

N. Amishiro et al. / Bioorg. Med. Chem. 8 (2000) 1195±1201
1197
Scheme 2. (a) HMT, TFA, rt, (57%); (b) 4-methoxycinnamic acid (70%) or 5,6,7-trimethoxy-2-indolecarboxylic acid (76%) or 3-(5,6,7-trimethoxy-
2-indole)acrylic acid (43%), EDCI, CH2Cl2, rt.
react with the corresponding carboxylic acid in the pre-
sence of EDCI to yield compounds 8, 9, 10.
alkylation reactivity of cyclopropane ring to DNA.
However, compounds 6b, 5d, 5g, 5h showed more
potent antitumor activity than 2c. Among them, com-
pound 5d showed excellent activity in vivo with T/C
values of 0.14. Moreover, the peripheral blood toxicity
(reduction of the number of peripheral blood platelets)
of compound 5d was lower than that of compound 2b
bearing Seg-B of natural type, and was equal to that
of compound 2c. Similarly, the other C7-substituted
A-ring pyrrole compounds bearing a 40-methoxy-
cinnamoyl group showed low peripheral blood toxicity.
Results and Discussion
The antitumor activity of some representative deriva-
tives was evaluated primarily by assays of the inhibition
of HeLa S3 cells growth (in vitro), and antitumor activ-
ity 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 represents means of tumor volume in treated and
control mice, respectively. All of the C7-substituted A-
ring pyrrole compounds bearing a 40-methoxycinnamoyl
group showed inferior anticellular activity to 2c.10b
Moreover, all of the C7-substituted A-ring pyrrole
compounds bearing a 40-methoxycinnamoyl group gen-
erally exhibited sucient ecacy in vivo at 10±20 times
higher dose than that of 2c.10b These results suggest that
the electron-withdrawing C7-substituents decrease the
We have expected that the four rings compounds 8, 9,
10 would increase biological activity by the binding of
these compounds to a di€erent position of DNA minor
groove. However, the four rings compounds 8, 9, 10
showed extremely low anticellular and antitumor activ-
ity. These results suggest that the four rings compounds
show low anity to the minor groove of DNA.
Table 1. Anticellular activity, antitumor activity and hematotoxicity
of duocarmycin derivatives
Conclusions
A series of the C7-substituted A-ring pyrrole derivatives
of duocarmycin were synthesized, and evaluated for in
vitro anticellular activity against HeLa S3 cells and in
vivo antitumor activity against murine sarcoma 180
in mice. All of the C7-substituted A-ring pyrrole com-
pounds decreased potency in vitro and in vivo. How-
ever, some showed strong antitumor activity with T/C
values less than 0.3. Among them, the 7-formyl com-
pound 5d showed remarkable potent in vivo antitumor
activity and low peripheral blood toxicity, which were
equal to 2c. Modi®cation at the C-7 position of Seg-A
was shown to be a promising approach for preparing
new candidates in DUM derivatives. Further detailed
studies of the structure±activity relationships (SAR) of
these derivatives are in progress.
No.
HeLa S3
IC50 (nM)a
Sarcoma
180 (s.c.±i.v.)b
Hematotoxicity
1 h
72 h
Dose
(mg/kg)
T/Cc
WBCd
(%)
PLe
(%)
5a
6a
6b
6c
5d
5e
6f
5g
5h
8
74
1600
800
84
190
35
3200
180
6.0
16
16
8
4
16
8
4
8
8
8
0.35
30
26
40
23
28
42
100
56
NTg
NTg
109
77
41
19
30
29
9.9
930
94
0.49
0.23
0.38
0.14
0.56
0.72
0.28(1)f
0.24
0.94
0.63
0.91
75
104
111
95
76
93
77
93
38
63
920
0.72
28
3500
>10000
10 >10000
57
2200
600
100
105
105
22
9
8
8
2b
2c
0.045
2.9±7.0
0.0052
0.26±0.94
0.25 0.21
0.83 0.34
50
Experimental
aDrug concentration required to inhibit the growth of HeLa S3 cells by
50%.
Infrared spectra (IR) were recorded on a JASCO IR-810
spectrometer. 1H NMR spectra were measured on
JEOL JNM-EX270 spectrometers and are reported in d
units. Mass spectra were measured with JEOL JMS-
DX303 and SHIMAZU QP-1000 spectrometers. Ele-
mental analyses were performed with a Perkin±Elmer
2400 C, H, N analyzer. For column chromatography,
silica gel (SiO2, Merck Kieselgel 60 F254) was used.
bMice (5 mice in 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).
fMortality (5 mice in group).
gNot tested.

1198
N. Amishiro et al. / Bioorg. Med. Chem. 8 (2000) 1195±1201
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 sol-
vents under reduced pressure.
to the mixture. Then, the whole was extracted with
CHCl3 and the usual work-up was conducted. There-
after, the residue was puri®ed by PTLC (CHCl3:MeOH,
1
15:1) to give 28 mg (36%) of 4e: H NMR (270 MHz,
CDCl3+CD3OD): d 4.70 (2H, s), 3.89 (1H, dd, J=12.5,
5.6 Hz), 3.80 (1H, d, J=12.5 Hz), 3.70 (3H, s), 3.55±3.62
(1H, m), 3.39 (3H, s), 2.47 (3H, s), 2.02 (1H, dd, J=7.9,
3.3 Hz), 0.99 (1H, dd, J=4.3, 4.0 Hz). FABMS: m/z 331
(M+H)+.
2-Methyl-3-methoxycarbonyl-7-bromo-A-ring pyrrole-
DUM-Segment A (4a). To a solution of 3a (20 mg,
0.078 mmol) in CCl4 (1 mL) was added NBS (15 mg,
0.085 mmol). The mixture was stirred at room tempera-
ture for 3 h 40min, and 0.01 M-phosphoric bu€er (pH 7)
was added to the mixture. Then, the whole was extracted
with CHCl3 and the usual work-up was conducted.
Thereafter, the residue was puri®ed by PTLC (CHCl3:
MeOH, 15:1) to give 18 mg (70%) of 4a: 1H NMR
(270 MHz, CDCl3+CD3OD): d 3.69 (1 H, dd, J=11.2,
5.3 Hz), 3.64 (3H, s), 3.57 (1H, d, J=10.9 Hz), 3.49±3.55
(1H, m), 2.38 (3H, s), 2.04 (1H, dd, J=7.8, 3.1 Hz), 1.04
(1H, dd, J=4.6, 3.6 Hz). FABMS: m/z 339, 337 (M+H)+.
2-Methyl-3-methoxycarbonyl-7-nitrosyl-A-ring pyrrole-
DUM-Segment A (4f). To a solution of 3a (20 mg,
0.078 mmol) in AcOH (0.4 mL) and 1,4-dioxane (0.4 mL)
was added NaNO2 (11 mg, 0.16 mmol). The mixture was
stirred at 20 ꢀC for 1 h 20 min, and aqueous NaHCO3
was added to the mixture. Then, the whole was extrac-
ted with CHCl3 and the usual work-up was conducted.
Thereafter, the residue was puri®ed by column chroma-
tography (CHCl3:MeOH, 20:1) to give 16 mg (70%) of
4f: 1H NMR (270 MHz, CDCl3+ CD3OD): d 4.02 (1H,
dd, J=14.8, 5.6 Hz), 3.93 (1H, dd, J=14.8, 1.3 Hz), 3.74
(3H, s), 3.64±3.71 (1H, m), 2.52 (3H, s), 2.14 (1H, dd,
J=7.9, 3.6 Hz), 1.00 (1H, dd, J=5.0, 4.0 Hz). FABMS:
m/z 288 (M+H)+.
2-Methyl-3-methoxycarbonyl-7-chloro-A-ring pyrrole-
(4b). Yield 63%; 1H NMR
DUM-Segment
A
(270 MHz, CDCl3+CD3OD): d 3.78 (1H, dd, J=10.9,
5.3 Hz), 3.72 (3H, s), 3.66 (1H, d, J=10.9 Hz), 3.55±3.61
(1H, m), 2.46 (3H, s), 2.15 (1H, dd, J=7.8, 3.1 Hz), 1.14
(1H, dd, J=5.0, 3.3 Hz). FABMS: m/z 293 (M+H)+.
2-Methyl-3, 7-diformyl-A-ring pyrrole-DUM-Segment A
(4g). Yield 15%; 1H NMR (270 MHz, CDCl3+
CD3OD): d 9.93 (1H, s), 9.67 (1H, s), 3.91 (1H, dd,
J=12.9, 5.6 Hz), 3.82 (1H, d, J=12.2 Hz), 3.56±3.60
(1H, m), 2.52 (3H, s), 2.06 (1H, dd, J=7.8, 3.5 Hz), 1.07
(1H, dd, J=4.6, 4.0 Hz). FABMS: m/z 257 (M+H)+.
2-Methyl-3-methoxycarbonyl-7-acetyl-A-ring pyrrole-
DUM-Segment A (4c). To a solution of BF3-Et2O
(0.057 mL, 0.47 mmol) in Ac2O (1.2 mL) was added 3a
(40 mg, 0.16 mmol). The mixture was stirred at room
temperature for 2 h, and aqueous NaHCO3 was added
to the mixture. Then, the whole was extracted with
CHCl3 and the usual work-up was conducted. There-
after, the residue was puri®ed by PTLC (CHCl3:MeOH,
2-Methyl-3-bromo-7-formyl-A-ring pyrrole-DUM-Segment
A (4h). Yield 16%; 1H NMR (270 MHz, CDCl3+
CD3OD): d 9.92 (1H, s), 3.93 (1H, dd, J=12.5, 5.6 Hz),
3.84 (1H, d, J=12.2 Hz), 3.26±3.33 (1H, m), 2.21 (3H,
s), 2.08 (1H, dd, J=7.8, 4.1 Hz), 1.06 (1H, dd, J=4.6,
4.6 Hz). FABMS: m/z 309, 307 (M+H)+.
1
15:1) to give 18 mg (39%) of 4c: H NMR (270 MHz,
CDCl3+CD3OD): d 3.91 (1H, dd, J=12.4, 5.8 Hz),
3.81 (1H, d, J=12.2 Hz), 3.76 (3H, s), 3.58±3.65 (1H,
m), 2.64 (3H, d, J=1.0 Hz), 2.52 (3H, d, J=1.0 Hz),
2.06 (1H, dd, J=7.8, 3.5 Hz), 1.02 (1H, dd, J=4.3,
4.0 Hz). FABMS: m/z 301 (M+H)+.
40-Methoxycinnamoyl 2-methyl-3-methoxycarbonyl-7-
bromo-A-ring pyrrole-DUMC2 (5a). Method A: to a
solution of 4a (66 mg, 0.20 mmol) in AcOEt (3 mL) was
added 6.86 N HCl in EtOH (0.085 mL, 0.59 mmol), and
the mixture was stirred at room temperature for 1 h.
After concentrating in vacuo, 4-methoxycinnamic acid
(104 mg, 0.585 mmol) and EDCI (112 mg, 0.585 mmol)
were added to a solution of the residue in DMF (3 mL),
and the mixture was stirred at room temperature for
22 h 15 min. 0.01 M phosphate bu€er (pH 7) was added
to the mixture. Then, the whole was extracted with
AcOEt and the usual work-up was conducted. There-
after, the residue was puri®ed by column chromato-
graphy (CHCl3:MeOH, 100:1±80:1) to give 47 mg (45%)
2-Methyl-3-methoxycarbonyl-7-formyl-A-ring pyrrole-
DUM-Segment
A (4d). To a solution of TiCl4
(0.025 mL, 0.35 mmol) and Cl2HCOMe (0.032 mL,
0.35 mmol) in CH2Cl2 (1.2 mL) was added 3a (30 mg,
0.12 mmol). The mixture was stirred at room tempera-
ture for 4 h 20 min, and aqueous NaHCO3 was added to
the mixture. Then, the whole was extracted with AcOEt
and the usual work-up was conducted. Thereafter, the
residue was puri®ed by PTLC (CHCl3:MeOH, 15:1)
to give 9.5 mg (29%) of 4d: 1H NMR (270 MHz,
CDCl3+CD3OD): d 9.92 (1H, s), 3.89 (1H, dd, J=12.5,
5.6 Hz), 3.80 (1H, d, J=12.5 Hz), 3.71 (3H, s), 3.58±3.62
(1H, m), 2.46 (3H, s), 2.10 (1H, dd, J=7.9, 3.3 Hz), 1.03
(1H, dd, J=4.6, 3.6 Hz). FABMS: m/z 287 (M+H)+.
1
of 5a: H NMR (270 MHz, CDCl3+CD3OD): d 7.59
(1H, d, J=15.5 Hz), 7.40 (2H, d, J=8.6 Hz), 6.79 (2H,
d, J=8.6 Hz), 6.58 (1H, d, J=15.8 Hz), 4.47 (1H, d,
J=10.6Hz), 4.11 (1H, dd, J=10.6, 6.6 Hz), 4.01 (1H, m),
3.97 (3H, s), 3.80 (1H, dd, J=9.2, 2.6 Hz), 3.78 (3H, s),
3.15 (1H, dd, J=10.6, 10.2 Hz), 2.56 (3H, s). IR (KBr):
1645, 1603, 1574, 1512, 1446, 1421, 1360, 1254, 1207,
1173, 1095 cm 1. FABMS: m/z 535, 533 (M+H)+.
2-Methyl-3-methoxycarbonyl-7-methoxymethylcarbonyl-
A-ring pyrrole-DUM-Segment A (4e). To a solution
of AlCl3 (62 mg, 0.47 mmol) and 3a (28 mg, 0.11 mmol)
in CH2Cl2 (2.4 mL) was added MeOCH2COCl
(0.043 mL, 0.47 mmol). The mixture was stirred at room
temperature for 1 h, and aqueous NaHCO3 was added
.
Anal. calcd for C24H22BrClN2O5 0.5H2O: C, 53.11; H,
4.27; N, 5.16; found C, 52.95; H, 4.15; N, 4.64.

N. Amishiro et al. / Bioorg. Med. Chem. 8 (2000) 1195±1201
1199
40-Methoxycinnamoyl 2-methyl-3-methoxycarbonyl-7-
bromo-A-ring pyrrole-DUMA (6a). To a solution of 5a
(11 mg, 0.020 mmol) in CH3CN (0.82 mL) was added
DBU (0.015 mL, 0.10 mmol). The mixture was stirred at
room temperature for 1 h 20 min, and 0.01 M phosphate
bu€er (pH 7) was added to the mixture. Then, the whole
was extracted with CHCl3 and the usual work-up was
conducted. Thereafter, the residue was puri®ed by column
chromatography (CHCl3:acetone, 12:1) to give 7.0 mg
(70%) of 6a: 1H NMR (270 MHz, CDCl3): d 11.56 (1H,
brs), 7.77 (1H, d, J=15.5 Hz), 7.51 (2H, d, J=8.9 Hz),
6.89 (2H, d, J=8.9 Hz), 6.51 (1H, d, J=15.5 Hz), 4.48
(1H, d, J=11.5 Hz), 4.16 (1H, dd, J=11.4, 4.5 Hz), 3.83
(3H, s), 3.83 (3H, s), 3.40±3.46 (1H, m), 2.72 (3H, s),
2.62 (1H, dd, J=7.4, 3.8 Hz), 1.34 (1H, dd, J=5.3,
4.0 Hz); IR (KBr): 1705, 1601, 1512, 1454, 1362, 1284,
Method A: yield 26%; H NMR (270 MHz, CDCl3+
CD3OD): d 7.68 (1H, d, J=15.5 Hz), 7.49 (2H, d,
J=8.6 Hz), 6.88 (2H, d, J=8.6 Hz), 6.74 (1H, d, J=
15.5 Hz), 4.49 (1H, d, J=9.9 Hz), 4.28 (2H, s), 4.20±4.33
(2H, m), 3.84 (3H, s), 3.78 (3H, s), 3.75 (1H, brd,
J=7.8 Hz), 3.29 (3H, s), 3.13 (1H, dd, J=10.6, 9.2 Hz),
2.62 (3H, s). IR (KBr): 1655, 1599, 1512, 1423, 1306,
1257, 1198, 1173, 1092, 825 cm 1. FABMS: m/z 527
(M+H)+. FAB±HRMS calcd for C27H2835ClN2O7
(M+H)+ m/z 527.1585, found 527.1599.
1
40 -Methoxycinnamoyl 2-methyl-3-methoxycarbonyl-7-
nitrosyl-A-ring pyrrole-DUMA (6f). Method B: to a
solution of NaH (60%, 6.4 mg, 0.16 mmol) in DMF
(0.3 mL) was added a DMF solution (0.5 mL) of 4f
(40 mg, 0.14 mmol). The mixture was stirred under Ar
atomosphere at 20 ꢀC for 2 h 20 min. Then, a solution
of the p-nitrophenyl ester of 4-methoxycinnamic acid
(44 mg, 0.15 mmol) in DMF (0.5 mL) was added and
stirred for 1 h 40 min. 0.01 M-phosphoric bu€er (pH 7)
was added to the mixture. Then, the whole was extrac-
ted AcOEt and the usual work-up was conducted.
Thereafter, the residue was puri®ed by PTLC (CHCl3:
MeOH, 15:1) to give 27 mg (44%) of 6f: 1H NMR
(270 MHz, CDCl3): d 11.35 (1H, brs), 7.88 (1H, d,
J=15.8 Hz), 7.52 (2H, J=8.9 Hz), 6.91 (2H, d, J=
8.6 Hz), 6.46 (1H, d, J=16.2 Hz), 4.36 (2H, d, J=
4.0 Hz), 3.85 (3H, s), 3.80 (3H, s), 3.32±3.36 (1H, m),
2.66 (3H, s), 2.18 (1H , dd, J=8.3, 3.6 Hz), 0.92 (1H, dd,
J=5.0, 4.0 Hz). IR (KBr): 1709, 1641, 1601, 1512, 1448,
1230, 1215, 1173, 1117, 1026 cm 1. FABMS: m/z 499, 497
+
.
(M+H) . Anal. calcd for C24H21BrN2O5 0.2CHCl3: C,
55.77; H, 4.10; N, 5.37; found C, 55.68; H, 4.06; N, 4.69.
40-Methoxycinnamoyl 2-methyl-3-methoxycarbonyl-7-
chloro-A-ring pyrrole-DUMA (6b). Method A: yield
1
47%; H NMR (270 MHz, CDCl3): d 11.96 (1H, brs),
7.78 (1H, d, J=15.2 Hz), 7.50 (2H, d, J=8.6 Hz), 6.88
(2H, d, J=8.9 Hz), 6.48 (1H, d, J=15.2 Hz), 4.46 (1H,
d, J=11.6 Hz), 4.15 (1H, dd, J=11.6, 4.3 Hz), 3.83 (3H,
s), 3.82 (3H, s), 2.71 (3H, s), 2.62 (1H, dd, J=7.3,
4.0 Hz), 1.33 (1H, dd, J=5.0, 4.3 Hz). IR (KBr): 1664,
1616, 1603, 1512, 1454, 1369, 1281, 1232, 1205, 1173,
1120 cm 1. FABMS: m/z 453 (M+H)+. Anal. calcd for
C24H21ClN2O5 0.5H2O: C, 62.41; H, 4.80; N, 6.06;
found C, 62.75; H, 4.59; N, 5.75.
1288, 1255, 1171, 1080, 962 cm 1. FABMS: m/z 448
.
(M+H) . Anal. calcd for C24H21N3O6 0.8H2O: C,
62.41; H, 4.93; N, 9.10; found C, 62.45; H, 4.89; N, 8.90.
.
+
40-Methoxycinnamoyl 2-methyl-3-methoxycarbonyl-7-meth-
ylcarbonyl-A-ring pyrrole-DUMA (6c). Method A: yield
40-Methoxycinnamoyl 2-methyl-3, 7-diformyl-A-ring
pyrrole-DUMC2 (5g). Method A: yield 28%; H NMR
1
1
25%; H NMR (270 MHz, CDCl3): d 11.54 (1H, brs),
7.73 (1H, d, J=15.2 Hz), 7.49 (2H, d, J=8.9Hz), 6.90
(2H, d, J=8.9Hz), 6.48 (1H, d, J=15.2 Hz), 4.33 (1H, dd,
J=10.1, 4.8 Hz), 4.21 (1H, d, J=10.2 Hz), 3.85 (3 H, s),
3.81 (3H, s), 3.55±3.61 (1H, m), 2.69 (3H, s), 2.62 (3H, s),
2.48 (1H, dd, J=7.4, 3.5 Hz), 1.27 (1H, dd, J=4.9,
4.3 Hz). IR (KBr): 1697, 1603, 1512, 1379, 1286, 1255,
1232, 1173, 1124, 1074 cm 1. FABMS: m/z 461 (M+H)+.
FAB±HRMS calcd for C26H25N2O6 (M+H)+ m/z
(270 MHz, CDCl3): d 12.26 (1H, brs), 10.05 (1H, s), 9.39
(1H, s), 9.55 (1H, brs), 7.81 (1H, d, J=15.5 Hz), 7.56
(2H, d, J=8.6 Hz), 6.94 (2H, d, J=8.6 Hz), 6.78 (1H, d,
J=15.2 Hz), 4.59 (1H, d, J=10.6 Hz), 4.39 (1H, dd,
J=10.2, 8.2 Hz), 4.25±4.32 (1H, m), 3.86 (3H, s), 3.85
(1H, brd, J=9.4 Hz), 3.39 (1H, dd, J=10.2, 8.6 Hz),
2.78 (3H, s). IR (KBr): 1653, 1601, 1512, 1473, 1421,
1360, 1304, 1254, 1173 cm 1. FABMS: m/z 453
(M+H)+. FAB±HRMS calcd for C24H2235ClN2O5
(M+H)+ m/z 453.1217, found 453.1232. Anal. calcd
.
461.1712, found 461.1735. Anal. calcd for C26H24N2O6
0.4CHCl3: C, 62.39; H, 4.84; N, 5.51; found C, 62.60; H,
4.75; N, 5.26.
.
for C24H21ClN2O5 1.0H2O: C, 61.21; H, 4.92; N, 5.95;
found C, 60.91; H, 5.21; N, 5.41.
40-Methoxycinnamoyl 2-methyl-3-methoxycarbonyl-7-form-
yl-A-ring pyrrole-DUMC2 (5d). Method A: yield 50%;
1H NMR (270 MHz, CDCl3): d 12.29 (1H, s), 9.89 (1H,
s), 9.80 (1H, brs), 7.81 (1H, d, J=15.2 Hz), 7.54 (2H, d,
J=8.6 Hz), 6.93 (2H, d, J=8.6 Hz), 6.77 (1H, d, J=
15.5 Hz), 4.58 (1H, d, J=10.6 Hz), 4.33 (1H, dd, J=
10.6, 7.6 Hz), 4.20±4.26 (1H, m), 3.92 (3H, s), 3.86 (1H,
dd, J=10.2, 3.0 Hz), 3.85 (3 H, s), 3.27 (1H, dd, J= 10.2,
10.2 Hz), 2.72 (3H, s). IR (KBr): 1701, 1662, 1655, 1601,
40-Methoxycinnamoyl 2-methyl-3-bromo-7-formyl-A-ring
pyrrole-DUMC2 (5h). Method A: yield 38%; H NMR
1
(270 MHz, CDCl3): d 12.55 (1 H, s), 9.92 (1H, s), 9.06 (1 H,
brs), 7.81 (1H, d, J=15.5 Hz), 7.54 (2H, d, J=8.3 Hz),
6.93 (2H, d, J=8.6 Hz), 6.76 (1H, d, J=15.5 Hz), 4.62
(1H, d, J=10.9 Hz), 4.37 (1H, dd, J=10.6, 7.6 Hz),
3.95±4.02 (2H, m), 3.85 (3H, s), 3.36 (1H, dd, J=
11.2Hz), 2.45 (3H, s). IR (KBr): 1655, 1601, 1541, 1510,
1512, 1421, 1352, 1281, 1254, 1173 cm 1. FABMS: m/z
1421, 1348, 1252, 1171 cm 1. FABMS: m/z 505, 503
+
+
.
483 (M+H) . Anal. calcd for C25H23ClN2O6 1.5H2O: C,
58.88; H, 5.14; N, 5.49; found C, 59.26; H, 4.99; N, 5.24.
.
(M+H) . Anal. calcd for C23H20BrClN2O4 0.5H2O: C,
53.87; H, 4.13; N, 5.46; found C, 53.88; H, 4.17; N, 5.10.
40-Methoxycinnamoyl 2-methyl-3-methoxycarbonyl-7-
methoxymethylcarbonyl-A-ring pyrrole-DUMC2 (5e).
2-Methyl-3-methoxycarbonyl-6,7-(methanoiminomethano)-
A-ring pyrrole-DUM-Segment-A (7). To a solution of 3a

1200
N. Amishiro et al. / Bioorg. Med. Chem. 8 (2000) 1195±1201
(236 mg, 0.915 mmol) in TFA (7 mL) was added HMT
(257 mg, 1.83 mmol). The mixture was stirred at room
temperature for 3 h 40 min, and aqueous NaHCO3 was
added to the mixture. Then, the whole was extracted
with CHCl3 and the usual work-up was conducted.
Thereafter, the residue was puri®ed by column chroma-
tography (CHCl3:MeOH, 15:1) to give 155 mg (57%) of
1200, 1122 cm 1. FABMS: m/z 559 (M+H)+. Anal.
.
calcd for C30H30N4O7 1.0H2O: C, 62.49; H, 5.59; N,
9.72; found C, 62.64; H, 5.57; N, 9.48.
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 37ꢀC in a humidi®ed atmosphere of
5% CO2. For the pulse exposure experiment, cells were
treated with each compound for 1 h, washed with Dul-
1
7: H NMR (270 MHz, CDCl3): d 11.73 (1H, brs), 4.21
(1H, d, J=11.9 Hz), 4.02 (1H, d, J=10.9 Hz), 3.83 (1H,
d, J=16.8 Hz), 3.78 (3H, s), 3.75 (1H, d, J=14.2 Hz),
3.47±3.55 (2H, m), 3.39±3.45 (1H, m), 2.59 (3H, s), 2.28
(1H, dd, J=7.6, 2.6 Hz), 1.21 (1H, dd, J=4.6, 3.0 Hz).
FABMS: m/z 300 (M+H)+.
40-Methoxycinnamoyl 2-methyl-3-methoxycarbonyl-6,7-
(methanoiminomethano)-A-ring pyrrole-DUMA (8). 4-
Methoxycinnamic acid (27 mg, 0.15 mmol) and EDCI
(29 mg, 0.15 mmol) were added to a solution of 7
(15 mg, 0.050 mmol) in CH2Cl2 (1 mL). The mixture was
stirred at room temperature for 3 h 40 min, and 0.01 M
phosphate bu€er (pH 7) was added to the mixture.
Then, the whole was extracted with CHCl3 and the
usual work-up was conducted. Thereafter, the residue
was puri®ed by PTLC (CHCl3:MeOH, 20:1) to give
16 mg (70%) of 8: 1H NMR (270 MHz, CDCl3): d 10.97
(1H, brs), 7.70 (1H, d, J=15.5 Hz), 7.50 (2H, d, J=
8.9 Hz), 6.90 (2H, d, J=8.6 Hz), 6.89 (1H, d, J=
17.8 Hz), 5.53 (1H, d, J=10.6 Hz), 4.75 (1H, d, J=
15.5 Hz), 4.49 (1H, d, J=15.5 Hz), 4.24 (1H, d, J=
10.9 Hz), 3.84 (3H, s), 3.78 (3H, s), 3.69 (1H, d, J=
9.9 Hz), 3.46±3.57 (2H, m), 2.58 (3H, s), 2.34 (1H, dd,
J=7.3, 3.0 Hz), 1.36 (1H, dd, J=4.0, 3.6 Hz). IR (KBr):
1601, 1576, 1512, 1456, 1427, 1281, 1252, 1173, 1120,
1084 cm 1. FABMS: m/z 460 (M+H)+. FAB±HRMS
calcd for C26H26N3O5 (M+H)+ m/z 460.1873, found
becco's phosphate-bu€ered saline [Ca2+- and Mg2+
-
free, PBS(-)], and further incubated in fresh medium for
71 h. For the continuous exposure experiment, 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
adenocarcinoma) 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
.
460.1894. Anal. calcd for C26H25N3O5 1.0H2O: C,
65.40; H, 5.70; N, 8.80; found C, 65.50; H, 5.73; N, 8.24.
50,60,70-Trimethoxyindole-20-carboxyl 2-methyl-3-methoxy-
carbonyl-6,7-(methanoiminomethano)-A-ring pyrrole-
1
DUMA (9). Yield 76%; H NMR (270 MHz, CDCl3):
tumor volume ꢀmm3† ˆ length ꢀmm†  ‰width ꢀmm†Š2=2
according to the method of the National Cancer Institute.13
d 11.92 (1H, brs), 9.42 (1H, brs), 6.89 (1H, s), 6.79 (1H,
s), 5.51 (1H, d, J=10.6 Hz), 5.13 (1H, d, J=15.8 Hz),
4.68 (1H, d, J=14.8 Hz), 4.34 (1H, d, J=10.9 Hz), 4.03
(3H, s), 3.91 (3H, s), 3.90 (3H, s), 3.76 (3H, s), 3.66 (1H,
d, J=9.2 Hz), 3.46±3.54 (2H, m), 2.54 (3H, s), 2.33 (1H,
dd, J=7.3, 2.3 Hz), 1.32 (1H, dd, J=4.3, 3.2 Hz). IR
(KBr): 1701, 1635, 1579, 1464, 1429, 1306, 1257, 1223,
1196, 1119 cm 1. FABMS: m/z 533 (M+H)+. Anal.
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).14
.
calcd for C28H28N4O7 0.5H2O: C, 62.10; H, 5.40; N,
10.35; found C, 61.82; H, 5.46; N, 9.68.
ꢀ-(50,60,70-Trimethoxy-20-indole) acryloyl 2-methyl-3-
methoxycarbonyl-6,7-(methanoiminomethano)-A-ring
1
pyrrole-DUMA (10). Yield 43%; H NMR (270 MHz,
CDCl3): d 11.10 (1H, brs), 10.35 (1H, brs), 7.80 (1H, d,
J=15.2 Hz), 7.14 (1H, d, J=15.5 Hz), 6.79 (1H, s), 6.66
(1H, d, J=2.0 Hz), 5.49 (1H, d, J=10.9 Hz), 4.88 (1H,
d, J=15.5 Hz), 4.55 (1H, d, J=15.5 Hz), 4.20 (1H, d,
J=10.9 Hz), 4.06 (3H, s), 3.90 (3H, s), 3.87 (3H, s), 3.72
(3H, s), 3.56 (1H, d, J=9.2 Hz), 3.41±3.51 (2H, m), 2.52
(3H, s), 2.28 (1H, dd, J=7.3, 2.6 Hz), 1.23 (1H, m). IR
(KBr): 1647, 1630, 1618, 1578, 1466, 1448, 1313, 1213,
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

N. Amishiro et al. / Bioorg. Med. Chem. 8 (2000) 1195±1201
1201
normal male ddY mice (mean weight 20‹1 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
control (percent of control).
34, 2179. (f) Boger, D. L.; Johnson, D. S.; Yun, W. J. Am.
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4. (a) Hanka, L. J.; Dietz, A.; Gerpheide, S. A.; Kuentzel, S.
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Hurley, L. H. J. Antibiot. 1986, 39, 319.
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G. L.; Scahill, T. A. Science 1984, 226, 843. (b) Reynolds, V.
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E€ect on PB white blood cell counts. Drugs were admi-
nistered 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).
Acknowledgements
8. (a) Boger, D. L.; Ishizaki, T. Tetrahedron Lett. 1990, 31,
793. (b) Boger, D. L.; Mesini, P.; Tarby, C. M. J. Am. Chem.
Soc. 1994, 116, 6461. (c) Boger, D. L.; McKie, J. A.; Han, N.;
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Bioorg. Med. Chem. Lett. 1996, 6, 1955. (e) Boger, D. L.;
Boyce, C.; Johnson, D. S. Bioorg. Med. Chem. Lett. 1997, 7,
233.
We express our gratitude to Ms. Mikiko Saito and Ms.
Kumiko Masunaga for their excellent technical assis-
tance. We also thank Ms. Mariko Yoshida, Ms. Atsuko
Kobayashi and Ms. Hideko Yokoyama for measuring
Elemental analyses and MS spectra.
9. (a) Chidester, C. G.; Krueger, W. C.; Mizsak, S. A.;
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