Synthesis and antitumor activity of duocarmycin derivatives: Modification at C-8 position of A-ring pyrrole compounds bearing the simplified DNA-binding groups

Article abstract of DOI:10.1016/S0968-0896(99)00293-X

A series of the 8-O-substituted A-ring pyrrole derivatives of duocarmycin bearing the simplified DNA-binding moieties such as cinnamoyl or heteroarylacryloyl groups were synthesized, and evaluated for in vitro anticellular activity against HeLa S₃ cells and in vivo antitumor activity against murine sarcoma 180 in mice. In addition, the stability of the 8-O- substituted analogues in aqueous solution and the conversion to their active form (cyclopropane compound) from the 8-O-substituted analogues in mice or human serum were examined. The 8-O-substituted A-ring pyrrole derivatives bearing the simplified DNA-binding moieties showed remarkably potent in vivo antitumor activity and low peripheral blood toxicity compared with the 8-O- substituted A-ring pyrrole derivatives having the trimethoxyindole skeleton in segment-B (Seg-B), which were equal to 8-O-[(N-methylpiperazinyl)carbonyl] derivatives of 4'-methoxycinnamates and 4'-methoxy-β-heteroarylacrylates. Moreover, among 8-O-substituted analogues, several compounds can be chemically or enzymatically converted to their active form in human serum. This result indicated that new 8-O-substituted derivatives were different prodrugs from KW-2189 and 8-O-substituted analogues being the same type of prodrug as KW-2189. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00293-X

Bioorganic & Medicinal Chemistry 8 (2000) 381±391
Synthesis and Antitumor Activity of Duocarmycin Derivatives:
Modi®cation at C-8 position of A-Ring Pyrrole Compounds
Bearing the Simpli®ed DNA-Binding Groups
Nobuyoshi Amishiro,* Satoru Nagamura, Chikara Murakata, Akihiko Okamoto,
Eiji Kobayashi, Masao Asada, Katsushige Gomi, Tatsuya Tamaoki, Masami Okabe,
Naoko Yamaguchi, Kazuo Yamaguchi and Hiromitsu Saito
Pharmaceutical Research Institute, Kyowa Hakko Kogyo Company, Ltd., 1188 Shimotogari, Nagaizumi, Sunto, Shizuoka,
411-8731, Japan
Received 9 September 1999; accepted 29 October 1999
AbstractÐA series of the 8-O-substituted A-ring pyrrole derivatives of duocarmycin bearing the simpli®ed DNA-binding moieties
such as cinnamoyl or heteroarylacryloyl groups were synthesized, and evaluated for in vitro anticellular activity against HeLa S3
cells and in vivo antitumor activity against murine sarcoma 180 in mice. In addition, the stability of the 8-O-substituted analogues
in aqueous solution and the conversion to their active form (cyclopropane compound) from the 8-O-substituted analogues in mice
or human serum were examined. The 8-O-substituted A-ring pyrrole derivatives bearing the simpli®ed DNA-binding moieties
showed remarkably potent in vivo antitumor activity and low peripheral blood toxicity compared with the 8-O-substituted A-ring
pyrrole derivatives having the trimethoxyindole skeleton in segment-B (Seg-B), which were equal to 8-O-[(N-methylpiper-
azinyl)carbonyl] derivatives of 40-methoxycinnamates and 40-methoxy-b-heteroarylacrylates. Moreover, among 8-O-substituted
analogues, several compounds can be chemically or enzymatically converted to their active form in human serum. This result
indicated that new 8-O-substituted derivatives were di€erent prodrugs from KW-2189 and 8-O-substituted analogues being the
same type of prodrug as KW-2189. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
compound in analogues of A-ring pyrrole derivatives of
duocarmycin B2, showed good stability in the culture
medium and aqueous solubility greater than 10 mg/
mL.30±35 It showed strong activities against murine
ascitic and human solid tumors.27 KW-2189 (2b) is cur-
rently under phase II clinical evaluation.
Duocarmycin(DUM)s (A, 1a; SA, 1b; B1, 1e; B2, 1c;
C1, 1f; C2, 1d) are novel antitumor antibiotics isolated
from the culture broth of Streptomyces sp. (Fig. 1).1±8
DUMs are known to exhibit potent growth-inhibitory
activity against human uterine cervix carcinoma HeLa
S3 in vitro, and also exhibit modest broad antitumor
spectrum against murine transplantable solid tumor.9±13
Since DUMB1 (1e), B2 (1c), C1 (1f), and C2 (1d) readily
yield DUMA (1a) in aqueous solution, DUMA is
thought to be an active form among these antibiotics.
DUMA and DUMSA (1b) have a unique cyclopropane
ring responsible for the sequence-selective alkylation of
double-stranded DNA resulting in N3 adenine covalent
adduct formation.14±19 This mechanism is similar to that
of CC-1065 (1g) which has been reported to show high
cytotoxicity.20±25 KW-2189 (2b),26±29 selected as the best
The segment-A (Seg-A) has the electrophilic cyclopro-
pane ring necessary for the formation of covalent
bonding with DNA.38 On the other hand, the segment-
B (Seg-B) of DUM has been considered to play an
important role for noncovalent binding to the minor
groove of DNA.36±38 With the objective to identify
novel promising candidates, we have previously synthe-
sized a series of DUM analogues bearing the simpli®ed
DNA-binding moieties.39±41 Among these Seg-B deri-
vatives, some of A-ring pyrrole compounds bearing
cinnamoyl40 or heteroarylacryloyl41 groups as Seg-B
showed strong antitumor activity and low peripheral
blood toxicity.41 Moreover, the 8-O-(N,N-dialkyl-
carbamoyl) cinnamates having an amino group at the
30-position, and 8-O-[(N-methylpiperazinyl)carbonyl]
Keywords: antibiotics; antitumor compounds; DNA; natural products;
prodrug.
*Corresponding author.
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00293-X

382
N. Amishiro et al. / Bioorg. Med. Chem. 8 (2000) 381±391
Figure 1. Structure of duocarmycins, CC-1065, and duocarmycins derivatives.
derivatives of 6-membered N-heteroarylacrylates were
found to possess adequate water solubility in excess of
10 mg/mL.40,41
describe the evaluation of their anticellular and anti-
tumor activities, hematotoxicity. In addition, we exam-
ined the stability and solubility of their analogues under
aqueous conditions, and in vitro metabolism of their
analogues in mice or human serum.
In the previous papers,42 we have reported the synthesis
of A-ring pyrrole derivatives modi®ed to the various
substituent at the C-8 phenolic hydroxyl group of Seg-
A. We also have studied the structure±activity relation-
ships (SAR) of their analogues, and the stability and
solubility of their analogues under aqueous conditions.
The results suggest that 8-O-substituted A-ring pyrrole
analogues need to release an active compound 2a (DU-
86) chemically or enzymatically to exhibit any sig-
ni®cant in vivo antitumor activity. It seems that DU-86
(2a) as a metabolite of A-ring pyrrole analogues is
responsible for the in vivo antitumor activity of KW-
2189 (2b). KW-2189 itself seems not to be dominant in
its antitumor potency.42±46 KW-2189 was activated by
carboxyl esterase.43,45,46 However, the conversion of
KW-2189 to its active form was in¯uenced by carboxyl
esterase activity. As an example, KW-2189 was not
activated in human serum, but in mouse serum. For
prediction of the clinical e€ect, it is necessary that new
prodrug be able to release to an active form in human
serum. Unlike KW-2189 (2b), A-ring pyrrole derivatives
bearing the simpli®ed DNA-binding moieties further
modi®ed at the C-8 phenolic hydroxyl group are expec-
ted to release an active form in human serum. In this
paper, we describe the synthesis of A-ring pyrrole deri-
vatives bearing a cinnamoyl or heteroarylacryloyl group
modi®ed at the C-8 phenolic hydroxyl group to thio-
carbonate, ester, and hydrazinocarboxylate, and also
Chemistry
The 2-methyl-3-methoxycarbonyl A-ring pyrrole com-
pound 2a (DU-86) was initially prepared by employing
the Wagner±Meerwein type rearrangement of the
8-O-protected-3-hydroxyduocarmycin B2 followed by
deprotection of the protecting group under basic condi-
tions.40,41,47,48 Compound 2a was treated with NaOMe
in MeOH to quantitatively a€ord compound 3 (Seg-A)
and methyl trimethoxyindole-2-carboxylate as shown in
Scheme 1. The obtained compound 3 was allowed to
react with substituted-cinnamic acid or substituted-
heteroarylacrlic acid p-nitrophenyl esters in the pre-
sence of NaH to yield the corresponding cyclopropane
compounds 4a±4c in reasonable yield, as described
previously.40,41
The 8-O-thiocarbonate (5) was prepared by the reaction
of 4a with 48% HBr in CH3CN followed by the addi-
tion of methyl chlorothioformate in the presence of
triethylamine (see Scheme 2). The 8-O-acetate (the free
base of 6) and the 8-O-carboxylmethylsulfanylacetate
(7) were prepared by the reaction of 4a or 4b with 48%
HBr in CH3CN followed by the addition of acetic
anhydride or thioglycolic anhydride in the presence of

N. Amishiro et al. / Bioorg. Med. Chem. 8 (2000) 381±391
383
Scheme 1.
17 using HBr a€orded the aminomethylbenzoate (18).
The free bases of compound 6, 8, 13±16, 18 were con-
verted to the HCl salts (6, 8) or HBr salts (13±16, 18)
upon treatment with HCl or HBr.
Compounds 4a±4c were converted to the 4-nitrophenyl
carbonate by the reaction with 4-nitrophenyl chloro-
formate in the presence of triethylamine, which was
treated with various hydrazine, 1-amino-4-methylpiper-
azine and 1-amino-4-piperidinopiperidine to produce
carbazoyl (21, the free base of 25, 22), piper-
azinylaminocarbonyl (the free base of 19, 23, 24) and
piperidinylaminocarbonyl (the free base of 20) deriva-
tives in reasonable yield (see Scheme 3). The free bases
of 19, 20, 23±25 were converted to HBr salts (19, 20, 23±
25) by the same method as shown in Scheme 2.
Results and Discussion
The aqueous solubility of HCl or HBr salts was shown
in Table 1. The HCl or HBr salts (6, 14±16, 23±25) of
8-O-substituted derivatives of 4b and 4c were found to
possess adequate water solubility in excess of 10 mg/
mL. In contrast, the HCl or HBr salts (8, 13, 18) of 8-O-
substituted derivatives of 4a exhibited poor aqueous
solubility below 0.1 mg/mL. For improving aqueous
solubility, formation of organic acid salts of the free
base of 13 was examined. The results was found that
the conversion of the free base of 13 to 2MeSO3H salt
has improved aqueous solubility (>5 mg/mL). Further-
more, HBr salts (19, 20) of piperazinylaminocarbonyl
and piperidinylaminocarbonyl derivatives of 4a were
more aqueous soluble than HCl salt (2c) of piper-
azinylcarbamoyl derivative of 4a.
Scheme 2. (a) (1) HBr, CH3CN, (2) CH3SCOCl, Et3N, CH2Cl2; (b) (1)
HBr, CH3CN, (2) Ac2O, 4-dimethylaminopyridine (DMAP), CH2Cl2,
(3) HCl; (c) (1) HBr, CH3CN, (2) thiodiglycolic anhydride, DMAP,
CH2Cl2, (d) (1) HBr, CH3CN, (2) R1CO2H, DCC, DMAP, CH2Cl2,
(3) HCl for 8 or HBr for 13±16; (e) (1) HBr, ClCH2CH2Cl, 60 ꢀC, (2) HBr.
The stability of compounds was measured in 0.05 M
phosphate bu€er (pH 7) containing 50% DMF by
HPLC analysis (Table 1). The thioester (5), carboxyl-
methylsulfanylacetate (7), and heteroaryl esters (8, 9)
mainly decomposed to a cyclopropane compound (4a),
which was di€erent from the substituted benzoates (10,
11±13). Similarly the heterocyclic aminocarbonyl (19,
20) and carbazoyl (21, 22) compounds mainly decom-
posed to a cyclopropane compound (4a).
4-dimethylaminopyridine (DMAP).42 Further, we pre-
pared the 8-O-benzoates (10±12, 17, the free base of 13,
14, 16, 18) and the 8-O-heteroaryl esters (9, the free base
of 8, 15) with a hydrophilic moiety by the reaction with
the corresponding carboxylic acid in the presence of
dicyclohexylcarbodiimide (DCC) and 4-dimethylamino-
pyridine (DMAP).42 Deprotection of the Boc group of
Next, we examined in vitro metabolism of 8-O-sub-
stituted derivatives in mice or human serum (Table 3).

384
N. Amishiro et al. / Bioorg. Med. Chem. 8 (2000) 381±391
KW-2189, which showed a similar tendency in A-ring
pyrrole derivatives having the simpli®ed DNA-binding
moieties.41
Several analogues demonstrating a di€erent activated
form from KW-2189 and adequate water solubility were
evaluated in vivo for ecacy in nude mice bearing
human xenograft St-4 (poor di€erentiated stomach
adenocarcinoma) (Table 2). Compounds 13, 19, 20, 23,
24 showed excellent activity in vivo (T/C <0.3). These
activities against St-4 human stomach tumor xeno-
garaft were nearly comparable to our clinical candidate
KW-2189.
Conclusions
A series of the 8-O-substituted A-ring pyrrole deriva-
tives bearing the simpli®ed DNA-binding moieties such
as cinnamoyl or heteroarylacryloyl groups were pre-
pared and evaluated for in vitro anticellular activity
against HeLa S3 cells and in vivo antitumor activity
against sarcoma 180 murine solid tumor and St-4
human stomach tumor xenograft. In addition, the sta-
bility of the 8-O-substituted analogues in aqueous
solution and the conversion to their active form (cyclo-
propane compound) from the 8-O-substituted analo-
gues in mice or human serum were examined. Some
showed remarkably potent in vivo antitumor activity
and low peripheral blood toxicity compared with deri-
vatives bearing the trimethoxyindole skeleton in Seg-B,
which were equal to 8-O-[(N-methylpiperazinyl)car-
bonyl] derivatives of 40-methoxycinnamates and 40-
methoxy-b-heteroarylacrylates. Moreover, among 8-O-
substituted analogues, several compounds can be che-
mically or enzymatically converted to their active form
in human serum. This result indicated that new 8-O-
substituted derivatives were di€erent prodrugs from
KW-2189 and 8-O-substituted analogues being the same
type of prodrug as KW-2189.
Scheme 3. (a) (1) HBr, CH3CN, (2) 4-nitrophenyl chloroformate,
Et3N, CH2Cl2, (3) The free base of R1H; (b) HBr.
Compounds 2b (KW-2189), 6, 13, 19, 20, 25 were trea-
ted with mice or human serum, respectively. The results
indicated that compounds 6, 13, 19, 20, 25 released the
corresponding cyclopropane compounds in mice and
human serum, which were di€erent from KW-2189 (2b).
Enzymatic conversion ratio of these analogues in mice
serum was nearly equal. In contrast, the enzymatic
conversion ratio of these analogues in human serum
was di€erent, and the order of conversion was
6>19>20>13ꢁ25. No conversion to cyclopropane
compound (DU-86, 2a) from KW-2189 (2b) in human
serum was observed at all. These results indicated that
new 8-O-substituted analogues were di€erent prodrugs
from KW-2189.
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. A compound 2c having the
same 8-O-substituent as KW-2189 (2b) showed weak
anticellular activity, about 1000 times inferior to that of
4a (1 h exposure), which was the same tendency that
showed KW-2189 (2b) was less potent than 2a (DU-86).
On the other hand, it was assumed that new 8-O-sub-
stituted analogues, of which the IC50 values at 1 h
exposure were equal to that of the corresponding active
form, converted rapidly to cyclopropane compounds in
the culture medium. Moreover, all of new 8-O-sub-
stituted analogues exhibited potent antitumor activity in
vivo. This result also indicated that these compounds
were chemically or enzymatically converted to their
active form in vivo. Furthermore, most of the deriva-
tives showed lower peripheral blood toxicity (reduction
of the number of peripheral blood platelets) than that of
Experimental
Infrared spectra (IR) were recorded on a JASCO IR-810
spectrometer. 1H NMR spectra were measured on
JEOL JNM-EX270 spectrometer. Mass spectra were
measured with JEOL JMS-DX303 and SHIMAZU QP-
1000 spectrometers. Elemental analyses were performed
with a Perkin±Elmer 2400 C, H, N analyzer. For col-
umn 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
.
40-Methoxycinnamoyl 8-O-methylthiocarbonyl-A-ring
pyrrole-duocarmycin B2 (5). Hydrobromic acid (48%,
0.0054 mL) was added to a solution of 4a (10 mg,
0.024 mmol) in CH3CN (0.61 mL), and the mixture was
stirred at room temperature for 40 min. The mixture
was poured into 1 N HBr, and the whole was extracted
with CHCl3. The combined organic extracts were
washed with brine and concentrated in vacuo. Methyl

N. Amishiro et al. / Bioorg. Med. Chem. 8 (2000) 381±391
385
Table 1. Anticellular activity, antitumor activity, hematotoxicity, stability, and water solubility of duocarmycin derivatives
No
HeLa S3 IC50 (nM)a
Sarcoma 180 (s.c.±i.v.)b
Hematotoxicity
WBCd (%) PLe (%)
Stability t1/2 in
aq. soln.f (h)
Water solubility
(mg/mL)
1 h
72 h
0.43
0.54
0.33
0.40
0.38
2.7
Dose (mg/kg)
T/Cc
5
6
7
8
5.5
3.2
5.8
7.7
3.9
1
1
2
2
4
2
2
2
2
1
1
2
2
4
8
8
2
4
4
4
2
0.36
0.20
0.17
0.33
0.17
0.21
0.13
0.31
0.21
0.25
0.28
0.21
0.28
0.35
0.21
0.10
0.22
0.14
0.15
0.15
0.07
49
93
22i
N.T.j
8i
4i
1i
22
35
4
30
N.T.
N.T.
N.T.
N.T.
N.T.
<1i
<1i
12i
15i
N.T.
N.T.
N.T.
<0.1
>10
N.T.
<0.1
N.T.
N.T.
N.T.
N.T.
<0.1
>10
>10
>10
N.T.
<0.1
0.3
27
28
21
19
29
21
22
25
32
33
19
28
17
30
22
30
22
20
41
20
84
79
82
68
83
79
82
85
69
75
29
85
56 (2)g
97
61
89
70
83
76
69
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
52
770
91
11
5.5
0.43
0.83
1.3
9.3
7.6
7.9
0.43
1.1
59
0.52
1.0
0.91
0.8
0.94
1.5
1.6
11
12
11
15
18
14
12
23
3
N.T.
N.T.
>10
>10
>10
0.94
1.2
2a
2b
2c
4a
4b
4c
0.045
53
1800
0.0052
1.6
37
0.26±0.94
0.92
0.90
0.25
0.63
4
0.83
0.50
2
0.21
0.15
0.20
0.34
0.22
0.45
22
24
25
50
41
28
38
10
44h
63
86
86
N.T.
N.T.
23
N.T.
N.T.
N.T.
N.T.
>10
<0.1
N.T.
N.T.
2.9±7.0
2.5
2.5
N.T.
aDrug concentration required to inhibit the growth of HeLa S3 cells by 50%.
bMice (5 mice/group) were implanted subcutaneously (s.c.) with tumor cells, and the drug was dosed (mg/kg) intravenously (i.v.).
cT and C are the values of the mean of tumor volume of treated and control mice, respectively.
dNumber of white blood cells of tumor-bearing mice on day 4 (percentage of control).
eNumber of peripheral platelets of nomial mice on day 7 (percentage of control).
fA half-life at 35 ꢀC. Drug concentration was 0.05 mg/mL. See Experimental.
gMortality (5 mice/group).
hDose of 5.33 mg/kg.
iCyclopropane compound was detected as a main decomposition product.
jNot tested.
Table 2. Antitumor activity against human xenografted solid tumor
CHCl3, and washed with aqueous NaHCO3 and brine.
The organic extracts were concentrated in vacuo. The
residue was puri®ed by PTLC (CHCl3:CH3OH, 15:1) to
No.
St-4 (s.c.±i.v.)a
1
T/Cb
0.31
0.23
0.34
0.24 (1)c
0.12
0.11
0.19
0.17
give 12 mg (84%) of 5: H NMR (270 MHz, CDCl3): d
Dose (mg/kg)
8.80 (1 H, s), 8.37 (1 H, brs), 7.80 (1H, d, J=15.2 Hz),
7.56 (2 H, d, J=8.6 Hz), 6.93 (2H, d, J=8.9 Hz), 6.79
(1H, d, J=15.2 Hz), 4.50±4.59 (1H, m), 4.47 (1H, d,
J=10.6 Hz), 4.30 (1H, dd, J=9.2, 9.2 Hz), 3.95 (3H, s),
3.85 (3H, s), 3.79 (1H, dd, J=10.2, 3.4 Hz), 3.22 (1H,
dd, J=10.2, 10.2 Hz), 2.68 (3H, s), 2.44 (3H, s). IR
(KBr): 1701, 1610, 1506, 1404, 1294, 1265, 1234, 1196,
1176, 1111 cm 1. FABMS: m/z 575, 573 (M+H)+.
FAB-HRMS calcd for C26H2679BrN2O6S (M+H)+ m/z
573.0695, found 573.0703. Anal. calcd for C26H25Br
6
2.3
4.0
2.4
2.7
18
12
12
12
13
15
16
19
20
23
24
25
2.7
0.44
2b
2c
0.63
12
0.12
0.15
.
N2O6S 0.5 H2O: C, 53.61; H, 4.50; N, 4.81; found C,
53.75; H, 4.50; N, 4.51.
aMice (5 mice/group) were implanted subcutaneously (s.c.) with tumor
cells, and the drug was dosed (mg/kg) intravenously (i.v.).
bT and C are the values of the mean of tumor volume of treated and
control mice, respectively.
30-(Dimethylamino)-40-methoxycinnamoyl 8-O-acetyl-A-
ring pyrrole-duocarmycin B2 hydrochoride (6). Hydro-
bromic acid (48%, 0.017 mL) was added to a solution of
4b (23 mg, 0.050 mmol) in CH3CN (2 mL), and the
mixture was stirred at room temperature for 40 min.
The reaction mixture was concentrated in vacuo. Acetic
anhydride (0.015 mL, 0.16 mmol) and DMAP (20 mg,
0.16 mmol) were added to a stirred solution of the resi-
due in dry CH2Cl2 (2 mL) at 0 ꢀC. Then, the resulting
cMortality (5 mice/group).
chlorothiolformate (0.0064 mL, 0.074 mmol) and tri-
ethylamine (0.010 mL, 0.072 mmol) were added to a
stirred solution of the residue in dry CH2Cl2 (1.1 mL) at
78 ꢀC. Then, the resulting mixture was stirred at
78 ꢀC for 1 h 20 min. The mixture was diluted with

386
N. Amishiro et al. / Bioorg. Med. Chem. 8 (2000) 381±391
Table 3. In vitro metabolism of duocarmycin derivatives in serum
(M+H)+ m/z 631.0750, found 631.0725. Anal. calcd
.
for C28H27BrN2O8S 0.4 CHCl3: C, 50.22; H, 4.06; N,
4.12; found C, 50.07; H, 4.26; N, 3.95.
Compound
Metabolite
Enzymatic conversion
(non-enzymatic conversion)a
40-Methoxycinnamoyl 8-O-(pyridine-2-carbonyl)-A-ring
pyrrole-duocarmycin B2 hydrochloride (8). Hydrobromic
acid (48%, 0.011 mL) was added to a solution of 4a
(20 mg, 0.048 mmol) in CH3CN (1.2 mL), and the mix-
ture was stirred at room temperature for 50 min. The
mixture was poured into 1 N HBr, and the whole was
extracted with CHCl3. The combined organic extracts
were washed with brine and concentrated in vacuo.
DCC (30 mg, 0.14 mmol), DMAP (18 mg, 0.14 mmol)
and nicotinic acid (18 mg, 0.14 mmol) were added to a
stirred solution of the residue in dry CH2Cl2 (1.1 mL) at
20 ꢀC. Then, the resulting mixture was stirred at room
temperature for 18 h 30 min. The mixture was diluted
with CHCl3, and washed with aqueous NaHCO3 and
brine. The organic extracts were concentrated in vacuo.
The residue was puri®ed by PTLC (CHCl3:CH3OH,
30:1) to give 19 mg (65%) of the free base of 8. A solu-
tion of the free base of 8 (20 mg, 0.033 mmol) in AcOEt
(1.6 mL) was treated with anhydrous 6.86 N HCl in
ethanol (0.0096 mL) at room temperature for 3 h. The
mixture concentrated under reduced pressure to give
Mouse serum
Human serum
6
4b
4a
4a
4a
4b
2a
122 (23)
118 (4)
114 (31)
14 (4)
13
19
20
25
2b
116 (
108 (
)
)
89 (
27 (
)
)
102 (2)
98 (
2 (2)
± (
)
)
apmol/30 min/mg protein.
mixture was stirred at 0 ꢀC for 10 min. The mixture was
diluted with CHCl3, and washed with 0.01 M phosphate
bu€er (pH 7) and brine. The organic extracts were con-
centrated in vacuo. The residue was puri®ed by PTLC
(CHCl3:CH3OH, 15:1) to give 28 mg (95%) of the free
base of 6. A solution of the free base of 6 (20 mg,
0.035 mmol) in AcOEt (1.7 mL) was treated with anhy-
drous 6.86 N HCl in ethanol (0.010mL) at room tem-
perature for 3 h. The mixture concentrated under reduced
pressure to give 25 mg of 6: 1H NMR (270 MHz,
DMSO-d6): d 12.07 (1H, s), 8.06 (1H, brs), 7.95 (1H,
br), 7.80±7.83 (1H, br), 7.63 (1H, d, J=15.5 Hz), 7.27
(1H, d, J=8.6 Hz), 7.15 (1H, d, J=15.2 Hz), 4.43±4.53
(3H, br), 3.98 (3H, s), 3.85 (3H, s), 3.79 (1H, br), 3.09
1
20 mg of 8: H NMR (270 MHz, DMSO-d6): d 12.14
(1H, brs), 9.37 (1H, s), 8.96 (1H, brd, J=4.0 Hz), 8.58
(1H, dt, J=7.9, 2.0 Hz), 8.23 (1H, s), 7.75 (2H, d,
J=8.6 Hz), 7.71±7.74 (1H, m), 7.60 (1 H, d, J=15.5 Hz),
7.07 (1H, d, J=15.5 Hz), 7.00 (2H, d, J=8.6 Hz), 4.41±
4.59 (3H, m), 3.86 (4H, s), 3.81 (3H, s), 2.64 (3H, s). IR
(KBr): 1697, 1645, 1601, 1512, 1408, 1281, 1252, 1217,
1174, 1097 cm 1. FABMS (the free base): m/z 606, 604
(M+H)+. FAB-HRMS (the free base) calcd for
C30H2779BrN3O6 (M+H)+ m/z 604.1083, found
(6H, s), 2.66 (3H, s), 2.39 (3 H, s). IR (KBr): 1759, 1693,
1
1651, 1514, 1437, 1414, 1277, 1203, 1090, 1014 cm
.
FABMS (the free base): m/z 586, 584 (M+H)+. FAB-
HRMS (the free base) calcd for C28H3179BrN3O6
(M+H)+ m/z 584.1396, found 584.1400. Anal. calcd
.
.
for C28H30BrN3O6 HCl 1.5 H2O: C, 51.90; H, 5.29; N,
6.49; found C, 51.73; H, 5.31; N, 6.02.
.
.
604.1069. Anal. calcd for C30H26BrN3O6 HCl 1.0 H2O: C,
54.68; H, 4.44; N, 6.38; found C, 54.39; H, 4.44; N, 5.68.
40-Methoxycinnamoyl 8-O-carboxylmethylsulfanylacetyl-
A-ring pyrrole-duocarmycin B2 (7). Hydrobromic acid
(48%, 0.034 mL) was added to a solution of 4a (25 mg,
0.060 mmol) in CH3CN (1.5 mL), and the mixture was
stirred at room temperature for 1 h. The mixture was
poured into 1 N HBr, and the whole was extracted with
CHCl3. The combined organic extracts were washed
with brine and concentrated in vacuo. Thioglycolic
anhydride (25 mg, 0.19 mmol) and DMAP (23 mg,
0.19 mmol) were added to a stirred solution of the resi-
due in dry CH2Cl2 (1.5 mL) at 0 ꢀC. The resulting mix-
ture was stirred at 0 ꢀC for 3 h. 1 N HBr was added to
the reaction mixture. The mixture was diluted with
CHCl3, and washed with saturated NaHCO3 and brine.
Then, the organic extracts were concentrated in vacuo.
The residue was puri®ed by PTLC (CHCl3:CH3OH:
AcOH, 4:1:0.1) to give 26 mg (69%) of 7: 1H NMR
(270 MHz, CDCl3): d 10.40 (1H, brs), 8.02 (1H, s), 7.66
(1H, d, J=15.2 Hz), 7.49 (2H, d, J=8.3 Hz), 6.88 (2H,
d, J=8.3 Hz), 6.63 (1H, d, J=15.5 Hz), 4.36±4.46 (1H,
m), 4.29 (1H, d, J=10.6 Hz), 4.11 (1H, dd, J=9.9,
9.2 Hz), 3.89 (3H, s), 3.82 (3H, s), 3.64 (1H, dd, J=9.9,
2.3 Hz), 3.59 (2H, s), 3.40 (1H, d, J=14.8 Hz), 3.33
(1H, d, J=14.8 Hz), 3.07 (1H, dd, J=10.2, 9.9 Hz), 2.50
(3H, s). IR (KBr): 1697, 1637, 1603, 1512, 1437, 1416,
1252, 1219, 1174, 1105 cm 1. FABMS: m/z 633, 631
(M+H)+; FAB-HRMS calcd for C28H2879BrN2O8S
40-Methoxycinnamoyl 8-O-(3-amino-pyrazine-2-carbon-
yl)-A-ring pyrrole-duocarmycin B2 (9). Yield 64%; H
1
NMR (270 MHz, CDCl3+CD3OD): d 8.54 (1H, s), 8.29
(1H, d, J=2.0 Hz), 7.92 (1H, d, J=2.0 Hz), 7.73 (1H, d,
J=15.5 Hz), 7.53 (2H, d, J=8.6 Hz), 6.89 (2H, d,
J=8.9 Hz), 6.76 (1H, d, J=14.8 Hz), 4.48±4.59 (1H, m),
4.44 (1H, d, J=10.2 Hz), 4.29 (1H, dd, J=9.9, 8.6 Hz),
3.91 (3H, s), 3.81 (3H, s), 3.79 (1H, dd, J=8.6, 2.0 Hz),
3.21 (1H, dd, J=10.2, 9.9 Hz), 2.65 (3H, s). IR (KBr):
1716, 1697, 1647, 1597, 1512, 1408, 1296, 1248, 1174,
1092 cm 1. FABMS: m/z 622, 620 (M+H)+. FAB-
HRMS calcd for C29H2779BrN5O6 (M+H)+ m/z
620.1145, found 620.1138. Anal. calcd for C29H26
.
.
BrN5O6 1.0 H2O 0.6 CHCl3: C, 50.07; H, 4.06; N, 9.86;
found C, 50.38; H, 4.06; N, 9.61.
40-Methoxycinnamoyl 8-O-(3,4-diaminobenzoyl)-A-ring
pyrrole-duocarmycin B2 (10). Yield 36%; 1H NMR
(270 MHz, DMSO-d6): d 12.06 (1H, brs), 8.07 (1H, brs),
7.75 (2H, d, J=8.6 Hz), 7.59 (1H, d, J=15.5 Hz), 7.37
(1H, s), 7.35 (1H, dd, J=8.2, 2.0 Hz), 7.06 (1H, d,
J=15.5 Hz), 6.99 (2H, d, J=8.6 Hz), 6.63 (1H, d,
J=7.9 Hz), 5.51 (2H, brs), 4.82 (2H, br), 4.39±4.57 (3H,
m), 3.85 (3H, s), 3.83 (1H, brd, J=11.2 Hz), 3.81 (3H,
s), 3.45 (1H, dd, J=8.9, 8.9 Hz), 2.62 (3H, s). IR (KBr):

N. Amishiro et al. / Bioorg. Med. Chem. 8 (2000) 381±391
387
1
1701, 1693, 1645, 1593, 1512, 1410, 1306, 1250, 1205,
1092 cm 1. FABMS m/z 635, 633 (M+H)+. FAB-
HRMS calcd for C31H3079BrN4O6 (M+H)+ m/z
633.1349, found 633.1363. Anal. calcd for C31H29
BrN4O6 1.5 H2O: C, 56.37; H, 4.88; N, 8.48; found C,
56.07; H, 4.80; N, 8.09.
B2 hydrobromide (14). Yield 50%; H NMR (270 MHz,
DMSO-d6): d 12.15 (1H, brs), 9.49 (1H, br), 9.08 (2H,
s), 8.22 (2H, d, J=8.3 Hz), 8.16 (1H, s), 7.63 (2H, d,
J=8.3 Hz), 7.60 (1H, d, J=15.2 Hz), 7.37 (1H, d,
J=15.8 Hz), 4.41±4.50 (3H, m), 3.97 (3H, s), 3.86 (3H,
s), 3.82 (1H, brd, J=9.4 Hz), 2.95±3.20 (3H, br), 2.82
(3H, s), 2.63 (3H, s). IR (KBr): 1736, 1697, 1653, 1593,
1475, 1435, 1412, 1338, 1265, 1219, 1092 cm 1. FABMS
(the free base): m/z 719, 717 (M+H)+; FAB-HRMS
(the free base) calcd for C35H3879BrN6O6 (M+H)+
m/z 717.2036, found 717.2063. Anal. calcd for
.
40-Methoxycinnamoyl 8-O-(4-dimethylaminobenzoyl)-A-
1
ring pyrrole-duocarmycin B2 (11). Yield 58%; H NMR
(270 MHz, CDCl3): d 9.44 (1H, brs), 8.28 (1H, brs), 7.92
(2H, d, J=8.9 Hz), 7.75 (1H, d, J=15.2 Hz), 7.54 (2H,
d, J=8.6 Hz), 6.92 (2H, d, J=8.2 Hz), 6.77 (1H, d,
J=15.5 Hz), 6.51 (2H, d, J=8.6 Hz), 4.47±4.58 (1H, m),
4.43 (1H, d, J=10.2 Hz), 4.25 (1H, dd, J=9.2, 8.2 Hz),
3.94 (3H, s), 3.84 (3H, s), 3.82 (1H, brd, J=10.6 Hz),
3.19 (1H, dd, J=10.2, 9.9 Hz), 3.01 (6H, s), 2.50 (3H, s);
R (KBr): 1697, 1647, 1603, 1512, 1406, 1267, 1174,
1155, 1088 cm 1. FABMS m/z 648, 646 (M+H)+.
FAB-HRMS calcd for C33H3379BrN3O6 (M+H)+ m/z
646.1553, found 646.1541. Anal. calcd for C33H32
.
.
C35H37BrN6O6 2 HBr 1.0 H2O: C, 46.84; H, 4.60; N,
9.36; found C, 47.05; H, 5.10; N, 9.20.
30-Dimethylamino-40-methoxycinnamoyl 8-O-(pyridine-2-
carbonyl)-A-ring pyrrole-duocarmycin B2 hydrobromide
(15). Yield 84%; 1H NMR (270 MHz, DMSO-d6): d
12.16 (1H, s), 9.37 (1H, s), 8.96 (1H, d, J=4.3 Hz), 8.57
(1H, dt, J=7.9, 2.0 Hz), 8.23 (1H, brs), 8.12 (1H, brs),
7.93 (1H, d, J=8.3 Hz), 7.73 (1H, dd, J=7.9, 5.0 Hz),
7.65 (1H, d, J=15.2 Hz), 7.35 (1H, d, J=8.9 Hz), 7.22
(1H, d, J=15.2 Hz), 4.47±4.60 (3H, br), 4.02 (3H, s),
3.86 (3H, s), 3.84 (1H, dd, J=10.6, 2.6 Hz), 3.51 (1H,
dd, J=9.6, 8.6 Hz), 3.18 (6H, s), 2.64 (3H, s). IR (KBr):
1686, 1647, 1516, 1466, 1458, 1437, 1414, 1279, 1219,
1097 cm 1. FABMS (the free base): m/z 649, 647
(M+H)+; FAB-HRMS (the free base) calcd for C32
H3279BrN4O6 (M+H)+ m/z 647.1505, found 647.1526.
.
BrN3O6 1.5 H2O: C, 58.84; H, 5.25; N, 6.24; found C,
59.19; H, 4.98; N, 5.92.
40-Methoxycinnamoyl 8-O-(3-dimethylaminobenzoyl)-A-
1
ring pyrrole-duocarmycin B2 (12). Yield 51%; H NMR
(270 MHz, CDCl3): d 9.57 (1H, brs), 8.30 (1H, brs), 7.73
(1H, d, J=15.2 Hz), 7.53 (2H, d, J=8.6 Hz), 7.42 (1H,
d, J=7.9 Hz), 7.34 (1H, brs), 7.18 (1H, dd, J=7.9,
7.9 Hz), 6.92 (2 H, d, J=8.6 Hz), 6.83 (1H, dd, J=7.9,
2.6 Hz), 6.72 (1H, d, J=15.2 Hz), 4.45±4.58 (1H, m),
4.40 (1H, d, J=10.2 Hz), 4.25 (1H, dd, J=9.2, 8.2 Hz),
3.95 (3H, s), 3.84 (3H, s), 3.80 (1H, dd, J=9.6, 2.0 Hz),
3.18 (1H, dd, J=10.2, 9.9 Hz), 2.87 (6H, s), 2.56 (3H, s).
. .
.
Anal. calcd for C32H31BrN4O6 2 HBr 1.0 H2O: C,
46.46; H, 4.26; N, 6.77; found C, 46.31; H, 4.51; N, 6.58.
30-Dimethylamino-40-methoxycinnamoyl 8-O-[4-(4-methyl-
1-piperazinylmethyl)benzoyl]-A-ring pyrrole-duocarmycin
1
IR (KBr): 1734, 1697, 1653, 1603, 1512, 1458, 1406,
1
B2 hydrobromide (16). Yield 61%; H NMR (270 MHz,
1250, 1173, 1093 cm
.
FABMS: m/z 648, 646
DMSO-d6): d 12.14 (1H, s), 9.51 (1H, br), 8.21 (2H, d,
J=8.3 Hz), 8.19 (1H, d, J=15.2 Hz), 7.75±7.89 (2H, br),
7.63 (2H, d, J=8.9 Hz), 7.60 (1H, d, J=5.9 Hz), 7.24
(1H, d, J=8.6 Hz), 7.16 (1H, d, J=15.5 Hz), 4.45±4.58
(3H, br), 3.97 (3H, s), 3.86 (3H, s), 3.83 (1H, brd,
J=9.7 Hz), 3.06 (8H, br), 2.81 (3H, s), 2.63 (3H, s). IR
(KBr): 1734, 1695, 1647, 1616, 1516, 1437, 1412, 1263,
1217, 1092, 1018 cm 1. FABMS (the free base): m/z
760, 758 (M+H)+; FAB-HRMS (the free base) calcd
for C39H4579BrN5O6 (M+H)+ m/z 758.2553, found
(M+H)+; FAB-HRMS calcd for C33H3379BrN3O6
(M+H)+ m/z 646.1553, found 646.1523. Anal. calcd
.
for C33H32BrN3O6 1.0 H2O: C, 59.64; H, 5.16; N, 6.32;
found C, 59.68; H, 4.91; N, 6.21.
40-Methoxycinnamoyl 8-O-[4-(4-methyl-1-piperazinyl-
methyl)benzoyl]-A-ring pyrrole-duocarmycin B2 hydro-
bromide (13). The free base of 13: yield 61%; a solution
of the free base of 13 (13 mg, 0.018 mmol) in AcOEt
(1.2 mL) was treated with 5% HBr in methanol (172 mg)
at room temperature for 1 h 30 min. The mixture con-
centrated under reduced pressure to give 16 mg of 13:
1H NMR (270 MHz, DMSO-d6): d 12.14 (1H, s), 9.40±
9.80 (1H, br), 8.24 (2H, d, J=8.3 Hz), 8.17 (1H, s), 7.76
(2H, d, J=8.6 Hz), 7.67 (2H, d, J=7.3 Hz), 7.59 (1H, d,
J=15.5 Hz), 7.08 (1H, d, J=15.2 Hz), 7.00 (2H, d,
J=8.6 Hz), 4.44±4.54 (3H, m), 3.86 (3H, s), 3.81 (3H, s),
3.49 (2H, d, J=6.6 Hz), 3.06±3.21 (3H, m), 2.84 (3H, s),
2.63 (3H, s). IR (KBr): 1734, 1697, 1653, 1601, 1512,
1437, 1412, 1252, 1217, 1174, 1093 cm 1. FABMS (the
free base): m/z 717, 715 (M+H)+; FAB-HRMS (the
free base) calcd for C37H4079BrN4O6 (M+H)+ m/z
715.2131, found 715.2126. Anal. calcd for C37H39
.
.
758.2527. Anal. calcd for C39H44BrN5O6 2 HBr 5.5
H2O: C, 45.94; H, 5.63; N, 6.87; found C, 45.92; H,
5.24; N, 6.77.
40-Methoxycinnamoyl 8-O-[4-[(tert-butoxycarbonyl)amino-
methyl]benzoyl]-A-ring pyrrole-duocarmycin B2 (17).
Yield 54%; H NMR (270 MHz, CDCl3): d 9.44±9.76
(1H, brs), 8.30 (1H, brs), 8.00 (2H, d, J=7.3 Hz), 7.75
(1H, d, J=15.2 Hz), 7.55 (2H, d, J=8.6 Hz), 7.22 (2H,
d, J=7.3 Hz), 6.92 (2H, d, J=8.9 Hz), 6.76 (1H, d,
J=15.2 Hz), 5.25 (1H, brs), 4.47±4.59 (1H, m), 4.43
(1H, d, J=10.9 Hz), 4.21±4.38 (3H, m), 3.94 (3H, s),
3.84 (3H, s), 3.81 (1H, brd, J=10.9 Hz), 3.20 (1H, dd,
1
J=10.2, 10.2 Hz), 1.45 (9H, s). IR(KBr): 1705, 1695,
1
.
.
BrN4O6 HBr 3.5 H2O: C, 51.70; H, 5.51; N, 6.52; found
C, 51.66; H, 5.19; N, 6.32.
1645, 1512, 1410, 1261, 1217, 1173, 1090, 1016 cm
.
FABMS: m/z 734, 732 (M+H)+. FAB-HRMS calcd
for C37H3979BrN3O8 (M+H)+ m/z 732.1921, found,
ꢀ-(40-Methoxy-30,50-pyrimidinyl)acryloyl 8-O-[4-(4-methyl-
1-piperazinylmethyl)benzoyl]-A-ring pyrrole-duocarmycin
732.1920. Anal. calcd for C37H39BrN3O8 1.5 H2O: C,
58.50; H, 5.44; N, 5.53; found C, 58.34; H, 5.69; N, 4.97.
.

388
N. Amishiro et al. / Bioorg. Med. Chem. 8 (2000) 381±391
40-Methoxycinnamoyl
8-O-(4-aminomethylbenzoyl)-A-
bromide (20). Yield 43%; H NMR (270 MHz, DMSO-
d6): d 12.04 (1H, brs), 9.25 (1H, s), 9.00 (1H, br), 8.02
(1H, s), 7.75 (2H, d, J=8.6 Hz), 7.59 (1H, d, J=15.2 Hz),
7.06 (1H, d, J=15.2 Hz), 7.00 (2H, d, J=8.9 Hz), 4.36±
4.53 (3H, m), 3.85 (3H, s), 3.82 (3H, s), 3.78 (1H, m),
3.17±3.27 (2H, m), 2.86±3.04 (1H, m), 2.69±2.79 (2H,
m), 2.65 (3H, s), 1.99±2.07 (2H, br), 1.60±1.91 (9H, m),
1.34±1.49 (2H, m). IR (KBr): 1749, 1695, 1645, 1601,
1512, 1456, 1412, 1250, 1217, 1174 cm 1. FABMS (the
free base): m/z 710, 708 (M+H)+. Anal. calcd for
1
ring pyrrole-duocarmycin B2 hydrobromide (18). A solu-
tion of 17 (65 mg, 0.072 mmol) in ClCH2CH2Cl (2 mL)
was treated with 5% HBr in methanol (1.45 g) at 60 ꢀC
for 5 h 45 min. Aqueous NaHCO3 was added to the
reaction mixture. The mixture was extracted with
CHCl3, and washed with saturated NaHCO3 and brine.
Then, the organic layer was dried over Na2SO4 and
concentrated in vacuo. The residue was puri®ed by
PTLC (CHCl3:CH3OH, 4:1) and column chromato-
graphy (CHCl3:CH3OH, 10:1) to give 15 mg (33%) of
the free base of 18. The obtained free base (11 mg) was
employed in the same procedure as that of preparation
.
.
C35H42BrN5O6 2HBr 0.3 H2O: C, 48.00; H, 5.13; N,
8.00; found C, 48.16; H, 5.83; N, 7.83.
40-Methoxycinnamoyl 8-O-(2-methylcarbazoyl)-A-ring
pyrrole-duocarmycin B2 (21). Yield 71%; 1H NMR
(270 MHz, CDCl3): d 10.21 and 9.81 (1H, brs), 8.24
(1H, brs), 7.81 (1H, d, J=15.2 Hz), 7.57 (2H, d, J=
8.3 Hz), 6.93 (2H, d, J=8.6 Hz), 6.82 (1H, d, J=
15.5 Hz), 4.47±4.55 (1H, br), 4.46 (1H, d, J=10.2 Hz),
4.33 (1H, dd, J=9.2, 8.9 Hz), 3.94 (3H, s), 3.85 (3H, s),
3.77 (1H, dd, J=9.4, 2.1 Hz), 3.29 (3H, brs), 3.19 (1H,
1
of HBr salt to give 15 mg of 18: H NMR (270 MHz,
DMSO-d6): d 12.15 (1H, brs), 8.32 (2H, br), 8.27 (2H, d,
J=8.2 Hz), 8.18 (1H, brs), 7.75 (2H, d, J=8.3 Hz), 7.72
(2H, d, J=7.6 Hz), 7.58 (1H, d, J=15.5 Hz), 7.08 (1H, d,
J=15.2 Hz), 7.00 (2H, d, J=8.6Hz), 4.50±4.60 (1H, m),
4.41±4.48 (2H, br), 4.21±4.23 (2H, m), 3.86 (3H, s), 3.83
(1H, br), 3.81 (3H, s), 2.63 (3H, s). IR (KBr): 1734, 1697,
1
1635, 1601, 1514, 1437, 1417, 1259, 1174, 1093 cm
.
FABMS (the free base): m/z 634, 632 (M+H)+. FAB-
HRMS (the free base) calcd for C32H3179BrN3O6
(M+H)+ m/z 632.1428, found, 632.1426. Anal. calcd
dd, J=9.9, 9.9 Hz), 2.32 (3H, s). IR (KBr): 1697, 1647,
1
1599, 1512, 1433, 1410, 1248, 1217, 1173, 1111 cm
.
FABMS (the free base): m/z 573, 571 (M+H)+. FAB-
HRMS calcd for C26H2879BrN4O6 (M+H)+ m/z
571.1192, found 571.1192. Anal. calcd for C26H27
.
.
for C32H30BrN3O6 HBr 7.0 H2O: C, 45.78; H, 5.40; N,
5.01; found C, 45.81; H, 4.45; N, 4.91.
.
BrN4O6 1.0 H2O: C, 52.98; H, 4.96; N, 9.51; found C,
52.74; H, 4.78; N, 9.33.
40-Methoxycinnamoyl 8-O-[(4-methyl-1-piperazinyl)amino]-
carbonyl-A-ring pyrrole-duocarmycin B2 hydrobromide
(19). Hydrobromic acid (48%, 0.041 mL) was added to
a solution of 4a (30 mg, 0.072 mmol) in CH3CN
(1.7 mL), and the mixture was stirred at room tempera-
ture for 2 h. The mixture was poured into 1N HBr, and
the whole was extracted with CHCl3. The combined
organic extracts were washed with brine and con-
centrated in vacuo. p-Nitrophenyl chloroformate
(43 mg, 0.22 mmol) and triethylamine (0.030 mL,
0.22 mmol) were added to a stirred solution of the resi-
due in dry CH2Cl2 (1.7 mL) at 78 ꢀC. Then, the result-
ing mixture was stirred at 78 ꢀC for 40 min. 1-Amino-
4-methylpiperazine (0.043 mL, 0.36 mmol) was added to
the solution, and the mixture was stirred at 0 ꢀC for 24h.
The mixture was diluted with CHCl3, and washed with
aqueous NaHCO3 and brine. The organic extracts were
concentrated in vacuo. The residue was puri®ed by
PTLC (CHCl3:CH3OH, 9:1) to give 20.4 mg (44%) of
the free base of 19. The obtained free base (20 mg) was
employed in the same procedure as that of preparation
40-Methoxycinnamoyl 8-O-(2,3-dimethylcarbazoyl)-A-ring
pyrrole-duocarmycin B2 (22). Yield 46%; 1H NMR
(270 MHz, CDCl3): d 9.65 (1H, brs), 9.39 (1H, brs), 8.24
(1H, s), 7.80 (1H, d, J=15.2 Hz), 7.57 (2H, d, J=
8.9 Hz), 6.94 (2H, d, J=8.9 Hz), 6.81 (1H, d, J=
15.5 Hz), 4.50±4.57 (1H, m), 4.46 (1H, d, J=10.2 Hz),
4.31 (1H, dd, J=9.6, 8.9 Hz), 3.94 (3H, s), 3.86 (3 H, s),
3.78 (1H, dd, J=9.7, 2.5 Hz), 3.27 (3H, brs), 3.20 (1H,
dd, J=10.2, 9.9 Hz), 2.71 (3 H, brs), 2.44 (3 H, brs). IR
(KBr): 1716, 1697, 1686, 1647, 1601, 1512, 1410, 1252,
1219, 1173, 1159, 1109 cm 1. FABMS: m/z 587, 585
(M+H)+. FAB-HRMS calcd for C27H3079BrN4O6
(M+H)+ m/z 585.1349, found 585.1345. Anal. calcd
.
for C27H29BrN4O6 1.0 H2O: C, 53.74; H, 5.18; N, 9.28;
found C, 53.62; H, 4.90; N, 9.13.
ꢀ-(40-Methoxy-30,50-pyrimidinyl)acryloyl 8-O-[(4-methyl-
1-piperazinyl)amino]carbonyl-A-ring pyrrole-duocarmycin
1
B2 hydrobromide (23). Yield 31%; H NMR (270 MHz,
1
of HBr salt to give 23 mg of 19: H NMR (270 MHz,
DMSO-d6): d 12.09 (1H, brs), 9.58 (1H, brs), 9.48 (1H,
brs), 9.08 (2H, s), 8.04 (1H, s), 7.60 (1H, d, J=15.8 Hz),
7.37 (1H, d, J=15.5 Hz), 4.37±4.57 (2H, m), 4.47 (1H,
d, J=10.2 Hz), 3.97 (3H, s), 3.85 (3H, s), 3.79 (1H, brd,
J=9.6 Hz), 3.00±3.30 (7H, br), 2.80 (3H, s), 2.66 (3H, s).
IR (KBr): 1747, 1697, 1653, 1595, 1475, 1437, 1414,
1340, 1219, 1093 cm 1. FABMS (the free base): m/z
644, 642 (M + H)+. FAB-HRMS (the free base) calcd
for C28H3379BrN7O6 (M+H)+ m/z 642.1676, found
DMSO-d6): d 12.07 (1H, brs), 9.49 (2H, brs), 8.04 (1H,
brs), 7.76 (2H, d, J=8.3 Hz), 7.59 (1H, d, J=15.2 Hz),
7.07 (1H, d, J=15.5 Hz), 7.00 (2H, d, J=7.3 Hz), 4.38±
4.56 (3H, br), 3.85 (3H, s), 3.82 (3H, s), 3.77 (1H, br),
3.02±3.30 (5H, m), 2.82 (3H, s), 2.66 (3H, s). IR (KBr):
1697, 1647, 1601, 1512, 1437, 1412, 1250, 1217, 1174,
1093 cm 1. FABMS (the free base): m/z 642, 640
(M+H)+. FAB-HRMS (the free base) calcd for C30
H3579BrN5O6 (M+H)+ m/z 640.1771, found 640.1769.
.
.
642.1694. Anal. calcd for C28H32BrN7O6 HBr 4.0 H2O: C,
42.28; H, 5.19; N, 12.33; found C, 42.43; H, 5.19; N, 12.11.
.
.
Anal. calcd for C30H34BrN5O6 2HBr 1.0 H2O: C, 43.92;
H, 4.67; N, 8.54; found C, 44.01; H, 4.78; N, 8.45.
30-Dimethylamino-40-methoxycinnamoyl 8-O-[(4-methyl-
1-piperazinyl)amino]carbonyl-A-ring pyrrole-duocarmycin
40-Methoxycinnamoyl 8-O-[1-(4-piperidinopiperidinyl)-
amino]carbonyl-A-ring pyrrole-duocarmycin B2 hydro-
1
B2 hydrobromide (24). Yield 47%; H NMR (270 MHz,

N. Amishiro et al. / Bioorg. Med. Chem. 8 (2000) 381±391
389
DMSO-d6): d 12.09 (1H, s), 9.53 (1H, br), 9.48 (1H, s),
8.03 (2H, brs), 7.85 (1H, d, J=7.9 Hz), 7.63 (1H, d,
J=15.2 Hz), 7.31 (1H, d, J=8.3 Hz), 7.18 (1H, d,
J=15.2 Hz), 4.39±4.58 (3H, m), 4.00 (3H, s), 3.85 (3H,
s), 3.80 (1H, dd, J=9.4, 2.8 Hz), 3.45 (4H, br), 3.14 (6H,
s), 3.08±3.25 (5H, br), 2.82 (3H, d, J=4.3 Hz), 2.66 (3H,
s). IR (KBr): 1743, 1689, 1649, 1516, 1464, 1435, 1416,
1279, 1219, 1190, 1094 cm 1. FABMS (the free base):
m/z 685, 683 (M+H)+. FAB-HRMS (the free base)
calcd for C32H4079BrN6O6 (M+H)+ m/z 683.2192,
and control mice. The length and width of the tumors
were measured, and tumor volume was calculated as
tumor volume ꢀmm3† ˆ length ꢀmm†  ‰width ꢀmm†Š2=2
according to the method of the National Cancer Institute.59
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).50
.
found 683.2219. Anal. calcd for C32H39BrN6O6 2
.
HBr 3.0 H2O: C, 42.73; H, 5.27; N, 9.34; found C,
42.53; H, 5.32; N, 9.94.
30-Dimethylamino-40-methoxycinnamoyl 8-O-(2-methyl-
carbazoyl)-A-ring pyrrole-duocarmycin B2 hydrobromide
(25). Yield 74%; 1H NMR (270 MHz, DMSO-d6): d
12.02 (1H, brs), 8.12 (1H, brs), 7.97 (1H, brs), 7.83 (1H,
d, J=8.2 Hz), 7.63 (1H, d, J=14.8 Hz), 7.29 (1H, d,
J=8.6 Hz), 7.17 (1H, d, J=15.2 Hz), 4.40±4.58 (3H, br),
3.99 (3H, s), 3.85 (3H, s), 3.81 (1H, brd, J=10.0 Hz),
3.11 (6H, s), 2.67 (3H, s). IR (KBr): 1650, 1645, 1516,
1464, 1435, 1416, 1279, 1219, 1190, 1159, 1107,
1092 cm 1. FABMS (the free base): m/z 616, 614
(M+H)+; FAB-HRMS (the free base) calcd for
C28H3379BrN5O6 (M+H)+ m/z 614.1614, found
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 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 con-
trol (percent of control).
.
.
614.1596. Anal. calcd for C28H32BrN5O6 2HBr 1.0
H2O: C, 42.34; H, 4.57; N, 8.82; found C, 42.23; H,
4.87; N, 8.64.
Biological studies. Human uterine cervix carcinoma
HeLa S3 cells were obtained from American Type Cul-
ture Collection through Dainippon Pharmaceutical Co.
(Osaka, Japan). The cells (2Â104/well) were precultured
in the culture medium in 24-well multidishes (Nunc,
Roskilde, Denmark) for 24 h at 37 ꢀC in a humidi®ed
atmosphere of 5% CO2. For the pulse exposure experi-
ment, cells were treated with each compound for 1 h,
washed with Dulbecco'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.
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).
Stability of drug in aqueous solution
The stablilty of the 8-substituted derivatives under aque-
ous conditions was examined by chromatogaphy on a
UNISIL pack 5C18 reversed-phase HPLC column (GL
Science, Co., Ltd., Tokyo, Japan) or L-column ODS
4.6Â150 mm (Chemicals Inspection and Testing Insti-
tute, Tokyo, Japan). The compound (1 mg) was dis-
solved in DMF (10 mL). This solution (0.1 mL) was
diluted with aqueous solution (0.1 mL). The aqueous
solution was composed of 0.05 M phosphate bu€er
(pH 7). The resulting solution was incubated at 35 ꢀC.
Samples were removed at intervals and injected directly
into a HPLC injection port. The compound was eluted
with 0.05 M phosphate bu€er (pH 5.9): CH3CN (30:70)
and detected by measuring absorbance at 330 nm.
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 adults male
ddY mice. Human xenografts were passaged and used
in adult male BALB/c-nu/nu mice. Murine solid tumor
was inoculated subcutaneously (s.c.) at the axillary
region of mice. Human xenografts were inoculated s.c.
in the ¯ank of nude mice. Drugs were administered
intravenously (iv) beginning 1 day after tumor inocu-
lation. Antitumor ecacy is expressed as T/C, where T
and C are the values of mean tumor volume of treated
In vitro metabolism of duocarmycin derivatives in serum
Serum was prepared by evacuated serum collection
tubes (Venoject II, Terumo Co., Tokyo, Japan) from

390
N. Amishiro et al. / Bioorg. Med. Chem. 8 (2000) 381±391
the adult male BALB/c mice and the adult male human.
Then serum was diluted to 25% with Tris±HCl Bu€er
(20 mM, pH 7.4). After incubation of 25% serum
(198 mL) at 37 ꢀC for 3 min, drugs (2 mL, 200 mM) were
added to 25% serum in the dark. After 30 min, acetoni-
trile was added, and the mixture was centrifuged at
4 ꢀC for 5 min (15000 Â g) and ®ltered through an ultra-
free-C3-LG (Millipore Co., Bedford, MA, USA). The
®ltrate (100 mL) was analyzed by HPLC at 300 nm.
Contribution of non-enzymatic reaction was examined
by performing the same procedure after 25% serum was
boiled at 96 ꢀC for 5 min.
17. Boger, D. L.; Ishizaki, T.; Zarrinmayeh, H. J. Am. Chem.
Soc. 1991, 113, 6645.
18. Sugiyama, H.; Ohmori, K.; Chan, K. L.; Hosoda, M.;
Asai, A.; Saito, H.; Saito, I. Tetrahedron Lett. 1993, 34,
2179.
19. Boger, D. L.; Johnson, D. S.; Yun, W. J. Am. Chem. Soc.
1994, 116, 1635.
20. Hanka, L. J.; Dietz, A.; Gerpheide, S. A.; Kuentzel, S. L.;
Martin, D. G. J. Antibiot. 1978, 31, 1211.
21. Martin, D. G.; Chidester, C. G.; Duchamp, D. J.; Mizsak,
S. A. J. Antibiot. 1980, 33, 902.
22. Reynolds, V. L.; McGovren, J. P.; Hurley, L. H. J. Anti-
biot. 1986, 39, 319.
23. Hurley, L. H.; Reynolds, V. L.; Swenson, D. H.; Petzold,
G. L.; Scahill, T. A. Science 1984, 226, 843.
24. Reynolds, V. L.; Molineaux, I. J.; Kaplan, D. J.; Swensen,
D. H.; Hurley, L. H. Biochemistry 1985, 24, 6228.
25. Tang, M. S.; Lee, C. S.; Doisy, R.; Ross, L.; Needham-
VanDevanter, D. R.; Hurley, L. H. Biochemistry 1988, 27,
893.
26. Nagamura, S.; Asai, A.; Kanda, Y.; Kobayashi, E.; Gomi,
K.; Saito, H. Chem. Pharm. Bull. 1996, 44, 1723.
27. Kobayashi, E.; Okamoto, A.; Asada, M.; Okabe, M.;
Nagamura, S.; Asai, A.; Saito, H.; Gomi, K.; Hirata, T. Can-
cer Res. 1994, 54, 2404.
Acknowledgements
We express our gratitude to Mikiko Saito, and Etsuko
Koshimura for their excellent technical assistance. We
are grateful to Dr. Satoshi Kobayashi, Fumiko Oiji for
accurate suggestions and valuable discussions. We also
thank Mariko Yoshida, Atsuko Kobayashi and Hideko
Yokoyama for measuring elemental analyses and MS
spectra.
28. Asai, A.; Nagamura, S.; Saito, H. J. Am. Chem. Soc. 1994,
116, 4171.
29. Nagamura, S.; Kobayashi, E.; Gomi, K.; Saito, H. Bioorg.
Med. Chem. Lett. 1996, 6, 2147.
References and Notes
30. Nagamura, S.; Kanda, Y.; Kobayashi, E.; Gomi, K.;
Saito, H. Chem. Pharm. Bull. 1995, 43, 1530.
31. A direct relationship between solvolysis stability and in
vitro cytotoxic potency with the more sable agents exhibiting
the more potent activity is demonstrated.31±35 Boger, D. L.;
Ishizaki, T. Tetrahedron Lett. 1990, 31, 793.
32. Boger, D. L.; Mesini, P.; Tarby, C. M. J. Am. Chem. Soc.
1994, 116, 6461.
33. Boger, D. L.; McKie, J. A.; Han, N.; Taby, C. M.; Riggs,
H. W.; Kitos, P. A. Bioorg. Med. Chem. Lett. 1996, 6, 659.
34. Boger, D. L.; Goldberg, J.; McKie, J. A. Bioorg. Med.
Chem. Lett. 1996, 6, 1955.
1. Takahashi, I.; Takahashi, K.; Ichimura, M.; Morimoto, M.;
Asano, K.; Kawamoto, I.; Tomita, F.; Nakano, H. 1988, 41,
1915.
2. Yasuzawa, T.; Iida, T.; Muroi, K.; Ichimura, M.; Takaha-
shi, K.; Sano, H. Chem. Pharm. Bull. 1988, 36, 3728.
3. Ichimura, M.; Muroi, K.; Asano, K.; Kawamoto, I.;
Tomita, F.; Morimoto, M.; Nakano, H. J. Antibiot. 1988, 41,
1285.
4. Ogawa, T.; Ichimura, M.; Katsumata, S.; Morimoto, M.;
Takahashi, K. J. Antibiot. 1989, 42, 1299.
5. Ichimura, M.; Ogawa, T.; Takahashi, K.; Kobayashi, E.;
Kawamoto, I.; Yasuzawa, T.; Takahashi, I.; Nakano, H. J.
Antibiot. 1990, 43, 1037.
6. Ichimura, M.; Ogawa, T.; Katumata, S.; Takahashi, K.;
Takahashi, I.; Nakano, H. J. Antibiot. 1991, 44, 1045.
7. Yasuzawa, T.; Saitoh, Y.; Ichimura, M.; Takahashi, I.;
Sano, H. J. Antibiot. 1991, 44, 445.
8. Yasuzawa, T.; Muroi, K.; Ichimura, M.; Takahashi, I.;
Takahashi, K.; Sano, H.; Saitoh, Y. Chem. Pharm. Bull. 1995,
43, 378.
35. Boger, D. L.; Boyce, C.; Johnson, D. S. Bioorg. Med.
Chem. Lett. 1997, 7, 233.
36. Chidester, C. G.; Krueger, W. C.; Mizsak, S. A.;
Duchamp, D. J.; Martin, D. G. J. Am. Chem. Soc. 1981,
103, 7629.
37. Warpehoski, M. A.; Gebnard, I.; Kelly, R. C.; Krueger,
W. C.; Li, L. H.; McGovren, J. P.; Prairie, M. D.; Wicnienski,
N.; Wierenga, W. J. Med. Chem. 1988, 31, 590.
38. Boger, D. L.; Tun, W. J. Am. Chem. Soc. 1994, 116,
5523.
9. Gomi, K.; Kobayashi, E.; Miyoshi, K.; Ashizawa, T.; Oka-
moto, A.; Ogawa, T.; Katsumata, S.; Mihara, A.; Okabe, M.;
Hirata, T. Jpn. J. Cancer Res. 1992, 83, 113.
10. A number of reviews on the duocarmycins are available
from references.10±13 Boger, D. L. CHEMTRACTS: Org.
Chem. 1991, 4, 329.
39. Asai, A.; Nagamura, S.; Kobayashi, E.; Gomi, K.; Saito,
H. Bioorg. Med. Chem. Lett. 1996, 6, 1215.
40. Nagamura, S.; Asai, A.; Amishiro, N.; Kobayashi, E.;
Gomi, K.; Saito, H. J. Med. Chem. 1997, 40, 972.
41. Amishiro, N.; Nagamura, S.; Kobayashi, E.; Gomi, K.;
Saito, H. J. Med. Chem. 1999, 42, 669.
11. Boger, D. L. Acc. Chem. Res. 1995, 28, 20.
12. Boger, D. L.; Johnson, D. S. Proc. Natl. Acad. Sci. USA
1995, 92, 3642.
13. Boger, D. L.; Johnson, D. S. Angew. Chem., Int. Ed. Engl.
1996, 35, 1438.
42. Nagamura, S.; Kobayashi, E.; Gomi, K.; Saito, H. Bioorg.
Med. Chem. 1996, 4, 1379.
43. Ogasawara, H.; Nishio, K.; Takeda, Y.; Ohmori, T.;
Kubota, N.; Funayama, Y.; Ohira, T.; Kuraishi, Y.; Isogai,
Y.; Saijo, N. Jpn. J. Cancer Res. 1994, 85, 418.
44. Okamoto, A.; Asai, A.; Saito, H.; Okabe, M.; Gomi,
K.Jpn. J. Cancer Res. 1994, 85, 1304.
14. Sugiyama, H.; Hosoda, M.; Saito, I.; Asai, A.; Saito, H.
Tetrahedron Lett. 1990, 31, 7197.
15. Boger, D. L.; Ishizaki, T.; Zarrinmayeh, H. J. Org. Chem.
1990, 55, 4499.
16. Boger, D. L.; Ishizaki, T.; Zarrinmayeh, H.; Munk, S. A.;
Kitos, P. A.; Suntornwat, O. J. Am. Chem. Soc. 1990, 112,
8961.
45. Ogasawara, H.; Nishio, K.; Kanzawa, F.; Lee, Y. S.;
Funayama, Y.; Ohmori, T.; Kuraishi, Y.; Isogai, Y.; Saijo, N.
Jpn. J. Cancer Res. 1995, 86, 124.
46. Ogasawara, H.; Nishio, K.; Ishida, T.; Arioka, H.;
Fukuoka, K.; Saijo, N. Jpn. J. Cancer Res. 1997, 88, 1033.

N. Amishiro et al. / Bioorg. Med. Chem. 8 (2000) 381±391
391
47. Nagamura, S.; Asai, A.; Kanda, Y.; Kobayashi, E; Gomi,
K.; Saito, H. Chem. Pharm. Bull. 1996, 44, 933.
48. Berner, D.; Cox, D. P.; Dahn, H. J. Am. Chem. Soc. 1982,
104, 2631.
Schumacher, A. M.; Abbott, B. J. Cancer Chemother. Rep.
1972, 3, 1.
50. Inaba, M.; Kobatashi, T.; Tashiro, T.; Sakurai, Y.;
Maruo, K.; Ohnishi, Y.; Ueyama, Y.; Momura, T. Cancer
1989, 64, 1577.
49. Geran, R. I.; Greenberg, N. H.; MacDonald, M. M.;