Synthesis and antitumor activity of duocarmycin derivatives: Modification of segment-A of A-ring pyrrole compounds

Article abstract of DOI:10.1021/jm990094r

A series of 3-substituted A-ring pyrrole compounds 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. These compounds were ev

Full text of DOI:10.1021/jm990094r

2946
J . Med. Chem. 1999, 42, 2946-2960
Syn th esis a n d An titu m or Activity of Du oca r m ycin Der iva tives: Mod ifica tion of
Segm en t-A of A-Rin g P yr r ole Com p ou n d s
Nobuyoshi Amishiro,* Akihiko Okamoto, Chikara Murakata, Tatsuya Tamaoki, Masami Okabe, and
Hiromitsu Saito
Pharmaceutical Research Institute, Kyowa Hakko Kogyo Company, Ltd., 1188 Shimotogari, Nagaizumi, Sunto,
Shizuoka 411-8731, J apan
Received March 1, 1999
A series of 3-substituted A-ring pyrrole compounds 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. These compounds were evaluated on the peripheral blood
toxicity and delayed lethal toxicity. Further, to expand our investigation of their peripheral
blood toxicity, the toxicity to bone marrow cells (CFU-GM, CFU-Meg) was investigated. Among
3-substituted A-ring pyrrole compounds of duocarmycin bearing a 5′,6′,7′-trimethoxy-2′-
indolecarboxyl group as segment-B (Seg-B), several analogues showed remarkably potent
antitumor activity with low peripheral blood toxicity. The 3-formyl compound 12h , one of such
analogues, showed stronger antitumor activity with lower toxicity to bone marrow cells
compared to DU-86 (2a ), an active metabolite of KW-2189 (2b). However, compound 12h caused
delayed death. On the other hand, the 3-bromo compound 15f, one of the 3-substituted A-ring
pyrrole derivatives bearing a 4′-methoxycinnamoyl group as Seg-B, showed the most potent
antitumor activity among the 4′-methoxycinnamate analogues with low toxicity to bone marrow
cells. Furthermore, compound 15f did not cause delayed death similarly to 2d . These results
would indicate the importance of the C-3 substituents of A-ring pyrrole duocarmycin derivatives
for exhibiting antitumor activity and decreasing toxicity.
In tr od u ction
phase HPLC analysis. The results indicate that the
electrophilicity of the cyclopropane of DUMs is as
follows: DUMA > 2a > DUMSA ) 2c. Further, we have
observed that the DNA-alkylating reaction of DUMA
(1a ), SA (1b), and analogues is reversible.11 The rate of
regeneration of these compounds from the covalent DNA
adducts (2c > DUMSA > 2a . DUMA) was correlated
to the electrophilicity of these compounds. Moreover,
DUMA (1a ) is unstable in the culture medium to give
the inactive hydrolytic product.12,13 This hydrolytic
conversion seems to decrease the potency of analogues
having Seg-A of the natural type. Among the several
modification product of Seg-A, KW-2189 (2b)10 having
an A-ring pyrrole demonstrated excellent in vivo anti-
tumor activity and good stability in the culture medium.
KW-2189 (2b)10 is currently under phase II clinical
evaluation. In addition, several modifications of the
alkylation subunits on duocarmycin or CC-1065 (1g)
analogues have been previously reported.14
It is expected that our approach to synthesize the new
Seg-A analogues may enhance the potency and decrease
the toxicity by structural modifications of the A-ring
pyrrole moiety which influences the association between
DNA and drugs. From this viewpoint, new candidates
for antitumor drugs would be found by further modifica-
tion of the A-ring pyrrole moiety. Our study showed a
new approach in the modification at the C-3 position of
the A-ring pyrrole moiety in Seg-A for investigating
anticellular and antitumor activities, peripheral blood
toxicity, and structure-activity relationships of these
derivatives. Furthermore, we studied the evaluation of
delayed lethal toxicity and toxicity to bone marrow cells
(CFU-GM, CFU-Meg) of these derivatives in order to
investigate their peripheral blood toxicity in detail.
Duocarmycins (DUM) (A, 1a ; SA, 1b; B1, 1e; B2, 1c;
C1, 1f; C2, 1d ) are novel antitumor antibiotics isolated
from the culture broth of Steptomyces sp. (Chart 1).1
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.2,3
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 have a unique cyclopropane ring
responsible for the sequence-selective alkylation of
double-stranded DNA mediating N3 adenine covalent
adduct formation.4 This mechanism is similar to that
of CC-1065 (1g) which has been reported to show high
cytotoxicity.5,6 The segment-B (Seg-B) of DUM has been
considered to play an important role for noncovalent
binding to the minor groove of DNA.7 With the objective
to identify novel promising candidates, we have previ-
ously synthesized a series of DUM analogues bearing
simplified DNA-binding moieties.8,9 Among these Seg-B
derivatives, some of the A-ring pyrrole DUMs bearing
a cinnamoyl group as Seg-B showed strong antitumor
activity and low peripheral blood toxicity.9
On the other hand, Seg-A has the electrophilic cyclo-
propane ring necessary for the formation of covalent
bonding to DNA. In previous papers,10c we have reported
that the A-ring structure influences the electrophilicity
of the cyclopropane. To investigate the reactivity of the
cyclopropane subunit of DUMA (1a ), DUMSA (1b), 2a ,
and 2c, we evaluated the chemical stability of these
drugs under aqueous neutral conditions by reverse-
10.1021/jm990094r CCC: $18.00 © 1999 American Chemical Society
Published on Web 07/13/1999

Synthesis, Activity of Duocarmycin Derivatives
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 2947
Ch a r t 1. Structures of Duocarmycins, CC-1065, and Duocarmycin Derivatives
Sch em e 1a
a
(a) Allyl alcohol or benzyl alcohol, K2CO3, 0 °C; (b) NaBH4, allyl alcohol, 0 °C; (c) BF3-Et2O, CHCl3, rt; (d) DIBAL-H, THF, rt; (e) R2
) allyl: Pd(PPh3)4, dimedone, THF, rt, or R2 ) Bzl: Pd/C, 25% HCO2NH4(aq), THF, rt; (f) method 1: DECP, Et3N, THF, 0 °C-rt, method
2: EDCI, CH2Cl2, rt, method 3: EDCI, DMAP, CH2Cl2, rt.
Ch em istr y
prepared by employing the same type of rearrangement
of the corresponding 3-ester compounds 3b and 3c
obtained by the treatment of compound 3a with alcohols
in the presence of K2CO3 (Scheme 1). Compound 4a was
reduced with DIBAL-H in THF at room temperature to
afford the 3-methyl and 3-hydroxymethyl compounds 5a
and 5b. The production ratio of 5a and 5b was greatly
In a previous paper, we reported that the 2-methyl-
3-methoxycarbonyl A-ring pyrrole compound of duocar-
mycin (4a ) was prepared by employing the Wagner-
Meerwein type rearrangement of the 8-O-protected-3-
hydroxyduocarmycin B2.15 The 3-allyloxycarbonyl com-
pound 4b and 3-benzyloxycarbonyl compound 4c were

2948 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15
Amishiro et al.
Sch em e 2a
a
(a) nBu4NF, THF, rt; (b) 4 N HCl in AcOEt, CH2Cl2, rt.
affected by the equivalent of DIBAL-H and the reaction
time [5a (56%), 5b (7%): DIBAL-H (12 equiv), 23 h; 5a
(17%), 5b (35%): DIBAL-H (4 equiv), 1.3 h]. The
3-carboxyl compound 6 was prepared by deprotection
of the allyl group of 4b using Pd(PPh3)4/dimedone16 or
the benzyl group of 4c using palladium on carbon in the
presence of 25% aqueous HCO2NH4. Compound 6 was
converted to the 3-amides, 3-esters, and 3-thioesters 7f-
7p by the reaction with various amines, alcohols, and
thiols using diethyl cyanophosphonate (DECP) in the
presence of Et3N (method 1) or only 1-(3-dimethylami-
nopropyl)-3-ethylcarbodiimide hydrochloride (EDCI)
(method 2) or EDCI in the presence of 4-dimethylami-
nopyridine (DMAP) (method 3). The desilylation of
3-substituted compounds 4b, 4c, 5a , 5b, 6, and 7f-7p
was carried out with nBu4NF in THF to give 8a -8p ,
respectively (Scheme 2). Compounds 8h and 8i were
treated with 4 N HCl in AcOEt to afford 9h and 9i
carrying a chloromethyl group in the C-ring part. The
aqueous solubility of these HCl salts (9h , 9i) was found
to be more than 10 mg/mL.
methyl compound 11g, and 3-formyl compound 11h .
Treatment of 11h with 1,3-dithiopropane in the pres-
ence of Amberlyst-15 afforded the 3-dithioacetal 11i.18
The 3-oximes 11j and 11k were synthesized by the
reaction of 11h with hydroxyamine or methoxyamine
in EtOH. Compounds 11a -11k were converted to the
cyclopropane compounds 12a -12k by the same method
as Scheme 2. Compound 12g was converted to the HCl
salt 13 with high aqueous solubility (>10 mg/mL).
The preparation of the 3-substituted A-ring pyrrole
derivatives bearing a 4′-methoxycinnamoyl group is
outlined in Scheme 4. Synthesis of compounds 14a -14h
(Seg-A) was performed by the desilylation of 8-O-
TBDMS-3-substituted compounds with nBu4NF, fol-
lowed by methanolysis with sodium methoxide without
isolation. The obtained compounds 14a -14h were al-
lowed to react with 4′-methoxy-trans-cinnamic p-nitro-
phenyl ester in the presence of NaH to yield the
corresponding 3-substituted A-ring pyrrole derivatives
bearing a 4′-methoxycinnamoyl group (15a -15h ) in
reasonable yield, as described previously.9 Compound
15a was treated with 4 N HCl in AcOEt for protection
of the cyclopropane ring to afford an intermediate
compound carrying a chloromethyl group in the C-ring
part. After Mannich reaction, the obtained product was
then treated with DBU to yield 3-dimethylaminomethyl
compound 16. The HCl salt (17) of 16 was also prepared
by the same procedure as that of 9h and 9i. The aqueous
solubility of compound 17 was found to be more than
10 mg/mL.
We have already reported that the 2-decarbomethoxy-
3-hydroxy compound treated with camphorsulfonic acid
(CAS) affords the dehydration product 10.10a Further,
we have found another synthesis of the 3-hydro com-
pound 10 (Scheme 3). The 3-carboxyl compound 6 was
decarboxylated at reflux in bromobenzene to give the
3-hydro compound 10. Compound 10 was converted to
the 3-halogeno compounds 11a -11c by the reaction
with NCS or NBS or NIS in the presence of silica gel
under darkness.17 Moreover, the 3-hydro compound 10
was reacted with various electrophiles (Friedel-Crafts
acylation, Mannich reaction, Vilsmeier reaction) to yield
the corresponding 3-acyls 11d -11f, 3-dimethylamino-
Resu lts a n d Discu ssion
Tables 1 and 2 show anticellular activity of 3-substi-
tuted A-ring pyrrole derivatives against HeLa S3 cells

Synthesis, Activity of Duocarmycin Derivatives
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 2949
Sch em e 3a
a
(a) PhBr, reflux; (b) NCS or NBS or NIS, silica gel, CH2Cl2, rt; (c) Ac2O or (nPrO)2O or (PhO)2O, BF3-Et2O or TiCl4, CH2Cl2, rt or 50
°C; (d) Me2NCH2NMe2, TFA, -10 °C; (e) POCl3, DMF, rt; (f) HS(CH2)3SH, Amberlyst-15, THF, rt; (g) H2NOH or H2NOMe, EtOH, rt; (h)
nBu4NF, THF, rt; (i) 4 N HCl in AcOEt, CH2Cl2, rt.
Sch em e 4a
a
(a) (1) nBu4NF, THF, rt, (2) NaOMe, MeOH, rt; (b) (1) NaH, (2) 4-methoxycinnamic p-nitrophenyl ester, DMF, -20 °C; (c) (1) 4 N HCl
in AcOEt, CH3CN, rt, (2) Me2NCH2NMe2, TFA, -10 °C; (d) DBU, CH3CN, rt; (e) 4 N HCl in AcOEt, CH2Cl2, rt.
in vitro, antitumor activity against sarcoma 180 solid
tumor in mice, and hematotoxicity (WBC, PL) in vivo.
The efficacy in vivo is expressed as T/C, where T and C
represent means of tumor volume in treated and control
mice, respectively.
Among 3-substituted A-ring pyrrole analogues bear-
ing a 5′,6′,7′-trimethoxy-2′-indolecarbonyl group as Seg-B
of the natural type, 3-methyl (8c), 3-hydroxymethyl
(8d ), 3-halogeno (12a -12c), and 3-formyl (12h ) deriva-
tives showed strong anticellular activity comparable to
the 3-methoxycarbonyl compound 2a (DU-86), a cyclo-
propane compound of KW-2189 (2b).10 In contrast, the
3-amides, 3-esters, and 3-thioesters showed a tendency
to decrease anticellular activity. It is assumed that their
3-substituted groups are a steric hindrance to the DNA
minor groove.
On the other hand, the relationship between the in
vitro activity and the in vivo activity is somewhat

2950 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15
Amishiro et al.
Ta ble 2. Anticellular Activity, Antitumor Activity, and
Hematotoxicity of Duocarmycin Derivatives Bearing a
4′-Methoxycinnamoyl Group
Ta ble 1. Anticellular Activity, Antitumor Activity, and
Hematotoxicity of Duocarmycin Derivatives Bearing a
5′,6′,7′-Trimethoxy-2′-indolecarboxyl Group
sarcoma
sarcoma
180 (sc-iv)b
hematotoxicity
180 (sc-iv)b
hematotoxicity
HeLa S3
HeLa S3
IC50 (nM)a
IC50 (nM)a
dose
WBCd
(%)
PLe
(%)
dose
WBCd
(%)
PLe
(%)
no.
1 h
72 h
(mg/kg) T/Cc
no.
1 h
72 h
(mg/kg) T/Cc
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
9h
0.43
0.74
0.098
0.093
14
0.26
0.18
0.022
0.010
0.28
3.6
5.3
3.5
3.0
0.12
8.1
1.4
18
0.22
1.1
12
0.13
0.13
0.031
0.031
0.25
4.0
2.0
0.13
2.0
0.13
4.0
0.5
2.0
0.25
0.5
8.0
0.25
8.0
0.063
0.031
0.016
0.25
4.0
0.24
0.20
0.31
0.38
0.26
0.18
0.25
0.58
0.38
0.22
0.18
0.43
0.60
0.23
0.30
0.49
0.59
0.45
0.22
0.34
0.24
0.24
0.24
0.31
0.24
0.18
0.36
0.39
0.25
0.30
0.21
43
53
35
75
67
32
38
62
37
28
35
83
130
56
75
36
93
33
31
27
55
32
17
32
53
86
38
88
22
47
22
73
75
66
toxic
107
NTf
55
84
83
59
85
94
91
53
105
81
93
84
36
70
NT
64
26
90
81
56
77
98
59
82
38
15a
15b 11
7.0
0.17
1.1
0.5
1.0
0.5
0.34
0.51
0.15
0.41
0.21
0.11
0.16
0.37
0.35
0.37
0.34
30
42
21
59
19
24
23
52
65
48
50
63
98
83
80
64
67
70
86
83
88
63
15c
15d
15e
15f
15g
15h
16
7.5
0.22
0.37
0.029
0.065
0.63
0.92
0.39
0.57
0.96
1.4
2.9
2.4
3.3
3.6
7.8
0.13
0.25
0.21
0.25
1.0
0.25
1.0
0.83
16
24
39
34
0.21
19
4.0
130
0.72
2.7
30
9.1
60
17
2d
2.9-7.0 0.26-0.94
a
Drug concentration required to inhibit the growth of HeLa S3
cells by 50%. b Mice (5 mice/group) were implanted subcutaneously
(sc) with tumor cells, and the drug was dosed (mg/mg) intrave-
nously (iv). c T and C are the values of the mean tumor volume of
d
treated and control mice, respectively. Number of peripheral
0.78
4.1
platelets of normal mice on day 7 (percent of control). e Number
of white blood cells of tumor-bearing mice on day 4 (percent of
control).
9i
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
13
0.018
0.025
0.092
0.27
3.0
0.009
0.014
0.039
0.39
1.4
0.78
0.10
0.046
1.1
0.082
1.7
0.16
0.0052
marrow cells. Modification at the C-3 position of the
A-ring pyrrole resulted in a decreased toxicity not only
to peripheral blood toxicity but also to bone marrow
cells. Furthermore, the evaluation of delayed toxicity
was performed (Table 3).10b No toxic death was observed
in mice on and after day 30 by the administration of
compound 2d , indicating that compound 2d did not
cause delayed death in ddY mice. However, compounds
2a and 12h caused delayed death even when adminis-
tered below the maximum tolerated dose predetermined
in the evaluation of antitumor activity against murine
sarcoma 180 in ddY mice. It was assumed that delayed
death by 2a and 12h was caused by the strong nonco-
valent interaction between DNA and Seg-B of the
natural type.
2.5
1.0
0.25
0.08
3.1
0.13
8.7
0.13
0.031
0.5
0.13
1.0
0.28
0.045
0.5
0.25
2a
a
Drug concentration required to inhibit the growth of HeLa S3
cells by 50%. b Mice (5 mice/group) were implanted subcutaneously
(sc) with tumor cells, and the drug was dosed (mg/mg) intrave-
nously (iv). c T and C are the values of the mean tumor volume of
d
treated and control mice, respectively. Number of peripheral
platelets of normal mice on day 7 (percent of control). e Number
of white blood cells of tumor-bearing mice on day 4 (percent of
control). f Not tested.
Table 2 shows the anticellular and antitumor activity
and hematotoxicity of the 3-substituted A-ring pyrrole
derivatives bearing a 4′-methoxycinnamoyl group. We
expected that 4′-methoxycinnamates would show lower
toxicity to bone marrow toxicity than the derivatives
bearing a trimethoxyindole skeleton in Seg-B. The
3-chloro (15e) and 3-bromo (15f) compounds exhibited
the most remarkable anticellular activity of all the
derivatives having a 4′-methoxycinnamoyl group as
Seg-B with IC50 values below 0.1 nM at 72-h exposure.9
Compound 15f showed the strongest antitumor activity
among 4′-methoxycinnamates with a T/C value of 0.11.
Among 4′-methoxycinamates, compounds 15d and 15f
were studied on efficacy against human xenografted
carcinoma (LC-6) and toxicity to bone marrow cells
(Figure 2). Compound 15f showed excellent activity in
vivo with a T/C value of 0.099 (0.25 mg/kg dose);
however, compound 15d showed weak antitumor activ-
ity. Bone marrow toxicity of 15d and 15f was lower than
that of 2a bearing Seg-B of the natural type and was
equal to that of 4′-methoxycinnamate (2d ) (Figures 1
and 2). Moreover, compound 15f did not cause delayed
death at a dose below the maximum tolerated dose in
the evaluation of antitumor activity against murine sar-
coma 180 in ddY mice (Table 3). These results indicate
different. Although the analogues having a steric hin-
drance group at the C-3 position of the A-ring pyrrole
indicated lower anticellular activity than 2a , several
analogues showed efficient antitumor activity in vivo
(T/C < 0.3). It is conceivable that good stability in the
culture medium and the association between DNA and
drugs may cause strong antitumor activity.13 Moreover,
many 3-substituted A-ring pyrrole DUMs having a
trimethoxyindole skeleton in Seg-B showed lower pe-
ripheral blood toxicity than 2a , an active metabolite of
KW-2189 (2b). We evaluated the toxicity to bone mar-
row cells (CFU-GM, CFU-Meg) of duocarmycin deriva-
tives in order to investigate their peripheral blood
toxicity in detail. First, comparative examination of
compound 2a bearing a 5′,6′,7′-trimethoxy-2′-indolecar-
bonyl group and compound 2d bearing a 4′- methoxy-
cinnamoyl group was performed (Figure 1). The results
indicated that the toxicity of compound 2d to bone
marrow cells was lower and its recovery was also more
rapid than that of compound 2a , like the tendency of
peripheral blood toxicity. Next, the 3-formyl compound
12h , demonstrating potent antitumor activity with low
peripheral blood toxicity, was evaluated in vivo on its
toxicity to bone marrow cells (Figure 2). Compound 12h
showed lower toxicity than 2a in the level of bone

Synthesis, Activity of Duocarmycin Derivatives
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 2951
F igu r e 1. Suppression and recovery of hematopoietic functions after drug administration. Drug was administered iv in ddY
mice on day 0, and the ratios of colony-forming unit-granulocytes and macrophages (CFU-GM) and colony-forming unit-
megakaryocytes (CFU-Meg) per femur to those of control were calculated on the days indicated.
F igu r e 2. Suppression and recovery of hematopoietic functions after drug administration. Drug was administered iv in ddY
mice on day 0, and the ratios of colony-forming unit-granulocytes and macrophages (CFU-GM) and colony-forming unit-
megakaryocytes (CFU-Meg) per femur to those of control were calculated on the days indicated.
that compound 15f is as promising a candidate as
compound 2d for a novel antitumor agent.
In addition, compounds 9g, 9h , 13, and 17, having
an amino group at the C-3 position of the A-ring pyrrole,
were found to possess adequate water solubility in
excess of 10 mg/mL.
remarkably potent antitumor activity with low periph-
eral blood toxicity and low bone marrow toxicity.
Moreover, compound 15f did not cause delay death
similarly to 2d . Modification at the C-3 position was
shown to be a promising approach for preparing new
candidates for antitumor drugs.
Exp er im en ta l Section
Con clu sion s
Infrared spectra (IR) were recorded on a J ASCO IR-810
spectrometer. 1H-NMR spectra were measured on J EOL J NM-
EX270 and Bruker AM-400 spectrometers and are reported
in δ units. Mass spectra were measured with J EOL J MS-
DX303 and SHIMAZU QP-1000 spectrometers. 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
A series of 3-substituted A-ring pyrrole compounds
of duocarmycin were prepared and evaluated for their
anticellular activity against Hela S3 cells and antitumor
activity against sarcoma 180 murine solid tumor and
LC-6 human lung tumor xenograft. Moreover, we per-
formed the evaluation on these derivatives regarding
delayed lethal toxicity and bone marrow toxicity in order
to investigate their peripheral blood toxicity in detail.
The 3-bromo A-ring pyrrole compound 15f of duocar-
mycin bearing a 4′-methoxycinnamoyl group showed
F
254s. Usual workup refers to washing of organic layers with
brine, drying over anhydrous Na2SO4, and evaporating off the
solvents under reduced pressure.

2952 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15
Amishiro et al.
Ta ble 3. Lethal Toxicity of Duocarmycin Derivatives in Mice
mixture was stirred under Ar atmosphere at room temperature
for 23 h; 0.5 N HCl was added, and the whole was extracted
with CHCl3, then washed with aqueous NaHCO3, and worked
up as usual. The residue was purified by PTLC (hexane-
AcOEt, 3:2) to give 26 mg (56%) of 5a and 3.4 mg (7%) of 5b.
5a : 1H-NMR (270 MHz, CDCl3) δ 9.45 (1 H, brs), 7.79 (1 H,
brs), 7.76 (1 H, s), 6.96 (1 H, d, J ) 2.3 Hz), 6.88 (1 H, s), 4.74
(1 H, d, J ) 10.9 Hz), 4.54 (1 H, dd, J ) 10.2, 8.3 Hz), 4.08-
4.15 (1 H, m), 4.07 (3 H, s), 3.94 (3 H, s), 3.92 (3 H, s), 3.71 (1
H, dd, J ) 10.4, 1.8 Hz), 3.25 (1 H, dd, J ) 10.9, 10.6 Hz),
2.38 (3 H, s), 2.32 (3 H, s), 1.06 (9 H, s), 0.36 (3 H, s), 0.34 (3
H, s); FAB-MS m/z 644, 642 (M + H)+. 5b: 1H-NMR (270 MHz,
CDCl3) δ 9.44 (1 H, brs), 7.94 (1 H, s), 7.85 (1 H, brs), 6.96 (1
H, d, J ) 2.0 Hz), 6.88 (1 H, s), 4.89 (1 H, d, J ) 12.2 Hz), 4.74
(1 H, d, J ) 12.5 Hz), 4.73 (1 H, d, J ) 10.9 Hz), 4.54 (1 H, dd,
J ) 10.2, 8.3 Hz), 4.13-4.20 (1 H, m), 4.06 (3 H, s), 4.00 (1 H,
dd, J ) 10.2, 2.0 Hz), 3.94 (3 H, s), 3.91 (3 H, s), 3.29 (1 H, dd,
J ) 10.6, 10.6 Hz), 2.48 (3 H, s), 1.06 (9 H, s), 0.36 (3 H, s),
0.35 (3 H, s); FAB-MS m/ z 660, 658 (M + H)+.
8-O-(ter t-Bu tyld im eth silyl)-2-m eth yl-3-ca r boxy-A-r in g-
p yr r ole-DUMB2 (6). Th e ca se of 4b: To a solution of 4b
(60 mg, 0.085 mmol) in THF (3 mL) were added dimedone (59.4
mg, 0.424 mmol) and Pd(PPh3)4 (20 mg, 0.017 mmol), and the
mixture was stirred under Ar atmosphere at room temperature
for 1 h; 0.01 M phosphoric buffer (pH 7) was added, and the
whole was extracted with CHCl3 and then worked up as usual.
The residue was purified by PTLC (hexane-AcOEt, 3:2) to give
56 mg (99%) of 6.
dose
(mg/kg)
no.
deceased day
mortalitya
2a
0.17
0.20
0.24
0.29
0.35
0.42
0.50
0.58
0.69
0.83
1.0
65
1/10
5/10
6/10
10/10
10/10
10/10
10/10
0/10
46, 48, 48, 49, 66
5, 12, 12, 58, 58, 62
5, 5, 5, 5, 5, 5, 5, 6, 7, 7
5, 5, 5, 5, 5, 5, 5, 5, 5, 5
2, 2, 2, 2, 5, 6, 6, 6, 6, 6
2, 2, 2, 2, 2, 2, 2, 2, 2, 2
not deceased
2d
not deceased
not deceased
0/10
0/10
5, 5, 5, 5, 5, 5, 5, 6, 6, 9
5, 5, 5, 5, 5, 5, 5, 5, 5, 6
2, 2, 2, 2, 2, 3, 5, 5, 5, 5
2, 2, 2, 2, 2, 2, 2, 2, 2, 2
49
10/10
10/10
10/10
10/10
1/10
3/10
7/10
10/10
10/10
0/10
0/10
0/10
8/10
10/10
10/10
1.2
1.4
1.7
12h
15f
0.031
0.044
0.063
0.088
0.13
0.099
0.15
0.22
0.33
0.50
0.75
59, 60, 65
49, 50, 59, 60, 64, 65, 65
4, 4, 4, 4, 4, 7, 7, 7, 7, 7
3, 3, 3, 3, 3, 4, 4, 4, 4, 4
not deceased
not deceased
not deceased
5, 5, 5, 5, 6, 6, 6, 29
2, 2, 4, 4, 4, 4, 4, 4, 4, 4
2, 2, 2, 2, 2, 2, 2, 2, 2, 2
a
Observed for 60-71 days. Each compound was administered
Th e ca se of 4c: To a solution of 4c (48 mg, 0.062 mmol) in
THF (2 mL) were added 25% HCO2NH4(aq) (0.46 mL) and 10%
Pd/C (30 mg), and the mixture was stirred under Ar atmo-
sphere at room temperature for 30 min. The mixture was
filtered through Celite. Then, the filrate was poured into 0.01
M phosphoric buffer (pH 7). The whole was extracted with
CHCl3 and worked up as usual. The residue was purified by
PTLC (hexane-AcOEt, 3:2) to give 33 mg (80%) of 6: 1H-NMR
(270 MHz, CDCl3) δ 9.59 (1 H, brs), 8.52 (1 H, s), 8.00 (1 H,
brs), 7.01 (1 H, d, J ) 2.3 Hz), 6.89 (1 H, s), 4.74 (1 H, d, J )
10.2 Hz), 4.70-4.75 (1 H, m), 4.54 (1 H, dd, J ) 9.9, 8.9 Hz),
4.06 (3 H, s), 3.95 (3 H, s), 3.91 (3 H, s), 3.90 (1 H, br), 3.29 (1
H, dd, J ) 9.9, 9.6 Hz), 2.82 (3 H, s), 1.07 (9 H, s), 0.39 (3 H,
s), 0.38 (3 H, s); FAB-MS m/z 674, 672 (M + H)+; IR (KBr)
3458, 2856, 1630, 1495, 1466, 1443, 1419, 1389, 1308, 1228,
iv in male ddY mice on day 0.
8-O-(ter t-Bu tyld im eth ylsilyl)-2-m eth yl-3-(a llyloxyca r -
b on yl)-A-r in g-p yr r ole-DUMB2 (4b ). To a solution of 3a
(3.58 g, 5.10 mmol) in allyl alcohol (100 mL) was added K2-
CO3 (2.01 g, 15.2 mmol), and the mixture was stirred at 0 °C
for 41 h 40 min; 0.5 N HCl was added, and the whole was
extracted with AcOEt and then washed with aqueous NaHCO3.
Usual workup afforded 5.70 g of 3b. To a solution of 3b (5.70
g) in allyl alcohol (39 mL) was added NaBH4 (577 mg, 15.2
mmol), and the mixture was stirred at 0 °C for 1 h 45 min; 0.5
N HCl was added, and the whole was extracted with CHCl3,
washed with aqueous NaHCO3, and worked up as usual. The
residue was purified by column chromatography (hexane-
AcOEt, 4:1-2:1) to give 1.29 g (35%) of 8-O-(tert-butyldimeth-
ylsilyl)-3-hydroxy-DUMB2. To a solution of 8-O-(tert-butyldim-
ethylsilyl)-3-hydroxy-DUMB2 (200 mg) in CHCl3 (6 mL) was
added BF3-Et2O (0.101 mL, 0.822 mmol), and the mixture was
stirred at room temperature for 17 h 30 min. Aqueous NaHCO3
was added, and the whole was extracted with CHCl3, then
washed with aqueous NaHCO3, and worked up as usual. The
residue was purified by PTLC (hexane-AcOEt, 3:1) to give
139 mg (71%) of 4b: 1H-NMR (270 MHz, CDCl3) δ 9.44 (1 H,
brs), 8.36 (1 H, brs), 7.98 (1 H, brs), 6.99 (1 H, d, J ) 2.3 Hz),
6.89 (1 H, s), 6.12 (1 H, ddt, J ) 19.6, 10.4, 5.9 Hz), 5.44 (1 H,
ddd, J ) 17.2, 1.6, 1.3 Hz), 5.31 (1 H, ddd, J ) 10.2, 1.3, 1.3
Hz), 4.82-4.98 (2 H, m), 4.73 (1 H, d, J ) 9.2 Hz), 4.55-4.62
(1 H, m), 4.52 (1 H, dd, J ) 8.3, 8.3 Hz), 4.06 (3 H, s), 3.94 (3
H, s), 3.92 (3 H, s), 3.80 (1 H, dd, J ) 7.6, 3.0 Hz), 3.21 (1 H,
dd, J ) 9.9, 9.9 Hz), 2.76 (3 H, s), 1.06 (9 H, s), 0.38 (3 H, s),
0.36 (3 H, s); FAB-MS m/z 714, 712 (M + H)+.
8-O-(ter t-Bu t yld im et h ylsilyl)-2-m et h yl-3-(b en zyloxy-
ca r bon yl)-A-r in g-p yr r ole-DUMB2 (4c): yield 21%; 1H-NMR
(270 MHz, CDCl3) δ 9.45 (1 H, brs), 8.40 (1 H, brs), 7.98 (1 H,
s), 7.48-7.52 (2 H, m), 7.31-7.43 (3 H, m), 6.98 (1 H, d, J )
2.3 Hz), 6.89 (1 H, s), 5.54 (1 H, d, J ) 12.2 Hz), 5.35 (1 H, d,
J ) 12.2 Hz), 4.71 (1 H, d, J ) 9.9 Hz), 4.56 (1 H, m), 4.49 (1
H, dd, J ) 9.6, 8.6 Hz), 4.06 (3 H, s), 3.94 (3 H, s), 3.92 (3 H,
s), 3.80 (1 H, dd, J ) 9.9, 2.3 Hz), 3.21 (1 H, dd, J ) 9.9, 9.9
Hz), 2.71 (3 H, s), 1.05 (9 H, s), 0.37 (3 H, s), 0.36 (3 H, s);
FAB-MS m/z 764, 762 (M + H)+.
8-O-(ter t-Bu tyld im eth ylsilyl)-2,3-d im eth yl-A-r in g-p yr -
r ole-DUMB2 (5a ) a n d 8-O-(ter t-Bu tyld im eth ylsilyl)-2-
m eth yl-3-(h yd r oxym eth yl)-A-r in g-p yr r ole-DUMB2 (5b).
To a solution of 4a (50 mg, 0.073 mmol) in THF (2 mL) was
added DIBAL-H (0.98 M, 0.89 mL, 0.87 mmol), and the
1203, 1111, 845 cm-1
.
8-O-(ter t-Bu tyld im eth ylsilyl)-2-m eth yl-3-(N-p h en ylca r -
ba m oyl)-A-r in g-p yr r ole-DUMB2 (7f). Meth od 1: To a solu-
tion of 6 (60 mg, 0.089 mmol) in THF (3 mL) were added
aniline (0.016 mL, 0.18 mmol), DECP (0.020 mL, 0.13 mmol),
and Et3N (0.025 mL, 0.18 mmol), and then the mixture was
stirred at 0 °C for 1 h. After the mixture stirred at room
temperature for 12 h, 0.01 M phosphoric buffer (pH 7) was
added, and the whole was extracted with AcOEt and then
worked up as usual. The residue was purified by PTLC
(hexane-AcOEt, 1:1) to give 41 mg (61%) of 7f: 1H-NMR (270
MHz, CDCl3) δ 9.40 (1 H, brs), 8.17 (1 H, s), 8.00 (1 H, s), 7.65
(2 H, d, J ) 8.6 Hz), 7.50 (1 H, s), 7.40 (2 H, dd, J ) 7.9, 7.6
Hz), 7.11-7.20 (1 H, m), 6.92 (1 H, d, J ) 2.3 Hz), 6.87 (1 H,
s), 4.51-4.58 (2 H, m), 4.39 (1 H, m), 4.06 (3 H, s), 3.94 (3 H,
s), 3.90 (3 H, s), 3.73 (1 H, dd, J ) 9.9, 3.0 Hz), 3.26 (1 H, dd,
J ) 9.6, 9.6 Hz), 2.67 (3 H, s), 1.07 (9 H, s), 0.38 (3 H, s), 0.37
(3 H, s); FAB-MS m/z 749, 747 (M + H)+.
8-O-(ter t-Bu tyld im eth ylsilyl)-2-m eth yl-3-(N-n -p r op yl-
ca r ba m oyl)-A-r in g-p yr r ole-DUMB2 (7g). Meth od 2: To a
solution of 6 (30 mg, 0.045 mmol) in CH2Cl2 (1.2 mL) were
added nPrNH2 (0.011 mL, 0.13 mmol) and EDCI (26 mg, 0.13
mmol), and the mixture was stirred at room temperature for
9 h; 0.01 M phosphoric buffer (pH 7) was added, and the whole
was extracted with CHCl3 and then worked up as usual. The
residue was purified by PTLC (hexane-AcOEt, 1:1) to give
28 mg (89%) of 7g: 1H-NMR (270 MHz, CDCl3) δ 9.41 (1 H,
brs), 8.19 (1 H, s), 7.95 (1 H, brs), 6.90 (1 H, d, J ) 2.0 Hz),
6.87 (1 H, s), 5.83 (1 H, t, J ) 5.8 Hz), 4.56 (1 H, dd, J ) 10.6,
2.6 Hz), 4.48 (1 H, dd, J ) 10.6, 8.6 Hz), 4.28-4.42 (1 H, m),
4.05 (3 H, s), 3.93 (3 H, s), 3.89 (3 H, s), 3.65 (1 H, dd, J ) 9.9,

Synthesis, Activity of Duocarmycin Derivatives
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 2953
3.0 Hz), 3.38-3.58 (2 H, m), 3.19 (1 H, dd, J ) 9.6, 9.6 Hz),
2.56 (3 H, s), 1.71 (2 H, dt, J ) 7.6, 7.3 Hz), 1.05 (9 H, s), 1.02
(3 H, t, J ) 7.3 Hz), 0.35 (3 H, s), 0.34 (3 H, s); FAB-MS m/z
715, 713 (M + H)+.
8-O -(t er t -B u t y ld im e t h y ls ily l)-2-m e t h y l-3-[N -(2-d i-
m et h yla m in oet h yl)ca r b a m oyl]-A-r in g-p yr r ole-DUMB2
(7h ): method 2; yield 72%; 1H-NMR (270 MHz, CDCl3) δ 9.49
(1 H, brs), 8.21 (1 H, s), 7.95 (1 H, brs), 6.93 (1 H, d, J ) 1.7
Hz), 6.88 (1 H, s), 6.60 (1 H, br), 4.51-4.65 (2 H, m), 4.35-
4.48 (1 H, m), 4.05 (3 H, s), 3.93 (3 H, s), 3.90 (3 H, s), 3.60-
3.71 (3 H, m), 3.18 (1 H, dd, J ) 10.2, 9.9 Hz), 2.60-2.68 (2 H,
m), 2.60 (3 H, s), 2.33 (6 H, s), 1.05 (9 H, s), 0.35 (3 H, s), 0.34
(3 H, s); FAB-MS m/z 744, 742 (M + H)+.
8-O-(ter t-Bu t yld im et h ylsilyl)-2-m et h yl-3-(4-m et h yl-1-
p ip er a zin ylca r bon yl)-A-r in g-p yr r ole-DUMB2 (7i): method
2; yield 71%; 1H-NMR (270 MHz, CDCl3) δ 9.47 (1 H, brs), 8.17
(1 H, s), 7.93 (1 H, brs), 6.96 (1 H, d, J ) 2.0 Hz), 6.88 (1 H, s),
4.59 (2 H, brd, J ) 5.6 Hz), 4.20-4.70 (1 H, br), 4.05 (3 H, s),
3.93 (3 H, s), 3.90 (3 H, s), 3.41-3.56 (7 H, br), 3.19 (1 H, dd,
J ) 10.2, 9.9 Hz), 2.45 (3 H, s), 2.33 (3 H, s), 2.29-2.52 (2 H,
br), 1.05 (9 H, s), 0.36 (3 H, s), 0.35 (3 H, s); FAB-MS m/ z
756, 754 (M + H)+.
2.83 (3 H, s), 1.05 (9 H, s), 0.37 (3 H, s), 0.36 (3 H, s); FAB-MS
m/ z 780, 778 (M + H)+.
8-O-(ter t-Bu t yld im et h ylsilyl)-2-m et h yl-3-(5-h yd r oxy-
1,2-dith ian -4-yloxycar bon yl)-A-r in g-pyr r ole-DUMB2 (7o):
method 1; yield 51%; 1H-NMR (270 MHz, CDCl3) δ 9.44 (1 H,
brs), 8.46 (1 H, brs), 8.00 (0.5 H, brs), 7.98 (0.5 H, brs), 6.98 (1
H, d, J ) 2.0 Hz), 6.88 (1 H, s), 5.16-5.26 (1 H, m), 4.73 (0.5
H, d, J ) 8.9 Hz), 4.71 (0.5 H, d, J ) 9.2 Hz), 4.51-4.57 (2 H,
m), 4.06 (1.5 H, s), 4.05 (1.5 H, s), 4.03 (1 H, br), 3.94 (3 H, s),
3.91 (3 H, s), 3.80 (0.5 H, brd, J ) 10.6 Hz), 3.72 (0.5 H, dd, J
) 9.5, 2.5 Hz), 3.04-3.42 (5 H, m), 2.77 (3 H, s), 1.06 (9 H, s),
0.37 (3 H, s), 0.36 (3 H, s); FAB-MS m/ z 808, 806 (M + H)+.
8-O-(ter t-Bu tyld im eth ylsilyl)-2-m eth yl-3-[2-(m eth ylsu l-
fon yl)eth oxycar bon yl]-A-r in g-pyr r ole-DUMB2 (7p): method
1; yield 56%; 1H-NMR (270 MHz, CDCl3) δ 9.45 (1 H, brs), 8.47
(1 H, s), 7.99 (1 H, s), 6.99 (1 H, d, J ) 2.0 Hz), 6.89 (1 H, s),
4.88 (1 H, td, J ) 12.5, 6.3 Hz), 4.81 (1 H, td, J ) 12.5, 6.3
Hz), 4.72 (1 H, d, J ) 9.2 Hz), 4.49-4.56 (2 H, m), 4.06 (3 H,
s), 3.94 (3 H, s), 3.92 (3 H, s), 3.76 (1 H, brd, J ) 8.9 Hz),
3.54-3.58 (2 H, m), 3.19 (1 H, dd, J ) 9.9, 9.9 Hz), 3.02 (3 H,
s), 2.75 (3 H, s), 1.05 (9 H, s), 0.38 (3 H, s), 0.36 (3 H, s); FAB-
MS m/z 780, 778 (M + H)+.
8-O-(t er t -Bu t yld im e t h ylsilyl)-2-m e t h yl-3-(2-b r om o-
eth oxyca r bon yl)-A-r in g-p yr r ole-DUMB2 (7j): method 2;
yield 63%; H-NMR (270 MHz, CDCl3) δ 9.45 (1 H, brs), 8.44
2-Met h yl-3-(a llyloxyca r b on yl)-A-r in g-p yr r ole-DUMA
(8a ). To a solution of 4b (40 mg, 0.056 mmol) in THF (1.5 mL)
was added nBu4NF (1.0 M in THF, 0.19 mL, 0.17 mmol), and
the mixture was stirred at room temperature for 45 min; 0.01
M phosphoric buffer (pH 7) was added, and the whole was
extracted CHCl3 and then worked up as usual. The residue
was purified by PTLC (CHCl3-MeOH, 15:1) to give 24 mg
(83%) of 8a : 1H-NMR (270 MHz, CDCl3) δ 11.92 (1 H, brs),
9.52 (1 H, brs), 7.13 (1 H, s), 6.95 (1 H, d, J ) 2.0 Hz), 6.80 (1
H, s), 6.01 (1 H, ddt, J ) 19.5, 10.6, 5.6 Hz), 5.36 (1 H, dq, J
) 17.2, 1.3 Hz), 5.27 (1 H, dq, J ) 10.4, 1.2 Hz), 4.73 (1 H, d,
J ) 5.6 Hz), 4.44 (2 H, br), 4.06 (3 H, s), 3.93 (3 H, s), 3.89 (3
H, s), 3.67 (1 H, m), 2.65 (3 H, s), 2.37 (1 H, dd, J ) 7.3, 3.3
Hz), 1.36 (1 H, dd, J ) 4.3, 4.0 Hz); FAB-MS m/z 518 (M +
H)+; FAB-HRMS calcd for C28H28N3O7 (M + H)+ m/z 518.1927,
found 518.1917; IR (KBr) 1701, 1645, 1605, 1583, 1489, 1385,
1263, 1211, 1105 cm-1. Anal. (C28H27N3O7‚1.0H2O) C, H, N.
2-Me t h y l-3-(b e n z y lo x y c a r b o n y l)-A-r i n g -p y r r o le -
1
(1 H, brs), 7.99 (1 H, s), 7.00 (1 H, d, J ) 2.0 Hz), 6.89 (1 H, s),
4.53-4.75 (5 H, m), 4.06 (3 H, s), 3.94 (3 H, s), 3.92 (3 H, s),
3.80 (1 H, dd, J ) 10.2, 2.6 Hz), 3.72 (1 H, td, J ) 5.6, 1.7 Hz),
3.21 (1 H, dd, J ) 9.9, 9.9 Hz), 2.78 (3 H, s), 1.06 (9 H, s), 0.38
(3 H, s), 0.36 (3 H, s); FAB-MS m/ z 782, 780, 778 (M + H)+.
8-O-(ter t-Bu t yld im et h ysilyl)-2-m et h yl-3-(p h en ylt h io-
ca r bon yl)-A-r in g-p yr r ole-DUMB2 (7k ): method 1; yield
1
61%; H-NMR (270 MHz, CDCl3) δ 9.43 (1 H, brs), 8.42 (1 H,
s), 8.04 (1 H, s), 7.47-7.61 (5 H, m), 6.97 (1 H, d, J ) 2.0 Hz),
6.89 (1 H, s), 4.64 (1 H, d, J ) 8.6 Hz), 4.54 (1 H, dd, J ) 8.6,
8.3 Hz), 4.45-4.51 (1 H, m), 4.06 (3 H, s), 3.94 (3 H, s), 3.91 (3
H, s), 3.60 (1 H, dd, J ) 8.6, 3.0 Hz), 3.19 (1 H, dd, J ) 9.6,
9.2 Hz), 2.94 (3 H, s), 1.07 (9 H, s), 0.39 (3 H, s), 0.38 (3 H, s);
FAB-MS m/z 766, 764 (M + H)+.
8-O-(ter t-Bu tyld im eth ylsilyl)-2-m eth yl-3-(4-p yr id in yl-
th ioca r bon yl)-A-r in g-p yr r ole-DUMB2 (7l): method 1; yield
1
DUMA (8b): yield 78%; H-NMR (270 MHz, CDCl3) δ 11.84
1
(1 H, brs), 9.47 (1 H, brs), 7.31-7.42 (5 H, m), 7.11 (1 H, s),
6.93 (1 H, d, J ) 2.0 Hz), 6.80 (1 H, s), 5.26 (2 H, s), 4.36-4.40
(2 H, br), 4.06 (3 H, s), 3.94 (3 H, s), 3.89 (3 H, s), 3.61-3.67
(1 H, m), 2.62 (3 H, s), 2.35 (1 H, dd, J ) 7.4, 3.5 Hz), 1.33 (1
H, d, J ) 4.6, 3.6 Hz); FAB-MS m/z 568 (M + H)+; FAB-HRMS
calcd for C32H30N3O7 (M + H)+ m/ z 568.2084, found 568.2059;
IR (KBr) 1701, 1638, 1603, 1583, 1489, 1396, 1292, 1265, 1211,
1105 cm-1. Anal. (C32H29N3O7‚1.7H2O) C, N; H: calcd, 5.46;
found, 5.01.
77%; H-NMR (270 MHz, CDCl3) δ 9.50 (1 H, brs), 9.00 (1 H,
brs), 8.70 (2 H, d, J ) 4.6 Hz), 8.05 (1 H, brs), 7.55 (2 H, d, J
) 5.0 Hz), 6.97 (1 H, d, J ) 1.7 Hz), 6.88 (1 H, s), 4.65 (1 H, d,
J ) 9.9 Hz), 4.54 (1 H, dd, J ) 10.6, 8.6 Hz), 4.42-4.50 (1 H,
m), 4.04 (3 H, s), 3.93 (3 H, s), 3.91 (3 H, s), 3.59 (1 H, dd, J )
9.9, 2.6 Hz), 3.17 (1 H, dd, J ) 9.6, 9.2 Hz), 2.93 (3 H, s), 1.04
(9 H, s), 0.37 (3 H, s), 0.36 (3 H, s); FAB-MS m/ z 767, 765 (M
+ H)+.
8-O-(ter t-Bu t yld im et h ylsilyl)-2-m et h yl-3-(1-n a p h t h a -
len ylth ioca r bon yl)-A-r in g-p yr r ole-DUMB2 (7m ): method
1; yield 55%; 1H-NMR (270 MHz, CDCl3) δ 9.44 (1 H, brs), 8.46
(1 H, brs), 8.12 (1 H, s), 8.04 (1 H, brs), 7.88-7.96 (3 H, s),
7.51-7.64 (3 H, m), 6.96 (1 H, d, J ) 2.0 Hz), 6.88 (1 H, s),
4.63 (1 H, d, J ) 8.6 Hz), 4.54 (1 H, dd, J ) 8.6, 7.3 Hz), 4.45-
4.54 (1 H, m), 4.06 (3 H, s), 3.94 (3 H, s), 3.91 (3 H, s), 3.63 (1
H, dd, J ) 8.6, 2.6 Hz), 3.21 (1 H, dd, J ) 9.6, 9.2 Hz), 2.98 (3
H, s), 1.07 (9 H, s), 0.40 (3 H, s), 0.38 (3 H, s); FAB-MS m/ z
816, 814 (M + H)+.
2,3-Dim eth yl-A-r in g-p yr r ole-DUMA (8c): yield 97%; 1H-
NMR (270 MHz, CDCl3) δ 10.64 (1 H, brs), 9.51 (1 H, brs),
6.95 (1 H, s), 6.92 (1 H, d, J ) 2.3 Hz), 6.78 (1 H, s), 4.39 (2 H,
br), 4.05 (3 H, s), 3.92 (3 H, s), 3.88 (3 H, s), 2.90-2.99 (1 H,
m), 2.26 (3 H, s), 1.98 (1 H, dd, J ) 7.4, 2.5 Hz), 1.92 (3 H, s),
1.34 (1 H, dd, J ) 4.6, 4.6 Hz); FAB-MS m/ z 448 (M + H)+;
IR (KBr) 1637, 1606, 1578, 1560, 1468, 1385, 1306, 1265, 1230,
1109 cm-1. Anal. (C25H25N3O5‚1.8H2O) C, N; H: calcd, 6.01;
found, 5.50.
2-Me t h yl-3-(h yd r oxym e t h yl)-A-r in g-p yr r ole -DUMA
(8d ): yield 47%; 1H-NMR (270 MHz, CDCl3 + CD3OD) δ 6.86
(1 H, s), 6.82 (1 H, s), 6.71 (1 H, s), 4.42 (1 H, dd, J ) 10.2, 4.6
Hz), 4.36 (1 H, d, J ) 12.5 Hz), 4.32 (1 H, d, J ) 9.6 Hz), 4.28
(1 H, d, J ) 12.5 Hz), 3.96 (3 H, s), 3.86 (3 H, s), 3.82 (3 H, s),
3.08-3.15 (1 H, m), 2.27 (3 H, s), 2.09 (1 H, dd, J ) 7.4, 4.1
Hz), 1.30 (1 H, dd, J ) 4.6, 4.6 Hz); FAB-MS m/ z 464 (M +
H)+; FAB-HRMS calcd for C25H26N3O6 (M + H)+ m/ z 464.1822,
found 464.1830; IR (KBr) 3234, 1637, 1618, 1603, 1578, 1560,
1527, 1466, 1389, 1306, 1267, 1107 cm-1. Anal. (C25H25N3O6‚
2.0H2O) C, N; H: calcd, 5.85; found, 5.41.
8-O-(ter t-Bu t yld im et h ylsilyl)-2-m et h yl-3-(ben zylt h io-
ca r bon yl)-A-r in g-p yr r ole-DUMB2 (7n ). Meth od 3: To a
solution of 6 (50 mg, 0.074 mmol) in CH2Cl2 (2 mL) were added
benzyl mercaptan (0.0262 mL, 0.223 mmol), EDCI (71 mg,
0.372 mmol), and DMAP (45.4 mg, 0.372 mmol), and then the
mixture was stirred at room temperature for 69 h 40 min; 0.01
M phosphoric buffer (pH 7) was added, and the whole was
extracted with CHCl3 and then worked up as usual. The
residue was purified by PTLC (CHCl3-MeOH, 70:1) to give
44 mg (75%) of 7n : 1H-NMR (270 MHz, CDCl3) δ 9.44 (1 H,
brs), 8.31 (1 H, s), 8.01 (1 H, s), 7.42-7.46 (2 H, m), 7.28-7.37
(3 H, m), 7.00 (1 H, d, J ) 2.0 Hz), 6.90 (1 H, s), 4.67-4.75 (1
H, m), 4.51-4.58 (2 H, m), 4.44 (1 H, d, J ) 13.9 Hz), 4.32 (1
H, d, J ) 13.5 Hz), 4.06 (3 H, s), 3.94 (3 H, s), 3.92 (3 H, s),
3.61 (1 H, brd, J ) 9.9 Hz), 3.13 (1 H, dd, J ) 9.9, 9.2 Hz),
2-Met h yl-3-ca r b oxy-A-r in g-p yr r ole-DUMA (8e): yield
1
79%; H-NMR (270 MHz, DMSO-d6) δ 12.29 (1 H, brs), 12.23
(1 H, brs), 11.58 (1 H, brs), 7.01 (1 H, s), 6.93 (1 H, s), 6.48 (1
H, s), 4.45 (1 H, dd, J ) 10.7, 4.8 Hz), 4.27 (1 H, d, J ) 10.9

2954 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15
Amishiro et al.
Hz), 3.90 (3 H, s), 3.80 (3 H, s), 3.79 (3 H, s), 3.51-3.53 (1 H,
m), 2.47 (3 H, s), 2.18 (1 H, dd, J ) 7.3, 2.6 Hz), 1.43 (1 H, dd,
J ) 3.6, 3.3 Hz); FAB-MS m/ z 478 (M + H)+; IR (KBr) 3460,
2837, 1643, 1636, 1616, 1581, 1489, 1466, 1387, 1267, 1230,
1180, 1109 cm-1. Anal. (C25H23BrN3O7‚1.3H2O) C, H, N.
2-Me t h y l-3-(N -p h e n y lc a r b a m o y l)-A-r in g -p y r r o le -
DUMA (8f): yield 76%; 1H-NMR (270 MHz, CDCl3 + CD3-
OD) δ 7.50 (2 H, d, J ) 7.6 Hz), 7.30 (2 H, dd, J ) 8.3, 7.6 Hz),
7.08 (1 H, dd, J ) 7.6, 7.3 Hz), 6.94 (1 H, s), 6.86 (1 H, s), 6.74
(1 H, s), 4.41 (1 H, dd, J ) 10.2, 4.6 Hz), 4.33 (1 H, d, J ) 10.2
Hz), 3.99 (3 H, s), 3.86 (3 H, s), 3.81 (3 H, s), 3.40-3.46 (1 H,
m), 2.51 (3 H, s), 2.16 (1 H, dd, J ) 7.3, 3.6 Hz), 1.30-1.36 (1
H, m); FAB-MS m/z 553 (M + H)+; IR (KBr) 1653, 1647,
1635, 1601, 1578, 1525, 1489, 1389, 1263, 1107 cm-1. Anal.
(C31H28N4O6‚1.5H2O) C, H, N.
2-Me t h yl-3-(N -n -p r op ylca r b a m oyl)-A-r in g-p yr r ole -
DUMA (8g): yield 80%; 1H-NMR (270 MHz, CDCl3 + CD3-
OD) δ 6.92 (1 H, s), 6.86 (1 H, s), 6.74 (1 H, s), 6.33 (1 H, br),
4.39 (1 H, br), 4.32 (1 H, d, J ) 10.2 Hz), 3.96 (3 H, s), 3.85 (3
H, s), 3.81 (3 H, s), 3.42 (1 H, br), 3.23-3.28 (2 H, m), 2.40 (3
H, s), 2.09 (1 H, br), 1.52-1.55 (2 H, m), 1.29 (1 H, br), 0.90 (3
H, t, J ) 7.6 Hz); FAB-MS m/ z 519 (M + H)+; IR (KBr) 1639,
1618, 1579, 1527, 1466, 1389, 1304, 1263, 1109 cm-1. Anal.
(C28H30N4O6‚1.5H2O) C, H, N.
FAB-MS m/ z 571 (M + H)+; FAB-HRMS calcd for C30H27N4O6S
(M + H)+ m/z 571.1651, found 571.1641; IR (KBr) 1626, 1606,
1579, 1481, 1427, 1385, 1306, 1265, 1228, 1107 cm-1. Anal.
(C30H26N4O6S‚ 0.6H2O‚0.4CHCl3) C, H, N.
2-Meth yl-3-(1-n a p h th a len ylth ioca r bon yl)-A-r in g-p yr -
1
r ole-DUMA (8m ): yield 78%; H-NMR (270 MHz, CDCl3) δ
12.14 (1 H, brs), 9.45 (1 H, brs), 8.00 (1 H, brs), 7.79-7.88 (3
H, m), 7.47-7.56 (3 H, m), 7.20 (1 H, s), 6.90 (1 H, d, J ) 2.0
Hz), 6.79 (1 H, s), 4.41 (1 H, dd, J ) 10.2, 4.6 Hz), 4.35 (1 H,
d, J ) 9.9 Hz), 4.07 (3 H, s), 3.93 (3 H, s), 3.88 (3 H, s), 3.53-
3.59 (1 H, m), 3.00 (3 H, s), 2.28 (1 H, dd, J ) 7.6, 3.3 Hz),
1.30 (1 H, dd, J ) 4.6, 3.6 Hz); FAB-MS m/ z 620 (M + H)+;
IR (KBr) 1647, 1616, 1481, 1427, 1385, 1304, 1265, 1176, 1109,
858 cm-1. Anal. (C35H29N3O6S‚1.5H2O) C, H, N.
2-Me t h y l-3-(b e n zy lt h io c a r b o n y l)-A-r in g -p y r r o le -
1
DUMA (8n ): yield 57%; H-NMR (270 MHz, CDCl3) δ 11.78
(1 H, brs), 9.39 (1 H, brs), 7.23-7.37 (5 H, m), 7.14 (1 H, s),
6.95 (1 H, d, J ) 2.0 Hz), 6.81 (1 H, s), 4.42-4.51 (2 H, m),
4.26 (2 H, s), 4.07 (3 H s), 3.94 (3 H, s), 3.89 (3 H, s), 3.67-
3.70 (1 H, br), 2.77 (3 H, s), 2.31 (1 H, dd, J ) 7.6, 3.3 Hz),
1.35 (1 H, dd, J ) 4.3, 4.0 Hz); FAB-MS m/ z 584 (M + H)+;
IR (KBr) 1633, 1605, 1576, 1481, 1429, 1385, 1304, 1263, 1228,
1109 cm-1. Anal. (C32H29N3O6S‚0.7H2O) C, H, N.
2-Meth yl-3-(5-h yd r oxy-1,2-d ith ia n -4-yloxyca r bon yl)-A-
r in g-p yr r ole-DUMA (8o): yield 71%; 1H-NMR (270 MHz,
CDCl3 + CD3OD) δ 6.91 (1 H, s), 6.87 (1 H, d, J ) 0.7 Hz),
6.73 (1 H, s), 4.92-5.01 (1 H, m), 4.40 (1 H, dd, J ) 10.2, 4.3
Hz), 4.34 (1 H, d, J ) 10.2 Hz), 3.98 (1.5 H, s), 3.97 (1.5 H, s),
3.85 (1.5 H, s), 3.82 (1.5 H, s), 3.78-3.89 (1 H, m), 3.55-3.64
(1 H, m), 3.47 (3 H, s), 3.12-3.23 (2 H, m), 2.92-3.05 (2 H,
m), 2.53 (3 H, s), 2.34 (0.5 H, dd, J ) 7.8, 3.5 Hz), 2.25 (0.5 H,
dd, J ) 7.4, 3.5 Hz), 1.28-1.30 (1 H, m); FAB-MS m/ z 612 (M
+ H)+; FAB-HRMS calcd for C29H30N3O8S (M + H)+ m/ z
612.1475, found 612.1475; IR (KBr) 3205, 2941, 1643, 1612,
1527, 1446, 1387, 1304, 1265, 1105 cm-1. Anal. (C29H29N3O8S2‚
0.5H2O) C, H, N.
2-Meth yl-3-[2-(m eth ylsu lfon yl)eth oxycar bon yl]-A-r in g-
p yr r ole-DUMA (8p ): yield 61%; 1H-NMR (270 MHz, CDCl3)
δ 11.46 (1 H, brs), 9.40 (1 H, brs), 7.11 (1 H, s), 6.87 (1 H, d,
J ) 2.3 Hz), 6.76 (1 H, s), 4.66-4.77 (2 H, m), 4.46 (1 H, dd,
J ) 10.6, 4.6 Hz), 4.42 (1 H, d, J ) 10.6, Hz), 4.03 (3 H, s),
3.92 (3 H, s), 3.88 (3 H, s), 3.66-3.67 (1 H, m), 3.60 (1 H, brt,
J ) 6.1 Hz), 3.50 (1 H, brt, J ) 5.8 Hz), 3.03 (3 H, s), 2.60 (3
H, s), 2.31 (1 H, dd, J ) 7.4, 3.1 Hz), 1.32 (1 H, dd, J ) 4.3,
4.0 Hz); FAB-MS m/ z 584 (M + H)+; IR (KBr) 1645, 1537,
1616, 1489, 1398, 1302, 1265, 1103 cm-1. Anal. (C28H29N3O9S‚
1.0H2O) C, H, N.
2-Met h yl-3-[N-(2-d im et h yla m in oet h yl)ca r b a m oyl]-A-
r in g-p yr r ole-DUMA (8h ): yield 50%; 1H-NMR (270 MHz,
CDCl3 + CD3OD) δ 6.88 (1 H, s), 6.85 (1 H, s), 6.72 (1 H, s),
4.39 (1 H, dd, J ) 10.2, 4.6 Hz), 4.30 (1 H, d, J ) 10.2 Hz),
3.96 (3 H, s), 3.83 (3 H, s), 3.80 (3 H, s), 3.31-3.44 (1 H, m),
3.39 (2 H, t, J ) 6.1 Hz), 2.45 (2 H, t, J ) 6.1 Hz), 2.41 (3 H,
s), 2.21 (6 H, s), 2.09 (1 H, dd, J ) 7.6, 3.6 Hz), 1.29 (1 H, dd,
J ) 4.3, 4.3 Hz); FAB-MS m/ z 548 (M + H)+; FAB-HRMS calcd
for C29N34N5O6 (M + H)+ m/ z 548.2509, found 548.2504; IR
(KBr) 1633, 1616, 1581, 1525, 1464, 1385, 1304, 1265, 1230,
1107 cm-1. Anal. (C29H33N5O6‚0.9H2O‚0.6CHCl3) C, H, N.
2-Meth yl-3-[(4-m eth yl-1-p ip er a zin yl)ca r bon yl]-A-r in g-
1
p yr r ole-DUMA (8i): yield 70%; H-NMR (270 MHz, CDCl3)
δ 11.45 (1 H, s), 9.41 (1 H, s), 7.04 (1 H, s), 6.93 (1 H, d, J )
2.3 Hz), 6.80 (1 H, s), 4.45 (1 H, dd, J ) 10.1, 4.5 Hz), 4.38 (1
H, d, J ) 10.2 Hz), 4.06 (3 H, s), 3.93 (3 H, s), 3.89 (3 H, s),
3.40-3.70 (3 H, br), 2.30-2.60 (6 H, br), 2.36 (3 H, s), 2.32 (3
H, s), 2.27 (1 H, br), 1.44 (1 H, br); FAB-MS m/ z 560 (M +
H)+; FAB-HRMS calcd for C30H34N5O6 (M + H)+ m/ z 560.2509,
found 560.2502; IR (KBr) 1633, 1614, 1585, 1470, 1392, 1298,
1261, 1228, 1138, 1109, 999 cm-1. Anal. (C30H33N5O6‚0.8H2O‚
0.5CHCl3) C, H; N: calcd, 11.05; found, 10.59.
2-Meth yl-3-(2-br om oeth oxyca r bon yl)-A-r in g-p yr r ole-
1
DUMA (8j): yield 82%; H-NMR (270 MHz, CDCl3) δ 11.55
2-Met h yl-3-[N-(2-d im et h yla m in oet h yl)ca r b a m oyl]-A-
r in g-p yr r ole-DUMC2 H yd r och lor id e (9h ). A solution of
8h (19 mg, 0.034 mmol) in CH2Cl2 (1.74 mL) was treated
with 4 N HCl in AcOEt (0.026 mL) at room temperature for
30 min. The mixture was concentrated under reduced pressure
to give 23 mg of 9h : 1H-NMR (270 MHz, DMSO-d6) δ 11.45 (1
H, s), 11.32 (1 H, s), 10.16 (1 H, br), 9.98 (1 H, brs), 8.00 (1 H,
t, J ) 5.3 Hz), 7.66 (1 H, brs), 6.97 (1 H, s), 6.96 (1 H, s), 4.57
(1 H, dd, J ) 10.9, 8.6 Hz), 4.35 (1 H, d, J ) 11.2 Hz), 4.10-
4.23 (1 H, m), 3.93 (3 H, s), 3.81 (3 H, s), 3.79 (3 H, s), 3.60-
3.72 (3 H, m), 3.38 (1 H, dd, J ) 9.9, 9.6 Hz), 3.13-3.33 (2 H,
m), 2.84 (6 H, t, J ) 5.3 Hz), 2.55 (3 H, s); IR (KBr) 1525,
(1 H, brs), 9.39 (1 H, brs), 7.13 (1 H, s), 6.96 (1 H, d, J ) 2.0
Hz), 6.81 (1 H, s), 4.56 (2 H, t, J ) 5.6 Hz), 4.46 (2 H, br), 4.08
(3 H, s), 3.94 (3 H, s), 3.90 (3 H, s), 3.67-3.69 (1 H, m), 3.63 (2
H, t, J ) 5.6 Hz), 2.69 (3 H, s), 2.39 (1 H, dd, J ) 7.3, 3.3 Hz),
1.38 (1 H, dd, J ) 4.3, 4.0 Hz); FAB-MS m/ z 586, 584 (M +
H)+; FAB-HRMS calcd for C27H2779BrN3O7 (M + H)+ m/ z
554.1927, found 554.1907; IR (KBr) 1643, 1605, 1487, 1387,
1294, 1265, 1230, 1209, 1105 cm-1. Anal. (C27H26BrN3O7‚
0.5H2O) C, H, N.
2-Me t h y l-3-(p h e n y lt h io c a r b o n y l)-A-r in g -p y r r o le -
1
DUMA (8k ): yield 78%; H-NMR (270 MHz, CDCl3) δ 12.00
1495, 1466, 1429, 1390, 1315, 1224, 1128, 1107, 1045 cm-1
.
(1 H, brs), 9.40 (1 H, s), 7.43-7.52 (5 H, m), 7.18 (1 H, s), 6.92
(1 H, d, J ) 2.0 Hz), 6.81 (1 H, s), 4.42 (1 H, dd, J ) 10.6, 4.6
Hz), 4.37 (1 H, d, J ) 9.9 Hz), 4.07 (3 H, s), 3.94 (3 H, s), 3.89
(3 H, s), 3.57-3.63 (1 H, m), 2.94 (3 H, s), 2.28 (1 H, dd, J )
7.4, 3.5 Hz), 1.31 (1 H, dd, J ) 4.6, 4.0 Hz); FAB-MS m/z
570 (M + H)+; FAB-HRMS calcd for C31H28N3O6S (M + H)+
m/ z 570.1699, found 570.1689; IR (KBr) 1647, 1630, 1479,
1427, 1396, 1385, 1304, 1263, 1228, 1109, 858 cm-1. Anal.
(C31H27N3O6S‚0.8H2O) C, H, N.
Anal. (C29H34ClN5O6‚HCl‚1.5H2O) C, H; N: calcd, 10.82; found,
9.72.
2-Meth yl-3-[(4-m eth yl-1-p ip er a zin yl)ca r bon yl]-A-r in g-
p yr r ole-DUMC2 h yd r och lor id e (9i): 1H-NMR (270 MHz,
DMSO-d6) δ 11.51 (1 H, s), 11.33 (1 H, brs), 10.88 (1 H, br),
10.01 (1 H, brs), 7.67 (1 H, brs), 6.96 (2 H, brs), 4.62 (1 H, dd,
J ) 10.2, 9.2 Hz), 4.33 (1 H, d, J ) 11.2 Hz), 4.17-4.30 (1 H,
m), 3.93 (3 H, s), 3.81 (3 H, s), 3.79 (3 H, s), 3.00-3.75 (10 H,
br), 2.81 (3 H, brs), 2.42 (3 H, s); IR (KBr) 1612, 1495, 1466,
2-Meth yl-3-(4-p yr id in ylth ioca r bon yl)-A-r in g-p yr r ole-
1427, 1390, 1315, 1223, 1117, 1047, 974 cm-1. Anal. (C30H34
-
1
DUMA (8l): yield 64%; H-NMR (270 MHz, CDCl3) δ 12.38
ClN5O6‚HCl‚4.0H2O) C, H, N.
(1 H, brs), 9.48 (1 H, brs), 8.65 (2 H, d, J ) 5.0 Hz), 7.42 (2 H,
d, J ) 5.0 Hz), 7.21 (1 H, s), 6.91 (1 H, brs), 6.80 (1 H, s), 4.42
(2 H, br), 4.06 (3 H, s), 3.94 (3 H, s), 3.89 (3 H, s), 3.57 (1 H,
m), 2.93 (3 H, s), 2.24 (1 H, dd, J ) 7.4, 3.4 Hz), 1.32 (1 H, br);
8-O-(ter t-Bu tyld im eth ylsilyl)-2-m eth yl-A-r in g-p yr r ole-
DUMB2 (10). A solution of 6 (138 mg, 0.205 mmol) in
bromobenzene (11.4 mL) was stirred at reflux (165 °C) for 10

Synthesis, Activity of Duocarmycin Derivatives
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 2955
8-O-(ter t-Bu tyldim eth ylsilyl)-2-m eth yl-3-(dim eth ylam i-
h. Then the resulting mixture was concentrated in vacuo. The
residue was purified by PTLC (hexane-AcOEt, 2:1) to give
77 mg (60%) of 10: 1H-NMR (400 MHz, CDCl3) δ 9.44 (1 H,
brs), 7.90 (1 H, s), 7.83 (1 H, s), 6.95 (1 H, d, J ) 2.4 Hz), 6.88
(1 H, s), 6.14 (1 H, q, J ) 1.1 Hz), 4.85 (1 H, m), 4.50 (2 H, m),
4.06 (3 H, s), 3.94 (3 H, s), 3.91 (3 H, s), 3.90 (1 H, m), 3.38 (1
H, dd, J ) 10.3, 10.3 Hz), 2.48 (3 H, s), 1.07 (9 H, s), 0.36 (6
H, s); EI-MS m/ z 629, 627 (M)+.
8-O-(ter t-Bu tyldim eth ylsilyl)-2-m eth yl-3-ch lor o-A-r in g-
p yr r ole-DUMB2 (11a ). To a solution of 10 (150 mg, 0.239
mmol) in CH2Cl2 (6 mL) were added NCS (38.3 mg, 0.287
mmol) and silica gel (49.5 mg), and the mixture was stirred
under darkness at room temperature for 3 h; 0.01 M phos-
phoric buffer (pH 7) was added, and the whole was extracted
with CHCl3 and then worked up as usual. The residue was
purified by PTLC (hexane-AcOEt, 2:1) to give 113 mg (71%)
of 11a : 1H-NMR (270 MHz, CDCl3) δ 9.53 (1 H, brs), 8.12 (1
H, s), 7.89 (1 H, brs), 6.95 (1 H, d, J ) 1.7 Hz), 6.88 (1 H, s),
4.72 (1 H, d, J ) 10.6 Hz), 4.54 (1 H, dd, J ) 10.6, 8.6 Hz),
4.16-4.25 (1 H, m), 4.04 (3 H, s), 3.97 (1 H, dd, J ) 10.2, 3.0
Hz), 3.93 (3 H, s), 3.90 (3 H, s), 3.30 (1 H, dd, J ) 10.2, 10.2
Hz), 2.42 (3 H, s), 1.05 (9 H, s), 0.35 (6 H, s); FAB-MS m/ z
664, 662 (M + H)+.
8-O-(ter t-Bu tyld im eth ysilyl)-2-m eth yl-3-br om o-A-r in g-
p yr r ole-DUMB2 (11b): yield 62%; 1H-NMR (270 MHz,
CDCl3) δ 9.46 (1 H, brs), 8.10 (1 H, s), 7.87 (1 H, brs), 6.97 (1
H, d, J ) 2.3 Hz), 6.89 (1 H, s), 4.75 (1 H, d, J ) 10.9 Hz), 4.56
(1 H, dd, J ) 10.2, 8.9 Hz), 4.27-4.33 (1 H, m), 4.06 (3 H, s),
4.01 (1 H, dd, J ) 10.2, 2.6 Hz), 3.94 (3 H, s), 3.92 (3 H, s),
3.27 (1 H, dd, J ) 10.6, 10.2 Hz), 2.45 (3 H, s), 1.06 (9 H, s),
0.36 (3 H, s), 0.35 (3 H, s); FAB-MS m/ z 710, 708, 706 (M +
H)+.
8-O-(ter t-Bu t yld im et h ylsilyl)-2-m et h yl-3-iod o-A-r in g-
p yr r ole-DUMB2 (11c): yield 47%; 1H-NMR (270 MHz,
CDCl3) δ 9.48 (1 H, brs), 8.33 (1 H, brs), 7.88 (1 H, brs), 6.96
(1 H, d, J ) 2.3 Hz), 6.89 (1 H, s), 4.76 (1 H, d, J ) 10.9 Hz),
4.55 (1 H, dd, J ) 10.6, 8.3 Hz), 4.37-4.43 (1 H, m), 4.05 (3 H,
s), 4.02 (1 H, dd, J ) 10.9, 1.8 Hz), 3.94 (3 H, s), 3.91 (3 H, s),
3.22 (1 H, dd, J ) 10.9, 10.2 Hz), 2.48 (3 H, s), 1.05 (9 H, s),
0.36 (3 H, s), 0.35 (3 H, s); FAB-MS m/ z 755, 753 (M + H)+.
8-O-(ter t-Bu tyld im eth ylsilyl)-2-m eth yl-3-a cetyl-A-r in g-
p yr r ole-DUMB2 (11d ). To a solution of 6 (15 mg, 0.024 mmol)
in CH2Cl2 (0.8 mL) were added Ac2O (0.0068 mL, 0.072 mmol)
and BF3-Et2O (0.0088 mL, 0.072 mmol), and the mixture was
stirred at room temperature for 14 h 40 min. Aqueous NaHCO3
was added, and the whole was extracted with CHCl3 and then
worked up as usual. The residue was purified by PTLC
(hexane-AcOEt, 1:1) to give 7.6 mg (47%) of 11d : 1H-NMR
(270 MHz, CDCl3) δ 9.46 (1 H, brs), 8.50 (1 H, brs), 8.02 (1 H,
s), 6.99 (1 H, d, J ) 1.3 Hz), 6.88 (1 H, s), 4.70 (1 H, d, J ) 9.9
Hz), 4.65-4.75 (1 H, br), 4.51 (1 H, dd, J ) 9.9, 9.6 Hz), 4.05
(3 H, s), 3.93 (3 H, s), 3.91 (3 H, s), 3.74 (1 H, dd, J ) 9.2, 2.6
Hz), 3.18 (1 H, dd, J ) 9.6, 9.6 Hz), 2.77 (3 H, s), 2.56 (3 H, s),
1.05 (9 H, s), 0.37 (3 H, s), 0.36 (3 H, s); FAB-MS m/z 672, 670
(M + H)+.
8-O-(ter t-Bu tyldim eth ylsilyl)-2-m eth yl-3-bu tyr yl-A-r in g-
p yr r ole-DUMB2 (11e): yield 47%; 1H-NMR (270 MHz,
CDCl3) δ 9.43 (1 H, brs), 8.28 (1 H, brs), 8.02 (1 H, s), 7.00 (1
H, s), 6.89 (1 H, s), 4.71-4.77 (1 H, m), 4.69 (1 H, d, J ) 10.2
Hz), 4.54 (1 H, dd, J ) 10.2, 8.9 Hz), 4.06 (3 H, s), 3.94 (3 H,
s), 3.92 (3 H, s), 3.64 (1 H, dd, J ) 9.6, 3.0 Hz), 3.17 (1 H, dd,
J ) 9.9, 9.6 Hz), 2.83 (2 H, t, J ) 7.4), 2.77 (3 H, s), 1.83 (2 H,
tq, J ) 7.3, 7.3 Hz), 1.06 (9 H, s), 1.04 (3 H, t, J ) 7.6 Hz),
0.38 (3 H, s), 0.36 (3 H, s); FAB-MS m/z 700, 698 (M + H)+.
8-O-(ter t-Bu tyldim eth ylsilyl)-2-m eth yl-3-ben zoyl-A-r in g-
p yr r ole-DUMB2 (11f): yield 51%; 1H-NMR (270 MHz, CDCl3)
δ 9.54 (1 H, brs), 8.43 (1 H, brs), 8.04 (1 H, brs), 7.78 (2 H, d,
J ) 6.9 Hz), 7.56-7.58 (1 H, m), 7.48 (2 H, dd, J ) 7.6, 6.9
Hz), 6.96 (1 H, d, J ) 2.0 Hz), 6.86 (1 H, s), 4.58 (1 H, dd, J )
10.6, 2.3 Hz), 4.50 (1 H, dd, J ) 10.6, 8.6 Hz), 4.06 (3 H, s),
3.94 (3 H, s), 3.90 (3 H, s), 3.87-3.93 (1 H, m), 3.51 (1 H, dd,
J ) 10.1, 3.1 Hz), 3.11 (1 H, dd, J ) 9.9, 9.9 Hz), 2.27 (3 H, s),
1.07 (9 H, s), 0.41 (3 H, s), 0.39 (3 H, s); FAB-MS m/ z 734,
732 (M + H)+.
n om eth yl)-A-r in g-p yr r ole-DUMB2 (11g). Me2
NCH2NMe2
(0.0434 mL, 0.318 mmol) was added to TFA (1 mL), and the
mixture was stirred under Ar atmosphere at -10 °C for 20
min. Then, 27 (40 mg, 0.064 mmol) was added, and stirring
was continued at -10 °C for 50 min. Aqueous NaHCO3 was
added, and the whole was extracted with CHCl3 and then
worked up as usual. The residue was purified by PTLC
(CHCl3-MeOH, 8:1) to give 31.1 mg (71%) of 11g: 1H-NMR
(270 MHz, CDCl3) δ 9.47 (1 H, s), 7.89 (1 H, brs), 7.82 (1 H,
brs), 6.95 (1 H, d, J ) 2.0 Hz), 6.87 (1 H, s), 4.71 (1 H, d, J )
10.9 Hz), 4.53 (1 H, dd, J ) 9.9, 8.3 Hz), 4.16-4.27 (2 H, m),
4.05 (3 H, s), 3.93 (3 H, s), 3.91 (3 H, s), 3.58 (1 H, d, J ) 13.2
Hz), 3.36 (1 H, d, J ) 13.2 Hz), 3.20 (1 H, dd, J ) 10.6, 10.2
Hz), 2.42 (3 H, s), 2.29 (6 H, s), 1.06 (9 H, s), 0.36 (3 H, s), 0.34
(3 H, s); FAB-MS m/ z 687, 685 (M + H)+.
8-O-(ter t-Bu tyldim eth ylsilyl)-2-m eth yl-3-for m yl-A-r in g-
p yr r ole-DUMB2 (11h ). To a solution of 10 (905 mg, 1.44
mmol) in DMF (36 mL) was added POCl3, (0.161 mL, 1.73
mmol), and the mixture was stirred at room temperature for
1 h 15 min; 0.01 M phosphoric buffer (pH 7) was added, and
the whole was extracted with AcOEt and then worked up as
usual. The residue was purified by column chromatography
(hexane-AcOEt, 3:1-1:1) to give 808 mg (85%) of 11h : 1H-
NMR (270 MHz, CDCl3) δ 10.06 (1 H, s), 9.43 (1 H, brs), 8.56
(1 H, s), 8.02 (1 H, s), 7.00 (1 H, d, J ) 2.3 Hz), 6.89 (1 H, s),
4.69-4.75 (1 H, m), 4.54-4.58 (2 H, m), 4.06 (3 H, s), 3.94 (3
H, s), 3.92 (3 H, s), 3.80 (1 H, brd, J ) 9.2 Hz), 3.27 (1 H, dd,
J ) 9.2, 9.2 Hz), 2.77 (3 H, s), 1.06 (9 H, s), 0.39 (3 H, s), 0.37
(3 H, s); FAB-MS m/z 658, 656 (M + H)+.
8-O-(ter t-Bu tyld ieth ylsilyl)-2-m eth yl-3-(1,3-d ith ia n yl)-
A-r in g-p yr r ole-DUMB2 (11i). To a solution of 11h (70 mg,
0.11 mmol) in THF (2.8 mL) were added 1,3-propanedithiol
(0.0215 mL, 0.214 mmol) and Amberlyst-15 (12 mg), and then
the mixture was stirred at room temperature for 17 h. After
addition of 1,3-propanedithiol (0.0215 mL, 0.214 mmol) and
Amberlyst-15 (12 mg), the mixture was stirred for 5 h 15 min.
Aqueous NaHCO3 was added, and the whole was extracted
with CHCl3 and then worked up as usual. The residue was
purified by column chromatography (hexane-AcOEt, 4:1) to
give 52 mg (65%) of 11i: 1H-NMR (270 MHz, CDCl3) δ 9.44 (1
H, brs), 7.94 (1 H, s), 7.82 (1 H, brs), 6.96 (1 H, d, J ) 2.0 Hz),
6.89 (1 H, s), 5.58 (1 H, s), 4.71 (1 H, d, J ) 10.9 Hz), 4.59 (1
H, dd, J ) 10.6, 7.9 Hz), 4.28 (1 H, br), 4.60-4.12 (1 H, m),
4.06 (3 H, s), 3.94 (3 H, s), 3.92 (3 H, s), 3.25 (1 H, dd, J )
10.9, 10.9 Hz), 3.10-3.19 (2 H, td, J ) 12.5, 1.0 Hz), 2.94 (2
H, dd, J ) 13.9, 1.0 Hz), 2.70 (3 H, s), 2.21 (1 H, brd, J ) 13.9
Hz), 1.92-2.02 (1 H, m), 1.05 (9 H, s), 0.36 (3 H, s), 0.34 (3 H,
s); FAB-MS m/ z 748, 746 (M + H)+.
8-O-(ter t-Bu tyld im eth ylsilyl)-2-m eth yl-3-(h yd r oxyim i-
n om eth yl)-A-r in g-p yr r ole-DUMB2 (11j). To a solution of
11h (25 mg, 0.038 mmol) in ethanol (1 mL) was added
hydroxyamine hydrochloric acid (5.3 mg, 0.076 mmol), and
then the mixture was stirred at room temperature for 1 h 40
min. After addition of hydroxyamine hydrochloric acid (5.3 mg,
0.076 mmol), the mixture was stirred for 15 h; 0.01 M
phosphoric buffer (pH 7) was added, and the whole was
extracted with CHCl3 and then worked up as usual. The
residue was purified by PTLC (hexane-AcOEt, 1:1) to give
15 mg (57%) of 11j: 1H-NMR (270 MHz, CDCl3) δ 9.46 (1 H,
brs), 8.34 (1 H, s), 8.16 (1 H, brs), 7.95 (1 H, brs), 7.20 (1 H,
br), 6.97 (1 H, d, J ) 2.0 Hz), 6.88 (1 H, s), 4.72 (1 H, d, J )
9.9 Hz), 4.45 (1 H, dd, J ) 10.9, 9.9 Hz), 4.41-4.52 (1 H, m),
4.05 (3 H, s), 3.95 (1 H, br), 3.94 (3 H, s), 3.91 (3 H, s), 3.29 (1
H, dd, J ) 10.2, 9.9 Hz), 2.53 (3 H, s), 1.06 (9 H, s), 0.37 (3 H,
s), 0.36 (3 H, s); FAB-MS m/z 673, 671 (M + H)+.
8-O-(ter t-Bu tyld im eth ylsilyl)-2-m eth yl-3-(m eth oxyim i-
n om eth yl)-A-r in g-p yr r ole-DUMB2 (11k ): yield 87%; 1H-
NMR (270 MHz, CDCl3) δ 9.46 (1 H, brs), 8.29 (1 H, d, J ) 1.0
Hz), 8.20 (1 H, brs), 7.97 (1 H, brs), 6.99 (1 H, d, J ) 1.6 Hz),
6.88 (1 H, s), 4.78 (1 H, d, J ) 9.9 Hz), 4.57-4.62 (1 H, m),
4.53 (1 H, dd, J ) 9.9, 8.6 Hz), 4.07 (3 H, s), 4.06 (3 H, d, J )
1.0 Hz), 3.94 (3 H, s), 3.92 (3 H, s), 3.90 (1 H, dd, J ) 10.2, 2.3

2956 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15
Amishiro et al.
Hz), 3.17 (1 H, dd, J ) 10.2, 9.9 Hz), 2.51 (3 H, s), 1.06 (9 H,
s), 0.38 (3 H, s), 0.36 (3 H, s); FAB-MS m/ z 687, 685 (M +
H)+.
The synthesis of compounds 12a -12k was performed ac-
cording to the same procedure as for 8a .
2-Meth yl-3-ch lor o-A-r in g-p yr r ole-DUMA (12a ): yield
78%; 1H-NMR (270 MHz, CDCl3) δ 11.59 (1 H, brs), 9.55 (1 H,
brs), 7.00 (1 H, s), 6.93 (1 H, s), 6.78 (1 H, s), 4.43 (2 H, m),
4.05 (3 H, s), 3.93 (3 H, s), 3.88 (3 H, s), 3.14 (1 H, br), 2.33 (3
H, s), 2.28 (1 H, dd, J ) 7.6, 4.3 Hz), 1.35 (1 H, dd, J ) 4.6,
4.0 Hz); FAB-MS m/ z 468 (M + H)+; FAB-HRMS calcd for
C24H23ClN3O5 (M + H)+ m/ z 468.1326, found 468.1328; IR
(KBr) 1637, 1608, 1581, 1527, 1473, 1385, 1304, 1265, 1105
cm-1. Anal. (C24H22ClN3O5‚1.0H2O) C, H; N: calcd, 8.65; found,
8.04.
s), 9.47 (1 H, brs), 7.17 (1 H, s), 6.96 (1 H, d, J ) 2.4 Hz), 6.82
(1 H, s), 4.48 (2 H, br), 4.07 (3 H, s), 3.94 (3 H, s), 3.90 (3 H,
s), 3.65 (1 H, m), 2.70 (3 H, s), 2.36 (1 H, dd, J ) 9.0, 3.3 Hz),
1.38 (1 H, dd, J ) 4.3, 4.1 Hz); FAB-MS m/ z 462 (M + H)+;
FAB-HRMS calcd for C25H24N3O6 (M + H)+ m/ z 462.1665,
found 462.1649; IR (KBr) 1647, 1610, 1585, 1527, 1487, 1468,
1387, 1265, 1228, 1109 cm-1. Anal. (C25H23N3O6‚1.0H2O) C,
H, N.
2-Meth yl-3-(1,3-d ith ia n yl)-A-r in g-p yr r ole-DUMA (12i):
yield 78%; 1H-NMR (270 MHz, CDCl3) δ 10.81 (1 H, brs), 9.42
(1 H, brs), 6.99 (1 H, s), 6.93 (1 H, d, J ) 2.3 Hz), 6.80 (1 H, s),
5.04 (1 H, s), 4.47 (1 H, dd, J ) 10.2, 4.3 Hz), 4.40 (1 H, d, J
) 10.2 Hz), 4.06 (3 H, s), 3.93 (3 H, s), 3.90 (3 H, s), 3.36 (1 H,
br), 3.01 (2 H, td, J ) 12.2, 0.8 Hz), 2.87 (2 H, dd, J ) 12.0,
0.8 Hz), 2.55 (3 H, s), 2.31 (1 H, br), 2.16 (1 H, brd, J ) 14.2
Hz), 1.85-1.94 (1 H, m), 1.43 (1 H, dd, J ) 4.6, 4.6 Hz);
FAB-MS m/ z 552 (M + H)+; IR (KBr) 1637, 1618, 1579, 1527,
1466, 1396, 1383, 1304, 1261, 1109 cm-1. Anal. (C28H29N3O5S)
C, H, N.
2-Meth yl-3-br om o-A-r in g-p yr r ole-DUMA (12b): yield
78%; 1H-NMR (270 MHz, CDCl3 + CD3OD) δ 6.87 (1 H,
s), 6.85 (1 H, s), 6.74 (1 H, s), 4.33-4.42 (2 H, m), 4.00 (3
H, s), 3.87 (3 H, s), 3.82 (3 H, s), 3.19 (1 H, m), 2.30 (1 H, dd,
J ) 7.4, 4.1 Hz), 2.25 (3 H, s), 1.31 (1 H, dd, J ) 4.6, 4.6
Hz); FAB-MS m/z 514, 512 (M + H)+; FAB-HRMS calcd for
2-Me t h yl-3-(h yd r oxyim in om e t h yl)-A-r in g-p yr r ole -
1
DUMA (12j): yield 94%; H-NMR (270 MHz, CDCl3 + CD3-
24H2379BrN3O5 (M + H)+ m/ z 512.0821, found 512.0840; IR
OD) δ 7.96 (1 H, s), 6.87 (1 H, s), 6.85 (1 H, s), 6.72 (1 H, s),
4.34 (1 H, dd, J ) 10.2, 4.3 Hz), 4.28 (1 H, d, J ) 10.2 Hz),
3.97 (3 H, s), 3.84 (3 H, s), 3.80 (3 H, s), 3.43-3.49 (1 H, m),
2.34 (1 H, dd, J ) 7.6, 3.3 Hz), 2.31 (3 H, s), 1.19 (1 H, dd, J
) 4.6, 4.0 Hz); FAB-MS m/ z 477 (M + H)+; FAB-HRMS calcd
for C25H25N4O6 (M + H)+ m/ z 477.1774, found 477.1767; IR
(KBr) 3463, 1579, 1527, 1491, 1468, 1425, 1385, 1306, 1263,
1198, 1109 cm-1. Anal. (C25H24N4O6‚0.7H2O‚0.2CHCl3) C, H;
N: calcd, 10.92; found, 10.32.
C
(KBr) 1633, 1608, 1581, 1470, 1387, 1304, 1263, 1111, 1051
cm-1. Anal. (C24H22BrN3O5‚0.5H2O) C, H; N: calcd, 8.05; found,
7.45.
2-Meth yl-3-iod o-A-r in g-p yr r ole-DUMA (12c): yield 83%;
1H-NMR (270 MHz, CDCl3) δ 11.66 (1 H, s), 9.50 (1 H, s), 7.02
(1 H, s), 6.94 (1 H, d, J ) 1.7 Hz), 6.79 (1 H, s), 4.39-4.51 (2
H, m), 4.06 (3 H, s), 3.93 (3 H, s), 3.89 (3 H, s), 3.36 (1 H,
m), 2.37 (3 H, s), 2.30-2.45 (1 H, br), 1.32 (1 H, dd, J ) 4.6,
4.6 Hz); FAB-MS m/ z 560 (M + H)+; FAB-HRMS calcd for
C24H23IN3O5 (M + H)+ m/ z 560.0682, found 560.0673; IR (KBr)
2-Me t h yl-3-(m e t h oxyim in om e t h yl)-A-r in g-p yr r ole -
DUMA (12k ): yield 90%; 1H-NMR (270 MHz, CDCl3) δ 11.74
(1 H, s), 9.55 (1 H, s), 8.01 (1 H, s), 7.09 (1 H, s), 6.94 (1 H, d,
J ) 2.0 Hz), 6.78 (1 H, s), 4.40-4.44 (2 H, m), 4.06 (3 H, s),
3.93 (3 H, s), 3.92 (3 H, s), 3.89 (3 H, s), 3.60-3.67 (1 H, m),
2.48 (1 H, dd, J ) 7.4, 3.5 Hz), 2.42 (3 H, s), 1.27 (1 H, dd, J
) 4.0, 3.7 Hz); FAB-MS m/ z 491 (M + H)+; IR (KBr) 1603,
1633, 1605, 1581, 1466, 1385, 1304, 1261, 1109, 1039 cm-1
Anal. (C24H22IN3O5) C, H, N.
.
2-Meth yl-3-acetyl-A-r in g-pyr r ole-DUMA (12d): yield 70%;
1H-NMR (270 MHz, CDCl3 + CD3OD) δ 6.97 (1 H, s), 6.88 (1
H, s), 6.75 (1 H, s), 4.37 (2 H, m), 3.99 (3 H, s), 3.86 (3 H, s),
3.82 (3 H, s), 3.65-3.75 (1 H, m), 2.59 (3 H, s), 2.38 (3 H, s),
2.29 (1 H, dd, J ) 7.4, 3.1 Hz), 1.23 (1 H, dd, J ) 4.6, 3.6 Hz);
FAB-MS m/ z 476 (M + H)+; IR (KBr) 1649, 1603, 1483, 1460,
1387, 1302, 1265, 1228, 1109 cm-1. Anal. (C26H25N3O6‚0.5H2O)
C, H, N.
2-Met h yl-3-bu t yr yl-A-r in g-p yr r ole-DUMA (12e): yield
63%; 1H-NMR (270 MHz, CDCl3) δ 12.10 (1 H, brs), 9.44 (1 H,
brs), 7.18 (1 H, s), 6.95 (1 H, d, J ) 2.0 Hz), 6.82 (1 H, s), 4.45
(2 H, br), 4.07 (3 H, s), 3.94 (3 H, s), 3.89 (3 H, s), 3.83 (1 H,
m), 2.74 (3 H, s), 2.67 (2 H, t, J ) 7.1 Hz), 2.33 (1 H, dd, J )
7.3, 3.3 Hz), 1.68 (2 H, tq, J ) 7.6, 7.3 Hz), 1.28 (1 H, dd, J )
4.6, 3.6 Hz), 0.96 (3 H, t, J ) 7.4 Hz); FAB-MS m/ z 504 (M +
H)+; IR (KBr) 1647, 1618, 1605, 1439, 1396, 1385, 1303, 1265,
1230, 1109 cm-1. Anal. (C28H29N3O6‚0.6H2O) C, H, N.
2-Met h yl-3-ben zoyl-A-r in g-p yr r ole-DUMA (12f): yield
40%; 1H-NMR (270 MHz, CDCl3) δ 11.96 (1 H, brs), 9.45 (1 H,
brs), 7.68 (2 H, d, J ) 7.6 Hz), 7.54-7.59 (1 H, m), 7.46 (2 H,
dd, J ) 7.9, 6.9 Hz), 7.14 (1 H, s), 6.95 (1 H, s), 6.80 (1 H, s),
4.50 (1 H, dd, J ) 10.4, 4.5 Hz), 4.43 (1 H, d, J ) 10.2 Hz),
4.06 (3 H, s), 3.93 (3 H, s), 3.88 (3 H, s), 3.50-3.52 (1 H,
m), 2.19 (1 H, dd J ) 7.4, 3.5 Hz), 2.09 (3 H, s), 1.41 (1 H, dd,
J ) 4.0, 4.0 Hz); FAB-MS m/ z 538 (M + H)+; IR (KBr) 1641,
1614, 1601, 1470, 1385, 1302, 1263, 1230, 1107 cm-1. Anal.
(C31H27N3O6‚1.5H2O) C, H, N.
1583, 1527, 1489, 1466, 1385, 1306, 1263, 1109, 1051 cm-1
.
Anal. (C26H26N4O6‚0.8H2O) C, H; N: calcd, 11.10; found, 10.38.
The synthesis of compound 13 was performed according to
the same procedure as for 9h .
2-Me t h yl-3-(d im e t h yla m in om e t h yl)-A-r in g-p yr r ole -
DUMC2 h yd r och lor id e (13): 1H-NMR (270 MHz, DMSO-
d6) δ 11.50 (1 H, brs), 11.38 (1 H, brs), 10.01 (1 H, s), 9.61 (1
H, br), 7.62 (1 H, br), 6.98 (1 H, s), 6.96 (1 H, s), 4.57 (1 H, dd,
J ) 10.6, 7.3 Hz), 4.45 (1 H, d, J ) 11.2 Hz), 4.38-4.46 (1 H,
m), 4.11-4.16 (1 H, m), 3.95-4.04 (1 H, m), 3.92 (3 H, s), 3.83-
3.88 (1 H, br), 3.82 (3 H, s), 3.79 (3 H, s), 3.51 (1 H, dd, J )
9.6, 9.5 Hz), 2.81 (3 H, d, J ) 4.3 Hz), 2.71 (3 H, d, J ) 3.6
Hz), 2.46 (3 H, s); IR (KBr) 1637, 1608, 1527, 1495, 1466, 1427,
1390, 1315, 1223, 1107, 1047 cm-1. Anal. (C27H31ClN4O5‚HCl‚
1.0H2O) C, H; N: calcd, 9.63; found, 8.76.
2-Meth yl-A-r in g-p yr r ole-DUM-Seg-A (14a ). To a solution
of 10 (600 mg, 0.954 mmol) in THF (40.9 mL) was added
nBu4NF (1.0 M in THF, 3.17 mL, 2.86 mmol), and then the
mixture was stirred at room temperature for 30 min. The
mixture was concentrated under reduced pressure, and NaOMe
(28 wt % in MeOH, 0.551 mL) was added to a solution of the
residue in MeOH (40.9 mL). After the mixture stirred at room
temperature for 60 min, 0.01 M phosphoric buffer (pH 7) was
added, and the whole was extracted with CHCl3 and then
worked up as usual. The residue was purified by column
chromatography (CHCl3-MeOH, 40:1-20:1) to give 170 mg
(89%) of 14a : 1H-NMR (270 MHz, CDCl3 + CD3OD) δ 5.60 (1
H, s), 5.41 (1 H, s), 3.77 (1 H, dd, J ) 10.9, 5.3 Hz), 3.59 (1 H,
d, J ) 9.2 Hz), 2.73-2.80 (1 H, m), 2.32 (3 H, s), 1.58 (1 H, dd,
J ) 7.9, 3.6 Hz), 1.28 (1 H, dd, J ) 5.0, 3.6 Hz); FAB-MS m/z
201 (M + H)+.
2-Me t h yl-3-(d im e t h yla m in om e t h yl)-A-r in g-p yr r ole -
1
DUMA (12g): yield 55%; H-NMR (270 MHz, CDCl3) δ 9.88
(1 H, brs), 9.30 (1 H, brs), 6.95 (1 H, s), 6.93 (1 H, d, J ) 2.3
Hz), 6.78 (1 H, s), 4.41 (1 H, dd, J ) 10.4, 4.5 Hz), 4.35 (1 H,
d, J ) 10.2 Hz), 4.07 (3 H, s), 3.93 (3 H, s), 3.89 (3 H, s), 3.18-
3.22 (1 H, m), 3.13 (1 H, d, J ) 13.5 Hz), 3.07 (1 H, d, J ) 13.5
Hz), 2.48 (1 H, dd, J ) 7.4, 3.8 Hz), 2.31 (3 H, s), 2.17 (6 H, s),
1.31 (1 H, dd, J ) 4.6, 4.3 Hz); FAB-MS m/ z 491 (M + H)+;
IR (KBr) 1641, 1610, 1579, 1527, 1468, 1427, 1396, 1385, 1304,
1263, 1155, 1109 cm-1. Anal. (C27H30N4O5‚1.0H2O) C, H; N:
calcd, 11.02; found, 10.25.
2-Met h yl-3-(a llyloxylca r b on yl)-A-r in g-p yr r ole-DUM-
Seg-A (14b ): yield 74%; 1H-NMR (270 MHz, CDCl3
+
CD3OD) δ 5.93 (1 H, ddt, J ) 19.6, 10.6, 5.6 Hz), 5.36 (1 H, s),
5.28 (1 H, ddd, J ) 17.3, 1.7, 1.3 Hz), 5.19 (1 H, ddd, J ) 10.4,
1.3, 1.0 Hz), 4.63 (1 H, brd, J ) 5.9 Hz), 3.68 (1H, dd, J )
10.9, 5.6 Hz), 3.53 (1 H, d, J ) 10.9 Hz), 3.44-3.50 (1 H, m),
2-Meth yl-3-for m yl-A-r in g-p yr r ole-DUMA (12h ): yield
72%; 1H-NMR (270 MHz, CDCl3) δ 12.27 (1 H, brs), 9.83 (1 H,

Synthesis, Activity of Duocarmycin Derivatives
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 2957
2.47 (3 H, s), 2.08 (1 H, dd, J ) 7.9, 3.0 Hz), 1.02 (1 H, dd, J
) 4.8, 3.1 Hz); FAB-MS m/ z 285 (M + H)+.
3.84 (3 H, s), 2.26 (3 H, s), 2.01 (1 H, dd, J ) 6.9, 4.6 Hz), 1.92
(3 H, s), 1.29 (1 H, dd, J ) 4.6, 4.6 Hz); FAB-MS m/ z 375 (M
+ H)+; FAB-HRMS calcd for C23H23N2O3 (M + H)+ m/ z
375.1708, found 375.1703; IR (KBr) 1664, 1618, 1603, 1572,
1560,1512,1468,1390,1281,1240,1173 cm-1.Anal.(C23H22N2O3‚
0.3H2O) C, H, N.
2,3-Dim eth yl-A-r in g-p yr r ole-DUM-Seg-A (14c): yield
77%; 1H-NMR (270 MHz, CDCl3 + CD3OD) δ 5.38 (1 H, s),
3.69 (1 H, dd, J ) 10.6, 5.3 Hz), 3.54 (1 H, d, J ) 10.6 Hz),
3.39 (3 H, s), 2.88-2.94 (1 H, m), 2.16 (3 H, s), 1.82 (3 H, s),
1.78 (1 H, dd, J ) 7.6, 3.6 Hz), 1.06 (1 H, dd, J ) 4.6, 4.0 Hz);
FAB-MS m/ z 215 (M + H)+.
4′-Met h oxycin n a m oyl 2-m et h yl-3-for m yl-A-r in g-p yr -
r ole-DUM (15d ): yield 38%; 1H-NMR (270 MHz, CDCl3
+
CD3OD) δ 9.66 (1 H, s), 7.62 (1 H, d, J ) 15.2 Hz), 7.41 (2 H,
d, J ) 8.6 Hz), 6.79 (2 H, d, J ) 8.9 Hz), 6.60 (1 H, d, J ) 15.5
Hz), 4.13 (1 H, d, J ) 10.9 Hz), 4.04 (1 H, dd, J ) 10.9, 4.6
Hz), 3.72 (3 H, s), 3.37-3.43 (1 H, m), 2.49 (3 H, s), 2.24 (1 H,
dd, J ) 7.6, 3.6 Hz), 1.22 (1 H, dd, J ) 5.0, 3.6 Hz); FAB-MS
m/ z 389 (M + H)+; IR (KBr) 1664, 1601, 1578, 1512, 1479,
1394, 1390, 1242, 1173 cm-1. Anal. (C23H20N2O4‚0.6H2O) C,
H, N.
4′-Meth oxycin n am oyl 2-m eth yl-3-ch lor o-A-r in g-pyr r ole-
DUM (15e): yield 41%; 1H-NMR (270 MHz, CDCl3 + CD3OD)
δ 7.66 (1 H, d, J ) 15.5 Hz), 7.44 (2 H, d, J ) 8.3 Hz), 6.83 (2
H, d, J ) 8.3 Hz), 6.65 (1 H, d, J ) 15.2 Hz), 6.45 (1 H, br),
4.17 (1 H, d, J ) 11.2 Hz), 4.06 (1 H, dd, J ) 11.1, 4.8 Hz),
3.76 (3 H, s), 2.96-3.09 (1 H, m), 2.25 (1 H, dd, J ) 7.6, 4.3
Hz), 2.21 (3 H, s), 1.24 (1 H, dd, J ) 4.6, 4.3 Hz); FAB-MS
m/ z 395 (M + H)+; IR (KBr) 1601, 1574, 1512, 1475, 1392,
1286, 1240, 1225, 1171, 1061 cm-1. Anal. (C22H19ClN2O3‚
0.8H2O) C, H, N.
2-Meth yl-3-for m yl-A-r in g-pyr r ole-DUM-Seg-A(14d): yield
48%; 1H-NMR (270 MHz, CDCl3) δ 12.43 (1 H, brs), 9.71 (1 H,
s), 5.94 (1 H, brs), 5.60 (1 H, s), 3.75 (1 H, dd, J ) 10.1, 5.4
Hz), 3.57 (1 H, d, J ) 10.6 Hz), 3.45-3.47 (1 H, m), 2.55 (3 H,
s), 2.09 (1 H, dd, J ) 7.6, 2.6 Hz), 1.07 (1 H, dd, J ) 4.7, 4.3
Hz); FAB-MS m/ z 229 (M + H)+.
2-Meth yl-3-ch lor o-A-r in g-pyr r ole-DUM-Seg-A(14e): yield
95%; 1H-NMR (270 MHz, CDCl3 + CD3OD) δ 5.35 (1 H, s),
3.69 (1 H, dd, J ) 10.9, 5.3 Hz), 3.54 (1 H, d, J ) 10.9 Hz),
3.03-3.10 (1 H, m), 2.18 (3 H, s), 2.02 (1 H, dd, J ) 7.8, 3.5
Hz), 1.04 (1 H, dd, J ) 4.3, 4.3 Hz); FAB-MS m/ z 235 (M +
H)+.
2-Meth yl-3-br om o-A-r in g-pyr r ole-DUM-Seg-A (14f): yield
92%; 1H-NMR (270 MHz, CDCl3 + CD3OD) δ 5.37 (1 H, s),
3.71 (1 H, dd, J ) 10.7, 5.4 Hz), 3.56 (1 H, d, J ) 10.9 Hz),
3.12-3.19 (1 H, m), 2.21 (3 H, s), 2.09 (1 H, dd, J ) 7.9, 3.6
Hz), 1.05 (1 H, dd, J ) 4.6, 4.0 Hz); FAB-MS m/ z 281, 279 (M
+ H)+.
4′-Meth oxycin n am oyl 2-m eth yl-3-br om o-A-r in g-pyr r ole-
DUM (15f): yield 62%; 1H-NMR (270 MHz, CDCl3 + CD3OD)
δ 7.70 (1 H, d, J ) 15.5 Hz), 7.47 (2 H, d, J ) 8.6 Hz), 6.86 (2
H, d, J ) 8.9 Hz), 6.68 (1 H, d, J ) 15.2 Hz), 6.47 (1 H, br),
4.20 (1 H, d, J ) 10.9 Hz), 4.09 (1 H, dd, J ) 11.2, 5.0 Hz),
3.79 (3 H, s), 3.10-3.16 (1 H, m), 2.34 (1 H, dd, J ) 7.4, 4.1
Hz), 2.25 (3 H, s), 1.26 (1 H, dd, J ) 5.0, 4.3 Hz); FAB-MS m/z
441, 439 (M + H)+; FAB-HRMS calcd for C22H2079BrN2O3 (M
+ H)+ m/ z 439.0657, found 439.0673; IR (KBr) 1666, 1597,
1574, 1512, 1470, 1390, 1284, 1240, 1171, 1051 cm-1. Anal.
(C22H19BrN2O3) C, H, N.
4′-Meth oxycin n a m oyl 2-m eth yl-3-iod o-A-r in g-p yr r ole-
DUM (15g): yield 63%; 1H-NMR (270 MHz, CDCl3 + CD3OD)
δ 7.62 (1 H, d, J ) 15.5 Hz), 7.41 (2 H, d, J ) 8.9 Hz), 6.80 (2
H, d, J ) 8.9 Hz), 6.62 (1 H, d, J ) 15.5 Hz), 6.45 (1 H, br),
4.14 (1 H, d, J ) 11.2 Hz), 4.04 (1 H, dd, J ) 11.2, 4.6 Hz),
3.73 (3 H, s), 3.18-3.23 (1 H, m), 2.33 (1 H, dd, J ) 7.6, 4.3
Hz), 2.21 (3 H, s), 1.17 (1 H, dd, J ) 5.0, 4.3 Hz); FAB-MS
m/ z 487 (M + H)+; IR (KBr) 1666, 1601, 1574, 1464, 1392,
1240, 1171, 1038, 822 cm-1. Anal. (C22H19IN2O3) C, H, N.
4′-Meth oxycin n am oyl 2-m eth yl-3-acetyl-A-r in g-pyr r ole-
DUM (15h ): yield 33%; 1H-NMR (270 MHz, CDCl3 + CD3OD)
δ 7.68 (1 H, d, J ) 15.2 Hz), 7.46 (2 H, d, J ) 8.6 Hz), 6.85 (2
H, d, J ) 8.6 Hz), 6.67 (1 H, br), 6.65 (1 H, d, J ) 15.2 Hz),
4.16 (1 H, d, J ) 10.9 Hz), 4.07 (1 H, dd, J ) 10.9, 4.6 Hz),
3.59-3.66 (1 H, m), 2.58 (3 H, s), 2.37 (3 H, s), 2.31 (1 H, dd,
J ) 7.4, 3.1 Hz), 1.17 (1 H, dd, J ) 4.3, 4.0 Hz); FAB-MS
m/ z 403 (M + H)+; IR (KBr) 1662, 1601, 1576, 1512, 1390,
1240, 1225, 1173, 1006, 825 cm-1. Anal. (C24H22N2O4‚0.5H2O)
C, H, N.
2-Meth yl-3-iod o-A-r in g-p yr r ole-DUM-Seg-A (14g): yield
70%; 1H-NMR (270 MHz, CDCl3 + CD3OD) δ 5.36 (1 H, s),
3.71 (1 H, dd, J ) 10.9, 5.3 Hz), 3.56 (1 H, d, J ) 10.6 Hz),
3.25-3.31 (1 H, m), 2.22 (3 H, s), 2.12 (1 H, dd, J ) 7.6, 3.6
Hz), 1.02 (1 H, dd, J ) 4.3, 4.3 Hz); FAB-MS m/ z 327 (M +
H)+.
2-Meth yl-3-acetyl-A-r in g-pyr r ole-DUM-Seg-A(14h ): yield
69%; 1H-NMR (270 MHz, CDCl3 + CD3OD) δ 5.38 (1 H, s),
3.66 (1 H, dd, J ) 10.6, 5.3 Hz), 3.57-3.62 (1 H, m), 3.52 (1 H,
d, J ) 10.6 Hz), 2.52 (3 H, s), 2.33 (3 H, s), 2.08 (1 H, dd, J )
7.6, 2.6 Hz), 0.94 (1 H, dd, J ) 4.5, 2.8 Hz); FAB-MS m/z 243
(M + H)+.
4′-Meth oxycin n a m oyl 2-Meth yl-A-r in g-p yr r ole-DUM
(15a ). To a solution of 60% NaH (6.0 mg, 0.15 mmol) in DMF
(0.36 mL) was added a DMF solution (0.48 mL) of 14a (25 mg,
0.12 mmol), and the mixture was stirred under Ar atmosphere
at -20 °C for 2 h. Then, a solution of the p-nitrophenyl ester
of 4-methoxycinnamic acid (41 mg, 0.14 mmol) in DMF (0.48
mL) was added and stirred for 50 min; 0.01 M phosphoric
buffer (pH 7) was added, and the whole was extracted with
AcOEt and then worked up as usual. The residue was puri-
fied by column chromatography (CHCl3-MeOH, 100:1) to give
40 mg (89%) of 15a : 1H-NMR (270 MHz, CDCl3) δ 9.44 (1 H,
brs), 7.76 (1 H, d, J ) 15.5 Hz), 7.52 (2 H, d, J ) 8.6 Hz), 6.91
(2 H, d, J ) 8.6 Hz), 6.79 (1 H, d, J ) 15.5 Hz), 6.41 (1 H, br),
5.67 (1 H, d, J ) 1.7 Hz), 4.22 (1 H, d, J ) 11.6 Hz), 4.10 (1 H,
dd, J ) 11.2, 5.0 Hz), 3.85 (3 H, s), 2.58-2.62 (1 H, m), 2.36
(3 H, s), 1.71 (1 H, dd, J ) 7.8, 4.1 Hz), 1.47 (1 H, dd, J ) 4.6,
4.6 Hz); FAB-MS m/z 361 (M + H)+; IR (KBr) 1666, 1601,
1574, 1510, 1479, 1242, 1227, 1173 cm-1. Anal. (C22H20N2O3)
C, H, N.
4′-Meth oxycin n a m oyl
2-Meth yl-3-(d im eth yla m in o-
4′-Meth oxycin n a m oyl 2-m eth yl-3-(a llyloxylca r bon yl)-
A-r in g-p yr r ole-DUM (15b): yield 80%; 1H-NMR (270 MHz,
CDCl3 + CD3OD) δ 7.70 (1 H, d, J ) 15.5 Hz), 7.47 (2 H, d, J
) 8.9 Hz), 6.86 (2 H, d, J ) 8.9 Hz), 6.67 (1 H, d, J ) 15.2 Hz),
6.62 (1 H, br), 5.96 (1 H, ddt, J ) 19.5, 10.2, 5.6 Hz), 5.32 (1
H, ddd, J ) 17.3, 1.7, 1.3 Hz), 5.23 (1 H, ddd, J ) 10.4, 1.3,
1.0 Hz), 4.67 (2 H, dt, J ) 5.6, 1.3 Hz), 4.18 (1 H, d, J ) 10.9
Hz), 4.09 (1 H, dd, J ) 10.9, 4.6 Hz), 3.49-3.56 (1 H, m), 2.54
(3 H, s), 2.34 (1 H, dd, J ) 7.6, 3.3 Hz), 1.26 (1 H, dd, J ) 5.0,
3.6 Hz); FAB-MS m/ z 445 (M + H)+; IR (KBr) 1554, 1512,
1489, 1390, 1292, 1242, 1225, 1173, 1111, 1072 cm-1. Anal.
(C26H24N2O5‚1.0H2O) C, H, N.
4′-Meth oxycin n am oyl 2,3-dim eth yl-A-r in g-pyr r ole-DUM
(15c): yield 51%; 1H-NMR (270 MHz, CDCl3) δ 10.30 (1 H,
brs), 7.75 (1 H, d, J ) 15.2 Hz), 7.51 (2 H, d, J ) 8.6 Hz), 6.90
(2 H, d, J ) 8.9 Hz), 6.79 (1 H, d, J ) 15.5 Hz), 6.43 (1 H, br),
4.24 (1 H, d, J ) 11.2 Hz), 4.10 (1 H, dd, J ) 11.2, 4.6 Hz),
m eth yl)-A-r in g-p yr r ole-DUM (16). To a solution of 15a (47
mg, 0.13 mmol) in THF (1.8 mL) was added 4 N HCl in AcOEt
(0.098 mL, 0.39 mmol), and the mixture was stirred at room
temperature for 45 min. The mixture was concentrated under
reduced pressure, and a solution of Me2NCH2NMe2 (0.089 mL,
0.65 mmol) in TFA (0.5 mL) was stirred at -10 °C for 25 min
and then was added to a solution of the residue in TFA (0.8
mL) at -10 °C. After the mixture stirred for 1 h 45 min,
aqueous NaHCO3 was added, and the whole was extracted
with CHCl3 and then worked up as usual. The residue was
purified by PTLC (CHCl3-acetone, 10:1) to give 22 mg (36%)
of 4′-methoxycinnamoyl 2-methyl-3-(dimethylaminomethyl)-
A-ring-pyrrole-DUMC2. To a solution of 4′-methoxycinnamoyl
2-methyl-3-(dimethylaminomethyl)-A-ring-pyrrole-DUMC2 (22
mg, 0.047 mmol) in CH3CN (1.5 mL) was added DBU (0.0354
mL, 0.0237 mmol), and the mixture was stirred at room
temperature for 40 min; 0.01 M phosphoric buffer (pH 7) was

2958 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15
Amishiro et al.
added, and the whole was extracted with CHCl3 and then
worked up as usual. The residue was purified by PTLC
(CHCl3-acetone, 10:1) to give 17 mg (87%) of 16: 1H-NMR
(270 MHz, CDCl3) δ 10.45 (1 H, brs), 7.76 (1 H, d, J ) 15.5
Hz), 7.51 (2 H, d, J ) 8.6 Hz), 6.91 (2 H, d, J ) 8.6 Hz), 6.80
(1 H, J ) 15.5 Hz), 6.46 (1 H, br), 4.21 (1 H, d, J ) 11.2 Hz),
4.09 (1 H, dd, J ) 11.4, 4.8 Hz), 3.84 (3 H, s), 3.08-3.14 (1 H,
m), 2.54 (1 H, dd, J ) 7.3, 3.6 Hz), 2.31 (3 H, s), 2.15 (6 H, s),
1.24 (1 H, dd, J ) 4.6, 4.0 Hz); FAB-MS m/ z 418 (M + H)+;
FAB-HRMS calcd for C25H28N3O3 (M + H)+ m/z 418.2131,
found 418.2106; IR (KBr) 1616, 1601, 1576, 1512, 1473, 1394,
1284, 1240, 1171, 1018 cm-1. Anal. (C25H27N3O3‚0.2H2O) C,
H, N.
ddY mice (mean weight 20 ( 1 g). After 7 days, PB was
obtained from the orbital vein to measure the platelet counts
using a microcell counter (CC-180A, Toa Medical Electronics
Co., Ltd., Kobe, J apan). Results are presented as percentage
of the absolute value of the treated group versus that of control
(percent of control).
Effect on P B w h ite blood cell cou n ts: Drugs were
administered iv beginning 1 day after tumor inoculation. After
4 days, PB 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,
J apan). Results are presented as percentage of the absolute
value of the treated group versus that of control (percent of
control).
4′-Meth oxycin n a m oyl
2-Meth yl-3-(d im eth yla m in o-
m eth yl)-A-r in g-p yr r ole-DUMC2 Hyd r och lor id e (17). A
solution of 16 (11 mg, 0.026 mmol) in CH2Cl2 (1 mL) was
treated with 4 N HCl in AcOEt (0.0326 mL) at room temper-
ature for 30 min. The mixture was concentrated under reduced
pressure to give 12 mg of 17: 1H-NMR (270 MHz, DMSO-d6)
δ 11.46 (1 H, s), 9.97 (1 H, brs), 9.50-9.53 (1 H, br), 7.81 (1 H,
s), 7.75 (2 H, d, J ) 8.6 Hz), 7.59 (1 H, d, J ) 14.9 Hz), 7.08
(1 H, d, J ) 15.5 Hz), 7.00 (2 H, d, J ) 8.3 Hz), 4.29-4.49 (3
H, m), 3.96-4.13 (2 H, m), 3.81 (3 H, s), 3.78 (3 H, br), 3.52-
3.56 (1 H, br), 2.81 (3 H, d, J ) 4.0 Hz), 2.69 (3 H, d, J ) 4.0
Hz), 2.45 (3 H, s); FAB-MS m/ z 454 (M + H)+; IR (KBr)
1633, 1601, 1512, 1495, 1441, 1427, 1252, 1174 cm-1. Anal.
(C25H28ClN3O3‚HCl‚2.5H2O) C, H, N.
Biologica l Stu d ies. Human uterine cervix carcinoma HeLa
S3 cells were obtained from American Type Culture Collection
through Dainippon Pharmaceutical Co. (Osaka, J apan). The
cells (2 × 104/well) were precultured in the culture medium
in 24-well multidishes (Nunc, Roskilde, Denmark) for 24 h at
37 °C in a humidified atmosphere of 5% CO2. For the pulse
exposure experiment, cells were treated with each compound
for 1 h, washed with Dulbecco’s phosphate-buffered 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, J apan) and counted by
using a Microcell Counter (CC-180A, Toa Medical Electronics
Co., Ltd., Kobe, J apan). The IC50 values (drug concentration
required for 50% inhibition of the cell growth) were deter-
mined.
Leth a l Toxicity. For the evaluation of delayed lethal
toxicity, each drug was given by single iv administration in
adult male ddY mice (mean weight 20 ( 1 g), and their
survival days were observed over 60 days.
Eva lu a tion of Bon e Ma r r ow Toxicity. Bon e m a r r ow
cells: Male ddY mice received a single iv dose of the drug by
tail vein injection on day 0 of the experiment. Then bone
marrow (BM) cells were harvested by flushing from the limbs
of mice with Iscove’s modified Dulbecco’s medium (IMDM,
Gibco, Grand Island, NY) using a 23-G needle on a 1-mL
syringe and were prepared at the concentration of 1.0 × 106
cells/mL using a microcell counter.
Colon y-for m in g u n it-gr a n u locytes a n d m a cr op h a ges
(CF U-GM) a ssa y: BM cells (2.5 × 104) were plated in 0.3%
agar (agar noble, Difco Lab., Detroit, MI) with IMDM, 20%
FBS (Filtron, Brooklyn, Australia), and 100 units/mL GM-CSF
(Boehringer Mannheim, Tokyo, J apan). Cultures were incu-
bated in humidified air containing 5% CO2 at 37 °C, and
colonies (>40 cells/colony) were counted after 7 days of
incubation after staining using a Wright’s eosin methylene
blue solution (Omron, Tokyo, J apan).
Colon y-for m in g u n it-m ega k a r yocytes (CF U-Meg) a s-
sa y: BM cells (1.0 × 105) were plated in 0.3% agar (agar noble,
Difco Lab., Detroit, MI) with IMDM, 10% FBS (Filtron,
Brooklyn, Australia), 50 units/mL IL-3 (Boehringer Mannheim,
Tokyo, J apan), and 1000 units/mL IL-6 (Boehringer Man-
nheim, Tokyo, J apan). Cultures were incubated in humidified
air containing 5% CO2 at 37 °C, and colonies (>4 cells/colony)
were counted after 7 days of incubation after staining for the
presence of acetylcholinesterase.
Sarcoma 180 cells were kindly supplied by the National
Cancer Center (Tokyo, J apan). Sarcoma 180 cells were pas-
saged and used for the experiment in adults male ddY mice.
Murine solid tumor was inoculated subcutaneously (sc) at the
axillary region of mice. Drugs were administered intravenously
(iv) beginning 1 day after tumor inoculation. Antitumor
efficacy 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 measured, and tumor volume
was calculated as:
Ack n ow led gm en t. We express our gratitude to
Mikiko Saito, Kumiko, Masunaga, and Etsuko Ko-
shimura for their excellent technical assistance. We also
thank Mariko Yoshida, Atsuko Kobayashi, and Hideko
Yokoyama for measuring elemental analyses and MS
spectra.
Su p p or tin g In for m a tion Ava ila ble: HPLC analytical
data of compounds 8b-8d , 8i, 9h , 12a , 12b, 12g, 12j, 12k ,
and 13. This material is available free of charge via the
Internet at http://pubs.acs.org.
tumor volume (mm3) ) length (mm) × [width (mm)]2/2
Refer en ces
according to the method of the National Cancer Institute.19
The criteria for effectiveness against murine solid tumors
were the percentage T/C values with 42% and less, and
statistical significance was determined by the Mann-Whitney
U-test (p < 0.05). Drug efficacy 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 effectiveness were T/C
values with 50% and less, and statistical significance was
determined by the Mann-Whitney U-test (p < 0.01, one-
sided).20
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