Synthesis and antitumor activity of duocarmycin derivatives: A-ring pyrrole compounds bearing cinnamoyl groups

Article abstract of DOI:10.1021/jm9606094

A series of N-cinnamates of the A-ring pyrrole compound 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. The 4'-methoxy- and 4'-BocNH-cinnamates exhibited strong in vitro anticellular activity among the synthesized compounds. The ortho substitution of the 4'-methoxycinnamate did not affect the anticellular activity and contributed to an enhancement of water solubility. Most of the 8-O-(N,N- dialkylcarbamoyl) derivatives of the 4'-methoxycinnamates displayed remarkably superior in vivo antitumor activity to duocarmycin A or B2. Moreover, it is noteworthy that these 8-O-(N,N-dialkylcarbamoyl) derivatives exhibited significant antitumor activity at wider range of doses as compared with the A-ring pyrrole derivatives having the trimethoxyindole skeleton in segment B.

Full text of DOI:10.1021/jm9606094

972
J . Med. Chem. 1997, 40, 972-979
Syn th esis a n d An titu m or Activity of Du oca r m ycin Der iva tives: A-Rin g P yr r ole
Com p ou n d s Bea r in g Cin n a m oyl Gr ou p s
Satoru Nagamura,*,†,‡ Akira Asai,† Nobuyoshi Amishiro,§ Eiji Kobayashi,§ Katsushige Gomi,§ and
Hiromitsu Saito§
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6, Asahi-machi, Machida-shi, Tokyo 194, J apan, and
Pharmaceutical Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 1188, Shimotogari, Nagaizumi, Sunto,
Shizuoka 411, J apan
Received August 23, 1996X
A series of N-cinnamates of the A-ring pyrrole compound 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. The 4′-methoxy- and 4′-BocNH-cinnamates
exhibited strong in vitro anticellular activity among the synthesized compounds. The ortho
substitution of the 4′-methoxycinnamate did not affect the anticellular activity and contributed
to an enhancement of water solubility. Most of the 8-O-(N,N-dialkylcarbamoyl) derivatives of
the 4′-methoxycinnamates displayed remarkably superior in vivo antitumor activity to
duocarmycin A or B2. Moreover, it is noteworthy that these 8-O-(N,N-dialkylcarbamoyl)
derivatives exhibited significant antitumor activity at wider range of doses as compared with
the A-ring pyrrole derivatives having the trimethoxyindole skeleton in segment B.
In tr od u ction
the delayed irreversible toxicity of CC-1065, and several
analogs of CC-1065 were taken into clinical trials.12
Therefore, we have synthesized a series of duocarmycin
analogs bearing the simplified moieties such as acetyl,
indol-2-ylcarbonyl, benzofuran-2-ylcarbonyl, cinnamoyl,
and phenoxyacetyl group. These compounds exhibited
varied in vitro anticellular activities. In order to
enhance in vivo antitumor activity, the compounds that
showed potent in vitro anticellular activity were con-
verted to the 8-O-(N,N-dialkylcarbamoyl) derivatives,
in view of our acquired information.7 Among them, the
8-O-(N,N-dialkylcarbamoyl) derivatives introduced into
the cinnamoyl derivatives exhibited sufficient in vivo
activity against sarcoma 180 over a wide range of doses
without detectable toxic effects.13 With the goal of
finding new candidates having greater activity or less
toxicity than that of conventional duocarmycins or their
derivatives, a series of the N-cinnamates of A-ring
pyrrole compound of duocarmycin were synthesized and
evaluated for their anticellular and antitumor activities.
The 4′-methoxy- and 4′-BocNH-cinnamates seemed to
be especially suitable for evaluation of in vitro activity,
and the 4′-methoxycinnamate minimized the decreased
in vitro activity caused by the substitution at the 3′-
position.6d,14 In this paper, we report our investigation
into the synthesis, anticellular and antitumor activities,
and structure-activity relationships of these deriva-
tives.
Duocarmycins (A, 1a ; SA, 1c; B2, 1d ; C2, 1e; B1, 1f;
C1, 1g) are novel antitumor antibiotics isolated from
the culture broth of Streptomyces sp. (Figure 1).1 Duo-
carmycin A (1a ),1a-c which is considered as an active
form among these duocarmycins, possesses a unique
cyclopropane ring with the ability to alkylate DNA.
Duocarmycin A (1a ) has been reported to show its
cytotoxicity through a sequence-selective minor groove
alkylation of double-stranded DNA resulting in N3
adenine covalent adduct formation2 as in the case of the
antitumor antibiotic CC-1065 (1b).3,4 Duocarmycins are
known to exhibit potent growth-inhibitory activity
against human uterine cervix carcinoma HeLa S3 in
vitro and modest broad antitumor spectrum against
murine transplantable solid tumors.5,6 However, their
marginal activity against human solid tumors, their
poor stability, and their insolubility in water dissuaded
us from considering them for further evaluation. We
were interested in synthesizing analogs in order to
enhance and broaden their spectrum of antitumor
activity and to improve their stability and solubility.7,8
One such analog, KW-2189 (2b), was prepared by the
modification of the segment A in duocarmycin B2 (1d ),
demonstrating excellent in vivo antitumor activity, good
stability in the culture medium, and aqueous solubility
greater than 10 mg/mL.9 It was designed as a prodrug
which requires enzymatic hydrolysis followed by regen-
eration of DU-86 (2a ) as an active metabolite.10 KW-
2189 (2b) is currently under going phase I clinical
evaluation.
Ch em istr y
Initially, the 2-methyl-3-methoxycarbonyl A-ring pyr-
role compound of duocarmycin (DU-86, 2a ) was pre-
pared by employing the Wagner-Meerwein type rear-
rangement of the 8-O-protected-3-hydroxyduocarmycin
B2 followed by deprotection of the protecting group
under basic conditions.15 Compound 2a was treated
with NaOMe in MeOH to afford compound 3 and
methyltrimethoxyindole-2-carboxylate quantitatively, as
shown in Scheme 1. The obtained compound 3 was
allowed to react with 4′-substituted-trans-cinnamic acid
On the other hand, segment B of duocarmycins has
been considered to play an important role in the
noncovalent binding to DNA.11 Moreover, the modifica-
tion of the noncovalent binding segments may contrib-
ute to a decrease in toxicity. These strategies overcame
† Tokyo Research Laboratories.
‡ Present address: Technical Research Laboratories, Kyowa Hakko
Kogyo Co., Ltd., 1-1, Kyowa-machi, Hofu-shi, Yamaguchi 747, J apan.
§ Pharmaceutical Research Laboratories.
X Abstract published in Advance ACS Abstracts, February 1, 1997.
S0022-2623(96)00609-7 CCC: $14.00 © 1997 American Chemical Society

Duocarmycin Derivatives
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6 973
F igu r e 1. Structures of duocarmycins, CC-1065, and duocarmycin derivatives.
Sch em e 1
p-nitrophenyl esters in the presence of NaH to yield the
corresponding 4′-substituted cinnamates 4a -j in rea-
sonable yields, as described previously.13 The 4′-
substituted-trans-cinnamic acids were prepared from
the available 4′-substituted benzaldehyde and malonic
acid in pyridine. Their p-nitrophenyl esters were then
synthesized from the corresponding 4′-substituted cin-
namic acid and p-nitrophenol using the Mukaiyama
reagent in good yields.16 Compound 4k was prepared
in 47% yield by the reaction of 4j with 48% HBr and
TFA in CH3CN followed by the treatment of triethyl-
amine. The 4′-methoxy-3′-substituted-cinnamates 5a -g
were also prepared by the same procedure as that of
4a -j. Conversion of 5a to 5h was achieved in accept-
able yield by the method of PPh3 reduction. Compound
5i was synthesized by the same method as that of 4k .
In order to enhance in vivo antitumor activities of
these cinnamates (4, 5), the 8-O-(N,N-dialkylcarbamoyl)
series were prepared as described in preceding papers.7,9
The cinnamates were treated with HBr, followed by
conversion to the p-nitrophenyl carbonate by the reac-
tion with p-nitrophenyl chloroformate in the presence

974 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6
Nagamura et al.
Ta ble 3. Anticellular Activity and Antitumor Activity of
Ta ble 1. Anticellular Activity of 4′-Substituted-cinnamates of
Duocarmycin
Cinnamates of Duocarmycin
HeLa S3 IC50 (nM)a
sarcoma 180 (sc-iv)b
1 h
72 h
37
224
61
280
52
30
mg/kg
T/Cc
6a
6b
6c
6d
6e
6f
1800
1000
1200
2100
430
290
290
10000
4
4
8
8
2
0.13
8
4
0.2
0.17
0.12
0.2
0.13
0.77
0.39
0.72
HeLa S3 IC50 (nM)a
6g
6h
30
>1000
no.
R
1 h
72 h
4a
4b
4c
4d
4e
4f
OCH3
2.9-7.0
0.26-0.94
8.3
7.5
a
O(CH2)2CH3
OCH2CHdCH2
O(CH2)4CH3
OCH2Ph-p-OMe
OH
51
Drug concentration required to inhibit the growth of HeLa S3
b
39
>100
13
cells by 50%. Mice (five mice/group) were implanted subcutane-
50
ously (sc) with tumor cells, and the drug was dosed (mg/mg)
intravenously (iv). c T and C are the values of mean tumor volume
of treated and control mice, respectively.
0.81
3.5
19
4g
4h
4i
4j
4k
2a
N(CH3)2
CH3
CH2CH3
NHBoc
NH2
12
33
51
1.6
2.3
3.8
0.56
2.6
(4a ); however, they exhibited about 10 times decreased
anticellular activity compared to 4a .
4.0
32
0.39-0.50
On the other hand, compounds 5a -i, bearing an ortho
substituent, showed anticellular activity similar to that
of 4a . It is suggested, therefore, that the 3′-substituent
of the 4′-methoxycinnamates did not seriously influence
the association between DNA and drug.
0.15-0.16
a
Drug concentration required to inhibit the growth of HeLa S3
cells by 50%.
Ta ble 2. Anticellular Activity of
The in vivo activity of selected compounds was evalu-
ated against sarcoma 180 murine solid tumor. The in
vivo efficacy was expressed as T/ C, which is defined as
treated versus control value of tumor volume. Tumor
volume was calculated according to the method de-
scribed previously.7,9 As shown in Table 3, all of the
8-O-(N,N-dialkylcarbamoyl) derivatives of the 4′-meth-
oxycinnamates showed 102 -103 times inferior anticel-
lular activity to that of the corresponding cyclopropane
derivatives. However, compounds 6a -e exhibited po-
tent antitumor activity in vivo. In contrast, compounds
6f-h having hydrophilic moieties at the 3′-position were
somewhat less active than 6a -e, suggesting that a
charged group in these analogs may reduce cell perme-
ability and therefore antitumor activity.17 Moreover,
compound 6a -e also showed efficient in vivo antitumor
activity even at the second highest doses (a half of
maximally tolerated dose). Indeed, the cinnamoyl de-
rivatives generally exhibited sufficient efficacy over a
wide range of doses without detectable toxic effect.13 At
the same time, the in vivo antitumor activities of some
representative cyclopropane compounds (4a -c) were
also evaluated, but they did not demonstrate significant
antitumor activity (T/ C ) 0.38-0.47), and the effective
range of doses was narrow, similar to that of duocar-
mycin A or SA or DU-86 which is an active metabolite
of KW-2189 (data not shown).9,18
4′-Methoxy-3′-substituted-cinnamates of Duocarmycin
HeLa S3 IC50 (nM)a
no.
R
1 h
2.0
3.0
7.4
1.3
9.5
2.5
5.9
72 h
0.74
1.6
2.3
0.86
2.3
0.92
1.4
0.53
0.26-0.94
5a
5b
5c
5d
5e
5f
5h
5i
4a
O(CH2)3N3
O(CH2)3NMe2
OCH2CO2-t-Bu
OCH2CO2CH3
NEt2
NMe2
O(CH2)3NH2
NH2
2.1
2.9-7.0
H
a
Drug concentration required to inhibit the growth of HeLa S3
cells by 50%.
of triethylamine. The carbonates were allowed to react
with the corresponding secondary amines to produce the
8-O-(N,N-dialkylcarbamoyl) derivatives (6a -h ) in rea-
sonable yields.
Resu lts a n d Discu ssion
Tables 1 and 2 show the in vitro anticellular activity
of those cinnamates against HeLa S3 cells. As can be
seen, the anticellular activity of these cinnamates
depends on the substituent at the 4′-position. The
methoxy (4a ) and the BocNH (4j) moiety as a substitu-
ent impact increased in vitro activity in this series. They
exhibited strong anticellular activities with the IC50
values below 1.0 nM at 72 h exposure. Among the 4′-
alkoxycinnamates, the anticellular potency decreased
with increasing size of the alkoxy moiety (4a -d ). Many
compounds having polar functional groups (e.g., OH,
NH2, N(CH3)2) at the 4′-position did not show sufficient
activity superior to that of 4a or 4j. Noteworthy, the
4′-methyl and -ethyl compounds (4h and 4i) have
similar bulkiness to that of the 4′-methoxycinnamate
Compound 6a exhibits poor solubility, below 0.1 mg/
mL in water or phosphate buffer (pH 7). In contrast,
the 8-O-(N,N-dialkylcarbamoyl)cinnamates having an
amino group at the 3′-position (6d -g) were found to
possess adequate water solubility in excess of 10 mg/
mL. Several analogs demonstrating both potent anti-
tumor activity against sarcoma 180, and adequate water
solubility were evaluated in vivo for efficacy in nude
mice bearing human xenograft St-4 (poorly differenti-
ated stomach adenocarcinoma). Compound 6a and 6d
showed excellent activity in vivo with T/ C values of 0.15
(12 mg/kg dose) and 0.29 (18 mg/kg dose), respectively.
They caused significant tumor size regression with less
toxicity as judged by body weight loss. These activities
against St-4 human stomach tumor xenograft were

Duocarmycin Derivatives
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6 975
nearly comparable to our clinical candidate KW-2189.9
Consequently, compound 6d is one of the candidates for
the next generation of duocarmycin derivatives. Fur-
ther studies on antitumor spectra and toxicity of these
derivatives are in progress.19
s), 3.56 (1 H, m), 2.62 (3 H, s), 2.39 (1 H, dd, J ) 7.6, 3.5 Hz),
1.31 (1 H, dd, J ) 4.9, 3.5 Hz); IR (KBr) 1702, 1601, 1512,
1292, 1225, 1173, 1110, 1072 cm-1; EIMS m/ z 418 (M)+. Anal.
(C24H22N2O5‚0.5H2O) C, H, N.
4′-P r op oxycin n a m oyl A-r in g p yr r ole d u oca r m ycin 4b:
yield 80%; 1H NMR (400 MHz, CDCl3) δ 10.01 (1 H, br s), 7.77
(1 H, d, J ) 15.7 Hz), 7.32-6.93 (4 H, m), 6.84 (1 H, d, J )
15.7 Hz), 6.67 (1 H, br s), 4.24 (1 H, dd, J ) 11.0, 11.0 Hz),
4.15 (1 H, dd, J ) 11.0, 4.7 Hz), 3.94 (2 H, t, J ) 6.3 Hz), 3.82
(3 H, s), 3.55 (1 H, m), 2.59 (3 H, s), 2.39 (1 H, dd, J ) 7.5, 3.7
Hz), 1.82 (2 H, m), 1.31 (1 H, dd, J ) 4.9, 3.7 Hz), 1.05 (3 H,
t, J ) 7.4 Hz); IR (KBr) 1697, 1654, 1596, 1437, 1388, 1292,
1246, 1213, 1110 cm-1; SIMS m/ z 447 (M + H)+, 259. Anal.
(C26H26N2O5‚0.5H2O) C, H, N.
Con clu sion s
A series of 8-O-(N,N-dialkylcarbamoyl) derivatives of
the 4′-methoxycinnamates were prepared, and some of
them were effective against sarcoma 180 murine solid
tumor and St-4 human stomach tumor xenograft, nearly
comparable to our clinical candidate KW-2189, a novel
derivative of duocarmycin B2. Moreover, the effective
range of doses was significantly wider than the A-ring
pyrrole derivatives, bearing a trimethoxyindole skeleton
in segment B. It was shown that the methoxy group at
the 4′-position of these cinnamates plays a significant
role for the biological activity, and the substituent at
the 3′-position contributes to an enhancement of water
solubility.
4′-(P r op en yloxy)cin n a m oyl A-r in g p yr r ole d u oca r m y-
cin 4c: yield 87%; 1H NMR (400 MHz, CDCl3) δ 10.51 (1 H, br
s), 7.77 (1 H, d, J ) 15.6 Hz), 7.33-6.95 (4 H, m), 6.84 (1 H,
d, J ) 15.6 Hz), 6.77 (1 H, br s), 6.07 (1 H, m), 5.43 (1 H, dd,
J ) 17.4, 1.8 Hz), 5.31 (1 H, dd, J ) 10.5, 1.2 Hz), 4.57 (2 H,
dt, J ) 5.1, 1.5 Hz), 4.25 (1 H, dd, J ) 11.0, 10.9 Hz), 4.15 (1
H, dd, J ) 11.0, 4.6 Hz), 3.82 (3 H, s), 3.57 (1 H, m), 2.60 (3 H,
s), 2.40 (1 H, dd, J ) 7.6, 3.7 Hz), 1.32 (1 H, dd, J ) 4.7, 3.7
Hz); IR (KBr) 1701, 1603, 1486, 1445, 1388, 1292, 1246, 1215,
1109 cm-1; SIMS m/ z 445 (M + H)+, 259. Anal. (C26H24
N2O5‚0.5H2O) C, H, N.
-
Exp er im en ta l Section
4′-(P en tyloxy)cin n a m oyl A-r in g p yr r ole d u oca r m ycin
4d : yield 82%; 1H NMR (400 MHz, CDCl3) δ 10.34 (1 H, br s),
7.78 (1 H, d, J ) 15.4 Hz), 7.52-6.93 (4 H, m), 6.85 (1 H, d, J
) 15.4 Hz), 6.67 (1 H, br s), 4.24 (1 H, dd, J ) 11.0, 11.0 Hz),
4.15 (1 H, dd, J ) 11.0, 4.6 Hz), 3.99 (2 H, t, J ) 6.6 Hz), 3.82
(3 H, s), 3.55 (1 H, m), 2.61 (3 H, s), 2.39 (1 H, dd, J ) 7.6, 3.7
Hz), 1.81 (2 H, m), 1.44 (4 H, m), 1.31 (1 H, dd, J ) 4.9, 3.7
Hz), 0.94 (3 H, t, J ) 7.0 Hz); IR (KBr) 1701, 1628, 1599, 1457,
All melting points were measured on a Yanagimoto micro
melting point apparatus and are uncorrected. Infrared spectra
(IR) were recorded on a J ASCO IR-810. 1H spectra were
measured on a Varian EM-390, a J EOL J NM-GX270, and a
Bruker AM-400 spectrometer. Chemical shifts were reported
in parts per million (ppm) downfield from tetramethylsilane.
Elemental analyses were performed with a Perkin-Elmer 2400
C, H, N analyzer. Mass spectra were measured with a Hitachi
B-80 and a Shimadzu QP-1000 spectrometer. For column
chromatography, silica gel (SiO2, Wako C-200) was used.
Analytical thin-layer chromatography (TLC) was performed
on silica gel 60 F254 plates (Merck). All organic solvent extracts
were dried over anhydrous sodium sulfate prior to concentra-
tion in vacuo.
Meth yl 6-Meth yl-1,2,8,8a -tetr a h yd r ocyclop r op [1,2-c]-
p yr r olo[3,2-e]in d ol-4(5H)-on e-7-ca r boxyla te (3). Sodium
methoxide (25 wt % solution in methanol; 0.38 mL, 1.76 mmol)
was added to a solution of DU-86 (2a ; 285 mg, 0.58 mmol) in
MeOH (10 mL), and the mixture was stirred at room temper-
ature for 3 h. Then, 0.01 M phosphate buffer (pH 7) was added
to the reaction mixture, and the mixture was extracted with
CHCl3. The combined extracts were washed with brine. The
organic layer was dried over Na2SO4 and concentrated in
vacuo. The residue was subjected to column chromatography
(CHCl3-MeOH, 20:1) to give 113 mg (76%) of 3 as a light-tan
powder: 1H NMR (400 MHz, DMSO-d6) δ 11.72 (1 H, br s),
7.81 (1 H, br s), 6.92 (1 H, br s), 5.34 (1 H, s), 4.15 (1 H, dd, J
) 5.4, 5.4 Hz), 3.72 (3 H, s), 3.67 (1 H, dd, J ) 10.6, 5.4 Hz),
3.41 (1 H, m), 2.65 (3 H, s), 1.98 (1 H, dd, J ) 7.7, 2.8 Hz),
0.99 (1 H, dd, J ) 4.5, 2.8 Hz); IR (KBr) 1682, 1607, 1573,
1458, 1305, 1229, 1108, 1083 cm-1; SIMS m/ z 259 (M + H)+.
Anal. (C14H14N2O3) C, H, N.
Gen er a l Syn t h et ic Met h od for Typ e 4 Com p ou n d s
(4a -j). 4′-Meth oxycin n a m oyl A-Rin g P yr r ole Du oca r -
m ycin 4a . NaH (60%; 23mg, 0.57 mmol) was added to a
solution of 3 (96 mg, 0.37 mmol) in DMF (2 mL) at argon
atmosphere, and the mixture was stirred for 3 h at -50 °C.
The p-nitrophenyl ester of 4-methoxycinnamic acid (166 mg,
0.56 mmol) dissolved in DMF (2 mL) was added to a stirred
solution at -50 °C. Then, the resulting mixture was stirred
at the same temperature for 1 h. The mixture was diluted
with AcOEt, and the combine was washed with 0.01 M
phosphate buffer (pH 7) and brine. The organic layer was
dried over Na2SO4 and concentrated in vacuo. The residue
was chromatographed on silica gel with CHCl3-MeOH (50:1)
to give 132 mg (85%) of 4a as a white powder: 1H NMR (400
MHz, CDCl3) δ 11.14 (1 H, br s), 7.79 (1 H, d, J ) 15.4 Hz),
7.52 (2 H, d, J ) 8.8 Hz), 7.26 (1 H, s), 6.91 (2 H, d, J ) 8.8
Hz), 6.75 (1 H, d, J ) 15.4 Hz), 4.24 (1 H, dd, J ) 10.9, 10.9
Hz), 4.15 (1 H, dd, J ) 10.9, 4.8 Hz), 3.85 (3 H, s), 3.82 (3 H,
1389, 1255, 1216, 1109 cm-1
Anal. (C28H30N2O5‚1.5 H2O) C, H, N.
; SIMS m/ z 475 (M + H)+, 259.
4′-[(Meth oxyp h en yl)m eth yl]cin n a m oyl A-r in g p yr r ole
d u oca r m ycin 4e: yield 66%; 1H NMR (270 MHz, CDCl3) δ
10.78 (1 H, br s), 7.77 (1 H, d, J ) 15.4 Hz), 7.52 (2 H, d, J )
8.9 Hz), 7.36 (2 H, d, J ) 8.4 Hz), 6.97 (2 H, d, J ) 8.9 Hz),
6.92 (2 H, d, J ) 8.4 Hz), 6.75 (1 H, d, J ) 15.4 Hz), 6.62 (1 H,
br s), 5.03 (2 H, s), 4.22 (1 H, dd, J ) 10.9, 10.9 Hz), 4.14 (1 H,
dd, J ) 10.9, 4.5 Hz), 3.82 (6 H, s), 3.55 (1 H, m), 2.61 (3 H, s),
2.39 (1 H, dd, J ) 7.4, 3.4 Hz), 1.31 (1 H, dd, J ) 4.5, 4.5 Hz);
IR (KBr) 1701, 1512, 1390, 1242, 1225, 1171 cm-1; SIMS m/ z
525 (M + H)+. Anal. (C31H28N2O6‚0.5H2O) C, H, N.
4′-Hyd r oxylcin n a m oyl A-r in g p yr r ole d u oca r m ycin 4f:
1
yield 23%; H NMR (270 MHz, DMSO-d6) δ 12.40 (1 H, br s),
10.05 (1 H, s), 7.62 (1 H, d, J ) 14.9 Hz), 7.60 (2 H, d, J ) 8.4
Hz), 6.87 (1 H, d, J ) 14.9 Hz), 6.83 (2 H, d, J ) 8.4 Hz), 6.77
(1 H, br s), 4.35 (1 H, dd, J ) 11.4, 11.4 Hz), 4.21 (1 H, dd, J
) 11.2, 4.9 Hz), 3.76 (3 H, s), 3.47 (1 H, m), 2.49 (3 H, s), 2.11
(1 H, dd, J ) 7.5, 3.5 Hz), 1.32 (1 H, dd, J ) 4.5, 3.5 Hz); IR
(KBr) 1701, 1603, 1489, 1395, 1240, 1169, 1088 cm-1; SIMS
m/ z 405 (M + H)+. Anal. (C23H20N2O5‚3.5H2O) C, H, N.
4′-(N,N-Dim eth ylam in o)cin n am oyl A-r in g pyr r ole du o-
ca r m ycin 4g: yield 64%; 1H NMR (270 MHz, CDCl3) δ 10.73
(1 H, br s), 7.77 (1 H, d, J ) 15.2 Hz), 7.46 (2 H, d, J ) 9.0
Hz), 6.59 (2 H, d, J ) 9.0 Hz), 6.64 (1 H, d, J ) 15.2 Hz), 6.63
(1 H, br s), 4.21 (1 H, dd, J ) 10.9, 10.9 Hz), 4.14 (1 H, dd, J
) 10.9, 4.6 Hz), 3.82 (3 H, s), 3.53 (1 H, m), 3.04 (6 H, s), 2.61
(3 H, s), 2.36 (1 H, dd, J ) 7.3, 4.3 Hz), 1.30 (1 H, dd, J ) 4.9,
4.3 Hz); IR (KBr) 1701, 1593, 1525, 1389, 1360, 1242, 1217,
1169 cm-1; SIMS m/ z 432 (M + H)+. Anal. (C25H25N3O4‚
0.5H2O) C, H, N.
4′-[(ter t-Bu toxyca r bon yl)a m in o]cin n a m oyl A-r in g p yr -
r ole d u oca r m ycin 4h : yield 65%; 1H NMR (270 MHz, CDCl3)
δ 11.32 (1 H, br s), 7.76 (1 H, d, J ) 15.5 Hz), 7.51 (2 H, d, J
) 8.9 Hz), 7.41 (2 H, d, J ) 8.9 Hz), 6.80 (1 H, d, J ) 15.5 Hz),
6.62 (1 H, br s), 4.23 (1 H, dd, J ) 11.2, 11.2 Hz), 4.12 (1 H,
dd, J ) 11.2, 7.3 Hz), 3.82 (3 H, s), 3.59 (1 H, m), 2.62 (3 H, s),
2.40 (1 H, dd, J ) 7.6, 4.3 Hz), 2.05 (1 H, s), 1.54 (9 H, s), 1.30
(1 H, dd, J ) 4.3, 4.0 Hz); IR (KBr) 1707, 1620, 1587, 1525,
1520, 1394, 1294, 1240, 1159 cm-1; SIMS m/ z 504 (M + H)+.
Anal. (C28H29N3O6‚0.5H2O) C, H, N.
4′-Meth ylcin n a m oyl A-r in g p yr r ole d u oca r m ycin 4i:
yield 83%; 1H NMR (270 MHz, CDCl3) δ 10.49 (1 H, br s), 7.79

976 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6
Nagamura et al.
(1 H, d, J ) 15.5 Hz), 7.46 (2 H, d, J ) 8.2 Hz), 7.20 (2 H, d,
J ) 8.2 Hz), 6.83 (1 H, d, J ) 15.5 Hz), 6.67 (1 H, br s), 4.23
(1 H, dd, J ) 10.9, 10.9 Hz), 4.13 (1 H, m), 3.82 (3 H, s), 3.56
(1 H, m), 2.61 (3 H, s), 2.39 (3 H, s), 2.39 (1 H, m), 1.30 (1 H,
dd, J ) 7.6, 3.6 Hz); IR (KBr) 1701, 1606, 1487, 1294, 1242,
1215, 1109 cm-1; SIMS m/ z 403 (M + H)+. Anal. (C24H22N2O4‚
0.2H2O) C, H, N.
Hz), 6.83 (1 H, d, J ) 8.5 Hz), 6.60 (1 H, d, J ) 15.5 Hz), 6.58
(1 H, br s), 4.52 (2 H, s), 4.14 (1 H, dd, J ) 10.9, 10.9 Hz), 4.09
(1 H, m), 3.86 (3 H, s), 3.75 (3 H, s), 3.49 (1 H, m), 2.54 (3 H,
s), 2.31 (1 H, dd, J ) 7.6, 3.4 Hz), 1.55 (9 H, s), 1.25 (1 H, dd,
J ) 4.6, 3.4 Hz); IR (KBr) 1751, 1701, 1616, 1512, 1458, 1392,
1294, 1142 cm-1; SIMS m/ z 549 (M + H)+. Anal. (C30H32N2O8‚
0.5H2O) C, H, N.
4′-Eth ylcin n a m oyl A-r in g p yr r ole d u oca r m ycin 4j:
yield 62%; 1H NMR (270 MHz, CDCl3) δ 10.39 (1 H, br s), 7.79
(1 H, d, J ) 15.6 Hz), 7.49 (2 H, d, J ) 8.1 Hz), 7.23 (2 H, d,
J ) 8.1 Hz), 6.84 (1 H, d, J ) 15.6 Hz), 6.65 (1 H, br s), 4.23
(1 H, dd, J ) 11.2, 11.2 Hz), 4.14 (1 H, dd, J ) 11.2, 4.6 Hz),
3.82 (3 H, s), 3.54 (1 H, m), 2.68 (2 H, q, J ) 7.6 Hz), 2.60 (3
H, s), 2.39 (1 H, dd, J ) 7.6, 3.7 Hz), 1.31 (1 H, dd, J ) 5.3,
3.7 Hz), 1.26 (3 H, t, J ) 7.6 Hz); IR (KBr) 1701, 1618, 1609,
1458, 1390, 1244, 1109 cm-1; SIMS m/ z 417 (M + H)+. Anal.
(C25H24N2O4‚1.5H2O) C, H, N.
3′-[[(Me t h oxyca r b on yl)m e t h yl]oxy]-4′-m e t h oxycin -
n a m oyl A-r in g p yr r ole d u oca r m ycin 5d : yield 88%; 1H
NMR (270 MHz, CDCl3) δ 10.69 (1 H, br s), 7.65 (1 H, d, J )
15.5 Hz), 7.15 (1 H, dd, J ) 8.2, 1.7 Hz), 6.96 (1 H, d, J ) 2.0
Hz), 6.83 (1 H, d, J ) 8.6 Hz), 6.63 (1 H, d, J ) 15.5 Hz), 6.60
(1 H, br s), 4.66 (2 H, s), 4.14 (1 H, dd, J ) 10.9, 10.9 Hz), 4.07
(1 H, m), 3.86 (3 H, s), 3.76 (3 H, s), 3.74 (3 H, s), 3.48 (1 H,
m), 2.52 (3 H, s), 2.31 (1 H, dd, J ) 7.6, 3.4 Hz), 1.25 (1 H, dd,
J ) 5.0, 3.4 Hz); IR (KBr) 1699, 1653, 1616, 1516, 1458, 1396,
1219 cm-1; SIMS m/ z 507 (M + H)+. Anal. (C27H26N2O8) C,
H, N.
3′-(Dieth yla m in o)-4′-m eth oxycin n a m oyl A-r in g p yr -
r ole d u oca r m ycin 5e: yield 87%; 1H NMR (270 MHz, CDCl3)
δ 10.39 (1 H, br s), 7.76 (1 H, d, J ) 15.5 Hz), 7.25 (1 H, dd, J
) 8.6, 1.9 Hz), 7.11 (1 H, d, J ) 2.0 Hz), 6.86 (1 H, d, J ) 8.6
Hz), 6.70 (1 H, d, J ) 15.5 Hz), 6.67 (1 H, br s), 4.22 (1 H, dd,
J ) 10.9, 10.9 Hz), 4.15 (1 H, dd, J ) 10.9, 4.6 Hz), 3.90 (3 H,
s), 3.82 (3 H, s), 3.54 (1 H, m), 3.19 (4 H, q, J ) 7.3 Hz), 2.60
(3 H, s), 2.36 (1 H, dd, J ) 7.6, 3.6 Hz), 1.32 (1 H, dd, J ) 5.3,
3.6 Hz), 1.05 (6 H, t, J ) 7.0 Hz); IR (KBr) 1701, 1616, 1508,
1389, 1255, 1109 cm-1; FABMS m/ z 490 (M + H)+. Anal.
(C28H31N3O5‚1.8H2O) C, H, N.
3′-(Dim eth yla m in o)-4′-m eth oxycin n a m oyl A-r in g p yr -
r ole d u oca r m ycin 5f: yield 64%; 1H NMR (270 MHz, CDCl3)
δ 10.73 (1 H, br s), 7.77 (1 H, d, J ) 15.5 Hz), 7.22 (1 H, dd, J
) 8.6, 2.0 Hz), 7.11 (1 H, d, J ) 2.1 Hz), 6.87 (1 H, d, J ) 8.5
Hz), 6.73 (1 H, d, J ) 15.5 Hz), 4.23 (1 H, dd, J ) 11.0, 10.9
Hz), 4.15 (1 H, dd, J ) 11.0, 4.6 Hz), 3.94 (3 H, s), 3.82 (3 H,
s), 3.55 (1 H, m), 2.82 (6 H, s), 2.61 (3 H, s), 2.11 (1 H, dd, J )
7.6, 3.3 Hz), 1.32 (1 H, dd, J ) 4.9, 3.3 Hz); IR (KBr) 1705,
1614, 1576, 1387, 1240, 1219, 1109 cm-1; FABMS m/ z 462
(M + H)+. Anal. (C26H27N3O5‚1.7H2O) C, H, N.
3′-[(t er t -B u t o x y c a r b o n y l)a m i n o ]-4′-m e t h o x y c i n -
n a m oyl A-r in g p yr r ole d u oca r m ycin 5g: yield 97%; 1H
NMR (270 MHz, CDCl3) δ 10.76 (1 H, br s), 8.37 (1 H, br s),
7.78 (1 H, d, J ) 15.5 Hz), 7.17 (1 H, dd, J ) 8.6, 2.3 Hz), 7.11
(1 H, br s), 6.84 (1 H, d, J ) 8.6 Hz), 6.79 (1 H, br s), 6.74 (1
H, d, J ) 15.5 Hz), 4.24 (1 H, dd, J ) 10.5, 10.5 Hz), 4.16 (1
H, dd, J ) 10.5, 3.9 Hz), 3.92 (3 H, s), 3.82 (3 H, s), 3.54 (1 H,
m), 2.60 (3 H, s), 2.36 (1 H, dd, J ) 7.5, 3.3 Hz), 1.54 (9 H, s),
1.32 (1 H, dd, J ) 4.0, 3.3 Hz); IR (KBr) 1705, 1614, 1531,
1390, 1261, 1219, 1157 cm-1; FABMS m/ z 534 (M + H)+.
Anal. (C29H31N3O7) C, H; N: calcd, 7.88; found, 8.30.
3′-(Am in op r op oxy)-4′-m eth oxycin n a m oyl A-Rin g P yr -
r ole Du oca r m ycin (5h ). A solution of 5a (15 mg, 0.029
mmol) in dry THF (1.5 mL) was stirred at room temperature.
PPh3 (23 mg, 0.088 mmol) was added, and the mixture was
stirred for 24 h. Then, aqueous NaHCO3 was added to the
resulting mixture, and the whole was extracted with CHCl3.
The organic layer was washed with brine, dried over Na2SO4,
and concentrated in vacuo. The residue was chromatographed
on silica gel with CHCl3-MeOH-Et3N (200:10:1) to give 4 mg
(28%) of 5h as a yellowish powder: 1H NMR (270 MHz, DMSO-
d6) δ 7.75 (1 H, d, J ) 15.2 Hz), 7.54 (1 H, br s), 7.49 (1 H, br
d, J ) 8.6 Hz), 7.18 (1 H, d, J ) 8.6 Hz), 7.09 (1 H, d, J ) 15.2
Hz), 7.03 (1 H, br s), 4.15 (1 H, br d, J ) 11.2 Hz), 4.39 (1 H,
m), 4.27 (2 H, t, J ) 3.6 Hz), 3.96 (3 H, s), 3.87 (3 H, s), 3.61
(1 H, m), 3.12 (2 H, t, J ) 7.2 Hz), 2.61 (3 H, s), 2.23 (1 H, m),
2.16 (2 H, m), 1.46 (1 H, m); IR (KBr) 1647, 1610, 1512, 1458,
1394, 1385, 1294, 1219 cm-1; SIMS m/ z 492 (M + H)+. Anal.
(C27H29N3O6) C, H, N.
4′-Am in ocin n a m oyl A-Rin g P yr r ole Du oca r m ycin 4k .
HBr (1 N, 0.5 mL) and TFA (0.2 mL) were added to a solution
of 4h (8 mg, 0.016 mmol) in CH3CN (5 mL), and the reaction
mixture was stirred for 4 h at room temperature. Then, the
mixture was concentrated in vacuo. The residue was dissolved
in CH3CN (1 mL), H2O (0.2 mL), and Et3N (0.2 mL), and the
mixture was stirred for 24 h. Then, 0.2 M phosphate buffer
(pH 7) was added to the resulting mixture, and the whole was
extracted with CHCl3. The organic layer was washed with
brine, dried over Na2SO4, and concentrated in vacuo. The
residue was chromatographed on silica gel with CHCl3-MeOH
(10:1) to give 3 mg (47%) of 4k as a white powder: 1H NMR
(270 MHz, CDCl3) δ 10.28 (1 H, br s), 7.74 (1 H, d, J ) 15.5
Hz), 7.39 (2 H, d, J ) 8.6 Hz), 6.68 (1 H, d, J ) 15.5 Hz), 6.65
(2 H, d, J ) 8.6 Hz), 6.62 (1 H, br s), 4.21 (1 H, dd, J ) 11.2,
11.2 Hz), 4.11 (1 H, dd, J ) 11.2, 6.3 Hz), 3.82 (3 H, s), 3.56 (1
H, m), 2.60 (3 H, s), 2.36 (1 H, dd, J ) 7.6, 3.4 Hz), 1.32 (1 H,
dd, J ) 4.5, 3.4 Hz); IR (KBr) 1697, 1595, 1518, 1443, 1392,
1242, 1219, 1174 cm-1; SIMS m/ z 404 (M + H)+. Anal.
(C23H21N3O4‚1.0H2O) C, H; N: calcd, 9.97; found, 10.43.
Gen er a l Syn t h et ic Met h od for Typ e 5 Com p ou n d s
(5a -g). 3′-(Azid op r op oxy)-4′-m eth oxycin n a m oyl A-Rin g
P yr r ole Du oca r m ycin 5a . NaH (60%; 12 mg, 0.3 mmol) was
added to a solution of 3 (60 mg, 0.23 mmol) in DMF (0.6 mL)
at argon atmosphere, and the mixture was stirred for 2 h at
-50 °C. The p-nitrophenyl ester of 3-(3-azidopropoxy)-4-
methoxycinnamic acid (124 mg, 0.31 mmol) dissolved in DMF
(1.5 mL) was added to a stirred solution at -50 °C. Then, the
resulting mixture was stirred at the same temperature for 2
h. The mixture was diluted with AcOEt, and the combine was
washed with 0.2 M phosphate buffer (pH 7) and brine. The
organic layer was dried over Na2SO4 and concentrated in
vacuo. The residue was chromatographed on silica gel with
CHCl3-MeOH (50:1) to give 77 mg (65%) of 5a as a white
powder: 1H NMR (270 MHz, CDCl3) δ 9.81 (1 H, br s), 7.68 (1
H, d, J ) 15.5 Hz), 7.11 (1 H, dd, J ) 8.3, 2.0 Hz), 7.01 (1 H,
d, J ) 2.0 Hz), 6.81 (1 H, d, J ) 8.2 Hz), 6.66 (1 H, d, J ) 15.5
Hz), 6.56 (1 H, br s), 4.15 (1 H, dd, J ) 11.2, 11.2 Hz), 4.07 (2
H, t, J ) 3.6 Hz), 4.06 (1 H, m), 3.84 (3 H, s), 3.76 (3 H, s),
3.50 (2 H, t, J ) 6.6 Hz), 3.46 (1 H, m), 2.52 (3 H, s), 2.31 (1
H, dd, J ) 7.3, 3.4 Hz), 2.05 (2 H, m), 1.25 (1 H, dd, J ) 5.3,
3.4 Hz); IR (KBr) 2098, 1697, 1622, 1608, 1516, 1392, 1263,
1217 cm-1; SIMS m/ z 518 (M + H)+. Anal. (C27H27N5O6‚
1.5H2O) C, H, N.
3′-[(N ,N -D im e t h y a m in o )p r o p o x y ]-4′-m e t h o x y c in -
n a m oyl A-r in g p yr r ole d u oca r m ycin 5b: yield 52%; 1H
NMR (270 MHz, DMSO-d6) δ 12.38 (1 H, br s), 7.59 (1 H, d, J
) 15.6 Hz), 7.39 (1 H, br s), 7.28 (1 H, br d, J ) 8.5 Hz), 7.00
(1 H, d, J ) 8.6 Hz), 6.94 (1 H, d, J ) 15.6 Hz), 6.90 (1 H, br
s), 4.34 (1 H, br d, J ) 10.8 Hz), 4.27 (1 H, m), 4.06 (2 H, t, J
) 6.2 Hz), 3.81 (3 H, s), 3.76 (1 H, m), 3.73 (3 H, s), 3.12 (2 H,
m), 2.59 (3 H, s), 2.23 (1 H, m), 2.16 (2 H, m), 1.46 (1 H, m),
3′-Am in o-4′-m eth oxycin n a m oyl A-Rin g P yr r ole Du o-
ca r m ycin 5i. The procedure was the same as that employed
for the preparation of 4k . 5g (30 mg, 0.056 mmol) was
subjected to the reaction to afford 12 mg (49%) of 5i as a light-
tan powder: 1H NMR (270 MHz, DMSO-d6) δ 12.40 (1 H, br
s), 7.53 (1 H, d, J ) 15.5 Hz), 7.03 (1 H, d, J ) 1.7 Hz), 6.96
(1 H, dd, J ) 8.3, 1.8 Hz), 6.88 (1 H, d, J ) 8.3 Hz), 6.83 (1 H,
br s), 6.76 (1 H, d, J ) 15.5 Hz), 4.88 (2 H, s), 4.30 (1 H, dd, J
1.23 (6 H, s); IR (KBr) 1684, 1601, 1443, 1437, 1385, 1263 cm-1
;
FABMS m/ z 520 (M + H)+. Anal. (C29H33N3O6‚1.5H2O) C,
H, N.
3′-[[(ter t-Bu toxyca r bon yl)m eth yl]oxy]-4′-m eth oxycin -
n a m oyl A-r in g p yr r ole d u oca r m ycin 5c: yield 52%; 1H
NMR (270 MHz, CDCl3) δ 10.61 (1 H, br s), 7.65 (1 H, d, J )
15.5 Hz), 7.15 (1 H, dd, J ) 8.2, 1.6 Hz), 6.92 (1 H, d, J ) 2.0

Duocarmycin Derivatives
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6 977
) 10.5, 10.5 Hz), 4.22 (1 H, dd, J ) 10.5, 4.9 Hz), 3.85 (3 H, s),
3.76 (3 H, s), 3.48 (1 H, m), 2.50 (3 H, s), 2.11 (1 H, dd, J )
7.3, 4.6 Hz), 1.35 (1 H, dd, J ) 4.6, 3.3 Hz); IR (KBr) 1703,
1612, 1514, 1446, 1390, 1271, 1217, 1111 cm-1; FABMS m/ z
434 (M + H)+. Anal. (C24H23N3O5‚0.5H2O) C, H, N.
s),7.58 (1 H, d, J ) 15.2 Hz), 7.46 (2 H, br s), 7.04 (1 H, d, J )
15.2 Hz), 7.03 (1 H, br s), 4.49 (4 H, m), 4.18 (1 H, m), 3.86 (3
H, s), 3.85 (3 H, s), 3.79 (1 H, br d, J ) 9.9 Hz), 3.48 (3 H, br
s), 2.85 (10 H, br s), 2.68 (3 H, s), 2.50 (3 H, s); IR (KBr) 1716,
1697, 1647, 1510, 1434, 1414, 1246, 1217, 1095 cm-1; FABMS
(the free base) m/ z 670 668 (M + H)+. Anal. (C32H38BrN5O6‚
1.0HCl‚1.0H2O) C, H, N.
Gen er a l Syn t h et ic Met h od for Typ e 6 Com p ou n d s
(6a -e). 4′-Meth oxycin n a m oyl 8-O-[(N-Meth ylp ip er a zi-
n yl)ca r bon yl] A-Rin g P yr r ole Du oca r m ycin B2 Hyd r o-
ch lor id e 6a . Hydrobromic acid (48%, 2.5 mL) was added to
a solution of 4a (50 mg, 0.12 mmol) in CH3CN (5 mL), and the
mixture was stirred for 1 h at room temperature. The
resulting mixture was poured into 1 N HBr, and the combine
was extracted with CHCl3. The organic layer was washed with
brine, dried over Na2SO4, and concentrated in vacuo. p-
Nitrophenyl chloroformate (61 mg, 0.30 mmol) and triethyl-
amine (0.042 mL, 0.30 mmol) were added to a stirred solution
of the residue in dry methylene chloride (5 mL) at -78 °C.
Then, N-methylpiperazine (0.040 mL, 0.36 mmol) was added
to the solution, and the mixture was stirred at 0 °C for 0.5 h.
The mixture was diluted with CHCl3 and washed with 0.2 M
phosphate buffer (pH 7) and brine. The organic layer was
dried over Na2SO4 and concentrated in vacuo. The residue
was chromatographed on silica gel with CHCl3-MeOH (20:1)
to give 67 mg (89%) of the free base of 6a . A solution of the
free base (45 mg, 0.072 mmol) in ethanol (2 mL) and methanol
(4 mL) was treated with anhydrous 5.8 N HCl in EtOH (0.025
mL) at room temperature for 1 h. The resulting mixture was
evaporated in vacuo to give 47 mg (99%) of 6a as a white
crystalline compound: 1H NMR (400 MHz, CDCl3) δ 12.07 (1
H, br s), 10.57 (1 H, br s), 8.10 (1 H, s), 7.74 (2 H, d, J ) 8.7
Hz), 7.58 (1 H, d, J ) 15.3 Hz), 7.06 (1 H, d, J ) 15.3 Hz), 7.00
(2 H, d, J ) 8.7 Hz), 4.50 (1 H, m), 4.42 (3 H, br s), 4.17 (1 H,
br s), 3.85 (3 H, s), 3.82 (3 H, s), 3.79 (1 H, br s), 3.58 (3 H, br
s), 3.50 (4 H, br s), 2.86 (3 H, s), 2.68 (3 H, s). IR (KBr) 1705,
1648, 1599, 1511, 1405, 1218, 1173, 1095, 1023 cm-1; SIMS
3′-(Am in op r op oxy)-4′-m et h oxycin n a m oyl
8-O-[(N-
m eth ylp ip er a zin yl)ca r bon yl] A-Rin g P yr r ole Du oca r -
m ycin B2 Hyd r och lor id e 6f. Hydrobromic acid (48%, 1 mL)
was added to a solution of 5a (50 mg, 0.096 mmol) in CH3CN
(5 mL), and the mixture was stirred for 1 h at room temper-
ature. The resulting mixture was poured into 1 N HBr, and
the whole was extracted with CHCl3. The organic layer was
washed with brine, dried over Na2SO4, and concentrated in
vacuo. p-Nitrophenyl chloroformate (58 mg, 0.29 mmol) and
triethylamine (0.04 mL, 0.29 mmol) were added to a stirred
solution of the residue in dry methylene chloride (5 mL) at
-78 °C. Then, the resulting mixture was stirred at the same
temperature for 0.5 h. N-Methylpiperazine (0.038 mL, 0.34
mmol) was added to the solution, and the mixture was stirred
at 0 °C for 1 h. The mixture was diluted with CHCl3, and the
combine was washed with 0.2 M phosphate buffer (pH 7) and
brine. The organic layer was dried over Na2SO4 and concen-
trated in vacuo. The residue was chromatographed on silica
gel with CHCl3-MeOH (20:1) to give 51 mg of the 8-O
derivative. A solution of the 8-O derivative (35 mg, 0.049
mmol) with 10% Pd/BaSO4 (22 mg) in acetone (1 mL) and CH3-
OH (5 mL) was stirred under 1 atm of H2 at room temperature
for 4 h. The reaction mixture was filtered and concentrated
in vacuo. The residue was chromatographed on silica gel using
CHCl3-CH3OH-NH4OH (10:1:1) as an eluent to afford 12 mg
of the free base of 6f. The obtained free base was employed
in the same procedure as that for the preparation of HCl salt
to give 10 mg of 6f in 41% yield from 5a : 1H NMR (270 MHz,
DMSO-d6) δ 12.18 (1 H, br s), 10.93 (1 H, br s), 8.09 (1 H, s),
7.89 (2 H, br s), 7.56 (1 H, d, J ) 15.3 Hz), 7.43 (1 H, br s),
7.33 (1 H, d, J ) 8.2 Hz), 7.06 (1 H, d, J ) 15.3 Hz), 7.03 (1 H,
d, J ) 8.3 Hz), 4.50 (1 H, m), 4.40 (2 H, m), 4.15 (2 H, t, J )
5.6 Hz), 3.85 (3 H, s), 3.83 (3 H, s), 3.46 (6 H, br s), 3.26 (4 H,
br s), 2.99 (2 H, m), 2.84 (3 H, s), 2.68 (3 H, s), 2.05 (2 H, m);
(the free base) m/ z 627 625 (M + H)+. Anal. (C30H33
BrN4O6‚1.0HCl‚3.0H2O) C, H, N.
-
4′-Meth oxycin n a m oyl 8-O-[(4-p ip er id in op ip er id in yl)-
ca r bon yl] A-r in g p yr r ole d u oca r m ycin B2 h yd r och lor id e
6b: yield 87%; 1H NMR (270 MHz, DMSO-d6) δ 12.02 (1 H, s),
9.96 (1 H, br s), 8.05 (1 H, s), 7.74 (2 H, d, J ) 8.9 Hz), 7.57 (1
H, d, J ) 15.4 Hz), 7.05 (1 H, d, J ) 15.4 Hz), 6.99 (2 H, d, J
) 8.9 Hz), 4.46 (2 H, m), 4.41 (2 H, br s), 4.19 (1 H, br d, J )
13.3 Hz), 3.84 (3 H, s), 3.80 (3 H, s), 3.77 (1 H, br s), 3.47 (5 H,
br s), 3.13 (1 H, br d, J ) 12.9 Hz), 2.95 (2 H, br s), 2.66 (3 H,
s), 2.15 (2 H, br s), 1.82 (6 H, br s), 1.70 (2 H, br s); IR (KBr)
IR (KBr) 1716, 1647, 1509, 1437, 1408, 1263, 1140 cm-1
;
FABMS m/ z 700 698 (M + H)+. Anal. (C33H40BrN5O7‚
2.0HCl‚1.0H2O) C, H, N.
3′-Am in o-4′-m eth oxycin n a m oyl 8-O-[(N-Meth ylp ip er -
a zin yl)ca r bon yl] A-Rin g P yr r ole Du oca r m ycin B2 Hy-
d r och lor id e 6g. The procedure was the same as that
employed for the preparation of 5i. 5g (29 mg, 0.054 mmol)
was subjected to the reaction to afford 16 mg of 6g in 44%
yield from 5g: 1H NMR (270 MHz, DMSO-d6) δ 12.09 (1 H, br
s), 10.64 (1 H, br s), 8.10 (1 H, br s), 7.49 (1 H, d, J ) 15.2 Hz),
7.22 (1 H, br s), 7.16 (1 H, d, J ) 8.6 Hz), 6.95 (1 H, d, J ) 2.9
Hz), 6.90 (1 H, d, J ) 15.2 Hz), 4.42 (2 H, m), 4.19 (1 H, m),
3.86 (3 H, s), 3.85 (3 H, s), 3.79 (1 H, br s), 3.49 (9 H, br s),
2.89 (3 H, s), 2.68 (3 H, s); IR (KBr) 1699, 1645, 1514, 1439,
1412, 1281, 1219 cm-1; FABMS m/ z 642 640 (M + H)+. Anal.
(C30H34BrN5O6‚2.0HCl‚3.5H2O) C, H, N.
3′-[(Ca r b oxym et h yl)oxy]-4′-m et h oxycin n a m oyl 8-O-
(Dim eth ylca r ba m oyl) A-Rin g P yr r ole Du oca r m ycin B2
(6h ). Hydrobromic acid (48%, 0.16 mL) was added to a
solution of 5c (99 mg, 0.18 mmol) in CH3CN (5 mL), and the
mixture was stirred for 1 h at room temperature. The
resulting mixture was poured into 1 N HBr, and the whole
was extracted with CHCl3. The organic layer was washed with
brine, dried over Na2SO4, and concentrated in vacuo. p-
Nitrophenyl chloroformate (109 mg, 0.54 mmol) and triethyl-
amine (0.076 mL, 0.54 mmol) were added to a stirred solution
of the residue in dry methylene chloride (5 mL) at -78 °C.
Then, the resulting mixture was stirred at the same temper-
ature for 0.5 h. Dimethylamine (50%, 0.162 mL, 1.8 mmol)
was added to the solution, and the mixture was stirred at 0
°C for 1 h. The mixture was diluted with CHCl3, and the
combine was washed with 0.2 M phosphate buffer (pH 7) and
brine. The organic layer was dried over Na2SO4 and concen-
trated in vacuo. The residue was chromatographed on silica
gel with CHCl3-MeOH (20:1) to give 44 mg of the 8-O
1688, 1646, 1598, 1514, 1407, 1252, 1213, 1093, 1023 cm-1
FABMS (the free base) m/ z 695 693 (M + H)+. Anal. (C35H41
BrN4O6‚1.0HCl‚3.5H2O) C, H, N.
;
-
4′-Meth oxycin n a m oyl 8-O-[[[[(N-isop r op yla m in o)ca r -
bon yl]m eth yl]p ip er a zin yl]ca r bon yl] A-r in g p yr r ole d u o-
1
ca r m ycin B2 h yd r och lor id e 6c: yield 70%; H NMR (270
MHz, DMSO-d6) δ 12.21 (1 H, s), 10.39 (1 H, br s), 8.60 (1 H,
br s), 8.09 (1 H, s), 7.75 (2 H, d, J ) 8.4 Hz), 7.57 (1 H, d, J )
14.3 Hz), 7.06 (1 H, d, J ) 14.3 Hz), 6.99 (1 H, d, J ) 8.4 Hz),
4.41 (3 H, m), 4.12 (1 H, m), 3.91 (4 H, m), 3.84 (3 H, s), 3.81
(3 H, s), 3.77 (1 H, m), 3.69 (6 H, m), 2.68 (3 H, s), 1.22 (1 H,
m), 1.11 (6 H, d, J ) 6.4 Hz); IR (KBr) 1678, 1643, 1599, 1515,
1409, 1251, 1212, 1095, 1032 cm-1; FABMS (the free base) m/ z
712 710 (M + H)+. Anal. (C34H40BrN5O7‚1.0HCl‚3.0H2O) C,
H, N.
3′-(Diet h yla m in o)-4′-m et h oxycin n a m oyl
8-O-[(N-
m eth ylp ip er a zin yl]ca r bon yl] A-r in g p yr r ole d u oca r m y-
cin B2 h yd r och lor id e 6d : yield 50%; 1H NMR (270 MHz,
DMSO-d6) δ 12.19 (1 H, br s), 10.88 (1 H, br s), 8.10 (2 H, br
s), 7.60 (1 H, br d, J ) 8.6 Hz), 7.40 (1 H, br s), 7.24 (1 H, br
s), 7.00 (1 H, br s), 4.58 (3 H, m), 3.85 (6 H, s), 3.81-3.26 (2 H,
m), 3.21 (4 H, q, J ) 7.2 Hz), 2.88 (4 H, br s), 2.69 (3 H, s),
2.51 (4 H, br s), 2.37 (3 H, s), 1.07 (6 H, t, J ) 7.0 Hz); IR
(KBr) 1714, 1645, 1435, 1417, 1410, 1255, 1219 cm-1; FABMS
(the free base) m/ z 698 696 (M + H)+. Anal. (C34H42BrN5O6‚
2.0HCl‚3.0H2O) C, H, N.
3′-(Dim et h yla m in o)-4′-m et h oxycin n a m oyl
8-O-[(N-
m eth ylp ip er a zin yl)ca r bon yl] A-r in g p yr r ole d u oca r m y-
cin B2 h yd r och lor id e 6e: yield 55%; 1H NMR (270 MHz,
DMSO-d6) δ 12.09 (1 H, br s), 10.55 (1 H, br s), 8.10 (1 H, br

978 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6
Nagamura et al.
SA, A Potent Antitumor Antibiotic. J . Antibiot. 1991, 44, 445-
447. (g) Ichimura, M.; Ogawa, T.; Katsumata, S.; Takahashi, K.;
Takahashi, I.; Nakano, H. Duocarmycins, New Antibiotics
Produced by Streptomyces; Producing Organisms and Improved
Production. J . Antibiot. 1991, 44, 1045-1053. (h) Yasuzawa, T.;
Muroi, K.; Ichimura, M.; Takahashi, I.; Ogawa, T.; Takahashi,
K.; Sano, H; Saitoh, Y. Duocarmycins, Potent Antitumor Anti-
biotics by Streptomyces sp. Structures and Chemistry. Chem.
Pharm. Bull. 1995, 43, 378-391.
derivative. A solution of the 8-O derivative (30 mg, 0.043
mmol) in CH2Cl2 (1 mL) and TFA (0.051 mL) was stirred at
80 °C for 24 h. The mixture was diluted with CHCl3, and the
combine was washed with 1 N HBr and brine. The organic
layer was dried over Na2SO4 and concentrated in vacuo. The
residue was chromatographed on silica gel using CHCl3-CH3-
OH-CH3COOH (100:10:1) as an eluent to afford 23 mg of 6h
in 29% yield from 5c: 1H NMR (270 MHz, DMSO-d6) δ 12.01
(1 H, br s), 8.02 (1 H, br s), 7.51 (1 H, d, J ) 15.1 Hz), 7.26 (1
H, br d, J ) 8.5 Hz), 7.17 (1 H, br s), 6.97 (1 H, d, J ) 15.1
Hz), 6.96 (1 H, d, J ) 8.5 Hz), 4.50 (1 H, m), 4.38 (2 H, m),
4.30 (2 H, m), 3.84 (3 H, s), 3.81 (3 H, s), 3.78 (2 H, br s), 3.15
(3 H, s), 2.96 (3 H, s), 2.65 (3 H, s); IR (KBr) 1701, 1585, 1437,
1416, 1317, 1267, 1169 cm-1; FABMS m/ z 645 643 (M + H)+.
Anal. (C29H29BrN3O9) C, N; H: calcd, 4.53; found, 4.12.
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+-, Mg2+-free; PBS(-)], and further incu-
bated 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 (Toa Medical Electronics Co., Ltd.,
Kobe, J apan). The IC50 values (drug concentration required
for 50% inhibition of the cell growth) were determined.
Sarcoma 180 cells were kindly supplied by the National
Cancer Center (Tokyo, J apan). Sarcoma 180 cells were
passaged and used for the experiment in adult male ddY mice.
Murine solid tumor was inoculated subcutaneously (sc) at the
axillary region of mice. Drugs were administered intrave-
nously (iv) beginning 1 day after tumor inoculation. Antitumor
efficacy was 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
(2) (a) Boger, D. L.; Ishizaki, T.; Zarrinmayeh, H. Synthesis and
Preliminary Evaluation of Agents Incorporating the Pharma-
cophore of the Duocarmycin/Pyrindamycin Alkylation Subunit:
Identification of the CC-1065/Duocarmycin Common Pharma-
cophore. J . Org. Chem. 1990, 55, 4499-4502. (b) Sugiyama, H.;
Hosoda, M.; Saito, I.; Asai, A.; Saito, H. Covalent Alkylation of
DNA with Duocarmycin A. Identification of Abasic Site Struc-
ture. Tetrahedron Lett. 1990, 31, 7197-7200. (c) Boger, D. L.;
Ishizaki, T.; Zarrinmayeh, H.; Munk, S. A.; Kitos, P. A.;
Suntornwat, O. Duocarmycin-Pyrindamycin DNA Alkylation
Properties and Identification, Synthesis, and Evaluation of
Agents Incorporating the Pharmacophore of the Duocarmycin-
Pyrindamycin Alkylation Subunit. Identification of the CC-1065-
Duocarmycin Common Pharmacophore. J . Am. Chem. Soc. 1990,
112, 8961-8971. (d) Boger, D. L.; Ishizaki, T.; Zarrinmayeh, H.
Isolation and Characterization of the Duocarmycin-Adenine
DNA Adduct. J . Am. Chem. Soc. 1991, 113, 6645-6649. (e)
Sugiyama, H.; Ohmori, K.; Chan, K. L.; Hosoda, M.; Asai, A.;
Saito, H.; Saito, I. A Novel Guanine N3 Alkylation by Antitumor
Antibiotic Duocarmycin A. Tetrahedron Lett. 1993, 34, 2179-
2182. (f) Boger, D. L.; J ohnson, D. S.; Yun, W. (+)- and ent-(-)-
Duocarmycin SA and (+)- and ent-(-)-N-Boc-DSA DNA Alkyla-
tion Properties. Alkylation Site Models That Accommodate the
Offset AT-Rich Adenine N3 Alkylation Selectivity of the Enan-
tiomeric Agents. J . Am. Chem. Soc. 1994, 116, 1635-1656.
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Chemistry, Mechanism of Action and Biological Properties of CC-
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L.; Scahill, T. A. Reaction of the Antitumor Antibiotic CC-1065
with DNA: Structure of a DNA Adduct with DNA Sequence
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Reaction of the Antitumor Antibiotic CC-1065 with DNA.
Location for the Site of Thermally Induced Strand Breakage and
Analysis of DNA Sequence Specificity. Biochemistry 1985, 24,
6228-6237. (c) Tang, M. S.; Lee, C. S.; Doisy, R.; Ross, L.;
Needham-VanDevanter, D. R.; Hurley, L. H. Recognition and
Repair of the CC-1065 -(N3-Adenine)-DNA Adduct by the
UVRABC Nuclease. Biochemistry 1988, 27, 893-901.
tumor volume (mm3) ) length (mm) × [width (mm)]2/2
according to the method of the National Cancer Institute.20
The criteria for effectiveness against murine solid tumors
were the percentage T/ C value with 42% and less, and
statistical significance 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 at the day
of evaluation and V0 is the tumor volume at the day of initial
drug treatment. The criteria for effectiveness were T/ C value
with 50% and less and statistical significance determined by
the Mann-Whitney U test (p < 0.01, one-sided).21
(5) Gomi, K.; Kobayashi, E.; Miyoshi, K.; Ashizawa, T.; Okamoto,
A.; Ogawa, T.; Katsumata, S.; Mihara, A.; Okabe, M.; Hirata,
T. Anticellular and Antitumor Activity of Duocarmycins, Novel
Antitumor Antibiotics. J pn. J . Cancer Res. 1992, 83, 113-120.
(6) A number of reviews on the duocarmycins are available from
the following: (a) Boger, D. L. Duocarmycins: A New Class of
Sequence Selective DNA Minor Groove Alkylating Agents.
CHEMTRACTS: Org. Chem. 1991, 4, 329-349. (b) Boger, D.
L. The Duocarmycins: Synthetic and Mechanistic Studies. Acc.
Chem. Res. 1995, 28, 20-29. (c) Boger, D. L.; J ohnson, D. S.
CC-1065 and the Duocarmycins: Unraveling the Keys to a New
Class of Naturally Derived DNA Alkylating Agents. Proc. Natl.
Acad. Sci. U.S.A. 1995, 92, 3642-3649. (d) Boger, D. L.; J ohnson,
D. S. CC-1065 and the Duocarmycins: Understanding Their
Biological Function through Mechanistic Studies. Angew. Chem.,
Int. Ed. Engl. 1996, 35, 1438-1474.
Ack n ow led gm en t. We thank Dr. Mayumi Yoshida
and Mr. Shingo Kakita for measuring NMR spectra.
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reported else in the near future.
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J M9606094