New water-soluble duocarmycin derivatives: Synthesis and antitumor activity of A-ring pyrrole compounds bearing β-heteroarylacryloyl groups

Article abstract of DOI:10.1021/jm980559y

A series of A-ring pyrrole compounds of duocarmycin bearing 4'-methoxy- β-heteroarylacryloyl groups were synthesized and evaluated for in vitro anticellular activity against HeLa S₃ cells and in vivo antitumor activity against murine sarcoma 18

Full text of DOI:10.1021/jm980559y

J . Med. Chem. 1999, 42, 669-676
669
New Wa ter -Solu ble Du oca r m ycin Der iva tives: Syn th esis a n d An titu m or Activity
of A-Rin g P yr r ole Com p ou n d s Bea r in g â-Heter oa r yla cr yloyl Gr ou p s
Nobuyoshi Amishiro,* Satoru Nagamura, Eiji Kobayashi, Katsushige Gomi, and Hiromitsu Saito
Pharmaceutical Research Institute, Kyowa Hakko Kogyo Company, Ltd., 1188, Shimotogari, Nagaizumi, Sunto,
Shizuoka 411-0943, J apan
Received October 2, 1998
A series of A-ring pyrrole compounds of duocarmycin bearing 4′-methoxy-â-heteroarylacryloyl
groups were synthesized and evaluated for in vitro anticellular activity against HeLa S3 cells
and in vivo antitumor activity against murine sarcoma 180 in mice. Most of the 4′-methoxy-
â-heteroarylacrylates displayed in vitro anticellular activity equivalent to that of 4′-methoxy-
cinnamates. Among the 8-O-[(N-methylpiperazinyl)carbonyl] derivatives of 4′-methoxy-â-
heteroarylacrylates, compound 15b having a (4-methoxy-3,5-pyrimidinyl)acryloyl as segment-B
(Seg-B) showed remarkably potent in vivo antitumor activity and low peripheral blood toxicity
compared with the A-ring pyrrole derivatives having the trimethoxyindole skeleton in Seg-B,
which were equal to 8-O-[(N-methylpiperazinyl)carbonyl] derivatives of 4′-methoxycinnamates.
Moreover, these 8-O-[(N-methylpiperazinyl)carbonyl] derivatives of 4′-methoxy-â-heteroaryl-
acrylates had high aqueous solubility.
In tr od u ction
derivative, the natural type of Seg-B.12 In previous
papers,13 we have reported the structure-activity re-
lationship of 4′-substituted-trans-cinnamoyl derivatives.
Among derivatives modified at Seg-B of KW-2189 (3b),
compound 4b was found to exhibit sufficient in vivo
activity against sarcoma 180 with low peripheral blood
toxicity.12 The reduction of toxicity of compound 4b, of
which Seg-B is 4-methoxycinnamoyl, is considered to
result from the decrease of noncovalent binding to DNA
and the increase of reversibility of covalent bonding to
DNA.7c,14 In the same manner, it is shown that the
derivatives modified at the noncovalent binding moiety
of CC-1065 have excellent broad-spectrum in vivo
antitumor activity without causing delayed deaths.15
Duocarmycins (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).1-3 Since
duocarmycins B1 (1e), B2 (1c), C1 (1f), and C2 (1d )
readily yield duocarmycin A (1a )1a-c in aqueous solution,
duocarmycin A is thought to be an active form among
these antibiotics. Duocarmycins A and SA (1b) have a
unique cyclopropane ring responsible for the sequence-
selective alkylation of double-stranded DNA mediating
N3 adenine covalent adduct formation.4 This mecha-
nism is similar to that of CC-1065 (2) which has been
reported to show a high cytotoxicity.5,6 KW-2189 (3b),7
selected as the best compound in analogues of A-ring
pyrrole derivatives of duocarmycin B2, showed good
stability in the culture medium and aqueous solubility
greater than 10 mg/mL.8,9 It also showed strong activi-
ties against murine ascitic and human solid tumors.7b
KW-2189 (3b) was designed as a prodrug which requires
enzymatic hydrolysis followed by regeneration of DU-
86 (3a ) as an active metabolite.10 KW-2189 (3b) is
currently under phase II clinical evalution.
However, the aqueous solubility of compound 4b is
below 0.1 mg/mL. Due to its poor solubility, compound
4b is difficult to use in clinical studies. We have
previously synthesized a variety of analogues by modi-
fying the substituent on the aromatic ring of the
4′-methoxycinnamoyl group.3d,16 The 8-O-(N,N-dialkyl-
carbamoyl)cinnamates having an amino group at the
3′-position were found to possess adequate water solu-
bility in excess of 10 mg/mL.13
The segment-A (Seg-A) containing a spirocyclopropyl-
hexadienone moiety is necessary for the formation of
covalent binding with DNA. Our previous results indi-
cate that the Seg-A structure will influence the electro-
philicity of cyclopropane.7c On the other hand, the
segment-B (Seg-B) of duocarmycin has been considered
to play an important role for noncovalent binding to the
minor groove of DNA.11 We have previously synthesized
a series of duocarmycin analogues bearing a simplified
DNA noncovalent binding segment such as acetyl, indol-
2-ylcarbonyl, benzofuran-2-ylcarbonyl, cinnamoyl, and
phenoxyacetyl groups to increase antitumor activity and
decrease toxicity. Among them, the cinnamoyl and
phenoxyacetyl derivatives were more potent and had
lower toxicity than benzofuran-2-ylcarbonyl or indol-2-
ylcarbonyl derivatives including the trimethoxyindole
Recently, it has been reported that several efforts are
being made to modify DNA sequence selectivity of the
distamycin molecule, one of the DNA minor groove
binders. Structural modifications have been based on
replacement of N-methylpyrrole by an N-methylimid-
azole to accommodate hydrogen bonding to the 2-amino
group of guanine. The result was that imidazole-
containing analogues of distamycin changed AT se-
quence specificity to GC in the DNA minor groove.17
It is expected that our approach to synthesize new
Seg-B analogues having â-heteroarylacryloyl groups
may enhance potency and decrease toxicity by non-
covalent binding site changes attributable to an interac-
tion between nitrogen on the heteroaryl and DNA.
Moreover, it is expected that these new analogues could
10.1021/jm980559y CCC: $18.00 © 1999 American Chemical Society
Published on Web 02/03/1999

670 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4
Amishiro et al.
Ch a r t 1. Structures of Duocarmycins, CC-1065, and Duocarmycin Derivatives
Sch em e 1
improve aqueous solubility. In this paper, we report our
investigation regarding the synthesis, anticellular and
antitumor activities, hematotoxicity, and structure-
activity relationships (SAR) of these derivatives.
conditions using potassium carbonate as a base and
tetrabutylammonium chloride as the phase-transfer
agent.20 The â-heteroarylacrylic methyl esters were
treated with 4 N KOH to afford â-heteroarylacrylic acid
derivatives, which were converted to p-nitrophenyl
esters 11a -11d by the reaction with p-nitrophenol
using 1-methyl-2-chloropyridinium salt (Mukaiyama’s
method)21 or condensing agent (DCC). We obtained
better results by Mukaiyama’s methods.
The â-heteroarylacrylates of duocarmycin (13a -13d ,
14a -14d , 15a -15d ) were prepared as follows (Scheme
2). The 2-methyl-3-methoxycarbonyl A-ring pyrrole
compound of duocarmycin (DU-86, 3a ) was prepared by
employing the Wagner-Meerwein type rearrangement
of the 8-O-protected-3-hydroxyduocarmycin B2 followed
by deprotection of the protecting group under basic
conditions.13,22 Compound 3a was treated with NaOMe
in MeOH to afford Seg-A (12). The reaction of 12 and a
Ch em istr y
Initially, the various â-heteroarylacrylic acids were
synthesized according to Scheme 1. The synthetic
intermediate for 11a was (E)-3-(6-methoxypyridinyl)-
acrylic acid, which was prepared by Nishikawa’s meth-
ods.18 The synthetic intermediates (6, 8, 10) of the other
â-heteroarylacrylic acids were synthesized from starting
materials 5, 7, and 9. The coupling reaction of com-
pounds 6, 8, and 10 and methyl acrylate was carried
out by Pd-Heck reaction.19 We tried Pd-Heck reaction
under various conditions to find that the reaction would
successfully proceed in the presence of a catalytic
amount of palladium acetate under phase-transfer

New Water-Soluble Duocarmycin Derivatives
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4 671
Sch em e 2
Ta ble 1. Anticellular Activity, Antitumor Activity,
Hematotoxicity, and Water Solubility of Duocarmycin
Derivatives
suitable acylating agent, p-nitrophenyl esters 11a -11d ,
was carried out with base (NaH) in DMF to give 13a -
13d, respectively.12,13 Compounds 13a-13d were treated
with 48% HBr followed by addition of p-nitrophenyl
chloroformate in the presence of triethylamine in
CH2Cl2-toluene at -78 °C to give carbonates as an
intermediate. By treating carbonates as an intermediate
with N-methylpiperazine, the 8-O-[(N-methylpiper-
azinyl)carbonyl] derivatives 14a -14d were obtained.
When CH2Cl2 was used as the solvent, the reaction yield
was low due to the heterogeneous system. We tried
various solvent systems. The result was that com-
pounds 14a -14d were obtained in high yield by using
CH2Cl2-toluene as solvent, which was a homogeneous
system.
sarcoma 180
(sc-iv)b
hematotoxicity
HeLa S3
water
solubility
(mg/mL)
IC50 (nM)a
dose
(mg/kg) T/Cc (%)
WBCd
PLe
(%)
no.
1 h
72 h
13a 3.3
15a 900
13b 2.5
15b 1300
13c 0.5
15c 1100
13d 9.7
15d 1300
0.42
110
0.90
170
0.28
90
0.25
0.60
0.36
0.45
0.26
50
38
28
26
84
1
2
2
92
86
69
>20
>20
>20
>20
1
0.31
51
115 (2) f
2.9
150
4
0.5
38
105
4a
4b
2.9-7.0 0.26-0.94
1800 37
0.83
4
0.34
0.2
50
25
63
44g
<0.1
Formation of salts of 14a -14d was examined for
aqueous solubility. Treatment of 14b with 48% HBr
resulted in a degradation product, 4′-hydroxy-3′,5′-
pyrimidinyl acrylate, which was formed by demethyl-
ation on the pyrimidine ring of Seg-B and detected with
1H NMR and FAB-MS. It was considered that the 4′-
hydroxy-3′,5′-pyrimidinyl acrylate was obtained by H2O
in 48% HBr. Treatment of 14a -14d with 6.86 N HCl
in EtOH provided the salts 15a -15d without decom-
position. The aqueous solubility of the salts 15a -15d
was found to be more than 20 mg/mL.
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 of tumor volume
d
of 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 Mortality (5 mice/group). Dose of 5.33 mg/kg.
g
is obtained as an active metabolite by enzymatic hy-
drolysis of compound 4b, which is the same action as
that of DU-86 (3a ), an active metabolite of KW-2189 (3b)
(data not shown).7d,10 The same tendency was shown
between 13a -13d and 15a -15d . Compounds 13a -13c
exhibited strong anticellular activity with IC50 values
below 1.0 nM at 72-h exposure, which was almost
equivalent to that of compound 4a . Compound 13d , in
which the position of nitrogen in the â-heteroarylacryl-
oyl of Seg-B was different from that in 13a , showed
anticellular activity inferior to that of 13a . This result
supported the results we have shown previously that
the 3′-position of the 4′-methoxycinnamates did not
seriously influence the association between DNA and
Resu lts a n d Discu ssion
The antitumor activity of some representative deriva-
tives was evaluated primarily by assays of the inhibition
of HeLa S3 cell growth (in vitro) and antitumor activity
against murine sarcoma 180 (in vivo). As shown in Table
1, the efficacy in vivo is expressed as T/C, where T and
C represent means of tumor volume in treated and
control mice, respectively. Compound 4b showed 100
times weaker anticellular activity compared with com-
pound 4a (72-h exposure). It appears that compound 4a

672 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4
Amishiro et al.
drug.13 The biological activity indicated that the hetero-
aryl substituents did not influence improvement of
potency.
was added N-iodosuccinimide (3.96 g, 16.9 mmol), and the
mixture was stirred at reflux (80 °C) for 11 h. Then water was
added, and the resulting mixture was extracted with CHCl3,
washed with aqueous NaHCO3 and 5% Na2S2O3, and worked
up as usual. The residue was purified by column chromatog-
raphy (hexane-AcOEt, 3:1) to give 523 mg (26%) of 2-methoxy-
5-iodopyrimidine (6): 1H NMR (90 MHz, CDCl3) 8.64 (2 H, s),
3.99 (3 H, s).
Peripheral blood toxicity (reduction of the number of
peripheral blood platelets) of 4′-methoxy-â-heteroaryl-
acrylates was lower than that of the derivatives bearing
Seg-B of natural type and was equal to that of 4′-
methoxycinnamates. Among new Seg-B derivatives,
compound 15b showed antitumor activity against mu-
rine solid tumor with T/C values less than 0.3, which is
comparable to that of compound 4b [15b: T/C ) 0.26;
KW-2189: T/C ) 0.15 (0.63 mg/kg dose)].7 Indeed,
compound 15b exhibited sufficient efficacy with low
peripheral blood toxicity.12,13 The other compounds
showed a tendency to decrease antitumor activity in
vivo. Compound 15b was evaluated for in vivo anti-
tumor activity against nude mice bearing human xeno-
graft St-4 (poorly differentiated stomach adenocarcin-
oma). Compound 15b showed excellent activity in vivo
with T/C values of 0.28 (12 mg/kg dose). Its potency
against St-4 human stomach tumor xenograft was
nearly comparable to the potency of our clinical candi-
date KW-2189 (3b) [T/C ) 0.12 (0.63 mg/kg dose)].7
Compound 4b exhibits poor solubility below 0.1 mg/
mL in water or phosphate buffer (pH 7). However, the
8-O-[(N-methylpiperazinyl)carbonyl] derivatives of 4′-
methoxy-â-heteroarylacrylates (15a -15d ) were found
to possess adequate water solubility in excess of 20 mg/
mL. Compounds 15a -15d were over 200-fold more
soluble in aqueous solution than compound 4b.
3-Meth oxy-6-iod op yr id a zin e (8). To a solution of 3,6-
dichloropyridazine (7) (2.00 g, 13.4 mmol) in acetone (60 mL)
was added NaI (20.1 g, 268 mmol), and the mixture was heated
under reflux (70 °C) for 2 h. Water was added, and the
resulting mixture was extracted with AcOEt. Usual workup
gave 2.73 g (61%) of 3,6-diiodopyridazine. To a solution of 3,6-
diiodopyridazine (2.73 g, 8.23 mmol) in methanol (80 mL) was
added 28 wt % NaOMe in methanol (3.17 g, 16.5 mmol), and
the mixture was stirred at room temperature for 13 h. Then
water was added; the whole was extracted with AcOEt. Usual
workup and purification by column chromatography (hexane-
AcOEt, 8:1) gave 1.661 g (86%) of 3-methoxy-6-iodopyridazine
1
(8): H NMR (90 MHz, CDCl3) 7.64 (1 H, d, J ) 9.1 Hz), 8.31
(1 H, d, J ) 9.1 Hz), 4.09 (3 H, s).
3-Meth oxy-6-iod op yr id in e (10). To a methanol (80 mL)
solution of 3-hydroxypyridine (9) (3.00 g, 31.5 mmol), NaI (4.72
g, 31.5 mmol), and NaOH (1.26 g, 31.5 mmol) was added
dropwise 4% NaOCl in water (58.5 g) at 0 °C over 100 min.
Then the mixture was stirred at 0 °C for 3 h; 5% Na2S2O3 (10
mL), 0.1 N HCl was added, and the whole was extracted with
AcOEt. Usual workup and purification by column chromatog-
raphy (hexane-AcOEt, 4:1) afforded 2.38 g (34%) of 3-hydroxy-
6-iodopyridine. To a solution of 60% NaH (647 mg, 16.2 mmol)
in DMF (20 mL) was added a DMF solution (20 mL) of
3-hydroxy-6-iodopyridine (2.38 g, 10.8 mmol), and the mixture
was stirred under Ar atomosphere at room temperature. After
2 h, NaI (2.14 g, 15.1 mmol) was added and stirred for 1 h;
0.01 M phosphoric buffer (pH 7) was added; the whole was
extracted AcOEt and then worked up as usual. The residue
was purified by column chromatography (hexane-AcOEt, 5:1-
4:1) to give 2.57 g (100%) of 3-methoxy-6-iodopyridine (10): 1H
NMR (90 MHz, CDCl3) 8.03 (1 H, m), 7.22-7.30 (2 H, m), 3.96
(3 H, s).
(E)-p-Nit r op h en yl 3-(2-Met h oxy-5-p yr id in yl)a cr yla t e
(11a ). To a solution of (E)-3-(2-methoxy-5-pyridinyl)acrylic
acid16 (182 mg, 1.02 mmol) in CH2Cl2 (15 mL) were added
p-nitrophenol (241 mg, 1.73 mmol), trimethylamine (0.483 mL,
3.47 mmol), and 2-chloro-1-methylpyridinium iodide (443 mg,
1.73 mmol), and the mixture was heated under reflux (50 °C)
for 2 h. The reaction mixture was added to aqueous NaHCO3
and extracted with CHCl3. Usual workup and purification by
column chromatography (CHCl3-MeOH, 100:1) gave 220 mg
(72%) of 11a : 1H NMR (270 MHz, CDCl3) 8.35 (1 H, d, J ) 2.3
Hz), 8.31 (2 H, d, J ) 8.9 Hz), 7.86 (1 H, d, J ) 16.2 Hz), 7.85
(1 H, dd, J ) 8.6, 2.3 Hz), 7.37 (2 H, d, J ) 8.9 Hz), 6.82 (1 H,
d, J ) 8.6 Hz), 6.52 (1 H, d, J ) 16.2 Hz), 4.00 (3 H, s).
Con clu sion s
A series of A-ring pyrrole compounds of duocarmycin
bearing 4′-methoxy-â-heteroarylacryloyl groups were
prepared and evaluated for their anticellular activity
against HeLa S3 cells and for antitumor activity against
sarcoma 180 murine solid tumor and St-4 human
stomach tumor xenograft. Some showed potent anti-
tumor activity nearly comparable to that of our clinical
candidate KW-2189. Moreover, these compounds had
lower peripheral blood toxicity than the derivatives
bearing a trimethoxyindole skeleton in Seg-B and higher
aqueous solubility than 4′-methoxycinnamates.
Exp er im en ta l Section
All melting points were measured on a Yanagimoto micro-
melting point apparatus without correction. Infrared spectra
(IR) were recorded on a J ASCO IR-810 spectrometer. 1H NMR
spectra were measured on J EOL J NM-EX270 and HITACHI
R-90H spectrometers and are reported in δ units. Mass spectra
were measured with J EOL J MS-DX303, J MS-SX102, and
SHIMAZU QP-1000 spectrometers. Elemental analyses were
performed with a Perkin-Elmer 2400 C, N, N analyzer. For
column chromatography, silica gel (SiO2, Merck Kieselgel 60
(E)-p -Nit r op h en yl 3-(2-Met h oxy-5-p yr im id in yl)a cr y-
la te (11b). To a solution of 2-methoxy-5-iodopyrimidine (6)
(1.74 g, 7.36 mmol) in DMF (36 mL) were added Pd(OAc)2 (99
n
mg), K2CO3 (2.54 g, 18.4 mmol), Bu4NCl (2.05 g, 7.36 mmol),
and methyl acrylate (3.17 g, 36.8 mmol), and the mixture was
heated at 80 °C under Ar atmosphere for 1 h. After the mixture
was cooled, water was added; the whole was extracted with
AcOEt. Usual workup and purification by column chromatog-
raphy (hexane-AcOEt, 2:1-1:1) gave 1.26 g (88%) of (E)-
methyl 3-(2-methoxy-5-pyrimidinyl)acrylate. To a solution of
(E)-methyl 3-(2-methoxy-5-pyrimidinyl)acrylate (1.26 g, 6.49
mmol) in methanol (40 mL) was added 4 N KOH (3.25 mL),
and the mixture was heated at 50 °C for 2 h. After the mixture
was cooled, 0.5 N HCl was added; the whole was extracted
with AcOEt. Usual workup afforded 1.12 g (96%) of (E)-3-(2-
methoxy-5-pyrimidinyl)acrylic acid. The synthesis of compound
11b was performed according to the same procedure as for 11a .
11b: yield 90%; 1H NMR (270 MHz, CDCl3) 8.69 (2 H, s), 8.25
(2 H, d, J ) 9.4 Hz), 7.74 (1 H, d, J ) 16.3 Hz), 7.31 (2 H, d,
J ) 8.9 Hz), 6.58 (1 H, d, J ) 15.8 Hz), 4.03 (3 H, s).
F
254) was used. Preparative TLC (PTLC) was carried out on
glass plates coated with Merck Kieselgel 60 F254s. Usual
workup refers to washing of organic layers with brine, drying
over anhydrous Na2SO4, and evaporating off the solvents under
reduced pressure.
2-Meth oxy-5-iod op yr im id in e (6). To a solution of 2-chlo-
ropyrimidine (5) (970 mg, 8.47 mmol) in methanol (7 mL) was
added 28 wt % NaOMe in methanol (3.40 g, 10.9 mmol), and
the mixture was stirred at room temperature for 30 min. Then
water was added, and the resulting mixture was extracted
with CHCl3 and worked up as usual. To a solution of the
residue in TFA (14 mL) and trifluoroacetic anhydride (3 mL)

New Water-Soluble Duocarmycin Derivatives
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4 673
(E)-p -Nit r op h en yl 3-(3-Met h oxy-6-p yr id a zin yl)a cr y-
la te (11c). To a solution of 3-methoxy-6-iodopyridazine (8)
(1.66 g, 7.04 mmol) in DMF (34 mL) were added Pd(OAc)2 (99
The synthesis of compounds 13b-13d was performed ac-
cording to the same procedure as for 13a .
Met h yl (7b R,8a S)-2-[2-(4-m et h oxy-3,5-p yr im id in yl)-
eth en -1-ylca r bon yl]-6-m eth yl-4(5H)-oxo-1,2,8,8a -tetr a h y-
d r ocyclop r op a [c]p yr r olo[3,2-e]in d ole -7-ca r b oxyla t e
(13b): yield 74%; mp 280-290 °C; 1H NMR (270 MHz, DMSO-
d6) 12.39 (1 H, brs), 9.04 (2 H, s), 7.65 (1 H, d, J ) 15.8 Hz),
7.21 (1 H, d, J ) 15.8 Hz), 6.91 (1 H, br), 4.38 (1 H, d, J )
10.9 Hz), 4.21 (1 H, dd, J ) 10.9, 4.6 Hz), 3.97 (3 H, s), 3.74 (3
H,s), 3.46-3.52 (1 H, m), 2.47 (3 H, s), 2.11 (1 H, dd, J ) 7.6,
3.0 Hz), 1.32 (1 H, dd, J ) 4.0, 4.0 Hz); IR (KBr, cm-1) 1701,
1676, 1624, 1618, 1595, 1477, 1400, 1340, 1250, 1111; FAB-
MS m/z 421 (M + H)+; FAB-HRMS calcd for C22H21N4O5 (M +
H)+ m/z 421.1500, found 420.1512. Anal. (C22H20N4O5‚0.3H2O)
C, H, N.
n
mg), K2CO3 (1.06 g, 7.67 mmol), Bu4NCl (1.96 g, 7.05 mmol),
and methyl acrylate (12.1 g, 141 mmol), and the mixture was
heated at 80 °C under Ar atmosphere for 4.5 h. After the
mixture had cooled, water was added; the whole was extracted
with AcOEt, CHCl3. Usual workup and purification by column
chromatography (hexane-AcOEt, 3:1-2:1) gave 828 mg (60%)
of (E)-methyl 3-(3-methoxy-6-pyridazinyl)acrylate. To a solu-
tion of (E)-methyl 3-(3-methoxy-6-pyridazinyl)acrylate (818
mg, 4.21 mmol) in methanol (25 mL) was added 4 N KOH (4.22
mL), and the mixture was heated at 50 °C for 8 h. After the
mixture was cooled, 0.5 N HCl was added; the whole was
extracted with AcOEt, CHCl3. Usual workup afforded 668 mg
(88%) of (E)-3-(3-methoxy-6-pyridazinyl)acrylic acid. The syn-
thesis of compound 11c was performed according to the same
procedure as for 11a . 11c: yield 60%; 1H NMR (270 MHz,
CDCl3) 8.30-8.33 (2 H, m), 8.04 (1 H, d, J ) 16.2 Hz), 7.62 (1
H, d, J ) 9.2 Hz), 7.38-7.41 (2 H, m), 7.05 (1 H, d, J ) 8.9
Hz), 7.01 (1 H, d, J ) 16.2 Hz), 4.22 (3 H, s).
(E)-p-Nit r op h en yl 3-(3-Met h oxy-6-p yr id in yl)a cr yla t e
(11d ). To a solution of 3-methoxy-6-iodopyridine (10) (500 mg,
2.13 mmol) in DMF (6 mL) were added Pd(OAc)2 (29 mg),
K2CO3 (736 mg, 5.33 mmol), nBu4NCl (592 mg, 2.13 mmol),
and methyl acrylate (917 mg, 10.7 mmol), and the mixture
was heated in a sealed tube at 120 °C for 12 h. After the
mixture was cooled, 0.01 M phosphoric buffer (pH 7) was
added; the whole was extracted with AcOEt. Usual workup
and purification by column chromatography (hexane-AcOEt,
4:1-3:1) afforded 168 mg (41%) of (E)-methyl 3-(3-methoxy-
6-pyridinyl)acrylate. To a solution of (E)-methyl 3-(3-methoxy-
6-pyridinyl)acrylate (429 mg, 2.22 mmol) in methanol (12 mL)
was added 4 N KOH (1.11 mL), and the mixture was heated
at room temperature for 20 h. After the mixture was cooled, 1
N HCl was added; the whole was extracted with AcOEt, CHCl3.
Usual workup afforded 277 mg (70%) of (E)-3-(3-methoxy-6-
pyridinyl)acrylic acid. To a solution of (E)-3-(3-methoxy-6-
pyridinyl)acrylic acid (268 mg, 1.50 mmol) in CH2Cl2 (28 mL)
was added DCC (620 mg, 3.00 mmol), and the mixture was
stirred at 0 °C for 45 min. Then, p-nitrophenol (418 mg, 3.00
mmol) and DMAP (367 mg, 3.00 mmol) were added to the
reaction mixture, and the mixture was stirred at room tem-
perature for 2 h 10 min; 1 N HCl was added; the whole was
extracted with CHCl3, washed with aqueous NaHCO3, and
worked up as usual. The whole was added to AcOEt and was
obtained as a crystalline material. Recrystallization from
EtOH gave 136 mg (30%) of 11d : 1H NMR (270 MHz, CDCl3)
8.31 (1 H, d, J ) 15.2 Hz), 8.27-8.33 (3 H, m), 7.36-7.40 (2
H, m), 7.32 (1 H, d, J ) 4.3 Hz), 7.29 (1 H, d, J ) 1.7 Hz), 7.20
(1 H, d, J ) 15.8 Hz), 3.94 (3 H, s).
Meth yl (7bR,8a S)-2-[2-(4-Meth oxy-3-p yr id in yl)eth en -
1-ylca r b on yl]-6-m et h yl-4(5H )-oxo-1,2,8,8a -t et r a h yd r o-
cyclop r op a [c]p yr r olo[3,2-e]in d ole-7-ca r boxyla te (13a ). To
a solution of 60% NaH (4.7 mg, 0.12 mmol) in DMF (0.38 mL)
was added a DMF solution (0.5 mL) of 12 (Seg-A) (25.0 mg,
0.0970 mmol), and the mixture was stirred under Ar atomo-
sphere at -20 °C for 2 h 20 min. Then a solution of 11a (32.1
mg, 0.107 mmol) in DMF (0.5 mL) was added and stirred for
1 h 20 min; 0.01 M phosphoric buffer (pH 7) was added; the
whole was extracted with AcOEt, and then worked up as usual.
The residue was purified by column chromatography (CHCl3-
MeOH, 100:1-70:1) to give 31.8 mg (78%) of 13a : mp 245-
250 °C; 1H NMR (270 MHz, CDCl3) 11.52 (1 H, br), 8.32 (1 H,
d, J ) 2.0 Hz), 7.81 (1 H, dd, J ) 2.3, 8.6 Hz), 7.77 (1 H, d, J
) 15.2 Hz), 6.77 (1 H, d, J ) 15.5 Hz), 6.77 (1 H, d, J ) 8.9
Hz), 6.74 (1 H, br), 4.24 (1 H, d, J ) 10.9 Hz), 4.16 (1 H, dd,
J ) 10.9, 4.6 Hz), 3.98 (3 H, s), 3.81 (3 H, s), 3.55-3.61 (1 H,
m), 2.62 (3 H, s), 2.40 (1 H, dd, J ) 7.4, 3.5 Hz), 1.31 (1 H, dd,
J ) 4.6, 3.6 Hz); IR (KBr, cm-1) 1701, 1668, 1614, 1601, 1495,
1462, 1389, 1292, 1244, 1219, 1111; FAB-MS m/z 420 (M +
H)+; FAB-HRMS calcd for C23H22N3O5 (M + H)+ m/z 420.1559,
found 420.1552. Anal. (C23H21N3O5‚1.5H2O) H, N; C: calcd,
61.88; found, 62.60.
Met h yl (7b R,8a S)-2-[2-(4-m et h oxy-2,3-p yr id a zin yl)-
eth en -1-ylca r bon yl]-6-m eth yl-4(5H)-oxo-1,2,8,8a -tetr a h y-
dr ocyclopr opa[c]pyr r olo[3,2-e]in dole-7-car boxylate (13c):
yield 73%; mp 210-215 °C; 1H NMR (270 MHz, CDCl3) 11.48
(1 H, brs), 7.84 (1 H, d, J ) 15.2 Hz), 7.52 (1 H, d, J ) 9.2 Hz),
7.44 (1 H, d, J ) 15.2 Hz), 7.00 (1 H, d, J ) 9.2 Hz), 6.93 (1 H,
br), 4.29 (1 H, d, J ) 10.9 Hz), 4.20 (1 H, dd, J ) 10.9, 4.6 Hz),
4.19 (3 H, s), 3.81 (3 H, s), 3.54-3.66 (1 H, m), 2.62 (3 H, s),
2.39 (1 H, dd, J ) 7.6, 3.6 Hz), 1.31 (1 H, dd, J ) 5.0, 3.6 Hz);
IR (KBr, cm-1) 1701, 1610, 1411, 1396, 1294, 1248, 1217, 1109,
1072; FAB-MS m/z 421(M + H)+; FAB-HRMS calcd for
C
22H21N4O5 (M + H)+ m/z 421.1500, found 421.1512. Anal.
(C22H20N4O5‚1.5 H2O) C, H; N: calcd, 12.52; found, 12.11.
Meth yl (7bR,8a S)-2-[2-(4-m eth oxy-2-p yr id in yl)eth en -
1-ylca r b on yl]-6-m et h yl-4(5H )-oxo-1,2,8,8a -t et r a h yd r o-
cyclop r op a [c]p yr r olo[3,2-e]in d ole-7-ca r boxyla te (13d ):
yield 83%; mp 235-240 °C; 1H NMR (270 MHz, CDCl3) 11.80
(1 H, br), 8.23 (1 H, d, J ) 15.2 Hz), 8.22 (1 H, dd, J ) 4.1, 1.5
Hz), 7.46 (1 H, d, J ) 15.2 Hz), 7.21-7.30 (2 H, m), 6.92 (1 H,
br), 4.31 (1 H, d, J ) 10.9 Hz), 4.20 (1 H, dd, J ) 10.6, 4.6 Hz),
3.90 (3 H,s), 3.81 (3 H, s), 3.55-3.62 (1 H, m), 2.62 (3 H, s),
2.36 (1 H, dd, J ) 7.6, 3.3 Hz), 1.29 (1 H, dd, J ) 4.6, 3.6 Hz);
IR (KBr, cm-1) 1701, 1672, 1618, 1578, 1450, 1390, 1296, 1252,
1217, 1113; FAB-MS m/z 420 (M + H)+; FAB-HRMS calcd for
C
23H22N3O5 (M + H)+ m/z 420.1559, found 420.1561. Anal.
(C22H21N3O5‚1.0H2O) C, H; N: calcd, 9.61; found, 9.08.
Meth yl (1S)-1-(Br om om eth yl)-7-m eth yl-5-[(4-m eth ylpip-
er azin yl)car bon yloxy]-3-[2-(4-m eth oxy-3-pyr idin yl)eth en -
1-ylca r bon yl]-1,2-d ih yd r o-3H-p yr r olo[3,2-e]in d ole-8-ca r -
boxyla te (14a ). To a solution of 13a (21.2 mg, 0.0510 mmol)
in CH3CN (1.27 mL) was added 48% HBr (0.012 mL), and the
mixture was stirred at room temperature for 30 min. Then
the mixture was concentrated under reduced pressure. p-
Nitrophenyl chloroformate (29.6 mg, 0.158 mmol) and triethyl-
amine (0.0210 mL, 0.153 mmol) were added to the residue in
dry CH2Cl2 (1.07 mL) and toluene (0.42 mL) under cooling at
-78 °C. The mixture was stirred at -78 °C for 30 min. Then
N-methylpiperazine (0.0196 mL, 0.179 mmol) was added, and
stirring was continued at 0 to -78 °C for 20 min. The mixture
was extracted with CHCl3, washed with aqueous NaHCO3, and
worked up as usual. The residue was purified by PTLC
(CHCl3-MeOH, 9:1) to give 25 mg (78%) of 14a : mp 150-155
°C; 1H NMR (270 MHz, CDCl3) 9.20 (1 H, brs), 8.34 (1 H, d, J
) 2.3 Hz), 8.21 (1 H, brs), 7.89 (1 H, dd, J ) 8.6, 2.3 Hz), 7.79
(1 H, d, J ) 15.2 Hz), 6.82 (1 H, d, J ) 15.2 Hz), 6.81 (1 H, d,
J ) 8.6 Hz), 4.49-4.60 (1 H, m), 4.45 (1 H, d, J ) 10.2 Hz),
4.30 (1 H, dd, J ) 9.6, 8.9 Hz), 3.98 (3 H, s), 3.95 (3 H, s), 3.79
(1 H, dd, J ) 9.6 Hz), 3.76 (2 H, br), 3.63 (2 H, br), 3.21 (1 H,
dd, J ) 10.2, 9.9 Hz), 2.53 (3 H, s), 2.49 (4 H, br), 2.36 (3 H, s);
IR (KBr, cm-1) 1701, 1697, 1649, 1495, 1435, 1410, 1381, 1290,
1217, 1153, 1095; FAB-MS m/z 628, 626 (M + H)+; FAB-HRMS
calcd for C29H3379BrN5O6 (M + H)+ m/z 626.1614, found
626.1642.
The synthesis of compounds 14b-14d was performed ac-
cording to the same procedure as for 14a .
Meth yl (1S)-1-(br om om eth yl)-7-m eth yl-5-[(4-m eth ylpip-
er a zin yl)ca r bon yloxy]-3-[2-(4-m eth oxy-3,5-p yr im id in yl)-
eth en -1-ylca r bon yl]-1,2-d ih yd r o-3H-p yr r olo[3,2-e]in d ole-
8-ca r boxyla te (14b): yield 65%; mp 160-165 °C; 1H NMR

674 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4
Amishiro et al.
(270 MHz, CDCl3) 9.01 (1 H, s), 8.75 (2 H, s), 8.21 (1 H, s),
7.71 (1 H, d, J ) 15.5 Hz), 6.93 (1 H, d, J ) 15.5 Hz), 4.53-
4.63 (1 H, m), 4.45 (1 H, d, J ) 10.2 Hz), 4.31 (1 H, dd, J )
9.6, 9.6 Hz), 4.07 (3 H, s), 3.95 (3 H, s), 3.80 (1 H, dd, J ) 7.3,
2.6 Hz), 3.74 (2 H, br), 3.64 (2 H, br), 3.23 (1 H, dd, J ) 10.2,
9.9 Hz), 2.61 (3 H, s), 2.50 (4 H, brs), 2.37 (3 H, s); IR (KBr,
cm-1) 1714, 1701, 1653, 1473, 1435, 1412, 1338, 1219, 1153,
1095; FAB-MS m/z 629, 627 (M + H)+; FAB-HRMS calcd for
C28H3279BrN6O6 (M + H)+ m/z 627.1567, found 627.1538.
Meth yl (1S)-1-(br om om eth yl)-7-m eth yl-5-[(4-m eth ylpip-
er a zin yl)ca r bon yloxy]-3-[2-(4-m eth oxy-2,3-p yr id a zin yl)-
eth en -1-ylca r bon yl]-1,2-d ih yd r o-3H-p yr r olo[3,2-e]in d ole-
8-ca r boxyla te (14c): yield 70%; mp 165-170 °C; 1H NMR
(270 MHz, CDCl3) 9.15 (1 H, brs), 8.24 (1 H, s), 7.84 (1 H, d,
J ) 15.2 Hz), 7.57 (1 H, d, J ) 15.2 Hz), 7.54 (1 H, d, J ) 8.9
Hz), 7.01 (1 H, d, J ) 8.9 Hz), 4.53-4.60 (1 H, m), 4.52 (1 H,
d, J ) 9.9 Hz), 4.33 (1 H, dd, J ) 9.6, 9.6 Hz), 4.20 (3 H, s),
3.95 (3 H, s), 3.79 (1 H, dd, J ) 10.2, 2.6 Hz), 3.75 (2 H, br),
3.61 (2 H, br), 3.21 (1 H, dd, J ) 10.2, 9.9 Hz), 2.57 (3 H, s),
2.49 (4 H, br), 2.36 (3 H, s); IR (KBr, cm-1) 1714, 1705, 1699,
1653, 1466, 1412, 1294, 1217, 1153, 1093, 1005; FAB-MS m/z
629, 627 (M + H)+; FAB-HRMS calcd for C28H3279BrN6O6 (M
+ H)+ m/z 627.1567, found 627.1539.
Meth yl (1S)-1-(br om om eth yl)-7-m eth yl-5-[(4-m eth ylpip-
er azin yl)car bon yloxy]-3-[2-(4-m eth oxy-2-pyr idin yl)eth en -
1-ylca r bon yl]-1,2-d ih yd r o-3H-p yr r olo[3,2-e]in d ole-8-ca r -
boxyla te (14d ): yield 58%; mp 230-235 °C; 1H NMR (270
MHz, CDCl3) 9.47 (1 H, brs), 8.28 (1 H, d, J ) 15.2 Hz), 8.27
(1 H, dd, J ) 4.0, 1.3 Hz), 7.55 (1 H, d, J ) 14.9 Hz), 7.21-
7.30 (2 H, m), 4.54 (1 H, br), 4.52 (1 H, d, J ) 9.6 Hz), 4.33 (1
H, dd, J ) 9.9, 9.2 Hz), 3.93 (3 H, s), 3.89 (3 H, s), 3.76 (1 H,
dd, J ) 9.9, 2.6 Hz), 3.74 (2 H, br), 3.60 (2 H, br), 3.21 (1 H,
dd, J ) 9.9, 9.9 Hz), 2.51 (3 H, s), 2.46 (4 H, br), 2.34 (3 H, s);
IR (KBr, cm-1) 1722, 1701, 1697, 1433, 1408, 1292, 1259, 1217,
1153, 1093; FAB-MS m/z 628, 626 (M + H)+; FAB-HRMS calcd
for C29H3379BrN5O6 (M + H)+ m/z 626.1614, found 626.1609.
cm-1) 1722, 1699, 1655, 1614, 1437, 1416, 1255, 1219. Anal.
(C29H32BrN5O6‚2.0 HCl‚3.5H2O) C, H; N: calcd, 9.18; found,
8.41.
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 compond
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.
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 adult 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:
tumor volume (mm3) ) length (mm) × [width (mm)]2/2
according to the method of the National Cancer Institute.23
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 is
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).24
Hem a totoxicity (effect of com p ou n d s on p er ip h er a l
blood (P B) p la telet cou n ts a n d w h ite blood cell cou n ts).
Effect on P B p la telet cou n ts: Each drug was dissolved with
saline and was administered into the tail vein of normal male
ddY mice (mean weight 20 ( 1 g). After 7 days, peripheral
blood was obtained from the orbital vein to measure the
platelet counts using a microcell counter (CC-180A, Toa
Medical Electronics Co., Ltd., Kobe, J apan). Results are
presented as percentage of the absolute value of the treated
group versus that of control (percent of control).
14a Hyd r och lor id e (15a ). A solution of 14a (20.9 mg,
0.0334 mmol) in ethanol (0.91 mL) and methanol (0.46 mL)
was treated with anhydrous 6.86 N HCl in ethanol (0.0146
mL) at room temperature for 4 h. The mixture was concen-
trated under reduced pressure to give 22.1 mg of 15a : mp 235-
240 °C; 1H NMR (270 MHz, DMSO-d6) 12.12 (1 H, s), 10.70 (1
H, br), 8.50 (1 H, d, J ) 2.3 Hz), 7.35 (1 H, dd, J ) 8.6, 2.3
Hz), 8.10 (1 H, br), 7.61 (1 H, d, J ) 15.5 Hz), 7.19 (1 H, d, J
) 15.2 Hz), 6.92 (1 H, d, J ) 8.9 Hz), 4.36-4.51 (4 H, m), 4.12-
4.18 (2 H, br), 3.91 (3 H, s), 3.85 (3 H, s), 3.79 (1 H, brd, J )
8.3 Hz), 2.85 (3 H, s), 2.68 (3 H, s); IR (KBr, cm-1) 1714, 1695,
1657, 1651, 1435, 1414, 1219, 1173, 1095. Anal. (C29H32
-
BrN5O6‚2.0HCl‚2.5H2O) C, H, N.
The synthesis of compounds 15b-15d was performed ac-
cording to the same procedure as for 15a .
14b Hyd r och lor id e (15b): mp 250-260 °C; 1H NMR (270
MHz, DMSO-d6) 12.06 (1 H, s), 10.44 (1 H, br), 9.08 (2 H, s),
8.11 (1 H, s), 7.59 (1 H, d, J ) 15.8 Hz), 7.36 (1 H, d, J ) 15.8
Hz), 4.40-4.49 (3 H, m), 4.10-4.24 (1 H, br), 3.98 (3 H, s),
3.85 (3 H, s), 3.76-3.84 (1 H, m), 2.86 (3 H, s), 2.68 (3 H, s);
IR (KBr, cm-1) 1705, 1701, 1659, 1477, 1433, 1412, 1336, 1215,
1186, 1095. Anal. (C28H31BrN6O6‚2.0HCl‚0.7H2O) C, H; N:
calcd, 11.79; found, 10.68.
14c Hyd r och lor id e (15c): mp 145-150 °C; 1H NMR (270
MHz, DMSO-d6) 12.12 (1 H, s), 10.54 (1 H, br), 8.31 (1 H, d, J
) 8.9 Hz), 7.74 (1 H, d, J ) 15.2 Hz), 7.54 (1 H, d, J ) 15.2
Hz), 7.34 (1 H, d, J ) 8.6 Hz), 4.34-4.60 (4 H, m), 4.02-4.24
(1 H, br), 4.09 (3 H, s), 3.85 (3 H, s), 3.76-3.82 (1 H, m), 2.85
(3 H, br), 2.69 (3 H, s); IR (KBr, cm-1) 1716, 1705, 1699, 1417,
1435, 1414, 1252, 1219, 1093. Anal. (C28H31BrN6O6‚2.0HCl‚
5.5H2O) C, H, N.
Effect on P B w h ite blood cell cou n ts: Drug were
administered intravenously (iv) beginning 1 day after tumor
inoculation. After 4 days, peripheral blood was obtained from
the orbital vein of tumor-bearing mice to measure the white
blood cell counts using a microcell counter (CC-180A, Toa
Medical Electronics Co., Ltd., Kobe, J apan). Results are
presented as percentage of the absolute value of the treated
group versus that of control (percent of control).
Ack n ow led gm en t. We express gratitude to Mikiko
Saito and Etsuko Koshimura for their excellent techni-
cal assistance. We also thank Mariko Yoshida, Atsuko
Kobayashi, and Hideko Yokoyama for measuring ele-
mental analyses and MS spectra.
14d Hyd r och lor id e (15d ): mp 230-235 °C; 1H NMR (270
MHz, DMSO-d6) 12.15 (1 H, s), 10.75 (1 H, br), 8.28 (1 H, d, J
) 3.6 Hz), 8.11 (1 H, s), 7.96 (1 H, d, J ) 15.2 Hz), 7.62 (1 H,
d, J ) 8.6 Hz), 7.54 (1 H, d, J ) 15.5 Hz), 7.45-7.50 (1 H, m),
4.30-4.58 (4 H, m), 4.08-4.24 (1 H, br), 3.94 (3 H, s), 3.85 (3
H, s), 3.81 (1 H, br), 2.85 (3 H, br), 2.69 (3 H, s); IR (KBr,
Su p p or tin g In for m a tion Ava ila ble: HPLC analytical
data of compounds 13a , 13c, 13d , 15b , and 15d . This ma-

New Water-Soluble Duocarmycin Derivatives
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4 675
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.
terial is available free of charge via the Internet at
http://pubs.acs.org.
Refer en ces
(1) (a) Takahashi, I.; Takahashi, K.; Ichimura, M.; Morimoto, M.;
Asano, K.; Kawamoto, I.; Tomita, F.; Nakano, H. Duocarmycin
A, a New Antibiotic from Streptomyces. J . Antibiot. 1988, 41,
1915-1917. (b) Yasuzawa, T.; Iida, T.; Muroi, K.; Ichimura, M.;
Takahashi, K.; Sano, H. Structure of Duocarmycins, Novel
Antitumor Antibiotics Produced by Streptomyces sp. Chem.
Pharm. Bull. 1988, 36, 3728-3731. (c) Ichimura, M.; Muroi, K.;
Asano, K.; Kawamoto, I.; Tomita, F.; Morimoto, M.; Nakano, H.
DC89-A1, A New Antibiotic from Streptomyces. J . Antibiot. 1988,
41, 1285-1288. (d) Ogawa, T.; Ichimura, M.; Katsumata, S.;
Morimoto, M.; Takahashi, K. New Antitumor Antibiotics, Duo-
carmycin B1 and B2. J . Antibiot. 1989, 42, 1299-1301. (e)
Ichimura, M.; Ogawa, T.; Takahashi, K.; Kobayashi, E.; Kawa-
moto, I.; Yasuzawa, T.; Takahashi, I.; Nakano, H. Duocarmycin
SA, a New Antitumor Antibiotic from Streptomyces sp. J .
Antibiot. 1990, 43, 1037-1038. (f) Ichimura, M.; Ogawa, T.;
Katumata, S.; Takahashi, K.; Takahashi, I.; Nakano, H. Duo-
carmycins, New Antitumor Antibiotics Produced by Streptomy-
ces; Producing Organisms and Improved Production. J . Antibiot.
1991, 44, 1045-1053. (g) Yasuzawa, T.; Saitoh, Y.; Ichimura,
M.; Takahashi, I.; Sano, H. Structure of Duocarmycin SA, a
Potent Antitumor Antibiotic. J . Antibiot. 1991, 44, 445-447. (h)
Yasuzawa, T.; Muroi, K.; Ichimura, M.; Takahashi, I.; Taka-
hashi, K.; Sano, H.; Saitoh, Y. Duocarmycins, Potent Antitumor
Antibiotics by Streptomyces sp. Structure and Chemistry. Chem.
Pharm. Bull. 1995, 43, 378-391.
(2) 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 Antibiotic. J pn. J . Cancer Res. 1992, 52, 113-120.
(3) 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: Synthesis and Mechanistic Studies. Acc.
Chem. Res. 1995, 28, 20-29. (c) Boger, D. L.; J ohnson, D. S.
CC-1065 and 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.
(7) (a) Nagamura, S.; Asai, A.; Kanda, Y.; Kobayashi, E.; Gomi, K.;
Saito, H. Synthesis and Antitumor Activity of Duocarmycin
Derivatives: Modification of Segment A Duocarmycin B2. Chem.
Pharm. Bull. 1996, 44, 1723-1730. (b) Kobayashi, E.; Okamoto,
A.; Asada, M.; Okabe, M.; Nagamura, S.; Asai, A.; Saito, H.;
Gomi, K.; Hirata T. Characteristics of Antitumor Activity of KW-
2189, a Novel Water-soluble Derivative of Duocarmycin, Against
Murine and Human Tumors. Cancer Res. 1994, 54, 2404-2410.
(c) Asai, A.; Nagamura, S.; Saito, H. A Novel Property of
Duocarmycin and its Analogues for Covalent Reaction with DNA.
J . Am. Chem. Soc. 1994, 116, 4171-4177. (d) Nagamura, S.;
Kobayashi, E.; Gomi, K.; Saito, H. Studies on the Active
Metabolite (DU-86) of KW-2189, a Novel Derivative of Duocar-
mycin. Bioorg. Med. Chem. Lett. 1996, 6, 2147-2150.
(8) Nagamura, S.; Kanda, Y.; Kobayashi, E.; Gomi, K.; Saito, H.
Synthesis and Antitumor Activity of Duocarmycin Derivatives.
Chem. Pharm. Bull. 1995, 43, 1530-1535.
(9) A direct relationship between solvolysis stability and in vitro
cytotoxic potency with the more stable agents exhibiting the
more potent activity is demonstrated. (a) Boger, D. L.; Ishizaki,
T. Resolution of A CBI Precursor and Incorporation into the
Synthesis of (+)-CBI, (+)-CBI-CDPI1, (+)-CBI-CDPI2: Enhanced
Functional Analogues of (+)-CC-1065. A Critical Appraisal of a
Proposed Relationship between Electrophile Reactivity, DNA
Binding Properties, and Cytotoxic Potency. Tetrahedron Lett.
1990, 31, 793-796. (b) Boger, D. L.; Mesini, P.; Tarby, C. M.
Chemical and Structural Comparison of N-Boc-CBQ and N-Boc-
CBI: Identification and Structural Origin of an Unappreciated
but Productive Stability of the CC-1065 and Duocarmycin SA
Alkylation Subunits. J . Am. Chem. Soc. 1994, 116, 6461-6462.
(c) Boger, D. L.; McKie, J . A.; Han, N.; Taby, C. M.; Riggs, H.
W.; Kitos, P. A. A Hammett Correlation for CC-1065 and
Duocarmycin Analogues: Magnitude of Substituent Electronic
Effects on Functional Reactivity. Bioorg. Med. Chem. Lett. 1996,
6, 659-664. (d) Boger, D. L.; Goldberg, J .; McKie, J . A. A
Comparative Study of the Solvolysis Reactivity, Regioselectivity,
and Stereochemistry of the Duocarmycin A and SA Alkylation
Subunits. Bioorg. Med. Chem. Lett. 1996, 6, 1955-1960. (e)
Boger, D. L.; Boyce, C.; J ohnson, D. S. pH Dependence of the
Rate of DNA Alkylation for (+)-Duocarmycin SA and (+)-CBI-
TMI. Bioorg. Med. Chem. Lett. 1997, 7, 233-238.
(4) (a) Sugiyama, H.; Hosoda, M.; Saito, I.; Asai, A.; Saito, H.
Covalent Alkylation of DNA with Duocarmycin A. Identification
of Abasic Site Structure. Tetrahedron Lett. 1990, 31, 7197-7200.
(b) Boger, D. L.; Ishizaki, T.; Zarrinmayeh, H. Synthesis and
Preliminary Evaluation of Agents Incorporating the Phama-
cophore of the Duocarmycin/Pyrindamycin Alkylation Subunit:
Identification of the CC-1065/Duocarmycin Common Pharma-
cophore. J . Org. Chem. 1990, 55, 4499-4502. (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-(-)-Duocar-
mycin SA and (+)- and ent-(-)-N-Boc-DSA DNA Alkylation
Properties. Alkylation Site Models that Accommodate the Offset
AT-rich Adenine N3 Alkylation Selectivity of the Enantiomeric
Agents. J . Am. Chem. Soc. 1994, 116, 1635-1656.
(5) (a) Hanka, L. J .; Dietz, A.; Gerpheide, S. A.; Kuentzel, S. L.;
Martin, D. G. CC-1065 (NSC-298223), a New Antitumor Anti-
biotic. Production, in vitro Biological Activity, Microbiological
Assays, and Taxonomy of the Producing Microorganism. J .
Antibiot. 1978, 31, 1211-1217. (b) Martin, D. G.; Chidester, C.
G.; Duchamp, D. J .; Mizsak, S. A. Structure of CC-1065 (NSC-
298223), a New Antitumor Antibiotic. J . Antibiot. 1980, 33, 902-
903. (c) Reynolds, V. L.; McGovren, J . P.; Hurley, L. H. The
Chemistry, Mechanism of Action and Biological Properties of CC-
1065, a Potent Antitumor Antibiotic. J . Antibiot. 1986, 39, 319-
334.
(10) (a) Nagamura, S.; Kobayashi, E.; Gomi, K.; Saito, H. Synthesis
and Antitumor Activity of Duocarmycin Derivatives: A-ring
Pyrrole Analogues of Duocarmycin B2. Bioorg. Med. Chem. 1996,
4, 1379-1391. (b) Ogasawara, H.; Nishio, K.; Takeda, Y.;
Ohmori, T.; Kubota, N.; Funayama, Y.; Ohira, T.; Kuraishi, Y.;
Isogai, Y.; Saijo, N. A Novel Antitumor Antibiotics, KW-2189 is
Activated by Carboxyl Esterase and Induces DNA Strand Breaks
in Human Small Cell Lung Cancer Cells. J pn. J . Cancer Res.
1994, 54, 418-425. (c) Okamoto, A.; Asai, A.; Saito, H.; Okabe,
M.; Gomi, K. Differential Effect of Duocarmycin A and its Novel
Derivatives DU-86 on DNA Strand Breaks in HeLa S3 Cells. J pn.
J . Cancer Res. 1994, 85, 1304-1311. (d) Ogasawara, H.; Nishio,
K.; Kanzawa, F.; Lee, Y. S.; Funayama, Y.; Ohmori, T.; Kuraishi,
Y.; Isogai, Y.; Saijo, N. Intracellular Carboxyl Esterase Activity
is A Determinant of Cellular Sensitivity to the Antineoplastic
Agent KW-2189 in Cell lines Resistant to Cisplatin and CPT-
11. J pn. J . Cancer Res. 1995, 55, 124-129. (e) Ogasawara, H.;
Nishio, K.; Ishida, T.; Arioka, H.; Fukuoka, K.; Saijo, N. In vitro
Enhancement of Antitumor Activity of a Water-soluble Duocar-
mycin Derivative, KW-2189, by Caffeine-mediated DNA-repair
Inhibition in Human Lung Cancer Cells. J pn. J . Cancer Res.
1997, 88, 1033-1037.
(11) (a) Chidester, C. G.; Krueger, W. C.; Mizsak, S. A.; Duchamp,
D. J .; Martin, D. G. The Structure of CC-1065,
a Potent
Antitumor Agent, and its Binding to DNA. J . Am. Chem. Soc.
1981, 103, 7629-7635. (b) Warpehoski, M. A.; Gebnard, I.; Kelly,
R. C.; Krueger, W. C.; Li, L. H.; McGovren, J . P.; Prairie, M. D.;
Wicnienski, N.; Wierenga, W. Stereoelectronic Factors Influenc-
ing the Biological Activity and DNA Interaction of Synthetic
Antitumor Agents Modeled on CC-1065. J . Med. Chem. 1988,
31, 590-603. (c) Boger, D. L.; Tun, W. Role of the CC-1065 and
Duocarmycin N2 Substituent: Validation of A Direct Relation-
ship Between Solvolysis Chemical Stability and in vitro Biologi-
cal Potency. J . Am. Chem. Soc. 1994, 116, 5523-5524.
(12) Asai, A.; Nagamura, S.; Kobayashi, E.; Gomi, K.; Saito, H.
Syntesis and Antitumor Activity of Novel Duocarmycin Deriva-
tives. Bioorg. Med. Chem. Lett. 1996, 6, 1215-1220.
(13) Nagamura, S.; Asai, A.; Amishiro, N.; Kobayashi, E.; Gomi, K.;
Saito, H. Synthesis and Antitumor Activity of Duocrmycn
Derivatives: A-ring Pyrrole Compounds Bearing Cinnamoyl
Groups. J . Med. Chem. 1997, 40, 972-979.
(6) (a) Hurley, L. H.; Reynolds, V. L.; Swenson, D. H.; Petzold, G.
L.; Scahill, T. A. Reaction of the Antitumor Antibiotic CC-1065
with DNA: Structure of A DNA Adduct with DNA Sequence
Specificity. Science 1984, 226, 843-844. (b) Reynolds, V. L.;
Molineaux, I. J .; Kaplan, D. J .; Swensen, D. H.; Hurley, L. H.
Reaction of the Antitumor Antibiotic CC-1065 with DNA.

676 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4
Amishiro et al.
(14) (a) McGovren, J . P.; Clarke, G. L.; Pratt, E. A.; DeKoning, T. F.
Preliminary Toxicity Studies with the DNA-binding Antibiotics,
CC-1065. J . Antibiot. 1984, 37, 63-70. (b) Warpehoski, M. A.;
Harper, D. E.; Mitchel, M. A.; Monroe, T. J . Reversibility of the
Covalent Reaction of CC-1065 and Analogues with DNA. Bio-
chemistry 1992, 31, 2502-2508. (c) Boger, D. L.; Yun, W.
Reversibility of the Duocarmycin A and SA DNA Alkylation
Reaction. J . Am. Chem. Soc. 1993, 115, 9872-9873. (d) Asai,
A.; Nagamura, S.; Saito, H.; Takahashi, I.; Nakano, H. The
Reversible DNA-alkylating of Duocarmycin and its Analogues.
Nucleic Acids Res. 1994, 22, 83-93.
36, 863-870. (b) Wyatt, M. D.; Lee, M.; Hartley, J . A. The
Sequence Specificity of Alkylation for A Series of Benzoic Acid
Mustard and Imidazole-containing Distamycin Analogues: The
Importance of Local Sequence Conformation. Nucleic Acid Res.
1997, 25, 2359-2364.
(18) Nishikawa, Y.; Shindo, T.; Ishii, K.; Nakamura, H.; Kon, T.; Uno,
H. Acrylamide Derivatives as Antiallergic Agents. 2. Synthesis
and Structure-activity Relationships of N-[4-(4-(diphenyl-
methyl)-1-piperazinyl]butyl-3-(3-pyridyl)acryl Amides. J . Med.
Chem. 1989, 32, 583-593.
(19) (a) Heck, R. F.; Nolley, J . P. Palladium-catalyzed Vinylic
Hydrogen Substitution Reaction with Aryl, Benzyl, and Styryl
Halides. J . Org. Chem. 1972, 37, 2320-2322. (b) Dieck, H. A.;
Heck, R. F. Organophosphinepalladium Complexes as Catalysts
for Vinylic Hydrogen Substitution Reactions. J . Am. Chem. Soc.
1974, 96, 1133-1136. (b) Heck, R. F. Palladium-catalyzed
Vinylation of Organic Halides. Org. React. 1982, 27, 345. (c)
Wada, A.; Yamamoto, J .; Hase, T.; Nagai, S.; Kanatomo, S. A
Convenient Synthesis of Methyl (E)-Pyrimidinyacrlates. Syn-
thesis 1986, 7, 555-556. (d) Wada, A.; Yamamoto, J .; Hase, T.;
Nagai, S.; Kanatomo, S. A Convenient Synthesis Methyl (E)-
Pyrimidinylacrylates. Synthesis 1986, 555-556.
(20) (a) J effery, T. Palladium-catalyzed Vinylation of Organic Halides
under Solid-liquid-Phase Transfer Conditions. J . Chem. Soc.,
Chem. Commun. 1984, 1287-1289. (b) J effery, T. Highly Stereo-
specific Palladium-catalysed Vinylation of Vinylic Halides under
Solid-liquid-Phase Transfer Conditions. Tetrahedron Lett. 1985,
26, 2667-2670.
(21) Mukaiyama, T.; Usui, M.; Shimada, E.; Saigo, K. A Convenient
Method for the Synthesis of Carboxylic Esters. Chem. Lett. 1975,
1045-1048.
(22) (a) Nagamura, S.; Asai, A.; Kanda, Y.; Kobayashi, E.; Gomi, K.;
Saito, H. Wagner-Meerwein Rearrangement of Duocarmycins.
Chem. Pharm. Bull. 1996, 44, 933-939. (b) Berner, D.; Cox, D.
P.; Dahn, H. On the Migration of a HOOC Group in A Wagner-
Meerwein Rearrangement in Superacid Solution: Proof by
Double Labeling with Carbon-13. J . Am. Chem. Soc. 1982, 104,
2631-2632.
(23) Geran, R. I.; Greenberg, N. H.; MacDonald, M. M.; Schumacher,
A. M.; Abbott, B. J . Protocols for Screening Chemical Agents
and Natural Products Against Animal Tumor and Other Biologi-
cal Systems. Cancer Chemother. Rep. 1972, 3, 1-103.
(24) Inaba, M.; Kobatashi, T.; Tashiro, T.; Sakurai, Y.; Maruo, K.;
Ohnishi, Y.; Ueyama, Y.; Momura, T. Evaluation of Antitumor
Activity in A Human Breast Tumor/Nude Mouse Model with A
Special Emphasis on Treatment Dose. Cancer 1989, 64, 1577-
1582.
(15) CC-1065 analogues are promising candidates in clinical develop-
ments. (a) Li, L. H.; Kelly, R. C.; Warpehoski, M. A.; McGovren,
J . P.; Gebhard, I.; Dekoning, T. F. Adozelesin, a Selected Lead
among Cyclopropylpyrroloindole Analogues of the DNA-binding
Antibiotic, CC-1065. Invest. New Drugs 1991, 9, 137-148. (b)
Li, L. H.; Dekoning, T. F.; Kelly, R. C.; Krueger, W. C.;
McGovren, J . P.; Pudbury, G. E.; Petzold, G. L.; Wallace, T. L.;
Ouding, R. J .; Prairie, M. D.; Gebhard, I. Cytotoxicity and
Antitumor Activity of Carzelesin, A Prodrug Cyclopropylpyrrolo-
indole Analogue. Cancer Res. 1992, 52, 4904-4913. (c) Burris,
H. A.; Dieras, V. C.; Tunca, M.; Earhart, R. H.; Eckardt, J . R.;
Rodriguez, G. I.; Shaffer, D. S.; Fields, S. M.; Campbell, E.;
Schaaf, L.; Kasunic, D.; Von Hoff, D. D. Phase I Study with the
DNA Sequence-Specific Agent Adozelesin. Anti-Cancer Drugs
1997, 8, 588-596. (d) Wolff, I.; Bench, K.; Beijnen, J . H.;
Bruntsch, U.; Cavalli, F.; J ong, J .; Groot, Y.; Tellingen, O.;
Wanders, J .; Sessa, C. Phase I Clinical and Pharmacokinetic
Study of Carzelesin (U-80244) Given Daily for Five Consecutive
Days. Clin. Cancer Res. 1996, 2, 1717-1723. (e) Tellingen, O.;
Punt, C. J . A.; Awada, A.; Wagener, D. J . T.; Piccart, M. J .; Groot,
Y.; Schaaf, L. J .; Henrar, R. E. C.; Nooijen, W. J .; Beijnen, J . H.
A Clinical Pharmacokinetics Study of Carzelesin Given by Short-
Term Intravenous Infusion in a Phase I Study. Cancer Chemo-
ther. Pharmacol. 1998, 41, 377-384. (f) Tellingen, O.; Nooijen,
W. J .; Schaaf, L. J .; Valk, M.; Asperen, J .; Henrar, R. E. C.;
Beijnen, J . H. Comparative Pharmacology of the Novel Cyclo-
propylpyrroloindole-Prodrug Carzelesin in Mice, Rats, and
Humans. Cancer Res. 1998, 58, 2410-2416.
(16) (a) Boger, D. L.; Bollinger, B.; J ohnson, D. S. Examination of
the Role of the Duocarmycin SA Methoxy Substituents: Iden-
tification of the Minimum, Fully Potent DNA Binding Subunit.
Bioorg. Med. Chem. Lett. 1996, 6, 2207-2210. (b) Boger, D. L.;
Yun, W.; Han, N.; J ohnson, D. S. CC-1065 CBI Analogues: An
Example of Enhancement of DNA Alkylation Efficiency through
Introduction of Stabilizing Electrostatic Interactions. Bioorg.
Med. Chem. 1995, 3, 611-621. (c) Boger, D. L.; Yun, W.; Han,
N. 1,2,9,9a-Tetrahydrocyclopropa[c]benz[e]indol-4-one-(CBI) Ana-
logues of CC-1065 and the Duocarmycins: Synthesis and Evalu-
ation. Bioorg. Med. Chem. 1995, 3, 1429-1453.
(17) (a) Lee, M.; Rhodes, A. L.; Wyatt, M. D.; D′Incalci, M.; Forrow,
S.; Hartley, J . A. In Vitro Cytotoxicity of GC Sequence Directed
Alkylating Agents Related to Distamycin. J . Med. Chem. 1993,
J M980559Y