Novel combretastatin analogues effective against murine solid tumors: Design and structure-activity relationships

Article abstract of DOI:10.1021/jm980101w

A series of combretastatin A-4 (CA-4) analogues were synthesized, and their cytotoxic effects against murine Colon 26 adenocarcinoma and inhibitory activity on tubulin polymerization were evaluated. Since CA-4 has limited aqueous solubility, the target co

Full text of DOI:10.1021/jm980101w

3022
J . Med. Chem. 1998, 41, 3022-3032
Novel Com br eta sta tin An a logu es Effective a ga in st Mu r in e Solid Tu m or s:
Design a n d Str u ctu r e-Activity Rela tion sh ip s
Koji Ohsumi,* Ryusuke Nakagawa, Yumiko Fukuda, Toshihiro Hatanaka, Yoshihiro Morinaga, Yukio Nihei,
Kazuo Ohishi, Yasuyo Suga, Yukio Akiyama, and Takashi Tsuji
Central Research Laboratories, Ajinomoto Company Ltd., 1-1 Suzuki-cho, Kawasaki, Kawasaki 210, J apan
Received February 13, 1998
A series of combretastatin A-4 (CA-4) analogues were synthesized, and their cytotoxic effects
against murine Colon 26 adenocarcinoma and inhibitory activity on tubulin polymerization
were evaluated. Since CA-4 has limited aqueous solubility, the target compounds were designed
to improve solubility by introduction of a nitrogen-containing group. Among the compounds
synthesized, those with an amino moiety in place of the phenolic OH of CA-4 showed potent
antitubulin activity and cytotoxicity against murine Colon 26 adenocarcinoma in vitro. Some
of the compounds which were potent in vitro were evaluated in the murine tumor model Colon
26 in vivo. Among these, 13bHCl, 21a HCl, and 21bHCl showed significant antitumor activity
in the animal model, while CA-4 was ineffective. 13bHCl and 21a HCl were further evaluated
in two murine tumor models (Colon 38 and 3LL) and human xenografts HCT-15. These
compounds showed potent antitumor activity comparable or superior to that of CDDP. The
structure-activity relationships of this series of compounds are also discussed.
Ch a r t 1
In tr od u ction
The microtubule system of eukaryotic cells is an
important target for the development of anticancer
agents. Chemicals which attack microtubules through
tubulin disrupt cellular microtubule structure and func-
tion resulting in mitotic arrest. Examples of clinically
used antimitotic agents are vincristine, which inhibits
microtubule polymerization, and paclitaxel, which pro-
motes microtubule assembly and inhibits microtubule
disassembly. However, despite their potent antitumor
activities, these drugs have undesirable side effects and
are subject to multidrug resistance (MDR). Thus, it is
essential to develop new anticancer agents with fewer
side effects and activity against cancers not effectively
treated by existing anticancer drugs.
Combretastatins are mitotic agents isolated from the
bark of the South African tree Combretum caffrum
(Chart 1).1-3 The most potent of these is combretastatin
A-4 (CA-4) (i), which has been found to be a potent
cytotoxic agent and which strongly inhibits the polym-
erization of brain tubulin by binding to the colchicine
site. CA-4 shows potent cytotoxicity against a wide
variety of human cancer cell lines including MDR cancer
cell lines.4,5 Combretastatin A-4 (i) is thus attractive
as a lead compound for development of anticancer drugs.
In our laboratory, however, the antitumor activity of
combretastatin A-4 against murine Colon 26 adenocari-
noma was evaluated in vivo, and it did not show any
antineoplastic effects. Since CA-4 is highly lipophilic
and has limited aqueous solubility, its lack of efficacy
in vivo was considered to be due to its poor pharmaco-
kinetics. A prodrug formulation and derivatization of
introduced an oxygen or nitrogen-containing group into
combretastatin A-4. A number of studies have been
reported on structure-activity relationships of CA-
4.13-19 These studies showed that the cis orientation
of the two benzene rings is essential and 3,4,5-tri-
methoxy substituents on the A-ring of CA-4 are indis-
pensable for potent cytotoxicity.17,18 Thus we mainly
examined the substituents on the olefin site and the
B-ring.
In this paper, we describe the syntheses and biological
activities of novel derivatives of combretastatin A-4 with
improved solubility. The cytotoxic effects against the
murine Colon 26 adenocarcinoma, inhibitory activity on
tubulin polymerization, and antitumor activity in vivo
of these compounds were evaluated.
CA-4 to improve its aqueous solubility have been
Ch em istr y
6-12
reported,
but no studies have confirmed the anti-
tumor activity of CA-4 or its derivatives in vivo.
To obtain compounds with pharmaceutically accept-
able properties and improved antitumor activity, we
The syntheses of 3 and 5-7 are shown in Scheme 1.
Base-catalyzed condensation of 3,4,5-trimethoxybenzal-
dehyde with 4-methoxyphenylacetic acid (1a ) in the
S0022-2623(98)00101-0 CCC: $15.00 © 1998 American Chemical Society
Published on Web 07/09/1998

CA-4 Analogues Effective against Murine Tumors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 16 3023
Sch em e 1a
ammonium chloride and NaOH at room temperature
gave (Z)-acrylonitriles 9a -e in good yields.21 Since the
active form of combretastatins was reported to be the
cis form, we attempted to isomerize the (Z)-acrylonitriles
to the corresponding (E)-acrylonitriles in which two
benzene rings were located in the cis orientation. The
(Z)-acrylonitriles 9a -e were dissolved in CH3CN at high
dilution (10 mmol/L) and irradiated for 30 min. This
reaction gave a 1:1 mixture of E- and Z-isomers.
Prolonged reaction time resulted in a decrease in yield
due to formation of byproducts. E- and Z-isomers were
separated easily by crystallization from EtOAc/hexane.
E- or Z-geometries of these compounds were determined
by comparison of the chemical shifts of B-ring protons
adjacent to the olefin site (protons on the B-ring of
Z-form derivatives resonate 0.5-1.0 ppm downfield from
the corresponding protons of the E-form by the aniso-
tropic effect of the nitrile group). 10b was deprotected
with HCl to give phenol 13a . Nitro compounds (9c,d ,
10c-e) were reacted with Zn in acetic acid/CH2Cl2 to
give anilino compounds (11a ,b, 13b-d ) in good yields.
The obtained anilino compounds were reacted with 4 N
HCl-dioxane to give corresponding hydrochloride salts
(13b-d HCl). Acetylation of aniline 13b with Ac2O gave
acetamide 14. (Z)-Acrylonitrile 11a was hydrogenated
on 10% Pd-C to give saturated compound 12.
a
Reagents: (a) (1) Ac2O, Et3O, 140 °C, (2) NaOH(aq); (b) MeI,
K2CO3, DMF, rt; (c) LiAlH4, THF, 0 °C; (d) phthalimide, diethyl
azodicaboxylate, PPh3, rt; (e) hydrazine, EtOH, reflux; (f) MnO2,
CH2Cl2, rt; (g) MeI, K2CO3, DMF, rt.
presence of triethylamine and acetic anhydride at 140
°C gave the carboxylic acid 2a . In this reaction, only
the E-form was obtained.20 Reaction of 2a with MeI at
room temperature gave methyl ester 2b. 2b was then
reacted with LiAlH4 to give alcohol 3. Mitsunobu
reaction of 3 with phthalimide gave 4. 4 was depro-
tected by hydrazine to give the aminomethyl compound
5. Reaction of 5 with MeI gave trimethylamino iodide
salt 6. Oxidation of the alcohol 3 with MnO2 gave
ketone 7.
The syntheses of 18 and 19 are shown in Scheme 3.
Condensation of 3,4,5-trimethoxybenzaldehyde with
4-methoxy-3-nitrophenylacetic acid (1b ) in triethyl-
amine and acetic anhydride at 140 °C for 12 h gave
acrylic acid 15. The acid 15 was converted to amide 16
by treatment with SOCl2 followed by aqueous NH3
treatment. The acrylamide 16 was reacted with SOCl2
to give acrylonitrile 17. Compounds 16 and 17 were
reduced with Zn in acetic acid to give corresponding
anilino compounds 18 and 19, respectively.
The syntheses of 9a ,c, 10a , 11a ,b, 12, 13a -d , 14, and
13b-d HCl are shown in Scheme 2. Base-catalyzed
condensation of 3,4,5-trimethoxyphenylacetonitrile with
benzaldehyde 8a -e in the presence of methyltrioctyl-
Sch em e 2a
a
Reagents: (a) NaOH, methyltrioctylammonium chloride, CH2Cl2, rt; (b) CH3CN, irradiation; (c) HCl, AcOH, rt; (d) Zn, AcOH, rt; (e)
H2, 10% Pd-C, MeOH, rt; (f) 4 N HCl-dioxane, rt; (g) Ac2O, Py, rt.

3024 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 16
Ohsumi et al.
Sch em e 3a
Sch em e 5a
a
Reagents: (a) NaOH, methyltrioctylammonium chloride,
a
Reagents: (a) (1) Ac2O, Et3N, 140 °C, (2) NaOH(aq); (b) (1)
CH2Cl2, rt; (b) CH3CN, irradiation; (c) H2, 10% Pd-C, MeOH, rt.
SOCl2, rt, (2) 28% aq NH3, rt; (c) SOCl2, Py, rt; (d) Zn, AcOH, rt.
Sch em e 6a
Sch em e 4a
a
Reagents: (a) NaH, toluene, rt; (b) Zn, AcOH; rt; (c) 4 N HCl-
dioxane, CH2Cl2, rt.
Syntheses of 21a -c and 21a ,bHCl are shown in
Scheme 4. Wittig reaction of 3,4,5-trimethoxyben-
zylphosphonium bromide with nitrobenzaldehyde 8c-e
in the presence of sodium hydride in toluene gave a 1:1
mixture of (E)- and (Z)-stilbenes.18 20a was separated
from the mixture by crystallization (from EtOAc, then
EtOH). 20b,c were purified by silica gel column chro-
matography. The cis and trans geometries of the
a
Reagents: (a) Ac2O, Et3N, 140 °C; (b) (1) SOCl2, rt, (2) 28%
1
stilbenes were assigned by the characteristic H NMR
aq NH3, rt; (c) Zn, AcOH, rt; (d) NaOH, methyltrioctylammonium
chloride, CH2Cl2, rt; (e) CH3CN, irradiation.
coupling constants of the olefinic protons (about 12 Hz
for cis and 16 Hz for trans isomers).22 The obtained (Z)-
nitro compounds 20a -c were reacted with Zn in acetic
acid-CH2Cl2 at room temperature to give anilino com-
pounds 21a -c. The anilino compounds were reacted
with 4 N HCl-dioxane to give hydrochloride salts
21a ,bHCl, respectively.
uct was obtained. Reduction of 23 with 10% Pd-C gave
saturated compound 25.
The syntheses of 27b and 31a -c are shown in
Scheme 6. Condensation of 4-methoxy-3-nitrobenzal-
dehyde (8c) with 3,4,5-trimethoxyphenylacetic acid in
triethylamine and acetic anhydride at 140 °C for 12 h
gave acrylic acid 26. The acrylic acid 26 was converted
to acrylamide 27a by SOCl2 followed by aqueous NH3
treatment. 27a was reacted with Zn in acetic acid to
give anilino compound 27b. Condensation of phenylac-
etonitriles 28a -c and 4-methoxy-3-nitrobenzaldehyde
(8c) in the presence of NaOH gave (Z)-acrylonitriles
29a -c. The acrylonitriles 29a -c were isomerized by
irradiation to give a 1:1 mixture of (E)- and (Z)-
The syntheses of 23 and 25, in which the B-ring was
replaced with 2-methoxypyridine to mimic 3-amino-4-
methoxybenzene, are shown in Scheme 5. Base-
catalyzed condensation of 3,4,5-trimethoxyphenylaceto-
nitrile and pyridylaldehyde 22 gave (Z)-acrylonitrile 23.
To obtain (E)-acrylonitrile, 23 was irradiated with
visible light; however, this reaction did not give the
desired (E)-acrylonitrile 24, and only decomposed prod-

CA-4 Analogues Effective against Murine Tumors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 16 3025
Ta ble 1. Modification of the Olefin and B-Ring Substituents
Colon 26a
IC50 (nM)
antitubulin activityb
compd
R1
R2
R3
IC50 (µM)
3
5
6
7
10a
10c
13a
13b
13c
13d
14
CH2OH
CH2NH2
CH2N(CH3)4
CHO
CN
CN
CN
CN
CN
H
H
H
H
H
NO2
OH
NH2
NH2
NH2
NHAc
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
CH3
363
>3000
>3000
213
>20
>20
>20
4
6
5
5
10
10
6
10
4
18.0
135
23.5
5.9
61.7
58.1
18.3
18.0
CN
CN
Cl
OCH3
combretastatin A-4
a
b
Drug concentration required to inhibit the growth of Colon 26 cells by 50%. Tubulin polymerization was determined as described in
the text.
Ta ble 2. Modification of the Olefin and B-Ring Substituents
Colon 26a
IC50 (nM)
antitubulin activityb
compd
R1
R2
R3
IC50 (µM)
18
19
H
H
CONH2
CN
H
H
H
OCH3
OCH3
OCH3
OCH3
CH3
223
235
195
5.1
43.5
68.9
5.9
nt
5
3
4
4
3
10
4
27b
21a
21b
21c
13b
CONH2
H
H
H
H
H
Cl
OCH3
CN
combretastatin A-4
18.0
a
b
Drug concentration required to inhibit the growth of Colon 26 cells by 50%. Tubulin polymerization was determined as described in
the text. nt, not tested.
acrylonitriles. (E)-Acrylonitriles 30a -c were purified
by silica gel column chromatography and reacted with
Zn in acetic acid to give anilino compounds 31a -c.
First, substituents were introduced into the olefin site
adjacent to the A-ring (Table 1). The Purdue group
reported that the 3-hydroxyl group on the B-ring of CA-4
is not necessary for potent activity.17 Therefore, we used
a 4-methoxyphenyl group as the B-ring for ease of
preparation (3, 5-7, 10a ). Introduction of a hydroxy-
methyl group (3) resulted in loss of antimitotic activity
(IC50 > 20 µM) and a 20-fold decrease in cytotoxicity
compared to that of CA-4. Compounds 5 and 6 dimin-
ished antimitotic and cytotoxic activities. These func-
tional groups were bulky, so next we introduced smaller
groups into the olefin site (7, 10a ). Aldehyde 7 retained
antimitotic activity but showed a 10-fold decreased
cytotoxicity. Nitrile 10a showed slightly decreased
antimitotic activity (IC50 6 µM) but strong cytotoxicity
(IC50 18.0 nM) comparable to CA-4. Insertion of a CN
group did not affect the activity of CA-4. These results
demonstrate that larger substituents on the olefin site
adjacent to the A-ring result in weaker activity and a
nitrile group is about the maximum tolerable size.
Resu lts a n d Discu ssion
A series of 28 newly synthesized stilbene analogues
were evaluated for their cytotoxic effects against murine
Colon 26 adenocarcinoma and for tubulin polymeriza-
tion inhibitory activity (Tables 1-4).
The Purdue group has studied structure-activity
relationships of combretastatin A-4 (i) extensively.17,18
They synthesized a large number of A-ring-substituted
analogues and found that a 3,4,5-trimethoxy group on
the A-ring was essential for strong cytotoxicity and
antimitotic activity. They introduced several ester
groups on the olefin site, but these alterations resulted
in complete loss of antitubulin activity or significant
decrease of cytotoxic activity. They examined 4-sub-
stituents of the B-ring of CA-4, but other positions of
the B-ring have not been examined in detail. Therefore,
we examined smaller substituents on the olefin site and
substituents on the B-ring other than at the 4-position.
The presence of a nitrile group is not sufficient to
improve solubility or physicochemical property of the

3026 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 16
Ta ble 3. Modification of the Aryl Substituents
Ohsumi et al.
cytotoxicity (IC50 5.9 nM), more potent than those of 10a
and CA-4. Acetamide (14) retained cytotoxicity but
showed decreased antimitotic activity (IC50 10 µM). By
introducing an amino group into the 3-position of the
B-ring of 10a , we obtained the new potent cytotoxic
agent 13b.
Next, 4-substituents of the B-ring of 13b were exam-
ined. Replacement of 4-OMe of the B-ring with a
4-methyl group (13c) or 4-chloro group (13d ) resulted
in a 10-fold decrease in cytotoxicity. The antitubulin
activity of 13d (IC50 6 µM) was more potent than that
of 13b. A 3-amino-4-methoxy substituent on the B-ring
seemed to be optimal for strong cytotoxicity.
bond
type
Colon 26a
IC50 (nM)
antitubulin activityb
compd
X
IC50 (µM)
13b showed greater cytotoxicity than CA-4, but its
antimitotic activity was significantly decreased (IC50 10
µM). The presence of an amino group on the B-ring in
addition to a CN group on the olefin site of cis-stilbene
seemed to decrease antimitotic activity. To obtain
compounds with both potent cytotoxicity and antimitotic
activity, we examined substituents on the olefin site
again (18, 19, 27b, 21a ) (Table 2). Compound 19, a
nitrile regioisomer of 13b, showed a 40-fold decrease in
cytotoxicity, but potent antitubulin activity (IC50 5 µM).
The amide compounds 18 and 27b, intermediates of
nitrile compounds, showed approximately 30-fold de-
creases in cytotoxicity. Only 27b showed strong anti-
tubulin activity (IC50 3 µM). These results indicated
that the position of the nitrile group is critical for
cytotoxicity and antitubulin activity.18
9a
9c
11a
11b
12
A
B
C
D
C
E
E
double
double
double
double
single
double
single
63.7
>3000
12.6
722
22.5
2370
>3000
>20
>20
>20
>20
7
>20
>20
23
25
a
Drug concentration required to inhibit the growth of Colon
b
26 cells by 50%. Tubulin polymerization was determined as
described in the text.
Ta ble 4. Modifications of the Aryl Substituents
Next, the requirement of the nitrile group on the
olefin site for strong cytotoxicity was examined. (Z)-
Stilbene 21a which lacked a CN group on the olefin site
of 13b showed strong cytotoxicity (IC50 5.1 nM) and
potent antitubulin activity (IC50 4 µM). This result
demonstrated that the CN group on the olefin site is
not necessary for potent cytotoxicity, and the presence
of the CN group weakened the antitubulin activity. The
strong cytotoxicity of 21a seemed to be due to replace-
ment of the phenolic OH of combretastatin A-4 by an
NH2 group.
Colon 26a antitubulin activityb
compd
R1
R2
R3
IC50 (nM)
IC50 (µM)
31a
31b
31c
13b
H
H
H
H
H
H
36.0
1430
8.1
8
9
7
OCH3
OCH3 OCH3
OCH3 OCH3 OCH3
5.9
10
a
Drug concentration required to inhibit the growth of Colon
26 cells by 50%. Tubulin polymerization was determined as
b
described in the text.
Next, 4-substituents on the B-ring of 21a were
examined. Replacement of the 4-methoxy group of 21a
with a 4-methyl (21b) or 4-chloro (21c) group resulted
in an order of magnitude decrease in cytotoxicity. On
the other hand, 21b,c showed strong antitubulin activi-
ties (IC50 3-4 µM). There seemed to be a discrepancy
between the potency of cytotoxicity and antitubulin
activity.
Table 3 shows the activities of (Z)-acrylonitriles,
which were obtained as intermediates of (E)-acryloni-
triles and saturated analogues (9a ,c, 11a ,b, 12, 23, 25)
All the (Z)-acrylonitriles lost their antitubulin activity.
Only 9a and 11a showed potent cytotoxicity (IC50 63.7
and 12.6 nM, respectively). Compound 12, a saturated
analogue of 11a , showed strong cytotoxicity (IC50 22.5
nM) and moderate antitubulin activity (IC50 7 µM). The
two benzene rings of 12 can adapt a favorable orienta-
tion that could mimic (E)-acrylonitrile (13b) to exert
potent activity. (Z)-Acrylonitrile 11a was more cytotoxic
than the saturated compound 12 suggesting that 11a
exerts its strong cytotoxicity in the Z-form rather than
on isomerization to the E-form. 11a may exert its
potent cytotoxic effect by a mechanism other than
inhibition of tubulin polymerization. Compounds in
mother compound, so we synthesized a series of com-
pounds with a nitrile substituent on the olefin site and
substituents on the B-ring were examined (Table 1). A
4-methoxy or 4-methyl group was required in the B-ring
for strong activity.18 Combretastatin A-1 (ii) which has
an additional 2-OH group on the B-ring of Combret-
astatin A-4 (i) showed significantly decreased cytotox-
icity suggesting that a 2-substituent is unacceptable for
potent cytotoxicity.14 So, we examined substituents on
the 3-position of the B-ring (10c, 13a ,b). Introduction
of a NO2 group (10c) resulted in a 7-fold decrease in
cytotoxicity compared to 10a . Introduction of OH
resulted in a compound (13a , IC50 23.5 nM) which was
as cytotoxic as the parent compound 10a . The antimi-
totic activity of 13a was also comparable to that of 10a .
The presence of a hydroxyl group on the 3-position of
the B-ring did not affect the activity of 10a . This
relationship of 10a and 13a was identical to that
observed when the 3-hydroxyl group of CA-4 was
deleted.17 These results validate our initial strategy to
use a 4-methoxyphenyl group as the B-ring in place of
the 3-hydroxyl-4-methoxyphenyl group of CA-4. Intro-
duction of an amino group (13b) significantly decreased
antimitotic activity (IC50 10 µM), but 13b showed strong

CA-4 Analogues Effective against Murine Tumors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 16 3027
Ta ble 5. Antitumor Activities of the Anilino Compounds
Ta ble 6. Antitumor Activities of 13bHCl and 21a HCl
compd
13bHCl
13cHCl
13d HCl
21a HCl
21bHCl
combretastatin A-4
dosesa (mg/kg) IR (%)b Colon 26 (sc-iv)c
IR (%)a (sc-iv)b
10
5
77
11
compd
Colon 38 (dose)c
3LL (dose)c
HCT-15 (dose)c
13bHCl
21a HCl
cisplatin
75 (10)
72 (10)
43 (5)
59 (5)
65 (40)
33 (4)
28 (10)
53 (40)
26 (8)
5
-23
40
40
160
69
62
31
a
IR (%) ) (1 - T/C) × 100; T, tumor volume (treated); C, tumor
b
volume (untreated). Mice were implanted subcutaneously (sc)
a
b
Maximum tolerable doses were administered. IR (%) ) (1 -
T/C) × 100; T, tumor volume (treated); C, tumor volume (un-
treated). c Mice were implanted subcutaneously (sc) with tumor
cells, and the drug was administered intravenously (iv).
with tumor cells, and the drug was administered intravenously
(iv). c Numbers in parentheses show maximum tolerable doses (mg/
kg).
respectively), while 13c,d HCl did not show antitumor
activity (IR ) 11% at 5 mg/kg, -23% at 5 mg/kg,
respectively). Combretastatin A-4 also did not show
antitumor activity (IR ) 31% at 160 mg/kg). 13b-
d HCl, each of which had a nitrile group on the olefin
site, seemed to be more toxic (MTD 5-10 mg/kg) than
21a ,bHCl, which had no substituent on the olefin site
of cis-stilbene (MTD 40 mg/kg). The toxic effects of
13c,d HCl prevented their evaluation at elevated doses.
13bHCl and 21a HCl, which showed potent activity
in the Colon 26 murine tumor model, were further
evaluated in the Colon 38 and 3LL murine tumor
models and HCT-15 xenografts (Table 6). 13bHCl
showed potent antitumor activity against Colon 38 (IR
) 75% at 10 mg/kg) and moderate antitumor activity
against 3LL (IR ) 59% at 5 mg/kg) but did not show
antitumor effects against HCT-15 xenografts (IR ) 28%
at 10 mg/kg). 21a HCl showed potent antitumor activity
against Colon 38 (IR ) 72% at 10 mg/kg), 3LL (IR )
65% at 40 mg/kg), and HCT-15 xenografts (IR ) 53%
at 40 mg/kg). Cisplatin did not show antitumor activity
against Colon 38, 3LL, or HCT-15. 21a HCl was supe-
rior to cisplatin in antitumor activity in these animal
models. Thus, we obtained compounds with potent
antitumor activity by introducing an amino group in
place of the phenolic OH of CA-4. This is the first report
of combretastatin A-4 analogues showing potent anti-
tumor activity in vivo.
which the B-ring was replaced with 2-methoxypyridine
were synthesized to mimic a 3-amino-4-methoxyphenyl
group and evaluated. Since we could not synthesize the
E-form of 23, we synthesized a saturated analogue to
evaluate the activities of the pyridine compounds.
However, the pyridine compounds 23 and 25 did not
show cytotoxicity or antitubulin activity. Since the
saturated compound 25 did not show cytotoxicity, the
E-form of compound 25 would not show strong cytotox-
icity if synthesized.
Finally, we examined the 3,4,5-trimethoxy group in
13b on the A-ring (31a -c) The 3,4-dimethoxyphenyl
compound 31c showed strong cytotoxicity (IC50 8.1 nM)
comparable to that of 13b and moderate antimitotic
activity (7 µM). 4-Methoxyphenyl 31b and nonsubsti-
tuted compound 31a showed decreased cytotoxicity. In
combretastatin A-4, the 3,4,5-trimethoxy group on the
A-ring was thought to be indispensable for its strong
cytotoxicity.18 However, our results indicated that a
methoxy group on the 3-position of A-ring is not neces-
sary for potent cytotoxicity of amino acrylonitriles.
The discrepancy between cytotoxicity and antitubulin
activity was observed in several compounds. This has
been noticed in other classes of antimitotic agents.23
Since strong cytotoxicity was observed in the amino
compounds with weaker antimitotic activity such as
13b,c, there might be another mechanism for exertion
of cytotoxicity. The strong cytotoxicity of amino com-
pound 11a , a trans-olefin with complete loss of antitu-
bulin activity, supports this hypothesis. Some other
compounds such as 19 and 27b showed strong antimi-
totic activity but weak cytotoxicity. Poor permeability
into cells is one possibility; however, the decrease in
activity other than antitubulin activity is the most
possible explanation. The amino group in the 3-position
plays no important role in the tubulin binding as the
3-hydroxyl group of combretastatin A-4 does,17 but it
seems to be important for the strong cytotoxicity caused
by the other mechanism.
13b-d and 21a ,b, which showed potent cytotoxicity
against Colon 26 in vitro, were selected for in vivo
testing. These compounds have an amino group on the
3-position of the B-ring, and hydrochloride salts were
formulated to improve aqueous solubility. The obtained
hydrochloride salts of each compound were water-
soluble. 13b-d HCl and 21a ,bHCl were evaluated for
their antitumor activities against murine Colon 26
adenocarcinoma in vivo (Table 5). The maximum toler-
able dose (MTD) of each compound was injected into
mice intravenously, and compounds which showed IR
(%) > 50 were defined as effective. 13bHCl and
21a ,bHCl showed marked tumor regression (IR ) 77%
at 10 mg/kg, 69% at 40 mg/kg, 62% at 40 mg/kg,
The marked antitumor activity of 21a HCl in contrast
to combretastatin A-4 may be due to increased hydro-
philicity of the compound. 21a HCl is more soluble (10.0
mg/mL) than CA-4 (0.11 mg/mL) in phosphate-buffered
saline solution, and different pharmacokinetics were
expected for both drugs. Delivery to the tumor site or
duration of contact with the tumor cells of 21a HCl may
be favorable for exerting the antitumor activity in mice.
Con clu sion
To improve in vivo antineoplastic activity of combre-
tastatin A-4, we synthesized a series of stilbene com-
pounds with oxygen- or nitrogen-containing groups. By
replacing phenolic OH of combretastatin A-4 by amine,
compounds with potent cytotoxicity and antitubulin
activity were obtained. Among these, 13bHCl and
21a HCl showed potent antitumor activity against Colon
26, Colon 38, and 3LL murine tumor models in mice.
21a HCl also showed antitumor activity against HCT-
15 human xenografts in nude mice. 21a HCl was
superior to cisplatin in these tumor models (Colon 38,
3LL, and HCT-15). 21a HCl showed improved solubil-
ity, and further studies are required to clarify the reason
for its superior in vivo efficacy over combretastatin A-4.
Further experiments to evaluate the potential of 21aHCl

3028 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 16
Ohsumi et al.
(39 mg, 0.12 mmol) in DMF (2 mL) were added K2CO3 (100
mg, 0.72 mmol) and MeI (15µL, 0.36 mmol), and the solution
was stirred at room temperature for 2 h. The reaction mixture
was diluted with CH2Cl2, washed with brine, and then dried
over Na2SO4. After concentration, the residue was purified
by alumina column chromatography (5% MeOH/CH2Cl2) to
give product 6 as an oil (33.6 mg, 56%): 1H NMR (CDCl3) δ
7.27 (1H, d, J ) 3.6), 7.00 (2H, d, J ) 8.7), 6.66 (2H, d, J )
8.7), 6.61 (2H, s), 5.09 (2H, brs), 3.86 (3H, s), 3.79 (6H, s), 3.75
(3H, s), 3.33 (9H, s); MS (FD) 372 (M+); formula (C22H30N1O4)+
I-; HRMS calcd 372.2175, found 372.2167.
as an anticancer agent for solid tumors are now under-
way in our laboratory.
Exp er im en ta l Section
Ch em ica l P r oced u r es. Column chromatography was
performed using silica gel (Merck, particle size 0.063-0.200
mm). TLC analyses were performed on silica gel plates
(Merck, Art. 5715). All melting points were determined on a
Yanaco micromelting point apparatus and are shown uncor-
rected. NMR spectra were recorded on a Varian EM-390 300-
MHz spectrometer with tetramethylsilane as the internal
standard; J values are given in hertz (Hz). Mass spectra (MS)
were measured on J EOL J MS-DX300 (FD, ESI, and FAB) and
J EOL J MS-HX110/HX110 (HRMS) instruments. Analytical
results indicated by elemental symbols were within (0.4% of
the theoretical values. For photoisomerization, high-pressure
mercury lamp (UVL-400HA, Riko-Kagaku Sangyo Co., Ltd.)
was used. Combretastatin A-4 was prepared by the method
reported by Pettit.24 Cisplatin was purchased from Nihon
Kayaku Co., Ltd.
(E)-3-(4-Met h oxyp h en yl)-2-(3,4,5-t r im et h oxyp h en yl)-
a cr ylic Acid (2a ). A mixture of 3,4,5-trimethoxyphenylacetic
acid (30.0 g, 0.13 mol), 4-methoxy benzaldehyde (18.9 g, 0.13
mol), and triethylamine (20 mL) in Ac2O (100 mL) was heated
at 140 °C for 12 h. After cooling, the mixture was evaporated
to dryness. The residue was diluted with aqueous NaOH for
saponification. Then, the solution was acidified with AcOH
and extracted with CH2Cl2. The extract was dried over Na2SO4
and evaporated to dryness. The residue was crystallized from
EtOAc-hexane to give product 2a as a white solid (16.5 g,
36%): 1H NMR (CDCl3) δ 7.87 (1H, s), 7.06 (2H, d, J ) 8.4),
6.73 (2H, d, J ) 8.4), 6.47 (2H, s), 3.90 (3H, s), 3.78 (6H, s),
3.77 (3H, s); MS (FD) 344 (M+); HRMS found 344.1260, calcd
344.1251.
(E)-3-(4-Met h oxyp h en yl)-2-(3,4,5-t r im et h oxyp h en yl)-
p r op en a l (7). To a solution of 3 (2.5 g, 7.6 mmol) in CH2Cl2
(25 mL) was added MnO2 (20 g). The reaction mixture was
stirred vigorously at room temperature for 8 h and filtered
over Celite, and the filtrate was evaporated to dryness. The
residue was purified by silica gel column chromatography (33%
EtOAc/hexane) to give product 7 (1.71 g, 5.2 mmol, 68%) as
1
white crystals: mp 109-110 °C; H NMR (CDCl3) δ 9.71 (1H,
s), 7.31 (1H, s), 7.20 (2H, d, J ) 7.8), 6.78 (2H, d, J ) 7.8),
6.41 (2H, s), 3.91 (3H, s), 3.79 (9H, s); MS (FD) 328 (M+);
HRMS (FAB) calcd 328.1311, found 328.1312. Anal. (C19H20O5)
C, H.
Gen er a l P r oced u r e for th e P r ep a r a tion of 9a -e a n d
23. A mixture of 3,4,5-trimethoxyphenylacetonitrile (16.6
mmol), benzaldehyde or pyridylaldehyde (16.6 mmol), NaOH
(19.9 mmol)-H2O (15 mL), and methyltrioctylammonium
chloride (2.4 mmol) in CH2Cl2 (30 mL) was stirred at room
temperature for 4 h. The solution was poured into brine,
extracted with CH2Cl2, and then dried over Na2SO4. After
concentration, the residue was crystallized from EtOAc to give
pure product.
(Z)-3-(4-Met h oxyp h en yl)-2-(3,4,5-t r im et h oxyp h en yl)-
a cr ylon itr ile (9a ): 8.4 g, 75%, yellow crystals, mp 82-83 °C;
1H NMR (CDCl3) δ 7.87 (2H, d, J ) 8.7), 7.38 (1H, s), 6.98
(2H, d, J ) 8.7), 6.85 (2H, s), 3.92 (6H, s), 3.86 (6H, s); MS
(FD) 325 (M+); HRMS calcd 325.1314, found 325.1339. Anal.
(C19H19N1O4) C,H, N.
(E)-Met h yl 3-(4-Met h oxyp h en yl)-2-(3,4,5-t r im et h oxy-
p h en yl)a cr yla te (2b). To a solution of carboxylic acid 2a (6.5
g, 18.2 mmol) in DMF (60 mL) were added K2CO3 (6.5 g, 47.1
mmol) and methyl iodide (1.35 mL, 21.8 mmol). The reaction
mixture was stirred at room temperature for 3 h and filtered,
and the filtrate was evaporated to dryness. The residue was
dissolved in EtOAc, washed with brine, and then dried over
Na2SO4. After concentration, the residue was used for the next
reaction without further purification.
(Z)-3-(3-((Met h oxym et h yl)oxy)-4-m et h oxyp h en yl)-2-
(3,4,5-tr im eth oxyp h en yl)a cr ylon itr ile (9b): oil, 5.87 g,
95%; 1H NMR (CDCl3) δ 7.71 (1H, d, J ) 2.4), 7.37 (1H, s),
7.03 (1H, d, J ) 2.4, 8.7), 6.98 (1H, d, J ) 8.7), 6.85 (2H, s),
5.29 (2H, s), 3.95 (3H, s), 3.93 (6H, s), 3.89 (3H, s), 3.56 (3H,
s); MS (FD) 385 (M+).
(E)-3-(4-Met h oxyp h en yl)-2-(3,4,5-t r im et h oxyp h en yl)-
p r op -2-en -1-ol (3). To a solution of methyl ester 2b (10.5 g,
29.3 mmol) in anhydrous THF (60 mL) was added 1.0 M
LiAlH4-THF (30 mL, 30 mmol). The solution was stirred at
0 °C for 1 h and poured into cold brine. The solution was
extracted with CH2Cl2 and dried over Na2SO4. After concen-
tration, the residue was purified by silica gel column chroma-
tography (33% EtOAc/hexane) to give pure product 3 as an oil
(6.0 g, 18.2 mmol, 62%): 1H NMR (CDCl3) δ 6.98 (2H, d, J )
9.0), 6.69 (2H, d, J ) 9.0), 6.59 (1H, s), 6.46 (2H, s), 4.43 (2H,
s), 3.87 (3H, s), 3.75 (9H, s); MS (FD) 330 (M+); HRMS found
330.1459, calcd 330.1467. Anal. (C19H22O5‚0.3H2O) C, H.
(E)-3-(4-Met h oxyp h en yl)-2-(3,4,5-t r im et h oxyp h en yl)-
a llyla m in e (5). To a solution of 3 (384 mg, 1.16 mmol) in
THF (4 mL) were added diethyl azodicarboxylate (200 mg, 1.16
mmol), triphenylphosphine (300 mg, 1.16 mmol), and phthal-
imide (170 mg, 1.16 mmol), and the reaction mixture was
stirred at room temperature for 5 h. After concentration, the
residue was purified by silica gel column chromatography (33%
EtOAc/hexane) to give (E)-N-3-(4-methoxyphenyl)-2-(3,4,5-
trimethoxyphenyl)prop-2-ene phthalimide (4) (540 mg, 87%).
A mixture of 4 (540 mg, 1.0 mmol) and hydrazine hydrate (71
mg) in ethanol (6 mL) was stirred at 100 °C for 1 h. After
concentration, a portion of the residue was purified by silica
gel column chromatography (5% MeOH/CH2Cl2) to give product
5 as an oil (44 mg, 0.13 mmol): 1H NMR (CDCl3) δ 6.94 (2H,
d, J ) 8.4), 6.65 (2H, d, J ) 8.4), 6.53 (1H, s), 6.43 (2H, s),
3.86 (3H, s), 3.74 (9H, s), 3.69 (2H, s); MS (FD) 329 (M+). Anal.
(C19H23N1O4) C, H, N.
(Z)-3-(4-Met h oxy-3-n it r op h en yl)-2-(3,4,5-t r im et h oxy-
p h en yl)a cr ylon itr ile (9c): yellow crystals, 4.42 g, 71%, mp
191-192 °C; H NMR (CDCl3) δ 8.30 (1H, dd, J ) 2.4, 9.0),
8.21 (1H, d, J ) 2.4), 7.38 (1H, s), 7.21 (1H, d, J ) 9.0), 6.86
(2H, s), 4.05 (3H, s), 3.94 (6H, s), 3.89 (3H, s); MS (FAB) 370
(M+); HRMS calcd 370.1165 (M+), found 370.1174. Anal.
(C19H18N2O6) C, H, N.
1
(Z)-3-(4-Me t h yl-3-n it r op h e n yl)-2-(3,4,5-t r im e t h oxy-
p h en yl)a cr ylon itr ile (9d ): yellow crystals, 2.0 g, 31%, mp
1
162-163 °C; H NMR (CDCl3) δ 8.35 (1H, J ) 1.5), 8.18 (1H,
J ) 1.5, 8.1), 7.47 (1H, J ) 8.1), 7.44 (1H, s), 6.88 (2H, s), 3.95
(6H, s), 3.90 (3H, s), 2.67 (3H, s); MS (FAB) 354 (M+); HRMS
calcd 354.1216, found 354.1204. Anal. (C19H18N2O5) C, H, N.
(Z)-3-(4-Ch lor o-3-n it r op h e n yl)-2-(3,4,5-t r im e t h oxy-
p h en yl)a cr ylon itr ile (9e): yellow crystals, 4.9 g, 48%, mp
198-199 °C; 1H NMR (CDCl3) δ 8.23 (1H, d, J ) 2.1), 8.15
(1H, dd, J ) 2.1, 8.4), 7.67 (1H, d, J ) 8.4), 7.41 (1H, s), 6.88
(2H, s), 3.94 (6H, s), 3.91 (3H, s); MS (FAB) 374 (M+). Anal.
(C18H15N2O5Cl1) C, H, N.
(Z)-3-(2-Met h oxy-5-p yr id yl)-2-(3,4,5-t r im et h oxyp h en -
yl)a cr ylon itr ile (23): yellow crystals, 609 mg, 61%, mp 119-
120 °C; 1H NMR (CDCl3) δ 8.44 (1H, s), 8.42 (1H, d, J ) 9.6),
7.36 (1H, s), 6.86 (2H, s), 6.86 (1H, d, J ) 9.6), 4.01 (3H, s),
3.95 (6H, s), 3.89 (3H, s); MS (FAB) 327 (MH+); HRMS calcd
327.1345 (MH+), found 327.1344. Anal. (C18H18N2O4) C, H,
N.
Gen er a l P r oced u r e for th e P r ep a r a tion of 10a -e. A
solution of (Z)-acrylonitrile 9a (5.4 mmol) in 500 mL of CH3CN
(CH3CN was bubbled with N2 gas to get rid of oxygen before
use) was irradiated with a RICO 400-W high-pressure mercury
(E)-[3-(4-Meth oxyp h en yl)-2-(3,4,5-tr im eth oxyp h en yl)-
a llyl]tr im eth yla m m on iu m Iod id e (6). To a solution of 5

CA-4 Analogues Effective against Murine Tumors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 16 3029
lamp equipped with a Pyrex filter for 30 min. This reaction
gave a 1:1 mixture of E- and Z-isomers. The mixture was
concentrated to dryness. The residue was purified by silica
gel column chromatography (25% EtOAc/hexane) to give pure
product.
(E)-3-(3-Am in o-4-m eth oxyp h en yl)-2-(3,4,5-tr im eth oxy-
p h en yl)a cr ylon itr ile (13b): yellow crystals, 2.2 g (68% from
10c), mp 144-145 °C; H NMR (CDCl3) δ 7.16 (1H, s), 6.65
(4H, s), 6.56 (1H, s), 3.88 (3H, s), 3.84 (3H, s), 3.77 (6H, s); MS
(FAB) 340 (M+); HRMS calcd 340.1423, found 340.1402. Anal.
(C19H20N2O4) C, H, N.
1
(E)-3-(4-Met h oxyp h en yl)-2-(3,4,5-t r im et h oxyp h en yl)-
a cr ylon itr ile (10a ): 800 mg (40% from 9a ), yellow crystals,
(E)-3-(3-Am in o-4-m et h ylp h en yl)-2-(3,4,5-t r im et h oxy-
p h en yl)a cr ylon itr ile (13c): yellow crystals, 57.2 mg (60%
1
mp 126-127 °C; H NMR (CDCl3) δ 7.23 (1H, s), 7.17 (2H, d,
1
from 10d ), mp 161-162 °C; H NMR (CDCl3) δ 7.20 (1H, s),
J ) 8.4), 6.77 (2H, d, J ) 8.4), 6.61 (2H, s), 3.88 (3H, s), 3.79
(3H, s), 3.76 (6H, s); MS (FD) 325 (M+); HRMS calcd 325.1314,
found 325.1340. Anal. (C19H19N1O4) C, H, N.
6.92 (1H, d, J ) 7.5), 6.62 (2H, s), 6.56 (1H, dd, J ) 0.9, 7.5),
6.51 (1H, s), 3.87 (3H, s), 3.75 (6H, s), 2.13 (3H, s); MS (FAB)
324 (M+); HRMS calcd 324.1474, found 324.1481. Anal.
(C19H20N2O3) C, H, N.
(E)-3-(3-((Met h oxym et h yl)oxy)-4-m et h oxyp h en yl)-2-
(3,4,5-tr im eth oxyp h en yl)a cr ylon itr ile (10b): E- and Z-
isomers were not separated, and the mixture was used for the
next reaction.
(E)-3-(4-Met h oxy-3-n it r op h en yl)-2-(3,4,5-t r im et h oxy-
p h en yl)a cr ylon itr ile (10c): 1.34 g (50% from 9c), yellow
crystals, mp 158-159 °C; 1H NMR (CDCl3) δ 7.74 (1H, d, J )
2.1), 7.35 (1H, dd, J ) 2.1, 9.0), 7.19 (1H, s), 6.94 (1H, d, J )
9.0), 6.58 (2H, s), 3.95 (3H, s), 3.89 (3H, s), 3.78 (6H, s); MS
(FAB) 370 (M+); HRMS calcd 370.1165, found 370.1174. Anal.
(C19H18N2O6) C, H, N.
(E )-3-(4-Me t h yl-3-n it r op h e n yl)-2-(3,4,5-t r im e t h oxy-
p h en yl)a cr ylon itr ile (10d ): white crystals, 420 mg (42%
from 9d ), mp 169-170 °C; 1H NMR (CDCl3) δ 7.84 (1H, d, J )
1.8), 7.29 (1H, dd, J ) 1.8, 8.1), 7.26 (1H, s), 7.22 (1H, d, J )
8.1), 6.56 (2H, s), 3.89 (3H, s), 3.75 (6H, s), 2.57 (3H, s); MS
(FAB) 354 (M+); HRMS calcd 354.1216, found 354.1216. Anal.
(C9H18N2O5) C, H, N.
(E)-3-(3-Am in o-4-ch lor op h en yl)-2-(3,4,5-t r im et h oxy-
p h en yl)a cr ylon itr ile (13d ): white crystals, 102 mg (33%
1
from 10e), mp 150-151 °C; H NMR (CDCl3) δ 7.17 (1H, s),
7.12 (1H, d, J ) 8.1), 6.61 (1H, d, J ) 1.8), 6.59 (2H, s), 6.53
(1H, dd, J ) 1.8, 8.1), 3.88 (3H, s), 3.75 (6H, s); MS (FAB) 344
(M+); HRMS calcd 344.0928, found 344.0943. Anal.
(C18H17N2O3Cl1) C, H, N.
(E)-3-(3-(Acet yla m in o)-4-m et h oxyp h en yl)-2-(3,4,5-t r i-
m eth oxyp h en yl)a cr ylon itr ile (14). A mixture of 13b (24
mg, 0.07 mmol) and Ac2O (1 mL) in pyridine (1 mL) was stirred
at room temperature for 1 h. The solution was evaporated to
dryness, and the residue was purified by preparative TLC
(CH2Cl2) to give 14 (26.4 mg, 98%) as white crystals: 230-
1
231 °C; H NMR (CDCl3) δ 8.34 (1H, s), 7.62 (1H, brs), 7.25
(1H, s), 6.87 (1H, dd, J ) 1.8, 8.7), 6.68 (1H, d, J ) 8.7), 3.88
(3H, s), 3.87 (3H, s), 3.76 (6H, s), 2.16 (3H, s); MS (FAB) 382
(M+); HRMS calcd 382.1529, found 382.1542. Anal.
(C21H22N2O5) C, H, N.
(E )-3-(4-Ch lor o-3-n it r op h e n yl)-2-(3,4,5-t r im e t h oxy-
p h en yl)a cr ylon itr ile (10e): yellow crystals, mp 181-182 °C,
(E)-2-(4-Met h oxy-3-n it r op h en yl)-3-(3,4,5-t r im et h oxy-
p h en yl)a cr ylic Acid (15). A mixture of 3,4,5-trimethoxy-
benzaldehyde (930 mg, 4.7 mmol), 4-methoxy-3-nitrophenyl-
acetic acid (1b), and triethylamine (1 mL) in Ac2O (10 mL)
was heated at 140 °C for 12 h. After cooling, the mixture was
concentrated to dryness. The residue was diluted with water,
and the solution was extracted with CH2Cl2. The extract was
dried over Na2SO4 and evaporated to dryness. The residue
was purified by silica gel column chromatography (5% MeOH/
CH2Cl2) to give 15 as a solid (990 mg, 54%): 1H NMR (CDCl3)
δ 7.89 (1H, s), 7.78 (1H, d, J ) 1.8), 7.39 (1H, d, J ) 7.8), 7.09
(1H, d, J ) 7.8), 6.34 (2H, s), 3.95 (3H, s), 3.83 (3H, s), 3.59
(6H, s); MS (FAB) 389 (M+); HRMS calcd 389.1111, found
389.1091.
(E)-2-(4-Met h oxy-3-n it r op h en yl)-3-(3,4,5-t r im et h oxy-
p h en yl)a cr yla m id e (16). To a solution of 15 (700 mg, 1.82
mmol) in CH2Cl2 (9 mL) was added SOCl2 (0.70 mL) in DMF
(0.1 mL). The solution was stirred at room temperature for 1
h; then the mixture was evaporated to dryness. The residue
was dissolved with CH2Cl2 (5 mL), and the solution was added
to well-stirred 28% aqueous NH3 (30 mL) at room temperature.
After 30 min, the reaction mixture was extracted with CH2Cl2
and dried over Na2SO4. After concentration, the residue was
purified by preparative TLC (33% EtOAc/hexane) to give 16
(386 mg, 54%): yellow crystals, mp 193-194 °C; 1H NMR
(CDCl3) δ 7.85 (1H, d, J ) 2.1), 7.78 (1H, s), 7.49 (1H, dd, J )
2.1), 7.20 (1H, d, J ) 2.1, 8.4), 6.28 (2H, s), 3.99 (3H, s), 3.81
(3H, s), 3.60 (6H, s); MS (FAB) 388 (M+); HRMS calcd
388.1271, found 388.1269.
(E)-2-(4-Met h oxy-3-n it r op h en yl)-3-(3,4,5-t r im et h oxy-
p h en yl)a cr ylon itr ile (17). To a solution of 16 (116 mg, 0.3
mmol) in pyridine (5 mL) was added SOCl2 (0.10 mL). The
reaction mixture was stirred at room temperature for 6 h. The
mixture was concentrated to dryness, and the residue was
purified by preparative TLC (33% EtOAc/hexane) to give 17
as yellow crystals (48 mg, 43%): 1H NMR (CDCl3) δ 7.92 (1H,
d, J ) 2.4), 7.60 (1H, dd, J ) 2.4, 8.7), 7.31 (1H, s), 7.11 (1H,
d. J ) 8.7), 6.42 (2H, s), 3.99 (3H, s), 3.86 (3H, s), 3.66 (6H,
s); MS (FAB) 370 (M+); HRMS calcd 370.1165, found 370.1174.
Gen er a l P r oced u r e for th e P r ep a r a tion of 18 a n d 19.
To a solution of nitro compound 16 (162 mg, 0.42 mmol) in
AcOH (2 mL) was added zinc powder (300 mg). The reaction
mixture was stirred at room temperature for 1 h. The mixture
1
600 mg (40% from 9e); H NMR (CDCl3) δ 7.74 (1H, d, J )
2.1), 7.44 (1H, d, J ) 8.7), 7.32 (1H, dd, J ) 2.1, 8.7), 7.23
(1H, s), 6.55 (2H, s), 3.89 (3H, s), 3.77 (6H, s); MS (FAB) 374
(M+).
Gen er a l P r oced u r e for th e P r ep a r a tion of 11a ,b. To
a solution of 9c,d (13.6 mmol) in AcOH (200 mL) was added
zinc powder (23 g). The reaction mixture was stirred at room
temperature for 2 h and filtered over Celite; then the filtrate
was evaporated to dryness. After concentration, the residue
was purified by silica gel column chromatography (CH2Cl2) to
give pure product.
(Z)-3-(3-Am in o-4-m eth oxyp h en yl)-2-(3,4,5-tr im eth oxy-
p h en yl)a cr ylon it r ile (11a ): 3.90 g (83% from 9c), yellow
crystals, mp 106-107 °C; 1H NMR (CDCl3) δ 7.40 (1H, d, J )
2.1), 7.30 (1H, s), 7.21 (1H, dd, J ) 2.1, 8.4), 6.83 (2H, s), 6.82
(1H, d, J ) 8.4), 3.92 (6H, s), 3.92 (3H, s), 3.88 (3H, s); MS
(FAB) 340 (M+); HRMS calcd 340.1423, found 340.1412. Anal.
(C19H20N2O4) C, H, N.
(Z)-3-(3-Am in o-4-m et h ylp h en yl)-2-(3,4,5-t r im et h oxy-
p h en yl)a cr ylon itr ile (11b): oil, 95.3 mg (95% from 9d ); 1H
NMR (CDCl3) δ 7.34 (1H, s), 7.31 (1H, s), 7.13 (2H, s), 6.85
(2H, s), 3.93(6H, s), 3.88 (3H, s), 2.21 (3H, s); MS (FAB) 324
(M+); HRMS calcd 324.1474, found 324.1479. Anal.
(C19H20N2O3) C, H, N.
(E)-3-(3-Hydr oxy-4-m eth oxyph en yl)-2-(3,4,5-tr im eth oxy-
p h en yl)a cr ylon itr ile (13a ): E- and Z-mixture of 10b (5.5 g,
14.3 mmol); concentrated HCl (5 mL) in AcOH (55 mL) was
stirred at room temperature for 20 min; after concentration,
the residue was crystallized from Et2O to give 13a (1.95 g,
1
40%) as white crystals; mp 188-189 °C; H NMR (CDCl3) δ
7.18 (1H, s), 6.82 (1H, d, J ) 2.1), 6.77 (1H, dd, J ) 8.7, 2.1),
6.72 (1H, d, J ) 8.7), 5.52 (1H, s), 3.88 (6H, s), 3.77 (6H, s);
MS (FD) 341 (M+); HRMS calcd 341.1263, found 341.1240.
Anal. (C19H19N1O5) C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of 13b -d . To
a solution of nitro compounds 10c-e (8.6 mmol) in AcOH (160
mL) was added zinc powder (32 g). The reaction mixture was
stirred at room temperature for 1 h. The reaction mixture was
filtered over Celite, and the filtrate was evaporated to dryness.
After concentration, the residue was purified by silica gel
column chromatography (25% Et2O/hexane) to give pure
product.

3030 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 16
Ohsumi et al.
was filtered over Celite, and the filtrate was concentrated to
dryness. The residue was purified by preparative TLC (5%
MeOH/CH2Cl2) to give pure product.
(E)-2-(3-Am in o-4-m eth oxyp h en yl)-3-(3,4,5-tr im eth oxy-
p h en yl)a cr yla m id e (18): yellow crystals, 76 mg (50% from
16), mp 148-149 °C; 1H NMR (CDCl3) δ 7.72 (1H, s), 6.87 (1H,
s, J ) 7.9), 6.64 (1H, d, d, J ) 2.0, 7.9), 6.62 (1H, d, J ) 2.0),
6.38 (2H, s), 5.73 (2H, br), 5.15 (2H, br), 3.88 (3H, s), 3.80 (3H,
s), 3.58 (6H, s); MS (FAB) 358 (M+); HRMS calcd 358.1529,
found 358.1530. Anal. (C19H22N2O5) C, H, N.
(E)-2-(3-Am in o-4-m eth oxyp h en yl)-3-(3,4,5-tr im eth oxy-
p h en yl)a cr ylon itr ile (19): yellow crystals, 21 mg (76% from
17), mp 128-129 °C; 1H NMR (CDCl3) δ 7.12 (1H, s), 6.77 (3H,
brs), 6.49 (2H, s), 3.86 (3H, s), 3.84 (3H, s), 3.64 (6H, s); MS
(FAB) 340 (M+); HRMS calcd 340.1423, found 340.1402. Anal.
(C19H20N2O4) C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of 20a-c. NaH
(10.0 mmol, washed with hexane) was added to a stirred
suspension of phosphonium bromide (8.4 mmol) and benzal-
dehyde 8c-e (8.4 mmol) in toluene (60 mL), and the mixture
was stirred at room temperature for 12 h. AcOH (5 mL) was
added to the reaction mixture, which was then poured onto
ice-water and extracted with CH2Cl2. The extract was dried
over Na2SO4 and evaporated to dryness. The residue was
crystallized from EtOH. The obtained solid was recrystallized
from EtOAc/hexane to give the desired Z-form product.
(Z)-1-Meth oxy-2-n itr o-4-[2-(3,4,5-tr im eth oxyp h en yl)vi-
n yl]ben zen e (20a ): 1.27 g, 43%, yellow crystals, mp 123-
124 °C; 1H NMR (CDCl3) δ 7.79 (1H, d, J ) 2.1), 7.42 (1H, dd,
J ) 2.1, 8.7), 6.93 (1H, d, J ) 8.7), 6.58 (1H, d, J ) 12.9), 6.47
(2H, s), 6.44 (1H, d, J ) 12.9), 3.93 (3H, s), 3.85 (3H, s), 3.71
(6H, s); MS (FAB) 345 (M+); HRMS calcd 345.1212, found
345.1196. Anal. (C18H19N1O6) C, H, N.
(Z)-1-Meth yl-2-n itr o-4-[2-(3,4,5-tr im eth oxyph en yl)vin yl]-
ben zen e (20b): 990 mg, 47%, oil; 1H NMR (CDCl3) δ 7.89
(1H, d, J ) 1.8), 7.40 (1H, dd, J ) 1.8, 7.8), 7.19 (1H, d, J )
7.8), 6.63 (1H, d, J ) 12.3), 6.50 (1H, d, J ) 12.3), 6.46 (2H,
s), 3.85 (3H, s), 3.69 (6H, s), 2.55 (3H, s); MS (FAB) 329 (M+).
(Z)-1-Ch lor o-2-n itr o-4-[2-(3,4,5-tr im eth oxyph en yl)vin yl]-
ben zen e (20c): yellow crystals, mp 73-74 °C, 950 mg, 50%;
1H NMR (CDCl3) δ 7.79 (1H, s), 7.39 (2H, s), 6.70 (1H, d, J )
12.0), 6.47 (1H, d, J ) 12.0), 6.44 (2H, s) 3.86 (3H, s), 3.72
(6H, s); MS (FAB) 349 (M+).
The residue was crystallized from EtOH/Et2O to give the
corresponding HCl salt.
(E)-3-(3-Am in o-4-m eth oxyp h en yl)-2-(3,4,5-tr im eth oxy-
p h en yl)a cr ylon itr ile h yd r och lor id e (13bHCl): white crys-
tals (from EtOH/Et2O), mp 154-155 °C, 356 mg (71% from
1
13b); H NMR (DMSO-d6), δ 7.51 (1H, s), 7.04-7.14 (2H, m),
6.66 (2H, s), 3.84 (3H, s), 3.71 (3H, s), 3.69 (6H, s). Anal.
(C19H20N2O4‚HCl) C, H, N.
(E)-3-(3-Am in o-4-m et h ylp h en yl)-2-(3,4,5-t r im et h oxy-
p h en yl)a cr ylon itr ile h yd r och lor id e (13cHCl): white crys-
tals (from EtOH/Et2O), mp 162-163 °C, 250 mg (72% from
13c); 1H NMR (DMSO-d6) δ 7.56 (1H, s), 7.15 (1H, d, J ) 7.5),
7.08 (1H, brs), 6,89 (1H, d, J ) 7.5), 6.65 (2H, s), 3.71 (3H, s),
3.68 (6H, s), 2.30 (3H, s). Anal. (C19H20N2O3‚HCl) C, H, N.
(E)-3-(3-Am in o-4-ch lor op h en yl)-2-(3,4,5-t r im et h oxy-
p h en yl)a cr ylon itr ile h yd r och lor id e (13d HCl): white crys-
tals (from EtOH/Et2O), mp 149-150 °C, 364 mg (75% from
13d ); 1H NMR (DMSO-d6) δ 7.48 (1H, s), 7.12 (1H, d, J ) 8.4),
6.75 (1H, d, J ) 1.8), 6.65 (2H, s), 6.38 (1H, dd, J ) 1.8, 8.4),
3.70 (3H, s), 3.68 (6H, s). Anal. (C18H17N2O3Cl1‚HCl) C, H,
N.
(Z)-2-Me t h oxy-5-[2-(3,4,5-t r im e t h oxyp h e n yl)vin yl]-
p h en yla m in e h yd r och lor id e (21a HCl): white crystals (from
EtOH/Et2O), mp 117-118 °C, 8.4 g (82% from 21a ); 1H NMR
(CDCl3) δ 7.58 (1H, d, J ) 2.1), 7.28 (1H, dd, J ) 2.1, 8.7),
6.79 (1H, d, J ) 8.7), 6.52 (1H, d, J ) 12.0), 6.48 (2H, s), 6.45
(1H, d, J ) 12.0), 3.91 (3H, s), 3.83 (3H, s), 3.73 (6H, s). Anal.
(C18H21N1O4‚HCl) C, H, N.
(Z)-2-Me t h y l-5-[2-(3,4,5-t r im e t h o x y p h e n y l)v in y l]-
p h en yla m in e h yd r och lor id e (21bHCl): oil, 254 mg (72%
1
from 21b); H NMR (DMSO-d6) δ 7.31 (1H, d, J ) 1.5), 7.23
(1H, d, J ) 7.8), 7.23 (1H, dd, J ) 1.5, 7.8), 6.52 (2H, d, J )
12.3), 3.63 (3H, s), 3.57 (3H, s), 3.56 (3H, s), 2.31 (3H, s). Anal.
(C18H21N1O3‚HCl) C, H, N.
Gen er a l P r oced u r e of th e P r ep a r a tion of 12 a n d 25.
A mixture of acrylonitrile (11.5 mmol) and 10% Pd-C (3.0 g)
in MeOH (120 mL) was stirred at room temperature under
hydrogen atmosphere for 3 h. The mixture was filtered over
Celite, and the filtrate was evaporated to dryness. The residue
was purified by silica gel column chromatography (CH2Cl2) to
give pure product.
3-(3-Am in o-4-m e t h oxyp h e n yl)-2-(3,4,5-t r im e t h oxy-
p h en yl)p r op a n en itr ile (12): white crystals, mp 126-127 °C,
1
Gen er a l P r oced u r e for th e P r ep a r a tion of 21a -c. To
a solution of nitro compounds 20a -c (2.03 mmol) in AcOH
(160 mL) was added zinc powder (32 g). The reaction mixture
was stirred at room temperature for 1 h. The reaction mixture
was filtered over Celite, and the filtrate was evaporated to
dryness. After concentration, the residue was purified by silica
gel column chromatography (CH2Cl2) to give pure product.
(Z)-2-Me t h oxy-5-[2-(3,4,5-t r im e t h oxyp h e n yl)vin yl]-
1.81 g (46% from 11a ); H NMR (CDCl3) δ 6.69 (1H, d, J )
7.8), 6.51 (1H, s), 6.50 (1H, dd, J ) 2.4, 7.8), 6.44 (2H, s), 3.84
(3H, s), 3.83 (3H, s), 3.82 (6H, s), 3.84 (1H, m), 3.06 (1H, dd,
J ) 8.1, 13.5), 2.97 (1H, dd, J ) 8.1, 13.5); MS (FAB) 343
(MH+); HRMS calcd 343.1658 (MH+), found 343.1636. Anal.
(C19H22N2O4) C, H, N.
3-(2-Met h oxy-5-p yr id yl)-2-(3,4,5-t r im et h oxyp h en yl)-
p r op a n en itr ile (25): white crystals, mp 67-68 °C, 76.7 mg
1
1
p h en yla m in e (21a ): oil, 314 mg (49% from 20a ); H NMR
(76% from 23); H NMR (CDCl3) δ 7.91 (1H, d, J ) 2.4), 7.34
(CDCl3) δ 6.69 (1H, s), 6.67 (2H, s), 6.55 (2H, s), 6.45 (1H, d,
J ) 12.0), 6.36 (1H, d, J ) 12.0), 3.84 (3H, s), 3.82 (3H, s),
3.69 (6H, s); MS (FAB) 315 (M+); HRMS calcd 315.1471, found
315.1462. Anal. (C18H21N1O4) C, H, N.
(1H, d, J ) 2.4, 8.4), 6.68 (1H, d, J ) 8.4), 6.41 (2H, s), 3.92
(3H, s), 3.89 (1H, t, J ) 7.2), 3.84 (3H, s), 3.82 (6H, s), 3.10
(1H, dd, J ) 2.7, 7.2); MS (FAB) 329 (MH+); HRMS calcd
329.1501, found 329.1483. Anal. (C18H20N2O4) C, H, N.
(E)-3-(4-Met h oxy-3-n it r op h en yl)-2-(3,4,5-t r im et h oxy-
p h en yl)a cr ylic Acid (26). A mixture of 3,4,5-trimethoxy-
phenylacetic acid (1.01 g, 4.7 mmol), 4-methoxy-3-nitroben-
zaldehyde (850 mg, 4.7 mmol), and triethylamine (1 mL) in
Ac2O (10 mL) was heated at 140 °C for 12 h. After cooling,
the mixture was evaporated to dryness. The residue was
diluted with water, and the solution was extracted with
CH2Cl2. The extract was dried over Na2SO4 and evaporated
to dryness. The residue was crystallized from EtOAc/hexane
to give 26 (635 mg, 36%): yellow crystals, mp 217-218 °C; 1H
NMR (CDCl3) δ 7.81 (1H, s), 7.65 (1H, d, J ) 2.7), 7.21 (1H,
dd, J ) 2.7, 9.0), 6.89 (1H, d, J ) 9.0), 6.45 (2H, s), 3.93 (3H,
s), 3.91 (3H, s), 3.79 (6H, s); MS (FAB) 389 (M+); HRMS calcd
389.1111, found 389.1099. Anal. (C19H19N1O8) C, H, N.
(E)-3-(4-Met h oxy-3-n it r op h en yl)-2-(3,4,5-t r im et h oxy-
p h en yl)a cr yla m id e (27a ). To a solution of carboxylic acid
26a (101 mg, 0.26 mmol) in CH2Cl2 (1 mL) were added SOCl2
(38 µL) and pyridine (0.1 mL). The reaction mixture was
(Z)-2-Me t h y l-5-[2-(3,4,5-t r im e t h o x y p h e n y l)v in y l]-
1
p h en yla m in e (21b): oil, 620 mg (82% from 20b); H NMR
(CDCl3) δ 6.93 (1H, d, J ) 7.5), 6.65 (1H, dd, J ) 1.8, 7.5),
6.63 (1H, d, J ) 1.8), 6.53 (2H, s), 6.49 (1H, d, J ) 12.3), 6.40
(1H, d, J ) 12.3), 3.83 (3H, s), 3.68 (6H, s), 2.13 (3H, s); MS
(FD) 299 (M+); HRMS calcd 299.1521, found 299.1523. Anal.
(C18H21N1O3‚0.2H2O) C, H, N.
(Z)-2-C h lo r o -5-[2-(3,4,5-t r im e t h o x y p h e n y l)v in y l]-
p h en yla m in e (21c): oil, 52 mg (66% from 20c); 1H NMR
(CDCl3) δ 7.12 (1H, d, J ) 7.8), 6.71 (1H, d, J ) 1.8), 6.62 (1H,
dd, J ) 1.8, 7.8), 6.49 (2H, s), 6.45 (2H, s), 3.84 (3H, s), 3.69
(6H, s); MS (FAB) 319 (M+); HRMS calcd 319.0975, found
319.0987. Anal. (C17H18N1O3Cl1‚0.1H2O) C, H, N.
Gen er a l P r oced u r e of th e P r ep a r a tion of 13b-d HCl
a n d 21a ,bHCl. To a solution of anilino compounds (13b-d
and 21a ,b) (1.58 mmol) in CH2Cl2 (10 mL) was added 4 N
HCl-dioxane (1.0 mL). The reaction mixture was stirred at
room temperature for 1 h and then concentrated to dryness.

CA-4 Analogues Effective against Murine Tumors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 16 3031
stirred at room temperature for 2 h. The mixture was
concentrated to dryness. The residue was dissolved with
CH2Cl2 (2 mL), and the solution was added to well-stirred 28%
aqueous NH3 (2 mL). After 1 h, the mixture was diluted with
water, extracted with CH2Cl2, and dried over Na2SO4. After
concentration, the residue was purified by preparative TLC
(5% MeOH/CH2Cl2) to give 27a (72.8 mg, 73%): yellow
crystals, mp 202-203 °C; 1H NMR (CDCl3) δ 7.75 (1H, s), 7.51
(1H, d, J ) 2.4), 7.26 (1H, dd, J ) 2.4), 6.92 (1H, d, J ) 2.4,
9.0), 6.48 (2H, s), 5.59 (2H, br), 3.93 (6H, s), 3.82 (6H, s); MS
(FAB) 389 (MH+); HRMS calcd 388.1271, found 388.1254.
Anal. (C19H20N2O7) C, H, N.
(E)-3-(3-Am in o-4-m eth oxyp h en yl)-2-(3,4,5-tr im eth oxy-
p h en yl)a cr yla m id e (27b). To a solution of 27a (204 mg, 0.52
mmol) in AcOH (10 mL) was added zinc powder (2.0 g) at room
temperature. The reaction mixture was stirred vigorously for
2 h. The mixture was filtered over Celite, and the filtrate was
evaporated to dryness. The residue obtained was purified by
preparative TLC (EtOAc) to give 27b (54 mg, 29%): yellow
crystals, mp 182-183 °C; 1H NMR (CDCl3) δ 7.72 (1H, s), 6.61
(1H, d, J ) 8.4), 6.51 (2H, s), 6.52 (1H, dd, J ) 2.4, 8.4), 6.39
(1H, dd, J ) 2.4, 8.4), 5.49 (2H, br), 3.92 (3H, s), 3.82 (9H, s);
MS (FAB) 358 (M+). Anal. (C19H22N2O5) C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of 29a -c. A
mixture of phenylacetonitriles (15.0 mmol), 4-methoxy-3-
nitrobenzaldehyde (15.0 mmol), NaOH (15.0 mmol)-H2O (15
mL), and methyltrioctylammonium chloride (2.4 mmol) in
CH2Cl2 (30 mL) was stirred at room temperature for 4 h. The
solution was poured onto brine, extracted with CH2Cl2, and
then dried over Na2SO4. After concentration, the residue was
crystallized from EtOAc to give pure product.
(Z)-3-(4-Me t h oxy-3-n it r op h e n yl)-2-p h e n yla cr ylon i-
tr ile (29a ): yellow crystals, mp 151-152 °C; 1H NMR (CDCl3)
δ 8.30 (1H, dd, J ) 2.4, 8.7), 8.22 (1H, d, J ) 2.4), 7.64-7.70
(2H, m), 7.40-7.50 (3H, m), 7.46 (1H, s), 7.21 (1H, d, J ) 8.7),
4.05 (3H, s); MS (ESI) 267 (MH+).
(Z)-3-(4-Meth oxy-3-n itr op h en yl)-2-(4-m eth oxyp h en yl)-
a cr ylon itr ile (29b): yellow crystals, mp 168-169 °C; 1H NMR
(CDCl3) δ 8.27 (1H, dd, J ) 2.1, 8.7), 8.19 (1H, d, J ) 2.1),
7.60 (2H, d, J ) 9.0), 7.34 (1H, s), 7.19 (1H, d, J ) 8.7), 6.97
(2H, d, J ) 9.0); MS (ESI) 311 (MH+).
(E )-3-(4-Me t h oxy-3-n it r op h e n yl)-2-(3,4-d im e t h oxy-
p h en yl)a cr ylon itr ile (29c): yellow crystals, mp 205-206 °C;
1H NMR (CDCl3) δ 8.28 (1H, dd, J ) 2.7, 9.0), 8.19 (1H, d, J
) 2.7), 7.35 (1H, s), 7.25 (1H, dd, J ) 2.1, 8.4), 7.22 (1H, d, J
) 9.0), 7.12 (1H, d, J ) 2.1), 6.93 (1H, d, J ) 8.4); MS (ESI)
341 (MH+).
Gen er a l P r oced u r e for th e P r ep a r a tion of 30a -c. A
solution of (Z)-acrylonitrile 29a -c (5.0 mmol) in CH3CN (500
mL) was irradiated with a RICO 400-W high-pressure mercury
lamp equipped with a Pyrex filter for 30 min. This reaction
gave a 1:1 mixture of E- and Z-isomers. The mixture was
evaporated to dryness, and the residue was purified by silica
gel column chromatography (25% EtOAc/hexane) to give pure
product.
(E)-3-(4-Meth oxy-3-n itr oph en yl)-2-ph en ylacr ylon itr ile-
(30a ): yellow crystals, mp 152-153 °C, 405 mg (40% from
29a ); 1H NMR (CDCl3) δ 7.64 (1H, d, J ) 2.4), 7.34-7.50 (5H,
m), 7.30 (1H, dd, J ) 2.4, 9.0), 7.25 (1H, s), 6.92 (1H, d, J )
9.0); MS (FAB) 280 (M+); HRMS calcd 280.0848, found
280.0861. Anal. (C16H12N2O3) C, H, N.
Gen er a l P r oced u r e of th e P r ep a r a tion of 31a -c. To a
solution of nitro compounds 30a -c (8.6 mmol) in AcOH (160
mL) was added zinc powder (32 g). The reaction mixture was
stirred at room temperature for 1 h. The reaction mixture was
filtered over Celite, and the filtrate was concentrated to
dryness. After concentration, the residue was purified by silica
gel column chromatography (25% Et2O/hexane) to give pure
product.
(E )-3-(3-Am in o -4-m e t h o x y p h e n y l)-2-p h e n y la c r y l-
a m id e (31a ): oil, 820 mg (71% from 30a ); 1H NMR (CDCl3) δ
7.34-7.46 (5H, m), 7.20 (1H, s), 6.63 (1H, d, J ) 8.1), 6.58
(1H, dd, J ) 2.1, 8.1), 6.46 (1H, d, J ) 2.1), 3.83 (3H, s), 3.70
(2H, br); MS (FAB) 250 (M+); HRMS calcd 250.1106, found
250.1107. Anal. (C16H14N2O1) C, H, N.
(E)-3-(3-Am in o-4-m eth oxyp h en yl)-2-(4-m eth oxyp h en -
yl)a cr yla m id e (31b): yellow crystals, mp 121-122 °C, 214
1
mg (51% from 30b); H NMR (CDCl3) δ 7.33 (2H, d, J ) 9.0),
7.13 (1H, s), 6.88 (2H, d, J ) 9.0), 6.60-6.66 (2H, m), 6.52
(1H, d, J ) 1.8), 3.83 (6H, s), 3.70 (2H, br); MS (FAB) 280
(M+); HRMS calcd 280.1212, found 280.1217. Anal.
(C17H16N2O2) C,H, N.
(E)-3-(3-Am in o-4-m et h oxyp h en yl)-2-(3,4-d im et h oxy-
p h en yl)a cr yla m id e (31c): yellow crystals, mp 87-88 °C, 588
mg (98% from 30c); 1H NMR (CDCl3) δ 7.14 (1H, s), 7.01 (1H,
dd, J ) 2.1, 8.1), 6.90 (1H, d, J ) 2.1), 6.84 (1H, d, J ) 8.1),
6.63 (2H, s), 6.55 (1H, s), 3.90 (3H, s), 3.83 (3H, s), 3.77 (3H,
s); MS (FAB) 310 (M+); HRMS calcd 310.1301, found 310.1324.
Anal. (C18H18N2O3) C, H, N.
Tu bu lin P olym er iza tion In h ibition Assa y. Electro-
phoretically homogeneous tubulin was purified from bovine
brain.25 Determination of IC50 values for the polymerization
of purified tubulin was performed as follows.26 Tubulin was
preincubated at 37 °C with test compounds at various con-
centrations, and reaction mixtures were chilled on ice. GTP
(guanosine 5′-triphosphate; required for the polymerization
reaction) was added, and polymerization was followed at 37
°C by turbidimetry measurement at 350 nm in a Gliford
recording spectrometer equipped with an electronic temper-
ature controller. The extent of polymerization after 20-min
incubation was determined. IC50 values were determined
graphically. Compounds were examined in three independent
assays, and the values shown for these compounds are the
averages of three determinations.
Cell Gr ow th In h ibition Assa y. Murine Colon 26 adeno-
carcinoma cells were maintained in plastic dishes in RPMI-
1640 supplemented with 10% fetal bovine serum. For in vitro
treatment, tumor cells were seeded in 50 µL of culture
medium/well in 96-well plates to a final cell density of 1 × 105
cells/ml and incubated in a CO2 incubator at 37 °C for 24 h.
The cells were treated with various concentrations of test
compounds and incubated in a CO2 incubator at 37 °C for 48
h. The number of viable cells was estimated using the
tetrazolium dye reduction assay (MTT assay).27,28 Cytotoxicity
of the test compounds was expressed in terms of IC50 values.
Compounds were examined in three independent assays, and
the values shown for these compounds are the averages of
three determinations.
In Vivo An titu m or Activity. In transplanted solid tumor
models, fragments of Colon 26 tumor (5 mg) were inoculated
sc into CD2F1 mice, those of Colon 38 tumor (5 mg) or 3LL (5
mg) were inoculated sc into BD2F1 mice, and fragments of
HCT-15 (5 mg) were inoculated sc into ICR nu/nu mice on day
0. Test compounds were given iv on days 7, 11, and 15. On
day 21, two diameters of each tumor were measured using
calipers. The tumor weights were calculated using the for-
mula: tumor weight (mg) ) [length (mm) × width (mm)2]/2.
The inhibition ratio was evaluated as (1 - T/C) × 100 (%)
(where T is the mean tumor weight of the treated group and
C is the mean tumor weight of the control group). Each group
consisted of 5 mice.
(E)-3-(4-Meth oxy-3-n itr op h en yl)-2-(4-m eth oxyp h en yl)-
a cr ylon itr ile (30b): yellow crystals, mp 95-96 °C, 487 mg
(35% from 29b); 1H NMR (CDCl3) δ 8.01 (1H, d, J ) 2.1), 7.66
(1H, dd, J ) 2.1, 8.7), 7.61 (2H, d, J ) 9.0), 7.48 (1H, s), 7.26
(1H, d, J ) 8.7), 7.22 (2H, d, J ) 9.0), 4.26 (3H, s), 4.16 (3H,
s); MS (FAB) 310 (M+). Anal. (C17H14N2O4) C, H, N.
(E )-3-(4-Me t h oxy-3-n it r op h e n yl)-2-(3,4-d im e t h oxy-
p h en yl)a cr ylon itr ile (30c): yellow crystals, mp 141-142 °C,
1
Refer en ces
732 mg (41% from 29c); H NMR (CDCl3) δ 7.74 (1H, d, J )
2.4), 7.34 (1H, dd, J ) 2.4, 9.0), 7.17 (1H, s), 6.92-6.99 (2H,
m), 6.86 (1H, d, J ) 9.0), 6.83 (1H, d, J ) 2.1); MS (FAB) 340
(M+). Anal. (C18H16N2O5) C, H, N.
(1) Pettit, G. R.; Cragg, G. M.; Herald, D. L.; Schmidt, J . M.;
Lohavanijaya, P. Isolation and Structure of Combretastatin.
Can. J . Chem. 1982, 60, 1374-1376.

3032 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 16
Ohsumi et al.
(2) Pettit, G. R.; Singh, S. B.; Hamel, E.; Lin, C. M.; Alberts, D. S.;
Garcia-Kendall, D. Isolation and Structure of the Strong Cell
Growth and Tubulin Inhibitor Combretastatin A-4. Experientia
1989, 45, 209-211.
(16) Lin, C. M.; Ho, H. H.; Pettit, G. R.; Hamel, E. Antimitotic Natural
Products Combretastatin A-4 and Combretastatin A-2: Studies
on the Mechanism of Their Inhibition of the Binding of Colchi-
cine to Tubulin. Biochemistry 1989, 28, 6984-6991.
(17) Cushman, M.; Nagarathnam, D.; Gopal, D.; Chakraborti, A. K.;
Lin, C. M.; Hamel, E. Synthesis and Evaluation of Stilbene and
Dihydrostilbene Derivatives as Potential Anticancer Agents That
Inhibit Tubulin Polymerization. J . Med. Chem. 1991, 34, 2579-
2588.
(18) Cushman, M.; Nagarathnam, D.; Gopal, D.; He, H.-M.; Lin, C.
M.; Hamel, E. Synthesis and Evaluation of Analogues of (Z)-1-
(4-Methoxyphenyl)-2-(3,4,5-trimethoxyphenyl) ethene as Poten-
tial Cytotoxic and Antimitotic Agents. J . Med. Chem. 1992, 35,
2293-2306.
(19) Woods, J . A.; Hadfield, J . A.; Pettit, G. R.; Fox, B. W.; McGown,
A. T. The Interaction with Tubulin of a Series of Stilbenes Based
on Combretastatin A-4. Br. J . Cancer 1995, 71, 705-711.
(20) R-Phenylcinnamic Acid. Organic Syntheses; Wiley: New York,
1963; Collect. Vol. IV, pp 777.
(21) Suh, J . T.; Puma, B. M. A Novel Synthesis of 2,3-Disubstituted
Indoles, A Study of the Reductive Cyclization of Some 3-Sub-
stituted 2-(4,5-dimethoxy-2-nitrophenyl)acrylonitrile. J . Org.
Chem. 1965, 30, 2253.
(22) Silverstein, E. M.; Bassler, G. C.; Morrill, T. C. Spectrometric
Identification of Organic Compounds; J ohn Wiley and Sons: New
York, 1981; pp 264-265.
(23) Goldbrunner, M.; Loidl, G.; Polossek, T.; Mannschreck, A.;
Angerer, E. Inhibition of Tubulin Polymerization by 5,6-Dihy-
droindolo[2,1-a]isoquinoline Derivatives. J . Med. Chem. 1997,
40, 3524-3533.
(24) Pettit, G. R.; Singh, S. B.; Boyd, M. R.; Hamel, E.; Pettit, R. K.;
Schmidt, J . M.; Hogan, F. Antineoplastic Agents. 291. Isolation
and Synthesis of Combretastatin A-4, A-5, and A-6. J . Med.
Chem. 1995, 38, 1666-1672.
(25) Hamel, E.; Lin, C. M. Separation of Active Tubulin and
Microtubule-Associated Proteins by Ultracentrifugation and
Isolation of a Component Causing the Formation of Microtubule
Bundles. Biochemistry 1984, 23, 4173-4184.
(3) Hamel, E.; Lin, C. M. Interactions of Combretastatin, A New
Plant-Derived Antimitotic Agent, with Tubulin. Biochem. Phar-
macol. 1983, 32, 3864-3867.
(4) McGown, A. T.; Fox, B. W. Differential Cytotoxicity of Com-
bretastatins A1 and A4 in Two Daunorubicin-Resistant P388
Cell Lines. Cancer Chemother. Pharmacol. 1990, 26, 79-81.
(5) El-Zayat, A. A. E.; Degen, D.; Drabek, S.; Clark, G. M.; Pettit,
G. R.; Von Hoff, D. D. In vitro Evaluation of the Antineoplastic
Activity of Combretastatin A-4, A Natural Product from Com-
bretum caffrum (arid shrub). Anti-Cancer Drugs 1993, 4, 19-
25.
(6) Bedford, S. B.; Quarteman, C. P.; Rathbone, D. L.; Slack, J . A.
Synthesis of Water-Soluble Prodrug of the Cytotoxic Agent
Combretastatin A-4. Bioorg. Med. Chem. Lett. 1996, 6, 157-
160.
(7) Pettit, G. R.; Temple, C., J r.; Narayanan, V. L.; Varma, R.; Boyd,
M. R.; Rener, G. A.; Bansal, N. Antineoplastic Agents 322.
Synthesis of Combretastatin A-4 Prodrugs. Anti-Cancer Drug
Des. 1995, 10, 299-309.
(8) Dorr, R. T.; Dvorakova, K.; Snead, K.; Alberts, D. S.; Salmon, S.
E.; Pettit, G. R. Antitumor Activity of Combretastatin A-4
Phosphate, A Natural Product Tubulin Inhibitor. Invest. New
Drugs 1996, 14, 131-137.
(9) Shirai, R.; Tokuda, K.; Koiso, Y.; Iwasaki, S. Synthesis and Anti-
Tublulin Activity of Aza-Combretastatins. Bioorg. Med. Chem.
Lett. 1994, 4, 699-704.
(10) Cushman, M.; He, H. M.; Lin, C. M.; Hamel, E. Synthesis and
Evaluation of a Series of Benzylaniline Hydrochlorides as
Potential Cytotoxic and Antimitotic Agents Acting by Inhibition
of Tubulin Polymerization. J . Med. Chem. 1993, 36, 2817-2821.
(11) Brown, R. T.; Fox, B. W.; Hadfield, J . A.; McGown, A. T.;
Mayalarp, S. P.; Pettit, G. R.; Woods, J . A. Synthesis of Water-
Soluble Sugar Derivatives of Combretastatin A-4. J . Chem. Soc.,
Perkin Trans. 1 1995, 5, 577-581.
(12) Fulvia, O.; Francesca, P.; Barbara, B.; Giuliana, M. Synthesis
of Biologically Active Polyphenolic Glycosides (Combretastatin
and Resveratrol series). Carbohydr. Res. 1997, 301, 95-109.
(13) Lin, C. M.; Singh, S. B.; Chu, P. S.; Dempcy, R. O.; Schmidt, J .
M.; Pettit, G. R.; Hamel, E. Interactions of Tubulin with Potent
Natural and Synthetic Analogues of the Antimitotic Agent
Combretastatin: a Structure-Activity Study. Mol. Pharmacol.
1988, 34, 200-208.
(14) Pettit, G. R.; Singh, S. B.; Niven, M. L.; Hamel, E.; Schmit, J .
M. Isolation, Structure, and Synthesis of Combretastatins A-1
and B-1, Potent New Inhibitors of Microtubule Assembly,
Derived from Combretum caffrum. J . Nat. Prod. 1987, 50, 119-
131.
(15) Talvitie, A.; Mannila, E.; Kolehmainen, E. Syntheses of Some
Biologically Active Compounds from Stilbenes Isolated from the
Bark of Picea abies. Liebigs Ann. Chem. 1992, 399-401.
(26) Muzaffar, A.; Brossi, A.; Lin, C. M.; Hamel, E. Antitubulin
Effects of Derivatives of 3-Demethylthiocolchicine, Methylthio
Ethers of Natural Colchicinoids, and Thioketones Derived from
Thiocolchicine. Comparison with Colchicinoids. J . Med. Chem.
1990, 33, 567-571.
(27) Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M. L.;
Czerwinski, M. J .; Fine, D. L.; Abbott, B. J .; Mayo, J . G.;
Shoemaker, R. H.; Boyd, M. R. Feasibility of Drug Screening
with Panels of Human Tumor Cell Lines Using a Microculture
Tetrazolium Assay. Cancer Res. 1988, 48, 589-601.
(28) Mossman, T. Rapid Colorimetric Assay for Cellular Growth and
Survival: Application to Proliferation and Cytotoxicity Assays.
J . Immunol. Methods 1983, 65, 55-63.
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