Synthesis of antimitotic polyalkoxyphenyl derivatives of combretastatin using plant allylpolyalkoxybenzenes (1)

Full text of DOI:10.1021/np1004278

1796
J. Nat. Prod. 2010, 73, 1796–1802
Synthesis of Antimitotic Polyalkoxyphenyl Derivatives of Combretastatin Using Plant
Allylpolyalkoxybenzenes1
Victor V. Semenov,*,†,‡ Alex S. Kiselyov,§ Ilia Y. Titov,† Irina K. Sagamanova,† Natalie N. Ikizalp,§ Natalia B. Chernysheva,†
Dmitry V. Tsyganov,† Leonid D. Konyushkin,† Sergei I. Firgang,† Roman V. Semenov,† Irina B. Karmanova,†
Mikhail M. Raihstat,† and Marina N. Semenova‡,⊥
Zelinsky Institute of Organic Chemistry RAS, 47 Leninsky Prospect, 119991 Moscow, Russian Federation, Chemical Block Ltd., 3 Kyriacou
Matsi, 3723 Limassol, Cyprus, CHDI Foundation, 6080 Center DriVe, Suite 100, Los Angeles, California 90045, United States, and Institute of
DeVelopmental Biology RAS, 26 VaViloV Street, 119334 Moscow, Russian Federation
ReceiVed June 28, 2010
Combretastatins A-1, A-2, and A-4 are natural antimitotic
compounds that interact with the colchicine binding site of tubulin.
A significant effort has been dedicated to the discovery of their
synthetic derivatives featuring improved safety-efficacy profiles.
In searching for a reliable natural source of building blocks for the
synthesis of novel combretastatin analogues, the Apiaceae family
of plants including dill and parsley species were of particular interst
since they produce diverse allylpolyalkoxybenzenes. These starting
materials were converted initially to isoapiol (2a), isodillapiol (2b),
isoallyltetramethoxybenzene (2c), and isomyristicin (2d). Com-
pounds 2a-d were used to access the corresponding aldehydes
(3a-d). The sequence included a key ozonolysis step conducted
in a CHCl3-CH3OH-pyridine system at low temperature (-15 to
0 °C) with controlled addition of O3. This procedure worked well
on a preparative scale (ca. 100 g). Subsequent synthetic steps
included either Wittig reaction of the resulting aldehydes or their
conversion to carboxylic acid intermediates to yield a series of novel
polyalkoxyaryl derivatives of combretastatins including both olefinic
and the respective ortho-substituted five-membered aza-heterocyclic
analogues. Compounds 5Z, 6Z, 7Z, 8Z, 9Z, 10Z, and 11Z exhibited
high antiproliferative effects in a sea urchin embryo assay.
Combretastatins A-1, A-2, and A-4 (Figure 1; CA1, CA2, CA4)
are natural antimitotic compounds isolated initially from the bark
of Combretum caffrum Kuntze (Combretaceae) in 1982.2 These
molecules are tubulin polymerization inhibitors that interact at the
colchicine binding site.3,4 The phosphorylated prodrugs CA4
disodium phosphate (CA4P, Zybrestat) and combretastatin A-1
phosphate (Oxi4503) are currently undergoing clinical evaluation
as antitumor vascular targeting agents.5,6 However, after CA4P
administration, side effects have been observed, particularly regard-
ing the cardiovascular system.7 A significant effort has been
dedicated to the discovery of synthetic combretastatins featuring
potenciessimilartotheparentmoleculesbutwithbettersafety-efficacy
profiles.8,9 Combretastatin analogues display homology with the
Figure 1
A and C rings of colchicine and are generally described as a tilted
biaryl system connected by a hydrocarbon linker (bridge) of variable
length (i.e., the A and B rings of combretastatins, Figure 1). The
bridge furnishes a cis-configuration of the biaryl template necessary
Examples of synthetic modifications of ring B with diverse
pharmacophores (e.g., OH, NH2, F) are quite abundant in the
literature.8,9 At the same time, structure-activity relationship (SAR)
for efficient interaction of a molecule with the colchicine binding
site of tubulin.10,11 Notably, five-membered heterocycles were
studies of substituents on ring A have been conducted to a lesser
extent, although several resultant derivatives were reported to be
reported to provide a nonisomerizable and metabolically stable
potent tubulin binders. This is presumably due to the lack of the
necessary building blocks substituted with more than three alkoxy
groups. In a representative example, a CA4 analogue modified with
the ortho-NH2 group in ring A exhibited a comparable activity to
the parent molecule.14 It was assumed that similar modification of
ring A with an additional OCH3 functionality could yield potent
antimitotic agents. Moreover, the tetraalkoxybenzene pharmacoph-
ore is featured in several natural products with reported antipro-
liferative activity, including Cactaceae tetrahydroisoquinolines15 and
flavonoids and isoflavonoids.16
isosteric replacement for the cis-styrene. In particular, 1,2-
substituted isoxazolines, oxadiazolines, and isoxazole derivatives
(I) were reported to be more active than the respective 3,5-
substituted analogues (II) (Figure 1).12,13
* To whom correspondence should be addressed. Tel: +7 (916) 620-
9584. Fax: +7 (499) 137-2966. E-mail: vs@zelinsky.ru.
† Zelinsky Institute of Organic Chemistry RAS.
‡ Chemical Block Ltd., Limassol, Cyprus.
§ CHDI Foundation, Los Angeles.
⊥ Institute of Developmental Biology RAS.
10.1021/np1004278  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 11/04/2010

Antimitotic Polyalkoxyphenyl DeriVatiVes
Journal of Natural Products, 2010, Vol. 73, No. 11 1797
Scheme 1. Synthesis of Combretastatin Analogues from Allylpolyalkoxybenzenesa
a Reagents and conditions: (i) KOH, 100 °C, 40 min; (ii) O3, CHCl3-MeOH-pyridine (80:20:3 v/v), -15 °C, 1-2 h; (iii) n-BuLi, rt, 10 h; (iv) TBAF ·3H2O, THF,
rt, 1 h. WR ) Wittig reagent.
In searching for a reliable natural source of building blocks for
combretastatin analogues, the Apiaceae family of plants became
of interest. These species have been reported to contain significant
amounts of pharmacologically active allyl polyalkoxybenzenes,
namely, apiol (1a), dillapiol (1b), allyltetramethoxybenzene (1c),
myristicin (1d), and elemicin (1e) (Figure 1).17 The distribution of
allylalkoxybenzenes varies greatly between species, plant organs,
and geographic regions of collection.18
and isomyristicin (2d) (Scheme 1). The resulting molecules were
evaluated in vivo using a sea urchin embryo assay.23
Results and Discussion
In the initial approach toward the respective aldehydes of apiol,
dillapiol, tetramethoxybenzene, and myristicin (3a-d, Scheme 1),
a published procedure describing ozonolysis of the respective
styrene precursors in acetic acid was followed.24 However, yields
of the targeted molecules did not exceed 20-40% versus the
reported 75-80% values. In order to improve both purity and yields
of the targeted molecules 3a-d, the literature protocol was
amended. Accordingly, the reaction was conducted in the solvent
system CHCl3-CH3OH-pyridine (4:1:1) at low temperatures (-15
to 0 °C). Addition of pyridine was found to be critical to the reaction
outcome, and presumably this facilitates conversion of the inter-
mediate molozonide into aldehydes 3a-d. In addition, excess of
O3 led to significant side reactions. These included oxidation of
the aldehyde group into the respective carboxylic acid, Bayer-Villiger
rearrangement to the corresponding phenols, and their further
oxidation to afford a biaryl system. In order to carefully control
the amount of O3, the ozonolysis step was conducted using a
custom-designed apparatus (Science and Technology Park, St.
Petersburg State Polytechnic University, Russia), equipped with an
IR detector, allowing for the accurate allotment of the reagent. The
targeted polyalkoxybenzoic acids (12a,b,d) were synthesized in high
yields(80-90%)fromthealdehydeprecursorsusingtheurea-hydrogen
peroxide complex in water. Polyalkoxybenzonitriles 24a,b,d were
prepared in >90% yields via oxidation of aldehydes by molecular
iodine (molar ratio 1:1.1) in aqueous NH4OH, as described
recently.25 A combination of the specialized equipment and
optimized protocols described above allowed for preparation of the
targeted aldehydes in yields of 60-85% on a 100 g scale (Scheme
1, step ii).
It was discovered that seeds of parsley, Petroselinum satiVum
Hoffm., cultivated in Russia, and dill, Anethum graVeolens L.,
grown in India, are both versatile sources of allylpolyoxybenzenes
that can be isolated by supercritical CO2 extraction followed by
high-efficiencylarge-scaledistillation(upto40kgofallylbenzenes).1a,19
Multiple SAR studies of combretastatin analogues converged on
the 3-hydroxy-4-methoxyphenyl group as the optimal substituent
for the ring B (e.g., CA2, CA4, Figure 1).8,9 This moiety was
incorporated into test molecules while varying the nature of both
ring A and the bridge. Compounds featuring a 1,3,4-oxadiazole
linker displayed good steric overlap with the isovanillin moiety of
combretastatins as reported earlier.20 Introduction of 3,5-disubsti-
tuted 1,2,4-oxadiazole21 and 2,5-diaryl-2,3-dihydro-1,3,4-oxadia-
zoline13 linkers into combretastatin derivatives yielded agents
selective against specific cancer cell lines. Analogues featuring the
triazole core moiety also displayed potent inhibition of tubulin
polymerization and cytotoxicity against cancer cell lines including
multi-drug-resistant cells.22 Considering evidence from the earlier
in-house studies as well as these literature data, a short synthetic
protocol was developed to access a series of new polyalkoxyaryl
derivatives of combretastatins. These molecules included the
respective ortho-substituted five-membered analogues that feature
the relevant spatial orientations of rings A and B. The starting
materials used for the synthesis of combretastatin analogues were
isoapiol (2a), isodillapiol (2b), isoallyltetramethoxybenzene (2c),

1798 Journal of Natural Products, 2010, Vol. 73, No. 11
SemenoV et al.
Scheme 2. Synthesis of Heterocyclic Combretastatin Analogues from Allylpolyalkoxybenzenesa
a Reagents and conditions: (i) CO(NH2)2 · H2O2, CH3OH, reflux, 1.5 h; (ii) (1) SOCl2, MeOH, reflux, 3 h; (2) 100% NH2NH2 · H2O, MeOH, reflux, 6 h; (iii)
MeOH-MeONa, reflux, 10 h; (iv) ArCOCl, pyr, rt, 12 h; (v) I2-NH4OH(28%)-H2O, rt, 16 h; (vi) NH2OH·H2O, EtOH, reflux, 12 h; (vii) 3-OH, 4-OCH3-C6H3COOH,
CDI, DMF, 120-125 °C, 3 h; (viii) (1) SOCl2, C6H6, 48-50 °C, 2.5 h; (2) KCN, CH3CN, 50 °C, 10 h. Compounds 14, 18, 21-23, and 29-31 were synthesized from
the corresponding commercial aldehydes, acids, and nitriles using the same procedures.
Polymethoxy analogues of combretastatin were synthesized
conveniently via Wittig reaction from aldehydes 3a-d (Scheme
1), as reported for the synthesis of CA4.26 n-BuLi was a base of
choice to ensure the preferential formation of a cis-alkene at -78
°C.27 Combined yields of the silylated derivatives 4a-g and 4′a
obtained as a mixture of Z-/E-isomers were in the 70-85% range.
These compounds were separated by column chromatography and
subsequently converted to the targeted molecules 5Z, 5′Z, 6-11Z
and 5E, 5′E, 6-11E (Scheme 2) with TBAF ·3H2O in THF
(85-90% yields). Notably, attempts to separate E- and Z-isomers
as their respective phenols were unsuccessful under a variety of
conditions. Chemical identity of the individual isomers was
carboxylic acid (12) or nitrile (24) precursors (Scheme 2). Synthetic
reactions leading to the 1,3,4-triazole (14-17)28 and 1,3,4-
oxadiazole (18-23)29 derivatives involved preparation of the
intermediate hydrazides (13). The respective 1,2,4-oxadiazole
molecules 26-28, 30, and 31 were accessed from 24 via this
modified procedure.21 A similar heterocyclization step was followed
in the synthesis of 29 (Scheme 2).
Synthetic derivatives of combretastatin synthesized as described
above (Schemes 1 and 2) were further evaluated for their antipro-
liferative and tubulin-destabilizing activity using a phenotypic sea
urchin embryo assay.23 This in vivo assay allows for the robust
and reliable identification of compounds targeting tubulin including
their antiproliferative, antimitotic, and cytotoxic effects. It features
(i) a fertilized egg test for antimitotic activity as displayed by
cleavage alteration/arrest and (ii) behavioral monitoring of a free-
swimming blastula treated immediately after hatching (9-12 h after
fertilization). Lack of forward movement, settlement to the bottom
of the culture vessel, and rapid spinning around the animal-vegetal
axis of the embryo suggest a tubulin-destabilizing effect caused
by a molecule.30 The results are presented in Table 1 (see also
Table S1, Supporting Information). Notably, the starting materials
inclusive of apiol, dillapiol,31 and myristicin on their own had no
effect on sea urchin embryo cleavage up to 80-100 µM. Isoapiol
1
confirmed unequivocally by H NMR spectroscopy. Specifically,
J values for each Z/E pair were ca. 12 and 16 Hz, respectively. A
detailed analysis of the NMR data was conducted for the Z-/E-
isomers of myristicin derivatives 4d and 8 (Figures S1 and S2,
Supporting Information). For the pair 4e and 9, chemical shifts for
the Z- and E-isomers overlapped. In order to assign chemical
structures to the individual compounds, we used the respective
vicinal constants of a strong-field 13C satellite (Figure S3, Sup-
porting Information).
The respective five-membered aza-heterocyclic analogues of
combretastatins were conveniently prepared from the respective

Antimitotic Polyalkoxyphenyl DeriVatiVes
Journal of Natural Products, 2010, Vol. 73, No. 11 1799
Table 1. Effects of Polyalkoxybenzene Analogues of
Combretastatins on Sea Urchin Embryos
bulin activity of a compound, while a methylenedioxy group
decreases such activity.3,11,33 Compounds CA4 and CA2 (8Z)
showed similar effects in the sea urchin embryo test. Azole
derivatives featuring a myristicin moiety (17, 19, 20, and 28) were
consistently more potent than other derivatives within the same
structural class.
EC, µMa
yield,
%
cleavage
alteration
cleavage
arrest
embryo
spinning
compound
2a
2b
mp, °C
56.5b
2
20
>5
8
80
>5
0.01
0.01
>11.2
0.1
4
>4
0.2
4
0.1
1
0.01
0.1
0.01
0.1
0.2
2
>10
>100
>5
1
0.05
>11.2
1
44c
Numerous studies have confirmed that the cis-orientation of the
aromatic substituents in combretastatin analogues is important for
their antimitotic activity. The corresponding trans-isomers were
reported to be significantly less potent or inactive.3,8-11,32,35 In
agreement with these observations, cis-stilbenes were consistently
more active than their corresponding trans-isomers (Table 1;
compare Z-/E-isomers in 5-11). Notably, the trans-isomer of CA2
(8E) still displayed good potency in the sea urchin embryo assay.
This was comparable with the reported cytotoxicity of 8E against
murine leukemia cells and in vitro inhibition of tubulin polymer-
ization.3 For Z-isomers 5-8 the antimitotic activity decreased in
the following order: CA4 ) myristicin derivative CA2 (8Z) >
dillapiol derivative (6Z) > apiol derivative (5Z) ) tetramethoxy-
benzene derivative (7Z). Replacement of the methylenedioxy moiety
in 8Z with an ethylenedioxy group (9Z) did not have any significant
effect on the compound’s potency. A comparative study of
ethylenedioxy derivatives 9-11 further confirmed the importance
of the 5-methoxy substituent in ring A, as both the respective
6-methoxy (10Z and 10E) and unsubstituted (11Z and 11E)
analogues were less active. A substitution of the p-OMe group with
a p-OH (Table 1, 5′E) moiety dramatically reduced the antiprolif-
erative effect of the resulting molecule.3,10 Nitro and amino
derivatives of combretastatins endowed with the p-OH group
completely lacked cytotoxicity and tubulin-depolymerizing activ-
ity.36 In the series reported, a related replacement also yielded
inactive molecule 5′E (compared to 5E), the analogue of isocom-
bretastatin A-4 derived from vanillin.37 A carboxylic group at the
olefin bridge was not tolerated (Figure 1, Table 1), as in CA4COOH,
synthesized from 3,4,5-trimethoxyphenylacetic acid and isovanil-
lin.38 A similar observation was made for the CA4 analogue,
whereby introduction of the COOH group into the bridge dramati-
cally decreased the cytotoxicity of the resulting compound.35
2d
42.5c
CA4Pd
CA4e
0.005
0.002
>11.2
0.02
0.5
>4
0.01
0.1
0.02
0.1
0.002
0.02
0.002
0.02
0.02
0.1
0.02
0.1
4
>4
4
2
>4
0.5
0.5
0.5
0.05
1
116e
CA4COOHg 237-239g
5Z
23-26
99
63
66
79
96
98
99
92
62
91
98
99
99
80
96
64
48
26
67
18
12
78
76
41
40
18
29
32
12
39
18
5E
5′E
6Z
6E
7Z
7E
8Z, CA2
8E
9Z
9E
10Z
10E
11Z
11E
14
140-145
145-147
oil
5
>5
0.5
5
1
5
0.05
1
0.05
2.00
1
88-90
80-82
97-101
oil
137-141
82-85
96-98
78-83
87-93
>5
1
2
oil
0.5
1
97-99
125
>4
>4
>4
>4
>4
2
2
10
4
>4
>4
>4
>4
>4
>4
>4
>4
>4
>4
>5
>4
>5
>5
5
15
16
17
18
192-194
218-220
192-195
222-225
235-238
175-178
176-178
155-157
153-155
164-168
168-172
180-183
168-170
178-180
143-146
19
20
21h
22
2
23
26
27
28
29
30
31
>5
>4
>4
>4
>4
>4
>5
>4
>4
4
4
>4
>4
a The sea urchin embryo assay was conducted as described in ref 23.
Duplicate measurements showed no differences in effective threshold
concentration (EC) values. b Ref 40. c Ref 39. d Obtained from
OXiGENE. e Synthesized according to ref 26c; mp value ref 26c.
g Synthesized from 3,4,5-trimethoxyphenylacetic acid and isovanillin
according to ref 38. h Synthesized according to ref 49.
A replacement of the olefin bridge with respective aromatic
isosteres including the 1,3,4-triazoles 14-17, 1,3,4-oxadiazoles
18-23, and 1,2,4-oxadiazoles 26-31 caused a dramatic drop in
antimitotic activity of the respective derivatives, with the 1,3,4-
oxadiazole derivatives 19-22 being the only notable exceptions.
These molecules did affect embryo cleavage alteration. However
their effect on either cleavage arrest or spinning was modest at
best. Agents 28 and 29 yielded cleavage alterations only at the
maximal tested concentration (4 µM). Compound 27 applied after
hatching at a concentration of 2-4 µM caused developmental
abnormalities; specifically it inhibited growth of the skeletal
spiculae. A CA2 analogue (19) and its respective derivative (20),
featuring three methoxy groups in the ring B, were determined to
be ca. 250 times less potent than CA2. Unfortunately, poor solubility
of these compounds in DMSO and/or seawater precluded the
accurate testing of their potency at higher concentrations. Molecule
23 showed weak antimitotic activity with an EC of 1 µM. Agents
15, 18, 26, 27, 30, and 31 failed to alter embryo development at
the maximal tested concentration (4 µM).
In conclusion, a variety of ring A moieties derived from apiol
(5), dillapiol (6), tetramethoxybenzene (7), and myristicin (8),
including the 5-methoxy-2,3-ethylenedioxy (9), 6-methoxy-2,3-
ethylenedioxy (10), and 2,3-ethylenedioxy (11) analogues, yielded
active compounds. A typical range of antiproliferative potencies
for these molecules in the sea urchin embryo assay was 0.002-0.1
µM (Table 1; 5-11). The difference in threshold concentrations
varied from 5-25 times for cleavage alteration to 10-40 times
for cleavage arrest. The most active compounds, 8Z (CA2) and
(2a) and isodillapiol (2b) displayed non-tubulin antiproliferative
activity, whereas isomyristicin (2c) was inactive up to 5 µM. Tests
of 2c at higher concentrations were not successful due to its low
solubility.
It has been reported that CA4 is considerably more cytotoxic
and more effective as a tubulin polymerization inhibitor than
CA2.3,26c,32 According to the literature data, CA4 and CA4P
showed similar cytotoxicity against a panel of cancer cell lines.5,11
CA2 exhibited a more pronounced cytotoxic effect than its
phosphorylated derivative CA2P.33 In the sea urchin embryo assay,
CA4P was somewhat less effective than CA4 (Table 1). This effect
could be attributed to a time-dependent conversion of prodrug CA4P
into active molecule CA4 mediated by intracellular phosphatases.
As evidenced from Table 1, compounds 5-9, 10Z, 11, 21, and
22 displayed significant cleavage alteration, arrest, and embryo
spinning, suggesting their tubulin-destabilizing activity. It was
assumed that the less potent compounds 10E, 19, and 20 were
tubulin destabilizers as well, due to the tuberculose shape of the
arrested eggs in the assay, although these molecules did not induce
embryo spinning.20,23,34 Agents 10E, 19, and 20 were not tested
at concentrations of >5 µM due to their limited solubility in DMSO
and/or seawater.
Literature data suggest that the presence of three methoxy
substituents in the A ring of combretastatins promotes the antitu-

1800 Journal of Natural Products, 2010, Vol. 73, No. 11
SemenoV et al.
concentrated in vacuo, and separated using column chromatography
(silica gel, mesh 5/40, 100 g), eluted with hexane-EtOAc mixtures
(9:1 to 4:1 gradient), affording 70-85% yields.
9Z, contained 5-methoxymethylenedioxy and 5-methoxyethylene-
dioxy substituents, respectively.
General Procedure 4 for the Synthesis of Combretastatin
Analogues 5-11. A solution of TBAF ·3H2O (0.525 mmol) in 1 mL
of dry THF was added to the solution of a stilbene (4a-g) (0.35 mmol)
in 2 mL of the same solvent, and the reaction mixture was stirred for
1 h at rt, treated with 20 mL of H2O, and extracted with 2 × 40 mL of
Et2O. The organic extracts were combined, washed with 2 × 20 mL
of H2O, dried over Na2SO4, and concentrated in vacuo to afford the
desired compounds in 85-99% yields.
Experimental Section
General Experimental Procedures. NMR spectra were collected
on a Bruker DR-500 instrument [working frequencies of 500.13 MHz
(1H) and 75.47 (13C)]. Mass spectra were obtained on a Finnigan MAT/
INCOS 50 instrument (70 eV) using direct probe injection. Elemental
analysis was accomplished with the automated Perkin-Elmer 2400 CHN
microanalyzer. Ozonolysis was conducted using a custom-designed
apparatus (Science and Technology Park, St. Petersburg State Poly-
technic University, Russia) equipped with an IR detector of O3
concentration (Japan) and an automated shut-down circuit. The device
allowed for the controlled generation of ozone, with a maximal capacity
of 10 g of O3 per hour from O2.
Apiol Acid (12a). Aqueous NaOH (1.6 mL of 6 M solution) was
added to a stirred solution of the urea-hydrogen peroxide complex
(1:1) (6 g, 63.4 mmol) and aldehyde 3a (0.9 g, 4.28 mmol) in 20 mL
of CH3OH at rt. The reaction mixture was stirred at reflux for 1 h
followed by the addition of the urea-hydrogen peroxide complex (1.5
g, 15.85 mmol) and reflux for 30 min. The reaction mixture was brought
to rt, and the pH was adjusted to 3 with 18% aqueous HCl. The
precipitate was filtered, washed with 2 × 50 mL of ice water, and dried
to afford 12a (0.85 g, 3.76 mmol, 88% yield) as an off-white solid:
mp 174 °C (lit.39 mp 175.5 °C).
Isolation of Plant Allylpolyalkoxybenzenes. 1a Liquid CO2 extrac-
tion of parsley and dill seeds was carried out earlier by Company
Karavan Ltd. (Krasnodar, Russia). Allylpolymethoxybenzenes 1a-d
with 98-99% purity were obtained by high-efficiency distillation using
a pilot plant device at N.D. Zelinsky Institute of Organic Chemistry
RAS (Moscow, Russia). The seed essential oils of parsley varieties
cultivated in Russia contained 70-75% of 1a (var. Sakharnaya), 21%
of 1c (var. Slavyanovskaya), and 40-46% of 1d (var. Astra). Indian
dill seeds were purchased from Vremya & Co. (St. Petersburg, Russia).
The dill seed essential oil contained 30-33% of 1b.
General Procedure 1 for the Synthesis of Styrenes 2a-d. These
were prepared according to a published procedure.1a,39,40 A mixture
of an allylbenzene (1a-d) (0.25 mol), freshly recrystallized tetrabu-
tylammonium bromide (2.5 g, 0.0075 mol), and powdered KOH (10
g, 0.18 mol) was heated for 40 min on a water bath. The reaction
mixture was cooled to rt and extracted with Et2O (2 × 250 mL), and
the combined organic extracts were washed with H2O (2 × 150 mL),
dried over Na2SO4, and concentrated in vacuo. The solid residue was
recrystallized from petroleum ether to furnish 85-90% overall yields
of targeted molecules as a mixture of trans- (ca. 80-85%) and cis-
isomers (15-20%).
Dillapiol Acid (12b). This was prepared as described for 12a (1.83
g, 8.1 mmol, 87% yield): off-white solid; mp 151 °C (lit.41 mp 151-152
°C).
Myristicin Acid (12d). This was prepared as described for 12a.
Aqueous NaOH (1.7 mL of 6M) was added dropwise to a stirred
solution of the urea-hydrogen peroxide complex (1:1) (6 g) and
myristicin aldehyde (0.9 g, 5 mmol) in 20 mL of MeOH at rt. The
resulting mixture was stirred at 66 °C for 1 h, cooled, treated with
1.5 g of the urea-hydrogen peroxide complex (1:1), and reheated at
66 °C for an additional 30 min. The pH of the reaction mixture was
adjusted to 3 with 18% HCl, and the resulting precipitate was collected,
washed with ice water (2 × 50 mL), and dried to afford 12d (0.8 g,
4.07 mmol, 81.6% yield) as an off-white solid: mp 212 °C (lit.39 mp
211 °C).
General Procedure 5 for the Synthesis of Polyalkoxybenzoic Acid
Methyl Esters. A mixture of acid 12 (5 mmol) and SOCl2 (6 mmol)
in 10 mL of MeOH was refluxed for 3 h and concentrated in vacuo,
and 100 mL of CH2Cl2 added. The organic extract was treated with
saturated aqueous NaHCO3 (5 mL) to neutral pH and water (3 × 5
mL), dried over Na2SO4, and concentrated in vacuo to yield the desired
methyl esters.
General Procedure 6 for the Synthesis of Polyalkoxybenzoic Acid
Hydrazides (13a,b,d). These were prepared as described in the
literature.44 A mixture of the methyl ester of 12 (5 mmol) and 100%
NH2NH2 ·H2O (25 mmol) in 5 mL of MeOH was refluxed for 6 h,
concentrated in vacuo, with the residue treated with ice water (5 mL),
and filtered. The filtrate was washed with 3 × 2 mL of ice water and
dried to afford the desired hydrazide.
3-Hydroxy-4-methoxybenzenecarboximidoyl Chloride. This was
synthesized according to the published procedure.45 Dry HCl was
bubbled through a suspension of 3-hydroxy-4-methoxybenzonitrile (1.86
g, 12.5 mmol) and absolute MeOH (0.58 g, 0.73 mL, 18 mmol) in 20
mL of dry Et2O at 0 °C for 10 min. The reaction mixture was sealed
and stirred for 6 days at +5 °C and for an additional 10 h at 0 to +4
°C and triturated with Et2O. The solid residue was washed with 3 × 5
mL of dry Et2O and dried in vacuo at 2 mmHg over P2O5 to furnish
the imidoyl chloride (2.585 g, 13.9 mmol, 95% yield) as a greenish
powder. This was used in the next step without further purification.
General Procedure 7 for the Synthesis of 3,5-Diaryl-1,2,4-
triazoles (14-17). A mixture of hydrazide 13 (2 mmol) and 3-hydroxy-
4-methoxybenzenecarboximidoyl chloride (3 mmol) in a flame-dried
flask was treated with 3 mL of absolute MeOH and MeONa (3 mL of
a 1 M solution in absolute MeOH) and stirred for 10-15 h at 45 °C
(monitoring the disappearance of the starting hydrazide by TLC),
refluxed for 10 h, treated with AcOH to neutral pH, and concentrated.
The residue was redissolved in MeOH, treated with silica gel (3 g),
and concentrated in vacuo at rt, followed by chromatographic purifica-
tion (silica gel, 35 g, mesh 5/40) with a benzene-EtOAc gradient (9:1
to 1:1).
General Procedure 2 for the Synthesis of Aldehydes 3a-d. O3
(6.24 g, 0.13 mol) was bubbled through a styrene (2a-d) (0.1 mol) in
a mixture of CHCl3-MeOH-pyridine (240:60:9 mL) for 1-2 h at
-15 °C. The resulting solution was kept for an additional 3 h at 0 °C
and concentrated in vacuo at 20 °C. The residue was treated with 150
mL of H2O, and the pH of the slurry was adjusted to ca. 3 with
concentrated HCl. The resulting solid was filtered, washed with 3 ×
70 mL of H2O, and dried to afford the desired aldehyde (60-80%).
2,5-Dimethoxy-3,4-methylenedioxybenzaldehyde (Apiol Alde-
hyde, 3a). Reaction of 175 g (787 mmol) of crude isoapiol 2a furnished
3a (111 g, 529 mmol, 67% yield) as an off-white solid: mp 102 °C
(lit.41 102 °C); H NMR (CDCl3, 500 MHz) δ 10.12 (1H, s, CHO),
1
7.00 (1H, s, H-6), 6.20 (2H, s, OCH2O), 3.99 (3H, s, OCH3), 3.83 (3H,
s, OCH3).
2,3-Dimethoxy-4,5-methylenedioxybenzaldehyde (Dillapiol Al-
dehyde, 3b): 16.8 g, 79.9 mmol, 80% yield; off-white solid; mp 75-77
°C (lit.41 75 °C); H NMR (CDCl3, 500 MHz) δ 10.08 (1H, s, CHO),
1
6.85 (1H, s, H-6), 6.13 (2H, s, OCH2O), 3.98 (3H, s, OCH3), 3.87 (3H,
s, OCH3).
2,3,4,5-Tetramethoxybenzaldehyde (3c): 66.8 g, 295 mmol, 70%
yield; off-white solid; bp 124-129/1-2 mmHg, mp 35-36 °C (EtOH)
(lit.42 37-39 °C) (lit.43 115-125 °C/2 mmHg); H NMR (DMSO-d6,
1
500 MHz) δ 10.19 (1H, s, CHO), 7.03 (1H, s, H-6), 3.90 (3H, s, OCH3),
3.88 (3H, s, OCH3), 3.86 (3H, s, OCH3), 3.81 (3H, s, OCH3).
3-Methoxy-4,5-methylenedioxybenzaldehyde (Myristicin Alde-
hyde) (3d): 11 g, 61.06 mmol, 61% yield; off-white solid; mp 133-135
°C (lit.39 132.5 °C); 1H NMR (CDCl3, 500 MHz) δ 9.78 (1H, s, CHO),
7.26 (1H, s, H-Ph), 7.05 (1H, s, H-Ph), 6.12 (2H, s, OCH2O), 3.95
(3H, s, OCH3-3).
General Procedure 3 for the Synthesis of Combretastatin
OTBDMS Analogues 4a-g and 4′a. These were synthesized according
to a published procedure.26 A solution of WR (2.136 g, 3.6 mmol) in
15 mL of dry THF at -78 °C under Ar was treated with n-BuLi (3.78
mmol, 1.5 mL of a 2.5 M solution in hexane). The mixture was stirred
for 30 min, and an aldehyde (3a-g) (3.0 mmol) in 7 mL of dry THF
was added. The reaction mixture was stirred for 10 h at ambient
temperature, treated with 20 mL of H2O, and extracted with 2 × 50
mL of EtOAc. The organic layers were combined, dried over Na2SO4,
General Procedure 8 for the Synthesis of 2,5-Diaryl-1,3,4-
oxadiazoles (18-23). A solution of hydrazide 13 (2 mmol) in 4 mL
of dry pyridine at -20 °C was treated with a solution of aroyl chloride
(prepared in situ by refluxing 2.2 mmol of acid and 1 mL of SOCl2 for

Antimitotic Polyalkoxyphenyl DeriVatiVes
Journal of Natural Products, 2010, Vol. 73, No. 11 1801
2 h) in 1.5-2 mL of dry CH2Cl2. The resulting mixture was stirred for
12 h at rt and concentrated in vacuo. The residue was treated with 2 ×
10 mL of H2O and filtered. The filtrate was washed with H2O followed
by 5% aqueous NH3 and 10% aqueous HCl to adjust the pH to neutral
and dried to afford the targeted diacylhydrazines as grayish crystals in
81-92% yield. The resulting diacylhydrazine was used in the next
step29 without further purification. Specifically, a suspension of
diacylhydrazine in 15 mL of dry CH2Cl2 was treated with TsCl (2.2
mmol) and Et3N (2.5 mmol); the resulting mixture was strirred overnight
at rt and concentrated in vacuo. The residue was treated with water,
and the solid was collected, washed with H2O followed by 5% aqueous
NH3 and 10% aqueous HCl, and dried. The resulting product was
deacetylated by treating the crystals with a mixture of Et3N (0.5 mL),
H2O (0.5 mL), and MeOH (2 mL) for 24 h at rt. The reaction mixture
was concentrated in vacuo, and the solid residue obtained was treated
with H2O followed by 5% of aqueous NH3 and 10% aqueous HCl, and
dried. It was then redissolved in EtOAc and purified by flash
chromatography (silica gel, 36 g, mesh 5/40), using a benzene-EtOAc
gradient (19:1 to 1:1) as solvent.
(5), 148 (11); anal. C 59.64, H 4.97, N 6.25%, calcd for C11H11NO4,
C 59.73, H 5.01, N 6.33%.
General Procedure 10 for the Synthesis of Myristicin, Apiol, and
Dillapiol Amidoximes (25a,b,d). Aqueous NH2OH·H2O (5 mL, 20
mmol) was added to a solution of nitrile 24 (10 mmol) in 20 mL of
EtOH. The reaction mixture was brought to reflux, treated portionwise
with solid NaHCO3 (20 mmol), refluxed for 12 h, and concentrated in
vacuo. The residue was recrystallized from EtOH to furnish the targeted
amidoximes (65-75% yields, 85-90% purity), which were used for
the next step without further purification.
General Procedure 11 for the Synthesis of 3,5-Diaryl-1,2,4-
oxadiazoles (26-31). These were synthesized by a modified proce-
dure.21 Solid CDI (3.6 mmol) was added to a stirred suspension of
crude amidoxime 25 (3 mmol) in 5 mL of dry MeCN. The resulting
mixture was stirred for 1 h at rt until the amidoxime was dissolved
completely. 3-Hydroxy-4-methoxybenzoic acid (3 mmol) was added
at once, and the mixture was stirred for 12 h and concentrated in vacuo.
The solid residue was redissolved in 5 mL of dry DMF, with the
resulting solution stirred for 3 h at 120-125 °C. Solvent was removed
in vacuo and the residue was purified by column chromatography (silica
gel, EtOAc-petroleum ether, 1:4).
Biological Evaluation Using a Sea Urchin Embryo Assay. Adult
sea urchins, Paracentrotus liVidus L. (Echinidae), were collected from
the Mediterranean Sea on the Cyprus coast in March-May and
October-December, 2009, and kept in an aerated seawater tank.
Gametes were obtained by intracoelomic injection of 0.5 M KCl. Eggs
were washed with filtered seawater and fertilized by adding drops of
diluted sperm. Embryos were cultured at room temperature under gentle
agitation with a motor-driven plastic paddle (60 rpm) in filtered
seawater. The embryos were observed with a Biolam light microscope
(LOMO, St. Petersburg, Russia). For treatment with the test compounds,
5 mL aliquots of embryo suspension were transferred to six-well plates
and incubated as a monolayer at a concentration up to 2000 embryos/
mL. Stock solutions of compounds were prepared either in 95% EtOH
at 5 mM or in DMSO at 5-20 mM concentrations followed by a 10-
fold dilution with 95% EtOH. This procedure enhanced solubility of
the test compounds in the salt-containing medium (seawater), as
evidenced by microscopic examination of the samples. The maximal
tolerated concentrations of DMSO and EtOH in the in vivo assay were
determined to be 0.05% and 1%, respectively. Higher concentrations
of either DMSO (g0.1%) or EtOH (>1%) caused nonspecific alteration
and retardation of the sea urchin embryo development independent of
the treatment stage. CA4P (OXiGENE) was prepared as 5 mM in
distilled water. The antiproliferative activity was assessed by exposing
fertilized eggs (10-25 min after fertilization, 45-60 min before the
first mitotic cycle completion) to 2-fold decreasing concentrations of
the compound. For the Z-/E-isomers, compound stock solutions and
treated embryo samples were protected from light. Cleavage alteration
and arrest were clearly detected 2.5-6 h after fertilization. The effects
were estimated quantitatively as an effective threshold concentration,
EC, resulting in cleavage alteration and embryo death before hatching
or full mitotic arrest. For tubulin-destabilizing activity, the compounds
were tested on free-swimming blastulae just after hatching (9-12 h
after fertilization), which originated from the same embryo culture.
Embryo spinning was observed after 0.5-20 h of treatment, depending
on the nature and concentration of the compound. Both spinning and
lack of forward movement were interpreted to be the result of the
tubulin-destabilizing activity of a molecule according to previous
studies.23,30
General Procedure 9 for the Synthesis of Polyalkoxybenzoic Acid
Nitriles (24a,b,d). A literature procedure was followed.25 A solution
of aldehyde 3 (42.04 g, 0.2 M) in THF (300 mL) was treated with I2
(56.85 g, 0.224 mol) in 28% aqueous NH4OH (1800 mL). The reaction
mixture was stirred overnight and extracted with CHCl3 (3 × 300 mL),
and the combined organic layers were washed with water (300 mL),
10% aqueous Na2S2O3 (300 mL), and brine (200 mL), dried over
Na2SO4, and concentrated in vacuo. The resulting residue was recrystal-
lized from EtOH to afford the respective nitriles in 65-90% yields.
2,5-Dimethoxy-3,4-methylenedioxybenzonitrile (24a): 3.7 g, 17.86
mmol, 89% yield; off-white solid; mp 134-135 °C (lit.46 135.5 °C);
1H NMR (CDCl3, 500 MHz) δ 6.71 (1H, s, H-6), 6.07 (2H, s, OCH2O),
4.07 (3H, s, OCH3), 3.86 (3H, s, OCH3); EIMS m/z 207 [M]+ (100),
192 (95), 162 (17), 134 (20); anal. C 58.06, H 4.47, N 6.69%, calcd
for C10H9NO4, C 57.97, H 4.38, N 6.76%.
2,3-Dimethoxy-4,5-methylenedioxybenzonitrile (24b): 24.2 g,
116.8 mmol, 91% yield; off-white solid; mp 92-94 °C; 1H NMR
(CDCl3, 500 MHz) δ 6.64 (1H, s, H-6), 6.03 (2H, s, OCH2O), 4.03
(3H, s, OCH3), 3.95 (3H, s, OCH3); EIMS m/z 207 [M]+ (100), 192
(93), 162 (13), 134 (15); anal. C 57.89, H 4.44, N 6.70%, calcd for
C10H9NO4, C 57.97, H 4.38, N 6.76%.
3-Methoxy-4,5-methylenedioxybenzonitrile (24d). For the margin-
ally soluble myristicin aldehyde, the amount of solvents was increased
by 15% to the overall volume of 345 mL of THF and 2070 mL of
aqueous NH4OH (28%). A reaction of myristicin aldehyde (57.6 g,
mol) and I2 (100.8 g, mol) in 3200 mL of 28% aqueous NH3 and THF
(480 mL) furnished 55 g of a crude mixture containing 24d (88%) and
unreacted aldehyde (12%). This was further oxidized with KMnO4 (6
g per 60 mL of H2O) in acetone (150 mL) at reflux for 4 h.
Concentration of the residue followed by its recrystallization from EtOH
afforded myristicin acid (12d) (4.5 g, 22.9 mmol, 7% yield; mp 212
°C; lit.39 mp 211 °C) and the targeted nitrile 24d (37 g, 208.9 mmol,
1
65% yield) as an off-white solid: mp 162-164 °C; H NMR (CDCl3,
500 MHz) δ 6.86 (1H, d, J ) 1.4 Hz, H-6), 6.80 (1H, d, J ) 1.4 Hz,
H-2), 6.08 (2H, s, OCH2O), 3.92 (3H, s, OCH3); EIMS m/z 177 [M]+
(100), 176 (77), 162 (15), 132 (42), 104 (26); anal. C 61.12, H 4.05,
N 7.83%, calcd for C9H7NO3, C 61.02, H 3.98, N 7.91%.
2,5-Dimethoxy-3,4-methylenedioxybenzyl Chloride. Neat SOCl2
(47 mL, 644 mmol) was added dropwise to a stirred solution of 2,5-
dimethoxy-3,4-methylenedioxybenzyl alcohol47 (66.5 g, 313 mmol) in
dry benzene (150 mL) at 10 °C. The resulting mixture was warmed to
48-50 °C, stirred for 2.5 h, and concentrated in vacuo. The resulting
crude benzyl chloride (68 g, 294.8 mmol, 94% yield) was used for the
synthesis of nitrile without further purification.
Compound substructure searching in the sea urchin embryo screening
database is available free on line.48
Acknowledgment. We thank Dr. Scott L. Young (OXiGENE, Inc.)
for providing combretastatin A4 disodium phosphate. This work was
supported by a grant from Chemical Block Ltd.
2,5-Dimethoxy-3,4-methylenedioxyphenylacetonitrile. A solution
of crude 2,5-dimethoxy-3,4-methylenedioxybenzyl chloride (68 g, 0.28
mol) in acetonitrile (300 mL) was treated with dibenzo-18-crown-6
(1.4 g, 3.9 mmol) followed by dried KCN (42 g, 0.645 M). The reaction
mixture was stirred for 10 h at 50 °C, concentrated in vacuo, and treated
with water (180 mL). The solid residue was filtered and recrystallized
from alcohol to yield a nitrile (57.5 g, 259.9 mmol, 88.2%) as an off-
Supporting Information Available: Experimental details regarding
selected syntheses, analytical data, and effects on sea urchin embryo
development. This material is available free of charge via the Internet
at http://pubs.acs.org.
1
References and Notes
white solid: mp 96-98 °C; H NMR (CDCl3, 500 MHz) δ 6.50 (1H,
s, H-6), 5.99 (2H, s, OCH2O), 3.97 (3H, s, OCH3), 3.86 (3H, s, OCH3),
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3.61 (2H, s, CH2); EIMS m/z 221 [M]+ (72), 206 (80), 176 (5), 166

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