Application of plant allylpolyalkoxybenzenes in synthesis of antimitotic phenstatin analogues

Article abstract of DOI:10.1016/j.bmcl.2011.01.124

Phenstatin and its derivatives with the modified ring A have been synthesized, using plant allylpolyalkoxybenzenes as a starting material. The targeted molecules were evaluated in a phenotypic sea urchin embryo assay for antiproliferative activity. It was found that phenstatin ring A modifications yielded antimitotic compounds. The most effective myristicin derivative 7d (combretastatin A-2 analogue) was determined to be ca. 10 times more potent than phenstatin, displaying antimitotic tubulin-destabilizing activity at the same concentration range as combretastatins. In contrast to combretastatins, 7d featured the steric stability with potential for further design as anticancer agent.

Full text of DOI:10.1016/j.bmcl.2011.01.124

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1578–1581
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Application of plant allylpolyalkoxybenzenes in synthesis of antimitotic
phenstatin analogues
Ilia Y. Titov a, Irina K. Sagamanova a, Roman T. Gritsenko a, Irina B. Karmanova a, Olga P. Atamanenko a,
a,c
Marina N. Semenova b,c, , Victor V. Semenov
⇑
a Zelinsky Institute of Organic Chemistry RAS, 47 Leninsky Prospect, 119991 Moscow, Russia
b Chemical Block Ltd, 3 Kyriacou Matsi, 3723 Limassol, Cyprus
c Institute of Developmental Biology RAS, 26 Vavilov Str., 119334 Moscow, Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
Phenstatin and its derivatives with the modified ring
A have been synthesized, using plant
Received 20 December 2010
Revised 26 January 2011
Accepted 27 January 2011
Available online 2 February 2011
allylpolyalkoxybenzenes as a starting material. The targeted molecules were evaluated in a phenotypic
sea urchin embryo assay for antiproliferative activity. It was found that phenstatin ring A modifications
yielded antimitotic compounds. The most effective myristicin derivative 7d (combretastatin A-2
analogue) was determined to be ca. 10 times more potent than phenstatin, displaying antimitotic
tubulin-destabilizing activity at the same concentration range as combretastatins. In contrast to combre-
tastatins, 7d featured the steric stability with potential for further design as anticancer agent.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Allylpolyalkoxybenzenes
Antimitotic agents
Phenstatin
Tubulin
Sea urchin embryo
Combretastatins are natural antimitotic stilbenes derived from
the bark of Combretum caffrum Kuntze (Combretaceae).1 The three
most active molecules combretastatin A-1, A-2, and A-4 (CA1, CA2,
CA4; Fig. 1) have attracted a great attention as potent inhibitors of
cancer cell growth and tubulin polymerization.2–5 The phosphory-
lated prodrugs Zybrestat and Oxi4503 are currently undergoing
clinical evaluation as antitumor vascular targeting agents.4,6,7
Numerous studies suggest that the olefinic bond plays a significant
role in the orientation of benzene rings in stilbene molecule, opti-
mizing the distance and dihedral angle for targeting tubulin.8,9 The
anticancer activity of stilbenes strictly requires for Z-geometry,
however, the compounds are prone to isomerization during storage
and administration and in the course of metabolism in liver micro-
somes.10–12 In this regard, benzophenone analogues with biaryl
system connected by a carbonyl group have several advantages
including no need of controlling the geometric selectivity (Z- and
E-configuration) as well as the ease of synthetic design for increased
potency and stability. It was also suggested that the sp2-hybridized
carbonyl group in benzophenones constrains the two aryl rings in a
quasi ‘cis’ orientation necessary for efficient interaction of a
molecule with the colchicine binding site of tubulin.13
Phenstatin was designed from the CA4 skeleton by replacement
of the double bond linker with a carbonyl moiety (Fig. 1).13,14 It is
a potent microtubule destabilizing agent that interacts with colchi-
cine binding site of tubulin and displays significant anticancer and
antimitotic activities comparable to those of CA4.13 Phenstatin and
related compounds are currently under preclinical develop-
ment.15–18 Among them, the benzophenone derivative of CA1
hydroxyphenstatin (Fig. 1) is a potent inhibitor of tubulin polymer-
ization and displays pronounced cytotoxicity against a panel of can-
cer cell lines.15,16 Multiple SAR studies of phenstatin analogues
showed that the B-ring (substituted phenyl) could be successfully
modified without losing cytotoxicity and antitubulin properties by
substitution of m-OH for m-NH2 group,19–21 or by replacement with
thienyl,22 naphthyl,23,24 benzo[b]thiophenyl,25,26 benzo[b]furanyl,27
indolyl,28,29 carbazolyl,30 quinolyl,31 and indazolyl32 templates. The
carbonyl bridge of phenstatins is not essential for antimitotic behav-
ior, since isocombretastatins featuring a 1,1-diarylethylene scaffold
displayed potent inhibition of tubulin assembly and cytotoxicity
against cancer cell lines similar to those of the corresponding com-
bretastatins.33,34 In contrast, the reported modifications of the
3,4,5-trimethoxyphenyl A-ring in phenstatin analogues, namely,
changes in distribution or removal of the methoxy groups, dramat-
ically reduced the cytotoxic and antitubulin activity.23,25,30,33,35,36
Recently it was found that 2,3,4-trimethoxyphenyl A-ring in combi-
nation with N-methyl-5-indolyl B-ring yielded a potent cytotoxic
tubulin destabilizing compound, the only one exception reported
⇑
Corresponding author. Tel.: +7 499 793 7174; fax: +7 499 137 2966.
E-mail addresses: il_tit@bk.ru (I.Y. Titov), sagamanova@mail.ru (I.K. Sagamano-
va), romagree@gmail.com (R.T. Gritsenko), info@chemblock.com (I.B. Karmanova),
info@chemblock.com (O.P. Atamanenko), ms@chemical-block.com (M.N. Semeno-
va), vs@zelinsky.ru (V.V. Semenov).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.124

I. Y. Titov et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1578–1581
1579
apiol (1b), dillapiol (1c), myristicin (1d), and tetramethoxybenzene
(1e) (Fig. 1) that can be isolated by liquid CO2 extraction followed by
high-efficiency large-scale distillation (up to 40 kg of allylbenz-
enes).40 In our recent study compounds 1a–e have been successfully
used in the synthesis of combretastatin analogues with significant
antimitotic tubulin destabilizing activity.41–43
Previously phenstatin and related compounds were synthesized
by the reaction of Ar–Li with amides or aldehydes as a starting mate-
rial.13,19,37,44 Another efficient economical route of phenstatin syn-
thesis has been developed, providing overall yield of 60%.45 The
procedure included the reaction of guaiacol, protected by easily
cleaved chloroacetyl function, with 3,4,5-trimethoxybenzoic acid,
using polyphosphoric acid as condensing agent. Recently a nucleo-
philic addition of ArMgBr onto the Weinreb amides has been
described to produce phenstatin analogues.46 In the present study
a combination of the specialized ozonolysis equipment and opti-
mized synthetic protocol allowed for preparation of the targeted
aldehydes 3a–e from the respective styrene precursors 2a–e
(Scheme 1, step b) in yields of 60–85% on a 100 g scale.40,42 The
polyalkoxybenzoic acids 4a–f were obtained in high yields
(80–90%) from the aldehydes 3a–f using the urea–hydrogen perox-
ide complex in water (Scheme 1, step c). Phenstatin (7a), as well as
its methylenedioxy (7b–d), tetramethoxybenzene (7e), and ethyl-
enedioxy (7f, g) analogues were synthesized via transformation of
acids 4a–f to corresponding Weinreb amide precursors 5a–f
(Scheme 1, step d), using a procedure reported previously,47 with a
new modification involving carbonyl diimidazole as condensation
agent. Then amides 5a–f were treated with Li-salt of TBDMS-pro-
tected guaiacol followed by the removal of TBDMS-group (Scheme
1, steps e and f). Experimental details regarding syntheses and
analytical results are presented in Supplementary data.
Figure 1.
to date.37 However, the biological effects of benzophenones derived
from CA2 with methylenedioxy moiety in the ring A have never been
studied. Moreover, the methylenedioxybenzene pharmacophore is
featured frequently in antimitotic natural products interacting with
colchicine binding site of tubulin, such as cornigerine, podophyllo-
toxin, steganacin, and alkaloids isolated from Papaver and
Chelidonium.38,39
In searching for a reliable natural source of building blocks
containing methylenedioxy group for phenstatine analogues, the
Apiaceae family of plants became of interest. Previously it was
discovered that seeds of parsley, Petroselinum sativum Hoffm., culti-
vated in Russia, and dill, Anethum graveolens L., grown in India, are
both versatile sources of allylpolyalkoxybenzenes elemicin (1a),
Synthetic derivatives of phenstatin (7a–g, Scheme 1) were fur-
ther evaluated for their antiproliferative and tubulin-destabilizing
activity using a phenotypic sea urchin embryo assay48 with CA2
and CA4 as benchmark reference compounds (Table 1). 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
Scheme 1. Reagents and conditions: (a) powdered KOH, (n-Bu)4N+Brꢀ, without solvent, 100 °C, 40 min; (b) O3, CHCl3–MeOH–pyridine (80:20:3 v/v), ꢀ15 °C, 1–2 h; (c)
CO(NH2)2ꢁH2O2, CH3OH, reflux, 1.5 h; (d) N,N0-carbonyldiimidazole, MeNH(OMe)ꢁHCl, CH3CN–Et3N, 40 °C, 20 h; (e) Ar–Br, n-BuLi–hexane, THF, rt, 10 h; (f) TBAFꢁ3H2O, THF, rt,
1 h.

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I. Y. Titov et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1578–1581
Table 1
Acknowledgment
Effects of polyalkoxybenzene analogues of phenstatin on sea urchin embryos
Compound
ECa
(l
M)
This work was supported by a grant from Chemical Block Ltd.
Cleavage alteration
Cleavage arrest
Embryo spinning
CA4b
0.002
0.002
0.01
2
0.01
0.01
0.05
>4
4
0.01
>4
0.05
0.05
0.5
>5
>5
0.1
>5
Supplementary data
CA2c
7a, phenstatin
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.01.124. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
7b
7c
7d
7e
7f
1
0.001
2
0.1
0.2
1
>5
>5
7g
0.5
References and notes
a
The sea urchin embryo assay was conducted as described in Ref. 48. Duplicate
measurements showed no differences in effective threshold concentration (EC)
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7b, 7c, and 7e–f were not tested at concentrations of >4–5 lM
due to their limited solubility in DMSO and/or seawater.
Literature data suggest that the presence of three methoxy sub-
stituents in the A ring of combretastatins and phenstatin analogues
is crucial for the antitubulin activity.3,11,23,25,30,35,36,53 However, in
the sea urchin embryo test, CA2-related phenstatin derivative
containing myristicin moiety (7d) was consistently more potent
than phenstatin (7a), exhibiting the activity similar to those of CA4
and CA2. Ethylenedioxy derivatives (7f and 7g) were less active.
The presence of additional methoxy group in apiol, dillapiol, and
tetramethoxybenzene fragments of 7b, 7c, and 7e, respectively,
dramatically reduced the antimitotic effect. A similar observation
was made for the polyalkoxyphenyl analogues of combretastatins.42
Biological evaluation of ethylenedioxy derivatives (7f and 7g)
showed that the removal of hydroxy group in the ring B decreased
antimitotic activity. This was comparable with the reported antimi-
totic effect of (4-methoxyphenyl)(3,4,5-trimethoxyphenyl)metha-
none that was determined to be significantly less potent than
phenstatin (IC50 values of 0.4–0.6
lM in the sea urchin embryo
assay).18
In summary, a variety of phenstatin ring A modifications de-
rived from apiol (1b), dillapiol (1c), myristicin (1d), and ethylen-
edioxybenzaldehyde (3f), yielded antimitotic compounds. The
myristicin derivative 7d (CA2 analogue) was more effective than
the parent compound phenstatin (7a), displaying the antiprolifera-
tive tubulin-destabilizing activity at the same concentration range
as CA2 and CA4. Compound 7d was synthesized using the simple
starting material extracted from parsley seeds. In contrast to com-
bretastatins, 7d featured the steric stability with potential for fur-
ther design as anticancer agent.
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