Synthesis of polyalkoxy-3-(4-methoxyphenyl)coumarins with antimitotic activity from plant allylpolyalkoxybenzenes

Article abstract of DOI:10.1016/j.mencom.2013.05.009

Novel polyalkoxy-3-(4-methoxyphenyl)coumarins - analogues of natural antimitotic compounds - were synthesized from plant allylpolyalkoxybenzenes and tested in vivo in the phenotypic sea urchin embryo assay for antiproliferative antitubulin activity.

Full text of DOI:10.1016/j.mencom.2013.05.009

Mendeleev
Communications
Mendeleev Commun., 2013, 23, 147–149
Synthesis of polyalkoxy-3-(4-methoxyphenyl)coumarins
with antimitotic activity from plant allylpolyalkoxybenzenes
Dmitry V. Tsyganov,a Natalia B. Chernysheva,a Lev K. Salamandra,a Leonid D. Konyushkin,a
Olga P. Atamanenko,a Marina N. Semenovab and Victor V. Semenov*a
a N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991 Moscow,
Russian Federation. Fax: +7 499 135 5328; e-mail: vs@zelinsky.ru
b N. K. Kol’tsov Institute of Developmental Biology, Russian Academy of Sciences, 119334 Moscow,
Russian Federation
DOI: 10.1016/j.mencom.2013.05.009
Novel polyalkoxy-3-(4-methoxyphenyl)coumarins – analogues of natural antimitotic compounds – were synthesized from plant allyl-
polyalkoxybenzenes and tested in vivo in the phenotypic sea urchin embryo assay for antiproliferative antitubulin activity.
Pharmacologically active allylpolyalkoxybenzenes elemicin 1a,
myristicin 1b, apiol 1c, and dillapiol 1d (Figure 1) are the
widespread metabolites of plants belonging to the Umbelliferae
family.1 Previously, we have developed a technology for isolation
of these compounds from parsley and dill seeds by liquid CO2-
extraction and high-performance rectification.2
Synthesis of analogues of natural cytostatic agents containing
polyalkoxybenzene moieties, in particular, 3-arylcoumarins, can
be a promising approach in synthetic application of allylpoly-
alkoxybenzenes.
Coumarins with three and four alkoxy groups at the benzene
ring and the pyran ring (Figure 1, I) are widespread in nature.3–6
Some of them possess strong fluorescent properties.3 These com-
pounds are also known to inhibit the growth and stimulate the
differentiation of myeloid promonocyte cells U-937.7,8 Certain di-
and trialkoxy-3-phenylcoumarins demonstrate an antiproliferative
effect toward human promyelocytic leukemia HL-60 and human
lung adenocarcinoma epithelial A549 cells.9 Active cytostatic
agents have been found among polyalkoxy-3,4-dihydro-4-phenyl-
coumarins that suppress the growth of tumour cells,10–14 induce
apoptosis,12 and inhibit tubulin polymerization.12,13
It is interesting that dicoumarol (Figure 1) lacking methoxy
groups blocked the first division of the fertilized eggs of sea
urchins Strongylocentrotus purpuratus, S. franciscanus, and
15
Lytechinus pictus at mitosis with IC50 = 10 mm by a different
mechanism. It has been shown that the cytostatic effect of this
compound resembled the effect of taxol, resulting from binding
with tubulin and alteration (stabilization) of the microtubules
dynamics, while inhibition of tubulin polymerization was not
observed. Similar results for dicoumarol obtained in our assay
with embryos of sea urchin Paracentrotus lividus (see below)
suggested that active cytostatics acting both by microtubule
destabilization and stabilization mechanisms could be found
among polyalkoxy-substituted coumarin analogues.
Considering 3-arylcoumarins as bioisosteric analogues of
combretastatins (Figure 1), introduction of a 4-methoxyphenyl
substituent to 3-position can lead to highly active structures since
4-methoxyphenyl pharmacophore is essential for the antimitotic
antitubulin effect of combretastatin.16
Polyalkoxyphenols 4 required for the synthesis of intermediate
salicyl aldehydes 8 were synthesized by rearrangement of alde-
hydes 2 by the Baeyer–Villiger reaction at –20°C (Scheme 1).†
However, the reaction was complicated by a number of side
oxidation reactions yielding quinonoid structures 5.17 According
to our data, carrying out the reaction in a glass vessel caused
partial decomposition of hydrogen peroxide, possibly due to the
presence of admixtures in the glass, so a part of the aldehyde
remained unconsumed. In a plastic reactor, the aldehyde was com-
O
O
O
O
R4
R3
R2
CH2
OH
OH
Dicoumarol
R1
1a–d
R4
†
Polyalkoxybenzaldehydes were synthesized by the known procedure.23
R3
O
O
Synthesis of polyalkoxyphenols (general procedure). Hydrogen peroxide
(35%, 14.3 ml) was added to formic acid (99%, 29 ml), stirred for 1 h at
room temperature, cooled to –10–15°C, followed by dropwise addition
of aldehyde 2a–d (47.6 mmol) in formic acid (100 ml). The reaction
mixture was kept for 24 h at –20°C and diluted with water (200 ml).
Excess H2O2 was quenched with Na2S2O3. The mixture was extracted
with CH2Cl2 (4×100 ml), washed with water (3×40 ml), and dried with
Na2SO4 to afford crude aryl formates 3a–d. A solution of aryl formate
(18.0 mmol) and KOH (2.6 g, 67 mmol) in methanol (50 ml) was stirred
for 2 h at room temperature and acidified with 2 n HCl. Methanol was
evaporated; the residue was diluted with water (100 ml) and extracted
with CH2Cl2 (3×100 ml). The solvent was dried and evaporated to afford
the target phenol 4.
a R1 = R2 = R3 = OMe, R4 = H
b R1 = OMe, R2 + R3 = OCH2O, R4 = H
c R1 = R4 = OMe, R2 + R3 = OCH2O
d R1 + R2 = OCH2O, R3 = R4 = OMe
R2
R1
I
RO
RO
OMe
OH
OMe
3,4,5-Trimethoxyphenol 4a: yield 4.38 g (50%), mp 144–145°C (AcOEt).
(lit.,24 mp 145–147°C). 1H NMR (500 MHz, DMSO-d6) d: 3.55 (s, 3H,
OMe), 3.69 (s, 6H, OMe), 6.05 (s, 2H, HAr), 9.18 (s, 1H, OH). EI-MS,
m/z (%): 184 [M]+ (73), 169 (100), 141 (28). Found (%): C, 58.73; H, 6.44.
Calc. for C9H12O4 (%): C, 58.69; H, 6.57.
Combretastatin A2: R + R = CH2
Combretastatin A4: R = Me
Figure 1
© 2013 Mendeleev Communications. All rights reserved.
– 147 –

Mendeleev Commun., 2013, 23, 147–149
R4
R4
R4
R4
R3
R3
R2
R3
R2
R3
R2
OH
OH
CH2
Me
i
i
i
O
R2
R1
R1
R1
R4
R1
1a–d
4a–d
8a–d
R4
R4
HO
O
R3
R2
R3
R2
R3
R2
O
R4
O
OH
ii
iii
O
R3
R2
O
O
OMe
R1
R1
3a–d
R1
4a–d
ii
R1
2a–d
OMe
9a–d
iv
OMe
Scheme 2 Reagents and conditions: i, [PCl5, HCOOEt], SnCl4, CH2Cl2,
5 h, 20°C; ii, CDI, MeCN, reflux, 3 h.
OH
O
R4
OMe
OMe
R3
O
O
n
molecules affecting cell division and provides information on the
mechanism of antimitotic activity. The experimental procedure
includes (i) fertilized egg test for antimitotic activity displayed by
cleavage alteration/arrest, and (ii) behavioral monitoring of a free-
swimming blastulae treated immediately after hatching. The lack
of forward movement, settlement to the bottom of the culture
vessel, and rapid spinning of embryos around the animal–vegetal axis
suggests a microtubule destabilizing activity caused by a molecule
(video illustrations are available at http://www.chemblock.com).
The effective concentrations of compounds causing cleavage
alteration of sea urchin embryos are comparable with IC50 values
for mammalian and human cancer cell lines.
+
O
O
OMe
O
HO
OMe
5a R3 = OMe, R4 = H
6 n = 0
7 n = 1
5c R3 = OH, R4 = OMe
Scheme 1 Reagents and conditions: i, see ref. 23; ii, HCOOH, H2O2,
–20°C; iii, KOH, MeOH, 2 h, room temperature; iv, AcOH, H2O2, 24 h;
room temperature, 8 days.
pletely consumed. However, subsequent oxidation of the inter-
mediate o-acylphenols 3 occurred at room temperature to give side
quinones and dimerization products.
‡
Upon detailed studying the oxidation of apiol aldehyde 2c
under Baeyer–Villiger rearrangement conditions at room tempe-
rature as an example, we have shown that considerable amounts
of side products were formed: phenol was oxidized to quinone 5c
with opening of the dioxolane ring and formaldehyde extrusion.
Subsequently, oxidative dimerization of phenol 4c led to poly-
methoxybiaryl 6, and diarylmethane 7 was formed in a reaction
with formaldehyde.
The required salicylic aldehydes 8a–d were prepared by
the treatment of phenols 4 with the formylating mixture PCl5–
HCOOEt at room temperature. Formylation of myristicin deri-
vative 4b resulted in a mixture of regioisomers 8b and 8'b in a
ratio of 3:1. The structure of aldehyde 8b isolated by column
chromatography was confirmed by comparison with samples
obtained by alternative methods.6,18
The target polyalkoxy-3-phenylcoumarins 9a–d were syn-
thesized by the Perkin reaction19 using carbonyldiimidazole (CDI) by
heating equimolar amounts of aldehyde 8 and 4-methoxyphenyl-
acetic acid (Scheme 2).‡
The compounds obtained were tested on embryos of
Mediterranian sea urchin Paracentrotus lividus.
The sea urchin embryos are widely used as a model object
to identify compounds with antiproliferative activity. This study
was carried out using the phenotypic sea urchin embryo assay
developed in our lab.20–22 The assay allows for identification of
Synthesis of polyalkoxysalicylic aldehydes 8a–d (general procedure).
(i) Synthesis of the formylation reagent (dichlorodimethyl ether). Ethyl
formate (1.32 g, 17.9 mmol) was added at room temperature to a suspen-
sion of PCl5 (3.38 g, 16.28 mmol) in dry CH2Cl2 (20 ml). The resulting
mixture was refluxed for 2 h with stirring. (ii) Formylation. A mixture of
the formylation reagent and phenol (10.85 mmol) was added dropwise to
a solution of SnCl4 (1.3 g, 43.4 mmol) in dry CH2Cl2 (20 ml) at room
temperature. The reaction mixture was stirred for 5 h and diluted with
ice-water, followed by extraction with CH2Cl2 (3×30 ml). The organic
extract was washed with water (4×50 ml), dried and concentrated in vacuo.
The residual salicylic aldehyde had the purity of about 85%, and was
further purified by crystallization or column chromatography (SiO2) to
afford the pure target aldehyde (20–80% yield, Rf 0.6–0.7, AcOEt: light
petroleum = 1:6).
6-Hydroxy-2,3,4-trimethoxybenzaldehyde 8a: yield 0.9 g (column chro-
matography, 45%), mp 54–55°C (AcOEt–light petroleum) (lit.,25 55–57°C).
1H NMR (500 MHz, DMSO-d6) d: 3.69 (s, 3H, OMe), 3.87 (s, 3H, OMe);
3.98 (s, 3H, OMe), 6.37 (s, 1H, HAr), 9.98 (s, 1H, CHO) 11.98 (s, 1H,
OH). EI-MS, m/z (%): 212 [M]+ (100), 197 (91), 195 (1), 182 (5), 179 (10),
169 (58), 167 (12), 137 (48), 109 (10), 95 (8), 69 (51).
Synthesis of 3-arylcoumarins 9a–d (general procedure). A solution of
4-methoxyphenylacetic acid (3.0 mmol) in dry MeCN (5 ml) was treated
with carbonyl diimidazole (3.92 mmol) at 20°C and stirred for 30 min
at room temperature. Three portions of a salicylic aldehyde (3.3 mmol)
solution in dry MeCN (3 ml) were added in succession. The reaction
mixture was refluxed for 1 h after addition of each portion. The target
3-arylcoumarin (yield 3–35%) was separated by column chromatography
(SiO2, Rf 0.4–0.5, AcOEt:light petroleum = 1:4).
5,6,7-Trimethoxy-3-(4-methoxyphenyl)-2H-chromen-2-one 9a: yield
0.36 g (35%), mp 112–114°C (AcOEt–light petroleum, 1:4). H NMR
1
Reaction of apiolaldehyde 2c with H2O2–AcOH. A solution of aldehyde
2c (2.12 g, 10.1 mmol) in a mixture of AcOH (20 ml) and H2O2 (35%,
2.35 ml) was kept for 8 days at room temperature. The reddish-violet
deposit of quinone 5c was filtered off, washed with AcOH (2.3 ml) and
dried. The filtrate was evaporated in vacuo and dried to afford 1.42 g of a
dark oil. Three compounds, namely phenol 4c (0.14 g), biphenyl 6 (0.44 g)
and diarylmethane 7 (0.33 g), were separated from the oil by column
chromatography (SiO2, benzene–AcOEt).
(500 MHz, DMSO-d6) d: 3.78 (s, 3H, OMe), 3.81 (s, 3H, OMe), 3.91 (s,
3H, OMe), 3.98 (s, 3H, OMe), 6.91 (s, 1H, HAr), 7.01 (d, 1H, HAr, J 9.0 Hz),
7.66 (d, 1H, HAr, J 9.0 Hz), 7.98 (s, 1H, C=CH). EI-MS, m/z (%): 342 [M]+
(100), 327 (16), 312 (2), 299 (13), 284 (16), 271 (8), 256 (37), 243 (14),
241 (32), 213 (32), 211 (13), 185 (26), 171 (18), 169 (12), 157 (30), 142 (27),
135 (17), 128 (27), 126 (11), 121 (26), 114 (11), 101 (8), 77 (7), 69 (28).
Found (%): C, 66.72; H, 5.38. Calc. for C19H18O6 (%): C, 66.66; H, 5.30.
For characteristics of compounds 8b–d and 9b–d, see Online Supple-
mentary Materials.
For characteristics of compounds 4b–d, 5a,c, 6 and 7, see Online Sup-
plementary Materials.
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Mendeleev Commun., 2013, 23, 147–149
Table 1 Effects of dicoumarol and polyalkoxy-3-phenylcoumarins on sea
This work was supported by a grant from Chemical Block Ltd.
We thank the National Cancer Institute (NCI) (Bethesda,
MD, USA) for screening compound 9a under the Developmental
Therapeutics Program at NCI (Anti-cancer Screening Program;
http://dtp.cancer.gov).
urchin embryos.
a
EC/mm
Compound
Cleavage alteration Cleavage arrest Embryo spinning
Dicoumarol 10
40
4
>50
>5
>4
>4
>4
Online Supplementary Materials
Supplementary data associated with this article can be found
in the online version at doi:10.1016/j.mencom.2013.05.009.
9a
9b
9c
9d
0.2
4
>4
>4
>4
1
>4
References
a The sea urchin embryo assay was conducted as described in ref. 18.
Fertilized eggs/embryos were exposed continuously to 2-fold decreasing
concentrations of a compound. Duplicate measurements showed no dif-
ferences in effective threshold concentration (EC) values. At these concen-
trations, all tested molecules caused 100% cleavage alteration and embryo
death before hatching, whereas at 2-fold lower concentrations, the com-
pounds failed to produce any effect.
1 http://www.ars-grin.gov/duke/
2 V.V. Semenov,V.A. Rusak, E. M. Chartov, M. I. Zaretsky, L. D. Konyushkin
S. I. Firgang, A. O. Chizhov, V. V. Elkin, N. N. Latin, V. M. Bonashek and
O. N. Stas’eva, Russ. Chem. Bull., Int. Ed., 2007, 56, 2448 (Izv. Akad.
Nauk, Ser. Khim., 2007, 2364).
,
3 R. E. Reyes and A. G. Gonzalez, Phytochemistry, 1970, 9, 833.
4 A. A. Semenov, Ocherki khimii prirodnykh soedinenii (Essays on Chemistr
y
of Natural Compounds), Nauka, Novosibirsk, 2000, p.664 (in Russian).
5 T. S. Wu, Z. J. Tsang, P. L. Wu, F. W. Lin, C. Y. Li, C. M. Teng and
K. H. Lee, Bioorg. Med. Chem., 2001, 9, 77.
The results obtained in the sea urchin embryo assay for the
target compounds are shown in Table 1. Figure 2 illustrates mor-
phological changes of arrested eggs caused by dicoumarol and
3-phenylcoumarin 9a.
Dicoumarol caused cleavage alteration and arrest; in this case,
a mitotic spindle was distinctly visible as a light rounded spot in
the egg center [Figure 2(b)]. The results confirmed the previously
described mechanism of dicoumarol cytostatic effect based on
cell cycle arrest in mitosis due to microtubule stabilization and
alteration of the mitotic spindle dynamics.15
6 D. Maes, S. Vervisch, S. Debenedetti, C. Davio, S. Mangelinckx,
N. Giubellina and N. De Kimpe, Tetrahedron, 2005, 61, 2505.
7 M. E. Riveiro, C. Shayo, F. Monczor, N. Fernandez,A. Baldi, N. De Kimpe,
J. Rossi, S. Debenedetti and C. Davio, Cancer Lett., 2004, 210, 179.
8 M. E. Riveiro, D. Maes, R. Vazquez, M. Vermeulen, S. Mangelinckx,
J. Jacobs, S. Debenedetti, C. Shayo, N. De Kimpe and C. Davio, Bioorg.
Med. Chem., 2009, 17, 6547.
9 J. Yang, G.-Y. Liu, F. Dai, X.-Y. Cao, Y. Kang, L.-M. Hu, J.-J. Tang,
X.-Z. Li, Y. Li, X.-L. Jin and B. Zhou, Bioorg. Med. Chem. Lett., 2011,
21, 6420.
Among 3-(4-methoxyphenyl)coumarins 9a–d, only the com-
pound with three methoxy groups, i.e. 9a, had a noticeable
antiproliferative activity (EC = 0.2 mm). The structures with
a myristicin (9b) or apiol (9c) moieties altered cell division
at higher concentrations of 4 or 1 mm, respectively, whereas
dillapiol derivative 9d was found to be inactive. None of the
compounds tested caused changes in embryo motility typical
of microtubule destabilizers. Meanwhile, the antimitotic effect of
compound 9a could be considered as a result of targeting tubulin,
since cleavage arrest at a concentration of 4 mm was accompanied
by formation of tuberculate eggs [Figure 2(c)] characteristic of
10 P. Singh, U. Faridi, S. Srivastava, J. K. Kumar, M. P. Darokar, S. Luqman,
K. Shanker, C. S. Chanotiya,A. Gupta, M. M. Gupta andA. S. Negi, Chem.
Pharm. Bull., 2010, 58, 242.
11 J. T. Pierson, A. Dumetre, S. Hutter, F. Delmas, M. Laget, J. P. Finet,
N. Azas and S. Combes, Eur. J. Med. Chem., 2010, 45, 864.
12 C. Rappl, P. Barbier, V. Bourgarel-Rey, C. Gre’goire, R. Gilli, M. Carre,
S. Combes, J.-P. Finet and V. Peyrot, Biochemistry, 2006, 45, 9210.
13 C. Bailly, C. Bal, P. Barbier, S. Combes, J. P. Finet, M. P. Hildebrand,
V. Peyrot and N. Wattez, J. Med. Chem., 2003, 46, 5437.
14 S. Combes, P. Barbier, S. Douillard, A. McLeer-Florin, V. B. Bourgarel-
Rey, J. T. Pierson, A. Y. Fedorov, J. P. Finet, J. Boutonnat and V. Peyrot,
J. Med. Chem., 2011, 54, 3153.
15 H. Madari, D. Panda, L. Wilson and R. S. Jacobs, Cancer Res., 2003,
m
icrotubule destabilizers.20 Thus, both dicoumarol and trimethoxy-
63, 1214.
3-(4-methoxyphenyl)coumarin 9a exhibited antimitotic activity
affecting tubulin/microtubules. However, unlike dicoumarol,
compound 9a most probably displayed its antiproliferative pro-
perties due to microtubule destabilization.
The most active coumarin with an elemicin moiety, 9a, was
further tested in NCI60 cytotoxicity screen against 60 human
cancer cell lines at the National Cancer Institute (NCI, USA).
It was found that 9a caused the inhibition of cancer cell growth
with the mean GI50 = 3.981 mm. The screening results are
presented in Online Supplementary Materials.
16 N.-H. Nam, Curr. Med. Chem., 2003, 10, 1697.
17 F. Dallacker, Monatsh. Chem., 1969, 100, 742.
18 B. A. McKittrick and R. Stevenson, J. Chem. Soc., Perkin Trans. 1, 1984,
709.
19 L. M. Kabeya, A. A. de Marchi, A. Kanashiro, N. P. Lopes, C. H. T. P.
da Silva, M. T. Pupo and Y. M. Lucisano-Valim, Bioorg. Med. Chem.,
2007, 15, 1516.
20 M. N. Semenova, A. S. Kiselyov and V. V. Semenov, BioTechniques,
2006, 40, 765.
21 L. D. Konyushkin, T. I. Godovikova, S. K. Vorontsova, D. V. Tsyganov,
I. B. Karmanova, M. M. Raihstat, S. I. Firgang, M. A. Pokrovskii, A. G.
Pokrovskii, M. N. Semenova and V. V. Semenov, Russ. Chem. Bull., Int.
Ed., 2010, 59, 2268 (Izv. Akad. Nauk, Ser. Khim., 2010, 2212).
22 A. B. Sheremetev, D. E. Dmitriev, N. K. Lagutina, M. M. Raihstat, A. S.
Kiselyov, M. N. Semenova, N. N. Ikizalp and V. V. Semenov, Mendeleev
Commun., 2010, 20, 132.
(a)
(b)
(c)
23 V. V. Semenov, A. S. Kiselyov, I.Y. Titov, I. K. Sagamanova, N. N. Ikizalp,
N. B. Chernysheva, D. V. Tsyganov, L. D. Konyushkin, S. I. Firgang, R. V.
Semenov, I. B. Karmanova, M. M. Raihstat and M. N. Semenova, J. Nat.
Prod., 2010, 73, 1796.
24 J. Gong, K. Huang, F. Wang, L.Yang,Y. Feng, H. Li, X. Li, S. Zeng, X. Wu,
J. Stockigt, Y. Zhao and J. Qu, Bioorg. Med. Chem., 2009, 17, 3414.
25 O. Garcia, E. Nicolas and F. Albericio, Tetrahedron Lett., 2003, 44, 4961.
Figure 2 Effects of dicoumarol and compound 9a on the sea urchin embryo
development. (a) Intact early blastula. (b),(c) Arrested eggs. Fertilized eggs
were exposed continuously to (b) 40 mm of dicoumarol or (c) 4 mm of 9a.
Time after fertilization, 6 h. The average embryo diameter is 115 mm. In (b),
note a light rounded spot in the egg center marked by an arrow.
Received: 14th February 2013; Com. 13/4067
– 149 –