Synthesis and biotests of 2-aryl-5-arylmethylidene-substituted 1,3-oxazol-5(4H)-ones and N-methyl-3,5-dihydro-4H-imidazol-4-ones as combretastatin A-4 analogs

Article abstract of DOI:10.1007/s11172-015-1041-0

A series of 2-aryl-5-arylmethylidene-1,3-oxazol-5(4H)-ones and 2-aryl-5-arylmethylidene- N-methyl-3,5-dihydro-4H-imidazol-4-ones was synthesized as structural analogs of combret- astatin A-4 (a compound possessing antitumor activity). (5Z)-5-[(4-Methoxyph

Full text of DOI:10.1007/s11172-015-1041-0

Russian Chemical Bulletin, International Edition, Vol. 64, No. 7, pp. 1560—1563, July, 2015
1560
Synthesis and biotests of 2ꢀarylꢀ5ꢀarylmethylideneꢀsubstituted
1,3ꢀoxazolꢀ5(4H)ꢀones and Nꢀmethylꢀ3,5ꢀdihydroꢀ4Hꢀimidazolꢀ4ꢀones
as combretastatin Aꢀ4 analogs
E. S. Barskaia,a A. A. Beloglazkina,a B. Wobith,b N. A. Zefirov,a A. G. Majouga,a E. K. Beloglazkina,a
N. V. Zyk,a S. A. Kuznetsov,b and O. N. Zefirovaa,c
aM. V. Lomonosov Moscow State University, Department of Chemistry,
Build. 3, 1 Leninskie Gory, 119991 Moscow, Russian Federation.
Fax: +7 (459) 939 0290. Eꢀmail: bel@org.chem.msu.ru; olgaz@org.chem.msu.ru
bInstitute of Biological Sciences, University of Rostock,
18106 Rostock, Germany.
Eꢀmail: sergei.kuznetsov@uniꢀrostock.de
cInstitute of Physiologically Active Compounds, Russian Academy of Sciences,
1 Severnyi prꢀd, 142432 Chernogolovka, Moscow Region, Russian Federation.
Eꢀmail: kolaz92@gmail.com
A series of 2ꢀarylꢀ5ꢀarylmethylideneꢀ1,3ꢀoxazolꢀ5(4H)ꢀones and 2ꢀarylꢀ5ꢀarylmethylideneꢀ
Nꢀmethylꢀ3,5ꢀdihydroꢀ4Hꢀimidazolꢀ4ꢀones was synthesized as structural analogs of combretꢀ
astatin Aꢀ4 (a compound possessing antitumor activity). (5Z)ꢀ5ꢀ[(4ꢀMethoxyphenyl)methylꢀ
idene]ꢀ3ꢀmethylꢀ2ꢀ(4ꢀmethylphenyl)ꢀ3,5ꢀdihydroꢀ4Hꢀimidazolꢀ4ꢀone was found to exhibit the
highest cytotoxicity against cells of human A549 lung carcinoma line (EC50 = 6 0.8 mol L–1).
Key words: combretastatin analogs, oxazolones, imidazolones, colchicine domain, tubulin,
cytotoxicity.
Development of efficient compounds with simple
cases with the replacement of phenyl rings with aromatic
heterocycles or their analogs).1—5 In the course of transꢀ
formation of such compounds, the distance between the
aromatic groups frequently remains the same as in the
starting molecule. However, lately a tendency to the deꢀ
velopment of combretastatin analogs with longer linkers
becomes increasingly popular: there are literature examꢀ
ples of several successful types of such compounds with
high cytotoxicity against different lines of tumor cells (see,
for example, Refs 6—8). In this work, we suggested strucꢀ
tures (A) as analogs of CAꢀ4, in which the aromatic groups
are connected with an elongated linker as compared to
that in the starting molecule. This linker is a combination
of a heterocyclic fragment and a double bond, which was
not studied earlier for this purpose.
chemical structure, which makes affordable the correꢀ
sponding therapy, is an important aspect in the design of
antitumor agents. The natural combretastatin Aꢀ4 (CAꢀ4)
due to its relatively simple structure, very high cytotoxiciꢀ
ty, and ability to cause vascular disruption in tumors beꢀ
came a lead compound in the development of numerous
series of analogs as potential antitumor agents.1—5 Comꢀ
bretastatin Aꢀ4 binds to a certain site of the dimeric cell
protein tubulin at the colchicine domain. That leads to the
inhibition of the polymerization process of this protein
and prevents the formation of microtubules, which play
a key role in the cellular division process.
Most analogs of CAꢀ4 were obtained by the replaceꢀ
ment of the bridging group in the starting molecule with
heterocyclic and other fragments (usually together with
variations of substituents in the phenyl rings and in some
Initially, we studied easily synthetically available
(Scheme 1) 5ꢀarylmethylideneoxazolꢀ4ꢀones 5—9. An auꢀ
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 7, pp. 1560—1563, July, 2015.
1066ꢀ5285/15/6407ꢀ1560 © 2015 Springer Science+Business Media, Inc.

Combretastatin analogs
Russ.Chem.Bull., Int.Ed., Vol. 64, No. 7, July, 2015
1561
tomated docking of these compounds into the colchicine
binding site using the CLC Drug Discovery Workbench
program showed that in the case of Zꢀconfiguration of the
C=C double bond, their position is close enough to that of
CAꢀ4 except a slight "shift" of ring B (Fig. 1).
Compound 9 without substituents in the aryl fragments
was obtained as a control.
5ꢀArylmethylideneoxazolꢀ4ꢀones 5—9 were syntheꢀ
sized from substituted hippuric acids 1—4, which were
obtained from aromatic carboxylic acyl halides and glyꢀ
cine according to the known procedures9—13 (in the case
of 3,4,5ꢀtrimethoxyhippuric acid 1, 3,4,5ꢀtrimethoxyꢀ
benzoic acid was additionally synthesized by trimethylaꢀ
tion of gallic acid with excess of dimethyl sulfate13).
NꢀAcylglycines 1—4 were involved in the condensation
reaction with aromatic aldehydes, using potassium aceꢀ
tate as a base (see Scheme 1), which increased the yield of
the desired Zꢀisomer14 (the signal for the vinyl proton is in
the region  7.1—7.3). A wide range of the target comꢀ
pound yields (see Scheme 1) is apparently explained by
their different solubilities, as well as by a possibility of side
hydrolysis reaction of oxazolone 5 during column chroꢀ
matography.
Scheme 1
The testing of compounds 5—9 on the human epitheꢀ
lial lung carcinoma cells A549 in a standard colorimetric
test with 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenylꢀ2Hꢀ
tetrazolyl bromide (MTT)15 showed that their cytotoxiciꢀ
ty is low (EC50 > 50 mol L–1) and does not depend on the
type of substituents in the aryl rings. This can possibly
result from the mentioned above and, by all accounts,
unfavorable "shift" of ring B in 5ꢀarylmethylidenoxazolꢀ4ꢀ
ones (see Fig. 1).
Reagents and conditions: i. Ac2O, CH3COOK, heating.
Compound
1, 5
2, 6
3, 7, 8
4, 9
R
OMe
H
H
H
R´
OMe
Me
F
R
OMe
H
H
H
H
Product
Ar
Yield (%)
To confirm this conclusion, we synthesized another
two compounds related to the suggested structural type A,
namely, Nꢀmethylꢀ3,5ꢀdihydroꢀ4Hꢀimidazolꢀ4ꢀones 10
and 11 with different positions of rings B, according to the
computer molecular modeling data (Fig. 2, a, b). As it is
seen, in the structure 10 ring B is closer to that in CAꢀ4,
while in the structure 11, it is "shifted" as compared to
rings B in the starting molecule and in 2ꢀarylꢀ5ꢀarylmethꢀ
ylidenoxazolꢀ4ꢀones.
5
6
7
8
9
4ꢀMeOꢀC6H4
4ꢀMeOꢀC6H4
4ꢀMeOꢀC6H4
3,4ꢀ(MeO)2ꢀC6H3
Ph
27
46
22
21
99
The choice of substituents in the aryl fragments of
oxazolones 5—8 was based on both the results of the
computer molecular modeling (the scoring function
values) and the literature data on the bioisostericity of
paraꢀfluorophenyl and 3,4,5ꢀtrimethoxyphenyl groups in
some active analogs of CAꢀ4 with an elongated linker.8
The synthesis of compounds 10 and 11 was accomꢀ
plished by reflux of oxazolones in solution of methylamine
in aqueous ethanol in the presence of potassium carbonate
1
as a base (Scheme 2).16 The H NMR spectra of final
compounds exhibit the only signal for the vinyl proton in
the region  6.6 and 7.04, that confirm the presence of
only one geometric isomer (Zꢀisomer17). A singlet for the
protons of the CH3N group is observed at  3.65 and 3.45,
respectively.
The examination of compounds 10 and 11 in the
MTTꢀtest showed that imidazolone 11 is inactive
(EC50 > 50 mol L–1), while imidazolone 10 exhibits a noꢀ
ticeable cytotoxicity (EC50 = 6 0.8 mol L–1) against the
A549 cell culture. These data indirectly confirm a concluꢀ
sion that the activity of compounds of this type is sensitive
to the insignificant changes in the position of ring B.
(5Z)ꢀ5ꢀ[(4ꢀMethoxyphenyl)methylidene]ꢀ3ꢀmethylꢀ2ꢀ(4ꢀ
methylphenyl)ꢀ3,5ꢀdihydroꢀ4Hꢀimidazolꢀ4ꢀone obtained
in this work can become a lead compound in further search
Fig. 1. Position of structure 5 in the colchicine domain of tubulin
(for comparison, a CAꢀ4 molecule is shown in light; hydrogen
atoms are omitted).

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Russ.Chem.Bull., Int.Ed., Vol. 64, No. 7, July, 2015
a
Barskaia et al.
for more efficient analogs of combretastatin as potential
antitumor agents.
Experimental
Reaction progress and purity of compounds were monitored
by TLC on SilufolꢀUV 254 plates. Chromatographic separation
was carried out on columns with Acros silica gel (40—60 m).
1H and 13C NMR spectra were recorded on a Bruker Avance 400
spectrometer (400 and 100 MHz, respectively) at 28 C. Chemiꢀ
cal shifts are given relative to the residual signals of the solvent.
Elemental analysis was performed on a Vario micro cube
CHNꢀanalyzer. IR spectra were recorded on an IRꢀ200 Therꢀ
moNicolet spectrophotometer in KBr pellets. Electron ionizaꢀ
tion mass spectra were obtained on a Finnigan MAT SSQ 7000
GLCꢀMS spectrometer (ionization energy 70 eV, an OVꢀI quartz
capillary column, 25 m). Automated docking in the 3D model of
tubulin complex with NꢀdeacetylꢀNꢀ(2ꢀmercaptoacetyl)colꢀ
chicine (PDB ID: 1SA0) was performed using the CLC Drug
Discovery Workbench program (Version 1.5): Evaluation license
(2014).
b
(4Z)ꢀ4ꢀ[(4ꢀMethoxyphenyl)methylidene]ꢀ2ꢀ(3,4,5ꢀtrimethꢀ
oxyphenyl)ꢀ1,3ꢀoxazolꢀ5(4H)ꢀone (5). A mixture of 4ꢀmethoxyꢀ
benzaldehyde (0.285 g, 2.1 mmol), acid 1 (0.5 g, 2.1 mmol), and
potassium acetate (0.21 g, 2.1 mmol) in (CH3CO)2O (5 mL) was
refluxed with stirring for 12 h. The solvent was evaporated, the
residue was subjected to chromatography (eluent ethyl acetate—
light petroleum ether (40—70 C), 1 : 1). Dried in vacuo of an oil
pump. Compound 5 (0.21 g, 27%) was obtained as brown oily
1
Fig. 2. Position of structures 10 (a) and 11 (b) in the colchicine
domain of tubulin (for comparison, a CAꢀ4 molecule is shown in
light; hydrogen atoms are omitted).
liquid. Rf = 0.18. H NMR (CDCl3), : 7.63 (s, 2 H, Ar); 7.45
(d, 2 H, Ar, J = 8.8 Hz); 7.27 (s, 1 H, HC=); 6.98 (d, 2 H, Ar,
J = 8.8 Hz); 3.88 (s, 3 H, CH3O); 3.80 (s, 9 H, CH3O). MS, m/z
(Irel (%)): 369.4 [M]+ (10). Found (%): C, 64.98; H, 5.09;
N, 3.78. C20H19NO6. Calculated (%): C, 65.04; H, 5.15; N, 3.79.
(4Z)ꢀ4ꢀ[(4ꢀMethoxyphenyl)methylidene]ꢀ2ꢀ(4ꢀmethylpheꢀ
nyl)ꢀ1,3ꢀoxazolꢀ5(4H)ꢀone (6). A mixture of anisaldehyde
(0.37 mL, 3 mmol), 4ꢀmethylhippuric acid (0.75 g, 3 mmol),
and potassium acetate (0.3 g, 3 mmol) in acetic anhydride (5 mL)
was refluxed for 2 h. A precipitate formed was filtered and washed
with ethanol to obtain compound 6 (0.389 g, 46%), yellow crysꢀ
tals, m.p. 186—187 C (Ref. 18: m.p. 186 C). 1H NMR (CDCl3),
: 8.18 (d, 2 H, Ar, J = 7.8 Hz); 8.04 (d, 2 H, Ar, J = 8.4 Hz);
7.31 (d, 2 H, Ar, J = 7.8 Hz); 7.24—7.18 (m, 3 H, Ar, HC=);
3.88 (s, 3 H, CH3O); 2.44 (s, 3 H, CH3). IR, /cm–1: 1792
(C=O), 1651 (C=N). Found (%): C, 73.66; H, 5.19; N, 4.62.
C18H15NO3. Calculated (%): C, 73.72; H, 5.12; N, 4.78.
(4Z)ꢀ2ꢀ(4ꢀFluorophenyl)ꢀ4ꢀ[(4ꢀmethoxyphenyl)methylidene]ꢀ
1,3ꢀoxazolꢀ5(4H)ꢀone (7) was obtained according to the proceꢀ
dure described earlier19 using potassium acetate. 1H NMR specꢀ
trum agrees with that reported in the literature (see Ref. 20,
supporting information).
Scheme 2
(4Z)ꢀ4ꢀ[(3,4ꢀDimethoxyphenyl)methylene]ꢀ2ꢀ(4ꢀfluoropheꢀ
nyl)ꢀ1,3ꢀoxazolꢀ5(4H)ꢀone (8). A mixture of 3,4ꢀdimethoxybenzꢀ
aldehyde (0.3 g, 2.5 mmol), 4ꢀfluorohippuric acid (0.5 g,
2.5 mmol), and potassium acetate (0.25 g, 2.5 mmol) in (CH3CO)2O
(5 mL) was refluxed for 3 h with stirring. A precipitate formed
was filtered and recrystallized from ethanol to obtain compound 8
(0.19 g, 21%), yellow crystals, m.p. 188—190 C. 1H NMR
(CDCl3), : 8.12—8.15 (m, 3 H, Ar); 7.57 (d, 1 H, Ar, J = 8.4 Hz);
7.18—7.23 (m, 3 H, Ar, HC=); 6.94 (d, 1 H, Ar, J = 8.4 Hz);
Reagents and conditions: i. MeNH2, K2CO3, heating.

Combretastatin analogs
Russ.Chem.Bull., Int.Ed., Vol. 64, No. 7, July, 2015
1563
3.01 (s, 3 H, CH3); 3.96 (s, 3 H, CH3). Found (%): C, 60.98;
H, 4.79; N, 3.93. C18H14NO4F•1.5H2O. Calculated (%): C, 61.02;
H, 4.84; N, 3.95.
4. R. Singh, H. Kaur, Synthesis, 2009, 15, 2471.
5. R. K. Gill, R. Kaur, G. Kaur, R. K. Rawal, A. K. Shah,
J. Bariwal, Curr. Org. Chem., 2014, 18, 2462.
(4Z)ꢀ2ꢀPhenylꢀ4ꢀ(phenylmethylidene)ꢀ1,3ꢀoxazolꢀ5(4H)ꢀone
(9) was obtained according to the procedure described earlier19
using potassium acetate. 1H NMR spectrum agrees with that
reported in the literatures (see Ref. 21, supporting information).
(5Z)ꢀ5ꢀ[(4ꢀMethoxyphenyl)methylidene]ꢀ3ꢀmethylꢀ2ꢀ(4ꢀmeꢀ
thylphenyl)ꢀ3,5ꢀdihydroꢀ4Hꢀimidazolꢀ4ꢀone (10). A 40% aqueous
solution of methylamine (5 mL) and potassium carbonate (0.1 g,
0.7 mmol) were added to a solution of oxazolone 6 (0.2 g,
0.7 mmol) in ethanol with stirring, the mixture was refluxed for
8 h with stirring. The solvent was evaporated, the residue was
subjected to chromatography (eluent ethyl acetate—light petroꢀ
leum ether (40—70 C), 1 : 3, then ethyl acetate) to obtain comꢀ
pound 10 (0.031 g, yield 15%), orange oily liquid, Rf = 0.53
(ethyl acetate—light petroleum ether, 1 : 3). 1H NMR (DMSOꢀd6),
: 7.85 (d, 2 H, Ar, J = 8.0 Hz); 7.51 (d, 2 H, Ar, J = 7.9 Hz);
6.90—7.05 (m, 4 H, Ar); 6.60 (s, 1 H, HC=); 3.89 (s, 3 H,
CH3O); 3.50 (s, 3 H, CH3N); 2.13 (s, 3 H, CH3). MS, m/z
(Irel (%)): 307.30 [M + H]+ (60). Found (%): C, 74.47; H, 5.87;
N, 9.12. C18H15NO3. Calculated (%): C, 74.49; H, 5.92; N, 9.14.
(5Z)ꢀ5ꢀ[(3,4ꢀDimethoxyphenyl)methylidene]ꢀ2ꢀ(4ꢀfluorophenꢀ
yl)ꢀ3ꢀmethylꢀ3,5ꢀdihydroꢀ4Hꢀimidazolꢀ4ꢀone (11) was syntheꢀ
sized similarly to compound 10 from oxazolone 8 (0.15 g,
0.4 mmol), 40% aqueous solution of methylamine (5 mL), and
potassium carbonate (0.1 g, 0.7 mmol) to obtain compound 10
(0.02 g, 12%), orange oily liquid, Rf = 0.6 (ethyl acetate—light
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1
petroleum ether, 1 : 3). H NMR (DMSOꢀd6), : 7.81 (d, 2 H,
Ar, J = 8.8 Hz); 7.48 (d, 1 H, Ar, J = 8.2 Hz); 7.38 (dd, 2 H, Ar,
J1 = 8.8 Hz, J2 = 1.0 Hz); 7.12 (s, 1 H, Ar); 7.04 (s, 1 H, HC=);
7.00 (d, 1 H, Ar, J = 8.2 Hz); 3.83 (s, 3 H, OCH3); 3.78 (s, 3 H,
OCH3); 3.45 (s, 3 H, NCH3). Found (%): C, 66.99; H, 5.01;
N, 8.22. C18H15NO3. Calculated (%): C, 67.05; H, 5.03; N, 8.23.
MTTꢀtest on cytotoxicity was carried out on human epitheꢀ
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the procedures described in the works.22,23
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This work was financially supported by the Russian
Foundation for Basic Research (Project No. 15ꢀ03ꢀ04894),
the Division of Chemistry and Material Sciences of the
Russian Academy of Sciences, and the German Academic
Exchange Service, DAAD.
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Received January 20, 2015;
in revised form April 24, 2015