Synthesis and antiproliferative activity of combretastatin derivatives with adamantane fragment

Article abstract of DOI:10.1007/s11172-015-1146-5

The work describes the synthesis of 2-adamantyl 7-[(2-{[(2E)-3-(3-hydroxy-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)prop-2-enoyl]amino}ethyl)amino]-7-oxoheptanoate and 7-[(2-{[(2E)-2-(3,4-dimethoxyphenyl)-3-(3-hydroxy-4-methoxyphenyl)prop-2-enoyl]amino}e

Full text of DOI:10.1007/s11172-015-1146-5

2248
Russian Chemical Bulletin, International Edition, Vol. 64, No. 9, pp. 2248—2252, September, 2015
Synthesis and antiproliferative activity
of combretastatin derivatives with adamantane fragment
a
a
a,b
c
a,b
E. V. Nurieva, N. A. Zefirov, N. S. Zefirov, S. A. Kuznetsov, and O. N. Zefirova
aM. V. Lomonosov Moscow State University, Department of Chemistry,
Build. 3, 1 Leninskie Gory, 119991 Moscow, Russian Federation.
Fax: +7 (459) 939 0290. Eꢀmail: olgaz@org.chem.msu.ru
b
Institute of Physiologically Active Compounds, Russian Academy of Sciences,
1
Severnyi prꢀd, 142432 Chernogolovka, Moscow Region, Russian Federation.
Eꢀmail: kolaz92@gmail.com
c
Institute of Biological Sciences, University of Rostock,
1
8106 Rostock, Germany.
Eꢀmail: sergei.kuznetsov@uniꢀrostock.de
The work describes the synthesis of 2ꢀadamantyl 7ꢀ[(2ꢀ{[(2E)ꢀ3ꢀ(3ꢀhydroxyꢀ4ꢀmethoxyꢀ
phenyl)ꢀ2ꢀ(3,4,5ꢀtrimethoxyphenyl)propꢀ2ꢀenoyl]amino}ethyl)amino]ꢀ7ꢀoxoheptanoate and
7
ꢀ[(2ꢀ{[(2E)ꢀ2ꢀ(3,4ꢀdimethoxyphenyl)ꢀ3ꢀ(3ꢀhydroxyꢀ4ꢀmethoxyphenyl)propꢀ2ꢀenoyl]amino}ꢀ
ethyl)amino]ꢀ7ꢀoxoheptanoate. The latter compound exhibits moderate cytotoxicity
(
EC50 = 4.8 mol L–1) against the human epithelial lung carcinoma cells A549.
Key words: combretastatin, adamantane, colchicine, tubuloclustin, tubulin, cytotoxicity,
carcinoma A549.
Earlier,1—3 in the search for compounds with antiꢀ
tumor activity we obtained C(7)ꢀsubstituted derivatives of
natural colchicine, among which were compounds (for
example, tubuloclustin (1)) with very high cytotoxicity
3
,4
against different types of tumor cells in vitro.
Attempted replacement of the colchicine moiety in
compounds of the type 1 with other analogues possessing
lower general toxicity, such as nocodazole and 2ꢀmethoxyꢀ
extradiol, was unsuccessful because of either synthetic
difficulties or instability and inactivity of the comꢀ
pounds obtained. The purpose of the present work is the
synthesis and biotesting of analogues of 1 based on the
natural combretastatin Aꢀ4 (CAꢀ4), whose antitumor acꢀ
tivity is also related to the interaction with the colchicine
binding site with its cell target protein tubulin.9
Colchicine fragment
5
,6
7
8
According to a pharmacophore model of the tubulin—
colchicine site ligand complexes,1
0,11
the nitrogen atom
of the colchicine amide group is on a certain distance
~3—3.5 Å) from both carbon atoms of the combretastaꢀ
(
tin double bond. This necessitates the elongation of the
linker connecting the adamantane core with CAꢀ4, as
compared to that of tubuloclustin (1). Among known CAꢀ4
derivatives with substituents at the double bond carbon
atoms appropriate for modification, the ability to cause
the effect typical of colchicine,12 viz., the inhibition of
tubulin polymerization approximately in the same conꢀ
centrations as for colchicine, was confirmed only for comꢀ
R = H (CAꢀ4), C(O)NH2 (2)
pound 2. Therefore, we decided to synthesize compounds
3a,b, in which a substituted adamantane group is attached
to the amide group of 2. We assumed that the introduction
of two additional (as compared to 1) methylene groups
into the structures 3a,b would provide the linker elongaꢀ
tion mentioned above. In these studies, 3b was the target
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 9, pp. 2248—2252, September, 2015.
066ꢀ5285/15/6409ꢀ2248 © 2015 Springer Science+Business Media, Inc.
1

Adamanatane combretastatin derivatives
Russ.Chem.Bull., Int.Ed., Vol. 64, No. 9, September, 2015 2249
R = H (3a), OMe (3b)
compound, whereas 3a was used as a model for the studies
of the role of trimethoxyphenyl group in 3b in exhibiting
cytotoxic properties.
of monoprotected NꢀBocꢀethylenediamine (5) and pimelic
13
polyanhydride gave amide 6. The C NMR spectrum of
this compound exhibits three signals characteristic of the
carbonyl carbon atoms: the tertꢀbutoxycarbonyl, the carbꢀ
oxy, and the amide ones at  157.02, 174.25, and 177.29,
respectively. Then, pimelic acid monoamide 6 was esteriꢀ
The starting combretastatin derivatives 4a and 4b were
obtained by Perkin condensation1
2,13
from 3,4ꢀdimethoxyꢀ
or 3,4,5ꢀtrimethoxyphenylacetic acid and 3ꢀhydroxyꢀ4ꢀ
methoxybenzaldehyde in the presence of acetic anhydride
and triethylamine (Scheme 1).
The synthesis of target compounds 3a,b was carried
out according to Scheme 2. In the first step, the reaction
1
fied with adamantanꢀ2ꢀol to ester 7. In the H NMR specꢀ
trum of compound 7, the signal for the proton at atom
C(2) of the adamantane group is downfield shifted ( 4.89)
as compared to the starting adamantanꢀ2ꢀol ( 3.83). A simꢀ
Scheme 1
R = H (4a, 34%), OMe (4b, 43%)
i. NEt , Ac O, heating.
3
2
Scheme 2

2
250
Russ.Chem.Bull., Int.Ed., Vol. 64, No. 9, September, 2015
Nurieva et al.
ilar shift is also observed for atom C(2) in the 13C NMR
spectrum ( 76.82 for 7 and  74.52 for Adꢀ2ꢀOH).
It should be noted that an alternative order of assemꢀ
bling molecule 7, namely, the addition of NꢀBocꢀethylꢀ
enediamine to monoester of adamantanꢀ2ꢀol and pimelic
results using computer molecular modeling. The results of
the automatic docking of ligand 3b into the 3D model of
1
0
tubulin in the colchicine binding site using the CLC
Drug Discovery Workbench program showed that in one
of the most favorable variants of arrangement of strucꢀ
ture 3b in the colchicine site (among several different variꢀ
ants with similar to each other and to compound 1 scorꢀ
ing functions), the combretastatin group of 3b was placed
into the region similar to that occupied by free CAꢀ4
(Fig. 1).
3
acid 8 (obtained according to procedure ) in the presence
of 2ꢀethoxyꢀNꢀethoxycarbonylꢀ1,2ꢀdihydroquinoline
(
EEDQ) was preparatively unsuccessful because of very
low yield of product 7 (see Experimental).
The protecting group in compound 7 was removed
according to a standard procedure, the resulting amine 9
was involved in the reaction with combretastatin carboxy
The binding of 3b with protein involves both methoxy
groups in metaꢀpositions of the aromatic ring. The oxygen
atom of one of them forms a hydrogen bond with the
hydrogen atom of the amide group of the main chain
Leu255, whereas the methyl group of the other one is
involved in hydrophobic interactions with the side chains
Ala354, Ala316, Val318 and the alkyl fragment of the
side chain Lys352. This explains a decrease in the activiꢀ
ty for analogue 3b without one metaꢀmethoxy substituent
in the aromatic ring (3a). A decrease in cytotoxicity of
conjugate 3b as compared to tubuloclustin (1) can be exꢀ
plained by a noticeable difference in the position of free
CAꢀ4 and that in conjugate 3b (see Fig. 1), apparently,
caused either by too high conformational "rigidity" or too
long diamide chain of 3b. Therefore, the further search for
the tubulin ligands of the type under consideration should
be focused on the analogues of 3b with more "flexible" and
shorter group between the CAꢀ4 fragment and the substiꢀ
tuted adamantane core. The approaches to the synthesis
of such compounds are currently under study.
13
derivatives 4a,b in the presence of EEDQ. The C NMR
spectra of the target products exhibit the signals for three
carbonyl carbon atoms in the range of  168—174 and the
signals for the aromatic atoms of combretastatin fragment
1
within  106—155. The H NMR spectroscopy and eleꢀ
mental analysis data also confirm the structure of comꢀ
pounds 3a and 3b.
The biotesting of conjugates 3a and 3b in the standard
colorimetric MTT test1 against the culture of human
lung carcinoma cells A549 showed that compound 3b
possesses a noticeable, but a moderate cytotoxicity:
4
–
1
EC50 = 4.8± 0.1 mol L
(for colchicine, EC50 =
–
1
=
0.04±0.09 mol L ). Compound 3a is low active
(
EC50 > 50 mol L– ).
1
Based on the assumption that the obtained values of
cytotoxicity are determined only by the binding with proꢀ
tein (without considering metabolism, the ease of peneꢀ
tration into the cell, etc.), we attempted to explain the
Fig. 1. One of the most energetically favorable variants of the arrangement of structure of 3b in the tubulin dimer (the ꢀsubunit is on
the left) according to the results of automatic docking (CLC Drug Discovery Workbench). The molecule of free combretastatin Aꢀ4 is
shown for comparison, hydrogen bonds are depicted in dashed lines (hydrogen atoms are omitted).

Adamanatane combretastatin derivatives
Russ.Chem.Bull., Int.Ed., Vol. 64, No. 9, September, 2015 2251
Experimental
(0.494 g, 2.4 mmol), adamantanꢀ2ꢀol (0.357 g, 2.3 mmol), and
a catalytic amount of DMAP were added to a solution of acid 6
(0.638 g, 2.1 mmol) in CH Cl (10 mL). The reaction mixture
was stirred for 20 h at 20 C, concentrated, diluted with ethyl
acetate (10 mL), and allowed to stand for 3 h at 0—5 C. The
crystals formed were filtered off, the solution was concentrated,
the residue was subjected to chromatography (eluent ethyl aceꢀ
tate—light petroleum ether (40—60 C), 1 : 1) to obtain comꢀ
pound 7 (0.187 g, 21%), a colorless oily liquid.
Automatic docking into the 3D model of the tubulin comꢀ
plex with NꢀdeacetylꢀNꢀ(2ꢀmercaptoacetyl)colchicine (a predetꢀ
ermined radius 16 Å) was performed using the CLC Drug Disꢀ
covery Workbench program (Version 1.5): Evaluation license
2014).
Reaction progress and purity of compounds were monitored
2
2
(
by TLC on SilufolꢀUV254 plates. Chromatographic separation
was carried out on a column with Acros silica gel (40—60 m).
H and C NMR spectra were recorded on a Bruker Avance 400
Method B. A solution of 7ꢀ(2ꢀadamantyloxy)ꢀ7ꢀoxoheptanoic
acid 8 (0.298 g, 0.001 mol), amine 5 (0.131 mL, 0.83 mmol),
EEDQ (0.329 g, 1.33 mmol) in CH Cl (5 mL) was stirred for
1
13
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 CHNꢀ
analyzer. IR spectra were recorded on a IRꢀ200 ThermoNicolet
spectrophotometer in KBr pellets.
2
2
24 h at 20 C; the mixture was concentrated, the residue was
subjected to chromatography (eluent ethyl acetate—light petroꢀ
leum ether (40—60 C), 1 : 2; then methanol—dichloromethane,
1
1 : 15) to obtain compound 7 (0.020 g, 5%). H NMR (CDCl ),
3
t
(
2E)ꢀ3ꢀ(3ꢀHydroxyꢀ4ꢀmethoxyphenyl)ꢀ2ꢀ(3,4ꢀdimethoxyꢀ
phenyl)acrylic acid (4a). 3,4ꢀDimethoxyphenylacetic acid (1.73 g,
.82 mmol) was added to a solution of 3ꢀhydroxyꢀ4ꢀmethoxyꢀ
: 1.38 (m, 2 H, C H ); 1.46 (s, 9 H, Bu ); 1.56—1.59 (m, 2 H,

2
Ad); 1.64—1.72 (m, 4 H, C H ); 1.75—1.80 (m, 4 H, Ad); 1.85

2
8
(m, 4 H, Ad); 1.89—2.03 (m, 4 H, Ad); 2.16 (t, 2 H, C H ,

2
3
3
benzaldehyde (0.67 g, 4.4 mmol) in Ac O (4 mL, 42.4 mmol)
Ja = 7.8 Hz); 2.31 (t, 2 H, C H , J = 7.4 Hz); 3.25 (m, 2 H,
2
 ,
2
and NEt (2 mL, 14.29 mmol). The mixture was stirred for 6 h at
BocNH—CH ); 3.33 (m, 2 H, CH NHC=O); 4.89 (m, 1 H,
CAd(2)H); 5.21 (br.s, 1 H, NH); 6.49 (br.s, 1 H, NH). C NMR
3
2
2
13
1
20 C, poured onto ice (20 g), and acidified with concentrated
HCl. A precipitate was filtered, washed with cold CH Cl , and
(CDCl ), : 24.76, 25.28, 26.98, 27.21, 28.35 (Me); 28.71
2
2
3
recrystallized from EtOH to obtain compound 4a (0.5 g, 34%),
(ꢀCH ); 31.77 (CAd); 31.86 (CAd); 34.61 (CH CO ); 36.31 (CAd);
2
2
2
1
yellow crystals, m.p. 173—175 C. H NMR (CDCl ), : 3.83
36.39 (CH CONH); 37.36, 40.29 (CH NHCO); 40.68
2 2
3
t
(
s, 3 H, Me); 3.88 (s, 3 H, Me); 3.94 (s, 3 H, Me); 6.69 (s, 1 H,
(CH NHBoc); 76.82 (C (2)); 79.60 (CMe ); 158.18 (CO Bu );
2 Ad 3 2
C (2)H); 6.70 (m, 2 H, C (5)H and C (6)H); 6.77 (d, 1 H,
173.10 (C=O); 173.63 (C=O). Found (%): C, 66.09; H, 9.19;
N, 6.40. C H N O . Calculated (%): C, 66.03; H, 9.23; N, 6.42.
Ar
Ar
Ar
CAr(2)H, J = 1.7 Hz); 6.83 (dd, 1 H, C (5)H, J = 8.4 Hz,
Ar
24 40
2
5
J = 1.7 Hz); 6.93 (d, 1 H, CAr(6)H, J = 8.4 Hz); 7.83 (s, 1 H,
2ꢀAdamantyl 7ꢀ(2ꢀaminoethylamino)ꢀ7ꢀoxoheptanoate (9).
Trifluoroacetic acid (0.5 mL, 6.52 mmol) was added to protectꢀ
13
C=CH). C NMR (DMSOꢀd ), : 55.87 (Me); 55.93 (Me);
6
5
5.98 (Me); 111.99, 112.31, 113.68, 117.61, 122.23 (CAr(6));
ed amine 7 (0.187 g, 0.429 mmol) in CH Cl2 (2.5 mL). The
2
1
23.26 (C (6)); 127.74 (C(2)); 129.34 (C (1)); 130.77 (C (1));
mixture was stirred for 3 h at 20 C and concentrated. The resiꢀ
Ar
Ar
Ar
1
1
1
3
39.39 (C(3)); 146.29 (C (3)); 146.78 (C (3)); 148.63 (C (4));
due was diluted with CH Cl (10 mL), washed with saturated
Ar
Ar
Ar
2
2
–
1
49.18 (CAr(4)); 169.29 (C=O). IR (KBr), /cm : 808, 1024,
136—1144, 1255, 1439, 1514, 1603, 1666 (C=O), 2837, 2953,
417 (OH). Found (%): C, 65.52; H, 5.51. C H O . Calculatꢀ
aqueous NaHCO , dried with MgSO , and concentrated to obꢀ
3 4
1
tain compound 9 (0.155 g, 95%), a colorless oily liquid. H NMR
(CDCl ), : 1.36 (m, 2 H, C H ); 1.38—1.48 (br.s, 2 H, NH );
18
18
6
3

2
2
ed (%): C, 65.45; H, 5.49.
1.55 (m, 2 H, Ad); 1.65 (m, 4 H, 2 C H ); 1.72—1.76 (m, 4 H,

2
(
2E)ꢀ3ꢀ(3ꢀHydroxyꢀ4ꢀmethoxyphenyl)ꢀ2ꢀ(3,4,5ꢀtrimethoxyꢀ
Ad); 1.82 (m, 4 H, Ad); 1.96—2.00 (m, 4 H, Ad); 2.19 (t, 2 H,
3
phenyl)acrylic acid (4b) was obtained according to the procedure
described earlier.12 The yield was 43%, yellow crystals, m.p.
38—240 C (cf. Ref. 13: m.p. 237—239 C). Spectral data agree
with those given in the literature.
CH CONH, J = 7.6 Hz); 2.33 (m, 2 H, CH CO ); 2.84 (t, 2 H,
2
,
2
2
3
3
CH NH , J = 5.5 Hz); 3.30 (m, 2 H, CH NHCO, J = 5.5 Hz);
4.90 (m, 1 H, CAd(2)H); 6.09 (br.s, 1 H, NH). C NMR
2
2
2
1
3
2
1
2
(CDCl ), : 24.77, 25.53, 26.97, 27.20, 28.70, 31.76 (Ad); 31.87
3
7
ꢀ[2ꢀ(tertꢀButoxycarbonylamino)ethylamino]ꢀ7ꢀoxoheptanoꢀ
(Ad); 34.6, 36.31 (Ad); 36.41, 37.36, 41.24 (CH NH ); 41.55
2
2
ic acid (6). Amine 5 (0.64 g, 4 mmol) was added to a solution of
(CH NH); 76.83 (CAd(2)H); 173.14 (C=O); 173.39 (C=O).
2
pimelic anhydride (0.715 g, 5 mmol) in CH Cl (10 mL). After
Found (%): C, 67.76; H, 9.64; N, 8.39. C H N O . Calculatꢀ
2
2
19 32
2
3
stirring the mixture for 24 h at 20 C, it was diluted with Et O
ed (%): C, 67.82; H, 9.59; N, 8.33.
2
(
15 mL) and washed with water. The organic fraction was dried with
2ꢀAdamantyl 7ꢀ{2ꢀ[(2E)ꢀ2ꢀ(3,4ꢀdimethoxyphenyl)ꢀ3ꢀ(3ꢀhydrꢀ
oxyꢀ4ꢀmethoxyphenyl)propꢀ2ꢀenoylamino]ethylamino}ꢀ7ꢀoxoꢀ
heptanoate (3a) Acid 4a (0.030 g, 0.09 mmol) and EEDQ (0.15
g, 0.607 mmol) were added to a solution of amine 9 (0.123 g,
0.366 mmol) in CH Cl . The mixture was stirred for 24 h at
MgSO and concentrated, the residue was subjected to chromatoꢀ
graphy (eluent ethyl acetate—light petroleum ether (40—60 C),
1
pound 6 (0.638 g, 53%), a colorless oily liquid. H NMR
(
C H ); 2.19 (m, 2 H, C H ); 2.32 (m, 2 H, C H ); 3.29 (m, 2 H,
CH NHCO); 3.32 (m, 2 H, CH NHBoc); 5.38 (br.s, 1 H, NH);
6
(
4
4
: 1; then methanol—dichloromethane, 1 : 10) to obtain comꢀ
1
2
2
t
CDCl ), : 1.35 (m, 2 H, C H ); 1.41 (s, 9 H, Bu ); 1.62 (m, 4 H,
20 C and concentrated, the residue was subjected to chromatoꢀ
graphy (eluent ethyl acetate—light petroleum ether (40—60 C),
1 : 1; then methanol—dichloromethane, 1 : 10) to obtain comꢀ
pound 3a (0.050 g, 52%), a white solid substance, m.p. 130—135 C.
3

2

2

2

2
2
2
13
.80 (br.s, 1 H, NH). C NMR (CDCl ), : 24.37, 25.25, 28.36
3
1
Me); 28.52, 33.83 (C ); 36.02 (CH CONH); 36.21 (CH NHCO);
H NMR (CDCl ), : 1.35 (m, 2 H, C H ); 1.53—1.56 (m, 2 H,

2
2
3

2
t
0.16 (CH NHBoc); 79.62 (CMe ); 157.02 (CO Bu ); 174.25
Ad); 1.62—1.68 (m, 4 H, 2 C H ); 1.73—1.77 (m, 4 H, Ad); 1.82

2
2
3
2
(
CO H); 177.29 (CONH). Found (%): C, 55.60; H, 8.69; N, 9.25.
(m, 4 H, Ad); 1.97—2.00 (m, 4 H, Ad); 2.17 (t, 2 H, C H ,
J, = 7.6 Hz); 2.32 (t, 2 H, C H , J = 7.6 Hz); 3.34 (m, 2 H,
 ,
2
2

2
3
3
C H N O . Calculated (%): C, 55.61; H, 8.67; N, 9.26.
14
26
2
5
2
ꢀAdamantyl 7ꢀ[2ꢀ(tertꢀbutoxycarbonylamino)ethylamino]ꢀ7ꢀ
BocNHCH ); 3.43 (m, 2 H, CH NHC=O); 3.83 (s, 6 H,
2
2
oxoheptanoate (7). Method A. 1,3ꢀDicyclohexylcarbodiimide
2 OMe); 3.95 (s, 3 H, OMe); 4.90 (m, 1 H, CAd(2)H); 5.99 (s, 1 H,

2
252
Russ.Chem.Bull., Int.Ed., Vol. 64, No. 9, September, 2015
Nurieva et al.
OH); 6.02 (br.s, 1 H, NH); 6.53 (br.s, 1 H, NH); 6.57 (dd, 1 H,
Ar, J = 8.7 Hz, J = 1.7 Hz); 6.65 (d, 1 H, Ar, J = 8.7 Hz); 6.67
The authors are grateful to Yu. M. Rumyantseva and
D. V. Shishov.
This work was financially supported by the Russian
Foundation for Basic Research (Project Nos 15ꢀ03ꢀ04894
and 13ꢀ03ꢀ12460) and the Russian Academy of Sciences.
(
d, 1 H, Ar, J = 1.8 Hz); 6.72 (d, 1 H, Ar, J = 1.7 Hz); 6.80 (dd,
1
7
2
3
5
1
1
H, Ar, J = 8.2 Hz, J = 1.7 Hz); 6.96 (d, 1 H, Ar, J = 8.2 Hz);
.72 (s, 1 H, C=CHAr). 13C NMR (CDCl ), : 24.81, 25.31,
3
6.95, 27.19, 28.73, 31.75, 31.83, 34.61, 36.29, 36.36, 37.35,
9.83 (NH—CH ), 40.41 (NH—CH ), 55.78 (Me); 55.87 (Me),
2
2
5.99 (Me), 76.79 (CAd(2)), 110.36, 112.12, 112.60; 116.82,
22.14, 123.26, 127.74, 128.10, 131.63, 137.24, 145.24, 147.51,
49.18, 149.91, 169.12 (C=O), 173.11 (C=O), 173.6 (C=O).
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1
2
3
4
5
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Found (%): C, 68.45; 7.38; N, 4.35. C H N O . Calculatꢀ
3
7
48
2
8
ed (%): C, 68.50; H, 7.46; N, 4.32.
ꢀAdamantyl 7ꢀ{2ꢀ[(2E)ꢀ3ꢀ(3ꢀhydroxyꢀ4ꢀmethoxyphenyl)ꢀ2ꢀ
3,4,5ꢀtrimethoxyphenyl)propꢀ2ꢀenoylamino]ethylamino}ꢀ7ꢀoxoꢀ
2
(
heptanoate (3b) was obtained similarly to compound 3a from
amine 9 (0.123 g, 0.366 mmol), acid 4b (0.16 g, 0.444 mmol),
and EEDQ (0.15 g, 0.607 mmol). The yield was 0.037 g (16%),
1
a white waxꢀlike mass. H NMR (CDCl ), : 1.33—1.38 (m, 2 H,
3
C H ); 1.53—1.56 (m, 2 H, Ad); 1.62—1.65 (m, 4 H, C H );

2

2
1
.73—1.77 (m, 4 H, Ad); 1.82 (m, 4 H, Ad); 1.92—2.0 (m, 4 H,
3
Ad); 2.15—2.18 (t, 2 H, C H , J = 7.5 Hz); 2.31 (t, 2 H,
C H , J = 7.1 Hz); 3.34 (m, 2 H, BocNH—CH ); 3.44 (m, 2 H,

2
,
3

2
,
2
CH —NHC=O); 3.81 (s, 6 H, OMe); 3.84 (s, 3 H, OMe); 3.93
2
(
6
s, 3 H, OMe); 4.90 (m, 1 H, CAd(2)H); 6.05 (br.s, 2 H, 2 NH);
.45 (s, 2 H); 6.52 (m, 1 H); 6.55 (dd, 1 H, Ar, J = 8.4 Hz,
2
013, 68, 37 [Vestn. Mosk. unꢀta. Ser. 2. Khim., 2013, 54, 45].
J = 1.9 Hz); 6.65 (d, 1 H, Ar, J = 8.4 Hz); 6.69 (d, 1 H, Ar,
6
7
. N. S. Zefirov, Ya. S. Glazkova, I. V. Kuznetsova, E. V. Nuꢀ
rieva, O. N. Zefirova, Moscow University Chem. Bull., 2015,
J = 1.9 Hz); 7.7 (s, 1 H, C=CHAr). 13C NMR (CDCl ), : 24.8,
3
2
5.3, 26.97, 27.2, 28.74, 31.76, 36.3, 37.37, 39.9 (NH—CH );
2
7
0, 69 [Vestn. Mosk. unꢀta. Ser. 2. Khim., 2015, 56, 85].
4
0.45 (NH—CH ); 55.81 (Me); 56.27 (2 Me), 61.03 (Me); 76.81
2
. O. N. Zefirova, E. V. Nurieva, Ya. S. Glazkova, N. A. Zeꢀ
firov, A. V. Mamaeva, B. Wobith, V. I. Romanenko, N. A.
Lesnaya, E. M. Treschalina, S. A. Kuznetsov, Pharm. Chem. J.
(
1
(
CAd(2)); 106.56, 110.37, 116.81, 123.26, 127.92, 131.13, 131.80,
37.22, 138.18, 145.26, 147.54, 154.33, 168.72 (C=O); 173.09
C=O); 173.57 (C=O). IR (KBr), /cm–1: 1128, 1240, 1257,
(Engl. Transl.), 2014, 48, 373 [Khim. Farm. Zh., 2014, 48, 19].
1
412, 1452, 1512, 1581, 1651, 1725 (C=O); 2854, 2927,
8
9
. O. N. Zefirova, Ya. S. Glazkova, E. V. Nurieva, N. A. Zeꢀ
firov, A. V. Mamaeva, B. Wobith, N. S. Zefirov, S. A. Kuzꢀ
netsov, Russ. Chem. Bull. (Int. Ed.), 2014, 63, 1126 [Izv.
Akad. Nauk, Ser. Khim., 2014, 1126].
3
369 (OH and NH). Found (%): C, 67.20; H, 7.38; N, 4.07.
C H N O . Calculated (%): C, 67.24; H, 7.42; N, 4.13.
3
8
50
2
9
MTTꢀtest on cytotoxicity. The human epithelial lung carciꢀ
noma cells (line Aꢀ549, CCLꢀ185) were seeded in 96ꢀwell plates
~3000 cells per well) in DMEM culture medium (200 L) at 37 C.
. N. H. Nam, Curr. Med. Chem., 2003, 10, 1697.
(
1
1
0. T. L. Nguyen, C. McGrath, A. R. Hermone, J. C. Burnett,
D. W. Zaharevitz, B. W. Day, P. Wirf, E. Hamel, R. Gussio,
J. Med. Chem., 2005, 48, 6107.
1. O. N. Zefirova, A. G. Diikov, N. V. Zyk, N. S. Zefirov, Russ.
Chem. Bull. (Int. Ed.), 2007, 56, 680 [Izv. Akad. Nauk, Ser.
Khim., 2007, 655].
Solutions of tested compounds (or colchicine as a positive conꢀ
trol) in DMSO were added into the wells in the concentration
range 0.005—50 mol L–1 (eight wells for each concentration)
and this was matured for 24 h. A solution of 3ꢀ(4,5ꢀdimethylthiꢀ
azolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide (MTT) (5 mg mL–
1
)
was prepared in a phosphate buffer, filtered through a 0.22ꢀmm
filter, and 2 h before the end of maturing the tested compound,
the solution of MTT (20 L) was added into each well so that the
final concentration would be 0.45 mg mL– . After removal of
the cell medium, a solution of DMSO containing 10% of sodium
dodecylsulfate and 0.6% of acetic acid (100 L) was added into
each well, the crystals of formazane formed were solubilized by
stirring, using a shaker. Optical density was measured at 590 nm
with a 690ꢀnm reference filter on a EL808 Ultra Microplate
Reader instrument (BioꢀTek Instruments, USA).
1
1
1
2. C. Borrel, S. Thoret, X. Cachet, D. Guénard, F. Tillequin,
M. Koch, S. Michel, Bioorg. Med. Chem., 2005, 13, 3853.
3. K. Gaukroger, J. A. Hadfield, L. A. Hepworth, J. Org. Chem.,
1
2
001, 66, 8135.
4. T. Mosmann, J. Immunol. Methods, 1983, 65, 55.
Received April 16, 2015;
in revised form June 1, 2015