Adamantane acid esters with alkoxyaryl alcohols: Synthesis, antiproliferative activity, and influence on microtubule network of tumor cells

Article abstract of DOI:10.1007/s11172-017-1915-4

Adamantaneacetic and adamantanecarboxylic acid esters containing 3-hydroxy-4-methoxybenzyl, 3,4,5-trimethoxybenzyl, or 5-(hydroxymethyl)-2-methoxyphenyl groups were synthesized as unusual analogs of natural antitumor and anti-tubulin agents combretastatin A-4 and 2-methoxyestradiol. The compounds were found to possess noticeable cytotoxicity to epithelial human carcinoma cell line A549 (EC₅₀ = 4.3—81 μmol L^–1). An ability to cause complete depolymerization of microtubule network of A549 cells was demonstrated for 5-(hydroxymethyl)-2-methoxyphenyl adamantan-1-ylacetate (6a) at a concentration of 100 μmol L^–1. Ester 6a belongs to a new structural type, which is unusual for the ligands of the tubulin colchicine domain, and is an interesting lead compound for further structural optimization.

Full text of DOI:10.1007/s11172-017-1915-4

Russian Chemical Bulletin, International Edition, Vol. 66, No. 8, pp. 1503—1509, August, 2017
1503
Adamantane acid esters with alkoxyaryl alcohols:
synthesis, antiproliferative activity, and influence on microtubule network
of tumor cells*
N. A. Zefirov,a E. V. Nurieva,a Yu. A. Pikulina,b A. V. Ogon´kov,c B. Wobith,c S. A. Kuznetsov,c and O. N. Zefirovaa,d
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
bM. V. Lomonosov Moscow State University, Department of Fundamental Medicine,
Korp. 1, 27 Lomonosovsky prosp., 119991 Moscow, Russian Federation.
Eꢀmail: pikulina8053@gmail.com
cInstitute of Biological Sciences, University of Rostock,
18106 Rostock, Germany.
Eꢀmail: sergei.kuznetsov@uni_rostock.de
dInstitute of Physiologically Active Compounds, Russian Academy of Sciences,
1 Severnyi prꢀd, 142432 Chernogolovka, Moscow Region, Russian Federation.
Eꢀmail: kolaz92@gmail.com
Adamantaneacetic and adamantanecarboxylic acid esters containing 3ꢀhydroxyꢀ4ꢀmethꢀ
oxybenzyl, 3,4,5ꢀtrimethoxybenzyl, or 5ꢀ(hydroxymethyl)ꢀ2ꢀmethoxyphenyl groups were synꢀ
thesized as unusual analogs of natural antitumor and antiꢀtubulin agents combretastatin Aꢀ4
and 2ꢀmethoxyestradiol. The compounds were found to possess noticeable cytotoxicity to
epithelial human carcinoma cell line A549 (EC50 = 4.3—81 μmol L–1). An ability to cause
complete depolymerization of microtubule network of A549 cells was demonstrated for
5ꢀ(hydroxymethyl)ꢀ2ꢀmethoxyphenyl adamantanꢀ1ꢀylacetate (6a) at a concentration of
100 μmol L–1. Ester 6a belongs to a new structural type, which is unusual for the ligands of the
tubulin colchicine domain, and is an interesting lead compound for further structural optimiꢀ
zation.
Key words: adamantane, combretastatin Aꢀ4, 2ꢀmethoxyestradiol, colchicine domain of
tubulin, depolymerization of microtubules, antiproliferative activity.
When developing the design of structures for pharmaꢀ
ceutical agents, sometimes cage and bridging groups are
used to replace aromatic fragments1—4 or polycyclic skelꢀ
etons of parent molecules.1,2,5—8 The replacement of
a planar aromatic ring with the nonplanar cage core is
usually directed at maximization of hydrophobic conꢀ
tacts in the target protein cavity,9,10 while the replaceꢀ
ment of a polycyclic system, as a rule, is aimed at simpliꢀ
fication of the original molecule structure2,7 or improveꢀ
ment of its pharmacokinetic properties.4
to their ability to interact with the colchicine domain of
cell protein tubulin and inhibit its polymerization to
microtubules.11—13
In the present work, we consider a possibility of using
a cage group (adamantane) for the development of unꢀ
usual analogs of known natural antitumor agents comꢀ
bretastatin Aꢀ4 (CAꢀ4) and 2ꢀmethoxyestradiol (2ꢀME).
The anticancer activity of these compounds is largely due
To date, neither combretastatin Aꢀ4, nor 2ꢀmethoxyꢀ
estradiol, nor their numerous derivatives are used in antiꢀ
cancer therapy, and only a few of them are at different
stages of clinical trials.14 This requires the use of nonꢀ
standard approaches to the development of structural
analogs of the molecules under consideration, for example,
through the modification of their skeletons or the introꢀ
duction of unusual substituents. It is however important
* Based on the Materials of XX Mendeleev Congress on General
and Applied Chemistry (September 26—30, 2016, Ekaterinꢀ
burg, Russia).
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 8, pp. 1503—1509, August, 2017.
1066ꢀ5285/17/6608ꢀ1503 © 2017 Springer Science+Business Media, Inc.

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Zefirov et al.
that in the course of such modifications these compounds
retain their ability to cause depolymerization of the microꢀ
tubule network of tumor cells.
To test whether in principle such substances can inꢀ
hibit the polymerization of tubulin, we have suggested
a series of six readily available esters 4a,b—6a,b for synꢀ
thesis and biotesting (Scheme 2). In these compounds,
the alkoxyaryl groups are attached to adamantane through
a linker with the length equal to or close to that in moleꢀ
cule 1 (which has better antitubulin activity than CAꢀ4).15
It was assumed that the greater than in the original comꢀ
pounds 1 and 2 flexibility of the chain linking the aryl
ring to the adamantane core will provide the latter with
the opportunity to adopt the optimal position in the protein.
As aromatic fragments, we have chosen methoxypheꢀ
nyl (4a,b) and trimethoxyphenyl groups (5a,b) present in
the combretastatin and/or 2ꢀmethoxyestradiol molecule,
as well as 5ꢀ(hydroxymethyl)ꢀ2ꢀmethoxyphenyl group
(6a,b). In compounds 6a,b, the adamantane framework
is attached through the linker to the phenol hydroxyl of
5ꢀ(hydroxymethyl)ꢀ2ꢀmethoxyphenol (in contrast to esꢀ
ters 4a,b with the attachment of the alcohol group to
atom C(5)).
In the process of construction of new structural anaꢀ
logs of CAꢀ4 and 2ꢀME, we relied on the known data on
high antitubulin activity and cytotoxicity of CAꢀ4 vinylog,
namely, compound 1 (see Ref. 15), as well as on that of
CAꢀ4 derivatives, in which ring B of the parent molecule
is replaced with the unsubstituted naphthalene core (2)
or with benzothiophene attached through the atom C(2)
of thiophene16 (Scheme 1). Both cytotoxicity and antituꢀ
bulin activity are also retained by 2ꢀmethoxyestradiol deꢀ
rivatives without hydroxyl group at atom C(17) (3).13
Comparison of these facts leads to the conclusion that for
the efficient binding to the colchicine domain of tubulin,
it is sufficient to have in the structure a specifically subꢀ
stituted aromatic moiety in combination with a bulky
lipophilic fragment (although, according to the classical
pharmacophore model of the ligand of the colchicine doꢀ
main, rings A in CAꢀ4 and in 2ꢀME have different locaꢀ
tions in the protein17). The presented data led to the idea
to develop new structures through the combination of
alkoxyaryl groups with a lipophilic adamantane cage, reꢀ
placing the aromatic fragment in the structures 1 and 2 or
the polycyclic skeleton in the structure 3. Note that this
structural type is not characteristic of tubulin ligands and
have not been studied previously for this purpose.
The synthesis of all the suggested compounds was carꢀ
ried out similarly (see Scheme 2), namely, by esterificaꢀ
tion of adamantaneacetic (7a) and adamantanecarboxylꢀ
ic (7b) acids with protected alcohol 8 (see Ref. 18), or
with (3,4,5ꢀtrimethoxyphenyl)methanol, or 5ꢀ(hydroxyꢀ
methyl)ꢀ2ꢀmethoxyphenol in the presence of N,N´ꢀdiꢀ
cyclohexylcarbodiimide (DCC) and 4ꢀN,Nꢀdimethylꢀ
aminopyridine (DMAP).
1
In the H NMR spectra of the intermediate esters 9a
Scheme 1
and 9b, the signals for the protons of the methylene group
are downfield shifted (δ 5.00 in 9a and δ 5.01 in 9b) as
compared to that for alcohol 8 (δ 4.57). The protecting
group in compounds 9a,b was removed according to the
standard procedure in the presence of 1,8ꢀdiazabicycloꢀ
[5.4.0]undecꢀ7ꢀene (DBU). The composition and the
structure of the target compounds 4a,b, 5a,b, and 6a,b
were confirmed by NMR and IR spectroscopy, mass spectroꢀ
metry, and elemental analysis (see Experimental section).
The biotesting of the series of synthesized compounds
on human lung carcinoma cells A549 in a standard test
with 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazole
hydrobromide (MTT)19 showed (Table 1) that all the
compounds possess a noticeable cytotoxicity, although
less than that of CAꢀ4 and 2ꢀME. Compounds 4b, 6a,
and 6b were the most active, their EC50 values lie in the
submicromolar range of concentrations and are close to
that for 2ꢀmethoxyestradiol. In addition, we studied the
influence of some compounds (4a, 4b, 6a) on the growth
of A549 cells by direct counting under a microscope 24,
48, and 72 h after treatment with a test compound at
a concentration of 15 or 100 μmol L–1. In the first case,
after 48 h of treatment the amount of cells almost did not
decrease (data not shown). However, at a concentration
of 100 μmol L–1 compounds 4a, 4b, and 6a completely
inhibited not only the proliferation of the cells, but also
1
3
2

Esters with Adꢀfragment as ligands of tubulin
Russ. Chem. Bull., Int. Ed., Vol. 66, No. 8, August, 2017
1505
Scheme 2
n = 1 (a), 0 (b)
Compound Yield (%)
Compound Yield (%)
Compound Yield (%)
Compound Yield (%)
4a
4b
88
90
5a
5b
40
35
6a
6b
51
42
9a
9b
96
77
the number of adherent cells, which gradually decreased
from the time of treatment until the end of the experiꢀ
ment to near zero (Fig. 1).
The results of studying the effect of all the substances
obtained on the morphology of A549 cells and the dyꢀ
namics of their microtubules (carried out by immunoꢀ
Table 1. Biotesting results for compounds 4a,b, 5a,b, and 6a,b on carcinoma cells A549
Compound
Cytotoxicity*
EC50/μmol L–1
(c = 100 μmol L–1
Effect on cells
Effect
on microtubules
(c = 100 μmol L–1
)
)
4а
4b
5a
5b
6a
6b
17.8 0.5
4.9 0.5
Rounding, loss of adhesion
Rounding, loss of adhesion
Rounding, loss of adhesion
Rounding, loss of adhesion
Rounding, loss of adhesion, vesiculation
Rounding, loss of adhesion
No effect
No effect
No effect
No effect
No effect
22
81
4
2
4.3 0.2
6.6 0.1
N.d.
Depolymerization
No effect
5ꢀ(Hydroxymethyl)ꢀ
2ꢀmethoxyphenol
AdCH2COOH и
AdCH2COOСН3
САꢀ4
No effect
N.d.
No effect
No effect
0.01 0.1
0.8 0.1
No effect
Rounding, loss of adhesion
Depolymerization
Depolymerization
2ꢀМЕ
* The results of 3—4 independent experiments. N.d. means not determined.

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Russ. Chem. Bull., Int. Ed., Vol. 66, No. 8, August, 2017
Zefirov et al.
N (%)
250
fluorescence microscopy, see Table 1 and Fig. 2) indicate
that at high concentrations (100 μmol L–1) all of them
strongly alter the morphology of the cells, causing roundꢀ
ing and loss of adhesion, as well as a reduction in the
surface (see Fig. 2, c—e), but esters 4a,b, 5a,b and 6b have
no effect on the microtubule network. Compound 6a,
however, exhibits a pronounced ability to cause complete
depolymerization of microtubules within 72 h (Fig. 2, f).
Note that at this concentration, compound 6a causes
a noticeable fragmentation of A549 cell nuclei, which is
characteristic of apoptosis.
200
150
100
50
4
3
1
2
24
48
72 t/h
The interesting result obtained for compound 6a stimꢀ
ulated additional experiments. Although the hydrolysis of
the ester bond in molecule 6a with nonspecific cell esꢀ
terases is hindered by the presence of a bulky adamantane
substituent, we studied the effect of the products of this
Fig. 1. Influence of compounds 4a (1), 4b (2), and 6a (3)
(at a concentration of 100 μmol L–1) on the cell growth in
culture (counted using phase contrast microscopy; the control
was 0.5% DMSO, N is the number of cells, t is the duration of
experiment).
a
c
e
b
d
f
10 μm
Fig. 2. Immunofluorescence microscopy data for A549 cells treated with CAꢀ4 at a concentration of 1 μmol L–1 (a, positive control,
depolymerization of microtubules); with 0.5% DMSO (b, negative control; microtubule network is normal); compounds 4a or 4b at
a concentration of 100 μmol L–1 (c) and compounds 5a or 5b at a concentration of 100 μmol L–1 (d, reduction of the cell surface and
decrease of the cell density); with compound 6b at a concentration of 100 μmol L–1 (e, reduction of the cell surface and, as
a consequence, the formation of the cell membrane “protrusions”); with compound 6a at a concentration of 100 μmol L–1
(f, complete depolymerization of microtubules; cell vesiculation). The scale is 10 μm.

Esters with Adꢀfragment as ligands of tubulin
Russ. Chem. Bull., Int. Ed., Vol. 66, No. 8, August, 2017
1507
possible hydrolysis on the microtubule network of A549
cells. However, at a concentration of 100 μmol L–1 neiꢀ
ther 5ꢀ(hydroxymethyl)ꢀ2ꢀmethoxyphenol, nor adamanꢀ
taneacetic acid, nor its methyl ester with better permeꢀ
ability through the cell membrane had any effect on
microtubules (see Table 1). Thus, all the results obtained
confirm that compound 6a has a reliable cytotoxic effect
on A549 carcinoma cells, while its antiproliferative propꢀ
erties are at least partially associated with the influence
on the microtubular network.
ability to cause depolymerization of the microtubule netꢀ
work for the compound with the adamantane moiety as
a basic structural unit is important and interesting in terms
of expanding the structural diversity of such substances.
5ꢀ(Hydroxymethyl)ꢀ2ꢀmethoxyphenyl adamantanꢀ1ꢀ
ylacetate (6a) itself is promising as a new original lead
compound for further optimization.
Experimental
The automatic computer docking of ligand 6a to the
threeꢀdimensional model of the tubulin colchicine doꢀ
main (PDB ID: 1SA0) shows that in one of the most
favorable arrangement of this structure in protein, the
aryl fragment of compound 6a is located in the region
close to that occupied by ring B (CAꢀ4) and ring A (2ꢀME)
(Fig. 3).
Automatic docking in a threeꢀdimensional model of tubuꢀ
lin complex with NꢀdeacetylꢀNꢀ(2ꢀmercaptoacetyl)colchicine
(PDB ID: 1SA0) was carried out using the CLC Drug Discovꢀ
ery Workbench program (Version 1.5): Evaluation license
(2014). The preset radius value was 16 Å, the number of iteraꢀ
tions was 500. The ligand—tubulin complexes with the best
values of scoring functions calculated according to this proꢀ
gram were selected.
In this case, the hydroxy group in the structure 6a
forms two hydrogen bonds with the carbonyl groups of
the main chain of the amino acid moieties βVal315 and
βAsn350. The adamantane cage is located above the plane
of the trimethoxyphenyl group of CAꢀ4 and the steroid
skeleton of 2ꢀME, but it falls into the hydrophobic region
formed by the side chains of the residues βVal318, βIle378,
and βLeu255. Note that the arrangement of molecule 6a
in the protein shown in Fig. 3 has been chosen from
several variants with the minimum values of scoringꢀfuncꢀ
tion (according to the automatic docking data) as the
closest to that for the original CAꢀ4 and 2ME molecules.
In conclusion, we emphasize that only separate exꢀ
amples of adamantaneꢀcontaining tubulin ligands are deꢀ
scribed in the literature, in which the framework plays the
role of a substituent attached (frequently through a linkꢀ
er) to the molecules of combretastatin Aꢀ4, colchicine or
podophyllotoxin.20—23 Therefore, the found in this study
All the solvents for extraction and chromatography were
purified and dried according to the standard procedures. Adaꢀ
mantaneacetic and adamantanecarboxylic acids, 3,4,5ꢀtriꢀ
methoxyphenylbenzaldehyde, 3ꢀhydroxyꢀ4ꢀmethoxybenzaldeꢀ
hyde were purchased from SigmaꢀAldrich. The starting alcoꢀ
hols, 5ꢀ(hydroxymethyl)ꢀ2ꢀmethoxyphenol and (3,4,5ꢀtriꢀ
methoxyphenyl)methanol, were synthesized according to the
standard procedure by the reduction of the corresponding aldeꢀ
hydes with sodium borohydride, [3ꢀ(3ꢀtertꢀbutyldimethylsilyꢀ
loxy)ꢀ4ꢀmethoxyphenyl]methanol (8) was synthesized from
5ꢀ(hydroxymethyl)ꢀ2ꢀmethoxyphenol according to the proceꢀ
dure described earlier.18 Reaction progress and purity of comꢀ
pounds were monitored by thinꢀlayer chromatography on Siluꢀ
folꢀUV254 plates. Chromatographic separation was carried out
on columns with Acros silica gel (40—60 μm).1H and 13C NMR
spectra were recorded on a on Bruker Avance 400 spectrometer
(400 and 100 MHz, respectively) at 28 °C. Chemical shifts are
given relative to the residual signal of the solvent (CDCl3: δ 7.26
(1H NMR); 77.0 (13C NMR)). Elemental analysis was carried
out on a Vario Micro Cube CHNꢀanalyzer. IR spectra were
recorded on an IRꢀ200 ThermoNicolet spectrophotometer in
KBr pellets. MALDIꢀTOF mass spectra were recorded on
a VISIONꢀ2000 instrument.
Esterification (general procedure). A corresponding alcoꢀ
hol, N,N´ꢀdicyclohexylcarbodiimide (DCC), and a catalytic
amount of 4ꢀN,Nꢀdimethylaminopyridine (DMAP) (2—4 mg)
were added to a solution of a carboxylic acid in CH2Cl2 (10 mL).
The reaction mixture was stirred for 24 h at room temperature,
the solvent was evaporated in vacuo. Then, EtOAc (20 mL) was
added and the mixture was allowed to stand for 2—3 h at 4 °C.
The crystals of N,N´ꢀdicyclohexylurea were filtered off and
washed with cold EtOAc (2×10 mL), the solvent was evapoꢀ
rated in vacuo. The residue was subjected to chromatography
(eluent: ethyl acetate—light petroleum ether, 40—70 °C, gradiꢀ
ent 1 : 9—1 : 6).
3ꢀ[tertꢀButyl(dimethyl)silyloxy]ꢀ4ꢀmethoxybenzyl adamanꢀ
tanꢀ1ꢀyl acetate (9a) was obtained according to the general
esterification procedure from alcohol 8 (0.240 g, 0.90 mmol),
adamantaneacetic acid (0.2 g, 1.03 mmol), and DCC (0.22 g,
1.07 mmol). The yield of compound 9a was 0.384 g (96%),
a colorless oily liquid. 1H NMR (CDCl3), δ: 0.17 (s, 6 H,
Si(Me)2); 1.01 (s, 9 H, Si(But)); 1.61—1.64 (m, 9 H); 1.68—1.71
Fig. 3. One of the most energetically favorable arrangement of
structure 6a in the tubulin dimer (β subunit is on the left) based
on the results of automatic docking (the CLC Drug Discovery
Workbench program). Hydrogen bonds are shown by dashed
lines (hydrogen atoms are omitted). For comparison, thin lines
show molecules of combretastatin Aꢀ4 and 2ꢀmethoxyestradiol.

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Russ. Chem. Bull., Int. Ed., Vol. 66, No. 8, August, 2017
Zefirov et al.
(m, 3 H); 1.96 (m, 3 H); 2.11 (s, 2 H, AdCH2); 3.81 (s, 3 H,
OMe); 5.00 (s, 2 H, ArCH2); 6.82 (d, 1 H, C(5)H, J = 8.2 Hz);
6.88 (d, 1 H, C(2)H, J = 1.9 Hz); 6.92 (dd, 1 H, C(6)H, J = 8.2 Hz,
J = 1.9 Hz). 13C NMR (CDCl3), δ: –4.64 (Si(Me)2),
18.45 (SiC(Me)3), 25.72 (SiC(Me)3), 28.61, 32.87 (C(1ꢀAd)),
36.72, 42.41, 49.00 (AdCH2), 55.51 (OMe), 65.65 (ArCH2),
111.81 (C(5)), 121.38 (C(2)), 122.06 (C(6)), 128.81
(C(1)), 144.95 (C(3)), 150.95 (C(4)), 171.69 (C=O). IR (KBr,
ν/cm–1): 1513, 1731 (C=O); 2902, 2850. MS, m/z: 467
[M + Na]+.
(3ꢀHydroxyꢀ4ꢀmethoxybenzyl) adamantanꢀ1ꢀylacetate (4a).
1,8ꢀDiazabicyclo[5.4.0]undecꢀ7ꢀene (DBU) (0.120 g, 0.79 mmol)
was added to a solution of ester 9a (0.350 g, 0.79 mmol) in
a mixture of MeCN—H2O (99 : 1, 10 mL). The mixture was
stirred for 2 h at room temperature, diluted with water (20 mL),
and extracted with CH2Cl2 (3×20 mL), the organic layers were
combined, dried with Na2SO4, the solvent was evaporated
in vacuo. The residue was subjected to chromatography (eluent:
light petroleum ether (40—70 °C), then ethyl acetate—light
petroleum ether (40—70 °C) 1 : 8—1 : 6). The yield of comꢀ
pound 4a was 0.229 g (88%), a colorless oily liquid. 1H NMR
(CDCl3), δ: 1.61—1.65 (m, 9 H); 1.69—1.72 (m, 3 H); 1.96 (m,
3 H); 2.11 (s, 2 H, AdCH2); 3.89 (s, 3 H, OMe); 5.00 (s, 2 H,
ArCH2); 5.72 (s, 1 H, OH); 6.83 (d, 1 H, C(5)H, J = 8.2 Hz);
6.87 (dd, 1 H, C(6)H, J = 8.2 Hz, J = 2.0 Hz); 6.96 (d, 1 H,
C(2)H, J = 2.0 Hz). 13C NMR (CDCl3), δ: 28.56, 32.80 (C(1ꢀAd)),
36.67, 42.33, 48.86 (AdCH2), 55.92 (OMe), 65.62 (ArCH2),
110.42 (C(5)), 114.75 (C(2)), 120.26 (C(6)), 129.39 (C(1)),
145.58 (C(3)), 146.46 (C(4)), 171.65 (C=O). IR (KBr, ν/cm–1):
1513, 1729 (C=O); 2902. Found (%): C, 72.69; H, 7.95.
C20H26O4. Calculated (%): C, 72.70; H, 7.93.
3ꢀ[tertꢀButyl(dimethyl)silyloxy]ꢀ4ꢀmethoxybenzyl adamanꢀ
taneꢀ1ꢀcarboxylate (9b) was obtained according to the general
esterification procedure from alcohol 8 (0.240 g, 0.90 mmol),
adamantanecarboxylic acid (0.180 g, 1 mmol), and DCC (0.206
g, 1 mmol). The yield of compound 9b was 0.300 g (77%),
a colorless oily liquid. 1H NMR (CDCl3), δ: 0.17 (s, 6 H,
Si(Me)2); 1.02 (s, 9 H, SiBut); 1.68—1.76 (m, 6 H); 1.92 (m, 6 H);
2.02 (m, 3 H); 3.81 (s, 3 H, OMe); 5.01 (s, 2 H, ArCH2); 6.82
(d, 1 H, C(5)H, J = 8.2 Hz); 6.86 (d, 1 H, C(2)H, J = 2.1 Hz);
6.89 (dd, 1 H, C(6)H, J = 8.1 Hz, J = 2.1 Hz). 13C NMR (CDCl3),
δ: –4.66 (Si(Me)2), 18.43 (SiC(Me)3), 25.69 (SiC(Me)3), 27.92,
36.48, 38.81, 40.70 (C(1ꢀAd)), 55.48 (OMe), 65.43 (ArCH2),
111.79 (C(5)), 120.69 (C(2)), 121.35 (C(6)), 129.12 (C(1)),
144.92 (C(3)), 150.76 (C(4)), 177.43 (C=O). IR (KBr, ν/cm–1):
1513, 1725 (C=O); 2854, 2906, 2929. MS, m/z: 453 [M + Na]+,
469 [M + K]+.
(3ꢀHydroxyꢀ4ꢀmethoxybenzyl) adamantaneꢀ1ꢀcarboxylate
(4b) was obtained similarly to compound 4a from ester 9b (0.250 g,
0.581 mmol) and DBU (0.250 g, 0.581 mmol). The yield of
compound 4b was 0.165 g (90%), white crystals, m.p. 107—109 °C.
1H NMR (CDCl3), δ: 1.68—1.76 (m, 6 H); 1.92—1.93 (m, 6 H);
2.02 (m, 3 H); 3.90 (s, 3 H, OMe); 5.01 (s, 2 H, ArCH2); 5.66
(s, 1 H, OH); 6.82—6.86 (m, 2 H, C(5)H, C(6)H); 6.93 (d, 1 H,
C(2)H, J = 1.2 Hz). 13C NMR (CDCl3), δ: 27.96, 36.51, 38.83,
40.75 (C(1ꢀAd)), 55.97 (OMe), 65.58 (ArCH2), 110.45 (C(5)),
114.20 (C(2)), 119.75 (C(6)), 129.83 (C(1)), 145.61 (C(3)),
146.35 (C(4)), 177.52 (C=O). IR (KBr, ν/cm–1): 1590, 1708
(C=O); 2921. Found (%): C, 72.15; H, 7.67. C19H24O4. Calcuꢀ
lated (%): C, 72.13; H, 7.65.
acid 7a (0.503 g, 2.59 mmol), (3,4,5ꢀtrimethoxyphenyl)ꢀ
methanol (0.426 g, 2.15 mmol), and DCC (0.532 g, 2.58 mmol).
The yield was 0.328 g (40%), white crystals, m.p. 35—37 °C.
1H NMR (CDCl3), δ: 1.47—1.50 (m, 9 H); 1.55—1.58 (m, 3 H);
1.82 (m, 3 H); 1.99 (s, 2 H, AdCH2); 3.70 (s, 3 H, OMe); 3.72
(s, 6 H, 2 OMe); 4.91 (s, 2 H, ArCH2); 6.48 (s, 2 H, Ar).
13C NMR (CDCl3), δ: 28.04, 32.28 (C(1ꢀAd)), 36.12, 41.80,
48.22 (AdCH2), 55.39 (2OMe), 60.04 (OMe), 65.28 (ArCH2),
104.85 (C(2), C(6)), 131.37 (C(4)), 137.23 (C(1)), 152.67 (C(3),
C(5)), 170.69 (C=O). IR (KBr, ν/cm–1): 1592, 1731 (C=O);
2902. MS, m/z: 374 [M]+, 397 [M + Na]+, 413 [M + K]+.
Found (%): C, 70.53 H, 8.03. C22H30O5. Calculated (%):
C, 70.56; H, 8.07.
3,4,5ꢀTrimethoxybenzyl adamantaneꢀ1ꢀcarboxylate (5b) was
obtained according to the general esterification procedure from
acid 7b (0.420 g, 2.33 mmol), (3,4,5ꢀtrimethoxyphenyl)ꢀ
methanol (0.416 g, 2.09 mmol), and DCC (0.480 g, 2.33 mmol).
The yield was 0.267 g (35%), white crystals, m.p. 74—76 °C.
1H NMR (CDCl3), δ: 1.67—1.75 (m, 6 H); 1.92—1.93 (m, 6 H);
2.02 (m, 3 H); 3.84 (s, 3 H, OMe); 3.86 (s, 6 H, 2 OMe); 5.03
(s, 2 H, ArCH2); 6.55 (s, 2 H, Ar). 13C NMR (CDCl3), δ:
27.78, 36.32, 38.70, 39.12 (C(1ꢀAd)), 55.93 (2 OMe), 60.66
(OMe), 65.64 (ArCH2), 104.55 (C(2), C(6)), 132.13 (C(4)),
137.45 (C(1)), 153.10 (C(3), C(5)), 177.21 (C=O). MS, m/z:
360 [M]+, 383 [M + Na]+, 399 [M + K]+. Found (%): C, 70.04;
H, 7.78. C21H28O5. Calculated (%): C, 69.98; H, 7.83.
5ꢀ(Hydroxymethyl)ꢀ2ꢀmethoxyphenyl adamantanꢀ1ꢀylacetꢀ
ate (6a) was obtained according to the general esterification
procedure from acid 7a (0.450 g, 2.32 mmol), 5ꢀ(hydroxyꢀ
methyl)ꢀ2ꢀmethoxyphenol (0.324 g, 2.10 mmol), and DCC
(0.481 g, 2.33 mmol). The yield of compound 6a was 0.353 g
(51%), a waxy solid compound. 1H NMR (CDCl3), δ: 1.68—1.78
(m, 13 H, H(Ad) + OH); 2.03 (m, 3 H); 2.34 (s, 2 H, AdCH2);
3.83 (s, 3 H, OMe); 4.62 (s, 2 H, CH2OH); 6.95 (d, 1 H,
C(3)H, J = 8.3 Hz); 7.05 (d, 1 H, C(6)H, J = 2.0 Hz); 7.19 (dd,
1 H, C(4)H, J = 2.0 Hz, J = 8.3 Hz). 13C NMR (CDCl3), δ:
28.65, 33.07 (C(1ꢀAd)), 36.76, 42.25, 48.54 (AdCH2), 55.83
(OMe), 64.62 (CH2OH), 112.30 (C(3)), 121.95 (C(6)), 125.39
(C(4)), 133.57 (C(5)), 139.73 (C(1)), 150.62 (C(2)), 169.70
(C=O). IR (KBr, ν/cm–1): 1592, 1731 (C=O); 2902. MS, m/z:
353 [M + Na]+, 369 [M + K]+. Found (%): C, 72.74; H, 7.89.
C20H26O4. Calculated (%): C, 72.70; H, 7.93.
5ꢀ(Hydroxymethyl)ꢀ2ꢀmethoxyphenyl adamantaneꢀ1ꢀcarbꢀ
oxylate (6b) was obtained according to the general esterificaꢀ
tion procedure from acid 7b (0.420 g, 2.33 mmol), 5ꢀ(hydrꢀ
oxymethyl)ꢀ2ꢀmethoxyphenol (0.324 g, 2.10 mmol), and DCC
(0.481 g, 2.33 mmol). The yield of compound 6b was 0.284 g
(42%), white crystals, m.p. 105—107 °C. 1H NMR (CDCl3), δ:
1.78 (m, 6 H); 2.08 (m, 9 H); 2.40 (br.s, 1 H, OH); 3.78 (s, 3 H,
OMe); 4.53 (s, 2 H, CH2OH); 6.90 (d, 1 H, C(3)H, J = 8.4 Hz);
6.99 (d, 1 H, C(6)H, J = 2.0 Hz); 7.13 (dd, 1 H, C(4)H, J = 2.0 Hz,
J = 8.4 Hz). 13C NMR (CDCl3), δ: 27.87, 36.40, 38.73, 40.95
(C(1ꢀAd)), 55.96 (OMe), 64.25 (CH2OH), 112.31 (C(3)),
121.66 (C(6)), 125.04 (C(4)), 133.73 (C(5)), 140.00 (C(1)),
150.46 (C(2)), 175.80 (C=O). IR (KBr, ν/cm–1): 1511, 1733
(C=O); 2903. MS, m/z: 339 [M + Na]+, 355 [M + K]+. Found (%):
C, 72.10; H, 7.63. C19H24O4. Calculated (%): C, 72.13; H, 7.65.
MTT test for cytotoxicity was carried out on epithelial huꢀ
man carcinoma cells (line Aꢀ549, CCLꢀ185) according to the
procedures described in the works.24,25
3,4,5ꢀTrimethoxybenzyl adamantanꢀ1ꢀylacetate (5a) was
obtained according to the general esterification procedure from
Study of cell growth. The A549 cells were plated into 96ꢀwell
plates (density about 100 cells per well). The cells were treated

Esters with Adꢀfragment as ligands of tubulin
Russ. Chem. Bull., Int. Ed., Vol. 66, No. 8, August, 2017
1509
during 72 h with a solution of compound 6a in DMSO at conꢀ
centrations 15 or 100 μmol L–1 or 0.5% DMSO, which was
used as a negative control. The cells labelled with Hoechst dye
(0.8 mmol L–1 in phosphateꢀbuffered solution, PBS) were
counted under a microscope directly on the cell counter.
Test with immunofluorescently labelled microtubules. For laꢀ
belling microtubules, the cell A549 were cultured on small covꢀ
erslips 11 mm in diameter placed into plates with 12 wells (denꢀ
sity about 100 cells per coverslip). The cells were incubated
during 8 h with test compounds or colchicine as a positive
control at concentrations of 50 and 100 μmol L–1 at 37 °C and
5% CO2. A negative control was 0.5% DMSO. Fixed cells were
stained with monoclonal antibodies of mice to αꢀtubulin (Sigma,
St. Louis. USA) in dilution 1 : 400, followed by incubation with
fluorescently labelled AlexaFlour488 goat secondary antibodies
against mouse immunoglobulins (IgG) (Molecular Probes,
Eugene, USA) at dilution 1 : 200. Fixed cells were analyzed
using a Nikon Diaphot 300 microscope (Nikon GmbH, Dusꢀ
seldorf, Germany) equipped with a SenSys camera (Photometꢀ
rics, Munich, Germany).
6. O. N. Zefirova, E. V. Nurieva, D. V. Shishov, I. I. Baskin,
F. Fuchs, H. Lemcke, F. Schrцder, D. G. Weiss, N. S.
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This work was financially supported by the Russian
Foundation for Basic Research (Project No. 15ꢀ03ꢀ04894)
and the Division of Chemistry and Material Sciences of
the Russian Academy of Sciences (Program OKhNM
RAN No. 9), as well as by the German Academic
Exchange Service (DAAD).
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20. O. N. Zefirova, E. V. Nurieva, B. Wobith, V. V. Gogol,
N. A. Zefirov, A. V. Ogonkov, D. V. Shishov, N. S. Zefirov,
S. A. Kuznetsov, Mol. Divers., 2017, 21; DOI: 10.1007/
s11030ꢀ017ꢀ9739ꢀ6.
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Received April 3, 2017;
in revised form May 15, 2017