Synthesis and biological testing of tubuloclustin analogs containing alicyclic groups and 2-methoxyestradiol moiety

Article abstract of DOI:10.1007/s11172-014-0559-x

A number of analogs of tubuloclustin, N-[7-(2-adamantyloxy)-7-oxoheptanoyl]-N-deacetylcolchicine, were obtained. In these analogs, the colchicine moiety is formally replaced by the cyclohexane, adamantane, and 2-methoxyestradiol moieties (the steroid is attached through the hydroxy group at the C(17) atom). MTT assays revealed that the conjugates obtained are much less cytotoxic against A549 lung carcinoma cells than the lead compound.

Full text of DOI:10.1007/s11172-014-0559-x

Russian Chemical Bulletin, International Edition, Vol. 63, No. 5, pp. 1126—1129, May, 2014
1126
Synthesis and biological testing of tubuloclustin analogs
containing alicyclic groups and 2ꢀmethoxyestradiol moiety*
O. N. Zefirova,a,b Ya. S. Glazkova,a E. V. Nurieva,a N. A. Zefirov,a A. V. Mamaeva,a
B. Wobith,c N. S. Zefirov,a,b and S. A. Kuznetsovc
aDepartment of Chemistry, M. V. Lomonosov Moscow State University,
1 Vorob´evy Gory, 119991 Moscow, Russian Federation.
Fax: +7 (459) 939 0290. Eꢀmail: olgaz@org.chem.msu.ru
bInstitute of Physiologically Active Compounds, Russian Academy of Sciences,
1 Severnyi pr., 142432 Chernogolovka, Moscow Region, Russian Federation.
Eꢀmail: kolaz92@gmail.com
cInstitute of Biological Sciences, University of Rostock, 18106 Rostock, Germany.
Eꢀmail: sergei.kuznetsov@uniꢀrostock.de
A number of analogs of tubuloclustin, Nꢀ[7ꢀ(2ꢀadamantyloxy)ꢀ7ꢀoxoheptanoyl]ꢀNꢀdeꢀ
acetylcolchicine, were obtained. In these analogs, the colchicine moiety is formally replaced by
the cyclohexane, adamantane, and 2ꢀmethoxyestradiol moieties (the steroid is attached through
the hydroxy group at the C(17) atom). MTT assays revealed that the conjugates obtained are
much less cytotoxic against A549 lung carcinoma cells than the lead compound.
Key words: adamantane, colchicine, tubuloclustin, alicyclic compounds, 2ꢀmethoxyestraꢀ
diol, cytotoxicity.
The antitumor activity of natural colchicine is due to
its interaction with the cellular protein tubulin, which inꢀ
hibits tubulin polymerization into microtubules1 (this process
is essential in cell division). Many compounds able to inꢀ
teract with the colchicine binding site in tubulin are curꢀ
rently available. However, the overwhelming majority of
them is not used in cancer therapy because of their high
general toxicity or inadequate efficiency. This gives impetus
to various structural modifications of colchicine and its analꢀ
ogs with the aim of making them more active and less toxic.2
to form unusual tubulin clusters, compound 1a was named
tubuloclustin.4 Early structure—activity investigations of
analogs of compound 1a revealed that the adamantane
fragment and the linker of strictly specified length (five (1a)
or six methylene units (1b)) are both critical for these
compounds to be highly cytotoxic and capable of forming
clusters.3 In the present work, we modified compounds 1a
and 1b in different ways involving replacement of the
colchicine moiety and studied the biological activity of
the resulting derivatives.
To prove the important contribution of colchicine to
the cytotoxic properties of conjugates 1, first we replaced
colchicine by alicyclic cyclohexane and adamantane
groups (Scheme 1). For this purpose, we esterified dicarbꢀ
oxylic polyanhydrides with adamantanꢀ2ꢀol and isolated
not only monoesters 2a,b (see Ref. 3) but also diesters 3a,b
in the individual state. Ester 2a was used in a reaction with
cyclohexylamine in the presence of ethyl 2ꢀethoxyꢀ1,2ꢀ
dihydroquinolineꢀ1ꢀcarboxylate (EEDQ), which affords
target compound 4 in high yield.
1: n = 5 (a), 6 (b)
Compounds 3a,b and 4 failed in a standard coloriꢀ
metric assay involving 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀ
diphenylꢀ2Hꢀtetrazolium bromide (MTT) as a dye5 against
A549 human lung carcinoma cells. Their low cytotoxicity
(IC50 > 10 mol L–1) provides evidence for the decisive
role of colchicine in binding conjugates 1a,b to tubulin,
although their mechanisms of action differ from that of
free colchicine.4
Earlier, we have obtained a colchicine analog (1a),
which is more cytotoxic in vitro to various cancer cell
strains than is the parent molecule.3,4 Because of its ability
* Based on the materials of the First Russian Conference on
Medicinal Chemistry ("MedChem Russiaꢀ2013") with Internaꢀ
tional Participation (September 8—12, 2013, Moscow).
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 5, pp. 1126—1129, May, 2014.
1066ꢀ5285/14/6305ꢀ1126 © 2014 Springer Science+Business Media, Inc.

Tubuloclustin analogs
Russ.Chem.Bull., Int.Ed., Vol. 63, No. 5, May, 2014
1127
Scheme 1
Reagents: i. [OC(O)(CH2)nC(O)]m, DMAP, CH2Cl2; ii. 2a, C6H11NH2, EEDQ, CH2Cl2.
Compound
2a
2b
n
5
6
Yield (%)
70
Compound
3a
3b
n
5
6
Yield (%)
6
9
42
Then we studied the possibility of replacing colchicine
in lead compounds by 2ꢀmethoxyestradiol, another known
ligand to the colchicineꢀbinding site of tubulin with a lower
general toxicity. Since the exact location of 2ꢀmethoxyꢀ
estradiol at the colchicineꢀbinding site of the protein reꢀ
mains unknown and has been only hypothesized in a model,1
when selecting a position for an adamantane substituent
in the steroid, we used the literature data on the activity of
2ꢀmethoxyestradiol derivatives containing bulky groups.
Specifically, the position at the C(17) atom has been reꢀ
ported6,7 to be suitable for introduction of such substituꢀ
ents without changing the cytotoxicity of the starting molꢀ
ecule. As a result, we proposed analogs of lead compounds
in which ester linkers are attached through the hydroxy
group at the C(17) atom of the steroid (Scheme 2). To
examine how the cytotoxicity of such conjugates depends
on the linker length, we obtained a compound containing
the same chain as in 1a and another compound with
a longer chain consisting of seven methylene units.
In the first step, 7ꢀ(2ꢀadamantyloxy)ꢀ7ꢀoxoheptanoic
acid (2a) and 9ꢀ(2ꢀadamantyloxy)ꢀ9ꢀoxononanoic acid
(2c) (prepared3 by reactions of adamantanꢀ2ꢀone with
pimelic and azelaic polyanhydrides, respectively) were esꢀ
terified with 2ꢀmethoxyestradiol 5 containing the Bnꢀprotꢀ
ected phenolic OH group in the presence of DCC and
DMAP. The 1H NMR spectra of the resulting esters 6a,b
show characteristic signals for the hydrogen atom at the
C(17) atom (C(17)HOC(O)) of the steroid at  4.72 and
Scheme 2
n = 5 (2a, 6a, 7a), 7 (2c, 6b, 7b)
Reagents: i. DCC, DMAP, CH2Cl2; ii. 5% Pd/C, H2, EtOH.
Compound
6a
6b
Yield (%)
80
Compound
7a
7b
Yield (%)
79
78
65

1128
Russ.Chem.Bull., Int.Ed., Vol. 63, No. 5, May, 2014
Zefirova et al.
4.70, respectively. In the 13C NMR spectra of compounds
6a,b, the signal for the C(17) atom appears at  82, while
the signals for two carbonyl C atoms of the linker appear
at  173. Debenzylation of esters 6a,b by hydrogenation on
5% Pd/C gave the target conjugates 7a,b. The molecular
formulas and structures of products 7a,b were confirmed
by data from NMR spectroscopy, elemental analysis, and
MALDIꢀTOF mass spectrometry (m/z 578 [M]+ and
606 [M]+, respectively). The IR spectrum of comꢀ
pound 7a features absorption bands at 1729 (C=O) and
3463—3538 cm–1 (O—H).
In MTT assays against the A549 cell culture, steroid
conjugates 7a and 7b proved to be substantially less cytoꢀ
toxic (EC50 > 50 mol L–1) than tubuloclustin 1a. Since
both compounds 7 exhibit nearly equal poor cytotoxicity
regardless of the linker length, this failure may be attributꢀ
ed to a wrong position the substituted adamantane is atꢀ
tached to (the hydroxy group at the C(17) atom). This
calls for further investigations aimed at introducing this
fragment into other positions of 2ꢀmethoxyestradiol.
Approaches to the synthesis of such derivatives are curꢀ
rently under study.
1.55—1.58 (m, 4 H, Ad); 1.65—1.68 (m, 4 H, H()); 1.75—1.79
(m, 8 H); 1.84—1.87 (m, 8 H); 2.00—2.03 (m, 8 H, Ad); 2.34
(t, 4 H, H(), J = 7.4 Hz); 4.93 (m, 2 H, C(2)AdH). 13C NMR
(CDCl3), : 24.62, 26.95, 27.20, 31.75, 31.84, 34.46, 36.29, 37.35,
76.83 (C(2)Ad); 172.72 (C=O). Found (%): C, 76.12; H, 9.39.
C28H42O4. Calculated (%): C, 75.98; H, 9.56. Further elution
gave monoester 2b.
2ꢀAdamantyl 7ꢀcyclohexylaminoꢀ7ꢀoxoheptanoate (4). Cycloꢀ
hexylamine (0.050 g, 0.51 mmol) and ethyl 2ꢀethoxyꢀ1,2ꢀdihydꢀ
roquinolineꢀ1ꢀcarboxylate (0.090 g, 0.36 mmol) were added to
a solution of acid 2a (0.100 g, 0.34 mmol) in CH2Cl2 (5 mL). The
reaction mixture was stirred at 25 C for 24 h and concentrated.
The residue was chromatographed with ethyl acetate—light peꢀ
troleum (b.p. 40—70 C, 1 : 3) as an eluent. The yield of comꢀ
pound 4 was 0.114 g (89%), clear oily liquid. 1H NMR (CDCl3),
: 1.00—1.18 (m, 2 H); 1.35—1.42 (m, 4 H); 1.56—1.59 (m, 2 H,
Ad); 1.63—1.70 (m, 8 H); 1.75—1.80 (m, 4 H); 1.85—1.88 (m, 4 H);
1.90—1.94 (m, 4 H, Cy, J = 3.4 Hz, J = 12.4 Hz); 1.99—2.03
(m, 4 H, Ad); 2.15 (t, 2 H, H(), J = 7.5 Hz); 2.35 (t, 2 H, H(),
J = 7.4 Hz); 3.78 (m, 1 H, CHN); 4.94 (m, 1 H, C(2)AdH); 5.33
(d, 1 H, NH, J = 7.0 Hz). 13C NMR (CDCl3), : 24.95, 25.14,
25.31, 26.37, 26.99, 28.38, 28.54, 31.87, 31.98, 32.85, 33.67,
34.63, 37.39, 43.60 (CHN); 76.85 (C(2)Ad); 173.41 (C=O); 175.08
(C=O). Found (%): C, 75.42; H, 9.64; N, 3.70. C23H37NO3.
Calculated (%): C, 75.56; H, 9.93; N, 3.73.
2ꢀAdamantyl 3ꢀbenzyloxyꢀ2ꢀmethoxyestraꢀ1,3,5(10)ꢀtrienꢀ
17ꢀyl pimelate (6a). 7ꢀ(2ꢀAdamantyloxy)ꢀ7ꢀoxoheptanoic acid
(2a) (0.068 g, 0.23 mmol), DCC (0.060 g, 0.29 mmol), and
a catalytic amount of DMAP (0.01 g) were added to a solution of
3ꢀbenzyloxyꢀ2ꢀmethoxyestraꢀ1,3,5(10)ꢀtrienꢀ17ꢀol (5) (0.075 g,
0.19 mmol; prepared in three steps8,9 from estradiol) in CH2Cl2
(10 mL). The reaction mixture was stirred at 25 C for 24 h and
concentrated. Then EtOAc (20 mL) was added, and the resulting
mixture was kept at –5 C for 2—3 h. The crystals of N,N´ꢀdiꢀ
cyclohexylurea that formed were filtered off and washed with
cooled EtOAc. The solvent was removed, and the residue was
chromatographed with ethyl acetate—light petroleum (b.p.
40—60 C, 1 : 9) as an eluent. The yield of compound 6a was
0.100 g (80%), clear oily liquid. 1H NMR (CDCl3), : 0.85 (s, 3 H,
C(18)H3); 1.26—1.59 (m, 12 H); 1.65—1.85 (m, 15 H);
2.01—2.04 (m, 4 H, Ad); 2.22 (m, 2 H); 2.31—2.38 (m, 4 H,
CH2C(O)O, J = 7.4 Hz, J = 7.6 Hz); 2.68—2.80 (m, 2 H,
C(6)H2); 3.86 (s, 3 H, OMe); 4.72 (dd, 1 H, C(17)H, J = 8.0 Hz,
J = 8.8 Hz); 4.95 (m, 1 H, C(2)AdH); 5.12 (s, 2 H, OCH2Ph);
6.64 (s, 1 H, C(4)H); 6.86 (s, 1 H, C(1)H); 7.24—7.47 (m, 5 H,
Ph). 13C NMR (CDCl3), : 12.08 (C(18)); 23.19, 24.69, 24.75,
26.32, 26.93, 27.17, 27.24, 27.57, 28.57, 29.04, 31.73, 31.82,
34.59, 36.26, 36.91, 37.32, 38.42, 42.89, 44.04, 49.71, 56.23
(OMe); 71.02 (OCH2Ph); 76.69 (C(2)Ad); 82.42 (C(17)); 109.72
(C(1)); 114.58 (C(4)); 127.21, 127.63, 128.40, 128.70, 132.72,
137.38, 146.28 (C(2)); 147.53 (C(3)); 172.92 (C(17)O(O)C),
173.57 (C(O)OAd). Found (%): C, 77.51; H, 8.29. C43H56O6.
Calculated (%): C, 77.21; H, 8.44.
Experimental
The course of the reactions was monitored and the purity of
the compounds obtained was checked by TLC on SilufolꢀUV254
plates. Chromatographic separation was carried out on Acros
columns packed with silica gel (40—60 m). H and 13C NMR
1
spectra were recorded on a Bruker Avance 400 spectrometer
(400 and 100 MHz, respectively) in CDCl3 at 28 C. Chemical
shifts  are referenced to a residual signal of CDCl3 at  7.28 and
77.0. Elemental analysis was carried out on a Vario micro cube
CHNꢀanalyzer. IR spectra were recorded on an IRꢀ200 spectroꢀ
photometer (ThermoNicolet) in KBr pellets. MALDIꢀTOF mass
spectra were measured on a VISIONꢀ2000 instrument.
Di(2ꢀadamantyl) pimelate (3a). Pimelic polyanhydride (0.190 g,
1.33 mmol) and 4ꢀdimethylaminopyridine (DMAP, 0.01 g) were
added to a solution of adamantanꢀ2ꢀol (0.200 g, 1.32 mmol) in
CH2Cl2 (20 mL). The reaction mixture was refluxed for 12 h and
concentrated. The residue was chromatographed with ethyl
acetate—light petroleum (b.p. 40—70 C, 1 : 10) as an eluent.
The yield of compound 3a was 0.072 g (6%), white crystals, m.p.
69—73 C. 1H NMR (CDCl3), : 1.32—1.39 (m, 2 H, H());
1.51—1.54 (m, 4 H, Ad); 1.65 (quintet, 4 H, H(), J = 7.6 Hz,
J = 7.4 Hz); 1.70—1.75 (m, 8 H); 1.80—1.87 (m, 8 H); 1.95—1.98
(m, 8 H, Ad); 2.32 (t, 4 H, H(), J = 7.4 Hz); 4.90 (m, 2 H,
C(2)AdH). 13C NMR (CDCl3), : 24.69, 26.88, 27.11, 31.65,
31.75, 34.52, 36.19, 37.26, 76.86 (C(2)Ad); 172.82 (C=O).
Found (%): C, 75.56; H, 9.38. C27H40O4. Calculated (%):
C, 75.66; H, 9.41. Further elution gave monoester 2a.
2ꢀAdamantyl 3ꢀbenzyloxyꢀ2ꢀmethoxyestraꢀ1,3,5(10)ꢀtrienꢀ
17ꢀyl azelate (6b) was obtained from alcohol 5 (0.090 g,
0.23 mmol) and 9ꢀ(2ꢀadamantyloxy)ꢀ9ꢀoxononanoic acid (2c)
(0.096 g, 0.30 mmol) in the presence of DCC (0.071 g, 0.34 mmol)
and DMAP as described for compound 6a. The yield of comꢀ
pound 6b was 0.124 g (78%), clear oily liquid. 1H NMR (CDCl3),
: 0.80 (s, 3 H, C(18)H3); 1.26—1.59 (m, 16 H); 1.64—1.87
(m, 15 H); 1.96—2.06 (m, 4 H, Ad); 2.23 (m, 2 H); 2.31—2.37
Di(2ꢀadamantyl) suberate (3b) was obtained from adamanꢀ
tanꢀ2ꢀol (0.250 g, 1.64 mmol) and suberic polyanhydride (0.260 g,
1.67 mmol) as described for compound 3a. The solvent was reꢀ
moved, and the residue was chromatographed with ethyl acetꢀ
ate—light petroleum (b.p. 40—70 C, 1 : 10) as an eluent. The
yield of compound 3b was 0.124 g (9%), white crystals, m.p.
54—56 C. 1H NMR (CDCl3), : 1.36—1.39 (m, 4 H, H());

Tubuloclustin analogs
Russ.Chem.Bull., Int.Ed., Vol. 63, No. 5, May, 2014
1129
(m, 4 H, CH2C(O)O, J = 7.2 Hz, J = 7.3 Hz); 2.72—2.78 (m, 2 H,
C(6)H2); 3.87 (s, 3 H, OMe); 4.70 (dd, 1 H, C(17)H, J = 7.8 Hz,
J = 8.7 Hz); 4.93 (m, 1 H, C(2)AdH); 5.11 (s, 2 H, OCH2Ph);
6.63 (s, 1 H, C(4)H); 6.85 (s, 1 H, C(1)H); 7.26—7.46 (m, 5 H,
Ph). 13C NMR (CDCl3), : 12.11 (C(18)); 23.22, 25.00, 25.09,
26.35, 26.96, 27.20, 27.59, 28.95, 29.08, 31.76, 33.85, 34.81,
36.29, 36.92, 37.36, 38.45, 42.92, 44.06, 49.73, 56.27 (OMe);
71.04 (OCH2Ph); 76.68 (C(2)Ad); 82.38 (C(17)); 109.74 (C(1));
114.58 (C(4)); 127.24, 127.66, 128.43, 132.75, 137.40, 146.29
(C(2)); 147.53 (C(3)); 173.19 (C(17)O(O)C); 173.83 (C(O)OAd).
Found (%): C, 77.38; H, 8.79. C45H60O6. Calculated (%):
C, 77.55; H, 8.68.
2ꢀAdamantyl 3ꢀhydroxyꢀ2ꢀmethoxyestraꢀ1,3,5(10)ꢀtrienꢀ
17ꢀyl pimelate (7a). Hydrogen was bubbled through a mixture
of benzyl ether 6a (0.075 g, 0.11 mmol) and 5% Pd/C (0.100 g)
in ethanol (5 mL) at 25 C for 8 h. The precipitate that formed was
filtered off. The filtrate was concentrated to give compound 7a
(0.051 g, 79%) as a yellow oily liquid. 1H NMR (CDCl3), : 0.84
(s, 3 H, C(18)H3); 1.25—1.59 (m, 12 H); 1.65—1.89 (m, 15 H);
2.00—2.04 (m, 4 H, Ad); 2.22 (m, 2 H); 2.33—2.38 (m, 4 H,
CH2C(O)O, J = 7.3 Hz, J = 7.4 Hz); 2.76—2.80 (m, 2 H,
C(6)H2); 3.86 (s, 3 H, OMe); 4.72 (dd, 1 H, C(17)H, J = 8.0 Hz,
J = 8.8 Hz); 4.93 (m, 1 H, C(2)AdH); 5.50 (br.s, 1 H, C(3)OH);
6.64 (s, 1 H, C(4)H); 6.79 (s, 1 H, C(1)H). 13C NMR (CDCl3),
: 12.10 (C(18)); 23.21, 24.72, 24.78, 26.48, 26.96, 27.20, 27.25,
27.59, 28.60, 28.91, 31.76, 31.85, 34.34, 34.63, 36.29, 36.94,
37.35, 38.51, 42.92, 44.06, 49.76, 56.02 (OMe); 76.69 (C(2)Ad);
82.47 (C(17)), 108.07 (C(1)); 114.58 (C(4)); 129.41, 131.55,
143.46 (C(3)OH); 144.57 (C(2)); 172.97 (C(17)O(O)C);
173.63 (C(O)OAd). IR (KBr), /cm–1: 985, 1025, 1101, 1118,
1211, 1241, 1265, 1357, 1454, 1511, 1590, 1617, 1729 (C=O);
2856—2923 (CH); 3463—3538 (OH). Found (%): C, 74.47;
H, 8.82. C36H50O6. Calculated (%): C, 74.71; H, 8.71. MS
(MALDIꢀTOF), m/z: 578 [M]+.
37 C. The culture medium (200 L) consisted of DMEM and
10% FBS. The cells were kept for 24 h with solutions of test
compounds (or colchicine as a positive control) in DMSO. The
concentration of the test compounds was varied from 0.005 to
50 mol L–1; each concentration was present in eight wells.
A phosphateꢀbuffered solution of MTT (C = 5 mg mL–1) was
prepared and filtered through a filter with a pore diameter of
0.22 mm. Two hours before the exposure time of the test comꢀ
pounds elapsed, a sterile solution of MTT (20 L) had been
added to each well so that its final concentration was 0.45 mg mL–1
.
The culture medium was removed, and a lysis buffer (100 L)
consisting of 10% sodium dodecyl sulfate and 0.6% acetic acid in
DMSO was added to each well. The resulting formazan crystals
were solubilized by thorough mixing on a plate shaker. Optical
density was measured at 590 nm with a 690ꢀnm reference filter
on an EL808 Ultra Microplate Reader instrument (BioꢀTek
Instruments, USA).
This work was financially supported by the Russian
Foundation for Basic Research (Project Nos 12ꢀ03ꢀ00720
and 13ꢀ03ꢀ12460), the Russian Academy of Sciences, and
the German Academic Exchange Service (Deutscher Akaꢀ
demischer Austauschdienst (DAAD)) in accordance with
the Cooperation Agreement between the M. V. Lomonosov
Moscow State University and the University of Rostock.
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17ꢀyl azelate (7b) was obtained from benzyl ether 6b (0.120 g,
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cells were cultured in 96ꢀwell plates (~3000 cells per well) at
Received October 31, 2013;
in revised form April 29, 2014