Tubuloclustin analogues with ether moiety: synthesis and evaluation of tubulin clustering and antimitotic activity in cancer cells

Article abstract of DOI:10.1016/j.mencom.2020.01.035

New analogues of anticancer agent tubuloclustin N-[7-(adamantan-1-yloxy)-7-heptanoyl]-N-deacetylcolchicine with ether moiety in the linker between colchicine and adamantine fragments were synthesized from w-(adamantan-1-yloxy)alkan- 1-ols. These compounds effectively inhibited growth of human lung carcinoma cell line A549 (IC₅₀ = 5–15.5 nM), induced both apoptosis and formation of tubulin clusters. The conjugates lacking ester carbonyl in the linker exhibit improved metabolic stability and are promising for further cytotoxicity studies in vivo.

Full text of DOI:10.1016/j.mencom.2020.01.035

Mendeleev
Communications
Mendeleev Commun., 2020, 30, 106–108
Tubuloclustin analogues with ether moiety: synthesis and evaluation
of tubulin clustering and antimitotic activity in cancer cells
Nikolay A. Zefirov,*a,b Leonie Gädert,c Artemy R. Fatkulin,a Vadim M. Shibilev,a
Gennady M. Butov,d Vladimir M. Mokhov,e Sergei A. Kuznetsovc and Olga N. Zefirovaa,b
a Department of Chemistry, M. V. Lomonosov Moscow State University, 119991 Moscow, Russian Federation.
E-mail: kolaz92@gmail.com
b Institute of Physiologically Active Compounds, Russian Academy of Sciences, 142432 Chernogolovka,
Moscow Region, Russian Federation. E-mail: olgaz@med.chem.msu.ru
c Institute of Biological Sciences, University of Rostock, D-18059 Rostock, Germany.
E-mail: sergei.kuznetsov@uni-rostock.de
d Volzhsky Polytechnic Institute (branch), Volgograd State Technical University, 404121 Volzhsky,
Russian Federation. E-mail: butov@post.volpi.ru
e Volgograd State Technical University, 400131 Volgograd, Russian Federation. E-mail: mvtons@yandex.ru
DOI: 10.1016/j.mencom.2020.01.035
New analogues of anticancer agent tubuloclustin N-[7-
(adamantan-1-yloxy)-7-heptanoyl]-N-deacetylcolchicine with
ether moiety in the linker between colchicine andadamantane
fragments were synthesized from w-(adamantan-1-yloxy)-
alkan-1-ols. These compounds effectively inhibited growth of
human lung carcinoma cell line A549 (IC50 = 5–15.5 nm),
induced both apoptosis and formation of tubulin clusters.
The conjugates lacking ester carbonyl in the linker exhibit
improved metabolic stability and are promising for further
cytotoxicity studies in vivo.
ꢅ
MeO
MeO
N
ꢆCꢅ2ꢇn
O
O
MeO
O
OMe
n ꢀ ꢁꢂ ꢃꢂ ꢄ
Keywords: adamantane, dehydroadamantane, colchicine, tubuloclustin, metabolic stability, tubulin, colchicine binding site, depoly-
merization of microtubules, carcinoma A549 cell line.
Antiproliferative activity of colchicine is based on its ability to
depolymerize cell microtubules (MT) into a,b-tubulin dimers.1
Numerous colchicine (1a) C7 derivatives were synthesized in
order to improve anticancer activity and reduce systemic toxicity
of colchicine2 (for recent examples, see structures 1b–f,3–7
Figure 1). Compound 1d (tubuloclustin) and its equipotent
analogue 1e are more active than colchicine in vitro, have
different toxicological profile in vivo and induce formation of
tubulin clusters in cultured cells.5,8 Importantly, clustering
strength correlates with antiproliferative activity for many
MeO
R
ꢄ
MeO
MeO
O
OMe
ꢀdꢈf R ꢀ ꢁN
OAd
ꢀa R ꢀ NꢁCꢂOꢃMe
ꢂCꢁ2ꢃn
O
O
ꢎꢏR 22ꢌ
O
O
ꢂCꢁ2ꢃꢆ
ꢀd n ꢀ 2ꢉ Ad ꢀ 2ꢊadamantyl
ꢀe n ꢀ ꢆꢉ Ad ꢀ ꢋꢊadamantyl
ꢀf n ꢀ ꢌꢉ Ad ꢀ ꢋꢊadamantyl
O
ꢁN
ꢀb R ꢀ
ꢂCꢁ2ꢃꢅ
O
O
O
OAd
A R ꢀ
ꢇ
ꢁN
O
ꢂCꢁ2ꢃn
N
N
ꢀc R ꢀ N
O
O
ꢂCꢁ2ꢃk
ꢁ R ꢀ
O
ꢁN
ꢂCꢁ2ꢃn
O
ꢍAd
Ad ꢀ ꢋꢊadamantyl
Figure 1 Colchicine (1a) and some of its C7 derivatives, including tubuloclustin (1d) and its close analogue 1e. A and B – general structures of tubuloclustin
analogues studied in the present work. Right panel shows predicted binding modes for conjugates A (n = 1 shown in red and n = 3 shown in green) in
a,b-tubulin colchicine binding site.
© 2020 Mendeleev Communications. Published by ELSEVIER B.V.
on behalf of the N. D. Zelinsky Institute of Organic Chemistry of the
Russian Academy of Sciences.
– 106 –

Mendeleev Commun., 2020, 30, 106–108
Table 1 Results of biological activity tests for compounds 4a–c and 6.
Microtubules
disassemblya
Tubulin clusteringb
Cell growth
inhibition
IC50/nm
Cytotoxicity
EC50/nm
Compound
50 nm
100 nm
50 nm
100 nm
200 nm
400 or 800 nm
1200 nm
4a
4b
4c
6
1d
1e
1a
+
+
+
+
+
+
+
+
++
++
–
++
+++
–
+++
+++
+/–
+
+++
+++
+
+++
+++
++
6.4
5.0
1
1
3
2
7.8
6.7
2
2
4
4
+
+/–
+
15.5
13.4
6.1
18.4
15.8
7.0
+/–
++
n.d.
–
+
++
++
+
+++
n.d.
–
+++
n.d.
–
+++
+++
–
+++
+++
–
+
n.d.
n.d.
11
n.d.
30
a Effect on microtubules of A549 lung carcinoma cells at marked concentration after 24 h treatment; full (+) or partial (+/–) MT depolymerization is indicated
[see Figure 2(b),(c)]. b Relative clustering intensity at marked concentration (at 2400 nm the intensity does not change, data not shown): no effect (–), point-
like clusters (+), moderate clusters (++), long clusters (+++) [see Figure 2(d)–(f)]; n.d. – not determined.
derivatives of 1d and 1e.6,9 We have recently demonstrated that
i
ii
upon shifting the ester group in conjugate 1e, both clustering
ability and activity are retained.9 This observation led to an idea
of tubuloclustin analogues with improved metabolic stability
that lack ester carbonyl in the linker (see Figure 1, general
formula A). The aim of the present work was to synthesize and
test conjugates with an ether bond.
O(CH2)nCH2OH
2a–c
O(CH2)nCO2H
3a–c
H
MeO
MeO
N
(CH2)n
Linker length in the compounds of general formula A was
chosen based on a molecular docking study carried out using 3D
modelofcolchicinebindingsiteina,b-tubulin(PDBID:4O2B),10
AutoDock Vina 1.1.2 software11 and USCF Chimera 1.13.1
program.12 The proposed binding mode of ligands A (n = 1–3)
was close to that of tubuloclustin (see ref. 5 and refs. herein), but
without a hydrogen bond to aTyr224 residue (see Figure 1).
Interestingly, this binding mode was not observed for ligands A
with longer linker chain (n = 4, 5). Based on these data we
selected compounds A (n = 1, 3 and 4 as a control) for synthesis.
To enhance structural diversity of tubuloclustin analogues we
also included one compound with both ether and ester groups in
the linker – structure B (n = 3, k = 2, X = O) with length equal to
that of recently studied and very active conjugates B (n = 4,
k = 1, X = CH2 and n = 5, k = 0, X = CH2).8
O
iii
O
MeO
a n = 1
b n = 3
c n = 4
O
OMe
4a–c
Scheme 1 Reagents and conditions: i, alkanediol, Et2O, 30–40 oC, 2–3 h;13
ii, Jones reagent, THF, room temperature, 5 min; iii, N-deacetylcolchicine,
EEDQ, CH2Cl2, room temperature, 12 h.
i
HO
(CH2)3
(CH2)2
2a
O
O
O
Target compounds were synthesized as outlined in Schemes 1
and 2. Using recently described procedure,13 1,3-dehydro-
adamantane was treated with the corresponding alkanediols to give
w-(adamantan-1-yloxy)alkan-1-ols 2a–c which were further
oxidized by chromium(vi) oxide to afford corresponding acids 3a–c
in fair yields (52–61%).† Amidation of acids 3a–c with
N-deacetylcolchicine produced by a described procedure14 was
carried out in the presence of N-ethoxycarbonyl-2-ethoxy-1,2-
dihydroquinoline (EEDQ, see Scheme 1).Yields of target conjugates
4a–c were 28–36%. In 1H NMR spectrum of compounds 4a–c, the
C7–proton resonance is shifted downfield (4.63–4.68 ppm)
5
H
N
MeO
MeO
(CH2)2
(CH2)3
ii
O
O
O
MeO
O
6
OMe
Scheme 2 Reagents and conditions: i, glutaric anhydride, 4-DMAP,
CH2Cl2, room temperature, 24 h; ii, N-deacetylcolchicine, EEDQ, CH2Cl2,
room temperature, 12 h.
1
compared to that in N-deacetylcolchicine (3.71 ppm). H and 13C
NMR spectra, mass spectra (m/z 549 [M]+, 577 [M]+, and 591 [M]+
for 4a, 4b and 4c, respectively) and elemental analysis confirm the
formation of target compounds 4a–c.
Biological activity tests for compounds 4a–c and 6 were
carried out using human epithelial lung carcinoma cell line
A549. Microtubule morphology was studied by immunofluores-
cence microscopy as described previously.15 Antimitotic activity
was evaluated using MTT assay16 and cell growth inhibition test
(direct cell counting by microscopy over 24 h of culturing).15
The ability of compounds to induce apoptosis was also
investigated.17 Test results are presented in Table 1 and
Figure 2(a)–(f) (for the experimental data on apoptosis, see
Online Supplementary Materials).
At 50 nm concentrations, the compounds caused total (4a, 4b,
6) or partial (4c) MT depolymerization in A549 cells after 24 h
of treatment. Cluster formation efficacy decreased in the
following sequence: 4b > 4a > 6 >> 4c. The structure–clustering
activity relationship (SAR) in series 4a, 4b and 4c correlates
Conjugate 6 was prepared from alcohol 2c which was acylated
with glutaric anhydride in the presence of N,N-dimethylamino-
pyridine (DMAP) to give the monoester 5 (see Scheme 2).
Reaction of the latter with N-deacetylcolchicine in the presence
of EEDQ led to the target conjugate 6. Characteristic signals at
C7 atom in compound 6 have chemical shifts of 52.16 and
4.63 ppm in 13C and 1H NMR and spectra, respectively. 1H and
13C NMR spectra and mass spectra (m/z 650 [M]+) confirm the
formation of target conjugate 6.
†
For synthetic details and characteristics of all novel compounds, see
Online Supplementary Materials.
– 107 –

Mendeleev Commun., 2020, 30, 106–108
ꢀaꢁ
ꢀbꢁ
ꢀcꢁ
carried out within the framework of the basic part of the state
task for 2017–2019 (project no. 4.7491.2017/BCh) with the
support from the Ministry of Science and Higher Education of
the Russian Federation. The authors are grateful to the DAAD
(German Academic Exchange Service) and to Birgit Wobith for
technical assistance.
Online Supplementary Materials
ꢀfꢁ
ꢀdꢁ
ꢀeꢁ
Supplementary data associated with this article can be found
in the online version at doi: 10.1016/j.mencom.2020.01.035.
References
1 B. Kumar, R. Kumar, I. Skvortsova and V. Kumar, Curr. Cancer Drug
Targets, 2017, 17, 357.
ꢂ0 µm
2 Y. Lu, J. Chen, M. Xiao, W. Li and D. D. Miller, Pharm. Res., 2012, 29,
2943.
Figure 2 Tubulin in A549 cells treated with (a) 0.5% DMSO (intact MT);
(b) 50 nm of 4c (partial depolymerization of MT, no clustering); (c) 100 nm
of 4c (complete depolymerization of MT, no tubulin clustering); (d) 100 nm
of 6 (point-like clustering); (e) 400 nm of 6 (moderate clustering); (f) 1200 nm
of 4b (strong clustering).
3 C. Vilanova, S. Díaz-Oltra, J. Murga, E. Falomir, M. Carda and
J. A. Marco, Bioorg. Med. Chem. Lett., 2015, 25, 3194.
4 P. Thomopoulou, J. Sachs, N. Teusch, A. Mariappan, J. Gopalakrishnan
and H.-G. Schmalz, ACS Med. Chem. Lett., 2016, 7, 188.
5 O. N. Zefirova, H. Lemcke, M. Lantow, E. V. Nurieva, B. Wobith,
G. E. Onishchenko, A. Hoenen, G. Griffiths, N. S. Zefirov and
S. A. Kuznetsov, ChemBioChem, 2013, 14, 1444.
6 O. N. Zefirova, E. V. Nurieva, B. Wobith, V. V. Gogol, N. A. Zefirov,
A. V. Ogonkov, D. V. Shishov, N. S. Zefirov and S. A. Kuznetsov,
Mol. Divers., 2017, 21, 547.
with the molecular docking data (see above), which predicted
different tubulin binding modes for a pair of structurally close
conjugates 4b/4c (but not for a shorter 4a/4b pair). Namely, the
biotest data for compounds 4a,b indicated that tubuloclustin
analogues with ether bond in the linker could induce formation
of strong tubulin clusters [see Figure 2(f)].
7 N. A. Zefirov,Yu. A. Evteeva, A. R. Fatkulin, S. Schulz, S. A. Kuznetsov
and O. N. Zefirova, Pharm. Chem. J., 2019, 53, 423 [Khim.-Farm. Zh.,
2019, 53 (5), 13].
Biological testing revealed that tubulin clustering ability of
novel compounds correlates with cytotoxicity, cell growth
inhibition (see Table 1) and earlier SAR studies for tubuloclustin
derivatives.6,9 Conjugate 6 activity was close to that of its
analogues B (n = 4, k = 1, X = CH2 and n = 5, k = 0, X = CH2),9
suggesting that clustering ability is negligibly affected by ester
bond position. The best IC50 and EC50 values in low nanomolar
concentration range were observed for compounds 4a and 4b.
The latter was equally active to tubuloclustin in both
antiproliferative assays. Each of conjugates 4a–c and 6 induced
nucleus fragmentation in A549 cells which is typical for the cells
undergoing apoptosis.18 The ability to cause apoptosis was close
for all new compounds, but there was no exact correlation
between apoptotic index (highest for 4c) and tubulin clustering
activity and cytotoxicity (see Online Supplementary Materials).
In conclusion, novel tubuloclustin analogues with ether
moiety in the linker between colchicine and adamantane
fragments were synthesized and tested in a series of bioassays.
The data obtained unambiguously prove that these conjugates
retain the ability to induce strong tubulin clustering. Compound
4b represents tubuloclustin analogue with extremely high
antiproliferative activity and improved metabolic stability and
therefore it is promising for further in vivo studies.
8 O. N. Zefirova, E.V. Nurieva,Ya. S. Glazkova, N.A. Zefirov,A.V. Mamaeva,
B. Wobith, V. I. Romanenko, N. A. Lesnaya, H. M. Treshchalina and
S. A. Kuznetsov, Pharm. Chem. J., 2014, 48, 373 [Khim.-Farm. Zh.,
2014, 48 (6), 19].
9 N.A. Zefirov, M. Hoppe, I.V. Kuznetsova, N.A. Chernyshov,Yu. K. Grishin,
O. A. Maloshitskaya, S. A. Kuznetsov and O. N. Zefirova, Mendeleev
Commun., 2018, 28, 308.
10 A. E. Prota, F. Danel, F. Bachmann, K. Bargsten, R. M. Buey,
J. Pohlmann, S. Reinelt, H. Lane and M. O. Steinmetz, J. Mol. Biol.,
2014, 426, 1848.
11 O. Trott and A. J. Olson, J. Comput. Chem., 2010, 31, 455.
12 E. F. Pettersen, T. D. Goddard, C. C. Huang, G. S. Couch,
D. M. Greenblatt, E. C. Meng and T. E. Ferrin, J. Comput. Chem., 2004,
25, 1605.
13 G. M. Butov and V. M. Mokhov, Russ. J. Org. Chem., 2018, 54, 1760
(Zh. Org. Khim., 2018, 54, 1746).
14 J. D. Bagnato, A. L. Eilers, R. A. Horton and C. B. Grissom, J. Org.
Chem., 2004, 69, 8987.
15 N. A. Zefirov, Yu. A. Evteeva, B. Wobith, S. A. Kuznetsov and
O. N. Zefirova, Struct. Chem., 2019, 30, 465.
16 D. Gerlier and N. Thomasset, J. Immunol. Methods, 1986, 94, 57.
17 C. S. Potten, Br. J. Cancer, 1996, 74, 1743.
18 N. Zamzami and G. Kroetmer, Nature, 1999, 401, 127.
This work was supported by the Russian Science Foundation
Received: 5th August 2019; Com. 19/6009
(project no. 19-13-00084). Synthesis of initial alcohols 2a–c was
– 108 –