Design, synthesis, and bioactivity of putative tubulin ligands with adamantane core

Article abstract of DOI:10.1016/j.bmcl.2008.07.116

Several adamantane-based taxol mimetics were synthesized and found to be cytotoxic at micromolar concentrations and to cause tubulin aggregation. The extent of the aggregation is maximal for N-benzoyl-(2R,3S)-phenylisoseryloxyadamantane (5) and is very sensitive to the structural modifications. A hybrid compound (15), combining adamantane-based taxol mimetic with colchicine was synthesized and found to possess both microtubule depolymerizing and microtubule bundling activities in A549 human lung carcinoma cells.

Full text of DOI:10.1016/j.bmcl.2008.07.116

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5091–5094
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and bioactivity of putative tubulin ligands with
adamantane core
a
b
c
a
Olga N. Zefirova a,c, , Evgeniya V. Nurieva , Heiko Lemcke , Andrei A. Ivanov , Dmitrii V. Shishov ,
*
Dieter G. Weiss b, Sergei A. Kuznetsov b, Nikolay S. Zefirov a,c
a Department of Chemistry, M. V. Lomonosov Moscow State University, 119992 Moscow, Russian Federation
b Institute of Biological Sciences, Cell Biology and Biosystems Technology, University of Rostock, D-18059 Rostock, Germany
c Institute of Physiologically Active Compounds, 142432, Chernogolovka, Noginsk Area, Moscow Region, Russian Federation
a r t i c l e i n f o
a b s t r a c t
Article history:
Several adamantane-based taxol mimetics were synthesized and found to be cytotoxic at micromolar
concentrations and to cause tubulin aggregation. The extent of the aggregation is maximal for N-ben-
zoyl-(2R,3S)-phenylisoseryloxyadamantane (5) and is very sensitive to the structural modifications. A
hybrid compound (15), combining adamantane-based taxol mimetic with colchicine was synthesized
and found to possess both microtubule depolymerizing and microtubule bundling activities in A549
human lung carcinoma cells.
Received 10 June 2008
Revised 30 July 2008
Accepted 31 July 2008
Available online 3 August 2008
Keywords:
Taxol
Ó 2008 Elsevier Ltd. All rights reserved.
Taxoter
Taxol mimics
Tubulin ligands
Adamantane derivatives
Taxol (paclitaxel, 1a, Fig. 1), isolated from the bark extracts of
Taxus brevifolia, and its synthetic analogue taxotere (docetaxel,
1b) possess high antitumor activity due to their ability to cause
spontaneous polymerization of the intracellular protein tubulin
into stable microtubules and to stabilize preformed microtubules,
thereby preventing cell division.1,2 The most important contribu-
tion to tubulin binding is provided by the C13 side chain taxol
and taxotere (i.e., N-benzoyl- or N-tert-butoxycarbonyl-(2R,3S)-
phenylisoseryl), while C2 (–OBz), C4 (–OAc) substituents, and the
oxetan fragment also play a role in this binding.1,2
The intricate molecular structure of taxane compounds and the
necessity to obtain them semi-synthetically from natural sources
make the development of taxol and taxotere analogues with a sim-
pler structure important. During the last ten years there appeared
several publications about such attempts.3–9
In 2002, based on the hypothesis, that the main function of
the taxane skeleton is to provide proper orientation of the sub-
stituents important for tubulin binding,10 we suggested a general
theoretical model of a ‘simplified’ taxol analogue with a bicy-
clo[3.3.1]nonane core (structurally similar to the AB rings of
the parent molecule).11 Later, we synthesized a number of such
compounds, including structure 2, and demonstrated that they
possess weak cytotoxicity and can cause slight tubulin aggrega-
tion, but not polymerization to microtubules.12 Because one of
the reasons for this result might be improper substituents posi-
tions in structure 2 and similar compounds due to the relative
conformational freedom of the bicyclo[3.3.1]nonane framework,
we suggested to synthesize their analogues with a rigid adaman-
tane core. Here, we present the results of molecular modeling
and synthesis of structure 3 and the data of biological tests of
compound 3 and two other adamantane-based taxol mimetics
4 and 5 (Fig. 1).
The model of tubulin structure (kindly granted to us by Prof. J.
Snyder, USA)13 was used for the molecular docking of compound
3 to the taxol binding site. The obtained binding mode of ester 3
is presented in Figure 2.14,15 According to the modeling results, in
case of an identical location of taxol and compound 3 side chains
in the protein, the oxetan oxygen of 3 can be hydrogen bonded
to the Thr 276 amino group. This interaction was not observed
for the compound 2,12 but it corresponds exactly to the bond
formed by the oxetan oxygen in taxol.13 Moreover, the carbonyl
oxygen of structure 3 ester linker can be hydrogen bonded with
Arg284 (Fig. 2).
For the synthesis of compound 3 double esterification was per-
formed for the TMS-protected 1-hydroxyadamantane-5-carboxylic
acid 6, first by 3-hydroxyoxetan (obtained in five steps from glyc-
erin) with the formation of ester 7,16 and then by an oxazolidine-
protected amino acid (Scheme 1).17 The subsequent opening of
the oxazolidine ring in the product 8 led to the ester 3.18
* Corresponding author. Tel.: +7 495 939 48 78; fax: +7 495 939 02 90.
E-mail address: olgaz@org.chem.msu.ru (O.N. Zefirova).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.116

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O. N. Zefirova et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5091–5094
RO
O
NHBoc O
OH
NHR1
H
OH
O
10
9
Rh
O
7
C
13
A
1
Ph
O
B
2
OH
2
O
4
H
HO
D
H
Taxol R=Ac, R1=Bz
O
CO
O
BzO
1a
1b
AcO
1
Taxotere R=H, R =Boc
R
NHBz
NHBoc
O
O
O
Ph
O
Ph
O
O
OH
OH
O
3
4
5
R=H
R= OBz
Figure 1. Structures of taxoids and their ‘simplified’ analogues.
O
Ph
NBoc
O
Ph
C
OH
OH
O
O
BocN
O
a
b
4, 5
R
R
R= O or H, H
Scheme 2. Reagents and conditions: (a) DCC, DMAP, CH2Cl2, 25 °C, 12 h, 99% from
kemantane, 82% from adamantanol; (b) for 4: 1—NaBH4, MeOH, Et2O, 0 °C, 99%; 2—
BzCl, Et3N, DMAP, CH2Cl2, 25 °C, 12 h, 74%; 3—HCO2H, 25 °C, 2 h, then BzCl, NaHCO3,
EtOAc, H2O, 25 °C, 15 min, 83%. For 5: HCO2H, 25 °C, 2 h, then BzCl, NaHCO3, EtOAc,
H2O, 25 °C, 15 min, 44%.
carcinoma cell line. The results are presented in Table 1. These data
indicate that compounds 3–5 exhibit cytotoxicity at the micromo-
lar level, that is, the same as the bicyclo[3.3.1]nonane esters (e.g.,
2)12 and other simplified taxol analogues (e.g., indolizidinones7
and macrocycles9).
To determine if the ‘adamantane taxoids’ 3–5 are able to cause
tubulin polymerization, the samples of tubulin purified from bo-
vine brain23 were incubated with compounds 3–5 or with taxol
as a positive control and studied by means of video-enhanced con-
trast light microscopy (AVEC-DIC microscopy).24 This study
showed that all tested taxol mimetics were not able to promote
microtubule assembly of isolated tubulin (no microtubule bundles
were observed). However, sedimentation assays25 revealed that
these compounds induced tubulin aggregation (Fig. 3A). Remark-
ably, ester 5 showed the highest ability to promote tubulin sedi-
mentation—57%, whereas for all other compounds (including 2
and similar structures)12 it varied between 25% and 33%
(Fig. 3B).
Figure 2. Structure 3 inside the taxol binding site of b-tubulin (hydrogen atoms are
not indicated).
Compounds 4 and 5 were synthesized by esterification of
kemantane (1-hydroxy-adamantane-4-one)19 and adamantane-1-
ol20 correspondingly using slightly different oxazolidine amino
acid protections21 (general Scheme 2). It should be noted that this
type of protection is unsuitable for the synthesis of compound 3
due to the destruction of the oxetan ring during amino acid regen-
eration by formic acid.
The synthesized adamantane-based taxol mimetics were evalu-
ated for their in vitro cytotoxicity against the A549 human lung
O
Ph
NBoc
OH
OTMS
OH
O
N
O
O
OCH3
2 steps
O
Ph
Boc
b
a
3
COOH
OCH3
C
C
O
O
O
O
O
O
8
6
7
Scheme 1. Reagents and conditions: (a) DCC, DMAP, CH2Cl2, 25 °C, 12 h, 49%; (b) TsOH, MeOH, 25 °C, 2 h, 89%.

O. N. Zefirova et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5091–5094
5093
Table 1
mantane (compound 14) maintains the high level of tubulin aggre-
gation.
In vitro cytotoxicities against the A549 cell line
The interesting ability of structure 5 enabled us to chose taxol/
taxotere side chain substituted adamantane as a structural frag-
ment in a hybrid compound 15 analogous to recently obtained hy-
brid taxol-colchicine ligand colchitaxel.26 (It is known that taxol
and colchicine interact with tubulin at different binding sites and
the latter inhibit the protein polymerization and microtubule for-
mation.)27 We hypothesized that binding of a ‘colchicine part’ of
compound 15 with colchicine binding site might induce the ‘ada-
mantane-based taxoid mimic part’ to interact effectively with the
taxol binding site of tubulin.
Compound
IC50
(
l
M)
Taxol
2
0.002
3.8 (Ref. 12)
2.5
9.5
5.6
3
4*
5
15
0.0006
*
Ester 4 was tested as a mixture of trans- and cis-isomers (in the ratio 2:1).22
The synthesis of the structure 15 is presented in Scheme 3. The
esterification of kemantane by an oxazolidine-type-protected
N-tert-butoxycarbonylphenylisoserine led to the ester 16, and sub-
sequent reduction of its keto-group by NaBH4 gave the correspond-
ing alcohol 17 (trans-/cis-isomer ratio 2:1). The further
esterification of 17 by the polyanhydride of pimelinic acid led to
isomeric esters 18. Finally, deacetylcolchicine (obtained in three
steps from colchicine)27 was attached to the carboxylic group of
compound 18, and the following deprotection of amino acid in
the product 19 led to compound 15 (trans-/cis-isomers ratio 2:1).
Compound 15 was found to possess very high cytotoxicity
against human lung carcinoma cell line A549 (Table 1). Though it
is caused mostly by interaction with colchicine binding site, it is
interesting to mention that immuno-fluorescent microscopy of
microtubules in the A549 cells28 revealed the existence of a small
amount of microtubule bundles, characteristic for the taxol action
among microtubules and their shortened aggregates in cells trea-
Figure 3. Sedimentation analysis of the tubulin after incubation in the presence of
compounds 2–5 and 12. (A) SDS–PAGE of tubulin pellets. (B) Estimation of the
relative amount of tubulin in the sediment (100% corresponds to the amount of
tubulin pelleted after incubation with taxol; DMSO was used as a negative control).
ted with 5 lM of 15. This observation needs, however, additional
investigation, because visually similar structures might be a result
of compound 15 interacting with a vinca-alkaloid binding site on
tubulin. These investigations are currently in progress.
Although we were unable to observe aggregates of tubulin by
AVEC-DIC microscopy in all tested adamantane ester samples (3–
5), electron microscopy of these samples stained with uranyl ace-
tate showed the presence of amorphous aggregates (data not
shown). Thus, the results of the sedimentation assay and electron
microscopy indicate that compounds 3–5 induce tubulin aggrega-
tion, and this ability is remarkably high for ester 5. It is interesting
that both unsubstituted adamantane core and taxol side chain
seem to play a part in this ability, because specially synthesized es-
ters 9–12 as well as 13a–c demonstrate a relative amount of tubu-
lin in the sedimentation assay of less than 35% (e.g., 12 in Fig. 3). At
the same time, the taxol side chain shift to the 2-position of ada-
In summary, the synthesized adamantane derivatives, though
being cytotoxic, turned out to be unable to promote microtubule
assembly of purified tubulin in vitro like most of the known simpli-
fied taxol analogues (e.g., Refs. 4,6,7,9,12). This indicates that the
‘simplification’ in the presented structures is too drastic in compar-
ison with the parent molecule. Nevertheless, the N-benzoyl-
(2R,3S)-phenylisoseryloxyadamantane (5) demonstrated the
ability to cause significant aggregation of the protein, whereas
the hybrid compound 15 revealed high and rather unusual cyto-
toxicity profile and may be considered as a structural clue for the
further studies.
O
OR
OR
OCCH2CH2NHBz
R1
R2
OR
a R1,R2: =O
H
H
b R1=R2= -CH3
c R1=H; R2= -CH3
RO
CH3
RO
H3C
9
10
12
13 a-c
14
11
R = taxol side chain
O
Ph
NH
O
O
O
OCH3
O
BocNH
O
OH
OCH3
CH3O
15
CH3O

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O. N. Zefirova et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5091–5094
Ph
O
Ph
O
O
Ph
OH
O
O
OH
BocN
O
O
BocN
O
BocN
HO
p-MeO-Ph
b
a
O
O
p-MeO-Ph
16
17
p-MeO-Ph
H2N
Ph
OH
O
O
OCH3
(OOC (CH2)5 COO)n
O
O
O
O
BocN
OCH3
CH3O
c
CH3O
d
O
18
p-MeO-Ph
Ph
HN
O
O
O
O
OCH3
O
BocN
e
15
O
O
OCH3
CH3O
p-MeO-Ph
CH3O
19
Scheme 3. Reagents and conditions: (a) DCC, DMAP, CH2Cl2, rt, 12 h, 46%; (b) NaBH4, Et2O, rt, 10 h, 96%; (c) DMAP, CH2Cl2, rt, 24 h, 46 %; (d) EEDQ, CH2Cl2, rt, 24 h, 76%; (e)
TsOH, MeOH, rt, 2 h, 63%.
10. Zefirova, O. N.; Selunina, E. V.; Averina, N. V.; Zyk, N. V.; Zefirov, N. S. Zh. Org.
Acknowledgments
Khim. 2002, 38, 1176 (Russ. J. Org. Chem. 2002, 38, 1125).
11. Averina, N. V.; Lapina, T. V.; Zefirova, O. N.; Zefirov, N. S. Vestnik Mosk. Univ. 2.
We are grateful to Dr. Walter Steffen for electron microscopy.
This work was done in the frame of a Scientific Cooperation Agree-
ment between Moscow and Rostock Universities and supported in
part by the German organization DAAD (the Leonard Euler Stu-
dentship program), by the Russian Foundation for Basic Research
(Project No. 07-03-12110-ofi), and by grants of the Russian Acad-
emy of Sciences and Russian Federation Ministry of Education.
We thank the laboratory of Dr. Kenneth A. Jacobson (National
Institute of Diabetes & Digestive & Kidney Diseases, NIH, Bethesda,
MD, USA) for useful collaboration on the molecular modeling stud-
ies, and for reading the manuscript.
Khim. 2002, 43, 244 (in Russian).
12. Zefirova, O. N.; Nurieva, E. V.; Lemcke, H.; Ivanov, A. A.; Zyk, N. V.; Weiss, D. G.;
Kuznetsov, S. A.; Zefirov, N. S. Mendeleev Commun. 2008, 18, 183.
13. Snyder, J. P.; Nettles, J. H.; Cornett, B.; Downing, K. H.; Nogales, E. Proc. Natl.
Acad. Sci. U.S.A. 2001, 98, 5312.
14. The molecular docking of compound 3 was performed automatically with the
Glide program of the MacroModel package.15 The binding site was defined as a
box with a side of 29 Å with a centre in the centroid of taxol atoms. For all
other parameters their default values were used. The full flexibility of the
ligand was allowed.
15. Mohamadi, F. N.; Richards, G. J.; Guida, W. C.; Liskamp, R.; Lipton, M.;
Caufield, C.; Chang, G.; Hendrickson, T.; Still, W. C. J. Comput. Chem. 1990, 11,
440.
16. Kiryukhin, M. V.; Nurieva, E. V.; Shishov, D. V.; Nuriev, V. N.; Zyk, N. V.; Zefirov,
N. S.; Zefirova, O. N. Vestnik Mosk. Univ. 2. Khim. 2007, 48, 342 (Moscow
University Chem. Bull. 2007, 48, 281).
Supplementary data
17. Didier, E.; Fouque, E.; Taillepied, I.; Commerçon, A. Tetrahedron Lett. 1994, 35,
2349.
18. For the synthetic details and the characteristics of all the compounds see
Online Supplementary Materials.
19. Zefirova, O. N.; Selunina, E. V.; Nuriev, V. N.; Zyk, N. V.; Zefirov, N. S. Zh. Org.
Khim. 2003, 39, 880 (Russ. J. Org. Chem. 2003, 39, 831).
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.07.116.
20. Selunina, E. V.; Zefirova, O. N.; Zyk, N. V.; Zefirov, N. S. Vestnik Mosk. Univ. 2.
Khim. 2002, 43, 237 (in Russian).
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28. In this assay A549 cells were treated with compound 15, fixed with poly-
formaldehyde and stained with antibodies (primary mouse anti-
secondary fluorescent labelled rabbit anti mouse-ALEXA 488).
a-tubulin