Compounds combining aminoadamantane and monoterpene moieties: Cytotoxicity and mutagenic effects

Article abstract of DOI:10.2174/1573406411666150518110053

A series of secondary amines combining monoterpenoid and aminoadamantane moieties have been synthesized. Their cytotoxic activity against human cancer cells CEM-13, MT-4, and U-937 has been studied for the first time. Most of the obtained compounds exhibited a significant cytotoxic activity with the median cytotoxic dose (CTD₅₀) ranging from 6 to 84 μM. The most promising results were obtained for compound 2b which was synthesized from 1-aminoadamantane and (-)-myrtenal and revealed a high activity against all tumor lines used (CTD₅₀= 12/21 μM) along with low toxicity with respect to MDCK cells (CTD₅₀= 1500 μM). The synthesized amines do not exert the genotoxic effect on cells of the biosensor strain based on recombinant E. coli cells bearing the pRAC-gfp plasmid.

Full text of DOI:10.2174/1573406411666150518110053

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Medicinal Chemistry, 2015, 11, 629-635
629
Compounds Combining Aminoadamantane and Monoterpene Moieties:
Cytotoxicity and Mutagenic Effects
Evgeniy V. Suslova,*, Konstantin Yu. Ponomareva, Artem D. Rogacheva,b, Michail A. Pokrovskyb,
Andrey G. Pokrovskyb, Maria B. Pykhtinac, Anatoly B. Beklemishevc, Dina V. Korchaginaa,
Konstantin P. Volchoa,b and Nariman F. Salakhutdinova,b
aN.N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch, Russian Academy of
b
Sciences, Novosibirsk, 630090, Russia; Novosibirsk State University, Novosibirsk, 630090, Russia;
cResearch Institute for Biochemistry, Siberian Branch of the Russian Academy of Medical Sciences,
Novosibirsk, Russia
Abstract: A series of secondary amines combining monoterpenoid and aminoadamantane moieties
have been synthesized. Their cytotoxic activity against human cancer cells CEM-13, MT-4, and U-937
has been studied for the first time. Most of the obtained compounds exhibited a significant cytotoxic
activity with the median cytotoxic dose (CTD50) ranging from 6 to 84 ꢀM. The most promising results
were obtained for compound 2b which was synthesized from 1-aminoadamantane and (ꢁ)-myrtenal and revealed a high
activity against all tumor lines used (CTD50 = 12÷21 ꢀM) along with low toxicity with respect to MDCK cells (CTD50
=
1500 ꢀM). The synthesized amines do not exert the genotoxic effect on cells of the biosensor strain based on recombinant
E. coli cells bearing the pRAC-gfp plasmid.
Keywords: Adamantane, amine, anticancer activity, cytotoxicity, genotoxicity, monoterpene.
INTRODUCTION
the latter [6]. Some of the synthesized compounds were
found to exhibit sufficiently high cytotoxicity against
MDCK cells. For example, CTD50 (the concentration causing
death of 50% of cells in the culture) for compound 2a (a
mixture of cis- and trans-isomers at the 1:1 ratio, Fig. 1) is
20.5 ꢀM.
The presence of the adamantyl moiety in a molecule is
known to promote its exhibiting diverse biological activity
[1-3]. Thus, one of the first antiviral drugs acting on the in-
fluenza virus was 1-aminoadamantane hydrochloride 1
(amantadine) (Fig. 1) [4]. Unfortunately, almost all isolated
O
O
O
OH
.
NH2 HCl
HO
NH
3
4
N
H
HO
OH
Adaphostin (NSC680410)
1
2a
Amantadine
Adarotene (ST1926)
Fig. (1). Structures of biologically active adamantane-containing compounds 1, 2a, 3 and 4.
strains of the influenza H3N2 and H1N1 viruses are resistant
to this drug [5]. Recently, we have demonstrated that the
addition of a monoterpenoid moiety to the 1-amino-
adamantane molecule reconstitutes the antiviral activity of
Some adamantane derivatives are known to have high cy-
totoxicity with respect to different cancer cell types [1, 7].
Thus, adaphostin 3 (NSC680410), which is currently under-
going preclinical trials, exhibits activity against leukemia,
glioblastoma, and nonsmall cell lung cancer cell line [1, 8].
Adarotene 4 (ST1926) is active against ovarian carcinoma,
neuroblastoma, and leukemia cells (Fig. 1) [1, 9]. Moreover,
monoterpenoids are known to possess the cytotoxic activity
against some cancer cell lines [10]. For example, citral in-
*Address correspondence to this author at the Novosibirsk Institute of
Organic Chemistry, Siberian Branch of the Russian Academy of Sciences,
630090, Novosibirsk, Russia; Tel: +73833308870; Fax: +73833309752;
E-mail: redfox@nioch.nsc.ru
1ꢀ7ꢁ-ꢂꢂꢃꢀ/15 $58.00+.00
© 2015 Bentham Science Publishers

630 Medicinal Chemistry, 2015, Vol. 11, No. 7
Suslov et al.
R
.
NH2 HCl
.
HN
NH2 HCl
H
N
R
1
O
H
5
R
(a), (b)
(a), (b)
or (c) (for 6d)
or (c) (for 6d)
6a-d
7a-d
2a-d
2a (61%), 6a, 7a (48%) R =
2c (58%), 6c, 7c (48%) R =
2b (84%), 6b, 7b (64%) R =
2d (45%), 6d, 7d (38%) R =
HO
O
H
Scheme 1. Synthesis of compounds 2a-d and 7a-d ((a) MeOH, Et3N; (b) NaBH4, MeOH; (c) H2, 10% Pd/C, H-Cube reactor).
18
17
20
19
12
14
16
15
13
11
O
N
H
1
8
(d)
9
2d, 52%
7
2
10
6
3
O
5
9, 87%
)
4
, (c
1
H
3 h
(a), (b)
O
5
, (
O
c
)
(-)-a-pinene 8
45
(d)
m
6d, 46%
N
i
n
H
7d, 47%
10, 81%
Scheme 2. Synthesis of compounds 2d, 6d, 7d, 9 and 10 ((a) O3, -80°...-60°C, MeOH; (b) Me2S, -60°C; (c) MeOH, Et3N, 20°C; (d) H2, 10%
Pd/C, H-Cube reactor).
duced apoptosis in several hematopoietic cancer cell lines in
concentration of 22.25 ꢂM [11]. However, due to their meta-
bolic instability, such monoterpenoids can not be considered
as promising antitumor agents for practical use.
[13, 14]. In the present study, compounds 2a-c and 7a-c
were synthesized in accordance with the procedures [6, 12];
the yields of the desired amines per starting aminoadaman-
tanes ranged from 38% to 84%.
These findings prompted us to study the anticancer activ-
ity of the derivatives of 1- and 2-aminoadamantanes contain-
ing the monoterpenoid moiety which was the objective of
this work.
Moreover, expanding a set of adamantylamine deriva-
tives possessing monoterpenoid moieties, we have synthe-
sized compounds 2d and 7d for the first time. Ketoaldehyde
6d required for the synthesis of these substances was ob-
tained by ozonolysis of (ꢁ)-ꢃ-pinene 8 (Scheme 2) in accor-
dance with the procedure [15].
RESULTS AND DISCUSSION
Chemistry
Imine 9 was produced by a reaction of 1-amino-
adamantane 1 with aldehyde 6d in methanol in the presence
of Et3N for 3 hours with the yield of 87% (Scheme 2). If 2-
aminoadamantane hydrochloride 5 was used, the reaction
proceeded slightly faster, and complete conversion of com-
pound 6d was achieved in 45 min; the yield of imine 10 was
81%. Note that in terms of its reactivity with respect to hy-
drochlorides 1 and 5, aldehyde 6d is similar to (ꢁ)-myrtenal
6b, which reacted with compound 5 faster than with adaman-
tylamine 1 (20 min and 90 min, respectively). Interestingly,
the inverse relationship was observed for citral 6a. For this
Previously [6, 12], we described the synthesis of com-
pounds 2a-c and 7a-c. These compounds were obtained by a
reaction of 1- and 2-aminoadamantane hydrochlorides 1 and
5 with citral 6a, (ꢁ)-myrtenal 6b, and (1R,6R)-2-(2,2-
dimethylcyclopent-3-enyl)-2-hydroxypropanal 6c, respec-
tively, in the presence of triethylamine followed by the re-
duction of imines with NaBH4 (Scheme 1). Monoterpenoids
6a and 6b are the commercially available compounds; alde-
hyde 6c was produced by isomerization of verbenol epoxide

Compounds Combining Aminoadamantane and Monoterpene Moieties
Medicinal Chemistry, 2015, Vol. 11, No. 7 631
Table 1. Cytotoxic activity of compounds 2a-d and 7a-d against lymphoblastoid cell lines and MDCK cells.
Cytotoxicity Against Cell Line (CTD50, ꢀM)a
Compound
CEM-13
U-937
MT-4
MDCK [6]
2a
5.9 2.1
11.9 0.6
>100
27.9 3.0
21.1 1.6
>100
>100
11.9 1.1
62.6 9.4
>100
20.5
1506.1
145.6
n.d. b
980.8
157.6
731.5
n.d.
2b
2c
2d
>100
>100
7a
16.7 8.2
42.1 6.6
>100
83.6 22.2
>100
25.4 4.3
42.1 4.3
>100
7b
7c
7d
>100
69.2 29.5
3.4 2.1
49.5 6.8
0.17 0.06
>100
Doxorubicin
2.8 1.8
n.d.
a CTD50 - concentration (ꢄM) causing the death of 50% of the cells in culture
b n.d. – not determined
aldehyde, a complete conversion of starting materials was
achieved one day after the start of the reaction with 2-
aminoadamantane hydrochloride 5 and in 5 hours, when re-
acted with compound 1.
The most promising results were obtained for compound
2b synthesized from 1-aminoadamantane 1 and the bicyclic
monoterpenoid myrtenal 6b. Compound 2b exhibited sig-
nificant activity against all used tumor cell lines with CTD50
ranging from 12 ꢄM to 21 ꢄM, being two orders of magni-
tude less toxic to non-cancerous MDCK cells (Table 1).
The imine bond in compounds 9 and 10 was reduced us-
ing flow hydrogenation reactor “H-Cube Pro”. The yield of
the desired amines 2d and 7d after purification by column
chromatography was 52% and 47%, respectively.
Compound 2c demonstrated an appreciable activity
against the MT-4 cell line only, and amine 2d revealed no
cytotoxic effect in all experiments.
After producing the set of compounds 2a-d and 7a-d, we
studied their cytotoxic activity.
The substitution of the 1-aminoadamantane moiety with
the isomeric 2-aminoadamantane one when switching from
type 2 compounds to type 7 compounds changed the ob-
served patterns (Table 1). Compound 7a with an acyclic sub-
stituent appeared to be least toxic to non-cancerous MDCK
cells, whereas a derivative of (ꢁ)-myrtenal, compound 7b,
exhibited appreciable toxicity with respect to this cell line.
Both compounds, 7a and 7b, demonstrated significant cyto-
toxicity against cancer cells CEM-13 and MT-4, with amine
7a being almost two times more active.
Study of Biological Activity
Cytotoxicity
The cytotoxic activity of compounds 2a-d and 7a-d was
studied on human cancer cells CEM-13, MT-4 (T-cellular
human leucosis cells), and U-937 (human monocytes). The
cytotoxic activity (Table 1) was determined by measuring
the concentration inhibiting tumor cell viability by 50%
(CTD50) using the conventional MTT assay, which allows
estimating the number of survived cells [16]. Compounds
with the CTD50 values less 100 ꢄM were considered to be
active. Doxorubicin was used as a positive control (see con-
centrations in Table 1). For comparison, Table 1 presents the
previously obtained data on cytotoxicity of the tested com-
pounds with respect to non-cancer MDCK cells [6].
Compound 7c was not active against all cancer cell lines
studied, and compound 7d revealed a moderate activity
against U-937 and CEM-13. It is interesting that an isomer
of amine 7d, compound 2d, was not active against all cancer
cell lines used.
Thus, it was found that the compounds combining ami-
noadamantane and monoterpenoid moieties can possess sig-
nificant cytotoxicity with respect to cancer cells. The most
promising was compound 2b obtained from 1-
aminoadamantane 1 and (-)-myrtenal 6b, which demon-
strated a significant activity against all the tumor lines used
along with low toxicity to MDCK cells.
Compound 2a, obtained from 1-aminoadamantane hy-
drochloride 1 and acyclic monoterpenoid citral 6a, exhibited
a high cytotoxic activity against the CEM-13 cell line, which
was comparable to that of the reference drug, doxorubicin,
and much lower activity against U-937 cells (Table 1). The
MT-4 cell line was resistant to the action of this amine. It
should be noted that compound 2a having high cytotoxicity
with respect to both MDCK and cancer cells looks like un-
promising for further studies.
Note that additional studies are needed to elucidate the
mechanisms of cancer cell death caused by the action of
compound 2b.

632 Medicinal Chemistry, 2015, Vol. 11, No. 7
Suslov et al.
Evaluation of Mutagenic Effects of the Compounds on
Bacterial Biosensor Cells
analyzed samples, while that in positive control samples con-
taining 86 ꢅM nalidixic acid was 3.4. These results indicate
that none of the studied compounds activates the SOS re-
sponse system in cells of the biosensor strain (i.e. provides
the genotoxic effect at the tested concentrations). The data
obtained during the experiments and used to calculate the
induction factors of GFP synthesis are shown in the supple-
mentary materials.
Mutagenicity is an important feature of potential drugs
(including anticancer ones) that largely determines their
prospects. In the present work, the potential genotoxicity of
compounds 2a-d and 7a-d was investigated using a whole-
cell biosensor based on recombinant E. coli cells.
Chromosomal DNA repair in bacteria is induced by acti-
vation of different systems, depending on the nature of DNA
damage. One of the main ways to DNA repair involves the
recA-mediated SOS response. At the first stage, expression
of the recA gene is activated, which leads to synthesis of
RecA protein. This protein initiates self-cleavage of the
LexA repressor that inhibits the SOS response genes under
normal conditions. RecA plays a key role in several DNA
repair pathways in bacteria. The creation of artificial genetic
constructs, in which the genes of reporter proteins (chro-
mogenic, fluorescent, chemiluminescent) are linked to the
regulatory regions of the recA genes of various bacteria, un-
derlies the development of most of sensitive whole-cell bio-
sensor systems designated to determine genotoxicity of com-
pounds under study. A large number of similar whole-cell
bacterial biosensors for rapid and sensitive detection of the
genotoxic effect of mutagens of chemical and physical
nature on cells have been developed to date [17-20].
CONCLUSION
A number of compounds combining monoterpenoid and
aminoadamantane moieties were synthesized; their cytotoxic
activity against human cancer cells CEM-13, MT-4 (T-
cellular human leucosis cells), and U-937 (human mono-
cytes) was studied for the first time. Most of the obtained
compounds exhibited a significant cytotoxic activity with
CTD50 ranging from 6 to 84 ꢅM. The most promising results
were obtained for compound 2b synthesized from 1-
aminoadamantane hydrochloride 1 and (ꢁ)-myrtenal 6b,
which demonstrated a significant activity against all cancer
cell lines used with CTD50 ranging from 12 ꢅM to 21 ꢅM,
being two orders of magnitude less toxic to non-cancer
MDCK cells. None of the tested compounds was found to
activate the SOS response in cells of the biosensor strain
based on recombinant E. coli cells bearing the pRAC-gfp
plasmid, i.e. they did not have the genotoxic effect at the
studied concentrations.
In this study, the possible genotoxicity was investigated
using a whole-cell biosensor based on E. coli recombinant
cells bearing the pRAC-gfp plasmid derived from the previ-
ously constructed expression vector pREB-gfp [19]. The
pREB-gfp and pRAC-gfp plasmids provided expression of
the gene encoding the reporter green fluorescent protein
(GFP) in biosensor cells under the control of the regulatory
region of the recA gene from Proteus mirabilis. It was previ-
ously shown that after the biosensor cell strains are treated
with chemical mutagens [16, 19], the cells responded by an
appropriate level of GFP synthesis. There was a direct corre-
lation between the effect of different doses and the exposure
time of the biosensor cells to mutagens of physical (UV irra-
diation) and chemical nature (nalidixic acid, mitomycin C, 5-
bromo-2-deoxyuridine, actinomycin D, and 1,1-dimethyl-
hydrazine) and the intensity of fluorescence emitted by the
indicator strain culture [19]. In preliminary experiments, it
was found that nalidixic acid had a dose-dependent geno-
toxic effect on E. coli cells, bearing the pRAC-gfp plasmid,
at concentrations ranging from 4.3 to 215 ꢅM.
EXPERIMENTAL METHODS
Chemistry
Reagents were purchased from commercial suppliers and
used as received. Compounds 2a-c, 7a-c were synthesized in
accordance with [6, 12], the experimental procedures and
spectral data for these compounds are provided in the sup-
porting materials. Solvents were purified by distillation be-
fore use. Column chromatography (CC) was performed on
silica gel (60-230 ꢅ, Macherey-Nagel). Optical rotation: po-
lAAr 3005 spectrometer; MeOH soln. concentration g/100
mL, specific rotation is expressed as (deg mL)/(g dm). GC:
7820A gas chromatograph (Agilent Tech., USA); flame-
ionization detector; HP-5 capillary column (ꢀꢁ0.25 mm ꢀ 30
m ꢀ 0.25 ꢅm), He as a carrier gas (flow rate 2 mL/min, flow
1
division 99:1). H- and 13C-NMR spectra: Bruker DRX-500
spectrometer (500.13 MHz (1H) and 125.76 MHz (13C) in
CDCl3); chemical shifts ꢆ in ppm rel. to residual chloroform
[ꢆ(H) 7.24, ꢆ(C) 76.90 ppm], J in Hz. The structures of the
Concentrations of compounds 2a-d and 7a-d, at which
studies of their genotoxicity were carried out, were chosen
individually for each substance, based on the data obtained
in a study of cytotoxicity of these compounds on CEM-13,
MT-4, and U-937 cell lines, and ranged from 56 to 200 ꢅM.
A compound can be considered to be genotoxic if the induc-
tion factor of GFP synthesis is 2 or higher [21]. The positive
control samples contained nalidixic acid as a mutagen; the
negative control samples contained water.
1
products were determined by analyzing their H NMR spec-
1
tra, H,1H double- resonance spectra, J-modulated 13C NMR
spectra (JMOD) and 13C,1H-type 2D heteronuclear correla-
tion with one bond and long-range spin-spin coupling con-
1
2,3
stants (COSY, J(C,H) = 135 Hz, COLOC, J(C,H) = 10
Hz). Numeration of atoms in the compounds is given for
assigning the signals in the NMR spectra and does not coin-
cide with that for the names according to the nomenclature
of the compounds. HR-MS: DFS Thermo Scientific spec-
trometer in a full scan mode (0-500 m/z, 70 eV electron im-
pact ionization, direct sample injection).
None of the tested compounds 2a-d and 7a-d at the stud-
ied doses induced GFP synthesis in cells of the BL-21(DE3)
biosensor E. coli strain bearing the pRAC-gfp plasmid. The
induction factor of GFP synthesis was less than 2.0 in all the

Compounds Combining Aminoadamantane and Monoterpene Moieties
Medicinal Chemistry, 2015, Vol. 11, No. 7 633
Hydrogenation of imines 9 and 10 to corresponding
amines 2d and 7d was performed in flow reactor “H-Cube
Pro” (ThalesNano, Inc) using 10% Pd/C as catalyst (Cat-
CartTM catalyst cartridge system, 30 mm). Hydrogenation
conditions: solvent – MeOH; concentration of imine 0.05 M;
temperature 40oC, the reactor pressure 1 bar, flow rate 0.5
mL/min.
6, C-10), 38.20 (t, C-11), 31.57 (t, C-12), 40.10 (d, C-13),
43.15 (s, C-14), 54.12 (d, C-15), 23.03 (t, C-16), 17.03 (q, C-
17), 30.34 (q, C-18), 207.68 (s, C-19), 29.85 (q, C-20). HR-
MS: found M 303.2549, calculated M 303.2557, C20H33NO.
1-((1R,3R)-3-(2-adamantan-2-yl)amino)ethyl)-2,2-
dimethylcyclobutyl)ethan-1-one (7d)
The purity of the target compounds was determined by
gas chromatography methods. All of the target compounds
reported in this paper have a purity of more than 95%.
Triethylamine (0.100 g, 0.99 mmol) and aldehyde 6d (0.1
g, 0.59 mmol) were added to a solution of 2-adamantylamine
hydrochloride 5 (0.100 g, 0.53 mmol) in methanol (2 mL).
The mixture was stirred for 45 min, then the solvent was
distilled off and the residue was suspended in hexane (15
mL). The suspension was filtered, precipitation was dis-
carded and hexane was evaporated from the filtrate. This
gave 1-((1R,3R)-3-(2-((adamantan-2-yl)imino)ethyl)-2,2-di-
methylcyclobutyl)ethan-1-one 10 (0.130 g (81%)).
Spectral and analytical investigations were carried out at
Collective Chemical Service center of Siberian Branch of the
Russian Academy of Sciences.
Synthesis and Characterization
1-((1R,3R)-3-(2-adamantan-1-yl)amino)ethyl)-2,2-
dimethylcyclobutyl)ethan-1-one (2d)
1H-NMR (500 MHz, CDCl3): ꢁ = 0.78 (s; 3H, Me-17),
1.19 (s; 3H, Me-18), 1.38 (dm; 2H, 2J ~ 12.5 Hz, H-4, H-9),
1.48-1.54 (m; 2H, H-1, H-3), 1.91 (s; 3H, Me-20), 2.04-2.22
Triethylamine (0.100 g, 0.99 mmol) and aldehyde 6d
(0.098 g, 0.58 mmol) were added to a solution of 1-
adamantylamine hydrochloride 1 (0.100 g, 0.53 mmol) in
methanol (2 mL). The mixture was stirred for 3 h, then the
solvent was distilled off and the residue was suspended in
hexane (15 mL). The suspension was filtered, precipitation
was discarded and hexane was evaporated from the filtrate.
This gave 1-((1R,3R)-3-(2-((adamantan-1-yl)imino)ethyl)-
2,2-dimethylcyclobutyl)ethan-1-one 9 (0.139 g, 87%).
(m; 5H, H-4’, H-9’, 2H-12, H-13), 2.71 (dd; 1H, J15,16
=
10.2, J15,16’ = 7.4 Hz, H-15), 3.02 (br.t, 1H, J2,1(3) ~ 2.5 Hz,
H-2), 7.53 (dd, 1H, J11,12 = 5.1, J11,12’ = 4.0 Hz, H-11). The
signals of the other protons appeared in 1.60-1.95 ppm in the
form of overlapping multiplets. 13C-NMR (125 MHz,
CDCl3): ꢁ = 34.96 and 35.09 (d, C-1, C-3), 74.44 (d, C-2),
31.65 and 31.68 (t, C-4, C-9), 27.90 (d, C-5), 37.93 (t, C-6),
27.19 (d, C-7), 37.18 and 37.23 (t, C-8, C-10), 159.25 (d, C-
11), 36.76 (t, C-12), 38.91 (d, C-13), 43.19 (s, C-14), 54.08
(d, C-15), 22.86 (t, C-16), 17.33 (q, C-17), 30.51 (q, C-18),
206.33 (s, C-19), 29.76 (q, C-20). HR-MS: found M
301.2395, calculated M 301.2400, C20H31NO.
1H-NMR (500 MHz, CDCl3): ꢁ = 0.78 (s; Me-17), 1.18
2
(s; 3H, Me-18), 1.75 (ddd; 1H, J = 11.0, J16,13 = J16,15 = 7.4
Hz, H-16), 1.92 (s; 3H, Me-20), 1.98-2.02 (m; 3H, H-3, H-5,
H-7), 2.05-2.21 (m; 3H, 2H-12, H-13), 2.72 (dd; 1H, J15,16’
10.2, J15,16 = 7.4 Hz, H-15), 7.42 (dd; 1H, J11,12 = 5.3, J11,12’
=
=
Hydrogenation of imine 10 (0.13 g, 0.43 mmol) in the
flow reactor “H-Cube Pro” and further separation using col-
4.3 Hz, H-11). The signals of the other protons appeared in
1.44-1.65 and 1.85-1.96 ppm in the form of overlapping
multiplets. 13C-NMR (125 MHz, CDCl3): ꢁ = 56.56 (s, C-1),
42.84 (t, C-2, C-8, C-9), 29.24 (d, C-3, C-5, C-7), 36.26 (t,
C-4, C-6, C-10), 156.60 (d, C-11), 37.31 (t, C-12), 39.25 (d,
C-13), 43.33 (s, C-14), 53.98 (d, C-15), 22.64 (t, C-16),
17.23 (q, C-17), 30.27 (q, C-18), 207.35 (s, C-19), 29.48 (q,
C-20). HR-MS: found M 301.2397, calculated M 301.2400,
C20H31NO.
[ꢀ]3D1
umn chromatography led to amine 7d (0.062 g, 47%).
= 1.3 (C 0.47, MeOH).
1H-NMR (500 MHz, CDCl3): ꢁ = 0.83 (s; 3H, Me-17),
1.25 (s; 3H, Me-18), 1.45 (br.d; 2H, 2J = 12.3 Hz, H-4, H-9),
2
1.45-1.53 (m; 1H, H-12’), 1.35 (dtd; 1H, J = 13.0, J12,11
=
8.7, J12,13 = 6.0 Hz, H-12), 1.63-1.69 (m; 4H, 2H-6, H-8, H-
10), 1.70-1.75 (m; 1H, H-5), 1.77-1.96 (m; 10H, H-1, H-3,
H-4’, H-7, H-8’, H-9’, H-10’, H-13, 2H-16), 1.99 (s; 3H,
Me-20), 2.41-2.51 (m; 2H, 2H-11), 2.64 (br.s; 1H, H-2), 2.77
(dd; 1H, J15,16 = 10.0, J15,16’ = 7.6 Hz, H-15). 13C-NMR (125
MHz, CDCl3): ꢁ = 31.97 and 32.00 (d, C-1, C-3), 61.78 (d,
C-2), 31.25 and 31.27 (t, C-4, C-9), 27.55 (d, C-5), 37.87 (t,
C-6), 27.75 (d, C-7), 37.51 (t, C-8, C-10), 45.08 (t, C-11),
31.03 (t, C-12), 40.27 (d, C-13), 43.25 (s, C-14), 54.27 (d, C-
15), 23.20 (t, C-16), 17.11 (q, C-17), 30.42 (q, C-18), 207.84
(s, C-19), 29.94 (q, C-20). HR-MS: found [M-H] 302.2479,
calculated [M-H] 302.2478, C20H32NO.
Hydrogenation of 0.125 g (0.42 mmol) of imine 9 in the
flow reactor “H-Cube Pro” and further separation using col-
[ꢀ]3D1
umn chromatography led to amine 2d (0.065 g, 52%).
= -12.5 (C 1.75, MeOH).
1H-NMR (500 MHz, CDCl3): ꢁ = 0.78 (s; 3H, Me-17),
1.21 (s; 3H, Me-18), 1.20-1.31 (m; 1H, H-12), 1.39 (dddd;
2
1H, J = 13.2, J12’,11’ = 9.3, J12’,11 = 6.0, J12’,13 = 5.5 Hz, H-
12’), 1.53 and 1.59 (br.d; 6H, J = 12 Hz, 2H-4, 2H-6, 2H-
10), 1.52-1.56 (m; 6H, 2H-2, 2H-8, 2H-9), 1.73-1.91 (m; 3H,
H-13, 2H-16), 1.95 (s; 3H, Me-20), 1.96-2.01 (m; 3H, H-3,
H-5, H-7), 2.38 (ddd; 1H, 2J = 10.6, J11,12 = 9.3, J11,12’ = 6.0
Hz, H-11), 2.44 (ddd; 1H, 2J = 10.6, J11’,12’ = 9.3, J11’,12 = 5.7
Hz, H-11’), 2.73 (dd; 1H, J15,16 = 10.0, J15,16’ = 7.4 Hz, H-
15). 13C-NMR (125 MHz, CDCl3): ꢁ = 50.04 (s, C-1), 42.68
(t, C-2, C-8, C-9), 29.41 (d, C-3, C-5, C-7), 36.59 (t, C-4, C-
Biological Study
Cell Viability Assays
The human cancer cells MT-4, CEM-13 (the cells of T-
cellular human leucosis), and U-937 (human monocytes)
were used in this study. The cells were cultured in the RPMI-
1640 medium that contained 10% embryonic calf serum, L-

634 Medicinal Chemistry, 2015, Vol. 11, No. 7
Suslov et al.
The results of genotoxicity evaluation for the analyzed
compounds and different concentrations of DMSO are pro-
vided in the supplementary materials.
glutamine (2 mmol/L), gentamicin (80 ꢇg/mL), and linco-
mycin (30 mg/mL) in a CO2 incubator at 37°C. The com-
pounds were dissolved in DMSO and added to the cellular
culture at the required concentrations. Cells (0.5ꢀ106 cells
per mL) were placed on 96-well microliter plates and culti-
vated at 37 °C in 5% CO2/95% air for 72 h. Three wells were
used for each concentration. The cells which were incubated
with the same final concentration of DMSO without com-
pounds were used as a control. Cell viability was assessed
through an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-phenyl-
2H-tetrazolium bromide] conversion assay. 1% MTT was
added to each well. Four hours later DMSO was added and
mixed for 15 min. Finally, the optical density values were
monitored at 570 nm.
CONFLICT OF INTEREST
The authors confirm that this article content has no con-
flict of interest.
ACKNOWLEDGEMENTS
The work was partly supported by the Ministry of Educa-
tion and Science of the Russian Federation and Ministry of
Education, Science and Innovation Policy of the Novosibirsk
Region, as well as by RFBR, research project No. 15-33-
20198.
Genotoxicity Assays
SUPPLEMENTARY MATERIAL
Evaluation of possible genotoxicity of the compounds
under study was carried out using a whole-cell biosensor
based on the recombinant BL-21(DE3) E. coli strain bearing
the pRAC-gfp plasmid. Experiments were performed accord-
ing to the following scheme. The sample (3mL) of the over-
night culture of the biosensor E. coli strain was inoculated
into a flask containing 100 mL of fresh Luria-Bertani (LB)
Additional supporting information may be found in the
online version of this article at the publisher’s web-site
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Received: September 04, 2014
Revised: May 14, 2015
Accepted: May 14, 2015