Synthesis and cytotoxic activity of substituted hexahydro-2H-4,6-(epoxymethano)chromen-8(5H)-ones obtained from (–)-verbenone

Article abstract of DOI:10.1007/s11172-015-1148-3

A number of compounds with a substituted hexahydro-2H-4,6-(epoxymethano)chromen8(5H)-one framework was synthesized starting from monoterpenoid (–)-verbenone and aromatic aldehydes containing methoxy and hydroxy groups. Cytotoxic activity of these compounds on human tumor cell lines was studied for the first time. Compounds containing three methoxy groups in each aromatic ring were found to be the most promising for further studies.

Full text of DOI:10.1007/s11172-015-1148-3

Russian Chemical Bulletin, International Edition, Vol. 64, No. 9, pp. 2257—2260, September, 2015
2257
Synthesis and cytotoxic activity
of substituted hexahydroꢀ2Hꢀ4,6ꢀ(epoxymethano)chromenꢀ8(5H)ꢀones
obtained from (–)ꢀverbenone
I. V. Il´ina,a,b M. A. Pokrovsky,b O. S. Mikhalchenko,a D. V. Korchagina,a K. P. Volcho,a,b
A. G. Pokrovsky,b and N. F. Salakhutdinova,b
aN. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of Sciences,
9 prosp. Akad. Lavrent´eva, 630090 Novosibirsk, Russian Federation.
Eꢀmail: volcho@nioch.nsc.ru
bNovosibirsk State University,
2 ul. Pirogova, 630090 Novosibirsk, Russian Federation
A number of compounds with a substituted hexahydroꢀ2Hꢀ4,6ꢀ(epoxymethano)chromenꢀ
8(5H)ꢀone framework was synthesized starting from monoterpenoid (–)ꢀverbenone and aroꢀ
matic aldehydes containing methoxy and hydroxy groups. Cytotoxic activity of these comꢀ
pounds on human tumor cell lines was studied for the first time. Compounds containing three
methoxy groups in each aromatic ring were found to be the most promising for further studies.
Key words: monoterpene, cytotoxicity, verbenone, clay, heterocyclic compounds.
Chemical therapy of oncologic diseases is one of the
key problems of modern medicine, which is due to both
the unsatisfactory efficiency of many available drugs and
the side effects1 which accompany their application. Apart
from that, the tumor cells survived the chemotherapy freꢀ
quently exhibit the drug resistance to a wide range of mediꢀ
cines,2 that makes the search for pharmaceutical agents of
new structural types an important and actual problem.
Many anticancer agents used in chemotherapy are natural
compounds, or their derivatives, or analogues,3—5 thereꢀ
fore, the search for new cytotoxic agent among these
groups of compounds attracts attention of many reꢀ
searches.6,7 It is known that some compounds combining
an aromatic and a monoterpenoid fragments possess subꢀ
stantial activity against cancer cell lines, for example, comꢀ
pounds A—C were found to possess considerable cytoꢀ
toxicity.8—10 It is important to note that all these comꢀ
pounds are distinguished by the presence of hydroxy and
methoxy groups in the aromatic ring.
Earlier, we have found11—14 that the reaction of diol 1
obtained from (–)ꢀverbenone (2) with aromatic aldehydes
3a—f bearing electronꢀdonating substituents in the presꢀ
ence of montmorillonite clay K10 led to the formation of
unusual compounds 4a—f with substituted hexahydroꢀ2Hꢀ
4,6ꢀ(epoxymethano)chromenꢀ8(5H)ꢀone framework, which
with the chromeneꢀtype framework were the major prodꢀ
ucts in these reactions in all the cases, a stereoselective
formation of complex polycyclic compounds 4 with earꢀ
lier unknown framework in one preparative step makes
them a unique object for the study of biological properties.
are the products of the addition of two aldehyde molecules
to monoterpenoid 1 (Scheme 1). Though compounds 5a—f
* Dedicated to Academician of the Russian Academy of Sciences
N. S. Zefirov on the occasion of his 80th birthday.
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 9, pp. 2257—2260, September, 2015.
1066ꢀ5285/15/6409ꢀ2257 © 2015 Springer Science+Business Media, Inc.

2258
Russ.Chem.Bull., Int.Ed., Vol. 64, No. 9, September, 2015
Il´ina et al.
Scheme 1
i. 1) H2O2/NaOH, 2) LiAlH4, 3) clay K10; ii. clay K10.
3—5
R1
H
H
H
OMe
R2
H
OMe
OMe
OMe
R3
R4
H
H
OMe
H
3—5
e
f
R1
H
H
R2
OMe
OH
R3
OH
OMe
OH
R4
OMe
H
a
b
c
d
OMe
OMe
OMe
OMe
g
H
OMe
H
The structure of compounds 4a—f resembles the strucꢀ
ture of many natural cytostatics with antitubulin effect,7
and the studies of their antitumor properties can be very
promising. The purpose of the present work is the syntheꢀ
sis and studies of cytotoxic activity of compounds 4 with
substituted hexahydroꢀ2Hꢀ4,6ꢀ(epoxymethano)chromenꢀ
8(5H)ꢀone framework.
At the beginning of our studies, we synthesized comꢀ
pounds 4a—f according to the procedures described earꢀ
lier,11—14 the yields of the target products were within 7—11%.
One more compound was synthesized by the reaction of
monoterpenoid 1 with vanillin (3g) on clay K10 for 3 days
at room temperature; the target compound 4g was obꢀ
tained in 11% yield, compound 5g with the chromeneꢀ
type framework was obtained in 20% yield.
cytotoxicity on tumor cell lines CEMꢀ13. In contrast to
this, almost all the tested compounds with CTD50 within
64—99 mol L–1 turned out to be active against the cell
line MTꢀ4, an exception was compound 4g exhibiting no
any cytotoxic activity.
A comparison of the test results on the human monoꢀ
cytes tumor line Uꢀ937 showed that the activity depended
on the amount of methoxy groups in the studied comꢀ
pounds: the most active were compounds 4c and 4d conꢀ
taining three methoxy groups in each aromatic ring inꢀ
dependent of their positions; the least active was comꢀ
pound 4b with two methoxy groups; whereas compound 4a
with one methoxy group or compounds 4e—g also conꢀ
taining a hydroxy group did not exhibit noticeable cytoꢀ
toxic activity.
Cytotoxic activity of compounds 4a—g was studied on
the tumor cells of the human Tꢀcell leukosis CEMꢀ13 and
MTꢀ4 and the tumor line of human monocytes Uꢀ937.
The activity was determined by measurement of the
concentration causing the death of 50% of tumor cells
(CTD50), using MTTꢀanalysis,* which allows one to evaluꢀ
ate amount of the survived cells.15 The results are given
in Table 1. The compounds with the CTD50 below
100 mol L–1 were considered as active.
Table 1. Cytotoxic activity (CTD50) of compounds 4a—g
Compound
CTD50/mol L–1
CEMꢀ13
MTꢀ4
Uꢀ937
4a
4b
4c
4d
4e
4f
4g
137.4 24.4
130.4 11.4
188.4 10.7
114.2 5.31
101.0 44.1
175.8 7.71
>200
64.0 11.8
99.4 3.71
86.6 24.9
77.3 16.9
69.9 11.1
94.5 11.6
>200
113.7 1.7
191.1 13.3
70.0 5.3
170.0 25.6
122.3 6.8
164.8 40.0
—
The results show that only compound 4c obtained from
3,4,5ꢀtrimethoxybenzaldehyde (3c) exhibits a noticeable
* MTTꢀanalysis is used to evaluate the viability of cells with
3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide.
Doxorubicin
3.44 2.11
2.76 1.76
10.17 0.06

Cytotoxic activity of (ꢀ)ꢀverbenone derivatives
Russ.Chem.Bull., Int.Ed., Vol. 64, No. 9, September, 2015 2259
based on the ratio of the peak areas for proton H3a in the 1H NMR
spectrum.
In conclusion, a series of compounds with substituted
hexahydroꢀ2Hꢀ4,6ꢀ(epoxymethano)chromenꢀ8(5H)ꢀone
framework was synthesized, starting from available monoꢀ
terpenoid (–)ꢀverbenone (2) and a number of aromatic
aldehydes containing methoxy and hydroxy groups, their
cytotoxic activity on human tumor cell lines was studied
for the first time. Compounds 4c and 4d containing three
methoxy groups in each aromatic ring were shown to be
the most promising for further studies.
Compound 4g. A light brown dense oil. []30 –27 (c 0.28,
D
CHCl3). 1H NMR (CDCl3), : 1.06 (d, 3 H, C(18)H3, J18,9
=
= 7.5 Hz); 1.39 (s, 3 H, C(17)H3); 1.71 (dd, 1 H, H(4a),
2J = 13.8 Hz, J4a,3a = 12.1 Hz); 1.80 (m, 1 H, H(8e), all J  3.0 Hz);
1.97 (dd, 1 H, H(4e), 2J = 13.8 Hz, J4e,3a = 2.5 Hz); 2.22 (ddd,
1 H, H(7a), 2J = 14.2 Hz, J7a,6e = 3.3 Hz, J7a,8e = 3.0 Hz); 2.29
(dddd, 1 H, H(6e), J6e,1a = 5.9 Hz, J6e,7a = 3.3 Hz, J6e,7e = 3.0 Hz,
2
J6e,8e = 0.5 Hz); 2.39 (dddd, 1 H, H(7e), J = 14.2 Hz, J7e,6e
=
= 3.0 Hz, J7e,8e = 3.0 Hz, J7e,9e = 1.8 Hz); 2.51 (qdd, 1 H, H(9e),
J9e,18 = 7.5 Hz, J9e,8e = 2.2 Hz, J9e,7e = 1.8 Hz); 3.84 (s, 3 H,
Experimental
C(19)H3); 3.86 (s, 3 H, C(27)H3); 4.39 (d, 1 H, H(1a), J1a,6e
=
= 5.9 Hz); 5.00 (d, 1 H, H(20), J20,8e = 2.1 Hz); 5.05 (dd, 1 H,
H(3a), J3a,4a = 12.1 Hz, J3a,4e = 2.5 Hz); 5.87 (br.s, 2 H, 2 OH);
6.68 (dd, 1 H, H(26), J26,25 = 8.1 Hz, J26,22 = 1.8 Hz); 6.78 (d, 1 H,
H(22), J22,26 = 1.8 Hz); 6.828 (d, 1 H, H(15), J15,16 = 8.1 Hz);
6.833 (d, 1 H, H(25), J25,26 = 8.1 Hz); 6.86 (dd, 1 H, H(16),
J16,15 = 8.1 Hz, J16,12 = 1.8 Hz); 6.98 (d, 1 H, H(12), J12,16 = 1.8 Hz).
13C NMR, : 76.12 (d, C(1)); 69.14 (d, C(3)); 45.82 (t, C(4));
72.79 (s, C(5)); 40.98 (d, C(6)); 22.21 (t, C(7)); 42.32 (d, C(8));
43.19 (d, C(9)); 209.92 (s, C(10)); 133.80 (s, C(11)); 109.01
(d, C(12)); 146.30 (s, C(13)); 144.79 (s, C(14)); 113.88 (d, C(15));
118.70 (d, C(16)); 21.85 (q, C(17)); 17.33 (q, C(18)); 55.67
(q, C(19)); 75.48 (d, C(20)); 132.00 (s, C(21)); 108.81 (d, C(22));
146.22 (s, C(23)); 144.49 (s, C(24)); 113.99 (d, C(25)); 118.37
(d, C(26)); 55.65 (q, C(27)). MS, found m/z: 454.1983 [M]+.
1
H and 13C NMR spectra were recorded on a Bruker DRXꢀ500
(500.13 and 125.76 MHz, respectively) for solutions of comꢀ
pounds in CDCl3. Signals of chloroform ( 7.24,  76.90)
H
C
were used as references. The structure of obtained compounds
was established based on the analysis of 1H NMR spectra, using
a 1H—1H double resonance procedure, as well as on the analysis
of 13C NMR spectra, using 2D heteronuclear correlation spectroꢀ
scopy 13C—1H on the direct (1JC,H = 160 Hz) and remote (2,3JC,H
=
= 10 Hz) spinꢀspin coupling constants. The splitting pattern of
signals in the 13C NMR spectra was determined using the specꢀ
tra recorded in the Jꢀmodulation mode (JMOD). The atom numꢀ
bering system in compound 4g is given for the convenience of
the signal assignment in the NMR spectra and does not correꢀ
spond to that required by nomenclature. High resolution mass
spectra were recorded on a DFS Thermo Scientific spectromꢀ
eter in the full scan mode in the m/z 15—500 range, electron
impact (EI) ionization 70 eV with the direct injection of the
sample. Specific rotation []D was determined on a polAAr 3005n
spectrometer for solutions in CHCl3. IR spectra were recorded
on a Vectorꢀ22 Fourierꢀtransform spectrometer in KВr pellets.
The spectroꢀanalytical studies were carried out in the Multiꢀ
Access Chemical Service Center of the Siberian Branch of the
Russian Academy of Sciences.
Calculated for C26H30O7: M = 454.1986. IR (KBr), /cm–1
:
3433 (OH); 1710 (C=O); 1517 (Ar); 1271, 1031 (C—O); 756
(CH(Ar)).
Assessment of cytotoxicity. Cytotoxicity of compounds under
study was determined on the human tumor cell lines MTꢀ4,
CEMꢀ13 (cells of human Tꢀcell leukosis), and Uꢀ937 (cells of
human monocytes). The cell were cultured in the RPMIꢀ1640
medium containing 10% blood serum of cattle embryos,
Lꢀglutamine (2 mmol L–1), gentamicin (80 g mL–1), and lincoꢀ
mycin (30 mg mL–1) at 37 C in the 5% CO2 atmosphere in the
incubator. A tested compound was dissolved in DMSO and added
to the cell culture in a required concentration. The tests were
carried out in triplicate for each final concentration: 0.1, 1, 10,
and 100 g mL–1. The cell incubated without addition of tested
compounds were used as a control. Doxorubicin was used as
a comparison agent.
(1R,2R,6S)ꢀ3ꢀMethylꢀ6ꢀ(propꢀ1ꢀenꢀ2ꢀyl)cyclohexꢀ3ꢀenꢀ
1,2ꢀdiol 1 ([]26 –62 (c 1.33, CHCl3)) was synthesized from
D
(–)ꢀverbenone (2) ([]30D –186 (c 0.37, CHCl3)) according to the
procedures published earlier.16,17 Compounds 4a—g were obꢀ
tained by the reaction of monoterpenoid 1 with aldehydes 3a—g
in the presence of clay K10 according to the described proceꢀ
dures11—14 in the following yields: 4a (11%), 4b (10%), 4c (9%),
4d (9%), 4e (8%), 4f (8%).
The chemical part of the work was financially supꢀ
ported by the Russian Foundation for Basic Research
(Project No. 13ꢀ03ꢀ00206), the biological part by the Minꢀ
istry of Education and Science of the RF (State Assignꢀ
ment No. 17.1891.2014/K).
(2R,4S,4aR,6S,7R,8aR,9S)ꢀ2,9ꢀBis(4ꢀhydroxyꢀ3ꢀmethoxyꢀ
phenyl)ꢀ4,7ꢀdimethylhexahydroꢀ2Hꢀ4,6ꢀ(epoxymethano)chromꢀ
enꢀ8(8aH)ꢀone (4g). Vanillin 3g (1.00 g, 6.58 mmol) and a soluꢀ
tion of compound 1 (0.800 g, 4.76 mmol) in CH2Cl2 (20 mL)
were sequentially added to a suspension of clay K10 (3.0 g) (calꢀ
cined for 3 h at 105 C immediately before use) in CH2Cl2
(10 mL), which was passed through the column with Al2O3.
The solvent was evaporated. The mixture was allowed to stand at
room temperature for 3 days. After addition of EtOAc (20 mL),
the catalyst was filtered off, the solvent was evaporated, the
residue was separated on a column with SiO2 (60—200 
(Macherey—Nagel), eluent hexane with EtOAc gradient from
0 to 100%) to obtain compounds 4g (0.240 g, 11%) and 5g
((S) : (R) = 1.5 : 1) (0.302 g, 20%). Spectral characteristics of
compounds (4S(R))ꢀ5g are similar to those given in the
work.11 The ratio of (S) : (R)ꢀdiastereoisomers was determined
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Received April 28, 2015;
in revised form June 25, 2015