Catalytic cyclometallation in steroid chemistry V: Synthesis of hybrid molecules based on steroid oximes and (5Z,9Z)-tetradeca-5,9-dienedioic acid as potential anticancer agents

Article abstract of DOI:10.1016/j.steroids.2018.06.002

Synthetic analogues of natural 5Z,9Z-dienoic acids - hybrid molecules based on the oximes of cholesterol, pregnenolone, and androsterone with 1,14-tetradeca-5Z,9Z-dienedicarboxylic acid - were synthesized for the first time and studied for antitumor activity in vitro. The acid was prepared using catalytic cyclomagnesiation of O-containing 1,2-dienes with Grignard reagent in the presence of Cp₂TiCl₂ as the key step. Using flow cytometry, it was shown for the first time that the new molecules are efficient apoptosis inducers in the HeLa, Hek293, U937, Jurkat, and K562.

Full text of DOI:10.1016/j.steroids.2018.06.002

Accepted Manuscript
Catalytic cyclometallation in steroid chemistry V: Synthesis of hybrid molecules
based on steroid oximes and (5Z,9Z)-tetradeca-5,9-dienedioic acid as potential
anticancer agents
Vladimir A. D'yakonov, Regina A. Tuktarova, Lilya U. Dzhemileva, Svetlana
R. Ishmukhametova, Milyausha M. Yunusbaeva, Usein M. Dzhemilev
PII:
S0039-128X(18)30109-0
https://doi.org/10.1016/j.steroids.2018.06.002
STE 8271
DOI:
Reference:
Steroids
To appear in:
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Revised Date:
Accepted Date:
13 March 2018
31 May 2018
2 June 2018
Please cite this article as: D'yakonov, V.A., Tuktarova, R.A., Dzhemileva, L.U., Ishmukhametova, S.R.,
Yunusbaeva, M.M., Dzhemilev, U.M., Catalytic cyclometallation in steroid chemistry V: Synthesis of hybrid
molecules based on steroid oximes and (5Z,9Z)-tetradeca-5,9-dienedioic acid as potential anticancer agents,
Steroids (2018), doi: https://doi.org/10.1016/j.steroids.2018.06.002
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Catalytic cyclometallation in steroid chemistry V1: Synthesis of hybrid molecules based on
steroid oximes and (5Z,9Z)-tetradeca-5,9-dienedioic acid as potential anticancer agents
,
,
*
Vladimir A. D'yakonov,a Regina A. Tuktarova,a Lilya U. Dzhemileva,a * Svetlana R.
Ishmukhametova,a Milyausha M. Yunusbaeva,a Usein M. Dzhemileva
aInstitute of Petrochemistry and Catalysis of RAS,
141 Prospekt Oktyabrya, 450075, Ufa, Russian Federation.
ABSTRACT Synthetic analogues of natural 5Z,9Z-dienoic acids - hybrid molecules based on
the oximes of cholesterol, pregnenolone, and androsterone with 1,14-tetradeca-5Z,9Z-
dienedicarboxylic acid - were synthesized for the first time and studied for antitumor activity in
vitro. The acid was prepared using catalytic cyclomagnesiation of О-containing 1,2-dienes with
Grignard reagent in the presence of Cp2TiCl2 as the key step. Using flow cytometry, it was
shown for the first time that the new molecules are efficient apoptosis inducers in the HeLa,
Hek293, U937, Jurkat, and K562.
Key words: Cross-cyclomagnesiation; Grignard reagents; Steroid oximes; 5Z,9Z-Dienoic acids;
Anticancer activity.
1. Introduction
According to published data [1], aliphatic 5Z,9Z-dienolic acids isolated from marine
invertebrates possess a broad spectrum of biological activities such as antimalarial, antibacterial,
and antifungal activities. In addition, 5Z,9Z-dienoic acids exhibit high inhibitory activity against
human topoisomerases I and IIα [2-6], which are considered as intracellular targets of
chemotherapeutic agents that prevent the break repair and thus cause accumulation of damaged
DNA molecules and promote tumor cell death [7].
In recent years, more and more publications have addressed the use of cytotoxic steroids
for the treatment of cancer; most of these steroids are hybrid molecules in which the cytotoxic
substituent in linked to one functional group of the steroid [8-10]. In these molecules, the steroid
moiety functions as a cytostatic transport, owing to its lipophilic properties and the ability to
easily penetrate the lipid bilayer of biological membranes, thus providing access to definite
target proteins and increasing the selectivity of drug action. The results indicate that the direct
1 Steroids, 2016, 108, 77-84.
* Corresponding authors. Tel./Fax: +7 347 2842750; e-mail addresses: DyakonovVA@gmail.com (DVA),
Dzhemilev@mail.ru (DLU).

action of steroid molecules on the genetic apparatus of the cell is the key mechanism of their
functioning.
Previously [11], we have demonstrated the possibility of synthesis of hybrid molecules
based on steroids and 1,14-tetradeca-5Z,9Z-dienedicarboxylic acid, which was prepared using
the catalytic cross-cyclomagnesiation of 1,2-dienes with Grignard reagents in the presence of
Cp2TiCl2 [12,13]. It has been shown that steroid-containing 5Z,9Z-dienoic acids obtained by
catalytic esterification of (5Z,9Z)-tetradeca-5,9-dienoic acid with hydroxy steroid derivatives in
the presence of DCC/DMAP [14] can efficiently induce apoptosis in tumor cell lines of various
etiology and inhibit relaxation of supercoiled DNA by topoisomerase I in vitro.
In view of the fact that 5Z,9Z-dienoic acids can selectively inhibit cell cycle enzymes,
topoisomerases I and II, and simultaneously exhibit antitumor, anti-inflammatory, antibacterial,
and antifungal activities and as continuation of the studies dealing with the synthesis of
analogues of natural 5Z,9Z-dienoic acids, we synthesized for the first time hybrid molecules
based on cholesterol, prеgnenolone, and androsterone oximes and 1,14-tetradeca-5Z,9Z-
dienedicarboxylic acid and studied their cytotoxic activity against the Jurkat, K562, U937,
Hek293, and HeLa tumor cells.
2. Experimental
2.1. Chemistry
Cholesterol, 5-pregnen-β-ol-20-on, 5-androsten-β-ol-17-on, 4-dimethylaminopyridine
(DMAP), N-[3-(methylamino)propyl]-N'-ethylcarbodiimide hydrochloride (EDC·HCl) were
obtained from Sigma-Aldrich and Acros Organics. Dichloromethane was freshly distilled before
use. Optical rotations were measured on a Perkin–Elmer 341 polarimeter. IR spectra were
recorded on Bruker VERTEX 70V using KBr discs over the range of 400–4000 cm-1. Mass
spectra of MALDI TOF/TOF positive ions (matrix of sinapic acid) are recorded on a mass
spectrometer Bruker AutoflexTM III Smartbeam. 1H and 13C NMR spectra were obtained using a
1
13
Bruker Ascend 500 spectrometer in CDCl3 operating at 500 MHz for H and 125 MHz for C.
Elemental analyses were measured on 1106 Carlo Erba apparatus. Individuality and purity of the
synthesized compounds were controlled using TLC on Sorbfil plates; anisic aldehyde in acetic
acid was used as a developer. Column chromatography was carried out on Acrus silica gel
(0.060–0.200 мм).

13
2.1.1. 1H NMR (CDCl3, 400 MHz) and C NMR (CDCl3, 100 MHz) spectral data and synthesis
method for 2,2'-[(5Z,9Z)-tetradeca-5,9-diene-1,14-diylbis(oxy)]bistetrahydro-2H-pyran 3 are
described in the literature [15].
1
13
2.1.2. H NMR (CDCl3, 500 MHz) and C NMR (CDCl3, 125 MHz) spectral data for (5Z,9Z)-
tetradeca-5,9-dienedioic acid 4 are described in the literature [11].
2.1.3. Reaction of steroid oximes with (5Z,9Z)-tetradeca-5,9-dienedioic acid (4).
To a solution of steroid oxime (1.0 mmol) in dichloromethane (50 ml) the (5Z,9Z)-
tetradeca-5.9-dienedioic acid 4 (0.51 g, 2.0 mmol) was added followed by N-[3-
(methylamino)propyl]-N'-ethylcarbodiimide hydrochloride (0.48 g, 2.5 mmol) and 4-
dimethylaminopyridine (18 mg, 0.15 mmol) under argon. The mixture was stirred at room
temperature for 12 h until the reaction was complete (TLC monitoring, hexane/ethyl acetate).
The mixture was diluted with H2O (10 ml) and the CH2Cl2 layer was separated, dried over
MgSO4, and concentrated. The crude product was purified by column chromatography (silica
gel) using hexane/ethyl acetate = 5/1 as the elution solvent to afford 5Z,9Z-dienoic acids of
steroid.
2.1.3.1. (5Z,9Z)-14-({[(3β)-3-(Acetyloxy)cholest-4-en-6E-ylidene]amino}oxy)-14-oxotetradeca-
5,9-dienoic acid (8a). White waxy solid, 0.45 g, 65% yield. [α] 19 + 4.1 (c 0.54, CHCl3); IR
D
(KBr) νmax 2946, 2868, 1737, 1708, 1459, 1375, 1238, 1122, 1042, 1021, 911, 867, 822, 610 cm-
1; 1H NMR (CDCl3, 500 MHz) δ 5.91 (1H, s, H-4), 5.41 (2H, m, H-6', H-9'), 5.36 (2H, m, H-5',
H-10'), 5.29 (1H, t, J = 7.5 Hz, H-3), 3.21 (1H, d, J = 7.5 Hz, H-7β), 2.45 (2H, t, J = 7.5 Hz, H-
13'), 2.36 (2H, t, J = 7.5 Hz, H-2'), 2.11 (4H, m, H-4', H-11'), 2.09 (4H, m, H-7', H-8'), 2.06 (3H,
s, COCH3), 2.04‒1.00 (25H, m), 1.77 (2H, m, H-12'), 1.71 (2H, m, H-3'), 1.03 (3H, s, H-19),
13
0.92 (3H, d, J = 6.0 Hz, H-21), 0.88 (6H, d, J = 6.5 Hz, H-26, H-27), 0.69 (3H, s, H-18); C
NMR (CDCl3, 125 MHz) δ 178.8 (C-1'), 171.4 (C-14'), 171.0 (COCH3), 165.4 (C-6), 141.9 (C-
5), 130.3 (C-6', C-9'), 128.9 (C-5', C-10'), 126.3 (C-4), 69.5 (C-3), 56.4 (C-14), 55.9 (C-17), 52.5
(C-9), 42.8 (C-13), 39.5 (C-24), 39.3 (C-12), 38.1 (C-10), 36.1 (C-22), 35.7 (C-20), 34.3 (C-1),
34.2 (C-8), 33.3 (C-2'), 32.4 (C-13'), 32.3 (C-7), 28.0 (C-16, C-25), 27.3 (C-7', C-8'), 26.6, 26.5
(C-4', C-11'), (24.8 (C-12'), 24.6 (C-3'), 24.2 (C-2), 24.1 (C-15), 23.8 (C-23), 22.8 (C-27), 22.6
(C-26), 21.3 (COCH3), 20.8 (C-11), 18.7 (C-19), 18.6 (C-21), 11.9 (C-18); anal. calcd for
C43H67NO6: C, 74.42; H, 9.73; found C, 74.36; H, 9.67. MALDI TOF: m/z 716.465 ([M+Na]+,
calcd 716.487), 732.427 ([M+K]+, calcd 732.461).

2.1.3.2. (5Z,9Z)-14-({[(3β)-3-(Acetyloxy)cholest-5-en-7Z-ylidene]amino}oxy)-14-oxotetradeca-
5,9-dienoic acid (8b). White waxy solid, 0.44 g, 63% yield. [α] 19 ‒ 148.6 (c 0.44, CHCl3); IR
D
(KBr) νmax 2950, 2869, 1736, 1709, 1639, 1457, 1439, 1375, 1366, 1241, 1125, 1035, 914, 878,
803, 737, 609 cm-1; 1H NMR (CDCl3, 500 MHz) δ 6.44 (1H, m, H-6), 5.40 (2H, m, H-6', H-9'),
5.36 (2H, m, H-5', H-10'), 4.70 (1H, m, H-3), 2.60‒1.00 (26H, m), 2.45 (2H, m, H-13'), 2.36
(2H, t, J = 7.5 Hz, H-2'), 2.12 (4H, m, H-4', H-11'), 2.08 (4H, m, H-7', H-8'), 2.06 (3H, s,
COCH3), 1.76 (2H, m, H-12'), 1.71 (2H, m, H-3'), 1.16 (3H, s, H-19), 0.94 (3H, d, J = 6.5 Hz, H-
13
21), 0.88 (6H, d, J = 6.5 Hz, H-26, H-27), 0.72 (3H, s, H-18); C NMR (CDCl3, 125 MHz) δ
178.8 (C-1'), 171.7 (C-14'), 170.5 (COCH3), 163.0 (C-7), 156.2 (C-5), 130.4, 130.2 (C-6', C-9'),
129.0, 128.8 (C-5', C-10'), 114.2 (C-6), 72.8 (C-3), 54.6 (C-17), 49.9 (C-14), 49.3 (C-9), 42.9 (C-
13), 39.5 (C-24), 38.6 (C-8), 38.5 (C-12), 38.4 (C-10), 38.2 (C-4), 36.2 (C-1), 36.1 (C-22), 35.6
(C-20), 33.3 (C-2'), 32.4 (C-13'), 28.3 (C-16), 28.0 (C-25), 27.3 (C-2, C-7', C-8'), 27.1 (C-15),
26.6, 26.5 (C-4', C-11'), 24.8 (C-12'), 24.6 (C-3'), 23.7 (C-23), 22.8 (C-27), 22.6 (C-26), 21.3
(COCH3), 20.8 (C-11), 18.9 (C-21), 17.8 (C-19), 12.1 (C-18); anal. calcd for C43H67NO6: C,
74.42; H, 9.73; found C, 74.37; H, 9.68. MALDI TOF: m/z 716.487 ([M+Na]+, calcd 716.487),
732.448 ([M+K]+, calcd 732.461).
2.1.3.3. (5Z,9Z)-14-({[(3β)-3-(Acetyloxy)pregn-5-en-20E-ylidene]amino}oxy)-14-oxotetradeca-
5,9-dienoic acid (8c). White waxy solid, 0.40 g, 66% yield. [α] 19 ‒ 9.7 (c 0.85, CHCl3); IR
D
(KBr) νmax 3006, 2962, 2937, 2855, 1732, 1631, 1438, 1368, 1250, 1130, 1037, 802, 756, 611
1
cm-1; H NMR (CDCl3, 400 MHz) δ 5.39 (2H, m, H-6', H-9'), 5.36 (3H, m, H-5', H-6, H-10'),
4.62 (1H, m, H-3), 2.44 (2H, t, J = 7.5 Hz, H-13'), 2.37 (2H, m, H-2'), 2.38‒1.00 (20H, m), 2.14
(4H, m, H-4', H-11'), 2.08 (4H, m, H-7', H-8'), 2.04 (3H, s, COCH3), 1.96 (3H, s, H-23), 1.77
13
(2H, J = 7.5 Hz, H-12'), 1.70 (2H, m, H-3'), 1.03 (3H, s, H-19), 0.69 (3H, s, H-18); C NMR
(CDCl3, 100 MHz) δ 178.9 (C-1'), 171.7 (C-14'), 170.6 (COCH3), 166.7 (C-20), 139.6 (C-5),
130.4, 130.3 (C-6', C-9'), 128.9, 128.8 (C-5', C-10'), 122.4 (C-6), 73.9 (C-3), 56.9 (C-17), 56.2
(C-14), 49.9 (C-9), 44.1 (C-13), 38.6 (C-12), 38.1 (C-4), 36.9 (C-1), 36.6 (C-10), 33.3 (C-2'),
32.5 (C-13'), 31.9 (C-8), 31.7 (C-7), 27.7 (C-2), 27.3 (C-7', C-8'), 26.5, 26.6 (C-4', C-11'), 24.8
(C-12'), 24.6 (C-3'), 24.3 (C-15), 23.1 (C-16), 21.4 (COCH3), 20.9 (C-11), 19.3 (C-19), 17.1 (C-
21), 13.3 (C-18); anal. calcd for C37H55NO6: C, 72.87; H, 9.09; found C, 72.69; H, 9.03. MALDI
TOF: m/z 632.395 ([M+Na]+, calcd 632.393), 648.370 ([M+K]+, calcd 648.367).
2.1.3.4.
(5Z,9Z)-14-({[(3β)-3-(Acetyloxy)androst-5-en-17E-ylidene]amino}oxy)-14-
oxotetradeca-5,9-dienoic acid (8d). White waxy solid, 0.37 g, 63% yield. [α] 19 ‒ 36.9 (c 0.81,
D

CHCl3); IR (KBr) νmax 3003, 2963, 2942, 2857, 1732, 1628, 1441, 1363, 1247, 1132, 1033, 805,
754, 612 cm-1; 1H NMR (CDCl3, 400 MHz) δ 5.40 (2H, m, H-6', H-9'), 5.37 (1H, m, H-6), 5.37
(2H, m, H-5', H-10'), 4.62 (1H, m, H-3), 2.68‒1.06 (19H, m), 2.42 (2H, t, J = 7.5 Hz, H-13'),
2.36 (2H, m, H-2'), 2.13 (4H, m, H-4', H-11'), 2.08 (4H, m, H-7', H-8'), 2.05 (3H, s, COCH3),
1.74 (2H, m, H-12'), 1.71 (2H, m, H-3'), 1.06 (3H, s, H-19), 1.01 (3H, s, H-18); 13C NMR
(CDCl3, 100 MHz) δ 178.6 (C-17, C-1'), 171.6 (C-14'), 170.6 (COCH3), 139.9 (C-5), 130.4,
130.3 (C-6', C-9'), 128.9 (C-5', C-10'), 121.8 (C-6), 73.8 (C-3), 53.9 (C-14), 49.9 (C-9), 44.9 (C-
13), 38.1 (C-4), 36.9 (C-1), 36.7 (C-10), 33.5 (C-12), 33.2 (C-2'), 32.4 (C-13'), 31.3 (C-7, C-8),
27.7 (C-2), 27.3 (C-7', C-8'), 27.2 (C-16), 26.6, 26.5 (C-4', C-11'), 24.8 (C-12'), 24.6 (C-3'), 23.1
(C-15), 21.4 (COCH3), 20.4 (C-11), 19.3 (C-19), 16.8 (C-18); anal. calcd for C35H51NO6: C,
72.26; H, 8.84; found C, 72.14; H, 8.80. MALDI TOF: m/z 620.319 ([M+K]+, calcd 620.335).
2.2. Biological assays
2.2.1. Cell culturing
Cells (Jurkat, K562, U937) were purchased from Russian Cell Culture Collection
(Institute of Cytology of the Russian Academy of Sciences) and cultured according to standard
mammalian tissue culture protocols and sterile technique. Human cancer cell lines HEK293 and
HeLa were obtained from the HPA Culture Collections (UK). All cell lines used in the study
were tested and shown to be free of mycoplasma and viral contamination.
HEK293, HeLa cell lines and fibroblasts were cultured as monolayers and maintained in
Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL) supplemented with 10% foetal
bovine serum and 1% penicillin-streptomycin solution at 37 °C in a humidified incubator under a
5% CO2 atmosphere.
Cells were maintained in RPMI 1640 (Jurkat, K562, U937) (Gibco) supplemented with 4
mM glutamine, 10% FBS (Sigma) and 100 units/ml penicillin-streptomycin (Sigma). All types
of cells were grown in an atmosphere of 5 % CO2 at 37 °C. The cells were subcultured at 2-3
days intervals. Adherent cells (HEK293, HeLa, fibroblasts) were suspended using trypsin/EDTA
and counted after they have reached 80% confluency. Cells were then seeded in 24 well plates at
5x104 cells per well and incubated overnight. Jurkat, K562, U937 cells were subcultured at 2 day
intervals with a seeding density of 1×105cells per 24 well plates in RPMI with 10% FBS.

2.2.2. Cytotoxicity assay
Viability (Live/dead) assessment was performed by staining cells with 7-AAD (7-
Aminoactinomycin D) (Biolegend). Cells after treatment with compounds 7a-d, 8a-d at various
concentrations (2, 1, 0.5, 0.25, 0.1 μМ) and incubated in an atmosphere of 5 % CO2 at 37 °C
during 24 hours were harvested, washed 1-2 times with phosphate-buffered saline (PBS) and
centrifuged at 400g for 5 minutes. Cell pellets were resuspended in 200 uL of flow cytometry
staining buffer (PBS without Ca2+and Mg2+, 2,5% FBS) and stained with 5 uL of 7-AAD
staining solution for 15 minutes at room temperature in the dark. Samples were acquired on
NovoCyteTM 2000 FlowCytometry System (ACEA) equipped with 488 nm argon laser.
Detection of 7-AAD emission was collected through a 675/30 nm filter in FL4 channel.
2.2.3. Viability and apoptosis
Apoptosis was determined by flow cytometric analysis of Annexin V and 7-
aminoactinomycin D staining. K562 Tumor cells after treatment with compound 8d at
concentrations (0.04, 0.03, 0.02, 0.01 μМ) during 24 hours were harvested, washed 1-2 times
with phosphate-buffered saline (PBS) and centrifuged at 400g for 5 minutes. Cell pellets were
resuspended in 200 uL of flow cytometry staining buffer (PBS without Ca2+and Mg2+, 2,5%
FBS). Then, 200 μl of Guava Nexin reagent (Millipore, Bedford, MA, USA) was added to 5 ×
105 cells in 200 μl, and the cells were incubated with the reagent for 20 min at room temperature
in the dark. At the end of incubation, the cells were analyzed on NovoCyteTM 2000
FlowCytometry System (ACEA).
2.2.4. Cell cycle analysis
Cell cycle was analyzed using the method of propidium iodide staining. K562 Tumor
cells after treatment with compound 8d at concentrations (0.04, 0.03, 0.02, 0.01 μМ) during 24
hours were harvested, washed 1-2 times with phosphate-buffered saline (PBS) and centrifuged at
400g for 5 minutes. Cell pellets were resuspended in 200 uL of flow cytometry staining buffer
(PBS without Ca2+and Mg2+, 2,5% FBS). Then, cells were plated in 24-well round bottom plates
at a density 10×105 cells per well, centrifuged at 450× g for 5 minutes, and fixed with ice-cold
70% ethanol for 24 hour at 0 °C. Cells were then washed with PBS and incubated with 250 μl of

Guava Cell Cycle Reagent (Millipore) for 30 minutes at room temperature in the dark. Samples
were analyzed on NovoCyteTM 2000 FlowCytometry System (ACEA).
3. Results and Discussion
Tetradeca-5Z,9Z-diene-1,14-dicarboxylic acid was synthesized in two steps using the
previously developed [15,16] homo-cyclomagnesiation of tetrahydropyran 5,6-heptadien-1-ol
ether 1 with EtMgBr in the presence of the Cp2TiCl2 catalyst (5 mol. %) to give
magnesacyclopentane 2. The subsequent hydrolysis of organomagnesium compound 2 and
oxidation of the resulting 1,14-bis-tetrahydropyranyl-5Z,9Z-diene-1,14-diol 3 with the Jones
reagent gives target dicarboxylic acid 4 in an overall yield of ~52 % (Scheme 1).
Scheme 1. Synthesis of (5Z,9Z)-tetradeca-5,9-dienedioic acid 4. (a): EtMgBr, Mg, Cp2TiCl2 (5
mol%), diethyl ether; (b): H3O+; (c): H2CrO4/H2SO4, acetone, CH2Cl2.
As the initial stage of our studies dealing with the synthesis of new hybrid molecules
based on 1,14-tetradeca-5Z,9Z-dienedicarboxylic acid 4 and steroid oximes, we prepared these
compounds from appropriate keto steroids 6a-d.
Keto derivative 6a, needed for the synthesis of oxime 7a, was prepared by successive
reactions including epoxidation of (3β)-cholest-5-en-3yl acetate 5, oxidation, and elimination of
the hydroxyl group at С-5 by treatment with thionyl chloride in pyridine [17]. Then keto steroid
6a was reacted with hydroxylamine hydrochloride in dry pyridine to give (6E)-
hydroximinocholest-4-en-3β-yl acetate 7а (Scheme 2). The structure of compound 7а was
13
confirmed by 1D (1H, C, Dept 135) and 2D (HSQC, HMBC and HH COSY, NOESY) NMR
1
experiments. The H NMR spectrum exhibits a signal at 5.68 ppm for the proton at the double
bond and a signal at 3.34 ppm (dd, J = 15.0, 4.5 Hz) for the H-7β proton; together with the C-6
13
carbon signal at 158.2 ppm in the С NMR spectrum, are in agreement with those reported in
the literature for the E-configuration of oxime 7а [17].

Scheme 2. Synthesis of steroid oximes 7a,b. (a): mCPBA, CH2Cl2 (97%); (b): CrO3, H2O (90%);
(c) SOCl2, Py (92%); (d): NH2OH·HCl, Py (~96%); (e): Celite, PDC, t-BuOH, benzene (81%).
The target (7Z)-hydroximinocholest-5-en-3β-yl acetate 7b was synthesized by oxidation
of (3β)-cholest-5-en-3yl acetate 5 with tert-butyl hydroperoxide-pyridinium dichromate [18]
followed by the reaction of keto derivative 6b with hydroxylamine hydrochloride (Scheme 2)
[19].
The configurations of oximes, (3β)-acetoxy-20-hydroxyimino-5-pregnen-20E-one 7с and
(3β)-17E-(hydroximino)-5-androsten-3-ol acetate 7d (Scheme 3), synthesized by the procedure
presented above for oximes 7a,b are consistent with published 1Н and 13С NMR data [20-22].
Scheme 3. Synthesis of steroid oximes 7с,d. (a): NH2OH·HCl, Py (~96%).
Having prepared the key steroid oximes 7a-d and 1,14-tetradeca-5Z,9Z-
dienedicarboxylic acid 4, we synthesized the desired hybrid molecules 8a-d using N-[3-
(methylamino)propyl]-N'-ethylcarbodiimide hydrochloride (EDC·HCl) and catalytic amounts of
4-dimethylaminopyridine (DMAP) (Scheme 4).

Scheme 4. Synthesis of 5Z,9Z-dienoic acids 8a-d. (a): DMAP, EDC·HCl, CH2Cl2.
Acids 8a-d and the steroid oxime precursors 7a-d were tested for the first time for
antitumor activity in vitro against the Jurkat, K562, Hek293, HeLa, and U937 cell lines with
determination of cytotoxicity CC50. Compounds that exhibited the highest activity were
additionally studied for the cell viability and effect on the cell cycle by flow cytometry using the
Guava Nexin Reagent, Guava Cell Cycle, and Guava ViaCount kits (Millipore).
The in vitro cytotoxic activities of compounds 7а-d and 8а-d against the Jurkat, K562,
U937, and HeLa cultures and the Hek293 human embryonic kidney cells are summarized in
Table 1.
Table 1. Inhibition of tumor cell viability by 5Z,9Z-dienoic acid derivatives 8a-d and steroid
oximes 7a-d, CC50 (μМ) ± SE.
Jurkat
K562
U937
Hek293
HeLa
Fibrobl.
1.36±0.026 1.14±0.027 0.81±0.035 0.92±0.033 0.73±0.028 1.52±0.034
1.12±0.029 0.61±0.023 0.71±0.029 1.24±0.037 1.04±0.027 1.39±0.045
0.82±0.015 1.01±0.022 0.74±0.018 0.62±0.021 0.64±0.024 1.19±0.032
0.67±0.012 0.53±0.017 0.45±0.025 0.74±0.021 0.71±0.015 1.12±0.021
0.68±0.018 0.27±0.027 0.38±0.035 0.49±0.023 0.30±0.034 1.17±0.047
0.51±0.026 0.33±0.021 0.49±0.035 0.76±0.023 0.58±0.017 1.13±0.043
0.32±0.035 0.34±0.012 0.41±0.028 0.13±0.011 0.29±0.036 0.79±0.019
0.02±0.004 0.02±0.003 0.04±0.003 0.03±0.007 0.17±0.006 0.55±0.023
1.12±0.012 2.10±0.013 1.32±0.011 3.21±0.017 2.18±0.016 4.19±0.067
CC50 (7a)
CC50 (7b)
CC50 (7c)
CC50 (7d)
CC50 (8a)
CC50 (8b)
CC50 (8c)
CC50 (8d)
CC50 (camp.)
The highest activities against chronic myeloid leukemia cells (K562) and Т-cell leukemia
cells (Jurkat) were found for acid 8d (CC50 = 0.025 and CC50 = 0.015, respectively), with the

cytotoxic effect being dose-dependent and markedly exceeding that of camptothecin for the same
cell lines. The lowest activities against all of the studied cell lines were found for compounds 8a
and 8b derived from cholesterol (for CC50, see Table 1). It is also noteworthy that acid 8с has a
rather high cytotoxic activity (CC50 = 0.13 and 0.031, respectively) against the HEK293 human
embryonic kidney cells and HeLa cells.
We found that the cytotoxicity of hybrid molecules 8a-d substantially exceeds the
cytotoxicity of steroid oxime precursors 7a-d (Table 1). In the CC50 study for compounds 8a-d
in normal fibroblast cultures, it has been shown that the cytotoxicity of hybrid molecules is
higher than that of tumor cells, indicating a higher sensitivity of tumor lines to synthesized
hybrids.
It should be noted that the cytotoxic activity of compound 8d was 5 times higher than the
cytotoxic activity of previously synthesized (5Z,9Z)-dienoic acid on the basis of pregnenolone
against the cells of kidney cancer HEK293, and almost 10 times the T cell lines of Jurkat
leukemia and myelogenous leukemia K562 [14].
In view of the pronounced cytotoxic effect found for hybrid molecule 8d, we investigated
the apoptosis and cell cycle in the culture cells.
According to published data, steroid hormones have a differentiating effect on various
types of cells, namely, prevent apoptosis of some types of cells and induce apoptosis of other cell
types. For example, neuroactive steroids, or neurosteroids, can induce apoptosis in neuroglia
tissues [23]. Also neurosteroids are actively used to treat multiple sclerosis patients, because the
neural tissue of neurodegenerative disease patients was found to be sharply deficient in these
steroids [24].
Previously, we found an antiproliferative effect of 3-keto-diosgenin oxime against the
cervical carcinoma (HeLa), colon carcinoma (HCT-116), osteosarcoma 1547, hepatocellular
carcinoma (HepG2, C3A, and HUH-7), and breast carcinoma (MCF-7) cells [25-29].
In view of our results, we studied in more detail the apoptosis-inducing activities of
compounds 8d on myeloid and T-cell leukemia cells. The highest percentage of apoptosis after
the addition of compound 8d to K562 cell culture was attained for 0.2 µL concentration and
amounted to 72.8% (Fig. 1). The percentage of late apoptosis for acid 8d was 80% for Jurcat
cells, 78% for HEK293 cells, 92% for U937 cells, and 65% for HeLa cells.

A
B
C
E
D
Fig. 1. K562 tumor cells treated with compound 8d in different concentrations and stained with
annexin V/7AAD. Flow cyrometry data: (A) control; (B) 8d (0.01 µM); (C) 8d (0.02 µM); (D)
8d (0.03 µM), (E) 8d (0.04 µM).
C
A
B
E
D

Fig. 2. Cell cycle phases of K562 cells treated with compound 8d. (A) Control; (B) 8d (0.01
µM); (C) 8d (0.02 µM); (D) 8d (0.03 µM); (E) 8d (0.04 µM) (The incubation time of 8d with the
cells was 24 hours).
Figure 2 shows the results of a study of the cell cycle phases of cells K562 line, determined
by DNA flow cytometry, after treatment with acid 8d at different concentrations (0.04, 0.03,
0.02, 0.01 μМ) after 24 hours.
The cell cycle characteristics of the K562 and Jurkat cells in control samples showed
considerable predominance of cells in the G0-G1 phase and a balance between the synthesis (S-
phase) and apoptosis (sub-G0-G1 range) processes.
Two days after the exposure to compound 8d, apoptosis processes predominated (the sub-
G0-G1 range increased), while the ability of cells to DNA synthesis (S-phase) was retained or
even increased. The percentage of G0-G1 cells decreased, while the proliferation block increased
and the proliferation index decreased due to decreasing amount of G2 + M cells.
All the foregoing may be indicative of the cytotoxic activity of acid 8d against chronic
myeloid leukemia cells and Т-cell leukemia cells, caused by the ability of this compound to
induce apoptosis.
4. Conclusion
Thus, we prepared previously unknown synthetic analogues of natural 5Z,9Z-dienoic
acids - hybrid molecules based on cholesterol, pregnenolone, and androsterone oximes and 1,14-
tetradeca-5Z,9Z-dienedicarboxylic acid. Using flow cytometry, it was shown for the first time
that the new molecules are efficient apoptosis inducers in the HeLa, Hek293, U937, Jurkat, and
K562 cell cultures.
Acknowledgments
The work was financially supported by the Russian Science Foundation (Grant No. 16-
13-10172), the Russian Foundation for Basic Research (Grant No. 16-03-00543_A) and the
Grant of the RF President (Sci. Sh. - 5240.2018.3). The structural studies of the synthesized
compounds were performed with the use of Collective Usage Centre “Agidel” at the Institute of
Petrochemistry and Catalysis of RAS. The biological studies of Cu complexes were done in the
Center for Molecular Design and Drug Bioscreening at the Institute of Petrochemistry and
Catalysis of RAS that was created with the financial support of the Russian Science Foundation.

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Highlights
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−
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Synthesis of steroid containing 5Z,9Z-dienoic acids have been developed.
New molecules are efficient apoptosis inducers in cancer cells.
The structures of all novel compounds were confirmed by NMR measurements.

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Catalytic cyclometallation in steroid chemistry V:
Synthesis of hybrid molecules based on steroid
oximes and (5Z,9Z)-tetradeca-5,9-dienedioic acid as
potential anticancer agents
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V.A.D’yakonov, L.U.Dzhemileva, R.A.Tuktarova, S.R. Ishmukhametova, M.M. Yunusbaeva, U.M.Dzhemilev