Catalytic cyclometallation in steroid chemistry VI: Targeted synthesis of hybrid molecules based on steroids and tetradeca-5Z,9Z-diene-1,14-dicarboxylic acid and study of their antitumor activity

Article abstract of DOI:10.1016/j.steroids.2018.06.004

Hybrid molecules based on a number of steroids (cholesterol, pregnenolone, androsterone) and 1,14-tetradeca-5Z,9Z-dienedicarboxylic acid linked via mono- and diethylene glycol spacers were synthesized for the first time and studied for antitumor activity in vitro. The acid was prepared using catalytic cyclomagnesiation of oxygenated 1,2-dienes with Grignard reagent in the presence of Cp₂TiCl₂ as the key synthetic step. Using flow cytometry, the new molecules were shown for the first time to be efficient apoptosis inducers in the HeLa, Hek293, U937, Jurkat, and K562 cell cultures and to have dose-dependent effect on the S and G2 phases of the cell cycle.

Full text of DOI:10.1016/j.steroids.2018.06.004

Accepted Manuscript
Catalytic cyclometallation in steroid chemistry VI: Targeted synthesis of hybrid
molecules based on steroids and tetradeca-5Z,9Z-diene-1,14-dicarboxylic acid
and study of their antitumor activity
Vladimir A. D'yakonov, Regina A. Tuktarova, Lilya U. Dzhemileva, Svetlana
R. Ishmukhametova, Milyausha M. Yunusbaeva, Usein M. Dzhemilev
PII:
S0039-128X(18)30111-9
https://doi.org/10.1016/j.steroids.2018.06.004
STE 8273
DOI:
Reference:
Steroids
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 April 2018
1 June 2018
4 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 VI: Targeted synthesis of
hybrid molecules based on steroids and tetradeca-5Z,9Z-diene-1,14-dicarboxylic acid and study of their antitumor
activity, Steroids (2018), doi: https://doi.org/10.1016/j.steroids.2018.06.004
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Catalytic cyclometallation in steroid chemistry VI1: Targeted synthesis of hybrid molecules
based on steroids and tetradeca-5Z,9Z-diene-1,14-dicarboxylic acid and study of their
antitumor activity
Vladimir A. D'yakonov,* Regina A. Tuktarova, Lilya U. Dzhemileva,* Svetlana R.
Ishmukhametova, Milyausha M. Yunusbaeva, and Usein M. Dzhemilev
Institute of Petrochemistry and Catalysis, Russian Academy of Sciences
pr. Oktyabrya 141, 450075 Ufa, Russian Federation.
Fax: +7 (347) 284 2750. E-mail: DyakonovVA@rambler.ru
Abstract: Hybrid molecules based on a number of steroids (cholesterol, pregnenolone,
androsterone) and 1,14-tetradeca-5Z,9Z-dienedicarboxylic acid linked via mono- and diethylene
glycol spacers were synthesized for the first time and studied for antitumor activity in vitro. The
acid was prepared using catalytic cyclomagnesiation of oxygenated 1,2-dienes with Grignard
reagent in the presence of Cp2TiCl2 as the key synthetic step. Using flow cytometry, the new
molecules were shown for the first time to be efficient apoptosis inducers in the HeLa, Hek293,
U937, Jurkat, and K562 cell cultures and to have dose-dependent effect on the S and G2 phases
of the cell cycle.
Keywords: Сross-cyclomagnesiation, Grignard reagent, Steroids, Hybrid molecules, Antitumor
activity
1. Introduction
The synthesis of new hybrid molecules for the design of modern efficient
pharmacophores and the modeling of existing and well-proven therapeutic agents is a vigorously
developing area of medicinal chemistry, as indicated by the avalanche growth of the number of
relevant publications in the world literature in the last 10‒15 years [1-15].
Steroid molecules attract close attention of researchers, which is largely caused by their
high biological activity and participation in the most important cell processes that occur in
human and animal bodies. The beginning of steroid application as therapeutic agents dates back
to the end of the 1940s. Back in the late 1930s, German chemists demonstrated that the adrenal
cortex produces steroid hormones. In 1937, first, deoxycorticosterone and then cortisone and
1 Steroids, 2018, in press.

hydrocortisone were isolated from the adrenal cortex. The broad range of biological activities of
hydrocortisone and cortisone predetermined the possible use of these compounds as therapeutic
agents. A typical feature of most natural steroids is the presence of an oxygenated substituent in
the С-3 position, which opens up a simple and efficient way for building-up and modification of
the molecule.
The broad range of biological activity, the presence of various functional groups arranged
around a rigid tetracyclic core, and the clear-cut ability of the steroid molecules to penetrate
through the cell membrane and to bind to definite hormonal receptors make this class of
compounds a convenient platform for the design of therapeutic agents with unique
pharmacological properties.
For the development of new therapeutic drugs meant for the prevention and treatment of
a broad range of cardiovascular and autoimmune diseases, cancer, states of shock of any type
(post-traumatic, operative, toxic, anaphylactic, burn, and cardiogenic shock) and for
transplantation of organs and tissues to suppress the rejection, a lot of hope is now placed on
hybrid compounds, in which the pharmacophores of optically active components are linked to
steroid residues [6-16].
Recently [17], we have shown that the hybrid molecules synthesized from steroids and
tetradeca-5Z,9Z-diene-1,14-dicarboxylic acid, prepared using the catalytic cross-
cyclomagnesiation of 1,2-dienes with Grignard reagents in the presence of Cp2TiCl2 [18], show
high antitumor and cytotoxic activities in vitro against a number of tumor cell lines and are
efficient inhibitors of human topoisomerase I [19,20].
In view of the above and the practical value and relevance of development of new
efficient, low-toxic, and selective antitumor agents, in this communication, we present the results
of studies dealing with the synthesis of new hybrid molecules composed of cholesterol,
pregnenolone, androsterone and tetradeca-5Z,9Z-diene-1,14-dicarboxylic acid linked via a
mono- or diethylene glycol spacer.
This choice of spacers is caused by the fact that ethylene glycols show anchoring
behavior for biological receptors and ligands, which reduces non-specific binding of proteins and
other biologically active molecules [21,22].
Also, the hybrid molecules we synthesized were studied for cytotoxicity against several
tumor cell lines (HeLa, Hek293, U937, Jurkat, and K562) and for the ability to induce apoptosis
and the effect on the cell cycle using advanced flow cytometry and fluorescence microscopy
methods.

2. Experimental
2.1. Chemistry
Cholesterol, 5-pregnen-β-ol-20-on, 5-androsten-β-ol-17-on, hepta-5,6-dien-1-ol, 4-
dimethylaminopyridine (DMAP), N-[3-(methylamino)propyl]-N'-ethylcarbodiimide
hydrochloride (EDC·HCl) were obtained from Sigma-Aldrich and Acros organics.
Dichloromethane was and freshly distilled before use. Optical rotations were measured on a
Perkin–Elmer 341 polarimeter. Melting points were recorded on Stuart SMP3. IR spectra were
recorded on Bruker VERTEX 70V using KBr discs over the range of 400–4000 cm_1. 1H and 13C
NMR spectra were obtained using a Bruker Ascend 500 spectrometer in CDCl3 operating at 500
MHz for 1H and 125 MHz for C and a Bruker AVANCE 400 spectrometer in CDCl3 operating
13
1
at 400 MHz for H and 100 MHz for 13C. Mass spectra of MALDI TOF/TOF positive ions
(matrix of sinapic acid) are recorded on a mass spectrometer Bruker AutoflexTM III Smartbeam.
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).
2.1.1. General procedure for the synthesis of mono- and diethylene glycol derivatives of steroids
3a-c, 4a-c
Steroids tosylates 2a-c (1 equiv) prepared from steroids and tosyl chloride was refluxed
for 6h with diol (6 equiv), in dry dioxane [23]. The insolubles were removed by filtration. The
filtrate was concentrated in vacuo, the residue redissolved in dichoromethane, washed with brine,
dried over anhydrous Na2SO4 and concentrated on rotary evaporator. The residue was purified
on a silica gel chromatography column to get 3a-c, 4a-c, yield ~70‒80%.
2.1.1.1. 2-[(3β)-Cholest-5-en-3-yloxy]ethanol (3a)
White crystals, 78% yield; m.p. 96–98 °С; [α]D19.5 ‒ 29.0 (c 0.72, CHCl3); IR (KBr) νmax
2930, 2868, 1459, 1375, 1365, 1238, 1163, 1100, 1053, 911, 811, 756 cm-1; 1H NMR (500 MHz,
CDCl3) δ 5.34 (1H, m, H-6), 3.70 (2H, m, СH2OH), 3.57 (2H, m, СH2O), 3.20 (1H, m, H-3),
2.59 (1H, br s, OH), 2.38‒0.90 (28H, m), 0.99 (3H, s, H-19), 0.92 (3H, d, J = 6.5 Hz, H-21), 0.87
(6H, d, J = 6.5 Hz, H-26, H-27), 0.67 (3H, s, H-18); 13C NMR (CDCl3, 125 MHz) δ 140.6 (C-5),
121.7 (C-6), 79.4 (C-3), 69.1 (СH2O), 61.9 (СH2OH), 56.8 (C-14), 56.2 (C-17), 50.2 (C-9), 42.3
(C-13), 39.8 (C-12), 39.5 (C-24), 38.1 (C-4), 37.2 (C-1), 36.8 (C-10), 36.2 (C-22), 35.8 (C-20),

31.9 (C-7, C-8), 28.4 (C-2), 28.4 (C-16), 27.9 (C-25), 24.3 (C-15), 23.9 (C-23), 22.8 (C-27), 22.6
(C-26), 21.1 (C-11), 19.4 (C-19), 18.7 (C-21), 11.9 (C-18); anal. calcd for C29H50O2: C, 81.87;
H, 11.70; found C, 81.79; H, 11.68. MALDI TOF: m/z 453.354 ([M+Na]+, calcd 453.371).
2.1.1.2. (3β)-3-(2-Hydroxyethoxy)pregn-5-en-20-one (3b)
18
White crystals, 75% yield; m.p. 96–98 °С; [α]D ‒ 5.1 (c 1.09, CHCl3); IR (KBr) νmax
1
2961, 2933, 2871, 1704, 1451, 1400, 1383, 1256, 1111, 1068, 1020, 954, 884, 593 cm-1; H
NMR (CDCl3, 500 MHz) δ 5.36 (1H, m, H-6), 3.73 (2H, m, СH2OH), 3.59 (2H, m, СH2O), 3.20
(1H, m, H-3), 2.57‒1.00 (20H, m), 2.13 (3H, s, H-21), 1.01 (3H, s, H-19), 0.64 (3H, s, H-18);
13C NMR (CDCl3, 125 MHz) δ 209.6 (C-20), 140.7 (C-5), 121.4 (C-6), 79.3 (C-3), 69.0 (СH2O),
63.7 (C-17), 62.1 (СH2OH), 56.9 (C-14), 50.0 (C-9), 43.9 (C-13), 39.0 (C-4), 38.8 (C-12), 37.2
(C-1), 36.9 (C-10), 31.8 (C-7, C-8), 31.5 (C-21), 28.4 (C-2), 24.5 (C-15), 22.8 (C-16), 21.1 (C-
11), 19.4 (C-19), 13.2 (C-18); anal. calcd for C23H36O3: C, 76.62; H, 10.06; found C, 76.56; H,
10.01. MALDI TOF: m/z 383.255 ([M+Na]+, calcd 383.256), 399.220 ([M+K]+, calcd 399.230).
2.1.1.3. (3β)-3-(2-Hydroxyethoxy)-17,2'-spiro([1,3]-dioxolane)androst-5-en (3c)
White crystals, 74% yield; m.p. 122–124 °С; [α]D19 ‒ 69.6 (c 1.19, CHCl3); IR (KBr) νmax
2948, 2904, 2866, 1457, 1400, 1433, 1382, 1306, 1226, 1169, 1101, 1061, 1040, 961, 882, 869,
760, 579 cm-1; 1H NMR (CDCl3, 500 MHz) δ 5.31 (1H, m, H-6), 3.88‒3.81 (4H, m, H-4', H-5'),
3.67 (2H, m, СH2OH), 3.54 (2H, m, СH2O), 3.16 (1H, m, H-3), 2.67 (1H, br s, OH), 2.36‒0.92
13
(19H, m), 0.97 (3H, s, H-19), 0.82 (3H, s, H-18); C NMR (CDCl3, 125 MHz) δ 140.6 (C-5),
121.4 (C-6), 119.4 (C-17), 79.3 (C-3), 69.1 (СH2O), 65.1, 64.5 (C-4', C-5'), 61.9 (СH2OH), 50.6
(C-14), 49.9 (C-9), 45.7 (C-13), 39.0 (C-4), 37.2 (C-1), 36.8 (C-10), 34.2 (C-16), 32.1 (C-8),
31.2 (C-7), 30.5 (C-12), 28.3 (C-2), 22.7 (C-15), 20.5 (C-11), 19.4 (C-19), 14.2 (C-18); anal.
calcd for C23H36O4: C, 73.37; H, 9.64; found C, 73.28; H, 9.62. MALDI TOF: m/z 399.231
([M+Na]+, calcd 399.251), 415.219 ([M+K]+, calcd 415.225).
2.1.1.4. 2-{2-[(3β)-Cholest-5-en-3-yloxy]ethoxy}ethanol (4a)
22
Colorless waxy solid, 72% yield; [α]D ‒ 28.0 (c 1.28, CHCl3).; IR (KBr) νmax 2929,
2863, 1464, 1379, 1365, 1235, 1160, 1100, 1049, 921, 863, 811, 754 cm-1; 1H NMR (CDCl3, 500
MHz) δ 5.36 (1H, H-6), 3.74 (2H, m, СH2OH), 3.69 (2H, m, СH2O), 3.66 (2H, m, СH2O), 3.64
(2H, m, СH2O), 3.21 (1H, m, H-3), 2.60 (1H, br s, OH), 2.42‒0.93 (28H, m), 1.01 (3H, s, H-19),
13
0.92 (3H, d, J = 6.5 Hz, H-21), 0.88 (6H, d, J = 7.0 Hz, H-26, H-27), 0.69 (3H, s, H-18); C
NMR (CDCl3, 125 MHz) δ 140.8 (C-5), 121.7 (C-6), 79.6 (C-3), 72.6 (СH2O), 70.8 (СH2O),
67.4 (СH2O), 61.9 (СH2OH), 56.8 (C-14), 56.2 (C-17), 50.2 (C-9), 42.3 (C-13), 39.8 (C-12),

39.5 (C-24), 38.9 (C-4), 37.2 (C-1), 36.9 (C-10), 36.2 (C-22), 35.8 (C-20), 31.9 (C-7, C-8), 28.3
(C-2), 28.2 (C-16), 28.0 (C-25), 24.3 (C-15), 23.8 (C-23), 22.8 (C-27), 22.6 (C-26), 21.1 (C-11),
19.4 (C-19), 18.7 (C-21), 11.9 (C-18); anal. calcd for C31H54O3: C, 78.43; H, 11.46; found C,
78.34; H, 11.43. MALDI TOF: m/z 497.381 ([M+Na]+, calcd 497.397), 513.364 ([M+K]+, calcd
513.371).
2.1.1.5. (3β)-3-[2-(2-Hydroxyethoxy)ethoxy]pregn-5-en-20-one (4b)
Colorless waxy solid, 71% yield; [α]D23 ‒ 3.0 (c 0.71, CHCl3); IR (KBr) νmax 2933, 2869,
1
1703, 1509, 1451, 1437, 1383, 1357, 1229, 1132, 1100, 1069, 953, 894, 594 cm-1; H NMR
(CDCl3, 500 MHz) δ 5.28 (1H, m, H-6), 3.67 (2H, m, СH2OH), 3.60 (2H, m, СH2O), 3.58 (2H,
m, СH2O), 3.56 (2H, m, СH2O), 3.15 (1H, m, H-3), 3.05 (1H, br s, OH), 2.50‒0.90 (20H, m),
2.08 (3H, s, H-21), 0.94 (3H, s, H-19), 0.57 (3H, s, H-18); 13C NMR (CDCl3, 125 MHz) δ 209.4
(C-20), 140.7 (C-5), 121.3 (C-6), 79.4 (C-3), 72.5 (СH2O), 70.7 (СH2O), 67.4 (СH2O), 63.6 (C-
17), 61.7 (СH2OH), 56.8 (C-14), 49.9 (C-9), 43.9 (C-13), 38.9 (C-4), 38.8 (C-12), 37.1 (C-1),
36.8 (C-10), 31.8 (C-8), 31.7 (C-7), 31.5 (C-21), 28.2 (C-2), 24.4 (C-15), 22.8 (C-16), 21.0 (C-
11), 19.3 (C-19), 13.2 (C-18); anal. calcd for C25H40O3: C, 74.22; H, 9.97; found C, 74.09; H,
9.95. MALDI TOF: m/z 427.265 ([M+Na]+, calcd 427.282), 443.249 ([M+K]+, calcd 443.256).
2.1.1.6. (3β)-3-[2-(2-Hydroxyethoxy)ethoxy]androst-5-en-17-one (4c)
Colorless waxy solid, 70% yield; [α]D19 + 4.6 (c 0.85, CHCl3); IR (KBr) νmax 2965, 2945,
2931, 2871, 2836, 1747, 1666, 1464, 1451, 1398, 1368, 1245, 1180, 1131, 1085, 1027, 997, 964,
1
899, 799, 584 cm-1; H NMR (CDCl3, 500 MHz) δ 5.33 (1H, m, H-6), 3.66 (2H, m, СH2OH),
3.61 (2H, m, СH2O), 3.58 (2H, m, СH2O), 3.56 (2H, m, СH2O), 3.15 (1H, m, H-3), 3.00 (1H, br
s, OH), 2.44‒0.92 (19H, m), 0.97 (3H, s, H-19), 0.83 (3H, s, H-18); 13C NMR (CDCl3, 125
MHz) δ 220.9 (C-17), 140.9 (C-5), 120.9 (C-6), 79.3 (C-3), 72.5 (СH2O), 70.7 (СH2O), 67.4
СH2O), 61.7 (СH2OH), 51.7 (C-14), 50.2 (C-9), 47.5 (C-13), 38.9 (C-4), 37.1 (C-1), 36.9 (C-10),
35.8 (C-16), 31.4 (C-8, C-12), 30.8 (C-7), 28.2 (C-2), 21.8 (C-15), 20.3 (C-11), 19.4 (C-19), 13.5
(C-18); anal. calcd for C23H36O4: C, 73.37; H, 9.64; found C, 73.23; H, 9.61. MALDI TOF: m/z
399.248 ([M+Na]+, calcd 399.251), 415.208 ([M+K]+, calcd 415.225).
1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) spectral data for (5Z,9Z)-
tetradeca-5.9-dienedioic acid (8) are in agreement with the literature data [17].

2.1.2. General procedure for the synthesis of steroidal derivatives of 5Z,9Z-dienoic acid 9a-c,
10a-c
To a solution of mono-or diethylene glycol derivatives of steroids 3a-c (or 4a-c) (1.0
mmol) in dichloromethane (10 ml) was added the (5Z,9Z)-tetradeca-5.9-dienedioic acid (8) (0.51
g, 2.0 mmol) 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 18 h until the reaction was complete (TLC monitoring,
hexane/ethyl acetate = 1/1). 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.
2.1.2.1. (5Z,9Z)-14-{2-[(3β)-Cholest-5-en-3-yloxy]ethoxy}-14-oxotetradeca-5,9-dienoic acid
(9a)
19
Colorless waxy solid, 0.49 g, 74% yield; [α]D ‒ 15.3 (c 0.26, CHCl3); IR (KBr) νmax
2932, 2854, 1738, 1708, 1622, 1456, 1431, 1384, 1323, 1238, 1180, 1151, 1116, 1077, 1025,
1
878, 800 cm-1; H NMR (CDCl3, 500 MHz) δ 5.41 (2H, m, H-6', H-9'), 5.37 (2H, m, H-5', H-
10'), 5.35 (1H, m, H-6), 4.22 (2H, m, СH2O), 3.69 (2H, m, СH2O), 3.19 (1H, m, H-3), 2.36 (2H,
m, H-2'), 2.35 (2H, m, H-13'), 2.38‒0.90 (28H, m), 2.10 (4H, m, H-4', H-11'), 2.08 (4H, m, H-7',
H-8'), 1.71 (2H, m, H-3'), 1.70 (2H, m, H-12'), 1.01 (3H, s, H-19), 0.92 (3H, d, J = 6.5 Hz, H-
13
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
178.9 (C-1'), 173.9 (C-14'), 140.7 (C-5), 130.4, 130.2 (C-6', C-9'), 129.0, 128.8 (C-5', C-10'),
121.7 (C-6), 79.6 (C-3), 65.9 (СH2O), 63.9 (СH2O), 56.8 (C-14), 56.2 (C-17), 50.2 (C-9), 42.3
(C-13), 39.8 (C-12), 39.5 (C-24), 38.9 (C-4), 37.2 (C-1), 36.8 (C-10), 36.2 (C-22), 35.8 (C-20),
33.6 (C-13'), 33.3 (C-2'), 31.9 (C-7, C-8), 28.3 (C-2), 28.2 (C-16), 28.0 (C-25), 27.3 (C-7', C-8'),
26.6 (C-11'), 26.5 (C-4'), 24.9 (C-12'), 24.6 (C-3'), 24.3 (C-15), 23.8 (C-23), 22.8 (C-27), 22.6
(C-26), 21.1 (C-11), 19.4 (C-19), 18.7 (C-21), 11.9 (C-18); anal. calcd for C43H70O5: C, 77.43;
H, 10.58; found C, 77.36 H, 10.55. MALDI TOF: m/z 689.415 ([M+Na]+, calcd 689.512),
705.377 ([M+K]+, calcd 705.486).
2.1.2.2. (5Z,9Z)-14-Oxo-14-(2-{[(3β)-20-oxopregn-5-en-3-yl]oxy}ethoxy)tetradeca-5,9-dienoic
acid (9b)
18
Colorless waxy solid, 0.43 g, 72% yield; [α]D ‒ 0.45 (c 0.88, CHCl3); IR (KBr) νmax
2938, 2870, 1737, 1709, 1455, 1436, 1384, 1307, 1240, 1219, 1170, 1109, 1042, 960, 800, 753
1
cm-1; H NMR (CDCl3, 500 MHz) δ 5.41 (2H, m, H-6', H-9'), 5.35 (3H, m, H-5', H-10', H-6),

4.21 (2H, m, СH2O), 3.68 (2H, m, СH2O), 3.19 (1H, m, H-3), 2.55‒0.99 (20H, m), 2.37 (2H, m,
H-2'), 2.34 (2H, m, H-13'), 2.13 (3H, s, H-21), 2.10 (4H, m, H-4', H-11'), 2.07 (4H, m, H-7', H-
13
8'), 1.71 (2H, m, H-3'), 1.68 (2H, m, H-12'), 1.00 (3H, s, H-19), 0.63 (3H, s, H-18); C NMR
(CDCl3, 125 MHz) δ 209.8 (C-20), 179.6 (C-1'), 173.9 (C-14'), 140.7 (C-5), 130.4, 130.2 (C-6',
C-9'), 129.0, 128.8 (C-5', C-10'), 121.4 (C-6), 79.5 (C-3), 65.9 (СH2O), 63.8 (СH2O), 63.7 (C-
17), 56.9 (C-14), 50.0 (C-9), 44.0 (C-13), 38.9 (C-4), 38.8 (C-12), 37.2 (C-1), 36.8 (C-10), 33.6
(C-13'), 33.3 (C-2'), 31.8 (C-7, C-8), 31.5 (C-21), 28.3 (C-2), 27.3 (C-7', C-8'), 26.6 (C-11'), 26.5
(C-4'), 24.9 (C-12'), 24.6 (C-3'), 24.5 (C-15), 22.8 (C-16), 21.1 (C-11), 19.3 (C-19), 13.2 (C-18);
anal. calcd for C37H56O6: C, 74.46; H, 9.46; found C, 74.41; H, 9.44. MALDI TOF: m/z 619.348
([M+Na]+, calcd 619.397), 635.317 ([M+K]+, calcd 635.371).
2.1.2.3.
(5Z,9Z)-14-Oxo-14-(2-{[(3β)-17,2'-spiro([1,3]-dioxolane)androst-5-en-3-
yl]oxy}ethoxy)tetradeca-5,9- dienoic acid (9c)
Colorless waxy solid, 0.45 g, 73% yield; [α]D
19.5
‒ 24.0 (c 0.94, CHCl3); IR (KBr) νmax
2938, 2870, 1737, 1709, 1455, 1436, 1384, 1307, 1240, 1219, 1170, 1109, 1042, 960, 837, 800,
753 cm-1; 1H NMR (CDCl3, 500 MHz) δ 5.42 (2H, m, H-6', H-9'), 5.36 (3H, m, H-5', H-10', H-
6), 4.22 (2H, m, СH2O), 3.93‒3.87 (4H, m, H-4'', H-5''), 3.69 (2H, m, СH2O), 3.22 (1H, m, H-3),
2.38‒0.99 (19H, m), 2.36 (2H, m, H-2'), 2.35 (2H, m, H-13'), 2.11 (4H, m, H-4', H-11'), 2.09
(4H, m, H-7', H-8'), 1.71 (2H, m, H-3'), 1.69 (2H, m, H-12'), 0.99 (3H, s, H-19), 0.88 (3H, s, H-
18); 13C NMR (CDCl3, 125 MHz) δ 178.3 (C-1'), 173.9 (C-14'), 140.7 (C-5), 130.4, 130.2 (C-6',
C-9'), 129.0, 128.9 (C-5', C-10'), 121.5 (C-6), 119.5 (C-17), 79.5 (C-3), 65.9 (СH2O), 65.2, 64.6
(C-4'', C-5''), 63.9 (СH2O), 50.6 (C-14), 50.0 (C-9), 45.7 (C-13), 38.9 (C-4), 37.2 (C-1), 36.9 (C-
10), 34.2 (C-16), 33.6 (C-13'), 33.2 (C-2'), 32.2 (C-8), 31.3 (C-7), 30.6 (C-12), 28.3 (C-2), 27.3
(C-7', C-8'), 26.6 (C-11'), 26.5 (C-4'), 24.9 (C-12'), 24.6 (C-3'), 22.8 (C-15), 20.5 (C-11), 19.4
(C-19), 14.2 (C-18); anal. calcd for C37H56O7: C, 72.51; H, 9.21; found C, 72.46; H, 9.19.
MALDI TOF: m/z 635.327 ([M+Na]+, calcd 635.392).
2.1.2.4.
(5Z,9Z)-14-(2-{2-[(3β)-Cholest-5-en-3-yloxy]ethoxy}ethoxy)-14-oxotetradeca-5,9-
dienoic acid (10a)
22
Colorless waxy solid, 0.49 g, 70% yield; [α]D ‒ 8.5 (c 1.20, CHCl3); IR (KBr) νmax
1
2934, 2867, 1737, 1712, 1619, 1458, 1382, 1260, 1138, 1107, 1024, 956, 874, 801 cm-1; H
NMR (CDCl3, 500 MHz) δ 5.41 (2H, m, H-6', H-9'), 5.36 (2H, m, H-5', H-10'), 5.36 (1H, m, H-
6), 4.24 (2H, m, СH2O), 3.72 (2H, m, СH2O), 3.65 (4H, m, СH2O), 3.20 (1H, m, H-3), 2.36 (2H,
m, H-2'), 2.35 (2H, m, H-13'), 2.37‒0.93 (28H, m), 2.10 (4H, m, H-4', H-11'), 2.08 (4H, m, H-7',
H-8'), 1.72 (2H, m, H-3'), 1.70 (2H, m, H-12'), 1.01 (3H, s, H-19), 0.92 (3H, d, J = 6.4 Hz, H-

13
21), 0.88 (6H, d, J = 6.4 Hz, H-26, H-27), 0.69 (3H, s, H-18); C NMR (CDCl3, 125 MHz) δ
178.1 (C-1'), 173.8 (C-14'), 140.9 (C-5), 130.4, 130.2 (C-6', C-9'), 128.9 (C-5', C-10'), 121.6 (C-
6), 79.6 (C-3), 70.8 (СH2O), 69.2 (СH2O), 67.3 (СH2O), 63.5 (СH2O), 56.8 (C-14), 56.2 (C-17),
50.2 (C-9), 42.3 (C-13), 39.8 (C-12), 39.5 (C-24), 39.0 (C-4), 37.2 (C-1), 36.9 (C-10), 36.2 (C-
22), 35.8 (C-20), 33.6 (C-13'), 33.2 (C-2'), 31.9 (C-7, C-8), 28.3 (C-2), 28.2 (C-16), 28.0 (C-25),
27.3 (C-7', C-8'), 26.6 (C-11'), 26.5 (C-4'), 24.8 (C-12'), 24.6 (C-3'), 24.3 (C-15), 23.8 (C-23),
22.8 (C-27), 22.6 (C-26), 21.1 (C-11), 19.4 (C-19), 18.7 (C-21), 11.9 (C-18); anal. calcd for
C45H74O6: C, 76.01; H, 10.49; found C, 75.96 H, 10.46. MALDI TOF: m/z 733.425 ([M+Na]+,
calcd 733.538), 749.378 ([M+K]+, calcd 749.512).
2.1.2.5. (5Z,9Z)-14-Oxo-14-[2-(2-{[(3β)-20-oxopregn-5-en-3-yl]oxy}ethoxy)ethoxy]tetradeca-
5,9-dienoic acid (10b)
26
Colorless waxy solid, 0.44 g, 69% yield; [α]D ‒ 0.1 (c 0.99, CHCl3); IR (KBr) νmax
2933, 2870, 1736, 1705, 1455, 1417, 1383, 1359, 1242, 1179, 1169, 1139, 1108, 1045, 954, 800,
736 cm-1; 1H NMR (CDCl3, 500 MHz) δ 5.40 (2H, m, H-6', H-9'), 5.36 (3H, m, H-5', H-10', H-
6), 4.24 (2H, m, СH2O), 3.72 (2H, m, СH2O), 3.65 (4H, m, СH2O), 3.19 (1H, m, H-3), 2.56‒0.95
(20H, m), 2.37 (2H, m, H-2'), 2.33 (2H, m, H-13'), 2.13 (3H, s, H-21), 2.09 (4H, m, H-4', H-11'),
2.06 (4H, m, H-7', H-8'), 1.71 (2H, m, H-3'), 1.68 (2H, m, H-12'), 1.00 (3H, s, H-19), 0.63 (3H, s,
13
H-18); C NMR (CDCl3, 125 MHz) δ 209.8 (C-20), 178.5 (C-1'), 173.8 (C-14'), 140.9 (C-5),
130.2, (C-6', C-9'), 128.9 (C-5', C-10'), 121.3 (C-6), 79.5 (C-3), 70.8 (СH2O), 69.2 (СH2O), 67.3
(СH2O), 63.7 (C-17), 63.4 (СH2O), 56.9 (C-14), 50.0 (C-9), 44.0 (C-13), 38.9 (C-4), 38.8 (C-12),
37.2 (C-1), 36.9 (C-10), 33.6 (C-13'), 33.3 (C-2'), 31.8 (C-7, C-8), 31.5 (C-21), 28.3 (C-2), 27.3
(C-7', C-8'), 26.6 (C-11'), 26.5 (C-4'), 24.8 (C-12'), 24.6 (C-3'), 24.5 (C-15), 22.8 (C-16), 21.1
(C-11), 19.4 (C-19), 13.2 (C-18); anal. calcd for C39H60O7: C, 73.09; H, 9.44; found C, 72.97; H,
9.43. MALDI TOF: m/z 633.351 ([M+Na]+, calcd 633.424), 679.305 ([M+K]+, calcd 679.398).
2.1.2.6. (5Z,9Z)-14-oxo-14-[2-{[(3β)-17-oxoandrost-5-en-3-yl]oxy}ethoxy)ethoxy]tetradeca-5,9-
dienoic acid (10c)
23
Colorless waxy solid, 0.42 g, 68% yield; [α]D + 1.0 (c 1.02, CHCl3); IR (KBr) νmax
1
2933, 2859, 1737, 1454, 1376, 1245, 1180, 1141, 1105, 1059, 800 cm-1; H NMR (CDCl3, 500
MHz) δ 5.40 (2H, m, H-6', H-9'), 5.37 (2H, m, H-5', H-10', H-6), 5.36 (1H, m, H-6), 4.23 (2H,
m, СH2O), 3.72 (2H, m, СH2O), 3.64 (4H, m, СH2O), 3.19 (1H, m, H-3), 2.38‒1.00 (19H, m),
2.37 (2H, m, H-2'), 2.33 (2H, m, H-13'), 2.10 (4H, m, H-4', H-11'), 2.08 (4H, m, H-7', H-8'), 1.70
13
(2H, m, H-3'), 1.68 (2H, m, H-12'), 1.02 (3H, s, H-19), 0.89 (3H, s, H-18); C NMR (CDCl3,
125 MHz) δ 221.3 (C-17), 177.9 (C-1'), 173.8 (C-14'), 141.1 (C-5), 130.3, 130.2 (C-6', C-9'),

128.9 (C-5', C-10'), 120.8 (C-6), 79.4 (C-3), 70.8 (СH2O), 69.3 (СH2O), 67.3 (СH2O), 63.4
(СH2O), 51.8 (C-14), 50.3 (C-9), 47.6 (C-13), 39.0 (C-4), 37.1 (C-1), 36.9 (C-10), 35.8 (C-16),
33.6 (C-13'), 33.2 (C-2'), 31.5 (C-8), 31.4 (C-7), 30.8 (C-12), 28.2 (C-2), 27.3 (C-7', C-8'), 26.6
(C-11'), 26.5 (C-4'), 24.8 (C-12'), 24.6 (C-3'), 21.9 (C-15), 20.3 (C-11), 19.4 (C-19), 13.5 (C-18);
anal. calcd for C37H56O7: C, 72.51; H, 9.21; found C, 72.47; H, 9.18. MALDI TOF: m/z 635.382
([M+Na]+, calcd 635.392), 651.341 ([M+K]+, calcd 651.366).
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 9a-с, 10a-с 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. U937 tumor cells after treatment with compounds 9b or 10b at
concentrations (0.3, 0.2, 0.1 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. U937 tumor cells
after treatment with compound 9b at concentrations (0.4, 0.3, 0.2, 0.1 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
3.1. Chemistry
The investigation program was started with the synthesis of mono- and diethylene glycol
derivatives of cholesterol, pregnenolone and androsterone 3a-c, 4а-с by refluxing the
corresponding steroid tosylates 2а-с with an excess (6 eq.) of mono- or diethylene glycol in 1,4-
dioxane (Scheme 1) [23]. It is noteworthy that under these conditions, androsterone tosylate 2с
reacts with ethylene glycol via both the tosyl group and the oxo group at С-17 to give the
monoethylene glycol derivative of androsterone with spiro-connected dioxolane moiety 3с.

Scheme 1. Synthesis of mono(di)ethylene glycol derivatives of steroids 3a-c, 4a-c: (a) TsCl, anhydrous
pyridine, 0 °C; (b) ethylene glycol (or diethylene glycol), dioxane, reflux.
The initial tetradeca-5Z,9Z-diene-1,14-dicarboxylic acid 8 was synthesized in two steps
using the previously developed [24,25 ] homo-cyclomagnesiation of the tetrahydropyran ether of
5,6-hepta-5,6-dien-1-ol 5 with EtMgBr in the presence of the Cp2TiCl2 catalyst (5 mol. %),
which was followed by hydrolysis of magnesacyclopentane 6 and oxidation of thus formed 1,14-
bis-tetrahydropyranyl-5Z,9Z-diene-1,14-diol 7 with the Jones reagent. The overall yield was
~60% (Scheme 2).
Scheme 2. Synthesis of (5Z,9Z)-tetradeca-5,9-dienedioic acid 8. (a) EtMgBr, Mg, Cp2TiCl2 (5 mol%),
diethyl ether; (b) H3O+; (c) H2CrO4/H2SO4, acetone, CH2Cl2.
The subsequent reaction of tetradeca-5Z,9Z-diene-1,14-dicarboxylic acid 8 with mono-
(3a-c) and diethylene glycol (4а-с) steroid derivatives takes place in the presence of N-(3-
dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC·HCl) and catalytic amounts of
dimethylaminopyridine (DMAP) and gives the target 5Z,9Z-dienoic acids 9a-c, 10a-c (Scheme
3).

Scheme 3. Synthesis of steroid-containing 5Z,9Z-dienoic fatty acids 9a-c, 10a-c: (а) EDC·HCl, DMAP,
CH2Cl2.
The structures of hybrid molecules 9a-c and 10a-c were established by 1H and C NMR
13
spectroscopy, and 2D heteronuclear correlation experiments (HSQC, HMBC).
3.2. Biological evaluation
All of the synthesized hybrid molecules 9a-c and 10a-c were tested for the first time for
the antitumor activity in vitro using the Jurkat, K562, Hek293, HeLa, and U937 cell lines; the
study included determination of CC50, the apoptosis-inducing activity, and the effect on the cell
cycle using flow cytometry.
The cytotoxic activities of compounds 9a-c and 10a-c in vitro against the Jurkat, K562,
U937, and HeLa tumor cultures and the cell line obtained from the Hek293 human embryonic
kidney cells are summarized in Table 1.

Table 1
Cytotoxic activities of synthesized hybrid steroids 9a-c and 10a-c measured on tumor cell cultures
(Jurkat, K562, Hek293, HeLa, U937) and on normal fibroblasts (CC50, µM).
Сompound
9a
Jurkat
K562
Hek293
HeLa
U937
Fibroblasts
1.17 0.011
0.67 0.033
0.69 0.038
0.82 0.031
0.77 0.015
0.64 0.028
3.64 0.017
0.18 0.019
0.06 0.023
0.13 0.011
0.20 0.029
0.14 0.012
0.33 0.037
1.03 0.015
0.15 0.011
0.15 0.012
0.14 0.012
0.39 0.043
0.11 0.017
0.21 0.024
1.29 0.019
0.51 0.015
0.42 0.014
0.03 0.043
0.48 0.012
0.41 0.038
0.62 0.011
1.41 0.015
0.79 0.047
0.15 0.017
0.68 0.031
0.75 0.036
0.53 0.012
0.74 0.048
1.19 0.049
0.26 0.048
0.10 0.011
0.16 0.034
0.30 0.042
0.13 0.028
0.21 0.024
0.98 0.025
9b
9c
10a
10b
10c
Camp.
The cytotoxic effect of the hybrid molecules determined by flow cytometry against five
tumor cell lines and human fibroblasts follows a clear-cut dose-dependent pattern, which is
specific to each compound (Table 1 and Fig. 1 and Fig. 2). It can be seen from the Table that the
inhibitory concentrations CC50 for acids 9b and 10b, found by exposure of the test compounds to
the above-mentioned cell lines followed by cell staining with the dye 7AAD, generally varies
depending on the cell culture, but the lowest values were found for two lines, Jurkat and U937.
The Jurkat value was the lowest, being 0.06 µM for compound 9b and 0.14 µM for compound
10b. In the case of U937 culture, CC50 was 0.10 µM for compound 9b and 0.13 µM for 10b. The
CC50 values for normal fibroblasts were 0.67 µM for 9b and 0.77 µM for 10b. Thus, the half
maximal inhibitory concentration of 9b is almost 10 times lower for the Jurkat cells and 5 times
lower for the U937 cells than for normal fibroblasts. A similar situation is observed for
compound 10b (Table 1). Simultaneously, it have been shown that mono- (3a-c) and diethylene
glycol (4a-c) steroid derivatives to have a 5-7-fold lower cytotoxic effect on tumor cell lines than
the corresponding hybrid molecules.
Determination of the apoptosis induced by compounds 9b and 10b using the vital double
staining with AnnexinV/7AAD and flow cytometry detection was performed in relation to Jurkat
and U937 tumor cells, with the test compound concentrations varying from 0.1 µM to 0.3 µM.
Compounds 9b and 10b were found to induce the tumor cell death for the given cell lines by
apoptotic mechanism (see Fig. 1).

1
3
4
2
7
6
5
Fig. 1. Tumor cell line U937 treated with compounds 9b (2-4) and 10b (5-7) in various
concentrations, stained by annexin V/7AAD, and analyzed by flow cytometry. (1) control; (2) 9b (0.1
µM); (3) 9b (0.2 µM); (4) 9b (0.3 µM), (5) 10b (0.1 µM). (6) 10b (0.2 µM). (7) 10b (0.3 µM).
Table 2
Results of measurement of apoptosis in the Jurkat and U937 cell cultures induced by acids 9b and 10b.
Jurkat
U937
Сompound
Early
apoptosis (%) apoptosis (%) apoptosis (%)
Late
Early
Late
apoptosis (%)
9b (0.1 µM)
9b (0.2 µM)
9b (0.3 µM)
10b (0.1 µM)
10b (0.2 µM)
10b (0.3 µM)
Campt. (1 µM)
4.5
2.4
1.2
2.2
4.7
2.2
5.1
35.8
54.8
74.2
20.1
38.9
78.9
78.9
5.7
3.3
0.2
2.9
5.1
2.2
5.7
37.7
75.4
83.6
21.2
40.9
80.8
83.1
As the concentration of hybrid steroids 9b and 10b is increased, the number of cells that
have reached early and late apoptosis stages statistically significantly increases. The highest

amount of apoptotic cells occurring at early and late apoptosis stages, which were overally
AnV+7AAD-, AnV+7AAD+, and AnV-7AAD+ positive, was found for U937 cells and was 83.6%
at 0.3 µM concentration of 9b. Thus, the obtained results attest to the ability of compounds 9b
and 10b to exert selective cytotoxic action on tumor cells and induce apoptotic cell death.
Figure 2 shows the results of analysis of kinetic characteristics of the cell cycle for U937
cells determined by DNA flow cytometry after treating the cells with compound 9b in various
concentrations and incubation for 24 h.
2
1
3
5
4
Fig. 2. Phases of the cell cycle for the U937 cells treated with compound 9b. (1) control; (2) 9b (0.1 µM);
(3) 9b (0.2 µM); (4) 9b (0.3 µM); (5) 9b (0.4 µM). The incubation time of 9b with the cells is 24 h.
It can be seen from Fig. 2 that hybrid product 9b affects mainly the S and G2 cell cycle
phases, with this effect being dose-dependent. The most pronounced changes in the S and G2
phases arise when the test compound is taken in 0.4 µM concentration (Fig. 2). Also, with
increasing concentration of compound 9b, a subG0 peak appears, indicating the apoptotic death
of tumor cells. The most pronounced enrichment of subG0 cell population is induced by 0.4 µM
concentration.

4. Conclusion
Synthetic analogues of natural 5Z,9Z-dienoic acids, namely, hybrid molecules based on
cholesterol, progesterone, and androsterone and 1,14-tetradeca-5Z,9Z-dienedicarboxylic acid
linked by mono- and diethylene glycol spacers were synthesized and studied for the antitumor
activity in vitro. Using flow cytometry, the new hybrid molecules were shown to have high
cytotoxicity in vitro against HeLa, Hek293, U937, Jurkat, and K562 tumor cells, to exert phase-
specific cytotoxic action on the S and G2 phase cells, and to be efficient apoptosis inducers.
These results open up prospects for the development of new highly efficient antitumor drugs.
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 hybrid molecules were done in
the Center for Molecular Design and Drug Bioscreening at the Institute of Petrochemistry and
Catalysis of RAS.
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Graphical Abstract

Highlights
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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.