Synthesis and Cytostatic Activity of New Mepregenol 17-Acetate Derivatives with Respect to Hela Cancer Cell Culture

Article abstract of DOI:10.1007/s11094-020-02167-1

New mepregenol 17-acetate derivatives with potential cytotoxicity were synthesized. Mepregenol 17-acetate 3-acrylate (III) was shown to exhibit potent cytotoxicity although the ester of N-benzylpyrrolidine-3-carboxylic acid (IV) had weaker activity that w

Full text of DOI:10.1007/s11094-020-02167-1

DOI 10.1007/s11094-020-02167-1
Pharmaceutical Chemistry Journal, Vol. 54, No. 2, May, 2020 (Russian Original Vol. 54, No. 2, February, 2020)
SEARCH FOR NEW DRUGS
SYNTHESIS AND CYTOSTATIC ACTIVITY
OF NEW MEPREGENOL 17-ACETATE DERIVATIVES
WITH RESPECT TO HELA CANCER CELL CULTURE
T. A. Fedotcheva,1 E. D. Sveshnikova,1 N. I. Sheina,1 M. N. Sokolov,2
K. V. Kudryavtsev,2 N. I. Fedotcheva,3 and N. L. Shimanovskii1
Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 54, No. 2, pp. 17 – 23, February, 2020.
Original article submitted November 30, 2019.
New mepregenol 17-acetate derivatives with potential cytotoxicity were synthesized. Mepregenol 17-acetate
3-acrylate (III) was shown to exhibit potent cytotoxicity although the ester of N-benzylpyrrolidine-3-carboxy-
lic acid (IV) had weaker activity that was comparable with those of megestrol acetate and progesterone. The
IC50 values for III, IV, progesterone, and megestrol acetate were 30, 200, 480, and 130 mM with respect to na-
tive HeLa culture and 9.1, 180, 5.6, and 115 mm for an estradiol-stimulated culture of HeLa, respectively. The
cytotoxicities of steroids III and IV correlated with their DNA-damaging effect in a DNA comet assay. Thus,
III was promising for further research as an antitumor compound with respect to estradiol-dependent tumors.
Keywords: pregnane steroids, gestagens, estradiol, HeLa, cervical cancer.
EXPERIMENTAL CHEMICAL PART
Esters of 17a-acetoxy-3b-hydroxy-6-methylpregna-4,6-
dien-20-one with gestagenic and antiproliferative activities
Commercially available reagents and solvents were used
after peroral administration that exceed significantly those of
in the syntheses. Melting points were determined in open
progesterone were reported [1 – 3]. The main metabolic cen-
capillaries and are uncorrected. PMR and 13C NMR spectra
ters on C3, C6, and C17 are blocked in 17a-acetoxy-b-hyd-
were recorded in CDCl and DMSO-d solutions with resid-
3
6
roxy-6-methylpregna-4,6-dien-20-one esters, which prolongs
the in vivo residence time and the pharmacological effects
[4]. However, the structure—activity relationship of
17a-acetoxy-b-hydroxy-6-methylprena-4,6-dien-20-one es-
ters cannot be fully analyzed because of their limited num-
ber. The goals of the present work were to prepare and study
the cytotoxicities of new 17a-acetoxy-b-hydroxy-6-methyl-
pregna-4,6-dien-20-one esters as compared to megestrol ace-
tate and progesterone using HeLa native human cervical can-
cer cell culture and an estradiol-stimulated culture
(HeLa-ESC).
ual solvent resonances as standards on Bruker AM-400 and
Agilent 400 MR instruments at operating frequency
400 MHz and 293 – 303 K. Chemical shifts (d) were given in
ppm; SSCC (J), in Hz. Structural data for mepregenol 17-ac-
etate 3-acrylate (III) were obtained using a STOE diffracto-
meter, Pilatus 100K semiconducting detector, microfocused
Cu Ka beam (1.54086 Å), and a multilayer thin-film focus-
ing monochromator (M. V. Lomonosov MSU, Chemistry
Department). X-ray data were processed using the STOE
X-AREA 1.67 program suite (STOE & Cie GmbH,
Darmstadt, Germany, 2013). Specific rotation was measured
at 589 nm (Na D-line) on a PerkinElmer 341 MC polari-
meter. Elemental analyses of the compounds were deter-
mined on a Vario microcube Elementar automated analyzer.
Mepregenol 17-acetate (II, AMOL). A solution of
megestrol 17-acetate (I, 1.000 g) in THF (50 mL) was stirred
1
N. I. Pirogov Russian National Research Medical University, Moscow,
117997 Russia.
2
M. V. Lomonosov Moscow State University, Moscow, 119991 Russia.
3
Institute of Theoretical and Experimental Biophysics, Russian Academy
of Sciences, Pushchino, Moscow Oblast, 142290 Russia.
119
0091-150X/20/5402-0119 © 2020 Springer Science+Business Media, LLC

120
T. A. Fedotcheva et al.
and treated at room temperature with NaBH (0.100 g) and
12 h. The solvent was removed at reduced pressure in a ro-
tary evaporator. The solid was purified by column chroma-
tography over silica gel with elution by CH Cl —MeOH
4
then distilled H O (5 mL). Additional NaBH (0.030 g) was
2
4
added after 2 h. The mixture was stirred for 1 h, treated with
2
2
distilled H O (150 mL), and stirred for 2 h until a precipitate
(100:1(50:1) to afford IV (1.140 g, 88%) as a colorless oil,
C H NO . Calc, %: C 75.36; H 8.26; N 2.44. Found, %: C
2
formed. The precipitate was filtered off, rinsed with distilled
36 47
5
75.48; H 8.29; N 2.51. [a]2D0 – 88.4 (c 1.18, MeOH). PMR
H O, and dried to constant mass to afford II (0.965 g, 97%)
2
as a colorless solid, mp 195°C. PMR spectrum (400 MHz;
spectrum (400 MHz; CDCl ), d, ppm: 7.41 – 7.17 (m, 5H),
3
CDCl ), d, ppm: 5.55 (s, 1H), 5.49 (s, 1H), 4.29 (br.s, 1H),
3
5.51 (s, 1H), 5.41 (d, J 2.9 Hz, 1H), 3.64 (d, J 6.1 Hz, 1H),
3.13 – 2.85 (m, 3H), 2.67 (dd, J 14.1, 7.0 Hz, 1H), 2.56 (d, J
9.0 Hz, 1H), 2.06 (d, J 14.9, 8H), 2.22 – 2.01 (m, 4H), 1.96
(d, J 4.2 Hz, 1H), 1.92 – 1.63 (m, 10H), 1.57 (t, J 3.2 Hz,
1H), 1.54 (d, J 3.9 Hz, 1H), 1.50 – 1.22 (m, 4H), 1.12 (br.s,
1H), 0.98 (s, 3H), 0.69 (s, 3H). 13C NMR spectrum
2.95 (dd, J 13.9, 11.3 Hz, 1H), 2.16 – 2.08 (m, 1H), 2.06 (s,
3H), 2.08 – 2.05 (m, 1H), 2.04 (s, 3H), 1.92 (dd, J 12.8,
4.1 Hz, 1H), 1.90 – 1.82 (m, 2H), 1.80 (s, 3H), 1.73 (s, 1H),
1.71 – 1.49 (m, 5H), 1.49 – 1.24 (m, 4H), 1.17 – 1.04 (m,
1H), 0.97 (s, 3H), 0.69 (s, 3H). 13C NMR spectrum
(100 MHz; CDCl ), d, ppm: 14.37, 18.08, 20.08, 20.31,
3
(100 MHz; CDCl ), d, ppm: 204.14, 174.98, 170.71, 146.79,
3
21.14, 23.29, 26.33, 28.67, 30.30, 31.24, 33.69, 35.01, 36.83,
47.55, 49.39, 51.06, 68.25, 96.60, 123.88, 128.32, 131.40,
145.16, 170.70, 204.20.
131.26, 128.85, 128.69, 128.39, 128.22, 126.94, 119.52,
96.62, 71.18, 60.02, 56.78, 56.68, 53.80, 50.89, 49.38, 47.57,
47.41, 42.29, 36.83, 35.01, 33.43, 31.26, 30.34, 27.76, 27.59,
26.34, 24.32, 23.32, 21.18, 21.03, 20.27, 20.07, 17.93, 14.38.
(3S,8R,9S,10R,13S,14S,17R)-17-Acetoxy-17-acetyl-6,
10,13-trimethyl-2,3,8,9,10,11,12,13,14,15,16,17-dodecahyd-
ro-1H-cyclopenta[a]phenanthrenyl-3-yl acrylate (III).
Mepregenol 17-acetate (II, 9.60 g) in CH Cl (150 mL) was
EXPERIMENTAL BIOLOGICAL PART
2
2
cooled to –5°C, treated with Et N (3.50 mL), stirred under
3
HeLa cell culture (human epithelial cervical cancer) was
obtained from the Russian Cell Culture Collection. Cells
were cultivated under sterile conditions using an LB-V lami-
Ar, treated in portions with a solution of acryloyl chloride
(2.70 g) in CH Cl (30 mL) without allowing the tempera-
2
2
ture of the reaction mixture to rise, stirred at room tempera-
ture for 12 h after the addition of acryloyl chloride was fin-
nar flow hood and incubated at 37°C with 5% CO . Standard
2
DMEM medium with added thermally inactivated fetal bo-
vine serum (20%, PanEco, Russia), L-glutamine
(100 mg/mL) and antibiotics gentamycin sulfate and strepto-
mycin sulfate (40 mg/mL) was used for cell culture.
HeLa-ESC cells were grown at 37°C in the presence of
estradiol (10–8 M) (Sigma, USA) in DMEM medium con-
taining fetal bovine serum (20%).
ished, and washed with H O and saturated NaCl solution.
2
The organic layer was dried over anhydrous Na SO . The
2
4
solvent was removed at reduced pressure in a rotary evapora-
tor. The solid was purified by column chromatography over
silica gel with elution by petroleum-ether—EtOAc (5:1) to
afford III (7.50 g, 73%) as
a colorless solid, mp
184 – 185°C. [a]2D0 – 79.7 (c 0.98, CH Cl ). Calc, %,
2
2
Cells were grown in 25-mL tubes, trypsinized after form-
ing a monolayer, and placed (200 mL) in wells of a COSTAR
flat-bottomed plate. Solutions of compounds in DMEM me-
dium were added to final concentrations of 10–7 – 10–5 M.
The plates were incubated for 72 h, after which the viability
of the cultures was assayed using the standard MTT test [5].
Medium was removed from the plate wells after the cells
were incubated with the compounds. DMEM/F12 medium
(1:1, 200 mL) and starting MTT solution (10 mg/mL, 10 mL
each) were placed into the wells. Cells were incubated at
C H O : C 73.61; H 8.24. Found, %: C 73.72; H 8.29.
27 36
5
PMR spectrum (400 MHz; CDCl ), d, ppm: 6.41 (dd, J 17.3,
3
1.5 Hz, 1H), 6.13 (dd, J 17.3, 10.4 Hz, 1H), 5.82 (dd, J 10.4,
1.5 Hz, 1H), 5.49 (s, 1H), 5.46 (d, J 3.1 Hz, 1H), 2.96 (dd, J
15.2, 1.3 Hz, 1H), 2.93 (dd, J 15.8, 2.4 Hz, 1H), 2.15 – 2.07
(m, 2H), 2.06 (s, 3H), 2.02 (s, 3H), 1.94 (t.d, J 12.7, 4.2 Hz,
1H), 1.90 – 1.79 (m, 3H), 1.77 (dd, J 1.9, 1.3 Hz, 3H),
1.75 – 1.67 (m, 2H), 1.64 – 1.57 (m, 1H), 1.56 – 1.50 (m,
1H), 1.46 – 1.33 (m, 2H), 1.30 (ddd, J 16.5, 10.8, 3.7 Hz,
1H), 1.16 – 1.06 (m, 1H), 0.97 (s, 3H), 0.67 (s, 3H). 13C
37°C for 3 h in a moist atmosphere with 5% CO . When the
NMR spectrum (100 MHz; CDCl ), d, ppm: 198.92, 165.51,
2
3
incubation was finished, the medium was removed from the
wells. DMSO (150 mL) was added to each well to dissolve
the formed formazan crystals with shaking for 15 min at
room temperature. The color was recorded by determining
the optical density at 530 nm on a plate photometer (Uniplan
AIFR-01, Russia). The ratio of optical densities of this com-
pound concentration to the average for the control was taken
as the fraction of surviving cells.
160.91, 141.72, 126.07, 125.40, 123.72, 123.63, 114.25,
66.04, 45.72, 44.19, 42.38, 31.65, 29.83, 28.25, 26.08, 25.15,
21.15, 19.16, 18.14, 15.99.
(8R,10R,13S,14S,17R)-17-Acetoxy-17-acetyl-6,10,13-
trimethyl-2,3,8,9,10,11,12,13,14,15,16,17-dodecahydro-
1H-cyclopenta[a]phenanthren-3-yl-1-benzylpyrrolidine-
3-carboxylate (IV). A solution of acrylate III (1.000 g) in
toluene (100 mL) was stirred, cooled to 0°C under Ar,
treated sequentially with N-methoxymethyl-N-(trimethylsi-
lylmethyl)benzylamine (0.670 g) and CF CO H solution
Apoptotic activity of progesterone, megestrol acetate,
and the new mepregenol 17-acetate derivatives (III and IV)
was analyzed using a DNA comet assay. For this, an HeLa
3
2
(0.1 mL, 1 M) in CH Cl , and stirred at room temperature for
2
2

Synthesis and Cytostatic Activity
121
Fig. 1. Molecular structure of pregnane steroid III from an x-ray crystal structure analysis (CCDC 1909285).
cell suspension was prepared after incubation with the com-
pounds. HeLa cells were incubated with the steroids at
slides were placed onto an ice-cold surface and left for
10 min to harden the new layer. Then, the cover glass was
carefully removed using forceps along the edge.
5 ´ 10–5 M, which was equal to one half of IC for steroid
50
Lysis and gel electrophoresis. Cover glasses with two
agarose layers were placed into a 2-L flask, treated with pre-
viously cooled lysing buffer, capped with foil, and placed
into a refrigerator (³ 1 h but <24 h). Then, the glasses were
removed. The liquid was allowed to drain off at an angle.
The slides were placed on the surface of a horizontal electro-
phoresis chamber (Khelikon, Russia). The slides should lay
perpendicular to the flask edge with the rough edge toward
the cathode; the smooth, toward the anode. The chamber was
filled with electrophoresis solution to 3 – 4 mm above the
slides. Also, the slides should not change positions in the
chamber. First, the slides were stored for 20 min in alkaline
III as the most active compound, and with cyclophosphami-
de (CPS) at 20 mM (Baxter, Germany) as a positive control.
The incubation time was 20 h. The incubation medium was
decanted. Each tube was rinsed with phosphate-buffered sa-
line (PBS, 5 – 10 mL, pH 7.4; PanEco, Russia) and treated
with trypsin solution (1 mL, 0.25%) while shaking the tube.
The liquid was decanted. Cells were removed by DMEM
medium (5 mL) with 10% FBS by tapping on the sides of the
tube.
Preparation of gel slides. A solution (1%) of universal
agarose was prepared on a water bath (<65°C) until fully
liquified. A clean slide was held with forceps or by the sides.
The first layer was deposited. Agarose (150 mL) was pipetted
onto the edge of the glass opposite the rough or previously
labeled edge. The gel was spread by the pipette tip over the
whole smooth surface and 3 – 5 mm of the rough surface.
The prepared glasses were placed on a hot plate to solidify
the first agarose layer. The resulting gel-slides were stored in
a dark dry place.
solution (300 mM NaOH, 1 mM Na EDTA, pH > 13) with-
2
out switching on the electrophoresis apparatus. The electro-
phoresis lasted 20 min at field potential 1 V/cm and current
300 mA. When the procedure was finished, the slides were
removed with forceps, allowed to drain at an angle, placed
into a preliminary fixing solution for ³ 15 min. The prepara-
tions were dried at room temperature for 1 – 2 h and stored in
a dark dry place until analysis.
Preparation of microtubes. A solution of low-melting
agarose (1%) was prepared in PBS on a water bath (<42°C).
The resulting mixture was aliquoted (240 mL) into Eppendorf
microcentrifuge tubes that were placed into a Gnom
microthermostat (Russia) at 42°C to prevent solidification of
the mixture. The microcentrifuge tubes with agarose gel
were treated with previously prepared cell suspension
(60 mL) and mixed 2 – 3 times with the pipette. The resulting
mixture (60 mL) was placed onto the central part of a slide
and immediately covered with a cover glass. The prepared
Staining preparations using intercalating stain SYBR
Green I and analysis of the results. A working solution of
SYBR Green I (140 mL; Syntol, Russia) in TE-buffer (1´,
10 mM Tris, 1 mM EDTA, pH 8.0; AppliChem, Spain) was
placed onto half of a slide and stained for 20 min. The slides
were analyzed on an epi-fluorescence microscope (Axiostar,
Zeiss, Germany) at 200´ magnification. The DNA content in
the comet head (%) and in the comet tail (TDNA, Tail DNA,
%); the tail length (mm); the tail moment (product of comet

122
T. A. Fedotcheva et al.
à – III
b – IV
c – Progesterone
d – Megestrol acetate
e – Negative control
f – Positive control, CPS
Fig. 2. Estimate of DNA damage at the single-cell level of HeLa-ESC after incubation with mepregenol derivatives at concentrations equal to
their IC values for 20 h. Note: negative control, native cells; positive control, cells incubated with cyclophosphamide (CPS) (20 mM).
50
length and TDNA, %); Olive tail moment (OTM, product of
TDNA in % and the distance between the center of the comet
head and center of gravity of the comet tail). Slides were ana-
(III) was established by an x-ray crystal structure analysis
(Fig. 1). The complete crystallographic parameter set for III
was deposited in the Cambridge Crystallographic Data Cen-
tre under identifier No. CCDC 1909285. Compound III
could be further modified at the C3-position of the pregnane
scaffold, primarily via cycloaddition, because of the acti-
vated double bond. Acrylate III underwent 1,3-dipolar
cycloaddition with an azomethine ylide generated in situ
from N-methoxymethyl-N-(trimethylsilylmethyl)benzylami-
ne (Scheme 1) [8]. Oligopeptides constructed of monomeric
fragments of functionalized pyrrolidine-3-carboxylic acids
were found to have high antiproliferative activity with re-
spect to hormone-resistant prostate cancer cell lines [9, 10].
As a result, the ester of N-benzylpyrrolidine-3-carboxylic
acid (IV) was obtained in good yield.
lyzed using the CASP 1.2.2 program. IC values were calcu-
50
lated using Excel software as before [6].
RESULTS AND DISCUSSION
Scheme 1 illustrates the syntheses of the studied com-
pounds. The starting compound was megestrol 17-acetate
(I), which was selectively reduced at the pregnane backbone
C3-position by NaBH [7]. In contrast with methods reported
4
in the scientific and patent literature for reducing pregnane
steroid I, mepregenol 17-acetate (II or AMOL) was prepared
in a medium without alcohol solvents, which produced spec-
trally and chromatographically pure product using precipita-
The purities of the new mepregenol 17-acetate esters III
and IV (>98%) were confirmed by elemental analyses.
HeLa cells were incubated for 14 d in the presence of
estradiol (10–8 M) to produce HeLa-ESC. Survival of control
tumor cells with estradiol stimulation was 22.5% greater
(p < 0.05) than for the basal proliferation control. This indi-
tion from the reaction mixture by H O without additional pu-
2
rification. The C3-b-OH was acylated by carboxylic acid
chlorides in the presence of a tertiary amine to produce
mepregenol 17-acetate 3-acrylate (III) (Scheme 1). The mo-
lecular structure of the new ester of mepregenol 17-acetate
TABLE 1. Half-maximum Steroid Concentrations IC (mM) Inhibiting by 50% Viability of HeLa and HeLa-ESC Cells After 72-h Incubation
50
Steroid
Cell culture
progesterone
480 ± 20
megestrol acetate
130 ± 30
III
IV
HeLa IC50, mM
30 ± 1.03
9.1 ± 0.8
200 ± 26
180 ± 0.7
HeLa-ESC IC50, mM
5.6 ± 0.73
115 ± 5

Synthesis and Cytostatic Activity
123
O
O
CH3
CH3
CH3
CH3
OCOCH3
OCOCH3
NaBH4
CH3
H
CH3
H
H
H
H
H
THF / H2O
HO
O
II
I
CH3
CH3
97%
Megestrol 17-acetate
Mepregenol acetate (AMOL)
acryloyl chloride
O
Et3N/CH2Cl2
CH3
CH3
73%
OCOCH3
CH3
H
O
CH3
CH3
O
H
H
OCOCH3
TMS
MeO
N
O
CH3
H
CH3
O
N
H
H
O
CF3CO2H/toluene
CH3
IV
88%
III
Scheme 1. Preparation of the new mepregenol 17-acetate esters.
cated that estradiol stimulated tumor cell growth and that the
cell culture met the requirements for biotest systems for ana-
lyzing hormone activity.
breast cancer or endometrial cancer at a dose of 160 mg de-
spite its moderate cytotoxicity [11].
The antitumor effect of Megace was related to inhibition
of gonadotropin synthesis by the pituitary gland followed by
reduced estrogen synthesis. This gestagen blocks estradiol
from stimulating proliferation at the receptor level in hor-
mone-dependent breast cancer cells and also has local effects
on cancerous cells by transforming actively growing stroma
into highly differentiated decidua cells [12].
Table 1 presents results for the cytostatic activities of the
steroids.
Table 1 demonstrates that the mepregenol 17-acetate de-
rivatives, in contrast to progesterone and megestrol acetate,
inhibited the survival of native and HeLa-ESC cells whereas
megestrol acetate and IV were significantly inferior to them
in cytotoxicity with respect to HeLa-ESC. Compound III
had the most potent cytotoxicity with respect to both cultures
while megestrol acetate and IV had weak activity.
A preparation based on megestrol acetate, Megace, was
indicated for palliative treatment of inoperable or metastatic
The mechanism of cytotoxicity of III in vitro could be
related to the DNA-damaging action of the steroid. As a rule,
DNA in cells dying of apoptosis undergoes internucleosomal
degradation, which is a characteristic marker of the process.
Apoptotic cells appear as weakly fluorescing DNA-comets
TABLE 2. Quantitative Estimates of DNA-damaging Effects of Steroids on HeLa Cell Culture
DNA content, %
Olive tail moment
(OTM)
Compound
Tail moment (TM)
Comet length, mm
in comet head
96.2 ± 2.1
in comet tail
3.8 ± 0.7
Progesterone, 5 mM
Megestrol acetate, 5 mM
Cyclophosphamide, 20 mM
III, 5 mM
43.35 ± 4.63
49.12 ± 4.1
174 ± 10.5**
84.57 ± 9.6**
46.2 ± 1.3
0.04 ± 0.01
0.06 ± 0.02
60.5 ± 10.21*
16.3 ± 2.2*
1.05 ± 0.011
0.03 ± 0.01
0.2 ± 0.06
0.11 ± 0.03
45.2 ± 7.3*
11.8 ± 3.62*
2.17 ± 0.04
0.15 ± 0.04
91.04 ± 4.32
49.15 ± 12.01*
58.3 ± 10.3**
97.4 ± 2.44
8.96 ± 4.32
50.85 ± 12.01*
41.7 ± 10.3**
2.6 ± 2.44
IV, 5 mM
Control
98.71 ± 1.36
1.06 ± 0.54
40.13 ± 2.75
*
Parameter statistically significantly different from the negative control (p < 0.05) (DMEM medium with 0.1% DMSO); ** p < 0.01.

124
T. A. Fedotcheva et al.
a – III
b – IV
c – Progesterone
d – Megestrol acetate
e – Negative control
f – Positive control, CPS
Fig. 3. Estimate of DNA damage at the single-cell level of HeLa-ESC after incubation with mepregenol derivatives at concentrations equal to
their IC values for 20 h. Note: negative control, native cells; positive control, cells incubated with cyclophosphamide (CPS) (20 mM; Baxter,
Germany).
50
with a broad diffuse tail and practically no head. The DNA
comet assay is useful for detecting apoptotic cells in popula-
tions, especially when only a small amount of sample is
available. The DNA comet assay produced information not
only about the DNA content in apoptotic cells but also about
the sizes of DNA fragments released from cells through the
action of an electrical field [13].
control in the DNA comet assay (Figs. 2 and 3, Tables 2 and
3) [14].
Figures 2 and 3 demonstrate that III had the greatest
DNA-damaging effects on both cultures whereas IV and
megestrol acetate practically did not form comets. The
DNA-damaging effects of the steroids (Tail moment, TM)
for III and IV in the DNA comet assay correlated negatively
The mechanism of the potent cytotoxicity of III with re-
spect to HeLa and HeLa-ESC was related to apoptosis induc-
tion according to the increased percent contents of DNA in
the comet tail in the presence of a steroid as compared to the
with their IC values. For example, the correlation coeffi-
50
cient r for HeLa culture was 0.63; for HeLa-ESC, 0.5. Thus,
III possessed potent cytotoxicity, the mechanism of which
was related to a DNA-damaging apoptotic effect.
TABLE 3. Quantitative Estimates of DNA-damaging Effect of Steroids on HeLa Cell Culture Stimulated by Estradiol (HeLa-ESC)
DNA content, %
Comet length,
Tail moment
(TM)
Olive tail moment
(OTM)
Compound
mm
in comet head
98.4 ± 1.6
in comet tail
1.6 ± 1.6
Progesterone, 5 mM
Megestrol acetate, 5 mM
Cyclophosphamide, 20 mM
III, 5 mM
41.4 ± 2.2
47.2 ± 5.2
0.06 ± 0.02
0.055 ± 0.03
62.5 ± 10.21*
18.5 ± 3.4*
0.99 ± 0.02
0.05 ± 0.012
0.16 ± 0.08
0.09 ± 0.02
50.5 ± 4.6*
13.6 ± 2.3*
3.1 ± 0.05
97.2 ± 0.5
2.8 ± 0.5
45.5 ± 9.5*
54.2 ± 11.5**
97.5 ± 1.5
54.5 ± 9.5*
45.8 ± 11.5**
2.5 ± 1.5
180 ± 13.6**
86.64 ± 10.2**
47.5 ± 3.2
IV, 5 mM
Control
99.01 ± 0.4
0.99 ± 0.4
44.1 ± 1.5
0.18 ± 0.03
*
Parameter statistically significantly different from the negative control (p < 0.05) (DMEM medium with 0.1% DMSO); **p < 0.01.

Synthesis and Cytostatic Activity
ACKNOWLEDGMENTS
125
0023-1134-2018-52-3-17-20; Pharm. Chem. J., 52(3),
205 – 208 (2018); doi: 10.1007 / s11094-018-1791-8.
6. D. S. Stepanova, J. Chernoff, and N. L. Shimanovskiy, Pharm.
The work was sponsored by the Russian Foundation for
Basic Research (Projects No. 19-015-00195-a and No.
18-33-012-60).
Chem. J., 48(12), 777 – 782 (2015); doi: 10.1007
s11094-015-1194-z.
/
7. G. M. Kadatskii, G. S. Grienko, A. I. Terekhina, et al.,
Khim.-farm. Zh., 13(6), 63 – 68 (1979).
8. Y. Terao, H. Kotaki, N. Imai, et al., Chem. Pharm. Bull., 33(2),
896 – 898 (1985).
REFERENCES
9. K. V. Kudryavtsev, C. C. Yu, P. M. Ivantcova, et al., Chem.
1. T. A. Fedotcheva and N. L. Shimanovskii, Probl. Endokrinol.,
64(1), 54 – 61 (2018); doi: 10.14341 / probl201864154 – 61.
2. T. A. Fedotcheva, A. G. Kruglov, V. V. Teplova, et al., Biofizika,
57(6), 1014 – 1019 (2012); T. A. Fedotcheva, A. G. Kruglov,
V. V. Teplova, et al., Biophysics, 57(6), 792 – 795 (2012); doi:
10.1134 / S0006350912060061.
Asian J., 10(2), 383 – 389 (2015); doi: 10.1002
asia.201403171.
/
10. M.-L. Chan, C.-C. Yu, J.-L. Hsu, et al., Oncotarget, 8(57),
96668 – 96683 (2017); doi: 10.18632 / oncotarget.18040.
11. E. Kalogera, S. C. Dowdy, and J. N. Bakkum-Gamez, Int. J.
Women’s Health, 6, 691 – 701 (2014); doi: 10.2147 / IJWH.
S47232.eCollection 2014.
3. O. A. Zeinalov, V. V. Yaderets, T. S. Stytsenko, et al.,
Khim.-farm. Zh., 46(4), 7 – 10 (2012).
12. https: // www.drugs.com / monograph / megestrol-acetate.html,
in a monograph: American Society of Health-System Pharma-
cists, Drugs.com database, USA (2019).
4. O. A. Zeinalov, V. A. Andryushina, and K. G. Skryabin, Ross.
Khim. Zh. (Zh. Ross. Khim. Ob-va im. D. I. Mendeleeva), 49(1),
118 – 124 (2005).
13. U. B. Sorochinskaya and V. M. Mikhailenko, Onkologiya,
10(3), 303 – 309 (2008).
5. V. V. Knyazev, V. S. Rogovskii, and E. D. Sveshnikova,
Khim.-farm. Zh., 52(3), 17 – 20 (2018); doi: 10.30906 /
14. E. A. Kuznetsova, N. P. Sirota, and T. A. Zenchenko, Biofizika,
64(3), 457 – 467 (2019); doi: 10.1134 / S0006302919030050.