Novel steroidal 1,3,4-thiadiazines: Synthesis and biological evaluation in androgen receptor-positive prostate cancer 22Rv1 cells

Article abstract of DOI:10.1016/j.bioorg.2019.103142

A flexible approach to previously unknown spirofused and linked 1,3,4-thiadiazine derivatives of steroids with selective control of heterocyclization patterns is disclosed. (N-Arylcarbamoyl)spiroandrostene-17,6′ [1,3,4]thiadiazines and (N-arylcarbamoyl)17-[1′,3′,4′]thiadiazine-substituted androstenes, novel types of heterosteroids, were prepared from 16β,17β-epoxypregnenolone and 21-bromopregna-5,16-dien-20-one in good to high yields by the treatment with oxamic acid thiohydrazides. The synthesized compounds were screened for antiproliferative activity against the human androgen receptor-positive prostate cancer cell line 22Rv1. Most of (N-arylcarbamoyl)17-[1′,3′,4′]thiadiazine-substituted androstenes exhibit better antiproliferative potency (IC₅₀ = 2.1–6.6 μM) than the antiandrogen bicalutamide. Compounds 7d with IC₅₀ = 3.0 μM and 7j with IC₅₀ = 2.1 μM proved to be the most active in the series under study. Lead synthesized compound 7j downregulates AR expression and activity in 22Rv1 cells. NF-κB activity is also blocked in 7j-treated 22Rv1 cells. Apoptosis is considered as a possible mechanism of 7j-induced cell death.

Full text of DOI:10.1016/j.bioorg.2019.103142

Accepted Manuscript
Novel steroidal 1,3,4-thiadiazines: synthesis and biological evaluation in an-
drogen receptor-positive prostate cancer 22Rv1 cells
Anna S. Komendantova, Alexander M. Scherbakov, Alexander V. Komkov,
Viktoriya V. Chertkova, Alexey O. Gudovanniy, Elena I. Chernoburova, Danila
V. Sorokin, Yaraslau U. Dzichenka, Valerii Z. Shirinian, Yulia A. Volkova, Igor
V. Zavarzin
PII:
S0045-2068(19)30697-2
https://doi.org/10.1016/j.bioorg.2019.103142
103142
DOI:
Article Number:
Reference:
YBIOO 103142
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 May 2019
17 July 2019
19 July 2019
Please cite this article as: A.S. Komendantova, A.M. Scherbakov, A.V. Komkov, V.V. Chertkova, A.O. Gudovanniy,
E.I. Chernoburova, D.V. Sorokin, Y.U. Dzichenka, V.Z. Shirinian, Y.A. Volkova, I.V. Zavarzin, Novel steroidal
1,3,4-thiadiazines: synthesis and biological evaluation in androgen receptor-positive prostate cancer 22Rv1 cells,
Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103142
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Novel steroidal 1,3,4-thiadiazines: synthesis and biological evaluation in
androgen receptor-positive prostate cancer 22Rv1 cells
Anna S. Komendantova,a Alexander M. Scherbakov,b,* Alexander V. Komkov,a Viktoriya V.
Chertkova,a Alexey O. Gudovanniy,a Elena I. Chernoburova,a Danila V. Sorokin,b Yaraslau U.
Dzichenka,c Valerii Z. Shirinian,a Yulia A. Volkova,a,* Igor V. Zavarzina
a
N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 47 Leninsky
prosp., 119991 Moscow, Russia
b
Department of Experimental Tumor Biology, N.N. Blokhin National Medical Research Center
of Oncology, 24 Kashirskoe shosse, 115522 Moscow, Russia
с
Institute of Bioorganic Chemistry of NAS of Belarus, Laboratory of Protein Engineering,
Academician V.F. Kuprevich str. 5/2, Minsk, Republic of Belarus
ABSTRACT
A flexible approach to previously unknown spiro-fused and linked 1,3,4-thiadiazine derivatives
of steroids with selective control of heterocyclization patterns is disclosed. (N-
Arylcarbamoyl)spiro-androstene-17,6΄[1,3,4]thiadiazines
and
(N-arylcarbamoyl)17-
[1΄,3΄,4΄]thiadiazine-substituted androstenes, novel types of heterosteroids, were prepared from
16β,17β-epoxypregnenolone and 21-bromopregna-5,16-dien-20-one in good to high yields by
the treatment with oxamic acid thiohydrazides. The synthesized compounds were screened for
antiproliferative activity against the human androgen receptor-positive prostate cancer cell line
22Rv1. Most of (N-arylcarbamoyl)17-[1΄,3΄,4΄]thiadiazine-substituted androstenes exhibit better
antiproliferative potency (IC50 = 2.1–6.6 µM) than the antiandrogen bicalutamide. Compounds
7d with IC50 = 3.0 μM and 7j with IC50 = 2.1 μM proved to be the most active in the series under
study. Lead synthesized compound 7j downregulates AR expression and activity in 22Rv1 cells.
NF-κB activity is also blocked in 7j-treated 22Rv1 cells. Apoptosis is considered as a possible
mechanism of 7j-induced cell death.
Br
O
NHR
O
O
Lead compounds
S
N
O
N
N
N
7d
, R = CH2Ph,
IC50 (22Rv1) = 2.1 mkM
7j
NHR
S
steroid core
S
steroid core
H2N
NHR
O
H
OH
N
H
, R = p-MeOC6H4,
IC50 (22Rv1 ) = 3.0 mkM
H
O
H
H
H
H
HO
R = C6H5, p-MeC6H4, m-MeC6H4,
p-EtC6H4, p-OMeC6H4, o-FC6H4,
p-ClC6H4, p-BrC6H4, p-CF3C6H4,
o-NO2C6H4, CH2C6H4
AcO
6 examples
51-63%
10 examples
65-90%
1

Keywords:
steroids; heterosteroids; 1,3,4-thiadiazine; hydrazones; antiproliferative activity; prostate cancer;
apoptosis, androgen receptor
2

1. Introduction
Extranuclear heterosteroids have been considered over the years to be a privileged scaffold for
drug discovery due to their outstanding biological activity, which is especially true for D-ring
modified steroids [1-8]. Thus, the naturally occurring steroids cardenolides (i.e. digoxin,
ouabain) and bufadienolides (i.e. bufalin, marinobufogenin) are inhibitors of the plasma
membrane Na+, K+-ATPase and are of interest as potential drugs for cardiac failure and certain
types of arrhythmias [9, 10]. Spirostan saponins display hemolytic activity and high cytotoxicity
[11, 12]. Moreover, synthetic heterosteroids encompass a wide range of compounds with various
biological activities, e.g., reductase inhibitors such as finasteride [13], high-affinity agonist
ligands for the glucocorticoid receptor, e.g., cortivazol [14], neuromuscular junction blocking
agents such as rocuronium and pipecuronium [15], the anti-inflammatory and
immunosuppressive drug hydrocortamate [16], anticancer agents such as abiraterone [17].
Our interest in the preparation of D-ring heterosteroids [18, 19] suggested a need for a facile
flexible strategy, in which a common intermediate can be used in a conjunctive fashion to form
an array of structurally diverse N-heterocycles attached or fused to a steroid core. In this regard,
we studied oxamic acid thiohydrazides as simple versatile agents for the modification of steroids
bearing a carbonyl group. In contrast to classical hydrazones, three major Lewis structural
formulas provided by an extra C=S group contribute to the real structure of hydrazones of
oxamic acid thiohydrazides, thereby efficiently extending the number of possible chemical
transformations (Figure 1). The Lewis structural formulas of thione A and thiol B are defined by
the highly active competitive NH- and SH-nucleophilic sites, while the additional cyclic
thiadiazoline structure C is characterized by facilitated C-N bond oxidation. Structure B also can
be considered as 2,3-diazapolyene synthon.
3

Reactivity of monothiohydrazide hydrazones
CR2R1
CR2R1
S
N
N
R3
NH
R1
N
HN
R2
S
SH
R3
HN
O
N
O
R3
N
O
N
H
H
cleavable bond
C
SH-nucleophilic site
B
NH-nucleophilic site
A
this work
NHR
NHR
S
O
a)
O
electrophilic
O
N
N
HO
O
S
site
HN
O
NH2NHCSCONHR
N
-H2O
steroid core
steroid core
steroid core
NHR
b)
H
H
O
Br
O
S
S
NHR
HN
electrophilic
site
N
NH2NHCSCONHR
-H2O
N
N
O
-HBr
Br
steroid core
steroid core
steroid core
Fig. 1. Reactivity and application of monothiohydrazide hydrazones in the synthesis of
extranuclear heterosteroids
In this context, thiohydrazides serve as synthetic building blocks for diverse five- and six-
membered O,N,S-heterocycles, including such widely used pharmacophores as pyridazines [20],
1,3,4-thiadiazoles [21], and 4,5-dihydro-1,3,4-thiadiazoles [22]. In particular, monothiohydrazide
hydrazones are employed in the synthesis of steroidal pyrazolines [23], pyridazines [19], and
1,3,4-thiadiazoles [24].
Herein, we report the synthesis of unique derivatives of androstene series containing a 1,3,4-
thiadiazine motif using oxamic acid thiohydrazides (Figure 1). The relative spatial arrangement
of the electrophilic reaction site and the carbonyl moiety in the steroid structure defines the
reaction product. Spiro-androstene-17,6΄[1΄,3΄,4΄]thiadiazines were exclusively synthesized
employing 16β,17β-epoxypregnenolone (Figure 1, line a). Using 21-bromopregna-5,16-dien-20-
one as a substrate, 17-[1΄,3΄,4΄]thiadiazine-substituted androstenes were prepared (Figure 1, line
b). Their inhibitory activity was evaluated against human androgen receptor-positive prostate
cancer cells 22Rv1. Effects of two lead compounds on signaling pathways were analyzed.
2. Results and discussion
2.1. Chemistry
Based on our previous results [23] and the chemistry of hydrozones [25], we could envision the
possibility of assembling new steroidal pyrazole derivative 4 from 16β,17β-epoxypregnenolone 1
4

by the classical nucleophilic oxirane ring opening with an NH-nucleophile followed by the
dehydration driven by aromatization. However, by serendipity, we found that hydrazone 3a
generated from oxirane 1 and oxamic acid thiohydrazide 2a chemoselectively react with a sulfur
nucleophile, thus providing an approach to the enantiopure synthesis of previously unknown
16β-hydroxy-spiro-androstene-17,6΄[1,3,4]thiadiazines 5a (Scheme 1).
S
H
NH
N
N
O
O
O
O
H
H
acid
H
H
H
H
H
H
HO
HO
3a
1
+
NH2NHCSCONHPh
2a
-H2O
N
N
N
N
S
H
NH
S
H
H
O
OH
O
H
NH
HO
H
H
5a
4
HO
Scheme 1. Synthesis of spiro[1,3,4]thiadiazine 5a.
To study the scope of this approach, we performed the reaction of thiohydrazides 2a-f containing
both electron-donating and -withdrawing substituents on the benzoyl group with 16β,17β-
epoxypregnenolone 1 (Scheme 2). The reaction of thiohydrazides 2 with oxirane 1 in
o
1,4-dioxane in the presence of a catalytic amount of H2SO4 at 60 C gave, after 2 hours, the
corresponding spiro(androstene thiadiazines) 5 in good yield with virtually complete
stereoselectivity. Other solvents, such as ethanol or acetic acid, at variable temperatures were not
so effective (the reactions afforded complex mixtures). The use of 30 mol % TsOH, TFA, or
BF3*Et2O did not lead to the target products in higher yields. Product 5a derived from phenyl-
substituted thiohydrazide was obtained in 62% yield.
5

O
N
N
O
NH2NHCSCONHR
O
H
2a-f
S
NHR
OH
H
30 mol% H2SO4
dioxane
H
H
H
HO
1
5a-f
60 oC, 2h
MeO
N
N
N
N
N
N
NH
NH
NH
S
S
S
O
O
O
OH
OH
OH
H
H
H
5a
, 62%
5b
, 55%
5c
, 58%
F
Cl
F3C
N
N
N
N
N
N
NH
NH
NH
S
S
S
O
O
O
OH
OH
OH
5f
H
H
H
5d
5e
, 63%
, 51%
, 56%
Scheme 2. Synthesis of 16β-hydroxyspiro-androsteno-17,6΄[1,3,4]thiadiazines 5.
The investigation of the substrate scope by employing thiohydrazides 2 with electron-donating
and -withdrawing groups in the reaction showed that the electronic effects of substituents on the
aryl ring are not pronounced. Methyl, methoxy, fluoride, chloride, and trifluoromethane
derivatives 5b-f were obtained in 51–63% yields, respectively.
The solution-state structures of compounds 5d-f were derived by means of detailed NMR
studies, such as 2D NOESY, 1H–1H COSY, 13C–1H HMBC, 13C–1H RSQC, 1H–1H TOCSY, and
J-coupling constants (see the Supporting material).
Further, we made an attempt to generate novel D-ring androstene derivatives by treating 21-
bromopregna-5,16-dien-20-one 6 with oxamic acid thiohydrazides 2 under mild basic conditions
(Scheme 3). Having so far observed a seemingly general transformation pattern, we found that
the reaction affords 17-(6΄H-1΄,3΄,4΄-thiadiazine-2΄-carboxamide)androst-5,17-dienes 7 in
virtually quantitative yield. The structural assignments were confirmed by 2D NMR (1H–1H
COSY, 13C–1H HMBC, and 13C–1H HSQC) techniques and HRMS. We tested acid
thiohydrazides 2 containing electron-donating and -withdrawing groups. The reaction showed
high chemoselectivity and functional group tolerance. Thiohydrazides 2 bearing methyl, ethyl,
methoxy, fluoride, chloride, bromide, trifluoromethane, and nitro groups provided pyridazines
6

7a-i in high yields. Moreover, (N-benzylcarbamoyl)-17-[1΄,3΄,4΄]thiadiazine-substituted
androstene 7j was obtained in 79% yield.
O
NHR
Br
S
O
N
N
NH2NHCSCONHR
2a-k
H
1 equiv K2CO3
MeOH
25 oC, 6 h
H
H
H
AcO
6
7a-j
H
N
O
H
N
H
N
O
O
S
S
S
N
N
N
N
R
OMe
N
N
7a
, 85%
7d
, 90%
7b
7c
, R = Me, 73%
, R = Et, 76%
H
H
H
H
N
H
N
H
N
F
O
O
O
S
S
S
N
N
N
Cl
Br
N
N
N
7e
, 65%
7f
7g
, 88%
, 90%
H
H
H
H
N
H
N
H
N
NO2
O
O
O
Ph
S
S
S
N
N
N
CF3
N
N
N
7h
, 70%
7i
, 77%
7j
, 79%
H
H
H
Scheme 3. Synthesis of 17-(6΄H-1΄,3΄,4΄-thiadiazine-2΄-carboxamide)androst-5,17-dienes 7.
7

2.2. Biology
2.2.1. Cytotoxic effect against prostate cancer cells
Inspired by novel steroidal drugs effective in the treatment of prostate cancer [26, 27], we
evaluated the antiproliferative activity of all synthesized compounds against the human androgen
receptor-positive prostate cancer cell line 22Rv1 using the MTT assay.
In the late 90s, 22Rv1 prostate cancer cell line has been derived from a human prostatic
carcinoma xenograft, CWR22R [28]. These cells are derived from a xenograft that was serially
propagated in mice after castration-induced regression and relapse of the androgen-dependent
CWR22 xenograft. The AR mutation was detected subsequently in 22Rv1 cells, indicating their
differences from CWR22 xenograft. 22Rv1 cell line displayed both androgen-responsive and
androgen-insensitive features due, at least in part, to an insertional mutation of the AR as
described by Dr. Clifford G. Tepper in the work [29]. Summary, 22Rv1 cell line represents very
intriguing AR-positive prostate cancer model, demonstrating AR-dependent and AR-independent
growth.
The hormonal drug bicalutamide was used as the reference compound. (N-Arylcarbamoyl)spiro-
androstene-17,6΄[1,3,4]thiadiazines 5a-f showed weak activity with inhibitory concentrations
IC50 (IC50 is the half maximal inhibitory concentration) ranging between 20–25 μM. Meanwhile,
virtually all (N-arylcarbamoyl)17-[1΄,3΄,4΄]thiadiazine-substituted androstenes 7 exhibited
significant antiproliferative potency. The corresponding inhibitory concentrations of compounds
7 are enlisted in Table 1. All the tested compounds showed toxicity with IC50 lower than 10 μM;
the only exception was compound 7g with the IC50 value higher 25 μM. High toxicity was
observed for compounds 7a (IC50 = 3.1 μM) and 7d (IC50 = 3.0 μM) with phenyl and 4-
methoxyphenyl substituents. Compounds 7b,c,e,f,h containing methyl, ethyl, fluorine, chlorine,
or trifluoromethyl groups at the aryl moiety showed toxicity with IC50 ranging between 3.7–6.6
μM. Steroidal 1,3,4-thiadiazine 7j bearing a benzyl substituent at the carboxamide moiety was
the most active compound in the series of the tested compounds with IC50 = 2.1 μM (Table 1,
entry 9). Nonsteroidal antiandrogen bicalutamide showed low antiproliferative activity with the
IC50 value higher 25 μM, indicating partial AR-independent features of 22Rv1 cells [29].
8

Table 1
Antiproliferative activity of the synthesized steroidal 1,3,4-thiadiazines 7 and the reference drug
against the human prostate cancer cell line 22Rv1.
H
O
N
R
S
N
N
AcO
entry compound
R
IC50, μM
entry compound
R
IC50, μM
1
2
3
7a
7b
7c
3.1 ± 0.3
6.1 ± 0.7
6.6 ± 0.7
6
7
8
7f
7g
7h
7j
3.7 ± 0.5
Cl
Br
> 25
Me
Et
6.2 ± 0.7
CF3
4
5
7d
7e
3.0 ± 0.3
6.6 ± 0.7
9
2.1 ± 0.3
> 25
OMe
F
10
Bicalutamide
2.2.2. Effects of compounds 7d and 7j on the signaling pathways in 22Rv1 cells
Two most active compounds were selected for detailed studies. Androgen receptor is a well-
known driver of the hormone-dependent prostate cancers. 22Rv1 cells harbor two AR forms, a
full one and a C-terminally truncated, constitutively active form, both of which have been
investigated in various works [30-34]. We analyzed expression of full AR isoform (110 kDA) by
immunoblotting after 24-hour treatment of 22Rv1 cells with compounds 7d and 7j. Compound
7d did not cause significant changes in AR expression, whereas 7j inhibited AR expression in a
dose-dependent manner (Figure 2A).
9

(A)
(B)
Fig. 2. Immunoblotting for AR, PARP, α-tubulin, phosphorylated (ph) and unphosphorylated
NF-κB p65, Akt, ERK1/2 using lysates from 22Rv1 cells treated with the indicated
concentrations of compounds 7j and 7d. (A) 24 h treatment, (B) 48 h treatment. Densitometry
for immunoreactive proteins/α-tubulin (right diagrams) ratio was carried out using ImageJ
software with the densitometry protocol provided by The University of Queensland and the
recommendations from references [35, 36]. Density of the probes treated with vehicle control
was taken as one relative unit.
The NF-κB pathway actively supports the growth of tumor cells, including prostate cancer.
Many studies have shown that steroidal molecules can significantly affect this signaling
pathway. In the study [37] Mary Kaileh et al. showed that the leave extract of Withania
10

somnifera, as well as its major constituent steroidal lactone withaferin A, potently inhibits NFκB
pathway by preventing the tumor necrosis factor-induced activation of IκB kinase β via a
thioalkylation-sensitive redox mechanism. IκBα is a key constitutive inhibitor of NFκB activity.
It was described a pivotal role for glucocorticoids in inducing expression of IκBα to enhance the
cytosolic retention of NF-κB [38]. Dexamethasone induces the synthesis of IκBα mRNA in GR-
positive cells [39]. Corticosteroids can also partially inhibit transcriptional activation by NF-κB
but not through an increase in IκBα protein alone [40]. Analysis of NF-κB p65 and its
phosphorylated (ph) form using immunoblotting after 24-hour treatment with compounds 7d and
7j revealed that 7j inhibited phNF-κB p65 in dose-dependent manner, while 7d had no effects on
this protein. Both compounds did not block Akt phosphorylation and expression (Figure 2A).
The extracellular signal-regulated kinase (ERK) signaling pathway plays a crucial role in cell
growth and death. Depending on stimulus, ERK activity mediates different antiproliferative
events, such as apoptosis and autophagy [41-43]. Chemotherapeutic agents and DNA damage
stimuli, including etoposide, doxorubicin, ionizing irradiation, and ultraviolet irradiation (UV)
activate ERK1/2 protein kinase in various cells [41, 44]. TNFα plays interesting role in balance
between proliferation, survival and apoptosis. It was found out, that TNFα induces apoptosis or
supports survival through the FLIP-L-dependent activation of the ERK pathway [45-49].
Opposite and diverse effects of TNF-α and ERKs in malignant and normal cells are reported in
various works, showing intricate mechanisms underlying cell death [49, 50]. Moreover,
compounds with hormonal activity, including estradiol, tamoxifen, apigenin, and quercetin also
influence on cell death via ERK1/2 modulation [41, 43]. In our study ERK1/2 activation was
analyzed in 22Rv1 cells using immunoblotting. Both compounds showed high ERK1/2
phosphorylation (Figure 2A).
Interestingly, ERK1 was maximally activated by compound 7j at 2.5 µM concentration, whereas
at higher concentrations 7j was less active. Compound 7d caused increase in phERK1/2
expression in a dose-dependent manner. Thus, compound 7j has effects on ERKs activity
together with impact on AR and phNF-κB p65 expression, while only ERKs are involved in the
cell response to compound 7d.
To prove the effects of compound 7j on AR and NF-κB pathways additional experiments were
performed using gene reporter analysis. The luciferase reporter assay is commonly used as a tool
to study activity of transcription factors. In our study we carried out experiments with plasmids
containing luciferase gene under AR- or NF-kB-dependent promoter. To activate AR signaling
10 nM DHT was used. As shown in Figure 3A, compound 7j significantly inhibited DHT-
11

induced AR activity. Moreover, NF-kB activity was also blocked by compound 7j at 2 µM
concentration (Figure 3B). Targeting NF-κB signaling may restore sensitivity of castrate-
resistant prostate cancer cells to antiandrogens. Artemisinin derivatives, semisynthetic
compounds derived from Artemisia annua, are approved first-line antimalarial drugs. Dr. Jessica
J. Nunes and co-authors showed, that artesunate, clinically important artemisinin derivative, in
combination with bicalutamide inhibits NF-κB signaling and decreases AR and/or mutant AR
expression via ubiquitin-mediated proteasomal degradation [51].
Compound 7j exhibits influence on AR and NF-κB activity, as well as on NF-κB p65
phosphorylation and AR expression. Thus, targeting NF-κB signaling holds great promise for
therapies of hormone-dependent or castrate-resistant prostate cancers.
(A)
(B)
Fig. 3. Gene reporter analysis. Plasmids containing luciferase gene under AR- or NF-κB-
dependent promoter were used. (A) AR-mediated luciferase activity induced by DHT treatment
was assessed after incubation 22Rv1 cells with the indicated doses of compound 7j, * p < 0.05 –
versus 22Rv1 cells treated with 10 nM DHT alone. (B) NF-κB-mediated luciferase activity was
analyzed without additional inducer,* p < 0.05 – versus 22Rv1 cells treated with vehicle control.
12

Cleaved PARP is an apoptotic marker, which can be detected by immunoblotting. PARP
cleavage was analyzed after 48 h incubation with compounds 7j,d. As shown in Figure 2B, both
compounds induced PARP cleavage at 2.5 µM concentration. Thus, compounds 7j,d elicit
apoptosis in the AR-positive prostate cancer 22Rv1 cells.
3. Conclusion
This study demonstrates the utility of the modified strategy of hydrazone cyclization for
providing easy access to a variety of steroidal products containing functionalized D rings starting
from 16,17-epoxy/21-bromo-20-keto steroids and oxamic acid thiohydrazides as readily
available precursors. Due to operational simplicity, these protocols may be useful in
pharmacological research for chemoselective transformations of other complex substrates
bearing a simple carbonyl group. The evaluation of cytotoxic activity of the newly synthesized
compounds against the human androgen receptor-positive prostate cancer cells 22Rv1 showed
that 17-[1΄,3΄,4΄]thiadiazine-substituted androstenes display higher cytotoxicity than the spiro-
androstene-17,6΄[1,3,4]thiadiazines and the reference drug. Compounds 7d with IC50 = 3.0 μM
and 7j with IC50 = 2.1 μM proved to be the most active in the series under study. Our data
suggest that the modulation of the AR, NF-κB, ERK1/2, PARP pathways without decreasing Akt
phosphorylation is involved in compound 7j-induced 22Rv1 cells death. Targeting AR and NF-
κB by novel steroidal 1,3,4-thiadiazines are very promising but future preclinical and clinical
trials should confirm initial experimental observations. This work provides evidence that
steroidal 1,3,4-thiadiazines are lead compounds for developing novel and highly effective
anticancer drugs for treatment of prostate cancer.
4. Experimental section
4.1. Chemistry
4.1.1. General Information
NMR spectra were acquired on Bruker Avance 600, 500, 300 spectrometers at room
temperature; the chemical shifts δ were measured in ppm relative to the solvent (1Н: CDCl3, δ =
13
7.27 ppm, DMSO-d6, δ = 2.50 ppm; C: CDCl3, δ = 77.00 ppm, DMSO-d6, δ = 39.50 ppm).
Splitting patterns are designated as s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd,
double doublet; ddd, double double doublet. The coupling constants (J) are in Hertz. The
structures of all compounds were established using 1D NMR (1H, 13C, JMOD) and 2D NMR
1
1
(1H-1H COSY, H-1H TOCSY, H-1H ROESY, 13C-1H HMBC, 13C-1H HSQC, NOESY)
spectroscopy. High-resolution and accurate mass spectra were obtained on BrukermicrOTOF-
13

QTM ESI-TOF (Electrospray Ionization/Time of Flight) and Thermo Scientific* LTQ Orbitrap
mass spectrometers. Melting points (mp) are uncorrected and were measured on a Boetius
capillary melting point apparatus. Analytical thin layer chromatography (TLC) was carried out
on silica gel plates (silica gel 60 F254 aluminum supported plates); the visualization was
accomplished with an UV lamp (365 nm) and using chemical staining with [Ce(SO4)2/H2SO4].
Column chromatography was performed on silica gel 60 (230-400 mesh, Merck). Synthesis of
16β,17β-epoxy-17-isopregn-5-en-3β-ol-20-one (1), 3β-acetoxy-21-bromopregn-5,16-dien-20-one
(6), and oxamic acids thiohydrazides 2 described in Supporting Information. All reactions were
carried out using freshly distilled and dry solvents.
4.1.2. General procedure for the preparation of 5’-methyl-[2΄-(N-arylcarbamoyl)]-3β,16β-
dihydroxy-spiroandrost-5-ene-17,6΄[1,3,4]thiadiazines 5a-f
Sulfuric acid (20 µL, 30 mol%) was added to a solution of 16β,17β-epoxy-17-isopregn-5-en-3β-
ol-20-one (330 mg, 1 mmol) and oxamic acid thiohydrazide (1.2 mmol) in dry dioxane (15 mL).
The reaction mixture was heated at 60 °C for 2 h, cooled to room temperature, and quenched
with water (60 mL). The organic layer was separated. The aqueous layer was extracted with
chloroform (3 × 20 mL) and washed sequentially with a 5% Na2CO3 solution (3 × 25 mL) and
water (25 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated.
The crude product was purified by column chromatography using petroleum ether/ethyl acetate.
4.1.2.1.
5’-Methyl-[2΄-(N-Phenylcarbamoyl)]-3β,16β-dihydroxy-spiro-androst-5-ene-
17,6΄[1,3,4]-thiadiazine (5a)
o
1
Colorless solid; mp 188 - 190 C; yield 62% (315 mg). H NMR (600 MHz, DMSO-d6): δ 0.79
(ddd, J = 4.94, 12.41, 15.37 Hz, 1H, 9-CH), 0.85 (s, 3H, 18-CH3), 0.87 (s, 3H, 19-CH3), 0.89-
1.07 (m, 2H, 12-CH2), 0.98-1.03 (m, 2H, 1-CH2), 1.21-1.34 (m, 2H, 11-CH2), 1.28-1.59 (m, 2H,
2-CH2), 1.34-1.37 (m, 1H, 8-CH), 1.49-1.51 (m, 1H, 15-CH2), 1.54-1.92 (m, 2H, 7-CH2), 1.78
(ddd, J = 2.30, 10.43, 11.18 Hz, 1H, 14-CH), 1.88 (dd, J = 9.42, 11.67 Hz, 1H, 15-CH2), 2.05
(dd, J = 13.31 Hz, 1H, 4-CH2), 2.12 (dd, J = 4.92, 13.31 Hz, 1H, 4-CH2), 2.47 (s, 3H, 21-CH3),
3.19-3.20 (m, 1H, 3-CH), 4.55 (d, J = 5.14 Hz, 1H, OH), 5.22-5.25 (m, 1H, 6-CH), 5.23-5.25 (m,
1H, 16-CH), 5.67 (d, J = 4.11 Hz, 1H, OH), 7.12 (dd, J =7.20 Hz, 1H, 6’-CH), 7.34 (dd, J =
7.20, 8.05 Hz, 2H, 5’-CH+7’-CH), 7.86 (d, J = 8.05 Hz, 2H, 4’-CH+8’-CH), 10.46 (br.s, 1H,
NH). 13C NMR (125 MHz, DMSO-d6): δ 14.8 (18-CH3), 19.0 (19-CH3), 20.0 (11-CH2), 23.5
(21-CH3), 31.1 (8-CH), 31.2 (7-CH2), 31.3 (2-CH2), 33.7 (15-CH2), 34.7 (12-CH2), 36.0 (10-C),
14

36.6 (1-CH2), 42.0 (4-CH2), 46.0 (14-CH), 48.9 (9-CH), 51.2 (13-C), 62.2 (17-C), 69.9 (3-CH),
72.5 (16-CH), 120.0 (6-CH), 120.4 (4’-CH+C-8’-CH), 124.1 (6’-CH), 128.6 (5’-CH+7’-CH),
137.9 (3’-C), 141.2 (5-C), 152.9 (2’-C), 154.0 (20-C), 160.0 (1’-C). HRMS (ESI) for
C29H38N3O3S ([M+H]+): calcd 508.2628, found 508.2626.
4.1.2.2. 5’-Methyl-[2΄-(N-(3-methylphenylcarbamoyl)]-3β,16β-di-hydro-xyspiroandrost-5-ene-
17,6΄[1,3,4]thiadiazine (5b)
Colorless solid; mp 190 - 195 oC; yield 55% (300 mg). 1H NMR (600 MHz, DMSO-d6): δ 0.89-
0.91 (m, 1H, 9-CH), 0.93 (s, 3H, 18-CH3), 0.94 (s, 3H, 19-CH3), 0.97-1.07 (m, 2H, 1-CH2), 1.01-
1.12 (m, 2H, 12-CH2), 1.24-1.43 (m, 2H, 11-CH2), 1.40-1.42 (m, 1H, 8-CH), 1.45-1.79 (m, 2H,
2-CH2), 1.62-1.96 (m, 2H, 7-CH2), 1.67-1.70 (m, 1H, 15-CH2), 1.97 (dd, J = 9.25, 13.36 Hz, 1H,
15-CH2), 2.05-2.08 (m, 1H, 14-CH), 2.18 (dd, J = 12.94 Hz, 1H, 4-CH2), 2.28 (dd, J = 4.00,
12.94 Hz, 1H, 4-CH2), 2.38 (s, 3H, Me), 2.57 (s, 3H, 21-CH3), 3.49-3.51 (m, 1H, 3-CH), 5.30-
5.32 (m, 1H, 6-CH), 5.34 (dd, J = 3.30, 9.84 Hz, 1H, 16-CH), 6.98 (d, J = 7.80 Hz, 1H, 6’-CH),
7.26 (t, J = 7.80 Hz, 1H, 1H, 7’-CH), 7.50 (d, J = 7.80 Hz, 1H, 8’-CH), 7.60 (s, 1H, 4’-CH), 9.23
13
(br. s, 1H, NH), signals of OH groups were not observed. C NMR (125 MHz, DMSO-d6): δ
15.2 (18-CH3), 19.2 (19-CH3), 20.3 (11-CH2), 21.5 (Me), 23.9 (21-CH3), 31.4 (2-CH2), 31.5 (7-
CH2), 31.6 (8-CH), 34.2 (15-CH2), 34.8 (12-CH2), 36.3 (1-CH2), 36.8 (10-C), 42.1 (4-CH2), 46.0
(14-CH), 48.7 (9-CH), 52.2 (13-C), 62.4 (17-C), 71.6 (3-CH), 74.3 (16-CH), 116.0 (8’-CH),
120.3 (6-CH), 120.9 (4’-CH), 125.6 (6’-CH), 129.0 (7’-CH), 137.0 (5’-C), 139.2 (3’-C), 140.8
(5-C), 153.7 (2’-C), 154.3 (20-C), 158.8 (1’-C). HRMS (ESI) for C30H39N3O3SNa ([M+Na]+):
calcd 544.2604, found 544.2598.
4.1.2.3. 5’-Methyl-[2΄-(N-(4-methoxyphenylcarbamoyl)-3β,16β-dihydro-xyspiroandrost-5-ene-
17,6΄[1,3,4]thiadiazine (5c)
Colorless solid; mp 205 – 207 oC; yield 58% (312 mg). 1H NMR (600 MHz, DMSO-d6): δ 0.79
(ddd, J = 5.14, 11.31, 12.34 Hz, 1H, 9-CH), 0.84 (s, 3H, 18-CH3), 0.87 (s, 3H, 19-CH3), 0.89-
1.07 (m, 2H, 12-CH2), 0.91-1.03 (m, 2H, 1-CH2), 1.21-1.34 (m, 2H, 11-CH2), 1.28-1.59 (m, 2H,
2-CH2), 1.33-1.37 (m, 1H, 8-CH), 1.47-1.51 (m, 1H, 15-CH2), 1.52-1.91 (m, 2H, 7-CH2), 1.78
(ddd, J = 1.83, 10.28, 11.30 Hz, 1H, 14-CH), 1.86 (dd, J = 10.28, 13.36 Hz, 1H, 15-CH2), 2.05
(dd, J = 12.34 Hz, 1H, 4-CH2), 2.12 (dd, J = 4.11, 12.34 Hz, 1H, 4-CH2), 2.46 (s, 3H, 21-CH3),
3.18-3.21 (m, 1H, 3-CH), 3.73 (s, 3H, OMe), 5.20-5.25 (m, 1H, 6-CH), 5.24 (m, 1H, 16-CH),
6.91 (d, J = 9.01 Hz, 2H, 5’-CH+7’-CH), 7.77 (d, J = 9.01 Hz, 2H, 4’-CH+8’-CH), 10.42 (br. s,
1H, NH), signals of OH groups were not observed. 13C NMR (125 MHz, DMSO-d6): δ 14.8 (18-
CH3), 19.0 (19-CH3), 20.0 (11-CH2), 23.5 (21-CH3), 31.2 (8-CH), 31.3 (2-CH2), 31.4 (7-CH2),
15

33.7 (15-CH2), 34.6 (12-CH2), 36.0 (10-C), 36.6 (1-CH2), 42.1 (4-CH2), 46.0 (14-CH), 48.9 (9-
CH), 51.2 (13-C), 55.2 (OMe), 62.2 (17-C), 69.9 (3-CH), 72.5 (16-CH), 113.8 (5’-CH+7’-CH),
120.0 (6-CH), 121.8 (4’-CH+8’-CH), 131.1 (3’-C), 141.2 (5-C), 153.0 (2’-CH2), 153.8 (20-C),
155.8 (6’-C), 159.6 (1’-C). HRMS (ESI) for C30H40N3O4S ([M+H]+): calcd 538.2734, found
538.2747.
4.1.2.4. 5’-Methyl-[2΄-(N-(4-fluorophenylcarbamoyl)]-3β,16β-di-hydro-xyspiroandrost-5-ene-
17,6΄[1,3,4]thiadiazine (5d)
Colorless solid; mp 211 – 214 oC; yield 63% (345 mg). 1H NMR (600 MHz, DMSO-d6): δ 0.79
(ddd, J = 5.13, 8.30, 12.33 Hz, 1H, 9-CH), 0.85 (s, 3H, 18-CH3), 0.88 (s, 3H, 19-CH3), 0.92-1.05
(m, 2H, 12-CH2), 0.93-1.06 (m, 2H, 1-CH2), 1.22-1.37 (m, 2H, 11-CH2), 1.28-1.63 (m, 2H, 2-
CH2), 1.36-1.39 (m, 1H, 8-CH), 1.49-1.52 (m, 1H, 15-CH2), 1.52-1.93 (m, 2H, 7-CH2), 1.78
(ddd, J = 1.85, 11.31, 11.64 Hz, 1H, 14-CH), 1.85 (dd, J = 10.20, 10.34 Hz, 1H, 15-CH2), 2.04-
2.07 (dd, J = 12.56 Hz, 1H, 4-CH2), 2.10-2.14 (dd, J = 3.96, 12.56 Hz, 1H, 4-CH2), 2.47 (s, 3H,
21-CH3), 3.19-3.21 (m, 1H, 3-CH), 5.20-5.24 (m, 1H, 16-CH), 5.23-5.26 (m, 1H, 6-CH), 5.66
(br. s, 1H, OH), 7.18 (dd, J = 8.20 Hz, 2H, 5’-CH+7’-CH), 7.88 (dd, J = 5.14, 8.20 Hz, 2H, 4’-
13
CH+8’-CH), 10.58 (br. s, 1H, NH), signal of one OH group was not observed. C NMR (125
MHz, DMSO-d6): δ 14.8 (18-CH3), 19.0 (19-CH3), 20.0 (11-CH2), 23.6 (21-CH3), 31.1 (8-CH),
31.2 (7-CH2), 31.3 (2-CH2), 33.7 (15-CH2), 34.6 (12-CH2), 35.8 (10-C), 36.6 (1-CH2), 42.1 (4-
CH2), 46.0 (14-CH), 48.8 (9-CH), 51.2 (13-C), 62.2 (17-C), 69.9 (3-CH), 72.5 (16-CH), 115.1
(d, JC-F = 23.0 Hz, 5’-CH+7’-CH), 120.0 (6-CH), 122.3 (d, JC-F = 6.9 Hz, 4’-CH+8’-CH), 134.4
(3’-C), 141.2 (5-C), 152.8 (2’-C), 154.0 (20-C), 158.5 (d, JC-F = 240.9 Hz, 6’-CH), 160.0 (1’-C).
HRMS (ESI) for C29H36FN3O3SNa ([M+Na]+): calcd 548.2354, found 548.2345.
4.1.2.5. 5’-Methyl-[2΄-(N-(4-chlorophenylcarbamoyl)]-3β,16β-di-hydro-xyspiroandrost-5-ene-
17,6΄[1,3,4]thiadiazine (5e)
Colorless solid; mp 215 - 217 oC; yield 51% (276 mg). 1H NMR (600 MHz, DMSO-d6): δ 0.89-
0.92 (m, 1H, 9-CH), 0.94 (s, 3H, 18-CH3), 0.95 (s, 3H, 19-CH3), 1.03-1.11 (m, 2H, 12-CH2),
1.27-1.43 (m, 2H, 11-CH2), 1.40-1.42 (m, 1H, 8-CH), 1.43-1.80 (m, 2H, 2-CH2), 1.62-1.93 (m,
2H, 7-CH2), 1.67-1.98 (m, 2H, 1-CH2), 1.69-1.96 (m, 2H, 15-CH2), 2.05-2.08 (m, 1H, 14-CH),
2.20-2.30 (m, 2H, 4-CH2), 2.57 (s, 3H, 21-CH3), 3.49-3.51 (m, 1H, 3-CH), 5.30-5.33 (m, 1H, 6-
CH), 5.34 (dd, J = 3.38, 9.49 Hz, 1H, 16-CH), 7.34 (d, J = 9.25 Hz, 2H, 5’-CH+7’-CH), 7.67 (d,
J = 9.25 Hz, 2H, 4’-CH+8’-CH), 9.28 (br. s, 1H, NH), signals of OH groups were not observed.
13C NMR (125 MHz, DMSO-d6): δ 15.3 (18-CH3), 19.3 (19-CH3), 20.4 (11-CH2), 23.8 (21-
CH3), 31.5 (7-CH2), 31.6 (2-CH2), 31.8 (8-CH), 34.4 (15-CH2), 34.9 (12-CH2), 36.8 (10-C), 37.0
16

(1-CH2), 42.2 (4-CH2), 46.2 (14-CH), 48.9 (9-CH), 52.2 (13-C), 62.2 (17-C), 71.6 (3-CH), 74.4
(16-CH), 119.7 (6-CH), 120.9 (4’-CH+8’-CH), 129.3 (5’-CH-7’-CH), 129.4 (6’-C), 135.8 (3’-C),
140.9 (5-C), 153.1 (2’-C), 153.9 (20-C), 158.9 (1’-C). HRMS (ESI) for C29H37ClN3O3S
([M+H]+): calcd 542.2239, found 542.2246.
4.1.2.6.
5’-Methyl-[2΄-(N-(4-(trifluoromethyl-phenyl)carba-moyl)]-3β,16β-dihydroxyspiro-
androst-5-ene-17,6΄[1,3,4]thiadiazine (5f)
o
Colorless solid; mp 218 - 220 C; yield 56% (322 mg).1H NMR (600 MHz, DMSO-d6): δ 0.78
(ddd, J = 4.11, 11.30, 12.93 Hz, 1H, 9-CH), 0.84 (s, 3H, 18-CH3), 0.87 (s, 3H, 19-CH3), 0.89-
1.06 (m, 2H, 12-CH2), 0.90-1.03 (m, 2H, 1-CH2), 1.19-1.34 (m, 2H, 11-CH2), 1.27-1.60 (m, 2H,
2-CH2), 1.35-1.38 (m, 1H, 8-CH), 1.49-1.53 (m, 1H, 15-CH2), 1.53-1.92 (m, 2H, 7-CH2), 1.78
(ddd, J = 1.84, 9.24, 11.30 Hz, 1H, 14-CH), 1.88 (dd, J = 10.16, 12.01 Hz, 1H, 15-CH2), 2.04
(dd, J = 11.30 Hz, 1H, 4-CH2), 2.12 (dd, J = 3.92, 11.30 Hz, 1H, 4-CH2), 2.47 (s, 3H, 21-CH3),
3.19-3.23 (m, 1H, 3-CH), 4.55 (d, J = 4.11 Hz, 1H, OH), 5.20-5.25 (m, 1H, 6-CH), 5.24 (m, 1H,
16-CH), 5.69 (d, J = 5.13 Hz, 1H, OH), 7.71 (d, J = 8.80 Hz, 2H, 5’-CH+7’-CH), 8.10 (d, J =
13
8.80 Hz, 2H, 4’-CH+ 8’-CH), 10.86 (br. s, 1H, NH). C NMR (125 MHz, DMSO-d6): δ 14.7
(18-CH3), 18.9 (19-CH3), 19.9 (11-CH2), 23.4 (21-CH3), 31.1 (7-CH2), 31.2 (8-CH), 31.3 (2-
CH2), 33.7 (15-CH2), 34.6 (12-CH2), 35.9 (10-C), 36.6 (1-CH2), 42.0 (4-CH2), 46.0 (14-CH),
48.8 (9-CH), 51.1 (13-C), 62.2 (17-C), 69.8 (3-CH), 72.5 (16-CH), 119.8 (6-CH), 120.4 (4’-
CH+8’-CH), 124.1 (6’-C), 124.2 (CF3), 125.7 (5’-CH+7’-CH), 141.3 (3’-C), 141.7 (5-C), 152.5
(2’-C), 154.0 (20-C), 160.5 (1’-C). HRMS (ESI) for C30H37F3N3O3S ([M+]+): calcd 576.2502,
found 576.2509.
4.1.3. General procedure for the preparation of 17-(N-aryl-6’H-1’,3’,4’-thiadiazine-2’-
carboxamide)-3β-acetoxy-androst-5,17-dienes 7a-j
Oxamic acid thiohydrazide (0.3 mmol) and potassium carbonate (39 mg, 0.28 mmol) were added
to a solution of 3β-acetoxy-21-bromopregn-5,16-dien-20-one (122 mg, 0.28 mmol) in methanol
(1.5 mL). The reaction mixture was stirred at r.t. for 6 h and concentrated at reduced pressure.
The crude product was purified by column chromatography using petroleum ether/ethyl acetate
(5:1).
4.1.3.1. 17-(N-Phenyl-6’H-1’,3’,4’-thiadiazine-2’-carboxamide)-3β-acetoxy-androst-5,17-diene
(7a)
o
1
Yellow solid; mp
198 - 200 C; yield 85% (126 mg). H NMR (300 MHz, CDCl3): δ
(with dec)
0.81-0.97 (m, 2H, 1-СН2, 9-СН), 1.11 (s, 3Н, 19-СН3), 1.12 (s, 3Н, 18-СН3), 1.49-1.77 (m, 7H,
17

1-CH2, 2-CH2, 8-CH, 11-CH2, 14-CH), 1.81-1.96 (m, 2H, 7-CH2), 2.06 (s, 3H, OAc), 2.09-2.24
(m, 2H, 4-CH2), 2.29-2.44 (m, 2H, 12-CH2), 2.44-2.50 (m, 1H, 15-CH2), 2.61-2.71 (m, 1H, 15-
CH2), 3.18 (AB-system, J = 13.68 Hz, 1Н, 2’-CH2), 3.24 (AB-system, J = 13.68 Hz, 1Н, 2’-
CH2), 4.57-4.71 (m, 1H, 3-СH), 5.38-5.47 (m, 1H, 6-СH), 6.62 (s, 1H, 16-СH), 7.18 (dd, J =
7.35 Hz, 1Н, Ph), 7.39 (dd, J =7.35 Hz, 2Н, Ph), 7.70 (d, J = 7.35 Hz, 2Н, Ph), 9.29 (br.s, 1H,
NH). 13C NMR (75 MHz, CDCl3): δ 15.8 (18-СН3), 19.4 (19-СН3), 20.8 (2’-CH2), 20.8 (11-
СН2), 21.5 (COCH3), 27.8 (2-СH2), 30.5 (8-СH), 31.6 (7-СH2), 32.7 (12-CH2), 35.4 (15-CH2),
36.9 (10-С), 36.9 (1-CH2), 38.3 (4-СH2), 47.3 (13-С), 50.5 (9-СH), 57.1 (14-CH), 73.9 (3-СH),
119.8 (2×CH, Ph), 122.1 (CH, Ph), 124.9 (6-C), 129.3 (2×CH, Ph), 137.1 (16-CH), 140.4 (5-C),
141.9 (C, Ph), 146.0 (17-С), 150.9 (C), 152.4 (C), 158.3 (CO), 170.6 (CO). IR (KBr), ν/cm–1:
3355 (NH), 2932, 2894, 2866 (CH), 1728 (CО), 1690 (CO), 1519, 1446, 1242, 1036, 751.
HRMS (ESI) for C31H37N3O3SNa ([M+Na]+): calcd 554.2446, found 554.2448.
4.1.3.2.
17-(N-(p-Tolyl)-6’H-1’,3’,4’-thiadiazine-2’-carboxamide)-3β-acetoxy-androst-5,17-
diene (7b)
Yellow solid; mp (with dec) 186 - 188 oC; yield 73% (111 mg). 1H NMR (300 MHz, CDCl3): δ 1.11
(s, 3Н, 19-СН3), 1.13 (s, 3Н, 18-СН3), 1.14-1.30 (m, 2H, 1-СН2, 9-СН), 1.50-1.83 (m, 7H, 1-
CH2, 2-CH2, 8-CH, 11-CH2, 14-CH), 1.87-1.94 (m, 2H, 7-CH2), 2.06 (s, 3H, OAc), 2.09-2.21
(m, 2H, 4-CH2), 2.36 (s, 3H, Me), 2.36-2.42 (m, 2H, 12-CH2), 2.41-2.48 (m, 1H, 15-CH2), 2.64-
2.69 (m, 1H, 15-CH2), 3.18 (AB-system, J = 13.87 Hz, 1Н, 2’-CH2), 3.23 (AB-system, J = 13.87
Hz, 1Н, 2’-CH2), 4.60-4.68 (m, 1H, 3-СH), 5.41-5.45 (m, 1H, 6-СH), 6.61 (s, 1H, 16-СH), 7.19
(d, J = 8.20 Hz, 2Н, Ar), 7.58 (d, J = 8.20 Hz, 2Н, Ar), 9.24 (br.s, 1H, NH). 13C NMR (75 MHz,
CDCl3): δ 15.7 (18-СН3), 19.2 (19-СН3), 20.7 (CH3), 20.7 (2’-CH2), 20.9 (11-СН2), 21.4
(COCH3), 27.7 (2-СH2), 30.3 (8-СH), 31.5 (7-СH2), 32.5 (12-CH2), 35.3 (15-CH2), 36.8 (10-С),
36.9 (1-CH2), 38.1 (4-СH2), 47.2 (13-С), 50.3 (9-СH), 56.9 (14-CH), 73.9 (3-СH), 119.7 (2×CH,
Ar), 121.9 (6-CH), 129.7 (2×CH, Ar), 134.5 (C, Ar), 134.5 (C, Ar), 140.3 (16-CH), 141.7 (5-C),
146.9 (17-C), 150.9 (C), 152.4 (C), 158.0 (CO), 170.5 (CO). IR (KBr), ν/cm–1: 3355 (NH), 2932,
2894, 2866 (CH), 1728 (CО), 1690 (CO), 1519, 1446, 1242, 1036, 751. HRMS (ESI) for
C32H40N3O3S ([M+H]+): calcd 546.2786, found 546.2785.
4.1.3.3. 17-(N-(4’-Ethylphenyl)-6’H-1’,3’,4’-thiadiazine-2’-carboxamide)-3β-acetoxy-androst-
5,17-diene (7c)
o
1
Yellow solid; mp
159 - 161 C; yield 76% (120 mg). H NMR (300 MHz, CDCl3): δ
(with dec)
1.05-1.28 (m, 2H, 1-СН2, 9-СН), 1.10 (s, 3Н, 19-СН3), 1.12 (s, 3Н, 18-СН3), 1.25 (t, J = 7.56
Hz, 3H, CH2CH3), 1.48-1.83 (m, 7H, 1-CH2, 2-CH2, 8-CH, 11-CH2, 14-CH), 1.85-1.95 (m, 2H,
18

7-CH2), 2.05 (s, 3H, OAc), 2.07-2.21 (m, 2H, 4-CH2), 2.30-2.39 (m, 2H, 12-CH2), 2.39-2.47 (m,
1H, 15-CH2), 2.61-2.70 (m, 1H, 15-CH2), 2.65 (q, J = 7.56 Hz, 2H, CH2CH3), 3.17 (AB-system,
J = 13.88 Hz, 1Н, 2’-CH2), 3.22 (AB-system, J = 13.88 Hz, 1Н, 2’-CH2), 4.59-4.68 (m, 1H, 3-
СH), 5.38-5.45 (m, 1H, 6-СH), 6.60-6.61 (m, 1H, 16-СH), 7.21 (d, J = 8.20 Hz, 2Н, Ar), 7.58 (d,
13
J = 8.20 Hz, 2Н, Ar), 9.25 (br.s, 1H, NH). C NMR (75 MHz, CDCl3): δ 15.6 (CH2CH3), 15.7
(18-СН3), 19.2 (19-СН3), 20.6 (2’-CH2), 20.7 (11-СН2), 21.4 (COCH3), 27.7 (2-СH2), 28.4
(CH2CH3), 30.3 (8-СH), 31.5 (7-СH2), 32.5 (12-CH2), 35.3 (15-CH2), 36.8 (10-С), 36.9 (1-CH2),
38.1 (4-СH2), 47.1 (13-С), 50.3 (9-СH), 56.9 (14-CH), 73.9 (3-СH), 119.8 (2×CH, Ar), 121.9 (6-
CH), 128.5 (2×CH, Ar), 134.6 (C, Ar), 140.3 (16-CH), 140.9 (5-C), 141.7 (C, Ar), 145.9 (17-C),
150.8 (C), 152.4 (C), 158.0 (CO), 170.5 (CO). IR (KBr), ν/cm–1: 3339 (NH), 2963, 2935, 2897,
2858 (CH), 1734 (CО), 1674 (CO), 1522, 1374, 1248, 1034, 827, 670. HRMS (ESI) for
C33H41N3O3SK ([M+K]+): calcd 598.2499, found 598.2500.
4.1.3.4.
17-(N-(4’-Methoxyphenyl)-6’H-1’,3’,4’-thiadiazine-2’-carboxamide)-3β-acetoxy-
androst-5,17-diene (7d)
Yellow solid; mp (with dec) 207 - 209 oC; yield 90% (140 mg). 1H NMR (300 MHz, CDCl3): δ 0.81-
0.97 (m, 2H, 1-СН2, 9-СН), 1.11 (s, 3Н, 19-СН3), 1.12 (s, 3Н, 18-СН3), 1.51-1.72 (m, 7H, 1-
CH2, 2-CH2, 8-CH, 11-CH2, 14-CH), 1.84-1.96 (m, 2H, 7-CH2), 2.06 (s, 3H, OAc), 2.09-2.23
(m, 2H, 4-CH2), 2.28-2.44 (m, 2H, 12-CH2), 2.44-2.50 (m, 1H, 15-CH2), 2.61-2.71 (m, 1H, 15-
CH2), 3.17-3.23 (m, 2Н, 2’-CH2), 3.83 (s, 3Н, OCH3), 4.57-4.70 (m, 1H, 3-СH), 5.38-5.47 (m,
1H, 6-СH), 6.60-6.61 (m, 1H, 16-СH), 6.92 (d, J = 8.80 Hz, 2Н, Ar), 7.62 (d, J = 8.80 Hz, 2Н,
Ar), 9.20 (br.s, 1H, NH). 13C NMR (75 MHz, CDCl3): δ 15.8 (18-СН3), 19.3 (19-СН3), 19.4 (2’-
CH2), 20.8 (11-СН2), 21.5 (COCH3), 27.8 (2-СH2), 29.8 (8-СH), 30.5 (7-СH2), 31.2 (12-CH2),
36.6 (15-CH2), 36.9 (1-СH2), 37.0 (10-C), 38.2 (4-СH2), 47.8 (13-С), 50.5 (9-СH), 55.6 (OCH3),
57.1 (14-CH), 73.9 (3-СH), 114.3 (2×CH, Ar), 120.4 (6-C), 121.5 (2×CH, Ar), 130.0 (17-C),
138.3 (C, Ar), 140.2 (16-CH), 141.9 (5-C), 150.5 (C), 152.0 (C), 156.4 (С, Ar), 159.3 (CO),
170.7 (CO). IR (KBr), ν/cm–1: 3363 (NH), 2965, 2931, 2864, 2820 (CH), 1724 (CО), 1678 (CO),
1519, 1371, 1245, 1037, 827. HRMS (ESI) for C32H39N3O4SNa ([M+Na]+): calcd 584.2553,
found 584.2556.
4.1.3.5.
17-(N-(2’-Fluorophenyl)-6’H-1’,3’,4’-thiadiazine-2’-carboxa-mide)-3β-acetoxy-
androst-5,17-diene (7e)
o
1
Yellow solid; mp
198 - 200 C; yield 65% (100 mg). H NMR (300 MHz, CDCl3): δ
(with dec)
1.05-1.30 (m, 2H, 1-СН2, 9-СН), 1.10 (s, 3Н, 19-СН3), 1.11 (s, 3Н, 18-СН3), 1.47-1.83 (m, 7H,
1-CH2, 2-CH2, 8-CH, 11-CH2, 14-CH), 1.83-1.96 (m, 2H, 7-CH2), 2.04 (s, 3H, Ac), 2.07-2.22
19

(m, 2H, 4-CH2), 2.31-2.42 (m, 2H, 12-CH2), 2.43-2.48 (m, 1H, 15-CH2), 2.62-2.72 (m, 1H, 15-
CH2), 3.18-3.20 (m, 2H, 2’-CH2), 4.56-4.69 (m, 1H, 3-СH), 5.37-5.46 (m, 1H, 6-СH), 6.60-6.61
13
(m, 1H, 16-СH), 7.08-7.22 (m, 3Н, Ar), 8.38-8.47 (m, 1Н, Ar), 9.50 (br.s, 1H, NH). C NMR
(75 MHz, CDCl3): δ 15.6 (18-СН3), 19.3 (19-СН3), 20.2 (2’-CH2), 20.8 (11-СН2), 21.5
(COCH3), 27.8 (2-СH2), 30.3 (8-СH), 31.4 (7-СH2), 32.5 (12-CH2), 35.5 (15-CH2), 36.7 (1-
СH2), 36.9 (10-C), 38.2 (4-СH2), 46.9 (13-С), 50.3 (9-СH), 57.0 (14-CH), 74.0 (3-СH), 115.2 (d,
JC-F = 18.91 Hz, CH, Ar), 121.8 (d, JC-F = 25.87 Hz, CH, Ar), 122.2 (6-CH), 124.7 (d, JC-F = 3.65
Hz, CH, Ar), 125.6 (d, JC-F = 10.36 Hz, CH, Ar), 137.8 (16-CH), 140.2 (5-C), 142.8 (C, Ar),
146.9 (17-C), 150.4 (C), 151.6 (C), 152.9 (d, JC-F = 245.76 Hz, C, Ar), 158.6 (CO), 170.6 (CO).
IR (KBr), ν/cm–1: 3361 (NH), 2966, 2932, 2897, 2861 (CH), 1721 (CО), 1690 (CO), 1535, 1523,
1458, 1365, 1249, 1105, 1035, 870, 755. HRMS (ESI) for C31H36FN3O3SNa ([M+Na]+): calcd
572.2360, found 572.2354.
4.1.3.6.
17-(N-(4’-Chlorophenyl)-6’H-1’,3’,4’-thiadiazine-2’-carboxa-mide)-3β-acetoxy-
androst-5,17-diene (7f)
Yellow solid; mp (with dec) 162 - 164 oC; yield 90% (142 mg). 1H NMR (300 MHz, DMSO-d6): δ
0.76-1.13 (m, 2H, 1-СН2, 9-СН), 1.02 (s, 3Н, 19-СН3), 1.04 (s, 3Н, 18-СН3), 1.37-1.73 (m, 7H,
1-CH2, 2-CH2, 8-CH, 11-CH2, 14-CH), 1.76-1.87 (m, 2H, 7-CH2), 1.99 (s, 3H, Ac), 2.00-2.16
(m, 2H, 4-CH2), 2.26-2.32 (m, 2H, 12-CH2), 2.33-2.41 (m, 1H, 15-CH2), 2.58-2.66 (m, 1H, 15-
CH2), 3.37 (AB-system, J = 14.34 Hz, 1Н, 2’-CH2), 3.54 (AB-system, J = 14.34 Hz, 1Н, 2’-
CH2), 4.41-4.51 (m, 1H, 3-СH), 5.37-5.41 (m, 1H, 6-СH), 6.88-6.89 (m, 1H, 16-СH), 7.41 (d, J
13
= 8.85 Hz, 2Н, Ar), 7.89 (d, J = 8.85 Hz, 2Н, Ar), 10.82 (br.s, 1H, NH). C NMR (75 MHz,
DMSO-d6): δ 15.7 (18-СН3), 19.2 (19-СН3), 20.6 (2’-CH2), 20.8 (11-СН2), 21.4 (COCH3), 27.7
(2-СH2), 30.3 (8-СH), 31.5 (7-СH2), 32.6 (12-CH2), 35.3 (15-CH2), 36.8 (1-СH2), 36.9 (10-C),
38.1 (4-СH2), 47.1 (13-С), 50.3 (9-СH), 56.9 (14-CH), 73.9 (3-СH), 121.0 (2×CH, Ar), 121.9 (6-
CH), 129.2 (2×CH, Ar), 135.6 (16-CH), 140.3 (5-C), 142.1 (C, Ar), 145.6 (17-C), 150.8 (C),
153.1 (C), 163.3 (CO), 170.6 (CO), signal of one C group was not observed. IR (KBr), ν/cm–1:
3324 (NH), 2934, 2904, 2853 (CH), 1724 (CО), 1673 (CO), 1590, 1521, 1373, 1250, 1064,
1033, 826. HRMS (ESI) for C31H37ClN3O3S ([M+H]+): calcd 566.2238, found 566.2239.
4.1.3.7. 17-(N-(4’-Bromophenyl)-6’H-1’,3’,4’-thiadiazine-2’-carboxa-mide)-3β-acetoxy-androst-
5,17-diene (7g)
Yellow solid; mp (with dec) 191 - 193 oC; yield 88% (150 mg). 1H NMR (400 MHz, DMSO-d6): δ
0.78-1.09 (m, 2H, 1-СН2, 9-СН), 1.01 (s, 3Н, 19-СН3), 1.03 (s, 3Н, 18-СН3), 1.35-1.73 (m, 7H,
1-CH2, 2-CH2, 8-CH, 11-CH2, 14-CH), 1.74-1.87 (m, 2H, 7-CH2), 1.97 (s, 3H, OAc), 1.98-2.15
20

(m, 2H, 4-CH2), 2.24-2.31 (m, 2H, 12-CH2), 2.32-2.41 (m, 1H, 15-CH2), 2.56-2.64 (m, 1H, 15-
CH2), 3.33 (AB-system, J = 13.94 Hz, 1Н, 2’-CH2), 3.48 (AB-system, J = 13.94 Hz, 1Н, 2’-
CH2), 4.40-4.50 (m, 1H, 3-СH), 5.34-5.39 (m, 1H, 6-СH), 6.83-6.84 (m, 1H, 16-СH), 7.46 (d, J
13
= 9.05 Hz, 2Н, Ar), 7.82 (d, J = 9.05 Hz, 2Н, Ar), 10.75 (br.s, 1H, NH). C NMR (75 MHz,
DMSO-d6): δ 15.9 (18-СН3), 19.3 (19-СН3), 20.2 (2’-CH2), 20.8 (11-СН2), 21.5 (COCH3), 27.8
(2-СH2), 30.3 (8-СH), 31.4 (7-СH2), 32.5 (12-CH2), 35.5 (15-CH2), 36.7 (1-СH2), 36.9 (10-C),
38.2 (4-СH2), 46.9 (13-С), 50.3 (9-СH), 56.8 (14-CH), 73.6 (3-СH), 116.5 (C, Ar), 122.2 (6-
CH), 122.8 (2×CH, Ar), 131.8 (2×CH, Ar), 137.8 (16-CH), 140.3 (5-C), 142.8 (C, Ar), 146.9
(17-C), 150.4 (C), 151.6 (C), 159.8 (CO), 170.0 (CO). IR (KBr), ν/cm–1: 3434 (NH), 2937, 2903,
2867 (CH), 1720 (C=О), 1676 (C=O), 1585, 1521, 1373, 1248, 1064, 1034, 822. HRMS (ESI)
for C31H37BrN3O3S ([M+H]+): calcd 612.1712, found 612.1715.
4.1.3.8.
17-(N-(4’-(Trifluoromethyl)phenyl)-6’H-1’,3’,4’-thiadiazine-2’-carboxamide)-3β-
acetoxy-androst-5,17-diene (7h)
Yellow solid; mp (with dec) 186 - 188 oC; yield 70% (119 mg). 1H NMR (600 MHz, DMSO-d6): δ
0.72-0.86 (m, 2H, 1-СН2, 9-СН), 1.00 (s, 3Н, 19-СН3), 1.01 (s, 3Н, 18-СН3), 1.35-1.73 (m, 7H,
1-CH2, 2-CH2, 8-CH, 11-CH2, 14-CH2), 1.74-1.85 (m, 2H, 7-CH2), 1.96 (s, 3H, OAc), 1.98-2.14
(m, 2H, 4-CH2), 2.23-2.30 (m, 2H, 12-CH2), 2.32-2.38 (m, 1H, 15-CH2), 2.56-2.62 (m, 1H, 15-
CH2), 3.37 (AB-system, J = 13.94 Hz, 1Н, 2’-CH2), 3.54 (AB-system, J = 13.94 Hz, 1Н, 2’-
CH2), 4.39-4.47 (m, 1H, 3-СH), 5.34-5.40 (m, 1H, 6-СH), 6.87-6.88 (m, 1H, 16-СH), 7.70 (d, J
13
= 8.44 Hz, 2Н, Ar), 8.03 (d, J = 8.44 Hz, 2Н, Ar), 11.00 (br.s, 1H, NH). C NMR (150 MHz,
DMSO-d6): δ 16.3 (18-СН3), 19.6 (19-СН3), 20.3 (2’-CH2), 21.0 (11-СН2), 21.8 (COCH3), 28.1
(2-СH2), 30.6 (8-СH), 31.6 (7-СH2), 32.8 (12-CH2), 35.7 (15-CH2), 36.9 (10-С), 37.1 (1-CH2),
38.3 (4-СH2), 47.2 (13-С), 50.5 (9-СH), 57.1 (14-CH), 73.9 (3-СH), 121.3 (2×CH, Ar), 122.5 (6-
CH), 126.7 (2×CH, Ar), 136.0 (16-C), 140.6 (5-C), 143.5 (C, Ar), 147.5 (17-C), 150.6 (C), 151.7
(C), 160.5 (CO), 170.4 (CO), signals of two C were not observed. IR (KBr), ν/cm–1: 3350 (NH),
2964, 2938, 2897, 2860 (CH), 1734 (CО), 1686 (CO), 1532, 1324 (CF3), 1249, 1167, 1117,
1060, 839. HRMS (ESI) for C32H36F3N3O3SK ([M+K]+): calcd 638.2061, found 638.2061.
4.1.3.9. 17-(N-(2’-Nitrophenyl)-6’H-1’,3’,4’-thiadiazine-2’-carbo-xamide)-3β-acetoxy-androst-
5,17-diene (7i)
o
1
Yellow solid; mp
136 - 138 C; yield 77% (124 mg). H NMR (300 MHz, CDCl3): δ
(with dec)
0.79-1.11 (m, 2H, 1-СН2, 9-СН), 1.10 (s, 3Н, 19-СН3), 1.11 (s, 3Н, 18-СН3), 1.42-1.80 (m, 7H,
1-CH2, 2-CH2, 8-CH, 11-CH2, 14-CH), 1.81-1.96 (m, 2H, 7-CH2), 2.06 (s, 3H, OAc), 2.09-2.22
(m, 2H, 4-CH2), 2.26-2.43 (m, 2H, 12-CH2), 2.43-2.49 (m, 1H, 15-CH2), 2.69-2.76 (m, 1H, 15-
21

CH2), 3.18-3.20 (m, 2Н, 2’-CH2), 4.59-4.69 (m, 1H, 3-СH), 5.36-5.47 (m, 1H, 6-СH), 6.59-6.60
(m, 1H, 16-СH), 7.23-7.28 (m, 1Н, Ar), 7.67-7.76 (m, 1Н, Ar), 8.25-8.33 (m, 1Н, Ar), 8.84-8.91
(m, 1Н, Ar), 12.09 (br.s, 1H, NH). 13C NMR (75 MHz, CDCl3): δ 15.7 (18-СН3), 19.3 (19-СН2),
20.6 (2’-CH2), 20.7 (11-СН2), 21.5 (COCH3), 27.8 (2-СH2), 30.4 (8-СH), 31.6 (7-СH2), 32.6
(12-CH2), 35.2 (15-CH2), 36.8 (1-СH2), 36.9 (10-C), 38.2 (4-СH2), 47.2 (13-С), 50.4 (9-СH),
56.9 (14-CH), 73.9 (3-СH), 121.9 (6-CH), 122.3 (CH, Ar), 123.9 (CH, Ar), 126.0 (CH, Ar),
135.8 (CH, Ar), 137.2 (16-CH), 139.9 (5-C), 140.4 (C, Ar), 141.6 (C, Ar), 145.4 (17-C), 150.8
(C), 151.0 (C), 159.8 (CO), 170.6 (CO). IR (KBr), ν/cm–1: 3286 (NH), 2937, 2903, 2863 (CH),
1728 (CО), 1696 (CO), 1583, 1502, 1436, 1341, 1247, 1033, 969, 861, 744. HRMS (ESI) for
C31H37N4O5S ([M+H]+): calcd 577.2470, found 577.2479.
4.1.3.10. 17-(N-Benzyl-6’H-1’,3’,4’-thiadiazine-2’-carboxamide)-3β-acetoxy-androst-5,17-diene
(7j)
Yellow solid; mp (with dec) 169 - 171 oC; yield 79% (120 mg). 1H NMR (300 MHz, CDCl3): δ 1.08
(s, 3Н, 19-СН3), 1.09 (s, 3Н, 18-СН3), 1.11-1.20 (m, 2H, 1-СН2, 9-СН), 1.43-1.81 (m, 8H, 1-
CH2, 2-CH2, 8-CH, 11-CH2, 12-CH2, 14-CH), 1.85-1.93 (m, 2H, 7-CH2), 2.05 (s, 3H, OAc),
2.09-2.18 (m, 2H, 4-CH2), 2.30-2.38 (m, 1H, 12-CH2), 2.38-2.45 (m, 1H, 15-CH2), 2.58-2.64 (m,
1H, 15-CH2), 3.13 (AB-system, J =13.87 Hz, 1Н, CH2), 3.18 (AB-system, J =13.87 Hz, 1Н,
CH2), 4.57-4.64 (m, 1H, 3-СH), 4.60 (d, J = 5.99 Hz, 2H, CH2), 5.38-5.45 (m, 1H, 6-СH), 6.56-
6.57 (m, 1H, 16-СH), 7.30-7.37 (m, 5Н, Ar), 7.73 (t, 1H, J = 5.99 Hz, NH). 13C NMR (75 MHz,
CDCl3): δ 15.7 (18-СН3), 19.2 (19-СН3), 20.5 (2’-CH2), 20.6 (11-СН2), 21.4 (COCH3), 27.7 (2-
СH2), 30.3 (8-СH), 31.5 (7-СH2), 32.5 (12-CH2), 35.3 (15-CH2), 36.8 (10-С), 36.9 (1-CH2), 38.1
(4-СH2), 43.8 (CH2Ph), 47.1 (13-С), 50.3 (9-СH), 56.9 (14-CH), 73.9 (3-СH), 121.9 (6-CH),
127.8 (CH, Ar), 128.1 (2×CH, Ar), 128.8 (2×CH, Ar), 137.2 (16-CH), 140.3 (5-C), 141.3 (C,
Ar), 145.7 (17-С), 150.9 (C), 151.6 (C), 160.4 (CO), 170.5 (CO). IR (KBr), ν/cm–1: 3350 (NH),
2937, 2904, 2868 (CH), 1733 (CО), 1685 (CO), 1524, 1373, 1245, 1072, 1033, 911, 811, 729.
HRMS (ESI) for C32H40N3O3S ([M+H]+): calcd 546.2783, found 546.2785.
4.2. Biology
4.2.1. Cell line and evaluation of antiproliferative activity
The human prostate cancer cell line 22Rv1 (CRL-2505) was purchased from the ATCC
collection. Cells were cultured in standard RPMI-1640 medium (Gibco) supplemented with 10%
fetal calf serum (FCS) (HyClone), RPMI-1640 Vitamins (PanEco) and 0.1 mg/ml sodium
pyruvate (Santa Cruz) at 37 °C, 5% CO2 and 80–85% humidity (NuAir CO2 incubator). The cell
22

growth was evaluated by the modified MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide) (Applichem) test [52] as described in [53]. 22Rv1 cells were
seeded at a density of 105 cells per well in 24-well plates (Corning) in 900 μL of the medium.
The compounds were dissolved in DMSO (Applichem) to 5 mM before experiments and then
were diluted in the medium to the required concentrations. The tested compounds with different
concentrations in 100 μL of the medium were added 24 h after the seeding, and the cells were
grown for 72 h. After incubation with the compounds, the medium was removed, and the MTT
reagent dissolved in the medium was added to the final concentration of 0.2 mg/ml to each well
and incubated for 3 h. The cell supernatants were removed and the MTT formazan purple
crystals were dissolved in 100% DMSO (350 μL per well). Then the plates were gently shaken
and the absorbance was measured at 571 nm with a MultiScan reader (ThermoFisher). The
viability of the cells was assessed after subtraction of the blank value (the absorbance in the well
w/o cells) from all wells. Dose-response curves were analyzed by regression analysis using
sigmoidal curves (Log(concentration) vs normalized absorbance). The half maximal inhibitory
concentrations (IC50) were determined with GraphPad Prism.
4.2.2. AR and NF-κB activities
22Rv1 human prostate cancer cells were transfected with the NF-κB-LUC, ARE-LUC plasmids
containing the luciferase reporter gene under the AR- or NF-κB-dependent promoters, and co-
transfected with β-galactosidase plasmid. Cell transfection for reporter gene products was carried
out as described earlier in [54]. Briefly, 22Rv1 cells were plated (5 × 105 cells/well) onto 24-well
plate containing standard cell culture medium. After 24-h incubation, cells were co-transfected
with plasmids containing luciferase and β-galactosidase genes. Phenol-free RPMI-1640
supplemented with 10% steroid-free serum were used for ARE-LUC assay. The plasmids used in
this work were kindly provided by Prof. P.Hartig and Prof. A.Gasparian [55, 56]. The
transfection was carried out for 8 h at 37 °C using Lipofectamine 2000. Following transfection,
medium was changed and cells were treated with synthesized compound or vehicle control. To
induce AR transcriptional activity 22Rv1 cells were treated with 10 nM DHT.
The luciferase activity was measured according to a standard protocol (Promega) using a Tecan
Infinite M200 Pro, β-galactosidase activity was analyzed by standard colorimetric assay using
MultiScan FC (Thermo) as described in [57]. The luciferase/β-galactosidase activities were
normalized by the internal control values and represented as the mean ± SD for the three
independent experiments. NF-κB and AR activities calculated in arbitrary units as the ratio of
luciferase/galactosidase activity.
23

4.2.3. Western blot analysis
22Rv1 cells were seeded on a 60 mm dishes (Corning). After 24 h growth the compounds were
added in fresh medium as mentioned in the figure 2. To prepare cell extracts 22Rv1 cells were
twice washed in phosphate buffer, and incubated for 10 min on ice in the modified lysis buffer
containing 50 mM Tris-HCl, pH 7.5, 0.5% Igepal CA-630, 150 mM NaCl, 1 mM EDTA, 1 mM
DTT, 1 mM PMSF, 0.1 mM sodium orthovanadate and aprotinin, leupeptin, pepstatin (1 µg/ml
each) as described earlier in [58]. The protein content was determined by the Bradford method
[59].
Cell lysates (40 µg protein) were separated in 10% SDS-PAGE under reducing conditions,
transferred to a nitrocellulose membrane (SantaCruz), and processed according to a standard
protocol. To prevent nonspecific absorption, the membranes were treated with 5% nonfat milk
solution in TBS buffer (20 mM Tris, 500 mM NaCl, pH 7.5) with 0.1% Tween-20 and then
incubated with primary antibodies overnight at 4 °C.
AR, PARP, phosphorylated (ph) and unphosphorylated NF-κB p65, Akt and ERK1/2, antibodies
were obtained from Cell Signaling Technology; the antibodies against α-tubulin (Cell Signaling
Technology) were added to standardize loading. Goat anti-rabbit IgGs (Jackson
ImmunoResearch) conjugated to horseradish peroxidase were used as secondary antibodies.
Signals were detected using the ECL reagent as described in Mruk and Cheng’s protocol [60]
and an ImageQuant LAS4000 system (GE HealthCare). ImageJ software (NIH) was used for
densitometry.
4.2.4. Statistical tests
Statistical analysis was performed using Microsoft Excel and GraphPad Prism. Each biology
experiment was repeated three times and results were expressed as mean + S.D. (standard
deviation value). Student’s t-test was used to evaluate the significance of differences in
comparisons. P value of <0.05 was considered statistically significant.
Corresponding Authors
Dr. Yulia A. Volkova, E-mail: yavolkova@gmail.com, ORCID 0000-0001-8554-838X.
Dr. Alexander M. Scherbakov, E-mail: alex.scherbakov@gmail.com, ORCID 0000-0002-2974-
9555.
24

Abbreviations
DHT – dihydrotestosterone, RPMI-1640 - Roswell Park Memorial Institute medium 1640,
NF-κB - nuclear factor kappa-light-chain-enhancer of activated B cells, AR – androgen receptor,
ERK - extracellular signal-regulated kinase, ph – phosphorylated, PARP - poly (ADP-ribose)
polymerase, IC50 - half maximal inhibitory concentration, ECL- enhanced chemiluminescence.
Acknowledgments
The research is supported by the Russian Foundation for Basic Research, project Nos 18-33-
00913 mol_a and 17-53-45127 (chemistry) and the Russian Science Foundation, project 19-15-
00245 (biology). The authors would like to thank Dr. Olga Andreeva for her helpful advices on
luciferase assay and Yurii Shchegolev for his assistance in immunoblotting. The densitometry
protocol for immunoblotting “Using ImageJ to quantify blots” was provided by The University
of Queensland (https://di.uq.edu.au/community-and-alumni/).
References
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antileishmanial compounds from 2002 to late 2011, Chemical reviews 114(20) (2014) 10369-
10428.
[2] F. Le Bideau, S. Dagorne, Synthesis of transition-metal steroid derivatives, Chemical reviews
113(10) (2013) 7793-7850.
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