Synthesis and cytotoxic evaluation of novel hybrid estrane heterocycles as chemotherapeutic anti-cancer agents

Article abstract of DOI:10.1016/j.steroids.2021.108813

New synthesized hybrid steroidal heterocyclic compounds have received a lot of attention in view of their biological activities as anticancer agents. In this study, a novel class of hybrid estrane heterocyclic compounds were synthesized and evaluated by analytical and spectral data which proved the validity of these derivatives. The cytotoxicity of synthesized compounds 2a, 2b, 2c, 3b, 8, 10a, 10b, 13, 14, 16a and 19 against three human cell lines: breast cancer cells (MCF-7), prostate cancer cells (PC3), and liver cancer cells (HepG2) has been tested using MTT assay. Compounds 10a, 10b, 2c, and 14 revealed more inhibitory influence on MCF7, PC3 and HepG2 growth than the reference drug doxorubicin (Dox) after 24 h incubation. Noteworthy, the tested compounds 10a, 10b, 2c, and 14 exhibited the most pronounced effect in this respect. The results were confirmed by morphology study.

Full text of DOI:10.1016/j.steroids.2021.108813

Steroids 169 (2021) 108813
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and cytotoxic evaluation of novel hybrid estrane heterocycles as
chemotherapeutic anti-cancer agents
,
Doaa S. Nadaa, Dina S Elkadyb, Ghada H. Elsayedb *, Adel A.-H. Abdel-Rahmana,
Gamal A. Elmegeedb
a Department of Chemistry, Faculty of Science, Menofia University, Shebin El-Koam, Egypt
b Hormones Department, National Research Centre, 12622 Dokki, Giza, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
Anticancer
Cytotoxicity
Estrone
New synthesized hybrid steroidal heterocyclic compounds have received a lot of attention in view of their
biological activities as anticancer agents. In this study, a novel class of hybrid estrane heterocyclic compounds
were synthesized and evaluated by analytical and spectral data which proved the validity of these derivatives.
The cytotoxicity of synthesized compounds 2a, 2b, 2c, 3b, 8, 10a, 10b, 13, 14, 16a and 19 against three human
cell lines: breast cancer cells (MCF-7), prostate cancer cells (PC3), and liver cancer cells (HepG2) has been tested
using MTT assay. Compounds 10a, 10b, 2c, and 14 revealed more inhibitory influence on MCF7, PC3 and HepG2
growth than the reference drug doxorubicin (Dox) after 24 h incubation. Noteworthy, the tested compounds 10a,
10b, 2c, and 14 exhibited the most pronounced effect in this respect. The results were confirmed by morphology
study.
Heterocycles
Morphology
Steroids
1. Introduction
steroids with heterocyclic rings enhanced a change of their physiological
and various biological activities [9]. These heterocyclic steroid de-
Cancer is a multicellular disease in which abnormal cells divide
without control and can invade nearby tissues [1]. It is one of the leading
causes of death worldwide according to World health organization
(WHO) [2]. Although, chemotherapy has confirmed a new period of
molecularly targeted therapeutics but the effectiveness of existing drugs
for treating cancer is still limited [3]. Therefore, there is an urgent need
to synthesize new, efficient and less toxic anticancer agents to overcome
the current therapy inclusive the development of drug resistance [4,5].
steroids have always attracted great attention because of their remark-
able structural design and their impressive variety of biological activ-
ities. Accurate variation in the steroid molecule can result significant
therapeutic action [6]. The heterocyclic rings play the most important
role in the therapeutic function of compounds due to their potent re-
ceptor binding properties [7]. The proportional ease of heterocyclic
rings that can be modulated with additional substituent let them to
cover a broad area of chemical space, more over considering as excellent
starting point for anticancer drug development [8]. Combination of
rivatives appear to be promising in the treatment of various types of
cancer including liver cancer, breast cancer, prostate cancer, etc
[10–14]. The present investigation aimed to test the effect of newly
synthesized heterocyclic estrane derivatives on breast (MCF7), prostate
(PC3) and liver (HepG2) human cancer cell lines and the results were
confirmed by morphology study.
2. Materials and methods
2.1. Synthetic methods, analytical and spectral data
The hormone used as starting material (estrone) was purchased from
sigma company (Tables 1 and 2), St. Louis, MO, USA. The used solvents
were an hydrated by distillation prior. All the melting points of products
were identified by using electro thermal apparatus and are uncorrected.
The IR spectra were identified by (KBr discs) on a Shimadzu FT-IR 8201
PC spectrometer and measured by cmꢀ 1 unit. The 1H NMR was
Abbreviations: Dox, Doxorubicin; WHO, World health organization; TLC, Thin layer chromatography; VACSERA, The holding company for biological products &
vaccines; RPMI, Roswell Park Memorial Institute; DPBS, Dulbecco’s phosphate buffered saline; MTT, (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide;
DMSO, dimethyl sulfoxide; DMF-DMA, dimethyl formamide dimethyl acetal.
* Corresponding author.
E-mail address: ghadanrc@yahoo.com (G.H. Elsayed).
https://doi.org/10.1016/j.steroids.2021.108813
Received 24 November 2020; Received in revised form 30 January 2021; Accepted 22 February 2021
Available online 27 February 2021
0039-128X/© 2021 Elsevier Inc. All rights reserved.

D.S. Nada et al.
Steroids 169 (2021) 108813
1
–
–
–
(CH ), 2864 (CH ), 1718 (C N), 1595 (C C). H NMR (DMSO – d ,
Table 1
–
3
2
6
The in vitro cytotoxic activity of synthesized compounds on MCF-7 cancer cell
line.
ppm)δ = 0.811 (s, 3H, 18- CH3), 0.811–2.490 (m, estrane moiety), 1.292
(m,1H, 14-CH), 1.518 (s, 1H, SH), 1.719 (m, 1H, 16-CH), 2.483 (m, 2H,
15-CH2), 2.490 (d, 1H, 17-CH), 2.728 (d, 1H, 22-CH), 6.437 (S, 1H, NH,
D2O exchangeable), 7.029 (d, 2H, phenyl group), 7.326 (d, 2H, phenyl
group), 9.02 (s, 1H, OH, D2O exchangeable), Calc for C26H29FN2OS
(436.20): C, 71.5; H, 6.70; F, 4.35; N, 6.42; S, 7.34% found: C, 71.46; H,
6.63; F, 4.38; N, 6.37; S, 7.28%
Compd.No
IC50 (µg/ml)
Compd.No
IC50 (µg/ml)
2a
616.69
141.65
54.21
156.5
77.12
18.26
93.08
10b
13
18.27
172.07
24.15
92.53
26.61
2b
2c
14
3b
16a
19
8
10-mercapto-12-(4-methoxyphenyl)-8a-methyl-
10a
2,6b,7,8,8a,8b,9,12,12a,13,13a,13b-dodecahydro-1H-naphtho
Doxorubicin
Doxorubicin
93.08
[2′,1′:4,5]indeno[1,2-d]pyrimidin-4-ol (2c)
IC50: Concentration required to inhibit cell viability by 50%.
White powder from absolute ethanol, yield 0.43 g (95.5%); mp
0
70–72 C; IR (KBr, cmꢀ 1):
ν
3858 (SH), 3343 (NH), 3216 (OH), 2934
1
–
–
–
–
(CH ), 2862 (CH ), 1718 (C N), 1593 (C C). H NMR (DMSO – d ,
3
2
6
Table 2
ppm)δ = 0.813 (s, 3H, 18- CH3), 0.813–2.561 (m, estrane moiety), 1.310
(m, 1H, 14- CH), 1.481 (S,1H, SH), 1.75 (m, 1H, 16-CH), 2.231 (m, 2H,
15- CH2), 2.480 (d, 1H, 17-CH), 2.731 (d, 1H, 22-CH), 3.231(s, 3H,
CH3OH), 6.531 (s, 1H, NH, D2O exchangeable), 7.121 (d, 2H, phenyl
group), 7.282 (d, 2H, phenyl group)9.02 (s, 1H, OH, D2O exchangeable),
MS (EI) m/z (%): 448.63 [M+1, 16.3%], 348 (25), 341 (32), 339 (95),
322 (90); Calc for C27H32N2O2S (448.22): C, 72.29; H, 7.19; N, 6.24; S,
7.15%; found: C, 72.13; H, 7.15; N, 6.29; S, 7.
The in vitro cytotoxic activity of synthesized compounds on PC3 cancer cell line.
Compd.No
IC50 (µg/ml)
Compd.No
IC50 (µg/ml)
2a
690.41
202.5
22.89
331.3
92.77
31.75
72.39
10b
13
38.4
2b
164.61
48.11
2c
14
3b
16a
19
164.86
111.17
8
10a
Doxorubicin
Doxorubicin
72.39
2.1.2. General procedure for synthesis of compounds3a, 3b and 3c
To a solution of a product 2a, 2b or 2c (0.104, 0.109 or 0.11 g, 0.4
mmol) in absolute ethanol (25–30 mL), phenacylbromide (0.049 g, 4
mmol) was added. The reaction mixture, in each case was heated under
reflux for 4 hrs until the starting material was disappeared as indicated
by TLC. The reaction mixture was treated with ice/water mixture. The
formed solid product was collected by filtration and crystallized from
absolute ethanol.
IC50: Concentration required to inhibit cell viability by 50%
identified by Joel instrument (Japan). At 300 MHz, in DMSO‑d6 as sol-
vent and chemical shifts were measured by ppm relative to TMS. The
spin multiplicities were abbreviated by the letters: s-singlet, d-doublet, t-
triplet, q-quartet and m (multiplet, more than quartet). Mass spectra
were recorded on GCMS-QP 1000ex spectra mass spectrometer working
at 70 EV and VPLCMs / MS “water” 3100 “USA” spectra mass spec-
trometer working at 30 EV. Elemental analysis was carried by the Micro
analytical data unit at Cairo University, Giza, Egypt and the Micro-
analytical data unit at the National Research Center, Giza, Egypt. All
reactions were observed by using thin layer chromatography (TLC)
which was executed using Merk 60 F254 aluminum sheets and visual-
ized by UV light (254 nm). The mixtures were separated by preparative
TLC and gravity chromatography.
8a-methyl-10,14-diphenyl-1,2,6b,7,8,8a,8b,14,14a,15,15a,15b-
dodecahydronaphtho[2′,1′:4,5]indeno[2,1-e]thiazolo[3,2-a]pyrimidin-
4-ol (3a)
White powder from absolute ethanol, yield 0.07 g (54.26%); mp
256–258 ◦C; IR (KBr, cmꢀ 1):
ν
3418 (OH), 2928 (CH3), 2867 (CH2), 1718
1
–
–
–
(C N), 1621 (C C). H NMR (DMSO – d , ppm): δ = 0.811(s, 3H, 18-
–
CH3), 0.811–2.740 (m, estrane moiety), 1.6341 (m, 1H, 14-CH), 1.46 (m,
1H, 16-CH), 2.303 (m, 2H, 15-CH2), 2.490 (d, 1H, 17-CH), 2.728 (d, 1H,
25-CH), 7.029 (d, 2H, phenyl ring), 7.046 (m, 3H, phenyl ring), 7320 (d,
2H, phenyl ring), 7.650 (m, 3H, phenyl ring), 9.015 (s, 1H, OH, D2O
exchangeable); MS (EI) m/z (%): 518 [M+, 5%], 466 (10), 414 (20), 307
(12), 254 (39); Calc for C34H34N2OS (518.72): C, 78.73; H, 6.61; N, 5.40;
S, 6.18%; found: C, 78.65; H, 6.52 N, 5.31; S, 6.11%
2.1.1. General procedure for synthesis of compounds 2a, 2b and 2c
A mixture of estrone (1) (0.27 g, 1 mmol) with benzaldehyde (0.106
g, 1 mmol), 4-fluorobenzaldehyde (0.124 g, 1 mmol) or 4-methoxyben-
zaldehyde (0.136 g, 1 mmol) and thiourea (0.078 g, 1 mmol) in absolute
ethanol was heated under reflux for 3–6 hrs until all the starting mate-
rials were disappeared as indicated by TLC. The reaction mixture was
treated with ice/water. The formed solid of each product was collected
by filtration and crystallized from absolute ethanol.
14-(4-fluorophenyl)-8-methyl-10-phenyl-
1,2,6b,7,8,8a,8b,14,14a,15,15a,15b-bodecahydonphtho[2′,1′:4,5]
ineno[2,1-e]thizolo[3,2-a]pyrimidin-4-ol (3b)
Colorless crystals from absolute ethanol, yield 0.07 g (52%); mp
10-mercapto-8a-methyl-12-phenyl-
248–250 0C; IR (KBr, cmꢀ 1):
ν
3343 (OH), 2938 (CH3), 2864 (CH2), 1718
2,6b,7,8,8b,9,12,12a,13,13a,13b-dodecahydro-1H-naphtho[2′,1′:4,5]
indeno[1,2-d]pyrimidin-4-ol (2a)
1
–
–
–
(C N), 1619 (C C). H NMR (DMSO – d ppm): δ = 0.807 (s, 3H, 18-
–
6
CH3), 0.807–2.831 (m, estrane moiety), 1.312 (m, 1H, 14-CH), 1.441
(m, 1H,16-CH), 2.185 (m, 2H, 15-CH2), 2.320 (d, 1H, 17-CH), 2.683 (d,
1H, 25-CH), 7.013 (d, 2H, phenyl ring), 7.026 (d, 2H, phenyl ring),
7.208 (d, 2H, phenyl ring), 7.613 (m, 3H, phenyl ring), 9.015 (s, 1H, OH,
D2O exchangeable); MS (EI) m/z (%): 440 [M+, 8%], 449 (10), 414 (8),
214 (30); Calc for C34H33FN2OS (536.23)C, 76.09; H, 6.20; F, 3.54;N,
5.22; S, 5.97%, found: C,76.13; H, 6.25;F, 3.51; N, 5.17; S, 5.84%
14(4-methoxyphenyl)-8a-methyl-10-phenyl-
White powder from absolute ethanol, yield 0.24 g (58.5%); mp
75–77 0C; IR (KBr, cmꢀ 1):
ν
3855–3751 (SH, NH), 2930 (CH3), 2869
1
–
–
–
(CH ), 1718 (C N), 1618 (C C). H NMR (DMSO – d , ppm): δ = 0.809
–
(s, 32H, 18- CH3), 0.809–2.739 (m, estrane moiety), 1.364 (m, 1H, 14- CH),
1.46 (S, 1H, SH), 1.72 (m, 1H, 16- CH), 2.065 (m, 2H, 15- CH2), 2.49 (d,
1H, 17- CH), 2.74 (d, 1H, 22– CH), 6.44 (s, 1H, NH, D2O exchangeable),
7.028 (d, 2H, phenyl group), 7.044 (m, 3H, phenyl group), 9.02 (s, 1H,
OH, D2O exchangeable) MS (EI) m/z (%): 418 [M+, 20%], 366 (15), 341
(30), 338 (17). Calc for C26H30N2O5 (418.2): C, 74.60; H, 7.22; N, 6.69;
S, 7.62%, found: C, 74.55; H, 7.25; N, 6.54; S, 7.65%
1,2,6b,7,8,8a,8b,14,14a,15,15a,15b,dodecahydronaphtho[2′,1′:4,5]
indeno[2,1-e]thiazolo[3,2-a]pyrimidin-4-ol (3c)
Pale yellow crystals from absolute ethanol, yield 0.05 g (36.5%); mp
12-(4-fluorophenyl)-10-mercapto-8a-methyl-
204–206 0C; IR (KBr, cmꢀ 1):
ν
3302 (OH), 2928 (CH3), 2868 (CH2), 1717
2,6b,7,8,8a,8b,9,12,12a,13,13a,13b-dodecahydro-1H-naphtho
1
–
–
–
[2′,1′,4,5]indeno[1,2-d]pyrimidin-4-ol (2b)
(C N), 1618 (C C); H NMR (DMSO – d , ppm): δ = 0.812 (s, 3H, 18-
–
6
CH3), 0.812–2.740 (m, estrane moiety), 1.341 (m, 1H, 14-CH), 1.468
(m, 1H, 16-CH), 2.359 (m, 2H, 15-CH2), 2.90 (d, 1H,17-CH), 2.740 (d,
2H, 25-CH), 3.327 (s, 3H, CH3CO), 7.029 (d, 2H, phenyl ring), 7.046 (d,
Pale yellow crystals from absolute ethanol, yield 0.41 g (95.3%);mp
62–64 ◦C; IR (KBr, cmꢀ 1):
ν 3855–3343 (SH, NH), 3213 (OH), 2937
2

D.S. Nada et al.
Steroids 169 (2021) 108813
2H, phenyl ring), 7.320 (d, 2H, phenyl ring), 7.650 (m, 3H, phenyl ring),
9.013 (s, 1H, OH, D2O exchangeable). Calc for C35H36N2O2S (548.25): C,
76.61; H, 6.61; N, 5.11; S, 5.4%; found: C, 76.58; H, 6.54; N, 503; S,
5.78%
Pale yellow crystals from absolute ethanol, yield 0.05 g (20%); mp
218–220 ◦C; IR (KBr, cmꢀ 1):
ν
3752 (NH), 3343 (OH), 2936 (CH3), 2864
1
–
–
–
(CH ), 1718 (C N), 1619 (C C); H NMR (DMSO – d , ppm): δ = 0.812
–
6
(s, 32H, 18-CH3), 0.812–2741 (m, estrane moiety), 1.343 (m, 1H, 14-CH),
1.360 (m, 1H, 16-CH), 2.483 (s, 1H, NH, D2O exchangeable), 2.487 (m,
2H, 15-CH2), 2.490 (d, 1H, 17-CH), 2.494 (d, 1H, 25-CH), 7.029 (m, 3H,
phenyl group),7.046 (d, 2H, phenyl group), 7.230 (m, 3H, phenyl
group), 7.242 (d, 2H, phenyl group), 7.359 (s, 1H, 21-CH), 9.291 (s, 1H,
OH, D2O exchangeable); MS (EI) m/z (%): 501 [M+4, 7], 399 (14), 353
(17), 324 (100);Calc. for C34H35N3O (501.28):C, 81.40; H, 7.03; N,
8.38%; found: C, 81.32; H, 7.03; N, 8.38%
2.1.3. General procedure for synthesis of compound 4a, 4b and 4d
A mixture of estrone 1 (0.279 g, 1 mmol), benzaldehyde (0.106 g, 1
mmol), 4-flourobenzaldehyde (0.124 g, 1 mmol), 4-methoxybenzalde-
hyde (0.13 g, 1 mmol) and guanidine (0.136 g, 1 mmol) in absolute
ethanol was heated under reflux for 4–6 hrs until all the starting mate-
rials were disappeared as indicated by TLC. The reaction mixture was
treated with ice/water, the formed solid of each product was collected
by filtration and crystallized from absolute ethanol.
14-(4-fourophenyl)-8a-methyl-10-phenyl-
2,6b,7,8,8a,8b,12,14,14a,15,15a,15b-dodecahydro-1H-imidazo[1.2-a]
10-amino-8a-methyl12-phenyl-2,6b,7,8,8a,8b,9,12,12a,13,13a,13b-
dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d]pyrimidin-4-ol (4a)
Pale yellow crystals from absolute ethanol, yield 0.37 g (92.2%); mp
naphtha[2′,1′:4,5]indeno[2,1-e]pyrimidin-4-ol (5b)
Yellow crystals from absolute ethanol, yield 0.05 g (19%); mp
194–196 ◦C; IR (KBr, cmꢀ 1):
ν
3751 (NH), 3344 (OH), 2936 (CH3), 2863
64–66 ◦C; IR (KBr, cmꢀ 1):
ν
3214 (NH2), 3117 (NH), 3043 (OH), 2978
(CH ), 1718 (C N), 1619 (C C); H NMR (DMSO – d ppm): δ = 0.811
1
–
–
–
–
(CH ), 2867 (CH ), 1716 (C N), 1596 (C C); H NMR (DMSO – d ,
(s, 3H, 18-CH3), 0.811–2.740 (m, estrane moiety), 16.343 (m, 1H, 14-
CH), 1.720 (m, 1H, 16-CH), 2.067 (s, 1H, NH, D2O exchangeable),
2.490 (m, 2H, 15-CH2), 2.740 (d, 1H, 17-CH), 2.764 (d, 1H, 25-CH),
7.029 (d, 2H, phenyl ring), 7.046 (d, 2H, phenyl ring), 7.242 (m, 3H,
phenyl ring), 7.342 (d, 2H, phenyl ring), 7.359 (s, 1H, 21-CH), 9.015 (s,
1H, OH, D2O exchangeable); MS (EI) m/z (%): 512 [M+, 25%], 399 (15),
387 (23), 347 (13); Calc. for C34H34FN3O (519.27): C, 78.58; H, 6.60; F,
3.66; N, 8.09%; found: C, 78.51; H, 6.53; F, 3.51; N, 8.01%
14-(4-methoxypheyl)-8a-methyl-10-phenyl-
2
1
–
–
–
–
ppm): δ = 0.809 (s, 3H, 18-CH3), 0.809–2.728 (m, estrane moiety6),
1.292 (m, 1H, 14-CH), 1.321 (m, 1H, 16-CH), 2.490 (m, 2H, 15-CH2),
2.728 (d, 1H, 17-CH), 2.813 (d, 1H, 22-CH), 4.507 (s, 1H, NH, D2O
exchangeable), 6.511 (s, 2H, NH2, D2O exchangeable), 7.127 (d, 2H,
phenyl ring), 7.333 (m, 3H, phenyl ring), 9.008 (s, 1H, OH, D2O
exchangeable); MS (EI) m/z (%): 373[M, 0.5%], 348 (0.7), 299 (0.3),
269 (7), 93 (100); Calc for C26H31N3O (401.25): C, 77.77; H, 7.78; N,
10.46%; found: C, 77.68; H, 7.5; N, 10.49%
3
2
10-amino-12(4-flourophenyl)-8a-methyl-
2,6a,7,8,8a,8b,12,14,14a,15,15a15b-dodecahydro-1H-imidazol[1,2-a]
2,6b,7,8,8a,8b,9,12,12a,13,13a,13b-dodecahydro-1
h-naphtho
naphtha[2′,1′:4,5]indeno[2,1-e]pyrimidin-4-ol (5c)
[2′,1′:4,5]indeno[1,2-d]pyrimidin-4-ol (4b)
Yellow crystals from absolute ethanol, yield 0.05 g (19%); mp
Pale yellow crystals from absolute ethanol, yield 0.39 g (93%); mp
194–196 0C; IR (KBr,cmꢀ 1):
ν
3751 (NH), 3344 (OH), 2936 (CH3), 2863
78–80 ◦C; IR (KBr, cmꢀ 1):
ν
3751 (NH2), 3678 (NH), 3290 (OH), 2929
(CH ), 1718 (C N), 1619 (C C); H NMR (DMSO – d , ppm): δ = 0.811
1
–
–
–
–
(CH ), 2868 (CH ), 1717 (C N), 1618 (C C); H NMR (DMSO – d ,
(s, 32H, 18-CH3), 0.811–2.740 (m, estrane moiety, 1.341 (m, 1H, 14-CH),
1.359 (m, 1H, 16-CH), 2.067 (s, 1H, NH, D2O exchangeable), 2.490 (m,
2H, 15-CH2), 2.740 (d, 1H, 17-CH), 2.763 (d, 1H, 16-CH), 2.764 (d, 1H,
25-CH), 3.213 (s, 3H,CH3OH), 7.029 (d, 2H, phenyl group), 7.046 (d,
2H, phenyl group), 7.180 (m, 3H, phenyl group), 7.242 (d, 2H, phenyl
group), 7.359 (s, 1H, 21-CH), 9.017 (s, 1H, OH, D2O exchangeable); MS
(EI) m/z (%): 531 [M+4, 30%], 403 (25), 347 (37), 29 (13); Calc for
6
1
–
–
–
–
3
2
ppm): δ = 0.808 (s, 3H, 18-CH3), 0.808–2.740 (m, estrane moiety6),
1.292 (m, 1H, 14-CH), 1.340 (m, 1H, 16-CH), 2.490 (m, 2H, 15-CH2),
2.740 (d, 1H, 17-CH), 2.820 (d, 1H, 22-CH), 4.509 (s, 1H, NH, D2O
exchangeable), 6.511 (s, 2H, NH2, D2O exchangeable), 7.028 (d, 2H,
phenyl group), 7.631 (d, 2H, phenyl group), 9.012 (s, 1H, OH, D2O
exchangeable); MS (EI) m/z (%): 419 [M+2, 0.7%], 299 (12), 269 (7),
228 (10); Calc. for C26H30FN3O (419.24): C, 74.43; H, 7.21; F, 4.53; N,
10.02%; found: C, 74.36; H, 7.25; F, 4.57; N, 9.98%
C
35H37N3O2 (531.29): C, 79.06; H, 7.01; N, 7.90%; found: C, 79.01; H,
6.96; N, 7.90%
10-amino-12-(4-methoxyphenyl)-8a-methyl-
2,6b,7,8,8a,8b,9,12,12a,13,13a,13b-dodecahydro-H-naphtho[2′,1′:4,5]
indeno[1,2-d]pyrimidin-4-ol (4c)
2.1.5. General procedure for synthesis of compounds 6a, 6b and 6c
A mixture of estrone1 (0.27 g, 1 mmol) benzaldehyde (0.106 g, 1
mmol), 4-flourobenzaldehyde (0.124 g, 1 mmol) or 4-methoxybenzalde-
hyde (0.136 g, 1 mmol) ad urea (0.06 g, 1 mmol) in absolute ethanol.
The reaction mixture was heated under reflux for 4–6 hrs until all the
starting materials were disappeared as indicated by TLC. The reaction
mixture was treated with ice/water. The formed solid of each product as
collected by filtration and crystallized from absolute ethanol.
White crystals from absolute ethanol, yield 0.40 g (92.8%); mp
60–62 ◦C; IR (KBr, cmꢀ 1):
ν
3751 (NH2), 3345 (NH), 3215 (OH), 2937
1
–
–
–
–
(CH ), 2863 (CH ), 1718 (C N), 1595 (C C); H NMR (DMSO – d ,
3
2
ppm): δ = 0.811 (s, 3H, 18-CH3), 0.811–2.720 (m, estrane moiety6),
1.252 (m, 1H, 14-CH), 1.320 (m, 1H, 16-CH), 2.421 (m, 2H,15-CH2),
2.738 (d, 1H, 17-CH), 2.791 (d,1H, 22-CH), 3.401 (s, 3H, CH3OH), 4.482
(s, 1H, NH, D2O exchangeable), 6.450 (s, 2H, NH2), 7.028 (d, 2H, phenyl
group), 7.620 (d, 2H, phenyl group), 9.012 (s, 1H, OH); MS (EI) m/z (%):
431 [M+, 5%], 324 (35), 307 (43), 299 (7), 269 (40); Calc. for
8a-methyl-12-phenyl-2,6b,7,8,8a,8b,9,12,12a,13,13a,13b-dodecahy-
dro-1H-naphtho[2′,1′:4,5]indeno[1,2-dpyrimidine-4,10-diol (6a)
White powder from absolute ethanol, yield 0.38 g (94.5%); mp
150–152 C; IR (KBr, cmꢀ 1):
ν
3751 (NH), 3051 (2OH), 2930 (CH3),
◦
C
27H33N3O2 (431.26): C, 75.14; H, 7.71; N, 9.74%; found: C, 75.19; H,
1
–
–
–
–
7.78; N, 9.78%
2868 (CH ), 1717 (C N), , 1618 (C C); H NMR (DMSO – d ppm): δ =
2
6
0.809 (s, 3H, 18-CH3), 0.809–2.740 (m, estrane moiety), 1.292 (m, 1H,
14-CH), 1.465 (m, 1H, 16-CH), 2.487 (m, 2H, 15-CH2), 2.490 (d, 1H, 17-
CH), 2.494 (d, 1H, 22-CH), 4.507 (s, 1H, NH, D2O exchangeable), 7.028
(d, 2H, phenyl group), 7.325 (m, 3H, phenyl group), 9.011 (s, 1H, OH,
D2O exchangeable), 11.051 (s, 1H, OH, D2O exchangeable); MS (EI) m/z
(%): 402 [M+4, 5], 325 (25), 269 (100), 228 (54); Calc. for C26H30N2O2
(402.54); c, 77.58; H, 7.51; N, 9.96%; found: C, 77.61; H, 7.54; N, 9.99%
12-(4-fluorophenyl)-8a-methyl-
2.1.4. General procedure for synthesis of compounds 5a, 5b and 5c
To a solution of a product 4a, 4b or 4c (0.200, 0.209 or 0.215 g, 2
mmol) in absolute ethanol (25–30 mL), phenacylbromide (0.99 g, 2
mmol) was added. The reaction mixture, in each case was heated under
reflux for 3 hrs until the starting materials were disappeared as indicated
by TLC. The reaction mixture was treated with ice/water mixture. The
formed solid product was collected by filtration and crystallized from
absolute ethanol.
2,6b,7,8,8a,8b,9,12,12a,13,13a,13b,dodecahydro-1H-naphtho
8a-methyl-10,14-diphenyl-2,6b,7,8,8a,8b,12,14,14a,15,15a,15b-
dodechyro-1H-imidazo[1,2-a]naphtha[2′,1′:4,5]indeno[2,1-e]pyr-
imidin-4-ol (5a)
[2′,1′:4,5]indeno[1,2-d]pyrimidine-4,10-diol (6b)
White powder from absolute ethanol, yield 0.25 g (59.5%); mp
210–212 ◦C; IR (KBr, cmꢀ 1):
ν 3751 (NH), 3302 (2OH), 2929 (CH3), 2868
3

D.S. Nada et al.
Steroids 169 (2021) 108813
1
(C N), 1619 (C C), 1155 (C O); 1H NMR (DMSO – d6, ppm): δ =
–
–
–
–
–
–
–
–
–
(CH ), 1717 (C N), 1620 (C C); H NMR (DMSO – d , ppm): δ = 0.801
2
6
(s, 3H, 18-CH3), 0.801–2.732 (m, estrane moiety), 1.286 (m, 1H, 14-
CH), 1.461 (m, 1H, 16-CH), 2.482 (m, 2H, 15-CH2), 2.485 (d, 1H, 17-
CH), 2.46 (d, 1H, 22-CH), 4.503 (s, 1H, NH, D2O exchangeable),
7.025 (d, 2H, phenyl group), 7.313 (d, 2H, phenyl group), 9.003 (s, 1H,
OH, D2O exchangeable), 11.031 (s, 1H, OH, D2O exchangeable); MS (EI)
m/z (%): 420[M+3, 34%], 346 (15), 313 (5); Calc for C26H29FN2O2
(420.22); c, 74.26; H, 6.26; F, 4.52; N, 6.66%; found: C, 74.21; H, 6.92;
F,4.47; N, 6.62%
0.812 (s, 3H, 18-CH3), 0.812–2.740 (m, estrane moiety), 1.326 (m, 1H,
14-CH), 1.341 (m, 1H, 16-CH), 3.324 (s, 3H, 29- CH3OH), 7.029 (s, 1H,
21-CH), 7.037 (d, 2H, phenyl group), 7.046 (d, 2H, phenyl group), 7.210
(m, 3H, phenyl group), 7.221 (d, 2H, phenyl group), 9.009 (s, 1H, OH,
D2O exchangeable); MS (EI) m/z (%): 532[M+3, 15], 400 (5), 357 (3),
293 (100), 254 (4); calc. for C35H36N2O3 (534.28): C, 78.92; H, 6.81; N,
5.26%; found: C, 78.97; H, 6.77; N, 5.25%
12-(4-methoxyphenyl)-8a-methyl-
2.1.7. Synthesis of 4-hydroxy-8a-methyl-9-phenyl-
2,6b,7,8,8a,8b,9,12,12a,13,13a,13b,dodecahydro-1H-naphtho[2′,1′:4,5]
indeno[1,2-d]pyrimidine-4,10-diol (6c)
1,2,6b,7,8,8a,9,12,12a,12b-decahydro-10H-naphtho[2′,1′:4,5]indeno
[1,2-d]thiazole-10-thione (8)
Pale brown powder from absolute ethanol, yield 0.29 g (67.13%); mp
To a solution of estrone(1) (0.135 g, 2 mmol) in absolute ethanol,
Sulphur (0.016 g, 2 mmol), phenylisothiocyanate (0.0675 g, 2 mmol)
and drop wise of triethylamine were added, the reaction mixture was
heated under reflux for 12 hrs until all the starting materials were dis-
appeared as indicated by TLC. The reaction mixture was treated with
ice/water mixture. The formed solid product was collected by filtration
and crystallized from absolute ethanol to form pale brown powder. yield
233–235 ◦C; IR (KBr, cmꢀ 1):
ν
3752 (NH), 3316 (2OH), 2928 (CH3), 2863
(CH ), 1718 (C N), 1621 (C C); MS (EI) m/z (%): 432[M+1, 14%], 350
–
–
–
–
2
(25), 348 (3); 1H NMR (DMSO – d6, ppm): δ = 0.813 (s, 3H, 18-CH3),
0.813–2.754 (m, estrane moiety), 1.291 (m, 1H, 14-CH), 1.464 (m, 1H,
16-CH), 2.493 (m, 2H, 15-CH2), 2.491(d, 1H, 17-CH), 2.49 (d, 1H, 22-
CH), 4.598 (s, 1H, NH, D2O exchangeable), 3.124 (s, 3H, CH3OH),
7.021(d, 2H, phenyl group), 7.318 (d, 2H, phenyl group), 9.003 (s, 1H,
OH, D2O exchangeable), 11.112 (s, 1H, OH, D2O exchangeable); MS (EI)
m/z (%): 432[M+1, 14%], 350 (25), 348 (3); Calc. for C27H32N2O3
(432.24); c, 74.97; H, 7.46; N, 6.48%; found: C, 74.92; H, 7.41; N, 6.43%
0.201 g (96.17%); mp 130–132 ◦C; IR (KBr, cmꢀ 1):
ν 3295 (OH), 2931
1
–
–
–
(CH ), 2869 (CH ), 1717 (C S), 1594 (C C); H NMR (DMSO – d ,
–
ppm): δ = 0.811 (s, 3H, 18-CH3), 0.811–2.741 (m, estrane moiety6),
1.292 (m, 1H, 14-CH), 2.491 (d, 2H, 15-CH2), 7.029 (d, 2H, phenyl
group), 7.325 (m, 2H, phenyl group), 9.011 (s, 1H, OH, D2O
exchangeable); MS (EI) m/z (%): 419 [M+1, 10], 403 (2), 368 (5), 284
(96), 226 (8), 213 (15); Calc for C25H25NOS2 (419.14): C,17.56; H, 6.01;
N, 3.34; S, 15.28%; found: C, 17.66; H, 6.11; N, 3.44; 15.32%
3
2
2.1.6. General procedure for synthesis of compounds 7a, 7b and 7c
To a solution of 6a, 6b or 6c (0.1005, 0.105 or 0.108 g, 4 mmol) in
absolute ethanol (25–30 mL), phenacylbromide (0.049 g, 4 mmol) was
added. The reaction mixture, in each case, was heated under reflux for 4
hrs until the starting materials were disappeared as indicated by TlC.
The reaction mixture was treated with ice/water mixture. The formed
solid product was collected by filtration and crystallized from absolute
ethanol
2.1.8. Synthesis of 16-bromo-3-hydroxy-13-methyl-
6,7,8,9,11,12,13,14,15,16-decahydro-17H-cyclopenta[a]phenanthren-17-
one (9)
To a solution of estrone (1) (0.135 g, 2 mmol) in absolute ethanol,
cupper bromide (0.112 g, 2 mmol) was added, the reaction mixture was
heated under reflux for 2 hrs until all the starting materials were dis-
appeared as indicated by TLC. The reaction mixture was treated with
ice/water mixture. The precipitate was filtered off and crystalized from
absolute ethanol as pale green powder, yield 0.171 g(98.27%); mp
8a-methyl-10,14-diphenyl-1,2,6b,7,8,8a,8b,14,14a,15,15a,15b-
dodecahydronaphtho[2′,1′:4,5]indeno[2,1-e]oxazolo[3,2-a]pyrimidin-
4-ol (7a)
White powder from absolute ethanol, yield 0.05 g (39.8%); mp210-
212 C; IR (KBr, cmꢀ 1):
ν 3409 (OH), 2929 (CH3), 2868 (CH2), 1718
◦
(C N), 1595 (C C), 1199 (C O); 1H NMR (DMSO – d6, ppm): δ =
176–178 ◦C; IR (KBr, cmꢀ 1):
ν
3403 (OH), 2928 (CH3), 2866 (CH2), 1722
–
–
–
–
–
1
–
–
–
0.811 (s, 3H, 18-CH3), 0.811–2.742 (m, estrane moiety), 1.340 (m, 1H,
14-CH), 1.361 (m, 1H, 16-CH), 2.487 (m, 2H, 15-CH2), 2.485 (d, 1H, 17-
CH), 2.493 (d, 1H, 25-CH), 7.029 (s, 1H, 21-CH), 7.038 (m, 3H, phenyl
group), 7.029 (s, 1H, 21-CH), 7.038 (m, 3H, phenyl group), 7.046 (d,
2H, phenyl group), 7.220 (m, 3H, phenyl group), 7.224 (d, 2H, phenyl
group), 9.011 (s, 1H, OH, D2O exchangeable); MS (EI) m/z (%): 502
[M+2, 12], 425 (0.5), 370 (0.7), 357 (15), 254 (3); Calc. for C34H34N2O2
(502.26): C, 81.24; H, 6.28; N, 5.57; %; found: C, 81.18; H, 6.23; N, 5.52;
%
(C O), 1612 (C C); H NMR (DMSO – d , ppm): δ = 0.802 (s, 3H, 18-
–
6
CH3), 0.802–2.490 (m, estrane moiety), 1.340 (m, 1H, 14-CH), 2.490
(m, 2H, 15-CH2), 5.1 (t, 1H, 16-CH), 9.852 (s, 1H, OH, D2O exchange-
able); MS (EI) m/z (%): 349 [M+1, 29%), 334 (5), 290 (8), 269 (10); Calc.
for C18H21BrO2 (349.27): C, 61.90; H, 6.06; Br, 22.88%; found: C, 61.86;
H, 6.02; Br, 22.83%
2.1.9. General procedure for synthesis of compounds 10a, 10band 10c
A mixture of (9) (0.116 g, 3 mmol), thiourea (0.025 g, 3 mmol),
guanidine (0.019 g, 3 mmol) or Urea (0.02 g, 3 mmol) and triethylamine
(2–3 drops) in absolute ethanol (20–30 mL). The reaction mixture was
heated and refluxed for 3–5 hrs until all the starting materials were
disappeared as indicated by TLC. The reaction mixture was treated with
ice/water mixture. The precipitate was filtered off and crystallized from
absolute ethanol.
14-(4-fluorophenyl)-8a-methyl-10-phenyl-
1,2,6b,7,8,8a,8b,14,14a,15,15a,15b-dodecahydronaphtho[2′,1′:4,5]
indeno[2,1-e]oxazolo[3,2-a]pyrimidin-4-ol (7b)
White powder from absolute ethanol, yield 0.178 g (57.4%); mp
232–234 ◦C; IR (KBr, cmꢀ 1):
ν
3396 (OH), 2928 (CH3), 2867 (CH2), 1718
1
–
–
–
–
(C N), 1622 (C C), 1155 (C O); H NMR (DMSO – d6 ppm): δ = 0.812
–
(s, 3H, 18-CH3), 0.811–2.740 (m, estrane moiety), 1.341 (m, 1H, 14-
CH), 1.359 (m, 1H, 16-CH), 2.068 (m, 2H, 15-CH2), 2.490 (d, 1H, 17-
CH), 2.740 (d, 1H, 25-CH), 7.029 (s, 1H, 21-CH), 7.037 (d, 2H, phenyl
group), 7.046 (d, 2H, phenyl group), 7.210 (m, , 3H, phenyl group),
7.223 (d, 2H, phenyl group), 9.012 (s, 1H, OH, D2O exchangeable); MS
(EI) m/z (%): 520 [M+, 10], 425 (12), 400 (15), 357 (5); Calc. for
10-amino-8a-methyl-2,6b,7,8,8a,12,12a,12b-octahydro-1H-naph-
tho[2′,1′:4,5]indeno[1,2-d]thiazol-4-ol (10a)
Yellow crystals from absolute ethanol, yield 0.152 g (93.25%); mp
42–44 ◦C; IR (KBr, cmꢀ 1):
ν
3411 (NH2), 3217 (OH), 2928 (CH3), 2865
1
–
–
(CH ), 1719 (C N), 1596 (C C); H NMR (DMSO – d , ppm): δ = 0.804
–
–
(s, 32H, 18-CH3), 0.804–2.490 (m, estroid moiety), 1.457 (m, 1H, 14-CH),
2.490 (d, 2H, 15-CH2), 4.510 (s, 2H, NH2, D2O exchangeable), 9.008 (s,
1H, OH, D2O exchangeable); MS (EI) m/z (%): 326 [M+, 25%], 293 (5),
268 (7), 214 (10); Calc. for C19H22N2OS (326.15): C, 73.76; H, 7.49; N,
13.58%; found: C, 73.71; H, 7.43; N, 13.54%
6
C
34H33FN2O2 (520.25): C, 78.44; H, 6.39; F, 3.65; N, 5.38%; found: C,
78.48; H, 6.35; F, 3.69; N, 5.32%
14-(4-methoxyphenyl)-8a-methyl-10-phenyl-
1,2,6b,7,8,8a,8b,14,14a,15,15a,15b-dodecahydronaphtho[2′,1′:4,5]
indeno[2,1-e]oxazolo[3,2-a]pyrimidin-4-ol (7C)
White powder from absolute ethanol, yield 0.06 g (45.11%); mp
10-amino-8a-methyl-1,2,6b,7,8,8a,11,12,12a,12b-decahydronaph-
tho[2′,1′:4,5]indeno[1,2-d]imidazole-4-ol (10b)
244–246 ◦C; IR (KBr, cmꢀ 1):
ν
3307 (OH), 2928 (CH3), 2868 (CH2), 1717
Yellow crystals from absolute ethanol, yield 0.09 g (87.37%); mp
4

D.S. Nada et al.
Steroids 169 (2021) 108813
44–46 ◦C; IR (KBr, cmꢀ 1):
ν
3752 (NH2), 3217 (NH), 3046 (OH), 2926
disappeared as indicated by TLC. The reaction mixture was treated with
ice/water. The formed solid of each product was collected by filtration
and crystallized from ethanol.
1
–
–
–
(CH ), 2862 (CH ), 1721 (C N), 1596 (C C); H NMR (DMSO – d ,
–
ppm): δ = 0.804 (s, 3H, 18-CH3), 0.804–2.730 (m, estrane moiety6),
2.490 (m, 1H, 14-CH), 2.730 (d, 2H, 15-CH), 7.631 (s, 2H, NH2, D2O
exchangeable), 9.013 (s, 1H, OH, D2O exchangeable), 11.058 (s, 1H,
NH, D2O exchangeable); MS (EI) m/z (%): 309 [M+, 3], 294 (5), 266 (4);
Calc. for C19H23N3O (309.41): C, 69.90; H, 6.79; N, 8.58; S, 9.82%;
found: C, 69.94; H, 6.76; N, 8.53; S, 9.78%
3
2
10-amino-4-hydroxy-8a-methyl-12-phenyl-
1,2,6b,7,8,8a,12,13,13a,13b-decahydronaphtho[2′,1′:4,5]indeno[1,2-
b]pyran-11-carbonitrile (13)
Pale brown powder from absolute ethanol, yield 0.17 g (41%): mp
216–218 ◦C; IR (KBr, cmꢀ 1):
ν 3303 (NH2), 3022 (OH), 2931 (CH3),
–
1
–
– –
2868 (CH2), 2180 (C N), 1619 (C C), 1288 (C O); H NMR (DMSO –
–
–
10-amino-8a-methyl-1,2,6b,7,8,8a,12,12a,12b-octahydro-1H-naph-
tho[2′,1′:4,5]indeno[1,2-d]oxazol-4-ol (10c)
d6, ppm): δ = 0.826 (s, 3H, 18-CH3), 0.826–2.752 (m, estrane moiety),
1.355 (m, 1H, 14-CH), 2.091 (d, 2H, 15-CH2), 3.298 (s, 1H, 22-CH),
6.453 (s, 1H, NH2, D2O exchangeable), 7.032 (d, 2H, phenyl group),
7.060 (m, 3H, phenyl group), 8.977 (s, 1H, OH, D2O exchangeable); MS
(EI) m/z (%): 395[M+, 12%], 369 (5), 398 (15); Calc. for C28H28,N2O2
(424.54): C, 79.22; H, 6.65; N, 6.60%; found: C, 79.18; H, 6.61; N, 6.56%
10-amino-4-hydroxy-8a-methyl-12-phenyl-
Yellow crystals from absolute ethanol, yield 0.097 g (94.17%); mp
46–48 ◦C; IR (KBr, cmꢀ 1):
ν
3418 (NH2), 3052 (OH), 2926 (CH3), 2861
1
–
–
–
–
(CH ), 1722 (C N), 1591 (C C), 1292 (C O); H NMR (DMSO – d6,
–
2
ppm): δ = 0.801 (s, 3H, 18-CH3), 0.801–2.725 (m, estrane moiety),
2.065 (m, 1H, 14-CH), 2.490 (d, 2H, 15-CH2), 7.325 (s, 2H, NH2, D2O
exchangeable), 9.850 (s, 1H, OH, D2O exchangeable); MS (EI) m/z (%):
293 [M+, 5], 228 (7); Calc. for C19H22N2O2 (310.40): C, 73.52; H, 7.14;
N, 9.03%; found: C, 73.55; H, 7.19; N, 8.07%
2,6b,7,8,8a,9,10,11,12,13,13a,13b-dodecahydro-1H-naphtho[2′,1′:4,5]
indeno[1,2-b]pyridine-11-carbonitrile (14)
Pale yellow powder from absolute ethanol, yield 0.423 g (94.5%):
2.1.10. Synthesis of 2-(4-hydroxy-8a-methyl-2,6b,7,8,8a,12,12a,12b-
octahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d]thiazol-10-yl)acetonitrile
(11)
mp 194–196 ◦C; IR (KBr, cmꢀ 1):
ν 3450 (NH2), 3316 (NH), 3027 (OH),
1
–
–
–
2930 (CH3), 2868 (CH2), 2221 (C N), 1619 (C C); H NMR (DMSO –
–
–
d6, ppm): δ = 0.822 (s, 3H, 18-CH3), 0.822–2.760 (m, estrane moiety),
1.351 (m, 1H, 14-CH), 2.493 (s, 1H, NH, D2O exchangeable), 2.498 (m,
2H, 15-CH2), 2.503 (d, 1H, 21-CH), 2.760 (d, 1H, 22-CH), 3.295 (d, 1H,
20-CH), 7.623 (d, 2H, phenyl group), 7.676, 7.967 (m, 3H, phenyl
group), 8.549 (s, 2H, NH2, D2O exchangeable), 8.975 (s, 1H, OH, D2O
exchangeable); MS (EI) m/z (%): 425[M+, 100], 144 (5); Calc. for
To a solution of (9) (0.1159 g, 3 mmol)in absolute ethanol, cyano-
thioacetamide (0.03 g, 3 mmol) was added, The reaction mixture was
heated under reflux for 4–5 hr until all the starting materials were dis-
appeared as indicated by TLC. The reaction mixture was treated with
ice/water mixture. The formed solid product was collected by filtration
and crystallized from ethanol to form dark brown powder,yield0.110 g
C28H31N3O (425.58): C, 79.02; H, 7.34; N, 9.87%; found: C, 78.97; H,
(86.2%); mp over 300 C; IR (KBr, cmꢀ 1):
ν
3416 (OH), 2926 (CH3),
7.31; N, 9.81%
◦
–
1
–
–
–
–
–
2862 (CH2), 2203 (C N), 1720 (C N), 1622 (C C); H NMR (DMSO –
–
d6, ppm): δ = 0.800 (s, 3H, 18- CH3), 0.800–2.724 (m, estrane moiety),
2.299 (m, 1H, 14-CH), 2.724 (m, 2H, 15-CH), 3.335 (s, 2H, 22-CH2,
acetonitrile), 9.013 (s, 1H, OH, D2O exchangeable); MS (EI) m/z (%):
350[M+,5], 335 (12); 294 (1.12), 292 (2.8); Calc. for C21H22N2OS
(350.48): C, 71.97; H, 6.33; N, 7.99; S, 9.15%; found: C, 71.92; H, 6.30;
N, 7.94; S, 8.98%
2.1.13. General procedure for synthesis of compounds 15a, 15b and 15c
To a solution of 14 (0.1057 g, 4 mmol) in absolute ethanol (30 mL),
formic acid (0.0115 g, 4 mmol), acetic acid (0.015 g, 4 mmol) or
chloroacetate (0.0236 g, 4 mmol) was added. The reaction mixture in
each case was heated under reflux for 4–6 hrs until the starting materials
were disappeared as indicated by TLC. The reaction mixture was treated
with ice/water mixture and neutralized by KOH, drop by drop in to ice
water. The formed solid product was collected by filtration and crys-
tallized from absolute ethanol
2.1.11. Synthesis of (E)-N-benzyl-4-hydroxy-8a-methyl-
2,6b,7,8,8a,12,12a,12b-octahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d]
thiazole-10-carbohydrazonoyl cyanide (12)
4-hydroxy-8a-methyl-14-phenyl-1,2,6a,7,8,8a,9,12,14,15,15a,15b-
dodecahydro-13H-naphtho[2′′,1′′:4′,5′]indeno[2′,1′:5,6]pyrido[2,3-d]pyr-
imidin-13-one (15a)
By using 3 tubes (Azo dye test), in the first tube solid salt 11 (0.1167
g, 3 mmol) and sodium hydroxide solution 0.0133 g, 3 mmol) in water,
in the second tube sodium nitrate (0.023 g, 3 mmol), in the third tube
aniline hydrochloride (0.043 g, 3 mmol), The three tubes are dunked
inice path for 10–15 min., the three tubes are poured in each other
respectively, The mixture was treated with ice/water mixture. The
formed product was extracted by chloroform (45 mL), sodium hydrogen
carbonate was added and the mixture was stirred for 45 min and filtered.
The chloroform layer was dried with anhydrous calcium chloride and
evaporated till dryness as brown gum, yield 0145 g(92.94%); Oily
Dark gray powder from absolute ethanol, yield 0.06 g (53%): mp
208–210 0C; IR (KBr, cmꢀ 1):
ν
3321 (NH), 3024 (OH), 2931 (CH3), 2864
1
–
–
–
–
(CH ), 1719 (C O), 1622 (C N), 1597 (C C);; H NMR (DMSO – d ,
–
–
ppm): δ = 0.824 (s, 3H, 18-CH3), 0.824–2.762 (m, estrane moiety6),
1.327 (m, 1H, 14-CH), 2.505 (d, 2H, 15-CH2), 4.433 (s, 1H, 26-CH), 6.53
(s, 1H, NH, D2O exchangeable), 7.031 (m, 1H, phenyl group), 7.503 (d,
2H, phenyl group), 7.509 (m, 2H, phenyl group), 7.562 (s, 1H, NH, D2O
exchangeable), 8.977 (s, 1H, OH, D2O exchangeable); MS (EI) m/z (%):
451[M+, 40], 408 (15), 396 (23), 342 (2), 324 (15); Calc. for
C29H29N3O2 (451.57): C, 77.14; H, 6.47; N, 9.31; O, 7.09%; found: C,
77.10; H, 6.43; N, 9.31%; found: C, 77.10; H, 6.43; N, 9.28%
4-hydroxy-8a,11-dimethyl-14-phenyl-
2
product; IR (KBr, cmꢀ 1):
ν
–
–
3433 (NH), 3201 (OH), 2362 (C N), 1940
–
(C S), 1726 (C N), 1597 (C C); 1H NMR (DMSO – d6, ppm): δ =
–
–
–
–
–
–
8.810 (s, 3H, 18-CH3), 0.810–2.497 (m, estrane moiety), 2.013 (m, 1H,
14-CH), 2.234 (d, 2H, 15-CH2), 3.319 (d, 2H, 25- CH2), 7.020 (d, 2H,
phenyl group), 7.358(m, 3H, phenyl group), 7.419 (d, 1H, NH, D2O
exchangeable), 9.003 (s, 1H, C-3, OH, D2O exchangeable); MS (EI) m/z
(%): 452[M+, 20%], 377 (13), 321(7); Calc. for C28H28N4OS (468.62): C,
71.77; H, 6.02; N, 11.96; S, 6.84%; found: C, 71.75; H, 6.01; N,11.94; S,
6.81%
1,2,6a,7,8,8a,9,12,14,15,15a,15b-dodecahydro-13H-naphtho[2′′,1′′:4′,5′]
indeno[2′,1′:5,6]pyrido[2,3-d]pyrimidin-13-one (15b)
Gray powder from absolute ethanol, yield 0.11 g (94%): mp
204–206 ◦C; IR (KBr, cmꢀ 1):
ν
3751 (NH), 3412 (OH), 2929 (CH3), 2868
1
–
–
–
–
–
(CH ), 1721 (C O), 1631 (C N), 1598 (C C); H NMR (DMSO – d ,
–
2
ppm): δ = 0.804 (s, 3H, 18-CH3), 0.804–2.732 (m, estrane moiety6),
1.292 (m, 1H, 14-CH), 2.499 (s, 3H, 22-CH3), 2.732 (d, 2H, 15-CH2),
4.203 (s, 1H, 26-CH), 6.653 (s, 1H, NH, D2O exchangeable), 7.031 (m,
1H, phenyl group), 7.045 (d, 2H, phenyl group), 7.059 (m, 2H, phenyl
group), 7.552 (s, 1H, NH, D2O exchangeable), 8.990 (s, 1H, OH, D2O
exchangeable); MS (EI) m/z (%): 465[M+, 33], 462 (67), 428 (100), 423
(5), 355 (28), 339, (67); Calc. for C30H31N3O2 (465.24): C, 77.39; H,
2.1.12. General procedure for synthesis of compounds 13, 14
A mixture of estrone1 (0.270 g, 1 mmol), benzaldehyde (0.106 g, 1
mmol) and malononitrile (0.066 g, 1 mmol) in absolute ethanol (30 mL)
in the presence of piperdine (0.085, 1 mmol) or 20% excess of ammo-
nium acetate (0.0785 g, 1 mmol + 20%, 0.0157 g). The reaction mixture
was heated under reflux for 4–5 hrs until all the starting materials were
5

D.S. Nada et al.
Steroids 169 (2021) 108813
1
–
6.71; N, 9.03%; found: C, 77.34; H, 6.68; N, 8.97%
1622 (C C); H NMR (DMSO – d ppm): δ = 0.820 (s, 3H, 18-CH ),
–
6
11-(chloromethyl)-4-hydroxy-8a-methyl-14-phenyl-
0.820–2.780 (m, estrane moiety), 1.326 (m, 1H, 14-CH), 2.730 (d, 23H,
15-CH), 3.300 (s, 6H, 2Me), 4.321 (s, 1H, 22-CH), 7.028 (m, 1H, phenyl
group), 7.056 (d, 2H, phenyl group), 7.134 (m, 2H, phenyl group), 8.012
(S, 1H, 24-CH), 8.537 (S, 1H, NH, D2O exchangeable), 8.975 (s, 1H, C-3,
OH, D2O exchangeable); MS (EI) m/z (%): 478[M+, 5], 422 (15), 385
(13), 341 (12), 325 (65); Calc for C31H34N4O (478.64): C, 77.79; H, 7.16;
N, 11.71%; found: C, 77.74; H, 7.12; N, 11.67%
1,2,6a,7,8,8a,9,12,14,15,15a,15b-dodecahydro-13H-naphtho[2′′,1′′:4′,5′]
indeno[2′,1′:5,6]pyrido[2,3-d]pyrimidin-13-one (15c)
Orange powder from absolute ethanol, yield 0.11 g (88%): mp
210–212 ◦C; IR (KBr, cmꢀ 1):
ν
3751 (NH), 3316 (OH), 2928 (CH3), 2867
1
–
–
–
–
(CH ), 1718 (C O), 1619 (C N), 1597 (C C); H NMR (DMSO – d ,
–
–
ppm): δ = 0.811 (s, 3H, 18-CH3), 0.811–2.740 (m, estrane moiety6),
1.341 (m, 1H, 14-CH), 2.490 (d, 2H, 15-CH2), 4.302 (s, 2H, 27-CH2,
chloromethyl), 4.621 (s, 1H, 26-CH), 7.424 (s, 1H, NH, D2O exchange-
able), 7.031 (m, 1H, phenyl group), 7.045 (d, 2H, phenyl group), 7.059
(m, 2H, phenyl group), 8.057 (s, 1H, NH, D2O exchangeable), 9.013 (s,
1H, OH, D2O exchangeable); MS (EI) m/z (%): 500[M+, 25%], 282 (12);
Calc. for C30H30ClN3O2 (500.04): C, 72.06; H, 6.05; Cl, 7.09; N, 8.40%;
found: C, 72.09; H, 6.09; Cl, 7.04; N, 8.35%
2
2.1.16. Synthesis of ethyl (z)-N-(11-cyano-4-hydroxy-8a-methyl-12-
phenyl-2,6b,7,8,8a,9,12,13,13a,13b-decahydro-1H-naphtho[2′,1′:4,5]
indeno[1,2-b]pyridin-10-yl)formimidate (18)
To a solution of 14 (0.1057 g, 4 mmol) and triorthoformate (0.037 g,
4 mmol) in absolute ethanol in the presence of acetic anhydride, The
reaction mixture was heated under refluxed for 5–7 hrs until all the
starting materials were disappeared as indicated by TLC. The reaction
mixture was treated with ice/water mixture. The precipitate was filtered
2.1.14. General procedure for synthesis of compounds 16a and 16b
To a solution of 14 (0.105 g, 4 mmol) in (ethanol: 10% potassium
hydroxide), carbon disulphide was added in the rate of (1:1) (0.105 g, 4
mmol:0.028 g, 4 mmol) or (1:2) (0.105 g, 4 mmol : 0.038 g , 2 mmol).
The reaction mixture was heated under reflux for 2–4 hrs until all the
starting materials were disappeared as indicated by TLC. The reaction
mixture was treated with ice/water. The formed solid of each product
was collected by filtration and crystallized from absolute ethanol.
4-hydroxy-8a-methyl-14-phenyl-11-thio-
off and crystallized from absolute ethanol as off white powder.
yield0.05 g (41.84%); mp 240–242 C; IR (KBr, cmꢀ 1):
ν 3751 (NH),
◦
–
–
–
3309 (OH), 2928 (CH3), 2864 (CH2), 2210 (C N), 1719 (C N), 1618
–
–
1
–
–
–
(C C), 1246 (C O); H NMR (DMSO – d6, ppm): δ = 0.811 (s, 3H, 18-
CH3), 0.811–2.739 (m, estrane moiety), 1.359 (m, 1H, 14-CH), 1.323 (t,
3H, 26-CH3), 2.490 (d, 2H, 15-CH2), 3.323 (q, 2H, 25-CH2), 4.321 (s, 1H,
22-CH), 7.028 (m, 1H, phenyl group), 7.458 (d, 2H, phenyl group),
7.473 (m, 2H, phenyl group), 7.498 (S, 1H, 24-CH), 7.511 (S, 1H, NH,
D2O exchangeable), 9.007 (s, 1H, C-3, OH, D2O exchangeable); MS (EI)
m/z (%): 479[M+2, 3], 403 (5); Calc for C31H33N4O2 (479.62): C, 77.63;
H, 6.94; N, 8.76%; found: C, 77.59; H, 6.40; N, 8.72%
1,2,6a,7,8,8a,9,10,11,12,14,15,15a,15b-tetradecahydro-13H-naphtho
[2′′,1′′:4′,5′]indeno[2′,1′:5,6]pyrido[2,3-d]pyrimidin-13-one (16a)
White powder from absolute ethanol, yield 0.067 g (55.8%): mp
224–226 ◦C; IR (KBr, cmꢀ 1):
ν
3855–3751 (3NH), 3309 (OH), 2930
1
–
–
–
–
(CH ), 2867 (CH ), 1718 (C S), 1620 (C N), 1598 (C C); H NMR
2.1.17. Synthesis of 12-amino-13-imino-8a-methyl-14-phenyl-
–
–
(DMSO – d6 ppm2): δ = 0.824 (s, 3H, 18-CH3), 0.824–2.751 (m, estrane
moiety), 1.328 (m, 1H, 14-CH), 2.353 (d,2H, 15-CH), 4.43 (s, 1H, 26-
CH), 5.83 (s, 1H, NH, D2O exchangeable), 6.526 (s, 1H, NH, D2O
exchangeable), 7.029 (m, 1H, phenyl group), 7.057 (d, 2H, phenyl
group), 7.324 (m, 2H, phenyl group), 7.442 (s, 1H, NH, D2O
exchangeable), 9.008 (S, 1H, OH, D2O exchangeable); MS (EI) m/z (%):
483[M+, 10%], 422 (48), 402 (20), 321 (7); Calc. for C29H29N3O2S
(483.63): C, 72.02; H, 6.04; N, 8.69; O, 6.62; S, 6.63%; found: C, 71.98;
H, 6.01; N, 8.64; O, 6.57; S, 6.60%
2,6b,7,8,8a,9,12,13,14,15,15a,15b- dodecahydro-1H-naphtho
3
[2′′,1′′:4′,5′]indeno[2′,1′:5,6]pyrido[2,3-d]pyrimidin-4-ol (19)
To a solution of 18 (0.119 g, 4 mmol) in absolute ethanol hydrazine
hydrate (0.008 g, 4 mmol) was added, The reaction mixture was heated
under reflux for 2–3 hrs until all the starting materials were disappeared
as indicated by TLC. The reaction mixture was treated with ice/water
mixture. The formed solid was collected by filtration and crystallized
from absolute ethanol as beige powder, yield 0.1 g (86.2%); mp
220–222 ◦C; IR (KBr, cmꢀ 1):
ν 3743 (NH2), 3302 (NH), 3114 (OH), 2928
–
–
–
4-hydroxy-8a-methyl-14-phenyl-1,2,6a,7,8,8a,9,10, 14,15,15a,15b-
dodecahydro-11H-naphtho[2′′,1′′:4′,5′]indeno[2′,1′:5,6]pyrido[2,3-d]pyr-
imidin-11,13(12H)-dithione (16b)
(CH ), 2859 (CH ), 1717 (C N), 1618 (C C); MS (EI) m/z (%): 465
–
3 2
[M+1, 47], 424 (70), 400 (65), 375 (5); Calc. for C29H31N5O (465.6): C,
74.81; H, 6.71; N, 15.04%; found: C, 74.78; H, 6.66; N, 15.01%
White powder from absolute ethanol, yield 0.053 g (64.12%): mp
220–222 ◦C; IR (KBr, cmꢀ 1):
ν 3855–3758 (3NH), 3022 (OH), 2929
2.2. In vitro cytotoxic assay
(CH ), 2867 (CH ), 1717 (C S), 1618 (C N), 1599(C C); 1H NMR
–
–
–
–
–
–
3
2
(DMSO – d6, ppm): δ = 0.822 (s, 3H, 18-, CH3), 0.824–2.751 (m, estrane
moiety), 1.329 (m, 1H, 14-CH), 2.730 (d,2H, 15-CH2), 4.321 (s, 1H, 26-
CH), 5.681 (s, 1H, NH, D2O exchangeable), 6.526 (s, 1H, NH, D2O
exchangeable), 7.028 (m, 1H, phenyl group), 7.056 (d, 2H, phenyl
group), 7.132 (m, 2H, phenyl group), 8.979 (s, 1H, NH, D2O
exchangeable), 9.071 (S, 1H, OH, D2O exchangeable); MS (EI) m/z (%):
422[M+, 5], 422 (5), 284 (7); Calc. for C29H29N3OS2 (499.69): C, 69.71;
H, 5.85; N, 8.41; O, 3.20; S, 12.83%; found: C, 69.66; H, 5.81; N, 8.37; O,
3.17; S, 12.79%
2.2.1. Cell propagation and maintenance
MCF-7 human breast cancer cells, PC3 human prostate cancer cells,
and HepG2 human liver cancer cells were obtained from VACSERA (The
holding company for biological products & vaccines) and maintained in
the proper conditions. The cells were cultured in RPMI (Roswell Park
Memorial Institute) medium supplemented by 2% serum (maintenance
medium) at 37˚C in a humidified incubator with 5% CO2. The cells
harvested after trypsinization (0.025% trypsin and 0.02% EDTA) and
washed twice with Dulbecco’s phosphate buffered saline (DPBS). When
the cell density reached approximately 80%, cells were split for further
culture. The experiments were made up when the cells were in the
logarithmic growth phase.
2.1.15. Synthesis of (z)-N-(11-cyano-4-hydroxy-8a-methyl-12-phenyl-
2,6b,7,8,8a,9,12,13,13a,13b-decahydro-1H-naphtho[2′,1′:4,5]indeno
[1,2-b]pyridin-10-yl)-N,N-dimethylformimidamide (17)
To a solution of 14 (0.1057 g, 4 mmol) and DMF-DMA (0.04 g, 4
mmol) in absolute ethanol in the presence of acetonitrile (0.01 g, 4
mmol), The reaction mixture was heated under reflux at 70 0C for 2 hrs
until all the starting materials were disappeared as indicated by TLC.
The reaction mixture was treated with ice/water mixture. The precipi-
tate was filtered off and crystallized from absolute ethanol as Pale brown
2.2.2. Cytotoxicity assay
Cell viability was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-
2,5 diphenyl tetrazolium bromide) assay. MTT assay is based on the
conversion of MTT into formazan crystals by living cells, which reflects
cytotoxicity based on mitochondrial activity [15]. The cytotoxic impact
of the tested compounds measured by MTT assay using MCF7, PC3 and
HepG2 cells respectively. The cells were incubated with various con-
centrations of the test compounds (31.25, 62.5, 125, 250, 500, 1000 µg/
powder. yield 0.1 g (83.68%); mp 230–232 ◦C; IR (KBr, cmꢀ 1):
ν 3393
–
–
–
(NH), 3311 (OH), 2929 (CH3), 2868 (CH2), 2372 (C N), 1718 (C N),
–
–
6

D.S. Nada et al.
Steroids 169 (2021) 108813
Scheme 1.
Scheme 2.
ml) for 24 h at a cell density of 104 cells/well of 96 well plate. After 24 h
incubation, MTT dissolved in PBS was added to each well at a final
concentration of 5 mg/ml, and the samples were incubated at 37 ◦C for
4 h. After 4 h, the medium was decanted and dimethyl sulfoxide (DMSO)
was added to each well and left for 30 min to dissolve formazan crystals
that formed during MTT cleavage in actively metabolizing cells.
Absorbance of formazan in each plate was measured at 570 nm, using a
microplate reader (Model 500; BIORad Instrument Inc., USA). Using the
relation between the used concentrations and the mitochondrial activity
(%), IC50 of the tested compound was calculated. For the untreated cells
(negative control), medium was added instead of the test compound. A
positive control Doxorubicin (Dox, Mr = 543.5) was used as a cytotoxic
natural agent giving 100% inhibition. Dimethyl sulfoxide (DMSO) was
the vehicle used for dissolution of tested compound and its final con-
centration on the cells was less than 0.2%. All tests and analyses were
done in triplicate and the results were averaged.
2.3. Statistical analysis
All the data are expressed as Mean ± standard error mean. Data were
calculated using Sigma plot ver. 125. Analysis of the data was done using
student t-test to detect the significant difference among the studied
compounds. Differences were considered to be statistically significant
when P less than 0.05.
7

D.S. Nada et al.
Steroids 169 (2021) 108813
Scheme 3.
3. Results and discussion
By the same way, the reaction of estrone (1) with benzaldehyde ,4-
fluorobenzaldehyde or 4-methoxybenzaldehyde and urea in the pres-
ence of absolute ethanol as a solvent produced pyrimidinylestrane de-
rivatives 6a, 6b and 6c respectively, which were reacted with
phenacylbromide to produce oxazolopyrimidinylestrone derivatives 7a,
7b and 7c (Scheme 4). When estrone (1) was allowed to react with
sulphur and phenyl isothiocyanate in refluxing absolute ethanol by
adding dropwise of triethylamine afforded thiazoloestrane derivative 8
(Scheme 4). Furthermore, estrone (1) was allowed to react with cupper
bromide in refluxing absolute ethanol to produce 16-bromo-estrone 9,
which allowed to react with thiourea, guanidine or urea in refluxing
absolute ethanol by adding 2–3 drops of triethylamine to produce
thiazolo, imidazolo and oxazoloestrane derivatives 10a, 10b and 10c
respectively, The 1HNMR and EIMS and all spectral data agreed well
with the assigned structures of the produced compounds (c.f experi-
mental part. When compound 9 was allowed to react with cyanothioa-
cetamide in boiling absolute ethanol produced the thiazoloestrane
derivative 11, which allowed to react with sodium hydroxide, sodium
nitrate and aniline hydrochloride (Azo dye test) to produce compound
12 (c.f experimental parts, Scheme 4).
3.1. Chemistry
Pyrimidine, thiazole, imidazole, pyran, pyridine and pyridopyr-
imidine rings represent molecular frameworks that serve as a platform
for developing pharmaceutical agents for various applications. Many
derivatives of these rings proved as antitumor agents [10–14]. The goal
of this research is to synthesis a variety of compounds in order to study
their function as anticancer agents, by using estrone (1) as starting
material. The development of the favored new compounds by the syn-
thetic pathways followed is shown in the schemes 1-5. When estrone (1)
was allowed to react with benzaldehyde, 4-fluorobenzaldehyde or 4-
mothoxy benzaldehyde and thiourea in refluxing absolute ethanol
afforded pyrimidinylestrane derivatives 2a, 2b and 2c respectively,
which reacted with phenacylbromide in equimolar amount by using
absolute ethanol as a solvent to afford thiazolopyrimidinylestrane de-
rivatives 3a, 3b and 3c respectively (Scheme l). The previous com-
pounds were determined by means of their IR, MS, 1HNMR and
analytical data. The IR spectra of the previous compounds revealed
–
–
absorption band at 1717, 1718 (C N) while compounds 2a, 2b and 2c
In this study, one pot multi-component reactions were attempted as a
straight forward method for the synthesis of heterocyclic estrane de-
rivatives. The reaction of estrone (1) with benzaldehyde and malono-
nitrile in refluxing absolute ethanol by adding piperidine or 20% excess
of ammonium acetate afforded aminopyranoestrane derivative 13 and
aminopyridinoestrane derivative 14 respectively (Scheme 5). The for-
mation of these derivatives can be explained by the possible mechanism
represented in our article Abd-Elhalim et al [16]. The IR spectra showed
strong absorption band at 3303, 3316 (NH2), 2180, 2221, (CN) respec-
tively, and 3750 (NH) of compound 14. The1HNMR and EIMS agreed
well with the assigned structure of experimental part. When compound
14 was allowed to react with formic acid, acetic acid or chloroacetate by
refluxing absolute ethanol produced pyridopyrimidinylestrane de-
rivatives 15a, 15b and 15c, respectively (c.f experimental part, Scheme
5). The reaction of compound 14 with equimolar amounts of carbon
disulfide or double molar of carbon disulfide in the presence of 10%
KOH with absolute ethanol produced compounds 16a and 16b respec-
tively. When compound 14 was allowed to react with DMF-DMA in the
revealed absorption bands at 3855, 3858 (SH) and 3343, 3751 (NH),
1HNMR and EIMS agreed well with the assigned structure of experi-
mental part (Scheme 1).
Similarly, the reaction of estrone (1) with benzaldehyde, 4 -fluo-
robenzaldehyde or 4- methoxy benzaldehyde and corresponding pyr-
imidinylestrane derivatives 4a, 4b and 4c which reacted with phenacyl
bromide in the presence of boiling absolute ethanol to produce imida-
zolopyrimidinylestrane derivatives 5a, 5b and 5c respectively, (Scheme
2). The inference of the produced estrone derivatives 4a , 4b and 4c can
be checked by IR, MS, 1HNMR spectra, The IR spectra showed the
appearance of the (NH2) band at 3214 and 3751 while (NH) band at
–
–
3117, 3345, 3678 and the absence of C O band in the IR spectra of
compound 1. The IR spectra of compounds 5a, 5b, and 5c also revealed
the presence of characteristic absorption bands corresponding to NH
group and the absence of NH2 band. Also all the analytical and spectral
data of these compounds are in accordance with the proposed structures
(c.f experimental part, Scheme 2).
8

D.S. Nada et al.
Steroids 169 (2021) 108813
Scheme 4.
9

D.S. Nada et al.
Steroids 169 (2021) 108813
Scheme 5.
10

D.S. Nada et al.
Steroids 169 (2021) 108813
Fig. 1. Effect of the tested compounds on the morphology of MCF-7 cells at different concentrations.
11

D.S. Nada et al.
Steroids 169 (2021) 108813
Fig. 2. Effect of the tested compounds on the morphology of PC3 cells at different concentrations.
12

D.S. Nada et al.
Steroids 169 (2021) 108813
Fig. 3. Effect of the tested compounds on the morphology of HepG2 cells at different concentrations.
13

D.S. Nada et al.
Steroids 169 (2021) 108813
Table 3
The in vitro cytotoxic activity of synthesized compounds on HepG2 cancer cell
line.
Compd.No
IC50 (µg/ml)
Compd.No
IC50 (µg/ml)
2a
479.83
357.08
24.64
91.28
59.42
19.07
98.55
10b
13
31.13
223.43
36.62
2b
2c
14
3b
16a
19
216.82
33.96
8
10a
Doxorubicin
Doxorubicin
98.55
IC50: Concentration required to inhibit cell viability by 50%.
presence of acetonitrile in refluxing absolute ethanol by heating at 70
0C, the compound 17 was afforded which established by means of their
spectral and analytical data. The reaction of compound 14 with tri-
orthoformate in absolute ethanol by refluxing in the presence of acetic
anhydride afforded compound 18 which reacted with hydrazine hydrate
in boiling absolute ethanol to produce pyridopyrimidinylestrane deriv-
ative 19.
3.2. Biological assays
3.2.1. In vitro evaluation of cytotoxic activity
The cytotoxic activity of the tested compounds 2a, 2b, 2c, 3b, 8,
10a, 10b, 13, 14, 16a and 19 were investigated individually as anti-
cancer agents a against human breast cancer MCF7, human prostate
cancer PC3 and human liver cancer HepG2 cells at different concen-
trations (31.25, 62.5, 125, 250, 500 and 1000 µg/ml) by using MTT
assay which is based on the diversion of MTT into formazan crystals by
living cells, that reflects cytotoxicity based on mitochondrial activity.
Data explained in (Figs. 1–3) showed average percentage of the toxicity
of cancer cells treated with different concentrations of these compounds
after 24 h. Doxorubicin used as reference drug with IC50 values of 93.08
µg/ml, 72.39 µg/ml and 98.55 µg/ml against the three tested cell lines
MCF7, PC3 and HepG2 respectively. The employment of DMSO as a
solvent had insignificant effect on MCF7, PC3 and HepG2 cells viability
when treated for 24 h. As compared to the control cells, all tested
compounds affected significantly on cell growth inhibition. Data in table
(1) and figures (4(A–C)) revealed that the cytotoxic activity of the tested
compounds was in the descending order of compound 10a > 10b > 14
> 19 > 2c > 8 > 16a > 2b > 3b > 13 > 2a against MCF-7 cancer cells.
At 24 h, compounds 10a, 10b, 14, 19, 2c, 8 and 16a (18.26, 18.27,
24.15, 26.61, 54.21, 77.12 and 92.52 µg/ml respectively) showed more
inhibitory effect than Dox (93.08 µg/ml). On the other hand, compounds
2b, 3b, 13 and 2a revealed moderate to slight cell growth inhibition of
MCF-7 with IC50 values of 141.65, 156.5, 172.07 and 616.69 µg/ml,
respectively. Data in table (2) and figures (5(A–C)) showed that the
cytotoxic activity from the highest to the lowest as follow compound 2c
> 10a > 10b > 14 > 8 > 19 > 13 > 16a > 2b > 3b > 2a contra PC3
cells. The treatment of PC3 cells with compounds 2c, 10a, 10b and 14
(22.89, 31.75, 38.4, 48.11 µg/ml respectively) showed the highest
cytotoxic activity as compared to Dox (72.39 µg/ml) after 24 h.
Furthermore, the other compounds 8, 19, 13, 16a, 2b, 3b and 2a with
IC50 values of 92.77, 111.17, 164.61, 164.86, 202.5, 331.3 and 690.41
µg/ml respectively showed moderate to slight inhibition growth against
PC3 cancer cells in comparison with control cells. From the data ob-
tained (Table 3, Fig. 6(A–C)), the most cytotoxic compounds contra
HepG2 cells was in the descending order of 10a > 2c > 10b > 19 > 14
> 8 > 3b > 16a > 13 > 2b > 2a. Compounds 10a, 2c, 10b, 19, 14, 8 and
3b with IC50 values (19.07, 24.64, 31.13, 33.96, 36.62, 59.42 and
91.28 µg/ml respectively) revealed more cytotoxic than Dox values
(98.55 µg/ml) when treated with HepG2 cells for 24 h. As compared to
control cells, compounds 16a, 13, 2b and 2a showed moderate inhibi-
tion effect of HepG2 cell growth with IC50 of 216.82, 223.43, 357.08
and 479.83 µg/ml, respectively. Finally, these results showed that the
Fig. 4. (A-C): Effects of tested compounds on MCF-7 cells at 24 h.
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D.S. Nada et al.
Steroids 169 (2021) 108813
Fig. 5. (A-C): Effects of tested compounds on PC3 cells at 24 h.
Fig. 6. (A-C): Effects of tested compounds on HepG2 cells at 24 h.
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D.S. Nada et al.
Steroids 169 (2021) 108813
tested compounds 10a, 10b, 2c, and 14 are better than Dox when
treated with cancer cell lines (MCF7, PC3 and HepG2 cells) after 24 h
incubation time.
Ethical Approval
This article does not contain any studies with human participants or
animals performed by any of the authors.
3.2.1.1. Structure activity relationship.. The analysis of the structure
activity relationships indicates that the combining (Figs. 4-6) of several
heterocyclic rings to estrone moiety contributed to changes in the
cytotoxic activity of these compounds. At 48 h incubation time amino
thiazoloestrane moiety, compound 10a is more cytotoxic compound
followed by amino imidazoleestrane moiety, compound 10b and phenyl
thiazoloestrane moiety, compound 8 on MCF7, PC3 and HepG2 cells.
The presence of methoxy phenyl group in compound 2c is more effective
than the presence of fluoro phenyl group in compound 2b and phenyl
group in compound 2a and also the existence of phenyl thiazolo group
attached to compound 2b made no change in the cytotoxic activity of
compound 3b when treated with three cell lines. The addition of amino
phenyl pyridine carbonitrile ring to estrone moiety to give compound 14
is more potent than the addition of amino phenyl pyran carbonitrile ring
to estrone moiety to give compound 13 and also the addition of amino-
imino phenyl pyrido pyrimidinyl ring to estrone moiety to give com-
pound 19 is more effective than the addition of phenyl pyrido thiopyr-
imidinone ring to estrone moiety to give compound 16a in all cell lines.
These results established the importance of the existence of pyrimidine,
thiazole, imidazole, pyran, pyridine and pyridopyrimidine rings as
pharmacophores for the anticancer activity.
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Declaration of Competing Interest
The authors declare that they have no known competing financial
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Acknowledgment
The authors acknowledge the financial support of National Research
Centre, Cairo, Egypt: Grant no. 10010105 for Prof Gamal A. Elmegeed.
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