Cu (I) catalyzed alkyne-azide 1,3-dipolar cycloaddition (CuAAC): Synthesis of 17α-[1-(substituted phenyl)-1,2,3-triazol-4-yl]-19-nor-testosterone-17β-yl acetates targeting progestational and antipro-liferative activities

Article abstract of DOI:10.1016/j.ejmech.2015.04.045

The progestational potency and selectivity of synthetic steroidal agonists can be enhanced by even larger chemical moieties at 17α-position of the steroid backbones. Hereby a series 5a-c and 6a-c of novel 17α-[1-(substituted phenyl)-1,2,3-triazol-4-yl]-19-nortestosterone-17β-yl acetates were designed and synthesized using click chemistry approach searching progestogenic derivatives with potential anticancer activity. Compounds 5a,b and 6a,c have affected to different extents the three histopatho-logical parameters considered for evaluation of their progestational activity. The compounds 5a,b and 6a,c showed modifications in rat uterus at 35.7-34.8 nM levels with privileged endometrial thickening effect and least change of uterine weight relative to NEA at 52.9 nM level. Up to 40 mg/kg dose compounds 5b and 6c were non-toxic. Molecular docking of the ligands in PR showed in the majority of cases a conformational fitting into the active site different from that of the reference steroid NEA. Compound 6b revealed about 46.4% growth inhibition of CNS cancer SNB-75 cell line, 56% growth inhibition of renal cancer A498 cell line and 56.7% growth inhibition of prostate cancer PC-3 cell line which was mediated by cell cycle arrest. Drugability of the screened compounds showed tolerated results after being challenged to diverse physicochemical parameters.

Full text of DOI:10.1016/j.ejmech.2015.04.045

Accepted Manuscript
Cu (I) catalyzed alkyne-azide 1,3-dipolar cycloaddition (CuAAC): Synthesis of 17α-[1-
(substituted phenyl)-1,2,3-triazol-4-yl]-19-nor- testosterone-17β-yl acetates targeting
progestational and antipro-liferative activities
Z.H. Mohamed, Nawal A. El-Koussi, Nadia M. Mahfouz, Adel F. Youssef, Gehad A.
Abdel Jaleel, Samia A. Shouman
PII:
S0223-5234(15)30008-8
10.1016/j.ejmech.2015.04.045
EJMECH 7860
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 December 2014
Revised Date: 30 March 2015
Accepted Date: 22 April 2015
Please cite this article as: Z.H. Mohamed, N.A. El-Koussi, N.M. Mahfouz, A.F. Youssef, G.A. Abdel
Jaleel, S.A. Shouman, Cu (I) catalyzed alkyne-azide 1,3-dipolar cycloaddition (CuAAC): Synthesis
of 17α-[1-(substituted phenyl)-1,2,3-triazol-4-yl]-19-nor- testosterone-17β-yl acetates targeting
progestational and antipro-liferative activities, European Journal of Medicinal Chemistry (2015), doi:
10.1016/j.ejmech.2015.04.045.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
1st author:
Z. H. Mohamed (Medicinal Chemistry Department, Faculty of Pharmacy, Assiut University,
Assiut 71526, Egypt) corresponding auther
2nd author:
Nawal A. El-Koussi (Medicinal Chemistry Department, Faculty of Pharmacy, Assiut
University, Assiut 71526, Egypt)
3rd author:
Nadia M. Mahfouz (Medicinal Chemistry Department, Faculty of Pharmacy, Assiut University,
Assiut 71526, Egypt)
4th author:
Adel F. Youssef (Medicinal Chemistry Department, Faculty of Pharmacy, Assiut University,
Assiut 71526, Egypt)
5th author:
Gehad A. Abdel Jaleel (Pharmacology Department, National Research Center, El-Bohoth St.,
Dokki, Cairo, Egypt)
6th author:
Samia A. Shouman (Cancer Biology Department, National Cancer Institute, Cairo University,
Cairo, Egypt)

ACCEPTED MANUSCRIPT
Cu (I) catalyzed alkyne-azide 1,3-dipolar cycloaddition (CuAAC):
Synthesis of 17α-[1-(substituted phenyl)-1,2,3-triazol-4-yl]-19-nor-
testosterone-17β-yl acetates targeting progestational and antipro-
liferative activities.
Abstract
The progestational potency and selectivity of synthetic steroidal agonists can be enhanced by even larger
chemical moieties at 17α-position of the steroid backbones. Hereby a series 5a-c and 6a-c of novel 17α-
[1-(substituted phenyl)-1,2,3-triazol-4-yl]-19-nortestosterone-17β-yl acetates were designed and
synthesized using click chemistry approach searching progestogenic derivatives with potential
anticancer activity. Compounds 5a,b and 6a,c have affected to different extents the three histopatho-
logical parameters considered for evaluation of their progestational activity. The compounds 5a,b and
6a,c showed modifications in rat uterus at 35.7-34.8 nM levels with privileged endometrial thickening
effect and least change of uterine weight relative to NEA at 52.9 nM level. Up to 40 mg/Kg dose
compounds 5b and 6c were non-toxic. Molecular docking of the ligands in PR showed in the majority of
cases a conformational fitting into the active site different from that of the reference steroid NEA.
Compound 6b revealed about 46.4% growth inhibition of CNS cancer SNB-75 cell line, 56% growth
inhibition of renal cancer A498 cell line and 56.7% growth inhibition of prostate cancer PC-3 cell line
which was mediated by cell cycle arrest. Drugability of the screened compounds showed tolerated
results after being challenged to diverse physicochemical parameters.
1. Introduction
angiogenesis, tubulin polymerization and the up
regulation of apoptotic pathways [1].
Steroidal derivatives in which ring D is modified
with exo-heterocycles exhibit numerous forms of
biological activity and are attractive for medicine.
A large number of steroid derivatives containing
five- or six-membered 17β-exo-heterocycles are
known to cause the efficient inhibition of 17α-
hydroxylase / C17, 20-lyase (P45017α), which can
block androgen synthesis at an early stage, and may
therefore be useful in the treatment of prostatic
carcinoma. Also some steroid compounds are
known to exert hormone receptor-independent
antiproliferative activity via the inhibition of
As previously reported progesterone receptor
pockets can rearrange to accommodate different
agonists [2]. Searching the progestational potency
and selectivity of synthetic steroid agonists were
enhanced by bulky chemical moieties at 17α-
position of the steroid backbones [3].
In drug discovery 1,4-disubstituted-1,2,3-triazole
moieties are attractive connecting units because
they are stable to metabolic degradation and
capable of hydrogen bonding, which can be
favorable in the binding with biomolecular targets
and can improve the solubility [4] .
1

Cu (I) catalyzed alkyne-azide 1,3-dipolar the resulting diazonium salts with sodium azide
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cycloaddition (CuAAC) was exploited by many [20]. Methyl azidobenzoates 3a-c were obtained via
authors for the synthesis of 1,4-disubstituted 1,2,3- esterification of the corresponding azidobenzoic
triazoles of antiproliferative [5-8], antiviral [9], acids 2a-c with methanol under reflux in presence
antitubercular [10,11], antifungal [12-15]
antibacterial agents [16,17].
and of sulfuric acid as catalyst. It was observed that the
o-azidobenzoic acid, 2a and its methyl ester, 3a
showed the lowest yield (83 and 63% respectively)
Our group applied CuAAC reaction for the
condensation of the terminal ethynyl group of
norethindrone enanthate and levonorgestrel with 3-
and 4-substituted phenyl azides to prepare 1,4-
disubstituted-1,2,3-triazole derivatives. In animal
experiments the new derivatives disclosed potent
progestational action at nM levels and marked
decrease in uterine contraction [18].
relative to the other two isomers that may be
attributed to the steric hindrance of the vicinal
moieties on the reacting substrates. The targeted
derivatives of NEA, 5a-c and 6a-c, were prepared
under click reaction conditions through 1,3-
cycloaddition of the terminal ethynyl group of
NEA, 4 and the respective azides, 2a-c and 3a-c
catalyzed by Cu (I). The reactions were performed
at room temperature under nitrogen and in presence
of ten equivalents of sodium ascorbate in (1:1)
aqueous t-BuOH milieu. Trial to accelerate the
synthesis of compound 5c by heating the reaction
In extension of our previous work we conjugated
17α-ethynyl group of norethindrone acetate (NEA)
with three positional isomers of azidobenzoic acid
and their methyl esters to prepare 17α-[1-
(substituted phenyl)-1,2,3-triazol-4-yl]-19-nortesto-
sterone-17β-yl acetates as potential candidates of
progestational and antiproliferative activities. In the
planned work we discussed the relation between the
observed biological activity of the prepared isomers
and in silico ligand receptor interaction abilities.
o
mixture up to 60 C was unsuccessful where
unidentified products were obtained. Sensitivity of
CuAAC reaction to steric hindrance was observed
through the decreased yields (45% and 50%) of the
triazoles 5a and 6a derived from o-azidobenzoic
acid, 2a and its methyl ester, 3a relative to the other
ligands prepared from the m- and p-isomers (55-
75%). All of the synthetic compounds were
characterized by IR, 1H-NMR, and elemental
analyses. 13C-NMR for representative compounds
was also carried out and was confirmed by DEPT
experiments at 135°. All compounds gave
satisfactory analytical and spectroscopic data,
which were in full accordance with their depicted
structures.
2. Results and discussion
2.1. Chemistry
Compounds 5a–c and 6a-c were synthesized by
conventional methods outlined in Scheme 1.
Different methods were reported for the synthesis
of organic azides [19]. In our work 2-,3-, and 4-
azidobenzoic acids 2a-c were prepared by
diazotization of the available 2-, 3-, and 4-
aminobenzoic acids 1a-c, followed by azidation of
2

2.2. Biological activity
reported hydrophilic
interactions
with
such
as
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hydrogen
bonding
the receptor,
2.2.1. In vivo progestational activity
particularly in sites close to the C-17 hydroxyl
function in the 19-nortestostenone series and at
C-3 and C-20 in progesterone derivatives [22].
Development of X-ray crystal structure of the
human progesterone receptor–ligand binding
domain (PR-LBD) complexed with progesterone,
norethisterone and other synthetic agonist and
antagonist progestins paved the way for the
development of novel clinically useful PR
The effect of hydrogen bonding interaction
forces was more influential either by their allowed
number or position within the ligands.
The effect of structure variations in the prepared
series on endometrial thickness can be directly
correlated with the number of hydrogen bond
interaction forces of ligands with the receptor.
Thus 5a, b and 6c with the strongest endometrial
thickening effect showed 1-2 hydrogen bonding
interaction forces more than any one of 5c, 6a, b
and NEA. The added number of hydrogen bonding
was accompanied by decreased binding energies
(ꢀG) values.
ligands. The binding of these ligands to the LBD
subsequently induces conformational changes
that lead to cascade of biological outcomes [21].
The preliminary in vivo progestational activity of
the synthesized compounds 5a-c and 6a-c was
evaluated using adult female Wistar rats. Induced
uterine histopathological changes displayed by
Table 1 and Fig. 1 were matched with the
reference NEA and the vehicle DMSO as a
control.
The prepared compounds, 5a-c and 6a, c and the
reference NEA showed varied magnitude of
pronounced endometrial thickening than the control
with maintained stronger effect than myometrial
thickening. The reversed picture displayed by
compound 6b was an exception since its
myometrial thickening effect was much more
prominent than the endometrial. Molecular docking
of 6b in the PR (Table 2 and Fig. 2A&B) revealed
the participation of the steroid C-3 and the m-
benzoate ester carbonyls in hydrogen bonding with
Asn719 and Arg766 respectively. Such orientation,
associated with decreased ꢀG score was different
from that displayed by the docked o- and p- esters,
6a and 6c where their C-3 carbonyls were hydrogen
bounded to the two amino acids Gln725 and
Differences observed in thickness of
endometrium, myometrium and height of
epithelial cells were correlated essentially with
the hydrogen bonding interaction forces between
the docked ligands and the progesterone receptor
displayed by Table 2.
It is well documented that hydrophobic
interaction is the main binding force of the steroid
ligands with the PRs. Our docked ligands on PR
showed contribution of 3-5 hydrophobic interaction
forces more than NEA that cope with their
relatively higher clog P values. Correlation between
hydrophobic forces variations and the observed
morphological modifications on the uterus cannot
be perceived. On the other hand Cunha et al
3

Arg766 similarly involved by docked NEA (Fig.
histopathological parameters
considered for
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2C). Compound 6c showed an additional hydrogen
evaluation of the progestational activity. Their role
bonding initiated between the ester carbonyl and was disclosed through their ability to modify the
Asn719. The evident differences between hydrogen hydrogen bonding interaction of the C-3 carbonyl
bonding attachment sites of the C-3 and ester group with the receptor amino acids in the active
carbonyls revealed by 6a (Fig. 2D) and 6c on one site. It was evident too that hydrogen bonding
side and those of 6b can explain the anomalous
interaction of Arg766 in the active site with either
C-3 or the polar functions on the phenyl ring is
essential for activity. On the other hand Gln725
may be replaced or coupled with Asn719 in the
binding process to the ligands with at least
conservation or enhancement of activity relative to
the reference.
thickening behavior of the latter.
According to the docking results, the presented
interpretation cannot satisfy the three fold
myometrial thickening effect of 6b relative to the
acidic 5c showing the same ligand receptor
interaction pattern. Here, other prevailing parameter
than their identical binding mode to PR must be
thought. Among relevant possibilities is the two
Relative uterine weights (RUW) of most of the
screened compounds were changed within the
fold cLog P values of the ester group 6a-c relative range (1.3 ± 0.1: 2.0 ± 0.13) which is insignificantly
to the carboxyl derivatives which correlates with different from that of the reference drug (1.7±
the observed higher pattern of their myometrial 0.05). One exception 5c was found to be
thickening effect relative to those displayed by the significantly different (3.9 ± 0.35) in RUW relative
acidic ones 5a-c .
to the references.
Furthermore the compounds 5a-c, 6b ,c showed
Distinct variations of the progestational activity
epithelial cell height values equal to or less than of the prepared compounds and those cited in Ref.
that of the control while 6a is the one that showed [18] (Fig. 3) are going to be correlated with the
significant higher value than the control and disparity of type and position of substituents on the
matching that of NEA. Actually 6a is the single
ligand in the series in which C-3 carbonyl was
bound to Gln725 and Arg766 via two hydrogen
bonds like in NEA with no further participation of
other bonding interaction.
phenyl ring.
Meta substitution on phenyl ring by the
electron withdrawing groups CO2Me and NO2 in
the interties 6b and 8 (Table 1), showed lower
progestational activity in relation to 6c and 9
isomers substituted in the para positions that was
allied with relative higher Hansch electronic σ
In the prepared series the substituted phenyl
bridged via the 1,2,3-triazole moiety to the steroidal
frame, have favorably altered interaction with the values of the para analogues [23].
receptor. Substituents in 5a,b and 6a,c have
On the other hand, the derivatives with
ionizable carboxyls 5b, 5c and 7 did not show a
affected to different extents the three
4

correlation between the progestational activity and results against the screened cancer cell lines are
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Hansch electronic σ values. It is worthy to note that listed in Table 3 and the whole results are provided
separation of the COOH group by CH2 distinctly in the supplementary data.
ameliorated the activity of 7 relative to 5c.
Krämer et al studied the effect on the
Under our experimental conditions 5a,b and 6a,c proliferation of HCC1500 cells incubated with
showed potent progestational activity (35.7-34.8
nM) with privileged endometrial thickening effect presence of growth factors.
and least change of uterine weight relative to NEA together with six progestins and testosterone
estradiol and steroidal compounds in absence and in
Norethendrone
(52.9 nM) .
showed to varying degrees antiproliferative effect
[26]. Another study carried out by Xu et al on the
effects of progestogens in human breast cancer cells
suggested that progestins exert different actions on
estrogen-metaboilizing enzymes depending on the
specific progestogen and regimen used [27].
2.2.2. Acute toxicity and lethality (LD50)
Acute toxicity of two active compounds 5b and
6c with prominent progestational activity was
determined according to Buck et al using albino
mice [24]. The animals were grouped (three per
group), separately received 5, 10, 15, 30 or 40
mg/kg dose and observed over 24 h for signs of
Our screened compounds showed varied growth
inhibitory effects on breast, prostate and other
toxicity and number of deaths. Control animals cancer cell lines. No clear difference in anticancer
receiving the vehicle (10% DMSO) were kept activity can be correlated with the positional
under the same conditions without addition of 5b or isomers of the carboxyl substituent. The attained
6c. All animals treated with the tested compounds best results was shown by compound 6b which
up to 40 mg /kg dose were alive during the 24 h of revealed 46.4% inhibition of SNB-75 cell line of
observation and did not show visible signs of acute CNS cancer and about 56 % inhibition of both
toxicity. Therefore these compounds were A498 cell line of renal cancer and PC-3 cell line of
considered to be non-toxic.
prostate cancer.
2.2.3. In vitro anticancer activity
The effects of compound 6b on cell cycle
distribution of PC-3 cell line were evaluated by
flow cytometry [28]. Treatment of PC-3 cell line
The newly synthesized 17α-(1-substituted-1,2,3-
triazol- 4-yl)-19-nortestosterone acetates, 5a-c and
6a-c (Conc. 10-5 M) were screened for their
anticancer activity according to NCI in vitro
protocols [25] against a panel consisting of 60
human tumor cell lines. These cell lines were
derived from eight cancer types: leukemia, non-
small cell lung, colon, CNS, ovarian, renal, prostate
and breast cancers. The most prominent activity
with compound 6b at the concentration 10-5
M
resulted in accumulation of 31.76 % of cells at S
phase as compared to 27.87 % in untreated cells
(Fig. 4). On the contrary the cell population at Go-
G1 phase was reduced to 53.94 % compared to the
untreated cells 59.01 %, while the cell percentage
of G2-M 14.75 % was slightly increased compared
5

3. Conclusion
to the untreated cells 13.12 %. The obtained results
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Anchored polar carboxyl moiety to NEA via
imply that the anticancer activity of 6b derivative
could be preceded by the accumulation of cells in
the S phase. It is clear that property of induction of
cell cycle arrest make this series of 17α-[1-(substit-
uted phenyl)-1,2,3-triazol-4-yl]-19-nortestosterone
acetates promising progestins with anticancer
potentials.
1,2,3-triazole ring provided the acid isomers 5a,b
and the esters 6a,c with potent progestational
activity at nM levels without exerting any toxicity
up to 40 mg/Kg dose. Molecular docking analysis
of the bound conformers to PR revealed that the
added moieties to the steroid nucleus strongly
affected the binding modes which can be correlated
with the biological activity. A number of the
prepared compounds disclosed varied activities
against certain cell lines of prostate, renal and CNS
cancers. Compound 6b exerted the most prominent
cytotoxic activity which was mediated by cell cycle
arrest. Our results strongly suggest that our agents
may provide a promising new avenue for the
development of progestogens with anticancer
activity.
2.3. Physicochemical calculations and
drugability
The effect of the structural modification on the
physicochemical properties of norethindrone
acetate was also attempted. Lipinski rule
parameters, solubility (logS), the topological polar
surface area (TPSA) and molecular volume
calculated using Molecular Operating Environment
(MOE®) version 10.2010 [29] and the molins-
piration server [30].
4. Experimental
Calculations displayed in Table 4 reveals: i) the
planned structural modification of NEA had
slightly increased the Lipinski parameters to the
extent not affecting the drugability of these targets.
ii) introduced modifications slightly diminished
predicted water solubility relative to NEA, however
the pendent ionization center carboxyl group
provides the possibility of salt formation with bases
that can enhance water solubility, iii) increased
polar surface area of the target compounds was
within the accepted limit [31] for orally active
drugs that are transported passively by the
transcellular route, iv) the increase in the molecular
volume of the targeted compounds did not hamper
interaction with the flexible PR active site that
accommodate bulky ligands.
4.1. General methods
All chemicals and reagents used in current study
were of analytical grade. TLC was performed on 60
F254 precoated sheets 20 X 20 cm, layer thickness
0.2 mm (E. Merck, Germany) and visualized in
UV light (254 nm). Column chromatography was
performed using Fluka silica gel 60 (particle size
0.063 – 0.02 mm.) eluting with ethyl acetate and
hexane. Melting points (uncorrected) were
determined on electrothermal apparatus (Stuart
Scientific, England). IR spectra were recorded as
KBr disk using Shimadzu IR 400-91527
Spectrophotometer (Shimadzu Corp., Kyoto,
1
Japan). All the H-NMR spectra were recorded on
Varian EM-360L NMR Spectrophotometer (60
6

MHz) (USA) and JEOl-JNM-LA400 FT-NMR
CDCl , δ ppm): 7.13-8.09 (m, 4H, H2,4-6); 9.16 (s,
3
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Spectrometer (400 MHz) (Japan) in CDCl3 and exchangable, 1H, COOH).
DMSO-d6. Chemical shifts were reported in δ
4.2.3. 4-Azidobenzoic acid (2c)
(ppm) with TMS as internal standard for protons
and solvent signals. 13C-NMR spectra were
performed on JEOl-JNM-LA400 FT-NMR
Spectrometer (400 MHz) (Japan) in CDCl3.
Elemental analyses were performed on Perkin
Elmer 2400 CHN elemental analyzer.
White, crystallized from water/ethanol, yield:
95% mp: 188-191○C; IR (KBr, cm-1) : 3755-3090
1
(OH); 2095 (N3); 1668 (C=O); H-NMR (60 MHz,
CDCl3, δ ppm, J Hz): 7.03-7.33 (d, 2H, J = 8,
H3,5); 7.93- 8.29 (d, 2H, J = 8, H2,6); 8.63 (s ,
exchangable , 1H, COOH)
.
4.2. General method of synthesis of azidobenzoic
acids (2a-c) [32]
4.3. General method for synthesis of methyl
azidobenzoates (3a-c)
A solution of sodium nitrite (1.06 g , 15.4 mmol)
in cold water (5○C) (3 mL) was added portion wise
to a stirred cold mixture of the respective amino
benzoic acid 1a-c (2 g, 14.5 mmol) , water (10 mL)
and concentrated sulfuric acid (3 mL) . To the
resultant clear mixture, a solution of sodium azide
(1.20 g, 18.45mol) in water (3 mL) was then added
with vigorous stirring. A white product was
precipitated; the stirring was continued for further
10 min. The precipitate was washed thoroughly
with water, filtered under suction, crystallized from
the proper solvents to afford the pure products 2a-c.
A mixture of the respective azidobenzoic acid
2a-c (2g, 0.0122mol), absolute methanol (5mL,
0.125mol) and concentrated sulfuric acid (0.36 g,
0.2 mL) was refluxed for 4 hours. The solvent was
evaporated under reduced pressure and allowed to
cool. The product was extracted with chloroform
(2×50 mL) and the organic layer washed with
sodium bicarbonate solution (20%) until
effervescence ceases, then with water, and dried
over anhydrous magnesium sulfate. The chloroform
was evaporated under vacuum and the product was
collected and used without further purification.
4.2.1. 2-Azidobenzoic acid (2a)
4.3.1. Methyl 2-azidobenzoate (3a)
White, crystallized from n-hexane, yield: 83%
mp: 141-143 ○C; IR (KBr, cm-1): 3670-3060 (OH);
2100 (N3); 1684 (C=O); 1H-NMR (60 MHz,
CDCl3, δ ppm, J Hz): 7.09-7.42 (m, 2H, H3,5);
7.42-7.81 (m, 1H, H4); 8.03-8.29 (d, 1H, J = 9,
H6); 11.21 (br.s, exchangable, 1H, COOH).
Dark red oil Yield: 63%; IR (KBr, cm-1): 2955 (-
C-H); 2095 (N3); 1713 (C=O); 1H-NMR (60 MHz,
CDCl3, δ ppm): 3.89 (s, 3H, CH3); 6.93-8.00 (m,
4H, H3-6).
4.3.2. Methyl 3-azidobenzoate (3b)
Dark red oil Yield: 75%; IR (KBr, cm-1): 2985 (-
1
4.2.2. 3-Azidobenzoic acid (2b)
C-H); 2090 (N3); 1715 (C=O); H-NMR (60 MHz,
White, crystallized from n-hexane, yield: 89%
CDCl3, δ ppm): 3.86 (s, 3H, CH3); 6.83-7.73 (m,
○
mp: 163-165 C; IR (KBr, cm-1): 3625-3380 (OH);
4H, H2, 4-6).
2345 (N3); 1647 (C=O); 1H-NMR (60 MHz,
4.3.3. Methyl 4-azidobenzoate (3c)
7

○
Reddish white crystals, Yield: 80%; mp: 38 C Hz): 8.03-8.01(d, 1H, Ar H6); 7.68-7.64 (m, 1H, Ar
ACCEPTED MANUSCRIPT
1
IR (KBr, cm-1): 2100 (N3); 1710 (C=O); H-NMR H4); 7.58-7.52 (m, 3H, Ar H3,5, triazole H5); 5.81
(60 MHz, DMSO-d6, δ ppm, J Hz): 3.86 (s, 3H,
CH3); 7.00- 7.33 (d, 2H, J = 8.5, H3, 5); 7.76-8.06
(d, 2H, J = 8.5, H2, 6).
(s,1H,steroidal C4-H); 3.05-0.64 (unresolved
multiplets of cycloaliphatic protons of the steroidal
nucleus in addition to acetyl and C18 methyl
protons); Anal.Calcd for C29H33N3O5: C, 69.17; H,
6.60; N, 8.34. Found: C, 69.21; H, 6.58; N, 8.49.
4.4. General method for synthesis of 17-α-(1-
substituted-1,2,3-triazol-4-yl)-19-nortestosterone
acetates (5a-c) and (6a-c).
4.4.2. 3-[4-(17-Acetoxy-13-methyl-3-oxo-2,3,6,7,8,
9,10,11,12,13,14,15,16,17-tetradecahydro-1H-
cyclopenta[a]phenanthren-17-yl)-1H-1,2,3-triazol-
1-yl]benzoic acid(5b)
Norethindrone acetate (4) (250 mg, 0.73 mmol)
and the respective azide 2a-c, and 3a-c (2.92 mmol)
were suspended in a mixture 1:1 of water and t-
butyl alcohol (12 mL). An aqueous solution of
sodium ascorbate (14.5 mg , 0.073 mmol, water
300 µL) was added, followed by copper (ΙΙ) sulfate
pentahydrate solution (1.8 mg, 0.0073 mmol, in
water 100 µL). The heterogeneous mixture was
stirred vigorously under nitrogen overnight, at
which point it cleared and TLC monitoring
White, Yield: 70%, mp: 151-153○C; IR (KBr,
cm-1): 2530-3435(OH); 1718(C=O acetyl); 1651
1
broad (C=O ketonic and COOH carbonyl); H-
NMR (400 MHz, CDCl3, δ ppm, J Hz) : 8.34
(s,1H,Ar H2); 8.14-8.12 (m, 2H, Ar H4,6) ; 7.84
(s,1H, triazole H5); 7.64-7.60 (t,1H, J=8, Ar H5);
5.82 (s,1H,steroidal C4-H); 3.13-0.66 (unresolved
multiplets of cycloaliphatic protons of the steroidal
nucleus in addition to acetyl and C18 methyl
protons); 13C-NMR (400 MHz, CDCl3) : 14.82
(C18); 21.73 (O=C-CH3); 24.07 (C11); 25.91(C15);
26.37 (C7); 30.70 (C6); 32.90 (C16); 35.47 (C2);
36.33 (C1,C12); 40.72 (C8); 42.41 (C10); 46.36
(C14); 47.61 (C13); 48.65 (C9); 88.11 (C17);
119.39 (Ar C2); 121.15 (Ar C4); 124.50 (Ar C5);
125.33 (C4); 130.02 (triazole C5); 130.11 (Ar C6);
131.15 (triazole C4); 137.12 (Ar C1); 150.58 (Ar
C3); 166.86 (C5); 170.51 (OCOCH3, COOH);
200.37 (C3); Anal. Calcd for C29H33N3O5: C, 69.17;
H, 6.60; N, 8.34. Found: C, 69.28; H, 6.69; N, 8.57.
(hexane/ethyl
acetate)
indicated
complete
consumption of the steroid. The reaction mixture
was diluted with water (50 mL), extracted with
ethyl acetate (3×20mL) , dried via filtering over
anhydrous magnesium sulfate. The organic layer
was evaporated under vacuum, and the residues
were purified by column chromatography using
gradient elution of (hexane/ethyl acetate) to afford
the target products.
4.4.1. 2-[4-(17-Acetoxy-13-methyl-3-oxo 2,3,6,7,8
,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-
cyclopenta[a]phenanthren-17-yl)-1H-1,2,3-triazol-
1-yl] benzoic acid (5a)
White, Yield: 45%, mp: 145-148○C; IR (KBr,
cm-1): 2590-3695 (OH); 1718 (C=O acetyl); 1651
broad (C=O ketonic overlapped with COOH
4.4.3. 4-[4-(17-Acetoxy-13-methyl-3-oxo-2,3,6,7,8,
9,10,11,12,13,14,15,16,17-tetradecahydro-1H-
cyclopenta[a]phenanthren-17-yl)-1H-1,2,3-triazol-
1-yl]benzoic acid(5c)
1
carbonyl); H-NMR (400 MHz, CDCl3, δ ppm, J
8

○
White, Yield: 55 %, mp: 184-186 C; IR (KBr, 4.4.5. Methyl 3-[4-(17-acetoxy-13-methyl-3-oxo-
ACCEPTED MANUSCRIPT
cm-1): 2565-3455 (OH); 1714 (C=O acetyl); 1645
2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradeca
hydro-1H-cyclopenta[a]phenanthren-17-yl)-1H-
1,2,3-triazol-1-yl] benzoate(6b)
1
(C=O ketonic); 1615 (C=O carboxyl); H-NMR
(400 MHz, CDCl3, δ ppm, J Hz) : 8.23-8.21 (d, 2H,
White,Yield: 75%, mp: 97-99 ○C; IR (KBr, cm -1):
J = 8.8, Ar H2,6); 7.89-7.86 (d, 2H, J = 8.8, Ar
1719 strong (acetyl and ester carbonyl group); 1657
H3,5); 7.82 (s, 1H, triazole H5); 5.81 (s, 1H,
1
(C=O ketonic); H-NMR (400 MHz, DMSO-d6, δ
steroidal C4-H); 3.75-0.65 (unresolved multiplets
ppm, J Hz) : 8.84 (s,1H,Ar H2); 8.44 (m,1H,
of cycloaliphatic protons of the steroidal nucleus in
triazole H5); 8.24-8.22 (m,1H, Ar H6); 8.03-8.02
addition to acetyl and C18 methyl protons);
(d, J =7.6 ,1H, Ar H4); 7.76-7.71 (m, 1H, Ar H5);
Anal.Calcd for C29H33N3O5: C, 69.17; H, 6.60; N,
5.67(s, 1H, steroidal C4-H); 3.90 (s, 3H, COOCH3);
8.34. Found: C, 69.24; H, 6.71; N, 8.62.
3.40-0.54 (unresolved multiplets of cycloaliphatic
4.4.4.
Methyl 2-[4-(17-acetoxy-13-methyl-3-oxo
protons of the steroidal nucleus in addition to acetyl
and C18 methyl protons). Anal.Calcd for
C30H35N3O5: C, 69.61; H, 6.82; N, 8.12. Found: C,
69.70; H, 6.88; N, 8.32.
2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradeca
hydro-1H-cyclopenta[a] phenanthren -17-yl)-1H-
1,2,3-triazol-1-yl] benzoate (6a)
White, Yield: 50%, mp: 93-96 ○C; IR (KBr,cm-1):
1722 strong (acetyl and ester carbonyl group); 1656
4.4.6. Methyl 4-[4-(17-acetoxy-13-methyl-3-oxo-
2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradeca
hydro-1H-cyclopenta[a]phenanthren-17-yl)-1H-
1,2,3-triazol-1-yl] benzoate(6c)
1
(C=O ketonic); H-NMR (400 MHz, CDCl3, δ
ppm, J Hz): 7.98-7.94 (m,1H,Ar H6); 7.64-7.49 (m,
4H, Ar H3,4,5+ triazole H5); 5.77 (s,1H,steroidal
C4-H); 3.67-3.60 (s,1H,COOCH3); 3.01-0.68
(unresolved multiplets of cycloaliphatic protons of
the steroidal nucleus in addition to acetyl and C18
methyl protons); 13C-NMR (400 MHz, CDCl3) : δ
14.52 (C18); 21.55 (OCOCH3); 24.07 (C11); 26.02
(C15); 26.57 (C7); 29.45 (C6); 30.83 (C16); 32.77
(C2); 35.47 (C12); 36.44 (C1); 40.75 (C8); 42.45
(C10); 46.13 (C14); 48.91 (C9,C13); 52.53
(COOCH3); 88.07 (C17); 118.32 (Ar C3); 123.47
(Ar C1); 124.57 (C4); 126.92 (Ar C6); 129.83 (Ar
C5,triazole C5); 131.27 (triazole C4); 132.63(Ar
C4); 150.95 (Ar C2); 166.46 (COOCH3);195.03
(OCOCH3), 218.26 (C3); Anal.Calcd for
C30H35N3O5: C, 69.61; H, 6.82; N, 8.12. Found: C,
69.68; H, 6.85; N, 8.27.
White, Yield: 60 %, mp: 119-122○C; IR (KBr,
cm-1): 1715 strong (acetyl and ester C=O group);
1
1655 (C=O ketonic); H-NMR (400 MHz, DMSO-
d6, δ ppm, J Hz) : 8.11-8.09 (d, 2H, J = 8.8, Ar
H2,6); 8.08-8.05 (d, 2H, J = 8.8, Ar H3,5); 8.78
(s,1H,triazole H5); 5.63(s, 1H, steroidal C4-H);
3.83 (s, 1H, COOCH3); 3.33-1.03(unresolved
multiplets of cycloaliphatic protons of the steroidal
nucleus in addition to acetyl and C18 methyl
protons).Anal.Calcd for C30H35N3O5: C, 69.61; H,
6.82; N, 8.12. Found: C, 69.57; H, 6.95; N, 8.33.
4.5. In vivo progestational activity
4.5.1. Animals
Adult female Wistar rats (weighing 200 -215g)
were housed into 8 groups 6 per cage at 20-22 °C
9

under controlled conditions of light, with free
Relative organ weight (kg) = [organ weight
ACCEPTED MANUSCRIPT
access to rat chow and tap water. Rats showing (g)/body weight (g)] × 1000
regular estrous cycle length (4–5 days). The phases
The uteri were fixed and stained, paraffin
of estrous cycle were determined by observing the
vaginal smear in the morning. Only those rats
showing at least two consecutive 4-days estrous
cycles were used. For all experiments the treatment
was started when the animals were in estrus phase.
sections were evaluated for histological changes.
The thickness endometrium, myometrium and the
uterine epithelial cell heights were measured using
an objective lens of magnification 10, and eye lens
10, the total magnification was 100 times. Ten
fields were chosen in each specimen and the mean
values were taken.
4.5.2. Reference standard
Norethindrone acetate was obtained as a gift
from Hi pharm pharmaceutical company, El Obour
City, Egypt.
4.5.5. Statistical analysis
The data were expressed as mean ± SEM and
analyzed using SPSS statistical software. One way
analysis of variance (ANOVA) was used to assess
the variation of the means among the treatments. If
the variation was greater than expected by chance
alone, Tukey multiple comparison tests were
performed to compare each treatment group with
the control and standard groups. Significance was
established when the p value was less than 0.05.
4.5.3. Morphometric measurements
Calculations were carried out using Lieca Qwin
500 Image Analyzer in Pathology Department,
National Research Centre, Cairo, Egypt. All the in
vivo investigational studies were carried out in
Pharmacology and Pathology Departments,
National Research Centre, Cairo, Egypt.
4.5.4. Methodology
4.5.6. Evaluation of the acute toxicity and lethality
Initial body weight before treatment and final
body weight at the time of sacrifice were recorded.
Then 1ml solution of the tested compounds 5a-c,
6a-c or reference standard (Norethindrone acetate)
in DMSO (0.018 mg/mL) was injected
subcutaneously daily for 8 days to rat groups, while
the control group received an equivalent amount of
the vehicle. Twenty-four hours after the final dose,
rats were killed, and their uteri were carefully
excised, trimmed of extraneous tissue, blotted filter
paper to remove excess fluid, weighed to calculate
the uterus weight as the following:
(LD50) for 5b and 6c
Test solutions: aliquots of the tested compounds
were dissolved separately in DMSO and diluted
with water to the required concentration to give
10% final dilution of DMSO.
Doses: the animals were injected subcutaneously
with a dose of 5, 10, 15, 30, 40 mg / kg mice.
Control animals: received the vehicle (10 %
DMSO).
Procedure: Groups of adult albino mice (25-30
g) of either sex, each of three animals (six in case
of 40 mg/kg dose) were injected with graded doses
10

of the tested compounds solutions and the vehicle norethindrone (PDB ID: 1SQN) was obtained from
ACCEPTED MANUSCRIPT
as control. Animals were observed per dose for 24h the Protein Data Bank through the internet
and signs of toxicity and number of deaths were (http://www.rcsb.org). All the molecular modeling
recorded.
calculations and docking simulation studies were
performed using Molecular Operating Environment
(MOE®) version 10.2010, Chemical Computing
Group (CCG) Inc., Montreal, Canada.
4.6. In vitro anticancer activity
4.6.1. Screening for anticancer activity
Newly synthesized 17α-(1-substituted-1, 2, 3-
triazol- 4-yl)-19-nortestosterone acetates 5a-c and
6a-c (conc. 10-5 M) were screened for their
anticancer activity according to NCI in vitro
protocols, against a panel consisting of 60 human
tumor cell lines.
Acknowledgment
We would like to acknowledge The National
Cancer Institute (NCI) for the in vitro anticancer
activity screening.
References and notes
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4.6.2. Cell cycle analysis for compound 6b
Prostate cancer cells PC-3 from the treated (10-5
M of 6b) and control cells were collected after 48
hours. Cell cycle distribution of the cell population
was analyzed using Cycle TEST TMPLUS DNA
Reagent kit (BD Biosciences, USA). Cells were
fixed with 70% ice-cold ethanol, washed and the
pellet was suspended in trypsin buffer and left for
10 min at room temperature. 1% RNAase buffer
was added after addition of trypsin inhibitor and
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100 ꢁg/ml propidium iodide. Samples were
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Distribution of cell-cycle phases with different
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12

Figure captions:ACCEPTED MANUSCRIPT
Fig. 1. Effect of compounds 5a-c, 6a-c and NEA on rat's uterus.
Fig. 2. (A) & (B) 2D & 3D representation of the binding mode of compound 6b
in the PR binding site. (C) 2D representation of the binding mode of NEA in the
PR binding site. (D) 2D representation of the binding mode of compound 6a in
the PR binding site.
Fig. 3. Previously prepared Norethindrone enenthate (NET-EN) derivatives
cited in Reference [18].
Fig. 4. Effect of compound 6b on cell cycle distribution in PC-3 cell line
compared with untreated cells.
13

ACCEPTED MANUSCRIPT
Tables
Table 1. Impact of the electronic character, positional isomerism and type of phenyl
substituents on the in vivo progestational activity of 5a-c, 6a-c and compounds cited in
Reference [18]
.
Endometrial
thickness
(µm)
Epithelial
cell height
(µm)
Myometrial
thickness
Substituent Hansh
on the
σ
Entry
phenyl ring values
(µm)
Vehicle
-
-
154.47±1.19
122.40±2.37
228.60±2.86*
57.80±0.92
20% DMSO
-
-
302.35±2.2*
68.22±0.71*
NEA
5a
452±12.67*,** 170.80±8.87*,** 45.50±1.063*,**
o-COOH
m-COOH
p- COOH
o-COOMe
m-COOMe
p-COOMe
NA
-0.1b
0.00 b
NA
375.5±13.99*,** 176±10.27*,**
277.5±10.42*
43.60±0.89*,**
5b
5c
113.60±3.78** 39.60±1.27*,**
64.60±1.31*
343.7±1.86*,** 197.70±7.92*,**
6a
214.04±2.21*,** 319.80±7.91*,** 59.10±0.75*,**
221.80±2.56*
0.37
0.45
6b
6c
417.6±7.17*,**
29.60±0.49*,**
NET-EN a
7 a
300±2*
225 ±7*
239± 4*
67± 1*
71± 2*
p-
-0.16 b
316±1.5*
CH2COOH
8 a
9 a
m-NO2
p-NO2
0.71
0.78
310± 4*
229± 1*
241± 3*
69 ±0.8*
78 ±1**
347± 24*
a
b
Results cited in Reference [18]
Values of the ionized functions NA : not available
* Significantly different from control group at P < 0.05 .
** Significantly different from reference drug group at P < 0.05.
14

ACCEPTED MANUSCRIPT
Table 2. Docked Ligands / PR interactions
Ligand binding groups / PR amino acids
Comp.
No.
Oxygen of C-3
carbonyl
(Distance A°)
Oxygen of carboxyl Oxygen of carboxyl
∆G
(Kcal/mole)
carbonyl
hydroxy
(Distance A°)
(Distance A°)
Gln725 (2.73)
Arg766 (2.67)
-
-
NEA
5a
-10.5131
Gln725 (2.77)
Arg766 (2.56)
Asn719 (2.82)
Asn719 (2.69)
Arg766 (2.49)
Arg766 (2.72)
-12.6971
-11.1299
Gln725 (2.72)
Arg766 (2.41)
5b
5c
Asn719 (2.65)
Arg766 (2.31)
-
-8.8336
Gln725 (2.78)
Arg766 (2.46)
-
-
-
-
6a
6b
6c
-9.7881
-8.0234
-11.3694
Asn719 (2.43)
Arg766 (2.31)
Asn719 (2.63)
Gln725 (2.52)
Arg766 (3.24)
15

ACCEPTED MANUSCRIPT
Table 3. In vitro anticancer activity for compounds 5a-c and 6a-c
Growth Percentage (%)
(10-5 M)
Cell line
5a
5b
5c
6a
6b
6c
94.11
102.31
95.55
91.60
102.35
95.73
83.16
103.61
84.99
77.11
70.41
72.24
68.57
90.00
87.71
87.89
RPMI-8226
Leukemia
92.69
92.02
SR
Non-Small
Cell Lung
cancer
A549/ATCC
90.41
117.20
110.02
66.54
93.50
72.64
97.72
75.69
79.16
87.48
NCI-H522
Colon cancer
(HT29)
102.06
100.28
76.79
104.44
91.28
72.11
103.90
98.03
68.32
92.85
96.41
66.46
89.47
90.77
53.60
74.94
74.93
73.88
93.66
82.72
SNB-75
CNS Cancer
SF-295
SNB-19
ovarian cancer
(OVCAR-4)
98.07
94.79
82.69
93.74
95.43
77.5
77.20
156.59
83.16
96.25
92.60
72.61
62.22
86.45
69.60
43.93
62.09
67.79
78.59
82.76
73.58
92.57
A498
80.60
96.31
UO-31
Renal cancer
CAKI-1
100.35
122.63
107.34
96.84
69.13
114.66
RXF393
Prostate cancer
(PC-3)
94.83
109.54
95.48
83.89
88.82
79.39
99.69
90.65
65.99
97.48
82.67
43.30
76.87
73.27
73.54
96.84
88.15
MCF7
Breast cancer
MDA-MB-
468
108.97
16

ACCEPTED MANUSCRIPT
Table 4. Calculated properties of the synthesized compounds 5a-c, 6a-c and NEA.
Compound
M.wt.
cLog P(O/W) Lip-don Lip-acc Volume TPSA
LogS
340.4630
503.5990
503.5990
503.5990
517.6260
517.6260
517.6260
3.7070
3.7990
3.8380
3.8010
4.0630
4.1020
4.0650
0
1
1
1
0
0
0
3
8
8
8
8
8
8
333.749 43.376 -5.4856
NEA
5a
458.363
111.393 -6.1313
458.363 111.393 -6.1313
458.363 111.393 -6.1313
475.891 100.399 -6.5436
475.891 100.399 -6.5436
475.891 100.399 -6.5436
5b
5c
6a
6b
6c
M.wt.: Molecular weight, Lip-don: Number of Lipinski hydrogen bond donors, Lip-acc :
Number of Lipinski hydrogen bond acceptors, Volume: Molecular volume, TPSA: Total polar
surface area, Log S: Aqueous solubility
17

ACCEPTED MANUSCRIPT
Figures
Fig. 1. Effect of compounds 5a-c, 6a-c and NEA on rat's uterus.
18

ACCEPTED MANUSCRIPT
Fig. 2. (A)&(B) 2D & 3D representation of the binding mode of compound 6b in the PR
binding site. (C) 2D representation of the binding mode of NEA in the PR binding site. (D)
2D representation of the binding mode of compound 6a in the PR binding site.
R
O
N
C6H13
O
N
R = p-CH2COOH (7)
N
m-NO2
p-NO2
(8)
(9)
O
Fig. 3. Previously prepared Norethindrone enanthate (NET-EN) derivatives cited in
Reference [18].
19

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Fig. 4. Effect of compound 6b on cell cycle distribution in PC-3 cell line compared with
untreated cells.
20

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Schemes
COOH
COOH
COOCH3
i
ii
NH2
N3
N3
(1 a-c)
(3 a-c)
(2 a-c)
O
O
iii
O
(4)
R
Entry
R
a
b
m-
m-
m-
m-
c
N
N
N
1
-NH2
- N3
o-
p-
p-
p-
(5a-c)
(6a-c)
O
2, 3
5
o-
o-
o-
O
-CO2H
-CO2CH3
6
p-
O
Scheme 1. Reagents and conditions: (i) H2SO4 , NaNO2 then NaN3 (ii) CH3OH, H2SO4,
Reflux,4h; (iii) CuSO4.5H2O , Sodium ascorbate H2O/t-BuOH (1:1), r.t.,N2,8hr.
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Highlights
• A series of 17α-1,2,3-triazolo-19-nortestosterone acetate
were designed.
• The designed compounds were synthesized using click
chemistry.
• These compounds were screened for their biological
activities.
• The synthesized compounds revealed varied progestational
and anticancer activities.

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1H NMR spectrum for 5a
1H NMR spectrum for 5b

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13C NMR spectrum for 5b
DEPT spectrum for 5b

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1H NMR spectrum for 5c
1H NMR spectrum for 6a

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13C NMR spectrum for 6a
1H NMR spectrum for 6b

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1H NMR spectrum for 6c