Synthesis of progesterone derivatives and evaluation of their efficiency as pneumococcal vaccines

Article abstract of DOI:10.1007/s00044-013-0894-8

Progesterone (1) was used as a template to develop new anticancer compounds. Ring D modification of 1, through its reaction with active methylene derivatives, gave the condensate derivatives 3a, b. The latter compounds underwent heterocyclization reaction

Full text of DOI:10.1007/s00044-013-0894-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0894-8
ORIGINAL RESEARCH
Synthesis of progesterone derivatives and evaluation of their
efficiency as pneumococcal vaccines
•
Rafat M. Mohareb Fatma O. Al Farouk
•
•
Sherif M. Sherif Konstantin Karaghiosoff
Received: 30 March 2013 / Accepted: 13 December 2013
Ó Springer Science+Business Media New York 2013
Abstract Progesterone (1) was used as a template to
develop new anticancer compounds. Ring D modification
of 1, through its reaction with active methylene derivatives,
gave the condensate derivatives 3a, b. The latter com-
pounds underwent heterocyclization reactions through the
reaction with either hydrazine hydrate or phenyl hydrazine
to give the pyrazole derivatives 6a–d, respectively. The
reaction of 1 with bromine gave the a-bromo derivative 7,
which in turn reacted with potassium cyanide to yield the
cyanoacetyl derivative 8. Compound 8 has been subjected
to a series of reactions that produced benzylidene, aryl
hydrazine, pyrazole, and pyran derivatives. The newly
synthesized products were tested for their efficiency as
pneumococcal vaccines and the results were promising.
Introduction
Found in animals, plants and microorganisms, steroids are
classified as organic compounds. Estradiol, Progesterone,
Epitestosterone, and Testosterone (sex hormones) are some
common steroids that are found in animals. It is found that a
steroidal skeleton is involved in the chemical structure of a
large number of therapeutic drugs, such as Dexamethasone
and Betamethasone. The positive effects of the adminis-
tration of exogenous steroids include muscle growth,
appetite stimulation, increased red blood cell production,
and bone density (Kintz et al., 2001; Bhasin et al., 1996;
Page et al., 2008). In the field of medicine, steroids have
been used in treating a variety of illnesses including tropical
diseases, inflammation, anemia, breast cancer, muscle
regeneration, and also osteoporosis in postmenopausal
women (Basaria et al., 2001). In spite of that, steroids
exhibit serious environmental toxicity and have several
health hazards (Pagonis et al., 2006). The fact that minor
structural changes in steroids can cause extensive changes
in their biological activity has for long aroused the curiosity
of medicinal chemists. Naturally occurring steroidal nuclei
have been modified in several ways with the aim of finding
more active compounds that are free from the undesirable
side effects, and of recognizing the structural and stereo-
chemical features required for the display of specific,
selective physiological activity. The replacement of one or
more carbon atoms in a steroid molecule with heteroatoms
results in substantial modifications of its biological activity
(Morand and Lyall, 1968; Huisman, 1971; Huisman and
Speckamp, 1976). Wolff and Zanati have reported that
some A-ring hetero-androstanes exhibit androgenic activity
similar to that of testosterone (Wolff and Zanati, 1970a, b).
A large number of compounds with oxygen, nitrogen, or
sulfur in the nucleus of the steroidal skeleton have been
Keywords Pneumococcal vaccines ꢀ Progesterone ꢀ
Pyrazole ꢀ Thiophene ꢀ Pyran ꢀ Arylhydrazone ꢀ
Benzylidene
R. M. Mohareb (&) ꢀ S. M. Sherif
The Chemistry Department, Faculty of Science,
Cairo University, Cairo, Egypt
e-mail: raafat_mohareb@yahoo.com
F. O. Al Farouk
The Chemistry Department, The American University in Cairo,
P.O. Box 74, New Cairo 11835, Egypt
K. Karaghiosoff
The Chemistry and Biochemistry Department,
Ludwig-Maximilians University, Butenandt Street,
5–13 (House D), 81377 Munich, Germany
123

Med Chem Res
prepared since 1970 and their activities studied (Zanati and
Wolff, 1971; Salvi et al., 1976; Rosen et al., 1980; Ouali,
2006, 2007; Ouali and Rocheblave, 2008). These modified
steroids have been found to possess various biological
activities, such as analgesic properties (Taylor and Lenard,
1967). These results have motivated organic chemists to
synthesize novel compounds that have therapeutic poten-
tial. It is known that progesterone completely prevents the
androgenic action of testosterone on the diameter of the
pigmented spot (Vermorken et al., 1980). In addition, pro-
gesterone stimulates the growth of sebaceous tissue from
flam organs in the same way that testosterone and dihy-
drotestosterone do (Vermorken et al., 1980).
The reaction of progesterone with bromine in acetic acid
gave the corresponding a-bromoacetyl derivative, 7. The
latter readily underwent nucleophilic displacement when
reacted with potassium cyanide to give the corresponding
a-cyanoacetyl derivative 8, with a high yield. This has
encouraged us to study the latter’s reactivity toward several
chemical reagents. Thus, its reaction with aromatic diazo-
nium salts 9a–c in basic ethanol at 0–5 °C, gave the aryl-
hydrazone derivatives 10a–c, respectively.
On the other hand, the reaction of compound 8 with
either hydrazine hydrate (5a) or phenylhydrazine (5b) gave
the pyrazole derivatives 11a and 11b, respectively
1
(Scheme 2). The H NMR spectrum was used to confirm
As a continuation of our previous work (Mohareb and
Hanaa, 2007; Mohareb and El-Omran, 2012; Mohareb
et al., 2012) on the synthesis of different bioactive fused
heterocyclic derivatives of progesterone and pregnenolone,
in this article we describe the employment of progesterone
in the formation of heterocyclic ring systems. Our strategy
involves the study of the use of the acetyl group in
attaching a heterocyclic ring system to ring D. Moreover,
we provide the experimental details of the newly synthe-
sized compounds followed by the results of testing their
efficiency as pneumococcal vaccines.
the structure of compound 11a which showed a singlet
(D2O exchangeable) at d 4.82 ppm, another at d 6.53 and a
third at d 8.22 indicating the presence of the NH2 group,
the pyrazole H-4 and the NH group, respectively. More-
over, the 13C NMR spectrum that showed beside the
expected peaks, a peak at d 119.2 indicating the CN group
and three peaks at d 114.6, 152.4, and 156.8 corresponding
to the pyrazole carbon atoms.
Compound 8 reacted with aromatic aldehydes 12a–c to
give the aromatic ylidene derivatives 13a–c, respectively, the
structures of which were based on analytical and spectral data
(see spectral data). Compounds 13a–c reacted with either of
the cyanomethylene derivatives 2a or 2b to give the pyran
derivatives 15a–d. Formation of the latter products took
place through the intermediate formation of 14a–d followed
by the intramolecular Michael addition of the enolic OH
Results and discussion
Recently, our research group was involved in a series of
heterocyclic transformations using pregnenolone, through
subjecting it to the Knoevenagel reaction with some cy-
anomethylene compounds to obtain reagents capable of
further heterocyclization (Mohareb et al., 2013). In this
paper, we carry out the same procedure, but using pro-
gesterone instead of pregnenolone. Thus, the reaction of
progesterone with either malononitrile (2a) or ethyl cya-
noacetate (2b), in the presence of ammonium acetate, gave
the condensate products 3a and 3b, respectively. Their
structures were verified based on analytical and spectral
data. Thus, the 13C NMR spectrum of 3a showed beside the
expected signals due to progesterone, two signals at d
116.5 and d 117.3 indicating the presence of the two CN
groups, and two signals at d 83.6, 178.0 corresponding to
the C=C group. Compounds 3a and 3b reacted with ele-
mental sulfur in the presence of triethylamine to give the
thiophene derivatives 4a and 4b, respectively. Formation
of the latter products follows the well-known pattern of
Gewald’s thiophene synthesis (McKibben et al., 1999;
Balamurugan et al., 2009). On the other hand, the reaction
of either 3a or 3b with either hydrazine hydrate or phenyl
hydrazine gave the pyrazole derivatives 6a–d, respectively.
Their analytical and spectral data were consistent with their
respective structures (Scheme 1).
group to the cyano group (Scheme 3). The 1H NMR and 13
C
NMR spectra provided proof of their respective structures.
Thus, the 1H NMR spectrum of 15a showed the presence of a
singlet at d 4.62 corresponding to the NH2 group, a singlet at
d 6.53 corresponding to the pyran H-4 and a multiplet at d
7.28–7.38 indicating the phenyl protons. Moreover, the 13C
NMR spectrum showed the presence of two signals at d
116.8 and 117.3 corresponding to the two CN groups.
Compounds 3, 5a–d, 7a, 7b, 9a–d, and 11b exhibit
stereoisomerism and could exist as E and Z forms due to
the presence of the p-electron conjugation. Trials to obtain
pure isomers were carried out using flash chromatography
of the crude compounds (0.5 mmol) in a hexane–acetone
mixture (3:1). Thin layer chromatography examination of
the reaction mixture revealed minor products probably due
to the presence of other isomers (Stulov et al., 2010).
Determination of antibacterial activity
Microorganisms
Different serotypes of Streptococcus pneumonia clinical
isolates were used; AV3 (serotype 3), AV6 (serotype 6B),
123

Med Chem Res
CN
X
O
C
H
H
NH4OAc
100 o
+
H2C CN
X
H
H
C
H
H
O
O
2a, X = CN
b, X = COOC2H5
3a, X = CN
b, X = COOC2H5
1
H2N
S
X
H
1,4-dioxan
Et3N
H
+
3
S8
H
O
4a, X = CN
b, X = COOC2H5
N
N
NH2
X
H
R = H
R=Ph
RNHNH2
3a,b
+
H
H
5a, R = H
b, R = Ph
O
Ph
N
N
6a, X = NH2
b, X = OH
NH2
X
H
H
H
6c, X = NH
d, X = O
O
Scheme 1 Synthesis of compounds 3a,b; 4a,b and 6a–d
´
AV14 (serotype 14), and AV23 (serotype 23F). The strains
were maintained at -70 °C in a Brain Heart Infusion (BHI)
containing 30 % (v/v) glycerol in CERELA (CONICET).
Bacterial inocula were prepared from an overnight culture in
a 5 % sheep blood Columbia Agar (BioMerieux Brazil S.A.)
at 37 °C until the log phase was reached. Then, each sus-
pension was diluted in a sterile 0.9 % NaCl solution to obtain
a cell suspension of 107 CFU/ml (colony forming units/ml).
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Med Chem Res
Br
O
H
AcOH
1
Br2
+
H
H
O
7
CN
O
H
EtOH
H
H
KCN
7
+
O
8
CN
N
O
Ar
NH
H
+
N
-
EtOH
NaOAc
NCl
Ar
+
8
H
H
O
9a, Ar = C6H5
b, 4-Cl-C6H4
c, 4-CH3-C6H4
10a, Ar = C6H5
b, Ar = 4-Cl-C6H4
c, Ar = 4-CH3-C6H4
R
N
NH2
N
RNHNH2
+
8
H
5a, R = H
b, R = Ph
H
H
O
11a, R = H
b, R = Ph
Scheme 2 Synthesis of compounds 7; 8; 10a–c and 11a–b
Determination of minimum inhibitory concentrations
(MIC)
pneumonia strains (Villena et al., 2010) by the agar macro
dilution method in accordance with the Clinical Laboratory
Standards Institute (CLSI, NCCLS) guidelines. Zuccagnia
punctata extract, HF, DHF, and DHC were sequentially
diluted in DMSO and added to Mueller–Hinton Agar
supplemented with a 5 % sheep blood medium at
Samples’ MIC-defined as the lowest concentration of
sample capable of inhibiting visible growth after incuba-
tion-, were determined against the different Streptococcus
123

Med Chem Res
CN
O
CHAr
1,4-dioxan
piperidine
+
H
ArCHO
8
H
H
12a, Ar = C6H5
O
b, Ar = 4-Cl-C6H4
c, Ar = 4-O-CH3
13a, Ar = C6H5
b, Ar = 4-Cl-C6H4
c, Ar = 4-O-CH3
NC
X
CH
CH
HO
Ar
CN
NC
1,4-dioxan
Et3N
13a-c
+
H2C
H
X
H
H
2a, X = CN
b, X = COOC2H5
O
14a-f
NH2
CN
X
O
Ar
H
H
H
O
X
15
Ar
C6H5
4-Cl-C6H4
4-OCH3-C6H4
C6H5
4-Cl-C6H4
4-OCH3-C6H4
a
b
c
d
e
f
CN
CN
CN
COOEt
COOEt
COOEt
Scheme 3 Synthesis of compounds 13a–c and 15a–b
concentrations of 12.5, 25, 50, 100, 200, and 400 lg GAE/
ml for the crude extract and concentrations of 0.1, 1, 10,
100, and 500 lg/ml for the isolated compounds.
values were also determined for commercial antibiotics
(amoxicillin, GlaxoSmithKline S.A.), and essays were per-
formed in triplicates.
After cooling and drying, the plates were inoculated in
spots with 2 ll of each bacterial cell suspension (5 9 104
CFU). A growth control of each tested strain and a solvent
control were prepared. The inoculated plates were incubated
for 20–24 h in a 5 % CO2 environment at 37 2 °C. MIC
Animals
Infant mice were selected for their high susceptibility to
pneumococcal respiratory infection. All experiments were
123

Med Chem Res
carried out in compliance with the Guide for Care and Use of
Laboratory Animals and approved by the Ethical Committee
of Animal Care at CERELA under the bioethical allowance
number BIOT-CRL/11. 3 weeks old Swiss albino mice, each
weighing 20 3 g, were used for the in vivo experiments.
They were kept in a temperature-controlled room under a
12-h luminous–12-h dark cycle. Animals had access to
commercial solid food and water ad libitum.
Table 1 Antibacterial activity (MIC) of obtained compound against
streptococcus pneumonia strains
Compd. no.
MIC (mg/ml)
AV3
AV6
AV14
AV23
3a
R
50
R
R
50
3b
100
R
100
R
100
100
100
100
100
100
R
4a
50
100
100
R
4b
500
200
R
R
Experimental streptococcus pneumonia infection
6a
300
100
200
100
100
100
100
100
400
300
200
500
100
200
300
200
100
200
R
6b
The therapeutic effects of the standardized Zuccagnia
punctata extract and the flavonoids isolated from them
were previously determined in a Streptococcus pneumonia
infection model in mice (Racedo et al., 2006). Freshly
grown colonies of the Streptococcus pneumonia strain AV6
(serotype 6B) were suspended in Todd Hewitt Broth (THB)
(Laboratorios Britania, Argentina) and incubated at 37 °C
until the log phase was reached. The pathogen was nasally
introduced to mice by dropping 25 ll of an inoculum
containing 106 CFU of Streptococcus pneumonia into each
nostril (Villena et al., 2010).
6c
500
R
100
R
6d
7
100
R
R
50
8
100
500
100
100
50
50
100
R
R
10a
10b
10c
11a
11b
13a
13b
13c
15a
15b
15c
15d
15e
15f
Amoxicillin
200
R
100
R
100
400
100
300
R
200
100
R
100
R
R
100
R
50
Treatment protocol
100
R
200
100
200
100
200
R
R
Mice were distributed into groups of nine mice each one: G1
control (C), G2 Streptococcus pneumoniae-infected (SI), G3
Streptococcus pneumoniae-infected ? Zuccagnia punctata
extract (0.25 mg/mice) (SIZp 0.25), G4 Streptococcus
R
R
50
100
R
100
200
R
300
R
R
pneumoniae-infected ? Zuccagnia
punctata
extract
R
R
(0.5 mg/mice) (SIZp 0.5), G5 Streptococcus pneumoniae-
infected ? Zuccagnia punctata extract (1.0 mg/mice) (SIZp
1.0), G6 Streptococcus pneumoniae-infected ? HF (1 mg/
mice) (SIHF), and G7 Streptococcus pneumoniae-infec-
ted ? DHC (1 mg/mice) (SIDHC). Mice without infections
treated with different concentrations of extract and flavo-
noids were included. The antibiotics (Zuccagnia punctata
extract or flavonoids) in DMSO were administered orally
once a day for 7 days postinfection (p.i.); and twice a day for
7 days p.i., Treatment of positive control was performed
with two doses of amoxicillin sodium salt (ClamoxylÒ 1 g;
GlaxoSmithKline S.A.): 0.5 and 2.0 mg/mice. Amoxicillin
was administered once a day for 7 days. In all groups, the
treatments of animals with drug started at 24 h p.i. At days 3,
5, and 7 p.i. three mice of each group were sacrificed, and
lung and blood samples were obtained for bacterial cell
counts. The serum was used by biochemical analysis. The
results are means of three independent experiments.
MIC Minimum inhibitory concentrations, compounds were diluted in
DMSO, R resistant until 400 or 500 lg/ml for Z. puntata extract,
Breakpoint for amoxicillin are less than -2.0 lg/l, 4.0 lg/ml (inter-
mediate) and [/8.0 lg/ml (resistant)
different serotypes of Streptococcus pneumonia strains, was
evaluated by the agar macro dilution method. The MIC values
of the samples against Streptococcus pneumonia strains are
shown in Table 1. All natural products showed in vitro anti-
microbial activity against each of the tested strains with MIC
values ranging from 50 to 500 lg/ml. Streptococcus pneu-
monia AV6 and AV23 strains were more sensitive to natural
products than the Streptococcus pneumonia AV14 strain.
The highest antibacterial activity against the various Strep-
tococcus pneumonia serotypes was achieved with DHC, fol-
lowed by HF and DHF. The MIC values of DHC were similar
to those observed with the Zuccagnia punctata extract.
The antibacterial activity (MIC) of the newly synthe-
sized compounds against the Streptococcus pneumonia
strains showed that compounds 3a, 4a, 6d, 8, 10b, 13b, and
15c are the most active compounds toward the four
In vitro antibacterial activity
The antimicrobial efficacy of Zuccagnia punctata extract,
DHC, HF, DHF, and commercial antibiotic, against the four
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Med Chem Res
Streptococcus pneumonia strains, and that such activities
are very close to that of the reference amoxicillin.
NMR (CDCl3): d 0.82 (s, 3H, CH3), 1.00 (s, 3H, CH3),
1.61–1.93 (m, 6H, 3CH2), 2.20–2.38 (m, 3H, 3CH), 2.66 (s,
3H, CH3), 2.82 (t, J = 8.6 Hz, 1H, CH), 3.01–3.49 (m,
10H, 5CH2), 5.83 (s, 1H, CH=C); 13C NMR (500 MHz,
CDCl3): d 13.6, 21.6 (C-18, C-19), 23.0 (ethylidene CH3),
31.9 (C-16), 37.2 (C-15), 43.8 (C-11), 45.1 (C-12), 49.3
(C-1), 49.3 (C-6), 50.1 (C-2), 57.2 (C-7), 71.5, 83.0, 84.2
(C-9, C-8, C-14), 116.5, 117.3 (C=C), 119.0, 120.6 (2 CN),
121.3 (C-4), 168.3 (C-5), 178.0 (C-17), 187.4 (C-3).
Experimental procedure
Synthetic methods, analytical and spectral data
The starting steroid, progesterone, was purchased from
Sigma Company, USA. All solvents were dried by distil-
lation prior to using. Melting points were recorded on a
Buchi melting point apparatus D-545, and IR spectra (KBr
disks) were recorded on a Bruker Vector 22 instrument.
NMR spectra were recorded on a Bruker DPX200 instru-
ment in CDCl3 and DMSO with TMS as internal standard
for protons and solvent signals as internal standard for
carbon spectra. Chemical shift values are provided in d
(ppm). Mass spectra were recorded on an EIMS (Shima-
dzu) and an ESI-esquire 3000 Bruker Daltonics instrument.
Elemental analyses were carried out at the Micro analytical
Data Unit at the National Research Center, Giza, Egypt and
the Micro analytical Data Unit at Cairo University, Giza,
Egypt. The progress of all reactions was monitored by TLC
on 2 9 5 cm pre-coated silica gel 60 F254 plates 0.25 mm
thick (Merck). The purity of the tested compounds was
evaluated using HPLC (Waters Associates, Milford, MA,
USA) employing an ultraviolet detector (215 nm). All
described compounds showed spectral data that are char-
acteristic of cyclopentanoperhydrophenanthrene nuclei of
Pregnene and Androstane series and were similar to those
reported in literature (IUPAC, 1989a, b).
Ethyl 2-cyano-3-((8S,9S,10R,13S,14S)-10,13-dimethyl-3-
oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-
1H-cyclopenta[a]phenanthren-17-yl)but-2-enoate (3b)
HPLC purity = 92 % (C-18 NovaPak column; MeCN:
MeOH:H2O/50:40:10) tr = 22 min; white crystals from
ethanol (73 %), m.p. 160–162 °C; IR (KBr) cm-1: 2936
1
(CH2), 2222 (CN), 1686, 1688 (2 CO), 1636 (C=C); H
NMR (CDCl3): d 0.80 (s, 3H, CH3), 1.00 (s, 3H, CH3), 1.12
(t, 3H, J = 6.77 Hz, ester CH3), 1.62–1.93 (m, 6H, 3CH2),
2.20–2.39 (m, 3H, 3CH), 2.84 (t, J = 7.25 Hz, 1H, CH),
3.02–3.48 (m, 10H, 5CH2), 4.21 (q, J = 6.77 Hz, 2H, ester
CH2), 5.84 (s, 1H, CH=C); 13C NMR (500 MHz, CDCl3):
d 13.6, 21.6 (C-18, C-19), 14.8 (ester CH3), 23.3 (ethyli-
dene CH3), 31.6 (C-16), 37.7 (C-15), 43.4 (C-11), 45.1
(C-12), 49.3 (C-1), 49.7 (C-6), 50.1 (C-2), 57.4 (C-7), 60.2
(ester CH2), 71.5, 83.0, 84.2 (C-9, C-8, C-14), 116.6, 119.2
(C=C), 120.6 (CN), 121.3 (C-4), 168.6 (C-5), 178.0) 187.4
(ester CO, C-3); MS: m/e = 409 (M?); Anal. Calcd. for
C26H35NO3: C, 76.25; H, 8.61; N, 3.42. Found C, 76.41; H,
8.48; N, 3.58.
Synthetic procedure of the thiophene derivatives 4a, b
Synthetic procedure for the etylidene derivatives 3a, b
To a solution of either 3a (0.362 g, 1 mmol) or 3b
(0.409 g, 1 mmol) in 1,4-dioxane (30 ml) containing a
catalytic amount of triethylamine, sulfur (0.032 g, 1 mmol)
was added. The reaction mixture was heated under reflux
for 1 h, left at room temperature overnight, poured onto an
ice-water mixture, and then the formed solid product was
collected by filtration.
To dry solid progesterone (1) (0.362 g, 1 mmol) either
malononitrile (0.066 g, 1 mmol) or ethyl cyanoacetate
(0.107 g, 1 mmol) was added, followed by the addition of
ammonium acetate (0.25 g). The reaction mixture was
heated at 80 °C for 4 h then left to cool down. The solid
product obtained upon trituration of the formed product by
ethanol was collected by filtration. The filtrate was then
dried and checked for purity through TLC. Thin layer
chromatography revealed a single spot proving the pre-
sence of a sole product.
2-Amino-4-((8S,9S,10R,13S,14S)-10,13-dimethyl-3-oxo-2,3,6,
7,8,9,10,11,12,13, 14,15,16,17-tetradecahydro-1H-cyclopenta
[a]phenanthren-17-yl)thiophene-3-carbonitrile(4a) HPLC
purity = 96 % (C-18 NovaPak column; MeCN:MeOH:
H2O/45:20:35), tr = 20 min; pale yellow crystals from
2-(1-((8S,9S,10R,13S,14S)-10,13-dimethyl-3-oxo-2,3,6,7,8,
9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]
phenanthren-17-yl)ethylidene)malononitrile (3a) HPLC
purity = 96 % (C-18 NovaPak column; MeCN:MeOH:
H2O/45:20:35), tr = 18 min; white crystals from EtOAc:
hexane (88 %), m.p. 220–223 °C; IR (KBr) cm-1: 2932
EtOAc: hexane (88 %), m.p. 220–223 °C; IR (KBr) cm-1
:
3484, 3438 (NH2), 3020 (CH thiophene), 2932 (CH2), 2227
(CN), 1687 (CO), 1639 (C=C); 1H NMR (DMSO-d6): d 0.83
(s, 3H, CH3), 1.00 (s, 3H, CH3), 1.62–1.93 (m, 6H, 3CH2),
2.20–2.39 (m, 3H, 3 CH), 2.81 (t, J = 7.12 Hz, 1H, CH),
1
(CH2), 2227, 2220 (2 CN), 1688 (CO), 1639 (C=C); H
123

Med Chem Res
3.20–3.45 (m, 10H, 5CH2), 4.38 (s, 2H, D2O exchangeable,
NH2), 5.85 (s, 1H, CH=C), 6.36 (s, 1H, thiophene H-5); 13
2.39 (m, 3H, 3CH), 2.83 (t, J = 7.12 Hz, 1H, CH), 3.01–
3.49 (m, 10H, 5CH2), 4.42, 4.81 (2 s, 4H, D2O exchangeable,
2NH2), 5.86 (s, 1H, CH=C); 13C NMR (DMSO-d6) d 13.0,
21.4 (C-18, C-19), 23.3 (ethyl CH3), 31.9 (C-16), 37.3
C-15), 43.8 (C-11), 45.0 (C-12), 49.0 (C-1), 49.6 (C-6),
50.2 (C-2), 56.5 (C-7), 71.8, 83.0, 84.4 (C-9, C-8, C-14),
121.7 (C-4), 102.8, 106.7 (ylidene C=C), 150.8, 153.2, 158.
2 (pyrazole C), 168.2 (C-5), 189.6 (C-3); MS: m/e = 396
(M?); Anal. Calcd. for C24H34N4O: C, 73.06; H, 8.69; N,
14.20. Found C, 72.96; H, 8.91; N, 14.39.
C
NMR (500 MHz, CDCl3): d 13.2, 21.6 (C-18, C-19), 31.9
(C-16), 37.3 (C-15), 43.8 (C-11), 45.1 (C-12), 49.0 C-1), 50.1
(C-2), 57.2 (C-7), 71.5, 83.0, 84.5 (C-9, C-8, C-14), 120.6
(CN), 121.3 (C-4), 126.2, 128.3, 132.8, 134.5, 140.2 (thio-
phene C), 168.0 (C-5), 184.3 (C-3); MS: m/e = 394 (M?);
Anal. Calcd. for C24H30N2OS: C, 73.06; H, 7.66; N, 7.10; S,
8.13. Found C, 72.86; H, 6.83; N, 7.31; S, 7.84.
Ethyl 2-amino-4-((8S,9S,10R,13S,14S)-10,13-dimethyl-3-
oxo-2,3,6,7,8,9,10,11,12,13, 14,15,16,17-tetradecahydro-
1H-cyclopenta[a]phenanthren-17-yl)thiophene-3-carbox-
ylate (4b) HPLC purity = 94 % (C-18 NovaPak column;
MeCN:MeOH:H2O/60:30:10) tr = 22 min; white crystals
(8S,9S,10R,13S,14S)-17-((E)-1-(3-amino-5-hydroxy-4H-
pyrazol-4-yl)ethyl)-10,13-dimethyl-6,7,8,9,10,11,12,13,
14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3
(2H)-one (6b) HPLC purity = 90 % (C-18 NovaPak
column; MeOH:H2O/80:20), tr = 26 min; yellow crystals
from ethanol (78 %), m.p. 122–124 °C; IR (KBr) cm-1
:
3488, 3422 (NH2), 3022 (thiophene CH), 2936, 2877 (CH3,
CH2), 1684, 1689 (2 CO), 1638 (C=C); 1H NMR (CDCl3):
d 0.82 (s, 3H, CH3), 1.00 (s, 3H, CH3), 1.14 (t, 3H, J =
7.58 Hz, ester CH3), 1.60–1.93 (m, 6H, 3CH2), 2.21–2.39
(m, 3H, 3 CH), 2.80 (t, J = 7.25 Hz, 1H, CH), 3.22–3.50
(m, 10H, 5CH2), 4.21 (q, J = 7.58 Hz, 2H, ester CH2),
4.58 (s, 2H, D2O exchangeable, NH2), 5.84 (s, 1H, CH=C),
6.36 (s, 1H, thiophene H-5), 13C NMR (500 MHz, CDCl3):
d 14.8 (ester CH3), 13.3, 21.6 (C-18, C-19), 31.9 (C-16),
37.9 (C-15), 43.4 (C-11), 45.1 (C-12), 49.4 (C-1), 49.7
(C-6), 50.4 (C-2), 58.5 (C-7), 71.5, 83.2, 84.2 (C-9, C-8,
C-14), 60.4 (ester CH2), 121.8 (C-4), 126.8, 128.6, 130.8,
135.4 (thiophene C), 168.2 (C-5), 178.3, 187.2 (ester CO,
C-3); MS: m/e = 441 (M?); Anal. Calcd. for C26H35NO3S:
C, 70.71; H, 7.99; N, 3.17; S, 7.26. Found C, 70.52; H,
8.31; N, 2.93; S, 7.06.
from ethanol (73 %), m.p. 166–168 °C; IR (KBr) cm-1
:
3477–3337 (OH, NH2), 2934, 2894 (CH3, CH2), 1688
1
(CO), 1640 (C=N); H NMR (DMSO-d6): d 0.81 (s, 3H,
CH3), 1.01 (s, 3H, CH3), 1.63–1.87 (m, 6H, 3CH2), 2.20–
2.39 (m, 3H, 3CH), 2.68 (s, 3H,CH3), 2.80 (t, J = 7.02 Hz,
1H, CH), 3.10–3.46 (m, 10H, 5CH2), 4.43 (s, 2H, D2O
exchangeable, NH2), 5.84 (s, 1H, CH=C), 10.05 (s, 1H,
D2O exchangeable, OH); 13C NMR (500 MHz, CDCl3): d
13.3, 21.6 (C-18, C-19), 23.3 (ethyl CH3), 31.9 (C-16),
37.3 (C-15), 43.8 (C-11), 45.0 (C-12), 49.3 (C-1), 49.6 (C-
6), 50.3 (C-2), 57.9 (C-7), 71.5, 83.0, 84.6 (C-9, C-8,
C-14), 103.0, 106.5 (C=C), 121.7 (C-4), 130.6, 144.2, 153.
6 (pyrazole C), 168.0 (C-5), 189.8 (C-3); MS: m/e = 395
(M?); Anal. Calcd. for C24H33N3O2: C, 72.88; H, 8.41; N,
10.62. Found C, 72.68; H, 8.22; N, 10.53.
(8S,9S,10R,13S,14S)-17-((E)-1-(3,5-diamino-1-phenyl-1H-
pyrazol-4(5H)-yl)ethyl)-10,13-dimethyl-6,7,8,9,10,11,12,13,
14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3
(2H)-one (6c) HPLC purity = 96 % (C-18 NovaPak
column; MeCN:MeOH:H2O/70:20:10), tr = 23 min; yel-
low crystals from ethanol (70 %), m.p. 152–154 °C; IR
(KBr) cm-1: 3582, 3420 (2NH2), 2930,1896 (CH3, CH2),
Synthetic procedure of the pyrazole derivatives 6a–d
To a solution of either 3a (0.362 g, 1 mmol) or 3b
(0.409 g, 1 mmol) in 1,4-dioxane (30 ml) either hydrazine
hydrate or phenyl hydrazine was added. The reaction
mixture was heated under reflux for 2 h, then poured onto
an ice-water mixture containing a few drops of concen-
trated hydrochloric acid. The formed solid product was
collected by filtration and crystallized from the appropriate
solvent.
1
1686 (CO), 1673 (C=N), 1636 (C=C); H NMR (DMSO-
d6): d 0.82 (s, 3H, CH3), 1.00 (s, 3H, CH3), 1.60–1.87 (m,
6H, 3CH2), 2.20–2.36 (m, 3H, 3CH), 2.67 (s, 3H,CH3),
2.82 (t, J = 6.12 Hz, 1H, CH), 3.10–3.46 (m, 10H, 5CH2),
3.54 (m, 1H, CH), 4.42, 5.21 (2 s, 4H, D2O exchangeable,
(8S,9S,10R,13S,14S)-17-(1-(3,5-diamino-4H-pyrazol-4-yl)
ethyl)-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-do-
decahydro-1H-cyclopenta[a]phenanthren-3(2H)-one (6a)
HPLC purity = 94 % (C-18 NovaPak column; MeOH:
H2O/65: 35), tr = 24 min; pale yellow crystals from eth-
anol (88 %), m.p. 180–183 °C; IR (KBr) cm-1: 3455–3421
(2NH2), 2938 (CH2), 1687 (CO), 1642 (C=N), 1622 (C=C);
1H NMR (DMSO-d6): d 0.80 (s, 3H, CH3), 1.00 (s, 3H,
CH3), 2.66 (s,3H,CH3), 1.60–1.91 (m, 6H, 3CH2), 2.22–
2NH2), 5.86 (s, 1H, CH=C), 7.32–7.44 (m, 5H, C6H5). 13
C
NMR (500 MHz, CDCl3): d 13.2, 21.8 (C-18, C-19), 23.3
(ethyl CH3), 31.6 (C-16), 37.4 (C-15), 44.0 (C-11), 44.9
(C-12), 49.3 (C-1), 49.6 (C-6), 50.3 (C-2), 57.2 (C-7), 71.5,
83.2, 84.8 (C-9, C-8, C-14), 121.6 (C-4), 124.3, 126.8,
128.3, 138.6 (C6H5), 156.3, 158.9 (pyrazole C), 168.2 (C-5),
177.7 (exocyclic C=N), 189.6 (C-3); MS: m/e = 472 (M?);
Anal. Calcd. for C30H38N4O: C, 76.56; H, 8.14; N, 11.90.
Found C, 76.52; H, 8.42; N, 12.15.
123

Med Chem Res
(8S,9S,10R,13S,14S)-17-(1-(5-amino-3-hydroxy-1-phenyl-
1H-pyrazol-4-yl)ethyl)-10,13-dimethyl-6,7,8,9,10,11,12,13,
14,15,16,17-dodecahydro-1H-cyclopenta[a]-phenanthren-
3(2H)-one (6d) HPLC purity = 92 % (C-18 NovaPak
column; MeCN:MeOH:H2O/55:35:10), tr = 20 min; yel-
low crystals from ethanol (66 %), m.p. 230–233 °C; IR
(KBr) cm-1: 3540, 3433 (OH, NH2), 2928, 2889 (CH3,
CH2), 1688 (CO), 1633 (C=N); 1H NMR (DMSO-d6): d 0.
81 (s, 3H, CH3), 1.00 (s, 3H, CH3), 1.62–1.87 (m, 6H, 4.44
(s, 2H, CH2), 2.68 (s, 3H, CH3), 3.10–3.48 (m, 10H, 5CH2),
4.48 (s, 2H, NH2), 5.86 (s, 1H, CH=C), 7.28–7.38 (m,5H,
C6H5), 8.22 (s, 1H, OH); 13C NMR (500 MHz, CDCl3): d
13.1, 21.8 (C-18, C-19), 23.4 (ethyl CH3), 31.6 (C-16),
37.6 (C-15), 43.9 (C-11), 45.0 (C-12), 49.3 (C-1), 49.6
(C-6), 50.6 (C-2), 57.2 (C-7), 71.5, 83.2, 84.8 (C-9, C-8,
C-14), 121.8 (C-4), 129.6, 130.4, 138.6, 143.8 (C6H5),
150.8, 168.4 (C-5), 190.2 (C-3); MS: m/e = 473 (M?);
Anal. Calcd. for C30H37N3O2: C, 76.40; H, 7.91; N, 8.91.
Found C, 76.26; H, 8.07; N, 9.04.
1 mmol) in water (1 ml) was added. The whole reaction
mixture was stirred at 60 °C for 2 h then poured onto an
ice-water mixture containing three drops of concentrated
hydrochloric acid, and the formed solid product was col-
lected by filtration.
HPLC purity = 93 % (C-18 NovaPak column;
MeOH:H2O/80:10), tr = 24 min; pale yellow crystals from
ethanol (58 %), m.p. 140–142 °C; IR (KBr) cm-1: 2926
1
(CH2), 2224 (CN), 1687, 1682 (2 CO), 1641 (C=C); H
NMR (DMSO-d6): d 0.80 (s, 3H, CH3), 1.00 (s, 3H, CH3),
1.60–1.89 (m, 6H, 3CH2), 2.21–2.34 (m, 3H, 3CH), 2.80 (t,
J = 6.52 Hz, 1H, CH), 3.10–3.48 (m, 10H, 5CH2), 3.52
(m, 1H, CH), 4.45 (s, 2H, CH2), 5.85 (s, 1H, CH=C); 13C
NMR (500 MHz, CDCl3): d 13.0, 21.6 (C-18, C-19), 31.4
(C-16), 37.6 (C-15), 43.5 (C-11), 44.9 (C-12), 49.4 (C-1),
50.8 (C-2), 57.0 (C-7), 63.8 (CH2CN), 71.6, 77.2, 83.2,
84.5 (C-9, C-8, C-14, C-17), 119.1 (CN), 121.8 (C-4),
168.0 (C-5), 177.6, 189.4 (acetyl CO, C-3); MS:
m/e = 339 (M?); Anal. Calcd. for C22H29NO2: C, 77.84;
H, 8.61; N, 4.13. Found C, 78.04; H, 8.41; N, 4.42.
(8S,9S,10R,13S,14S,17S)-17-(2-bromoacetyl)-10,13-
dimethyl-6,7,8,9,10,11,12,13,14, 15,16,17-dodecahydro-
1H-cyclopenta[a]phenanthren-3(2H)-one (7)
Synthesis of the arylhydrazo derivatives 10a–c
To a solution of compound 8 (0.339, 1 mmol) in ethanol
(30 ml) containing sodium acetate (2.5 g), either benze-
nediazonium chloride (0.01 mol), 4-chlorobenzenediazo-
nium chloride (1 mmol) or 4-methylbenzenediazonium
chloride (1 mmol) [prepared by adding sodium nitrite
solution (0.007 g, 1 mmol) to a cold solution of the
appropriate aniline or its derivative (1 mmol) in concen-
trated hydrochloric acid (3 ml, 18 M) with continuous
stirring] was added with stirring. The reaction mixture was
kept at room temperature for 1 h and the formed solid
product, in each case, was collected by filtration.
To a solution of progesterone (1) (0.362 g, 1 mmol) in
acetic acid (10 ml) at 60 °C, bromine (0.050 g, 1 mmol)
was added drop wise and stirred at room temperature for
1 h. The solvent was then evaporated under vacuum, and
the remaining product triturated with diethyl ether and
collected by filtration.
HPLC purity = 92 % (C-18 NovaPak column;
MeOH:H2O/90:10), tr = 22 min; pale yellow crystals from
ethanol (73 %), m.p. 66 °C; IR (KBr) cm-1: 2923 (CH2),
1
1689, 1684 (2CO) 1638 (C=C); H NMR (DMSO-d6): d
0.81 (s, 3H, CH3), 1.00 (s, 3H, CH3), 1.62–1.87 (m, 6H,
3CH2), 2.20–2.36 (m, 3H, 3CH), 2.82 (t, J = 7.23 Hz, 1H,
CH), 3.53 (m, 1H, CH), 3.13–3.51 (m, 10H, 5CH2), 3.88 (s,
2H, CH2), 5.82 (s, 1H, CH=C); 13C NMR (500 MHz,
CDCl3): d 13.0, 21.8 (C-18, C-19), 31.8 (C-16), 38.0 (C-
15), 37.6 (C-15), 44.4 (C-11), 44.9 C-12), 49.4 (C-1), 49.6
(C-6), 50.6 (C-2), 57.0 (C-7), 66.8 (CH2Br), 71.6, 77.2,
83.2, 84.5 (C-9, C-8, C-14, C-17), 121.6 (C-4), 168.2 (C-5),
177.8, 190.0 (acetyl CO, C-3); MS: m/e = 392 (M?); Anal.
Calcd. for C21H29BrO2: C, 64.12; H, 7.43; Br, 20.31.
Found C, 64.37; H, 7.68; Br, 20.63.
2-((8S,9S,10R,13S,14S,17S)-10,13-dimethyl-3-oxo-2,3,6,7,
8,9,10,11,12,13,14,15,16,
17-tetradecahydro-1H-cyclo-
penta[a]phenanthren-17-yl)-2-oxo-N0-phenylacetohydrazo-
noyl cyanide (10a) HPLC purity = 96 % (C-18 NovaPak
column; CH3CN:MeOH:H2O/35:55:10), tr = 22 min;
Orange crystals from ethanol (71 %), m.p. 190–193 °C; IR
(KBr) cm-1: 3463–3321 (NH), 3055 (CH aromatic), 2928
(CH2), 2220 (CN), 1710, 1686 (2CO), 1650 (C=N), 1636
(C=C); 1H NMR (DMSO-d6): d 0.81 (s, 3H, CH3), 1.00 (s,
3H, CH3), 1.60–1.88 (m, 6H, 3CH2), 2.21–2.36 (m, 3H,
3CH), 2.82 (t, J = 7.42 Hz, 1H, CH), 3.10–3.46 (m, 10H,
5CH2), 3.54 (m, 1H, CH), 5.88 (s, 1H, CH=C), 7.30–7.38
(m, 5H, C6H5), 8.42 (s, 1H, D2O exchangeable, NH): 13C
NMR (500 MHz, CDCl3): d 13.0, 21.6 (C-18, C-19), 31.4
(C-16), 37.6 (C-15), 44.7 (C-11), 44.6 (C-12), 49.8 (C-1),
49.9 (C-6), 50.8 (C-2), 57.2 (C-7), 71.8, 77.4, 83.0, 84.7
(C-9, C-8, C-14, C-17), 119.8 (CN), 121.7 (C-4), 120.8,
124.3, 126.3, 127.6 (C6H5), 158.3 (C=N), 168.2 (C-5),
3-((8S,9S,10R,13S,14S,17S)-10,13-dimethyl-3-oxo-
2,3,6,7,8,9,10,11,12,13,14, 15,16,17-tetradecahydro-1H-
cyclopenta[a]phenanthren-17-yl)-3-oxopropanenitrile (8)
A solution of compound 7 (0.392, 1 mmol) in ethanol
(30 ml) was heated in a boiling water bath to which a
concentrated solution of potassium cyanide (0.064 g,
123

Med Chem Res
177.7, 189.8 (acetyl CO, C-3); MS: m/e = 443 (M?); Anal.
Calcd. for C28H33N3O2: C, 75.81; H, 7.50; N, 9.47. Found
C, 75.64; H, 7.74; N, 9.21.
onto an ice-water mixture containing one drop of concen-
trated hydrochloric acid, and the formed solid product was
collected by filtration.
N0-(4-Chlorophenyl)-2-((8S,9S,10R,13S,14S,17S)-10,13-
dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetra-
decahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoaceto
hydrazonoyl cyanide (10b) HPLC purity = 90 % (C-18
NovaPak column; CH3CN:MeOH:H2O/40:50:10), tr = 24
min; Orange crystals from ethanol (66 %), m.p. 120–123 °C;
IR (KBr) cm-1: 3472–3329 (NH), 3058 (CH aromatic), 2932
(CH2), 2224 (CN), 1705, 1684 (2CO), 1638 (C=C); 1H NMR
(DMSO-d6): d 0.80 (s, 3H, CH3), 1.00 (s, 3H, CH3), 1.60–
1.88 (m, 6H, 3CH2), 2.23–2.37 (m, 3H, 3CH), 2.80 (t, J =
7.42 Hz, 1H, CH), 3.12–3.49 (m, 10H, 5CH2), 3.56 (m, 1H,
CH), 5.86 (s, 1H, CH=C), 7.26–7.36 (m, 4H, C6H4), 8.40 (s,
1H, D2O exchangeable, NH); 13C NMR (500 MHz, CDCl3):
d 13.3, 21.6 (C-18, C-19), 31.4 (C-16), 37.6 (C-15), 43.7
(C-11), 44.5 (C-12), 49.2 (C-1), 49.8 (C-6), 50.3 (C-2), 57.0
(C-7), 71.6, 77.7, 83.2, 84.9 (C-9, C-8, C-14, C-17), 119.4
(CN), 121.5 (C-4), 122.6, 124.3, 133.6, 134.8 (C6H4), 158.6
(C=N), 168.4 (C-5), 177.6, 189.6 (acetyl CO, C-3); MS:
m/e = 477 (M?); Anal. Calcd. for C28H32ClN3O2: C, 70.35;
H, 6.75; N, 8.79. Found C, 70.46; H, 6.55; N, 8.86.
(8S,9S,10R,13S,14S,17S)-17-(5-amino-1H-pyrazol-3-yl)-10,
13-dimethyl-6,7,8,9, 10,11,12,13,14,15,16,17-dodecahydro-
1H-cyclopenta[a]phenanthren-3(2H)-one
(11a) HPLC
purity = 91 % (C-18 NovaPak column; MeOH:H2O/90:
10), tr = 20 min; yellow crystals from ethanol (68 %),
m.p. 120–122 °C; IR (KBr) cm-1: 3471–3313 (NH2, NH),
3052 (CH pyrazole), 2930 (CH2), 1684 (CO), 1636 (C=C);
1H NMR (DMSO-d6): d 0.80 (s, 3H, CH3), 1.00 (s, 3H,
CH3), 1.61–1.86 (m, 6H, 3CH2), 2.20–2.37 (m, 3H, 3CH),
2.81 (t, J = 7.42 Hz, 1H, CH), 3.11–3.49 (m, 10H, 5CH2),
3.53 (m, 1H, CH), 4.82 (s, 2H, D2O exchangeable, NH2),
5.82 (s, 1H, C-4), 6.53 (s, 1H, pyrazole H-3), 8.22 (s, 1H,
D2O exchangeable, NH): 13C NMR (500 MHz, CDCl3): d
13.1, 21.4 (C-18, C-19), 31.5 (C-16), 37.6 (C-15), 43.8 (C-
11), 44.8 (C-12), 49.2 (C-1), 49.8 (C-6), 50.7 (C-2), 57.0
(C-7), 71.5, 77.7, 83.3, 84.5 (C-9, C-8, C-14, C-17), 119.2
(CN), 114.6, 152.4, 156.8 (pyrazole C), 168.8 (C-5), 190.0
(C-3); MS: m/e = 353 (M?); Anal. Calcd. for C22H31N3O:
C, 74.75; H, 8.84; N, 11.89. Found C, 74.84; H, 9.14; N,
12.09.
N0-(4-methylphenyl)-2-((8S,9S,10R,13S,14S,17S)-10,13-
dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetra-
decahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoaceto
hydrazonoyl cyanide (10c) HPLC purity = 93 % (C-18
NovaPak column; MeOH:H2O/80:20), tr = 18 min;
Orange crystals from ethanol (80 %), m.p. 144–146 °C; IR
(KBr) cm-1: 3465–3320 (NH), 3060 (CH aromatic), 2932
(8S,9S,10R,13S,14S,17S)-17-(5-amino-1-phenyl-1H-pyrazol-
3-yl)-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-do-
decahydro-1H-cyclopenta[a]phenanthren-3(2H)-one (11b)
HPLC purity = 93 % (C-18 NovaPak column; CH3CN/
MeOH:H2O/20:70:10), tr = 18 min; yellow crystals from
ethanol (73 %), m.p. 180–184 °C; IR (KBr) cm-1: 3477,
3323 (NH2), 3044 (CH pyrazole), 2928 (CH2), 1638 (C=
C); 1H NMR (DMSO-d6): d 0.82 (s, 3H, CH3), 1.00 (s, 3H,
CH3), 1.61–1.86 (m, 6H, 3CH2), 2.20–2.37 (m, 3H, 3CH),
2.83 (t, J = 7.89 Hz, 1H, CH), 3.09–3.49 (m, 10H, 5CH2),
4.82 (s, 2H, D2O exchangeable, NH2), 5.82 (s, 1H, CH=C),
6.53 (s, 1H, pyrazole H-3), 7.29–7.39 (m, 5H, C6H5); 13C
NMR (500 MHz, CDCl3): d 13.0, 21.4 (C-18, C-19), 31.5
(C-16), 37.6 (C-15), 43.8 (C-11), 44.9 (C-12), 49.4 (C-1),
49.8 (C-6), 50.7 (C-2), 57.2 (C-7), 71.8, 77.7, 83.5, 84.6
(C-9, C-8, C-14, C-17), 121.3 (C-4), 124.9, 133.6, 135.6
(C6H5), 142.3, 153.0, 156.2 (pyrazole C), 168.8 (C-5),
190.0 (C-3); MS: m/e = 429 (M?); Anal. Calcd. for
C28H35N3O: C, 78.28; H, 8.21; N, 9.78. Found C, 78.52; H,
8.04; N, 9.53.
1
(CH2), 2223 (CN), 1698, 1686 (2CO), 1660 (C=N); H
NMR (DMSO-d6): d 0.80 (s, 3H, CH3), 1.00 (s, 3H, CH3),
1.60–1.88 (m, 6H, 3CH2), 2.23–2.37 (m, 3H, 3CH), 2.80 (t,
J = 7.42 Hz, 1H, CH), 2.89 (s, 3H, CH3), 3.10–3.46 (m,
10H, 5CH2), 5.86 (s, 1H, CH=C), 7.28–7.44 (m, 4H, C6H4),
8.37 (s, 1H, D2O exchangeable, NH): 13C NMR (500 MHz,
CDCl3): d 13.0, 21.4, 24.3 (C-18, C-19, 4-CH3-phenyl),
31.4 (C-16), 37.6 (C-15), 43.8 (C-11), 44.8 (C-12), 49.3 (C-
1), 49.4 (C-6), 50.7 (C-2), 57.5 (C-7), 71.8, 77.5, 83.0, 84.5
(C-9, C-, C-14, C-17), 119.8 (CN), 121.7 (C-4), 124.3,
126.3, 133.6, 134.8 (C6H4), 168.6 (C-5), 173.5 (C=N), 177.
7, 189.4 (acetyl CO, C-3); MS: m/e = 457 (M?); Anal.
Calcd. for C29H35N3O2: C, 76.12; H, 7.71; N, 9.18. Found
C, 76.07; H, 7.59; N, 9.33.
Synthesis of the arylidene derivatives 13a–c
Synthesis of the pyrazole derivatives 11a, b
To a solution of compound 8 (0.339 g, 1 mmol) in 1,4-
dioxane (10 ml) containing piperidine (0.250 ml), either
benzaldehyde (0.106 g, 1 mmol), 4-chlorobenzaldehyde
(0.140 g, 1 mmol) or 4-methoxybenzaldehyde (0.136 g,
1 mmol) was added. The whole reaction mixture was
To a solution of compound 8 (0.339, 1 mmol) in ethanol
(30 ml), either hydrazine hydrate (0.050 g, 1 mmol) or
phenyl hydrazine (0.108 g, 1 mmol) was added. The
reaction mixture was heated under reflux for 4 h, poured
123

Med Chem Res
heated under reflux for 2 h, evaporated under vacuum, and
the remaining product triturated with ethanol and collected
by filtration.
(KBr) cm-1: 3058 (CH aromatic), 2927 (CH aromatic),
2220 (CN), 1687, 1686 (2CO), 1638 (C=C); 1H NMR
(DMSO-d6): d 0.80 (s, 3H, CH3), 1.00 (s, 3H, CH3),
1.60–1.88 (m, 6H, 3CH2), 2.20–2.36 (m, 3H, 3CH), 2.81 (t,
J = 6.22 Hz, 1H, CH), 3.02 (s, 3H, OCH3), 3.12–3.52 (m,
10H, 5CH2), 5.80 (s, 1H, CH=C), 6.01 (s, 1H, ylidene CH),
7.31–7.42 (m, 4H, C6H4); 13C NMR (500 MHz, CDCl3): d
13.0, 21.6, (C-18, C-19) 28.6 (OCH3), 31.5 (C-16), 37.6
(C-15), 43.6 (C-11), 44.9 (C-12), 49.4 (C-1), 49.8 (C-6),
50.7 (C-2), 57.3 (C-7), 71.6, 77.9, 83.3, 84.8 (C-9, C-8,
C-14, C-17), 106.9 (ylidene CH), 119.2 (CN), 122.6 (C-4),
124.9, 126.5, 134.2, 138.8 (C6H4), 149.3 (ylidene C), 168.2
(C-5), 166.4, 189.9 (acetyl CO, C-3); MS: m/e = 457
(M?); Anal. Calcd. for C30H35NO3: C, 78.74; H, 7.71; N,
3.06. Found C, 78.82; H, 7.41; N, 3.32.
2-((8S,9S,10R,13S,14S,17S)-10,13-dimethyl-3-oxo-2,3,6,7,
8,9,10,11,12,13,14,
15,16,17-tetradecahydro-1H-cyclo-
penta[a]phenanthrene-17-carbonyl)-3-phenylacrylonitrile
(13a) HPLC purity = 96 % (C-18 NovaPak column;
MeOH:H2O/80:20), tr = 20 min; yellow crystals from
ethanol (80 %), m.p. 166–168 °C; IR (KBr) cm-1: 3064
(CH aromatic), 2927 (CH2), 2220 (CN), 1687, 1683 (2CO),
1636 (C=C); 1H NMR (DMSO-d6): d 0.80 (s, 3H, CH3), 1.
00 (s, 3H, CH3), 1.60–1.88 (m, 6H, 3CH2), 2.20–2.38 (m,
3H, 3CH), 2.82 (t, J = 7.26 Hz, 1H, CH), 3.09–3.49 (m,
10H, 5CH2), 5.82 (s, 1H, CH=C), 6.01 (s, 1H, ylidene CH),
7.26–7.39 (s, 5H, C6H5); 13C NMR (500 MHz, CDCl3): d
13.0, 21.4 (C-18, C-19), 31.5 (C-16), 37.6 (C-15), 43.8 (C-
11), 49.4 (C-1), 49.9 (C-6), 50.7 (C-2), 57.2 (C-7), 71.8,
77.7, 83.5, 84.6 (C-9, C-8, C-14, C-17), 121.4 (C-4), 116.4
(ylidene CH), 119.8 (CN), 124.9, 127.9, 134.2, 135.6
(C6H5), 152.2 (ylidene C), 168.4 (C-5), 166.9, 189.6
(acetyl CO, C-3); MS: m/e = 427 (M?); Anal. Calcd. for
C29H33NO2: C, 81.46; H, 7.78; N, 3.28. Found C, 81.72; H,
7.94; N, 3.52.
Synthesis of the pyran derivatives 15a–f
To a solution of 13a (0.427 g, 1 mmol), 13b (0.461 g,
1 mmol) or 13c (0.427 g, 1 mmol) in 1,4-dioxane (20 ml)
containing triethylamine (0.50 ml), 2a (0.066 g, 1 mmol)
or 2b (0.107 g, 0.01 mol) was added. The reaction mixture
was heated under reflux for 2 h, poured into an ice-water
mixture, and the formed solid product collected by
filtration.
3-(4-Chlorophenyl)-2-((8S,9S,10R,13S,14S,17S)-10,13-
dimethyl-3-oxo-2,3,6,7,8,9, 10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthrene-17-car-
bonyl)acrylonitrile (13b) HPLC purity = 94 % (C-18
NovaPak column; CH3CN:MeOH:H2O/20:60:20), tr =
22 min; yellow crystals from ethanol (63 %), m.p. 80 °C;
IR (KBr) cm-1: 3052 (CH aromatic), 2927 (CH2), 2222
2-Amino-6-((8S,9S,10R,13S,14S)-10,13-dimethyl-3-oxo-2,
3,6,7,8,9,10,11,12,13,14,
15,16,17-tetradecahydro-1H-
cyclopenta[a]phenanthren-17-yl)-4-phenyl-4H-pyran-3,5-
dicarbonitrile (15a) HPLC purity = 93 % (C-18 Nov-
aPak column; CH3CN:MeOH:H2O/30:60:10), tr = 15 min;
yellow crystals from ethanol (73 %), m.p. 192–194 °C; IR
(KBr) cm-1: 3482, 3336 NH2), 3061 (CH aromatic), 2922
1
(CN), 1688, 1684 (2CO), 1636 (C=C): H NMR (DMSO-
1
d6): d 0.80 (s, 3H, CH3), 1.00 (s, 3H, CH3), 1.60–1.88 (m,
6H, 3CH2), 2.20–2.38 (m, 3H, 3CH), 2.82 (t, J = 7.83 Hz,
1H, CH), 3.03–3.51 (m, 10H, 5CH2), 5.83 (s, 1H, CH=C),
6.01 (s, 1H, ylidene CH), 7.30–7.38 (2d, 4H, J = 4.66 Hz,
C6H4); 13C NMR (500 MHz, CDCl3): d 13.0, 21.4 (C-18,
C-19), 31.5 (C-16), 37.6 (C-15), 43.8 (C-11), 44.4 (C-12),
49.3 (C-1), 49.8 (C-6), 50.7 (C-2), 57.3 (C-7), 71.8, 77.9,
83.6, 84.9 (C-9, C-8, C-14, C17), 106.8 (ylidine CH), 119.0
(CN), 121.6 (C-4), 124.9, 126.5, 127.9, 134.2, 141.8
(C6H4), 151.6 (ylidene C), 168.2 (C-5), 166.3, 189.8
(acetyl CO, C-3); MS: m/e = 461 (M?); Anal. Calcd. for
C29H32ClNO2: C, 75.39; H, 6.98; Cl, 7.67; N, 3.03. Found
C, 75.60; H, 7.25; Cl, 7.92; N, 3.27.
(CH2), 2222, 2218 (2CN), 1688 (CO), 1641 (C=C); H
NMR (DMSO-d6): d 0.80 (s, 3H, CH3), 1.00 (s, 3H, CH3),
1.60–1.86 (m, 6H, 3CH2), 2.20–2.36 (m, 3H, 3CH), 2.80
(t, J = 7.23 Hz, 1H, CH), 3.12–3.50 (m, 10H, 5CH2), 4.62
(s, 2H, D2O exchangeable, NH2), 5.80 (s, 1H, CH=C), 6.53
(s, 1H, pyran H-4), 7.28–7.38 (m, 5H, C6H5); 13C NMR
(500 MHz, CDCl3): d 13.0, 21.8 (C-18, C-19), 31.5 (C-16),
37.3 (C-15), 43.6 (C-11), 44.9 (C-12), 49.3 (C-1), 49.8
(C-6), 50.7 (C-2) 57.2 (C-7), 71.8, 77.3, 83.3, 84.9 (C-9,
C-8, C-14, C-17)), 116.8, 117.3 (2CN), 122.8 (C-4), 124.9,
126.5, 128.6, 134.8 (C6H5), 55.9, 106.9, 122.4, 154.9
(pyran C), 168.6 (C-5), 189.9 (C-3); MS: m/e = 493 (M?);
Anal. Calcd. for C32H35N3O2: C, 77.86; H, 7.15; N, 8.51.
Found C, 77.93; H, 7.42; N, 8.37.
2-((8S,9S,10R,13S,14S,17S)-10,13-dimethyl-3-oxo-2,3,6,7,
8,9,10,11,12,13,14,
15,16,17-tetradecahydro-1H-cyclo-
2-Amino-4-(4-chlorophenyl)-6-((8S,9S,10R,13S,14S)-10,
13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-4H-
pyran-3,5-dicarbonitrile (15b) HPLC purity = 95 % (C-
18 NovaPak column; MeOH:H2O/85:15), tr = 19 min;
penta[a]phenanthrene-17-carbonyl)-3-(4-methoxyphenyl)
acrylonitrile (13c) HPLC purity = 90 % (C-18 NovaPak
column; CH3CN:MeOH:H2O/20:60:20), tr = 20 min; yel-
low crystals from ethanol (68 %), m.p. 130–133 °C; IR
123

Med Chem Res
yellow crystals from ethanol (82 %), m.p. 111–113 °C; IR
NH2), 5.84 (s, 1H, CH=C), 6.28 (s, 1H, pyran H-4), 7.30–7.
44 (m, 5H, C6H5); 13C NMR (500 MHz, CDCl3): d 13.1,
21.6, (C-18, C-19), 16.2 (ester CH3), 32.5 (C-16, 37.8 (C-
15), 43.6 (C-11), 44.9 (C-12), 49.8 (C-1), 50.7 (C-6), 57.2
(C-7), 60.3 (ester CH2), 71.0, 77.7, 83.5, 84.9 (C-9, C-8,
C-14, C-17), 116.9, 119.0 (2CN), 55.2, 106.9, 122.6, 150.4
(pyran C), 122.3 (C-4), 124.4, 126.3, 129.3, 134.2, 139.2,
168.3 (C-5) 172.3, 189.8 (ester CO, C-3); MS: m/e = 540
(M?); Anal. Calcd. for C34H40N2O4: C, 75.53; H, 7.46; N,
5.18. Found C, 75.69; H, 7.21; N, 4.83.
(KBr) cm-1: 3476, 3321 (NH2), 3057 (CH aromatic), 2926
1
(CH2), 2224, 2220 (2CN), 1686 (CO), 1632 (C=C); H
NMR (DMSO-d6): d 0.80 (s, 3H, CH3), 1.00 (s, 3H, CH3),
1.60–1.86 (m, 6H, 3CH2), 2.20–2.36 (m, 3H, 3CH), 2.80 (t,
J = 7.03 Hz, 1H, CH), 3.12–3.50 (m, 10H, 5CH2), 4.62 (s,
2H, D2O exchangeable, NH2), 5.80 (s, 1H CH=C), 6.20 (s,
1H, pyran H-4), 7.26–7.42 (m, 4H, C6H4); 13C NMR
(500 MHz, CDCl3): d 13.0, 21.6 (C-18, C-19), 32.5 (C-16),
37.8 (C-15), 43.6 (C-11), 44.9 (C-12), 49.2 (C-1), 49.7
(C-6), 50.7 (C-2), 57.0 (C-7), 71.2, 77.9, 83.5, 84.6 (C-9,
C-8, C-14, C-17), 55.3, 106.9, 122.4, 154.2 (pyran C),
117.4, 119.1 (2CN), 122.9, 124.9, 127.5, 129.0, 138.4
(C6H5), 189.8 (C-3); MS: m/e = 527 (M?); Anal. Calcd.
for C32H34ClN3O2: C, 72.78; H, 6.49; Cl, 6.71; N, 7.96.
Found C, 72.42; H, 6.29; C, 7.06; N, 7.66.
Ethyl 2-amino-4-(4-chlorophenyl)-5-cyano-6-((8S,9S,10R,
13S,14S)-10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,
15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-
17-yl)-4H-pyran-3-carboxylate (15e) HPLC purity =
95 % (C-18 NovaPak column; MeOH:H2O/85:15), tr =
19 min; yellow crystals from ethanol (72 %), m.p.
172–175 °C; IR (KBr) cm-1: 3473, 3342 (NH2), 3060 (CH
aromatic), 2929, 2897 (CH3, CH2), 2222 (CN), 1689, 1686
2-Amino-6-((8S,9S,10R,13S,14S)-10,13-dimethyl-3-oxo-2,
3,6,7,8,9,10,11,12,13,14, 15,16,17-tetradecahydro-1H-cy-
clopenta[a]phenanthren-17-yl)-4-(4-methoxyphenyl)-4H-
pyran-3,5-dicarbonitrile (15c) HPLC purity = 96 % (C-
18 NovaPak column; MeOH:H2O/90:10), tr = 22 min;
yellow crystals from ethanol (73 %), m.p. 190–193 °C; IR
(KBr) cm-1: 3445, 3315 (NH2), 3054 (CH aromatic), 2928,
2889 (CH3, CH2), 2222, 2218 (2CN), 1688 (CO), 1635 (C=
C); 1H NMR (DMSO-d6): d 0.82 (s, 3H, CH3), 1.00 (s, 3H,
CH3), 1.60–1.88 (m, 6H, 3CH2), 2.20–2.36 (m, 3H, 3CH),
2.81 (t, J = 6.95 Hz, 1H, CH), 3.04 (s, 3H, CH3), 3.12–
3.52 (m, 10H, 5CH2), 4.62 (s, 2H, D2O exchangeable,
NH2), 5.83 (s, 1H, CH=C), 6.23 (s, 1H, pyran H-4), 7.28–
7.40 (m, 4H, C6H4); 13C NMR (500 MHz, CDCl3): d 13.0,
21.6 (C-18, C-19), 57.0 (OCH3), 31.5 (C-16), 37.8 (C-15),
43.6 (C-11), 44.9 (C-12), 49.3 (C-1), 49.8 (C-6), 50.7 (C-
2), 57.3 (C-7) 71.0, 77.5, 83.5, 84.7 (C-9, C-8, C-14, C-17),
117.5, 119.0 (2CN), 55.0, 106.9, 122.6, 150.3 (pyran C),
122.9 (C-4), 124.9, 126.3, 128.8, 136.8 (C6H4), 168.7 (C-
5), 189.6 (C-3); MS: m/e = 523 (M?); Anal. Calcd. for
C33H37N3O3: C, 75.69; H, 7.12; N, 8.02. Found C, 75.83;
H, 7.36; N, 7.99.
1
(2CO), 1634 (C=C); H NMR (DMSO-d6): d 0.80 (s, 3H,
CH3), 1.00 (s, 3H, CH3), 1.13 (t, J = 7.29 Hz, 3H, ester
CH3), 1.60–1.88 (m, 6H, 3CH2), 2.20–2.36 (m, 3H, 3CH),
2.83 (t, J = 7.29 Hz, 1H, CH), 3.12–3.50 (m, 10H, 5CH2),
4.19 (q, J = 7.29 Hz, 2H, ester CH2), 4.58 (s, 2H, D2O
exchangeable NH2), 5.86 (s, 1H, CH=C), 6.25 (s, 1H, pyran
H-4), 7.23-7.42 (2d, 4H, C6H4); 13C NMR (500 MHz,
CDCl3): d 13.0, 21.6 (C-18, C-19), 16.9 (ester CH3), 31.5
(C-16), 37.8 (C-15), 43.6 (C-11), 44.9 (C-12), 49.8 (C-1),
49.7 (C-6), 50.7 (C-2), 57.4 (C-7), 71.4, 77.8, 83.5, 84.6
(C-9, C-8, C-14, C-17), 57.0 (ester CH2), 116.8, 119.0 (2
CN), 55.6, 106.9, 122.2, 150.4 (pyran C), 122.6, 125.9,
126.3, 130.6 (C6H4), 168.3 (C-5), 172.0, 189.6 (ester CO,
C-3); MS: m/e = 574 (M?); Anal. Calcd. for
C34H39ClN2O4: C, 71.00; H, 6.83; Cl, 6.16; N, 4.87. Found
C, 70.83; H, 6.62; Cl, 5.93; N, 5.26.
Ethyl 2-amino-4-(4-methoxyphenyl)-5-cyano-6-((8S,9S,
10R,13S,14S)-10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,
13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenan-
thren-17-yl)-4H-pyran-3-carboxylate (15f) HPLC pur-
ity = 95 % (C-18 NovaPak column; MeOH:H2O/85:15),
tr = 22 min; yellow crystals from ethanol (67 %), m.p.
113–115 °C; IR (KBr) cm-1: 3469, 3322 (NH2), 3054 (CH
aromatic), 2945, 2922 (CH3, CH2), 2224 (CN), 1688, 1687
Ethyl
2-amino-5-cyano-6-((8S,9S,10R,13S,14S)-10,13-
dimethyl-3-oxo-2,3,6,7,8,9, 10,11,12,13,14,15,16,17-tetra-
decahydro-1H-cyclopenta[a]phenanthren-17-yl)-4-phenyl-
4H-pyran-3-carboxylate (15d) HPLC purity = 92 % (C-
18 NovaPak column; MeOH:H2O/90:10), tr = 22 min;
yellow crystals from ethanol (78 %), m.p. 222–224 °C; IR
(KBr) cm-1: 3458, 3322 (NH2), 3058 (CH aromatic), 2926,
28886 (CH3, CH2), 2228, 2220 (CN), 1684 (CO), 1632 (C–
C); 1H NMR (DMSO-d6): d 0.80 (s, 3H, CH3), 1.00 (s, 3H,
CH3), 1.12 (t, J = 7.55 Hz, 3H, 3CH), 1.60–1.88 (m, 6H.
3CH2), 2.20–2.36 (m, 3H, 3CH), 2.83 (t, J = 7.29 Hz, 1H,
CH), 3.04 (s, 3H, CH3), 3.12–3.48 (m, 10H, 5CH2), 4.22
(q, J = 7.29 Hz, 2H, CH2), 4.62 (s, 2H, D2O exchangeable,
1
(2CO), 1638 (C=C); H NMR (DMSO-d6): d 0.80 (s, 3H,
CH3), 1.00 (s, 3H, CH3), 1.11 (t, J = 7.22 Hz, 3H, ester
CH3), 1.60–1.88 (m, 6H, 3CH2), 2.20–2.36 (m, 3H, 3CH),
2.83 (t, J = 7.29 Hz, 1H, CH), 3.04 (s, 3H, OCH3), 3.09–3.
53 (m, 10H, 5CH2), 4.19 (q, J = 7.22 Hz, 2H, ester CH2),
4.61 (s, 2H, D2O exchangeable, NH2), 5.87 (s, 1H, CH=C),
6.28 (s, 1H, pyran H-4), 7.25–7.39 (m, 4H, C6H4); 13C
NMR (500 MHz, CDCl3): d 13.0, 21.6 (C-1, C-19), 16.7
(ester CH3), 57.7 (OCH3), 31.5 (C-16), 37.8 (C-15), 43.6
123

Med Chem Res
Mohareb RM, El-Omran F (2012) Reaction of pregnenolone with
cyanoacetylhydrazine: novel synthesis of hydrazide–hydrazone,
pyrazole, pyridine, thiazole, thiophene derivatives and their
cytotoxicity evaluations. Steroids 77:1551–1559
Mohareb RM, Hanaa H (2007) Synthesis of progesterone heterocyclic
derivatives of potential antimicrobial activity. Acta Pharm
58:29–42
Mohareb RM, Wardakhan WW, Elmegeed GA, Ashour RM (2012)
Hetero-cyclizations of pregnenolone: novel synthesis of thio-
semicarbazone, thiophene, thiazole, thieno[2,3-b]pyridine deriv-
atives and their cytotoxicity evaluations. Steroids 77:1560–1569
Mohareb RM, El-Sayed NN, Abdelaziz MA (2013) Knoevenagel
reactions of pregnenolone with cyanomethylene reagents: syn-
thesis of thiophene, thieno[2,3-b]pyridine, thieno[3,2-d]isoxaz-
ole derivatives of pregnenolone and their in vitro cytotoxicity
towards tumor and normal cell lines. Steroids 78:1209–1219
Morand P, Lyall J (1968) The steroidal estrogens. Chem Rev
68:85–124
Ouali MI (2006) Recent advances in thiasteroids chemistry. Steroids
71:1025–1044
Ouali MI (2007) Recent advances in oxasteroids chemistry. Steroids
72:475–508
Ouali MI, Rocheblave L (2008) Recent advances in azasteroids
chemistry. Steroids 73:375–407
Page ST, Marck BT, Tolliver JM, Matsumoto AM (2008) Tissue
selectivity of the anabolic steroid, 19-nor-4-androstenediol-
3b,17b-diol in male sprague dawley rats: selective stimulation
of muscle mass and bone mineral density relative to prostate
mass. Endocrinology 149:1987–1993
(C-11), 44.9 (C-12), 49.2 (C-1), 49.8 (C-6), 50.7 (C-2),
57.4 (C-7), 71.6, 77.8, 83.0, 84.3 (C-9, C-, C-14, C-17), 57.
4 (ester CH2), 116.9 (CN), 119.0 (C-4),), 55.9, 106.3,
122.2, 150.8 (pyran C), 123.8, 125.9, 132.6, 134.2 (C6H4),
168.3 (C-5), 172.1, 190.2 (ester CO, C-3); MS: m/e = 570
(M?); Anal. Calcd. for C35H42N2O5: C, 73.66; H, 7.42; N,
4.91. Found C, 73.81; H, 7.36; N, 5.19.
Conclusion
The objective of the present study was to synthesize a series
of heterocyclic derivatives of progesterone. The antibacte-
rial activity (MIC) of the newly synthesized compounds
against the Streptococcus pneumonia strains showed that
compounds 3a, 4a, 6b, 6d, 8, 10b, 13b, 15b, and 15c are the
most active compounds toward the four Streptococcus
pneumonia serotypes. The presence of either an OH, Cl, or
CN groups in any of such compounds is responsible for such
reactivity. Compounds, 3a and 4a showed high activity
against AV3 (serotype 3) and AV14 (serotype 14). On the
other hand, compound 6b, 13b, and 15b showed high activity
toward AV3, AV6 (serotype 6B), and AV23 (serotype 23F).
Pagonis TA, Angelopoulos NV, Koukoulis GN, Hadjichristodoulou
CS (2006) Psychiatric side effects induced by supraphysiological
doses of combinations of anabolic steroids correlate to the
severity of abuse. Eur Psychiatry 21:551–562
Racedo S,Villena J, Medina M, Aguero G, Rodrıguez V, Alvarez S
(2006) Lactobacillus casei administration reduces lung injuries
in a Streptococcus pneumoniae infection in mice. Microb Infect
8:2359–2366
Acknowledgments R. M. Mohareb would like to express his
deepest thank to the Alexander von Humboldt Foundation in Bonn for
affording his a fellowship in Germany, Mu¨nchen during summer,
2012 and the finance support together with completing this work.
Moreover, the author would like to thank Faculty of Science, Cairo
University for the funding support during this work.
Rosen P, Boris A, Oliva C (1980) Synthesis and biological-activity of
some 15-oxaestranes. J Med Chem 23:329–330
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