Synthesis of steroidal derivatives containing substituted, fused and spiro pyrazolines

Article abstract of DOI:10.1016/j.steroids.2014.05.013

An efficient and facile synthesis of fused, substituted and spiro pyrazoline steroid derivatives through a cycloaddition reaction of different α,β-unsaturated ketones with hydrazine acetate in acetic acid is reported. Depending on the starting material, the ring closure reaction provided a mixture of two steroidal pyrazoline epimers that were separated and studied by NMR techniques. In one case it was possible to isolate and characterize the hydrazone derivative as the reaction intermediate, which confirms the mechanism proposed in the literature [11,25,26].2014 Published by Elsevier Inc.

Full text of DOI:10.1016/j.steroids.2014.05.013

Steroids 87 (2014) 86–92
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis of steroidal derivatives containing substituted, fused and spiro
pyrazolines
⇑
⇑
Anabel Romero-López, Sara Montiel-Smith , Socorro Meza-Reyes , Penélope Merino-Montiel,
José Luis Vega-Baez
Benemérita Universidad Autónoma de Puebla, Facultad de Ciencias Químicas, Ciudad Universitaria, C.P. 72570 Puebla, Pue., Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient and facile synthesis of fused, substituted and spiro pyrazoline steroid derivatives through a
Received 26 January 2014
Received in revised form 3 May 2014
Accepted 25 May 2014
cycloaddition reaction of different
a,b-unsaturated ketones with hydrazine acetate in acetic acid is
reported. Depending on the starting material, the ring closure reaction provided a mixture of two steroi-
dal pyrazoline epimers that were separated and studied by NMR techniques. In one case it was possible to
isolate and characterize the hydrazone derivative as the reaction intermediate, which confirms the mech-
anism proposed in the literature [11,25,26].
Available online 11 June 2014
Keywords:
Pyrazoline
Ó 2014 Published by Elsevier Inc.
Unsaturated ketones
Hydrazine acetate
Cycloaddition
Hydrazone
1. Introduction
2. Experimental methods
Several types of steroids fused to heterocycles have been care-
fully studied since the 1960s and their chemistry has been pro-
gressing subsequently; based on these results, it was possible to
establish the structure–activity relationship between the steroidal
structure and their physiological properties [1,2]. In recent years,
the efforts have been undertaken towards the rational modification
of steroid molecules involving the incorporation of a heteroatom,
such as N or O. This kind of compounds has shown many different
biological activities including anti-microbial, anti-inflammatory,
hypotensive, hypocholesterolemic and diuretic activities [3–6].
Pyrazoline derivatives are electron rich nitrogen heterocycles
which show interesting pharmacological properties such as anal-
gesic, antipyretic, antirheumatic [7,8], anti-inflammatory [9], anti-
diabetic [10], anticancer [11–14] and antimicrobial activities [15].
Herein we report a very facile and high yielding synthesis of ste-
roidal pyrazolines located on D and E rings, starting from 20-keto
pregnane, trans-androsterone and diosgenin through a condensa-
2.1. General methods
NMR spectra 1D and 2D 1H and 13C (DEPT, COSY, NOESY, HSQC,
HMBC) were recorded on
a VARIAN Mercury spectrometer
(400 MHz for 1H, 100 MHz for 13C). Chemical shifts are stated in
ppm (d), and are referred to the 1H signal (d = 7.24) or to the central
13C triplet signal (d = 77.0) for CDCl3. Coupling constants (J) are
quoted in Hz. IR spectra were acquired on a Nicolet FT-IR 380 spec-
trophotometer (m
max, cmꢀ1). FAB-MS, EI-MS and high resolution
mass (HRMS) spectra were recorded on a Jeol-JMS-700 MS Station
spectrometer. Optical rotations were determined on a Perkin Elmer
241 polarimeter at room temperature using chloroform solutions.
Melting points were measured on a Mel-Temp apparatus and were
not corrected. Analytical TLC was performed on silica gel ALU-
GRAMÒ SIL G/UV254 plates and chromatographic columns were
carried out on silica gel Davisil™ grade 633 (200–425 mesh).
tion reaction between
a,b-unsaturated carbonyl compounds and
1,2-binucleophilic compounds such as hydrazine (Scheme 1) [16].
2.2. General procedure for the preparation of steroidal benzylidene
derivatives
A mixture of 1 or 5 (1 mmol) and benzaldehyde (1 mmol) was
dissolved in ethanol (5 mL) and an alcoholic solution of potassium
hydroxide (10%) was added drop wise. The mixture was stirred at
room temperature. After completion of the reaction, the precipi-
⇑
Corresponding authors. Tel.: +52 222 2295500x7382 or 2838; fax: +52 222
2443106.
E-mail addresses: maria.montiel@correo.buap.mx (S. Montiel-Smith), maria.
meza@correo.buap.mx (S. Meza-Reyes).
http://dx.doi.org/10.1016/j.steroids.2014.05.013
0039-128X/Ó 2014 Published by Elsevier Inc.

A. Romero-López et al. / Steroids 87 (2014) 86–92
87
4.68 (m, 1H, H-3), 5.40 (1H, dd, J = 12 Hz, J = 4 Hz, H-50), 7.15 (d,
2H, J = 8 Hz, H-200 and H-600), 7.22 (t, 1H, J = 8 Hz, H-400), 7.30 (t,
2H, J = 8 Hz, H-300and H-500). 13C NMR (CDCl3) d: 12.2 (C-19), 13.5
(C-18), 20.9 (3-Ac-CH3), 21.4 (N-Ac-CH3), 21.8 (C-11), 24.9 (C-15),
27.3 (C-2), 28.4 (C-6), 29.6 (C-16), 31.8 (C-7), 33.9 (C-4), 35.4 (C-
8), 35.5 (C-10), 36.7 (C-1), 38.4 (C-12), 44.1 (C-13), 44.5 (C-5),
45.9 (C-40), 51.9 (C-17), 54.1 (C-9), 56.0 (C-14), 59.0 (C-50), 73.5
(C-3), 125.2 (C- 200 and 600), 127.3 (C-400), 128.7 (C-300 and 500), 141.9
(C-100), 159.5 (C-30), 168.4 (N-Ac-CO), 170.7 (3-Ac-CO). HRMS-FAB
(m/z): calcd for C32H44N2O3: 504.3352, found 505.3365 [M+1]+.
The product 4b was obtained as a white solid powder; yield 50%;
ꢀ
1653. 1H NMR (CDCl3) d: 0.63 (s, 3H, CH3-18), 0.80 (s, 3H, CH3-
19), 2.01 (s, 3H, 3-Ac-CH3), 2.31 (s, 3H, N-Ac-CH3), 2.74 (dd, 1H,
J = 16 Hz, J = 4 Hz, H-40), 3.26 (dd, 1H, J = 16 Hz, J = 12 Hz, H-40),
4.67 (m, 1H, H-3), 5.40 (1H, dd, J = 12 Hz, J = 4 Hz, H-50), 7.15 (d,
2H, J = 8 Hz, H-200 and 600), 7.23 (t, 1H, J = 8 Hz, H-400), 7.31 (t, 2H,
J = 8 Hz, H-300 and 500). 13C NMR (CDCl3) d: 12.1 (C-19), 13.6 (C-18),
21.0 (3-Ac-CH3), 21.4 (N-Ac-CH3), 21.8 (C-11), 24.2 (C-15), 24.5
(C-2), 27.3 (C-6), 28.4 (C-16), 29.6 (C-7), 31.8 (C-4), 33.9 (C-8),
35.6 (C-10), 36.7 (C-1), 38.6 (C-12), 44.1 (C-13), 44.5 (C-5), 46.2
(C-40), 51.7 (C-17), 54.1 (C-9), 56.1 (C-14), 59.0 (C-50), 73.6 (C-3),
125.2 (C- 200 and 600), 127.3 (C-400), 128.8 (C-300 and 500), 142.2 (C-
100), 159.3 (C-30), 168.5 (N-Ac-CO), 170.7 (3-Ac-CO). HRMS-FAB
(m/z): calcd for C32H44N2O3: 504.3352, found 505.3365 [M+1]+.
R1
O
R2
HN NH2
N
+
-H2O
N
R
R1
R
R2
Scheme 1. General reaction for the formation of pyrazoline.
tated solid was filtered and washed with ethanol (10 mL) to afford
the product 2 or 6 as solid powder.
2.2.1. (21E)-21-Benzyliden-3b-hydroxypregn-20-one 2
mp. 149–152; [
a
]
D
ꢀ17.7° (c 1 CHCl3); IR
mmax: 2928, 2858, 1727,
Colorless solid powder; yield 80%; mp. 110–111 °C; [
a]
D +37.6°
max: 3411, 2926, 1660, 1607. 1H NMR (CDCl3) d:
ꢀ
m
(c 1, CHCl3); IR
0.61 (s, 3H, CH3-18), 0.79 (s, 3H, CH3-19), 2.82 (t, 1H, J = 8.5 Hz,
H-17), 3.55 (m, 1H, H-3), 6.75 (d, 1H, J = 16.0 Hz, H-21), 7.37 (over-
lapping multiplets, 3H, H-30, H-40, H-50), 7.55 (overlapping multi-
plets, 3H, H-20, H-60, H-22). 13C NMR (CDCl3) d: 12.5 (C-18), 13.9
(C-19), 21.5 (C-11), 22.9 (C-15), 24.8 (C-6), 28.8 (C-16), 31.6 (C-
2), 32.3 (C-7), 35.7 (C-10), 35.8 (C-1), 37.2 (C-8), 38.3 (C-4), 39.6
(C-12), 45.0 (C-5), 45.5 (C-13), 54.5 (C-9), 57.1 (C-14), 62.4 (C-
17), 71.20 (C-3), 126.7 (C-21), 128.5 (C-20,60), 129.1 (C-30,50),
130.4 (C-40), 135.0 (C-10), 141.3 (C-22), 200.4 (C-20). HREI: (m/z)
calcd for C28H38O2: 406.2872, found 406.2881.
2.2.2. (16E)-16-Benzyliden-3b-hydroxyl-5a-androstan-17-one 6
Colorless solid powder; yield 98%; mp. 163–165 °C. 1H NMR
(CDCl3) d: 0.86 (s, 3H, CH3-19), 0.95 (s, 3H, CH3-18), 2.40 (m, 1H,
H-15a), 2.86 (dd, 1H, H-15b), 3.59 (m, 1H, H-3), 7.37 (overlapping
multiplets, 4H, H-10, H-40, H-50, H-60), 7.53 (overlapping multiplets,
2H, H-30, H-70). 13C NMR (CDCl3) d: 12.3 (C-19), 14.4 (C-18), 20.5 (C-
11), 28.3 (C-6), 29.3 (C-15), 31.0 (C-7), 31.3 (C-12), 31.6 (C-2), 34.6
(C-4), 35.7 (C-1), 36.8 (C-10), 37.9 (C-8), 44.7 (C-5), 47.5 (C-13),
49.4 (C-14), 54.4 (C-9), 71.1 (C-3), 128.6 (C-40 and C-60), 129.1 (C-
50), 130.2 (C-30 and C-70), 132.9 (C-1), 135.5 (C-20), 136.1 (C-16),
210.0 (C-17). The spectroscopy data of the compound 6 was com-
pared with that reported in the literature [17].
2.3.2. (50R)- and (50S)-17b-(10-Acetyl-50-phenyl-1H-pyrazolin-30-yl)-
5a
-androstan-3b-ol (3a and 4a)
The individual compounds 3b or 4b (100 mg) were dissolved in a
solution (10%) of KOH in methanol (15 ml). The mixture was allowed
to stand at room temperature, and the progress of the reaction was
monitored by TLC. After completion of the transformation, the reac-
tion mixture was diluted with water. The resulting precipitate was
filtered off, washed with water and dried under vacuum to afford
3a as a white solid powder; yield 85%; mp. 239–241 °C; [
a]
D
ꢀ
+0.57° (c 0.1, CHCl3). IR
m
max: 3408, 2920, 2855, 1648. 1H NMR
(CDCl3) d: 0.63 (s, 3H, CH3-18), 0.81 (s, 3H, CH3-19), 2.31 (s, 3H, N-
Ac-CH3), 2.66 (dd, 1H, Jgem = 20 Hz, J4 a-5 = 4 Hz H-40), 3.36 (dd, 1H,
0
0
Jgem = 20 Hz, J4 b-5 = 12 Hz, H-40), 3.59 (m, 1H, H-3), 5.40 (1H, dd,
2.3. General process for the preparation of steroidal pyrazoline
derivatives
0
0
J5 -4 b = 12 Hz, J5 -4 a = 4 Hz, H-50), 7.14 (d, 2H, J = 8 Hz, H-200 and 600),
7.22 (t, 1H, J = 8 Hz, H-400), 7.30 (t, 2H, J = 8 Hz, H-300 and 500). 13C
NMR (CDCl3) d: 12.3 (C-19), 13.3 (C-18), 21.0 (C-11), 21.8 (N-Ac-
CH3), 24.3 (C-15), 24.4 (C-6), 28.5 (C-16), 29.6 (C-2), 31.4 (C-7),
31.9 (C-1), 35.5 (C-10), 35.6 (C-8), 36.9 (C-4), 38.0 (C-12), 38.5 (C-
13), 44.8 (C-5), 45.8 (C-40), 51.9 (C-17), 54.2 (C-9), 56.1 (C-14), 59.0
(C-50), 71.2 (C-3), 125.2 (C- 200 and 600), 127.3 (C-400), 128.7 (C-300 and
500), 141.9 (C-100), 159.5 (C-30), 168.4 (N-Ac-CO). HRMS-FAB
(m/z): calcd for C32H44N2O3: 462.3246, found 462.3219. 4a: white
ꢀ
0
0
0
0
Compounds 2, 6 or 10 (2.4 mmol) were dissolved in 10 ml of
acetic acid and then hydrazine acetate (4.8 mmol) was added.
The mixture was refluxed and monitored by TLC until complete
disappearance of starting material (ꢁ3 h). The product was precip-
itated by pouring the reaction mass into excessive amounts of ice-
cold water. The formed precipitate was filtered, washed with
water, and dried to afford the products 3a, 4a, 7, 12 and 16.
solid powder; yield 90%; mp. 149–152 °C; IR mmax: 3411, 2921, 2854,
2.3.1. (50S)- and (50R)-17b-(10-Acetyl-50-phenyl-1H-pyrazolin-30-yl)-
1644. 1H NMR (CDCl3) d: 0.63 (s, 3H, CH3-18), 0.78 (s, 3H, CH3-19),
2.31 (s, 3H, N-Ac-CH3), 2.71 (dd, 1H, Jgem = 16 Hz, J4 a-5 = 4 Hz, H-40),
0
0
5
a-androstan-3b-yl acetate (3b and 4b)
0
3.26 (dd, 1H, Jgem = 15.6 Hz, J4 b-5 = 12 Hz, H-40), 3.54 (m, 1H, H-3),
0
The mixture of epimers 3a and 4a was dissolved in CH2Cl2
5.40 (1H, dd, J4 b-5 = 12 Hz, J4 a-5 = 4 Hz, H-50), 7.15 (d, 2H, J = 8 Hz,
H-200 and 600), 7.23 (t, 1H, J = 8 Hz, H-400), 7.31 (t, 2H, J = 8 Hz, H-300
and 500). 13C NMR (d, ppm, CDCl3): 12.2 (C-19), 13.5 (C-18), 21.0
(C-11), 21.8 (N-Ac-CH3), 24.2 (C-15), 24.4 (C-6), 28.5 (C-16), 29.6
(C-2), 31.3 (C-7), 31.9 (C-1), 35.4 (C-10), 35.5 (C-8), 36.9 (C-4), 37.9
(C-12), 38.6 (C-13), 44.7 (C-5), 46.1 (C-40), 51.7 (C-17), 54.2 (C-9),
56.1 (C-14), 59.0 (C-50), 71.1 (C-3), 125.2 (C- 200 and 600), 127.3 (C-400),
128.7 (C-300 and 500), 142.2 (C-100), 159.4 (C-30), 168.6 (N-Ac-CO). HREI:
(m/z) calcd for C30H42N2O2: 462.3246, found 462.3219.
0
0
0
0
(5 ml), subsequently acetic anhydride (5 ml) and catalytic amounts
of DMAP were added, the solution was stirred at room temperature
during 15 min. The mixture was then diluted with water and the
organic phase was extracted with CH2Cl2 (50 mL). The residue
was washed with saturated solution of NaHCO3 (3 ꢂ 30 mL), and
water (40 mL). The resulting organic phase was dried over Na2SO4
and concentrated to dryness under vacuum. The acetylated crude
product was purified by column chromatography on silica gel (hex-
ane/EtOAc 8:2), the product 3b was eluted first and then 4b. The
product 3b was obtained as a white solid powder; yield 25%; mp.
ꢀ
184–186 °C; [
a]
+69.3° (c 1.8, CHCl3); IR
m
max: 2928, 2858, 1727,
2.3.3. 310-Acetyl-50-phenyl-40,50-dihydro[1,2]-diazolo[40,30:16,17]-5
a-
androstan-3b-yl acetate 7
D
1653. 1H NMR (CDCl3) d: 0.62 (s, 3H, CH3-18), 0.83 (s, 3H, CH3-
19), 2.02 (s, 3H, 3-Ac-CH3), 2.31 (s, 3H, N-Ac-CH3), 2.62 (dd, 1H,
J = 20 Hz, J = 4 Hz, H-40), 3.36 (dd, 1H, J = 20 Hz, J = 12 Hz, H-40),
White solid powder; yield 70%; mp. 145–148 °C; [
a]D ꢀ1.48° (c
0.1, CHCl3); IR
max: 2929, 2856, 1727, 1652. 1H NMR (CDCl3) d:
ꢀ
m

88
A. Romero-López et al. / Steroids 87 (2014) 86–92
0.59 (s, 3H, CH3-18), 0.77 (s, 3H, CH3-19), 1.99 (s, 3H, 3-Ac-CH3),
(3-Ac-CO). HREI: (m/z) calcd for C25H36N2O3: 412.2726, found
0
0
2.35 (s, 3H, N-Ac-CH3), 3.92 (m, 1H, H-16), 4.46 (d, 1H, J5 -4
412.2722.
= 12 Hz, H-50), 4.66 (m, 1H, H-3), 7.37 (m, 2H, H-300and 500), 7.71
(m, 3H, H-200, 400and 600). 13C NMR (CDCl3) d: 12.1 (C-19), 12.4
(C-18), 21.0 (C-11), 21.4 (N-Ac-CH3), 21.5 (C-15), 27.3 (C-2), 28.3
(C-6), 30.8 (C-15), 32.2 (C-7), 33.8 (4), 35.1 (C-8), 55.4 (C-10),
36.6 (C-1), 38.9 (C-12), 44.4 (C-5), 45.9 (C-13), 49.3 (C-16), 53.7
(C-9), 54.3 (C-14), 72.1 (C-50), 73.5 (C-3), 126.8 (C- 200 and 600),
128.5 (C-400), 129.8 (C-300 and 500), 131.0 (C-100), 157.4 (C-17),
169.29 (N-Ac-CO), 170.6 (3-Ac-CO). HREI: (m/z) calcd for
2.3.6. 6(22)-[10-acetyl-30-methyl-40(300-acetoxy-200-methyl)propyl]-
16b,22-epoxy-23,24-dinorspiro[22.50]-1H-pirazolinyl)-chol-6-en-3b-
yl acetate (16)
White solid powder; yield 70%; mp. 192–194 °C; [
a
]
D ꢀ16.3° (c
ꢀ
0.1, CHCl3); IR
m
max: 2943, 1725, 1706, 1238. 1H NMR (CDCl3) d:
0.93 (d, 1H, J = 4 Hz, CH3-27) 0.95 (s, 3H, CH3-18), 1.0 (s, 3H,
CH3-19), 1.2 (d, 1H, J = 8 Hz, H-21), 1.8 (s, 3H, N-Ac-CH3), 2.05 (s,
3H, 26-Ac-CH3), 2.0 (s, 3H, 3-Ac-CH3), 2.1 (s, 3H, H-300), 3.0 (m,
2H, H-20 and H-23), 3.8 (dd, 1H, J = 10 Hz, J = 4 Hz, H-26), 3.9
(dd, 1H, J = 10 Hz, J = 4 Hz, H-26), 4.58 (m, 1H, H-3), 4.8 (m, 1H,
C30H42N2O2: 476.6502, found 477.3176.
2.3.4. N-Acetyl-20-hydrazonopregna-5,16-dien-3b-yl acetate 11
13
ꢀ
m
H-16), 5.3 (d, 1H, J = 4 Hz, H-6). C NMR (CDCl3) d: 10.4 (C-300),
Yellow oil; yield 30%; [
a]
+0.51 (c 0.76, CHCl3) IR
max: 2941,
D
1718, 1667 1H NMR (CDCl3) d: 0.98 (s, 3H, CH3-18), 1.0 (s, 3H,
CH3-19), 1.90 (s, 3H, CH3-21), 2.0 (s, 3H, 3-Ac-CH3), 2.2 (s, 3H, N-
Ac-CH3), 4.60 (m,1H, H-3), 5.39 (d,1H, J = 4 Hz, H-6), 6.11 (dd, 1H,
J = 4 Hz, J = 4 Hz, H-16), 8.56 (s, 1H, NH). 13C NMR (CDCl3) d: 12.3
(C-21), 15.8 (C-18), 19.1 (C-19), 20.8 (N-Ac-CH3), 20.9 (C-11),
21.4 (3-Ac-CH3), 27.6 (C-2), 30.1 (C-8), 31.4 (C-7), 31.6 (C-15),
35.4 (C-12), 36.6 (C-10), 36.8 (C-1), 38.0 (C-4), 46.6 (C-13), 50.2
(C-9), 56.8 (C-14), 73.8 (C-3), 122.1 (C-6), 133.8 (C-16), 140 (C-5),
145.5 (C-20), 153.3 (C-17), 170.5 (3-Ac-CO), 173.6 (N-CO). HREI:
(m/z) calcd for C25H36N2O3: 412.2726, found 412.2813.
12.6 (C-18), 17.2 (C-27), 19.2 (C-19), 20.7 (C-11), 20.9 (26-Ac-
CH3), 21.3 (3-Ac-CH3), 21.6 (C-21), 27.2 (C-24), 27.6 (C-2), 28.0
(C-20), 31.3 (C-8), 31.5 (C-7), 34.0 (C-25), 35.0 (C-15), 36.5 (C-
10), 36.8 (C-1), 38.0 (C-4), 39.6 (C-12), 42.3 (C-13), 49.8 (C-9),
54.3 (C-14), 58.4 (C-17), 68.6 (C-26), 73.8 (C-3), 75.8 (C-16),
111.3 (C-22), 122.2 (C-6), 139.6 (C-5), 153.7 (C-30), 170.1 (-N-CO),
170.5 (3-Ac-CO), 171.0 (26-Ac-CO). HREI: (m/z) calcd for
C
25H36N2O3: 596.3825, found 597.3885 [M+1]+.
3. Results and discussion
2.3.5. 510-Acetyl-30-methyl-16,17-dihydro[1,2]-diazolo[50,40:16,17]-
androst-6-en-3b-yl acetate 12
The importance of different steroidal heterocycles is well
known. In our attempt to increase the biological activity we
White solid powder; yield 80% from 10 and 90% from 11; mp.
decided to build up pyrazoline rings from steroidal a,b-unsatu-
rated ketones and hydrazine. Three different types of compounds
ꢀ
170–173 °C; [
a]
+20.4° (c 0.1, CHCl3) IR
m
max: 1734, 1660, 1645.
D
1H NMR (CDCl3) d: 0.90 (s, 3H, CH3-18), 1.0 (s, 3H, CH3-19), 1.9
(s, 3H, CH3-300), 2.0 (s, 3H, 3-Ac-CH3), 2.2 (s, 3H, N-Ac-CH3), 3.0
(d,1H, J17-16 = 12 Hz, H-17), 4.5 (m, 1H, H-3), 4.71 (dd, 1H, J16-17
= 12 Hz, J16-15 = 4 Hz, H-16), 5.34 (d, 1H, J = 4 Hz, H-6). 13C NMR
(CDCl3) d,: 17.0 (C-300), 19.1 (C-19), 20.8 (C-18), 20.8 (3-Ac-CH3),
21.3 (N-Ac-CH3), 21.8 (C-11), 27.6 (C-1), 31.4 (C-7), 31.9 (C-8),
34.0 (C-15), 35.8 (C-12), 36.5 (C-10), 36.9 (C-4), 37.9 (C-12), 46.2
(C-13), 49.4 (C-9), 50.3 (C-14), 60.8 (C-16), 65.5 (C-17), 73.7 (C-
3), 122.2 (C-6), 139.2 (C-5), 156.6 (C-30), 168.3 (N-Ac-CO), 170.5
were synthesized as precursors of steroidal pyrazoline derivatives.
The first
a,b-unsaturated ketone was synthesized through the
aldolic condensation of pregnanolone acetate 1 with benzaldehyde
under basic conditions to give the corresponding benzylidene
derivative 2 (Scheme 2). It is important to mention that due to
the basic conditions used, the first step in the reaction was the
hydrolysis of the acetate on C-3 affording pregnanolone 1a and
then the condensation with the benzaldehyde took place yield a
single product. The 1H NMR spectrum of the steroidal benzyliden
O
O
i
AcO
H
HO
H
1
2
O
O
1'
1'
N
N
5'
5'
2'
2'
N
H
N
H
3'
3'
4'
4'
ii
+
RO
RO
H
H
3a: R = H
3b: R = Ac
4a: R = H
4b: R = Ac
iv
iii
iii
iv
Reagents and conditions: (i) EtOH, KOH, benzaldehyde. (ii) AcOH, hydrazine acetate, ref lux.
(iii) DMAP, acetic anhydride, rt. (iv) Methanol, KOH, rt.
Scheme 2. Synthesis of steroidal pyrazolines 3 and 4.

A. Romero-López et al. / Steroids 87 (2014) 86–92
89
C
3.70
3.65 3.60 3.55
3.50 3.45
3.40 3.35
3.30 3.25
3.20 3.15
3.10 3.05
3.00 2.95
2.90 2.85
2.80 2.75
2.70 2.65
2.60 2.55
2.50
B
3.65 3.60
3.55 3.50
3.45 3.40
3.35 3.30
3.25 3.20
3.15
3.10 3.05
3.00 2.95
2.90 2.85
2.80
2.75 2.70
2.65 2.60
2.55 2.50
A
3.70 3.65 3.60 3.55
3.50 3.45 3.40 3.35
3.30 3.25 3.20 3.15
3.10 3.05 3.00 2.95
2.90 2.85 2.80
2.75 2.70 2.65 2.60
2.55 2.50
Fig. 1. 1H NMR epimeric resolution of 3a and 4a.
O
O
2 showed two characteristic peaks around 6.75 and 7.55 ppm,
which can be assigned to the coupling protons of the double bond
at C-21 Their coupling constant of 16 Hz, indicates their trans rel-
ative position, therefore the configuration of the double bond is
E. The signals corresponding to the phenyl group (d = 7.56–
7.52 ppm) and a multiplet at 3.55 ppm assigned to H-3 were also
observed. In the 13C NMR spectrum of 2, it is observed at high fre-
quencies, the signals of the phenyl group (d = 128.5–135.0 ppm),
the vinylic carbons C-21 and C-22 (d = 126.7 and 141.3 ppm,
respectively) and the carbonyl group at 200.4 ppm.
i
HO
HO
H
H
5
6
O
H
N
N
To obtain the steroidal pyrazoline derivative and study the ste-
reochemistry of their formation, we treated (E)-21-benzyliden-3b-
hydroxy-pregn-20-one 2 with hydrazine acetate in acetic acid into
reflux during 3 h; the TLC indicated the presence of two products
3a and 4a, with a very close RF, in a ratio 1:2 found by 1H NMR
spectroscopy. For a more effective separation, the product mixture
was acetylated under standard conditions and the two products 3b
and 4b were fully separated by column chromatography. After sep-
aration, 2D NMR showed that the minor product was the 50R iso-
mer (3b), while the major product was the epimer 50S (4b). After
separation of the acetylated pyrazoline products (3b and 4b), they
were hydrolyzed to yield the 3b-hydroxy derivatives (3a and 4a).
The assignment of the stereochemistry of 50 position was
achieved by comparison with the steroidal pyrazoline analogue
described by Ivanyi et al. [11]. In the 1H NMR spectrum of the epi-
meric mixture (Fig. 1A) it is observed two signal groups at 3.39–
3.21 and 2.76–2.59 corresponding to the pyrazoline ring protons
H-40, the major one (Fig. 1B) belonging to the 50S diasteroisomer
(4a) and the minor one (Fig. 1C) assigned to the pyrazoline 50R (3a).
Pyrazolines 3a and 4a represent two examples of steroidal pyr-
azolines located on C-17; continuing with our study we decided to
synthesize some compounds where the heterocycle is fused to C-
ii
H
AcO
H
7
Reagents and conditions: (i) EtOH, KOH, benzaldehyde. (ii) AcOH, hydrazine acetate, reflux.
Scheme 3. Synthesis of pyrazoline steroidal derivative 7 from trans-androsterone.
were refluxed in acetic acid during 5 h to afford pyrazoline 7 as a
single product (Scheme 3).
The most remarkable signals of the 1H NMR spectrum of 7 are
multiplets (7.36–7.69 ppm) corresponding to Ar–H, a doublet at
4.46 ppm assigned to H-50 and a doublet of triplets signal at
3.92 ppm assigned to H-16 characteristic of pyrazoline heterocy-
cles, four singlets of methyl protons at 2.35, 1.99, 0.53 and
0.76 ppm corresponding to the NC(O)CH3, COOCH3, CH3-18 and
CH3-19, respectively. In the 13C NMR spectrum the characteristic
signals of pyrazoline heterocycle are observed at 157.4, 72.1 and
49.3 ppm corresponding to C-17, C-50 and C-16, respectively.
The stereochemistry of the new stereogenic center C-16 of com-
pound 7 was established considering the chemical shift of CH3-18
in the 1H NMR spectrum. According with the literature [19], when
a substituent on C-6 is placed with b orientation, the signal of the
CH3-18 is deshielded, and it can be observed at 0.78 ppm, approx-
imately. In the H1-NMR spectrum of 7, the signal of CH3-18 is
16 and C-17. For this purpose we obtained the
a,b-unsaturated
ketone 6 in quantitative yields through the reaction of trans-
androsterone 5 with benzaldehyde in the presence of potassium
hydroxide in ethanol [17,18]. Compound 6 and hydrazine acetate

90
A. Romero-López et al. / Steroids 87 (2014) 86–92
Table 1
Table 2
Calculated vicinal coupling constants 3J16H-5H (Hz) for the particular Cis configuration
Calculated vicinal coupling constants 3J16H-17H (Hz) for the particular Cis configuration
of 12 and their comparison with experimental vicinal coupling constants.
´
of 7 and their comparison with experimental vicinal coupling constants.
Methods
Methods
Compound 7
PM3
RHF/6-31+G(d)
B3LYP/6-31+G(d)
Compound 12
PM3
RHF/6-31+G(d)
B3LYP/6-31+G(d)
Energy (u.a.)
ꢀ0.1579
16.23
–
ꢀ1492.8676
23.94
ꢀ1502.5142
21.26
Energy (u.a.)
ꢀ0.1975
3.83
–
ꢀ1301.1908
13.14
ꢀ1309.5671
12.85
Dihedral angle
Dihedral angle
3 calculated
3 calculated
0
J
J
13.33
10.61
J
J
17.38
11.36
H16-H5
H16-H17
3 expimental
3 expimental
0
12.00
12.00
H16-H5
H16-H17
Additionally, we decided to use as starting material the
a,b-
observed at 0.53 ppm, such value is similar to the chemical shift
observed of some derivatives with the substituent on C-16 -ori-
entated [20,21]. Based on these observations we concluded that
C-16 in compound 7 has an S configuration. Furthermore, in the
analogue synthesized by Amr et al. [19] it was found that the value
unsaturated ketone 10 previously reported [22,23]; compound 10
was synthesized from commercial diosgenin 8 through an acetoly-
sis reaction affording pseudodiosgenin 9 [24] followed by an oxida-
tive cleavage of furan ring (Scheme 4). Fused pyrazoline 12 was
obtained by refluxing ketone 10 with hydrazine acetate in AcOH
during 5 h.
After one hour of reaction, the starting material was trans-
formed completely, and two new products were visualized by
TLC. The less polar was identified as the intermediate hydrazone
11 and the more polar corresponding to compound 12 (Scheme 4).
The presence of the hydrazone is consistent with the widely
a
0
of the coupling constant of J5 ,16b = 1.2 Hz, corresponding to a dihe-
dral angle close to 120°, evidencing the trans disposition of the pro-
tons H-50 and H-16; nevertheless, the value of the coupling
0
constant observed for compound 7 is J5 ,16b = 12 Hz, corresponding
a dihedral angle close to 0°, suggesting that H-50 is b-orientated
(50S). In addition, theoretical studies (PM3, RHF/6-31+G(d) and
B3LYP/6-31G(d) level of theory) were carried out to calculate the
Jvic values for the cis configuration of 7, this values are near to
experimental value (Table 1).
accepted mechanism for the formation of pyrazolines from
unsaturated ketone systems (Scheme 5) [11,25,26] involving the
nucleophilic attack of the hydrazine nitrogen on the carbonyl
a,b-
O
AcO
O
O
i
ii
HO
AcO
9
8
O
O
N
N
N
H
N
H
iii
H
O
AcO
AcO
AcO
12
11
10
Reagents and conditions: (i) BF3OEt2, AcOCOCF3, CH2Cl2. (ii) CrO3, AcOH, CH2Cl2. (iii) AcOH, hydrazine acetate, reflux.
Scheme 4. Synthesis of 12 from 3b-acetoxy-5,16-pregnadien-20-one (10).
H
H
H
O
H
O
O
O
O
O
H
H
H
N
N
N
N
H
H
NH2-NH2.AcOH
H
-H2O
AcO
10
A
O
O
N
HN
N
O
H
N
N
O
O
NH
N
H
H
N
N
N
H
Isomerization
+ H
- H
H
H
H
12
11
Scheme 5. Presumed mechanism for the formation of pyrazoline rings.

A. Romero-López et al. / Steroids 87 (2014) 86–92
91
O
O
O
O
O
O
O
i
ii
OAc
HO
HO
AcO
13
14
8
O
O
N
N
O
O
i
iii
OAc
OAc
AcO
15
AcO
16
Reagents and conditions: (i) Ac2O, BF3OEt2, rt. (ii) KOH (iii) AcOH, hydrazine acetate, reflux.
Scheme 6. Synthesis of steroidal pyrazoline 16.
group of the enone affording intermediate A; then a elimination of
a water molecule gave hydrazone 11. Intramolecular Michael addi-
tion of the nitrogen to the double bond led the pyrazoline 12.
In the 1H NMR spectrum of derivative 11 it is observed one
broad singlet around 8.56 ppm assigned to N-H proton, one
doublet of double signals at 6.11 and a doublet at 5.39 ppm corre-
sponding to the vinyl protons H-16 and H-6 respectively, three
singlets at 2.39, 2.00 and 1.90 ppm corresponding to NC(O)CH3,
COOCH3 and CH3-21, respectively. In 13C NMR spectrum the most
significant change compared with compound 10, is the disappear-
H3C
O
CH3
N
N
CH3
O H
H
H
OAc
H
Fig. 2. NOESY correlations of the steroidal pyrazoline 16.
ance of the signal at 196.6 ppm corresponding to the
a,b-unsatu-
rated ketone and the appearance of signal at 145.5 ppm
a
compound 15 under the cycloaddition conditions already estab-
lished gave the spiro-pyrazoline 16 (Scheme 6).
assigned to C-20; it also can be observed a disappearance of the
vinylic C-16 and C-17 at 133.8 and 153.3 ppm, respectively.
The hydrazone 11 was treated with AcOH and hydrazine acetate
at reflux during 3 h for complete transformation into pyrazoline
12. The 1H NMR showed an upfield shift of H-16 to 4.71 ppm and
the appearance of a signal at 3.01 ppm corresponding to the new
formed H-17. In the 13C NMR two signals are observed of the pyr-
azoline ring C-17 and C-16 at 60.8 and 65.6 ppm, respectively.
The configuration of C-16 and C-17 of compound 12, was estab-
lished considering the chemical shift of CH3-18 (0.90 ppm) in the
1H NMR spectrum, which is deshielded due to the influence of
the new pyrazoline ring place in b position [19,21]. The value of
the coupling constant of J16,17 = 12 Hz, confirms that both H-16
and H-17 are in cis position, concluding that the configuration of
C-16 and C-17 is R, S, respectively. Also the theoretical studies to
calculate the Jvic give the approximate value obtained experimen-
tally (Table 2).
In the 13C NMR spectrum of compound 16 it can be observed a
signal at 153.7 ppm characteristic of the C-30 of the pyrazoline ring.
Furthermore, C-50, involved in the spiranic junction, resonates at
111.3 ppm, whereas the same carbon resonates at roughly
60 ppm for fused pyrazolines, so confirming the formation of a
spiranic pyrazoline located on E ring.
The NOESY experiment of spiro compound 16 showed cross-
peaks from NOE interactions between the protons of AcN-20 of
the pyrazoline ring and CH3-18 (Fig. 2). These observations suggest
that the configuration of the spiro C-22 is R. Moreover, the config-
uration of C-23 is proposed as R since it is observed a dipolar cou-
pling between H-23 and CH3-21 (Fig. 2).
In conclusion, we accomplished the synthesis of different ste-
roidal pyrazolines, in good yields through the cycloaddition of dif-
ferent
a,b-unsaturated ketones and hydrazine acetate as the 1,2-
binucleofilic compound, under acidic conditions, containing the
pyrazoline scaffold on the C-17, fused to D ring or forming a first
spiranic junction at C-22.
Most of the steroidal heterocycles found in the literature are
located on the D ring; with the aim to generate a new group of ste-
roidal pyrazolines, we studied the synthesis of
a,b-unsaturated
systems on the E ring. Our research group has developed in recent
years new methodologies for the modification of the side chain of
sapogenins [27–29]; since our intention was to transform the E
ring, we decided to use the well known furostene derivative 15,
described by our research group [30]. The acetolysis reaction of
diosgenin gave the 22,26-epoxycholest-22-ene derivative 13 as a
major product, a basic hydrolysis of 13 afforded 23-acetyldiosgenin
14 which was treated with Ac2O and BF3ꢃOEt2 to form the desired
furostene 15 in quantitative yield [30] (Scheme 6). Treatment of
Acknowledgments
The authors thank CONACYT – Mexico for scholarships to ARL
and the Retention grant 190909 to PMM. Also we thank VIEP-BUAP
for financial support (grant to SMS). We are also grateful to the
anonymous reviewer for their comments and to Dr. Francisco J.
Meléndez-Bustamante (Lab. de Química Teórica, BUAP) for theo-
retical studies.

92
A. Romero-López et al. / Steroids 87 (2014) 86–92
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unsaturated carbonyls. 7th ed. Berlin: Springer; 2008.
a,b-
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