Synthesis and anti-cancer activity of chiral tetrahydropyrazolo[1,5-a]pyridine-fused steroids

Article abstract of DOI:10.1016/j.steroids.2017.03.006

Regio- and stereoselective synthesis of novel chiral 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine–fused steroids via [8π?+?2π] cycloaddition of diazafulvenium methides with steroidal scaffolds is reported. The biological evaluation of the new family of hexacyclic steroids as anti-cancer agents was also carried out. Hexacyclic steroids bearing a benzyl group at C-22, derived from 16-dehydropregnenolone and 16-dehydroprogesterone, show considerable cytotoxicity against EL4 (murine T-lymphoma) in contrast with the corresponding C-22-unsubstituted derivatives showing low cytotoxicity. Thus, results indicate that the presence of the benzyl group is important to ensure cytotoxicity.

Full text of DOI:10.1016/j.steroids.2017.03.006

Accepted Manuscript
Synthesis and anti-cancer activity of chiral tetrahydropyrazolo[1,5-a]pyridine-
fused steroids
Susana M.M. Lopes, Emanuel P. Sousa, Luísa Barreira, Cátia Marques, Maria
João Rodrigues, Teresa M.V.D. Pinho e Melo
PII:
S0039-128X(17)30041-7
http://dx.doi.org/10.1016/j.steroids.2017.03.006
STE 8088
DOI:
Reference:
Steroids
To appear in:
Received Date:
Revised Date:
Accepted Date:
16 January 2017
25 February 2017
20 March 2017
Please cite this article as: Lopes, S.M.M., Sousa, E.P., Barreira, L., Marques, C., Rodrigues, M.J., Pinho e Melo,
T.M.V., Synthesis and anti-cancer activity of chiral tetrahydropyrazolo[1,5-a]pyridine-fused steroids, Steroids
(2017), doi: http://dx.doi.org/10.1016/j.steroids.2017.03.006
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Synthesis and anti-cancer activity of
chiral tetrahydropyrazolo[1,5-a]pyridine-fused steroids
Susana M. M. Lopesa, Emanuel P. Sousaa, Luísa Barreirab, Cátia Marquesb, Maria João
Rodriguesb, Teresa M. V. D. Pinho e Meloa*
aCQC and Department of Chemistry, University of Coimbra, 3004-535 Coimbra, Portugal
bCCMAR – Centre of Marine Sciences, University of Algarve, Ed. 7, Campus de
Gambelas,8005-139 Faro, Portugal
E-mail: tmelo@ci.uc.pt
Abstract - Regio- and stereoselective synthesis of novel chiral 4,5,6,7-tetrahydropyrazolo[1,5-
a]pyridine–fused steroids via [8π+2π] cycloaddition of diazafulvenium methides with steroidal scaffolds
is reported. The biological evaluation of the new family of hexacyclic steroids as anti-cancer agents was
also carried out. Hexacyclic steroids bearing a benzyl group at C-22, derived from 16-
dehydropregnenolone and 16-dehydroprogesterone, show considerable cytotoxicity against EL4 (murine
T-lymphoma) in contrast with the corresponding C-22-unsubstituted derivatives showing low
cytotoxicity. Thus, results indicate that the presence of the benzyl group is important to ensure
cytotoxicity.
Keywords: Cycloadditions / Sigmatropic Shifts / Extended Dipoles / Steroids / Anti-cancer Activity /
Microwave Chemistry
1. Introduction
Steroidal compounds are widely abundant in the natural world and display a variety of
biological activities [1]. Therefore, chemical modification of the steroid scaffold has
been used as a strategy for drug discovery. The introduction of heterocycles often
results in changes of its biological properties [2,3]. Galeterone, is an example of a C-17
N-heterocycle steroid which has successfully completed phase II clinical development

in men prostate cancer [2a]. On the other hand, synthetic steroids that are fused to
heterocyclic compounds at positions 16 and 17 show also unique biological properties
[1b,3].
In this context, we have recently reported the first examples of [8π+2π] cycloaddition of
16-dehydropregnenolone acetate (16-DPA) with diazafulvenium methides 2, generated
from the SO2 extrusion of 2,2-dioxo-1H,3H-pyrazolo[1,5-c][1,3]thiazoles 1, leading to
chiral 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine–fused steroids 3 in a regio- and
stereoselective fashion [4a]. The reactivity of 1-methyl- and 1-benzyl-diazafulvenium
methides 4 towards 16-DPA was also explored affording selectively hexacyclic steroids
having an additional chiral centre (C-22). Quantum chemical calculations at DFT level
were carried out, using the cycloaddition of 1,7-dipoles 4 with N-phenylmaleimide as
model reactions, in order to rationalize the stereochemistry outcome. The results
indicate that endo cycloadditions of the more stable dipole conformation, having the 1-
substitutent pointing outward, are significantly more favourable than the alternative exo
cycloaddition. Therefore, we could conclude that 16-DPA reacted with diazafulvenium
methides to afford chiral 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine–fused steroids via
endo selective [8π+2π] cycloadditions, with the approach of the dipole for the less
hindered α-face of the steroid. Reactions with 1-substituted diazafulvenium methides 4
afford, along with the desired 1,7-cycloadduct, N-vinyl-1H-pyrazoles 6 resulting from
an allowed suprafacial sigmatropic [1,8]H shift (Scheme 1) [4].
2

Scheme 1. [8π+2π] cycloaddition of 16-dehydropregnenolone acetate (16-DPA) with diazafulvenium
methides [4].
Herein, the novel route to heterocycle-fused steroids based on [8π+2π] cycloadditions
of diazafulvenium methides was extended to other steroidal scaffolds, namely 16-
dehydropregnenolone (16-DHP) and 16-dehydroprogesterone. The biological evaluation
of the new family of hexacyclic steroids as anti-cancer agents was also carried out.
2. Experimental section
2.1. General methods
1H NMR spectra were recorded on an instrument operating at 400 MHz. 13C NMR
spectra were recorded on an instrument operating at 100 MHz. The solvent is
deuteriochloroform except where indicated otherwise. Chemical shifts are expressed in
parts per million related to internal TMS, and coupling constants (J) are in Hz. Infrared
spectra (IR) were recorded on a Fourier transform spectrometer. High-resolution mass
spectra (HMRS) were obtained on an electrospray (ESI) or electronic impact (EI) TOF
3

mass spectrometer. Melting points were determined in open glass capillaries and are
uncorrected. Optical rotations were measured on an Optical Activity AA-5 electrical
polarimeter. Thin-layer chromatography (TLC) analyses were performed using
precoated silica gel plates. Flash column chromatography was performed with silica gel
60 as the stationary phase. MW reactions were carried out in a microwave reactor CEM
Focused Synthesis System Discover S-Class using 10 mL microwave tubes. Dimethyl
2,2-dioxo-1H,3H-pyrazolo[1,5-c][1,3]thiazole-6,7-dicarboxylate (1a) [7], ethyl 6-
phenyl-2,2-dioxo-1H,3H-pyrazolo[1,5-c][1,3]thiazole-7-carboxylate (1b) [4a], methyl
2,2-dioxo-1H,3H-pyrazolo[1,5-c][1,3]thiazole-7-carboxylate (1c) [4a], dimethyl 3-
benzyl-2,2-dioxo-1H,3H-pyrazolo[1,5-c][1,3]thiazole-6,7-dicarboxylate
(10)
[4a],
methyl 2,2-dioxo-1H,3H-pyrazolo[1,5-c][1,3]thiazole-6-carboxylate (14) [4a], 16-DHP
(7) [5], 16-dehydroprogesterone (8) [6] and compounds 3a and 19 [4a], were prepared
as described in the literature.
2.1.1. General Procedure for the [8π+2π] Cycloadditions of Diazafulvenium Methides
with 16-DHP and 16-dehydroprogesterone
Method A. A suspension of 16-DHP (7) or 16-dehydroprogesterone (8) (0.32 mmol) and
the appropriate sulfone (1.5 or 2.5 equiv.) in 1,2,4-trichlorobenzene (1 mL) was
irradiated in the microwave reactor at 250 °C, for 10 minutes. After cooling to room
temperature, the reaction mixture was purified by flash chromatography [hexane] to
remove 1,2,4-trichlorobenzene followed by elution with ethyl acetate-hexane.
Method B. A suspension of 16-DHP (7) or 16-dehydroprogesterone (8) (0.32 mmol) and
the appropriate sulfone (1.5 or 2.5 equiv.) in 1,2,4-trichlorobenzene (1 mL) was heated
at reflux under dry nitrogen, for 4 hours. After cooling to room temperature, the mixture
was purified by flash chromatography [hexane] to remove 1,2,4-trichlorobenzene
followed by elution with ethyl acetate-hexane.
2.1.1.1. Dimethyl 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-2,3-dicarboxylate fused to
16-dehydropregnenolone (9)
Obtained
from
dimethyl
2,2-dioxo-1H,3H-pyrazolo[1,5-c][1,3]thiazole-6,7-
dicarboxylate (1a, 2.5 equiv.). Purification of the crude product by flash
chromatography [hexane, ethyl acetate/hexane (1:1)] gave compound 9 as a white solid
(Method A: 63 mg, 38%; Method B: 39 mg, 24%): mp 206.4-206.8 °C (from ethyl
4

acetate/hexane). [ꢀ]ꢂꢁꢃ -10 (c 1 in CH2Cl2). IR (KBr) ν 1086, 1219, 1256, 1493, 1699,
1736 and 2943 cm-1. 1H NMR: δ 0.76 (s, 3H, H-18), 0.84-0.89 (m, 2H), 1.00 (s, 3H, H-
19), 1.05-1.54 (m, 8H), 1.61-1.96 (m, 7H), 2.20 (s, 3H, H-21), 2.24-2.32 (m, 1H), 3.25
(d, 1H, J = 16.2 Hz, H-28), 3.36 (d, 1H, J = 16.2 Hz, H-28), 3.48-3.56 (m, 1H, H-3),
3.67-3.73 (m, 1H, H-16), 3.84 (dd, 1H, J = 13.4 and 6.8 Hz, H-22), 3.87 (s, 3H), 3.92 (s,
3H), 4.29 (dd, 1H, J = 13.4 and 6.3 Hz, H-22), 5.30 (d, 1H, J = 5.1 Hz, H-6). 13C NMR:
δ 16.6, 19.3, 20.8, 24.7, 27.7, 31.1, 31.5, 31.8, 31.9, 33.0, 36.2, 36.4, 37.1, 42.1, 46.2,
49.4, 51.5, 51.8, 52.4, 52.5, 66.3, 71.6, 110.4, 121.0, 140.7, 142.4, 143.5, 162.3, 163.2,
207.6. HRMS (ESI-TOF) m/z for C30H41N2O6 [M+H]+ calcd 525.29351, found
525.29557.
2.1.1.2.
Dimethyl
(S)-7-benzyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-2,3-
dicarboxylate fused to 16- dehydropregnenolone (11) and dimethyl 5-methyl-1-styryl-
1H-pyrazole-3,4-dicarboxylate (12) [4a]
Obtained from dimethyl 3-benzyl-2,2-dioxo-1H,3H-pyrazolo[1,5-c][1,3]thiazole-7-
carboxylate (10, 1.5 equiv.). Purification of the crude product by flash chromatography
[hexane, ethyl acetate/hexane (1:2) and (1:1)] gave, in order of elution, 12 as a white
solid (Method A: 37 mg, 26%; Method B: 18 mg, 12%) and 11 as a white solid (Method
A: 69 mg, 35%; Method B: 85 mg, 43%).
Compound 11: mp 131.4-133.0 °C (from ethyl acetate/hexane). [ꢀ]ꢂꢁꢃ -50 (c 0.5 in
CH2Cl2). IR (KBr) ν 1215, 1309, 1454, 1705, 1738 and 2935 cm-1. 1H NMR: δ 0.65 (s,
3H), 0.75-0.82 (m, 1H), 0.95 (s, 3H), 1.02-1.10 (m, 1H), 1.16-1.38 (m, 5H), 1.43-1.52
(m, 3H), 1.67-1.70 (m, 1H), 1.79-1.87 (m, 4H), 2.14 (s, 3H), 2.19-2.29 (m, 2H), 2.37 (d,
1H, J = 17.0 Hz), 3.04 (dd, 1H, J = 14.0 and 10.4 Hz), 3.35-3.38 (m, 1H), 3.47-3.54 (m,
1H), 3.89 (s, 3H), 3.94-4.01 (m, 1H), 3.96 (s, 3H), 4.13 (d, 1H, J = 17.0 Hz), 4.20-4.24
(m, 1H), 5.26 (d, 1H, J = 4.8 Hz), 7.28-7.30 (m, 3H), 7.34-7.38 (m, 2H). 13C NMR: δ
16.8, 19.5, 20.9, 23.5, 25.4, 27.6, 31.6, 31.8, 32.2, 32.9, 35.6, 36.5, 37.2, 40.8, 42.2,
46.7, 49.3, 51.9, 52.6, 60.7, 65.5, 71.5, 110.0, 121.1, 126.9, 128.7, 129.1, 136.9, 140.6,
142.6, 144.3, 162.9, 163.3, 207.6. HRMS (ESI-TOF) m/z for C37H47N2O6 [M+H]+ calcd
615.34286, found 615.34269.
2.1.1.3. Dimethyl 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-2,3-dicarboxylate fused to
16-dehydroprogesterone (13a)
5

Obtained
from
dimethyl
2,2-dioxo-1H,3H-pyrazolo[1,5-c][1,3]thiazole-6,7-
dicarboxylate (1a, 2.5 equiv.). Purification of the crude product by flash
chromatography [hexane, ethyl acetate/hexane (1:1)] gave compound 13a as a white
solid (Method A: 89 mg, 53%): mp 233.3-234.5 °C (from ethyl acetate/hexane). [ꢀ]ꢁꢂꢃ
+75 (c 1 in CH2Cl2). IR (KBr) ν 1088, 1180, 1217, 1493, 1672, 1697 and 1737 cm-1. 1H
NMR: δ 0.78 (s, 3H), 0.84-0.98 (m, 2H), 1.17 (s, 3H), 1.19-1.25 (m, 1H), 1.31-1.37 (m,
1H), 1.44-1.61 (m, 2H), 1.65-1.85 (m, 5H), 1.93-2.05 (m, 2H), 2.19 (s, 3H), 2.23-2.43
(m, 4H), 3.26 (d, 1H, J = 16.0 Hz), 3.33 (d, 1H, J = 16.0 Hz), 3.67-3.73 (m, 1H), 3.81-
3.86 (m, 1H), 3.84 (s, 3H), 3.91 (s, 3H), 4.29 (dd, 1H, J = 13.2 and 6.4 Hz), 5.71 (s,
1H). 13C NMR: δ 16.7, 17.3, 20.7, 24.6, 27.6, 30.9, 31.7, 32.5, 32.9, 33.9, 35.5, 35.6,
36.1, 38.4, 46.2, 51.3, 51.5, 51.8, 52.5, 52.9, 66.0, 110.5, 124.2, 142.4, 143.3, 162.3,
163.2, 169.9, 199.1, 207.4. HRMS (ESI-TOF) m/z for C30H49N2O6 [M+H]+ calcd
523.28026, found 523.28000.
2.1.1.4. Ethyl 2-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-3-carboxylate fused
to 16-dehydroprogesterone (13b)
Obtained from ethyl 6-phenyl-2,2-dioxo-1H,3H-pyrazolo[1,5-c][1,3]thiazole-7-
carboxylate (1b, 2.5 equiv.). Purification of the crude product by flash chromatography
[hexane, ethyl acetate/hexane (1:1)] gave compound 13b as a white solid (Method A: 85
mg, 48%): mp 168.5-169.8 °C (from ethyl acetate/hexane). [ꢀ]ꢂꢁꢃ +30 (c 0.5 in CH2Cl2).
1
IR (KBr) ν 1052, 1128, 1450, 1672 and 1701 cm-1. H NMR: δ 0.81 (s, 3H), 0.87-0.99
(m, 2H), 1.18 (s, 3H), 1.20 (t, 3H, J = 7.2 Hz), 1.25-1.30 (m, 1H), 1.36-1.42 (m, 1H),
1.45-2.07 (m, 9H), 2.23 (s, 3H), 2.27-2.48 (m, 4H), 3.35 (d, 1H, J = 16.4 Hz), 3.45 (d,
1H, J = 16.4 Hz), 3.70-3.75 (m, 1H), 3.86 (dd, 1H, J = 13.4 and 6.4 Hz), 4.21 (q, 2H, J
= 7.2 Hz), 4.28 (dd, 1H, J = 13.4 and 6.0 Hz), 5.72 (s, 1H), 7.35-7.39 (m, 3H), 7.58-7.60
(m, 2H). 13C NMR: δ 14.1, 16.7, 17.4, 20.7, 25.1, 27.8, 31.0, 31.8, 32.5, 32.9, 33.9,
35.5, 35.7, 36.3, 38.5, 46.3, 50.9, 51.5, 53.1, 59.9, 66.0, 107.5, 124.2, 127.6, 128.2,
129.5, 133.1, 143.5, 152.6, 163.8, 170.1, 199.2, 208.3. HRMS (ESI-TOF) m/z for
C35H42N2NaO5 [M+Na]+ calcd 577.30368, found 577.30298.
2.1.1.5.
Dimethyl
(S)-7-benzyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-2,3-
dicarboxylate fused to 16-dehydroprogesterone (13c) and dimethyl 5-methyl-1-styryl-
1H-pyrazole-3,4-dicarboxylate (12) [4a]
6

Obtained from dimethyl 3-benzyl-2,2-dioxo-1H,3H-pyrazolo[1,5-c][1,3]thiazole-7-
carboxylate (10, 1.5 equiv.). Purification of the crude product by flash chromatography
[hexane, ethyl acetate/hexane (1:2) and (1:1)] gave, in order of elution, 12 as a white
solid (Method A: 35 mg, 24%) and 13c as a white solid (Method A: 63 mg, 32%).
Compound 13c: mp 126.5-128.0 °C (from ethyl acetate/hexane). [ꢀ]ꢂꢁꢃ -15 (c 1 in
1
CH2Cl2). IR (KBr) ν 1215, 1452, 1668, 1707 and 1741 cm-1. H NMR: δ 0.68 (s, 3H),
0.70-0.79 (m, 2H), 0.83-0.90 (m, 1H), 1.12 (s, 3H), 1.16-1.22 (m, 1H), 1.26-1.32 (m,
1H), 1.36-1.45 (m, 2H), 1.62-1.71 (m, 4H), 1.86-1.89 (m, 2H), 1.97-2.02 (m, 1H), 2.13
(s, 3H), 2.18-2.37 (m, 4H), 3.03 (dd, 1H, J = 14.2 and 10.4 Hz), 3.33-3.37 (m, 1H), 3.87
(s, 3H), 3.97 (s, 3H), 4.00 (dd, 1H, J = 14.2 and 4.8 Hz), 4.21-4.26 (m, 1H), 5.70 (s,
1H), 7.27-7.30 (m, 3H), 7.35-7.38 (m, 2H). 13C NMR: δ 16.8, 17.3, 20.7, 23.2, 25.1,
27.4, 31.5, 32.6, 33.8, 35.4, 35.6, 35.63, 38.3, 40.6, 46.6, 51.6, 51.8, 52.6, 52.7, 60.6,
65.2, 110.0, 124.2, 127.0, 128.7, 129.1, 136.8, 142.6, 144.1, 162.9, 163.4, 170.0, 199.2,
207.4. HRMS (ESI-TOF) m/z for C37H45N2O6 [M+H]+ calcd 613.32721, found
613.32528.
2.1.1.6. Ethyl 2-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-3-carboxylate fused
to 16-dehydropregnenolone (15a/16a)
Obtained from ethyl 6-phenyl-2,2-dioxo-1H,3H-pyrazolo[1,5-c][1,3]thiazole-7-
carboxylate (1b, 2.5 equiv.). Purification of the crude product by flash chromatography
[hexane, ethyl acetate/hexane (1:1)] gave compounds 15a/16a as mixture of
regioisomers [Method A: 59 mg, 33% (87:13); Method B: 45 mg, 25% (81:19)].
Major Component (15a): IR (ATR) ν 1048, 1129, 1291, 1448, 1692, 1697 and 2930 cm-
1
1. H NMR: δ 0.78 (s, 3H), 0.91-0.96 (m, 1H), 1.00 (s, 3H), 1.04-1.11 (m, 1H), 1.22 (t,
3H, J = 7.2 Hz), 1.25-1.56 (m, 8H), 1.62-1.96 (m, 7H), 2.23 (s, 3H), 2.27-2.31 (m, 1H),
3.35 (d, 1H, J = 16.2 Hz), 3.46 (d, 1H, J = 16.2 Hz), 3.50-3.56 (m, 1H), 3.67-3.75 (m,
1H), 3.86 (dd, 1H, J = 13.2 and 6.5 Hz, H-22), 4.18-4.24 (m, 2H), 4.28 (dd, 1H, J = 13.2
and 6.5 Hz), 5.31 (d, 1H, J = 5.1 Hz), 7.35-7.39 (m, 3H), 7.58-7.61 (m, 2H). 13C NMR:
δ 14.3, 16.7, 19.5, 20.9, 22.5, 25.4, 28.0, 31.4, 31.7, 32.0, 33.2, 36.5, 36.6, 37.3, 42.3,
46.4, 49.6, 51.1, 52.5, 60.0, 66.4, 71.7, 107.6, 121.2, 127.7, 128.3, 129.6, 133.3, 140.9,
143.8, 152.8, 164.0, 208.3. HRMS (ESI-TOF) m/z for C35H45N2O4 [M+H]+ calcd
557.33738, found 557.33607.
7

2.1.1.7. Methyl 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-3-carboxylate fused to 16-
dehydropregnenolone (15b/16b)
Obtained from methyl 2,2-dioxo-1H,3H-pyrazolo[1,5-c][1,3]thiazole-7-carboxylate (1c,
2.5 equiv.). Purification of the crude product by flash chromatography [hexane, ethyl
acetate/hexane (1:2)] gave, in order of elution, 15b as a white solid (Method A: 54 mg,
37%; Method B: 57 mg, 38%) and 16b as a white solid (Method A: 13 mg, 9%; Method
B: 11 mg, 7%).
Compound 15b: mp 220.0-221.2 °C (from ethyl acetate/hexane). [ꢀ]ꢂꢁꢃ -30 (c 0.5 in
1
CH2Cl2). IR (KBr) ν 1053, 1207, 1238, 1562, 1695, 1722 and 2943 cm-1. H NMR: δ
0.75 (s, 3H), 0.85-0.94 (m, 1H), 0.99 (s, 3H), 1.02-1.18 (m, 3H), 1.28-1.53 (m, 6H),
1.77-1.96 (m, 6H), 2.19 (s, 3H), 2.23-2.30 (m, 2H), 3.29 (d, 1H, J = 16.0 Hz), 3.37 (d,
1H, J = 16.0 Hz), 3.48-3.53 (m, 1H), 3.65-3.68 (m, 1H), 3.77-3.81 (m, 1H), 3.82 (s,
3H), 4.25 (dd, 1H, J = 13.6 and 6.4 Hz), 5.29 (d, 1H, J = 5.2 Hz), 7.75 (s, 1H). 13C
NMR: δ 16.6, 19.3, 20.8, 24.6, 27.7, 31.2, 31.5, 31.8, 31.84, 33.1, 36.4, 36.44, 37.2,
42.1, 46.1, 49.4, 51.1, 51.12, 52.4, 66.5, 71.6, 109.8, 121.0, 140.2, 140.7, 142.3, 164.0,
208.1. HRMS (ESI-TOF) m/z for C28H39N2O4 [M+H]+ calcd 467.29043, found
467.28974.
Compound 16b: mp 147.5-148.8 °C (from ethyl acetate/hexane). [ꢀ]ꢂꢁꢃ -20 (c 0.5 in
1
CH2Cl2). IR (ATR) ν 1050, 1195, 1246, 1437, 1560, 1701 and 2930 cm-1. H NMR: δ
0.75 (s, 3H), 0.94-0.99 (m, 1H), 1.01 (s, 3H), 1.05-1.13 (m, 2H), 1.36-1.56 (m, 6H),
1.68-1.76 (m, 2H), 1.84-1.87 (m, 2H), 1.94-2.01 (m, 2H), 2.18 (s, 3H), 2.23-2.33 (m,
2H), 2.51 (dd, 1H, J = 16.0 and 8.4 Hz), 3.44-3.55 (m, 2H), 3.63 (dd, 1H, J = 16.0 and
8.0 Hz), 3.80 (s, 3H), 4.18 (d, 1H, J = 13.6 Hz), 4.59 (d, 1H, J = 13.6 Hz), 5.34 (d, 1H, J
= 5.2 Hz), 7.76 (s, 1H). 13C NMR: δ 16.6, 19.3, 20.6, 27.7, 28.3, 31.5, 31.7, 31.9, 32.8,
33.9, 34.2, 36.5, 37.2, 42.1, 45.9, 49.4, 49.5, 51.1, 52.3, 69.3, 71.6, 110.3, 121.0,
140.77, 140.78 143.6, 163.9, 207.5. HRMS (ESI-TOF) m/z for C28H39N2O4 [M+H]+
calcd 467.29043, found 467.28922.
2.1.1.8. Methyl 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-2-carboxylate fused to 16-
dehydropregnenolone (15c/16c)
Obtained from methyl 2,2-dioxo-1H,3H-pyrazolo[1,5-c][1,3]thiazole-6-carboxylate (14,
2.5 equiv.). Purification of the crude product by flash chromatography [hexane, ethyl
8

acetate/hexane (1:2)] gave compounds 15c/16c as mixture of regioisomers [Method A:
44 mg, 30% (79:21); Method B: 25 mg, 17% (79:21)].
1
Major Component (15c): IR (KBr) ν 1224, 1456, 1701, 1722, 2937 and 3421 cm-1. H
NMR: δ 0.75 (s, 3H), 0.86-0.95 (m, 1H), 0.99 (s, 3H), 1.03-1.21 (m, 3H), 1.31-1.71 (m,
9H), 1.83-1.93 (m, 3H), 2.12 (s, 3H), 2.20-2.31 (m, 2H), 2.89 (d, 1H, J = 15.6 Hz), 3.11
(d, 1H, J = 15.6 Hz), 3.48-3.55 (m, 1H), 3.63-3.70 (m, 1H), 3.87-3.92 (m, 1H), 3.90 (s,
3H), 4.32 (dd, 1H, J = 13.2 and 6.0 Hz), 5.29 (d, 1H, J = 4.8 Hz), 6.57 (s, 1H). 13C
NMR: δ 16.7, 19.3, 20.7, 25.5, 27.9, 31.2, 31.5, 31.8, 31.83, 33.1, 36.4, 36.7, 37.2, 42.1,
46.3, 49.4, 51.5, 51.9, 52.4, 66.6, 71.6, 105.3, 121.1, 139.1, 140.6, 142.1, 163.0, 208.0.
HRMS (ESI-TOF) m/z for C28H39N2O4 [M+H]+ calcd 467.29043, found 467.28937.
2.1.1.9. Methyl 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-3-carboxylate fused to 16-
dehydroprogesterone (17a/18a)
Obtained from methyl 2,2-dioxo-1H,3H-pyrazolo[1,5-c][1,3]thiazole-7-carboxylate (1c,
2.5 equiv.). Purification of the crude product by flash chromatography [hexane, ethyl
acetate/hexane (1:1)] gave, in order of elution, 17a as a white solid (Method A: 75 mg,
50%) and 18a as a yellow solid (Method A: 20 mg, 13%).
Compound 17a: mp 219.7-220.4 °C (from ethyl acetate/hexane). [ꢀ]ꢂꢁꢃ +70 (c 0.5 in
1
CH2Cl2). IR (KBr) ν 1024, 1238, 1437, 1496, 1676, 1695 and 1712 cm-1. H NMR: δ
0.77 (s, 3H, H-18), 0.84-0.97 (m, 2H), 1.17 (s, 3H, H-19), 1.19-1.2 (m, 1H), 1.31-1.37
(m, 1H), 1.47-1.86 (m, 7H), 1.94-1.99 (m, 1H), 2.01-2.06 (m, 1H), 2.20 (s, 3H, H-21),
2.23-2.43 (m, 4H), 3.30 (d, 1H, J = 16.0 Hz, H-28), 3.37 (d, 1H, J = 16.0 Hz, H-28),
3.66-3.72 (m, 1H, H-16), 3.79-3.84 (m, 1H, H-22), 3.82 (s, 3H), 4.26 (dd, 1H, J = 13.2
13
and 6.4 Hz, H-22), 5.69 (s, 1H, H-4), 7.73 (s, 1H, H-25). C NMR: δ 16.7, 17.3, 20.7,
24.5, 27.7, 31.0, 31.7, 32.5, 33.0, 33.9, 35.4, 35.6, 36.3, 38.4, 46.1, 51.0, 51.1, 51.5,
53.0, 66.3, 109.8, 124.1, 140.2, 142.1, 164.0, 170.1, 199.2, 207.8. HRMS (ESI-TOF)
m/z for C28H37N2O4 [M+H]+ calcd 465.27478, found 465.27370.
Compound 18a: mp 221.5-222.0 °C (from ethyl acetate/hexane). [ꢀ]ꢂꢁꢃ +50 (c 0.5 in
1
CH2Cl2). IR (ATR) ν 1049, 1193, 1245, 1439, 1560, 1665, 1701 and 1710cm-1. H
NMR: δ 0.78 (s, 3H, H-18), 0.94-1.06 (m, 2H), 1.18 (s, 3H, H-19), 1.44-1.53 (m, 2H),
1.60-1.84 (m, 8H), 1.96-2.06 (m, 2H), 2.17 (s, 3H, H-21), 2.30-2.43 (m, 3H), 2.51 (dd,
1H, J = 16.0 and 8.0 Hz, H-22), 3.44-3.51 (m, 1H, H-16), 3.63 (dd, 1H, J = 16.0 and 8.0
9

Hz, H-22), 3.79 (s, 3H), 4.15 (d, 1H, J = 13.8 Hz, H-28), 4.58 (d, 1H, J = 13.8 Hz, H-
28), 5.73 (s, 1H, H-4), 7.75 (s, 1H, H-25). 13C NMR: δ 16.7, 17.3, 20.6, 27.6, 28.3, 31.9,
32.5, 32.7, 33.7, 33.9, 34.1, 35.3, 35.6, 38.5, 45.8, 49.3, 51.1, 51.4, 53.1, 69.0, 110.4,
124.2, 140.8, 143.5, 163.8, 170.0, 199.2, 207.3. HRMS (ESI-TOF) m/z for C28H37N2O4
[M+H]+ calcd 465.27478, found 465.27377.
2.1.1.10. Methyl 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-2-carboxylate fused to 16-
dehydroprogesterone (17b/18b)
Obtained from methyl 2,2-dioxo-1H,3H-pyrazolo[1,5-c][1,3]thiazole-6-carboxylate (14,
2.5 equiv.). Purification of the crude product by flash chromatography [hexane, ethyl
acetate/hexane (2:1)] gave compounds 17b/18b as mixture of regioisomers [Method A:
43 mg, 29% (92:8)].
Major Component (17b): IR (KBr) ν 1226, 1454, 1666, 1701 and 1720 cm-1. 1H NMR:
δ 0.77 (s, 3H), 0.84-0.91 (m, 2H), 1.16 (s, 3H), 1.17-1.24 (m, 1H), 1.32-1.38 (m, 1H),
1.43-1.74 (m, 7H), 1.91-1.94 (m, 1H), 1.99-2.04 (m, 1H), 2.10 (s, 3H), 2.21-2.26 (m,
1H), 2.30-2.41 (m, 3H), 2.90 (d, 1H, J = 15.8 Hz), 3.07 (d, 1H, J = 15.8 Hz), 3.63-3.69
(m, 1H), 3.85-3.90 (m, 1H), 3.88 (s, 3H), 4.32 (dd, 1H, J = 13.2 and 6.0 Hz), 5.70 (s,
1H), 6.56 (s, 1H). 13C NMR: δ 16.8, 17.3, 20.6, 25.4, 27.9, 31.1, 31.8, 32.5, 32.9, 33.9,
35.4, 35.6, 36.7, 38.4, 46.2, 51.3, 51.4, 51.9, 52.9, 66.3, 105.4, 124.1, 138.9, 142.1,
162.9, 170.0, 199.2, 207.8. HRMS (ESI-TOF) m/z for C28H37N2O4 [M+H]+ calcd
465.27478, found 465.27374.
2.1.2. General Procedure for the hydrolysis of compounds 3a and 19:
A solution of compound 3a or 19 (0.176 mmol) in ethanol (5 mL) was treated with a
KOH satured solution at room temperature for the time indicated in each case. After the
reaction was complete (TLC control), the mixture was acidified with HCl 1M and
extracted with ethyl acetate. The organic phase was dried over anhydrous sodium
sulphate and the solvent evaporated off.
2.1.2.1. Dimethyl 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-2,3-dicarboxylic acid fused
to 16-dehydropregnenolone (20a)
10

Obtained from compound 3a (100 mg, 0.176 mmol) as described in the general
procedure (reaction time: 1,5 h). The product was obtained as a white solid which was
recrystallised with diethyl ether (56 mg, 64%): mp 260.2-261.5 °C (from diethyl ether).
[ꢀ]ꢂꢁꢃ -50 (c 0.5 in CH2Cl2). IR (KBr) ν 1207, 1238, 1498, 1600, 1703, 1720, 2933 and
1
3425 cm-1. H NMR (DMSO-d6): δ 0.66 (s, 3H), 0.84-0.89 (m, 1H), 0.94 (s, 3H), 1.24-
1.91 (m, 10H), 2.12 (s, 3H), 3.20-3.30 (m, 1H), 3.33 (d, 1H, J = 15.2 Hz), 3.53 (d, 1H, J
= 15.2 Hz), 3.81 (dd, 1H, J = 13.2 and 8.0 Hz), 4.33 (dd, 1H, J = 13.2 and 6.8 Hz), 5.25
(d, 1H, J = 3.2 Hz). 13C NMR (DMSO-d6): δ 16.0, 19.0, 20.3, 24.5, 27.6, 30.7, 31.2,
31.3, 32.2, 35.8, 36.0, 36.9, 42.1, 45.4, 48.9, 50.7, 51.1, 66.5, 69.9, 110.6, 120.0, 141.3,
142.9, 144.5, 164.1, 164.6, 208.0. HRMS (ESI-TOF) m/z for C28H37N2O6 [M+H]+ calcd
497.26461, found 497.26374.
2.1.2.2. (S)-7-Benzyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-2,3-dicarboxylic acid
fused to 16-dehydropregnenolone (20b)
Obtained from compound 19 (116 mg, 0.176 mmol) as described in the general
procedure (reaction time: 3 h). The product was obtained as a white solid which was
recrystallised with diethyl ether (52 mg, 50%): mp 250.0-250.4 °C (from diethyl ether).
[ꢀ]ꢂꢁꢃ -40 (c 0.25 in CH2Cl2). IR (KBr) ν 1433, 1496, 1637, 1699, 1716, 2937 and 3446
1
cm-1. H NMR (DMSO-d6): δ 0.38-0.40 (m, 1H), 0.54 (s, 3H), 0.58-0.61 (m, 1H), 0.88
(s, 3H), 0.92-1.11 (m, 4H), 1.21-1.34 (m, 4H), 1.56-1.81 (m, 6H), 1.97-2.03 (m, 1H),
2.10 (s, 3H), 2.72 (d, 1H, J = 16.4 Hz), 3.00 (dd, 1H, J = 13.8 and 9.6 Hz), 3.22-3.29
(m, 2H), 3.74 (dd, 1H, J = 13.8 and 4.8 Hz), 4.13 (d, 1H, J = 16.4 Hz), 4.36 (br s, 1H),
5.17 (s, 1H), 7.27-7.34 (m, 5H). 13C NMR (DMSO-d6): δ 16.3, 19.0, 20.3, 22.8, 25.1,
27.4, 31.3, 31.4, 32.3, 34.7, 35.9, 36.7, 40.4, 42.1, 46.0, 49.1, 52.4, 59.9, 65.5, 69.8,
110.4, 119.9, 126.5, 128.4, 129.2, 137.3, 141.2, 142.1, 145.6, 164.2, 164.7, 207.0.
HRMS (ESI-TOF) m/z for C35H43N2O6 [M+H]+ calcd 587.31156, found 587.31117.
2.2. Biology
2.2.1. Cell culture
Cytotoxicity of the compounds was tested on three tumoural cell lines: murine
lymphoma (EL4; ATCC® TIB-39™), human acute monocytic leukaemia (THP-1;
ATCC® TIB-202™) and human histiocytic lymphoma (U-937; ATCC® CRL-
11

1593.2™), and human hepatocarcinoma (HepG2; ATCC® HB-8065™) and one non-
tumoural murine bone marrow cell line (S17), kindly provided by D. Rawlings, UCLA,
Los Angeles, CA, to assess selectivity. EL4, THP-1, HepG2 and U-937 were cultured in
RPMI-1960 culture medium supplemented with 10% heat-inactivated fetal bovine
serum (FBS), 1% L-glutamine (2 mM), and 1% penicillin (50 U/ml)/streptomycin (50
µg/ml). S17 cells were maintained in DMEM culture medium, supplemented with 10%
heat-FBS, 1% L-glutamine (2 mM), and 1% penicillin (50 U/ml)/streptomycin (50
µg/ml). All cell lines were maintained at 37 ºC in humid atmosphere with 5% CO2.
2.2.2. Cytotoxicity assay
The cytotoxic activity of the new compounds was determined by the MTT colorimetric
assay [8]. Briefly, exponentially growing cells were plated in 96-well tissue plates at a
density of 5x103 cells/well (S17, HepG2) or 1x104 cells/well (THP-1, U-937, EL4).
Adherent cells (S17, HepG2) were previously incubated for 24 h to ensure adhesion to
the wells. The studied compounds were initially applied at 100 µM. Control cells were
treated with DMSO at the highest concentration used in test wells (0.5%). Cell viability
was determined after 72 h of incubation with the compounds/DMSO. Two hours prior
to the end of the incubation period, 20 µL of MTT (5 mg/mL in PBS) were added to
each well and further incubated at 37 ºC. 150 µL of DMSO were afterwards added to
each well in order to dissolve the formazan crystals and absorbance was measured at
590 nm (Biotek Synergy 4). For those compounds exhibiting cell viabilities lower than
50% at the 100 µM, the half maximal inhibitory concentration (IC50; µM) was
determined applying the compounds at concentrations ranging from 1 to 100 µM. The
selectivity of the extracts was estimated using the following equation: Selectivity =
(IC50 NT)/(IC50 VT), where NT and VT indicate the IC50 of the compound on non-
tumoural (S17) and tumoural cells (HepG2, THP-1, U-937 and EL4), respectively.
3. Results and discussion
3.1. Chemistry
16-Dehydropregnenolone (16-DHP, 7) and 16-dehydroprogesterone (8) were prepared
by known synthetic procedures (Scheme 2). Steroid 7 was synthesized from 16-DPA by
12

acetate group’s hydrolysis using potassium hydroxide in tert-butanol [5]. The Oppenaur
oxidation of 16-DHP, using aluminium isopropoxide and N-methylpiperidone, gave the
target 16-dehydroprogesterone in 72% yield [6].
O
O
O
Me
H
Me
H
Me
H
Al(OCH(CH3)2)3
KOH, t-BuOH
N-Methylpiperidone
Me
Me
Me
Toluene, reflux
6 h
r.t., 14 h
O
H
H
H
H
H
H
O
O
HO
16-DHP (7)
80%
16-DPA
16-Dehydroprogesterone (8)
72%
Scheme 2. Synthesis of 16-DHP (4) and 16-dehydroprogesterone (5) from 16-DPA.
The reactivity of 16-DHP (7) towards diazafulvenium methide 2a was first explored
(Table 1). The 2,2-dioxo-1H,3H-pyrazolo[1,5-c][1,3]thiazole 1a [7] underwent SO2
extrusion, under microwave irradiation or in refluxing 1,2,4-trichlorobenzene (TCB), to
give transient diazafulvenium methide 2a, which was trapped in situ by 16-DHP leading
to steroid derivative 9 as a single regio- and stereoisomer. Carrying out the reaction
under MW irradiation at 230 ºC for 10 min using 2.5 equivalents of sulfone 1a,
compound 6 was isolated in 32% yield (entry 1). The use of longer reaction time (15
min) did not lead to an improvement of the yield (entry 2). The cycloaddition was also
carried out at 240 ºC and 250 ºC for 10 min leading to the target compound in 32% and
38% yield, respectively (entries 3 and 4). The [8π+2π] cycloaddition of 16-DHP with
diazafulvenium methide 2a under conventional thermolysis, refluxing TCB for 4 h,
gave the hexacyclic steroid 9 in a lower yield (24%) (entry 5). Stereochemistry
assignment of compound 9 was made on the basis of two-dimensional COSY, NOESY,
HSQC and HMBC spectra (see Supplementary Material). In particular, cross peaks
were observed in the NOESY spectrum between protons of the acetyl group (H-21) and
the methyl group at C-18 and this methyl group also showed cross peaks with the proton
at the ring fusion (H-16).
13

Table 1. Optimization of the [8π+2π] cycloaddition of 16-DHP (7) with diazafulvenium methide 2a.
CO2Me
CO2Me
- SO2
CO2Me
CO2Me
TCB, ∆
N
N
O2S
N N
2a
1a
O
Me
H
Me
H
H
HO
16-DHP (7)
32
31
30
29
MeO2C
21
26
CO2Me
20
27
25
O
28
18 Me
12
N
N
17
24
11
19
23
13
16
Me H14
1
4
9
22
2
H
H 15
10
8
7
H
9
3
5
HO
6
Entry Reaction conditions Isolated yield, 9
1
2
3
4
5
MW, 230 ºC, 10 min
MW, 230 ºC, 15 min
MW, 240 ºC, 10 min
MW, 250 ºC, 10 min
Reflux, 4 h
32%
26%
32%
38%
24%
Sulfone 10 was used as precursor of the corresponding diazafulvenium methide, which
reacted with 16-DHP (7) yielding 1,7-cycloadduct 11, in a regio- and stereoselective
fashion (Scheme 3). Under MW irradiation at 250 ºC for 10 min steroid 11 was obtained
in 35% yield whereas conventional thermolysis afforded the target compound in 43%
yield (refluxing TCB for 4 h). From these experiments N-styryl-1H-pyrazole 12 was
also formed, a chemical outcome expected for 1-benzyl diazafulvenium methides [4].
14

Scheme 3. Reactivity of 2,2-dioxo-1H,3H-pyrazolo[1,5-c][1,3]thiazole 10 in presence of 16-DHP.
The work was extended to the reactivity of 16-dehydroprogesterone (8) towards 2,2-
dioxo-1H,3H-pyrazolo[1,5-c][1,3]thiazoles 1a, 1b and 10 under MW irradiation at 250
ºC for 10 min (Scheme 4). These sulfones underwent SO2 extrusion under these reaction
conditions to give the corresponding diazafulvenium methides followed by the
[8π+2π] cycloaddition with 16-dehydroprogesterone (8) giving steroids 13a, 13b and
13c in 53%, 48% and 32%, respectively. In the thermolysis of 3-benzyl-2,2-dioxide-
1H,3H-pyrazolo[1,5-c][1,3]thiazole 13, N-styryl-1H-pyrazole 10 was also isolated in
24% yield resulting from the competitive sigmatropic [1,8]H shift of the in situ
generated diazafulvenium methide. The novel hexacyclic steroids 13 were obtained
regio- and stereoselectively including compound 13c having an additional chiral center
(C-22).
15

O
Me
H
Me
H
H
O
8
CO2R2
R3
MW, 250 ºC
10 min
N N
O2S
R1
1a-b,10
R2O2C
R3
O
Me
N
17
N
22
16
Me
H
R1
H
H
H
O
13a R1 = H, R2 = Me, R3 = CO2Me 53%
13b R1 = H, R2 = Et, R3 = Ph
48%
13c R1 = Bn, R2 = Me, R3 = CO2Me 32%
Scheme 4. [8π+2π] cycloaddition of 16-dehydroprogesterone (8) with diazafulvenium methides.
The reactivity of 16-DHP (7) towards 2,2-dioxo-1H,3H-pyrazolo[1,5-c][1,3]thiazole 1b,
1c and 14 was also explored (Scheme 5). Carrying out the thermolysis of sulfone 1b in
presence of 16-DHP, under MW irradiation at 250 ºC for 10 min, an inseparable
mixture regioisomers 15a/16a was obtained in 33% overall yield (87:13 ration) whereas
the conventional heating was less efficient affording the mixture of 15a/16a in 25%
overall yield (81:19 ration). In both cases, derivative 15a was the major product. Similar
outcome was observed in the reaction of sulfone 14 with 16-DHP (7) where an
inseparable mixture of regioisomers 15c/16c, under microwave irradiation or
conventional heating in 30% and 17% overall yield, respectively. In both cases
1
hexacyclic steroids were obtained in 79:21 ratio, determined by H NMR, compound
15c being the major product. Regioisomer 16a and 16c were identified based on their
1
1H NMR spectra. In the H NMR spectrum of compound 16a signals were observed at
2.57 and 4.65 ppm corresponding to protons H-22 and H-28, respectively. On the other
1
hand, the H NMR spectrum of compound 16c showed two doublets at 4.25 and 4.65
corresponding to protons H-28, as expected for an AB system. Furthermore, in both
cases signals corresponding to H-6, H-18, H-19 and H-21 protons could also be
observed.
16

Sulfone 1c also underwent SO2 extrusion, either microwave-induced or in refluxing
TCB, to give the corresponding diazafulvenium methide which participated in [8π+2π]
cycloaddition with 16-DHP affording regioisomeric steroids 15b (37-38%) and 16b (7-
9%), separable by flash chromatography. In all cases, each regioisomer was obtained as
single stereoisomer.
Scheme 5. [8π+2π] cycloaddition of 16-DHP with diazafulvenium methides.
The MW-induced reactivity of sulfones 1c and 14 towards 16-dehydroprogesterone (8)
was studied (Scheme 6). Compounds 17a (50%) and 18a (13%) were obtained from the
reaction of sulfone 1c with steroid 8. The structural assignment of compounds 17a and
18a was supported by two-dimensional spectra (COSY, NOESY, HSQC and HMBC).
1
The H NMR spectra of compounds 17a and 18a had similar features to the ones of
steroids 15b and 16b, respectively. Furthermore, NMR spectra of compound 17a and
compound 9 were very similar, showing the same type of coupling and correlations. The
stereochemistry of compound 18a was established by cross peaks observed in the
NOESY spectrum, between the proton at ring fusion (H-16) with both methyl groups of
acetyl group (H-21) and at C-18 (See Supplementary Material).
17

Under the same reaction conditions, sulfone 14 afforded regioisomers 17b and 18b as
1
an inseparable mixture in 29% overall yield (92:8 ratio determined by H NMR),
compound 17b being the major product. Compound 18b was identified by two doublets
at 4.22 and 4.63 ppm corresponding to the H-28 protons and a doublet at 5.65 ppm
corresponding to the proton H-4, observed in the 1H NMR spectrum of the mixture.
CO2Me
MeO2C
21
N
O
N
18 Me
O
Me
N N
O2S
1c
N
+
CO2Me
N
16
Me
H
Me
H
MW, 250 ºC, 10 min
H
H
H
H
O
H
H
O
Me
O
18a 13%
17a 50%
Me
H
H
H
O
8
CO2Me
CO2Me
+
N
CO2Me
O
Me
N
O
Me
28
O2S
N N
N
N
14
MW, 250 ºC, 10 min
17b/18b 29% (92:8)
Me
H
Me
H
H
H
H
H
H
H
O
O
4
18b
17b
Scheme 6. Reactivity of 2,2-dioxo-1H,3H-pyrazolo[1,5-c][1,3]thiazoles 1c and 14 in presence of 16-
DHP.
Finally, the hydrolysis of the ester groups of hexacyclic steroids 3a and 19 [4] was
achieved by treatment with an excess of KOH in ethanol at room temperature giving the
corresponding diacids 20a and 20b in 64% and 50% yield, respectively (Scheme 7).
18

MeO2C
CO2Me
O
Me
N
N
Me
H
R
H
O
H
H
3a R = H
19 R = Bn
O
1.5-3 h
r.t.
KOH, EtOH
HO2C
CO2H
O
Me
N
N
Me
H
R
H
H
H
HO
20a R = H 64%
20b R = Bn 50%
Scheme 7. Hydrolysis of the ester groups to synthesize diacids 20.
3.2. Biology
The cytotoxicity of selected chiral hexacyclic steroids was evaluated in different
tumoural cell lines, namely EL4 (murine T-lymphoma), THP-1 (human acute
monocytic leukaemia), and HepG2 (hepatocellular carcinoma) and U-937 cells (human
histiocytic lymphoma). Compounds selectivity towards tumoural cells was assessed
determining their cytotoxicity with respect to one non-tumoural cell line: S17 (murine
bone marrow stromal cell line).
The IC50 values were calculated only for compounds inducing cellular viability lower
than 50% at 100 µM (Table 2). For the less toxic compounds (inducing cellular
viabilities between 50 and 100%) the IC50 was considered to be >100 µM. Compound
11, derived from 16-DHP, was considerably cytotoxic to both EL4 and THP-1 cells
with IC50 values of 11.1 and 15.7 µM, respectively, but not active against HepG2 (IC50
= 84.0 µM). Interestingly, compound 9, differing from compound 11 only by the
presence of a benzyl group at C-22, show significantly lower cytotoxicity against EL4
and THP-1 cells with an IC50 in the range 47.4-52.4 µM. Thus, the presence of the
benzyl group seams to lead to enhancement of anticancer activity. Compound 9 showed
also low cytotoxicity against HepG2 (IC50 = 38.8 µM). Steroid 13c, derived from 16-
dehydroprogesterone (8) and bearing a benzyl group at C-22, show also activity against
EL4 cells with IC50 = 11.9 µM in contrast with the corresponding derivative
19

unsubstituted at C-22, compound 13a, showing low cytotoxicity (IC50 = 51.3 µM). Once
more, the presence of the benzyl group leads to higher anticancer activity. Compounds
13c and 13a show low activity against THP-1 and HepG2 cells. Selectivity between
tumoural and non-tumoural cells is generally used as an approach to the effectiveness of
a compound in cancer therapies. Table 2, presents the IC50 of some of the compounds
towards S17 cells, which were derived from non-tumoural cells. Unfortunately, the
studied compounds were generally toxic towards these cells as well, and hence showed
low selectivity towards tumoural cells. Nonetheless, more studies should be performed
using non-tumoural primary cell cultures.
Cellular viability of EL4, THP-1, HepG2 and U-937 cells exposed to hexacyclic
steroids 3a, 3c, 15b, 19, 20a and 20b was also studied (Table S1). None of these
compounds could be considered toxic to the studied cell-lines, at the concentration
tested (100 µM) with one exception. Compound 19, derived from 16-DPA and bearing a
benzyl group at C-22, showed a value of 11.6% for the cellular viability for EL4 cells.
Notice that the C-22-unsubstituted steroid derived from 16-DPA, compound 3a, showed
a value of 53.0%. These results reinforce the conclusion that the presence of the benzyl
group is important to ensure cytotoxicity against EL4.
Table 2. Cytotoxicity of compounds 9, 11, 13a and 13b against three tumoural cell lines (EL4, THP-1
and HepG2) and a non-tumoural cell line (S17). Results are expressed as IC50 (µM) and in brackets
confidence interval (CI) are presented.
Cell line
EL4
9
52.4 (CI 43-63)
13a
51.3 (CI 30-87)
11
11.1 (CI 10-12)
13c
11.9 (CI 10-14)
THP-1
HepG2
47.3 (CI 33-68)
38.8 (CI 33-46)
44.6 (CI 34-58)
28.8 (CI 26-32)
>100
15.7 (CI 13-17)
84.0 (CI 56-125)
18.9 (CI 17-21)
>100
>100
N.D.
S17
N.D.
N.D. – not determined.
4. Conclusion
Novel chiral 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine–fused steroids derived from 16-
dehydropregnenolone and 16-dehydroprogesterone have been synthesized and their
biological evaluation as anti-cancer agents was carried out. The studied steroidal
scaffolds reacted as the 2π-component in the [8π+2π] cycloadditions with
20

diazafulvenium methides to afford the target compounds in a regio- and stereoselective
fashion. The anticancer activity screening of the hexacyclic steroids has shown that
compounds bearing a benzyl group at C-22, derived from 16-dehydropregnenolone and
16-dehydroprogesterone, show considerable cytotoxicity against EL4 (murine T-
lymphoma) with IC50 values in the range of 11.1-11.9 µM whereas the corresponding C-
22-unsubstituted steroids show low cytotoxicity (IC50 = 47.4-51.3 µM).
Acknowledgements
Thank are due to Coimbra Chemistry Centre (CQC), supported by the Portuguese
Agency for Scientific Research, Fundação para a Ciência e a Tecnologia (FCT),
through Project Nº 007630 UID/QUI/00313/2013, co-funded by COMPETE2020-UE.
Susana M. M. Lopes also acknowledge FCT for post-doctoral research grant
SFRH/BPD/84413/2012. We acknowledge the UC-NMR facility for obtaining the
NMR data (www.nmrccc.uc.pt).
Supplementary Material
Supplementary data associated with this article can be found, in the online version, at
http://.....
References
[1] (a) M. Ibrahim-Ouali, Synthesis of pentacyclic steroids, Steroids 73 (2008) 775-797;
(b) I. V. Zavarzin, V. V. Chertkova, I. S. Levina, E. I. Chernoburova, Steroids fused
to heterocycles at positions 16, 17 of the D-ring, Russ. Chem. Rev. 80 (2011), 661-
682;
(c) G. O. Ifere, E. Barr, A. Equan, K. Gordon, U. P. Singh, J. Chaudhary, J. U.
Igietseme, G. A. Ananaba, Differential effects of cholesterol and phytosterols on
cell proliferation, apoptosis and expression of a prostate specific gene in prostate
cancer cell lines, Cancer Detect. Prev. 32 (2009) 319-328;
(d) A. Gupta, B. Sathish, A. S. Negi, Current status on development of steroids as
anticancer agents, J. Steroid Biochem. Mol. Biol. 137 (2013) 242-270;
21

(e) J. A. R. Salvador, J. F. S. Carvalho, M. A. C. Neves, S. M. Silvestre, A. L.
Leitão, M. M. C. Silva, M. L. Sá e Melo, Anticancer steroids: linking natural and
semi-synthetic compounds, Nat. Prod. Rep. 30 (2013) 324-374.
[2] (a) V. C. O. Njar, A. M. H. Brodie, Discovery and Development of Galeterone
(TOK-001 or VN/124-1) for the Treatment of All Stages of Prostate Cancer, J.
Med. Chem. 58 (2015) 2077–2087;
(b) N.-J. Fan, Y.-B. Bai, F.-Y Zhang, B. Luo, J.-J. Tang, Q.-Z. Zhang, J.-M. Gao,
Synthesis and cytotoxicity of some novel 21E-benzylidene steroidal derivatives,
Steroids 78 (2013) 874-879;
(c) N.-J. Fan, J.-J. Tang, H. Li, X.-J. Li, B. Luo, J.-M. Gao, Synthesis and cytotoxic
activity of some novel steroidal C-17 pyrazolinyl derivatives, Eur. J. Med. Chem.
69 (2013) 182-190;
(d) N.-J. Fan, S.-P. Wei, J.-M. Gao, J.-J. Tang, Potential Insecticidal Activity of
Steroidal C-17 Pyrazolinyl Derivatives, J. Braz. Chem. Soc. 26 (2015) 389-392.
[3] (a) P. Chowdhury, J. M. Borah, M. Bordoloi, P. K. Goswami, A. Goswami, N. C.
Barua, P. G. Rao, A Simple Efficient Process for the Synthesis of 16-
Dehydropregnenolone Acetate(16-Dpa) - A Key Steroid Drug Intermediate from
Diosgenin, J. Chem. Eng. Process. Technol. 2 (2011) 117;
(b) M. Kumar, P. Rawat, M. Khan, A. K. Rawat, A. K. Srivastava, R. Maurya, Aza-
annulation on the 16-dehydropregnenolone, via tandem intermolecular Aldol
process and intramolecular Michael addition, Bioorg. Med. Chem. Lett. 21 (2011)
2232-2237;
(c) C.-Y. Duh, I-W. Lo, S.-K. Wang, C.-F. Dai, New cytotoxic steroids from the
soft coral Clavularia viridis, Steroids 72 (2007) 573-579.
[4] (a) S. M. M. Lopes, C. F. O. Correia, S. C. C. Nunes, N. A. M. Pereira, A. R. F.
Ferreira, E. P. Sousa, C. S. B. Gomes, J. A. R. Salvador, A. A. C. C. Pais, T. M. V.
D. Pinho e Melo, Synthesis of chiral hexacyclic steroids via [8π + 2π] cycloaddition
of diazafulvenium methides, Org. Biomol. Chem. 13 (2015) 9127-9139;
(b) For an overview on the generation and reactivity of aza- and diazafulvenium
methides see: T. M. V. D. Pinho e Melo, Chemistry of aza- and diazafulvenium
methides in heterocyclic synthesis, Pure Appl. Chem. 88 (2016) 457-475.
[5] H. Li, J. Fang, J. Li, Y. Wang, X. Tian, Y. Xiang, Synthesis and characterization of
new phenyl esters derived from 16-dehydropregnenolone acetate (16-DPA), Res.
Chem. Intermed. 39 (2013) 3887-3893.
22

[6] R. W. Hartmann, M. Hector, S. Haidar, P. B. Ehmer, W. Reichert, J. Jose, Synthesis
and Evaluation of Novel Steroidal Oxime Inhibitors of P450 17 (17α-
Hydroxylase/C17−20-Lyase) and 5α-Reductase Types 1 and 2, J. Med. Chem. 43
(2000) 4266-4277.
[7] O. B. Sutcliffe, R. C. Storr, T. L. Gilchrist, P. Rafferty, Cycloadditions to
Pyrrolo[1,2-c]thiazoles and Pyrazolo[1,5-c]thiazoles, Tetrahedron, 56 (2000)
10011-10021.
[8] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: Application
to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1983) 55–63.
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Highlights
•
Synthesis of novel chiral 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine–fused
steroids via a novel route to heterocycle-fused steroids based on [8π+2π]
cycloaddition reaction of diazafulvenium methides.
•
The biological evaluation of the new family of hexacyclic steroids as anti-
cancer agents is reported for the first time
•
•
Conclusions regarding structure-activity relationships could be withdrawn.
Hexacyclic steroids bearing a benzyl group at C24, derived from 16-
dehydropregnenolone and 16-dehydroprogesterone, show cytotoxicity
against EL4 whereas the corresponding C24-unsubstituted steroids show low
cytotoxicity.
24