Facile preparation and preliminary cytotoxicity evaluation of dehydroepiandrosterone C-16 spiro-pyrrolidine derivatives

Article abstract of DOI:10.1007/s11696-020-01346-4

A facile synthesis of dehydroepiandrosterones derived by C-16 spiro-pyrrolidine and their cytotoxic evaluation are reported. Seven derivatives, 3a–3g, were prepared by the [3 + 2] cycloaddition reaction of the 16-arylidene dehydroepiandrosterone and the a

Full text of DOI:10.1007/s11696-020-01346-4

Chemical Papers
https://doi.org/10.1007/s11696-020-01346-4
ORIGINAL PAPER
Facile preparation and preliminary cytotoxicity evaluation
of dehydroepiandrosterone C‑16 spiro‑pyrrolidine derivatives
Hong‑Wen Tao1 · Wen‑Yu Peng1 · Jiang‑Chun Yuan1 · Qiang Li1 · Lu‑Yao Zeng1 · Xian‑Yong Yu1 · Ping‑Gui Yi1
Received: 7 January 2020 / Accepted: 9 September 2020
© Institute of Chemistry, Slovak Academy of Sciences 2020
Abstract
A facile synthesis of dehydroepiandrosterones derived by C-16 spiro-pyrrolidine and their cytotoxic evaluation are reported.
Seven derivatives, 3a–3g, were prepared by the [3+2] cycloaddition reaction of the 16-arylidene dehydroepiandrosterone
and the azomethine ylide, where the azomethine ylide was generated in situ from 11H-indeno[1,2-b]quinoxalin-11-one and
sarcosine. Their cytotoxicity was evaluated on brine shrimp assay and the bioactivities represented by LC50 values were
found in the range of 6.19–27.2 µg/mL. Among them, the activities of 3d and 3g are the best, as manifested by LC50 values
less than 10 µg/mL. All products were confrmed by NMR, HR MS and X-ray analysis.
Graphic abstract
Keywords [3+2] cycloaddition · Dehydroepiandrosterone · Steroidal spiro-pyrrolidine · Azomethine ylide
Introduction
Chemical modifcation of natural steroids provides pharma-
ceutical chemists many products with amazing diversity in
structure and bioactivity (Singh and Panda 2013). Among
them, heterocyclic steroidal compounds belong to the promi-
nent candidates for their potential anti-infammatory, antiox-
idant, anti-cancer and anti-microbial activities (Gupta et al.
1996; Mohamed et al. 2012; Abdelhalim et al. 2007). The
steroidal materials with spiro-pyrrolidine moiety represent
an important family for the unique structure and signifcant
anti-cancer activity (Fig. 1) (Babu and Raghunathan 2008;
Yu et al. 2014, 2015; Gavaskar et al. 2016). For these novel
spiro-heterocyclic compounds (Liu et al. 2013; Yang et al.
2015; Singh et al. 2016; Zhang et al. 2017; Zeng et al. 2018),
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11696-020-01346-4) contains
supplementary material, which is available to authorized users.
* Hong-Wen Tao
hongwtao@hnust.edu.cn
1
Key Laboratory of Theoretical Organic Chemistry
and Functional Molecules, Ministry of Education, School
of Chemistry and Chemical Engineering, Hunan University
of Science and Technology, Xiangtan 411201, Hunan,
People’s Republic of China
Vol.:(0123456789)
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Chemical Papers
moderate yield (50‒65%), where the azomethine ylide was
generated in situ from 2 and sarcosine. As for the two raw
materials, 16-arylidene dehydroepiandrosterones (1) and
11H-indeno[1,2-b]quinoxalin-11-one (2), they were ef-
ciently prepared using Kanchithalaivan’s and Kaupp’s pro-
cedures, respectively (Kanchithalaivan et al. 2013; Kaupp
et al. 2002).
The dehydroepiandrosterone derivatives, 1b–1g, were
known compounds, of which 1g has no reported NMR data
except for this paper (Kanchithalaivan et al. 2013; Huang
et al. 2012; Christiansen et al. 1964). The positive HR-
ESIMS of 1a gave a [M+H]+ peak at m/z 423.2351, which is
consistent with the molecular formula of C27H34O2S. In the
deshielded feld of 1H NMR, 1a showed two group of dou-
blet protons, δ 7.47 (d, J=8.4 Hz) and 7.26 (d, J=8.4 Hz),
owning to the 1,4-substituted phenyl. The singlet proton (δ
7.39, s) was assigned to be characteristic aromatic methyl-
idene. The signals at 5.40 ppm, 3.55 ppm, and 2.52 ppm
were indicated as the olefn proton on C-6, the proton on
oxygenated C-3, and the methylthio group on aryl moiety,
respectively. All the spectral characteristics can confrm the
structure of the dipolarophile 1a.
Fig. 1 Reported steroidal spiro-pyrrolidine derivatives
the diverse structure will be expected to produce the diver-
sity of biological activity.
In our search for new structural and bioactive
entities (Tao et al. 2018, 2019), a series of dehy-
droepiandrosterone spiro-pyrrolidine derivatives,
1″-N-methyl-spiro[2′,11″]-indeno[1,2-b]quinoxaline-
spiro[3′,16]-dehydroepiandrosterone-4′-arylpyrrolidines
(Scheme 1), were obtained. Herein we report the synthesis,
structure, and bioassay of these steroidal derivatives. They
can be synthesized by the [3 + 2] cycloaddition reaction
without catalyst, and the relevant products can also be eas-
ily purifed using the solubility diference of products from
raw materials. The cytotoxicity evaluation on brine shrimp
bioassay showed that all products exhibited potent shrimp
lethality efect.
The structures of 3a–3g were confrmed by comprehen-
sive analysis of HR-ESIMS, IR, 1D and 2D NMR data. The
positive HR-ESIMS of 3a gave a quasi-molecular ion peak
at m/z 682.3461 [M+H]+, which indicated a molecular for-
mula of C44H47N3O2S. The IR spectrum of 3a displayed
νO–H and νC=O at 3417 and 1713 cm−1, respectively. The
1H NMR spectrum of 3a showed four methyl proton signals
at δ 2.50 (–SCH3), 1.94 (–NCH3), 0.74 (CH3-19) and 0.50
(CH3-18). The proton on oxygen-bearing C3 appears as a
multiple signal at δ 3.39. The olefn proton H6 was inter-
preted as a doublet at δ 4.89 (d, J=3.6 Hz). Twelve aromatic
protons appear as a doublet at δ 7.25 and six multiplets at δ
7.48–8.35. Obviously, the 1HNMR of 3a showed three more
groups of protons than those of 1a at δ 3.50–4.80, which
corresponded to the pyrrolidine ring newly introducted. The
two geminal protons on C5′ (CH2) adjacent to nitrogen on
pyrrolidine, showing strong 1H–1H COSY correlations each
other, exhibited two triplets at δ 4.34 (J=9.6 Hz, H5′a) and
3.71 (J = 8.3 Hz, H5′b). The benzyl proton H4′, showing
Results and discussion
Chemistry
The steroidal spiro-pyrrolidine derivatives 3 were prepared
using the [3 + 2] cycloaddition strategy using a modi-
fed literature method (Moemeni et al. 2012) (Scheme 1).
The cycloaddition involved a coupling of dipolarophiles
1 and the azomethine ylide to yield target products 3 in
1
strong H–1H COSY correlations with H5′b, occurs as a
Scheme 1 Synthesis of
dehydroepiandrosterone spiro-
pyrrolidine derivatives 3a–3g.
(i) In nitrogen atmosphere,
dry acetonitrile, refux, 15 h;
(ii) sarcosine, refux, 10–15 h,
50‒65%
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Chemical Papers
doublet doublet at δ 4.23 (dd, J=9,7, 8.1 Hz), rather than
singlet, which supported the regioselectivity of the cycload-
dition reaction as depicted in Scheme 1. The possible way
for azomethine ylide to approach dipolarophile 1 via path A
from the least obstructed side is shown in Scheme 2, which
is verifed by the single crystal data of 3c. The 13C NMR
spectrum showed two spiro-carbons signals at δ 69.36 (C16)
and δ 78.31 (C2′), a cyclopentanone carbonyl carbon sig-
nal at δ 222.23 (C17), respectively. The signal at δ 71.41
was assigned to the oxygen-bearing CH (C3), and those at δ
61.63 and 34.32 were located to nitrogen-bearing CH2 (C5′)
and CH3 (–NCH3), respectively, which coincided with the
correlations showed in the HMQC spectrum. The charac-
teristic HMBC correlations from the –NCH3 proton to C2′
(C11″) and C5′, from H5′b to C16 (C3′) and C2′ (C11″), and
from H4′ to C16 (C3′), C5′, C2‴ and C6‴ (δ 130.81 × 2)
can constructed N-methyl-4′-(4‴-methylthiophenyl)pyrro-
lidine ring. The HMBC correlations from CH3-18 to C14
(δ 47.87) and C17, and from H4′ to C17 and C16 (C3′) can
support the connectivity between the pyrrolidine heterocy-
cle and dehydroepiandrosterone skeleton via C16 (C3′). The
HMBC correlations from H15b (δ 0.66) to C14, C16 (C3′)
and C2′ (C11″), from H10″ (δ 7.53) to C2′ (C11″), and from
–NCH3 to C2′ (C11″) supported the connectivity between
the pyrrolidine heterocycle and indeno[1,2-b]quinoxaline
via C2′ (C11″). All the HMBC correlations confirmed
the spiro[2′,11″]-indeno[1,2-b]quinoxaline-spiro[3′,16]-
dehydroepiandrosterone structure (Fig. 2). Fortunately, the
Fig. 2 Partial HMBC correlations of 3a
crystal of 3c was obtained and analyzed (Fig. 3), which fur-
ther proved the relative structure of the product 3. Since the
CH3-18 and CH3-19 in dehydroepiandrosterone were both
naturally β-oriented, 9S and 13S were inferred as the abso-
lute confguration of C9 and C13 in products. The other chi-
ral centers in 3 are further determined according to the rela-
tive structure provided by the crystal data of 3c. As a result,
3a was named 3S,8R,9S,10R,13S,14S,16(3′)R,2′(11″)R,4′R-
1′-N-methyl-spiro[2′,11″]-indeno[1,2-b]quinoxaline-spiro[-
3′,16]-dehydroepiandrosterone-4′-(4‴-methylthiophenyl)-
pyrrolidine which is shown in Scheme 1 and Fig. 2.
Scheme 2 A plausible mechanism for the formation of dehydroepiandrosterone spiro-pyrrolidine derivatives
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Chemical Papers
cytotoxity with LC50 <100 µg/mL. The products exhibited
much higher cytotoxic activity than the 16-arylidene ster-
oids, which indicated that the introduction of the phenyl spi-
ropyrrolidene indeno quinoxaline signifcantly improved the
cytotoxic efect of the steroidal derivatives. 11H-indeno[1,2-
b]quinoxalin-11-one exhibited moderate cytotoxity with a
LC50 of 72.05 µg/mL which is stronger than other raw mate-
rials. The presence of indeno quinoxaline moiety contributed
to the cytotoxic activity of the steroidal spiro-heterocyclic
products to a certain extent.
Experimental
Materials
Fig. 3 ORTEP diagram of 3c (all H atoms have been omitted)
All the chemical reagents were commercially available and
used without further purifcation. All the solvents were dried
and redistilled before use. The thin-layer chromatography
(TLC) analysis was performed with silica gel GF254 plates.
The column chromatography was carried out using silica
gel (200–300 mesh). All NMR spectra were recorded on
a Bruker AV-II 500 MHz NMR spectrometer, operating
Taking products out of the reaction system or adding the
cheap reactant is an efective way to improve the conver-
sion rate of the substrate in equilibrium reaction. The equi-
librium shift of the [3+2] cycloaddition reaction between
16-(4-methoxyphenylmethylene) dehydroepiandrosterone
(1f) and the azomethine ylide from 11H-indeno[1,2-b]qui-
noxalin-11-one and sarcosine was studied (SI Part B). At
the beginning, the poor solubility of the product 3f leaded to
its low concentration in acetonitrile solution. As monitored
by UPLC-PDA-ESIMS, the reaction equilibrium tends to
form cycloaddition product 3f. But the change of the reac-
tion system will not be obvious when the concentration of
reactants dropped to a lower level after 15 h and the system
reached equilibrium in the solution. By adding sarcosine,
the reaction continues to aford the product smoothly until
the system reached another equilibrium at the 25th hour.
Then the conversion rate of reaction substrate is maintained
at a constant level, and 3f was obtained by fltration with a
moderate yield (55%) after 30 h.
1
at 500 MHz for H, and 126 MHz for 13C. TMS was used
as an internal reference for 1H and 13C chemical shifts and
CDCl3 was used as solvent. All the mass spectra and reac-
tion system monitoring data were obtained using a Waters
ACQUITY UPLC equipped a PDA detector and a successive
XEVO Q-TOF mass spectrometer. IR spectra were recorded
on a PerkinElmer spectrometer. Melting points were meas-
ured with a Yanaco MP500 melting point apparatus and
are uncorrected. The crystallographic data for compound
3c are summarized in the Table S1 in Supporting Informa-
tion. CCDC 1953655 contains the supplementary crystal-
lographic data, which can be obtained free of charge from
the Cambridge Crystallographic Data Centre via www.ccdc.
cam.ac.uk/data_request/cif.
Bioassay
Chemicals
All the starting materials including dehydroepian-
drosterone, 16-arylidene dehydroepiandrosterones
(1a–1g), 11H-indeno[1,2-b]quinoxalin-11-one (2), and
sarcosine, along with the synthesized 1′-N-methyl-
spiro[2′,11″]-indeno[1,2-b]quinoxaline-spiro[3′,16]-
dehydroepiandrosterone-4′-arylpyrrolidines (3a–3g) were
evaluated for their cytotoxicity on brine shrimp bioassay
(Meyer et al. 1982). The products showed cytotoxic efects
against Artemia salina with LC50 varying from 6.19 to
27.2 µg/mL. Compound 3d and 3g had the most signifcant
cytotoxic bioactivities with LC50 values of 6.19 and 9.92 µg/
mL, respectively. The dehydroepiandrosterone, 16-arylidene
dehydroepiandrosterones and sarcosine did not show
Typical procedure for the synthesis of 16‑arylidene
dehydroepiandrosterones (1a–1g)
The mixture of dehydroepiandrosterone (500 mg,
1.73 mmol) and 4-methylthio-benzaldehyde (263.9 mg,
1.73 mmol) was dissolved in EtOH (20 mL). The reaction
solution was refuxed about 2 h after addition of 2 mL aque-
ous NaOH solution (NaOH, 346.7 mg, 8.67 mmol), and the
reaction progress was monitored by TLC. Then the reaction
mixture was fltrated to give a pale yellow solid after being
slowly cooled to room temperature. The solid was washed
with 50% aqueous EtOH solution and dried to give pure
1a (670 mg, 94%). The synthesis and separation of other
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Table 1 Brine shrimp bioassay data of raw materials, products and
16-arylidene-17-ketosteroids (1b–1g) is the same as those
of 1a.
podophyllotoxin
Sample
LC50 (μg/mL)
1a
>200
>200
117.54
>200
120.42
>200
>200
72.05
20.86
27.20
20.36
6.19
24.59
14.30
9.92
>200
>200
2.16
Typical procedure for the synthesis of C‑16
dehydroepiandrosterone spiro‑pyrrolidine derivatives
(3a–3g)
1b
1c
1d
1e
The mixture of 1a (100 mg, 0.24 mmol), 2 (60 mg,
0.26 mmol) and sarcosine (32 mg, 0.36 mmol) was dissolved
in acetonitrile (5 mL). The reaction mixture was heated in
nitrogen atmosphere until the solids were completely dis-
solved, and target product 3a as yellow solid gradually pre-
cipitated within 15-h refux. To improve yield, sarcosine
(21 mg, 0.24 mmol) was added to above mixture. The reac-
tion was terminated until the product did not increase. And
then, the reaction was slowly cooled to room temperature
and fltrated to aford a yellow solid 3a (101 mg, 63% yield).
The other steroidal spiro-pyrrolidine derivatives (3b–3g)
were obtained according to the same procedure as that of 3a
with yields in a range of 50–65%.
1f
1 g
2
3a
3b
3c
3d
3e
3f
3g
Sarcosine
Dehydroepiandrosterone
Podophyllotoxin
DMSO
N.D.
Cytotoxicity study protocol (brine shrimp assay)
N.D. not detected
The bioassay procedure was performed as the literature
reported (Meyer et al. 1982). 50 mg Brine shrimp (Artemia
salina Leach) eggs were hatched for 48 h in artifcial sea-
water under constant aeration. The nauplii were collected
and subjected to cytotoxicity assay. To each well of 96-well
plates was made up to 195 μL with artifcial seawater after
ten nauplii were added. Sample solution was prepared with
DMSO at the initial concentration of 2.0 mg/mL and diluted
with DMSO to prepare sample solutions at fve concentra-
tion gradients, 2.0, 1.0, 0.5, 0.25 and 0.125 mg/mL. The
serial concentrations of positive control, podophyllotoxin,
were set to be 2.0, 1.0, 0.5, 0.25, 0.125, and 0.0625 mg/mL.
From each sample, 5 μL sample solution was transferred to
each well, and each sample was repeated three times. Each
negative control was added only 5 μL DMSO. After 24 h, the
number of survivors were counted and calculated by Bliss
method to determine the LC50 values (Bliss 1934). The brine
shrimp bioassay results of all raw materials, products and
positive control were listed in Table 1.
16‑(2,4‑Dichlorobenzylidene) dehydroepiandrosterone(1g)
White solid, yield 91%; mp: 199–201 °C. IR (KBr) (v
1
cm−1): 1712, 1625; H and 13C NMR data see SI Part C;
APIMS calc. for [C26H31Cl2O2]+ ([M+H]+): 445.2, Found:
445.1.
1′‑N‑Methyl‑spiro[2′,11″]‑indeno[1,2‑b]quinoxaline‑spiro[
3′,16]‑dehydroepiandrosterone‑4′(4‴‑methylthiophenyl)‑
pyrrolidine(3a)
Yellow solid, yield 63%; mp: 198–200 °C; IR (KBr) (v
1
cm−1): 3417, 1713; H and 13C NMR data see SI Part
C; HR-ESIMS calc. for [C44H48N3O2S]+ ([M+H]+):
682.3462, Found: 682.3461.
Spectroscopic data
1′‑N‑Methyl‑spiro[2′,11″]‑indeno[1,2‑b]quinoxaline‑spiro[
3′,16]‑dehydroepiandrosterone‑4′(4‴‑methylphenyl)‑pyrr
olidine(3b)
16‑(4‑Methylthiobenzylidene) dehydroepiandrosterone(1a)
Pale yellow solid, yield 94%; mp: 219–221 °C. IR (KBr)
(v cm−1): 1712, 1625; 1H and 13C NMR data see SI Part C;
HR-ESIMS calc. for [C27H35O2S]+ ([M+H]+): 423.2352,
Found: 423.2351.
Yellow solid, yield 50%; mp: 217–219 °C; IR (KBr) (v
1
cm−1): 3425, 1725; H and 13C NMR data see SI Part C;
HR-ESIMS calc. for [C44H48N3O2]+ ([M+H]+): 650.3741,
Found: 650.3743.
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Chemical Papers
1′‑N‑Methyl‑spiro[2′,11″]‑indeno[1,2‑b]quinoxaline‑spiro
[3′,16]‑dehydroepiandrosterone‑4′(4‴‑phenyl)‑pyrrolidin
e(3c)
in the range of 6.19–27.2 µg/mL. Among them, the activity
of 3d and 3g with LC50 value less than 10 µg/mL reached
the same level as the positive control. The results indicate
that the existence of spiro-pyrrolidine moiety signifcantly
increased the cytotoxic activity of steroids, which implies
another ideal choice in developing antitumor drugs.
Yellow solid, yield 57%; mp: 248–250 °C; IR (KBr) (v
1
cm−1): 3430, 1732; H and 13C NMR data see SI Part C;
HR-ESIMS calc. for [C43H46N3O2]+ ([M+H]+): 636.3585,
Found: 636.3588.
Acknowledgements This work was supported by the Scientific
Research Fund of Hunan Provincial Education Department (No.
15C0560), and the Open Project Programme of the Key Laboratory
of Theoretical Chemistry and Molecular Simulation of the Ministry
of Education (No. E21203), and the Student Research and Innova-
tion Program of Hunan University of Science and Technology (No.
SYZ2019050).
1′‑N‑Methyl‑spiro[2′,11″]‑indeno[1,2‑b]quinoxaline‑spiro[
3′,16]‑dehydroepiandrosterone‑4′(4‴‑fuorophenyl)‑pyrro
lidine(3d)
Yellow solid, yield 60%; mp: 246–248 °C; IR (KBr) (v
1
cm−1): 3416, 1727; H and 13C NMR data see SI Part C;
HR-ESIMS calc. for [C43H45FN3O2]+ ([M+H]+): 654.3490,
Found: 654.3488.
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