Discovery of methylpyrimidine ring-fused diterpenoid analogs as a novel testosterone synthesis promoter

Article abstract of DOI:10.1039/C9RA00702D

Herein we screened our small synthetic library of diterpenoid analogs for hit compounds on promoting testosterone synthesis and the methylpyrimidine ring-fused diterpenoid analog 7 was obtained as the hit. Based on the hit, a series of derivatives were de

Full text of DOI:10.1039/C9RA00702D

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Discovery of methylpyrimidine ring-fused
diterpenoid analogs as a novel testosterone
Cite this: RSC Adv., 2019, 9, 9709
synthesis promoter†
a
Jie Bai,‡a Jia Xie,‡b Li-Ting Wang,a Yajing Xing,b Qian-Ru Jiang,a Fan Yang,
a
b
a
*
*
Jie Tang, Zhengfang Yi and Wen-Wei Qiu
Herein we screened our small synthetic library of diterpenoid analogs for hit compounds on promoting
testosterone synthesis and the methylpyrimidine ring-fused diterpenoid analog 7 was obtained as the hit.
Based on the hit, a series of derivatives were designed, synthesized and evaluated for their effects on
testosterone secretion in mouse Leydig TM3 cells. Most of the derivatives showed better activity in
promoting testosterone synthesis than the positive control compound icariin, among which compound
17 has optimal activity and little cytotoxicity. Preliminary mechanism studies indicated that 17 significantly
promoted the expression of testosterone synthesis-related marker genes (StAR, 3b-HSD and CYP11A1).
Further studies showed that 17 provided sufficient steroid materials for testosterone synthesis by
stimulating autophagy in Leydig cells. Thus compound 17 emerged as a potential lead compound for
further development of therapeutics for late onset of hypogonadism (LOH).
Received 26th January 2019
Accepted 20th March 2019
DOI: 10.1039/c9ra00702d
rsc.li/rsc-advances
central body fat, forgetfulness, loss of memory, difficulty in
concentration, insomnia and erectile dysfunction (ED).14,15
Introduction
Testosterone replacement therapy (TRT), as the main clinical
treatment method for LOH at present, provides a wide range of
benets for hypogonadism, improving libido and sexual func-
tion, fertility, bone density, muscle mass, and quality of life.16,17
However the clinical efficacy and long-term safety of TRT
remains controversial. TRT involves the direct administration
of an exogenous hormone, and with this treatment, the
androgen level in the serum is superphysiological and
unstable.13 So, it has considerable side effects, for instance,
testicular atrophy, erythropoiesis, prostate cancer, cardiovas-
cular events, intrahepatic cholestasis, sleep apnea, liver failure
and worsening of lower urinary tract symptoms from an
enlarged prostate.18,19 Therefore, the discovery of novel agents
as anti-LOH agents is urgently needed.
Testosterone production is regulated by luteinizing hormone
(LH) and LH induces cAMP synthesis in Leydig cells by binding
to the membrane LH receptors.20 cAMP catalyzes the synthesis
of PKA, which transports cholesterol from the cytoplasmic pool
to mitochondria and promotes steroidogenesis by steroid-
generating enzymes (CYP11A1, 3b-HSD) and steroidogenic
acute regulatory (StAR) protein21 Additionally, autophagy in
Leydig cells promotes lipid metabolism and provides sources
such as triglycerides (TGs) and cholesterol for testosterone
production, which plays an important role in testosterone
synthesis.22,23
Diterpenoids are a major branch of natural products which
exert many different biological activities.1 For instance, anti-
proliferative,2 multi-drug resistance-reversing,3 antimicrobial,4
vasoactive,5 antidiabetic,6 anti-osteoporosis7 immunomodula-
tory,8 and anti-inammatory9 effects. Therefore, diterpenoids
play an important role in the discovery of novel bioactive agents.
Testosterone, which is primarily produced in Leydig cells as
the major circulating androgen, plays important roles in sexual
differentiation, secondary sex characteristics, reproductive
function and sexual function.10,11 Serum testosterone deciency
causes late onset of hypogonadism (LOH), which is a clinical
and biochemical syndrome.12 The incidence rates of LOH are
13%, 30% and 47% for men aged 40–49, 50–59 and over 70
respectively.13 Previous studies indicated that LOH-related
hypogonadal symptoms included sexual desire decrease,
muscle mass and strength reduction, osteoporosis, increased
aShanghai Engineering Research Center of Molecular Therapeutics and New Drug
Development, School of Chemistry and Molecular Engineering, East China Normal
University, 3663 North Zhongshan Road, Shanghai 200062, China. E-mail: wwqiu@
chem.ecnu.edu.cn; Tel: +86-21-54340103
bShanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences,
School of Life Sciences, East China Normal University, Shanghai 200241, China.
E-mail: zfyi@bio.ecnu.edu.cn; Tel: +86-21-54345016
† Electronic supplementary information (ESI) available: Spectrum of the
corresponding compounds. 1H and 13C NMR spectra of all nal compounds.
See DOI: 10.1039/c9ra00702d
Herein, we report the discovery of a hit compound as
testosterone synthesis promoter by screening our small
synthetic library of diterpenoid analogs. The methylpyrimidine
‡ These authors contributed equally.
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ring-fused diterpene analogue 7 was selected as the hit. Then
a series of analogs were synthesized based on the hit and their
activities in promoting testosterone production in mouse Ley-
dig TM3 cells were evaluated. Results showed that compound 17
(SH380) was the most potent testosterone synthesis promoter.
The mechanism by which the 17 increases testosterone
production was preliminary explored.
Results and discussion
Chemistry
The methylpyrimidine acyl derivatives 7–14 were synthesized as
shown in Scheme 1. Compound 6 was prepared according to
our previously reported procedure24,25 and details were as
follows. Coupling reaction of 6,7-epoxygeranyl acetate with (4-
methoxybenzyl) magnesium chloride yielded compound 1, Li2-
CuCl4 was added as catalyst. Key intermediate 2 was obtained by
the cyclization of 1 in presence of boron triuoride etherate,
and is a racemate according to the Stork–Eschenmoser
hypothesis.26,27 Therefore, all the synthesized diterpenoids
herein belong to racemates. Compound 3 was obtained by
Scheme 2 Reagents and conditions: (a) hydroxylamine hydrochloride,
K2CO3, EtOH, rt; (b) hydrazine hydrate (85%), TsOH, EtOH, reflux; (c)
methylmagnesium chloride, anhydrous THF, 0 ꢁC; (d) hydrazine
hydrate (85%), NaOH, triglycol, 120 ꢁC to 190 ꢁC; (e) NaBH4, DCM,
MeOH, 0 ꢁC; (f) TsOH, anhydrous THF, 60 ꢁC; (g) CrO3, AcOH, rt.
Activity and cytotoxicity evaluation of compound 7 and its
derivatives
oxidation of
2 with 2-iodoxybenzoic acid (IBX). Claisen
A hit was obtained by screening our small synthetic diterpe-
noids library. Mouse TM3 Leydig cells were treated with each
tested compound at concentration of 20 mM for 24 h, and the
testosterone levels in the culture medium were measured by
ELISA (enzyme-linked immuno sorbent assay) kit. Among those
analogs, compound 7 (QB364) was determined as a testosterone
synthesis promoter with a testosterone concentration of 271.7
mM Lꢀ1 and was more potent than the positive icariin.28
Therefore, it was selected as the hit compound for further
structure–activity relationship (SAR) optimization.
The rst-round synthetic compounds 8–14 were obtained by
Friedel–Cras acylation with various acyl chlorides respectively
and their activity in promoting testosterone production on
mouse Leydig TM3 cells was determined by ELISA kit. As shown
in Table 1, the activity decreased signicantly when the acetyl
group (7) was replaced by various acyl substituents. Thus,
condensation of 3 with ethyl formate in the presence of NaH
provided compound 4. Compound 6 was furnished by reaction
of 4 with piperidine in EtOH and then condensation with
acetamidine hydrochloride. Compound 7–14 were prepared by
Friedel–Cras acylation from 6 with various acyl chlorides
respectively.
The synthetic route of 2-methylpyrimidine derivatives 15–22
is outlined in Scheme 2. Compound 15 was prepared from 7
with hydroxylamine hydrochloride in the presence of K2CO3.
Condensation of 7 with hydrazine hydrate (85%) yielded 16.
Reaction of 7 with methylmagnesium chloride yielded 17.
Compound 18 was produced by Wolff–Kishner–Huang Min-lon
reduction of 7 with hydrazine hydrate (85%) in the presence of
NaOH. Reduction of 7 with sodium borohydride produced
compound 19, and 20 was provided by dehydration of 19 with p-
toluenesulfonic acid. Oxidation of 7 with CrO3 in AcOH
produced 21.
Scheme 1 Reagents and conditions: (a) (4-methoxybenzyl) magnesium chloride, LiCuCl4, anhydrous THF, 0 ꢁC; (b) boron trifluoride etherate,
anhydrous CH2Cl2, ꢀ78 ꢁC; (c) IBX, THF, DMSO, rt; (d) ethyl formate, NaH, anhydrous THF, rt; (e) piperidine, EtOH, reflux; (f) acetamidine
hydrochloride, sodium methoxide, EtOH, reflux; (g) acyl chloride, AlCl3, DCM, 0 ꢁC.
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Table 1 Testosterone levels and cytotoxicity of compounds 7–14 in Table 2 Testosterone levels and cytotoxicity of compounds 15–21 in
TM3 cellsa
TM3 cellsa
c
Compd
R2
R3
—
—
Testosterone Levelsb (mM/L)
271.7 ꢂ 10.0
IC50 (mM)
Compd
R1
Testosterone Levelsb (mM Lꢀ1
)
Control
Icariin
—
—
164.5 ꢂ 0.2
229.8 ꢂ 4.8
7
>100
>100
7
8
9
271.7 ꢂ 10.0
233.0 ꢂ 0.5
252.3 ꢂ 2.4
15
282.2 ꢂ 5.5
16
—
188.5 ꢂ 5.5
>100
17
18
—
—
303.5 ꢂ 5.5
237.0 ꢂ 4.0
>100
>100
10
242.6 ꢂ 6.0
19
20
—
—
222.8 ꢂ 7.0
227.1 ꢂ 3.0
>100
>100
11
12
245.4 ꢂ 4.9
217.1 ꢂ 9.9
21
O
259.5 ꢂ 6.5
>100
13
174.1 ꢂ 0.5
204.2 ꢂ 6.0
a
See Experimental section. b Testosterone levels in the culture medium
are measured by ELISA kit; data are expressed as mean ꢂ SD of three
14
c
independent assays. Activities of compounds against the growth of
TM3 cells are detected with the SRB assay and their IC50 data are an
average of at least three independent experiments.
a
See Experimental section. b Testosterone levels in the culture medium
are measured by ELISA kit; data are expressed as mean ꢂ SD of three
independent assays.
Table 3 IC50 of compound 24 against the growth of various normal
cellsa
compound 7 was selected for the second-round structure
modication.
Leydig
cell
Normal cellb
The second-round synthetic analogs 15–21 were obtained by
modication of the acetyl group of 7 and their activity in
promoting testosterone production was determined using
ELISA kit. The results (Table 2) showed that the activity
increased when the carbonyl group of 7 was substituted by
oxime (15), while decreased as hydrazine substituent (16) was
introduced into. If the carbonyl group of 7 was reduced to
hydroxy (19) the activity decreased obviously. To our delight, the
testosterone level in TM3 was increased signicantly as the
acetyl group was replaced by tert-butanol group (17). If the acetyl
group was converted to the alkyl substituent such as ethyl (18)
or vinyl (20), the activity of testosterone production was also
decreased. For derivative modied at the C-6 and C-7 positions,
the activity decreased as the carbonyl (21) substituent was
introduced into C7 position. Further cytotoxicity evaluation in
mouse TM3 cells with the SRB assay disclosed that all these
analogs showed very little cytotoxicity (IC50 > 100 mM). Consid-
ering the above results, the safe compound 17 exhibited most
potent activity in promoting testosterone production, thus it
was selected for further studies.
Cell line
TM3
HAF
NCM460
>200
L-02
>200
PTN1A
>200
IC50 (mM)c
112.6d
111.8d
a
See Experimental section. b HAF, human broblast cell line; NCM460,
human colon mucosal epithelial cell line; L-02, human fetal hepatocyte
cell line; PTN1A, human prostate epithelial cell line. c Various cells were
treated with indicated concentrations of compound 17 for 24 h; cell
proliferation was determined by SRB assay; data were expressed as
IC50 of three independent assays. d Variation ꢂ 10%.
Compound 17 has little cytotoxicity on TM3 and other normal
cells within the effective concentrations
To further determination of the in vitro safety of 17, we detected
its cytotoxicity in TM3 and several human normal cells
including NCM460, HAF, L-02 and PTN1A by SRB assay. As
shown in Table 3, compound 17 showed very little toxicity on
cell proliferation of TM3 (IC50 ¼ 112.6 mM) and HAF (IC50
¼
112.6 mM) cells. In addition, 17 has almost no toxic effects on
NCM460, L-02 and PTN1A cells (IC50 > 200 mM). Therefore,
compound 17 is a safe compound in vitro.
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Fig. 1 Compound 17 advanced the mRNA expression levels of testosterone synthetase in Leydig cells.a aSee Experimental section. Mouse Leydig
TM3 cells were treated with different concentrations of compound 17 for 24 h. mRNA levels of steroidogenesis-related genes StAR, 3b-HSD and
CYP11A1 were measured by quantitative-PCR. Data were expressed as mean ꢂ SD of three independent assays; Student's t-tests were per-
formed; *p < 0.05, **p < 0.01, ***p < 0.001.
mechanism studies indicated that 17 increased testosterone
levels in Leydig cells via promoting testosterone synthesis.
Compound 17 regulates the expression of autophagy-related
proteins in Leydig cells
Autophagy dysfunction played a key role in the loss of testos-
terone production in some LOH patients.21 Therefore, we tried
to identify if 17 promoted testosterone production via induction
of autophagy. LC3-II serves as a reliable marker for mature
autophagosomes and ATG5 is a gene product required for the
Fig. 2 Compound 17 advanced the protein expression levels of
testosterone synthetase in Leydig cells.a aSee Experimental section.
Mouse Leydig TM3 cells were treated with different concentrations of
17 for 24 h and the protein levels of StAR, 3b-HSD and CYP11A1 were formation of autophagosomes. We assessed the levels of the
measured by Western blotting. GAPDH was used as a control.
autophagy-related proteins by western blotting. As shown in
Fig. 3, the expressions of ATG5, LC3-II and the ratio of LC3-II/
LC3-I were increased signicantly aer treatment with 17. It
suggested that the compound could promoted testosterone
synthesis via activating autophagy in Leydig cells.
Conclusions
In this study, we designed and synthesized a number of 15
methylpyrimidine ring-fused tricyclic diterpene analogs. Most
Fig.
3 Compound 17 promotes the autophagy-related protein
of the derivatives showed potent activity in promoting testos-
terone production in mouse Leydig TM3 cells, which was much
better than that of the positive control icariin. Among these
analogs, compound 17 exhibited the most potent activity and
almost no toxic effect. Preliminary mechanism studies indi-
cated that 17 could signicantly enhance the expression of
testosterone synthesis-related marker genes (StAR, 3b-HSD and
CYP11A1) in a concentration-dependent manner. Further
studies disclosed that 17 could also stimulate autophagy in
Leydig cells and promote the expression of autophagy-related
genes (LC3 and ATG 5). In conclusion, compound 17 was
discovered as a promising compound, which could be further
developed as a potential therapeutic agent for LOH.
expression levels in TM3 cells.a aSee Experimental section. Mouse
Leydig TM3 cells were treated with different concentrations of 17 for
24 h. The expression of LC3 I/II and ATG5 were detected by western
blotting. GAPDH was used as a control.
Compound 17 enhanced the expression of testosterone
synthesis-regulating gens
For determination of the primary mechanism of 17 on testos-
terone synthesis, the mRNA and protein expression levels of
testosterone synthetase, including StAR, 3b-HSD and CYP11A1
were analyzed. The qPCR analysis showed that the mRNA
expression levels of StAR, 3b-HSD and CYP11A1 were increased
signicantly in a concentration-dependent manner (Fig. 1).
Furthermore, the protein expression levels of the three key
enzymes in TM3 cells were validated by western blotting.
Results indicated that 17 also increased the protein expression
levels of StAR, 3b-HSD and CYP11A1 obviously as the concen-
tration rised from 10 mM to 100 mM (Fig. 2). These primary
Experimental section
Chemistry
General methods. Unless otherwise specied, starting
materials, reagents, and solvents were purchased from
commercial suppliers and used without further purication.
Anhydrous THF was obtained by distillation over sodium wire.
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All non-aqueous reactions were run under a nitrogen atmo- reaction mixture was stirred for 6 h under reux. Then the
sphere and all reaction vessels were oven-dried. Thin-layer mixture was poured into water (30 mL) and extracted with
chromatography (TLC) was performed on silica gel plates EtOAc (10 mL ꢃ 3). The organic layer was washed with sodium
(QingDao) with a layer thickness of 0.25 mm and spots were bicarbonate solution (5%) and brine, dried over anhydrous
visualized by their quenching of the uorescence of an indicator Na2SO4, and concentrated under vacuum. The residue was
admixed to the SiO2 layer. Column chromatographic purica- puried by silica gel chromatography to give the product.
tion was carried out using silica gel (200–300 mesh) and an
Compound 7. This compound was obtained from compound
1
EtOAc/hexane mixture or gradient was used unless otherwise 6 employing method A. White solid. Yield 92%. H NMR (400
stated. 1H and 13C NMR spectra were recorded on a Bruker MHz, CDCl3) d 8.39 (s, 1H), 7.49 (s, 1H), 6.93 (s, 1H), 3.94 (s, 3H),
Advance III 400 spectrometer at 400 MHz (1H) and 100 MHz 3.16 (d, J ¼ 15.5 Hz, 1H), 2.98–2.93 (m, 1H), 2.85–2.75 (m, 2H),
(13C). Chemical shis are reported in d (ppm) using the 2.69 (s, 3H), 2.60 (s, 3H), 2.03–1.96 (m, 1H), 1.85 (d, J ¼ 12.3 Hz,
d 0 signal of tetramethylsilane (TMS) as internal standards. 1H), 1.78–1.71 (m, 1H), 1.36 (s, 3H), 1.34 (s, 3H), 1.16 (s, 3H). 13
C
High resolution mass spectra were performed using a Bruker NMR (100 MHz, CDCl3) d 199.41, 172.09, 166.09, 157.62, 157.34,
ESI-TOF high-resolution mass spectrometer. 151.81, 131.11, 127.87, 126.34, 123.29, 108.57, 55.57, 49.14,
General procedure for the Friedel–Cras acylation (method 40.65, 39.69, 37.52, 31.79, 31.22, 30.12, 25.82, 23.96, 23.16,
A). To a solution of compound 6 (100 mg, 0.27 mmol) in 20.44. HRMS (ESI): calcd for C23H29N2O2 [M + H]+, 365.2224;
anhydrous CH2Cl2 (20 mL) was added anhydrous AlCl3 (108 mg, found, 365.2233.
0.81 mmol) and respective acyl chloride (0.54 mmol) at 0 ꢁC
Compound 8. This compound was obtained from compound
1
under nitrogen. Then reaction mixture was stirred for 2 h at 6 employing method A. White solid. Yield 90%. H NMR (400
room temperature. Aer that the mixture was poured into water MHz, CDCl3) d 8.38 (s, 1H), 7.44 (s, 1H), 6.92 (s, 1H), 3.92 (s, 3H),
(50 mL) and extracted with CH2Cl2 (10 mL ꢃ 3). The organic 3.16 (d, J ¼ 15.5 Hz, 1H), 3.00–2.93 (m, 3H), 2.85–2.75 (m, 2H),
layer was washed with brine, dried over anhydrous Na2SO4, and 2.69 (s, 3H), 2.00–1.96 (m, 1H), 1.85 (d, J ¼ 12.4 Hz, 1H), 1.78–
concentrated under vacuum. The residue was puried by silica 1.71 (m, 1H), 1.36 (s, 3H), 1.33 (s, 3H), 1.16–1.13 (m, 6H). 13C
gel chromatography to afford the product.
NMR (100 MHz, CDCl3) d 203.11, 172.21, 166.11, 157.64, 156.97,
General procedure for the reduction reaction (method B). 151.25, 131.00, 127.94, 126.64, 123.42, 108.59, 55.65, 49.22,
NaBH4 (3 mmol) was added into a solution of compound 7 or 21 40.72, 39.75, 37.52, 36.95, 31.27, 30.20, 25.84, 24.02, 23.21,
(0.3 mmol) in anhydrous CH2Cl2 (10 mL) and MeOH (10 mL) 20.50, 8.48. HRMS (ESI): calcd for C24H31N2O2 [M + H]+,
with ice cooling under nitrogen. Aer stirring for 1 h at room 379.2380; found, 379.2384.
temperature, the reaction was quenched by saturated aqueous
Compound 9. This compound was obtained from compound
1
NH4Cl solution with ice cooling. The mixture was extracted with 6 employing method A. White solid. Yield 87%. H NMR (400
EtOAc (20 mL ꢃ 3). The organic layer was washed with brine, MHz, CDCl3) d 8.40 (s, 1H), 7.42 (s, 1H), 6.93 (s, 1H), 3.93 (s, 3H),
dried over Na2SO4 and concentrated under vacuum. The residue 3.17 (d, J ¼ 15.6 Hz, 1H), 2.99–2.92 (m, 3H), 2.86–2.76 (m, 2H),
was puried by silica gel chromatography to give the product.
2.70 (s, 3H), 2.02–1.97 (m, 1H), 1.87 (d, J ¼ 12.5 Hz, 1H), 1.80–
General procedure for the oxidation reaction (method C). 1.76 (m, 1H), 1.73–1.68 (m, 2H), 1.38 (s, 3H), 1.35 (s, 3H), 1.17 (s,
CrO3 (0.6 mmol) was added into a solution of compound 6 (0.3 3H), 0.97 (t, J ¼ 7.4 Hz, 3H). 13C NMR (100 MHz, CDCl3)
mmol) in AcOH (10 mL) with ice cooling. Aer stirring for 3 h at d 202.65, 172.22, 166.17, 157.66, 156.89, 151.18, 130.93, 128.00,
room temperature, the mixture was poured into water (50 mL) 127.08, 123.37, 108.65, 55.68, 49.29, 45.67, 40.78, 39.76, 37.54,
and extracted with EtOAc (20 mL ꢃ 3). The organic layer was 31.29, 30.22, 25.85, 24.01, 23.21, 20.53, 17.86, 13.95. HRMS
washed with sodium bicarbonate solution (5%) and brine, dried (ESI): calcd for C25H33N2O2 [M + H]+, 393.2537; found, 393.2560.
over anhydrous Na2SO4, and concentrated under vacuum. The
residue was puried by silica gel chromatography to give the compound 6 employing method A. White solid. Yield 88%. H
product. NMR (400 MHz, CDCl3) d 8.39 (s, 1H), 7.29 (s, 1H), 6.91 (s, 1H),
Compound 10. This compound was obtained from
1
General procedure for the Wolff–Kishner–Huang Min-lon 3.91 (s, 3H), 3.53–3.46 (m, 1H), 3.16 (d, J ¼ 15.5 Hz, 1H), 2.97–
reduction (method D). Hydrazine hydrate (85%) (1.2 mmol) 2.93 (m, 1H), 2.85–2.76 (m, 2H), 2.70 (s, 3H), 1.99 (dd, J ¼ 12.2,
was added into a stirred solution of compound 7 (0.3 mmol) in 4.5 Hz, 1H), 1.86 (d, J ¼ 12.3 Hz, 1H), 1.79–1.73 (m, 1H), 1.37 (s,
triglycol (10 mL) under nitrogen. The reaction mixture was 3H), 1.34 (s, 3H), 1.16–1.13 (m, 9H). 13C NMR (100 MHz, CDCl3)
stirred for 1 h at 120 ꢁC. Then removed the condensing tube and d 207.69, 172.18, 166.09, 157.63, 156.24, 150.62, 130.84, 127.96,
heated the mixture to 190 ꢁC. Aer the temperature is stable, the 127.07, 123.40, 108.42, 55.68, 49.20, 40.72, 39.94, 39.71, 37.45,
condensing tube was reinstalled and mixture was stirred for 3 h 31.25, 30.18, 25.85, 24.00, 23.18, 20.47, 18.68, 18.55. HRMS
at 190 C. Aer cooling, the mixture was poured into water (40 (ESI): calcd for C25H33N2O2 [M + H]+, 393.2537; found, 393.2557.
Compound 11. This compound was obtained from
ꢁ
mL) and extracted with EtOAc (15 mL ꢃ 3). The organic layer
1
was washed with brine, dried over anhydrous Na2SO4, and compound 6 employing method A. White solid. Yield 82%. H
concentrated. The residue was puried by silica gel chroma- NMR (400 MHz, CDCl3) d 8.39 (s, 1H), 7.41 (s, 1H), 6.92 (s, 1H),
tography to give the product.
3.92 (s, 3H), 3.16 (d, J ¼ 15.5 Hz, 1H), 2.97–2.93 (m, 3H), 2.85–
General procedure for the dehydration reaction (method E). 2.76 (m, 2H), 2.70 (s, 3H), 1.98 (dd, J ¼ 12.4, 5.1 Hz, 1H), 1.85 (d,
To a solution of compound 19 or 22 (0.3 mmol) in anhydrous J ¼ 12.4 Hz, 1H), 1.79–1.71 (m, 1H), 1.68–1.61 (m, 2H), 1.41–1.39
EtOH (10 mL) was added TsOH (0.6 mmol) under nitrogen. The (m, 2H), 1.37 (s, 3H), 1.34 (s, 3H), 1.16 (s, 3H), 0.92 (t, J ¼ 7.3 Hz,
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3H). 13C NMR (100 MHz, CDCl3) d 202.85, 172.13, 166.09, 30.26, 25.79, 24.05, 23.17, 20.54, 15.16. HRMS (ESI): calcd for
157.63, 156.79, 151.14, 130.91, 127.92, 126.93, 123.35, 108.53,
55.61, 49.18, 43.42, 40.69, 39.70, 37.48, 31.24, 30.16, 26.53,
C
23H30N3O2 [M + H]+, 380.2333; found, 380.2311.
Compound 16. To a solution of compound 7 (109 mg, 0.30
25.84, 23.98, 23.17, 22.51, 20.46, 13.97. HRMS (ESI): calcd for mmol) in anhydrous EtOH (10 mL) was added TsOH (63 mg,
26H35N2O2 [M + H]+, 407.2693; found, 407.2692.
0.33 mmol) and hydrazine hydrate (85%, 0.03 mL, 0.6 mmol).
Compound 12. This compound was obtained from Aer stirring for 6 h under reux, the mixture was poured into
C
1
compound 6 employing method A. White solid. Yield 84%. H water (50 mL) and pH was adjusted to 9 with sodium bicar-
NMR (400 MHz, CDCl3) d 8.40 (s, 1H), 7.42 (s, 1H), 6.92 (s, 1H), bonate solution. Then the mixture was extracted with DCM
3.93 (s, 3H), 3.17 (d, J ¼ 15.5 Hz, 1H), 2.99–2.93 (m, 3H), 2.86– (20 mL ꢃ 3). The organic layer was washed with brine, dried
2.76 (m, 2H), 2.70 (s, 3H), 1.99 (dd, J ¼ 12.7, 5.8 Hz, 1H), 1.86 (d, over anhydrous Na2SO4, and concentrated under vacuum. The
J ¼ 12.3 Hz, 1H), 1.80–1.65 (m, 5H), 1.37 (s, 2H), 1.35 (s, 6H), residue was puried by silica gel chromatography (petroleum
1.17 (s, 3H), 0.90 (t, J ¼ 6.9 Hz, 3H). 13C NMR (100 MHz, CDCl3) ether/EtOAc, 1/1, v/v) to afford compound 16 (85 mg, 75%) as
d 202.86, 172.15, 166.10, 157.64, 156.81, 151.15, 130.93, 127.93, a white solid. 1H NMR (400 MHz, CDCl3) d 8.40 (s, 1H), 7.29 (s,
126.93, 123.36, 108.54, 55.62, 49.19, 43.70, 40.70, 39.71, 37.49, 1H), 6.92 (s, 1H), 3.90 (s, 3H), 3.19 (d, J ¼ 15.6 Hz, 1H), 3.00–2.96
31.63, 31.25, 30.17, 25.85, 24.11, 23.99, 23.18, 22.53, 20.47, (m, 1H), 2.89–2.77 (m, 2H), 2.71 (s, 3H), 2.22 (s, 3H), 1.99 (dd, J
14.00. HRMS (ESI): calcd for C27H37N2O2 [M + H]+, 421.2850; ¼ 12.5, 5.5 Hz, 1H), 1.88 (d, J ¼ 12.4 Hz, 1H), 1.82–1.74 (m, 1H),
found, 421.2858.
1.61 (s, 2H), 1.38 (s, 3H), 1.36 (s, 3H), 1.18 (s, 3H). 13C NMR (100
Compound 13. This compound was obtained from MHz, CDCl3) d 172.36, 165.98, 158.51, 157.59, 156.13, 147.71,
1
compound 6 employing method A. White solid. Yield 91%. H 130.01, 127.89, 127.85, 123.67, 108.49, 55.81, 49.42, 40.88,
NMR (400 MHz, CDCl3) d 8.40 (s, 1H), 7.41 (s, 1H), 6.92 (s, 1H), 39.72, 37.28, 31.28, 30.33, 25.81, 24.08, 23.18, 20.59, 18.78.
3.93 (s, 3H), 3.17 (d, J ¼ 15.5 Hz, 1H), 2.97–2.94 (m, 3H), 2.86– HRMS (ESI): calcd for C23H31N4O [M + H]+, 379.2492; found,
2.76 (m, 2H), 2.70 (s, 3H), 1.99 (dd, J ¼ 12.8, 5.4 Hz, 1H), 1.86 (d, 379.2468.
J ¼ 12.5 Hz, 1H), 1.80–1.72 (m, 1H), 1.63–1.61 (m, 1H), 1.59–1.53
Compound 17. To a solution of compound 7 (109 mg, 0.30
(m, 2H), 1.37 (s, 3H), 1.35 (s, 3H), 1.17 (s, 3H), 0.92 (d, J ¼ 8.1 Hz, mmol) in anhydrous THF (15 mL) was added methylmagnesium
6H). 13C NMR (100 MHz, CDCl3) d 203.12, 172.17, 166.11, chloride dropwise at 0 ꢁC under nitrogen. Aer stirring for 6 h at
157.63, 156.76, 151.16, 130.97, 127.96, 126.94, 123.38, 108.53, room temperature, the reaction was quenched by saturated
55.60, 49.20, 41.80, 40.71, 39.73, 37.50, 33.33, 31.26, 30.18, aqueous NH4Cl solution with ice cooling. The mixture was
27.94, 25.85, 24.00, 23.19, 22.48 (2C), 20.48. HRMS (ESI): calcd extracted with EtOAc (20 mL ꢃ 3). The organic layer was washed
for C27H37N2O2 [M + H]+, 421.2850; found, 421.2876.
with brine, dried over Na2SO4 and concentrated under vacuum.
Compound 14. This compound was obtained from The residue was puried by silica gel chromatography (petro-
1
compound 6 employing method A. White solid. Yield 80%. H leum ether/EtOAc, 5/1, v/v) to afford compound 17 (98 mg, 86%)
NMR (400 MHz, CDCl3) d 8.40 (s, 1H), 7.41 (s, 1H), 6.92 (s, 1H), as white solid. 1H NMR (400 MHz, CDCl3) d 8.39 (s, 1H), 7.01 (s,
3.93 (s, 3H), 3.17 (d, J ¼ 15.4 Hz, 1H), 2.99–2.93 (m, 3H), 2.86– 1H), 6.89 (s, 1H), 4.16 (s, 1H), 3.95 (s, 3H), 3.15 (d, J ¼ 15.5 Hz,
2.76 (m, 2H), 2.70 (s, 3H), 1.99 (dd, J ¼ 12.5, 5.2 Hz, 1H), 1.86 (d, 1H), 2.94–2.81 (m, 2H), 2.76 (d, J ¼ 15.5 Hz, 1H), 2.70 (s, 3H),
J ¼ 12.3 Hz, 1H), 1.79–1.73 (m, 1H), 1.68–1.64 (m, 2H), 1.37 (s, 1.98 (dd, J ¼ 12.6, 5.8 Hz, 1H), 1.86 (d, J ¼ 12.4 Hz, 1H), 1.80–
3H), 1.34 (s, 6H), 1.31 (s, 3H), 1.17 (s, 3H), 0.88 (t, J ¼ 6.4 Hz, 1.73 (m, 1H), 1.60 (d, J ¼ 2.8 Hz, 6H), 1.37 (s, 3H), 1.34 (s, 3H),
3H). 13C NMR (100 MHz, CDCl3) d 202.88, 172.18, 166.10, 1.15 (s, 3H). 13C NMR (100 MHz, CDCl3) d 172.30, 165.98,
157.62, 156.81, 151.14, 130.93, 127.94, 126.96, 123.38, 108.55, 157.60, 155.40, 145.33, 133.94, 127.68, 126.45, 123.65, 108.27,
55.63, 49.21, 43.75, 40.71, 39.73, 37.49, 31.70, 31.26, 30.18, 72.24, 55.41, 49.39, 40.85, 39.66, 36.94, 31.25, 30.56, 29.76,
29.11, 25.84, 24.40, 24.00, 23.19, 22.54, 20.48, 14.06. HRMS 29.69, 25.83, 24.05, 23.14, 20.63. HRMS (ESI): calcd for
(ESI): calcd for C28H39N2O2 [M + H]+, 435.3006; found, 435.3025.
C
24H33N2O2 [M + H]+, 381.2537; found, 381.2514.
Compound 15. To a solution of compound 7 (100 mg, 0.27
Compound 18. This compound was obtained from
1
mmol) in anhydrous EtOH (10 mL) was added hydroxylamine compound 7 employing method D. White solid. Yield 67%. H
hydrochloride (38 mg, 0.54 mmol) and K2CO3 (151 mg, 1.10 NMR (400 MHz, CDCl3) d 8.39 (s, 1H), 6.88 (s, 1H), 6.82 (s, 1H),
mmol). Aer stirring for 12 h at room temperature, the mixture 3.86 (s, 3H), 3.17 (d, J ¼ 15.6 Hz, 1H), 2.93–2.81 (m, 2H), 2.76 (d,
was poured into water (50 mL) and extracted with DCM (20 mL J ¼ 15.8 Hz, 1H), 2.70 (s, 3H), 2.60 (qd, J ¼ 7.4, 2.4 Hz, 2H), 1.97
ꢃ 3). The organic layer was washed with brine, dried over (dd, J ¼ 12.7, 5.8 Hz, 1H), 1.87 (d, J ¼ 12.3 Hz, 1H), 1.81–1.73 (m,
anhydrous Na2SO4, and concentrated under vacuum. The 1H), 1.37 (s, 3H), 1.34 (s, 3H), 1.20 (t, J ¼ 7.5 Hz, 3H), 1.16 (s,
residue was puried by silica gel chromatography (petroleum 3H). 13C NMR (100 MHz, CDCl3) d 172.38, 165.89, 157.61,
ether/EtOAc, 2/1, v/v) to afford compound 15 (64 mg, 65%) as 155.92, 143.94, 130.78, 129.35, 127.23, 123.88, 107.18, 55.51,
a white solid. 1H NMR (400 MHz, CDCl3) d 8.40 (s, 1H), 7.04 (s, 49.52, 40.97, 39.66, 36.96, 31.27, 30.41, 25.86, 24.11, 23.15,
1H), 6.89 (s, 1H), 3.87 (s, 3H), 3.17 (d, J ¼ 15.6 Hz, 1H), 2.96–2.91 22.73, 20.69, 14.07. HRMS (ESI): calcd for C23H31N2O [M + H]+,
(m, 1H), 2.86–2.75 (m, 2H), 2.71 (s, 3H), 2.24 (s, 3H), 1.97 (dd, J 351.2431; found, 351.2417.
¼ 12.7, 5.8 Hz, 1H), 1.86 (d, J ¼ 12.4 Hz, 1H), 1.80–1.72 (m, 1H),
Compound 19. This compound was obtained from
1
1.37 (s, 3H), 1.35 (s, 3H), 1.16 (s, 3H). 13C NMR (100 MHz, CDCl3) compound 7 employing method B. White solid. Yield 93%. H
d 172.39, 166.00, 157.57, 157.13, 155.91, 147.70, 129.90, 127.74, NMR (400 MHz, CDCl3) d 8.38 (s, 1H), 7.05 (d, J ¼ 5.2 Hz, 1H),
125.16, 123.64, 108.29, 55.70, 49.39, 40.85, 39.72, 37.25, 31.27, 6.85 (s, 1H), 5.08–5.02 (m, 1H), 3.90 (s, 3H), 3.16 (d, J ¼ 15.5 Hz,
9714 | RSC Adv., 2019, 9, 9709–9717
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1H), 2.95–2.78 (m, 3H), 2.70 (s, 3H), 1.98 (dd, J ¼ 12.6, 5.6 Hz, brief, cells were plated at the appropriate cell densities in 96-
1H), 1.88–1.70 (m, 3H), 1.51 (dd, J ¼ 6.5, 2.8 Hz, 3H), 1.37 (s, well plates during the experiment. Aer incubation for 24 h, the
3H), 1.34 (s, 3H), 1.15 (s, 3H). 13C NMR (100 MHz, CDCl3) cells were treated with different concentrations of various
d 172.34, 165.98, 157.59, 155.16, 145.60, 131.58, 127.70, 126.80, compounds for 24 h. Control group were exposed to DMSO at
123.69, 107.50, 66.42, 55.42, 49.47, 40.94, 39.68, 37.08, 31.27, a concentration equivalent to that of the compound-treated
30.48, 25.82, 24.09, 23.15, 22.81, 20.61. HRMS (ESI): calcd for cells. Aer treatment, 25 mL of 50% TCA was added for cell
C
23H31N2O2 [M + H]+, 367.2380; found, 367.2382.
xation at 4 ꢁC. Aer 1 hour or more, the plates were washed by
Compound 20. This compound was obtained from water for ve times. The plates were allowed to dry using hair
compound 19 employing method E. White solid. Yield 80%. 1H dryer followed by being dyed with 100 mL 0.4% SRB for 10 min.
NMR (400 MHz, CDCl3) d 8.39 (s, 1H), 7.20 (s, 1H), 6.99 (dd, J ¼ Aer dying, the plates were washed by 1% acetic acid to remove
17.8, 11.2 Hz, 1H), 6.85 (s, 1H), 5.72 (dd, J ¼ 17.7, 1.6 Hz, 1H), the dye and allowed to dry using hair dryer. 100 mL of 10 mM
5.24 (dd, J ¼ 11.1, 1.6 Hz, 1H), 3.88 (s, 3H), 3.17 (d, J ¼ 15.5 Hz, Tris-based solution was added to each well, and absorbance was
1H), 2.98–2.92 (m, 1H), 2.87–2.74 (m, 2H), 2.70 (s, 3H), 1.98 (dd, measured using a 96-well plate reader at 515 nm. Three inde-
J ¼ 12.6, 5.7 Hz, 1H), 1.87 (d, J ¼ 13.3 Hz, 1H), 1.81–1.74 (m, 1H), pendent experiments were carried out in triplicate. The IC50 was
1.37 (s, 3H), 1.35 (s, 3H), 1.16 (s, 3H). 13C NMR (100 MHz, CDCl3) calculated using GraphPad Soware.
d 172.29, 165.96, 157.61, 155.33, 146.45, 131.29, 127.62, 127.03,
125.08, 123.69, 114.26, 107.97, 55.75, 49.43, 40.89, 39.69, 37.18,
31.27, 30.42, 25.85, 24.06, 23.17, 20.63. HRMS (ESI): calcd for
Western blot analysis
C
23H29N2O [M + H]+, 349.2274; found, 349.2270.
Cells were exposed to various concentrations of compounds for
Compound 21. This compound was obtained from indicated time, and total cellular protein was lysed in RIPA
1
compound 7 employing method C. White solid. Yield 70%. H buffer [50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA,
NMR (400 MHz, CDCl3) d 8.45 (s, 1H), 8.44 (s, 1H), 7.01 (s, 1H), 1% Triton X-100, 1% sodium deoxycholic acid, 0.1% SDS, 2 mM
4.05 (s, 3H), 3.30 (d, J ¼ 15.5 Hz, 1H), 2.91 (d, J ¼ 15.4 Hz, 1H), phenylmethanesulfonyl uoride (PMSF), 30 mM Na2HPO4,
2.81–2.75 (m, 2H), 2.71 (s, 3H), 2.61 (s, 3H), 2.43 (dd, J ¼ 13.5, 50 mM NaF, 1 mM Na3VO4] containing protease/phosphatase
4.1 Hz, 1H), 1.39 (s, 3H), 1.38 (s, 3H), 1.29 (s, 3H). 13C NMR (100 inhibitors (Roche). Lysates were combined with sample
MHz, CDCl3) d 198.40, 195.80, 170.80, 166.61, 162.65, 157.85, loading buffer and heated at 100 ꢁC. Protein concentrations
157.70, 130.59, 127.76, 124.13, 122.08, 107.23, 55.99, 47.33, were measured by a Bicinchoninic acid assay (Thermo Scien-
39.64, 39.43, 37.73, 36.46, 31.37, 30.72, 25.84, 23.70, 22.04. tic). Lysates were mixed with sample loading buffer and heated
HRMS (ESI): calcd for C23H27N2O3 [M + H]+, 379.2016; found, at 100 ꢁC. for 15 min. Aer separated by 8–15% SDS-PAGE,
379.2009.
extracted protein were transferred to nitrocellulose
membranes. Membranes were incubated in 5% (w/v) bovine
serum albumin (TBST/BSA) and stored overnight at 4 ꢁC on
a shaker with specic primary antibodies (1/1000 in TBST/BSA).
Then membranes were washed with TBST and incubated for
45 min with secondary antibody (1/10 000 in TBST/BSA) at room
temperature. Immunoreactive proteins were visualized using
the Odyssey Fluorescence Scanner (LI-COR Bioscience, Inc.,
Lincoln, NE, USA). The StAR (ab133657; 1 : 3000), HSD3B1
(ab65156; 1 : 1000) and CYP11A1 (BS6578; 1 : 1000) antibodies
were purchased from Abcam (Cambridge, MA) and Bioworld
Technology, Inc (St. Paul, MN). LC3A/B (12741; 1 : 3000) and
Atg5 (12994; 1 : 3000) antibodies were purchased from Cell
Signaling Technology (Danvers, MA). The b-actin and GAPDH
antibody (1 : 10 000) was purchased from Sigma (St Louis, MO).
The secondary antibody was conjugated with IRDye 680/800
(Millennium Science; 926-32221, 926-32210; 1 : 10 000).
Testosterone assays
TM3 cells were seeded in 24-well plates at a density of 1.0 ꢃ 105
cells per well and incubated overnight. The culture medium was
then replaced with fresh medium containing various
compounds (20 mM) respectively. Aer incubation for 24 h, the
culture medium was collected and centrifuged at 1000 ꢃ g for
5 min. Testosterone concentrations in the supernatant were
determined with enzyme-linked immunosorbent assay (EISA)
kits (Shanghai XinFan Biological Technology Co Ltd, Shanghai,
China).
Cell lines and culture conditions
The cell lines used in this study were obtained from the Amer-
ican Type Culture Collection (ATCC). NCM460, L-02 and PTN1A
cell lines were cultured in RPMI 1640 medium containing 10%
foetal bovine serum (FBS), and TM3 mouse Leydig cell line was
cultured in Dulbecco's modied eagle's medium (DMEM)/F12 Quantitative real-time polymerase chain reaction (qRT-PCR)
medium, which was supplemented with 2.5% FBS and 5%
Total RNA was extracted from cells with TRIZOL reagent (Invi-
horse serum. Moreover, HAF cell line was cultured in DMEM
trogen, Carlsbad, CA, USA) according to the manufacturer's
medium with 1% glutamine and 10% FBS. All cells were incu-
protocols. Total RNA (1 mg) was converted to cDNA using a Pri-
meScript reverse transcription Master Mix kit (TaKaRa, Japan)
ꢁ
bated at 37 C and 5% CO2 incubator.
according to the manufacturer's instructions. Quantitative RT-
PCR was performed using SYBR Premix Ex Taq™ Kit (TaKaRa,
Cell viability assay
The cell viability of cell lines in the presence of this series of Japan). The relative expression of StAR, 3b-HSD and CYP11A1
compounds was determined by SRB (Sigma Aldrich) assay. In were analyzed by RT-PCR with b-actin as an internal control.
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Three independent experiments were carried out in triplicate. Basic
Research
Development
Program
of
China
The gene-specic primers are listed in Table S1.†
(2015CB910400).
Statistical analysis
Grouped data are expressed as mean ꢂ s.d. Signicance References
between groups was analysed by one-way analysis of variance or
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Natural product-like
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CYP11A1 Cytochrome P450 family 11 subfamily A member 1
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Testosterone replacement therapy
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Enzyme-linked immuno sorbent assay
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Light chain 3
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