Synthesis and evaluation of novel D-ring substituted steroidal pyrazolines as potential anti-inflammatory agents

Article abstract of DOI:10.1016/j.steroids.2019.03.012

To identify new potential anti-inflammatory agents, a number of novel steroidal derivatives with nitrogen heterocyclic side chains 4a-4l were synthesized and evaluated for their anti-inflammatory effects in activated RAW 264.7 macrophage cells. The synthe

Full text of DOI:10.1016/j.steroids.2019.03.012

Accepted Manuscript
Synthesis and Evaluation of Novel D-ring Substituted Steroidal Pyrazolines as
Potential Anti-inflammatory Agents
Xiaorui Cai, Shulin Zhao, De Cai, Jinhong Zheng, Zhiwei Zhu, Duncan Wei,
Zhiwei Zheng, Huide Zhu, Yicun Chen
PII:
S0039-128X(19)30063-7
https://doi.org/10.1016/j.steroids.2019.03.012
STE 8393
DOI:
Reference:
Steroids
To appear in:
Received Date:
Revised Date:
Accepted Date:
12 January 2019
6 March 2019
27 March 2019
Please cite this article as: Cai, X., Zhao, S., Cai, D., Zheng, J., Zhu, Z., Wei, D., Zheng, Z., Zhu, H., Chen, Y.,
Synthesis and Evaluation of Novel D-ring Substituted Steroidal Pyrazolines as Potential Anti-inflammatory Agents,
Steroids (2019), doi: https://doi.org/10.1016/j.steroids.2019.03.012
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Synthesis and Evaluation of Novel D-ring Substituted Steroidal
Pyrazolines as Potential Anti-inflammatory Agents
Xiaorui Caia, Shulin Zhaoa, De Caib, Jinhong Zhengc, Zhiwei Zhua, Duncan Weib, Zhiwei
Zhenga, Huide Zhua, Yicun Chend,*
a Department of Pharmacy, The Affiliated Tumor Hospital of Shantou University Medical College, Shantou
515041, Guangdong, China
b Department of Pharmacy, The First Affiliated Hospital of Shantou University Medical College, Shantou
515041, Guangdong, China
c Department of Chemistry, Shantou University Medical College, Shantou 515041, Guangdong, China
d Department of Pharmacology, Shantou University Medical College, Shantou 515041, Guangdong, China
Xiaorui Cai and Shulin Zhao contributed equally to this article
* Corresponding author.
E-mail address: chenyicun@yeah.net (Y.Chen.).
A B S T R A C T
To identify new potential anti-inflammatory agents, a number of novel steroidal derivatives with nitrogen
heterocyclic side chains 4a-4l were synthesized and evaluated for their anti-inflammatory effects in activated
RAW 264.7 macrophage cells. The synthesis scheme involves two steps, Claisen-Schmidt condensation with the
corresponding pregnenolone and aromatic aldehydes as the first step followed by nucleophilic addition of
thiosemicarbazide across an α, β-unsaturated carbonyl as a later step. Compound structures were confirmed by
1H-NMR, 13C-NMR, HRMS, and IR. The compounds were assayed to test their anti-inflammatory effects in
activated RAW 264.7 cells. Compound 4g, 3β-hydroxy-pregn-5-en-17β-yl-5’-(m-fluorophenyl)-4’,
5’-dihydro-1’-carbothioic acid amido pyrazole , was identified as the most potent anti-inflammatory agent of the
analysed compounds, with an IC50 value of 0.86 µM on nitric oxide (NO) production in lipopolysaccharide
(LPS)-induced RAW 264.7 cells for 24 h compared to dexamethasone (IC50 = 0.62 µM) and low cytotoxicity
against RAW 264.7 cells. Compound 4g significantly inhibited NO produced by LPS-induced RAW 264.7 cells.
Further studies showed that compound 4g markedly inhibited the expression of pro-inflammatory factors,
including inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α) , and
cyclooxygenase-2 (COX-2) in LPS-induced RAW 264.7 cells. These results indicate that derivatives bearing
pyrazoline structure might be considered for further research and scaffold optimization in designing
anti-inflammatory drugs and compound 4g might be a promising therapeutic anti-inflammatory drug candidate.
Keywords: pyrazoline; pregnenolone; anti-inflammatory activity; RAW 264.7 cells
1

1. Introduction
Inflammatory diseases are widely prevalent throughout the world. Drugs used for the treatment of acute and
chronic inflammatory disorders suppress biological processes that contribute to the onset and symptoms of
inflammation [1, 2].
Glucocorticoids (GCs) represent the most important and frequently used class of anti-inflammatory drugs.
Glucocorticoids have been on the market for more than 60 years and represent the most effective therapy for both
acute and chronic inflammatory conditions, including allergic and non-allergic diseases such as asthma, chronic
obstructive pulmonary disease (COPD), rheumatoid arthritis and other systemic diseases, as well as in
allotransplantation. The therapeutic usage of GCs has risen continuously in recent years [3]. Although currently
marketed glucocorticoids (e.g., prednisolone, dexamethasone, budesonide, mometasone furoate) are very effective
for the treatment of inflammatory conditions, the chronic use of the anti-inflammatory drugs is limited due to their
severe side effects such as diabetes, gastrointestinal perforation, peptic ulceration, osteoporosis,
hypothalamic-pituitary-adrenal (HPA) axis suppression and glucose intolerance [4].
Generally, glucocorticoids are involved in two major gene transcription pathways, transrepression and
transactivation [3]. There is consensus that transrepression is the interaction of the glucocorticoid receptor-steroid
monomer complex with transcription factors such as AP-1 and NFκ-B and is believed to be the major contributor
to the anti-inflammatory activity of glucocorticoids. However, most of the side effects are due to the interaction of
glucocorticoid receptor-steroid dimer complex with DNA, predominantly mediated via transactivation (e.g.,
diabetes, glaucoma), while others are predominantly mediated via transrepression (e.g., suppression of the
hypothalamic-pituitary-adrenal axis). For a considerable number of side effects, the precise molecular mode either
is unknown or appears to involve both transactivation and transrepression (e.g., osteoporosis).
Steroids are an important group of compounds exhibiting important and diverse biological, chemical and
pharmaceutical applications. These compounds are extensively available in animals and plants. Many steroids have
been reported to display various bioactivities and great application potential. In particular, some steroids have been
used in both preclinical and clinical studies [5]. Recent years have seen an extensive focus of research directed
towards the rational modification of steroid molecules. This increased focus is probably because of the various
advantages associated with steroid-based chemotherapeutics [6]. Steroid-based chemotherapeutic drugs possess
various advantages such as low toxicity, less vulnerability to multi-drug resistance (MDR) and high bioavailability.
These properties may be because these drugs are capable of penetrating the cell wall and undergoing a predictable
and controllable one- or two-step transformation in vivo back to the inactive metabolite via known processes of
enzymatic deactivation [7]. Moreover, several studies have revealed that steroidal molecules incorporating
heteroatoms (N, O or S) into the A-and D-ring showed many different biological activities such as antitumour
[8-10], antimicrobial [11,12], anti-inflammatory [13], anti-mycobacterial [14], and hypotensive activities [15]. As
a result, a number of different heterocyclic systems have been introduced into the steroidal backbone with
pyrazoles, pyrazolines, isoxazoles, isoxazolines, thiazoles, thiadiazoles, pyridines, pyrimidines, imidazoles, etc.
Nitrogen-containing steroidal compounds have been well studied due to their less toxic effects; these
compounds possess a broad spectrum of biological activities such as antitumour [16-18], anti-inflammatory [19],
anti-mycobacterial [20], anti-HIV [21], anti-parasitic [22], antimalarial [23], 5α-reductase inhibition [24] and
antimicrobial [25,26] effects. The presence of the element nitrogen in multiple pharmacological agents has made it
an indispensable anchor for the design and development of new pharmacological agents. For example,
N-heterocycle-containing abiraterone (1) has been used for the treatment of advanced prostate cancer in the clinic
[27]; VN/124-1 galeterone (2) has successfully completed phase II clinical development for prostate cancer
treatment [28]; and VN/85-1 (3) reduces circulating androgen levels through inhibition of CYP17 [29] (Fig. 1).
2

At present, the antitumour properties of nitrogen-containing steroidal compounds have been well researched,
while the anti-inflammatory activities have been barely studied. This study provided a novel, efficient and simple
synthesis of D-ring pyrazoline derivatives of 20-keto pregnenanes and their anti-inflammatory biological activities
were evaluated preliminarily. Pregnenolone is an active ingredient in many traditional Chinese medicines with
good anti-inflammatory activity, such as Rhizoma ligustici wallichii, which shows great effects as an
anti-inflammatory and analgesic agent [30]. Therefore, we chose pregnenolone as the main synthetic substrate. In
this study, we successfully applied aldol condensations and cycloaddition to pregnenolone to produce a new family
of N-heterocycle-containing sterol derivatives 4a-4l. The anti-inflammatory capacities of these synthetic
compounds and their underlying mechanisms were assessed in RAW 264.7 macrophages cells.
Fig. 1 Structures of nitrogen-containing steroidal compounds
2. Experimental
2.1. Chemistry
2.1.1. Reagent and instrument
All reagents were purchased from commercial suppliers. Melting points were determined using an
Electrothermal 9100 melting point apparatus (Weiss-Gallenkamp, Loughborough, UK). Thin-layer
chromatography (TLC) was performed on silica gel F254 plates with visualization by UV light or iodine vapour.
IR spectra were recorded on an Is5 FT-IR spectrophotometer (Nicolet, Thermo, Waltham, MA, USA) as KBr
pellets or thin films. 1H-NMR spectra of CDCl3 (or DMSO-d6) solutions (tetramethylsilane as an internal standard)
were recorded on an AVANCE II-600 (600 MHz) spectrometer (Bruker, Karlsruhe, Germany). Mass spectra were
recorded on a SCIEX Triple Quad 6500+ LC/MS/MS system (SCIEX, Los Angeles, CA, USA). HRMS
(high-resolution mass spectrometry) was performed on a Q ExactiveTM system (Thermo ScientificTM, Waltham,
MA, USA). 13C-NMR spectra were measured at 150 MHz on a Bruker AVANCE III spectrometer. Chalcones were
prepared from substituted benzaldehyde and pregnenolone according to the procedure reported in the literature.
2.1.2. Chemical methods
2.1.2.1. General Synthesis of 3β-hydroxy-pregn-5-en-17β-yl-3’-phenylprop-2’-en-1’-one derivatives (3a–3l)
To a solution of pregnenolone 1 (3.16 g, 0.01 mol) in ethanol (30 ml) was added a conc. aq. solution of
NaOH (0.025 mol). Then, aldehyde 2 (0.012 mol) was charged into the reaction mixture and stirred at room
temperature for 48 h to obtain the corresponding benzylidine derivative 3. The progress of the reaction was
monitored by TLC (petroleum ether: ethyl acetate (2:1 v/v) as eluents). After completion, the reaction mixture
was poured into crushed ice water. The precipitate was filtered, dried and recrystallized from ethanol to give the
product as a solid white powder. It should be mentioned that when non-aromatic aldehydes were used, the
product was formed in a very minor quantity and was not sufficiently stable at ambient conditions. Thus, the
study was restricted to the use of aromatic aldehydes only, for which the obtained yields were 82–90%.
2.1.2.2.
General Procedure for the Synthesis of
3β-hydroxy-pregn-5-en-17β-yl-5’-phenyl-4’,
5’-dihydro-1’-carbothioic acid amidopyrazole derivatives (4a-4l)
A mixture of thiosemicarbazide (0.254 g, 0.02 mol), 3a-3l (0.01 mol) and sodium hydroxide (0.025 mol)
was added to ethanol (25 mL). The mixture was refluxed for 12 h at room temperature, and after completion of
3

the reaction, the solution was poured into ice water. The resulting precipitate was filtered and recrystallized from
absolute ethanol. Quantitative yields of 79–87% were obtained for compounds 4a–4l.
2.1.2.2.1 3β-hydroxy-pregn-5-en-17β-yl-5’-(p-benzyloxyphenyl)-4’,5’-dihydro-1’-carbothioic acid amido pyrazole
(4a).White solid powder (Yield 85.19%), M.p.: 139.4-141.0℃. IR (KBr, cm-1): 3447.20 (N-H), 3397 (N-H),
1
1613.88, 1585.50, 1241.08 (C=S), 1354.75 (C-H), 1044.59 (C-O), 1025.89 (C-O). H-NMR（600 M Hz, δ ppm,
CDCl3）： 7.29-7.41 (m, 5H, Ar-H), 7.03-7.12（m, 2H, Ar-H）, 6.79-7.00（m, 3H）, 5.93(s, 1H, exchangeable with
D2O, NH2), 5.80 (q, 1H, C5’-H), 5.35 (s, 1H, C6-H), 5.03 (s, 2H), 3.52 (m, 1H, C3α-H), 3.40 (m, 1H, C4’-H2), 2.74
(m, 1H, C4’-H2), 2.35 (m, 1H, C17α-H), 1.00 (s, 3H, C10-CH3), 0.63 (s, 3H, C13-CH3). 13C-NMR (ppm,
CDCl3):176.41, 162.43, 158.14, 140.83, 136.99, 134.44, 128.58(2C), 127.98(2C), 127.53, 126.55(2C), 121.29,
115.12(2C), 71.67, 70.06, 62.30, 56.48, 52.10, 51.82, 50.03, 46.89, 44.19, 42.24, 38.40, 37.26, 36.54, 32.04, 31.66,
24.64, 24.40, 20.95, 19.42, 13.37. MS (ESI) (m/z): 584.3 [M + H]+. HRMS (EI): m/z [M + Na]+ calcd for
C36H45N3O2S: 606.3130; found: 606.3112.
2.1.2.2.2 3β-hydroxy-pregn-5-en-17β-yl-5’-(p-methoxyphenyl)-4’,5’-dihydro-1’-carbothioic acid amido pyrazole
(4b). Yellow solid (Yield 80.88%), M.p.: 248.0-250.0℃. IR (KBr, cm-1): 3473.02 (N-H), 3351.78 (N-H), 1612.03,
1
1579.93, 1243.51 (C=S), 1353.42 (C-H), 1067.86 (C-O), 1026.07 (C-O). H-NMR (600 MHz, δ ppm, CDCl3):
7.07（d, 2H, J=6.0 Hz, Ar-H）, 6.90 (s, 1H, exchangeable with D2O, NH2), 6.85 (d, 2H, J=6.0 Hz, Ar-H), 5.95 (s,
1H, exchangeable with D2O, NH2), 5.80 (q, 1H, C5’-H), 5.35 (s, 1H, C6-H), 3.78 (s, 3H, C4’’-OCH3), 3.52 (m, 1H,
C3α-H), 3.45 (m, 1H, C4’-H2), 2.68 (m, 1H, C4’-H2), 2.36 (m, 1H, C17α-H), 1.01 (s, 3H, C10-CH3), 0.63 (s, 3H,
C13-CH3). 13C-NMR (ppm, CDCl3): 176.17, 162.53, 158.87, 140.85, 133.99, 126.53(2C), 121.27, 114.21(2C),
71.67, 62.37, 56.42, 55.25, 52.10, 50.04, 46.77, 44.23, 42.24, 38.34, 37.27, 36.55, 32.02, 31.71, 31.61, 24.44,
24.40, 20.93, 19.43, 13.24. MS (ESI) (m/z): 508.3 [M + H]+. HRMS (EI): m/z [M + Na]+ calcd for C30H41N3O2S:
530.2817; found: 530.2797.
2.1.2.2.3 3β-hydroxy-pregn-5-en-17β-yl-5’-p-tolyl-4’,5’-dihydro-1’-carbothioic acid amido pyrazole (4c).White
solid powder (Yield 79.76%), M.p.: 234.8-235.0℃. IR (KBr, cm-1): 3471.72 (N-H), 3345.43 (N-H), 1617.93,
1579.42, 1236.03 (C=S), 1348.31 (C-H), 1068.30 (C-O). 1H-NMR (600 MHz, δ ppm, CDCl3): 7.13 (d, 2H, J=12.0
Hz, Ar-H), 7.03 (d, 2H, J=6.0 Hz, Ar-H), 6.91 (s, 1H, exchangeable with D2O, NH2), 6.01 (s, 1H, exchangeable
with D2O, NH2), 5.80 (q, 1H, C5’-H), 5.35 (s, 1H, C6-H), 3.51 (m, 1H, C3α-H), 3.35 (m, 1H, C4’-H2), 2.79 (m, 1H,
C4’-H2), 2.32 (s, 3H, C4’’-CH3), 2.29 (m, 1H, C17α-H), 0.98 (s, 3H, C10-CH3), 0.63 (s, 3H, C13-CH3). 13C-NMR
(ppm, CDCl3):176.40, 162.23, 140.84, 139.26, 136.98, 129.57(2C), 125.12(2C), 121.27, 71.64, 62.51, 56.53, 51.81,
50.00, 47.01, 44.14, 42.22, 38.45, 37.25, 36.53, 32.04, 31.70, 31.58, 24.64, 24.34, 21.16, 20.95, 19.40, 13.44. MS
(ESI) (m/z): 492.3 [M + H]+. HRMS (EI): m/z [M + Na]+ calcd for C30H41N3OS: 514.2868; found: 514.2844.
2.1.2.2.4 3β-hydroxy-pregn-5-en-17β-yl-5’-(o,p-dichlorophenyl)-4’,5’-dihydro-1’-carbothioic acid amido pyrazole
(4d). Yellow solid powder (Yield 82.20%), M.p.: 222.7-224.1℃. IR (KBr, cm-1): 3442.66 (N-H), 3347.11 (N-H),
1575.74, 1268.76 (C=S), 1350.28 (C-H), 1049.21 (C-O). 1H-NMR (600 MHz, δ ppm, CDCl3): 7.41 (s, 1H, Ar-H),
7.21 (d, 1H, J=6.0 Hz, Ar-H), 6.98 (s, 1H, exchangeable with D2O, NH2), 6.94 (d, 1H, J=6.0 Hz, Ar-H), 6.12 (s,
1H, exchangeable with D2O,NH2), 6.09 (q, 1H, C5’-H), 5.35 (s, 1H, C6-H), 3.56 (m, 1H, C3α-H), 3.50 (m, 1H,
C4’-H2), 2.58 (m, 1H, C4’-H2), 2.32 (m, 1H, C17α-H), 1.01 (s, 3H, C10-CH3), 0.61 (s, 3H, C13-CH3). 13C-NMR
(ppm, CDCl3):176.50, 162.74, 140.86, 137.73, 133.73, 131.95, 129.85, 127.55, 126.92, 121.22, 71.63, 60.30, 56.43,
51.94, 51.70, 49.95, 45.45, 44.32, 44.15, 42.20, 38.48, 38.32, 37.26, 36.53, 32.00, 31.63, 24.42, 20.96, 19.39,
13.50. MS (ESI) (m/z): 546.2, 548.2 [M + H]+. 568.2, 570.2 [M + Na]+. HRMS (EI): m/z [M + Na]+ calcd for
C29H37Cl2N3OS: 568.1932; found: 568.1916.
4

2.1.2.2.5 3β-hydroxy-pregn-5-en-17β-yl-5’-p-benzamidol-4’,5’-dihydro-1’-carbothioic acid amido pyrazole
(4e).White solid (Yield 87.12%), M.p.: 216.2-219.2℃. IR (KBr, cm-1): 3448.66 (N-H), 3326.35 (N-H), 1663.99
(C=O), 1616.86, 1593.81, 1236.03 (C=S), 1355.07 (C-H), 1043.42 (C-O). 1H-NMR (600 MHz, δ ppm, DMSO-d6):
7.92 (s, 1H, NH2), 7.88 (s, 1H, NH2), 7.81 (d, 2H, J=6.0 Hz, Ar-H), 7.46 (s, 1H, NH2), 7.32(s, 1H, NH2), 7.12 (d,
2H, J=6.0 Hz, Ar-H）, 5.80 (q, 1H, C5’-H), 5.26 (s, 1H, C6-H), 3.53 (m, 1H, C4’-H2), 3.25 (m, 1H, C3α-H), 2.58 (m,
1H, C4’-H2), 2.34 (m, 1H, C17α-H), 0.90 (s, 3H, C10-CH3), 0.52 (s, 3H, C13-CH3). 13C-NMR (ppm, CDCl3):175.74,
167.62, 161.20, 146.34, 141.27, 132.86, 127.75(2C), 124.92(2C), 124.78, 120.18, 69.95, 61.71, 55.75, 50.79,
49.49, 46.04, 43.37, 42.16, 37.75, 36.88, 36.06, 31.55, 31.36, 31.17, 23.96, 20.50, 19.08, 13.20. MS (ESI) (m/z):
521.3 [M + H]+. HRMS (EI): m/z [M + Na]+ calcd for C30H40N4O2S: 543.2770; found: 543.2748.
2.1.2.2.6 3β-hydroxy-pregn-5-en-17β-yl-5’-(p-(methylthio) phenyl)-4’,5’-dihydro-1’-carbothioic acid amido
pyrazole (4f). Yellow solid (Yield 84.26%), M.p.: 251.5-251.7℃. IR (KBr, cm-1): 3453.42 (N-H), 3340.66 (N-H),
1614.76, 1579.15, 1244.96 (C=S), 1353.68 (C-H), 1066.77 (C-O). 1H-NMR (600 MHz, δ ppm, CDCl3): 7.20-7.22
（d, 2H, J=12.0 Hz, Ar-H), 7.06-7.07 (d, 2H, J=6.0 Hz, Ar-H), 6.92 (s, 1H, exchangeable with D2O, NH2), 5.94 (s,
1H, exchangeable with D2O, NH2), 5.81 (q, 1H, C5’-H), 5.35(s, 1H, C6-H), 3.52(m, 1H, C3α-H), 3.45 (m, 1H,
C4’-H2), 2.67 (m, 1H, C4’-H2), 2.46 (s, 3H, C4’’-SCH3), 2.35 (m, 1H, C17α-H), 1.01 (s, 3H, C10-CH3), 0.63 (s, 3H,
C13-CH3). 13C-NMR (ppm, CDCl3):176.23, 162.47, 140.84, 138.73, 137.60, 127.08(2C), 125.82(2C), 121.27,
71.68, 62.47, 56.41, 52.07, 50.03, 46.66, 44.25, 42.24, 38.34, 37.27, 36.55, 32.02, 31.70, 31.61, 24.45, 24.40,
20.93, 19.43, 15.85, 13.27. MS (ESI) (m/z): 524.3 [M + H]+. HRMS (EI): m/z [M + Na]+ calcd for C30H41N3OS2:
546.2589; found: 546.2568.
2.1.2.2.7 3β-hydroxy-pregn-5-en-17β-yl-5’-(m-fluorophenyl)-4’,5’-dihydro-1’-carbothioic acid amido pyrazole
(4g). White solid powder (Yield 85.42%), M.p.: 237.6-239.2℃. IR (KBr, cm-1): 3469.34 (N-H), 3334.02 (N-H),
1588.23, 1250.91 (C=S), 1357.13 (C-H), 1056.15 (C-O). 1H-NMR (600 MHz, δ ppm, CDCl3): 7.30 (m, 1H, Ar-H),
6.87-7.03 (m, 3H), 6.83 (d, 1H, J=12.0 Hz, Ar-H), 6.01 (s, 1H, exchangeable with D2O, NH2), 5.85 (q, 1H, C5’-H),
5.35 (s, 1H, C6-H), 3.52 (m, 1H, C3α-H), 3.47 (m, 1H, C4’-H2), 2.67(m, 1H, C4’-H2), 2.35 (m, 1H, C17α-H), 1.01 (s,
3H, C10-CH3), 0.63 (s, 3H, C13-CH3). 13C-NMR (ppm, CDCl3): 176.30, 162.36, 140.85, 130.51, 130.46, 121.24,
120.93, 114.53, 114.39, 112.35, 112.20, 71.64, 62.36, 56.40, 52.00, 50.01, 46.57, 44.27, 42.21, 38.33, 37.26, 36.54,
32.01, 31.69, 31.58, 24.44, 24.40, 20.92, 19.42, 13.25. MS (ESI) (m/z): 496.3, 497.3[M + H]+. HRMS (EI): m/z [M
+ Na]+ calcd for C29H38FN3OS: 518.2617; found: 518.2608.
2.1.2.2.8 3β-hydroxy-pregn-5-en-17β-yl-5’-(o-chlorophenyl)-4’,5’-dihydro-1’-carbothioic acid amido pyrazole
(4h). Light Yellow solid (Yield 86.87%), M.p.: 217.1-218.9℃. IR (KBr, cm-1): 3449.59 (N-H), 3377.31 (N-H),
1573.63, 1236.25 (C=S), 1351.31 (C-H), 1049.16 (C-O). 1H-NMR (600 MHz, δ ppm, CDCl3): 7.39 (m, 1H, Ar-H),
7.20-7.24 (m, 2H, Ar-H), 7.00 (d, 1H, J=6.0 Hz, Ar-H), 6.93 (s, 1H, exchangeable with D2O, NH2), 6.14 (q, 1H,
C5’-H), 6.05 (s, 1H, exchangeable with D2O, NH2), 5.35 (s, 1H, C6-H), 3.53 (m, 1H, C4’-H2), 3.50 (m, 1H, C3α-H),
2.62 (m, 1H, C4’-H2), 2.32 (m, 1H, C17α-H), 1.01 (s, 3H, C10-CH3), 0.61 (s, 3H, C13-CH3). 13C-NMR (ppm,
CDCl3):176.35, 162.83, 140.82, 138.66, 131.26, 129.99, 128.68, 127.18, 125.82, 121.24, 71.65, 60.71, 56.48,
51.97, 51.72, 49.97, 45.62, 44.21, 42.22, 38.46, 37.26, 36.55, 32.01, 31.65, 24.57, 24.34, 20.94, 19.38, 13.30. MS
(ESI) (m/z): 512.3, 514.2 [M + H]+. HRMS (EI): m/z [M + Na]+ calcd for C29H38ClN3OS: 534.2322; found:
534.2303.
5

2.1.2.2.9 3β-hydroxy-pregn-5-en-17β-yl-5’-(m-chlorophenyl)-4’,5’-dihydro-1’-carbothioic acid amido pyrazole
(4i). White solid powder (Yield 83.20%), M.p.: 248.5-251.5℃. IR (KBr, cm-1): 3473.21 (N-H), 3339.69 (N-H),
1
1584.42, 1247.23 (C=S), 1357.34 (C-H), 1055.48 (C-O). H-NMR(600 MHz, δ ppm, CDCl3): 7.20-7.25 (m, 2H,
Ar-H), 7.12 (s, 1H, Ar-H), 7.04 (d, 1H, J=6.0 Hz, Ar-H), 6.94 (s, 1H, exchangeable with D2O, NH2), 6.01(s, 1H,
exchangeable with D2O, NH2), 5.83 (q, 1H, C5’-H), 5.35 (s, 1H, C6-H), 3.52 (m, 1H, C3α-H), 3.47 (m, 1H, C4’-H2),
2.67 (m, 1H, C4’-H2), 2.35 (m, 1H, C17α-H), 1.01 (s, 3H, C10-CH3), 0.63 (s, 3H, C13-CH3). 13C-NMR (ppm,
CDCl3):176.36, 162.31, 143.83, 140.84, 134.75, 130.17, 127.74, 125.45, 123.53, 121.26, 71.67, 62.35, 56.41,
52.02, 50.01, 46.57, 44.27, 42.23, 38.36, 37.26, 36.55, 32.02, 31.69, 31.60, 24.44, 24.40, 20.92, 19.42, 13.26. MS
(ESI) (m/z): 512.3, 514.2 [M + H]+. HRMS (EI): m/z [M + Na]+ calcd for C29H38ClN3OS: 534.2322; found:
534.2301.
2.1.2.2.10 3β-hydroxy-pregn-5-en-17β-yl-5’-o-tolyl-4’,5’-dihydro-1’-carbothioic acid amido pyrazole (4j).Light
yellow solid (Yield 85.13%), M.p.: 279.1-279.4℃. IR (KBr, cm-1): 3479.65 (N-H), 3348.35 (N-H), 1619.83,
1
1560.88, 1277.69 (C=S), 1351.46 (C-H), 1055.77 (C-O). H-NMR (600 MHz, δ ppm, CDCl3): 7.14-7.20 (m, 3H,
Ar-H), 6.95 (s, 1H, exchangeable with D2O, NH2), 6.90 (m, 1H, Ar-H), 5.96 (q, 1H, C5’-H), 5.92 (s, 1H,
exchangeable with D2O, NH2), 5.35 (s, 1H, C6-H), 3.51 (m, 1H, C3α-H), 3.37 (m, 1H, C4’-H2), 2.67 (m, 1H,
C4’-H2), 2.40 (s, 3H, C2’’-CH3), 2.29 (m, 1H, C17α-H), 0.97 (s, 3H, C10-CH3), 0.62 (s, 3H, C13-CH3). 13C-NMR
(ppm, CDCl3):176.28, 162.29, 140.82, 140.22, 133.54, 130.69, 127.28, 126.62, 123.51, 121.28, 71.67, 59.91, 56.53,
51.83, 50.01, 45.85, 44.15, 42.23, 38.46, 37.25, 36.52, 32.03, 31.70, 31.59, 24.64, 24.34, 20.93, 19.52, 19.38,
13.38. MS (ESI) (m/z): 492.3 [M + H]+. HRMS (EI): m/z [M + Na]+ calcd for C30H41N3OS: 514.2868; found:
514.2849.
2.1.2.2.11 3β-hydroxy-pregn-5-en-17β-yl-5’-(o-methoxyphenyl)-4’,5’-dihydro-1’-carbothioic acid amido pyrazole
(4k). Yellow solid (Yield 86.38%), M.p.: 238.8-239.1℃. IR (KBr, cm-1): 3485.57 (N-H), 3341.24 (N-H), 1607.93,
1
1578.14, 1238.73 (C=S), 1347.51 (C-H), 1073.58 (C-O), 1049.12 (C-O). H-NMR (600 MHz, δ ppm, CDCl3):
7.21-7.23 (m, 1H), 6.83-7.03 (m, 4H), 6.06 (q, 1H, C5’-H), 6.04 (s, 1H, exchangeable with D2O, NH2), 5.35 (s, 1H,
C6-H), 3.84 (s, 3H, C2’’-OCH3), 3.51 (m, 1H, C3α-H), 3.42 (m, 1H, C4’-H2), 2.57 (m, 1H, C4’-H2), 2.32 (m, 1H,
C17α-H), 1.00 (s, 3H, C10-CH3), 0.60 (s, 3H, C13-CH3). 13C-NMR (ppm, CDCl3):176.09, 163.43, 155.72, 140.86,
129.14, 128.52, 125.61, 121.28, 120.58, 110.92, 71.65, 58.80, 56.42, 55.54, 52.06, 50.04, 45.68, 44.16, 42.23,
38.32, 37.27, 36.55, 32.02, 31.71, 31.59, 24.41, 24.36, 20.91, 19.42, 13.14. MS (ESI) (m/z): 508.3 [M + H]+.
HRMS (EI): m/z [M + Na]+ calcd for C30H41N3O2S: 530.2817; found: 530.2796.
2.1.2.2.12 3β-hydroxy-pregn-5-en-17β-yl-5’-(o-fluorophenyl)-4’,5’-dihydro-1’-carbothioic acid amido pyrazole
(4l). Light yellow solid (Yield 84.36%), M.p.: 201.2-203.8℃. IR (KBr, cm-1): 3442.79 (N-H), 3339.37 (N-H),
1596.03, 1566.91, 1222.91 (C=S), 1355.49 (C-H), 1045.16 (C-O). 1H-NMR (600 MHz, δ ppm, CDCl3): 7.24（m,
1H), 7.03-7.14 (m, 3H), 6.95 (s, 1H, exchangeable with D2O, NH2), 6.05 (s, 1H, exchangeable with D2O, NH2),
6.03 (q, 1H, C5’-H), 5.35 (s, 1H, C6-H), 3.54 (m, 1H, C3α-H), 3.42 (m, 1H, C4’-H2), 2.70 (m, 1H, C4’-H2), 2.35 (m,
1H, C17α-H), 1.00 (s, 3H, C10-CH3), 0.63 (s, 3H, C13-CH3). 13C-NMR (ppm, CDCl3):176.37, 162.54, 140.88,
129.08, 128.75, 128.46, 127.10, 124.22, 121.29, 115.87, 115.73, 71.58, 57.66, 56.45, 51.96, 51.75, 50.00, 45.68,
44.17, 42.20, 38.35, 37.27, 36.52, 32.08, 31.64, 24.66, 24.44, 24.15, 20.95, 19.43, 13.25. MS (ESI) (m/z): 496.3,
497.3 [M + H]+. HRMS (EI): m/z [M + Na]+ calcd for C29H38FN3OS: 518.2617; found: 518.2599.
2.2. Biological methods
2.2.1. Cell Culture and Treatment
6

The RAW 264.7 cells (murine macrophage cell line) were purchased from Chinese Type Tissue Culture
Collection (CTCC, Shanghai, China) and cultivated in H-DMEM medium containing 10% heat-inactivated 10%
foetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified
atmosphere of 95% air and 5% CO2. Stock solutions of compounds and dexamethasone were prepared in
dimethyl sulfoxide (DMSO) and then added to fresh culture medium to obtain final concentrations. Unless stated
otherwise, RAW 264.7 cells were plated at 1×104 cells per well in 96-well plates and incubated overnight to allow
adherence to the plate prior to each of the experiments described below. LPS (Escherichia coli 0111:B4) was
purchased from Sigma (St. Louis, MO, USA).
2.2.2. MTT Assay for Cell Viability
An MTT assay was performed to evaluate the compounds’ cytotoxicity, 3 µM concentrations of each
compound were used to identify the more effective compounds for further study. Peritoneal macrophages (2.5×104
cells/mL) were incubated in 96-well plates containing H-DMEM supplemented for 24 h. At the end of the
incubation period, the medium was removed, and cells were treated with 200 µL test compound 4g per well. After
24-h incubation at 37 °C, RAW 264.7 cells were stimulated with 1 μg/mL LPS for 24 h. The control group
received an equal amount of H-DMEM supplement. Test compounds were added to the culture medium 1 h prior
to the addition of LPS. Twelve hours later, 20 μL 5 mg/mL MTT was added to each well. Cells were incubated at
37 °C. After 4 h, the MTT solution was removed, and DMSO was added to each well (150 μL) to lyse the
formazan dye. After 10 min of shaking, the absorbance was measured at 570 nm using a FilterMax F3/F5
Microplate Reader (Molecular Devices, Sydney, Australia). The percent viability was calculated using the
following formula: Cell viability (%) = [(absorbance of cells treated with testing compounds) / absorbance of
control cells] × 100%.
2.2.3. Determination of Nitric Oxide/Nitrite
Nitrite as the end-point of NO generation by activated macrophages was measured by determining the NO
concentration in the cell culture supernatant. RAW 264.7 cells were seeded in 96-well plates at a density of 1 × 104
cells/well. The cells were pretreated with different concentrations of test compounds 4a, 4b, 4e, 4g, 4h, 4i and
dexamethasone (final concentrations= 0.1 µM, 0.3 µM, 1.0 µM, 3.0 µM, 10 µM, 30 µM) dissolved in H-DMEM
for 60 min before being stimulated with LPS (1 μg/mL) for 24 h. The cell culture medium was collected, and the
concentration of nitrite was measured using the NO assay kit (Griess reagent) according to the manufacturer’s
instructions (Jiancheng Bioengineering Institute, Nanking, China). The absorbance at 540 nm was measured with a
FilterMax F3/F5 Microplate Reader (Molecular Devices, Sydney, Australia).
2.2.4. Determination of TNF-α, IL-6, and PGE-2
RAW 264.7 cells were seeded in 96-well plates at a density of 1 × 104 cells/well. The cells were pretreated
with different concentrations of compound 4g (final concentrations= 0.1 µM, 0.3 µM, 1.0 µM, 3.0 µM, 10 µM, 30
µM) dissolved in H-DMEM for 60 min before being stimulated with LPS (1 μg/mL) for 24 h. Supernatants were
collected to analyse the levels of TNF-α, IL-6, and PGE-2, three well-known pro-inflammatory cytokines prevalent
in many different inflammatory diseases. Cytokine concentrations in the medium were measured using an
enzyme-linked immunosorbent assay (ELISA) kit: PGE-2 ELISA Kit (Elabscience Biotechnology, Wuhan, China),
mouse IL-6 ELISA Kit (Elabscience Biotechnology, Wuhan, China), mouse TNF-α ELISA Kit (Elabscience
Biotechnology, Wuhan, China) according to the manufacturer's instructions. The cytokine concentration was
measured by a FilterMax F3/F5 Microplate Reader (Molecular Devices, Sydney, Australia) at 450 nm and
calculated from a standard curve prepared with the corresponding recombinant mouse cytokine.
2.2.5. Quantitative Real-Time Polymerase Chain Reaction.
RAW 264.7 cells were seeded in 6-well plates at a density of 1 × 106 cells/well. The cells were preincubated
with or without compound 4g (0.1, 1.0, 10 µM) for 60 min and then stimulated with LPS (1 μg/mL). After
7

stimulation with LPS for 6 h, the cells were harvested, and total RNA was extracted using TRIzol reagent
(Invitrogen, Shanghai, China). cDNA was prepared from total RNA (1 μg) using the High-capacity cDNA Reverse
Transcription kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instruction. Real-time
qRT-PCR was performed using SYBR Green Supermix reagent (Thermo Fisher Scientific, USA) in a 7500 Real
Time PCR system (Applied Biosystems, Foster City, CA, USA). The relative quantification of gene expression
was reported as a relative quantity to the control value by using 2-△△Ct methods. The specific primers used for PCR
are the following: iNOS forward, TCT GCG CCT TTG CTC ATG AC; iNOS reverse, TAA AGG CTC CGG GCT
CTG; TNF-α forward, TAC TGA ACT TCG GGG TGA TCG GTC C; TNF-α reverse, CAG CCT TGT CCC TTG
AAG AGA ACC; IL-6 forward, GTA CTC CAG AAG ACC AGA G; IL-6 reverse, TGC TGG TGA CAA CCA
CGG C; COX-2 forward, GGG AAG CCT TCT CCA ACC; COX-2 reverse, GAA CCC AGG TCC TCG CTT;
and GAPDH forward, GGT GAA GGT CGG TGT GAA CG; GAPDH reverse, CTC GCT CCT GGA AGA TGG
TG.
2.2.6. Western Blots.
RAW 264.7 cells were seeded in 6-well plates at a density of 1 × 106 cells/well. The cells were preincubated
with or without compound 4g (0.1, 1.0, 10 µM) for 60 min and then stimulated with LPS (1 μg/mL). After
stimulation with LPS for 24 h, total protein was isolated with RIPA protein lysis buffer. The cell lysates (40 μg)
were separated by SDS–PAGE, transferred to a nitrocellulose membrane and probed with anti-iNOS or COX-2
antibody. β-Actin was used as an internal control. The following primary and second antibodies were used for
western blot analysis: antibodies against iNOS and COX-2, β-actin and horseradish peroxidase-conjugated
secondary antibodies purchased from Abcam (Cambridge, MA).
2.2.7. Statistical analysis
The experimental values are presented as Mean±SD, and statistical analysis were conducted by Statistical
Product and Service Solutions (SPSS) 17.0 (SPSS Inc., Chicago, IL, USA) through one-way ANOVA detected by
SNK-LSD test as the post-hoc test. P<0.05 was considered statistically significant.
3. Results and discussion
3.1. Chemistry
The general procedure for the synthesis of pyrazoline derivatives was carried out according to the steps shown
in Scheme 1. First, chalcone derivatives 3 were prepared via a Claisen-Schmidt condensation reaction with
corresponding pregnenolone 1 and aromatic aldehydes 2 [6]. NaOH was used as a catalyst in the reaction to obtain
3a-3l. Their further condensation with thiosemicarbazides in alcoholic media led to the formation of compounds 4
(4a–4l). The structures of compounds 4a–4l were elucidated by 1H-NMR, 13C-NMR, HRMS and IR analyses.
8

Scheme 1. General synthesis of compounds 4a–4l.Reagents and Conditions: (I) pregnenolone (0.01 mol),
substituted benzaldehyde (0.012 mol), absolute ethanol, sodium hydroxide, room temperature 48 h, yield 82–90%;
(II) absolute ethanol, sodium hydroxide, thiosemicarbazide, reflux 12 h, yield 79-87%.
3.2. Effects of the Testing Compounds on NO Generation in LPS-induced RAW264.7 cells and Cell Viability
Various inflammatory mediators such as NO, PGE-2, TNF-α and IL-6 are well known for their involvement
in the development of inflammation. It has been reported that NO exerts multiple modulating effects on
inflammation [31]. To identify the most promising anti-inflammatory agent among the synthesized derivatives
(4a–4l), their inhibitory activities on NO generation effects were tested on LPS-treated RAW 264.7 cells;
dexamethasone was used as a positive control. The results showed that most of the novel synthetic compounds
exhibited modest to strong inhibitory activity on NO generation at 3.0 μM (Table 1). We found that compounds 4g
and 4h displayed IC50 values lower than 2.0 µM against LPS-treated RAW264.7 cells at 24 h. The most effective
agent was compound 4g, with an IC50 value of 0.86 µM at 24 h; dexamethasone, the positive control, had an IC50
value of 0.62 µM at 24 h (Table 1 and Fig. 2). The testing compounds also displayed dose-dependent trends.
Among the derivatives 4a-4l, when the substitutions of R2 and R3 were the same (4h, 4j, 4k, 4l) and R1 was Cl,
CH3, OCH3 and F, respectively, the inhibitory activity of these compounds with different substituents decreased in
the following order: 4h > 4l > 4k > 4j. When the substitutions of R1 and R3 were the same (4g, 4i) and R2 was
either F or Cl, compound 4g with R2 group F showed stronger activity than compounds with a Cl R2 group.
The results showed that compounds with an aromatic ring bond with electron-withdrawing groups exhibited
moderate to strong inhibitory activity (4g, 4h, 4i). The introduction of a F or Cl group at the R1, R2 or R3 position
resulted in an increased inhibitory potency. However, compounds with an aromatic ring bond with
electron-donating groups showed inferior inhibitory activity (4b, 4c, 4f, 4j, 4k).
In addition, the cytotoxicity of the compounds was determined by MTT assay to exclude the possibility that
the reduced release of NO was caused by cytotoxic effects of the compounds (Table 1). An MTT assay was
performed to assess cell viability for all compounds at 3.0 μM. Compounds 4d, 4h and 4i exerted obvious toxicity,
in which R1, R2, or R3 was a Cl group, respectively. In the case where the R1, R2, and R3 substituent was changed
from benzyloxy to OCH3, CH3, and SCH3, respectively, the cytotoxicity decreased (4a, 4b, 4c, 4f, 4j, 4k). Overall,
we found that compounds with aromatic ring bond with electron-withdrawing groups exhibited strong inhibitory
activity but also toxicity, and compounds with aromatic ring bonds with electron-donating groups showed inferior
inhibitory activity but low toxicity.
Compounds 4a, 4g and 4h, which strongly suppressed LPS-induced NO generation and had low cytotoxicity
in RAW 264.7 cells (cell viability >90%), were chosen for a concentration-dependent experiment in LPS-induced
9

RAW 264.7 cells. Compounds 4a, 4g and 4h were found to be the most potent, with IC50 values of 1.05, 0.86, and
1.02 µM, respectively.
In terms of its strong inhibitory activity on NO generation and minimal cytotoxicity to RAW 264.7 cells,
compound 4g was selected for further evaluation. The dose-response study showed that compound 4g significantly
inhibited LPS-induced NO production in a dose-dependent manner in RAW 264.7 cells (Fig. 2-B). Compound 4g
did not affect cell viability at the tested concentration.
Table 1
Inhibitory Activity of Compounds 4a−4l (3 μM) on NO Generation in LPS-Induced RAW 264.7 cells1
compounds
%inhibition
82.27±4.80
49.32±6.36
34.33±5.58
41.85±4.27
88.10±0.37
66.70±3.74
94.72±0.96
93.80±0.78
75.26±4.80
18.63±5.66
33.09±8.13
53.66±4.54
92.34±2.06
%cell viability
91.17±1.83
97.91±1.09
87.32±5.26
78.22±1.26
106.06±0.46
95.66±1.56
98.32±1.76
85.06±1.83
78.43±1.32
90.18±1.93
97.18±1.32
86.62±3.15
95.09±1.78
IC50 (µM)
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
1.05±5.24
3.17±7.66
-
-
2.95±4.33
-
0.86±4.17
1.02±3.84
2.53±4.33
-
-
-
0.62±4.17
dexamethasone
1Each experiment was independently performed three times.
“—”: Not determined.
Fig.2. Concentration-dependent inhibition of NO production by compounds 4a, 4g, 4h, and dexamethasone in
LPS-induced RAW 264.7 cells after 24 h. RAW 264.7 cells were pretreated with the compound for 60 min prior to
LPS (1 μg/mL) stimulation for 24 h. The nitrite levels in the cell culture medium were detected using Griess
reagents. Data shown are the mean ± SD for each group (n=3) and are representative of three independent
experiments.
3.3. Inhibitory Effects of Compound 4g on LPS-induced NO Production in RAW 264.7 Cells
10

Fig.3. The effect of compound 4g on NO production in LPS-induced RAW 264.7 cells. RAW 264.7 cells were
pretreated with various concentrations of compound 4g (0, 0.1, 0.3, 1, 3, 10 and 30 μM/L) for 60 min and then
were stimulated with LPS (1 μg/ mL) for 24 h. Data shown are the mean ± SD for each group (n = 3) and are
representative of three independent experiments (*P <0.05 and **P < 0.01 vs. the LPS group; ##P < 0.01 vs. the
control group), data were analysed by ANOVA and Duncan ’s multiple range tests.
To determine whether compound 4g inhibits LPS-induced NO production, the cells were pretreated for 60
min with different concentrations of compound 4g and then stimulated with 1 μg/mL LPS. NO production was
almost undetectable in cells without LPS stimulation (Fig. 3). LPS (1 μg/mL) alone markedly induced NO
production compared to the control group, while compound 4g significantly reduced the level of NO production in
LPS-induced macrophages. This result indicates that compound 4g maximally inhibits NO generation by activated
RAW 264.7 macrophages.
3.4. Cell Viability of Compound 4g
Fig.4. Cell viability of compound 4g in RAW 264.7 cells. Cells were incubated with the indicated concentrations of
compound 4g (0, 0.1, 0.3, 1, 3, 10 and 30 μM/L) for 60 min and then stimulated with LPS (1 μg/mL) for 24 h. The
results are expressed as a percentage of the respective control. The values are presented as the mean ± S.D. (n=3) of
three independent experiments. (*P <0.05 and **P < 0.01 vs. the LPS group; ##P < 0.01 vs. the control group).
An MTT assay was performed to assess cell viability. The potential cytotoxic effects of compound 4g were
evaluated in RAW 264.7 cells in the presence of LPS. As shown in Fig. 4, compared to the control cells, 24 h LPS
treatment significantly decreased cell viability. However, compound 4g increased the cell viability of macrophages
with LPS-stimulation at concentrations of 0.1–30 μM/L compared to the group receiving LPS treatment alone.
11

Thus, the MTT assay showed that anti-inflammatory compound 4g was not cytotoxic at the concentrations of 0.1–
10 μM/L, while showing some cytotoxicity at the concentrations of 30 μM/L.
3.5. Inhibitory Effects of Compound 4g on LPS-induced IL-6, PGE-2, and TNF-α Production in RAW 264.7 Cells
Fig.5. Effect of compound 4g on IL-6 (A), PGE-2(B) and TNF-α (C) release in LPS-induced RAW 264.7 cells.
RAW 264.7 cells were preincubated with different concentrations of compound 4g (0, 0.1, 0.3, 1, 3, 10, 30 μM/L) for
60 min prior to LPS stimulation. After treatment with LPS for 24 h, cell culture medium was collected, and ELISA
was performed to assess the levels of inflammatory cytokines. The values are presented as the mean ± S.D. (n = 3) of
three independent experiments. (*P <0.05 and **P < 0.01 vs. the LPS group; ##P < 0.01 vs. the control group).
To investigate the anti-inflammatory effects of compound 4g in LPS-induced RAW 264.7 cells, the three
target cytokines (TNF-α, IL-6, and PGE-2) levels were determined. LPS treatment alone resulted in a significant
increase in cytokine production compared with the control group in RAW 264.7 cells (Fig. 5). In contrast,
pretreatment with higher concentrations of compound 4g (10 and 30 μM) significantly decreased IL-6 level, more
so than treatment at lower concentrations (0.1 μM). However, incubation with all doses of compound 4g
effectively downregulated the production of TNF-α and PGE-2 in dose-dependent manners. These results indicate
that compound 4g has a significant anti-inflammatory activity and might be a promising agent for the therapeutic
treatment of inflammatory disorders.
3.6. Inhibitory Effects of Compound 4g on iNOS, TNF-α, IL-6 and COX-2 Expression at the Transcriptional Level
in LPS-Induced RAW 264.7 Cells
12

Fig.6. Effects of compound 4g on mRNA levels of iNOS, TNF-α, IL-6 and COX-2 in LPS-induced RAW264.7
cells. RAW 264.7 cells were preincubated with different concentrations of compound 4g (0, 0.1, 1, 10 μM/L) for 60
min prior to LPS stimulation. After treatment with LPS (1 μg/mL) for 6 h, the total RNA was isolated and the iNOS
(A), the mRNA expression for TNF-α (B), IL-6 (C) and COX-2 (D) were determined by quantitative RT-PCR. The
data are representative from three independent experiments. (*P <0.05 and **P < 0.01 vs. the LPS group; ##P < 0.01
vs. the control group).
Inflammatory cytokines, including iNOS, TNF-α, IL-6 and COX-2, are also associated with inflammatory
disorders. Therefore, we tested whether compound 4g inhibited the transcriptional expression of these cytokines
and inflammatory enzymes using quantitative real-time (RT)-PCR analysis. The result showed that compound 4g
significantly reduced the expression of the genes for iNOS, TNF-α, IL-6, and COX-2 (Fig. 6). We also determined
their protein levels in order to investigate this anti-inflammatory effect further. The significant increases in iNOS,
TNF-α, IL-6 and COX-2 protein levels observed in LPS-induced RAW264.7 cells were markedly reduced by
treatment with 1.0 or 10 μM/L compound 4g. These findings indicated that compound 4g exerted significant
anti-inflammatory effects in LPS-induced RAW264.7 cells by suppressing mRNA and protein expression of iNOS,
TNF-α, IL-6 and COX-2.
3.7. Inhibitory Effects of Compound 4g on iNOS and COX-2 Expression at the Protein Level in LPS-Induced RAW
264.7 Cells
Fig.7. Effects of compound 4g on LPS-induced expression of iNOS and COX-2 proteins (A, B). RAW 264.7 cells
were preincubated with different concentrations of compound 4g (0, 0.1, 1, 10 μM/L) for 60 min prior to LPS
stimulation. After treatment with LPS (1 μg/mL) for 24 h, total protein was extracted and subjected to western blot
analysis to detect the iNOS and COX-2 proteins. The data are representative from three independent experiments.
(*P <0.05 and **P < 0.01 vs. the LPS group; ##P < 0.01 vs. the control group).
Together with NO, PGE-2 is also a well-known inflammatory mediator. PGE-2 and NO are major regulators
in the inflammatory pathogenesis. Because COX-2 and iNOS are the key enzymes for the production of PGE-2
and NO [32,33], we detected the expression level of COX-2 and iNOS employing western blotting. As revealed in
Fig. 7, in response to LPS, the levels of both proteins exhibited upregulation. In contrast, pretreatment with
compound 4g considerably alleviated their expression. These findings were consistent with NO and PGE-2
measurement. Our data demonstrate that compound 4g exerted a protective effect via the protein levels of iNOS
and COX-2, which consequently resulted in the inhibition of NO and PGE-2 generation.
13

4. Conclusion
We efficiently synthesized a series of novel steroidal derivatives with pyrazoline 4a-4l and investigated their
anti-inflammatory effects on RAW 264.7 cells. We found that compound 4g exhibited the most potent activity in
the inhibition of NO production on RAW 264.7 cells for 24 h, with an IC50 value of 0.86 µM, comparable to that of
the positive control dexamethasone with an IC50 of 0.62 µM. Together with NO, PGE-2 is also a well-known
inflammatory mediator. The production of NO and PGE-2 is catalyzed by iNOS and COX-2, respectively, which
are key inflammatory enzymes in the bodies’ response to inflammatory processes. Likewise, inflammatory
cytokines, including IL-6 and TNF-α, are also secreted by activated RAW 264.7 cells, which ultimately contributes
to the progression of inflammation. Therefore, we tested whether compound 4g inhibited the transcriptional
expression of these cytokines and inflammatory enzymes using quantitative real-time (RT)-PCR analysis. The
result showed that compound 4g significantly inhibited the LPS-induced expression of the genes for iNOS, TNF-α,
IL-6, and COX-2 mRNA in a dose-dependent manner in RAW 264.7 cells. We further examined the suppressive
effects of compound 4g on the production of iNOS, COX-2, TNF-α, IL-6, and PGE-2 at the protein level using
western blot analysis or ELISA. Consistent with the quantitative RT-PCR data, compound 4g significantly reduced
the levels of these proteins in LPS-induced RAW 264.7 cells.
Moreover, MTT assays of compound 4g showed that it had low cytotoxic effects on RAW 264.7 cells. These
results provide an important foundation for further development of compound 4g as a potential anti-inflammatory
agent. We found that compounds with a nitrogen heterocyclic ring containing electron-withdrawing groups such as
F and Cl exhibited better pharmacological activity than most other derivatives containing electron-donating groups
(e.g., CH3, OCH3) synthesized in this study. Compounds 4h and 4i will be tested further to improve the
characterization of their anti-inflammatory activities. These results indicate that derivatives bearing pyrazoline
structure might be considered for further research and scaffold optimization in designing anti-inflammatory drugs,
compound 4g might be a promising therapeutic anti-inflammatory drug candidate.
Appendix A. Supplementary Materials: The following are available online.
Acknowledgements: This work was supported by a grant from Guangdong province Natural Science Foundation of
China (No. 2015A030313432), and the teamwork projects funded by Guangdong collaborative innovation and
environment construction Foundation of China (No. 2016B090918129) and Shantou Science and Technology
Project (No. [2015] 132).
Author Contributions: Xiaorui Cai and Yicun Chen designed the research; Xiaorui Cai performed the synthetic
work, Yicun Chen and De Cai were responsible for the direction of the biological research, Xiaorui Cai, Shulin
Zhao performed the anti-inflammatory activity. Xiaorui Cai wrote the manuscript, and Yicun Chen was responsible
for the correspondence of the manuscript. All authors discussed, edited and approved the final version.
Conflicts of Interest: The authors declare no conflict of interest.
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Cai et al., Supplementary Table 1
Table 1
Inhibitory activity of compounds 4a−4l (3 μM) on NO generation in LPS-induced RAW 264.7 cells1
%inhibition
%cell viability
IC50(µM)
compounds
82.27±4.80
91.17±1.83
1.05±5.24
4a
4b
4c
4d
4e
4f
49.32±6.36
34.33±5.58
41.85±4.27
88.10±0.37
66.70±3.74
94.72±0.96
93.80±0.78
75.26±4.80
18.63±5.66
33.09±8.13
53.66±4.54
92.34±2.06
97.91±1.09
87.32±5.26
78.22±1.26
106.06±0.46
95.66±1.56
98.32±1.76
85.06±1.83
78.43±1.32
90.18±1.93
97.18±1.32
86.62±3.15
95.09±1.78
3.17±7.66
-
-
2.95±4.33
-
4g
4h
4i
0.86±4.17
1.02±3.84
2.53±4.33
4j
-
4k
4l
-
-
0.62±4.17
dexamethasone
1Each experiment was independently performed three times.
“—”: Not determined.
17

Cai et al., Supplementary Figure 1
Fig. 1 Structures of nitrogen-containing steroidal compounds
18

Cai et al., Supplementary Figure 2
Fig.2. Concentration-dependent inhibition of NO production by compounds 4a, 4g, 4h, and dexamethasone in
LPS-induced RAW 264.7 cells after 24 h. RAW 264.7 cells were pretreated with the compound for 60 min prior to
LPS (1 μg/mL) stimulation for 24 h. The nitrite levels in the cell culture medium were detected using Griess
reagents. Data shown are the mean ± SD for each group (n=3) and are representative of three independent
experiments.
19

Cai et al., Supplementary Figure 3
Fig.3. The effect of compound 4g on NO production in LPS-induced RAW 264.7 cells. RAW 264.7 cells were
pretreated with various concentrations of compound 4g (0, 0.1, 0.3, 1, 3, 10 and 30 μM/L) for 60 min and then
were stimulated with LPS (1 μg/ mL) for 24 h. Data shown are the mean ± SD for each group (n = 3) and are
representative of three independent experiments (*P <0.05 and **P < 0.01 vs. the LPS group; ##P < 0.01 vs. the
control group), data were analysed by ANOVA and Duncan ’s multiple range tests.
20

Cai et al., Supplementary Figure 4
Fig.4. Cell viability of compound 4g in RAW 264.7 cells. Cells were incubated with the indicated concentrations of
compound 4g (0, 0.1, 0.3, 1, 3, 10 and 30 μM/L) for 60 min and then stimulated with LPS (1 μg/mL) for 24 h. The
results are expressed as a percentage of the respective control. The values are presented as the mean ± S.D. (n=3) of
three independent experiments. (*P <0.05 and **P < 0.01 vs. the LPS group; ##P < 0.01 vs. the control group).
21

Cai et al., Supplementary Figure 5
Fig.5. Effect of compound 4g on IL-6 (A), PGE-2(B) and TNF-α (C) release in LPS-induced RAW 264.7 cells.
RAW 264.7 cells were preincubated with different concentrations of compound 4g (0, 0.1, 0.3, 1, 3, 10, 30 μM/L) for
60 min prior to LPS stimulation. After treatment with LPS for 24 h, cell culture medium was collected, and ELISA
was performed to assess the levels of inflammatory cytokines. The values are presented as the mean ± S.D. (n = 3) of
three independent experiments. (*P <0.05 and **P < 0.01 vs. the LPS group; ##P < 0.01 vs. the control group).
22

Cai et al., Supplementary Figure 6
Fig.6. Effects of compound 4g on mRNA levels of iNOS, TNF-α, IL-6 and COX-2 in LPS-induced RAW264.7
cells. RAW 264.7 cells were preincubated with different concentrations of compound 4g (0, 0.1, 1, 10 μM/L) for 60
min prior to LPS stimulation. After treatment with LPS (1 μg/mL) for 6 h, the total RNA was isolated and the iNOS
(A), the mRNA expression for TNF-α (B), IL-6 (C) and COX-2 (D) were determined by quantitative RT-PCR. The
23

data are representative from three independent experiments. (*P <0.05 and **P < 0.01 vs. the LPS group; ##P < 0.01
vs. the control group).
Cai et al., Supplementary Figure 7
Fig.7. Effects of compound 4g on LPS-induced expression of iNOS and COX-2 proteins (A, B). RAW 264.7 cells
were preincubated with different concentrations of compound 4g (0, 0.1, 1, 10 μM/L) for 60 min prior to LPS
stimulation. After treatment with LPS (1 μg/mL) for 24 h, total protein was extracted and subjected to western blot
analysis to detect the iNOS and COX-2 proteins. The data are representative from three independent experiments.
(*P <0.05 and **P < 0.01 vs. the LPS group; ##P < 0.01 vs. the control group).
24

Cai et al., Supplementary Scheme 1
Scheme 1. General synthesis of compounds 4a–4l.Reagents and Conditions: (I) pregnenolone (0.01 mol),
substituted benzaldehyde (0.012 mol), absolute ethanol, sodium hydroxide, room temperature 48 h, yield 82–90%;
(II) absolute ethanol, sodium hydroxide, thiosemicarbazide, reflux 12 h, yield 79-87%.
25

26

Highlights
► This study provided a novel, efficient and simple synthesis of D-ring pyrazoline derivatives of
20-keto pregnenanes and their anti-inflammatory biological activities were evaluated preliminarily a
new family of D-ring pyrazoline derivatives of 20-keto 3β-hydroxy-pregn-5-enes.
► A new family of steroidal pyrazoline derivatives, 3β-hydroxy-cholest-5-en-17β-yl-5’-phenyl-4’,
5’-dihydro-1’-carbothioic acid amidopyrazole derivatives were synthesized.
► The structures of all targeted synthesized compounds had been characterized.
► The anti-inflammatory biological activity of synthesized compounds was assayed.
► In vitro activity assay screened compound 4g with excellent anti-inflammatory biological activity
and low cytotoxicity against RAW 264.7 cells.
27