Synthesis and anti-inflammatory activity of novel steroidal chalcones with 3β-pregnenolone ester derivatives in RAW 264.7 cells in vitro

Article abstract of DOI:10.1016/j.steroids.2021.108830

To identify new potential anti-inflammatory agents, we herein report the synthesis of novel steroidal chalcones with 3β-pregnenolone esters of cinnamic acid derivatives using pregnenolone as the starting material. The structures of the newly synthesised c

Full text of DOI:10.1016/j.steroids.2021.108830

Steroids 171 (2021) 108830
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and anti-inflammatory activity of novel steroidal chalcones with
3β-pregnenolone ester derivatives in RAW 264.7 cells in vitro
,
,
Xiaorui Caia 1, Fei Shaa 1, Chuanyi Zhaob, Zhiwei Zhenga, Shulin Zhaoa, Zhiwei Zhua,
Huide Zhua, Jiaoling Chena, Yicun Chenb
,
*
a Department of Pharmacy, The Affiliated Tumor Hospital of Shantou University Medical College, Shantou 515041, Guangdong, China
b Department of Pharmacology, Shantou University Medical College, Shantou 515041, Guangdong, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
To identify new potential anti-inflammatory agents, we herein report the synthesis of novel steroidal chalcones
with 3β-pregnenolone esters of cinnamic acid derivatives using pregnenolone as the starting material. The
structures of the newly synthesised compounds were confirmed by 1H NMR, 13C NMR, HRMS and infrared
imaging. All the derivatives were examined to determine their in vitro anti-inflammatory profiles against LPS-
induced inflammation in RAW 264.7 cells; the derivates were evaluated by the quantification of the pro-
inflammatory mediator nitric oxide (NO) in the cell culture supernatant based on the Griess reaction, which
measures nitrite levels, followed by an in vitro cytotoxicity study. Among these novel derivatives, compound 11e
[3β-3-phenyl acrylate-pregn-5-en-17β-yl-3′ -(p-fluoro)-phenylprop-2′-en-1′-one] was identified as the most
potent anti-inflammatory agent, which showed significant anti-inflammatory activity by inhibiting the LPS-
induced pro-inflammatory mediator NO in a dose-dependent manner without any cytotoxicity. Moreover,
compound 11e markedly inhibited the expression of pro-inflammatory cytokines, including inducible nitric
Steroid
Pregnenolone
Chalcones
Cinnamic acid derivatives
Anti-inflammatory activity
RAW 264.7 cells
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. Further studies confirmed that compound 11e significantly suppressed the
transcriptional activity of NF-κB in activated RAW 264.7 cells. Molecular docking study revealed the strong
binding affinity of compound 11e to the active site of the pro-inflammatory proteins, which confirmed that
compound 11e acted as an anti-inflammatory mediator. These results indicated that steroidal chalcones with 3β-
pregnenolone esters of cinnamic acid derivatives might be considered for further research in the design of anti-
inflammatory drugs, and compound 11e might be a promising therapeutic anti-inflammatory drug candidate.
1. Introduction
inflammatory mediators [4,5]. However, severe or chronic inflamma-
tion is highly related to diseases, such as osteoarthritis, rheumatoid
Inflammation is the body’s self-defence system that protects organ-
isms from harmful stimuli, and it is characterized by pain, heat, swelling,
redness, and loss of function of the affected body part [1]. When in-
flammatory reactions are exaggerated or inadequate, they can lead to
physiological decompensation, organ dysfunction, and even death [2,3].
The inflammatory responses of organisms are triggered by various
stimuli, including physical, chemical and biological stimuli, resulting in
the recruitment of inflammatory and innate immune cells to the site of
injury or infection. In response to extracellular stimuli, macrophages can
produce and release pro-inflammatory mediators, such as NO, prosta-
arthritis and diabetes, because of the increased production of pro-
inflammatory mediators [6,7]. Therefore, macrophages may be a po-
tential therapeutic target for inflammatory diseases.
However, the chronic use of anti-inflammatory drugs is limited due
to their severe side effects, such as gastrointestinal injury, especially
gastrointestinal perforation, peptic ulceration or significant bleeding.
Thus, the development of new therapeutic agents that can avoid
gastrointestinal injury and lead to enhanced anti-inflammatory effects
has become an urgent need for patients with inflammatory diseases
[8,9].
glandin E2 (PGE-2), interleukin-1 beta (IL-1β), TNF-
α
and other
Lipopolysaccharide (LPS) is a component of the outer membrane of
* Corresponding author.
E-mail address: chenyicun@yeah.net (Y. Chen).
1
Xiaorui Cai and Fei Sha contributed equally to this article.
https://doi.org/10.1016/j.steroids.2021.108830
Received 14 January 2021; Received in revised form 5 March 2021; Accepted 28 March 2021
Available online 6 April 2021
0039-128X/© 2021 Elsevier Inc. All rights reserved.

X. Cai et al.
Steroids 171 (2021) 108830
Fig. 1. Structures of standard anti-inflammatory steroid drugs; dexamethasone (1), beclometasone dipropionate (2), fluticasone (3), budesonide (4), mometasone (5)
and ciclesonide (6).
Gram-negative bacteria and a potent inducer of the differentiation of
monocytes to M1-like, classically activated macrophages, which pro-
duce pro-inflammatory mediators, such as NO, PGE-2, iNOS, COX-2,
properties [30–32].
The coupling of two or more natural products to make hybrids leads
to an almost inexhaustible reservoir of new types of compounds with
diverse structures [25]. The underlying expectation is that a combina-
tion of structural features of two or more functionally active substances
into one molecule or their covalent coupling may either enhance or
modulate the desired characteristics of the individual components or
lead to new types of properties [33,34]. Previous literatures have been
reported that such structure like steroidal chalcones have been shown to
bear a lot of different biological activities such as antimicrobial, anti-
inflammatory, anticancer, antioxidant, and hypotensive activities
[35–38].
TNF-α, IL-6 and IL-1β [10,11]. Furthermore, LPS is known to activate
the nuclear factor-kappa B (NF-κB) and mitogen-activated protein ki-
nase (MAPK) signalling pathways through the activation of Toll-like
receptors (TLRs) [12,13].
MAPKs are known as upstream molecules of the NF-κB signalling
pathway that play a pivotal role in governing the inflammatory process
and are activated by LPS [14,15]. Consisting of three well-defined par-
allel modules, p38, extracellular signal-regulated kinase (ERK), and c-
Jun N-terminal kinase (JNK), MAPKs are implicated in the regulation of
COX-2 and iNOS expression [16]. MAPK activation contributes to the
activation of transcription factors such as NF-κB, which is largely
implicated in the expression of anti-inflammatory mediators, including
In this study, we prepared a novel class of modified steroids or
chalcone analogues or chalconoyl pregnenolones with 3β-pregnenolone
esters of cinnamic acid using pregnenolone as the steroid precursor. The
17-acetyl group of pregnenolone was used to form the enol using the
Claisen–Schmidt condensation method to condense different aromatic
aldehydes to synthesize a series of chalconoyl pregnenolones. In a later
step, we successfully applied Steglich esterification to produce a novel
class of steroidal chalcones with 3β-pregnenolone ester derivatives using
cinnamic acid. The anti-inflammatory capacities of these synthetic
compounds and their underlying mechanisms were assessed in RAW
264.7 macrophage cells. The results show that compound 11e exhibited
the most potent anti-inflammatory properties among the members of
this family. Compound 11e showed the most potent activity in the in-
hibition of NO production, significantly inhibited the expression of pro-
NO, PGE-2, IL-6, IL-1β, and TNF-α [17].
Steroidal compounds exhibit a variety of biological functions, play a
very important role in biology [18] and have attracted profound
attention in the development of potent pharmacological agents for the
treatment of various diseases, including cardiovascular disease, auto-
immune disorders, adrenal insufficiencies, and fungal and microbial
infections. Steroids are a very important class of anti-inflammatory
agents (SAIAs). Steroids suppress the immune response through the in-
hibition of NF-κB and the suppression of pro-inflammatory cytokines.
Steroids also inhibit the production of prostaglandins and leukotrienes.
Some of the notable steroidal anti-inflammatory drugs are dexametha-
sone (1), beclometasone dipropionate (2), fluticasone (3), budesonide
(4), mometasone (5), and ciclesonide (6) (Fig. 1). These drugs are used
for the management of various human disease conditions associated
with inflammation [19,20]. Steroid-based chemotherapeutic drugs
possess various advantages, such as low toxicity, low vulnerability to
multidrug resistance (MDR) and high bioavailability, because they are
capable of penetrating the cell wall [21].
inflammatory factors, such as IL-6, TNF-α and PGE-2, and suppressed the
transcriptional activity of NF-κB signalling in LPS-induced RAW 264.7
cells.
2. Materials and methods
2.1. Chemistry
Chalcones (1, 3-diphenyl-2-propen-1-one) are well known for their
diverse array of bioactivities. In particular, chalcones have been re-
ported to possess pharmacological activities, such as anticancer, anti-
oxidant, anti-inflammatory [22,23], and antimicrobial activities
[24,25]. The chalcone structure consists of three important components,
viz. two phenyl rings and an a,b-unsaturated carbonyl system that joins
them. Previous studies have revealed that the nature of the substituents
on the phenyl rings and their conformation in the core structure play a
vital role in determining their activities. In some specific studies, it was
found that variations leading to conformational changes, such as epox-
idation or substitution on the double bond, result in decreased bioac-
tivity [26].
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 chro-
matography (TLC) was performed on silica gel F254 plates and visual-
ized 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 solutions (tetrame-
thylsilane 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 spec-
trometry) was performed on a Q-FT-MS system (SolariX 7 T, Bruker,
USA). 13C NMR spectra were measured at 150 MHz on a Bruker AVANCE
III spectrometer. Chalcones were prepared from substituted benzalde-
hyde and pregnenolone according to the procedure reported in the
literature.
Pregnenolone is a naturally occurring neurosteroid that is synthe-
sized from cholesterol in the adrenal gland and the central nervous
system and is known as a precursor to other hormones, including
cortisone, oestrogen, testosterone and progesterone [27,28]. Pregnen-
olone is an active ingredient in many traditional Chinese medicines that
possess good anti-inflammatory activity, such as Rhizoma ligustici wall-
ichii, which shows strong anti-inflammatory and analgesic effects [29].
Cinnamic acid is one of the main chemical ingredients in Cinnamo-
mum cassia. It has been reported that cinnamic acid and its analogue,
styryl ketone, possess good antioxidative and anti-inflammatory
2

X. Cai et al.
Steroids 171 (2021) 108830
2.1.2. Chemical methods
39.13, 37.28, 36.54, 32.02, 31.86, 31.59, 24.70, 22.81, 21.13, 19.38,
14.71, 13.40. MS (ESI) (m/z): 423.3 [M + H]+
.
2.1.2.1.6. 3β-hydroxy-pregn-5-en-17β-yl-3′-(p-tolyl)-phenylprop-2′-
2.1.2.1. General synthesis of 3β-hydroxy-pregn-5-en-17β-yl-3′-phenylprop-
2′-en-1′-one derivatives (9a–9i). To a solution of pregnenolone 7 (3.16 g,
0.01 mol) in ethanol (30 mL), a conc. Aq. Solution of NaOH (1.0 g, 0.025
mol) was added. Then, aldehyde 8 (0.012 mol) was added to the reaction
mixture and stirred at room temperature for 48 h to obtain the corre-
sponding benzylidine derivative 9 (9a–9i). The progress of the reaction
was monitored by TLC (petroleum ether: ethyl acetate (2:1 v/v) as el-
uents). After completion, the reaction mixture was poured into crushed
ice water. The precipitate was filtered, dried and recrystallized from
ethanol to yield the product as a solid white powder [39,40]. 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
under ambient conditions. Thus, the study was restricted to the use of
only aromatic aldehydes, for which the obtained yields were 75–86%.
2.1.2.1.1. 3β-hydroxy-pregn-5-en-17β-yl-3′-(p-benzyloxy)-phenylprop-
2′-en-1′-one (9a).. White solid powder (Yield, 82.40%), M.p.:
218.4–219.4 ◦C. 1H NMR (400 MHz, δ ppm, CDCl3): 7.47–7.57 (m, 3H,
C = CH , Ar-H), 7.32–7.44 (m, 5H, Ar-H), 6.98 (d, 2H, J = 8.4 Hz, Ar-H),
6.65 (d, 1H, J = 16.0 Hz, CH = C), 5.37 (s, 1H, C6-H), 5.10 (s, 2H),
3.51–3.56 (m, 1H, C3α-H), 2.83 (m, 1H, C17α-H), 1.00 (s, 3H, C10-CH3),
0.63 (s, 3H, C13-CH3). 13C NMR (ppm, CDCl3): 200.34, 160.62, 141.46,
140.83, 136.47, 129.99(2C), 128.67(2C), 128.16, 127.45(3C), 124.82,
121.41, 115.30(2C), 71.68, 70.12, 61.99, 57.20, 50.12, 44.96, 42.26,
39.15, 37.29, 36.56, 32.04, 31.88, 31.61, 24.71, 22.81, 21.14, 19.40,
en-1′-one (9f).. Light yellow solid (Yield: 85.36%), M.p.:
1
◦
123.5–126.4 C. H NMR (400 MHz, δ ppm, CDCl3): 7.53 (d, 1H, J =
16.0 Hz, C = CH),7.46 (d, 2H, J = 8.0, Ar-H), 7.20 (d, 2H, J = 8.0 Hz, Ar-
H), 6.74 (d, 1H, J = 16.0 Hz, CH = C), 5.36 (s, 1H, C6-H), 3.51–3.54 (m,
′′
1H, C3α-H), 2.85 (m, 1H, C17α-H), 2.38 (s, 3H, C4 –CH3), 1.00 (s, 3H,
C10-CH3), 0.64 (s, 3H, C13-CH3). 13C NMR (ppm, CDCl3): 200.45,
141.58, 140.75, 132.07, 129.65(2C), 128.29(2C), 127.13, 125.94,
121.42, 71.70, 61.98, 57.21, 50.11, 44.97, 42.26, 39.14, 37.28, 36.55,
32.03, 31.86, 31.61, 24.70, 22.79, 21.48, 21.13, 19.38, 13.42. MS (ESI)
(m/z): 419.3 [M + H]+
.
2.1.2.1.7. 3β-hydroxy-pregn-5-en-17β-yl-3′-(m,p-dimethoxy)-phenyl-
prop-2′-en-1′-one (9 g).. Yellow solid powder (Yield, 83.27%), M.
p.:100.4–101.4 ◦C. 1H NMR (400 MHz, δ ppm, CDCl3): 7.52 (d, 1H, J =
16.0 Hz, C = CH),7.14–7.18 (m, 1H, Ar-H), 7.06–7.10 (m, 1H, Ar-H),
6.89 (d, 1H, J = 8.0 Hz, Ar-H), 6.67 (d, 1H, J = 16.0 Hz, CH = C),
′′
′′
5.38 (s, 1H, C6-H), 3.94 (s, 6H, C3 , 4 –OCH3), 3.51–3.56 (m, 1H, C3α-H),
2.89 (m, 1H, C17α-H), 1.01 (s, 3H, C10-CH3), 0.64 (s, 3H, C13-CH3). 13
C
NMR (ppm, CDCl3): 200.32, 151.22, 149.24, 141.66, 140.82, 127.76,
125.04, 122.73, 121.37, 111.17, 110.14, 71.64, 61.69, 57.21, 55.98
(2C), 50.12, 44.97, 42.23, 39.09, 37.29, 36.54, 32.02, 31.86, 31.57,
24.70, 22.90, 21.13, 19.38, 13.42. MS (ESI) (m/z): 465.4 [M + H]+
2.1.2.1.8. 3β-hydroxy-pregn-5-en-17β-yl-3′-(p-methylthio)-phenyl-
.
prop-2′-en-1′-one (9 h).. Yellow solid powder (Yield, 80.16%), M.
1
◦
13.42. MS (ESI) (m/z):511.3 [M + H]+
.
p.:155.2–156.4 C. H NMR (400 MHz, δ ppm, CDCl3): 7.44–7.60 (m,
3H, C = CH , Ar-H), 7.25 (d, 2H, J = 8.0 Hz, Ar-H), 6.76 (d, 1H, J = 16.0
Hz, CH = C), 5.39 (s, 1H, C6-H), 3.53–3.56 (m, 1H, C3α-H), 2.86 (m, 1H,
′′
2.1.2.1.2. 3β-hydroxy-pregn-5-en-17β-yl-3′-(p-methoxy)-phenylprop-
2′-en-1′-one (9b).. Yellow solid (Yield: 85.16%), M.p.: 102.8–105.8 ◦C.
1H NMR (400 MHz, δ ppm, CDCl3): 7.48–7.56 (m, 3H, C = CH , Ar-H),
6.93 (d, 2H, J = 8.4 Hz, Ar-H), 6.66 (d, 1H, J = 16.0 Hz, CH = C),
C
17α-H), 2.53 (s, 3H, C4 -SCH3),1.02 (s, 3H, C10-CH3), 0.66 (s, 3H, C13-
CH3). 13C NMR (ppm, CDCl3): 200.30, 141.95, 140.95, 131.35, 128.63
(3C), 126.07(2C), 125.86, 121.41, 71.70, 62.08, 57.20, 50.10, 45.00,
42.26, 39.14, 37.27, 36.54, 32.03, 31.86, 31.61, 24.69, 22.79, 21.13,
′′
5.39 (s, 1H, C6-H), 3.87 (s, 3H, C4 –OCH3), 3.53–3.56 (m, 1H, C3α-H),
2.86 (m, 1H, C17α-H), 1.02 (s, 3H, C10-CH3), 0.66 (s, 3H, C13-CH3). 13
C
19.38, 15.21, 13.43. MS (ESI) (m/z): 451.3 [M + H]+
.
NMR (ppm, CDCl3): 200.35, 161.45, 141.33, 140.84, 129.97(3C),
124.70, 121.37, 114.39(2C), 71.64, 61.95, 57.18, 55.39, 50.11, 44.95,
42.24, 39.12, 37.28, 36.54, 32.02, 31.86, 31.58, 24.70, 22.81, 21.13,
2.1.2.1.9. 3β-hydroxy-pregn-5-en-17β-yl-3′-(o-fluoro)-phenylprop-2′-
en-1′-one (9i).. White solid powder (Yield, 85.19%), M.p.:
1
◦
19.38, 13.40. MS (ESI) (m/z): 435.3 [M + H]+
.
140.6–142.2 C. H NMR (400 MHz, δ ppm, CDCl3): 7.67 (d, 1H, J =
16.0 Hz, C = CH),7.55–7.58 (m, 1H, Ar-H), 7.34–7.39 (m, 1H, Ar-H),
7.08–7.19 (m, 2H, Ar-H), 6.88 (d, 1H, J = 16.0 Hz, CH = C), 5.37 (s,
1H, C6-H), 3.48–3.60 (m, 1H, C3α-H), 2.88 (m, 1H, C17α-H), 1.00 (s, 3H,
2.1.2.1.3. 3β-hydroxy-pregn-5-en-17β-yl-3′-(p-bromo)-phenylprop-2′-
en-1′-one (9c).. White solid powder (Yield, 77.89%), M.p.:
144.8–145.6 ◦C. 1H NMR (400 MHz, δ ppm, CDCl3): 7.42–7.55 (m, 5H,
C = CH , Ar-H), 6.67 (d, 1H, J = 16.0 Hz, CH = C), 5.39 (s, 1H, C6-H),
3.50–3.56 (m, 1H, C3α-H), 2.83 (m, 1H, C17α-H), 1.02 (s, 3H, C10-CH3),
0.65 (s, 3H, C13-CH3). 13C NMR (ppm, CDCl3): 200.17, 140.79, 140.08,
133.77, 132.14(2C), 129.63(2C), 127.25, 124.48, 121.39, 71.69, 62.25,
57.19, 50.08, 45.05, 42.25, 39.16, 37.27, 36.54, 32.02, 31.84, 31.61,
24.67, 22.76, 21.12, 19.38, 13.45. MS (ESI) (m/z): 483.2, 485.2 [M +
C
10-CH3), 0.64 (s, 3H, C13-CH3). 13C NMR (ppm, CDCl3): 200.50,
162.48, 160.80, 140.80, 134.15, 131.60, 129.32, 124.45, 122.93,
121.40, 116.20, 71.69, 61.97, 57.19, 50.06, 44.99, 42.25, 39.07, 37.27,
36.54, 32.03, 31.85, 31.61, 24.68, 22.78, 21.14, 19.38, 13.47. MS (ESI)
(m/z): 423.3 [M + H]+
.
H]+
.
2.1.2.2. General synthesis of 3β-3-phenyl acrylates-pregn-5-en-17β-yl-3′-
phenylprop-2′-en-1′-one derivatives (11a-11i). A suspension of com-
pounds 9a-9i (0.01 mol), cinnamic acid 10 (2.96 g, 0.02 mol), EDCI
(0.38 g, 2 mmol) and DMAP (0.12 g, 1 mmol) in dry dichloromethane
(25 mL) was stirred at room temperature for 48 h and filtered. The
filtrate was washed with 5% NaHCO3 liquor (3 × 15 mL), dried over
MgSO4, filtered, and concentrated in vacuo. The residue was purified by
silica gel column chromatography (petroleum ether/ethyl acetate, 5:1)
to afford the corresponding ester [41]. Quantitative yields of 51–75%
were obtained for compounds 11a–11i.
2.1.2.1.4. 3β-hydroxy-pregn-5-en-17β-yl-3′-(p- chloro)-phenylprop-2′-
en-1′-one (9d). Light yellow solid (Yield: 81.48%), M.p.:
148.5–151.4 ◦C. 1H NMR (400 MHz, δ ppm, CDCl3): 7.41–7.53 (m, 3H,
C = CH , Ar-H), 7.36 (d, J = 8.0 Hz, 2H, Ar-H), 6.74 (d, 1H, J = 16.0 Hz,
CH = C), 5.36 (s, 1H, C6-H), 3.50–3.54 (m, 1H, C3α-H), 2.84 (m, 1H,
C
17α-H), 1.00 (s, 3H, C10-CH3), 0.64 (s, 3H, C13-CH3). 13C NMR (ppm,
CDCl3): 200.16, 140.79, 140.02, 136.15, 133.34, 129.41(2C), 129.18
(2C), 127.16, 121.39, 71.69, 62.24, 57.19, 50.08, 45.04, 42.25, 39.17,
37.27, 36.54, 32.02, 31.84, 31.61, 24.67, 22.76, 21.12, 19.38, 13.45.
MS (ESI) (m/z): 439.3 [M + H]+
.
2.1.2.2.1. 3β-3-phenyl acrylates-pregn-5-en-17β-yl-3′-(p-benzyloxy)-
2.1.2.1.5. 3β-hydroxy-pregn-5-en-17β-yl-3′-(p-fluoro)-phenylprop-2′-
en-1′-one (9e).. White solid powder (Yield, 75.02%), M.p.:
105.6–108.1 ◦C. 1H NMR (400 MHz, δ ppm, CDCl3): 7.43–7.56 (m, 3H,
C = CH , Ar-H), 6.89 (d, 2H, J = 8.0 Hz, Ar-H), 6.66 (d, 1H, J = 16.0 Hz,
CH = C), 5.36 (s, 1H, C6-H), 3.48–3.54 (m, 1H, C3α-H), 2.84 (m, 1H,
phenylprop-2′-en-1′-one (11a). White solid powder (Yield, 68.22%), M.
ꢀ 1
p.: 175.0–178.0 ◦C. IR (KBr, cm ): 1704.94 (C O), 1676.32, 1644.07,
–
–
–
–
1597.91, 1573.72, 1510.07, 1452.17, 1174.75 (C O), 1097.02 (C O).
1H NMR (600 MHz, δ ppm, CDCl3): 7.68 (d, 1H, J = 16.20 Hz, C = CH),
7.50–7.53 (m, 5H, C = CH, Ar-H), 7.33–7.46 (m, 8H, Ar-H), 6.98 (d, 2H,
J = 9.0 Hz, Ar-H), 6.67 (d, 1H, J = 15.60 Hz, CH = C), 6.43 (d, 1H, J =
16.20 Hz, CH = C), 5.42 (s, 1H, C6-H), 5.11 (s, 2H), 4.73–4.79 (m, 1H,
C
17α-H), 1.00 (s, 3H, C10-CH3), 0.63 (s, 3H, C13-CH3). 13C NMR (ppm,
CDCl3): 200.34, 160.86, 141.40, 140.81, 130.13, 129.97(2C), 127.31,
121.40, 114.87(2C), 71.67, 63.63, 61.95, 57.19, 50.12, 44.94, 42.25,
C
3α-H), 2.85 (m, 1H, C17α-H), 1.04 (s, 3H, C10-CH3), 0.65 (s, 3H, C13-
3

X. Cai et al.
Steroids 171 (2021) 108830
CH3). 13C NMR (ppm, CDCl3): 200.25, 166.41, 160.60, 144.48, 141.20,
139.72, 136.47, 134.53, 130.19, 129.98(2C), 128.87(2C), 128.68(2C),
128.16, 128.04(3C), 127.76, 127.45(2C), 124.86, 122.47, 118.68,
115.28(2C), 73.98, 70.11, 61.96, 57.15, 50.04, 44.94, 39.12, 38.21,
37.06, 36.68, 32.01, 31.89, 27.88, 24.71, 22.80, 21.10, 19.35, 13.42.
MS (ESI) (m/z): 641.3 [M + H]+ . HRMS (EI): m/z [M + Na]+ calcd for
14.73, 13.45. MS (ESI) (m/z): 553.2, 554.2 [M + H]+. HRMS (EI): m/z
[M + Na]+ calcd for C37H41FO3: 575.2937; found: 575.293141.
2.1.2.2.6. 3β-3-phenyl acrylates -pregn-5-en-17β-yl-3′-(p-tolyl)-phe-
nylprop-2′-en-1′-one (11f). Yellow solid powder (Yield: 58.37%), M.p.:
ꢀ 1
188.2–189.7 ◦C. IR (KBr, cm ): 1708.30(C O), 1679.80, 1644.25,
–
–
–
–
1604.25, 1585.0, 1487.06, 1450.53, 1174.51 (C O), 1097.16 (C O).
1H NMR (600 MHz, δ ppm, CDCl3): 7.68 (d, 1H, J = 16.20 Hz, C = CH),
7.49–7.56 (m, 3H, C = CH, Ar-H), 7.46 (d, 1H, J = 7.80 Hz, Ar-H),
7.33–7.41 (m, 3H, Ar-H),7.20 (d, 2H, J = 7.80 Hz, Ar-H), 6.75 (d, 1H,
J = 16.20 Hz, CH = C), 6.43 (d, 1H, J = 16.20 Hz, CH = C), 5.42 (s, 1H,
C6-H), 4.71–4.79 (m, 1H, C3α-H), 2.86 (m, 1H, C17α-H), 2.38 (s, 3H,
C
44H48O4: 663.3450; found: 663.344385.
2.1.2.2.2. 3β-3-phenyl acrylates -pregn-5-en-17β-yl-3′-(p-methoxy)-
phenylprop-2′-en-1′-one (11b). Yellow solid powder (Yield: 51.23%), M.
ꢀ 1
p.: 164.4–167.3 ◦C. IR (KBr, cm ): 1700.18 (C O), 1677.34, 1644.76,
–
–
–
–
1597.74, 1573.53, 1510.91, 1451.52, 1172.76 (C O), 1098.08 (C O).
1H NMR (600 MHz, δ ppm, CDCl3): 7.68 (d, 1H, J = 16.20 Hz, C = CH),
7.51–7.56 (m, 5H, C = CH, Ar-H), 7.33–7.41 (m, 3H, Ar-H), 6.91 (d, 2H,
J = 8.40 Hz, Ar-H), 6.67 (d, 1H, J = 16.20 Hz, CH = C), 6.43 (d, 1H, J =
16.20 Hz, CH = C), 5.42 (s, 1H, C6-H), 4.72–4.79 (m, 1H, C3α-H), 3.84 (s,
C4 –CH3), 1.04 (s, 3H, C10-CH3), 0.65 (s, 3H, C13-CH3).13C NMR (ppm,
′′
CDCl3): 200.38, 166.41, 144.48, 141.55, 139.72, 134.53, 132.09,
130.19, 129.66(2C), 128.87(2C), 128.30(2C), 128.04(2C), 127.12,
125.98, 122.46, 118.68, 73.98, 61.95, 57.15, 50.04, 44.96, 39.10,
38.21, 37.00, 36.64, 32.12, 31.95, 27.88, 24.70, 22.79, 21.49, 21.10,
19.35, 13.44. MS (ESI) (m/z): 549.3 [M + H]+. HRMS (EI): m/z [M +
Na]+ calcd for C38H44O3: 571.3188; found: 571.318145.
′′
3H, C4 –OCH3), 2.85 (m, 1H, C17α-H), 1.04 (s, 3H, C10-CH3), 0.65 (s, 3H,
C13-CH3). 13C NMR (ppm, CDCl3): 200.25, 166.4, 161.46, 144.47,
141.27, 139.72, 134.53, 130.19, 129.97(3C), 128.87(2C), 128.04(2C),
127.52, 124.76, 122.47, 118.68, 114.39(2C), 73.98, 61.93, 57.14,
55.40, 50.04, 44.93, 39.10, 38.21, 37.06, 36.68, 32.12, 31.95, 27.88,
2.1.2.2.7. 3β-3-phenyl acrylates -pregn-5-en-17β-yl-3′-(m,p-dime-
thoxy)-phenylprop-2′-en-1′-one (11 g). Yellow solid powder (Yield:
24.71, 22.81, 21.05, 19.33, 13.41. MS (ESI) (m/z): 565.3 [M + H]+
.
75.23%), M.p.: 153.1–153.7 ◦C. IR (KBr, cm ): 1701.73(C O),
ꢀ 1
–
–
–
HRMS (EI): m/z [M + Na]+ calcd for C38H44O4: 587.3137; found:
587.313051.
1676.37, 1638.22, 1595.80, 1513.00, 1450.95, 1171.51 (C O),
1
–
1098.15 (C O). H NMR (600 MHz, δ ppm, CDCl3): 7.68 (d, 1H, J =
2.1.2.2.3. 3β-3-phenyl acrylates -pregn-5-en-17β-yl-3′-(p-bromo)-phe-
nylprop-2′-en-1′-one (11c). Light yellow solid powder (Yield: 52.67%),
15.60 Hz, C = CH), 7.46–7.59 (m, 3H, C = CH, Ar-H), 7.34–7.43 (m, 3H,
Ar-H), 7.16 (d, 1H, J = 6.0 Hz, Ar-H), 6.87 (d, 1H, J = 7.80 Hz, Ar-H),
6.66 (d, 1H, J = 16.20 Hz, CH = C), 6.43 (d, 1H, J = 16.20 Hz, CH = C),
◦
–
–
M.p.: 169.3–172.6 C. IR (KBr, cm-1): 1708.13 (C O), 1678.78,
–
′′
′′
1644.75, 1603.46, 1562.25, 1486.96, 1450.74, 1175.02 (C O),
5.42 (s, 1H, C6-H), 4.72–4.78 (m, 1H, C3α-H), 3.92 (s, 6H, C3 , 4 –OCH3),
1
1096.08 (C O). H NMR (600 MHz, δ ppm, CDCl3): 7.68 (d, 1H, J =
2.88 (m, 1H, C17α-H), 1.04 (s, 3H, C10-CH3), 0.66 (s, 3H, C13-CH3). 13
C
–
15.60 Hz, C = CH), 7.46–7.52 (m, 5H, C = CH, Ar-H), 7.33–7.44 (m, 5H,
Ar-H), 6.76 (d, 1H, J = 15.60 Hz, CH = C), 6.43 (d, 1H, J = 16.20 Hz, CH
NMR (ppm, CDCl3): 200.24, 166.40, 151.22, 149.26, 144.48, 141.63,
139.71, 134.52, 130.19, 128.87(2C), 128.04(2C), 127.78, 125.11,
122.75, 122.45, 118.66, 111.16, 110.06, 73.96, 61.66, 57.17, 56.00
(2C), 50.06, 44.97, 39.08, 38.20, 37.07, 36.67, 32.01, 31.89, 27.88,
24.72, 22.91, 21.11, 19.35, 13.45. MS (ESI) (m/z):595.2 [M + H]+.
HRMS (EI): m/z [M + Na]+ calcd for C39H46O5: 617.3243; found:
617.323506.
= C), 5.41 (s, 1H, C6-H), 4.75–4.81 (m, 1H, C3α-H), 2.84 (m, 1H, C17α
-
H), 1.04 (s, 3H, C10-CH3), 0.64 (s, 3H, C13-CH3). 13C NMR (ppm, CDCl3):
200.09, 166.39, 144.49, 140.06, 139.71, 134.52, 133.78, 132.16(2C),
130.20, 129.65(2C), 128.88(4C), 128.04(2C), 127.28, 124.49, 122.42,
118.67, 73.95, 62.21, 57.13, 50.00, 45.03, 39.13, 38.21, 37.06, 36.66,
32.10, 31.93, 27.88, 24.68, 22.77, 21.09, 19.35, 13.47. MS (ESI) (m/z):
613.2, 615.3 [M + H]+. HRMS (EI): m/z [M + Na]+ calcd for
C37H41BrO3: 635.2137; found: 635.213063.
2.1.2.2.8. 3β-3-phenyl acrylates -pregn-5-en-17β-yl-3′-(p-methylthio)-
phenylprop-2′-en-1′-one (11 h). Light yellow solid powder (Yield:
ꢀ 1
72.44%), M.p.: 192.9–193.6 ◦C. IR (KBr, cm ): 1699.79(C O),
–
–
–
2.1.2.2.4. 3β-3-phenyl acrylates -pregn-5-en-17β-yl-3′-(p-chloro)-phe-
1677.15, 1638.28, 1600.68, 1492.25, 1450.81, 1173.95 (C O),
1
nylprop-2′-en-1′-one (11d). Light yellow solid powder (Yield: 58.10%),
1089.88 (C O). H NMR (600 MHz, δ ppm, CDCl3): 7.68 (d, 1H, J =
–
ꢀ 1
M.p.: 182.0–182.6 ◦C. IR (KBr, cm ): 1709.47 (C O), 1679.60,
16.20 Hz, C = CH), 7.44–7.57 (m, 5H, C = CH, Ar-H), 7.35–7.43 (m, 3H,
Ar-H),7.23 (d, 2H, J = 8.40 Hz, Ar-H), 6.73 (d, 1H, J = 16.20 Hz, CH =
C), 6.43 (d, 1H, J = 16.20 Hz, CH = C), 5.42 (s, 1H, C6-H), 4.73–4.78 (m,
–
–
–
1644.94, 1604.24, 1566.52, 1490.85, 1451.19, 1175.80 (C O),
1
–
1091.46 (C O). H NMR (600 MHz, δ ppm, CDCl3): 7.68 (d, 1H, J =
′′
16.20 Hz, C = CH), 7.46–7.57 (m, 5H, C = CH, Ar-H), 7.33–7.43 (m, 5H,
1H, C3α-H), 2.85 (m, 1H, C17α-H), 2.51 (s, 3H, C4 -SCH3), 1.04 (s, 3H,
Ar-H), 6.75 (d, 1H, J = 15.60 Hz, CH = C), 6.43 (d, 1H, J = 15.60 Hz, CH
C
10-CH3), 0.65 (s, 3H, C13-CH3). 13C NMR (ppm, CDCl3):200.24, 166.41,
= C), 5.42 (s, 1H, C6-H), 4.73–4.78 (m, 1H, C3α-H), 2.85 (m, 1H, C17α
-
144.48, 141.95, 140.93, 139.72, 134.53, 131.36, 130.19, 128.87(2C),
128.64(2C), 128.04(2C), 126.07(2C), 125.90, 122.45, 118.67, 73.97,
62.04, 57.14, 50.03, 44.99, 39.11, 38.20, 37.06, 36.67, 32.01, 31.88,
27.88, 24.72, 22.80, 21.10, 19.35, 15.22, 13.44. MS (ESI) (m/z): 581.2
[M + H]+. HRMS (EI): m/z [M + Na]+ calcd for C38H44O3S: 603.2909;
found: 603.290271.
H), 1.04 (s, 3H, C10-CH3), 0.65 (s, 3H, C13-CH3). 13C NMR (ppm, CDCl3):
200.10, 166.40, 144.49, 140.00, 139.55, 136.15, 134.52, 133.35,
130.20, 129.43(2C), 129.19(2C), 128.88(2C), 128.04(2C), 127.20,
122.42, 118.66, 73.95, 62.20, 57.13, 50.01, 45.03, 39.13, 38.20, 37.06,
36.67, 32.12, 31.90, 27.88, 24.68, 22.77, 21.09, 19.35, 13.47. MS (ESI)
(m/z): 569.2, 570.2 [M + H]+. HRMS (EI): m/z [M + Na]+ calcd for
2.1.2.2.9. 3β-3-phenyl acrylates -pregn-5-en-17β-yl-3′-(o-fluoro)-phe-
C
37H41ClO3: 591.2642; found: 591.263562.
nylprop-2′-en-1′-one (11i). Yellow solid powder (Yield: 66.21%), M.p.:
ꢀ 1
2.1.2.2.5. 3β-3-phenyl acrylates -pregn-5-en-17β-yl-3′-(p-fluoro)-phe-
174.5–177.5 ◦C. IR (KBr, cm ): 1703.24(C O), 1683.15, 1642.85,
–
–
nylprop-2′-en-1′-one (11e). Yellow solid powder (Yield: 66.57%), M.p.:
1612.38, 1577.17, 1482.15, 1455.34, 1173.27 (C O), 1097.89 (C O).
1H NMR (600 MHz, δ ppm, CDCl3):7.65–7.71 (m, 2H, C = CH), 7.57 (m,
1H, Ar-H), 7.48–7.53 (m, 2H, Ar-H), 7.34–7.41 (m, 4H, Ar-H), 7.17 (m,
1H, Ar-H), 7.10 (m, 1H, Ar-H), 6.88 (d, 1H, J = 15.60 Hz, CH = C), 6.43
–
–
ꢀ 1
174.8–177.2 ◦C. IR (KBr, cm ): 1703.61 (C O), 1677.03, 1643.59,
–
–
–
–
1597.65, 1573.65, 1510.26, 1451.13, 1173.80 (C O), 1097.14(C O).
1H NMR (600 MHz, δ ppm, CDCl3): 7.68 (d, 1H, J = 16.20 Hz, C = CH),
7.45–7.59 (m, 5H, C = CH, Ar-H), 7.34–7.42 (m, 3H, Ar-H), 6.89 (d, 2H,
J = 8.40 Hz, Ar-H), 6.67 (d, 1H, J = 16.20 Hz, CH = C), 6.43 (d, 1H, J =
15.60 Hz, CH = C), 5.42 (s, 1H, C6-H), 4.71–4.79 (m, 1H, C3α-H), 2.85
(m, 1H, C17α-H), 1.04 (s, 3H, C10-CH3), 0.65 (s, 3H, C13-CH3). 13C NMR
(ppm, CDCl3): 200.25, 166.41, 160.87, 144.47, 141.35, 139.72, 134.53,
130.18, 129.98(2C), 128.87(2C), 128.04(2C), 127.33, 124.63, 122.47,
118.68, 114.87(2C), 73.98, 63.64, 61.92, 57.15, 50.05, 44.93, 39.11,
38.21, 37.06, 36.68, 32.01, 31.89, 27.88, 24.71, 22.81, 21.10, 19.32,
(d, 1H, J = 15.60 Hz, CH = C), 5.42 (s, 1H, C6-H), 4.71–4.79 (m, 1H, C3α
-
H), 2.89 (m, 1H, C17α-H), 1.04 (s, 3H, C10-CH3), 0.64 (s, 3H, C13-CH3).
13C NMR (ppm, CDCl3): 200.41, 166.39, 162.49, 160.81, 144.47,
139.72, 134.53, 134.11, 131.61, 130.19, 129.33, 128.87(2C), 128.04
(2C), 124.47, 122.95, 122.44, 118.68, 116.22, 73.97, 61.97, 57.12,
49.99, 44.98, 39.03, 38.21, 37.05, 36.67, 32.01, 31.87, 27.88, 24.69,
22.78, 21.10, 19.35, 13.48. MS (ESI) (m/z): 553.3, 554.3 [M + H]+.
HRMS (EI): m/z [M + Na]+ calcd for C37H41FO3: 575.2937; found:
4

X. Cai et al.
Steroids 171 (2021) 108830
Scheme 1. General synthesis of compounds 11a–11i.Reagents and conditions: (I) pregnenolone (0.01 mol), substituted benzaldehyde (0.012 mol), absolute ethanol,
sodium hydroxide, room temperature 48 h, yield 75–86%; (II) dichloromethane, EDCI/DMAP, cinnamic acid, room temperature 48 h, yield 51–75%.
575.293075.
FilterMax F3/F5 Microplate Reader (Molecular Devices, Sydney,
Australia).
2.2. Biological methods
2.2.4. Quantification of TNF-α, IL-6, and PGE-2
RAW 264.7 cells were treated with the indicated concentrations of
2.2.1. Cell culture and treatment
compound 11e dissolved in DMEM for 60 min, followed by incubation
Primary cell culture was carried out as described previously [42].
RAW 264.7 cells (murine macrophage cell line) were purchased from
Chinese Type Tissue Culture Collection (CTCC, Shanghai, China) and
cultivated in H-DMEM (High Dulbecco modified Eagle medium, Sigma)
with 1
μg/mL LPS for 24 h. The cell culture medium was collected, and
the levels of TNF-
α
, IL-6, and PGE-2 in the supernatants were determined
by specific ELISA kits (Elabscience Biotechnology, Wuhan, China) ac-
cording to the manufacturer’s instructions. The cytokine concentration
was measured by a FilterMax F3/F5 Microplate Reader (Molecular De-
vices, Sydney, Australia) at 450 nm and calculated from a standard
curve prepared with the corresponding recombinant mouse cytokine.
containing 10% heat-inactivated 10% foetal bovine serum (FBS), 100 U/
◦
mL penicillin, and 100
μ
g/mL streptomycin at 37 C in a humidified
atmosphere of 95% air and 5% CO2. Stock solutions of the compounds
were prepared in dimethyl sulfoxide (DMSO) and then added to fresh
culture medium to obtain the final concentrations. Unless stated other-
wise, 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. For all experiments, LPS
2.2.5. Real-time quantitative PCR analysis
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 11e
(1, 3, or 10 μM) for 60 min and then stimulated with LPS (1 μg/mL).
was used at a final concentration of 1 μg/mL. LPS (Escherichia coli 0111:
After stimulation with LPS for 6 h, the cells were harvested, and total
RNA was extracted using TRIzol reagent (Invitrogen, Shanghai, China).
B4) was purchased from Sigma (St. Louis, MO, USA).
cDNA was prepared from total RNA (1 μg) using the High-capacity cDNA
2.2.2. Cell viability assay
Reverse Transcription kit (Thermo Fisher Scientific, USA) according to
the manufacturer’s instructions. Real-time qRT-PCR was performed
using the SYBR Green Supermix reagent (Thermo Fisher Scientific, USA)
in a 7500 Real Time PCR system (Applied Biosystems, Foster City, CA,
USA). The relative amount of gene expression was reported as a quantity
relative to the control value by using the 2ꢀ △△Ct method. The specific
primer sets used have been previously reported [42].
Cell viability was determined using the 3 [4, 5-dimethylthiazol-2-yl]-
2, 5-diphenyltetrazolium bromide (MTT) assay. The MTT assay was used
to assess cell viability as described in our previous report [42]. Three
micromolar concentrations of each compound were used to identify the
more effective compounds for further study. Cell growth was arrested by
incubation of the cells in 2% serum medium for 24 h before treatment.
The indicated cells were then seeded in 96-well culture plates at a
density of 5 × 103 cells/well and stimulated with different concentra-
2.2.6. Western blot analysis
tions of compound 11e (0, 0.1, 0.3, 1, 3, 10, or 30 μ
M/L) at 37 ◦C in 5%
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 11e
CO2 saturated humidity conditions for 24 h. Finally, the optical density
(OD) was measured at 570 nm with the aid of a FilterMax F3/F5
Microplate Reader (Molecular Devices, Sydney, Australia). The results
are expressed as the ratio by normalizing the targeted OD level to that of
the control OD.
(1, 3, or 10 μM) for 60 min and then stimulated with LPS (1 μg/mL).
After stimulation with LPS for 24 h, whole cell protein (for COX-2, IL-1β,
iNOS, and β-actin) and nuclear or cytoplasmic protein (for p65, IκBα,
IKKβ, and β-actin) were extracted from the cells using Cell Lysis Reagent
(Sigma) and ice cold lysis buffer (50 mM MHEPES, 400 mM KCl, 0.1 mM
2.2.3. Quantification of nitric oxide/nitrite
EDTA, 10% glycerol). The cell lysates (40
μg) were separated by
Nitrite that accumulated in the medium was measured as an indi-
cator of NO production based on the Griess reaction. 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 the test compounds
11a, 11b, 11c, 11d, and 11e and dexamethasone dissolved in H-DMEM
SDS–PAGE, transferred to a nitrocellulose membrane and probed with
anti-iNOS, COX-2 or IL-1β antibodies. β-actin was used as an internal
control. The following primary and secondary antibodies were used for
western blot analysis: primary antibodies against COX-2, IL-1β, iNOS,
total and phospho-IκB, phospho-IKK, and β-actin and horseradish
peroxidase-conjugated secondary antibodies. The antibodies were pur-
chased from Abcam (Cambridge, MA).
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
5

X. Cai et al.
Steroids 171 (2021) 108830
DMAP at room temperature (48 h) afforded moderate yields of the
corresponding esters 11 (11a–11i). The structures of compounds
11a–11i were determined by 1H NMR, 13C NMR, HRMS and IR analyses.
Table 1
Inhibitory activity of compounds 11a ¡ 11i (3
μM) on NO generation in LPS-
induced RAW 264.7 cells 1.
Compounds
%Inhibition
%Cell viability
IC50(µM)
3.2. Evaluation of the effects of compounds 11a-11i on NO generation in
11a
79.00 ± 1.34
63.68 ± 4.17
74.53 ± 6.81
79.06 ± 0.75
90.93 ± 0.85
44.94 ± 9.31
13.17 ± 3.80
6.84 ± 8.31
58.08 ± 2.61
92.34 ± 2.06
89.94 ± 4.11
98.59 ± 1.33
67.96 ± 1.64
88.57 ± 3.26
101.15 ± 2.93
88.00 ± 5.05
84.85 ± 2.94
88.07 ± 4.71
68.97 ± 1.00
95.09 ± 1.78
1.54 5.63
23.76 ± 4.37
13.75 ± 6.21
25.30 ± 8.25
1.46 3.66
–
LPS-induced RAW264.7 cells and cell viability in vitro
11b
11c
11d
NO has been identified as a pro-inflammatory molecule in the
development of various inflammatory diseases. The production of NO is
widely considered a hallmark of macrophage activation, which is
indispensable for the pathogenesis of inflammatory diseases [44].
Activated macrophages can stimulate iNOS expression to generate NO in
culture medium. To identify the most promising anti-inflammatory
agent among the synthesized derivatives (11a–11i), the inhibitory ef-
fects of these derivatives on NO generation were tested in LPS-induced
RAW 264.7 cells; dexamethasone was used as a positive control. The
results showed that most of the novel synthetic compounds exhibited
11e
11f
11 g
–
–
11 h
11i
6.84 5.17
dexamethasone
0.62 ± 4.17
note: 1Each experiment was independently performed three times.
note: “—”: Not determined.
2.2.7. Molecular docking
modest to strong inhibitory effects on NO generation at 3.0 μM (Table 1).
Compound 11e was docked with the target proteins (iNOS, COX-2,
Moreover, the cytotoxicity of the compounds was determined by MTT
assay to exclude the possibility that the reduced release of NO was
caused by the cytotoxic effects of the compounds (Table 1 and Fig. 2).
TNF-α, IL-1β, and IL-6) to confirm its anti-inflammatory potential.
AutoDock 4.2.6 and AutoDock Vina 1.1.2 were used for molecular
docking based on Lamarkian Genetic Algorithm [43]. Three-
dimensional structure for compound 11e was generated using Chem-
draw 3D. Three-dimensional structures of the target proteins, iNOS (PDB
Compounds 11a and 11e exhibited IC50 values lower than 2.0 μM
against LPS-induced RAW 264.7 cells at 24 h. The most effective agent
was compound 11e, with an IC50 value of 1.46 M at 24 h. The test
μ
ID: 4CX7), COX-2 (PDB ID: 5IKQ), TNF-α (PDB ID: 3ALQ), IL-1β (PDB ID:
compounds also displayed dose-dependent trends. The results showed
that compounds with an aromatic ring bond with electron-withdrawing
groups exhibited moderate to strong inhibitory activity (11d < 11c <
11i < 11e). The introduction of an F, Cl group at the R3 position resulted
in increased inhibitory potency. With the introduction of an F group at
the R1 position, the inhibitory potency decreased significantly (11i)
when compared to 11e. However, compounds with an aromatic ring
bond with electron-donating groups showed inferior inhibitory activity
(11b, 11f, 11 g, and 11 h).
1ITB), IL-6 (PDB ID: 1ALU), were retrieved from the Protein Data Bank
(PDB) (http://www.pdb.org). Compound 11e was docked to target
protein complexes with the molecule considered as a rigid body and the
ligands being flexible. The search was extended over the whole receptor
protein used as blind docking. PyMOL molecular graphics system was
applied to visualize the interactions between ligands and receptors.
2.2.8. 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.
No obvious toxicity was observed for compounds 11d and 11e
(Fig. 2), in which R3 was a Cl or F group. The R3 position with a ben-
zyloxy group brings obvious toxicity (11a). In the case in which the F
substituent was changed from the R3 position to the R1 position, cyto-
toxicity was also increased (11i). A halogen substituent group for R3 was
effective, as compounds 11c, 11e, and 11i all showed good inhibitory
activity. We also found that most of these 3β-pregnenolone esters of
cinnamic acid derivatives showed lower toxicity than the D-ring het-
erocycle derivatives that we previously reported [42], and this lower
toxicity may be related to the C-3 position (–OH) substituent.
3. Results and discussion
3.1. Chemistry
The synthesis of highly functional molecules from simple building
blocks has always attracted the attention of synthetic chemists. Here, we
report the convenient and efficient synthesis of novel steroidal chalcones
with 3β-pregnenolone esters of cinnamic acid derivatives involving aldol
condensation as the first step followed by Steglich esterification as a
later step. The synthesis of compounds 9a–9i and 11a–11i is presented
in Scheme 1. First, chalcone derivatives 9 were prepared via a Claisen-
Schmidt condensation reaction with the corresponding pregnenolone
7 and aromatic aldehyde 8. NaOH was used as a catalyst in the reaction
to obtain 9 (9a-9i). The further esterification of these molecules with
cinnamic acid 10 in dry dichloromethane in the presence of EDCI and
Compounds 11a, 11b, 11c, 11d, 11e and 11i, which strongly sup-
pressed LPS-induced NO generation and had low cytotoxicity in RAW
264.7 cells, were chosen for a concentration-dependent experiment in
LPS-induced RAW 264.7 cells (Fig. 2). Compounds 11a and 11e were
found to be the most potent, with an IC50 value of 1.54 and 1.46 μM,
respectively, but lower than dexamethasone, with IC50 values of 0.62
μ
M (Table 1 and Fig. 2). Due to its strong inhibitory activity on NO
generation and minimal cytotoxicity to RAW 264.7 cells, compound 11e
was selected for further evaluation.
Fig.2. The effects of compound 11a, 11b, 11c, 11d,
11e, 11i and dexamethasone on NO production and
cell viability in LPS-induced RAW 264.7 cells. RAW
264.7 cells were pretreated with various concentra-
tions (0.1, 0.3, 1, 3, 10 and 30 μM/L) of compound
11a, 11b, 11c, 11d, 11e, 11i and dexamethasone
for 60 min and then stimulated with LPS (1 μg/mL)
for 24 h. The MTT assays results are expressed as a
percentage of the respective control. Data shown are
the mean ± SD for each group (n = 3) and are
representative of three independent experiments.
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X. Cai et al.
Steroids 171 (2021) 108830
Fig.3. Cell viability of compound 11e in RAW 264.7 cells. Cells were incubated with the indicated concentrations of compound 11e (0, 0.1, 0.3, 1, 3, 10 and 30
μM/
L) for 60 min and then stimulated with LPS (1 g/mL) for 24 h. The results are expressed as a percentage of the respective control (A). The effect of compound 11e on
μ
NO production in LPS-induced RAW 264.7 cells. RAW 264.7 cells were pretreated with various concentrations of compound 11e for 60 min and then stimulated with
LPS (1
μ
g/mL) for 24 h (B). 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.
3.3. Cytotoxic effects of compound 11e on cell viability
can be important targets in the treatment or control of inflammation. To
investigate the anti-inflammatory effects of compound 11e in LPS-
induced RAW 264.7 cells, the NO levels were determined (Fig. 3B).
NO production was almost undetectable in cells without LPS stimulation
Inflammation can be defined as the orchestrated response of in-
flammatory mediators, such as pro-inflammatory cytokines (TNF-α, IL-
6, and IL-1), prostaglandin, and the free radical NO, against tissue
injury or infection. Prior to the determination of the potential anti-
inflammatory activity of compound 11e, the cytotoxic effect of com-
pound 11e on LPS-induced inflammation was assessed in RAW 264.7
cells. As shown in Fig. 3A, the determination of optical density by MTT
assay showed that 24 h LPS treatment significantly decreased the pro-
liferation of RAW 264.7 cells compared to that of control cells, and the
application of LPS with compound 11e had no obvious effect on the
(Fig. 3B). Exposure to 1 μg/mL LPS significantly increased NO produc-
tion in RAW 264.7 cells compared to the control group, and compound
11e significantly reduced the LPS-induced level of NO production in the
cells. Treatment with 0.3–30 μM compound 11e markedly reduced the
LPS-mediated NO production by 29.43 ± 2.14%, 48.23 ± 1.60%, 64.70
± 2.64%, 71.76 ± 4.51%, and 75.37 ± 5.90%. This result indicates that
compound 11e maximally inhibits NO generation by activated RAW
264.7 cells.
proliferation of RAW 264.7 cells. However, 0.1–30 μM/L compound 11e
increased the cell viability of LPS-stimulated RAW 264.7 cells compared
3.5. Effects of compound 11e on the production of IL-6, PGE-2, and TNF-
to the group receiving LPS treatment alone. As a result, compound 11e
α
did not show any cytotoxicity at concentrations up to 30 μM, whereas
10
μ
M and 30
μ
M compound 11e slightly reduced cell viability in the
The in vitro anti-inflammatory effect of compound 11e was evaluated
presence of 1
μ
g/mL LPS (Fig. 3A).
by determining the decrease in IL-6, PGE-2 and TNF-α in activated RAW
264.7 cells. The levels of these inflammatory cytokines were analysed
using ELISA kits. LPS treatment alone resulted in a significant increase
(P < 0.01) in cytokine production by RAW 264.7 cells compared with
the control group (Fig. 4). Furthermore, pretreatment with higher con-
3.4. Effects of compound 11e on the production of NO
Although NO is responsible for host defence mechanisms, a high
concentration of NO can cause toxicity and damage host cells. Excessive
NO production is associated with the pathogenesis of inflammatory
diseases, including atherosclerosis, vascular disease and septic shock
[44]. Nitric oxide is synthesized by iNOS and COX-2. Overexpression of
both iNOS and COX-2 is commonly associated with inflammation.
Therefore, the production of NO and the expression of iNOS and COX-2
centrations of compound 11e (3 and 10 μM) reduced the levels of IL-6
and PGE-2. As shown in Fig. 4, treatment with compound 11e led to a
decrease in PGE-2 from 451.86 ± 21.17 to 262.82 ± 22.04 pg/mL (P <
0.01) and 451.86 ± 21.17 to 254.20 ± 8.16 pg/mL (P < 0.01) , corre-
sponding to a decrease in IL-6 from 224.94 ± 6.03 to 180.76 ± 1.48 pg/
mL (P < 0.01) and 224.94 ± 6.03 to 167.78 ± 1.46 pg/mL (P < 0.01).
Fig.4. Effect of compound 11e on IL-6 (A), PGE-2 (B) and TNF-
concentrations of compound 11e (0, 1, 3, or 10
α (C) release in LPS-induced RAW 264.7 cells. RAW 264.7 cells were preincubated with different
μ
M/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).
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X. Cai et al.
Steroids 171 (2021) 108830
Fig.5. Effects of compound 11e on the mRNA levels of TNF-
α
, iNOS, IL-6 and COX-2 in LPS-induced RAW 264.7 cells. RAW 264.7 cells were preincubated with
different concentrations of compound 11e (0, 1, 3, or 10 μM/L) for 60 min prior to LPS stimulation. After treatment with LPS (1 μg/mL) for 6 h, total RNA was
isolated, and iNOS (B) and mRNA levels of TNF-α (A), IL-6 (C) and COX-2 (D) were determined by quantitative RT-PCR. The data are representative of three in-
dependent experiments. (*P < 0.05 and **P < 0.01 vs. the LPS group; ##P < 0.01 vs. the control group).
Incubation with high doses of compound 11e effectively downregulated
3.6. Effects of compound 11e on the expression of genes encoding pro-
the production of IL-6 and PGE-2 in a dose-dependent manner. However,
inflammatory proteins
compound 11e produced a much weaker inhibitory effect on TNF-
α
production. Briefly, compound 11e treatment exerted an inhibitory ef-
Inflammatory cytokines, including TNF-α, iNOS, IL-6 and COX-2, are
fect on the IL-6, PGE-2, and TNF-
264.7 cells.
α
expression levels in activated RAW
also associated with inflammatory disorders. Therefore, we tested
whether compound 11e inhibited the transcriptional expression of these
cytokines and inflammatory enzymes using quantitative real-time (RT)-
PCR analysis. The stimulation of RAW 264.7 macrophages with LPS
remarkably increased the mRNA levels of TNF-
α (Fig. 5A), iNOS
(Fig. 5B), IL-6 (Fig. 5C) and COX-2 (Fig. 5D). Similar to the ELISA
Fig.6. Effects of compound 11e on LPS-induced expression of COX-2, IL-1β and iNOS proteins. (A, B) Compound 11e reduced the protein expression of COX-2, IL-1β
and iNOS in LPS-induced RAW 264.7 cells. RAW 264.7 cells were preincubated with the indicated concentrations of compound 11e (0, 1, 3, or 10 μM/L) for 60 min
and then stimulated with LPS for 24 h. Total protein was extracted and subjected to western blot analysis to detect the COX-2, IL-1β and iNOS proteins. The data are
representative of three independent experiments. (*P < 0.05 and **P < 0.01 vs. the LPS group; #P < 0.05 and ##P < 0.01 vs. the control group).
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Steroids 171 (2021) 108830
Fig.7. Compound 11e suppressed the expression of inflammatory mediators by inhibiting NF-κB signalling in RAW 264.7 cells. (A, B) Compound 11e reduced LPS-
induced NF-κB signalling in RAW 264.7 cells. RAW 264.7 cells were preincubated with the indicated concentrations of compound 11e (0, 1, 3, or 10 μM/L) for 60 min
and then stimulated with LPS for 24 h. Nuclear extracts (NE) and cytosolic extracts (CE) were used for western blot analyses. The data are representative of three
independent experiments. (*P < 0.05 and **P < 0.01 vs. the LPS group; ##P < 0.01 vs. the control group).
results, compound 11e markedly prevented the LPS-induced production
Table 2
of TNF-α, iNOS, IL-6 and COX-2 protein in a dose-dependent manner.
Docking score (kcal/mol) of potential active compounds 11a-11i and standard
drug dexamethasone to five target proteins.
These findings indicated that compound 11e exerted significant anti-
inflammatory effects in LPS-induced RAW 264.7 cells by suppressing
Compounds
Target proteins
the mRNA and protein expression of TNF-α, iNOS, IL-6 and COX-2.
iNOS
COX-2
TNF-
α
IL-1β
IL-6
11a
ꢀ 13.2
12.7
ꢀ 10.4
ꢀ 10.1
ꢀ 10.3
ꢀ 10.4
ꢀ 11.1
ꢀ 10.9
ꢀ 9.7
ꢀ 8.1
ꢀ 7.6
ꢀ 8.2
ꢀ 8.0
ꢀ 8.6
ꢀ 8.1
ꢀ 7.7
ꢀ 7.9
ꢀ 7.8
ꢀ 6.7
ꢀ 9.2
ꢀ 8.9
ꢀ 8.9
ꢀ 9.3
ꢀ 9.3
ꢀ 9.2
ꢀ 8.9
ꢀ 8.6
ꢀ 9.0
ꢀ 6.5
ꢀ 9.1
ꢀ 8.2
ꢀ 8.2
ꢀ 8.5
ꢀ 8.1
ꢀ 8.4
ꢀ 8.3
ꢀ 8.4
ꢀ 8.7
ꢀ 6.6
3.7. Effects of compound 11e on COX-2, IL-1β and iNOS protein
11b
expression
11c
ꢀ 12.0
ꢀ 12.4
ꢀ 13.9
ꢀ 13.8
ꢀ 12.3
ꢀ 12.3
ꢀ 13.9
ꢀ 8.7
11d
11e
iNOS in macrophages may produce excessive NO to exert toxic ef-
fects on cells in response to inflammatory stimuli, such as LPS and cy-
tokines. It is accepted that macrophages may induce the expression of
inflammatory enzymes, such as iNOS and COX-2, during inflammatory
responses [44]. Compound 11e significantly reduced the expression of
COX-2, IL-1β and iNOS mRNAs, and we therefore determined their
protein levels to further investigate this anti-inflammatory effect. As
revealed in Fig. 6, in response to LPS, the levels of these proteins were
upregulated. In contrast, pretreatment with compound 11e obviously
inhibited the upregulation in COX-2, IL-1β and iNOS protein expression.
The significant increases in COX-2, IL-1β and iNOS protein levels
observed in LPS-induced RAW 264.7 cells were markedly reduced by
11f
11 g
11 h
ꢀ 10.0
ꢀ 11.5
ꢀ 8.2
11i
dexamethasone
results suggest that the LPS-stimulated activation of NF-κB might be
blocked by compound 11e.
3.9. Molecular docking analysis
treatment with 3 or 10 μM compound 11e (Fig. 6A-6B). These findings
The binding affinity of a ligand to a target is indispensable for tight
association, to attenuate the drug concentration and to lower the risk of
side effects engendered by nonspecific or nontarget binding during
treatment [46,47]. In vitro anti-inflammatory profiles of steroidal chal-
cones with 3β-pregnenolone esters were further confirmed by molecular
docking experiments. This molecular docking study aimed to rationalize
and verify the obtained biological data and to explain the possible in-
teractions of the derivatives with the crystal structures of the iNOS
indicated that compound 11e exerted significant anti-inflammatory ef-
fects in LPS-induced RAW 264.7 cells by suppressing the mRNA and
protein expression of COX-2, IL-1β and iNOS.
3.8. Effects of compound 11e on NF-κB signalling
NF-κB is an important transcription factor that regulates the
expression of inflammatory mediators. Nucleus translocation of p65-NF-
κB is considered a prerequisite for transcription [45]. The phosphory-
(4CX7), COX-2 (5IKQ), TNF-α (3ALQ), IL-1β (1ITB), and IL-6 (1ALU)
enzymes. The interaction study was compared with dexamethasone, a
standard steroidal anti- inflammatory drug.
lation of IκB
α is upstream of p65-NFκB translocation. The activation of
IKKβ is crucial for the phosphorylation of IκB
α
and the translocation of
The docking scores (kcal/mol) of potential active compounds 11a-
11i and the standard drug dexamethasone to five target proteins are
shown in Table 2. The autodock results of the synthesized compounds
into the binding site of iNOS revealed that compounds 11a, 11e, 11f and
11i exhibited the highest binding affinities through their lowest binding
free energies of ꢀ 13.2, ꢀ 13.9, ꢀ 13.8 and ꢀ 13.9 kcal/mol, respectively.
These derivatives also displayed at least fourteen hydrophobic bonds
with the key amino acids of iNOS. On the other hand, the autodock
results of the synthesized compounds into the binding site of COX-2
revealed that compounds 11e, 11f and 11i exhibited very high bind-
ing affinities through their very low binding free energies of ꢀ 11.1,
ꢀ 10.9 and ꢀ 11.5 kcal/mol, respectively. These compounds also dis-
played at least fifteen hydrophobic bonds with the key amino acids of
COX-2. Compounds 11e and 11i displayed one hydrogen bond with the
NFκB in a canonical pathway. To assess whether the inhibitory effects of
compound 11e on COX-2, IL-1β and iNOS expression were mediated by
NF-κB, we examined the effects of compound 11e on the LPS-induced
expression of NF-κB p65 and on the degradation of the upstream pro-
teins IκBα and IKKβ. The expression levels of NF-κB p65 were deter-
mined by western blot using the nuclear and cytosolic extracts of RAW
264.7 cells. The NF-κB p65 protein was markedly increased after
exposure to LPS. However, this increase was alleviated in the cells
treated with 3 or 10 μM compound 11e (Fig. 7A-7B). Exposure of RAW
264.7 cells to LPS significantly increased the phosphorylation of IκB
RAW 264.7 cells, whereas compound 11e markedly inhibited the LPS-
induced phosphorylation of IκB . Moreover, compound 11e attenu-
ated the LPS-induced degradation of IκB and IKKβ (Fig. 7A-7B). These
α in
α
α
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X. Cai et al.
Steroids 171 (2021) 108830
Table 3
Structure-activity relationship of potent compounds (11a, 11e, 11i) with inflammatory biomarkers iNOS, COX-2, TNF-
methasone, respectively.
α, IL-1β and IL-6 and standard drug dexa-
Target protein and (PDB
ID)
Ligand molecule
Amino acid residues on docked domains
iNOS (4CX7)
11a
11e
Ile462, Gln263, Glu377, Val352, Trp194, Asn370, Leu209, Tyr489, Phe369, Gly371, Pro350, Arg381, Pro467, Val465
Tyr489, Arg381, Glu377, Met374, Gly371, Asn370, Val352, Phe369, Leu209, Trp194, Arg199, Met120, Ile462, Trp463, Ile201,
Pro466, Pro467
11i
Leu209, Trp194, Gly371, Pro350, Glu377, Ile201, Met374, Pro467, Pro466, Trp463, Ile462, Arg381, Met120, Arg199, Cys200,
Tyr489, Asn370, Phe369, Val352
Dexamethasone
Glu377, Trp372, Ile201, Trp463, Met120, Arg381, Pro467, Cys200, Met374, Arg199
COX-2 (5IKQ)
11a
11e
Leu294, His214, His207, Val295, His386, Tyr385, Gln203, Ala199, Phe200, Leu391, Tyr404, Phe395, Val444, Ile408
Gln289, Glu290, Lys211, Thr212, His214, Phe210, Tyr385, Val447, His388, Trp387, Ala202, Thr206, His207, Gln203, Val291,
Ile274
11i
Gln289, Ile274, Glu290, Val291, Thr212, His388, His207, Val447, Gln203, Leu390, Ala202, Trp387, Tyr385, Thr206, Phe210,
Lys211
Dexamethasone
Arg376, Val538, Gly536, Asn375, Gly225, Val228, Gly227, Asn537, Tyr373, Gln374, Phe142
TNF-
α
(3ALQ)
11a
Ile136, Gln27, Asp45, Leu43, Arg31, Leu37, Glu135, Glu42, Asn30, Trp28, Asn46, Pro139, Leu26
Phe144, Ala145, Glu146, Ser147, Val150, Gly148, Val17, Arg32, Ala18, Pro20
11e
11i
Phe144, Ala145, Gln67, Leu142, Arg138, Tyr141, Asp140, Gly66, Ser65, Asp143, Glu23, Gln21, Pro20
Gly24, Asp143, Ser65, Tyr115, Phe144, Leu142, Gln67, Gly66, Asp140, Glu23
Dexamethasone
IL-1β (1ITB)
11a
Asp142, Phe133, Pro131, Gln81, Val132, Leu82, Ser84, Glu83, Tyr24, Leu80, Glu25, Ser125, Ala127
Pro131, Glu25, Tyr24, Ser84, Glu83, Leu82, Gln81, Leu80, Phe133, Val132
Pro131, Leu80, Gln81, Glu83, Ser84, Tyr24, Leu82, Glu25, Phe133, Val132
Leu80, Tyr24, Pro131, Gln81, Val132, Leu82, Glu25, Thr79, Phe133
11e
11i
Dexamethasone
IL-6 (1ALU)
11a
Arg30, Tyr31, Val115, Ala114, Gln111, Gly35, Asp34, Ser37, Lys171, Leu33
Lys27, Tyr31, Gln111, Gly35, Glu110, Ala114, Ser118, Val121, Gln28
Lys120, Ala114, Val115, Ile32, Tyr31, Gly35, Glu110, Met117, Gln111, Arg113, Ser118, Val121
Arg179, Arg182, Leu33, Leu178, Gln175, Arg30
11e
11i
Dexamethasone
key amino acids of COX-2, Gln289 (Table 3).
its binding potency to pro-inflammatory cytokines. The docked poses of
compound 11e with target proteins (iNOS, COX-2, TNF- , IL-1β, and IL-
The anti-inflammatory potential of compound 11e was confirmed by
α
Fig.8. The docked poses of compound 11e (colored
orange) on iNOS (PDB ID: 4CX7) and COX-2 (PDB ID:
5IKQ) proteins’ binding site. Targeted proteins are
shown in cartoon forms and portrayed in green.
Amino acid residues near this site are shown as sticks
and carbons are portrayed in blue. Fluorine is dis-
played in light blue. The yellow dashed lines represent
hydrogen bonds. Besides it, the 2D representations of
each docked pose and their interactions are shown.
(A) 3D docking diagram of compound 11e and iNOS.
(B) 3D docking diagram of compound 11e and COX-2.
(C) 2D docking diagram of compound 11e and iNOS.
(D) 2D docking diagram of compound 11e and COX-2.
10

X. Cai et al.
Steroids 171 (2021) 108830
Fig.9. The docked poses of compound 11e on TNF-
α
(PDB ID: 3ALQ), IL-1β (PDB ID: 1ITB) and IL-6 (PDB ID: 1ALU) proteins’ binding site. (A) 3D docking diagram of
compound 11e and TNF-
compound 11e and TNF-
α. (B) 3D docking diagram of compound 11e and IL-1β. (C) 3D docking diagram of compound 11e and IL-6. (D) 2D docking diagram of
α. (E) 2D docking diagram of compound 11e and IL-1β. (F) 2D docking diagram of compound 11e and IL-6.
Fig.10. The docked poses of dexamethaone (colored rose red) on iNOS (PDB ID: 4CX7), COX-2 (PDB ID: 5IKQ), TNF-α (PDB ID: 3ALQ), IL-1β (PDB ID: 1ITB) and IL-
6 (PDB ID: 1ALU) proteins’ binding site. (A) 3D docking diagram of dexamethaone and iNOS. (B) 3D docking diagram of dexamethaone and COX-2. (C) 3D docking
diagram of dexamethaone and TNF-
α. (D) 3D docking diagram of dexamethaone and IL-1β. (E) 3D docking diagram of dexamethaone and IL-6.
11

X. Cai et al.
Steroids 171 (2021) 108830
6) are shown in Fig. 8 and Fig. 9. The binding profile of compound 11e
docked with iNOS and COX-2 showed binding energies of ꢀ 13.9 and
ꢀ 11.1 kcal/mol, respectively. Ligand 11e docks into the active sites of
iNOS successfully, produces deep movement into the hydrophilic
(Tyr489) pockets of the receptor and binds to hydrophobic amino acids
(Arg381, Glu377, Met374, Gly371, Asn370, Val352, Phe369, Leu209,
Trp194, Arg199, Met120, Ile462, Trp463, Ile201, Pro466 and Pro467),
indicating that such interactions are almost essential for iNOS inhibitory
activity (Fig. 8A, 8C). Compound 11e also forms one hydrogen bond
with Gln289 of COX-2, and 15 binds to hydrophobic amino acids (with
residues Glu290, Lys211, Thr212, His214, Phe210, Tyr385, Val447,
His388, Trp387, Ala202, Thr206, His207, Gln203, Val291 and Ile274)
(Fig. 8B, 8D). The docking and binding affinity of compound 11e into
the iNOS active site are shown in Fig. 8, where 11e exhibited one
hydrogen bond between its F group and the OH of Tyr489.Additionally,
the docking and binding affinity of compound 11e into the COX-2 active
iNOS and COX-2 receptors and exhibit excellent anti-inflammatory ac-
tivities. It is worth mentioning that compound 11e may exert significant
anti-inflammatory activities by inhibiting the target proteins iNOS and
COX-2. The regulation of these cytokines was confirmed in in vitro
studies examining the immunomodulation potential of compound 11e.
4. Conclusion
In conclusion, we efficiently synthesized a series of novel steroidal
chalcones with 3β-pregnenolone esters of cinnamic acid derivatives 11a-
11i. Among the tested compounds, 3β-3-phenyl acrylate-pregn-5-en-
17β-yl-3′ -(p-fluoro)-phenylprop-2′-en-1′-one (11e) showed potent in-
hibition of inflammatory enzymes. Compound 11e bearing chalcone
analogues (1, 3-diphenyl-2-propen-1-one) and styryl ketone (trans-4-
phenyl-3-buten-2-one) pharmacophores in the side chain plays a vital
role in determining the preferences for the target sites. The significant
inhibitory action of compound 11e could be due to the presence of aryl
groups with strong electron withdrawing substituents such as fluorine.
Later, the potent NO inhibitor 11e was screened for its anti-
inflammatory activity against RAW 264.7 cells. We found that com-
pound 11e exhibited the most potent activity for the inhibition of NO
–
site exhibited one hydrogen bond between its C O group and the NH
–
of Gln289. Compound 11e showed better anti-inflammatory activitie2s
than the other synthesized compounds as they exhibited lower binding
free energies. The main interactions of compound 11e with the TNF-α,
IL-1β, and IL-6 enzymes were hydrophobic effects (Fig. 9). However,
compound 11e is highly actively docked with iNOS and COX-2, and this
high binding affinity may be due to the -F and -COOR chains. Previous
literatures have been reported that the introduction of fluorine atoms
may affect the binding strength of molecular ligands and substrate
proteins. Fluorine atoms can increase the lipophilicity of molecules,
which make it easier to pass through the lipid cell membrane, and then
improve the cell activity [48,49].
production, with an IC50 value of 1.46 μM, in LPS-induced RAW 264.7
cells. We also showed that compound 11e exhibited promising inhibi-
tory effects on the expression of pro-inflammatory factors, including
TNF-α, IL-6, and PGE-2, in vitro. Compound 11e significantly inhibited
the LPS-induced expression of iNOS, TNF-α, IL-6, and COX-2 mRNA in a
dose-dependent manner in RAW 264.7 cells. Furthermore, the anti-
inflammatory effects of compound 11e were likely achieved through
the inhibition of NF-κB signal transduction pathways in activated RAW
264.7 cells. The molecular binding of compound 11e to pro-
inflammatory and anti-inflammatory cytokines asserted the alteration/
function of the corresponding target proteins. The docked pose of
The main interactions of the standard drug dexamethasone with the
five enzymes were investigated. The hydroxy group of dexamethasone
exhibited three hydrogen bonds with the key amino acids of iNOS:
Glu377, Trp372 and Ile201. Additionally, other residues in the binding
pocket may be involved in such interactions, including Trp463, Met120,
Arg381, Pro467, Cys200, Met374 and Arg199 as shown in Fig. 10,
whereas, the main amino acids involved in COX-2 interaction with the
ligand dexamethasone are Asn375, Gly225, Val228, Gly227, Asn537,
Tyr373, Gln374 and Phe142. Arg376, Val538 and Gly536 form three
hydrogen bonds with the ligand dexamethasone. The main amino acids
compound 11e with the target proteins (iNOS, COX-2, TNF-α, IL-1β, and
IL-6) confirmed that compound 11e acted as an anti-inflammatory
mediator. Collectively, our initial pharmacological data revealed that
these novel structural compounds, especially compound 11e, might
serve as useful lead compounds for the development of therapeutic
agents for inflammation-related diseases.
involved in TNF-α, IL-1β, and IL-6 interactions with the ligand dexa-
methasone are shown in Fig. 10.
Acknowledgements
Table 3 depicts the structure–activity relationship of potent com-
pounds (11a, 11e, 11i) and the standard drug dexamethasone with the
This work was supported by a grant from Guangdong province
Special Funds for Science and Technology of China (Shantou Govern-
ment No. [2019] 113-66), and the teamwork projects were funded by
Guangdong collaborative innovation and environment construction
Foundation of China (No. 2016B090918129) and Shantou Science and
Technology Project (No. [2019] 77-5) and Shantou Science and Tech-
nology Project (No. [2018] 155-11).
inflammatory biomarkers iNOS, COX-2, TNF-α, IL-1β and IL-6. Hydro-
phobic interactions between ligand 11e and the active residues (with
residues Pro131, Glu25, Tyr24, Ser84, Glu83, Leu82, Gln81, Leu80,
Phe133, Val132) of the IL-1β protein are showed in Table 3. The ligand
docked with the IL-1β protein showed a ꢀ 9.3 kcal/mol binding energy.
The binding profile of compound 11e docked with TNF-α showed a
ꢀ 8.6 kcal/mol binding energy. The hydrophobic interactions between
the compound 11e and the active residues of the TNF- protein were
α
Author Contributions
Phe144, Ala145, Glu146, Ser147, Val150, Gly148, Val17, Arg32, Ala18
and Pro20. The pose of the docked ligand–protein IL-6 interaction was
found to have a ꢀ 8.1 kcal/mol binding energy. The hydrophobic in-
teractions between the ligand and the active residues of the protein were
Lys27, Tyr31, Gln111, Gly35, Glu110, Ala114, Ser118, Val121 and
Gln28. The docking and binding affinity of compound 11a for the IL-6
Xiaorui Cai and Yicun Chen designed the research; Xiaorui Cai per-
formed the synthetic work, Yicun Chen was responsible for the direction
of the biological research, Xiaorui Cai, Fei Sha and Chuanyi Zhao per-
formed the anti-inflammatory activity. Xiaorui Cai wrote the manu-
script, and Yicun Chen was responsible for the correspondence of the
manuscript. All authors discussed, edited and approved the final version.
–
active site exhibited one hydrogen bond between its C O group and
–
the NH2 of Arg30.The docking and binding affinity of compound 11i for
COX-2 and the TNF-
α
active site exhibited one or three hydrogen bonds
Conflicts of Interest
–
between its C O group and the NH of Gln289, Phe144, Ala145 and
–
2
Gln67, respectively.
The authors declare no conflict of interest.
References
Studies on the docking of compound 11e to pro-inflammatory cy-
tokines revealed the alteration and function of compound 11e on its
corresponding target proteins. The acquired structure–activity rela-
tionship shows that appropriately substituted derivatives have the
necessary geometry to provide potent and selective inhibition of the
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