Synthesis and biological evaluation of ursolic acid derivatives bearing triazole moieties as potential anti-Toxoplasma gondii agents

Article abstract of DOI:10.1080/14756366.2019.1584622

Ursolic acid (UA), a plant-derived compound, has many properties beneficial to health. In the present study, we synthesised three series of novel UA derivatives and evaluated their anti-Toxoplasma gondii activity both in vitro and in vivo. Most derivative

Full text of DOI:10.1080/14756366.2019.1584622

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Synthesis and biological evaluation of ursolic acid
derivatives bearing triazole moieties as potential
anti-Toxoplasma gondii agents
Tian Luan, Chunmei Jin, Chun-Mei Jin, Guo-Hua Gong & Zhe-Shan Quan
To cite this article: Tian Luan, Chunmei Jin, Chun-Mei Jin, Guo-Hua Gong & Zhe-Shan Quan
(2019) Synthesis and biological evaluation of ursolic acid derivatives bearing triazole moieties as
potential anti-Toxoplasmaꢀgondii agents, Journal of Enzyme Inhibition and Medicinal Chemistry,
34:1, 761-772, DOI: 10.1080/14756366.2019.1584622
To link to this article: https://doi.org/10.1080/14756366.2019.1584622
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2019, VOL. 34, NO. 1, 761–772
https://doi.org/10.1080/14756366.2019.1584622
RESEARCH ARTICLE
Synthesis and biological evaluation of ursolic acid derivatives bearing triazole
moieties as potential anti-Toxoplasma gondii agents
a
a
a
b,c
Tian Luan , Chunmei Jin , Chun-Mei Jin , Guo-Hua Gong and Zhe-Shan Quana
ꢀ
ꢀ
aKey Laboratory of Natural Resources and Functional Molecules of the Changbai Mountain, Affiliated Ministry of Education, College of
b
Pharmacy, Yanbian University, Yanji, Jilin, China; First Clinical Medical College of Inner Mongolia University for Nationalities, Tongliao, China;
cInner Mongolia Key Laboratory of Mongolian Medicine Pharmacology for Cardio-Cerebral Vascular System, Inner Mongolia University for
Nationalities, Tongliao, China
ABSTRACT
ARTICLE HISTORY
Received 16 January 2019
Revised 11 February 2019
Accepted 14 February 2019
Ursolic acid (UA), a plant-derived compound, has many properties beneficial to health. In the present
study, we synthesised three series of novel UA derivatives and evaluated their anti-Toxoplasma gondii
activity both in vitro and in vivo. Most derivatives exhibited an improved anti-T. gondii activity in vitro
when compared with UA (parent compound), whereas compound 3d exhibited the most potent anti-T.
gondii activity in vivo. Spiramycin served as the positive control. Additionally, determination of biochemical
parameters, including the liver and spleen indexes, indicated compound 3d to effectively reduce hepato-
toxicity and significantly enhance anti-oxidative effects, as compared with UA. Furthermore, our molecular
docking study indicated compound 3d to possess a strong binding affinity for T. gondii calcium-depend-
ent protein kinase 1 (TgCDPK1). Based on these findings, we conclude that compound 3d, a derivative of
UA, could act as a potential inhibitor of TgCDPK1.
KEYWORDS
Toxoplasma gondii; ursolic
acid; molecular docking;
TgCDPK1; in vivo; in vitro
1. Introduction
ursolic acid (UA) and its derivatives have been reported to possess
a wide range of biological activities, including anti-cancer6,7, anti-
diabetic8, anti-HIV9, anti-malarial10, anti-microbial, and anti-inflam-
matory activities11,12. Until recently, Choi et al. reported that UA
not only has strong anti-proliferative activity against T. gondii
activity as well as increases survival of T. gondii-infected mice but
also has the potential to be used as a promising anti-T. gondii
candidate for developing effective anti-parasitic drugs13. To the
best of our knowledge, studies related to anti-T. gondii activity of
any UA derivatives have not yet been reported. Besides, the
higher cytotoxicity in vitro and the low bioavailability in vivo of
UA restrict its clinical application14,15. Therefore, the present study
involved synthesis of different structurally modified compounds
of UA with significantly improved anti-T. gondii activity and
lower toxicity.
Recently, the chemistry of triazoles and their fused phenyl
derivatives has received considerable attention owing to their
effective biological and synthetic importance16–18. Sharling et al.
reported that a series of 1,2,3-triazoles conjugate phenyl deriva-
tives facilitated the development of potential anti-parasitic agents,
of which, five derivatives exhibited excellent in vitro selectivity for
T. gondii. Among these, compound 1 (Figure 1) exhibited the
most potent anti-T. gondii activity with a selectivity value of more
than 12018. Furthermore, Dzitko et al. reported anti-T. gondii activ-
ity of 3-(thiophen-2-yl)-1,2,4-triazole-5-thione (compound 2). The
Toxoplasma gondii is an opportunistic pathogen that causes infec-
tion in human beings and various animals, thereby severely
impairing their health. Congenital toxoplasmosis, caused by T.
gondii, is especially harmful to pregnant women as the infection
may result in abortion, stillbirth, and abnormality of foetus think-
ing barrier. The infection could also be fatal for immuno-compro-
mised patients1. Owing to the complexity of T. gondii life cycle, its
multifarious pathogenesis and different biological characteristics,
no preventive and medicine-specific treatment exists currently.
Traditional anti-T. gondii drugs have various disadvantages, such
as the inability to completely kill the protozoa and oocysts, high
toxicity, frequent recurrence, and failure in immuno-compromised
individuals2,3. Considering the increasing percentage of natural
product-based drugs in the market in the past years, researchers
have now focussed their attention to plant-based compounds
with anti-T. gondii activity. Moreover, several studies have shown
natural products and their derivatives to exert strong anti-T. gondii
effects, making these an attractive source of anti-T. gondii drugs4,5
In this regard, structural modifications of natural products to gen-
erate effective and less-toxic derivatives are considered to be very
promising for the development of anti-T. gondii drugs.
Pentacyclic triterpenes are a diverse and large class of natural
products that are widely distributed in the plant kingdom. Over
the decades, the synthesis of novel pentacyclic triterpenes has
.
gained much attention in medicinal chemistry. Among these, compound displayed significant and reproducible anti-parasitic
CONTACT Zhe-Shan Quan
zsquan@ybu.edu.cn; Chun-Mei Jin
cmjin@ybu.edu.cn
Key Laboratory of Natural Resources and Functional Molecules of the
Changbai Mountain, Affiliated Ministry of Education, College of Pharmacy, Yanbian University, Yanji, Jilin 133002, China; Guo-Hua Gong
gongguohua0211@163.com
These authors contributed equally to this work.
First Clinical Medical College of Inner Mongolia University for Nationalities, Tongliao, China
ꢀ
Supplemental data for this article can be accessed here.
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

762
T. LUAN ET AL.
R
N
N
N
OH
H
Cl
O
H
O
H
N
N
N
1a-1j
O
OH
O
HO
O
Molecular
O
N
O
N
Hybridization
H
N
H
UA
N
O
N
H
R
Compound I
HO
H
2a-2e
O
OH
N
HN
O
N
S
S
H
UA-1
O
Compound II
H
N
N
O
N
H
HO
H
R
3a-3e
Figure 1. Design of target compounds based on the combination principles.
effects in vitro, with selectivity values of 4.58 and 5.21 using 2.2. General procedure for synthesis of intermediates (UA-1,
3[H]uracil incorporation method and qRT-PCR, respectively19. Ia-Ij, IIa-IIe, IIIa-IIIe)
These studies indicate triazole-based compounds to have potential
The compound UA-1 was synthesised as per the protocol
described in a previous study 6. Ia-Ij (different 1-phenyl-1H-1,2,3-
triazole-4-carbaldehyde) and IIa-IIe (different 2-chloro-N-((1-phe-
nyl-1H-1,2,3-triazol-4-yl)methyl)acetamide) were prepared as previ-
ously described17,20. IIIa-IIIe were prepared as per Scheme 1:
different substitutions of benzoyl hydrazide (10 mmol) and N,N-
Dimethylformamide dimethyl acetal (DMFDMA; 1.31 g, 11 mmol)
were added to CH3CN (20 ml); the resulting mixture was stirred at
60 ꢂC for 1 h. Then, 2-aminoethanol (1.22 g, 20 mmol) and
CH3COOH (2.40 g, 40 mmol) were added, and the resulting mixture
was stirred at 90 ꢂC for 8–12 h. After confirming the reaction pro-
gress by TLC, the solvent was evaporated in vacuo. The mixture
was then purified using silica gel column chromatography and
eluted using a gradient of dichloromethane:methanol (100:1–40:1)
to obtain different 2–(3-phenyl-4H-1,2,4-triazol-4-yl)ethanol deriva-
tives. These products were placed in CHCl3 (20 ml) and 5 molar
ratios of sulfoxide chloride was added. The mixture was stirred at
60 ꢂC for 3 h. Upon completion, the solvent and excessive sulfox-
ide chloride was evaporated in vacuo to obtain different inter-
mediates, which were used in the next step without further
purification.
inhibitory activity against anti-T. gondii.
The aforementioned findings stimulated our interest in design-
ing and synthesising three series of novel UA derivatives by link-
ing different fragments containing 1,2,3-triazole and 1,2,4-triazole
and studying their effects against T. gondii, initially at the cellular
level. We next tested each of these derivatives for the strongest
anti-T. gondii activity in vivo, since in vivo effects are an important
factor in evaluating anti-parasitic activity. Finally, we aimed to
gain a better understanding of the molecular basis of inhibitory
potency of compounds against T. gondii. For this, we identified
three enzymes through literature search as reasonable targets for
discovering anti-T. gondii agents, and by using the molecular
docking approach, we aimed at finding the possible target.
2. Materials and methods
2.1. General procedures
All reactions were monitored by thin-layer chromatography (TLC)
performed on silica gel plates. Melting points were determined in
open capillary tubes and were uncorrected. Purity of final prod-
ucts was determined using a preparative high-performance liquid
chromatography (HPLC) system (HP-Q-P050; Agela Technologies)
with a C-18 column as the stationary phase (Agela Technologies,
2.3. General procedure for synthesis of compound (1a–1j)
Venusil PrepG, 120 Å, 10 lm, 10 mm ꢁ 250 mm). The nuclear mag- A mixture of UA-1 (90.8mg, 0.20 mmol), KOH (112 mg, 2.0mmol)
netic resonance (1H-NMR and 13C-NMR) spectra were recorded and different 1-phenyl-1H-1,2,3-triazole-4-carbaldehydes (0.21 mmol)
with AV-300 spectrometers (Bruker BioSpin, Switzerland); all chem- was prepared in CH3CH2OH (10 ml) and stirred at 30ꢂC for 3–5 h.
ical shifts were expressed in ppm relative to tetramethylsilane Progress of reaction was confirmed by TLC, following which the
(TMS), used as the internal standard. High-resolution mass spectra solvent was evaporated in vacuo. The mixture was neutralised with
were recorded using the Thermo Scientific LTQ Orbitrap XL in the hydrochloric acid, extracted with 15 ml ethyl acetate, and then
electrospray ionisation (ESI) mode. Major chemicals were pur- washed thrice with saline (5ml). The final products were purified
chased from Aldrich Chemical Corporation (Milwaukee, WI, USA). using preparative HPLC equipped with a C-18 column. A gradient
All other chemicals were of analytical grade.
elution was performed with tetrahydrofuran and water as the

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
763
N
O
N
N
N
OH
N
N
N3
a
b
R
R
R
Ia-Ij
Cl
N
HN
N
N
N
NH2
N
N
O
N3
c
d
R
R
R
IIa-IIe
O
O
N
N
N
N
NH2
N
N
g
N
N
e
f
R
R
H
H
N
N
R
R
OH
Cl
IIIa-IIIe
.
Scheme 1. Reagents and conditions: (a) propargyl alcohol, CuSO4 5H2O, sodium ascorbate, t-BuOH/H2O (1:1), 30 ꢂC. (b) MnO2, EtOAc, 70 ꢂC. (c) propynylamine, CuSO4
5H2O, sodium ascorbate, t-BuOH/H2O (1:1), 30 ꢂC. (d) chloroacetyl chloride, Et3N, CH2Cl2, 30 ꢂC. (e) DMFDMA, CH3CN, 60 ꢂC. (f) 2-aminoethanol, CH3COOH, 90 ꢂC. (g)
sulfoxide chloride, CHCl3, 60 ꢂC.
mobile phase and was monitored at 220 nm and 254 nm. 1H and (m/z): calculated for C39H51FN3O3
13C-NMR spectra of all the target compounds are available in the found: 628.3907.
Supplementary materials.
[M þ H]þ: 628.3909,
þ
(1S,2R,4aS,6aS,6bR,8aR,12aR,12bR,14bS,E)-11-((1–(3-fluorophenyl)-
1H-1,2,3-triazol-4-yl)methylene)-1,2,6a,6b,9,9,12a-heptamethyl-10-
oxo-1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahy-
dropicene-4a(2H)-carboxylic acid (1c)
(1S,2R,4aS,6aS,6bR,8aR,12aR,12bR,14bS,E)-1,2,6a,6b,9,9,12a-hepta-
methyl-10-oxo-11-((1-phenyl-1H-1,2,3-triazol-4-yl)methylene)-
1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydropi-
cene-4a(2H)-carboxylic acid (1a)
White solid; yield, 80%; m.p. >250 ꢂC; 1H-NMR (CDCl3, 300 MHz,
White solid; yield, 78%; m.p. >250 ꢂC; 1H-NMR (CDCl3, 300 MHz, ppm):
d
8.08 (s, 1H, triazole-H), 7.60 ꢃ 7.50 (m, 4H, Ar–H,
ppm):
d
8.09 (s, 1H, triazole-H), 7.80 ꢃ 7.77 (m, 2H, Ar-H), –CO–C ¼ CH–), 7.23 ꢃ 7.18 (m, 1H, Ar–H), 5.34 (s, 1H, C12–H), 3.54
7.61 ꢃ 7.47 (m, 4H, Ar–H, –CO–C ¼ CH–), 5.35 (s, 1H, C12–H), 3.54 (d, J ¼ 16.5 Hz, 1H, C1–He), 2.48 (d, J ¼ 16.8 Hz, 1H, C1–Ha),
(d, J ¼ 17.4 Hz, 1H, C1–He), 2.48 (d, J ¼ 18.0 Hz, 1H, C1–Ha), 2.16 ꢃ 2.19 (m, 2H), 2.09 ꢃ 2.01 (m, 2H), 1.93 ꢃ 1.90 (m, 1H),
2.27 ꢃ 2.18 (m, 2H), 2.10 ꢃ 2.00 (m, 2H), 1.94 ꢃ 1.89 (m, 1H), 1.83 ꢃ 1.69 (m, 4H), 1.57 ꢃ 1.52 (m, 4H), 1.45 ꢃ 1.27 (m, 5H),
1.83 ꢃ 1.69 (m, 4H), 1.58 ꢃ 1.52 (m, 4H), 1.45 ꢃ 1.27 (m, 5H), 1.19 ꢃ 1.15 (m, 9H), 0.99 ꢃ 0.94 (m, 9H), 0.87 (s, 4H). 13C-NMR
1.19 ꢃ 1.15 (m, 9H), 0.99 ꢃ 0.92 (m, 9H), 0.89 ꢃ 0.85 (m, 4H). 13C- (CDCl3, 125 MHz, ppm): d 207.65, 183.40, 163.15, 145.41, 137.91,
NMR (CDCl3, 75 MHz, ppm): d 207.63, 183.98, 145.17, 137.87, 137.81, 136.14, 131.38, 125.82, 123.28, 122.76, 115.97, 115.89,
136.69, 135.66, 129.89 (2C), 129.04, 125.82, 123.69, 122.94, 120.59 108.39, 53.07, 52.76, 48.10, 45.14, 45.09, 44.94, 42.22, 39.40, 39.17,
(2C), 53.04, 52.70, 48.11, 45.10 (2C), 44.94, 42.19, 39.38, 39.18, 38.87, 36.73, 36.06, 32.09, 30.65, 29.73, 28.02, 24.14, 23.71, 23.47,
38.86, 36.73, 36.04, 32.09, 30.65, 29.73, 28.03, 24.11, 23.70, 23.46, 22.62, 21.16, 20.þ39, 17.14, 16.73, 15.77. ESI-HRMS (m/z): calculated
22.61, 21.17, 20.37, 17.15, 16.75, 15.75. ESI-HRMS (m/z): calculated for C39H51FN3O3 [M þ H]þ: 628.3909, found: 628.3906.
for C39H52N3O3 [M þ H]þ: 610.4003, found: 610.4001.
þ
(1S,2R,4aS,6aS,6bR,8aR,12aR,12bR,14bS,E)-11-((1–(4-fluorophenyl)-
(1S,2R,4aS,6aS,6bR,8aR,12aR,12bR,14bS,E)-11-((1–(2-fluorophenyl)-
1H-1,2,3-triazol-4-yl)methylene)-1,2,6a,6b,9,9,12a-heptamethyl-10-
oxo-1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahy-
dropicene-4a(2H)-carboxylic acid (1b)
1H-1,2,3-triazol-4-yl)methylene)-1,2,6a,6b,9,9,12a-heptamethyl-10-
oxo-1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahy-
dropicene-4a(2H)-carboxylic acid (1d)
White solid; yield, 82%; m.p. >250 ꢂC; 1H-NMR (CDCl3, 300 MHz,
White solid; yield, 82%; m.p. >250 ꢂC; 1H-NMR (CDCl3, 300 MHz, ppm): d 8.04 (s, 1H, triazole-H), 7.79 ꢃ 7.74 (m, 2H, Ar–H), 7.51 (s,
ppm):
d
8.20 (s, 1H, triazole-H), 8.05 ꢃ 7.99 (m, 1H, Ar–H) 1H, –CO–C ¼ CH–), 7.31 ꢃ 7.25 (m, 2H, Ar–H), 5.34 (s, 1H, C12–H),
7.54 ꢃ 7.33 (m, 4H, Ar–H, –CO–C ¼ CH–), 5.35 (s, 1H, C12–H), 3.50 3.53 (d, J ¼ 16.8 Hz, 1H, C1–He), 2.47 (d, J ¼ 18.6 Hz, 1H, C1–Ha),
(d, J ¼ 17.7 Hz, 1H, C1–He), 2.47 (d, J ¼ 18.0 Hz, 1H, C1–Ha), 2.26 ꢃ 2.18 (m, 2H), 2.10 ꢃ 2.01 (m, 2H), 1.94 ꢃ 1.89 (m, 1H),
2.27 ꢃ 2.18 (m, 2H), 2.10 ꢃ 2.01 (m, 2H), 1.94 ꢃ 1.89 (m, 1H), 1.83 ꢃ 1.69 (m, 4H), 1.58 ꢃ 1.50 (m, 4H), 1.46 ꢃ 1.28 (m, 5H),
1.83 ꢃ 1.69 (m, 4H), 1.58 ꢃ 1.52 (m, 5H), 1.46 ꢃ 1.31 (m, 4H), 1.19 ꢃ 1.15 (m, 9H), 1.05 ꢃ 0.92 (m, 9H), 0.87 (s, 4H). 13C-NMR
1.27 ꢃ 1.15 (m, 10H), 0.99 ꢃ 0.92 (m, 10H), 0.90 ꢃ 0.87 (m, 2H). 13C- (CDCl3, 75 MHz, ppm): d 207.62, 183.80, 145.28, 137.91, 135.85,
NMR (CDCl3, 75 MHz, ppm): d 207.55, 183.78, 154.98, 151.66, 132.94, 126.92, 125.78, 123.50, 123.04, 122.65, 122.54, 117.05,
144.91, 137.86, 135.75, 130.36, 125.83, 125.42, 124.72, 123.68, 116.74, 53.05, 52.71, 48.10, 45.11, 45.08, 44.92, 42.19, 39.38, 39.17,
117.30, 117.04, 53.02, 52.71, 48.10, 45.14, 45.11, 44.99, 42.19, 38.86, 36.73, 36.04, 32.08, 30.64, 29.72, 28.03, 24.11, 23.70, 23.46,
39.38, 39.18, 38.85, 36.73, 36.04, 32.09, 30.65, 29.71, 28.02, 24.12, 22.61, 21.16, 20.þ36, 17.14, 16.75, 15.75. ESI-HRMS (m/z): calculated
23.65, 23.45, 22.58, 21.17, 20.36, 17.10, 16.75, 15.77. ESI-HRMS for C39H51FN3O3 [M þ H]þ: 628.3909, found: 628.3907.

764
T. LUAN ET AL.
(1S,2R,4aS,6aS,6bR,8aR,12aR,12bR,14bS,E)-11-((1–(2-chlorophenyl)- (1S,2R,4aS,6aS,6bR,8aR,12aR,12bR,14bS,E)-11-((1–(2-iodophenyl)-
1H-1,2,3-triazol-4-yl)methylene)-1,2,6a,6b,9,9,12a-heptamethyl-10-
oxo-1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahy-
dropicene-4a(2H)-carboxylic acid (1e)
1H-1,2,3-triazol-4-yl)methylene)-1,2,6a,6b,9,9,12a-heptamethyl-10-
oxo-1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahy-
dropicene-4a(2H)-carboxylic acid (1h)
White solid; yield, 81%; m.p. >250 ꢂC; 1H-NMR (CDCl3, 300 MHz, White solid; yield, 75%; m.p. >250 ꢂC; 1H-NMR (CDCl3, 300 MHz,
ppm): d 8.12 (s, 1H, triazole-H), 7.70 ꢃ 7.62 (m, 2H, Ar–H), 7.56 (s, ppm): d 8.07 ꢃ 8.01 (m, 2H, Ar-H, triazole-H), 7.59 ꢃ 7.49 (m, 3H,
1H, –CO–C ¼ CH–), 7.53 ꢃ 7.49 (m, 2H, Ar–H), 5.34 (s, 1H, C12–H), Ar–H, –CO–C ¼ CH–), 7.32 ꢃ 7.29 (m, 1H), 5.34 (s, 1H, C12–H), 3.44
3.45 (d, J ¼ 16.8 Hz, 1H, C1–He), 2.45 (d, J ¼ 17.4 Hz, 1H, C1–Ha), (d, J ¼ 17.7 Hz, 1H, C1–He), 2.46 (d, J ¼ 17.7 Hz, 1H, C1–Ha),
2.26 ꢃ 2.16 (m, 2H), 2.11 ꢃ 2.01 (m, 2H), 1.94 ꢃ 1.89 (m, 1H), 2.27 ꢃ 2.19 (m, 2H), 2.11 ꢃ 2.00 (m, 2H), 1.95 ꢃ 1.89 (m, 1H),
1.82 ꢃ 1.68 (m, 4H), 1.58 ꢃ 1.52 (m, 4H), 1.49 ꢃ 1.27 (m, 5H), 1.82 ꢃ 1.68 (m, 4H), 1.58 ꢃ 1.52 (m, 4H), 1.46 ꢃ 1.27 (m, 5H),
1.20 ꢃ 1.15 (m, 9H), 0.99 ꢃ 0.96 (m, 5H), 0.94 ꢃ 0.92 (m, 3H), 1.20 ꢃ 1.15 (m, 9H), 1.08 ꢃ 0.97 (m, 7H), 0.93 ꢃ 0.91 (m, 3H), 0.87
0.90 ꢃ 0.85 (m, 5H). 13C-NMR (CDCl3, 75 MHz, ppm): d 207.56, (s, 3H). 13C-NMR (CDCl3, 75 MHz, ppm): d 207.61, 183.68, 144.22,
183.51, 144.29, 137.91, 135.65, 134.60, 130.93 (2C), 128.52, 128.07, 140.43, 139.71, 137.94, 135.66, 131.64, 129.41, 127.80, 126.64,
127.63, 126.74, 125.79, 123.87, 53.01, 52.73, 48.10, 45.18, 45.11, 125.74, 124.03, 93.56, 52.96, 52.73, 48.11, 45.21, 45.12(2C),
45.08, 42.21, 39.38, 39.18, 38.85, 36.71, 36.03, 32.09, 30.65, 29.72, 42.21, 39.38, 39.16, 38.85, 36.71, 36.05, 32.08, 30.65, 29.73, 28.00,
28.02, 24.13, 23.69, 23.45, 22.55, 21.15, 20.36, 17.09, 16.75, 15.82. 24.11, 23.76, 23.46, 22.54, 21.15, 20.35, 1þ7.14, 16.75, 15.87. ESI-
ESI-HRMS (m/z): calculated for C39H51ClN3O3 [M þ H]þ: 644.3613, HRMS (m/z): calculated for C39H51IN3O3 [M þ H]þ: 736.2970,
þ
found: 644.3609.
found: 736.2968.
(1S,2R,4aS,6aS,6bR,8aR,12aR,12bR,14bS,E)-11-((1–(3,4-dichloro-
phenyl)-1H-1,2,3-triazol-4-yl)methylene)-1,2,6a,6b,9,9,12a-hepta-
methyl-10-oxo-1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-
octadecahydropicene-4a(2H)-carboxylic acid (1f)
(1S,2R,4aS,6aS,6bR,8aR,12aR,12bR,14bS,E)-11-((1–(2-methoxy-
phenyl)-1H-1,2,3-triazol-4-yl)methylene)-1,2,6a,6b,9,9,12a-hepta-
methyl-10-oxo-1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-
octadecahydropicene-4a(2H)-carboxylic acid (1i)
White solid; yield, 79%; m.p. >250 ꢂC; 1H-NMR (CDCl3, 300 MHz, White solid; yield, 77%; m.p. >250 ꢂC; 1H-NMR (CDCl3, 300 MHz,
ppm): d 8.06 (s, 1H, triazole-H), 7.95 (s, 1H, Ar–H), 7.67 (s, 2H, ppm): d 8.31 (s, 1H, triazole-H), 7.90 (dd, J ¼ 7.8, 1.5 Hz, 1H, Ar–H),
Ar–H), 7.48 (s, 1H, –CO–C ¼ CH–), 5.34 (s, 1H, C12–H), 3.54 (d, 7.59 (s, 1H, –CO–C ¼ CH–), 7.47 (td, J ¼ 8.4, 1.5 Hz, 1H, Ar–H),
J ¼ 15.7 Hz, 1H, C1–He), 2.47 (d, J ¼ 15.4 Hz, 1H, C1–Ha), 2.27 ꢃ 2.16 7.19 ꢃ 7.12 (m, 2H, Ar–H), 5.35 (s, 1H, C12–H), 3.93 (s, 3H,
(m, 2H), 2.10 ꢃ 2.00 (m, 2H), 1.95 ꢃ 1.89 (m, 1H), 1.83 ꢃ 1.69 (m, Ar–OCH3), 3.43 (d, J ¼ 17.4 Hz, 1H, C1–He), 2.43 (d, J ¼ 16.5 Hz, 1H,
4H), 1.58 ꢃ 1.50 (m, 4H), 1.45 ꢃ 1.27 (m, 5H), 1.19–1.52 (m, 9H), C1–Ha), 2.27 ꢃ 2.19 (m, 2H), 2.11 ꢃ 2.02 (m, 2H), 1.94 ꢃ 1.89 (m,
1.07 ꢃ 1.04 (m, 1H), 1.00 ꢃ 0.97 (m, 3H), 0.94 ꢃ 0.92 (m, 5H), 1H), 1.84 ꢃ 1.69 (m, 4H), 1.58 ꢃ 1.52 (m, 4H), 1.46 ꢃ 1.27 (m, 5H),
0.87–0.81 (m, 4H). 13C-NMR (CDCl3, 75 MHz, ppm): d 207.58, 1.20 ꢃ 1.15 (m, 9H), 1.07 ꢃ 0.96 (m, 7H), 0.93 ꢃ 0.91 (m, 3H), 0.88
183.48, 145.60, 137.93, 136.39, 135.67, 134.19, 133.24, 131.62, (s, 3H). 13C-NMR (CDCl3, 75 MHz, ppm): d 207.51, 183.73, 150.77,
125.76, 123.02, 122.59, 122.35, 119.46, 53.05, 52.73, 48.10, 45.14, 143.93, 138.04, 134.74, 130.18, 126.93, 125.92, 125.70, 125.05,
45.09, 44.94, 42.21, 39.38, 39.18, 38.85, 36.72, 36.05, 32.08, 30.64, 124.74, 121.41, 112.32, 55.92, 52.96, 52.76, 48.12, 45.27, 45.18,
29.70, 28.01, 24.12, 23.69, 23.45, 22.61, 21.15, 20.35, 17.12, 16.75, 45.06, 42.24, 39.37, 39.15, 38.84, 36.70, 35.94, 32.12, 30.66, 29.77,
15.75. ESI-HRMS (m/z): calculated for C39H50Cl2N3O3 [M þ H]þ: 28.01, 24.13, 23.71, 23.44, 22.54, 21.15, 20.34, 17.04, 16.73, 15.82.
þ
þ
678.3224, found: 678.3225.
ESI-HRMS (m/z): calculated for C40H54N3O4 [M þ H]þ: 640.4109,
found: 640.4106.
(1S,2R,4aS,6aS,6bR,8aR,12aR,12bR,14bS,E)-11-((1–(2-bromo-
phenyl)-1H-1,2,3-triazol-4-yl)methylene)-1,2,6a,6b,9,9,12a-hepta-
methyl-10-oxo-1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-
octadecahydropicene-4a(2H)-carboxylic acid (1g)
(1S,2R,4aS,6aS,6bR,8aR,12aR,12bR,14bS,E)-1,2,6a,6b,9,9,12a-hepta-
methyl-10-oxo-11-((1–(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-4-
yl)methylene)-1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-
octadecahydropicene-4a(2H)-carboxylic acid (1j)
White solid; yield, 76%; m.p. >250 ꢂC; 1H-NMR (CDCl3, 300 MHz,
ppm):
d
8.09 (s, 1H, triazole-H), 7.82 ꢃ 7.80 (m, 1H, Ar–H) White solid; yield, 76%; m.p. >250 ꢂC; 1H-NMR (CDCl3, 300 MHz,
7.64 ꢃ 7.41 (m, 4H, Ar–H, –CO–C ¼ CH–), 5.34 (s, 1H, C12–H), 3.46 ppm): d 8.04 (s, 1H, triazole-H), 7.50 (s, 1H, –CO–C ¼ CH–), 6.99 (s,
(d, J ¼ 17.1 Hz, 1H, C1–He), 2.45 (d, J ¼ 18.0 Hz, 1H, C1–Ha), 2H, Ar–H), 5.33 (s, 1H, C12–H), 3.97 ꢃ 3.92 (m, 9H, Ar–OCH3), 3.55
2.27 ꢃ 2.18 (m, 2H), 2.10 ꢃ 2.01 (m, 2H), 1.94 ꢃ 1.89 (m, 1H), (d, J ¼ 17.7 Hz, 1H, C1–He), 2.48 (d, J ¼ 17.4 Hz, 1H, C1–Ha),
1.82 ꢃ 1.66 (m, 4H), 1.58 ꢃ 1.47 (m, 6H), 1.42 ꢃ 1.32 (m, 3H), 2.26 ꢃ 2.19 (m, 2H), 2.10 ꢃ 2.01 (m, 2H), 1.94 ꢃ 1.89 (m, 1H),
1.21 ꢃ 1.16 (m, 9H), 1.08 ꢃ 0.97 (m, 6H), 0.94 ꢃ 0.92 (m, 3H), 1.83 ꢃ 1.69 (m, 4H), 1.58 ꢃ 1.52 (m, 4H), 1.45 ꢃ 1.27 (m, 5H),
0.88 ꢃ 0.82 (m, 4H). 13C-NMR (CDCl3, 75 MHz, ppm): d 207.62, 1.19 ꢃ 1.15 (m, 9H), 1.06 ꢃ 0.91 (m, 10H), 0.87 (s, 3H). 13C-NMR
183.67, 145.42, 137.90, 137.52, 136.15, 135.76, 130.97, 129.10, (CDCl3, 75 MHz, ppm): d 207.63, 183.75, 154.01(2C), 145.15, 138.56,
125.79, 123.26, 122.74, 120.86, 118.52, 53.05, 52.72, 48.10, 45.13, 137.93, 135.76, 132.46, 125.75, 123.49, 123.21, 98.44(2C), 61.07,
45.09, 44.94, 42.10, 39.39, 39.18, 38.85, 36.73, 36.05, 32.08, 30.64, 56.45(2C), 53.00, 52.73, 48.11, 45.17, 45.10, 45.06, 42.22, 39.37,
29.71, 28.02, 24.12, 23.70, 23.46, 22.61, 21.15, 20.36, 17.12, 16.75, 39.17, 38.84, 36.71, 35.96, 32.10, 30.65, 29.74, 28.01, 24.12, 23.72,
15.75. ESI-HRMS (m/z): calculated for C39H51BrN3O3 [M þ H]þ: 23.44, 22.58, 21.15, 20.36, 17.05, 16.74, 15.78. ESI-HRMS (m/z): cal-
þ
þ
688.3108, found: 688.3106.
culated for C42H58N3O6 [M þ H]þ: 700.4320, found: 700.4318.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
765
2.4. General procedure for synthesis of compound (2a–2e)
(1S,2R,4aS,6aS,6bR,8aR,12aR,12bR,14bS)-2-((1–(4-methoxyphenyl)-
1H-1,2,3-triazol-4-yl)methylamino)-2-oxoethyl-10-hydroxy-1,2,6a,
6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,
12b,13,14b-icosahydropicene-4a-carboxylate (2c)
A mixture of UA (91.2 mg, 0.20 mmol), K2CO3 (41.5 mg, 0.30 mmol)
and different phenyl 1,2,3-triazole chloroacetamides (0.21 mmol)
in CH3CN (15 ml) was stirred at 60 ꢂC for 2–3 h. After
confirming the reaction progress by TLC, the solvent was evapo-
rated in vacuo. The mixture was dissolved in 15 ml ethyl
acetate, and then washed thrice with saline (5 ml). The final prod-
ucts were purified using preparative HPLC equipped with a C-18
column. A gradient elution was performed with tetrahydrofuran
and water as the mobile phase and monitored at 220 nm
and 254 nm.
White powder; yield, 80%; m.p. 180–181 ꢂC; 1H-NMR (CDCl3,
300 MHz, ppm): d 7.94 (s, 1H, triazole-H), 7.65 (d, J ¼ 9.0 Hz, 2H,
Ar–H), 7.04 (d, J ¼ 9.0 Hz, 2H, Ar–H), 6.87 (t, J ¼ 6.0 Hz, 1H,
–CO–NH–), 5.27 (s, 1H, C12–H), 4.74 ꢃ 4.66 (m, 3H, –CO–O–CH2–,
–CO–NH–CHe), 4.40 (d, J ¼ 15.9 Hz, 1H, –CO–NH–CHa), 3.89 (s, 3H,
ph–OCH3), 3.23 ꢃ 3.17 (m, 1H, C3–OH), 2.24 (d, J ¼ 10.5 Hz, 1H),
2.09 ꢃ 2.03 (m, 1H), 1.93 ꢃ 1.85 (m, 1H), 1.75 ꢃ 1.68 (m, 4H),
1.60 ꢃ 1.45 (m, 9H), 1.42 ꢃ 1.27 (m, 6H), 1.15 ꢃ 1.13 (m, 1H), 1.09
(s, 3H), 1.00 ꢃ 0.98 (m, 7H), 0.90 ꢃ 0.88 (m, 3H), 0.80 (s, 3H), 0.75 (s,
3H), 0.71 ꢃ 0.67 (m, 1H), 0.63 (s, 2H). 13C-NMR (CDCl3, 75 MHz,
ppm): d 176.03, 167.82, 160.02, 144.53, 139.15, 130.21, 125.47,
122.02(2C), 120.88, 114.83(2C), 78.93, 62.79, 55.65, 55.12, 52.88,
48.40, 47.38, 42.14, 39.43, 39.13, 38.77, 38.70, 38.46, 36.87, 36.69,
34.49, 32.78, 30.51, 28.10, 27.85, 27.15, 24.41, 23.61, 23.11, 21.10,
18.20, 16.97, 16.90, 15.59, 15.29. ESI-HRMS (m/z): calculated for
(1S,2R,4aS,6aS,6bR,8aR,12aR,12bR,14bS)-2-oxo-2-((1-phenyl-1H-
1,2,3-triazol-4-yl)methylamino)ethyl-10-hydroxy-1,2,6a,6b,9,9,12a-
heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-
icosahydropicene-4a-carboxylate (2a)
C42H61N4O5 [M þ H]þ: 701.4636, found: 701.4640.
þ
White powder; yield, 80%; m.p. 170–172 ꢂC; 1H-NMR (CDCl3,
300 MHz, ppm): d 8.04 (s, 1H, triazole-H), 7.76 (d, J ¼ 7.8 Hz, 2H,
Ar–H), 7.57 ꢃ 7.44 (m, 3H, Ar–H), 6.88 (t, J ¼ 6.0 Hz, 1H, –CO–NH–),
(1S,2R,4aS,6aS,6bR,8aR,12aR,12bR,14bS)-2-oxo-2-((1-p-tolyl-1H-
5.27 (s, 1H, C12–H), 4.75 ꢃ 4.68 (m, 3H, –CO–O–CH2–, 1,2,3-triazol-4-yl)methylamino)ethyl-10-hydroxy-1,2,6a,6b,9,9,12a-
–CO–NH–CHe), 4.40 (d, J ¼ 15.6 Hz, 1H, –CO–NH–CHa), 3.22 ꢃ 3.17
heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-
icosahydropicene-4a-carboxylate (2d)
(m, 1H, C3–OH), 2.24 (d, J ¼ 11.4 Hz, 1H), 2.12 ꢃ 2.02 (m, 1H),
White powder; yield, 81%; m.p. 186–187 ꢂC; 1H-NMR (CDCl3,
1.93 ꢃ 1.84 (m, 1H), 1.75 ꢃ 1.67 (m, 4H), 1.64 ꢃ 1.62 (m, 2H),
1.58 ꢃ 1.44 (m, 6H), 1.41 ꢃ 1.22 (m, 6H), 1.15 ꢃ 1.13 (m, 1H), 1.09
300 MHz, ppm): d 7.99 (s, 1H, triazole-H), 7.62 (d, J ¼ 8.4 Hz, 2H,
(s, 3H), 0.98 ꢃ 0.97 (m, 7H), 0.90 ꢃ 0.88 (m, 4H), 0.77 ꢃ 0.74 (m, 5H), Ar–H), 7.33 (d, J ¼ 8.4 Hz, 2H, Ar–H), 6.88 (t, J ¼ 5.7 Hz, 1H,
0.70 ꢃ 0.66 (m, 1H), 0.63 (s, 3H). 13C-NMR (CDCl3, 75 MHz, ppm): d
176.02, 167.81, 144.81, 139.23, 136.82, 129.82(2C), 128.99, 125.45,
120.72, 120.38(2C), 78.94, 62.82, 55.11, 52.90, 48.42, 47.37, 42.15,
39.43, 39.14, 38.78, 38.70, 38.44, 36.85, 36.70, 34.52, 32.77, 30.51,
28.10, 27.84, 27.13, 24.42, 23.62, 23.08, 21.10, 18.20, 1þ6.98, 16.89,
15.57, 15.26. ESI-HRMS (m/z): calculated for C41H59N4O4 [M þ H]þ:
671.4531, found: 671.4528.
–CO–NH–), 5.26 (s, 1H, C12–H), 4.73 ꢃ 4.66 (m, 3H, –CO–O–CH2–,
–CO–NH–CHe), 4.40 (d, J ¼ 15.9 Hz, 1H, –CO–NH–CHa), 3.22 ꢃ 3.18
(m, 1H, C3–OH), 2.43 (s, 3H, ph–CH3), 2.24 (d, J ¼ 11.1 Hz, 1H),
2.12–2.02 (m, 1H), 1.93 ꢃ 1.83 (m, 1H), 1.76 ꢃ 1.66 (m, 5H),
1.63 ꢃ 1.45 (m, 7H), 1.39 ꢃ 1.21 (m, 6H), 1.13 ꢃ 1.12 (m, 1H), 1.08
(s, 3H), 1.03 ꢃ 0.97 (m, 7H), 0.89 ꢃ 0.87 (m, 4H), 0.78 (s, 3H), 0.74 (s,
3H), 0.70 ꢃ 0.66 (m, 1H), 0.62 (s, 2H). 13C-NMR (CDCl3, 75 MHz,
ppm): d 175.99, 167.73, 144.76, 139.19, 139.00, 134.62, 130.26(2C),
125.46, 120.50, 120.26(2C), 78.92, 62.81, 55.11, 52.89, 48.40, 47.36,
42.14, 39.42, 39.13, 38.77, 38.69, 38.44, 36.85, 36.69, 34.63, 32.76,
30.50, 28.09, 27.82, 27.14, 24.42, 23.61, 23.08, 21.09(2C), 18.19,
16.97, 16.88, 15.55, 15.26. ESI-HRMS (m/z): calculated for
(1S,2R,4aS,6aS,6bR,8aR,12aR,12bR,14bS)-2-((1–(4-chlorophenyl)-
1H-1,2,3-triazol-4-yl)methylamino)-2-oxoethyl-10-hydroxy-
C42H61N4O4 [M þ H]þ: 685.4687, found: 685.4685.
þ
1,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,
12,12a,12b,13,14b-icosahydropicene-4a-carboxylate (2b)
(1S,2R,4aS,6aS,6bR,8aR,12aR,12bR,14bS)-2-((1–(3,4-dichloro-
phenyl)-1H-1,2,3-triazol-4-yl)methylamino)-2-oxoethyl-10-hydroxy-
1,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,
12,12a,12b,13,14b-icosahydropicene-4a-carboxylate (2e)
White powder; yield, 82%; m.p. 176–178 ꢂC; 1H-NMR (CDCl3,
300 MHz, ppm): d 8.02 (s, 1H, triazole-H), 7.72 (d, J ¼ 8.7 Hz, 2H,
Ar–H), 7.53 (d, J ¼ 8.7 Hz, 2H, Ar–H), 6.88 (t, J ¼ 6.0 Hz, 1H,
–CO–NH–), 5.28 (s, 1H, C12–H), 4.75 ꢃ 4.66 (m, 3H, –CO–O–CH2–,
–CO–NH–CHe), 4.39 (d, J ¼ 15.9 Hz, 1H, –CO–NH–CHa), 3.23 ꢃ 3.18
(m, 1H, C3–OH), 2.24 (d, J ¼ 11.1 Hz, 1H), 2.13 ꢃ 2.03 (m, 1H),
1.96 ꢃ 1.86 (m, 1H), 1.80 ꢃ 1.67 (m, 5H), 1.64 ꢃ 1.46 (m, 8H),
1.42 ꢃ 1.24 (m, 6H), 1.16 ꢃ 1.13 (m, 1H), 1.09 (s, 3H), 1.01 ꢃ 0.94
(m, 8H), 0.90 ꢃ 0.88 (m, 3H), 0.82 ꢃ 0.79 (m, 3H), 0.75 (s, 2H),
0.71 ꢃ 0.67 (m, 1H), 0.63 (s, 2H). 13C-NMR (CDCl3, 75 MHz, ppm): d
176.04, 167.96, 143.16, 139.26, 135.24, 134.94, 130.04(2C), 125.43,
121.59(2C), 120.89, 78.95, 62.77, 55.11, 52.91, 48.42, 47.38, 42.17,
39.44, 39.14, 38.78, 38.70, 38.48, 36.88, 36.70, 34.42, 32.79, 30.51,
28.10, 27.85, 27.13, 24.42, 23.62, 23.13, 21.11, 18.22, 16.98, 16.92,
White powder; yield, 86%; m.p. 203–204 ꢂC; 1H-NMR (CDCl3,
300 MHz, ppm): d 8.03 (s, 1H, triazole-H), 7.93 (s, 1H, Ar–H), 7.63 (s,
2H, Ar–H), 6.88 (t, J ¼ 6.3 Hz, 1H, –CO–NH–), 5.28 (s, 1H, C12–H),
4.76 ꢃ 4.66 (m, 3H, –CO–O–CH2–, –CO–NH–CHe), 4.39 (d,
J ¼ 15.9 Hz, 1H, –CO–NH–CHa), 3.23 ꢃ 3.18 (m, 1H, C3–OH), 2.24 (d,
J ¼ 11.1 Hz, 1H), 2.13–2.03 (m, 1H), 1.95 ꢃ 1.87 (m, 1H), 1.80 ꢃ 1.66
(m, 4H), 1.63 ꢃ 1.60 (m, 4H), 1.53 ꢃ 1.47 (m, 4H), 1.42 ꢃ 1.23 (m,
7H), 1.14 ꢃ 1.10 (m, 4H), 1.03 ꢃ 0.98 (m, 6H), 1.08 (s, 3H),
0.90 ꢃ 0.85 (m, 5H), 0.80 ꢃ 0.76 (m, 5H), 0.72 ꢃ 0.68 (m, 1H), 0.63
(s, 2H), 0.70 ꢃ 0.66 (m, 1H), 0.62 (s, 2H). 13C-NMR (CDCl3, 75 MHz,
ppm): d 175.99, 167.91, 145.51, 139.39, 135.90, 134.12, 133.03,
131.50, 125.38, 122.17, 120.58, 119.23, 78.92, 62.79, 55.10, 52.94,
48.43, 47.37, 42.19, 39.44, 39.14, 38.81, 38.70, 38.49, 36.88, 36.72,
þ
15.58, 15.29. ESI-HRMS (m/z): calculated for C41H58ClN4O4
[M þ H]þ: 705.4141, found: 705.4146.

766
T. LUAN ET AL.
34.59, 32.78, 30.49, 28.09, 27.82, 27.15, 24.42, 23.62, 23.13, 21.09,
2
–(3-(4-fluorophenyl)-4H-1,2,4-triazol-4-
18.21, 16.98, 16.93, 15.56, 15.28. ESI-HRMS (m/z): calculated for
yl)ethyl(1S,2R,4aS,6aS,6bR,
8aR,12aR,12bR,14bS)-10-hydroxy-
þ
C41H57Cl2N4O4 [M þ H]þ: 739.3751, found: 739.3753.
1,2,6a,6b,9,9,12a-heptamethyl-
1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydropi-
cene-4a(2H)-carboxylate (3c)
White powder; yield, 85%; m.p. 221–222 ꢂC; 1H-NMR (CDCl3,
300 MHz, ppm): d 8.32 (s, 1H, triazole-H), 7.68–7.63 (m, 2H, Ar–H),
7.27–7.22 (m, 2H, Ar–H), 5.17 (s, 1H, C12–H), 4.28–4.25 (m, 4H,
–O–CH2CH2–N–), 3.25 ꢃ 3.21 (m, 1H, C3–OH), 2.16 (d, J ¼ 11.1 Hz,
1H), 2.04 ꢃ 1.98 (m, 1H), 1.91 ꢃ 1.85 (m, 2H), 1.69 ꢃ 1.63 (m, 4H),
1.56 ꢃ 1.43 (m, 7H), 1.36 ꢃ 1.25 (m, 6H), 1.08 ꢃ 1.05 (m, 4H),
1.00 ꢃ 0.97 (m, 7H), 0.91 (s, 3H), 0.87 ꢃ 0.84 (m, 3H), 0.79 (s, 3H),
0.72 (d, J ¼ 10.5 Hz, 1H), 0.62 (s, 3H). 13C-NMR (CDCl3, 75 MHz,
ppm): d 177.00, 160.28, 153.00, 144.16, 137.81, 131.25, 130.14,
125.97, 122.05, 116.62, 116.32, 78.94, 62.14, 55.16, 52.88, 48.30,
47.42, 44.18, 41.99, 39.48, 38.99, 38.85, 38.73, 38.54, 36.94, 36.70,
32.85, 30.43, 28.13, 27.87, 27.19, 24.22, 23.57, 23.22, 21.07, 18.26,
17.01, 16.96, 15.62, 15.40. ESI-HRMS (m/z): calculated for
2.5. General procedure for synthesis of compound (3a–3e)
A mixture of UA (91.2 mg, 0.20 mmol), K2CO3 (41.5 mg, 0.30 mmol)
and different 4–(2-chloroethyl)-3-phenyl-4H-1,2,4-triazoles (0.21 mmol)
in CH3CN (15 ml) was stirred at 60 ꢂC for 4–6 h. After confirming the
reaction progress by TLC, the solvent was evaporated in vacuo. The
mixture was dissolved in 15 ml ethyl acetate, and then washed thrice
with saline (5 ml). Final products were purified by preparative HPLC
equipped with a C-18 column. A gradient elution was performed
with tetrahydrofuran and water as the mobile phase and monitored
at 220 nm and 254 nm.
C40H57FN3O3 [M þ H]þ: 646.4378, found: 646.4373.
þ
2
–(3-phenyl-4H-1,2,4-triazol-4-
yl)ethyl(1S,2R,4aS,6aS,6bR,8aR,12aR,
12bR,14bS)-10-hydroxy-
2
–(3-(4-nitrophenyl)-4H-1,2,4-triazol-4-yl)ethyl(1S,2R,4aS,6aS,6bR,
1,2,6a,6b,9,9,12a-heptamethyl-1,3,4,5,6,6a,
8aR,12aR,12bR,14bS)-10-hydroxy-1,2,6a,6b,9,9,12a-heptamethyl-
1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydropi-
cene-4a(2H)-carboxylate (3d)
6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydropicene-4a(2H)-
carboxylate (3a)
White powder; yield, 77%; m.p. 243–244 ꢂC; 1H-NMR (CDCl3,
300 MHz, ppm): d 8.33 (s, 1H, triazole-H), 7.64 ꢃ 7.54 (m, 5H, Ar–H),
5.17 (s, 1H, C12–H), 4.31 ꢃ 4.24 (m, 4H, –O–CH2CH2–N–), 3.24 ꢃ 3.21
(m, 1H, C3–OH), 2.16 (d, J ¼ 11.7 Hz, 1H), 2.08 ꢃ 1.86 (m, 4H),
1.65 ꢃ 1.61 (m, 6H), 1.53 ꢃ 1.44 (m, 5H), 1.40 ꢃ 1.27 (m, 5H),
1.07 ꢃ 1.05 (m, 4H), 1.00 ꢃ 0.97 (m, 7H), 0.91 (s, 3H), 0.85 (d,
J ¼ 6.3 Hz, 3H), 0.79 (s, 3H), 0.72 (d, J ¼ 10.2 Hz, 1H), 0.63 (s, 3H).
13C-NMR (CDCl3, 75 MHz, ppm): d 177.06, 153.94, 144.18, 137.82,
130.34, 129.07 (2C), 128.97 (2C), 126.56, 125.97, 78.96, 62.39, 55.16,
52.88, 48.28, 47.44, 44.00, 41.98, 39.48, 39.00, 38.85, 38.73, 38.55,
36.95, 36.68, 32.85, 30.46, 28.13, 27.89, 27.20, 24.22, 23.58, 23.23,
21.09, 18.27, 17.01, 16.96, 15.62, 15.41. ESI-HRMS (m/z): calculated
1
White powder; yield, 82%; m.p. >250 ꢂC; H-NMR (CDCl3, 300 MHz,
ppm): d 8.43 ꢃ 8.39 (m, 3H, triazole-H, Ar–H), 7.93 (d, J ¼ 8.7 Hz,
2H, Ar–H), 5.15 (s, 1H, C12–H), 4.33 (dd, J ¼ 17.7, 5.1 Hz, 4H,
–O–CH2CH2–N–), 3.24 ꢃ 3.19 (m, 1H, C3–OH), 2.13 (d, J ¼ 11.1 Hz,
1H), 2.07 ꢃ 1.97 (m, 1H), 1.93 ꢃ 1.77 (m, 2H), 1.63 ꢃ 1.59 (m, 5H),
1.56 ꢃ 1.45 (m, 6H), 1.35 ꢃ 1.25 (m, 6H), 1.07 (s, 4H), 1.00 ꢃ 0.96
(m, 7H), 0.90 (s, 3H), 0.86 ꢃ 0.84 (m, 3H), 0.79 (s, 3H), 0.71 (d,
J ¼ 11.1 Hz, 1H), 0.60 (s, 3H). 13C-NMR (CDCl3, 75 MHz, ppm): d
176.97, 151.99, 148.83, 144.91, 137.76, 132.67, 129.82(2C), 126.00,
124.28(2C), 78.92, 62.08, 55.13, 52.90, 48.33, 47.39, 44.28, 41.98,
39.47, 38.97, 38.87, 38.72, 38.52, 36.92, 36.72, 32.82, 30.40, 28.12,
27.86, 27.16, 24.24, 23.56, 23.19, 21.06, 18.24, 17.02, 16.96, 15.62,
þ
for C40H58N3O3 [M þ H]þ: 628.4473, found: 628.4470.
15.39. ESI-HRMS (m/z): calculated for C40H57N4O5 [M þ H]þ:
þ
673.4323, found: 673.4320.
2
–(3-(4-chlorophenyl)-4H-1,2,4-triazol-4-
yl)ethyl(1S,2R,4aS,6aS,6bR,
8aR,12aR,12bR,14bS)-10-hydroxy-
2
–(3-(4-methoxyphenyl)-4H-1,2,4-triazol-4-yl)ethyl(1S,2R,4aS,6aS,
1,2,6a,6b,9,9,12a-heptamethyl-
6bR,8aR,12aR,12bR,14bS)-10-hydroxy-1,2,6a,6b,9,9,12a-hepta-
methyl-1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadeca-
hydropicene-4a(2H)-carboxylate (3e)
1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydropi-
cene-4a(2H)-carboxylate (3b)
White powder; yield, 79%; m.p. 245–246 ꢂC; 1H-NMR (CDCl3, White powder; yield, 76%; m.p. 233 ꢃ 235 ꢂC; 1H-NMR (CDCl3,
300 MHz, ppm): d 8.32 (s, 1H, triazole-H), 7.61 (d, J ¼ 8.7 Hz, 2H, 300 MHz, ppm): d 8.29 (s, 1H, triazole-H), 7.57 (d, J ¼ 9.0 Hz, 2H,
Ar–H), 7.53 (d, J ¼ 8.7 Hz, 2H, Ar–H), 5.16 (s, 1H, C12–H), 4.29 ꢃ 4.25 Ar–H), 7.04 (d, J ¼ 8.7 Hz, 2H, Ar–H), 5.17 (s, 1H, C12–H), 4.28 ꢃ 4.24
(m, 4H, –O–CH2CH2–N–), 3.24 ꢃ 3.19 (m, 1H, C3–OH), 2.14 (d, (m, 4H, –O–CH2CH2–N–), 3.89 (s, 3H, ph-OCH3), 3.24 ꢃ 3.19 (m, 1H,
J ¼ 11.1 Hz, 1H), 2.04–1.95 (m, 1H), 1.93 ꢃ 1.81 (m, 2H), 1.69 ꢃ 1.66 C3–OH), 2.16 (d, J ¼ 11.1 Hz, 1H), 2.03 ꢃ 1.97 (m, 1H), 1.91 ꢃ 1.84
(m, 3H), 1.63 ꢃ 1.55 (m, 6H), 1.51 ꢃ 1.43 (m, 4H), 1.36 ꢃ 1.27 (m, (m, 2H), 1.66 ꢃ 1.56 (m, 7H), 1.52 ꢃ 1.43 (m, 5H), 1.40 ꢃ 1.25 (m,
5H), 1.07 ꢃ 1.04 (m, 4H), 1.00 ꢃ 0.96 (m, 6H), 0.91 (s, 3H), 6H), 1.07 (s, 4H), 1.00 ꢃ 0.96 (m, 6H), 0.91 (s, 3H), 0.86 ꢃ 0.84 (m,
0.86 ꢃ 0.84 (m, 3H), 0.79 (s, 3H), 0.71 (d, J ¼ 11.7 Hz, 1H), 0.61 (s, 3H), 0.79 (s, 3H), 0.72 (d, J ¼ 11.4 Hz, 1H), 0.62 (s, 3H). 13C-NMR
3H). 13C-NMR (CDCl3, 75 MHz, ppm): d 177.00, 144.33, 144.29, (CDCl3, 75 MHz, ppm): d 176.98, 161.89, 152.04, 143.85, 137.84,
137.80, 137.07, 130.30(2C), 129.52(2C), 125.97, 124.43, 78.96, 62.12, 133.86, 130.79(2C), 125.93, 114.81(2C), 78.97, 61.95, 55.51, 55.15,
55.16, 52.89, 48.30, 47.42, 44.33, 41.99, 39.48, 38.99, 38.85, 38.73, 52.85, 48.29, 47.42, 44.17, 41.98, 39.46, 38.99, 38.81, 38.73, 38.54,
38.54, 36.94, 36.69, 32.84, 30.43, 28.13, 27.88, 27.19, 24.22, 23.57, 36.94, 36.67, 32.84, 30.43, 28.13, 27.87, 27.20, 24.21, 23.56, 23.21,
23.22, 21.08, 18.27, 17.02, 16.96, 15.62, 15.41. ESI-HRMS (m/z): cal- 21.08, 18.26, 17.02, 16.95, 15.63, 15.41. ESI-HRMS (m/z): calculated
þ
þ
culated for C40H57ClN3O3 [M þ H]þ: 662.4083, found: 662.4081.
for C41H60N3O4 [M þ H]þ: 658.4578, found: 658.4580.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
767
R
N
N
N
OH
H
a
O
O
OH
H
O
O
H
1a-1j
O
O
N
H
N
N
H
O
N
H
R
HO
H
2a-2e
b
O
OH
HO
O
H
N
N
O
N
H
HO
H
R
3a-3e
Scheme 2. Reagents and conditions: (a) Ia–Ij, KOH, CH3CH2OH, 30 ꢂC. (b) IIa–IIe, IIIa–IIIe, K2CO3, CH3CN, 60 ꢂC.
Table 1. In vitro T. gondii growth inhibition and cytotoxicity on HeLa cells.
Compounds
R
IC50 in HeLa cells (lM)
IC50 in T. gondii (lM)
SIc
a
b
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
2a
2b
2c
2d
2e
3a
-H
2-F
3-F
4-F
2-Cl
>1000
419.6
>1000
>1000
466.1
408.0
240.4
230.4
>1000
353.7
836.8
328.7
>1000
>1000
>1000
101.5
2.4
>1000
711.9
>1000
>1000
239.6
462.1
301.7
273.8
>1000
301.3
>1000
>1000
>1000
>1000
>1000
88.0
–
0.59
–
–
1.95
0.88
0.80
0.84
–
1.17
–
–
–
–
–
1.15
0.36
1.44
1.77
0.67
0.72
0.62
3,4-Cl
2-Br
2-I
2-OCH3
3,4,5-OCH3
-H
4-Cl
4-OCH3
4-CH3
3,4-Cl
-H
4-Cl
4-F
4-NO2
4-OCH3
–
3b
3c
3d
3e
6.7
61.4
128.0
116.5
262.2
72.2
88.2
226.7
78.2
189.0
44.8
Spiramycin
Ursolic Acid
–
aIC50 in HeLa cells: Median toxicity dose, a measure of cytotoxicity against host cells.
bIC50 in T. gondii: Median inhibitory concentration, a measure of tachyzoite inhibition.
cSI: Selectivity index, a measure of efficacy, calculated by IC50 in HeLa cells/IC50 in T. gondii.

768
T. LUAN ET AL.
2.6. In vitro anti-T. gondii activity
invasion by T. gondii RH strain tachyzoites in vitro was checked.
The cells were plated in 96-well plates at an appropriate density
to ensure exponential growth throughout the experimental period
(3 ꢁ 103 cells per well) and then allowed to adhere for 24 h at
37 ꢂC. The cells were infected with T. gondii (1.5 ꢁ 104 tachyzoites/
well), followed by incubation for 24 h. All compounds were pre-
pared in dimethyl sulfoxide (DMSO) at a stock concentration of
100 mM. Serial dilutions (1–1000 lM) of each compound were
tested. Spiramycin was used as a positive control. After 24 h of
incubation, 10 lL of MTT solution were added to each well and
cells were incubated for a further 2 h. The optical density (OD)
was read on a microplate reader at a wavelength of 492 nm. The
IC50 in HeLa cells, IC50 in T. gondii and selectivity index were calcu-
lated using Microsoft Excel.
The cytotoxicity of compounds was determined using the previ-
ously published thiazolyl blue-based colorimetric method. For this,
HeLa cells were used as host cells and their ability to resist
Table 2. In vivo anti-T. gondii activity.
Groups
Amount of tachyzoite (ꢁ104)
Toxoa
Spib
UA
1e
3d
225.0
97.2
77.9
141.8
66.6
aT. gondii-infected KM mice with no treatment.
bSpiramycin.
2.7. In vivo anti-T. gondii activity
Thirty female KM mice were used to establish an animal model of
acute T. gondii infection. These were randomly divided into five
groups: infected untreated, normal, infected with spiramycin treat-
ment, infected with 1e treatment and infected with 3d treatment.
Each group consisted of six mice. Four hours after infection,
100 mg/kg of the compounds was administered to the mice by
gavage, once a day for 4 consecutive days, whereas the untreated
group was administered the same dose of physiological saline. On
the fifth day, blood from the eyes of mice was collected and they
were sacrificed by cervical dislocation. Their abdominal cavity was
rinsed with sterile physiological saline to collect the parasites/
tachyzoites. These were counted under the light microscope, and
the inhibition rate of parasites was calculated. The liver and spleen
were dissected and liver and spleen indexes, serum alanine
Figure 2. Effect of compounds on the number of tachyzoites in KM mice, n ¼ 6,
#p < .05, ##p < .01, ###p < .001 compared with toxo group.
ꢀ
Figure 3. Effect of compounds on liver and spleen weights in T. gondii-infected KM mice, p < .05 compared with normal group; §p < .05 compared with spi group.
ꢀ
ꢀꢀ
Figure 4. Effect of compounds on ALT and AST levels in T. gondii-infected KM mice, p < .05, p < .01 compared with normal group; ##p < .01, ###p < .001 com-
pared with toxo group.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
769
aminotransferase (ALT), aspartate aminotransferase (AST), and liver UA. Similarly, compounds 1e, 1f, 1g, 1h, 1j and 3d displayed a
homogenate glutathione (GSH) and malonaldehyde (MDA)
were determined.
higher anti-T. gondii activity and less cytotoxicity when compared
with spiramycin.
Compounds 1a–1j are products of a reaction between UA-1
and Ia–Ij. The anti-T. gondii activity of these compounds with dif-
ferent substitutions on the benzene ring was found to be in the
following order: 2-Cl > 3,4,5-OCH3 > 3,4-Cl > 2-I > 2-Br > 2-F > 2-
OCH3=H. Based on an overall comparison, we hypothesised that
introduction of halogen substituents at the ortho position and
electron-donating group at the 3,4,5-position of benzene ring
could improve the anti-T. gondii activity. Compounds 2a–2e were
generated from UA and IIa–IIe. Unfortunately, all of these com-
pounds lost their anti-T. gondii activity. Among the compounds
3a–3e, which react with IIIa–IIIe, four compounds showed consid-
erably higher anti-T. gondii activity. It seems that the anti-T. gondii
2.8. Molecular docking
Molecular docking was performed using the Discovery Studio (DS)
2017 software. The protein and ligand samples were prepared,
water molecules were deleted, and a DS Server added hydrogen.
The docking result was treated with DS Client. In this study, three
crystal structures of the proteins were selected for docking, PDB
ID: 6BFA (calcium-dependent protein kinase 1)21, 1LII (adenosine
kinase)22 and 3MB8 (purine nucleoside phosphorylase)23. The dif-
ferent xyz coordinates and radii of these proteins were defined as
the binding site spheres. The output poses of the ligands gener-
ated were analysed using the LibDockScore function to find out ability was enhanced after the introduction of strong electron-
the best complimentary match between the ligand and the recep-
tor. The protocol, CDOCKER was used to perform the docking.
withdrawing group (–F, –NO2) to the para position of the benzene
ring. Based on these findings, we decided to conduct an in-depth
study of anti-T. gondii activity of compounds 1e and 3d in mice,
owing to their strong anti-T. gondii activity in vitro.
3. Results and discussion
3.1. Chemistry
3.3. Number of tachyzoites in vivo
Scheme 2 shows the procedure adopted to obtain target com-
pounds. UA-1 was obtained by Jones oxidation of UA at 0 ꢂC.
Compounds 1a–1j were prepared by Claisen Schmidt condensa-
tion of UA-1 with different aldehydes in the presence of ethanolic
KOH at 30 ꢂC. Good yields (76–82%) were obtained with this
method. All other UA derivatives (2a–2e and 3a–3e) were syn-
thesised from various chlorinated derivatives via nucleophilic sub-
stitution in good to excellent yields (76–86%). Before biological
evaluation, all target compounds were characterised via HRMS,
1H-NMR and 13C-NMR.
As shown in Table 2 and Figure 2, the number of intraperitoneal
tachyzoites in untreated KM mice was 225 ꢁ 104. After treatment
with 100 mg/kg of different compounds, this number decreased
to varying degrees in the ascitic fluid of spiramycin-, UA-, com-
pound 1e- and compound 3d-treated mice, with inhibitory rates
being 56.8%, 65.4%, 37.0% and 70.4%, respectively. It is clearly
interpreted from these data that treatment with compound 3d
could significantly decrease the number of tachyzoites in T. gon-
dii-infected KM mice (p < .001). It even showed better anti-T. gon-
dii activity than spiramycin and UA in vivo.
3.2. Evaluation of anti-T. gondii activity in vitro and preliminary
structure-activity relationship
3.4. Liver and spleen indexes
Liver and spleen indexes were used to evaluate the protective
effect of drugs on viscera. As shown in Figure 3, compared with
Selectivity index is a measure of specific resistance to T. gondii. As
shown in Table 1, the SI value of the lead compound UA (0.62)
was lower than that of the positive control drug spiramycin (0.72),
indicating a certain degree of anti-T. gondii activity of UA. Among
UA derivatives, nine compounds exhibited higher anti-T. gondii
activity than UA alone (1e, 1f, 1g, 1h, 1j, 3a, 3c, 3d and 3e), and
eight compounds exhibited an activity higher than spiramycin (1e,
1f, 1g, 1h, 1j, 3a, 3c and 3d). Besides, with the exception of com-
pound 3b, the IC50 value of all other compounds was higher than
that of UA, indicating these compounds to be less cytotoxic than
Table 3. Scores of UA and compound 3d docked to different enzymes.
CDOCKER interaction energy
Enzyme (PDB ID)
UA
Compound 3d
6BFA
1LII
3MB8
54.6825
No docking
No docking
58.0486
No docking
No docking
ꢀ
ꢀꢀ
Figure 5. Effect of compounds on MDA and GSH levels in T. gondii-infected KM mice, p < .05, p < .01 compared with normal group; #p < .05, ##p < .01 compared
with toxo group; §p < .05 compared with spi group.

770
T. LUAN ET AL.
the normal group, the liver index of the mice infected with T. gon- 3.6. MDA and GSH
dii increased only slightly. Although a significant increase in the
Free radicals generated within cells cause peroxidation of lipids,
spleen index was observed in mice infected with T. gondii, this
increase was subjugated by treatment with spiramycin, UA or
compound 3d. However, changes in spleen index did not show
any statistically significant differences.
resulting in the formation of MDA, which, in turn, causes cross-
linking and polymerisation of proteins, nucleic acids and other
macromolecules, thereby exerting cytotoxicity. As can be seen
from the data in Figure 5, the untreated group had a higher MDA
content compared with the normal group (p < .01), whereas levels
of MDA significantly decreased after treatment with spiramycin,
UA or compound 3d. GSH is an important antioxidant that scav-
enges the free radicals in the body. It combines with free radicals
and heavy metals, thereby converting them to harmless substan-
ces that are excreted from the body24. Compared with the normal
group, the GSH content in the untreated group was significantly
decreased (p < .05). However, compounds 3d and 1e could signifi-
cantly increase the GSH content as compared to the untreated
group, and had a similar efficacy to UA and spiramycin. These
3.5. ALT and AST
Levels of serum ALT and AST act as indicators of hepatotoxicity.
To further study the toxicity of these compounds, ALT and AST
levels in the serum of KM mice after infection with T. gondii were
measured (Figure 4). T. gondii infection resulted in a significant
elevation of serum ALT and AST levels as compared with the nor-
mal group. Treatment with UA, 1e and 3d led to a striking reduc-
tion in these levels as compared with the untreated group. These
results indicated UA, 1e and 3d could provide resistance against results implied that the anti-oxidative effects of UA and com-
T. gondii-mediated hepatotoxicity. pound 3d were comparable to that of spiramycin.
Figure 6. Computer modelling of compound 3d binding to calcium-dependent protein kinase 1 (6BFA). Compound 3d was coloured in yellow.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
771
Figure 7. Computer modelling of UA binding to calcium-dependent protein kinase 1 (6BFA). UA was coloured in yellow.
3.7. Molecular docking analysis
the ¼ NH moiety of Arg-A442 and the –CH2– moiety of Ser-A439
via hydrogen and carbon–hydrogen bonds, respectively, whereas
the –OH group of UA interacted with the –COOH group of Glu-
A135 and the –NH2 group of Lys-A338 via two hydrogen bonds.
In addition, the 1,2,4-triazole moiety formed one carbon–hydrogen
bond with Ser-A439 residue. We also observed that UA entered
into an alkyl interaction with amino acid residues Val-A56, Leu-
A345 and Leu-A438.
In order to better reflect the advantage of compound 3d, we
also performed the molecular docking analysis of UA. As shown in
Figure 7, three similar conventional hydrogen bonds are observed
with residue Gln-A393, Leu-A438 and Arg-A442. However, com-
pared with compound 3d, some significant chemical bonds such
as attractive charges are missing. This may explain why compound
3d has better anti-T. gondii activity than UA. These results indicate
compound 3d to possess a strong binding affinity for the enzyme
and therefore could act as a possible TgCDPK1 inhibitor.
TgCDPK1 plays a crucial role in the motility and gliding of T. gon-
dii, as well as the adenosine kinase and purine nucleoside phos-
phorylase are key purine metabolic enzymes from the T.
gondii21–23. Our in vivo study revealed compound 3d to signifi-
cantly inhibit the proliferation of tachyzoites in the abdominal
cavity of KM mice. Therefore, these three enzymes related to T.
gondii metabolism were selected for molecular docking study and
to determine possible targets with their specific modes of action
(Table 3). Interestingly, only TgCDPK1 (6BFA) could be docked and
expressed a high binding energy for the ligand. The CDOCKER
interaction energy of compound 3d was 58.0486, slightly higher
than that of UA, which was consistent with the result of in vivo.
Figure 6 illustrates the binding mode of compound 3d in its
active site; it was held in the active pocket of TgCDPK1 through a
combination of interactions with TgCDPK1þ. The nitro group of
compound 3d interacted with the –NH3 group of Lys-A59,
–COOH group of Asp-A210 and –COOH group of Glu-A64 via
three important attractive charges. These interactions may explain
the strong anti-T. gondii activity exhibited by compound 3d in
4. Conclusions
In the present study, 20 novel UA derivatives were synthesised
this series. Meanwhile, the carbonyl group of UA interacted with and examined for their anti-T. gondii properties. Most of these

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T. LUAN ET AL.
compounds displayed some anti-T. gondii activity, with a less cyto- 10. Keita FT, Gasquet M, Giorgio CD, et al. Antimalarial activity
toxicity than UA in vitro. The compound 3d exhibited the most
potent anti-T. gondii activity in vivo and was superior to UA and
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3d, as evident by the presence of three significant attractive
charges and three hydrogen bonds in it that play a crucial role in
its binding to the active site of TgCDPK1. Based on these findings,
we conclude that compound 3d may serve as a potential candi-
date for developing effective and anti-T. gondii drugs with fewer
side-effects.
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Disclosure statement
14. Liu MC, Yang SJ, Jin LH, et al. Synthesis and cytotoxicity of
novel ursolic acid derivatives containing an acyl piperazine
moiety. Eur J Med Chem 2012;58:128–35.
No potential conflict of interest was reported by the authors.
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Funding
This work was supported by the National Natural Science
Foundation of China (No. 21662036, 81160409 and 81260226).
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novel dehydroepiandrosterone analogues as potent antipro-
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