Evaluation of ursolic acid derivatives with potential anti-Toxoplasma gondii activity

Article abstract of DOI:10.1016/j.exppara.2020.107935

Toxoplasma gondii is an important pathogen that causes serious public health problems. Currently, therapeutic drugs for toxoplasmosis cause serious side effects, and more effective and novel substances with relatively low toxicity are urgently needed. Ursolic acid (UA) has many properties that can be beneficial to healthcare. In this study, we synthesized eight series of UA derivatives bearing a tetrazole moiety and evaluated their anti-T. gondii activity in vitro using spiramycin as a positive control. Most of the synthesized derivatives exhibited better anti-T. gondii activity in vitro than UA, among which compound 12a exhibited the most potent anti-T. gondii activity. Furthermore, the results of biochemical parameter determination indicated that 12a effectively restored the normal body weight of mice infected with T. gondii, reduced hepatotoxicity, and exerted significant anti-oxidative effects compared with the findings for spiramycin. Additionally, our molecular docking study indicated that the synthesized compounds could act as potential inhibitors of T. gondii calcium-dependent protein kinase 1 (TgCDPK1), with 12a possessing strong affinity for TgCDPK1 via binding to the key amino acids GLU129 and TYR131.

Full text of DOI:10.1016/j.exppara.2020.107935

Journal Pre-proof
Evaluation of ursolic acid derivatives with potential anti-Toxoplasma gondii activity
Lin-Hao Zhang, Li-Li Jin, Fang Liu, Chunmei Jin, Chun-Mei Jin, Zhi-Yu Wei
PII:
S0014-4894(19)30594-6
DOI:
https://doi.org/10.1016/j.exppara.2020.107935
YEXPR 107935
Reference:
To appear in: Experimental Parasitology
Received Date: 16 December 2019
Revised Date: 22 May 2020
Accepted Date: 2 June 2020
Please cite this article as: Zhang, L.-H., Jin, L.-L., Liu, F., Jin, C., Jin, C.-M., Wei, Z.-Y., Evaluation of
ursolic acid derivatives with potential anti-Toxoplasma gondii activity, Experimental Parasitology (2020),
doi: https://doi.org/10.1016/j.exppara.2020.107935.
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Lin-Hao Zhang：Software, Investigation, Data curation, Writing- Original draft
preparation.
Li-Li Jin: Methodology, Visualization, Writing - Original Draft，
Fang Liu: Formal analysis
Chunmei Jin: Validation,
Chun-Mei Jin: Resources, Supervision, Funding acquisition.
Zhi-Yu Wei: Conceptualization, Project administration, Writing - Review & Editing,
Funding acquisition.

Evaluation of ursolic acid derivatives with potential
anti-Toxoplasma gondii activity
Lin-Hao Zhang b,1, Li-Li Jin b,1, Fang Liu c, Chunmei Jin b, Chun-Mei Jin b,*, and Zhi-Yu Wei a,*

Evaluation of ursolic acid derivatives with potential
anti-Toxoplasma gondii activity
Lin-Hao Zhang b,1, Li-Li Jin b,1, Fang Liu c, Chunmei Jin b, Chun-Mei Jin b,*, and
Zhi-Yu Wei a,*
a Medical College of Dalian University, Dalian 116622, China
b Key Laboratory of Natural Resources of Changbai Mountain & Functional Molecules, Ministry
of Education, Yanbian University College of Pharmacy, Yanji 133002, China
c Department of Pharmacy, Yanbian University Hospital, Yanji 133002, China
* Corresponding authors:
E-mail addresses: weizhiyu@dlu.edu.cn (Z.-Y. Wei), cmjin@ybu.edu.cn (C.-M. Jin)
1 These authors contributed equally to this work.
1

Abstract
Toxoplasma gondii is an important pathogen that causes serious public health
problems. Currently, therapeutic drugs for toxoplasmosis cause serious side effects,
and more effective and novel substances with relatively low toxicity are urgently
needed. Ursolic acid (UA) has many properties that can be beneficial to healthcare. In
this study, we synthesized eight series of UA derivatives bearing a tetrazole moiety
and evaluated their anti-T. gondii activity in vitro using spiramycin as a positive
control. Most of the synthesized derivatives exhibited better anti-T. gondii activity in
vitro than UA, among which compound 12a exhibited the most potent anti-T. gondii
activity. Furthermore, the results of biochemical parameter determination indicated
that 12a effectively restored the normal body weight of mice infected with T. gondii,
reduced hepatotoxicity, and exerted significant anti-oxidative effects compared with
the findings for spiramycin. Additionally, our molecular docking study indicated that
the synthesized compounds could act as potential inhibitors of T. gondii
calcium-dependent protein kinase 1 (TgCDPK1), with 12a possessing strong affinity
for TgCDPK1 via binding to the key amino acids GLU129 and TYR131.
Keywords: ursolic acid; anti-T. gondii; in vitro; in vivo; docking; TgCDPK1 inhibitors
2

1. Introdiction
Toxoplasma gondii is a globally distributed Apicomplexa protozoan that infects a
wide range of intermediate hosts, including almost all warm-blooded animals and
humans (Ma et al., 2018;Hou et al., 2019). T. gondii can infect people through several
pathways, such as eating raw or undercooked meat and drinking the milk of infected
goats (Gazzonis et al., 2019). Toxoplasmosis can cause mild or no symptoms in
healthy individuals; however, it may cause serious diseases and even death in
immunocompromised patients, such as patients with AIDS or cancer (Grant et al.,
1990). In pregnant women, T. gondii infection can lead to mental or psychomotor
disability, chorioretinitis, microcephaly, and stillbirth in the fetus (Liesenfeld et al.,
2017). In addition, because of the prevalence of AIDS and cancer, as well as increased
pet ownership, T. gondii infection has increasingly become a serious public health
problem globally, and there are few reliable anti-T. gondii drugs available for
preventing and treating toxoplasmosis. Differing from common anti-cancer agents
that directly kill cancer cells, new anti-T. gondii drugs must be developed in full
consideration of host cell damage. Most currently used clinical anti-T. gondii drugs
are limited by their toxicity to host cells. Therefore, the discovery of more effective
and less toxic anti-T. gondii agents is awaited.
Ursolic acid (UA) is a pentacyclic triterpenoid found in most plant species (Lee
et al., 2001), and it is known to possess a number of bioactive properties (Wang et al.,
2017), such as anti-microbial (Zhang et al., 2013), anti-inflammatory (Nascimento et
al., 2014), immunomodulatory (Ramos et al., 2010), anti-oxidant (Soica et al., 2014),
and anti-cancer activities (Shanmugam et al., 2013). Recently, Choi et al. reported that
UA improved the survival time of T. gondii-infected mice, illustrating its promise as a
potential candidate for the development of anti-T. gondii drugs (Choi et a., 2018). We
3

also reported UA derivatives bearing triazole moieties as potential anti-T. gondii
agents, finding that compound A (Fig. 1A) displayed enhanced activity against T.
gondii and reduced hepatotoxicity compared with UA (Luan et al., 2019). Several
recent reports described various structural modifications to improve the bioactive
properties of UA. Indeed, structural modification of the UA C-28 carboxylic acid
group or C-3 hydroxyl group resulted in significant enhancement of its biological
activities and obvious decreases in toxicity (Nedopekina et al., 2017; Bai et al., 2012;
Gu et al., 2017; Yang et al., 2015; Chi et al., 2017). In addition, tetrazole is a mimetic
of carboxylic acid and a five-membered aromatic heterocyclic compound containing
four nitrogen atoms (Allen et al., et al., 2012). Its derivatives have diverse activities
including anti-microbial (Méndez et al., 2019; Andrejević et al., 2018), anti-oxidant
(Kumbar et al., 2018; Khan et al., 2018), anti-bacterial (Figueiredo et al., 2012;
Chernov’yants et al., 2016), anti-HIV (Bielenica et al., 2017), anti-cancer (Aziz et al.,
2018), and anti-parasitic activities (Fig. 1B and 1C) (Shaikh et al., 2017; Chen et al.,
2019; Hamid et al., 2018). Several tetrazole-containing drugs have been developed for
clinical use, including cefazolin (Allen et al., 2012), valsartan (Liu et al., 2019), and
pentetrazol (Lu et al., 2018) (Fig. 1). Some of these drugs possess a tetrazolium group
as the terminal moiety. Therefore, UA derivatives previously synthesized in our
laboratory containing a tetrazole moiety (as presented in Scheme 1) are likely to
possess anti-T. gondii activity and lower toxicity (Zhang et al., 2019). Thus, we report
the biological evaluation of these compounds in this work.
4

Figure 1. Structures of previously reported compounds as anti-parasitic agents and
clinical medication containing tetrazole moiety.
5

Scheme 1. Synthetic scheme for the synthesis of compounds 3a-d, 4a-d, 6a-d, 7a-d,
10a-d, 11a-d, 12a-b and 14a-e. Reagents and conditions: (a) 1,2-dibromoethane or
1,3-dibromopropane, K2CO3, KI, DMF, 50 °C, 6h; (b) K2CO3, KI, acetone, reflux, 8h;
(c) K2CO3, KI, acetone, reflux, 10h; (d) Jones’ reagent acetone, 0 °C, 5 h, 90%; (e)
1,2-dibromoethane or 1,3-dibromopropane, K2CO3, KI, DMF, 50 °C, 6h; (f) K2CO3,
KI, acetone, reflux, 10h; (g) SOCl2, DMF/DCM (1:1), trimethylamine; (h) Phenyl
hydrazine hydrochloride, acetic acid, ethanol, reflux,8h; (j) anhydride, DMAP, DCM,
trimethylamine, r.t., overnight; (k) 10%NaOH, 4NHCl, 50% method in water.
2. Materials and methods
2.1. Drugs and reagents
Spiramycin(spi) were purchased from Sigma Chemical Company (St. Louis, MO,
6

USA). UA derivatives were prepared according to the procedures we have been
reported. (Zhang et al., 2019). All sera,antibiotics and RPMI 1640 for cell culture
were obtained from Invitrogen (Biological Industry, Israel). All other chemicals were
of reagent grade.
2.2. Cells, parasites and animals
GES-1 cells were cultured in DMEM, supplemented with 0.01%
Penicillin-Streptomycin and 10% heat-inactivated FBS and maintained at 37
and
5% CO2. Cells were purchased from American Type Culture Collection (ATCC,
Manassas, VA, USA). Tachyzoites used in our study were from the virulent RH strain
of T. gondii (Yanbian University School of Medicine, China) and maintained by serial
intraperitoneal passage in KM female mice, which were purchased from Experiment
Center, Yanbian University. All experimental procedures were approved by animal
experimental center (Number of license: SCXK 2017-0003), Yanbian University and
complied with the relevant international animal ethics regulations for the care and use
of experimental animals. All mice were kept in a central animal care facility with free
access to water and rodent food during the experiment.
2.3. In vitro studies
The anti-T. gondii activity and cytotoxicity of compounds to host cells (GES-1)
in vitro were evaluated by the published method of (Jin et al., 2009). The cells were
plated in 96-well plates at an appropriate density to ensure exponential growth
throughout the experimental period and then allowed to adhere for 24 h at 37 . The
cells were infected with T. gondii, followed by incubation for 24 h. All compounds
were prepared in dimethyl sulfoxide (DMSO) at a stock concentration of 100 µM.
Serial dilutions (1–1000 µM) of each compound were tested. The aim of our
7

modification of structure was to obtain compounds with low toxicity and high
efficiency, therefore, spi with low toxicity was selected as a positive control. After
24h
of
incubation,
10
µL
of
3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide solution were added
to each well and cells were incubated for a further 4 h. The optical density (OD) was
read on a microplate reader at a wavelength of 492 nm. The IC50 in GES-1 cells, IC50
in T. gondii and selectivity index were calculated using Microsoft Excel.
2.4. In vivo studies
2.4.1. Inhibition rate of tachyzoites and body weight in vivo
Forty-two female KM mice (weighing 18–22g) were used to establish an animal
model of acute T. gondii infection. These were randomly divided into seven groups:
infected untreated, normal, infected with spi treatment, infected with UA treatment,
infected with 11d treatment, infected with 12a treatment, infected with 12b treatment
and infected with 14a treatment. Each group consisted of six mice. To unify the
various compounds to maintain the same effect comparison, and the main
consideration was the limited amount of drugs and the total usage of the mouse, we
almost always choose the concentration of 100 mg/kg. 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, the body weight of mice infected with T. gondii
from different groups was collect. Furthermore, 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.
8

2.4.2. Measurement of visceral weights and liver biochemical parameters
The liver and spleen were dissected and liver and spleen indexes, serum alanine
aminotransferase (ALT), aspartate aminotransferase (AST), and liver homogenate
glutathione (GSH) and malonaldehyde (MDA) were determined (Luan et al., 2019;
Guo et al., 2019; Zhang et al., 2016).
Serum levels of AST and ALT were measured by the method of (Zhang et al.,
2016). The quintuple substrate reaction of ALT or AST and serum was carried out
under incubation at 37 for 30 min, then added 1mmol/L 2, 4-DNPH and held for 20
min. Finally, 0.4 mol/L NaOH was added and allowed to react for 5 min. The
absorbance at 505 nm was measured.
The GSH was measured according to the method of (Zhang et al., 2016; Beutler
et al., 1963).The double volume liver homogenate was mixed with trichloroacetic acid
(20%, w/v) and centrifuged at 4000 rpm for 10 min. Then, 0.3 mol/L phosphate buffer
and DTNB (0.04%, w/v) were added to the separated supernatant and mixed
thoroughly. After 5 min at room temperature, the absorbance was measured at 412 nm.
MDA was measured by the standard method (Zhang et al., 2016; Ohkawa et al.,1979)
with minor modifications. The liver homogenate supernatant was mixed with
thiobarbituric acid (0.5%, w/v) and heated in the bath of boiling water for 60 min,
then cooled quickly and centrifuged at 3000 rpm for 10 min, the absorbance of pink
colored supernatant was measured at 532 nm. Tetraethoxypropane replaced the liver
homogenate in the standard sample.
2.5. Docking simulations
T. gondii calcium-dependent protein kinase 1 (TgCDPK1) plays a crucial role in
9

the motility and gliding of T. gondii, and the enzyme, together with adenosine kinase
and purine nucleoside phosphorylase, serves as a key purine metabolic enzyme for T.
gondii (Vidadala et al., 2016; Schumacher et al., 2000). Luan et al reported that UA
derivative was potential TgCDPK1 inhibitor (Luan et al., 2019). Therefore, the
development of molecular docking was determined. Molecular computation studies
were carried out by using Discovery Studio 2017 (DS, Accelrys, San Diego,CA,
USA). The X-ray crystal structure of Calcium-Dependent Protein Kinase 1 from T.
gondii (TgCDPK1) in complex with inhibitor UW1561 was obtained from protein
data bank (PDB: 4TZR). The water molecules, heavy atom and UW1561 in protein
were removed and the protein was prepared by adding hydrogen and correcting
incomplete residues using Clean Protein tool of DS. ChemBioDraw 14.0 was used to
draw the structure of the docking compounds and save them in the molar format. The
structures of UA, compound 12a and UW1561 were sketched in 2D and converted
into 3D using the DS molecule editor. LibDock was used to investigate whether the
synthesized compounds had potential TgCDPK1 inhibitory effects. CDDOCK studies
were carried out to investigate the binding mode of UA, compound 12a and UW1561
in the crystal structure of 4TZR from 2D images. The 3D image was chosen for
analyzing the binding features of UA, compounds 12a and UW1561 with TgCDPK1
(Zhao et al., 2018).
3. Results and discussion
3.1. In vitro results and discussion
The in vitro anti-T. gondii activities of the UA derivatives are summarized in
Tables 1–2. The selectivity index (SI) is usually applied to evaluate anti-T. gondii
10

agents. It is a measure of specific resistance to T. gondii, and it is generally believed
that larger SI values indicate stronger inhibitory activity against T. gondii and lower
cytotoxicity (Jin et al., 2009). As shown in Table 1, the SI of UA (0.6) was lower than
that of the positive control spi (0.8), indicating the poorer anti-T. gondii effects of UA.
Almost all of the UA derivatives exhibited stronger anti-T. gondii activity than the
parent compound, and 27 compounds exhibited similar or slightly greater activity than
spi. In addition, with the exception of compound 14b, the IC50 values of all other
compounds in GES-1 cells were higher than that of UA, indicating that these
compounds are less cytotoxic than UA. Regarding compounds 3a–3d and 4a–4d, a
3,4-(OCH2O) substituent on the benzene ring attached to the tetrazole moiety was
found to be more beneficial, and compounds 4c (SI = 0.9) and 4d (SI = 0.9) exhibited
potent growth-inhibiting effects on T. gondii in vitro. Compounds 6a–6d and 7a–7d,
which feature a shorter linkage (n = 1) between UA and the tetrazole moiety,
exhibited stronger inhibitory activities than compounds bearing longer linkages (n =
2). In these two series, compounds containing ortho-tetrazole moieties displayed
relatively good activity. Interestingly, compound 6d, which lacks a benzene ring
between the shorter linkage and tetrazole moiety, displayed enhanced potency
compared with the effects of compounds containing the benzene ring. However,
among compounds 7a–7d with a longer linkage, 7d exhibited lower inhibitory activity
than the other compounds (7b–7d). Compounds in series 10 and 11 exhibited slightly
more potent activity than those in series 6 and 7, in which the structures only differed
at position 3. The former series featured a ketone at this position, whereas compounds
in the latter series carried a hydroxyl group. Furthermore, in series 10 and 11, the
compounds featuring meta-tetrazole (10b, 11b) or those lacking a benzene group (10d,
11d) exhibited relatively good potencies, with compound 11d displaying the strongest
11

activity with an SI of 1.0. Compounds 12a and 12b also exhibited higher inhibitory
activities than other compounds with an SI of 1.0. Almost all of the compounds in
series 14 displayed good activities (SI = 1.0, 0.8, 0.8, and 0.9, respectively), excluding
14b. From the biological results for the compounds in series 12 and 14, in which the
tetrazole group was directly linked to a nitrogen atom of the C-28 amide group and
the C-3 hydroxyl group was changed to ketone, ester, or hydrazine, we can observed
that such a modification at the C-3 position results in enhanced activity of T.gondii
inhibition. It was found that the number of T. gondii invading host cells was
significantly reduced after administration, and it was difficult to observe T. gondii
extracellularly, and the growth status of the cells was no different from that of normal
cells.
Table 1. In vitro T. gondii growth inhibition and cytotoxicity of the series 3, 4, 6 and
7.
IC50 (µM)
SI c
Compound
R1
R2
GES-1cells a
T.gondii b
–
H
–
–
–
44.8
72.2
0.6
0.8
0.8
UA
3a
198.2
189.6
256.3
246.6
4-Cl
3b
12

4-CH3O
3,4-(OCH2O)
H
–
–
–
–
–
–
165.5
222.0
212.3
181.8
207.0
213.5
181.7
234.5
275.7
256.7
228.3
243.1
237.9
193.2
0.7
0.8
0.8
0.8
0.9
0.9
0.9
3c
3d
4a
4b
4c
4d
6a
4-Cl
4-CH3O
3,4-(OCH2O)
–
–
178.0
224.2
0.8
6b
–
–
–
169.6
205.0
147.2
199.9
227.5
186.9
0.8
0.9
0.8
6c
6d
7a
–
–
125.0
155.9
191.8
175.0
0.7
0.9
7b
7c
–
–
54.6
84.9
0.6
0.8
7d
–
207.8
250.5
spi
a IC50 in GES-1cells: Median toxicity dose, a measure of cytotoxicity against host cells. IC50 of UA
in HeLa cells. Values were shown as mean, n=3.
b IC50 in T. gondii: Median inhibitory concentration, a measure of tachyzoite inhibition. Values
13

were shown as mean, n=3.
c SI: Selectivity index, a measure of efficacy, calculated by IC50 in GES-1 cells/IC50 in T. gondii.
Values were shown as mean, n=3.
Table 2. In vitro T. gondii growth inhibition and cytotoxicity of the series 10, 11, 12
and 14.
H
H
N
N
H
N
O
N
N
H
R3
12a-b
IC50 (µM)
GES-1cells a T.gondii b
SI c
0.8
0.9
Compound
10a
R2
R3
–
R4
–
192.6
270.1
242.2
310.1
–
–
10b
–
–
–
–
–
–
264.3
161.1
152.8
315.3
179.7
187.6
0.8
0.9
0.8
10c
10d
11a
14

–
–
271.4
316.4
0.9
11b
–
–
–
–
–
–
246.2
188.2
213.2
245.0
130.3
─
306.2
192.8
218.6
248.5
136.8
─
0.8
1.0
1.0
1.0
1.0
─
11c
11d
12a
12b
14a
14b
14c
14d
14e
–
–
–
–
–
–
–
–
–
–
–
–
92.8
114.1
96.2
0.8
0.8
0.9
O
O
74.3
–
202.7
220.9
a IC50 in GES-1cells: Median toxicity dose, a measure of cytotoxicity against host cells. Values
were shown as mean, n=3.
b IC50 in T. gondii: Median inhibitory concentration, a measure of tachyzoite inhibition. Values
were shown as mean, n=3.
c SI: Selectivity index, a measure of efficacy, calculated by IC50 in GES-1 cells/IC50 in T. gondii.
Values were shown as mean, n=3.
3.2. In vivo results and discussion
15

We performed an in-depth study of the anti-T. gondii activities of compounds
11d, 12a, 12b, and 14a in mice due to their good anti-T. gondii activity in vitro and
their different structural characteristics.
3.2.1. Inhibition rate of tachyzoites and body weight in vivo
In order to study the degree of inhibition of tachyzoites by compounds, the
inhibition rate experiment was carried out. The results of inhibition rate of tachyzoites
are summarized in Table 3. After treatment with the compounds at a dosage of 100
mg/kg, the UA derivatives had different inhibitory effects on the number of T. gondii
specimens in the abdominal cavity of mice, among which 12a exerted the strongest
inhibitory effect (55.3%), exceeding that of spi (51.8%) in the maternal body.
However, the inhibitory activity of these four compounds showed different results in
vivo and in vitro. We speculate that the substituents at C-3 position of compounds 12b
(29.4%), 14a (31.8%) and 12a are different, and the substituents at C-28 position of
compounds 11d (16.5%) and 12a are different, which leads to their different
absorption, distribution, metabolism and excretion in vivo, thus leading to the
difference of activities.
Table 3. Effect of compounds on the inhibition rate of tachyzoites in mice.
spi
11d
12a
12b
14a
Inhibition rate
(%)
51.8%###
16.5%
55.3%###
29.4%#
31.8%#
Inhibition rate(%) of tachyzoites in mice peritoneal cavity treated with compounds, compared to
toxo group; n = 6, # P< 0.05 compared with toxo group and ### P< 0.001 compared with toxo
group.
16

Figure 2. Effect of compounds on body weights in T.gondii-infected KM mice,
**P<0.01compared with normal group; ***P<0.001compared with normal group;
#P<0.05 compared with toxo group; ###P<0.001 compared with toxo group;
§§P<0.01compared with spi group; §§§P<0.001compared with spi group.
Body weight is significantly decreased in mice following T. gondii infection
(Spano et al., 2002). As shown in Figure 2, the weight of mice treated with the test
compounds (spi, 14a, and 12b) was significantly lower than that of the normal group,
which weight was similar to toxo group, indicating that mouse weight was not
regained by treatment with these compounds to T. gondii-infected mice. However,
compared with the findings in T. gondii-infected mice, 12a and 11d significantly
increased body weight to the same levels observed in normal mice. Thus,
compounds 12a and 11d could alleviate the weight reduction caused by acute
Toxoplasma infection, leading to the speculation that these two compounds can affect
the gastrointestinal digestive system and restore the normal feeding status of mice or
that they can reduce the metabolic level and energy consumption of mice, thereby
restoring weight.
From the effect of these four compounds on body weights in T.gondii-infected
17

KM mice, we deduce that the carbonyl substitution at C-3 position of compounds 12b
and 11d are beneficial to weight recovery to mice. On the contrary, the hydrazone and
hydroxyl substituents at C-3 position of compounds 14a and 12b are not conducive to
weight recovery.
3.2.2. Liver and spleen indexes
The liver is the main part of histopathology, and the spleen plays an important
role in coordinating adaptation and innate immune responses (Khan et al.,
2017; Znalesniak et al., 2017). Therefore, liver and spleen indices are used to evaluate
the protective effects of drugs on viscera. As shown in Figure 3, liver and spleen
enlargement can occur after acute infection by T. gondii in mice, but the liver index
was unchanged compared with that in normal mice. It was noteworthy that the spleen
index was increased significantly by all UA derivatives compared with the normal
group findings, especially by 11d and 12a, which also significantly increased the
spleen index versus that observed in the infected group. Because splenomegaly is an
early sign of portal hypertension in cirrhosis, attention should be paid to
splenomegaly in mice infected by T. gondii following treatment.
From the effect of compounds on spleen and liver weights in T.gondii-infected
KM mice, we infer that natural product UA binding to tetrazolium moiety could
increase the spleen index evidently, especially the C-3 position was modified by
carbonyl group as well.
18

Figure 3. Effect of compounds on spleen and liver weights in T.gondii-infected KM
*
mice, P<0.05compared with normal group; **P<0.01compared with normal group;
***P<0.001compared with normal group; ###P<0.001 compared with toxo group;
§P<0.05compared with spi group; §§§P<0.001compared with spi group.
3.2.3. ALT and AST
The serum levels of ALT and AST are extremely sensitive indicators of liver
injury, and their elevation roughly reflects the degree of liver injury (Liu et al.,
2014). To further study the toxicity of these compounds, serum ALT and AST levels
were measured in mice infected with T. gondii. As shown in Figure 4, compared with
the normal group levels, acute infection by T. gondii resulted in significant increases
of serum ALT and AST levels. Serum ALT and AST levels were significantly higher
in mice treated with 11d, 12b, or 14a than in those in the normal and toxo groups,
indicating that these three derivatives caused liver damage without similar protective
effects to spi. However, compound 12a was linked to significantly decreased ALT
and AST levels in T. gondii-infected mice with similar protective effects to spi. The
results suggest that 12a can provide resistance to Toxoplasma-mediated
hepatotoxicity.
From the effect of compounds on ALT and AST levels in T.gondii-infected KM
19

mice, we conjecture that the C-28 position of UA is linked to tetrazole by
esterification and alkylation, and hydrazone formation or retention of hydroxyl group
at C-3 position has no regulation effect on AST and ALT indexes. Conversely,
oxidation at C-3 position of UA, and C-28 linked to tetrazole by amide bond can be
beneficial to protective effect on liver.
Figure 4. Effect of compounds on ALT and AST levels in T.gondii-infected KM mice,
**P<0.01compared with normal group; ***P<0.001compared with normal group;
##P<0.01 compared with toxo group; ###P<0.001 compared with toxo group;
§P<0.05compared with spi group; §§§P<0.001compared with spi group.
3.2.4. GSH and MDA
GSH is an important anti-oxidant and free radical scavenger in the human
anti-oxidant system that converts harmful free radicals and heavy metals into
harmless substances and facilitates their excretion from the body (Shaun et al.,
2008). MDA is a lipid peroxidation product formed by reactive oxygen radicals
oxidizing biological membranes after enhanced oxygen stress, which is an important
marker of lipid peroxidation (Gaweł et al., 2004). The levels of MDA and GSH in
20

liver can indirectly reflect the degree of cells damage with the degree of liver lipid
peroxidation in organism (Cederbaum et al., 2009). As shown in Figure 5, compared
with the normal group findings, GSH levels were significantly lower in T.
gondii-infected mice. Nevertheless, compared with the T. gondii-infected group
results, derivatives 12a and 11d significantly increased GSH content to levels
exceeding that in the toxo group, supporting their liver restored after exposure to
compounds 12a and 11d. Additionally, compared with the level in the normal group,
the MDA level was significantly elevated in T. gondii-infected mice, whereas MDA
content significantly decreased by treatment with compounds 12a, 12b, and 14a,
indicating that these compounds could reduce lipid peroxidation caused by acute T.
gondii infection.
From the effect of compounds on GSH and MDA levels in T.gondii-infected KM
mice, we believe that oxidation at C-3 position of UA benefits to enhance regulation
effect on GSH, the C-28 position linked to tetrazole via amide bond is to prejudice of
adjusting MDA index.
Figure 5. Effect of compounds on GSH and MDA levels in T.gondii-infected KM
*
mice, P<0.05compared with normal group; **P<0.01compared with normal group;
##
#P<0.05 compared with toxo group; P<0.01 compared with toxo group; ###P<0.001
compared with toxo group; §P<0.05compared with spi group; §§P<0.01compared with
spi group.
21

4. Molecular docking analysis
To investigate whether the synthesized compounds inhibit TgCDPK1, we used
the LibDock tool of DS software to investigate docking with TgCDPK1 protein
(4TZR). The results illustrated that all synthesized compounds could dock with 4TZR,
and the docking scores were higher than that of UA (see Supplementary material),
inferring that these compounds might be potential TgCDPK1 inhibitors, although
further validation is needed. Due to compound 12a with good T. gondii inhibitory
activity and the maximal increase in weight, splenomegaly, and alterations in the
enzymatic data, we used UA and inhibitor UW1561 as control to further analyzing the
docking results of it with 4TZR. Table 4 presents the docking results for 12a, UA and
UW1561 with 4TZR using the CDDOCK tools of DS. Compound 12a had the most
promising -CDDOCK Interaction Energy (98.89 k.cal/mol) among UA (54.77
k.cal/mol) and inhibitor UW1561 (57.74 k.cal/mol). That indicated that compound
12a may have better TgCDPK1 inhibitory activity than inhibitor UW1561, the
TgCDPK1 inhibitory activity of UA may lower than that of inhibitor, which of course
needs further validation. The preferred coordination modes of 12a, UA and UW1561
with the TgCDPK1 protein are presented in Fig. 6. It was observed that the tetrazole
fragment of compound 12a fit into a 4TZR cavity that was formed by GLU129,
ALA78, MET112, VAL65, LEU181, and TYR131 (Fig. 6A and 6B), which the ligand
of compound 12a displayed a similar orientation in the active site of TgCDPK1,
compared with inhibitor UW1561. The formed complex was stabilized by the
formation of hydrogen bonds (H-bonds) (Zhao et al., 2018). Furthermore, H-bonds
not only were formed with the key amino acids GLU129 and TYR131 (Gaweł et al.,
22

2004), but also with LYS338, which may be the reason that -CDDOCK Interaction
Energy of compound 12a is better than inhibitor UW1561. From the 3D image
of 12a bound to TgCDPK1, 12a was wrapped in the active pocket, and the tetrazole
part was buried deep in the pocket. UA formed H-bonds with TgCDPK1 amino acids
GLU135, GLU138, LYS338, and LYS185 (Fig. 6C and 6D), however, compared
to inhibitor UW1561, there is no H-bond formed with the key amino acid, which is
the difference observed from the binding of UA with the protein.
Table 4. The result of docking with TgCDPK1 (PDB ID: 4TZR).
Compound
12a
-CDDOCK Interaction Energy (k.cal/mol)
98.89
54.77
57.74
UA
UW1561
23

Figure 6. Docking result of compound 12a, UA and UW1561 with TgCDPK1 (PDB
ID: 4TZR). (A) 2D molecular docking modeling of compound 12a with 4TZR. (B)
Key residues in binding site surrounding 12a. (C) 2D molecular docking modeling of
compound UA with 4TZR. (D) Key residues in binding site surrounding UA. (E) 2D
molecular docking modeling of inhibitor UW1516 with 4TZR. (F) Key residues in
binding site surrounding UW1516.
5. Conclusions
24

Eight series of synthesized UA derivatives were evaluated for their anti-T.
gondii activities. Compared with spi, several compounds exhibited potent abilities to
restore the normal body weight of infected mice, better effects on liver and blood
indices, and stronger effects on GSH. Of the compounds tested, 12a displayed the
promising anti-T. gondii activity in vitro with an SI of 1.0, as well as the strong effect
against T. gondii in vivo, inhibiting tachyzoite growth by 55.3% in mice, which
exceeded the inhibitory effects of the traditional drug spi on the proliferation of T.
gondii. Molecular docking results showed that the ligand of compound 12a displayed
a similar orientation in the active site of TgCDPK1 binding to key amino acids
GLU129 and TYR131, compared to inhibitor UW1561. Thus, compound 12a can be
used as a hit for the development of more effective anti-T. gondii drugs.
Acknowledgement
The work was supported by the National Natural Science Foundation of China (No.
81160409) and The doctoral research project of dalian university (20181QL007).
We thank Joe Barber Jr., PhD, from Liwen Bianji, Edanz Editing China
(www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.
25

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