Design, synthesis, evaluation, and molecular docking of ursolic acid derivatives containing a nitrogen heterocycle as anti-inflammatory agents

Article abstract of DOI:10.1016/j.bmcl.2018.04.021

Ursolic acid derivatives containing oxadiazole, triazolone, and piperazine moieties were synthesized in an attempt to develop potent anti-inflammatory agents. Structures of the synthesized compounds were elucidated by ¹H NMR, ¹³C NMR, and HRMS. Most of the synthesized compounds showed pronounced anti-inflammatory effects at 100 mg/kg. In particular, compound 11b, which displayed the most potent anti-inflammatory activity of all of the compounds prepared, with 69.76% inhibition after intraperitoneal administration, was more potent than the reference drugs indomethacin and ibuprofen. The cytotoxicity of the compounds was also assessed by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, and no compounds showed any appreciable cytotoxic activity (IC₅₀ >100 μmol/L). Furthermore, molecular docking studies of the synthesized compounds were performed to rationalize the obtained biological results. Overall, the results indicate that compound 11b could be a therapeutic candidate for the treatment of inflammation.

Full text of DOI:10.1016/j.bmcl.2018.04.021

Accepted Manuscript
Design, synthesis, evaluation, and molecular docking of ursolic acid derivatives
containing a nitrogen heterocycle as anti-inflammatory agents
Zhi-Yu Wei, Ke-Qiang Chi, Ke-Si Wang, Jie Wu, Li-Ping Liu, Hu-Ri Piao
PII:
S0960-894X(18)30333-0
https://doi.org/10.1016/j.bmcl.2018.04.021
BMCL 25768
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
27 December 2017
8 April 2018
10 April 2018
Please cite this article as: Wei, Z-Y., Chi, K-Q., Wang, K-S., Wu, J., Liu, L-P., Piao, H-R., Design, synthesis,
evaluation, and molecular docking of ursolic acid derivatives containing a nitrogen heterocycle as anti-inflammatory
agents, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.04.021
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Design, synthesis, evaluation, and molecular docking of ursolic
acid derivatives containing a nitrogen heterocycle as
anti-inflammatory agents
Zhi-Yu Wei a, Ke-Qiang Chi a, Ke-Si Wang a, Jie Wu b, Li-Ping Liu a,*, and Hu-Ri
Piao b,*
a Medical College of Dalian University, Dalian, Liaoning Province, 116622, China
b Key Laboratory of Natural Resources and Functional Molecules of the Changbai
Mountain, Affiliated Ministry of Education; Yanbian University College of Pharmacy,
Yanji, Jilin Province, 133002, China
*Corresponding Author: Tel: + 86(433)243-6149; Fax: + 86(433)243-5026
E-mail: piaohr@ybu.edu.cn (H. R. Piao)
*Co-corresponding Author. Tel: +86 411 87402103; Fax: + 86(433)243-5026
E-mail: liuliping@ybu.edu.cn (L. P. Liu)
1

Abstract
Ursolic acid derivatives containing oxadiazole, triazolone, and piperazine
moieties were synthesized in an attempt to develop potent anti-inflammatory agents.
Structures of the synthesized compounds were elucidated by 1H NMR, 13C NMR, and
HRMS. Most of the synthesized compounds showed pronounced anti-inflammatory
effects at 100 mg/kg. In particular, compound 11b, which displayed the most potent
anti-inflammatory activity of all of the compounds prepared, with 69.76% inhibition
after intraperitoneal administration, was more potent than the reference drugs
indomethacin and ibuprofen. The cytotoxicity of the compounds was also assessed by
the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay,
and no compounds showed any appreciable cytotoxic activity (IC50 > 100 μmol/L).
Furthermore, molecular docking studies of the synthesized compounds were
performed to rationalize the obtained biological results. Overall, the results indicate
that compound 11b could be a therapeutic candidate for the treatment of
inflammation.
Keywords: Ursolic acid derivatives; Nitrogen heterocyclic; Anti-inflammatory activity;
Cytotoxicity; Molecular docking
2

Inflammation is a complex biological process for modulation of the immune
response against a diverse range of triggering factors, including infectious agents,
allergens, free radicals, highly refined foods, and a sedentary lifestyle.1 Exaggerated
and prolonged inflammation may cause various diseases, which can seriously threaten
human health, such as arthritis, sepsis, and even cancer.2,3 At present, non-steroidal
anti-inflammatory drugs (NSAIDs) are the most important class of widely used
therapeutics for the treatment of inflammation and account for 35% of the global
market for prescription pain medication.4-6 Common NSAIDs, such as aspirin and
indomethacin, can inhibit both cyclooxygenase-1 (COX-1) and cyclooxygenase-2
(COX-2), causing significant toxicity to the gastrointestinal tract and kidney.7-9 The
gastrointestinal irritation, bleeding, and ulceration side effects of classical NSAIDs
are caused by high COX-1 versus COX-2 selectivity, and may also be related to the
acidity of classical NSAIDs.10 The simple chemical derivatization (esterification or
amidation) of the carboxylic function of representative NSAIDs has been shown to
result, not only in reduction of the ulcerogenic effect, but also in increased
anti-inflammatory activity.11-17 Thus, there is a need to develop new anti-inflammatory
drugs with decreased acidity and increased specificity for targeting COX-2.
Pentacyclic triterpenes are a very powerful class of natural products because of
their wide range of biological activities and diversity of structures.18,19 Ursolic acid
(UA) is a well-known pentacyclic triterpene that is one of the major active
components of many traditional Chinese medicines.20 UA and its derivatives have
been reported to have antihepatodamage,21 anti-HIV,22 antimalarial,23
anti-inflammatory,24 antidiabetic,25 antimicrobial,26 and antitumor27 activity. However,
the low bioavailability of UA in vivo restricts its clinical application. In recent years,
chemical modification of UA has been widely investigated as a method of improving
its biological activity and bioavailability. Research has shown that keeping a polar
substituent at the C-3 position is essential for the pharmacological activity of UA.28
The introduction of a nitrogen-containing heterocycle has been shown to be a useful
tactic in the structural modification of natural products because the nitrogen atom can
carry a positive charge and act as a hydrogen-bond acceptor or donor that can strongly
3

influence the interaction between the molecule and its target.29 Recent studies have
used 1,3,4-oxadiazole as a pharmacophore because it is a good bioisostere of amides
and esters, which can influence the pharmacokinetic properties of drugs by increasing
the lipophilicity and thus the ability of drugs to reach their targets by transmembrane
diffusion.30 In addition, several 1,2,4-triazoles have been reported to possess potent
antimicrobial31 and anti-inflammatory32 activity. Piperazine-based drug discovery has
also attracted considerable attention in recent years. Studies have shown that the
incorporation of a piperazine moiety can occasionally provide unexpected
improvement in the bioactivity of compounds.33-35 Furthermore, it has been reported
that several ursolic acid, oxadiazole, triazole, and piperazine derivatives are COX-2
inhibitors and anti-inflammatory agents (Figure 1).36-39 These findings suggest that
introducing oxadiazole, triazole, and piperazine heterocycles into the C-28 position,
and keeping an hydroxy substituent at the C-3 position of the ursolic acid scaffold
may produce novel UA derivatives targeting COX-2 with improved anti-inflammatory
properties.
Figure 1. Chemical structures of some previous reported compounds as COX-2 inhibitors and
anti-inflammatory agents
On the basis of the abovementioned reports, the present work aimed to
synthesize novel derivatives of UA by introducing an 1,3,4-oxadiazole moiety, a
piperazine ring, or a 4-phenyl-1H-1,2,4-triazol-5(4H)-one moiety to the UA nucleus
with the objective of discovering potent anti-inflammatory agents that are devoid of
gastrointestinal side effects. The substituents on the phenyl ring were simultaneously
altered to investigate their contribution to the biological activity. Hence, four novel
4

series of ursolic acid derivatives were synthesized and evaluated for
anti-inflammatory activity. Furthermore, the cytotoxicity of the ursolic acid
derivatives was assessed by the MTT assay and the molecular docking with COX-2
was also studied.
Scheme 1. Synthetic scheme for the synthesis of compounds 4a-j, 6a-b, 7a-d and 11a-d. Reagents
and conditions: (a) MeOH, H2SO4; NH2NH2.H2O; (b) POCl3; (c) K2CO3, KI, acetone, reflux, 10 h;
(d) K2CO3, KI; (e) Br(CH2)nBr; K2CO3, KI, acetone, reflux, 10 h; (f) NH2NHCOOCH3,
CH(OC2H5)3; EtOH, MeONa, reflux, 48 h; (g) Br(CH2)nBr; K2CO3, KI; (h) K2CO3, KI, acetone,
reflux, 10 h
The synthesis of target compounds is outlined in Scheme 1. The appropriate
benzohydrazide 2 was prepared according to the reported method.40,41 An equimolar
mixture of chloroacetic acid and benzohydrazide 2 was refluxed in phosphorus
oxychloride (105–110 °C) for 5–6 h to afford compound 3. The resulting mixture was
cooled to room temperature and poured into ice water to give a solid, which was
filtered and washed several times with cold water.42 Then, reaction with anhydrous
piperazine in refluxing acetone in the presence of KI/K2CO3 afforded the key
intermediate 5. Compound 10 was synthesized by reacting aniline with methyl
5

hydrazinecarboxylate in refluxing ethanol, in the presence of triethyl orthoformate
and sodium methoxide, followed by reaction with a dibromoalkane in the presence of
potassium carbonate and potassium iodide in refluxing acetone, as previously
described.43 The target compounds 4a–j, 6a–b, 7a–d, and 11a–d were obtained in
60%–90% yields by reacting UA with the intermediates 3, 5, or 10 in refluxing
acetone in the presence of KI/K2CO3.35 The structures of the target compounds were
characterized by 1H NMR, 13C NMR, and HRMS.
The in vivo anti-inflammatory activities of the synthesized compounds were
evaluated using the in vivo para-xylene-induced mice ear-swelling model. Dimethyl
sulfoxide was used as the vehicle for the primary screening of the synthesized
compounds and all the target compounds were administered at a dose of 100 mg/kg.
Indomethacin (100 mg/kg) and ibuprofen (100 mg/kg) were used as reference drugs,
and UA (100 mg/kg) was used as a positive control. The anti-inflammatory activity
was assessed based on the ability of the tested compounds to prevent edema,44 and the
results are shown in Table 1.
Table 1. Anti-inflammatory activities of compounds 4a–j, 6a–b, 7a–d and 11a–d following i.p.
administration.
Number
Edema mean
± S.D. (mg)
Inhibition
rate (%)
—
Cytotoxic activity
IC50 (μM) a
NT b
R
Dose (mg/kg) of mice
DMSO
—
100
100
100
100
100
100
100
100
100
100
100
10
10
10
10
10
10
10
10
10
10
10
13.23 ± 0.81
9.68 ± 0.94***
9.90 ± 0.61***
5.60 ± 1.27***
4.98 ± 1.22***
8.10 ± 1.36***
4.61 ± 0.65***
13.30 ± 0.78**
5.90 ± 0.84***
6.12 ± 1.06***
4.13 ± 1.40***
Indometacin
—
26.83
25.17
57.67
62.31
38.77
65.44
—
NT
Ibuprofen
—
NT
UA
4a
4b
4c
4d
4e
4f
—
23.8
H
>100
2-Cl
3-Cl
4-Cl
2-Br
3-Br
4-Br
>100
>100
>100
55.27
53.74
68.86
>100
>100
>100
4g
6

2-CH3
3-CH3
4-CH3
H
100
100
100
100
100
100
100
100
100
100
100
100
100
10
10
10
10
10
10
10
10
10
10
10
10
10
4.56 ± 0.66***
13.52 ± 0.69*
5.53 ± 1.00***
5.41 ± 0.63
57.16
—
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
4h
4i
58.23
59.08
60.99
47.75
58.74
31.96
66.49
64.22
69.76
66.09
56.66
4j
6a
3-Br
H
5.16 ± 1.20***
6b
7a
6.90 ± 1.11
4-F
5.43 ± 1.50**
9.12 ± 1.50**
4.40 ± 1.06***
4.73 ± 0.82***
4.00 ± 0.74***
4.48 ± 0.41***
5.73 ± 0.85***
7b
7c
2-Cl
3-Br
－
7d
11a
11b
11c
11d
－
－
－
*: p < 0.05, **: p < 0.01, ***: p < 0.001 compared with a vehicle group.
—: no anti-inflammatory activity
a The cytotoxic activity of compounds was evaluated by MTT assay after 24 h treatment of HCT116 cells. Values
were shown as mean, n=3.
b Not tested.
Most of the synthesized compounds showed potent anti-inflammatory activities
at 100 mg/kg with inhibition rates ranging from 31.96% to 69.76%. It is noteworthy
that compound 11b exhibited the most pronounced anti-inflammatory activity of all
the compounds with an activity of 69.76%, which was higher than that of UA
(57.67%), ibuprofen (26.83%), and indomethacin (25.17%). Disappointingly,
compounds 4d and 4i did not exhibit any anti-inflammatory activity at the same dose,
and compound 7c only showed weak anti-inflammatory activity with an activity of
31.96%. Preliminary structure activity relationship (SAR) analysis can be drawn from
the results for anti-inflammatory activity. The in vivo anti-inflammatory activity of
compounds 4a, 4c, 4g and 4j was more potent than that of UA, which implies that
introduction of an oxadiazole group can improve activity. The presence of different
substituents on the phenyl ring of the 1,3,4-oxadiazole group was found to exert an
appreciable influence on the observed inhibitory effect on anti-inflammatory activity
in series 4, but no clear pattern could be found for the SAR. The unsubstituted
compound 4a exhibited potent inhibitory activity with an activity of 62.31%. For the
halogen substituted derivatives, the activity was in the order 3-Cl > 2-Cl > 4-Cl for
the chlorinated compounds (4c > 4b > 4d) and 4-Br > 2-Br > 3-Br for the brominated
7

compounds (4g > 4e > 4f). The compounds (4j, 4h, and 4i) bearing an
electron-donating group showed an activity order of 4-CH3 > 2-CH3 > 3-CH3 (4j >
4h > 4i). For the 6 and 7 series, there was an obvious enhancement in activity when a
piperazine ring was introduced. Compounds 6a (59.08%), 6b (60.99%), 7b (58.74%)
and 7d (66.49%) exhibited more potent inhibitory activity than UA (57.67%),
respectively. Comparing compounds 4f, 6b and 7d with 3-Br substitutions, the in vivo
anti-inflammatory activity was 7d > 6b > 4f. These results imply that in some cases
the incorporation of a piperazine moiety to ursolic acid could provide unexpected
improvement in the bioactivity of UA derivatives. For the compounds in series 11,
compounds 11a, 11b, 11c, and 11d exhibited significant anti-inflammatory activity
with inhibition rates ranging from 56.66% to 69.76%, 2-fold more potent than
ibuprofen and indomethacin and more potent than that of UA. In addition, the order of
activity for the different carbon chain lengths was C3 > C4 > C2 > C5 and the order
of in vivo anti-inflammatory activity was 11b > 11c > 11a > 11d. These results
indicate that incorporation of a triazolone moiety to ursolic acid can improve the
anti-inflammatory activity. In general, the in vivo anti-inflammatory activity was in
the order: series 11 > series 6 ~ series 7 > series 4, indicating that the triazolone
moiety was favorable for anti-inflammatory activity, and introducing a piperazine ring
to the oxadiazole structure can also enhance the biological activity of the UA
compounds.
Considering its promising anti-inflammatory activity, compound 11b was chosen
for further evaluation. A dose of 100 mg/kg was orally administered at different time
intervals (1, 2, 3, 4, 5, and 24 h) after xylene application. As shown in Table 2, the
activity profile of compound 11b was consistent with that of indomethacin. Notably,
the activity of compound 11b (41.17%) reached its peak at 2 h and was found to be
more potent than indomethacin at this time point (30.94%). The effect of the dosage
on the activity of compound 11b was also evaluated at concentrations of 25, 50, 100
and 125 mg/kg at 2 h after oral administration as shown in Table 3. The results showed
a maximal effect, with an ear inflammation inhibition rate of 50.12%, at 125 mg/kg,
which was more potent than indomethacin (38.74%). However, the inhibitory rate of
8

125 mg/kg doses was not be effectively measured due to the cytotoxicity of the
compound which resulted in the death of the test mice. The inhibition rate of 125
mg/kg doses did not increase significantly compare with 100 mg/kg group, but the
cytotoxicity was significantly increased. In a nutshell, these results indicated that the
100 mg/kg group of compound 11b had the best inhibition (47.86%) under the safe
dose.
Table 2. Anti-inflammatory activity of compound 11b administered orally at different times after
xylene application.
Time (h)
Dose (mg/kg)
Number of mice
Inhibition (%)
11b
Indometacin
11.96
30.94**
1
100
100
100
100
100
100
10
10
10
10
10
10
22.56
41.17**
2
3
29.73
18.65
4
20.25
15.63
5
16.37
10.28
24
—
—
**: p < 0.01 compared with a vehicle group.
—: no anti-inflammatory activity
Table 3. Anti-inflammatory activity of compound 11b administered orally at different doses.
Time (h)
Dose (mg/kg)
Number of mice
Inhibition (%)
11b
Indometacin
38.74*
33.32**
2
2
2
2
125
100
50
10
10
10
10
50.12
47.86**
35.24
19.73*
22.76
25
4.85*
*: 0.01 < p < 0.05 compared with a vehicle group. **: p < 0.01 compared with a vehicle group.
The cytotoxicity of these compounds was assessed in HCT116 cells by the MTT
assay, and the results are shown in Table 1. None of the compounds showed any
appreciable cytotoxic activity (IC50 > 100 μmol/L), except for UA (IC50 = 23.8
μmol/L). Furthermore, to determine whether the synthesized compounds were
selectively toxic towards normal or cancer cells, we evaluated the cytotoxicity of the
most potent compound 11b using the MTT assay. As shown in Table 4, compound 11b
gave IC50 values of 100, 200, and 200 µM against HCT116, AGS, and L02 cells,
9

respectively, which indicated that compound 11b showed no significant cytotoxicity
towards normal and cancer cells at concentrations less than 100 µM and did not show
much selectivity.
a
Table 4. Cytotoxic activity (IC50 , µg/mL) of compound 11b against human cell lines.
Substituents
In vitro cytotoxicity IC50 a (μM)
Compound
R
HCT116 b
AGS c
L02 d
> 200
－
> 100
> 200
11b
a IC50 is the concentration required to inhibit the cell growth by 50%. Data represent the average of three
independent experiments running in triplicate. Variation was generally between 5–10%.
b Human colon cancer cells.
c Human lung cancer cells.
d Human normal hepatic cells.
The superior anti-inflammatory activity and low cytotoxicity of the target
compounds led us to study the molecular docking with the COX-2 enzyme to
rationalize the observed anti-inflammatory activity and elucidate a possible
mechanism of action of these compounds. All the calculations were performed using
MOE 2008.10 software form Discovery studio 3.1 installed on 2.4G Core i5. The
crystal structure of the COX-2 enzyme obtained from the protein data bank (PDB ID:
5FDQ)45 was used for the molecular docking. Enzyme structures were checked for
missing atoms, bonds, and contacts. Hydrogen atoms were added to the enzyme
structure. Water molecules and bound ligands were manually deleted. The active site
of the enzyme was defined to include residues within a 10.0 Å radius to any of the
inhibitor atoms. The automated molecular docking program of MOE 2008.10 was
used to dock compounds 4a, 11b, and UA on the active site of the COX-2 enzyme.
The most stable molecular-docked model was selected according to the best scored
conformation predicted by the MOE scoring function. The results indicated that the
anti-inflammatory activities of the compounds might correlate with the results for
docking with COX-2 (Table 5). Compounds 4a and 11b with better anti-inflammatory
activities showed higher docking scores (150.19 and 151.72 kcal/mol) than UA
(126.69 kcal/mol).
10

Table 5. The Libdock scores of docking (compounds 4a, 11b and UA) with COX-2 (PDB ID:
5FDQ).
Compound
4a
Inhibition rate (%)
62.31
LibDock score (k.cal/mol)
150.19
151.72
126.69
69.76
11b
57.67
UA
The preferred coordination modes of 4a, 11b, and UA with the COX-2 protein
are presented in Figure 2. All the ligands showed a similar orientation in the COX-2
active site and the complex formed was stabilized by the formation of hydrogen bonds.
It was observed that the oxadiazole fragment of compound 4a fitted into the cavity
formed by Thr212 and Met458 that is required for COX-2 fitting (Fig. 2A, 2B).
H-bond interactions were formed between the nitrogen atom of the oxadiazole group
and the amino acid Thr212. Compound 11b, which had the highest docking score of
151.72 kcal/mol and the best anti-inflammatory activity (inhibition rate, 69.76%),
showed two hydrogen bonds and two other chemical bonds with the amino acids
Gln454, Val447, Ala446, and Ala450 of COX-2 (Fig. 2C, 2D). Only one hydrogen
bond, with Ala99, was formed between UA and COX-2 (Fig. 2E, 2F). The preliminary
docking results suggested that compounds 4a and 11b possibly cause their
anti-inflammatory activity through the interaction with the COX-2 protein by
targeting residues in the active cavities of COX-2. This may well explain why these
compounds showed selective activity against the COX-2 enzyme. To validate the
results of the molecular docking, further enzyme studies of these compounds with
COX-2 is currently underway in our laboratory.
11

A
B
C
D
F
E
Figure 2. Docking result of compound 4a, 11b and UA with COX-2 (PDB ID: 5FDQ). (A) Key
residues in binding site surrounding 4a. (B) 2D molecular docking modeling of compound 4a with
5FDQ. (C) Key residues in binding site surrounding 11b. (D) 2D molecular docking modeling of
compound 11b with 5FDQ. (E) Key residues in binding site surrounding UA. (F) 2D molecular
docking modeling of compound UA with 5FDQ.
In summary, 20 UA derivatives containing oxadiazole, triazolone, and piperazine
moieties were synthesized and evaluated for anti-inflammatory activity. Compound
11b showed the most potent inhibitory activity towards ear inflammation of all the
12

synthesized compounds (69.76%), which was higher than ibuprofen (25.17%) and
indomethacin (26.83%) at 100 mg/kg (i.p.), and was 1- and 2-fold more potent than
the standard drugs. The cytotoxicity of the compounds was assessed by the MTT
assay, and no compounds showed any appreciable cytotoxic activity (IC50 > 100
μmol/L), in contrast to UA. Furthermore, the molecular docking results indicated that
the UA derivatives exhibited high affinity for the COX-2 active site and possibly
exhibit their anti-inflammatory potency via inhibiting COX-2. Our findings might
provide information for the development of potentially new and safe COX-2
inhibitors as anti-inflammatory agents.
Acknowledgment
This work was supported by the National Natural Science Foundation of China
(Grant number 81260468) and the Natural Science Foundation of Jilin Province (No.
20160101218JC).
We thank Victoria Muir, PhD, from Liwen Bianji, Edanz Group China
(www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.
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In an attempt to develop novel and potent anti-inflammatory agents, four novel
series of ursolic acid derivatives containing oxadiazole, triazolone and piperazine
moieties were designed, synthesized, and evaluated for anti-inflammatory activity.
The cytotoxicity and molecular docking studies of these compounds was also
assessed.
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Highlights:
Ursolic acid derivatives containing nitrogen heterocyclic were synthesized.
The anti-inflammatory activity and cytotoxicity of compounds were evaluated.
Compound 11b is a promising anti-inflammatory agent.
All compounds did not exhibit any remarkable cytotoxic activity.
 The molecular docking studies of 11b was performed.
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