Design, synthesis, and biological evaluation of novel nitrogen heterocycle-containing ursolic acid analogs as antitumor agents

Article abstract of DOI:10.3390/molecules24050877

Nineteen ursolic acid analogues were designed, synthesized, and evaluated for their antiproliferative activity against the Hela and MKN45 cell lines. Some compounds containing a piperazine moiety displayed moderate to high levels of antitumor activities against the tested cancer cell lines. The most potent compound shares the IC₅₀ value of 2.1 μM and 2.6 μM for the Hela and MKN45 cell lines, respectively. Further mechanism studies and in vivo antitumor studies have shown that it decreased the apoptosis regulator (BCL2/BAX) ratio, disrupted mitochondrial potential and induced apoptosis, and suppressed the growth of Hela xenografts in nude mice.

Full text of DOI:10.3390/molecules24050877

molecules
Article
Design, Synthesis, and Biological Evaluation of
Novel Nitrogen Heterocycle-Containing Ursolic Acid
Analogs as Antitumor Agents
†
†
Wenzhi Wang , Lei Lei , Zhi Liu, Hongbo Wang * and Qingguo Meng *
School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University),
Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs
in Universities of Shandong, Yantai University, Yantai 264005, China; wxwnx123@163.com (W.W.);
1
8363855773@163.com (L.L.); zhi_liu0812@163.com (Z.L.)
*
Correspondence: hongbowangyt@163.com (H.W.) qinggmeng@163.com (Q.M.); Tel.: +86-535-6706022 (Q.M.);
Fax: +86-535-6706066 (H.W.)
†
Authors contributed equally.
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 21 January 2019; Accepted: 19 February 2019; Published: 1 March 2019
Abstract: Nineteen ursolic acid analogues were designed, synthesized, and evaluated for their
antiproliferative activity against the Hela and MKN45 cell lines. Some compounds containing a
piperazine moiety displayed moderate to high levels of antitumor activities against the tested cancer
cell lines. The most potent compound shares the IC50 value of 2.1 µM and 2.6 µM for the Hela and
MKN45 cell lines, respectively. Further mechanism studies and in vivo antitumor studies have shown
that it decreased the apoptosis regulator (BCL2/BAX) ratio, disrupted mitochondrial potential and
induced apoptosis, and suppressed the growth of Hela xenografts in nude mice.
Keywords: ursolic acid analogues; synthesis; nitrogen heterocycles; apoptosis; antitumor activity
1
. Introduction
Ursolic acid (3
naturally in many vegetables, fruits, and Chinese medicinal herbs [
possess extensive pharmacological effects, including antiviral [ ], antibacterial [
antiosteoporosis [ ], antidiabetic [ 10], anti-inflammatory, and hepatoprotective activity [11,
β
-hydroxy-12-urs-12-en-28-oic acid, UA) is a pentacyclic triterpenoid that occurs
]. UA and its derivatives
], anticancer [6,7],
12].
1,2
3
4,5
8
9,
In recent years, increasing attention has been focused on the antitumor activity and the possible
mechanisms of UA and its derivatives. It has been reported that they exert antitumor activity by a
number of different mechanisms. These include inducing apoptosis and differentiation, inhibition of
proliferation and angiogenesis, promotion of chemosensitization, and induction of cell cycle arrest [13].
Intense efforts have been made to endow UA with higher water solubility and to achieve an improved
antineoplastic effect [2,14–16].
Nitrogen heterocycles are significant structural components of most new synthetic molecules
that are endowed with significant biological activity [17,18] and pharmaceuticals. More than 59%
of unique small molecule drugs that are approved by the FDA contain nitrogen heterocycles [19].
Studies showed that 3-oxo-UA-triazolyl derivatives with o-bromo, o-chloro, or o-methoxy substitution
on the aromatic ring more potently inhibit the growth of MCF-7 and THP-1 cancer cell lines [20].
UA analogues containing an acyl piperazine moiety at C-28 exhibit high inhibitory activity against
MGC-803 and Bcap-37 cancer cell lines and promote apoptosis [21]. In addition, some UA derivatives
containing quinoline displayed potent antitumor activity against MDA-MB-231, Hela, and SMMC-7721
cell lines [22].
Molecules 2019, 24, 877; doi:10.3390/molecules24050877
www.mdpi.com/journal/molecules

Molecules 2019, 24, 877
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Using a drug discovery strategy that is based on nitrogen heterocycles, nineteen UA
analogues containing nitrogen heterocycles were designed and synthesized (Schemes 1–3). Their
antitumor activities were evaluated and the possible mechanism of Hela cell growth inhibition was
also investigated.
Scheme 1. Synthesis of compounds
3
–
7. Reagents and conditions: (
a
) PCC, CHCl , rt, 6 h
3
◦
(
b
) pyridinium tribromide, HOAc, rt, 6 h (
c) thiourea, 80 C, 10 h (
d
) 3-bromo-propyne, K CO ,
2
3
◦
N,N-dimethylformamide (DMF), 60 C 1 h (
e
) N -R, CuSO 5H O, sodium ascorbate, EtOH, H O, rt,
·
3
4 2 2
◦
◦
4
h (f) chloroacetonitrile, K CO , DMF, 60 C 1 h (g) NaN , ZnCl , isopropanol/H O, 85 C, 6 h.
2
3
3
2
2
Scheme 2. Synthetic pathways of compounds
8
–
14. Reagents and conditions: (
a
) 1,2-dibromoethane or
◦
1
,3-dibromopropane or 1,4-dibromobutane, K CO , DMF, 60 C, 1 h (
b
) piperazine or homopiperazine
2
3
◦
◦
or 1-methylpiperazine, K CO , DMF, 80 C, 30–60 min (
c
) Ac O, pyridine, 0 C~r.t, 15 h
2
3
2
◦
◦
(
d
) 2-chloroethanol, K CO , DMF 60 C, 3 h (
e
) MsCl, pyridine, 10 C~r.t, 3 h (
f
) piperazine
2
3
◦
◦
or homopiperazine or morpholine, K CO , DMF, 60 C, 30 min (
g
) PCC, CHCl , 0 C, 10 h
2
3
3
◦
h) 4-fluorobenzyl bromide or 2-nitrobenzyl bromide, K CO , DMF, 60 C 1 h.
2 3
(

Molecules 2019, 24, 877
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Scheme 3. Synthesis of 17 and 18. Reagents and conditions: (
a
) isoamyl nitrite, t-BuOK, t-BuOH,
◦
r.t, 2.5 h (
b
) hydroxylamine hydrochloride, pyridine, 110 C, 2 h (
c
) EtOH, NaOH, NaOCl, rt, 2 min
◦
◦
(
d
) 2-chloroethanol, K CO , DMF, 60 C, 3 h (
e
) MsCl, pyridine, 10 C~r.t, 3 h (
f
) piperazine, K CO ,
2
3
2
3
◦
DMF, 60 C, 30 min.
2
. Results and Discussion
2
.1. Chemistry
The compounds were synthesized, as outlined in the Schemes 1–3. The initial key intermediate
was synthesized according to published protocols [23]. As shown in Scheme 1, compound was
3
3
treated with 3-bromo-1-propyne or chloroacetonitrile in N,N-dimethylformamide (DMF), followed
by a click reaction with corresponding azides to introduce a triazole or tetrazole moiety (5a and ).
Compounds 8a were synthesized from UA and dibromoalkane, and then treated with piperazine,
homopiperazine, or 1-methylpiperazine to gain derivatives 9a with a moderate yield (38–43%).
Among them, compounds 9a and 9c have been reported as antitumor agents by M.C. Liu and X. Li,
respectively [21 24]. UA was subjected to acetylation and reacted with 2-chloroethanol to obtain
compound 10, followed by sulfonylation to generate derivative 11. Subsequently, compound 11 was
treated with piperazine, homopiperazine, or morpholine to afford derivatives 12a with a yield of
1–75%. Compound 13 was synthesized from compound 9a by oxidation with PCC in chloroform.
Compound 12a was treated with 4-fluorobenzyl or 2-nitrobenzyl bromide to generate derivatives
–e
7
–c
–
e
,
–
c
7
1
4a–b (Scheme 2).
Compound 17, bearing a furazan moiety at C-2 and C-3 of UA, was synthesized according to
the published method [25]. It was further spliced with an ethyl-piperazine to yield compound 18
1
(
Scheme 3). All of the target compounds were purified by silica gel column chromatography. H-NMR,
C-NMR and HR-MS spectra of compounds are shown in Supplementary Materials.
1
3
2
.2. Biological Evaluation
The target compounds were screened for antiproliferative activity against Hela and MKN45 cancer
cell lines using an MTT assay. Cisplatin and UA were used as positive controls and the preliminary
results are presented in Table 1. Compounds 5a and shared low to moderate antiproliferative
activity (inhibition rates from 1–45%) against Hela and MKN45 cancer cells at the tested concentration
10 M). Compounds 9a 9c 12a 12b 13 14a, and 18 displayed much higher activity than UA and
possessed IC50 values that were lower than 10 M. Nevertheless, derivatives 9e 12c, 14b, and 17
showed moderate to low activity against the two cancer cell lines.
–
e
7
(
µ
,
,
,
,
,
µ
,
The structure activity relationship studies revealed that the introduction of thiazole on A ring and
triazole or tetrazole moiety on C-28 have little help to improve antitumor activities. The introduction
of piperazine or homopiperazine can significantly improve the antitumor activity. The length of the
carbon chains between the carboxyl and piperazine moiety of compounds 9a
affected the antiproliferative activity. The two- or three-carbon chain linker appears to the optimum,
and 9c with a straight two- and three -carbon chain showed good antitumor activities against
, 9c, and 9d could have
9
a
the two cell lines. Compounds 12a and 12b possess more potent anticancer activity when compared
with 9a and 9b, which may be attributed to the introduced acetyl group. 14a showed much higher
antiproliferative activity than both UA and cisplatin with IC50 values of 2.1
µM and 2.6 µM for Hela
and MKN45, respectively. Compound 14b displayed weaker antiproliferative activity as compared

Molecules 2019, 24, 877
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with UA. These results demonstrated that 4-fluorobenzyl and piperazine moieties are the crucial
anticancer functional groups of these compounds.
Table 1. Cytotoxic activities of the compounds against Hela and MKN45 cell lines.
Inhibition Rates a
IC50 (µM) b
Compound
Hela
MKN45
Hela
MKN45
UA
13%
1%
2%
16%
3%
2%
18%
31%
35%
45%
31%
32%
35%
51%
29%
55%
10%
27%
77%
75%
0%
15.1 ± 2.7
16.7 ± 1.4
5
a
>20
>20
5
5
b
c
>20
>20
>20
NT
NT
>20
c
5
5
7
9
d
NT
e
a
NT
NT
1%
NT
59%
34%
61%
9%
24%
81%
89%
8%
9.8 ± 1.8
7.4 ± 1.9
9
9
b
c
>20
>20
9.3 ± 0.5
16.2 ± 0.8
NT
6.4 ± 0.3
7.3 ± 0.2
NT
7.9 ± 0.2
19.3 ± 0.3
NT
6.4 ± 0.1
6.2 ± 0.5
NT
9
9
d
e
1
1
1
2a
2b
2c
1
3
58%
89%
16%
3%
90%
77%
61%
84%
7%
3%
86%
8.5 ± 0.3
2.6 ± 1.1
NT
8.9 ± 0.1
2.1 ± 0.3
NT
1
1
4a
4b
1
1
7
8
NT
NT
3.9 ± 0.6
4.5 ± 0.2
d
CDDP
62%
15.1 ± 0.9
2.8 ± 0.1
a
Inhibition rate of cells treated with 10 µ µM) of compound that
M concentration for 72 h. b The concentration (
inhibited 50% of cell growth at 72 h after treatment. c Not tested. d Cisplatin, positive control.
Since the inhibition of apoptosis is one of hallmarks of cancer, the effects of compound 14a on cell
cycle progression were examined by the propidium iodide staining method. The results showed that
compound 14a, but not UA, at a concentration of 5 µM increased the proportion of cells in the sub-G1
phase (Figure 1A). This indicated that 14a induced cell death. Consistent with this, western blotting
showed that 14a increased the abundance of cleaved poly-ADP ribose polymerase (PARP), which is a
biomarker of apoptosis (Figure 1B). The disruption of the mitochondrial membrane potential, regulated
by BCL2 family of proteins, such as BCL2 and BAX, is a mechanism that triggers apoptosis following
treatment by anticancer drugs. Our results showed that treatment with 14a decreased the expression
of BCL2, one of the main mitochondrial-associated antiapoptotic proteins. 14a also increased the
expression of BAX, a key mitochondrial-associated apoptotic protein, in a dose-dependent manner
(Figure 1C). Therefore, our results suggest that compound 14a induces apoptosis via the intrinsic
mitochondrial pathway.

Molecules 2019, 24, 877
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Figure 1. Apoptotic profile of compound 14a in Hela cells. (A) DNA cell cycle analyses of Hela cells
exposed to 14a. The cells were treated with indicated concentrations of 14a for 24 h and stained with
propidium iodide (PI, 50
) and (
treated with 2
was used as an internal control. (
units by ImageJ software and was used to calculate the BAX/BCL2 ratio. (
µ
g/mL) to determine DNA fluorescence and cell cycle phase distribution.
5
(
B
C
) Effect of 14a on the expression of key apoptotic-related proteins. Cells (2
M or 5 M of 14a for 24 h before cell lysates were prepared and analyzed.
) The relative density of BCL2 and BAX was measured as arbitrary
) The relative density
×
10 ) were
µ
µ
β-actin
D
E
of cleaved poly-ADP ribose polymerase also was measured as arbitrary units by ImageJ software.
Data are representative of three experiments.
A xenograft model in nude mice was established and used to evaluate the in vivo antitumor
activity of 14a. The mice were treated with vehicle, cisplatin (3 mg/kg, i.p.) and 50 mg/kg of 14a
Our results showed that 14a could significantly suppress growth of Hela xenograft tumors and had a
similar effect to cisplatin treatment (Figure 2 and Table 2).
.
Figure 2. Effect of 14a on the growth of Hela in nude mice. Tumor diameter was serially measured
with a vernier caliper to calculate the relative tumor volume. Each experiment was conducted using 5
mice per group. Vehicle (
between the drug-treated group and the vehicle was determined by one-way ANOVA (* p < 0.05).
•
); 50 mg/kg 14a (N); and, 3 mg/kg cisplatin (ꢀ). The statistical difference

Molecules 2019, 24, 877
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Table 2. The anti-tumor activity of 14a against the growth of Hela in nude micea.
Number
Initial/End)
Body Weight (g)
Tumor Weight (g)
Group (mg/kg)
(
Initial
End
g
IR (%)
Control
Cisplatin 3
5/5
5/5
5/5
20.52 ± 0.99
20.80 ± 1.34
21.08 ± 1.04
20.56 ± 2.11
21.70 ± 1.32
20.48 ± 0.89
2.12 ± 0.40
0.88 ± 0.12 *
1.20 ± 0.59 *
58.49
43.40
1
4a 50
a
Data are expressed as the mean ±SD (n = 5). *p < 0.05, compared with control.
3
. Materials and Methods
3
.1. Materials
Ursolic acid (>95%) was purchased from Shaanxi Pioneer Biotech Co., Ltd (Hanzhong, China).
Reagents and solvents were either analytical or chemical purity. The silica gel GF254 plates and silica gel
200 to 300 mesh, Qingdao, Shandong, China) were used. The melting points were determined using
an XT3A micro-melting point apparatus and are uncorrected (Beijing Keyi Company, Beijing, China).
(
1
13
H NMR and C NMR were recorded on the Avance-400 spectrometer (Bruker, Ettlingen, Germany).
High resolution mass spectra (HR-MS) were obtained on an AutoSpec Ultima-Tof spectrometer with
an electrospray ionization (ESI) source (Micromass, Manchester, UK).
3
.2. Chemistry
3
.2.1. Preparation of Compounds 3, 4 and 6
Compound
3 was synthesized by the literature method with slight modifications [21]. The solution
of
3 (500 mg, 0.98 mmol), K CO (135 mg, 0.98 mmol), and 3-bromopropyne (0.11 mL, 1.28 mmol) or
2
3
◦
chloroacetonitrile (0.08 mL, 1.26 mmol) in DMF (20 mL) was stirred at 60 C for 1 h. After cooled to
room temperature, the mixture was poured into ice-cold water and filtered. The solid was dissolved
with ethyl acetate (100 mL) and washed with water (50 mL) and brine (50 mL
×
2), dried over Na SO
2 4
anhydrous, filtered, and concentrated to obtain
yield, respectively.
4 and 6, as yellowish-white powder, in 92% and 95%
3
.2.2. General Procedure for the Synthesis of Compounds 5a–e
To a solution of compound (200 mg, 0.36 mmol) in anhydrous alcohol (5 mL), CuSO4
37 mg, 0.15 mmol), sodium ascorbate (18 mg, 0.09 mmol), and corresponding benzyl azide (0.6 mmol)
were added. The mixture was stirred at room temperature for 4 h and then concentrated. The residue
was dissolved with ethyl acetate (80 mL), washed with water (40 mL) and brine (40 mL 2), and then
dried over Na SO anhydrous. After filtration, concentration and purification by silica gel column
4
·
5H O
2
(
×
2
4
chromatography with cyclohexane/methanol (3:1), compounds 5a–e were afforded.
1-(3-chlorobenzyl)-1H-1,2,3-triazol-5-yl]methyl-urs-12-en-[(3,2-d)thiazol-2-amino]-28-oate (5a). 26%, off-white
[
◦
1
powder. mp: 120.1–122.5 C. H NMR (400 MHz, CDCl ):
δ
7.51 (1H, s), 7.36–7.27 (2H, m), 7.23 (1H, s),
.14 (1H, d, J = 7.0 Hz), 5.53–5.42 (2H, m), 5.22 (1H, s), 5.19–5.09 (2H, m), 2.47 (1H, d, J = 15.4 Hz), 2.21
1H, d, J = 11.5 Hz), 2.16 (1H, d, J = 15.6 Hz), 1.25 (6H, s), 1.20 (3H, s), 1.12 (3H, s), 1.06 (3H, s), 0.92 (6H, s);
3
7
(
1
3
C NMR (100 MHz, CDCl3):
δ 177.64, 164.78, 152.08, 143.92, 138.17, 136.52, 135.20, 130.54, 129.20, 128.20,
126.21, 125.57, 124.04, 116.04, 57.45, 53.54, 53.04, 52.68, 48.32, 46.14, 42.34, 39.68, 39.26, 38.94, 38.89, 38.33,
3
6.95, 36.63, 32.50, 30.75, 30.49, 28.09, 24.30, 23.43, 23.31, 22.29, 21.25, 19.66, 17.08, 16.79, 15.72. HR-MS m/z
+
calcd. for C H ClN O S [M + H] 716.37595, found 716.37531.
41
55
5
2
[
1-(4-fluorobenzyl)-1H-1,2,3-triazol-5-yl]methyl-urs-12-en-[(3,2-d)thiazol-2-amino]-28-oate (5b). 26%, off-white
◦
1
powder. mp: 115.0–116.9 C; H NMR (400 MHz, CDCl ):
δ
7.47 (1H, s), 7.30–7.22 (2H, m), 7.05 (2H, t,
J = 8.5 Hz), 5.46 (2H, s), 5.21 (1H, s), 5.18–5.06 (2H, m), 2.47 (1H, d, J = 15.4 Hz), 2.20 (1H, d, J = 12.7 Hz),
.16 (1H, s), 1.24 (3H, s), 1.20 (3H, s), 1.11 (3H, s), 1.06 (3H, s), 0.92 (6H, s), 0.83 (3H, d, J = 6.4 Hz), 0.59
3
2

Molecules 2019, 24, 877
7 of 14
(
3H, s); 13C NMR (100 MHz, CDCl3):
δ
177.64, 164.75, 163.01 (d, J = 248.4 Hz), 152.13, 143.81, 138.17, 130.45
(d, J = 3.3 Hz), 130.13 (d, J = 8.4 Hz), 125.56, 123.84, 116.30 (d, J = 21.8 Hz), 57.47, 53.53, 53.05, 52.68, 48.32,
4
6.14, 42.34, 39.68, 39.26, 38.94, 38.91, 38.34, 36.96, 36.65, 32.51, 31.08, 30.76, 30.49, 28.08, 24.30, 23.44, 23.34,
+
22.26, 21.26, 19.64, 17.09, 16.77, 15.72. HR-MS m/z calcd. for C H FN O S [M + H] 700.40550, found
41
55
5
2
7
[
00.40482.
1-(2-nitrobenzyl)-1H-1,2,3-triazol-5-yl]methyl-urs-12-en-[(3,2-d)thiazol-2-amino]-28-oate (5c). 28%, off-white
◦
1
powder. mp: 130.3-132.5 C; H NMR (400 MHz, CDCl ):
δ
8.15 (1H, d, J = 8.1 Hz), 7.71 (1H, s), 7.53–7.60
2H, m), 7.09 (1H, d, J = 7.7 Hz), 5.91 (2H, s), 5.25 (1H, s), 5.23–5.13 (2H, m), 2.48 (1H, d, J = 15.3 Hz), 2.23
1H, d, J = 11.4 Hz), 2.17 (1H, d, J = 14.8 Hz), 1.20 (3H, s), 1.12 (3H, s), 1.07 (3H, s), 0.93 (6H, d, J = 1.0 Hz),
3
(
(
13
0
1
.65 (3H, s); C NMR (100 MHz, CDCl ):
δ
177.55, 164.60, 147.58, 143.85, 138.12, 134.44, 130.57, 129.88,
3
25.64, 125.54, 124.99, 116.11, 57.45, 5 .06, 52.66, 50.92, 48.32, 46.13, 42.32, 39.68, 39.24, 38.94, 38.89, 38.33,
3
3
6.95, 36.65, 32.49, 30.75, 30.53, 28.08, 24.28, 23.45, 23.34, 22.30, 21.24, 19.66, 17.07, 16.84, 15.70. HR-MS m/z
+
calcd. for C H N O S [M + H] 727.40000, found 727.39938.
41
55
6
4
[
1-(2,6-dichlorobenzyl)-1H-1,2,3-triazol-5-yl]methyl-urs-12-en-[(3,2-d)thiazol-2-amino]-28-oate (5d). 260%,
◦
1
off-white powder. mp: 136.0–138.8 C; H NMR (400 MHz, CDCl ):
δ 7.50 (1H, s), 7.40 (2H, d,
3
J = 7.9 Hz), 7.33–7.27 (1H, m), 5.83 (2H, s), 5.22 (1H, s), 5.17–5.07 (2H, m), 4.92 (2H, s), 2.48 (1H, d,
J = 15.4 Hz), 2.20 (1H, d, J = 11.7 Hz), 2.19–2.14 (2H, m), 1.20 (3H, s), 1.12 (3H, s), 1.06 (3H, s), 0.93
13
(
6H, s), 0.83 (3H, d, J = 6.4 Hz), 0.58 (3H, s); C NMR (100 MHz, CDCl3): δ 177.58, 164.64, 152.24, 143.14,
1
38.19, 136.95, 131.26, 130.05, 129.03, 125.55, 123.75, 116.09, 57.43, 53.03, 52.67, 49.15, 48.29, 46.13, 42.30,
3
9.66, 39.24, 38.96, 38.90, 38.33, 36.96, 36.65, 32.47, 30.76, 30.50, 28.06, 24.25, 23.45, 23.36, 22.28, 21.25,
+
1
9.68, 17.08, 16.67, 15.73. HR-MS m/z calcd. for C H Cl N O S [M + H] 750.33698, found 750.33651.
41
54
2
5
2
{
1-[4-(tert-butyl)benzyl]-1H-1,2,3-triazol-5-yl}methyl-urs-12-en-[(3,2-d)thiazol-2-amino]-28-oate (5e). 26%,
◦
1
off-white powder. mp: 149.1–151.3 C; H NMR (400 MHz, CDCl ):
δ
7.46 (1H, s), 7.38 (2H, d,
3
J = 8.3 Hz), 7.18 (2H, d, J = 8.3 Hz), 5.53–5.39 (2H, m), 5.22 (1H, s), 5.09–5.17 (2H, m), 4.86 (2H, s), 2.50
(
(
1
3
1H, d, J = 15.4 Hz), 1.30 (9H, s), 1.21 (3H, s), 1.12 (3H, s), 1.07 (3H, s), 0.97 (3H, s), 0.92 (3H, s), 0.83
3H, d, J = 6.4 Hz), 0.66 (3H, s); 13C NMR (100 MHz, CDCl3):
177.59, 164.59, 152.19, 152.10, 143.58,
38.17, 131.50, 128.04, 126.17, 125.59, 123.89, 116.19, 57.50, 54.00, 53.06, 52.70, 48.29, 46.18, 42.35, 39.71,
δ
9.26, 39.00, 38.91, 38.37, 36.97, 36.61, 32.53, 31.39, 30.77, 30.53, 28.10, 24.29, 23.46, 23.36, 22.32, 21.26,
+
1
9.68, 17.09, 16.85, 15.77. HR-MS m/z calcd. for C H N O S [M + H] 738.47752, found 738.48079.
45
64
5
2
(2H-tetrazol-5-yl) methyl-urs-12-en-[(3,2-d)thiazol-2-amino]-28-oate (
7). The solution of 6 (200 mg,
0
.36 mmol), sodium azide (48 mg, 0.74 mmol) and ZnCl (196 mg, 1.4 mmol) in isopropanol/water
2
◦
(
3:1, 8 mL) was added and stirred at 85 C for 6 h. After concentration, the residue was dissolved
with n-butanol (60 mL) and washed with water (30 mL) and brine (30 mL 2), dried over
Na SO anhydrous, filtered, evaporated and purified by silica gel column chromatography with
×
2
4
◦
1
chloroform/methanol (10:1) to give
DMSO-d6): 6.70 (2H, s), 5.28 (2H, s), 5.15 (1H, s), 2.40 (1H, d, J = 15.4 Hz), 2.16 (1H, d, J = 11.3 Hz),
.08 (1H, d, J = 15.5 Hz), 1.10 (3H, s), 1.04 (3H, s), 1.02 (3H, s), 0.91 (3H, s), 0.86 (3H, d, J = 7.7 Hz),
.81 (2H, d, J = 6.3 Hz), 0.51 (3H, s); 13C NMR (100 MHz, DMSO-d6):
175.83, 165.29, 153.23, 150.88,
7. 31%, white powder. mp: 256.0–258.0 C; H NMR (400 MHz,
δ
2
0
δ
137.41, 125.21, 112.23, 54.61, 52.58, 52.04, 47.63, 45.35, 39.00, 38.38, 38.36, 38.21, 37.78, 36.43, 35.98, 32.02,
3
0.39, 30.01, 27.45, 23.73, 23.10, 22.76, 22.12, 20.95, 19.14, 16.92, 16.30, 15.41. HR-MS m/z calcd. for
+
C H N O S [M + H] 593.36322, found 593.36123.
33
49
6
2
3
.2.3. General Procedure for Compounds 8a–c
To a solution of UA (1.10 g, 2.41 mmol), K CO (0.49 g, 3.55 mmol) and corresponding
2
3
◦
dibromoalkane (8.0 mmol) in 15 mL DMF, the mixture was stirred at 60 C for 1 h. This was
followed by the similar post-reaction treatment of compounds or , purified by silica gel column
4
5
chromatography with petroleum ether/ethyl acetate (5:1) to afford derivatives 8a–c, as yellowish-white
powder, 61–63% yield.

Molecules 2019, 24, 877
8 of 14
3
.2.4. General Procedure for Compounds 9a–e
K CO (1 equiv) and the corresponding nitric heterocyclic compounds (2–6 equiv) were added
2
3
◦
, 8b, or 8c (1 equiv) in DMF. The mixture was stirred at 80 C for 30–60 min and
to the solution of 8a
then poured into ice-cold water, stirred, and filtered. The crude product was dissolved in ethyl acetate,
washed with water and brine, dried over Na SO anhydrous, filtered, evaporated, and purified by
2
4
silica gel column chromatography to yield 9a–e.
[
2-(Piperazin-1-yl)ethyl] 3-hydroxy-urs-12-en-28-oate (9a).
40% (purified by silica gel column
◦
1
chromatography with dichloromethane/methanol, 10:1), white solid. mp: 180.0–182.0 C; H NMR
(
(
0
1
400 MHz, CDCl3):
4H, s), 2.65 (2H, t, J = 5.6 Hz), 2.20 (1H, d, J = 11.3 Hz), 1.08 (3H, s), 0.99 (3H, s), 0.95 (3H, d, J = 5.5 Hz),
.92 (3H, s), 0.86 (3H, d, J = 6.3 Hz), 0.79 (3H, s), 0.74 (3H, s); 13C NMR (100 MHz, CDCl3):
176.89,
37.61, 125.09, 78.47, 61.24, 56.67, 54.70, 54.06, 52.34, 47.48, 47.03, 45.52, 41.52, 39.04, 38.56, 38.37, 38.25,
δ 5.23 (1H, s), 4.19–4.07 (2H, m), 3.22 (1H, dd, J = 10.8, 4.6 Hz), 3.14 (4H, s), 2.75
δ
3
8.10, 36.46, 36.18, 32.53, 30.17, 27.65, 27.47, 26.73, 23.71, 23.05, 22.78, 20.70, 17.81, 16.65, 16.54, 15.15,
+
1
4.97. HR-MS m/z calcd. for C H N O [M + H] 569.46767, found 569.46555.
36
61
2
3
[
2-(homopiperazine-1-yl)ethyl] 3-hydroxy-urs-12-en-28-oate (9b). 39% (purified by silica gel column
◦
1
chromatography with dichloromethane/methanol, 10:1), white solid. mp: 189.2–192.0 C; H NMR
(
(
(
400 MHz, CDCl3):
δ 5.22 (1H, s), 4.14–4.02 (2H, m), 3.27–2.78 (9H, m), 2.18 (1H, d), 1.07 (3H, s), 0.98
13
3H, s), 0.93 (3H, d, J = 5.7 Hz), 0.91 (3H, s), 0.84 (3H, d, J = 6.4 Hz), 0.77 (3H, s), 0.73 (3H, s); C NMR
100 MHz, CDCl3):
δ
177.51, 138.17, 125.72, 79.09, 61.92, 56.43, 55.29, 54.00, 53.01, 51.03, 48.15, 47.61,
4
2
5
6.97, 44.67, 42.15, 39.63, 39.14, 38.96, 38.84, 38.70, 37.06, 36.86, 33.15, 30.72, 28.26, 28.09, 27.31, 25.36,
+
4.35, 23.62, 23.40, 21.26, 18.41, 17.27, 17.13, 15.77, 15.60. HR-MS m/z calcd. for C H N O [M + H]
37
63
2
3
83.48332, found 583.48116.
[
3-(piperazin-1-yl)propyl] 3-hydroxy-urs-12-en-28-oate (9c). 39% (purified by silica gel column
◦
1
chromatography with dichloromethane/methanol, 10:1), white solid. mp: 99.1–101.0 C; H NMR
(
(
0
400 MHz, CDCl3):
4H, s), 2.53–2.38 (2H, m), 2.20 (1H, d, J = 11.2 Hz), 1.07 (3H, s), 0.98 (3H, s), 0.93 (3H, d, J = 5.6 Hz),
.90 (3H, s), 0.84 (3H, d, J = 6.3 Hz), 0.77 (3H, s), 0.72 (3H, s); 13C NMR (100 MHz, CDCl3):
177.66,
38.37, 125.62, 79.14, 62.20, 55.31, 54.82, 52.97, 50.17, 48.21, 47.61, 43.76, 42.17, 39.65, 39.18, 38.99, 38.87,
δ 5.22 (1H, s), 4.11–3.90 (2H, m), 3.21 (1H, dd, J = 10.8, 4.6 Hz), 3.15 (4H, s), 2.68
δ
1
3
8.71, 37.08, 36.88, 33.14, 30.76, 28.27, 28.08, 27.31, 25.95, 24.34, 23.66, 23.42, 21.31, 18.43, 17.24, 17.15,
+
1
5.79, 15.62. HR-MS m/z calcd. for C H N O [M + H] 583.48332, found 583.48103.
37
63
2
3
[
4-(piperazin-1-yl)butyl] 3-hydroxy-urs-12-en-28-oate (9d).
chromatography with dichloromethane/methanol, 10:1), white solid. mp: 100.0–102.5 C; H NMR
400 MHz, CDCl3): 5.21 (1H, s), 4.06–3.91 (2H, m), 3.19–3.21 (5H, m), 2.70 (4H, s), 2.41 (2H, t,
J = 7.0 Hz), 2.20 (1H, d, J = 11.2 Hz), 1.07 (3H, s), 0.98 (3H, s), 0.93 (3H, d, J = 5.8 Hz), 0.90 (3H, s), 0.84
38% (purified by silica gel column
◦
1
(
δ
13
(
3H, d, J = 6.4 Hz), 0.77 (3H, s), 0.73 (3H, s); C NMR (100 MHz, CDCl3):
δ
177.69, 138.31, 125.62, 79.10,
6
3
3.88, 57.60, 55.29, 52.99, 49.92, 48.17, 47.61, 43.76, 42.16, 39.63, 39.16, 38.98, 38.85, 38.71, 37.06, 36.87,
3.16, 30.76, 28.26, 28.08, 27.30, 26.42, 24.33, 23.65, 23.40, 23.27, 21.30, 18.41, 17.24, 17.15, 15.78, 15.60.
+
HR-MS m/z calcd. for C H N O [M + H] 597.49897, found 597.49693.
38
65
2
3
[
2-(4-methylpiperazin-1-yl)ethyl] 3-hydroxy-urs-12-en-28-oate (9e). 43% (purified by silica gel column
◦
1
chromatography with petroleum ether/ethyl acetate, 3:1), white solid. mp: 100.0–103.0 C; H NMR
400 MHz, CDCl3): 5.22 (1H, s), 4.12 (2H, t, J = 6.0 Hz), 3.21 (1H, dd, J = 10.8, 4.7 Hz), 2.60 (10H, m),
.33 (3H, d, J = 9.8 Hz), 2.20 (1H, d, J = 11.2 Hz), 1.07 (3H, s), 0.98 (3H, s), 0.93 (3H, d, J = 6.1 Hz),
.90 (3H, s), 0.84 (3H, d, J = 6.4 Hz), 0.77 (3H, s), 0.74 (3H, s); 13C NMR (100 MHz, CDCl3):
177.48,
(
2
0
δ
δ
138.22, 125.72, 79.10, 61.97, 56.65, 55.33, 55.17, 53.29, 52.96, 48.10, 47.66, 46.05, 42.15, 39.67, 39.19, 39.00,
3
8.87, 38.74, 37.09, 36.79, 33.15, 30.79, 28.27, 28.10, 27.36, 24.34, 23.67, 23.41, 21.31, 18.43, 17.27, 17.16,
+
1
5.77, 15.59. HR-MS m/z calcd. for C H N O [M + H] 583.48332, found 583.48100.
37
63
2
3
3
.2.5. General Procedure for Compounds 10 and 11
Acetic anhydride (1.0 mL, 10.58 mmol) was added dropwise to the solution of UA (2.0 g,
◦
4
.38 mmol) in 15 mL pyridine under 0 C. Subsequently, the mixture was stirred at room

Molecules 2019, 24, 877
9 of 14
temperature for 15 h, poured into ice-cold water and filtered to give a white semisolid, 95% yield.
The semisolid was dissolved with ethyl acetate (150 mL) and washed with 1 mol/L hydrochloric
acid (60 mL), water (60 mL) and brine (60 mL), dried over Na SO anhydrous, filtered, and then
2
4
concentrated to afford 3-acetyl-UA. The mixture of 3-acetyl-UA (1.16 g, 2.33 mmol), K CO (0.49 g,
2
3
◦
3.55 mmol), and 2-chloroethanol (0.17 mL, 2.53 mmol) in 15 mL DMF was stirred at 60 C for 3 h.
Following the similar post-processing steps of 6, 10 was obtained as white powder, in 86% yield.
Methanesulfonyl chloride (0.5 mL, 6.46 mmol) in pyridine (5 mL) was dropwise added to the solution
◦
of 10 (1.10 g, 2.03 mmol) in 15 mL pyridine under 10 C. The mixture was stirred at room temperature
for 3 h and treated with the similar post reaction treatment of 3-acetyl-UA to provide 11, as off-white
powder, in 94% yield.
3
.2.6. General Procedure for the Synthesis of Compounds 12a–c
The mixture of 11 (1 equiv), K CO (1 equiv), and piperazine (6 equiv), homopiperazine (6 equiv)
2
3
◦
or morpholine (2 equiv) in DMF was stirred at 60 C for 30 min, poured into ice-cold water, stirred,
and filtered. The off-white solid was dissolved in ethyl acetate and washed with water and brine,
dried over Na SO anhydrous, filtered, evaporated, and purified by silica gel column chromatography
2
4
to yield 12a–c.
2-(piperazin-1-yl)ethyl] 3-acetoxy-urs-12-en-28-oate (12a). 73% (purified by silica gel column
[
◦
1
chromatography with dichloromethane/methanol, 10:1), white solid. mp: 80.1–82.9 C; H NMR
(
400 MHz, CDCl3):
2H, t, J = 5.6 Hz), 2.18 (1H, d, J = 12.5 Hz), 2.04 (3H, s), 1.06 (3H, s), 0.93 (6H, d, J = 5.8 Hz), 0.85 (9H,
177.51, 171.19, 138.26, 125.62, 81.03, 61.59,
8.52, 56.94, 55.39, 52.97, 52.78, 48.13, 47.56, 45.13, 42.14, 39.67, 39.16, 38.99, 38.39, 37.79, 36.97, 36.83,
δ 5.21 (1H, s), 4.52–4.44 (1H, m), 4.16–4.04 (2H, m), 3.10 (4H, s), 2.72 (4H, s), 2.63
(
d, J = 4.9 Hz), 0.72 (3H, s); 13C NMR (100 MHz, CDCl3)
δ
5
3
3.07, 30.75, 28.19, 28.07, 24.32, 23.66, 23.62, 23.41, 21.46, 21.29, 18.56, 18.31, 17.25, 17.18, 16.86, 15.65.
+
HR-MS m/z calcd. for C H N O [M + H] 611.47823, found 611.47836.
38
63
2
4
[
2-(Homopiperazine-1-yl)ethyl] 3-acetoxy-urs-12-en-28-oate (12b). 71% (purified by silica gel column
◦
1
chromatography with dichloromethane/methanol, 10:1), white solid. mp: 76.5–78.0 C. H NMR
(
(
400 MHz, CDCl3):
δ 5.22 (1H, s), 4.53–4.44 (1H, m), 4.16 (2H, s), 3.38 (4H, s), 3.20 (2H, s), 2.94–3.00
4H, m), 2.04 (3H, s), 1.06 (3H, s, CH), 0.94 (6H, d, J = 3.8 Hz), 0.85 (9H, d, J = 4.6 Hz), 0.72 (3H, s);
13
C NMR (100 MHz, CDCl3):
δ 177.52, 171.17, 138.22, 125.64, 81.01, 61.84, 56.44, 55.38, 53.97, 53.00,
5
2
1.16, 48.16, 47.55, 46.85, 44.47, 42.15, 39.65, 39.14, 38.97, 38.38, 37.79, 36.97, 36.87, 33.08, 30.72, 28.19,
8.08, 25.31, 24.35, 23.65, 23.60, 23.41, 21.45, 21.28, 18.31, 17.27, 17.18, 16.86, 15.67. HR-MS m/z calcd. for
+
C H N O [M + H] 625.49388, found 625.49126.
39
65
2
4
(
2-morpholinoethyl) 3-acetoxy-urs-12-en-28-oate (12c). 75% (purified by silica gel column chromatography
◦
1
with petroleum ether/ ethyl acetate, 3:1), white solid. mp: 63.1–65.1 C. H NMR (400 MHz, CDCl ):
3
δ
5.22 (1H, s), 4.54–4.44 (1H, m), 4.27 (2H, s), 3.82 (4H, s), 2.19 (1H, d, J = 11.2 Hz), 2.04 (3H, s), 1.07
13
(3H, s), 0.94 (6H, d, J = 5.5 Hz), 0.85 (9H, d, J = 4.9 Hz), 0.74 (3H, s); C NMR (100 MHz, CDCl3):
δ
177.37, 171.16, 138.24, 125.72, 81.00, 66.21, 56.95, 55.39, 53.52, 52.97, 48.17, 47.54, 42.15, 39.69, 39.16,
3
8.99, 38.38, 37.79, 36.98, 36.82, 33.06, 30.72, 29.82, 28.19, 28.04, 24.34, 23.65, 23.63, 23.39, 21.45, 21.28,
+
1
8.30, 17.32, 17.18, 16.86, 15.64. HR-MS m/z calcd. for C H NO [M + H] 612.46225, found 612.45986.
38
62
5
3
.2.7. Preparation of Compound 13
The mixture of 9a (500 mg, 0.88 mmol) and PCC (190 mg, 0.88 mmol) in 80 mL chloroform
◦
was stirred at 0 C for 10 h, then isopropanol (1.0 mL, 13.1 mmol) was added and stirred at room
temperature for 1 h. The mixture was washed with water (40 mL) and brine (2
×
40 mL), dried
over Na SO anhydrous, filtered, concentrated, and purified by silica gel column chromatography
2
4
with dichloromethane/methanol (10:1) to obtain [2-(Piperazin-1-yl)ethyl] 3-oxo-urs-12-en-28-oate (13).
◦
9%, off-white powder. mp:144.4–146.8 C; H NMR (400 MHz, CDCl ): δ 5.24 (1H, s), 4.18–4.04
3
1
6
(
(
2H, m), 3.19 (4H, s), 2.81 (4H, s), 2.65 (2H, t, J = 5.4 Hz), 2.59-2.47 (1H, m), 2.34–2.39 (1H, m), 2.23–2.14
1H, m), 1.08 (6H, s), 1.03 (6H, s), 0.94 (3H, d, J = 5.5 Hz), 0.85 (3H, d, J = 6.3 Hz), 0.77 (3H, s);

Molecules 2019, 24, 877
10 of 14
13C NMR (100 MHz, CDCl3):
δ
218.00, 177.42, 138.33, 125.48, 61.20, 56.39, 55.33, 53.06, 49.91, 48.20,
4
2
7.52, 46.82, 43.72, 42.27, 39.60, 39.38, 39.15, 38.95, 36.83, 36.77, 34.29, 32.65, 30.70, 28.06, 26.67, 24.30,
+
3.56, 21.62, 21.24, 19.69, 17.23, 17.13, 15.36. HR-MS m/z calcd. for C H N O [M + H] 567.45202,
36 59 2 3
found 567.44977.
3
.2.8. General Procedure for Synthesizing Compounds 14a and 14b
The mixture of 12a (300 mg, 0.49 mmol), K CO (70 mg, 0.51 mmol), and 4-fluorobenzyl bromide
2
3
(
6
120 mg, 0.63 mmol) or 2-nitrobenzyl bromide (130 mg, 0.60 mmol) in DMF (10 mL) was stirred at
0 C for 1 h, the mixture was poured into 50 mL ice-cold water and then filtered. The white solid
◦
was dissolved in ethyl acetate (100 mL) and washed with water (50 mL) and brine (2
×
50 mL), dried
over Na SO anhydrous, filtered, evaporated, and purified by silica gel column chromatography with
2
4
dichloromethane/methanol (20:1) to obtain 14a and 14b.
2-[4-(4-fluorobenzyl)piperazin-1-yl]ethyl} 3-acetoxy-urs-12-en-28-oate (14a).
{
45% as white solid.
◦
1
mp: 140.0–141.8 C; H NMR (400 MHz, CDCl ):
δ
7.76–7.58 (2H, m), 7.12 (2H t, J = 8.2 Hz), 5.16 (1H, s),
3
4
.51–4.43 (1H, m), 4.08 (2H, s), 2.73–3.90 (10H, m), 2.09 (1H, d, J = 11.3 Hz), 2.04 (3H, s), 1.04 (3H, d,
13
J = 7.4 Hz), 0.92 (6H, d, J = 10.4 Hz), 0.88–0.81 (9H, m), 0.65 (3H, d, J = 13.5 Hz); C NMR (100 MHz,
CDCl3): 176.88, 171.17, 164.24 (d, J = 252.6 Hz), 138.14, 136.01 (d, J = 8.5 Hz), 130.81 (d, J = 7.6 Hz),
25.92, 122.57, 116.74 (d, J = 21.6 Hz), 115.67 (d, J = 21.4 Hz), 80.89, 61.04, 57.23, 56.48, 55.33, 53.12,
δ
1
4
2
8.38, 47.42, 46.25, 46.17, 42.18, 39.65, 39.08, 39.04, 38.35, 37.77, 36.93, 36.78, 32.96, 30.50, 28.17, 27.97,
4.36, 23.56 (d, J = 4.5 Hz), 23.38, 21.43, 21.16, 18.22, 17.47, 17.17, 16.84, 15.62. HR-MS m/z calcd. for
+
C H FN O [M + H] 719.51576, found 719.51562.
45
68
2
4
{
8
2-[4-(2-nitrobenzyl)piperazin-1-yl]ethyl} 3-acetoxy-urs-12-en-28-oate (14b). 43%, white solid. mp:
◦
3.0–85.0 C; H NMR (400 MHz, CDCl ): δ 7.80 (1H, d, J = 7.9 Hz), 7.60–7.47 (2H, m), 7.40 (1H, t,
3
1
J = 7.5 Hz), 5.21 (1H, s), 4.55–4.41 (1H, m), 4.17 (2H, s), 3.80 (2H, s), 2.61 (8H, m), 2.19 (1H, d, J = 11.3 Hz),
2
.04 (3H, s), 1.06 (3H, s), 0.94 (6H, d, J = 4.9 Hz), 0.86 (9H, d, J = 4.0 Hz), 0.74 (3H, d, J = 10.5 Hz);
13
C NMR (100 MHz, CDCl3):
18.04, 81.04, 59.05, 56.49, 55.40, 53.35, 52.94, 48.12, 47.57, 42.14, 39.68, 39.17, 38.97, 38.39, 37.80, 36.98,
δ 177.44, 171.19, 149.98, 138.23, 132.49, 131.16, 128.21, 125.67, 124.57,
1
3
6.79, 33.07, 30.75, 28.21, 28.06, 24.32, 23.67, 23.63, 23.41, 21.48, 21.31, 18.31, 17.29, 17.19, 16.88, 15.66.
+
HR-MS m/z calcd. for C H N O [M + H] 746.51062, found 746.51035.
45
68
3
6
3
.2.9. General Procedure for Synthesizing Compounds 17 and 18
Compound 17 was synthesized from compound according to the literature method [25]. 18 was
1
prepared from 17 according to the synthetic procedure of 12a and then purified by silica gel column
chromatography with dichloromethane/methanol (15:1) to afford 18.
◦
1
Urs-12-en-[2,3-c][
1
,2
,5
]oxadiazole oxide-28-oic acid (17). 60%, white solid. mp: >286 C; H NMR
(
(
400 MHz, CDCl3): δ 5.29 (1H, s), 2.72 (1H, d, J = 16.6 Hz), 2.23 (1H, d, J = 11.6 Hz), 2.17 (3H, s), 1.41
3H, s), 1.30 (3H, s), 1.10 (3H, s), 0.95 (3H, d, J = 5.9 Hz), 0.91 (3H, s), 0.86 (3H d, J = 6.3 Hz), 0.82 (1H, s);
13
C NMR (100 MHz, CDCl3):
δ 184.01, 163.66, 138.25, 125.04, 112.03, 52.71, 52.65, 48.14, 46.01, 42.27,
3
9.63, 39.20, 38.90, 38.40, 36.75, 35.17, 34.72, 32.04, 30.71, 28.08, 24.10, 23.92, 23.55, 23.29, 21.27, 18.91,
+
1
7.09, 16.72, 16.30.HR-MS m/z calcd. for C H O N [M + H] 497.33738, found 497.33572.
30
45
4
2
[
1
2-(Piperazin-1-yl)ethyl)] urs-12-en-[2,3-c][
1
,
2
δ
,
5
]oxadiazole oxide-28-oate (18). 47%, white powder. mp:
◦
1
34.1–136.8 C; H NMR (400 MHz, CDCl ):
5.29 (1H, s, H-12), 4.12 (2H, t, J = 5.7 Hz, CH ), 3.02–3.05
3
2
(
(
4H, m), 2.64 (4H, d, J = 4.1 Hz), 2.62 (2H, d, J = 5.8 Hz), 2.24 (1H, d, J = 11.1 Hz), 1.41 (3H, s), 1.33
3H, s), 1.09 (3H, s), 0.94 (3H, d, J = 5.7 Hz), 0.91 (3H, s), 0.86 (3H, d, J = 6.3 Hz), 0.79 (3H, s); 1
3
C
NMR (100 MHz, CDCl3):
δ 177.38, 163.72, 138.49, 124.84, 112.02, 61.77, 57.17, 54.01, 53.02, 52.77, 48.16,
4
2
6
6.01, 45.79, 42.36, 39.69, 39.26, 38.96, 38.39, 36.72, 35.20, 34.73, 32.16, 30.75, 28.07, 24.27, 23.96, 23.48,
+
3.33, 21.26, 18.97, 17.13, 16.92, 16.32. HR-MS m/z calcd. for C H O N [M + H] 609.43743, found
36
57
4
4
09.43526.

Molecules 2019, 24, 877
11 of 14
3
.3. Biology
3
.3.1. Cell Culture and Treatment
MKN45, Hela cell (the Shanghai Institute of Life Sciences) were cultivated in DMEM media
Hyclone, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum
(
(
Life Technology, Thermo Fisher Scientific, Waltham, MA, USA), 100 U/mL penicillin, and l00
µg/mL
◦
streptomycin, and grown in a humidified incubator (37 C, 5% CO ). All of the cells were harvested in
2
the exponential growth phase for assays.
3
.3.2. MTT Assay
All of the tested compounds were dissolved in DMSO and subsequently diluted in the culture
medium before treatment of the cultured cells. When the cells were 80–90% confluent, they were
harvested by treatment with a solution containing 0.25% trypsin, thoroughly washed, and resuspended
3
in supplemented growth medium. Cells (2
9
×
10 /well) were plated in 100
µ
L of medium/well in
6-well plate. After incubations overnight, the cells were treated with different concentrations of the
compounds in DMEM with 10% FBS for 72 h. In parallel, the cells that were treated with DMSO
served as negative control and CDDP (cisplatin) as positive control. Finally, 20 L of MTT was added
and the cells were incubated for 4 h. The MTT-formazan that formed by metabolically viable cells
was dissolved in 100 L of DMSO for 10 min. The absorbance was then measured at 570 nm with
µ
µ
a microplate reader, in which the cell survival rates were calculated and the IC50 was calculated
following our previous method [26,27].
3
.3.3. Flow Cytometric Analysis
To determine the effect of compound 14a on the cell cycle, the cells were seeded in six-well plates
5
at a density of 2
×
10 cells/mL for 24 h. After incubation, the cells were treated with indicated
concentrations of compound 14a for 24 h. Subsequently, the cells were collected, washed, and fixed in
◦
70% ethanol in PBS at
−
20 C. After overnight, fixed cells were pelleted and stained with cell cycle
◦
analysis reagent propidium iodide solution as per the manufacturer instructions for 30 min at 37 C
in dark, and about 5000 events were analysed on a FACScan cytometry(C6 Plus, Becton Dickinson,
San Jose, CA, USA) following published protocol [28].
3
.3.4. Western Blotting Assay
The Hela cell were treated with 14a at three concentrations: 0, 2, 5
µ
M. After treatment, the cells
◦
were washed with PBS and kept in
−
80 C until further handing. Western blotting was then
performed, as previously described. Briefly, total cell extracts were collected by RIPA buffer and
electrophoresed on 8% or 10% SDS-polyacrylamide gels and then transferred to PVDF membranes
◦
(
Millipore, Temecula, CA, USA). The membranes were blocked and then incubated overnight at 4 C
with primary antibodies to Bax (2772, Cell Signaling Technology, Boston, MA, USA), Bcl2 (2876, Cell
Signaling Technology, Boston, MA, USA), PARP (46D11, Cell Signaling Technology, Boston, MA, USA),
and
β-actin (C4, Santa Cruz Biotechnology, Dallas, TX, USA). The member was developed and
visualized by Image Quant LAS4000 (GE Healthcare, Boston, MA, USA) [29].
3
.3.5. Xenograft Studies
Nude mice (6
−
8 weeks old, BALB/c, female) were used to establish the xenograft tumors
6
following the published protocol [30]. Briefly, Hela cells (5
×
10 ) were implanted in the dorsal region
3
of recipient mice by means of subcutaneous injection. Once a tumor had reached around 300 mm in
size, the mice were randomized into three groups as control, 14a (50 mg/kg) and cisplatin (3 mg/kg)
with five mice per group. The animals were administrated by intraperitoneal injection for cisplatin and
administrated by gavage for 14a. Tumor growth and body weight were measured every three days

Molecules 2019, 24, 877
12 of 14
during the treatment. At the end of the treatment, the mice were sacrificed and tumors were removed
and weighed. The use of animals was approved by the Animal Experimentation Ethics Committee of
Yantai University in accordance with the guidelines for ethical conduct in the care and use of animals
(Ethical code YT-YX-1817).
4
. Conclusions
In summary, nineteen nitrogen-bearing heterocyclic UA analogues have been synthesized in
this work. The antiproliferative activity against Hela and MKN45 cell lines in vitro were evaluated.
The results showed that compounds bearing thiazole, triazole or tetrazole groups (5a and
displayed moderate antitumor activity. 9a 9c 12a 12b 13 14a 18 (containing a piperazine or
homopiperazine moiety) exhibited promising results against the cancer cell lines that were tested
here, with IC50 values from 2.1–9.8 M. In addition, an analysis of the structure-activity relationships
revealed that a number of factors remarkably affected the cytotoxicity. These included acetylation of
-OH, the type of nitrogen heterocycle, the linkers’ length between 28-COOH and nitrogen heterocycles,
–
e
7)
,
,
,
,
,
,
µ
3
as well as different substituents in the piperazine. 14a, which is one of the most active compounds,
decreased the apoptosis regulator (BCL2/BAX) ratio, which subsequently disrupted the mitochondrial
potential and induced apoptosis and significantly suppressed the growth of Hela xenografts in
nude mice.
The anti-cancer activity of the newly synthesized ursolic acid analogs is encouraging and may
provide a starting point for further structural optimization. Further pharmacokinetic study for these
compounds is planned.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/24/5/877/s1,
1
13
H-NMR, C-NMR and HR-MS spectra of compounds.
Author Contributions: W.W., L.L., H.W. and Q.M. conceived and designed the experiments; W.W., L.L., and Z.L.
performed the experiments; H.W and Q.M. analyzed the data; W.W. and L.L. wrote the paper.
Funding: This work was supported by the National Natural Science Foundation of China (No. 81473104), Taishan
Scholar Project (No. 81728020), Key Research Project of Shandong Province (2017GSF18177), Shandong Graduate
Education Innovation Program (SDYY16132), Yantai University Graduate Innovation Fund (YDYB1825).
Conflicts of Interest: The authors have declared no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).