Synthesis and antitumor activity evaluation of ursolic acid derivatives

Article abstract of DOI:10.1080/10286020.2019.1571488

Eighteen uronic acid derivatives were designed and synthesized, and the cytotoxicities in vitro of two cancer cell lines (BEL7402 and SGC7901) were evaluated by MTT assay. The results showed that the inhibitory rate of the compounds on both cell lines was

Full text of DOI:10.1080/10286020.2019.1571488

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: https://www.tandfonline.com/loi/ganp20
Synthesis and antitumor activity evaluation of
ursolic acid derivatives
Yan-Qiu Meng, Chuan-Dong Xu, Ting-Ting Yu, Wei Li, Qian-Wen Li & Xiao-Xiao
Li
To cite this article: Yan-Qiu Meng, Chuan-Dong Xu, Ting-Ting Yu, Wei Li, Qian-Wen Li & Xiao-
Xiao Li (2019): Synthesis and antitumor activity evaluation of ursolic acid derivatives, Journal of
Asian Natural Products Research, DOI: 10.1080/10286020.2019.1571488
To link to this article: https://doi.org/10.1080/10286020.2019.1571488
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JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
https://doi.org/10.1080/10286020.2019.1571488
Synthesis and antitumor activity evaluation of ursolic acid
derivatives
Yan-Qiu Meng, Chuan-Dong Xu, Ting-Ting Yu, Wei Li, Qian-Wen Li and
Xiao-Xiao Li
Department of Pharmaceutical Engineering, Shenyang University of Chemical Technology, Shenyang
110142, China
ABSTRACT
ARTICLE HISTORY
Eighteen uronic acid derivatives were designed and synthesized,
and the cytotoxicities in vitro of two cancer cell lines (BEL7402
and SGC7901) were evaluated by MTT assay. The results showed
that the inhibitory rate of the compounds on both cell lines was
significantly higher than the parent compound. The IC50 of com-
pounds II , II , III , and III are comparable or stronger than the
Received 2 September 2018
Accepted 15 January 2019
KEYWORDS
Ursolic acid derivatives;
synthetic; anticancer
activity; NF-jB inhibitor
4
6
4
6
positive control drug, the interactions between compounds II
4 6
, II ,
III , III , and NF-jB were also studied by docking simulations.
4
6
1. Introduction
The involvement of NF-jB in many human diseases, including the pathogenesis of
tumors, is widely considered to be an anti-apoptotic factor, which plays an important
role in the pathogenesis and drug resistance of many tumors [1]. When the cells are
stimulated by external stimuli, such as apoptotic induction factor, cytokines, cell div-
ision, and proliferation, NF-jB is activated and the activated NF-jB binds specifically
to sequences in some gene promoters in the nucleus, thereby regulating or activating
transcription of downstream genes. Therefore, inhibiting the NF-jB pathway of
tumor cells can induce apoptosis, block cell cycle, and has an anti-tumor effect [2].
Ursolic acid (UA, 3b-hydroxy-urs-12-en-28-oic acid) is natural pentacyclic triterpe-
noids. It is widely found in oleanders, rosemary, and broadleaf holly [3]. Ursolic acid
has a variety of physiological activities [4–11]. The anti-tumor effect of ursolic acid is
mainly by inducing tumor cell apoptosis, inducing tumor cell differentiation,
CONTACT Yan-Qiu Meng
myq6581@163.com; mengyanqiu@hotmail.com
Department of Pharmaceutical
Engineering, Shenyang University of Chemical Technology, Shenyang 110142, China
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
Y.-Q. MENG ET AL.
inhibiting tumor angiogenesis, and inhibiting tumor invasion and metastasis [12–16].
Sun et al. [17] found that ursolic acid can strengthen mononuclear cell line THP-1
cells HMFB1 expression and activity of NF-jB. The expression of HMFB1 and the
activity of NF-jB were bidirectional in different concentrations of ursolic acid, which
1
lmol/L ursolic acid had the strongest effect.
In recent years, the structural modifications of UA have mainly focused on the
transformation of C-3 hydroxyl, C-11 allyl, C-28 carboxyl and A ring. Ma et al. [18]
modified the C-3 hydroxyl of UA, obtained compound 1, and evaluated the cytotoxic
effects of these derivatives by HL-60, BGC, Bel-7402, and Hela cell lines, the results
showed that compound 1 had a significant increase in anti-tumor activity through the
ꢀ
1
ED50 of compound 1 to the above 4 tumor cells was 2.0, 2.5, 1.7, and 2.4 mg L .
Meng et al. [19] introduced pyrazine and benzoparizine into the A ring of oleanolic
acid, and synthesized a series of compounds by esterifying C-28 carboxyl, which
showed a certain activity for the activity test of SGC7901 and A549 in vitro, of which
the IC50 of compound 2 to two cells were 2.6 and 8.7 lM, obviously higher than that
of control gefitinib and VP-16. Wang oxidized the C-3 position of UA into a carbonyl
and synthesized a series of compounds by esterifying the C-28 carboxyl. By analyzing
the IC50 value, it was found that enhancing the lipid solubility of UA was helpful to
improve the activity of the inhibition of A549 and HT-29 cells, and the length of the
alkyl side chain at the C-28 position has a great effect on its activity (Figure 1).
Based on the above, the activity of the drug was simulated by computer (Table 1
shows MVD scores for each compound, where the greater the relative score, the bet-
ter the activity), and we finally decided to oxidize the C-3 position of UA to carbonyl,
esterify the C-28 carboxyl, and react the carbonyl group of C-3 with phenylhydrazine.
In this article, we obtained a series of novel ursolic acid derivatives. The results were
confirmed by MS, elemental analysis and H-NMR spectrum (Tables 2 and 3). The anti-
tumor activity was evaluated in vitro, and the results showed that compounds II , II ,
4
6
Figure 1. The structures of compounds 1 and 2.
Table 1. MVD scores for each compound.
Compound
MVD score
Compound
MVD score
I
I
I
I
I
I
II
II
II
1
2
3
4
5
6
ꢀ120.60
ꢀ114.13
ꢀ125.06
ꢀ129.08
ꢀ127.26
ꢀ140.42
ꢀ134.26
ꢀ124.37
ꢀ144.29
ꢀ97.13
II
II
II
III
III
III
III
III
III
4
5
6
ꢀ129.73
ꢀ140.68
ꢀ144.41
ꢀ130.76
ꢀ126.70
ꢀ134.18
ꢀ137.23
ꢀ146.72
ꢀ141.43
1
2
3
4
5
6
1
2
3
UA

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
III , and III exhibited excellent antitumor activities. Furthermore, the results of docking
4
6
studies also showed that these UA derivatives are capable of interacting with NF-jB.
These results enlighten us to explore the effective derivatives of anti-tumor based on UA.
2. Results and discussion
2.1. Chemical synthesis
The synthetic route is outlined in Scheme 1. In this work, a series of novel UA com-
pounds were successfully synthesized. As shown in Scheme 1, the synthesis started by
the UA with Jones reagent in acetone, to give the compound UA . The compound
1
UA2a–2f was obtained via the reaction of compound UA with different halogenated
1
alkane in DMF and K CO . Finally, the intermediate products are reacted with phe-
2
3
nylhydrazine to obtain I –I , reacted with p-nitrophenylhydrazine to obtain II –II ,
1
6
1
6
interacted with p-chlorophenylhydrazine to get III –III .
1
6
2
.2. Biological activities
The anticancer potential and cytotoxicity of VP-16 and adriamycin (two listed anti-
cancer drugs) as positive control compounds and their parent compound UA on two
groups of human cancer lines (BEL-7402 and SGC-7901) were controlled by the
standard MTT assay. The inhibition rate and IC50 of different compounds on BEL-
7402 and SGC-7901 cells are shown in Table 4.
It can be seen from Table 4 that the target compounds have a certain inhibitory
effect on the growth of BEL-7402 and SGC-7901 cell lines, the IC50 values of com-
pounds II , III , and III against BEL-7402 are 7.08, 5.63, and 3.49 lM, respectively.
4
4
6
The IC50 values of Compounds II , III , and III against SGC-7901 were 6.30, 8.73,
6
4
6
and 7.01 lM, respectively, and the IC50 of these compounds on BEL-7402 and SGC-
Table 2. MS and elemental analysis data of target compounds.
Elemental analysis/found (Calcd.)
Num.
ESI-MS (m/z)/found (Calcd.)
C
H
O
N
Cl
I
I
I
I
I
I
II
II
II
II
II
II
III
III
III
III
III
III
1
2
3
4
5
6
573.4 (573.0)
587.5 (586.4)
601.5 (600.8)
601.5 (601.7)
615.4 (615.3)
629.3 (628.9)
618.5 (617.6)
632.6 (631.4)
646.4 (646.5)
646.4 (645.5)
660.6 (661.5)
674.3 (674.0)
607.5 (607.3)
621.5 (621.2)
634.5 (635.4)
635.5 (635.4)
649.5 (648.4)
663.6 (663.3)
79.60 (79.67)
79.63 (79.81)
80.02 (79.95)
80.05 (79.95)
80.24 (80.08)
80.11 (80.20)
73.96 (73.87)
74.34 (74.13)
74.17 (74.38)
74.22 (74.38)
74.83 (74.62)
74.69 (74.85)
75.23 (75.15)
75.44 (75.39)
75.70 (75.62)
75.69 (75.62)
75.77 (75.83)
75.98 (76.04)
9.90 (9.85)
9.87 (9.96)
10.13 (10.0)
10.24 (10.0)
10.12 (10.1)
10.28 (10.2)
9.05 (8.97)
9.21 (9.09)
9.14 (9.21)
9.16 (9.21)
9.20 (9.32)
9.45 (9.42)
9.20 (9.13)
9.41 (9.25)
9.28 (9.36)
9.28 (9.36)
9.43 (9.47)
9.62 (9.57)
5.63 (5.59)
5.60 (5.45)
5.30 (5.32)
5.25 (5.32)
5.29 (5.20)
5.04 (5.09)
10.21 (10.36)
10.09 (10.13)
10.03 (9.91)
9.97 (9.91)
9.66 (9.70)
9.54 (9.50)
5.19 (5.27)
5.08 (5.15)
5.12 (5.04)
5.13 (5.04)
5.05 (4.93)
4.88 (4.82)
4.76 (4.89)
4.80 (4.77)
4.53 (4.66)
4.59 (4.66)
4.61 (4.56)
4.52 (4.45)
6.87 (6.80)
6.75 (6.65)
6.50 (6.51)
6.53 (6.51)
6.42 (6.37)
6.20 (6.23)
4.60 (4.61)
4.63 (4.51)
4.47 (4.41)
4.45 (4.41)
4.30 (4.31)
4.31 (4.22)
1
2
3
4
5
6
1
2
3
4
5
6
5.76 (5.84)
5.67 (5.71)
5.61 (5.58)
5.62 (5.58)
5.41 (5.46)
5.29 (5.34)

4
Y.-Q. MENG ET AL.
1
Table 3. H NMR spectral data of target compounds.
1
Compounds
H-NMR
, 400 MHz) d: 10.83 (s, 1H, HN), 7.75–7.62 (m, 2H, H-Ph), 7.54–7.44 (m, 2H,
),
), 1.16 (s, 3H, CH
), 0.86 (d, J ¼ 7.5 Hz, 6H, CH ꢁ2).
, 400 MHz) d: 10.92 (d, J ¼ 8.9 Hz, 1H, HN), 7.82 (d, J ¼ 6.4 Hz, 2H, H-
Ph), 7.45 (d, J ¼ 7.1 Hz, 2H, H-Ph), 7.18 (d, J ¼ 5.7 Hz, 1H, H-Ph), 5.21 (s, 1H, H-12),
.07 (d, J ¼ 4.7 Hz, 2H, COOCH ), 2.64–2.55 (m, 1H, H-18), 1.83–1.70 (m, 2H,
COOCH CH ), 1.30–1.22 (m, 6H, CH , CH CH ), 1.16 (s, 3H, CH ), 1.08 (s, 3H, CH
s, 3H, CH ), 0.98 (s, 3H, CH ꢁ2).
), 0.87 (d, J ¼ 7.2 Hz, 6H, CH
H-NMR (CDCl
, 400 MHz) d: 10.64 (d, J ¼ 6.9 Hz, 1H, HN), 8.06 (d, J ¼ 7.1 Hz, 2H,
H-Ph), 7.87–7.72 (m, 2H, H-Ph), 7.13 (d, J ¼ 7.0 Hz, 1H, H-Ph), 5.16 (s, 1H, H-12),
.98–3.88 (m, 2H, COOCH
), 2.65 (s, 1H, H-18), 1.69 (dd, J ¼ 13.2, 8.7 Hz, 2H,
COOCH CH CH CH ), 1.27–1.13 (m, 3H, CH
), 1.31 (d, J ¼ 5.6 Hz, 2H, COOCH
.06 (s, 3H, CH ), 1.01 (s, 3H, CH ), 0.95 (s, 6H, CH ), 0.88 (d,
ꢁ2), 0.91 (s, 3H, CH
J ¼ 6.9 Hz, 6H, CH ꢁ2).
H-NMR (DMSO, 400 MHz) d: 10.83 (s, 1H, HN), 7.66 (s, 2H, H-Ph), 7.59–7.44 (m, 2H,
H-Ph), 7.36 (s, 1H, H-Ph), 5.21–5.12 (m, 1H, H-12), 4.08–3.99 (m, 2H, COOCH ),
.65–2.51 (m, 1H, H-18), 1.72–1.64 (m, 1H, COOCH CH), 1.23 (s, 3H, CH ), 1.10 (s, 3H,
CH ], 0.94 (s, 3H, CH ), 0.90 (s, 3H, CH ), 0.88 (d,
), 1.03 [d, J ¼ 8.1 Hz, 6H, CH(CH
J ¼ 7.3 Hz, 6H, CH ꢁ2), 0.83 (s, 3H, CH ).
H-NMR (CDCl , 400 MHz) d: 10.71 (s, 1H, HN), 7.88–7.59 (m, 2H, H-Ph), 7.62–7.44 (m, 2H,
1
I
1
H-NMR (CDCl
3
H-Ph), 7.30 (d, J ¼ 6.9 Hz, 1H, H-Ph), 5.28 (s, 1H, H-12), 4.10–4.06 (m, 2H, COOCH
2
2
.55 (dt, J ¼ 13.9, 7.2 Hz, 1H, H-18), 1.29–1.20 (m, 6H, CH
3
, CH
2
CH
3
3
),
1
.09 (s, 3H, CH ), 1.05 (s, 3H, CH ), 0.96 (s, 3H, CH
3
3
3
3
1
1
I
2
H-NMR (CDCl
3
4
2
2
2
3
2
3
3
3
), 1.01
(
3
3
3
I
3
3
3
2
2
2
2
2
2
2 3
CH ),
1
3
3
3
3
3
1
1
1
I
4
I
5
I
6
2
2
2
3
3
3
)
2
3
3
3
3
3
H-Ph), 7.06 (d, J ¼ 6.2 Hz, 1H, H-Ph), 5.28 (t, J ¼ 3.0 Hz, 1H, H-12), 4.04–3.96 (m, 2H,
COOCH
COOCH
2
2
), 2.65–2.31 (m, 1H, H-18), 1.93–1.66 (m, 2H, COOCH
CH (CH ], 1.21 (s, 3H, CH ), 1.13 (s, 3H, CH ), 1.06 (s, 3H, CH
CH ), 0.90 (s, 3H, CH ꢁ2).
), 0.83 (d, J ¼ 7.0 Hz, 6H, CH
H-NMR (CDCl , 400 MHz) d: 10.82 (s, 1H, HN), 7.79–7.71 (m, 2H, H-Ph), 7.50 (d,
J ¼ 4.6 Hz, 2H, H-Ph), 7.44–7.33 (m, 1H, H-Ph), 5.34–5.23 (m, 1H, H-12), 4.01 (d,
J ¼ 5.2 Hz, 2H, COOCH ), 2.64–2.47 (m, 1H, H-18), 1.69–1.56 [m, 8H, COOCH (CH
.30 (s, 3H, CH ), 1.22 (s, 3H, CH ), 1.15 (s, 6H, CH
ꢁ2), 1.02 (t, J ¼ 6.2 Hz, 3H,
CH CH ), 0.91 (s, 3H, CH ꢁ2).
), 0.85 (d, J ¼ 7.1 Hz, 6H, CH
H-NMR (CDCl
, 400 MHz) d: 10.05 (s, 1H, HN), 8.38 (dt, J ¼ 6.5, 3.1 Hz, 2H, H-Ph), 7.87
t, J ¼ 7.3 Hz, 2H, H-Ph), 5.28 (s, 1H, H-12), 4.10–4.02 (m, 2H, COOCH ), 2.55 (ddd,
J ¼ 13.9, 8.7, 6.0 Hz, 1H, H-18), 1.29–1.18 (m, 6H, CH , CH CH ), 1.07 (s, 3H, CH ), 1.02
s, 3H, CH ), 0.96 (s, 3H, CH ), 0.89 (s, 3H, CH ).
), 0.80 (d, J ¼ 7.3 Hz, 6H, CH
H-NMR (CDCl
, 400 MHz) d: 10.41 (d, J ¼ 6.7 Hz, 1H, HN), 8.15 (d, J ¼ 7.2 Hz, 2H, H-
Ph), 7.05 (d, J ¼ 6.9 Hz, 2H, H-Ph), 5.35 (d, J ¼ 4.8 Hz, H, H-12), 4.14–4.02 (m, 2H,
COOCH CH ), 1.30 (s, 3H,
), 2.45 (d, J ¼ 9.7 Hz, 1H, H-18), 1.83–1.76 (m, 2H, COOCH
CH ), 1.22 (s, 3H, CH ), 1.06 (m, 3H, CH CH ), 0.99 (s, 3H, CH ), 0.95 (s, 3H, CH ), 0.86
d, J ¼ 6.8 Hz, 6H, CH ꢁ2), 0.80 (s, 3H, CH ).
2
CH
2
), 1.35–1.30 [m, 4H,
2
2
)
2
3
3
3
), 0.99 (m, 6H,
3
CH , CH
2
3
3
3
3
2
2
2 4
) ],
1
3
3
3
2
3
3
3
1
1
II
1
2
3
(
2
3
2
3
3
(
3
3
3
3
II
3
2
2
2
3
3
2
3
3
3
(
3
3
1
1
1
1
1
II
II
II
II
3
4
5
6
H-NMR (DMSO, 400 MHz) d: 9.91 (d, J ¼ 8.2 Hz, 1H, HN), 8.06 (d, J ¼ 7.1 Hz, 2H, H-Ph),
7
2
3
.13 (d, J ¼ 7.1 Hz, 2H, H-Ph), 5.16 (s, 1H, H-12), 4.03–3.92 (m, 2H, COOCH
.70–2.60 (m, 1H, H-18), 1.33–1.26 [m, 4H, COOCH2(CH ], 1.23 (s, 3H, CH
H, CH ), 1.10 (s, 3H, CH ), 1.06–0.92 (m, 6H, CH , CH CH
), 0.85 (d, J ¼ 7.2 Hz, 6H,
ꢁ2), 0.74 (s, 3H, CH ).
H-NMR (CDCl , 400 MHz) d: 9.85 (s, 1H, HN), 7.72–7.45 (m, 2H, H-Ph), 7.18–7.02 (m,, 2H,
H-Ph), 5.25 (s, H, H-12), 4.04–3.89 (m, 2H, COOCH ), 2.60 (s, 1H, H-18), 2.12–2.02
m, 1H, COOCH CH), 1.31 (s, 3H, CH ), 1.26 (s, 3H, CH ), 1.15 (s, 3H, CH ), 1.05 [d,
J ¼ 8.1 Hz, 6H, CH(CH ], 0.94 (s, 3H, CH ), 0.90 (s, 3H, CH ) 0.82 (d, J ¼ 7.1 Hz,
H, CH
ꢁ2).
H-NMR (CDCl
, 400 MHz) d: 10.03 (s, 1H, HN), 8.06 (d, J ¼ 7.1 Hz, 2H, H-Ph), 7.14 (d,
J ¼ 7.1 Hz, 2H, H-Ph), 5.17 (s, 1H, H-12), 4.02–3.87 (m, 2H, COOCH ), 2.49 (s, 1H,
H-18), 1.58–1.46 [m, 6H, COOCH (CH ], 1.28 (s, 3H, CH ), 1.16 (s, 3H, CH ), 1.10
s, 3H, CH ), 1.05–0.96 (m, 6H, CH , CH CH ), 0.90 (s, 3H, CH
), 0.83 (d, J ¼ 7.0 Hz,
H, CH
ꢁ2).
H-NMR (CDCl
, 400 MHz) d: 10.03 (s, 1H, HN), 8.16 (d, J ¼ 7.2 Hz, 2H, H-Ph), 7.05 (d,
J ¼ 7.1 Hz, 2H, H-Ph), 5.28 (d, J ¼ 3.1 Hz, H, H-12), 4.06–3.98 (m, 2H, COOCH ), 2.50
dd, J ¼ 6.7, 3.2 Hz, 1H, H-18), 1.92–1.65 (m, 2H, COOCH CH ), 1.32–1.26 [m, 6H,
CH (CH ], 1.17 (s, 3H, CH ), 1.12 (s, 3H, CH ), 1.08 (s, 3H, CH
), 1.01 (t, J ¼ 7.8 Hz,
H, CH CH ), 0.92 (s, 3H, CH ꢁ2), 0.75 (s, 3H, CH ).
), 0.83 (d, J ¼ 7.2 Hz, 6H, CH
H-NMR (CDCl , 400 MHz) d: 9.44 (s, 1H, HN), 7.74–7.59 (m, 2H, H-Ph), 7.53–7.42 (m, 2H,
H-Ph), 5.28 (s, H, H-12), 4.07 (t, J ¼ 5.8 Hz, 2H, COOCH ), 2.59–2.38 (m, 1H, H-18),
2
),
2
)
2
3
), 1.17 (s,
3
3
3
2
3
CH
3
3
3
2
(
2
3
3
3
3
)
2
3
3
6
3
3
2
2
2
)
3
3
3
(
3
3
2
3
3
6
3
3
2
(
2
2
2
2
)
3
3
3
3
3
2
3
3
3
3
III
1
3
2
(continued)

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Table 3. Continued.
Compounds
1
H-NMR
1
0
.30 (s, 3H, CH
.91 (d, J ¼ 7.1 Hz, 6H, CH
, 400 MHz) d: 9.44 (d, J ¼ 8.7 Hz, 1H, HN), 7.73–7.64 (m, 2H, H-Ph), 7.48
3
), 1.22 (s, 3H, CH
3
), 1.15 (s, 3H, CH
3 2 3 3
), 1.08–0.99 (m, 6H, CH CH , CH ),
3
ꢁ2), 0.82 (s, 3H, CH
3
).
1
III
2
H-NMR (CDCl
3
(
d, J ¼ 6.3 Hz, 2H, H-Ph), 5.27 (d, J ¼ 3.5 Hz, H, H-12), 3.97 (d, J ¼ 5.3 Hz, 2H,
COOCH
2
), 2.55 (ddd, J ¼ 14.0, 8.6, 6.0 Hz, 1H, H-18), 1.74–1.65 (m, 2H, COOCH
2
CH
CH
2
),
),
1
0
.26 (s, 3H, CH
.93 (s, 3H, CH
3
3
), 1.21 (s, 3H, CH
3
), 1.09 (s, 3H, CH
ꢁ2).
3
), 1.01 (t, J ¼ 7.6 Hz, 3H, CH
2
3
), 0.84 (d, J ¼ 6.7 Hz, 6H, CH
3
1
1
1
III
III
III
3
4
5
H-NMR (DMSO, 400 MHz) d: 9.65 (s, 1H, HN), 7.74–7.56 (m, 4H, H-Ph), 5.44 (s, H, H-12),
.94 (d, J ¼ 5.1 Hz, 2H, COOCH ), 2.45–2.27 (m, 1H, H-18), 1.72–1.58 [m, 4H,
COOCH (CH ], 1.28 (s, 3H, CH ), 1.20 (s, 3H, CH ), 1.09 (s, 3H, CH ), 1.02–0.93 (m, 6H,
CH , CH CH ), 0.90 (s, 3H, CH ꢁ2)
), 0.83 (d, J ¼ 7.0 Hz, 6H, CH
H-NMR (CDCl
, 400 MHz) d: 9.92 (dd, J ¼ 9.1, 4.0 Hz, 1H, HN), 7.67–7.30 (m, 4H, H-Ph),
.38–5.24 (m, H, H-12), 3.98–3.91 (m, 2H, COOCH ), 2.58–2.36 (m, 1H, H-18), 1.94–1.85
m, 1H, CH CH), 1.29 (s, 3H, CH ), 1.20 (s, 3H, CH ), 1.14 (s, 3H, CH ), 1.07 (s, 3H, CH ),
.99 [t, J ¼ 7.9 Hz, 6H, CH(CH ], 0.90 (s, 3H, CH ), 0.83 (d, J ¼ 6.8 Hz, 6H, CH ꢁ2).
, 400 MHz) d: 9.92–9.83 (m, 1H, HN), 7.69–7.40 (m, 2H, H-Ph), 7.21 –6.99
m, 2H, H-Ph), 5.26 (d, J ¼ 2.6 Hz, 1H, H-12), 3.92–3.81 (m, 2H, COOCH ), 2.60 (d,
J ¼ 6.7 Hz, 1H, H-18), 1.66–1.60 (m, 2H, COOCH CH ), 1.56 [d, J ¼ 2.7 Hz, 4H,
COOCH CH (CH ], 1.23 (s, 3H, CH ), 1.19 (s, 3H, CH ), 1.13 (s, 3H, CH ), 1.09 (s, 3H,
CH ), 1.03–0.94 (m, 6H, CH , CH CH ꢁ2), 0.75 (s,
), 0.86 (d, J ¼ 6.5 Hz, 6H, CH
H, CH ).
H-NMR (CDCl3, 400 MHz) d: 9.72 (d, J ¼ 9.2 Hz, 1H, HN), 7.48 (d, J ¼ 9.2 Hz, 2H, H-Ph),
.39–7.30 (m, 2H, H-Ph), 5.28 (d, J ¼ 3.4 Hz, H, H-12), 4.00 (dt, J ¼ 7.8, 5.0 Hz, 2H,
COOCH ), 2.55–2.37 (m, 1H, H-18), 1.96–1.68 (m, 2H, COOCH CH
), 1.61 [h, J ¼ 4.7 Hz,
H, COOCH CH (CH ], 1.28 (s, 3H, CH ), 1.20 (s, 3H, CH ), 1.09 (s, 3H, CH ), 1.03–0.95
m, 6H, CH3, CH CH ꢁ2), 0.74 (s, 3H, CH ).
), 0.85 (d, J ¼ 7.2 Hz, 6H, CH
3
2
2
2
)
2
3
3
3
3
2
3
3
3
3
5
(
0
2
2
3
3
3
3
3
)
2
3
3
H-NMR (CDCl
(
3
2
2
2
2
2
2
)
2
3
3
3
3
3
2
3
3
3
3
1
III
6
7
2
2
2
4
(
2
2
2
)
3
2
3
3
3
2
3
3
Scheme 1. Synthetic routes of UA derivatives.

6
Y.-Q. MENG ET AL.
Table 4. Inhibitory activities of the target compounds on the BEL-7402 and SGC-7901 cell lines.
ꢂ
Inhibition rate (%)
IC50 (lM)
Compound
BEL-7402
SGC-7901
BEL-7402
SGC-7901
I
I
I
I
I
I
II
II
II
II
II
II
III
III
III
III
III
III
1
2
3
4
5
6
17.39
22.17
23.14
24.57
35.62
37.36
35.09
42.36
47.56
59.41
48.92
37.62
36.27
32.62
45.34
55.81
36.40
58.47
15.49
41.84
45.62
20.46
23.43
30.21
26.95
31.78
30.24
33.68
45.51
40.82
44.35
55.38
53.53
37.72
26.71
42.19
47.29
42.94
41.92
11.57
46.33
49.81
>50
>50
>50
>50
46.88
>50
23.45
27.17
14.62
16.45
18.52
15.62
9.66
47.83
44.74
19.94
18.73
16.83
22.39
10.61
7.08
10.72
8.57
22.36
31.12
15.53
5.63
1
2
3
4
5
6
6.30
1
2
3
4
5
6
19.15
25.27
13.62
8.73
9.35
7.01
>50
10.47
2.52
11.05
4.49
>50
15.61
3.68
UA
VP-16
Adriamycin
ꢂ
Compound concentration is 10 lM and cell inhibition rate after 48 h treatment.
Figure 2. Binding modes of compound II with NF-jB in 3D and 2D diagram. The H-bond interac-
4
tions with compounds are shown in green, alkyl-bonds are shown in pink, and sulfur-bonds are
shown in yellow.
7
901 cells was comparable or stronger than the control adriamycin and VP- 16. They
have a good pharmacological activity.
2.3. Docking study
The molecular docking of the above-designed new ursolic acid compound with the
NF-jB crystal structure (PDB:3rzf) showed that the compound was well embedded in
the active pocket of NF-jB, among which the key amino acids such as TYR98,
LEU167, GLU100, and GLY102 bind to small ligand molecules. The simulation shows
that the above two new compounds interact with NF-jB, indicating that the designed
compounds have strong affinity with the target.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
Figure 3. Binding modes of compound II with NF-jB in 3D and 2D diagram. The H-bond interac-
6
tions with compounds are shown in green, and alkyl-bonds are shown in pink.
Figure 4. Binding modes of compound III with NF-jB in 3D and 2D diagram. The H-bond interac-
4
tions with compounds are shown in green, and alkyl-bonds are shown in pink.
Figure 5. Binding modes of compound III with NF-jB in 3D and 2D diagram. The H-bond interac-
6
tions with compounds are shown in green, and alkyl-bonds are shown in pink.
The four best active compounds in cell experiments were respectively butted with
proteins to observe the interaction between them (Figures 2–5).
In summary, NF-jB plays a positive role in inhibiting tumor cell growth. A large
number of literatures had reported that UA can act as a NF-jB blocker and induce
apoptosis. In this study, we synthesized a series of new UA derivatives and evaluated
their anti-tumor activity by inhibiting two tumor cells in vitro, and investigated the

8
Y.-Q. MENG ET AL.
results of molecular docking. In general, virtual molecular docking has a guiding role
in the structural design and optimization of UA, laying a good foundation for the
future clinical anti-tumor drug research.
3. Experimental
3.1. Reagents and instruments
The melting points were measured with a B u€ chi B-540 melting apparatus produced
1
by Bruker Corporation (Flawil, Switzerland) without correction. H NMR spectra
were measured on BrukerARX-400 nuclear magnetic resonance analyzer from Bruker
Corporation (Ettlingen, Germany), using Me Si (TMS) as internal standard, CDCl3
4
and DMSO as solvent. Mass spectra were measured on Thermo-Finnigan LCQ mass
spectrometer from Thermo Finnigan (San Francisco, CA). All commercially available
solvents and reagents were used without further purification unless it was mentioned.
Routine thin-layer chromatography (TLC) was performed on silicagel plates (Silica
gel GF254 from VWR) and column chromatography was carried on silicagel
(200–300 mesh). Both of these are purchased from Qingdao Haiyang Chemical Co.,
Ltd. (Qingdao, China). The purity of ursolic acid is 98%, which is purchased from
Guangxi Changzhou Natural Products Co., Ltd. (Guangxi, China). All the reagents
are analytical or chemical pure.
3.2. Synthesis section
3.2.1. 3-Oxo-urs-12-ene-28-oic acid (UA1)
UA (200 mg, 0.44 mmol) was dissolved in 20 ml acetone, and Jones reagent was
added slowly under the condition of ice water bath. The resultant mixture was
stirred at room temperature for 1 h, monitoring the endpoint of the reaction with
TLC. After the reaction was completed, 4 ml of isopropanol was added dropwise
to quench the remaining oxidant, and the mixture was extracted with the solution
of saturated sodium chloride and ethyl acetate, and dried over anhydrous sodium
sulfate, to provide UA after vacuum filtration, rotary evaporation and column
1
ꢃ
chromatography with a yield of 80.42%, m.p. 233.3–234.6 C. ESI-MS: m/z
þ
4
54.5 [M þ H] .
3
.2.2. 3-Oxo-urs-12-ene-28-oic acid ester (UA2a–2f)
UA reacts with different bromo alkane at room temperature with DMF as solvent
1
and anhydrous potassium carbonate as catalyst, and the end of the reaction was
tested by TLC. After the reaction was completed, the mixture was extracted with the
solution of saturated sodium chloride and ethyl acetate, and dried over anhydrous
sodium sulfate, to provide UA2a–2f after vacuum filtration, rotary evaporation and col-
umn chromatography. The yields of compounds UA2a–2f are as follows: 76.32, 80.26,
7
3.91, 70.59, 76.56, and 79.17%. The m.p. and ESI-MS of the compounds UA
are
2a–2f
ꢃ
þ
ꢃ
þ
as follows: 153.3–155.6 C, m/z 482.4 [M þ H] ; 158.7–160.4 C, m/z 496.5 [M þ H] ;
ꢃ
þ
ꢃ
þ
1
1
67.2–169.8 C, m/z 510.5 [M þ H] ; 162.5–164.1 C, m/z 510.5 [M þ H] ;
þ þ
ꢃ
ꢃ
71.1–173.2 C, m/z 524.4 [M þ H] ; 184.1–186.7 C, m/z 538.4 [M þ H] .

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
9
3
.2.3. 3-Phenylhydrazone-urs-12-en-28-oic acid ester (I –I )
1 6
UA2a (200 mg, 0.41 mmol) and phenyl hydrazine (0.03 ml, 0.51 mmol) were dissolved
in 15 ml of methanol, and 0.2 ml of glacial acetic acid was added as a catalyst. The
ꢃ
mixture was refluxed under a water bath at 45 C, and the end of reaction was tested
by TLC. After the reaction was completed, the crude product was obtained by vac-
uum evaporation and then recrystallized with ethanol to get orange crystals (I ) with
1
ꢃ
a yield of 70.18% m.p. 168.5–170.3 C. According to the synthesis method of com-
pound I , compound UA reacted with phenyl hydrazine to get I with a yield of
1
2b
2
ꢃ
3.27%, m.p. 167.8–170.1 C; compound UA reacted with phenyl hydrazine to get I3
2c
7
ꢃ
with a yield of 69.89%, m.p. 169.8–172.6 C; compound UA2 reacted with phenyl
d
ꢃ
hydrazine to get I with a yield of 73.85%, m.p. 167.4–168.9 C; compound UA
4
2e
ꢃ
reacted with phenyl hydrazine to get I with a yield of 74.09%, m.p. 171.6–173.8 C;
5
compound UA2f reacted with phenyl hydrazine to get I with a yield of 70.36%,
6
ꢃ
m.p. 179.3–181.7 C.
0
3
.2.4. 3-(4 -Nitro-phenylhydrazone)-urs-12-en-28-oic acid ester (II –II )
1
6
UA2a (200 mg, 0.41 mmol) and 4-nitrophenylhydrazine (114 mg, 0.51 mmol) were dis-
solved in 15 ml of ethanol, and 0.2 ml of glacial acetic acid was added as catalyst. The
ꢃ
mixture was reflued under a water bath at 60 C, and tested the endpoint of reaction
by TLC. After the reaction was completed, the crude products were obtained by
decompression evaporation, then recrystallized by ethanol to get orange yellow crys-
ꢃ
tals (II ) with the yield is 73.28%, m.p. 178.1–179.8 C. According to the synthesis
1
method of compound II , compound UA reacted with 4-nitrophenylhydrazine to
1
2b
ꢃ
get II with a yield of 68.91%, m.p. 185.8–187.7 C; compound UA reacted with 4-
nitrophenylhydrazine to get II with a yield of 73.11%, m.p. 195.1–197.8 C; com-
2
2c
ꢃ
3
pound UA2d reacted with 4-nitrophenylhydrazine to get II with a yield of 73.11%,
4
ꢃ
m.p. 191.3–193.8 C; compound UA reacted with 4-nitrophenyl hydrazine to get II5
2
e
ꢃ
with a yield of 70.30%, m.p. 193.3–196.0 C; compound UA reacted with 4-nitrophe-
2f
ꢃ
nylhydrazine to get II with a yield of 74.04%, m.p. 195.5–198.1 C.
6
3
.2.5. 3-(4’-Chloridephenylhydrazone)-urs-12-en-28-oic acid ester (III –III )
1 6
To a solution of UA2a (200 mg, 0.41 mmol) in methanol (15 ml) was added p-chloro-
phenylhydrazine hydrochloride (94 mg, 0.51 mmol) and triethylamine (0.07 ml), then
stirred at room temperature for 1 h and used 0.2 ml glacial acetic acid as catalyst,
ꢃ
finally the mixture was reflued at 40 C water bath and tested the endpoint of reac-
tion by TLC. After the reaction was completed, extraction with saturated brine and
ethyl acetate, and dried and filtered, then evaporated to get crude product, the crude
product was recrystallized from ethanol to obtain orange yellow crystals (III ) with a
1
ꢃ
yield of 67.48%, m.p. 188.9–191.6 C. According to the synthesis method of com-
pound III , compound UA reacted with p-chlorophenyl hydrazine hydrochloride to
1
2b
ꢃ
get III with a yield of 69.03%, m.p. 196.9–198.5 C; compound UA reacted with p-
2
2c
chlorophenyl hydrazine hydrochloride to get III3 with a yield of 66.51%, m.p.
ꢃ
1
98.9–201.7 C; compound UA2 reacted with p-chlorophenyl hydrazine hydrochlor-
d
ꢃ
ide to get III with a yield of 64.86%, m.p. 197.3–199.5 C; compound UA reacted
4
2e
with p-chlorophenyl hydrazine hydrochloride to get III with a yield of 60.51%, m.p.
5

1
0
Y.-Q. MENG ET AL.
ꢃ
2
02.4–205.1 C; compound UA reacted with p-chlorophenyl hydrazine hydrochloride
2
f
ꢃ
to get III with a yield of 63.65%, m.p. 205.9–208.1 C.
6
3
.3. Cell proliferative assay
The anti-tumor activity of the synthesized target compounds was tested by MTT
method in vitro. The medium used for the activity test was RPMI-1640 (containing
10% fetal bovine serum, and 100 lg/ml penicillin–streptomycin from Shanghai Yuan-
Ye Biotechnology Co., Ltd., Shanghai, China). Proteinase K and bovine serum albu-
min (BSA) purchased from Shanghai Tucker Industrial Co. Ltd. (Shanghai, China).
The selected tumor cells were human hepatoma cell BEL-7402 and human gastric
cancer cell SGC-7901 that were provided by Department of Pharmacology, Shenyang
Pharmaceutical University. With VP-16 and adriamycin as positive control drugs
from AstraZeneca Pharmaceutical Co., Ltd. (Shanghai, China). The logarithmic
growth phase tumor cells were cultured in a 96-well culture plate, with 100 ll/well
ꢃ
(
ꢄ4000 cells), placed at 37 C in an atmosphere of 5% CO in incubator, and cultured
2
for 24 h. In the positive control group, five concentrations (33, 11, 3.6, 1.2, and
.4 mg/ml) were added, and at least three parallel holes were tested. In the negative
0
control group, an equal volume of solvent was added to the positive control group,
ꢃ
and set a blank control group containing only the medium, placed at 37 C in an
atmosphere of 5% CO in incubator. All liquid in the 96-wells plate was gently
2
throwed out after 48 h and 50 ll MTT solution (1 mg/ml) was added in each well.
ꢃ
After a further culture for 4 h at 37 C, 150 ll DMSO was then added in each well
and plates were gently stirred for 5 min on a mechanical stirrer. The optical density
(OD) was recorded on ELISA reader at 490 nm. Each test was performed three times.
3
.4. Docking study
Molecular docking of compounds II , II , III , and III into the X-ray crystallographic
4
6
4
6
structure of NF-jB (PDB:3rzf) was conducted by using the Molegro Virtual Docker.
Discovery Studio 4.0 and Chimera 1.10 was used for analysis and display.
Acknowledgments
We thank Shenyang Pharmaceutical University for the tool and technology assistance in dock-
ing studies.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the Natural Science Foundation of Liaoning Province [grant num-
ber 201605288]; the National Natural Science Foundation of China [grant number 21372156];
and Scientific Research Project of the Shenyang Science and Technology Board [grant number
F-16-230-6-00].

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
11
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