Conjugates of 18β-glycyrrhetinic acid derivatives with 3-(1H-benzo[d]imidazol-2-yl)propanoic acid as Pin1 inhibitors displaying anti-prostate cancer ability

Article abstract of DOI:10.1016/j.bmc.2017.08.002

Twenty-six conjugates of 18β-glycyrrhetinic acid derivatives with 3-(1H-benzo[d]imidazol-2-yl)propanoic acid were designed and synthesized as Pin1 inhibitors. Most of these semi-synthetic compounds showed improved Pin1 inhibitory activity and anti-proliferative effects against prostate cancer cells as compared to 3-(1H-benzo[d]imidazol-2-yl)propanoic acid and GA. Compounds 10a and 12i were the most potent to inhibit growth of prostate cancer PC-3 with GI₅₀ values of 7.80 μM and 3.52 μM, respectively. The enzyme inhibition ratio of nine compounds at 10 μM was over 90%. Structure-activity relationships indicated that both appropriate structure at ring C of GA and suitable length of linker between GA skeleton and benzimidazole moiety had significant impact on improving activity. Western blot assay revealed that 10a decreased the level of cell cycle regulating protein cyclin D1. Thus, these compounds might represent a novel anti-proliferative agent working through Pin1 inhibition.

Full text of DOI:10.1016/j.bmc.2017.08.002

Accepted Manuscript
Conjugates of 18β-glycyrrhetinic acid derivatives with 3-(1H-benzo[d]imida-
zol-2-yl)propanoic acid as Pin1 inhibitors displaying anti-prostate cancer ability
Kun Li, Tianyi Ma, Jingjing Cai, Min Huang, Hongye Guo, Di Zhou, Shenglin
Luan, Jinyu Yang, Dan Liu, Yongkui Jing, Linxiang Zhao
PII:
S0968-0896(17)30878-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.08.002
BMC 13905
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 April 2017
19 July 2017
2 August 2017
Please cite this article as: Li, K., Ma, T., Cai, J., Huang, M., Guo, H., Zhou, D., Luan, S., Yang, J., Liu, D., Jing,
Y., Zhao, L., Conjugates of 18β-glycyrrhetinic acid derivatives with 3-(1H-benzo[d]imidazol-2-yl)propanoic acid
as Pin1 inhibitors displaying anti-prostate cancer ability, Bioorganic & Medicinal Chemistry (2017), doi: http://
dx.doi.org/10.1016/j.bmc.2017.08.002
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Conjugates of 18β-glycyrrhetinic acid derivatives with 3-(1H-benzo[d]imidazol-2-yl)propanoic acid as Pin1
inhibitors displaying anti-prostate cancer ability
Kun Lia, Tianyi Maa, Jingjing Caia, Min Huanga, Hongye Guoa, Di Zhoua, Shenglin Luana, Jinyu Yanga, Dan Liua*,
Yongkui Jingb* and Linxiang Zhaoa*
a Key Laboratory of Structure-Based Drugs Design & Discovery of Ministry of Education, Shenyang Pharmaceutical
University, Shenyang 110016, China
b School of Life Sciences and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, China
*Corresponding authors.
Tel./Fax: +86 024 43520221.
E-mail address: linxiang.zhao@vip.sina.com (L. Zhao);
yongkjing@yahoo.com (Y. Jing);
sammyld@163.com (D. Liu)

Abstract
Twenty-six conjugates of 18β-glycyrrhetinic acid derivatives with 3-(1H-benzo[d]imidazol-2-yl)propanoic acid
were designed and synthesized as Pin1 inhibitors. Most of these semi-synthetic compounds showed improved Pin1
inhibitory activity and anti-proliferative effects against prostate cancer cells as compared to
3-(1H-benzo[d]imidazol-2-yl)propanoic acid and GA. Compounds 10a and 12i were the most potent to inhibit growth
of prostate cancer PC-3 with GI50 values of 7.80 µM and 3.52 µM, respectively. The enzyme inhibition ratio of nine
compounds at 10 µM was over 90%. Structure-activity relationships indicated that both appropriate structure at ring C
of GA and suitable length of linker between GA skeleton and benzimidazole moiety had significant impact on
improving activity. Western blot assay revealed that 10a decreased the level of cell cycle regulating protein cyclin D1.
Thus, these compounds might represent a novel anti-proliferative agent working through Pin1 inhibition.
Keywords: Pin1 inhibitors; 18β-glycyrrhetinic acid derivatives; anti-proliferative activity; cyclin D1

1. Introduction
Pin1, which belongs to the peptidyl-prolyl isomerase family, is the only enzyme that catalyzes isomerization of
specific phosphorylated motifs in a subset of proteins.1,2 Pin1 regulates diverse cellular processes, including
growth-signal response, cell-cycle regulation, cellular stress response, neuronal function and immune response.3 High
expression level of Pin1 is frequently observed in a variety of cancer cells, especially in prostate cancer. Studies
indicated that Pin1 could regulate the level of cancer-related signaling proteins, such as cyclin D1.4-6 The abnormal
increase of cyclin D1 can be attributed to many factors including transcription, translation and protein stability.7,8
Knockout of Pin1 results in mitotic arrest and apoptosis in tumor cell lines. Therefore, Pin1 has been used as a potential
therapeutic target for developing anticancer agent.9-11
There has been persistent interest for developing Pin1 inhibitors, and a number of promising entities have been
identified by several groups.12 Structure-guided design was adopted to design small molecule inhibitors, but only a few
of them were active in cancer cells due to their poor permeability.13-17 A couple of natural products have been found to
be able to inhibit Pin1 (Figure 1); for instance, Juglone and Epigallocatechin-3-gallate (EGCG).18,19 In our previous
studies, lots of triterpenoid derivatives have been reported with effective Pin1 inhibitory ability, including
Acetyl-11-keto-β-boswellic acid analogues and Elemonic acid derivatives.20,21
(Figure 1. should be listed here.)
Figure 1. The structures of natural products as Pin1 inhibitors.
The crystal structure of Pin1 protein indicated that PPIase domain consists of two hydrophobic domains and one

basic cluster. Based on Jonathan D. Moore’s research, the 3-(1H-benzo[d]imidazol-2-yl)propanoic acid moiety has been
identified to be a suitable core fragment for the prolyl pocket and basic cluster of Pin1 protein, and introducing an extra
hydrophobic fragment to access the shallow surface of Pin1 protein could enhance Pin1 inhibitory activity significantly.
However, most benzimidazole derivatives were not active in cell-based assays because of the poor permeability.14,15 As
continued research to discover new Pin1 inhibitors with anti-proliferative activity, the 3-(1H-benzo[d]imidazol-2-yl)
propanoic acid skeleton was adopted for conjugates. Considering the excellent hydrophobicity of triterpenoids,
18β-glycyrrhetinic acid derivatives were attached to N1 position of benzimidazole through different flexible linkers.22
The triterpenoid moiety was supposed to produce a hydrophobic interaction with the shallow surface of Pin1 PPIase
domain. It is reasonable to suggest that the construction of triterpenoid–benzimidazole hybrids might develop a series of
novel Pin1 inhibitors with anti-proliferative activity.
2. Results and discussion
2.1. Chemistry
The synthetic route adopted for the preparation of key intermediates 5a–5i was outlined in Scheme 1, which has
been reported previously.23 Briefly, the esterification of GA at C30 was performed by treatment with concentrated
sulfuric acid and methanol to give the intermediate 5a. Reduction of GA with sodium borohydride led to 1, which was
then refluxed in methanol and sulfuric acid to obtain 5b. The intermediate 2 with 12-en moiety at ring C was obtained
from GA by Clemmensen reduction. Then esterification of 2 was executed to give 5c, which continued to be acetylated
and oxidized resulting 3. A sequence of bromination/dehydrobromination of 3 provided 4 easily. The deacetylation of 3
and 4 gave 5d and 5e in high yield respectively. The acetylation and oxidization of hydroxyl at C3 position of GA and 2
provided 5f–5i smoothly.
(Scheme 1. should be listed here.)

o
Scheme 1. Synthesis of 5a–5i. Reagents and Conditions: (a) Concentrated H2SO4, methanol, 66 C; (b) Zn-Hg, HCl,
o
o
o
1,4-dioxane, 10 C; (c) NaBH4, NaOH(aq.), THF, 66 C; (d) Ac2O, pyridine, 115 oC; (e) 30% H2O2, AcOH, 100 C; (f)
KOH, methanol, 66 oC; (g) Br2, HBr, acetic acid, 40 oC; (h) Jones’ reagent, acetone, r.t..
The synthesis of compounds 10a–10j was described in Scheme 2. Intermediates 5a–5e were acylated using
2-chloroacetyl chloride or 3-chloropropionyl chloride with the presence of triethylamine or pyridine in dichloromethane
to generate 6a–6j, which were then reacted with 9. Compounds 10a–10j could be readily yielded by debenzylation with
H2/Pd-C or TiCl4 at ambient temperature.
(Scheme 2. should be listed here.)
Scheme 2. Synthesis of 10a–10j. Reagents and Conditions: (a) 2-chloroacetyl chloride or 3-chloropropionyl chloride,
triethylamine or pyridine, CH2Cl2, r.t.; (b) succinic anhydride, dioxane, reflux; (c) BnBr, K2CO3, DMF, r.t.; (d) Cs2CO3,

acetonitrile, 80 oC; (e) H2, 5% Pd/C, methanol, r.t. or TiCl4, CH2Cl2, r.t..
Compounds 12a–12p were prepared as depicted in Scheme 3. The coupling of GA, 2, 5f–5i with 11a–11d was
performed respectively, followed which an efficient debenzylation was carried out in good yield to provide compounds
12a–12p.
(Scheme 3. should be listed here.)
Scheme 3. Synthesis of 12a–12p. Reagents and Conditions: (a) 1,2-dibromoethane or 1,3-dibromopropane, Cs2CO3,
acetonitrile, 80 oC; (b) 2-chloroethylamine hydrochloride or 3-bromopropanamin hydrobromide, NaH, DMF, 80 oC; (c)
K2CO3, DMF, r.t.; (d) SOCl2, CH2Cl2, r.t.; (e) H2, 5% Pd/C, methanol, r.t. or TiCl4, CH2Cl2, r.t..
2.2. Biological activity
As Pin1 is often highly expressed in human prostate cancer cells, all target compounds were first evaluated for
their anti-proliferative activity against PC-3 and LNCaP cell lines using MTT assay. The results expressed as GI50
values were presented in Table 1, which were the average of three independent experiments.
As shown in Table 1, 3-(1H-benzo[d]imidazol-2-yl)propanoic acid (8) did not show cell growth inhibitory effects
might due to its poor permeability and weak activity against Pin1, whereas most of these hybrids exhibited superior
anti-proliferative activity to GA. This phenomenon indicated that the conjugation is necessary for significant
cytotoxicity. Generally, the structural modification of ring A and ring C together with the length of linker between GA
skeleton and benzimidazole moiety had a notable influence on the anti-proliferative activity. Compounds 10a and 12i
displayed the most potent inhibitory effects with GI50 values of 7.80/13.23 µM and 3.52/7.92 µM, respectively.
As for 10a–10j, compounds 10a (GI50 = 7.80 µM (PC-3), GI50 = 13.23 µM (LNCaP)) and 10b (GI50 = 15.31 µM
,
(PC-3)
GI50 = 21.37 µM (LNCaP)) with acetyl as linker showed more potent anti-proliferative activity than compounds 10f (GI50
=

16.05 µM (PC-3), GI50 = 26.61 µM (LNCaP)) and 10g (GI50 = 47.22 µM (PC-3), GI50 > 50 µM (LNCaP)) linked by propionyl,
indicating that two atoms length is favorable. A tiny tuning of the structure at ring C exerted a tremendous influence on
activity. Compounds 10c (GI50 = 29.21 µM (PC-3), GI50 = 30.01 µM (LNCaP)) and 10h (GI50 > 50 µM (PC-3), GI50 > 50 µM
(LNCaP)) with 12-oxo structure at ring C were inferior to corresponding derivatives with other distinct structures at ring C.
When the 11-oxo-12-en (10a) at ring C was replaced by 12-en (10b) or 9(11),12-dien (10e GI50 = 14.06/17.39 µM), the
inhibitory activities were decreased to about half.
Introducing the benzimidazole moiety to C30 carboxylic acid group of GA led to a significant decline of
anti-proliferative activity as exemplified by 12a (GI50 > 50 µM (PC-3), GI50 > 50 µM (LNCaP)) and 12b (GI50 > 50 µM (PC-3)
,
GI50 > 50 µM (LNCaP)). Replacing the hydroxy group at C3 position of ring A by acetoxyl group or carbonyl caused a
remarkable increase of anti-proliferation activity as exemplified by 12c (GI50 = 11.83 µM (PC-3), GI50 = 15.51 µM (LNCaP)),
12e (GI50 = 35.63 µM (PC-3), GI50 = 29.32 µM (LNCaP)) vs. 12a, which could be further confirmed by 12d (GI50 = 27.35
µM (PC-3), GI50 = 24.43 µM (LNCaP)), 12f (GI50 = 39.93 µM (PC-3), GI50 = 48.91 µM (LNCaP)) vs. 12b, suggesting a fine tuning
of functional groups at this position was beneficial. However, the anti-proliferative potency of these C30 coupled
derivatives was still a little less than 10a. Subsequently, a small set of C30 coupled analogues 12g–12l with 12-en at
ring C were synthesized. Among them, compounds 12i (GI50 = 3.52 µM (PC-3), GI50 = 7.92 µM (LNCaP)), 12j (GI50 = 10.91
µM (PC-3), GI50 = 13.34 µM (LNCaP)), 12k (GI50 = 12.98 µM (PC-3), GI50 = 19.71 µM (LNCaP)) and 12l (GI50 = 18.41 µM (PC-3)
,
GI50 = 25.80 µM (LNCaP)) were 2~3 fold more potent than their counterparts 12c, 12d, 12e and 12f, respectively. The
activities of compounds 12c and 12i were much better than 12d and 12j, indicating that for C30 coupled derivatives
three atoms length is favorable. In addition, 12i and 12j with acetoxyl group at C3 position of GA presented superior
activity to 12k and 12l with carbonyl; this phenomenon was appeared among 12c, 12d vs. 12e, 12f as well. Nonetheless,
once the ester of linker was replaced by amide (12m, 12n, 12o and 12p), the anti-proliferative effect decreased sharply.
(Table 1. should be listed here.)
Table 1. The anti-proliferative activities of target compounds against PC-3 and LNCaP cell lines.

GI50 (µM)a
GI50 (µM)a
Compd.
Compd.
PC-3
LNCaP
13.23 0.29
21.37 0.33
30.01 0.21
23.45 0.17
17.39 0.31
26.61 0.44
> 50
PC-3
LNCaP
48.91 0.41
42.66 0.33
> 50
7.80 0.31
15.31 0.35
29.21 0.59
21.22 0.35
14.06 0.25
16.05 0.35
47.22 0.29
> 50
39.93 0.22
25.63 0.33
> 50
10a
10b
10c
10d
10e
10f
12f
12g
12h
12i
3.52 0.17
10.91 0.28
12.98 0.39
18.41 0.30
> 50
7.92 0.69
13.34 0.19
19.71 0.52
25.80 0.77
> 50
12j
12k
12l
10g
10h
10i
> 50
12m
12n
12o
12p
8
21.39 0.38
20.41 0.37
> 50
28.95 0.78
25.32 0.39
> 50
> 50
> 50
> 50
> 50
10j
> 50
> 50
12a
12b
12c
12d
12e
> 50
> 50
> 50
> 50
11.83 0.33
27.35 0.19
35.63 0.27
15.51 0.68
24.43 0.47
29.32 0.55
95.72 0.66
4.77 0.29
-
GA
Juglone
-
a GI50 value is the concentration that inhibits 50% of cell growth compared with the untreated cells. Each value was
measured at least three times.
To investigate the Pin1 inhibitory potency of all synthesized compounds, the inhibition ratio at 10 µM was
determined using protease-coupled assay, and the results fitted well with the anti-proliferative activity (Table 2). The
bulk of these triterpenoid–benzimidazole conjugates displayed improved Pin1 inhibitory potency as compared to
3-(1H-benzo[d]imidazol-2-yl)propanoic acid (8) and GA. Among them, the inhibition ratio of nine compounds at 10
µM was over 90%. The IC50 values were then determined for those compounds. Compounds 10a (IC50 = 1.07 µM) and
12i (IC50 = 2.10 µM), which displayed the most potent growth inhibition effects, also proved to be outstanding in Pin1
inhibition. On the contrary, compounds 10h, 12a, 12m, 12n, 12o and 12p, which showed terrible anti-proliferative
activity, were found to be less potent ones. Therefore, 10a and 12i with both good Pin1 inhibitory activity and excellent
anti-proliferative effects were regarded as the most promising candidates among those target compounds.
(Table 2. should be listed here.)

Table 2. The Pin1 inhibitory activities of target compounds.
Pin1 inhibition
at 10 µM (%) a
Pin1 inhibition
at 10 µM (%) a
IC50 (µM)b
IC50 (µM)b
Compd.
Compd.
100
96
1.07
63
3
-
10a
10b
10c
10d
10e
10f
12f
12g
12h
12i
7
9
2
5
3
8
1
2.31
100
2
2.82
-
67
76
74
88
58
44
-
54 14
-
100
94
98
69
50
27
28
38
23
34
44
3
2.10
4.79
3.80
-
-
2
12j
-
-
7
5
6
4
2
9
6
3
7
12k
12l
10g
10h
10i
> 10
2.35
-
-
12m
12n
12o
12p
8
100
> 10
> 10
> 10
> 10
> 10
10.81
70
40
60
5
4
9
10j
12a
12b
12c
12d
12e
> 10
-
100
1.33
1.06
-
GA
Juglone
100
77
5
7
a Values are means of at least three independent experiments.
b All results represent the mean of duplicate experiments.
Pin1 directly targeted on the pThr-Pro motifs of cyclin D1 and stabilized cyclin D1 by preventing its nuclear export
and ubiquitin-mediated degradation.3 Thus to further demonstrate the Pin1 inhibitory effect of these compounds, the
expression level of cyclin D1 protein was determined by western blot assay in PC-3 cells treated with 10a at different
concentrations for 24 h. The results (Figure 2) indicated that 10a could efficiently decrease the level of cyclin D1 in a
concentration-dependent manner, which might be related to the decreased activity of Pin1.
(Figure 2. should be listed here.)

Figure 2. Compound 10a modulated cyclin D1 level in PC-3.
To demonstrate the binding modes of 10a and 12i, molecular docking was carried out using the glide protocol in
maestro software package (Figure 3). Docking studies revealed that these two compounds interacted with Pin1 in the
C-terminal PPIase domain. The catalytic center of PPIase domain composed of two hydrophobic pockets and a basic
cluster formed by Lys63, Arg68 and Arg69 residues.13-15 Analysis of the docked complexes (Figure 3) suggested that
10a and 12i occupied the active pocket completely and demonstrated interactions with key residues as a result of which
they exhibited a high binding affinity of -6.35 kcal/mol and -6.15 kcal/mol, respectively. The benzimidazole moiety of
these compounds fit well into the prolyl pocket with a π–π stack between benzene ring and His157. The nitrogen atom
on imidazole anchored with residue Ser154 via a crucial hydrogen bond. In addition, the carboxylate of propanoic acid
could interacted with the positive charged side chain of Lys63 and Arg69 through salt bridge, and the carbonyl oxygen
of carboxyl as a H-bond receptor formed a hydrogen bond with Ser114. Such interactions might have a great
contribution to their remarkable Pin1 inhibitory activity. As shown in Figure 3A, the pentacyclic triterpenoid scaffold
of 10a showed hydrophobic and van der Waal interactions with the shallow hydrophobic shelf embraces His59, Ala117,
Ala118, Leu122 and Ala124 residues, better yet, the carbonyl group at C11 position allowed the H-bond formation with
residue Ser114. For compound 12i, the pentacyclic triterpenoid scaffold connected with benzimidazole moiety at C30
carboxyl. In such circumstance, the acetyl group at C3 position was placed near residue Ala124, which was responsible
for the formation of an additional hydrogen bond. By analyzing the events mentioned above, we drew the conclusion
that both the benzimidazole moiety and pentacyclic triterpenoid scaffold were greatly helpful to enhance the Pin1
inhibition ability.
(Figure 3. should be listed here.)

Figure 3. The predicated binding modes of 10a (A, B) and 12i (C, D) in the PPIase domain of Pin1 (3KAI in PDB).
H-Bonding interactions were presented with green line.
3. Conclusion
We reported herein the discovery and synthesis of novel conjugates of 18β-glycyrrhetinic acid derivatives with
3-(1H-benzo[d]imidazol-2-yl)propanoic acid as potential Pin1 inhibitors. Several compounds were identified as
promising candidates with both good Pin1 inhibitory activity and excellent anti-proliferative effect against prostate
cancer PC-3 and LNCaP cell lines. Compounds 10a and 12i were found to be the most potent ones. SAR studies
indicated that the structural modification of ring A and ring C together with the length of linker between GA skeleton
and benzimidazole moiety had a notable influence on the anti-proliferative activity. Western blot assay revealed that
10a could down-regulate the expression level of cell cycle-related protein cyclin D1 in a concentration-dependent
manner, which further demonstrated the Pin1 inhibitory effect of these compounds. Molecular docking studies
displayed that both two components of these conjugates were essential for receptor binding. This study provides further
possibility to develop novel Pin1 inhibitors with anti-proliferative ability.

4. Experimental
4.1. Chemistry
18β-glycyrrhetinic acid (GA) was purchased from Haokang Chemicals Inc. (Shanghai China) with over 98% purity.
The other reagents were obtained commercially and were used with further purification if necessary. The melting points
(Mp) were determined on an electrically heated X4 digital visual melting point apparatus which were uncorrected. Mass
spectra (MS) were determined on Finnigan MAT/USA spectrometer (LC-MS). Infrared (IR) spectra were recorded on a
Bruker IR-27G spectrometer using KBr pellets. High-resolution mass spectra were measured with Bruker
microOTOF-Q in ESI mode (HR-MS). Nuclear magnetic resonance (1H and 13C NMR) spectra were measured on
Bruker AV-400 or ARX-600 MHz instrument. Chemical shifts were recorded in δ (ppm) with tetramethylsilane (TMS)
as the internal standard. All reactions were monitored by thin-layer chromatography (TLC, carried on fluorescent
precoated plates GF254, Qingdao Haiyang Chemical, China).
4.1.1. General procedure for the synthesis of 10a–10j
The preparations of key intermediates 5a–5i were based on our previous research, and the yields of target
compounds were quite similar to that reported before.23
To a solution of o-phenylenediamine 7 (46 mmol) in dioxane (110 mL) was added succinic anhydride (55.5 mmol)
at room temperature. Then the mixture was stirred at 80 oC for 4 h. When the mixture was cooled to room temperature,
the precipitate was filtered, washed with dioxane/water/aether, respectively. Finally the precipitate was dried to give 8
as white solid with 57% yield.
To a solution of 8 (5.26 mmol) in DMF (20 mL) was added K2CO3 (10 mmol). The mixture was stirred at room
temperature for 1 h, then benzyl bromide (6.32 mmol) was added dropwise and stirred for 24 h. The reaction solution
was poured into water (50 mL) and filtered. The precipitate was washed with water to PH 7 and dried to give 9 as white
solid with 80% yield.
To a solution of 5a-5e (2.1 mmol) in dry dichloromethane (60 mL), triethylamine or pyridine (4.2 mmol) was

added dropwise under ice-salt bath. Then 2-chloroacetyl chloride or 3-chloropropionyl chloride (3.2 mmol) was slowly
added. The mixture was allowed to warm up to room temperature and stirred for 5 h. The reaction was quenched with
water, and the water layer was separated, several times extracted with dichloromethane. The combined dichloromethane
solutions were dried with Na2SO4, filtered and evaporated. The resulting residue was then purified by column
chromatography on silica gel to give 6a–6j as white solid with 70~80% yield.
To a solution of 8 (3 mmol) in dry acetonitrile (50 mL) was added Cs2CO3 (6 mmol). The mixture was stirred at
room temperature for 1 h, then 6a–6j (3 mmol) was added slowly. The solution was warmed up to 82 oC and stirred for
additional 4 h. The solvent was evaporated to dryness and added water, extracted with ethyl acetate three times. The
organic layers were combined, washed with brine, dried with Na2SO4, filtered and evaporated. The resulting residue
was then purified by column chromatography on silica gel to give corresponding intermediates as white solid with
60~85% yield.
The corresponding intermediates (0.25 mmol) were dissolved in methanol (15 mL), and 5% Pd/C (20 mg) was
added slowly. The resulting mixture was carefully evacuated, flooded with hydrogen three times, stirred under a
hydrogen atmosphere at room temperature for 4 h. The reaction solution was filtered and concentrated under reduced
pressure. The crude product was purified by column chromatography on silica gel to give 10a-10j as white solid with
90~95% yield.
4.1.1.1. 3-(1-(2-((methyl 11-oxo-18β-olean-12-en-30-oate)-3β-oxy)-2-oxoethyl)-1H-benzo[d]imidazol-2-yl)
propanoic acid (10a)
Yield: 91.4%; White solid; Mp: 175-176 ºC; IR (KBr): 3422.3, 2926.5, 1730.5, 1630.3, 1464.7, 1384.4, 1218.1,
1
1147.4, 1111.9 cm-1; H NMR (400 MHz, DMSO-d6): δ 12.20 (s, 1H, -COOH), 7.36 (m, 4H, H-4’, H-5’, H-6’, H-7’),
5.26 (m, 2H, H-13’), 5.24 (s, 1H, H-12), 4.45 (m, 1H, H-3), 3.62 (s, 3H, -COOCH3), 3.01 (m, 2H, H-10’), 2.77 (m, 2H,
H-11’), 2.38 (s, 1H, H-9), 2.31 (m, 1H), 1.99 (m, 1H), 1.33 (s, 3H), 1.23 (s, 3H), 1.18 (s, 3H), 1.10 (s, 2H), 1.08 (s, 2H),
1.02 (s, 6H), 0.87 (m, 2H), 0.74 (s, 1H), 0.71 (s,3H), 0.64 (s, 1H), 0.58 (s, 3H); 13C NMR (DMSO-d6): δ 199.3 (C-11),

176.6 (C-30), 173.9 (C-12’), 170.0 (C-14’), 168.3 (C-13), 154.9 (C-2’), 142.4 (C-9’), 136.0 (C-8’), 127.7 (C-12), 122.1
(C-5’), 121.8 (C-6’), 118.8 (C-4’), 110.1 (C-7’), 81.8 (C-3), 61.1, 53.8, 52.1, 52.0, 48.3, 45.2, 45.1, 44.9, 43.9, 43.7,
43.3, 42.4, 40.8, 37.9, 36.8, 35.4, 31.9, 28.6, 28.0, 27.8, 26.4, 26.1, 23.3, 22.2, 20.9, 20.7, 18.6, 16.5, 16.4; LC-MS:
715.7 [M+H]+, 713.6 [M-H]-; HR-MS: m/z, calcd for C43H59N2O7 [M+H]+ 715.4322, found: 715.4324.
4.1.1.2. 3-(1-(2-((methyl 18β-olean-12-en-30-oate)-3β-oxy)-2-oxoethyl)-1H-benzo[d]imidazol-2-yl)propanoic acid
(10b)
Yield: 90.0%; White solid; Mp: 125-126 ºC; IR (KBr): 3428.5, 2927.1, 2853.9, 1732.4, 1518.1, 1464.8, 1383.0,
1
1315.2, 1217.0, 1157.2, 1088.6, 1015.2 cm-1; H NMR (400 MHz, DMSO-d6): δ 12.32 (s, 1H, -COOH), 7.56 (d, 1H,
H-4’), 7.47 (d, 1H, H-7’), 7.17 (m, 2H, H-5’, H-6’), 5.25 (m, 2H, H-13’), 5.17 (m, 1H, H-12), 4.45 (m, 1H, H-3), 3.61 (s,
3H, -COOCH3), 3.01 (m, 2H, H-10’), 2.80 (m, 2H, H-11’), 1.98 (m, 1H), 1.70 (s, 1H), 1.65 (m, 1H), 1.41 (s, 2H), 1.20
(s, 1H), 1.16 (s, 1H), 1.11 (s, 3H), 1.07 (s, 3H), 0.96 (m, 1H), 0.89 (s, 3H), 0.88 (s, 3H), 0.81 (s, 1H), 0.72 (s, 3H), 0.70
(s, 3H), 0.56 (s, 3H); 13C NMR (DMSO-d6): δ 176.9 (C-30), 173.9 (C-12’), 168.3 (C-14’), 154.8 (C-2’), 144.6 (C-13),
142.4 (C-9’), 136.0 (C-8’), 122.2 (C-5’), 122.2 (C-6’), 121.8 (C-12), 118.8 (C-4’), 110.0 (C-7’), 82.0 (C-3), 54.5, 51.8,
48.2, 47.1, 44.9, 44.0, 42.7, 41.5, 40.4, 38.3, 37.9, 37.6, 36.7, 32.0, 31.1, 28.4, 28.3, 27.9, 26.7, 26.7, 26.1, 25.9, 23.4,
23.3, 22.1, 18.0, 16.8, 16.5, 15.5; LC-MS: 701.4 [M+H]+, 699.2 [M-H]-; HR-MS: m/z, calcd for C43H61N2O6 [M+H]+
701.4530, found: 701.4537.
4.1.1.3. 3-(1-(2-((methyl 12-oxo-18β-olean-30-oate)-3β-oxy)-2-oxoethyl)-1H-benzo[d]imidazol-2-yl)propanoic acid
(10c)
Yield: 95.2%; White solid; Mp: 122-123 ºC; IR (KBr): 3428.5, 2926.3, 2853.3, 1731.4, 1518.7, 1465.8, 1082.2,
1
1013.7 cm-1; H NMR (400 MHz, DMSO-d6): δ 7.55 (d, 1H, H-4’), 7.46 (d, 1H, H-7’), 7.16 (m, 2H, H-5’, H-6’), 5.23
(m, 2H, H-13’), 4.43 (t, 1H, H-3), 3.58 (s, 3H, -COOCH3), 3.00 (m, 2H, H-10’), 2.78 (m, 2H, H-11’), 2.72 (m, 1H),
2.24 (m, 1H), 2.01 (m, 1H), 1.87 (m, 1H), 1.39(s, 3H), 1.23 (s, 8H), 1.09 (s, 3H), 1.05 (s, 3H), 0.85 (s, 3H), 0.79 (s, 6H),
0.69 (s, 3H), 0.56 (s, 3H); 13C NMR (DMSO-d6): δ 211.1 (C-12), 177.0 (C-30), 173.9 (C-12’), 168.2 (C-14’), 154.9

(C-2’), 142.4 (C-8’), 136.0 (C-9’), 122.1 (C-6’), 121.8 (C-5’), 118.8 (C-4’), 110.0 (C-7’), 81.8 (C-3), 54.1, 51.6, 49.6,
49.2, 44.9, 43.9, 42.0, 41.5, 40.4, 38.5, 37.7, 37.2, 36.6, 33.8, 32.1, 31.5, 31.3, 31.3, 31.0, 29.4, 28.7, 27.7, 27.3, 26.3,
25.9, 23.3, 22.2, 20.8, 18.1, 16.3, 16.0, 15.3; LC-MS: 717.5 [M+H]+, 715.2 [M-H]-; HR-MS: m/z, calcd for C43H61N2O7
[M+H]+ 717.4479, found: 717.4473.
4.1.1.4. 3-(1-(2-((methyl 12-oxo-18β-olean-9(11)-en-30-oate)-3β-oxy)-2-oxoethyl)-1H-benzo[d]imidazol-2-yl)
propanoic acid (10d)
Yield: 94.7%; White solid; Mp: 128-130 ºC; IR (KBr): 3426.7, 2926.4, 1731.9, 1660.3, 1597.7, 1518.8, 1465.3,
1
1382.6, 1326.3, 1220.4, 1196.1, 1157.5, 1087.1 cm-1; H NMR (400 MHz, DMSO-d6): δ 7.52 (m, 2H, H-4’, H-7’), 7.18
(m, 2H, H-5’, H-6’), 5.61 (s, 1H, H-11), 5.26 (m, 2H, H-13’), 4.45 (m, 1H, H-3), 3.62 (s, 3H, -COOCH3), 3.02 (m, 2H,
H-10’), 2.87 (m, 1H), 2.80 (m, 2H, H-11’), 2.09 (m, 1H), 1.69 (m, 2H), 1.58 (m, 4H), 1.24 (s, 6H), 1.14 (s, 3H), 1.05 (s,
3H), 0.91 (s, 3H), 0.87 (s, 5H), 0.75 (s, 3H), 0.60 (s, 3H); 13C NMR (DMSO-d6): δ 200.0 (C-12), 177.8 (C-30), 176.8
(C-12’), 173.8 (C-14’), 168.2 (C-9), 154.8 (C-2’), 142.1 (C-9’), 135.9 (C-8’), 122.5 (C-11), 122.3 (C-5’), 122.0 (C-6’),
118.7 (C-4’), 110.1 (C-7’), 81.2 (C-3), 51.7, 49.6, 47.6, 45.4, 44.9, 43.9, 41.8, 40.4, 38.5, 38.1, 37.9, 35.8, 33.5, 32.5,
32.0, 31.0, 30.9, 29.4, 28.6, 27.7, 27.4, 26.1, 24.1, 24.0, 23.7, 22.1, 21.9, 17.6, 16.5; LC-MS: 715.5 [M+H]+, 713.3
[M-H]-; HR-MS: m/z, calcd for C43H59N2O7 [M+H]+ 715.4322, found: 715.4317.
4.1.1.5. 3-(1-(2-((methyl 18β-olean-9(11),12-dien-30-oate)-3β-oxy)-2-oxoethyl)-1H-benzo[d]imidazol-2-yl)
propanoic acid (10e)
Yield: 91.3%; White solid; Mp: 118-119 ºC; IR (KBr): 3430.9, 2948.1, 2855.8, 1731.9, 1518.8, 1464.9, 1379.6,
1216.2, 1158.2, 1107.2, 1086.8 cm-1; 1H NMR (400 MHz, DMSO-d6): δ 7.56 (d, 1H, H-4’), 7.48 (d, 1H, H-7’), 7.17 (m,
2H, H-5’, H-6’), 5.59 (d, 1H, H-11), 5.51 (d, 1H, H-12), 5.25 (m, 2H, H-13’), 4.46 (m, 1H, H-3), 3.63 (s, 3H,
-COOCH3), 3.01 (m, 2H, H-10’), 2.79 (m, 2H, H-11’), 1.99 (m, 2H), 1.91 (m, 1H), 1.82 (m, 1H), 1.31 (m, 5H), 1.24 (s,
3H), 1.18 (m, 1H), 1.13 (s, 3H), 1.07 (s, 6H), 0.98 (s, 2H), 0.95 (s, 3H), 0.78 (s, 3H), 0.74 (s, 3H), 0.71 (s, 1H), 0.65 (s,
1H), 0.58 (s, 3H); 13C NMR (DMSO-d6): δ 176.9 (C-30), 173.8 (C-12’), 168.3 (C-14’), 154.8 (C-9), 154.3 (C-2’), 146.5

(C-13), 142.3 (C-9’), 136.0 (C-8’), 122.2 (C-5’), 121.9 (C-6’), 121.1 (C-4’), 118.8 (C-12), 115.8 (C-11), 110.1 (C-7’),
81.8 (C-3), 51.9, 50.9, 46.4, 44.9, 44.0, 43.7, 42.6, 38.6, 38.2, 37.8, 36.6, 34.6, 31.6, 31.0, 29.4, 28.6, 28.2, 28.0, 27.0,
25.3, 24.0, 22.1, 20.9, 20.3, 19.9, 19.7, 18.0, 16.6; LC-MS: 699.5 [M+H]+, 697.2 [M-H]-; HR-MS: m/z, calcd for
C43H59N2O6 [M+H]+ 699.4373, found: 699.4379.
4.1.1.6. 3-(1-(3-((methyl 11-oxo-18β-olean-12-en-30-oate)-3β-oxy)-3-oxopropyl)-1H-benzo[d]imidazol-2-yl)
propanoic acid (10f)
Yield: 93.5%; White solid; Mp: 145-147 ºC; IR (KBr): 3429.3, 2925.6, 2854.1, 1729.1, 1661.8, 1462.8, 1385.3,
1
1327.2, 1214.7, 1191.1, 1113.3 cm-1; H NMR (600 MHz, DMSO-d6): δ 7.52 (m, 2H, H-4’, H-7’), 7.16 (m, 2H, H-2’,
H-5’), 5.37 (d, 1H, H-12), 4.46 (t, 2H, H-13’), 4.39 (m, 1H, H-3), 3.62 (s, 3H, -COOCH3), 3.07 (m, 2H, H-14’), 2.85 (m,
2H, H-10’), 2.78 (m, 2H, H-11’), 2.30 (m, 1H), 1.35 (s, 3H), 1.34 (s, 3H), 1.23 (s, 3H), 1.19(s, 2H), 1.10 (s, 2H), 1.07 (s,
2H), 1.02 (s, 6H), 0.83 (m, 2H), 0.74 (s, 1H), 0.71 (s, 3H), 0.66 (s, 3H), 0.64 (s, 1H); 13C NMR (DMSO-d6): δ 199.3
(C-11), 178.4 (C-30), 176.7 (C-12’), 170.9 (C-15’), 170.0 (C-13), 154.5 (C-2’), 142.6 (C-9’), 135.1 (C-8’), 127.6 (C-12),
123.3 (C-5’), 121.9 (C-6’), 118.7 (C-4’), 110.4 (C-7’), 80.7 (C-3), 61.2, 59.9, 53.9, 52.0, 48.3, 45.2, 43.9, 43.7, 43.3,
42.4, 39.1, 37.8, 36.6, 35.4, 34.2, 31.9, 31.5, 28.6, 28.0, 27.9, 26.1, 23.3, 22.3, 20.9, 20.7, 18.5, 16.8, 16.7, 16.5, 16.0;
LC-MS: 729.5 [M+H]+, 727.2 [M-H]-; HR-MS: m/z, calcd for C44H61N2O7 [M+H]+ 729.4479, found: 729.4421.
4.1.1.7. 3-(1-(3-((methyl 18β-olean-12-en-30-oate)-3β-oxy)-3-oxopropyl)-1H-benzo[d]imidazol-2-yl)propanoic acid
(10g)
Yield: 96.7%; White solid; Mp: 215-216 ºC; IR (KBr): 3431.6, 2949.6, 2924.7, 2853.8, 1728.6, 1631.4, 1512.5,
1
1462.9, 1384.0, 1216.8, 1187.5, 1155.7, 1088.6 cm-1; H NMR (400 MHz, DMSO-d6): δ 7.51 (m, 2H, H-4’, H-7’), 7.14
(m, 2H, H-5’, H-6’), 5.32 (t, 1H, H-12), 4.45 (t, 2H, H-13’), 4.37 (m, 1H, H-3), 3.61 (s, 3H, -COOCH3), 3.06 (m, 2H,
H-14’), 2.83 (m, 2H, H-10’), 2.77 (m, 2H, H-11’), 2.37 (m, 1H), 1.34 (s, 3H), 1.32 (s, 3H), 1.22 (s, 3H), 1.17(s, 2H),
1.09 (s, 1H), 1.06 (s, 2H), 1.00 (s, 6H), 0.73 (m, 1H), 0.69 (s, 3H), 0.65 (s, 3H), 0.63 (s, 1H); 13C NMR (DMSO-d6): δ
177.0 (C-30), 174.0 (C-12’), 170.9 (C-15’), 154.3 (C-2’), 144.6 (C-13), 142.6 (C-9’), 135.2 (C-8’), 122.3 (C-5’), 122.0

(C-6’), 121.6 (C-12), 118.7 (C-4’), 110.4 (C-7’), 80.9 (C-3), 54.7, 51.9, 48.2, 47.1, 44.1, 42.7, 41.5, 39.1, 38.3, 38.0,
37.6, 36.7, 34.2, 32.3, 32.0, 31.1, 30.9, 29.4, 29.1, 28.4, 28.3, 27.9, 26.7, 26.1, 26.0, 23.3, 22.5, 22.2, 18.1, 16.9, 15.5;
LC-MS: 715.5 [M+H]+, 713.2 [M-H]-; HR-MS: m/z, calcd for C44H63N2O6 [M+H]+ 715.4686, found: 715.4639.
4.1.1.8. 3-(1-(3-((methyl 12-oxo-18β-olean-30-oate)-3β-oxy)-3-oxopropyl)-1H-benzo[d]imidazol-2-yl)propanoic
acid (10h)
Yield: 93.3%; White solid; Mp: 136-137 ºC; IR (KBr): 3421.7, 2946.1, 2875.1, 1734.0, 1719.4, 1656.8, 1257.4,
1
1232.8, 1166.2, 1019.1 cm-1; H NMR (400 MHz, DMSO-d6): δ 7.53 (m, 2H, H-4’, H-7’), 7.16 (m, 2H, H-5’, H-6’),
4.47 (t, 2H, H-13’), 4.37 (m, 1H, H-3), 3.59 (s, 3H, -COOCH3), 3.17 (s, 1H), 3.10 (m, 2H, H-14’), 2.86 (m, 2H, H-10’),
2.80 (m, 2H, H-11’), 2.73 (m, 1H), 2.25 (m, 1H), 2.02 (m, 1H), 1.89 (m, 1H), 1.78 (m, 1H), 1.72 (m, 1H), 1.62 (m, 2H),
1.50 (m, 2H), 1.41 (m, 4H), 1.33 (m, 2H), 1.24 (m, 5H), 1.10 (s, 3H), 1.07 (s, 3H), 0.93 (m, 2H), 0.87 (s, 3H), 0.81 (s,
3H), 0.80 (s, 3H), 0.71 (s, 3H), 0.67 (s, 3H); 13C NMR (DMSO-d6): δ 211.2 (C-12), 177.0 (C-30), 174.1 (C-12’), 170.9
(C-15’), 154.3 (C-2’), 142.6 (C-8’), 135.1 (C-9’), 122.0 (C-6’), 121.6 (C-5’), 118.7 (C-4’), 110.4 (C-7’), 80.7 (C-3),
54.3, 51.6, 49.6, 49.2, 43.9, 42.1, 41.5, 40.4, 39.1, 38.7, 38.5, 37.6, 37.3, 36.6, 34.1, 33.8, 32.1, 31.6, 31.2, 31.0, 28.7,
27.4, 27.2, 26.3, 25.9, 23.3, 22.2, 20.8, 18.1, 16.6, 16.0, 15.3; LC-MS: 731.5 [M+H]+, 729.3 [M-H]-; HR-MS: m/z, calcd
for C44H63N2O7 [M+H]+ 731.4635, found: 731.4634.
4.1.1.9. 3-(1-(3-((methyl 12-oxo-18β-olean-9(11)-en-30-oate)-3β-oxy)-3-oxopropyl)-1H-benzo[d]imidazol-2-yl)
propanoic acid (10i)
Yield: 94.0%; White solid; Mp: 110-111 ºC; IR (KBr): 3429.0, 2927.3, 2852.1, 1730.4, 1661.3, 1513.2, 1463.6,
1
1383.0, 1220.9, 1192.1, 1088.1 cm-1; H NMR (400 MHz, DMSO-d6): δ 7.53 (m, 2H, H-4’, H-7’), 7.15 (m, 2H, H-5’,
H-6’), 5.60 (s, 1H, H-11), 4.47 (t, 2H, H-13’), 4.38 (m, 1H, H-3), 3.61 (s, 3H, -COOCH3), 3.10 (t, 2H, H-14’), 2.88 (m,
2H, H-10’), 2.81 (m, 2H, H-11’), 2.09 (m, 1H), 1.88 (m, 2H), 1.79 (m, 2H), 1.58 (m, 4H), 1.39 (s, 9H), 1.33 (s, 3H),
1.23 (m, 3H), 1.13 (s, 3H), 1.06 (s, 3H), 0.92 (s, 3H), 0.86 (s, 3H), 0.75 (s, 3H), 0.71 (s, 3H); 13C NMR (DMSO-d6): δ
200.0 (C-12), 177.9 (C-30), 176.9 (C-12’), 174.0 (C-15’, C-9), 170.9 (C-2’), 154.3 (C-9’), 135.0 (C-8’), 122.5 (C-11),

122.1 (C-5’), 121.8 (C-6’), 118.6 (C-4’), 110.5 (C-7’), 80.0 (C-3), 51.7, 49.7, 47.6, 45.4, 43.9, 41.8, 40.4, 39.2, 38.5,
38.1, 37.9, 35.9, 34.1, 33.5, 32.6, 32.1, 31.2, 30.9, 28.7, 27.8, 27.4, 26.1, 26.0, 24.1, 24.0, 23.7, 22.1, 21.9, 17.7, 16.8;
LC-MS: 729.5 [M+H]+, 727.2 [M-H]-; HR-MS: m/z, calcd for C44H61N2O7 [M+H]+ 729.4479, found: 729.4468.
4.1.1.10. 3-(1-(3-((methyl 18β-olean-9(11),12-dien-30-oate)-3β-oxy)-3-oxopropyl)-1H-benzo[d]imidazol-2-yl)
propanoic acid (10j)
Yield: 92.1%; White solid; Mp: 154-155 ºC; IR (KBr): 3427.4, 2925.7, 2853.7, 1728.2, 1631.7, 1462.5, 1382.6,
1
1216.6, 1187.1, 1155.6 cm-1; H NMR (400 MHz, DMSO-d6): δ 7.53 (m, 2H, H-4’, H-7’), 7.16 (m, 2H, H-5’, H-6’),
5.59 (d, 1H, H-11), 5.51 (d, 1H, H-12), 4.47 (t, 2H, H-13’), 4.38 (m, 1H, H-3), 3.63 (s, 3H, -COOCH3), 3.11 (t, 2H,
H-14’), 2.87 (m, 2H, H-10’), 2.82 (m, 2H, H-11’), 1.12 (m, 2H), 1.13 (s, 3H), 1.08 (s, 3H), 1.07 (s, 3H), 1.04 (s, 1H),
0.99 (s, 1H), 0.96 (s, 3H), 0.82 (s, 1H), 0.78 (s, 3H), 0.73 (s, 3H), 0.70 (s, 3H), 0.67 (s, 1H), 0.65 (s, 1H); 13C NMR
(DMSO-d6): δ 176.9 (C-30), 174.0 (C-12’), 170.9 (C-15’), 154.4 (C-9), 154.3 (C-2’), 146.4 (C-13), 142.5 (C-9’), 135.1
(C-8’), 122.0 (C-5’), 121.7 (C-6’), 121.1 (C-4’), 118.7 (C-12), 115.7 (C-11), 110.4 (C-7’), 80.6 (C-3), 51.9, 51.0, 46.4,
44.0, 42.6, 39.1, 38.6, 38.2, 37.8, 36.8, 36.4, 34.6, 34.2, 31.6, 31.2, 30.8, 28.6, 28.1, 27.0, 25.5, 25.3, 23.9, 22.1, 20.9,
20.3, 19.9, 18.0, 16.9; LC-MS: 713.5 [M+H]+, 711.2 [M-H]-; HR-MS: m/z, calcd for C44H61N2O6 [M+H]+ 713.4530,
found: 713.4531.
4.1.2. General procedure for the synthesis of 12a–12p
To a solution of 9 (3 mmol) in dry acetonitrile (30 mL) was added Cs2CO3 (4.5 mmol). The mixture was stirred at
room temperature for 1 h, then 1,2-dibromoethane or 1,3-dibromopropane (9 mmol) was added slowly. The solution
o
was warmed up to 82 C and stirred for additional 4 h. The solvent was evaporated to dryness and added water,
extracted with ethyl acetate three times. The organic layers were combined, washed with brine, dried with Na2SO4,
filtered and evaporated. The resulting residue was then purified by column chromatography on silica gel to give 11a and
11b as colorless transparent oily liquid with 40~55% yield.
To a solution of 9 (3 mmol) in dry DMF (20 mL) was added sodium hydride (60% suspension in mineral oil, 6

mmol) in batches under ice-salt bath. The reaction mixture was allowed to warm up to room temperature and stirred for
0.5 h. Then added sodium hydride (6 mmol) in batches again followed by 2-chloroethylamine hydrochloride or
3-bromopropanamin hydrobromide (4.5 mmol) and the mixture was stirred at 80 oC for 1 h. Cooled to room
temperature, and poured into water, extracted with dichloromethane five times. The organic layers were combined,
washed with brine, dried with Na2SO4, filtered and evaporated. The resulting residue was then purified by column
chromatography on silica gel to give 11c and 11d as colorless transparent oily liquid with 30~48% yield.
To a solution of GA/5f/5g/2/5h/5i (0.5 mmol) in dry DMF (20 mL) was added K2CO3 (1 mmol). The mixture was
stirred at room temperature for 1 h, then 11a or 11b (0.5 mmol) was added and stirred for 4 h. The reaction solution was
poured into water (50 mL), and filtered. The precipitate was washed with water to PH 7 and dried to give corresponding
intermediates as white solid with 70~89% yield. The corresponding intermediates were debenzylated at the same
reaction condition as making 10a–10j to give 12a-12l as white solid with 90~95% yield.
To a solution of 5f/5g/5h/5i (0.5 mmol) in dry chloroform (25 mL) was added oxalyl chloride (2 mL). The mixture
was stirred at room temperature for 2 h, and evaporated to dryness. The crude product as white solid was directly used
in the next step without further purification. The corresponding acid chloride was dissolved in dry chloroform (20 mL),
then the solution of 11c or 11d in dry chloroform was added dropwise under ice-salt bath. The mixture was warmed up
to room temperature and stirred overnight. The solvent was evaporated to dryness, and the resulting residue was then
purified by column chromatography on silica gel to give corresponding intermediates as white solid with 50~58% yield.
The corresponding intermediates were debenzylated at the same reaction condition as making 10a–10j to yield
12m–12p as white solid with 90~95% yield.
4.1.2.1. 3-(1-(3-(3β-hydroxy-11-oxo-18β-olean-12-en-30-methanoyl)propyl)-1H-benzo[d]imidazol-2-yl)propanoic
acid (12a)
Yield: 91.6%; White solid; Mp: 100-101 ºC; IR (KBr): 3403.3, 2925.9, 2870.9, 1730.0, 1656.9, 1522.0, 1464.5,
1384.9, 1246.1, 1030.3 cm-1; 1H NMR (400 MHz, DMSO-d6): δ 12.20 (s, 1H, -COOH), 7.55 (d, 1H, H-4’), 7.48 (d, 1H,

H-7’), 7.16 (m, 2H, H-5’, H-6’), 5.43 (s, 1H, H-12), 4.30 (t, 2H, H-13’), 4.12 (t, 2H, H-15’), 3.76 (m, 1H, -OH), 3.59 (m,
1H, H-3), 3.06 (t, 2H, H-10’), 2.84 (t, 2H, H-11’), 2.58 (m, 1H), 2.34 (s, 1H), 2.06 (m, 2H), 1.99 (m, 2H), 1.53 (m, 2H),
1.36 (s, 3H), 1.24 (s, 2H), 1.10 (s, 3H), 1.03 (s, 6H), 0.91 (s, 3H), 0.75 (s, 3H), 0.69 (m, 3H); LC-MS: 701.7 [M+H]+,
699.4 [M-H]-; HR-MS: m/z, calcd for C43H61N2O6 [M+H]+ 701.4530, found: 701.4517.
4.1.2.2. 3-(1-(2-(3β-hydroxy-11-oxo-18β-olean-12-en-30-methanoyl)ethyl)-1H-benzo[d]imidazol-2-yl)propanoic
acid (12b)
Yield: 93.6%; White solid; Mp: 143-144 ºC; IR (KBr): 3424.2, 2926.8, 1730.1, 1657.3, 1643.0, 1384.9, 1115.4
1
cm-1; H NMR (400 MHz, DMSO-d6): δ 12.21 (s, 1H, -COOH), 7.54 (m, 2H, H-4’, H-7’), 7.15 (m, 2H, H-5’, H-6’),
5.39 (s, 1H, H-12), 4.54 (m, 2H, H-13’), 4.42 (m, 1H, -OH), 4.30 (t, 2H, H-14’), 3.12 (t, 2H, H-10’), 3.02 (m, 1H, H-3),
2.84 (t, 2H, H-11’), 2.58 (m, 1H), 2.31 (s, 1H), 1.33 (s, 1H), 1.31(s, 3H), 1.24 (m, 1H), 1.12 (m, 1H), 1.03 (s, 3H), 1.01
(s, 3H), 0.91 (s, 3H), 0.85 (s, 3H), 0.69 (s, 3H), 0.62 (m, 3H); 13C NMR (DMSO-d6): δ 199.4 (C-11), 176.1 (C-30),
173.9 (C-12’), 169.5 (C-13), 154.4 (C-2’), 142.5 (C-8’), 135.6 (C-9’), 127.8 (C-12), 121.9 (C-5’), 121.6 (C-6’), 118.8
(C-4’), 110.4 (C-7’), 70.0 (C-3), 62.9, 61.5, 54.4, 48.0, 45.2, 43.8, 43.2, 41.8, 40.6, 40.4, 39.1, 38.9, 37.5, 37.0, 32.5,
31.8, 31.1, 30.4, 28.5, 28.4, 27.6, 27.3, 26.4, 26.0, 23.3, 22.3, 18.7, 17.5, 16.5, 16.4; LC-MS: 687.7 [M+H]+, 685.5
[M-H]-; HR-MS: m/z, calcd for C42H59N2O6 [M+H]+ 687.4373, found: 687.4388.
4.1.2.3. 3-(1-(3-(3β-acetyloxy-11-oxo-18β-olean-12-en-30-methanoyl)propyl)-1H-benzo[d]imidazol-2-yl)propanoic
acid (12c)
Yield: 93.1%; White solid; Mp: 125-126 ºC; IR (KBr): 3432.3, 2927.8, 2853.4, 1729.2, 1658.9, 1512.8, 1463.4,
1
1385.8, 1326.3, 1246.6, 1153.6, 1029.6 cm-1; H NMR (400 MHz, CDCl3): δ 7.75 (m, 1H, H-4’), 7.30 (m, 3H, H-5’,
H-6’, H-7’), 5.79 (s, 1H, H-12), 4.50 (m, 1H, H-3), 4.30 (t, 2H, H-13’), 4.18 (t, 2H, H-15’), 3.21 (m, 2H, H-10’), 3.15
(m, 2H, H-11’), 2.75 (m, 1H), 2.37 (s, 1H), 2.05 (s, 3H, CH3COO-), 1.64 (m, 5H), 1.35 (s, 3H), 1.16 (s, 3H), 1.14 (s,
3H), 1.11 (s, 3H), 0.87 (s, 6H), 0.79 (s, 3H); 13C NMR (DMSO-d6): δ 211.1 (C-11), 177.0 (C-30), 174.1 (C-12’), 170.8
(CH3COO-), 154.5 (C-2’), 142.6 (C-13), 135.1 (C-9’), 129.9 (C-8’), 121.9 (C-5’), 121.6 (C-6’), 118.7 (C-12), 110.4

(C-7’), 80.7 (C-3), 54.3, 51.6, 49.6, 49.3, 43.9, 42.1, 41.5, 40.4, 39.1, 38.7, 38.5, 37.6, 37.3, 36.6, 34.2, 33.8, 32.1, 31.6,
31.0, 28.7, 27.7, 27.2, 26.3, 25.9, 23.3, 22.3, 20.8, 18.1, 16.6, 16.0, 15.3; LC-MS: 743.7 [M+H]+, 741.7 [M-H]-; HR-MS:
m/z, calcd for C45H63N2O7 [M+H]+ 743.4635, found: 743.4618.
4.1.2.4. 3-(1-(2-(3β-acetyloxy-11-oxo-18β-olean-12-en-30-methanoyl)ethyl)-1H-benzo[d]imidazol-2-yl)propanoic
acid (12d)
Yield: 95.7%; White solid; Mp: 114-115 ºC; IR (KBr): 3428.4, 2926.8, 2855.8, 1730.5, 1513.2, 1645.1, 1463.6,
1
1385.4, 1246.9, 1147.8, 1030.0 cm-1; H NMR (400 MHz, DMSO-d6): δ 12.08 (s, 1H, -COOH), 7.52 (m, 2H, H-4’,
H-7’), 7.13 (m, 2H, H-5’, H-6’), 5.38 (s, 1H, H-12), 4.53 (m, 2H, H-13’), 4.41 (m, 2H, H-14’), 4.31 (m, 1H, H-3), 3.11
(t, 2H, H-10’), 2.82 (m, 2H, H-11’), 2.62 (m, 1H), 2.37 (s, 1H), 2.22 (m, 2H), 1.99 (s, 3H, CH3COO-), 1.62 (m, 6H),
1.31 (s, 3H), 1.22 (m, 2H), 1.11 (m, 2H), 1.05 (s, 3H), 1.00 (s, 3H), 0.84 (s, 3H), 0.81 (s, 6H), 0.60 (s, 3H); 13C NMR
(DMSO-d6): δ 199.3 (C-11), 176.1 (C-30), 174.9 (C-12’), 173.9 (CH3COO-), 170.6 (C-13), 154.5 (C-2’), 135.5 (C-9’),
127.6 (C-8’), 122.0 (C-12), 121.8 (C-6’), 118.6 (C-5’), 110.6 (C-4’), 100.0 (C-7’), 80.1 (C-3), 67.5, 65.4, 62.9, 61.1,
60.3, 54.0, 51.8, 48.0, 45.2, 43.8, 43.2, 40.4, 37.9, 36.8, 31.8, 31.1, 30.7, 29.8, 28.4, 28.2, 28.0, 27.6, 26.3, 26.0, 23.3,
22.3, 21.3, 18.6, 16.9, 16.5; LC-MS: 729.7 [M+H]+, 727.5 [M-H]-; HR-MS: m/z, calcd for C44H61N2O7 [M+H]+
729.4479, found: 729.4482.
4.1.2.5. 3-(1-(3-(3,11-dioxo-18β-olean-12-en-30-methanoyl)propyl)-1H-benzo[d]imidazol-2-yl)propanoic acid
(12e)
Yield: 90.4%; White solid; Mp: 109-110 ºC; IR (KBr): 3427.3, 2927.0, 2853.0, 1727.8, 1705.1, 1658.2, 1511.3,
1461.8, 1280.3, 1247.7, 1210.6, 1154.0, 1087.0, 1032.2 cm-1; 1H NMR (400 MHz, DMSO-d6): δ 12.19 (s, 1H, -COOH),
7.55 (d, 1H, H-4’), 7.49 (d, 1H, H-7’), 7.16 (m, 2H, H-5’, H-6’), 5.48 (s, 1H, H-12), 4.31 (m, 2H, H-13’), 4.13 (m, 2H,
H-15’), 3.07 (t, 2H, H-10’), 2.85 (t, 2H, H-11’), 2.70 (m, 1H), 2.42 (m, 1H), 1.48 (m, 4H), 1.38 (s, 3H), 1.24 (s, 2H),
1.15 (s, 3H), 1.11 (s, 3H), 1.08 (s, 3H), 1.02 (s, 3H), 0.97 (s, 3H), 0.77 (s, 3H); 13C NMR (DMSO-d6): δ 216.2 (C-3),
198.9 (C-11), 176.0 (C-30), 173.9 (C-12’), 170.0 (C-13), 154.2 (C-2’), 142.5 (C-8’), 135.5 (C-9’), 127.7 (C-12), 121.9

(C-5’), 121.6 (C-6’), 118.9 (C-4’), 109.9 (C-7’), 68.6, 61.9, 60.6, 54.2, 48.4, 47.3, 45.1, 43.9, 43.4, 40.8, 39.3, 37.8,
36.5, 34.1, 31.9, 31.7, 31.1, 30.6, 29.0, 28.6, 28.0, 27.8, 26.5, 26.1, 23.3, 22.1, 21.3, 18.7, 18.4, 15.9; LC-MS: 699.8
[M+H]+, 697.7 [M-H]-; HR-MS: m/z, calcd for C43H59N2O6 [M+H]+ 699.4373, found: 699.4376.
4.1.2.6. 3-(1-(2-(3,11-dioxo-18β-olean-12-en-30-methanoyl)ethyl)-1H-benzo[d]imidazol-2-yl)propanoic acid (12f)
Yield: 91.1%; White solid; Mp: 114-115 ºC; IR (KBr): 3426.3, 2926.7, 2855.3, 1729.4, 1704.9, 1657.6, 1462.3,
1
1384.7, 1210.1, 1150.7, 1086.6 cm-1; H NMR (400 MHz, DMSO-d6): δ 12.22 (s, 1H, -COOH), 7.54 (m, 2H, H-4’,
H-7’), 7.15 (m, 2H, H-5’, H-6’), 5.44 (s, 1H, H-12), 4.54 (m, 2H, H-13’), 4.38 (m, 2H, H-14’), 3.13 (t, 2H, H-10’), 2.84
(t, 2H, H-11’), 2.70 (m, 1H), 2.48 (s, 1H), 2.31 (m, 1H), 1.62 (s, 2H), 1.59 (s, 1H), 1.47 (m, 4H), 1.33 (s, 3H), 1.15 (s,
3H), 1.05 (s, 3H), 1.01 (s, 3H), 0.97 (s, 3H), 0.91 (m, 1H), 0.86 (s, 3H), 0.64 (s, 3H); LC-MS: 685.4 [M+H]+, 683.5
[M-H]-; HR-MS: m/z, calcd for C42H57N2O6 [M+H]+ 685.4217, found: 685.4223.
4.1.2.7. 3-(1-(3-(3β-hydroxy-18β-olean-12-en-30-methanoyl)propyl)-1H-benzo[d]imidazol-2-yl)propanoic acid
(12g)
Yield: 91.2%; White solid; Mp: 119-120 ºC; IR (KBr): 3435.3, 2926.9, 1728.9, 1615.7, 1512.2, 1463.3, 1383.4,
1312.1, 1212.4, 1152.0, 1030.1 cm-1; 1H NMR (400 MHz, DMSO-d6): δ 12.19 (s, 1H, -COOH), 7.55 (d, 1H, H-4’), 7.48
(d, 1H, H-7’), 7.16 (m, 2H, H-5’, H-6’), 5.19 (t, 1H, H-12), 4.30 (m, 3H, H-13’, -OH), 4.11 (m, 2H, H-15’), 3.07 (t, 2H,
H-10’), 3.01 (m, 1H, H-3), 2.85 (t, 2H, H-11’), 2.05 (m, 2H), 1.23 (s, 2H), 1.13 (s, 3H), 1.08 (s, 3H), 0.91 (s, 6H), 0.87
(s, 3H), 0.74 (s, 3H), 0.68 (s, 3H); 13C NMR (DMSO-d6): δ 176.4 (C-30), 173.9 (C-12’), 154.2 (C-2’), 144.6 (C-13),
142.5 (C-9’), 135.4 (C-8’), 122.4 (C-12), 121.9 (C-5’), 121.6 (C-6’), 118.9 (C-4’), 109.9 (C-7’), 77.2 (C-3), 61.7, 55.1,
48.3, 47.4, 44.1, 42.6, 41.5, 40.4, 39.5, 38.8, 38.6, 38.3, 36.8, 32.6, 32.0, 31.0, 30.9, 29.0, 28.6, 28.4, 28.3, 27.4, 26.7,
26.1, 26.0, 23.4, 22.1, 18.3, 16.9, 16.4, 15.6; LC-MS: 687.8 [M+H]+, 685.6 [M-H]-; HR-MS: m/z, calcd for C43H63N2O5
[M+H]+ 687.4737, found: 687.4728.
4.1.2.8. 3-(1-(2-(3β-hydroxy-18β-olean-12-en-30-methanoyl)ethyl)-1H-benzo[d]imidazol-2-yl)propanoic acid (12h)
Yield: 94.9%; White solid; Mp: 120-121 ºC; IR (KBr): 3428.2, 2926.6, 1731.4, 1664.0, 1383.5, 1331.1, 1311.4,

1
1211.1, 1149.5, 1085.5, 1030.6 cm-1; H NMR (400 MHz, DMSO-d6): δ 12.21 (s, 1H, -COOH), 7.54 (m, 2H, H-4’,
H-7’), 7.15 (m, 2H, H-5’, H-6’), 4.89 (t, 1H, H-12), 4.53 (t, 2H, H-13’), 4.40 (m, 1H, -OH), 4.29 (m, 2H, H-14’), 3.13 (t,
2H, H-10’), 3.01 (m, 1H, H-3), 2.84 (t, 2H, H-11’), 1.40 (s, 1H), 1.24 (s, 2H), 1.05 (s, 3H), 0.90 (s, 3H), 0.88 (s, 3H),
0.87 (s, 3H), 0.86 (s, 3H), 0.68 (s, 3H), 0.59 (s, 3H); 13C NMR (DMSO-d6): δ 176.4 (C-30), 174.0 (C-12’), 154.4 (C-2’),
144.2 (C-13), 142.5 (C-9’), 135.6 (C-8’), 122.4 (C-12), 121.9 (C-5’), 121.6 (C-6’), 118.7 (C-4’), 110.4 (C-7’), 77.2
(C-3), 62.9, 55.1, 48.0, 47.4, 43.9, 42.4, 41.9, 41.3, 38.8, 38.6, 38.1, 36.8, 32.5, 31.8, 31.1, 30.7, 28.6, 28.2, 28.0, 27.4,
26.7, 26.6, 26.0, 25.9, 23.3, 22.3, 18.4, 16.9, 16.4, 15.6; LC-MS: 673.8 [M+H]+, 671.6 [M-H]-; HR-MS: m/z, calcdfor
C42H61N2O5 [M+H]+ 673.4580, found: 673.4578.
4.1.2.9. 3-(1-(3-(3β-acetyloxy-18β-olean-12-en-30-methanoyl)propyl)-1H-benzo[d]imidazol-2-yl)propanoic acid
(12i)
Yield: 90.4%; White solid; Mp: 212-213 ºC; IR (KBr): 3418.6, 2949.0, 2924.0, 1729.9, 1618.6, 1465.0, 1383.0,
1248.7, 1252.2, 1085.4, 1026.8 cm-1; 1H NMR (600 MHz, DMSO-d6): δ 12.10 (s, 1H, -COOH), 7.55 (d, 1H, H-4’), 7.48
(d, 1H, H-7’), 7.16 (m, 2H, H-5’, H-6’), 5.18 (t, 1H, H-12), 4.41 (m, 1H, H-3), 4.29 (m, 2H, H-13’), 4.09 (m, 2H,
H-15’), 3.07 (t, 2H, H-10’), 2.85 (m, 2H, H-11’), 2.05 (m, 2H), 2.00 (s, 3H, CH3COO-), 1.14 (s, 3H), 1.08 (s, 3H), 0.90
(s, 6H), 0.83 (s, 3H), 0.82 (s, 3H), 0.73 (s, 3H); LC-MS: 729.6 [M+H]+, 727.3 [M-H]-; HR-MS: m/z, calcd for
C45H65N2O6 [M+H]+ 729.4843, found: 729.4829.
4.1.2.10. 3-(1-(2-(3β-acetyloxy-18β-olean-12-en-30-methanoyl)ethyl)-1H-benzo[d]imidazol-2-yl)propanoic acid
(12j)
Yield: 93.7%; White solid; Mp: 168-169 ºC; IR (KBr): 3427.9, 2948.7, 1731.2, 1629.4, 1517.1, 1484.5, 1465.2,
1382.8, 1313.5, 1248.8, 1210.1, 1027.2 cm-1; 1H NMR (600 MHz, DMSO-d6): δ 7.95 (m, 1H, H-4’), 7.75 (m, 1H, H-7’),
7.51 (m, 2H, H-5’, H-6’), 4.84 (m, 2H, H-13’), 4.81 (t, 1H, H-12), 4.50 (m, 1H, H-3), 4.37 (m, 2H, H-14’), 3.41 (t, 2H,
H-10’), 3.30 (t, 2H, H-11’), 2.00 (s, 3H,CH3COO-), 1.04 (s, 3H), 0.90 (s, 3H), 0.89 (s, 3H), 0.85 (s, 3H), 0.82 (s, 6H),
0.58 (s, 3H); 13C NMR (DMSO-d6): δ 176.6 (C-30), 172.9 (C-12’), 171.0 (CH3COO-), 152.8 (C-2’), 144.0 (C-13),

131.5 (C-9’), 130.5 (C-8’), 126.5 (C-5’), 126.0 (C-6’), 122.6 (C-4’), 115.2 (C-12), 111.9 (C-7’), 80.9 (C-3), 61.4, 55.2,
48.0, 47.4, 44.1, 43.9, 42.5, 41.4, 39.7, 38.7, 38.2, 37.6, 36.7, 32.5, 31.8, 31.7, 31.0, 28.2, 28.0, 27.9, 26.7, 25.8, 23.5,
23.4, 21.2, 20.9, 18.1, 16.6, 16.5, 15.5, 15.4; LC-MS: 715.7 [M+H]+, 713.6 [M-H]-; HR-MS: m/z, calcd for C44H63N2O6
[M+H]+ 715.4686, found: 715.4687.
4.1.2.11. 3-(1-(3-(3-oxo-18β-olean-12-en-30-methanoyl)propyl)-1H-benzo[d]imidazol-2-yl)propanoic acid (12k)
Yield: 94.8%; White solid; Mp: 93-94 ºC; IR (KBr): 3427.4, 2926.2, 2855.3, 1728.3, 1704.9, 1512.2, 1461.3,
1
1383.2, 1311.6, 1212.9, 1155.3, 1085.9 cm-1; H NMR (400 MHz, DMSO-d6): δ 12.21 (s, 1H, -COOH), 7.55 (d, 1H,
H-4’), 7.49 (d, 1H, H-7’), 7.17 (m, 2H, H-5’, H-6’), 5.21 (t, 1H, H-12), 4.30 (t, 2H, H-13’), 4.11 (m, 2H, H-15’), 3.08 (t,
2H, H-10’), 2.86 (t, 2H, H-11’), 2.30 (m, 1H), 2.06 (m, 2H), 1.24 (s, 2H), 1.17 (s, 2H), 1.15 (s, 3H), 1.09 (s, 3H), 1.01 (s,
3H), 0.99 (s, 3H), 0.97 (s, 3H), 0.96 (s, 3H), 0.75 (s, 3H); 13C NMR (DMSO-d6): δ 216.5 (C-3), 176.4 (C-30), 173.9
(C-12’), 154.2 (C-2’), 144.6 (C-13), 142.5 (C-9’), 135.4 (C-8’), 122.3 (C-12), 122.0 (C-5’), 121.6 (C-6’), 118.9 (C-4’),
110.0 (C-7’), 61.7, 54.7, 48.4, 47.0, 46.5, 44.1, 42.6, 41.6, 40.4, 38.9, 38.3, 36.5, 34.1, 32.1, 32.0, 31.0, 30.9, 29.0, 28.4,
28.3, 26.7, 26.6, 26.1, 25.9, 23.5, 22.1, 21.5, 19.5, 19.0, 16.7, 15.2; LC-MS: 685.8 [M+H]+, 683.6 [M-H]-; HR-MS: m/z,
calcd for C43H61N2O5 [M+H]+ 685.4580, found: 685.4545.
4.1.2.12. 3-(1-(2-(3-oxo-18β-olean-12-en-30-methanoyl)ethyl)-1H-benzo[d]imidazol-2-yl)propanoic acid (12l)
Yield: 91.9%; White solid; Mp: 97-98 ºC; IR (KBr): 3428.8, 2925.9, 2854.6, 1730.4, 1704.1, 1616.1, 1514.2,
1
1462.7, 1383.4, 1210.8, 1085.6, 1009.8 cm-1; H NMR (400 MHz, DMSO-d6): δ 12.21 (s, 1H, -COOH), 7.55 (m, 1H,
H-4’, H-7’), 7.16 (m, 2H, H-5’, H-6’), 4.91 (t, 1H, H-12), 4.54 (t, 2H, H-13’), 4.31 (m, 2H, H-14’), 3.14 (t, 2H, H-10’),
2.84 (t, 2H, H-11’), 2.32 (m, 1H), 1.84 (m, 4H), 1.24 (s, 2H), 1.07 (s, 3H), 1.01 (s, 3H), 0.99 (s, 3H), 0.96 (s, 3H), 0.91
(s, 3H), 0.88 (s, 3H), 0.60 (s, 3H); LC-MS: 671.7 [M+H]+, 669.5 [M-H]-; HR-MS: m/z, calcd for C42H59N2O5 [M+H]+
671.4424, found: 671.4435.
4.1.2.13. 3-(1-(3-(3β-acetyloxy-11-oxo-18β-olean-12-en-30-carboxamido)propyl)-1H-benzo[d]imidazol-2-yl)
propanoic acid (12m)

Yield: 95.3%; White solid; Mp: 140-141 ºC; IR (KBr): 3427.8, 2869.5, 1728.4, 1653.1, 1461.7, 1385.2, 1330.7,
1
1281.5, 1258.4, 1211.6, 1169.5, 1085.5, 1038.0 cm-1; H NMR (400 MHz, DMSO-d6): δ 7.67 (t, 1H, -CONH-), 7.52 (d,
1H, H-4’), 7.47 (d, 1H, H-7’), 7.14 (m, 2H, H-5’, H-6’), 5.51 (s, 1H, H-12), 4.41 (m, 1H, H-3), 4.18 (m, 2H, H-13’),
3.18 (m, 2H, H-15’), 3.08 (m, 2H, H-10’), 2.91 (m, 2H, H-11’), 2.63 (m, 1H), 2.38 (s, 1H), 1.99 (s, CH3COO-), 1.87 (m,
4H), 1.74 (m, 1H), 1.64 (m, 4H), 1.38 (s, 3H), 1.36 (s, 3H), 1.16 (s, 2H), 1.14 (s, 2H),1.12 (s, 2H), 1.05 (s, 3H), 1.03 (s,
3H), 1.02 (s, 3H), 0.81 (s, 3H), 0.71 (s, 2H); 13C NMR (DMSO-d6): δ 199.4 (C-11), 175.7 (C-30), 173.9 (C-12’,
CH3COO-), 170.5 (C-13), 170.2 (C-2’), 154.1 (C-9’), 135.3 (C-8’), 127.8 (C-12), 122.0 (C-5’), 121.7 (C-6’), 118.7
(C-4’), 110.1 (C-7’), 80.1 (C-3), 68.6, 61.3, 54.1, 48.2, 45.2, 43.3, 41.3, 41.0, 40.4, 38.2, 37.9, 37.7, 36.8, 36.7, 32.3,
31.8, 30.9, 30.0, 29.1, 28.0, 27.8, 26.4, 26.3, 23.6, 23.4, 22.1, 21.4, 18.7, 17.3, 16.9, 16.5; LC-MS: 742.6 [M+H]+, 740.4
[M-H]-; HR-MS: m/z, calcd for C45H64N3O6 [M+H]+ 742.4795, found: 742.4768.
4.1.2.14. 3-(1-(2-(3β-acetyloxy-11-oxo-18β-olean-12-en-30-carboxamido)ethyl)-1H-benzo[d]imidazol-2-yl)
propanoic acid (12n)
Yield: 92.4%; White solid; Mp: 164-165 ºC; IR (KBr): 3418.5, 2926.0, 2855.6, 1731.1, 1658.4, 1516.6, 1464.5,
1
1384.9, 1246.5, 1116.2, 1029.9 cm-1; H NMR (400 MHz, DMSO-d6): δ 12.22 (s, 1H, -COOH), 7.77 (t, 1H, -CONH-),
7.50 (m, 2H, H-4’, H-7’), 7.14 (m, 2H, H-5’, H-6’), 5.47 (s, 1H, H-12), 4.41 (m, 1H, H-3), 4.21 (t, 2H, H-13’), 3.44 (m,
2H, H-14’), 3.08 (m, 2H, H-10’), 2.84 (m, 2H, H-11’), 2.63 (m, 1H), 2.37 (s, 1H), 1.99 (s, CH3COO-), 1.71 (m, 1H),
1.57 (m, 1H), 1.48 (m, 2H), 1.38 (s, 3H), 1.34 (s, 1H), 1.33 (s, 3H), 1.22 (s, 2H), 1.08 (s, 2H), 1.06 (s, 3H), 1.02 (s, 3H),
0.89 (s, 3H), 0.81 (s, 6H), 0.66 (s, 3H); 13C NMR (DMSO-d6): δ 199.4 (C-11), 176.3 (C-30), 172.4 (C-12’), 170.6
(CH3COO-), 170.2 (C-13), 154.0 (C-2’), 135.5 (C-9’), 129.4 (C-8’), 127.8 (C-12), 122.3 (C-5’), 122.0 (C-6’), 118.4
(C-4’), 110.5 (C-7’), 80.1 (C-3), 61.2, 60.4, 54.1, 47.9, 45.2, 43.3, 41.0, 40.4, 38.6, 37.9, 36.8, 32.3, 31.7, 31.1, 30.7,
28.9, 28.6, 28.0, 26.4, 26.2, 23.6, 23.4, 22.1, 21.3, 18.6, 17.4, 17.3, 16.9, 16.5, 14.4; LC-MS: 728.8 [M+H]+, 726.7
[M-H]-; HR-MS: m/z, calcd for C44H62N3O6 [M+H]+ 728.4639, found: 728.4618.
4.1.2.15. 3-(1-(3-(3,11-dioxo-18β-olean-12-en-30-carboxamido)propyl)-1H-benzo[d]imidazol-2-yl)propanoic acid

(12o)
Yield: 92.7%; White solid; Mp: 136-137 ºC; IR (KBr): 3420.8, 2926.5, 2868.5, 1704.7, 1656.7, 1518.2, 1462.2,
1
1385.1, 1328.0, 1259.4, 1167.9, 1113.2, 1012.3 cm-1; H NMR (400 MHz, DMSO-d6): δ 12.27 (s, 1H, -COOH), 7.71 (t,
1H, -CONH-), 7.54 (d, 1H, H-4’), 7.48 (d, 1H, H-7’), 7.16 (m, 2H, H-5’, H-6’), 5.56 (s, 1H, H-12), 4.20 (t, 2H, H-13’),
3.19 (m, 2H, H-15’), 3.06 (m, 2H, H-10’), 2.84 (m, 2H, H-11’), 2.72 (m, 1H), 2.30 (s, 1H), 2.11 (m, 2H), 1.86 (m, 4H),
1.63 (m, 2H), 1.40 (m, 4H), 1.38 (s, 3H), 1.24 (s, 2H), 1.15 (s, 3H), 1.07 (s, 3H), 1.05 (s, 3H), 1.02 (s, 3H), 0.97 (s, 3H),
0.75 (s, 3H); LC-MS: 698.7 [M+H]+, 696.5 [M-H]-; HR-MS: m/z, calcd for C43H60N3O5 [M+H]+ 698.4533, found:
698.4531.
4.1.2.16. 3-(1-(2-(3,11-dioxo-18β-olean-12-en-30-carboxamido)ethyl)-1H-benzo[d]imidazol-2-yl)propanoic acid
(12p)
Yield: 93.3%; White solid; Mp: 146-147 ºC; IR (KBr): 3419.2, 2926.1, 2868.8, 1705.1, 1657.4, 1516.9, 1463.0,
1385.2, 1329.0, 1248.7, 1115.2 cm-1; 1H NMR (400 MHz, DMSO-d6): δ 7.97 (t, 1H, -CONH-), 7.48 (m, 2H, H-4’, H-7’),
7.11 (m, 2H, H-5’, H-6’), 5.52 (s, 1H, H-12), 4.23 (t, 2H, H-13’), 3.38 (m, 2H, H-14’), 2.99 (m, 2H, H-10’), 2.70 (m,
2H, H-11’), 2.28 (m, 2H), 1.33 (s, 3H), 1.13 (s, 3H), 1.04 (s, 3H), 1.00 (s, 3H), 0.95 (s, 3H), 0.91 (s, 3H), 0.82 (m, 2H),
0.67 (s, 3H); 13C NMR (DMSO-d6): δ 216.3 (C-3), 198.9 (C-11), 176.2 (C-30), 174.0 (C-12’), 170.5 (C-13), 154.3
(C-2’), 142.5 (C-9’), 135.8 (C-8’), 127.8 (C-12), 121.9 (C-5’), 121.5 (C-6’), 118.8 (C-4’), 110.2 (C-7’), 60.6, 54.2, 47.9,
47.4, 45.0, 43.4, 43.3, 42.4, 41.0, 40.4, 39.3, 38.6, 37.5, 36.5, 34.0, 31.7, 31.2, 30.7, 28.9, 28.7, 26.6, 26.5, 26.3, 23.4,
22.3, 21.3, 18.7, 18.4, 15.9; LC-MS: 684.7 [M+H]+, 682.5 [M-H]-; HR-MS: m/z, calcd for C42H58N3O5 [M+H]+
684.4376, found: 684.4381.
4.2. Biological assays
4.2.1. Anti-proliferative assays 21
The human prostatic cancer PC-3 cell lines were cultured in RPMI1640 with 10% (v/v) heat-inactivated fetal
bovine serum. The anti-proliferative activities of semi-synthetic compounds were evaluated by the methyl

thiazolyltetrazolium bromide (MTT) assay. Cells (2×104 cells/well) were incubated in a humidified atmosphere with 5%
CO2 at 37 °C for 24 h, then various concentrations of each compound mixed in 100 µL medium were added to each well.
After incubated for another 96 h, MTT solution (50 µL of 2 mg/mL) was added to each well and incubated for an
additional 4 h. The medium was removed by aspiration and the cells were dissolved in 200 µL DMSO. The absorbance
at 570 nm was measured in the 96-well plate reader. Growth inhibition was calculated and expressed as the ratio of the
cell number in the treated group to that of the untreated group. The GI50 values were calculated according to inhibition
ratios. All experiments were performed three times independently, and the results were reported presented as mean.
4.2.2. Pin1 PPIase assay 20
PPIase inhibitory activities were measured at 10 ºC by protease-coupled assay as reported before.
Suc-Ala-Glu-cis-Pro-Phe-4-nitroanilide (Bachem) in 0.47 mol/L LiCl/trifluoroethanol was used as the substrate. The
assay buffer (860 µL of 35 mM HEPES at pH 7.8), Pin1 (20 µL, Bought from Sino Biological Inc.) and target
compounds (10 µL of varying concentrations in DMSO) were pre-equilibrated in the cuvette at 10 °C for 30 min. Then,
150 µL of ice-cooled chymotrypsin (60 mg/mL in 0.001 M HCl) was added and mixed immediately. Additional
substrate (40 µL) was added to start the assay and the reaction was monitored by absorbance at 390 nm for 90 s. The
data was analyzed by Graphpad Prism 6.0.
4.2.3. Western blot analysis
The human prostatic cancer PC-3 cells were incubated in presence of 10a at 10, 20, 30 µM or DMSO for 24 h,
harvested, and rinsed with ice-cold PBS. Total protein extracts (40 µg) were prepared with RIPA lysis buffer [50
mMTris-HCl, 150 mMNaCl, 0.1% sodium dodecyl sulfate (SDS), 1% NP-40, 0.5% sodium deoxycholate, 1
mMphenylmethylsulfonyl fluoride (PMSF), 100 µMleupeptin, and 2 µg/mL aprotinin (pH 8.0)]. Samples were loaded
on 10% SDS PAGE and transferred to nitrocellulose membranes. The membranes were stained with 0.2% Ponceau S
red to assure equal protein loading and transfer. After blocking with 5% nonfat milk, the membranes were incubated
with a specific antibody of cyclin D1 and β-actin overnight at 4 °C. Then they were incubated with secondary
antibodies at 37 °C for 1 h. Finally, detection was performed with an improved ECL reagent.

4.3. Molecular modeling
We performed a molecule docking using glide protocol in maestro software package based on crystal structure of
Pin1 (PDB code 3KAI). The crystal structure was retrieved from the Protein Data Bank. All of the water molecules and
other subunits were removed and hydrogen atoms were added. The 3D structures of 10a and 12i were constructed using
the Sketch Molecule module in maestro molecular modeling software package and subjected to energy minimization.
After the structures of ligands and protein were prepared, molecular docking was performed with glide protocol.
Default search parameters were used. The docking mode was chosen on the basis of docking scores.

Acknowledge
This work was supported by the National Natural Science Foundation of China (Grant No. 81273360).