Efficient synthesis and anti-fungal activity of oleanolic acid oxime esters

Article abstract of DOI:10.3390/molecules18033615

In order to develop potential glucosamine-6-phosphate synthase inhibitors and anti-fungal agents, twenty five oleanolic acid oxime esters were synthesized in an efficient way. The structures of the new compounds were confirmed by MS, HRMS, ¹H-N

Full text of DOI:10.3390/molecules18033615

Molecules 2013, 18, 3615-3629; doi:10.3390/molecules18033615
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Efficient Synthesis and Anti-Fungal Activity of Oleanolic Acid
Oxime Esters
Hanqing Zhao, Minjie Zhou, Lifeng Duan, Wei Wang, Jianjun Zhang *, Daoquan Wang and
Xiaomei Liang
Department of Applied Chemistry, China Agricultural University, Beijing 100193, China
* Author to whom correspondence should be addressed; E-Mail: zhangjianjun@cau.edu.cn;
Tel.: +86-10-6273-2219.
Received: 4 February 2013; in revised form: 14 March 2013 / Accepted: 15 March 2013 /
Published: 21 March 2013
Abstract: In order to develop potential glucosamine-6-phosphate synthase inhibitors and
anti-fungal agents, twenty five oleanolic acid oxime esters were synthesized in an efficient
way. The structures of the new compounds were confirmed by MS, HRMS, 1H-NMR and
13C-NMR. Preliminary studies based on means of the Elson-Morgan method indicated that
many compounds exhibited some inhibitory activity of glucosamine-6-phosphate synthase
(GlmS), and the original fungicidal activities results showed that some of the compounds
exhibited good fungicidal activities towards Sclerotinia sclerotiorum (Lib.) de Bary,
Rhizoctonia solani Kuhn and Botrytis cinerea Pers at the concentration of 50 µg/mL. These
compounds would thus merit further study and development as antifungal agents.
Keywords: oleanolic acid; oxime ester; glucosamine-6-phosphate synthase; anti-fungal activity
1. Introduction
Glucosamine-6-phosphate synthase (GlmS) is the first enzyme of the hexosamine biosynthetic
pathway [1]. This enzyme catalyzes the reaction of D-fructose-6P (Fru6P) with glutamine to afford
D-glucosamine-6P (GlcN6P) and glutamate. As a checkpoint of UDP-GlcNAc synthesis, it plays a key
role in the biosynthesis of the bacterial peptidoglycan, the lipopolysaccharide of Gram-negative
bacteria, chitin, and mannoproteins of the fungal cell wall [2,3].
The molecular mechanism of the reaction catalyzed by glucosamine-6-phosphate synthase is complex
and involves amide bond cleavage followed by ammonia channeling and sugar isomerization [4]. It is an

Molecules 2013, 18
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irreversible reaction and the sole biosynthetic route to GlcN-6-P known to date [5,6]. Although the
enzyme is also present in mammalian systems, there are substantial differences in physiological
consequences of GlcN-6-P synthase inhibition between fungi and mammals, thus it constitutes a firm
molecular basis for the selective toxicity of specific enzyme inhibitors. Recently, this enzyme has been
proposed as a good and promising target for new antifungal agents [7]. Like the most powerful GlmS
inhibitors such as arabinose-5-phosphate oxime, 5-methylenephosphono-D-arabino hydroximino-
lactone, N3-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid (FMDP) and 2-amino-2-deoxy-
D-glucitol-6-phosphate (ADGP), these compounds exhibit very poor, if any, antifungal activity because
of the restriction due to the highly inefficient uptake of these compounds by an unidentified active
transport system and apparent inability to cross the membrane by free diffusion [8].
Triterpenes are widely distributed in Nature, and they have attracted much attention due to their broad
spectrum of biological activities. Oleanolic acid (OA, Figure 1) is one of the most important triterpenes,
which has been in active clinical use as an anti-hepatitis drug in China for over 20 years, and possesses
some attractive biological activities, including protection of the liver against toxic injury [9–11],
anti-inflammation [12], anti-HIV [13,14], anti-tumor [15,16], anti-hyperglycemia [17] and
anti-cancer [18], etc.
Figure 1. Structure of the entagenic acid (EA) and target compounds (A, B).
OH
OH
H
H
COOH
COOH
HO
HO
Entagenic Acid (EA)
Oleanolic Acid (OA)
COOR1
COOR1
O
R2
N
HO
O
N
1
A
B
R1 = H
R1 = H, R2 = Substituted phenyl
Bn, R2 = Substituted phenyl
2
Bn
R1 =
R1 =
In 2011, Shimoga et al. reported that entagenic acid (EA, Figure 1) showed a high antibacterial
activity against B. cereus and B. subtilis, with a minimal inhibitory concentration of 200 μg mL−1 and
possessed good glucosamine-6-phosphate synthase inhibition activity in molecular docking studies with
minimum docking energy −9.22 kJ mol−1, binding energy −9.28 kJ mol−1 and inhibition constant
1.57e−007. The inhibition constant of streptomycin was 3.86e−005 [19]. As there is a good structural
similarity between entagenic acid and oleanolic acid, which possess various important bioactivities [17,20],
we rationalized that OA derivatives will have potential GlmS inhibitory activity on the basis of analog
synthesis and sub-structure ligation [21]. In an ongoing project for the discovery of novel
environmentally friendly antifungal agents from OA derivatives [22], we incorporated the structure of

Molecules 2013, 18
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oxime ester, an activity group in the field of pesticides, into oleanolic acid. Twenty five new oleanolic
acid oxime esters compounds (A/B, Figure 1) were efficiently synthesized, their enzyme inhibitory
activities towards Candida albicans GlcN-6-P synthase and fungicidal activities against Sclerotinia
sclerotiorum (Lib.) de Bary, Rhizoctonia solani Kuhn, Botrytis cinerea Pers, Phytophthora parasitica
Dast, Rice blas and Fusarium wilt were evaluated. We report herein the preliminary results of the study.
2. Results and Discussion
2.1. Chemistry
As shown in Figure 1, we envisioned that the target compounds A and B could be synthesized from
the intermediates 1 [23] or 2 [24]. As shown in Scheme 1, we envisioned that the target compounds A
and B could be synthesized from the synthon 2, and the benzyl group was chosen as the carboxylic acid
protective group in order to study the importance of the COOH-group in the biological activity and
avoid difficulties in the final deprotection to obtain A.
Scheme 1. Synthetic routes to the compounds A and B.
a
b
c
COOBn
COOBn
2
HO
O
3
4
d
OA
RCOOH
b
COOH
c
d
B
A
1
O
5
Reagents and conditions: (a) BnBr, K2CO3, DMF, r.t., 4 h, 98% for 3; (b) Ac2O, PDC, CH2Cl2, reflux
3–4 h, 95% for 4, 93% for 5; (c) NH2OH·HCl, Py, 80 °C, 1 h, 96% for 1, 94% for 2; (d) DCC, CH2Cl2,
reflux 8–14 h, 84%–93% for A, 70%–90% for B.
Firstly, benzylation of OA with benzyl bromide and K2CO3 in DMF provided the benzyl oleanolic
acid 3 quantitatively; then oxidation of C-3-OH of 3 with pyridinium dichromate (PDC) in CH2Cl2,
followed by oximation with NH2OH·HCl according to the reported method [23] afforded intermediate 2 in
94% yield. Condensation of 2 with substituted carboxylic acids provided the desired benzyl oleanolic
acid 3-oxime esters B. Initially, we tried to synthesize the target compound A from B with Pd/C in
MeOH/CH2Cl2 at 25 °C in the presence of hydrogen. However, instead of getting the desired compound
A, the compound 1 was obtained as the main product, as confirmed by its 1H-NMR spectrum, showing
the characteristic signals identical to the published data [23]. Later on, compound 1 was prepared
according to the reported procedures [23], and the target compounds A were obtained directly from 1 in
high yields.

Molecules 2013, 18
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The structures of A/B were confirmed from their 1H-NMR, 13C-NMR spectra and HRMS, showing
the characteristic signals such as a multiplet at  5.08 ppm for CH2C6H5 of B, a single peak at about 
5.29 ppm for H-12 of A/B. The physical data of the target compounds are given in Table 1.
Table 1. Physical Data of Compounds A and B.
Compd.
A-01
A-02
A-03
A-04
A-05
A-06
A-07
A-08
A-09
A-10
B-01
B-02
B-03
B-04
B-05
B-06
B-07
B-08
B-09
B-10
B-11
B-12
B-13
B-14
B-15
R2
Formula
Status
m.p./°C
98–100
78–80
58–60
78–80
73–75
70–72
88–90
120–122
160–162
96–98
72–76
134–136
68–72
71–74
136–138
54–56
—
Yield (%)
93
4-Cl-C6H4-
2,4-Cl2-C6H4-
3-Cl-C6H4-
4-NO2-C6H4-
1-Naphthyl-CH2-
4-Cl-C6H4OCH2-
2-F-C6H4-
C37H50ClNO4 White foamy solid
C37H49Cl2NO4 White foamy solid
C37H50ClNO4 White foamy solid
90
91
C37H50N2O6
C42H55NO4
White foamy solid
White foamy solid
84
86
C38H52ClNO5 White foamy solid
C37H50FNO4 White foamy solid
C37H50BrNO4 White foamy solid
87
80
4-Br-C6H4-
3-Pyridyl-
90
C36H50N2O4
C35H49NO5
White foamy solid
White foamy solid
91
2-Furan-
86
4-Cl-C6H4-
2,4-Cl2-C6H4-
3-Cl-C6H4-
4-NO2-C6H4-
1-Naphthyl-CH2-
4-Cl-C6H4OCH2-
2-F-C6H4-
C44H56ClNO4 White foamy solid
C44H55Cl2NO4 White foamy solid
C44H56ClNO4 White foamy solid
80
83
79
C44H56N2O6
C49H61NO4
White foamy solid
White foamy solid
85
75
C45H58ClNO5 White foamy solid
C44H56FNO4 Viscous liquid
C44H56BrNO4 White foamy solid
72
75
4-Br-C6H4-
3-Pyridyl-
74–78
70–72
68–70
76–80
68–70
116–118
80–84
78–81
78
C43H56N2O4
C42H55NO5
C44H56N2O6
C44H55N3O8
White foamy solid
White foamy solid
White foamy solid
White foamy solid
81
2-Furan-
71
3- NO2-C6H4-
3,5-( NO2) 2C6H4-
2-Cl-C6H4-
2-Pyridyl-
84
90
C44H56ClNO4 White foamy solid
76
C43H56N2O4
C44H57NO4
White foamy solid
White foamy solid
79
C6H5-
70
2.2. Bioassay of Enzyme Inhibitory Activities [25–28]
Inhibitory activity of all the synthesized compounds towards Candida albicans GlcN-6-P synthase
was evaluated using the further optimized Elson-Morgan method [25–27,29]. The absorption value of
the solution was measured at 585 nm, and then the concentration was counted by the specification curve
which was determined thanks to the relation between the absorption value and the concentration of
glucosamine-6-phosphate. Finally the enzyme inhibition rate was calculated according to formula (1):
M 0  M
I 
100%
(1)
M 0
where I is the inhibition rate,
is the average concentration of glucosamine-6-phosphate in the blank
M 0
test, and
is the average concentration of glucosamine-6-phosphate in the presence of target
M
compounds. The inhibition rates were given in Table 2 at 0.35 mM.

Molecules 2013, 18
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Many compounds of A series and B series exhibited better enzyme inhibitory activities than OA, but
this fact is not as obvious as possible since our work reveals that some compounds B exhibited less
activity. Compounds A-02, A-03, B-06, B-12 and B-13 are more active against
glucosamine-6-phosphate synthase than the other compounds. On the whole, the enzyme inhibitory
activity of A series of compounds is superior to the B series, which may be associated with a better
structural similarity between EA and the target compounds.
Table 2. Enzyme inhibition rates of compounds A and B at 0.35 mM.
Compd No.
OA
Inhibition Rate (%)
Compd No.
B-03
Inhibition Rate (%)
22.4
29.4
37.2
40.8
19.2
30.8
29.1
22.9
24.8
21.0
20.5
8.7
19.8
16.2
20.2
34.2
28.2
12.7
19.3
13.2
16.4
33.0
33.1
12.5
13.8
A-01
A-02
A-03
A-04
A-05
A-06
A-07
A-08
A-09
A-10
B-01
B-04
B-05
B-06
B-07
B-08
B-09
B-10
B-11
B-12
B-13
B-14
B-02
12.2
B-15
2.3. Bioassay of Fungicidal Activities [28]
Fungicidal activities of the target compounds against Sclerotinia sclerotiorum (Lib.) de Bary,
Rhizoctonia solani Kuhn, Botrytis cinerea Pers, Phytophthora parasitica Dast, rice blast and fusarium
wilt were evaluated using the mycelium growth rate test [28]. The diameter of the mycelia was measured
and the inhibition rate was calculated according to formula (2):
2
2
D1  D0
I 
100%
(2)
2
D1
where I is the inhibition rate,
is the average diameter of mycelia in the blank test, and
is the
D0
D1
average diameter of mycelia in the presence of target compounds: The inhibition rates of compounds A
and B against the six fungi at 50 µg/mL are given in Table 3.
Compounds A–B exhibited more fungicidal activity against R. solani, rice blast and S. sclerotiorum
than the other fungi. The fungicidal activity of the B series is better than that of the A series.
Compounds A-02, A-03, A-05, B-03, B-06, B-07 and B-09 exhibited good fungicidal activity,
consistent with their enzyme inhibitory activities.

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Table 3. Inhibition rates of compounds A–B against six fungi.
Inhibition rate (%)
Compd. No.
(CAU2012)
Phytophthora
S. sclerotiorum
B. cinerea
R. solani
Rice blast
Fusarium wilt
parasitica Dast
1.8
A-01
A-02
28.2
61.2
26.5
53.5
28.6
29.0
49.7
27.8
24.8
30.3
71.1
71.1
72.6
68.2
68.9
67.4
73.3
67.8
74.4
68.2
64.5
67.5
66.7
67.1
71.0
92.8
99.3
20.5
7.8
38.0
36.2
42.7
32.1
73.0
33.5
45.2
18.6
16.6
5.5
31.2
29.5
24.2
49.1
21.0
21.0
33.1
24.2
25.1
27.9
39.3
36.9
78.5
74.3
51.9
55.2
52.5
36.2
68.0
36.0
63.3
53.6
60.0
54.9
85.5
89.5
73.7
20.1
11.7
14.9
3.2
42.1
3.1
8.4
A-03
7.8
A-04
49.3
14.2
4.0
32.1
25.8
20.7
12.4
13.4
1.4
15.8
6.5
A-05
A-06
7.5
A-07
67.6
2.0
6.1
A-08
7.5
A-09
3.2
6.1
A-10
2.3
24.9
68.8
49.4
71.1
40.5
66.4
42.9
59.5
59.9
69.1
57.7
45.7
41.4
46.2
62.7
39.0
98.2
64.7
1.0
5.1
B-01
9.4
79.2
63.5
93.6
41.4
84.1
73.7
86.1
44.1
63.6
54.0
56.6
69.5
51.7
77.6
33.8
98.5
100
28.5
30.1
36.7
31.3
28.5
25.9
26.9
31.3
29.2
28.7
21.4
28.2
24.6
30.8
28.7
96.2
96.1
7.5
B-02
12.0
23.5
25.9
17.4
18.1
17.7
21.7
25.3
23.5
35.6
28.2
52.2
56.5
38.2
94.8
68.9
13.5
B-03
B-04
B-05
B-06
B-07
B-08
B-09
B-10
B-11
B-12
B-13
B-14
B-15
Chlorothalonil
Sanmate
OA
25.3
3. Experimental
3.1. General Methods
Solvents were purified in the usual way. All reactions were monitored by TLC analysis performed on
silica gel HF with detection by charring with 30% (v/v) H2SO4 in CH3OH or by UV detection. Column
chromatography was conducted by elution of a column (8 × 100, 16 × 240, 18 × 300, 35 × 400 mm) of
silica gel (200–300 mesh) with EtOAc-PE (petroleum ether, b.p. 60–90 °C) as the eluent. NMR spectra
(300/75 MHz, , ppm) were recorded on a Varian XL-300 spectrometer with TMS as the internal
standard. Elemental analysis was performed on a Yanaco CHN Corder MF-3 automatic elemental
analyzer. Mass spectra were recorded with a VG PLATFORM mass spectrometer using the electrospray
ionization (ESI) mode.

Molecules 2013, 18
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3.2. Chemical Synthesis
Oleanolic acid 3-oxime ester (A-01). 4-Chlorobenzoic acid (0.66 g, 4.2 mmol) and
N,N′-dicyclohexylcarbodiimide (DCC, 1 g, 5 mmol) were successively added to a soln. of oleanate
3-oxime 1 (1.68 g, 3.5 mmol) which was prepared according to the reported method [12] in CH2Cl2
(50 mL), Then the reaction mixture was refluxed for 8–14 h at the end of which time TLC (4:1
petroleum ether/EtOAc) indicated that the reaction was complete. The reaction mixture was filtered,
the soln. was concentrated, and the residue was subjected to column chromatography (6:1 petroleum
1
ether/EtOAc) to give the desired product A-01 (1.98 g, 93%) as a white foamy solid. H-NMR
(CDCl3):  8.00–7.42 (m, 4H, Ar-H), 5.28 (br s, 1 H, H-12), 3.05–3.01 (m, 1H), 2.85–2.80 (m, 1H),
2.48-2.41 (m, 1H), 1.34, 1.19, 1.13, 1.06, 0.93, 0.90, 0.80 (s, 7 × 3H, CH3); 13C-NMR (CDCl3): 184.0
(COOH), 176.4 (COONC), 163.4 (COONC), 143.8 (C-13), 139.4, 130.9, 130.9, 128.8, 128.8, 128.1
(aromatic carbons), 122.2 (C-12), 55.8, 47.1, 46.6, 45.8, 41.7, 41.0, 39.3, 38.7, 37.1, 33.8, 33.0, 32.4,
32.3, 30.6, 29.7, 27.6, 27.2, 25.8, 23.5, 23.4, 23.2, 22.9, 19.9, 18.9, 17.0, 15.1 (7 × CH3); Anal. Calcd for
C37H50ClNO4: C, 73.06; H, 8.29; N, 2.30. found: C, 73.27; H, 8.05; N, 2.51; HRMS calcd for
C37H50ClNO4 (M+H)+: 608.35011, found: 608.34985.
Oleanolic acid 3-oxime ester (A-02). The reaction was run similarly to that used to synthesize A-01. A
1
white foamy solid A-02 was obtained in 90% yield. H-NMR (CDCl3):  8.00–7.42 (m, 4H, Ar-H),
5.28 (br s, 1 H, H-12), 3.05–3.01 (m, 1H), 2.85–2.80 (m, 1H), 2.48–2.41 (m, 1H), 1.34, 1.19, 1.13,
1.06, 0.93, 0.90, 0.80 (s, 7 × 3H, CH3); 13C-NMR (CDCl3): 184.3 (COOH), 176.5 (COONC), 163.1
(COONC), 143.7 (C-13), 138.2, 134.3, 132.4, 130.8, 128.4, 127.0 (aromatic carbons), 122.2 (C-12),
55.8, 47.1, 46.5, 45.8, 41.6, 40.9, 39.3, 38.7, 37.0, 33.7, 33.0, 32.4, 32.2, 30.6, 29.6, 27.6, 27.0, 25.8,
23.5, 23.4, 23.0, 22.8, 20.2, 18.9, 17.0, 15.1 (7 × CH3); Anal. Calcd for C37H49Cl2NO4: C, 69.15; H,
7.68; N, 2.18. found: C, 69.35; H, 7.47; N, 2.33; HRMS calcd for C37H50ClNO4 (M+H)+: 642.31114,
found: 642.31079.
Oleanolic acid 3-oxime ester (A-03). The reaction was run similarly to that used to synthesize A-01. A
1
white foamy solid A-03 was obtained in 91% yield. H-NMR (CDCl3):  8.01–7.37 (m, 3H, Ar-H),
5.28 (br s, 1 H, H-12), 3.06–3.01 (m, 1H), 2.86–2.80 (m, 1H), 2.47–2.45 (m, 1H), 1.34, 1.19, 1.13,
1.07, 0.93, 0.90, 0.80 (s, 7 × 3H, CH3); 13C-NMR (CDCl3): 184.3 (COOH), 176.4 (COONC), 163.0
(COONC), 143.7 (C-13), 134.5, 133.0, 131.4, 129.7, 129.4, 127.6 (aromatic carbons), 122.2 (C-12),
55.8, 47.1, 46.5, 45.8, 41.6, 40.9, 39.3, 38.6, 37.0, 33.7, 33.0, 32.4, 32.2, 30.6, 29.6, 27.6, 27.0, 25.8,
23.5, 23.4, 23.0, 22.8, 20.2, 18.9, 17.0, 15.1 (7 × CH3); Anal. Calcd for C37H50ClNO4: C, 73.06; H, 8.29;
N, 2.30. found: C, 73.21; H, 8.43; N, 2.49; HRMS calcd for C37H50ClNO4 (M+H)+: 608.35011, found:
608.34937.
Oleanolic acid 3-oxime ester (A-04). The reaction was run similarly to that used to synthesize A-01. A
1
white foamy solid A-04 was obtained in 84% yield. H-NMR (CDCl3):  8.33–8.20 (m, 4H, Ar-H),
5.33 (br s, 1 H, H-12), 3.07–3.01 (m, 1H), 2.88–2.82 (m, 1H), 2.49–2.43 (m, 1H), 1.36, 1.19, 1.15,
1.07, 0.94, 0.92, 0.83 (s, 7 × 3H, CH3); 13C-NMR (CDCl3): 177.0 (COOH), 172.8 (COONC), 162.3
(COONC), 150.5, 143.3 (C-13), 135.1, 130.5, 130.5, 123.6, 123.6 (aromatic carbons), 122.6 (C-12),
55.8, 48.3, 47.1, 45.6, 41.8, 41.7, 39.4, 38.7, 37.0, 33.6, 32.9, 32.3, 31.9, 30.6, 29.6, 27.4, 27.1, 25.7,

Molecules 2013, 18
3622
23.5, 23.2, 22.9, 22.6, 20.0, 18.9, 17.1, 15.1 (7 × CH3); Anal. Calcd for C37H50N2O6: C, 71.82; H, 8.14;
N, 4.53. found: C, 71.97; H, 8.01; N, 4.69; HRMS calcd for C37H50ClNO4 (M+H)+: 619.37416,
found: 619.37708.
Oleanolic acid 3-oxime ester (A-05). The reaction was run similarly to that used to synthesize A-01. A
white foamy solid A-05 was obtained in 86% yield. 1H-NMR (CDCl3):  8.05–7.41(m, 7H, Ar-H), 5.27
(br s, 1 H, H-12), 4.20–4.18 (m, 2H, CH2), 2.84–2.78 (m, 1H), 2.58–2.53 (m, 1H), 1.25, 1.19, 1.09,
1.04, 0.93, 0.90, 0.74 (s, 7 × 3H, CH3); 13C-NMR (CDCl3): 184.2 (COOH), 175.5 (COONC), 169.3
(COONC), 143.6 (C-13), 133.7, 132.0, 130.2, 128.6, 127.9, 126.2, 125.7, 125.6, 125.3, 123.8 (aromatic
carbons), 122.2 (C-12), 65.4, 55.7, 47.0, 46.5, 45.7, 41.6, 41.3, 40.9, 39.2, 38.2, 36.8, 33.7, 33.0, 32.3,
32.2, 30.6, 29.6, 27.5, 26.9, 25.8, 23.5, 23.3, 22.8, 19.2, 18.7, 16.9, 14.9 (7 × CH3); Anal. Calcd for
C42H55NO4: C, 79.08; H, 8.69; N, 2.20. found: C, 79.24; H, 8.37; N, 2.42; HRMS calcd for
C37H50ClNO4 (M+H)+: 638.42039, found: 638.42004.
Oleanolic acid 3-oxime ester (A-06). The reaction was run similarly to that used to synthesize A-01. A
1
white foamy solid A-06 was obtained in 87% yield. H-NMR (CDCl3):  7.26–6.81 (m, 4H, Ar-H),
5.28 (br s, 1 H, H-12), 4.82 (s, 1H), 4.72(s, 1H), 2.87–2.82 (m, 1H), 2.30–2.21 (m, 1H), 1.26, 1.24,
1.11, 1.01, 0.93, 0.91, 0.78 (s, 7 × 3H, CH3); 13C-NMR (CDCl3): 183.9 (COOH), 175.8 (COONC),
167.8 (COONC), 156.4, 153.1, 143.6 (C-13), 129.3, 126.6, 122.1 (C-12), 116.0, 115.8 (aromatic
carbons), 65.4, 55.7, 47.0, 46.4, 45.7, 41.6, 40.9, 39.2, 38.5, 36.9, 33.7, 32.9, 32.5, 32.3, 30.5, 29.6, 27.5,
27.0, 25.7, 23.4, 23.3, 22.9, 22.8, 19.4, 18.8, 16.9, 15.0 (7 × CH3); Anal. Calcd for C38H52ClNO5: C,
71.51; H, 8.21; N, 2.19. found: C, 71.35; H, 8.39; N, 2.35; HRMS calcd for C37H50ClNO4 (M+H)+:
638.36068, found: 638.35919.
Oleanolic acid 3-oxime ester (A-07). The reaction was run similarly to that used to synthesize A-01. A
white foamy solid A-07 was obtained in 80% yield. 1H-NMR (CDCl3):  8.05–7.11(m, 4H, Ar-H), 5.29
(br s, 1 H, H-12), 3.12–3.07 (m, 1H), 2.85–2.82 (m, 1H), 2.49–2.38 (m, 1H), 1.34, 1.19, 1.13, 1.06, 0.93,
0.90, 0.80 (s, 7 × 3H, CH3); 13C-NMR (CDCl3): 184.5 (COOH), 176.1 (COONC), 163.1 (COONC),
143.6 (C-13), 134.3, 132.2, 124.0, 122.2 (C-12), 118.0, 116.9, 116.6 (aromatic carbons), 55.7, 47.0,
46.5, 45.7, 41.4, 40.9, 39.2, 38.6, 36.9, 33.7, 32.9, 32.3, 32.2, 30.5, 29.6, 27.5, 27.1, 25.7, 23.4, 23.3,
23.0, 22.7, 19.9, 18.8, 16.9, 15.0 (7 × CH3); Anal. Calcd for C37H50FNO4: C, 75.09; H, 8.52; N, 2.37.
found: C, 75.29; H, 8.38; N, 2.16; HRMS calcd for C37H50ClNO4 (M+H)+: 592.37966, found:
592.37909.
Oleanolic acid 3-oxime ester (A-08). The reaction was run similarly to that used to synthesize A-01. A
1
white foamy solid A-08 was obtained in 90% yield. H-NMR (CDCl3):  7.95–7.58 (m, 4H, Ar-H),
5.29 (br s, 1 H, H-12), 3.05–3.00 (m, 1H), 2.86–2.81 (m, 1H), 2.44–2.42 (m, 1H), 1.34, 1.19, 1.13, 1.06,
0.90, 0.88, 0.80 (s, 7 × 3H, CH3); 13C-NMR (CDCl3): 184.3 (COOH), 176.4 (COONC), 163.5
(COONC), 143.8 (C-13), 131.9, 131.8, 131.0, 131.0, 128.6, 128.1 (aromatic carbons), 122.3 (C-12),
55.8, 47.2, 45.8, 41.7, 41.6, 41.0, 39.4, 38.7, 37.1, 33.8, 33.0, 32.4, 31.9, 30.7, 29.7, 27.6, 27.2, 25.9,
23.6, 23.5, 23.2, 22.7, 19.9, 19.0, 17.0, 15.1 (7 × CH3); Anal. Calcd for C37H50BrNO4: C, 68.09; H, 7.72;
N, 2.15. found: C, 68.39; H, 7.46; N, 2.35; HRMS calcd for C37H50ClNO4 (M+H)+: 652.29960, found:
652.30011.

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Oleanolic acid 3-oxime ester (A-09). The reaction was run similarly to that used to synthesize A-01. A
white foamy solid A-09 was obtained in 91% yield. 1H-NMR (CDCl3):  9.25–7.44 (m, 4H, Ar-H), 5.30
(br s, 1 H, H-12), 3.08–3.03 (m, 1H), 2.90–2.84 (m, 1H), 2.48–2.41 (m, 1H), 1.35, 1.21, 1.14, 1.07, 0.94,
0.91, 0.82 (s, 7 × 3H, CH3); 13C-NMR (CDCl3): 183.1 (COOH), 176.7 (COONC), 162.7 (COONC),
153.0, 150.1, 143.9 (C-13), 137.3, 125.9, 123.6 (aromatic carbons), 122.0 (C-12), 55.7, 47.1, 46.4, 45.8,
41.6, 41.0, 39.2, 38.6, 37.0, 33.6, 33.0, 32.4, 32.2, 30.6, 29.6, 27.6, 27.1, 25.8, 23.5, 23.4, 23.1, 22.9,
19.9, 18.9, 16.9, 15.1 (7 × CH3); Anal. Calcd for C36H50N2O4: C, 75.22; H, 8.77; N, 4.87. found: C,
75.07; H, 8.64; N, 4.63; HRMS calcd for C37H50ClNO4 (M+H)+: 575.38433, found: 575.38373.
Oleanolic acid 3-oxime ester (A-10). The reaction was run similarly to that used to synthesize A-01. A
white foamy solid A-10 was obtained in 86% yield. 1H-NMR (CDCl3):  7.63–6.47 (m, 3H, Ar-H), 5.28
(br s, 1 H, H-12), 3.07–3.02 (m, 1H), 2.87–2.81 (m, 1H), 2.48–2.42 (m, 1H), 1.33, 1.17, 1.13, 1.05, 0.93,
0.90, 0.80 (s, 7 × 3H, CH3); 13C-NMR (CDCl3): 183.8 (COOH), 176.2 (COONC), 156.6 (COONC),
146.4, 143.8, 143.7 (C-13), 122.1(C-12), 118.0, 111.7 (aromatic carbons), 55.7, 47.1, 46.5, 45.8, 41.5,
40.9, 39.3, 38.6, 37.0, 33.7, 33.0, 32.5, 32.3, 30.6, 30.6, 27.6, 27.1, 25.5, 23.5, 23.4, 23.1, 22.8, 19.7,
18.9, 17.0, 15.0 (7 × CH3); Anal. Calcd for C35H49NO5: C, 74.57; H, 8.76; N, 2.48. found: C, 74.42; H,
8.91; N, 2.25; HRMS calcd for C37H50ClNO4 (M+H)+: 564.36835, found: 564.36804.
Benzyl oleanolic acid 3-oxime ester (B-01). 4-Chlorobenzoic acid (0.66 g, 4.2 mmol) and DCC (1 g,
5 mmol) were successively added to a soln. of benzyl oleanate 3-oxime 2 (2.00 g, 3.5 mmol) which
was prepared according to the reported method [13] in CH2Cl2 (50 mL), Then the reaction mixture was
refluxed for 8–14 h at the end of which time TLC (6:1 petroleum ether-EtOAc) indicated that the
reaction was complete. The reaction mixture was filtered, the soln was concentrated, and the residue
was subjected to column chromatography (8:1 petroleum ether-EtOAc) to give the desired product
B-01 (1.98 g, 80%) as a white foamy solid. 1H-NMR (CDCl3):  8.01–7.31 (m, 9H, Ar-H), 5.29 (br s, 1
H, H-12), 5.08 (dd, 2H, J = 12.5, 17.4 Hz, Ar-CH2), 3.04–2.89 (m, 2H), 2.47–2.33 (m, 1H), 1.34, 1.19,
1.12, 1.03, 0.92, 0.89, 0.64 (s, 7 × 3H, CH3); 13C-NMR (CDCl3): 177.3 (COOBn), 176.4 (COONC),
163.3 (COONC), 143.8 (C-13), 139.4, 134.4, 130.8, 130.8, 128.8, 128.8, 128.4, 128.4, 128.4, 128.1,
127.9, 127.9 (aromatic carbons), 122.1 (C-12), 65.9, 55.7, 47.1, 46.7, 45.8, 41.7, 41.5, 41.4, 39.3, 38.7,
36.9, 33.8, 33.0, 32.3, 32.3, 30.6, 27.5, 27.2, 25.7, 23.6, 23.4, 23.2, 23.0, 19.8, 18.9, 16.8, 15.1 (7 ×
CH3); Anal. Calcd for C44H56ClNO4: C, 75.67; H, 8.08; N, 2.01. found: C, 75.52; H, 8.33; N, 2.17;
HRMS calcd for C37H50ClNO4 (M+H)+: 698.39706, found: 698.39526.
Benzyl oleanolic acid 3-oxime ester (B-02). The reaction was run similarly to that used to synthesize
1
B-01. A white foamy solid B-02 was obtained in 83% yield. H-NMR (CDCl3):  7.80–7.29 (m, 8H,
Ar-H), 5.28 (br s, 1 H, H-12), 5.08 (dd, 2H, J = 12.5, 17.4 Hz, Ar-CH2), 3.04–2.89 (m, 2H), 2.48–2.33
13
(m, 1H), 1.32, 1.18, 1.12, 1.01, 0.92, 0.89, 0.64 (s, 7 × 3H, CH3); C-NMR (CDCl3): 177.4 (COOBn),
176.6 (COONC), 163.1 (COONC), 143.9 (C-13), 138.2, 136.4, 134.3, 132.4, 130.8, 128.5, 128.4,
128.4, 128.0, 128.0, 127.9, 127.1 (aromatic carbons), 122.1 (C-12), 65.9, 55.9, 47.1, 46.7, 45.8, 41.8,
41.7, 41.4, 39.3, 38.8, 37.0, 33.9, 33.1, 32.4, 32.3, 30.7, 27.6, 27.1, 25.8, 23.6, 23.5, 23.1, 23.0, 20.2,
19.0, 16.8, 15.1 (7 × CH3); Anal. Calcd for C44H55Cl2NO4: C, 72.11; H, 7.56; N, 1.91. found: C, 72.31;
H, 7.49; N, 1.77; HRMS calcd for C37H50ClNO4 (M+H)+: 732.35809, found: 732.35529.

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Benzyl oleanolic acid 3-oxime ester (B-03). The reaction was run similarly to that used to synthesize
1
B-01. A white foamy solid B-03 was obtained in 79% yield. H-NMR (CDCl3):  8.01–7.32 (m, 9H,
Ar-H), 5.30 (br s, 1 H, H-12), 5.08 (dd, 2H, J = 12.5, 17.4 Hz, Ar-CH2), 3.04–2.88 (m, 2H), 2.48–2.33
13
(m, 1H), 1.34, 1.19, 1.12, 1.02, 0.92, 0.89, 0.65 (s, 7 × 3H, CH3); C-NMR (CDCl3): 177.3 (COOBn),
176.6 (COONC), 163.0 (COONC), 143.8 (C-13), 136.3, 134.5, 133.0, 131.4, 129.8, 129.4, 128.4,
128.4, 127.9, 127.9, 127.9, 127.6 (aromatic carbons), 122.1 (C-12), 65.9, 55.7, 47.1, 46.7, 45.8, 41.7,
41.6, 41.4, 39.3, 38.7, 36.9, 33.8, 33.0, 32.3, 32.3, 30.6, 27.5, 27.1, 25.7, 23.6, 23.4, 23.2, 23.0, 19.9,
18.9, 16.8, 15.1 (7 × CH3); Anal. Calcd for C44H56ClNO4: C, 75.67; H, 8.08; N, 2.01. found: C, 75.52;
H, 8.27; N, 2.27; HRMS calcd for C37H50ClNO4 (M+H)+: 698.39706, found: 698.39697.
Benzyl oleanolic acid 3-oxime ester (B-04). The reaction was run similarly to that used to synthesize
1
B-01. A white foamy solid B-04 was obtained in 85% yield. H-NMR (CDCl3):  8.87–7.29 (m, 9H,
Ar-H), 5.31 (br s, 1 H, H-12), 5.08 (dd, 2H, J = 12.5, 17.4 Hz, Ar-CH2), 3.06–2.90 (m, 2H), 2.50–2.48
13
(m, 1H), 1.35, 1.21, 1.12, 1.04, 0.92, 0.90, 0.65 (s, 7 × 3H, CH3); C-NMR (CDCl3): 177.2 (COOBn),
177.1 (COONC), 162.1 (COONC), 148.2, 143.8 (C-13), 136.3, 135.1, 131.5, 129.7, 128.3, 128.3,
127.9, 127.9, 127.8, 127.3, 124.2 (aromatic carbons), 122.1 (C-12), 65.8, 55.7, 47.1, 46.7, 45.8, 41.7,
41.6, 41.4, 39.3, 38.6, 36.9, 33.8, 33.0, 32.3, 32.3, 30.6, 27.5, 27.2, 25.7, 23.5, 23.4, 23.2, 23.0, 20.0,
18.9, 16.8, 15.0 (7 × CH3); Anal. Calcd for C44H56N2O6: C, 74.55; H, 7.96; N, 3.95. found: C, 74.40; H,
7.79; N, 3.65; HRMS calcd for C37H50ClNO4 (M+H)+: 709.42111, found: 709.42096.
Benzyl oleanolic acid 3-oxime ester (B-05). The reaction was run similarly to that used to synthesize
1
B-01. A white foamy solid B-05 was obtained in 75% yield. H-NMR (CDCl3):  8.04–7.23 (m, 12H,
Ar-H), 5.28 (br s, 1 H, H-12), 5.06 (dd, 2H, J = 12.5, 17.4 Hz, Ar-CH2), 4.19 (s, 2 H, CH2C=O),
2.93–2.87 (m, 1H), 2.57–2.50 (m, 1H), 1.19, 1.08, 1.04, 0.92, 0.89, 0.89, 0.60 (s, 7 × 3H, CH3);
13C-NMR (CDCl3): 177.3 (COOBn), 175.6 (COONC), 169.3 (COONC), 143.7 (C-13), 136.3, 133.7,
132.0, 130.2, 128.6, 128.5, 128.3, 127.9, 127.9, 127.8, 127.8, 126.2, 125.7, 125.6, 125.3, 123.8
(aromatic carbons), 122.1 (C-12), 65.8, 55.7, 47.0, 46.6, 45.7, 41.7, 41.3, 41.3, 39.2, 38.5, 38.2, 36.8,
36.8, 33.8, 33.0, 32.2, 30.6, 27.5, 26.9, 25.7, 23.6, 23.3, 22.9, 22.9, 19.3, 18.8, 16.7, 15.0 (7 × CH3);
Anal. Calcd for C49H61NO4: C, 80.84; H, 8.45; N, 1.92. found: C, 80.69; H, 8.68; N, 1.69; HRMS calcd
for C37H50ClNO4 (M+H)+: 728.46734, found: 728.46869.
Benzyl oleanolic acid 3-oxime ester (B-06). The reaction was run similarly to that used to synthesize
1
B-01. A white foamy solid B-06 was obtained in 72% yield. H-NMR (CDCl3):  7.36–6.85 (m, 9H,
Ar-H), 5.29 (br s, 1 H, H-12), 5.08 (dd, 2H, J = 12.5, 17.4 Hz, Ar-CH2), 4.82 (s, 2 H, CH2C=O),
2.94–2.82 (m, 2H), 2.32–2.26 (m, 1H), 1.23, 1.12, 1.11, 0.98, 0.92, 0.90, 0.63 (s, 7 × 3H, CH3);
13C-NMR (CDCl3): 177.3 (COOBn), 176.0 (COONC), 167.9 (COONC), 156.5, 143.7 (C-13), 136.3,
129.4, 129.4, 128.4, 128.4, 127.9, 127.9, 127.9, 126.6, 122.1 (C-12), 116.1, 116.1 (aromatic carbons),
65.9, 65.4, 55.8, 47.1, 46.7, 45.7, 41.7, 41.5, 41.3, 39.2, 38.6, 36.9, 33.8, 33.0, 32.3, 30.6, 30.6, 27.5,
27.0, 25.7, 23.6, 23.4, 23.0, 23.0, 19.4, 18.9, 16.8, 15.0 (7 × CH3); Anal. Calcd for C45H58ClNO5: C,
74.20; H, 8.03; N, 1.92. found: C, 74.06; H, 8.29; N, 1.72; HRMS calcd for C37H50ClNO4 (M+H)+:
728.40763, found: 728.40668.

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Benzyl oleanolic acid 3-oxime ester (B-07). The reaction was run similarly to that used to synthesize
1
B-01. A white foamy solid B-07 was obtained in 75% yield. H-NMR (CDCl3):  8.80–7.27 (m, 9H,
Ar-H), 5.30 (br s, 1 H, H-12), 5.08 (dd, 2H, J = 12.5, 17.4 Hz, Ar-CH2), 3.04–2.89 (m, 2H), 2.47–2.33
13
(m, 1H), 1.33, 1.18, 1.11, 1.01, 0.92, 0.89, 0.64 (s, 7 × 3H, CH3); C-NMR (CDCl3): 177.0 (COOBn),
176.0 (COONC), 162.2 (COONC), 143.5 (C-13), 136.2, 132.6, 132.1, 128.2, 128.2, 128.2, 127.7,
127.7, 127.7, 123.9, 121.9 (C-12), 116.8, 116.5 (aromatic carbons), 65.6, 55.6, 46.9, 46.4, 45.5, 41.5,
41.2, 41.2, 39.0, 38.5, 36.7, 33.6, 32.9, 32.1, 32.1, 30.4, 27.3, 26.9, 25.5, 23.4, 23.2, 22.9, 22.8, 19.8,
18.7, 16.6, 14.8 (7 × CH3); Anal. Calcd for C44H56FNO4: C, 77.50; H, 8.28; N, 2.05. found: C, 77.41; H,
8.41; N, 2.25; HRMS calcd for C37H50ClNO4 (M+H)+: 682.42661, found: 682.42645.
Benzyl oleanolic acid 3-oxime ester (B-08). The reaction was run similarly to that used to synthesize
1
B-01. A white foamy solid B-08 was obtained in 78% yield. H-NMR (CDCl3):  7.93–7.26 (m, 9H,
Ar-H), 5.29 (br s, 1 H, H-12), 5.08 (dd, 2H, J = 12.5, 17.4 Hz, Ar-CH2), 3.04–2.89 (m, 2H), 2.44–2.33
13
(m, 1H), 1.33, 1.19, 1.11, 1.02, 0.91, 0.89, 0.64 (s, 7 × 3H, CH3); C-NMR (CDCl3): 177.2 (COOBn),
176.3 (COONC), 163.4 (COONC), 143.8 (C-13), 136.3, 131.7, 131.7, 130.9, 130.9, 128.6, 128.3,
128.3, 128.0, 127.9, 127.9, 127.8 (aromatic carbons), 122.1 (C-12), 65.8, 55.7, 47.0, 46.6, 45.7, 41.7,
41.5, 41.4, 39.3, 38.6, 36.9, 33.7, 33.0, 32.3, 32.3, 30.6, 27.5, 27.2, 25.7, 23.5, 23.4, 23.2, 23.0, 19.8,
18.9, 16.8, 15.0 (7 × CH3); Anal. Calcd for C44H56BrNO4: C, 71.14; H, 7.60; N, 1.89. found: C, 71.35;
H, 7.53; N, 1.60; HRMS calcd for C37H50ClNO4 (M+H)+: 742.34655, found: 742.34674.
Benzyl oleanolic acid 3-oxime ester (B-09). The reaction was run similarly to that used to synthesize
1
B-01. A white foamy solid B-09 was obtained in 81% yield. H-NMR (CDCl3):  9.25–7.28 (m, 8H,
Ar-H), 5.30 (br s, 1 H, H-12), 5.08 (dd, 2H, J = 12.5, 17.4 Hz, Ar-CH2), 3.06–2.89(m, 2H), 2.48–2.33
13
(m, 1H), 1.34, 1.20, 1.12, 1.03, 0.92, 0.90, 0.65 (s, 7 × 3H, CH3); C-NMR (CDCl3): 177.2 (COOBn),
176.7 (COONC), 162.8 (COONC), 153.3, 150.5, 143.7 (C-13), 136.9, 136.3, 128.3, 128.3, 127.9,
127.9, 127.8, 125.7, 123.4 (aromatic carbons), 122.0 (C-12), 65.8, 55.7, 47.0, 46.6, 45.7, 41.7, 41.6,
41.4, 39.2, 38.6, 36.9, 33.8, 33.0, 32.3, 32.2, 30.6, 27.5, 27.1, 25.7, 23.5, 23.4, 23.1, 23.0, 19.9, 18.9,
16.8, 15.0 (7 × CH3); Anal. Calcd for C43H56N2O4: C, 77.67; H, 8.49; N, 4.21. found: C, 77.73; H, 8.62;
N, 4.03; HRMS calcd for C37H50ClNO4 (M+H)+: 665.43128, found: 665.43182.
Benzyl oleanolic acid 3-oxime ester (B-10). The reaction was run similarly to that used to synthesize
1
B-01. A white foamy solid B-10 was obtained in 71% yield. H-NMR (CDCl3):  7.60–6.51(m, 8H,
Ar-H), 5.29 (br s, 1 H, H-12), 5.08 (dd, 2H, J = 12.5, 17.4 Hz, Ar-CH2), 3.06–2.88 (m, 2H), 2.43–2.33
13
(m, 1H), 1.32, 1.25, 1.11, 1.02, 0.92, 0.89, 0.64 (s, 7 × 3H, CH3); C-NMR (CDCl3): 177.3 (COOBn),
176.3 (COONC), 156.6 (COONC), 146.4, 143.8, 143.7 (C-13), 136.4, 128.4, 128.4, 128.0, 128.0,
127.9, 122.1 (C-12), 118.0, 111.8 (aromatic carbons), 65.9, 55.7, 47.1, 46.7, 45.8, 41.7, 41.5, 41.4, 39.3,
38.7, 36.9, 33.8, 33.0, 32.3, 32.3, 30.6, 27.5, 27.2, 25.7, 23.6, 23.4, 23.2, 23.0, 19.8, 18.9, 16.8, 15.1 (7 ×
CH3); Anal. Calcd for C42H55NO5: C, 77.15; H, 8.48; N, 2.14. found: C, 77.01; H, 8.30; N, 2.27; HRMS
calcd for C37H50ClNO4 (M+H)+: 654.41530, found: 654.41504.
Benzyl oleanolic acid 3-oxime ester (B-11). The reaction was run similarly to that used to synthesize
1
B-01. A white foamy solid B-11 was obtained in 84% yield. H-NMR (CDCl3):  8.86–7.28 (m, 9H,
Ar-H), 5.30 (br s, 1 H, H-12), 5.08 (dd, 2H, J = 12.5, 17.4 Hz, Ar-CH2), 3.06–2.90 (m, 2H), 2.50–2.48

Molecules 2013, 18
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13
(m, 1H), 1.35, 1.21, 1.12, 1.04, 0.92, 0.90, 0.65 (s, 7 × 3H, CH3); C-NMR (CDCl3): 177.2 (COOBn),
177.1 (COONC), 162.1 (COONC), 148.2, 143.7 (C-13), 136.3, 135.1, 131.4, 129.7, 128.3, 128.3,
127.9, 127.9, 127.8, 127.3, 124.2 (aromatic carbons), 122.0 (C-12), 65.8, 55.7, 47.0, 46.6, 45.7, 41.7,
41.6, 41.4, 39.2, 38.6, 36.9, 33.8, 33.0, 32.3, 32.2, 30.6, 27.5, 27.2, 25.7, 23.4, 23.4, 23.2, 22.9, 20.0,
19.0, 16.7, 15.0 (7 × CH3); Anal. Calcd for C44H56N2O6: C, 74.55; H, 7.96; N, 3.95. found: C, 74.30; H,
7.88; N, 3.68; HRMS calcd for C37H50ClNO4 (M+H)+: 709.42111, found: 709.42267.
Benzyl oleanolic acid 3-oxime ester (B-12). The reaction was run similarly to that used to synthesize
1
B-01. A white foamy solid B-12 was obtained in 90% yield. H-NMR (CDCl3):  9.24–7.28 (m, 8H,
Ar-H), 5.30 (br s, 1 H, H-12), 5.08 (dd, 2H, J = 12.5, 17.4 Hz, Ar-CH2), 3.04–2.88 (m, 2H), 2.54–2.51
13
(m, 1H), 1.35, 1.22, 1.13, 1.05, 0.92, 0.90, 0.65 (s, 7 × 3H, CH3); C-NMR (CDCl3): 178.0 (COOBn),
177.3 (COONC), 160.3 (COONC), 148.7, 143.8 (C-13), 136.3, 133.4, 129.2, 129.2, 128.4, 128.4,
128.0, 128.0, 127.9, 127.9, 122.3 (aromatic carbons), 122.0 (C-12), 65.9, 55.7, 47.1, 46.7, 45.8, 41.9,
41.7, 41.4, 39.3, 38.6, 37.0, 33.8, 33.0, 32.3, 32.3, 30.6, 27.5, 27.1, 25.7, 23.6, 23.4, 23.2, 23.0, 20.2,
18.9, 16.8, 15.1 (7 × CH3); Anal. Calcd for C44H55N3O8: C, 70.10; H, 7.35; N, 5.57. found: C, 70.43; H,
7.31; N, 5.85; HRMS calcd for C37H50ClNO4 (M+H)+: 754.40619, found: 754.40375.
Benzyl oleanolic acid 3-oxime ester (B-13). The reaction was run similarly to that used to synthesize
1
B-01. A white foamy solid B-13 was obtained in 76% yield. H-NMR (CDCl3):  7.82–7.30 (m, 9H,
Ar-H), 5.29 (br s, 1 H, H-12), 5.08 (dd, 2H, J = 12.5, 17.4 Hz, Ar-CH2), 3.07–2.88 (m, 2H), 2.42–2.32
13
(m, 1H), 1.32, 1.18, 1.12, 1.01, 0.92, 0.89, 0.64 (s, 7 × 3H, CH3); C-NMR (CDCl3): 177.3 (COOBn),
176.4 (COONC), 163.9 (COONC), 143.8 (C-13), 136.3, 133.1, 132.3, 131.3, 130.8, 130.2, 128.4,
128.4, 127.9, 127.9, 127.9, 126.6 (aromatic carbons), 122.1 (C-12), 65.9, 55.8, 47.1, 46.7, 45.7, 41.7,
41.6, 41.4, 39.2, 38.8, 36.9, 33.8, 33.0, 32.3, 32.3, 30.6, 27.5, 27.0, 25.7, 23.6, 23.4, 23.1, 23.0, 20.1,
18.9, 16.8, 15.1 (7 × CH3); Anal. Calcd for C44H56ClNO4: C, 75.67; H, 8.08; N, 2.01. found: C, 75.55;
H, 8.12; N, 2.32; HRMS calcd for C37H50ClNO4 (M+H)+: 698.39706, found: 698.39709.
Benzyl oleanolic acid 3-oxime ester (B-14). The reaction was run similarly to that used to synthesize
1
B-01. A white foamy solid B-14 was obtained in 79% yield. H-NMR (CDCl3):  8.80–7.27 (m, 9H,
Ar-H), 5.30 (br s, 1 H, H-12), 5.08 (dd, 2H, J = 12.5, 17.4 Hz, Ar-CH2), 3.04–2.89 (m, 2H), 2.47–2.33
13
(m, 1H), 1.34, 1.20, 1.12, 1.03, 0.92, 0.90, 0.64 (s, 7 × 3H, CH3); C-NMR (CDCl3): 177.1 (COOBn),
176.9 (COONC), 162.5 (COONC), 150.4, 150.4, 143.7 (C-13), 136.8, 136.2, 128.2, 128.2, 128.2, 127.8,
127.8, 127.7, 122.6 (aromatic carbons), 121.9 (C-12), 65.7, 55.6, 46.9, 46.5, 45.6, 41.6, 41.5, 41.2, 39.1,
38.5, 36.8, 33.6, 32.9, 32.1, 32.1, 30.5, 27.4, 27.0, 25.6, 23.4, 23.2, 23.0, 22.8, 19.8, 18.8, 16.6, 14.9 (7 ×
CH3); Anal. Calcd for C43H56N2O4: C, 77.67; H, 8.49; N, 4.21. found: C, 77.35; H, 8.53; N, 4.02;
HRMS calcd for C37H50ClNO4 (M+H)+: 665.43128, found: 665.43146.
Benzyl oleanolic acid 3-oxime ester (B-15). The reaction was run similarly to that used to synthesize
1
B-01. A white foamy solid B-15 was obtained in 70% yield. H-NMR (CDCl3):  8.07–7.32 (m, 10H,
Ar-H), 5.30 (br s, 1 H, H-12), 5.08 (dd, 2H, J = 12.5, 17.4 Hz, Ar-CH2), 3.08–2.88 (m, 2H), 2.47–2.31
13
(m, 1H), 1.35, 1.19, 1.12, 1.03, 0.92, 0.89, 0.65 (s, 7 × 3H, CH3); C-NMR (CDCl3): 177.3 (COOBn),
176.2 (COONC), 164.2 (COONC), 143.8 (C-13), 136.3, 132.9, 129.7, 129.4, 129.4, 128.4, 128.4,
128.4, 128.4, 127.9, 127.9, 127.9 (aromatic carbons), 122.1 (C-12), 65.9, 55.7, 47.1, 46.7, 45.7, 41.7,

Molecules 2013, 18
3627
41.5, 41.4, 39.3, 38.7, 36.9, 33.8, 33.0, 32.3, 32.3, 30.6, 27.5, 27.1, 25.7, 23.6, 23.4, 23.2, 23.0, 19.8,
18.9, 16.8, 15.1 (7 × CH3); Anal. Calcd for C44H57NO4: C, 79.60; H, 8.65; N, 2.11. found: C, 79.55; H,
8.83; N, 2.38; HRMS calcd for C37H50ClNO4 (M+H)+: 664.43606, found: 664.43500.
3.3. Enzyme Inhibitory Activities Bioassay
Inhibitory activity of all the synthesized compounds towards Candida albicans GlcN-6-P synthase
was determined. The Candida albicans GFA1 gene encoding the enzyme was PCR amplified and
cloned to a yeast expression vector pYES2.0, then induced expression using glactose in
Saccharomyces cerevisiae. We used the further optimized Elson-Morgan method [14–17] to determine
the activity of the enzyme from pyrophosphate extract in the presence of the synthesized compounds.
Assays were performed in potassium phosphate buffer (0.1M, pH7.0). Incubation mixture (0.4 mL
volume) consisted of 15 mM D-Fru-6-P, 10 mM L-glutamine, 1 mM EDTA, 0.35 mM compounds.
Following preincubation at 37 °C for 10 min, the enzymatic reaction was initiated by the addition of
0.02 unit of GlmS. The mixture was incubated at 37 °C for 30 min. Enzymatic reaction was terminated
by boiling for 1 min. Aliquots of 0.2 mL of saturated NaHCO3 solution and 0.1 mL acetic
anhydride/acetone mixture (10%v/v, prepared freshly before use) were added and the mixture was
incubated at room temperature for 3 min. The acetylation was stopped by boiling for 3 min, followed
by cooling on ice. An aliquot of 0.2 mL of 0.8 M K2B4O7 solution, pH 9.2, was added, the mixture was
incubated at 100 °C for 3 min and cooled on ice. A 5 mL portion of the Elson-Morgan reagent (1 g of
4-dimethylaminobenzaldehyde dissolved in 100 mL of glacial acetic acid, containing 1.25 mL of
concentrated HCl) was added and the resulting mixture was incubated for 20 min at 37 °C. Three
replicates were performed. Absorbance at λ = 585 nm was measured and GlcN-6-P concentration in the
sample was read from the standard curve [solutions of glucosamine-HCl (0.1–1 mM) were assayed
simultaneously, to obtain a standard line from the plot of extinction against concentration of
glucosamine]. In each experiment, two control samples, one without enzyme and one without
substrates, were assayed in the same way.
3.4. Fungicidal Activity Bioassay
The mycelium growth rate test was used [18]. The culture media, with known concentration of the
test compounds, were obtained by mixing the soln of compounds A–B in methanol with potato dextrose
agar (PDA), on which fungus cakes were placed. The blank test was made using methanol. The culture
was carried out at 24 ± 0.5 °C. Three replicates were performed.
4. Conclusions
Twenty five oleanolic acid 3-oxime esters were designed and efficiently synthesized. The bioassays
showed that they had inhibitory activities against glucosamine-6-phosphate synthase, and at the same
time, they also exhibited some fungicidal activity against six tested fungi. Although the enzyme
inhibitory activities of the target compound are not very obvious compared with the parent compound
(OA), they exhibited much more fungicidal activity than the latter. All the compounds exhibited better
fungicidal activity against R. solani and S. sclerotiorum. Further studies are in progress.

Molecules 2013, 18
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Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/18/3/3615/s1.
Acknowledgments
This work was partially supported by NSFC of China (21172257), the National Basic Research
Program of China (2010CB126105) and the National High Technology Research Program of China
(2011AA10A206, 2011BAE06B02-01, 2012BAK25B03-01), and the Chinese Universities Scientific
Fund (2011JS030).
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Sample Availability: Samples of the compounds A and B are available from the authors.
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