Synthesis of novel derivatives of esculentoside A and its aglycone phytolaccagenin, and evaluation of their haemolytic activity and inhibition of lipopolysaccharide-induced nitric oxide production

Article abstract of DOI:10.1002/cbdv.201000339

A series of 46 compounds derived from esculentoside A and its aglycone were synthesized and characterized. The effect of these compounds on lipopolysaccharide (LPS)-induced NO production, haemolytic activity, and cell viability was evaluated. Structure-ac

Full text of DOI:10.1002/cbdv.201000339

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
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Synthesis of Novel Derivatives of Esculentoside A and Its Aglycone
Phytolaccagenin, and Evaluation of Their Haemolytic Activity and Inhibition
of Lipopolysaccharide-Induced Nitric Oxide Production
by Wei Gonga), Zhihui Jianga), Peng Suna), Ling Lia), Yongsheng Jina), Lucheng Shaoa),
Wen Zhanga), Baoshu Liua), Hongwei Zhangb), Hua Tanga), Yufeng Chenc), Yanghua Yi*a), and
Dazhi Zhang*a)
a) School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433,
P. R. China (phone/fax: þ86-21-81871226; e-mail: zdzhang_yjhx@smmu.edu.cn)
b) Department of Pharmacy, the Eighth Hospital of PLA, 5 Western Zhufeng Road, Xigaze 857000,
P. R. China
c) Department of Pharmacy, the Shenyang North Hospital, 83 Wenhua Road, Shenyang 110016,
P. R. China
A series of 46 compounds derived from esculentoside A and its aglycone were synthesized and
characterized. The effect of these compounds on lipopolysaccharide (LPS)-induced NO production,
haemolytic activity, and cell viability was evaluated. Structureꢀactivity relationship was established by
comparing the derivatives of esculentoside A with its aglycone derivatives. Both the aglycone and its
derivatives showed higher inhibitory effects on LPS-induced NO production, and lower haemolytic
activities than esculentoside A and its derivatives.
Introduction. – Triterpenoid saponins, composed of aglycones and sugars, are
secondary metabolites produced by many plant species. There are several reports
stating that triterpenoid saponins have a variety of pharmacological properties such as
antitumor, anti-inflammatory, antifungal, and antibacterial activities [1]. Phytolacca
esculenta (Shang Lu, in Chinese) is an important medicinal plant used in Chinese
traditional medicine and listed in Chinese Pharmacopoeia [2]. A decoction of its root is
used to treat inflammation-related diseases such as lymphatic congestion, endome-
triosis, swollen lymph glands, edema, mastitis, abnormal congestion of all glands of the
body, and severe ascites of the abdomen. In our previous study, we found that
esculentosides, the triterpenoid saponins isolated from the root of Phytolacca esculenta,
strongly inhibit acute and chronic inflammation in cells and animal models [3–8]. In
particular, esculentoside A (EsA; 1), which is the main triterpenoid of total
esculentosides, has been reported to inhibit lipopolysaccharide (LPS)-induced
secretion of inflammatory mediators such as TNF, IL-1, IL-6, and PGE2 in several
types of cells, and to reduce phagocytosis and secretion of cytokines in overly activated
macrophages [9–11].
However, the administration of Phytolacca esculenta is restricted in clinic due to its
toxicity. EsA (1) has already been proved to have haemolytic activities that might result
in its toxicity. Therefore, the optimization of the structure of EsA (1) could be of great
ꢀ 2011 Verlag Helvetica Chimica Acta AG, Zꢁrich

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
importance for the discovery of potential bioactive semisynthetic compounds with
enhanced anti-inflammatory and decreased haemolytic activities.
Since 1 is the main triterpenoid of the total esculentosides, we have focused on the
development of 1 in the past three decades. On the other hand, the concentration of
phytolaccagenin (2), the aglycone of 1, is low in the root of Phytolacca esculenta, and it
is mainly obtained by hydrolysis of EsA (1). Thus, the research on phytolaccagenin (2)
was ignored for a long time, and no study on structureꢀactivity relationship (SAR) of
phytolaccagenin (2) has ever been reported. It is reported that the sugars of some
triterpenoid saponins can be easily cleaved off in organism by enzymes, allowing the
aglycone to be absorbed [12][13]. This enables the aglycones to insert into cell
membrane, modify the composition, and potentially affect the signaling of many
ligands and cofactors. This hints that phytolaccagenin (2) might be the key structure to
exert its bioactivity and worth further investigation.
Much work has been performed to study the SAR of triterpenoid saponins [14–17].
However, little information has been reported concerning the systematic comparison
between saponin derivatives and its aglycone derivatives. In this study, we focused on
the optimization of both EsA (1) and its aglycone. A preliminary study on SAR was
established by comparing 1 with its aglycone derivatives. Biological evaluation
indicated that the derivatives of the aglycone 2 show higher inhibitory activities
towards LPS-induced nitric oxide (NO) production and lower haemolytic activities
than EsA derivatives. We herein report the structure optimization of both EsA (1) and
its aglycone phytolaccagenin, and the SAR of their derivatives.
Results and Discussion. – 1. Synthesis. In our previous work [18], SAR study on
EsA (1) revealed that the conversion of C(28)OOH to an amide inhibited the activity
of COX-2 and reduced its haemolytic activity. In particular, the introduction of an
aromatic ring to 1 could promote higher potency than EsA and Celecoxib. Moreover,
an aliphatic chain, which contains 2–3 C-atoms, linking the aromatic ring to the amide
C(28) resulted in even higher activity. Based on these results, we performed further
modifications to 1 and its aglycone. A series of new compounds, 3, 5a–5k, and 7a–7k
derived from EsA (1), and compounds 4, 6a–6k, and 8a–8k derived from its aglycone
(2), were designed in the current work.
The starting materials are EsA (1) and phytolaccagenin (2). EsA (1) was extracted
from Phytolacca esculenta [19]. Phytolaccagenin (2) was prepared by hydrolyzing 1 in
5% HCl/MeOH. The structures of 1 and 2 were confirmed by MS and NMR. Propane-
1,3-diamine, employed as a flexible C3 aliphatic chain, was coupled with the C(28)OOH
group of 1 to give amide 3. The free NH2 group of 3 was modified by amidation with
various carboxylic acids to give amides 5a–5k. The above two coupling reactions were
catalyzed by HOBt and DCC, respectively. Another type of derivatives, 7a–7k, was
generated by alkylation of the NH2 group of 3 with various aldehydes in the presence of
NaBH3CN. Starting from 2, compounds 4, 6a–6k, 8a–8k were prepared by following
the above mentioned methods. Synthetic routes to prepare the derivatives are outlined
in the Scheme.
2. Biological Evaluation. The activation of macrophages plays a key role in
inflammatory events via the secretion of various factors such as NO, prostaglandins,
cytokines, etc. [20]. Excessive NO production damages tissue and causes transient

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1835
Scheme. Synthesis of the Target Compounds
a) 5% HCl/MeOH, r.t., 4 h; 99.5%. b) 1. 1-Hydroxybenzotriazole (HOBt), dicyclohexylcarbodiimide
(DCC), DMF/THF, r.t., 1 h; 2. propane-1,3-diamine, 608, 4 h; 95–97%. c) For 5a–5g, 6a–6g: 1.
carboxylic acid, HOBt, DCC, r.t., 1 h; 2. 3 or 4, 608, 4–8 h; 76–90%. For 5h–5k, 6h–6k: 1. Fmoc-
protected amino acid, HOBt, DCC, r.t., 1–3 h; 2. 3 or 4, 608, 4–8 h; 3. deprotection: Et2NH/DMF; 71–
88%. d) 1. Aromatic aldehyde, anh. MeOH, r.t., 4–8 h; 2. NaBH3CN, 2–8 h; 24–71%.
inflammation. In several studies, the anti-inflammatory effect of plant-based extract
was evaluated in LPS-stimulated macrophages [21–23]. In this study, the anti-
inflammatory effect of the designed derivatives was preliminarily evaluated by
determining the inhibitory effects (IC50 values) on LPS-induced NO production of
RAW 264.7 macrophages, which was performed as described by Wang et al. [24].
Another aim of our study was to reduce the toxicity of EsA (1) by structure
modification. The toxicity of the derivatives was evaluated by cell-viability assay and

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
haemolytic assay. RAW 264.7 Macrophages were used in cell-viability assay as
described by Mosmann [25]. Erythrocytes prepared from blood of healthy New
Zealand rabbit were used in haemolytic assay as described by Wang et al. [16].
2.1. Inhibition of LPS-Induced NO Production. The inhibitory effects (IC50 values)
of the desired compounds on NO production of LPS-stimulated macrophages were
compiled in the Table. The IC50 value of EsA (1) is 75.6ꢁ2.2 mm. In contrast, the IC50
value of aglycone phytolaccagenin (2) is 18.4ꢁ0.6 mm, i.e., fourfold lower than that of 1.
The intermediates 3 (IC50 ¼ 49.5ꢁ1.1 mm) and 4 (IC50 ¼ 16.1ꢁ0.7 mm), both derived
from 1 and 2 by coupling with propane-1,3-diamine, showed enhanced activities
compared with their corresponding parent compounds; moreover, the IC50 value of 4 is
lower than that of the EsA derivative 3. The SAR of the derivatives prepared by
introducing a series of groups to the free NH2 group of propane-1,3-diamine in 3 and 4
was established. Among 23 EsA derivatives, only four compounds are less active than 1
(slightly: 5h, 5j, and 7b; significantly: 7f), while the other 19 compounds have lower or
similar IC50 values compared with that of EsA (1), ranging from 14.1ꢁ0.7 to 66.0ꢁ
Table. In vitro Activities of the Target Compounds
Compound MTT Assay Haemolytic NO Assay
Compound MTT Assay Haemolytic NO Assay
[%, 100 mm] assay
IC50b) [mm]
[%, 100 mm] assay
IC50b) [mm]
HD50a) [mm]
HD50a) [mm]
1
3
0
0
0
0
0
0
0
0
178ꢁ6
75.6ꢁ2.2
49.5ꢁ1.1
2
4
39ꢁ1.4
>500
18.4ꢁ0.6
16.1ꢁ0.7
32.3ꢁ1.0
16.5ꢁ0.5
20.3ꢁ0.8
19.2ꢁ0.9
17.6ꢁ0.5
9.8ꢁ0.4
150ꢁ3
159ꢁ3
125ꢁ3
122ꢁ3
159ꢁ5
132ꢁ5
80ꢁ3
0
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
5a
5b
5c
5d
5e
5f
5g
5h
5i
60.6ꢁ1.4 6a
36.5ꢁ1.5 6b
32.8ꢁ1.1 6c
59.3ꢁ2.0 6d
51.5ꢁ1.1 6e
14.1ꢁ0.7 6f
51.9ꢁ1.9 6g
79.0ꢁ2.6 6h
22.8ꢁ0.9 6i
80.1ꢁ2.6 6j
29.8ꢁ0.9 6k
24.5ꢁ0.8 8a
76.5ꢁ2.2 8b
19.8ꢁ0.9 8c
33.9ꢁ0.8 8d
39.8ꢁ1.3 8e
23ꢁ0.9
12ꢁ0.8
11ꢁ1.1
12ꢁ0.6
0
0
0
6ꢁ0.5
150ꢁ5
222ꢁ7
99ꢁ3
24.3ꢁ1.0
27.2ꢁ1.1
12.4ꢁ0.4
30.3ꢁ0.9
18.1ꢁ0.8
15.0ꢁ0.6
22.1ꢁ0.8
7.6ꢁ0.5
2ꢁ0.3
3ꢁ0.6
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
5j
220ꢁ6
107ꢁ4
95ꢁ3
5k
7a
7b
7c
7d
7e
7f
7g
7h
7i
3ꢁ0.4
0
0
0
0
0
0
0
0
189ꢁ6
83ꢁ5
101ꢁ3
109ꢁ4
326ꢁ10
150ꢁ8
233ꢁ8
159ꢁ5
158ꢁ5
121ꢁ3
n.d.d)
24.4ꢁ0.7
28.9ꢁ1.2
>50
12.5ꢁ0.8
21.4ꢁ0.7
23.7ꢁ0.8
19.9ꢁ0.9
9.8ꢁ0.6
>100
8f
40.7ꢁ1.7 8g
66.0ꢁ2.3 8h
39.7ꢁ1.2 8i
40.1ꢁ1.3 8j
37.2ꢁ0.8 8k
0.5ꢁ0.2
0
0
0
7j
7k
A. G.c)
n.d.d)
a)b) Each value represents the meanꢁS.D. of three determinations. c) A. G. : Aminoguanidine, as positive
control. d) n.d.: Not determined.

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1837
2.3 mm. Among the 23 aglycone derivatives 2, 4, 6a–6k, and 8a–8k, ten compounds, i.e.,
4, 6b, 6e, 6f, 6i, 6k, 8a, 8c, 8g, and 8k have lower IC50 values than phytolaccagenin (2),
ranging from 9.8ꢁ0.4 to 18.1ꢁ0.8 mm; while the other 13 aglycone derivativess have
higher IC50 values ranging from 19.2ꢁ0.9 to >50 mm. In general, the modification of
the C(28)OOH moiety of EsA (1) and its aglycone resulted in high-potential
compounds. In paticular, the aglycone derivatives modified with the same substituent
R2 or R3 have lower IC50 values than the corresponding EsA derivatives, except that the
exact IC50 values of 7f and 8f were not determined. Thus, we can clearly conclude that
the aglycone phytolaccagenin (2) and its derivatives are more active than EsA and its
derivatives. The aglycone of EsA 2, phytolaccagenin, is worth further investigation for
its anti-inflammatory effect.
The 3-phenylpropanoyl group was introduced in 5f and 6f, while the 4-
bromophenyl group, the l-glutaminyl group, and the phenyl moieties were introduced
in 7c and 8c, 5i and 6i, 7a and 8a, respectively. Among the EsA derivatives, 5f, 7c, 5i, and
7a are the top four most active compounds with IC50 values of 14.1ꢁ0.7, 19.8ꢁ0.9,
22.8ꢁ0.9, and 24.5ꢁ0.8 mm, respectively. Interestingly, 8c, 6f, 6i, and 8a are among the
most active aglycone derivatives with IC50 values of 7.6ꢁ0.5, 9.8ꢁ0.4, 12.4ꢁ0.4, and
15.0ꢁ0.6 mm, respectively. These results strongly suggested that 3-phenylpropanoyl, 4-
bromophenyl, l-glutaminyl, and phenyl moieties can significantly enhance the
bioactivities of both EsA (1) and its aglycone 2. Moreover, compounds 5a and 6a, 5h
and 6h, 5j and 6j, 7b and 8b, 7f and 8f, and 7h and 8h exhibited quite low activities
within both kinds of derivatives, which also bear the same substituents, respectively.
These results indicate that the activities of the derivatives of EsA (1) and its aglycone 2
are strongly dependent on the variance of substituents (R2 or R3). The substituents R2
or R3 play important roles in determining the inhibitory effect on LPS-induced NO
production; therefore, further optimization is needed to obtain high-potential
compounds.
Substituents R2 led to amido derivatives, and R3 led to amino derivatives. Among
the aglycone derivatives, the amido derivatives 6f (IC50 ¼ 9.8ꢁ0.4 mm) and 6i (IC50
¼
12.4ꢁ0.4 mm), as well as the amino derivatives 8c (IC50 ¼ 7.6 ꢁ0.5 mm), 8g (IC50
¼
12.5ꢁ0.8 mm), and 8k (IC50 ¼ 9.8ꢁ0.6 mm) have the highest activities in their groups.
Among the EsA derivatives, the amido derivatives 5f and 5i also have similar potential
activities compared to the amino derivatives 7c and 7a. Overall, there is no significant
difference observed between the activities of the amido and amino derivatives.
We tried to shed some light on the SAR of the substituents of the free NH2 group of
propane-1,3-diamine, considering their electron-withdrawing/donating and hydrophil-
ic/hydrophobic properties. On one hand, our results showed that, among the amino
derivatives, both the electron-withdrawing group (Br in 8c and NO2 in 8g) and electron-
donating group (OH in 8k) increased the potential activity, suggesting that the electron-
withdrawing/donating property is not a controlling feature; on the other hand, 8e (3-
OH), 8f (4-Me2N), and 8k (2,3,4-(OH)3) all have hydrophilic groups, but showed
different reactive potencies, suggesting that hydrophilic/hydrophobic property is very
likely not a controlling feature either. Moreover, even a small change in structures
could have led to a large difference in IC50 values. For example, with a l-glutamine
moiety, amido derivative 6i exhibited a high IC50 value of 12.4ꢁ0.4 mm; however,
reducing just by one CH2 group compared to glutamine i.e., asparagine moiety,

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
dramatically lowered the activity of amido derivative 6j to 30.3ꢁ0.9 mm. A similar
comparison and result also applied to 5i and 5j in the EsA derivatives. So far, the SAR
of the substituents is still not clear to us. Further studies are required.
2.2. Haemolytic Assay. The results of haemolytic activity of the derivatives were
compiled in the Table. None of the phytolaccagenin derivatives showed haemolytic
effects even at a high concentration of 500 mm. However, EsA derivatives exhibited
haemolytic effects at concentrations from 80ꢁ3 mm to 326ꢁ10 mm. Both 2 and its
derivatives showed higher HD50 values compared to 1 and its derivatives, indicating a
lower haemolytic effect. We concluded that the sugars are important moieties for
haemolytic activities.
EsA Derivatives 5h, 5j, 7f, and 7h showed higher HD50 values than that of 1
(HD50 ¼ 178ꢁ6 mm), suggesting lower haemolytic toxicity. Unfortunately, all these
compounds also exhibited lower inhibitory effects on NO production in RAW 264.7
macrophages. On the contrary, compounds 5f, 7c, 7a, and 5i showed top four lowest
HD50 values (80ꢁ3, 83ꢁ5, 95ꢁ3, and 99ꢁ3 mm, resp.), indicating highest haemolytic
toxicities, but, at the same time, they were the most active compounds on inhibiting NO
production. Taken together, we concluded that it would be hard to simultaneously
increase the inhibitory effect on LPS-induced NO production and suppress its
haemolytic toxicity by the modification of EsA.
Past SAR studies on haemolytic activity of triterpenoid saponins indicated that
both the sugar chains and functional groups of aglycone play important roles in the
haemolytic activity [14–16][22][26]; however, it is hard to state which part contributes
more. Our results indicated that it is the sugar of EsA, instead of the modification of the
C(28)OOH group of the aglycone, which affected the haemolytic activities. The change
of substituents at C(28) only resulted in a very slight difference of haemolytic activity
among derivatives of both EsA (1) and its aglycone 2.
Based on these results mentioned above, we concluded that phytolaccagenin (2) is
much more worthy to be further investigated than EsA (1).
2.3. Cell Viability. Cell viability was determined by using the mitochondrial
respiration-dependent MTT (¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazo-
lium bromide) reduction method on the RAW 264.7 macrophages. As shown in the
Table, none of the EsA derivatives showed cytotoxicity to macrophages. At the
concentration of 100 mm, phytolaccagenin (2) inhibited the growth of macrophages by
39%, while compounds 6a, 6b, 6c, and 6d inhibited the growth of macrophages by 23,
12, 11, and 12%, respectively. The other phytolaccagenin derivatives showed no
cytotoxicity.
Conclusions. – In this study, we modified the structures of EsA (1) and its aglycone
phytolaccagenin (2). The modification of the C(28)OOH moiety in both 1 and 2
resulted in potential derivatives with high inhibitory effects on LPS-induced NO
production, suggesting that the C(28)OOH moiety plays an important role in their
bioactivity, and its modification is of great relevance. Most importantly, both the
aglycone 2 and its derivatives showed higher inhibitory effects on LPS-induced NO
production and much lower haemolytic activity than EsA (1) and its derivatives. This
result strongly suggested that the aglycone 2 of EsA is worthy further research in anti-
inflammatory drug development. The derivatives bearing 3-phenylpropanoyl, 4-

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1839
bromophenyl, l-glutaminyl, and phenyl moieties exhibited significantly enhanced
bioactivities compared to the other derivatives. The optimization of EsA is not perfect
yet due to the high haemolytic toxicity along with high inhibitory effect on NO
production. On one hand, the continuous modification and reconstruction of other
functional groups in EsA (1) and its aglycone 2 are currently in progress in our
laboratory, the results of which will be reported in near future. On the other hand,
though the molecular target of 1 and its aglycone 2 are still poorly understood, we
expect that the modifications and corresponding SAR study may greatly promote the
research on their action mechanisms, and this work is also in progress in our laboratory.
This research was supported by the grants from the Chinese Foundation for High-Tech Project ꢀ863ꢁ
(No. 2006AA090304), the National Natural Sciences Fund of China (No. 30100243; 30672533;
30772644), Shanghai Pujiang Program (No. 05PJ14003), and the ꢀ11th Five Year Planꢁ for scientific
Program of PLA (06 H013).
Experimental Part
General. Flash column chromatography (FC): silica gel (200–300 mesh) from Qing Dao Chemical
Co. Ltd., P.R. China. TLC: Precoated silica gel GF254 plates from Yan Tai Zhi Fu Chemical Co. Ltd, P.R.
China. HPLC: Agilent 1100 system equipped with a refractive index detector; Zorbax-300-SB-C18
column (9.4ꢂ250 mm, i.d.). M.p.: XT5-XMT Apparatus; uncorrected. 1H- and 13C-NMR spectra:
Bruker AVANCE 600 spectrometer, at 600 and 150 MHz, resp.; d in ppm; J in Hz. ESI-MS: Thermo
Fisher Finnigan LCQ XP Max mass spectrometer, in m/z.
Preparation of Phytolaccagenin (2). Esculentaside A (EsA; 1) was dissolved in 5% HCl/MeOH and
stirred for 4 h at r.t. Then, the soln. was neutralized to pH 7 with 5% NaOH soln., and was concentrated
in vacuo. The residue was subjected to a FC (silica gel; MeOH/CH2Cl2), and recrystallization from
MeOH provided 2. Its structure was confirmed by MS and NMR reported in [27].
Preparation of Compounds 3 or 4. EsA (1; 1. 239 g, 1.5 mmol) or 2 (798 mg, 1.5 mmol) was dissolved
in anh. DMF/THF 1:3 (v/v). To this soln., dicyclohexylcarbodiimide (DCC; 412.7 mg, 2 mmol) and 1-
hydroxybenzotriazole (HOBt; 270.3 mg, 2 mmol) were added. After stirring for 1 h at r.t., propane-1,3-
diamine (500 ml, 6 mmol) was added, and the mixture was stirred at 608 under N2 for 4 h. When the
reaction stopped, the mixture was cooled to r.t. The precipitate was filtered, and the filtrate was
concentrated in vacuo. The residue was subjected to FC (silica gel; MeOH/CH2Cl2, containing 1% Et3N)
and was repurified by RP-HPLC (MeOH/H2O) to provide 3 or 4.
Methyl (2b,3b)-28-[(3-Aminopropyl)amino]-3-{[4-O-(b-d-glucopyranosyl)-b-d-xylopyranosyl]-
oxy}-2,23-dihydroxy-28-oxoolean-12-en-30-oate (3). White solid powder (1.257 g, 95%). M.p. 196.7–
1
198.38. H-NMR (C5D5N): 6.50 (br. s, 1 H); 5.39 (t, J ¼ 3.6, 1 H); 5.01 (d, J¼7.8, 1 H); 4.96 (d, J¼7.2,
1 H); 4.79 (d, J¼1.2, 1 H); 4.67 (dd, J¼2.4, 12.0, 1 H); 4.33–4.28 (m, 3 H); 4.24–4.20 (m, 2 H); 4.18 (d,
J¼9, 1 H); 4.15(t, J¼9, 1 H); 4.08 (t, J¼8.4, 1 H); 4.03 (t, J¼8.4, 1 H); 4.01–3.96 (m, 1 H); 3.92 (t, J¼
8.4, 1 H); 3.76 (br. s, 2 H); 3.68 (d, J¼10.8, 1 H); 3.62 (s, 3 H); 3.58 (t, J ¼ 10.8, 1 H); 3.42–3.36 (m, 1 H);
3.25–3.23 (m, 2 H); 3.21–3.17 (m, 1 H); 3.09 (dd, J¼4.2, 13.2, 1 H); 2.98–2.92 (m, 2 H); 2.35 (dd, J ¼
2.4, 13.8, 1 H); 2.22 (d, J ¼ 13.2, 1 H); 2.16–1.76 (m, 12 H); 1.65–1.63 (m, 2 H); 1.61 (s, 3 H); 1.59–1.57
(m, 1 H); 1.53–1.50 (m, 2 H); 1.41 (td, J ¼ 4.2, 13.8, 1 H); 1.35 (s, 3 H); 1.33–1.30 (m, 2 H); 1.26 (s, 3 H);
1.19 (s, 3 H); 1.09 (d, J ¼ 13.2, 1 H); 0.86 (s, 3 H). 13C-NMR (C5D5N): 177.1; 175.0; 144.3; 123.5; 106.3;
103.5; 88.4; 78.8; 78.2; 77.5; 76.4; 75.0; 74.2; 71.8; 70.8; 65.4; 64.7; 62.7; 51.6; 48.5; 47.7; 45.9; 44.1; 44.0;
43.3; 43.0; 42.8; 42.3; 41.6; 39.9; 37.9; 36.9; 35.1; 32.9; 31.0; 28.4; 27.9; 26.1; 24.0; 24.0; 23.2; 18.0; 17.6; 17.3;
14.5. ESI-MS: 883.52 ([M þ H]þ ), 881.50 ([MꢀH]ꢀ ).
Methyl (2b,3b)-28-[(3-Aminopropyl)amino]-2,3,23-trihydroxy-28-oxoolean-12-en-30-oate (4).
1
White solid powder (855 mg, 97%). M.p. 142.5–144.18. H-NMR (C5D5N): 6.52 (br. s, 1 H); 5.54 (br.
s, 2 H); 4.26–4.22 (m, 1 H); 4.14 (d, J ¼ 10.2, 1 H); 3.71 (d, J ¼ 10.2, 1 H); 3.67 (s, 3 H); 3.43–3.40 (m,
2 H); 3.02 (d, J ¼ 10.2, 1 H); 2.98–2.92 (m, 2 H); 2.35 (d, J ¼ 14.4, 1 H); 2.20 (d, J ¼ 12.6, 1 H); 2.14–

1840
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
2.10 (m, 3 H); 2.07–2.01 (m, 4 H); 1.90–1.76 (m, 5 H); 1.73–1.70 (m, 1 H); 1.68–1.64 (m, 1 H); 1.63 (s,
3 H); 1.61–1.59 (m, 2 H); 1.47 (s, 3 H); 1.44–1.40 (m, 1 H); 1.38 (s, 3 H); 1.32–1.26 (m, 2 H); 1.23 (s,
3 H); 1.06 (d, J ¼ 13.8, 1 H); 0.86 (s, 3 H). 13C-NMR (C5D5N): 177.4; 177.1; 144.4; 123.3; 73.0; 71.5; 67.7;
51.6; 48.5; 48.1; 46.1; 44.8; 44.1; 43.3; 42.7; 42.4; 42.2; 39.8; 38.3; 37.9; 37.2; 34.5; 33.0; 30.8; 30.6; 28.4;
28.3; 26.2; 24.0; 23.9; 18.2; 17.4; 17.2; 14.5. ESI-MS: 589.42 ([M þ H]þ ), 611.40 ([M þ Na]þ ), 587.41
([MꢀH]ꢀ ).
General Procedure for Preparing Compounds 5a–5g or 6a–6g. A carboxylic acid (0.06 mmol) was
dissolved in dry DMF/THF 1:3 (v/v). To this soln., DCC (18.6 mg, 0.09 mmol) and HOBt (12.2 mg,
0.09 mmol) were added. After stirring for 1 h at r.t., 3 (44.1 mg, 0.05 mmol) or 4 (29.4 mg, 0.05 mmol) was
added, and the mixture was stirred at 608 under N2 for 4–8 h. The precipitate was filtered, and the filtrate
was concentrated in vacuo. The residue was subjected to a FC (silica gel; MeOH/CH2Cl2) and was
repurified by RP-HPLC (MeOH/H2O) to provide 5a–5g or 6a–6g.
Methyl (2b,3b)-28-{[3-(Benzoylamino)propyl]amino}-3-{[4-O-(b-d-glucopyranosyl)-b-d-xylopyra-
nosyl]oxy}-2,23-dihydroxy-28-oxoolean-12-en-30-oate (5a). White powder (41.2 mg, 83.4%). M.p.
1
224.5–226.38. H-NMR (C5D5N): 7.94 (br. s, 1 H); 7.77 (dd, 2 H); 7.46–7.42 (m, 3 H); 6.52 (br. s, 1 H);
5.39 (t, J ¼ 3.6, 1 H); 5.01 (d, J ¼ 7.8, 1 H); 4.96 (d, J ¼ 7.2, 1 H); 4.79 (d, J ¼ 1.2, 1 H); 4.67 (dd, J ¼ 2.4,
12.0, 1 H); 4.33–4.28 (m, 3 H); 4.24–4.20 (m, 2 H); 4.18 (d, J ¼ 9, 1 H); 4.15 (t, J ¼ 9, 1 H); 4.08 (t, J ¼
8.4, 1 H); 4.03 (t, J ¼ 8.4, 1 H); 4.01–3.96 (m, 1 H); 3.92 (t, J ¼ 8.4, 1 H); 3.68 (d, J ¼ 10.8, 1 H); 3.62 (s,
3 H); 3.58 (t, J ¼ 10.8, 1 H); 3.42–3.36 (m, 1 H); 3.21–3.17 (m, 1 H); 3.09 (dd, J ¼ 4.2, 13.2, 1 H); 2.35
(dd, J ¼ 2.4, 13.8, 1 H); 2.22 (d, J ¼ 13.2, 1 H); 2.16–1.76 (m, 12 H); 1.65–1.63 (m, 2 H); 1.61 (s, 3 H);
1.59–1.56 (m, 1 H); 1.53–1.50 (m, 2 H); 1.41 (td, J¼4.2, 13.8, 1 H); 1.35 (s, 3 H); 1.33–1.30 (m, 2 H); 1.26
(s, 3 H); 1.19 (s, 3 H); 1.09 (d, J¼13.2, 1 H); 0.86 (s, 3 H). 13C-NMR (C5D5N): 177.1; 175.8; 166.9; 144.6;
134.9; 131.2; 128.3; 126.8; 123.5; 106.3; 103.5; 83.0; 78.8; 78.2; 77.5; 76.4; 75.0; 74.2; 71.8; 70.8; 65.4; 64.7;
62.7; 51.7; 48.5; 47.7; 45.9; 44.1; 44.0; 43.3; 43.0; 42.8; 42.3; 41.6; 39.9; 37.9; 36.9; 35.1; 32.9; 31.0; 28.4; 27.9;
26.1; 24.1; 24.0; 23.3; 21.4; 21.0; 18.1; 17.6; 17.3; 14.5. ESI-MS: 987.51 ([M þ H]þ ), 1009.42 ([M þ Na]þ ),
985.52 ([MꢀH]ꢀ ).
Methyl (2b,3b)-3-{[4-O-(b-d-Glucopyranosyl)-b-d-xylopyranosyl]oxy}-2,23-dihydroxy-28-({3-[(4-
methylbenzoyl)amino]propyl}amino)-28-oxoolean-12-en-30-oate (5b). White powder (40.5 mg, 81.0%).
M.p. 136.7–138.18. 1H-NMR (C5D5N): 7.91 (br. s, 1 H); 7.69–7.65 (m, 2 H); 7.18–7.14 (m, 2 H); 6.52 (br.
s, 1 H); 5.39 (t, J ¼ 3.6, 1 H); 5.01 (d, J ¼ 7.8, 1 H); 4.96 (d, J ¼ 7.2, 1 H); 4.79 (d, J ¼ 1.2, 1 H); 4.67 (dd,
J ¼ 2.4, 12.0, 1 H); 4.33–4.28 (m, 3 H); 4.24–4.20 (m, 2 H); 4.18 (d, J ¼ 9, 1 H); 4.15 (t, J ¼ 9, 1 H); 4.08
(t, J ¼ 8.4, 1 H); 4.03 (t, J ¼ 8.4, 1 H); 4.00–3.96 (m, 1 H); 3.92 (t, J ¼ 8.4, 1 H); 3.68 (d, J ¼ 10.8, 1 H);
3.62 (s, 3 H); 3.58 (t, J ¼ 10.8, 1 H); 3.42–3.36 (m, 1 H); 3.27–3.23 (m, 4 H); 3.21–3.16 (m, 1 H); 3.09
(dd, J ¼ 4.2, 13.2, 1 H); 2.65 (dd, J ¼ 2.4, 13.8, 1 H); 2.35 (s, 3 H); 2.22 (d, J ¼ 13.2, 1 H); 2.16–1.76 (m,
12 H); 1.66–1.63 (m, 2 H); 1.61 (s, 3 H); 1.59–1.56 (m, 1 H); 1.53–1.50 (m, 2 H); 1.41 (td, J ¼ 4.2, 13.8,
1 H); 1.35 (s, 3 H); 1.33–1.30 (m, 2 H); 1.26 (s, 3 H); 1.19 (s, 3 H); 1.09 (d, J ¼ 13.2, 1 H); 0.86 (s, 3 H).
13C-NMR (C5D5N): 177.1; 175.8; 166.7; 144.3; 141.6; 132.4; 129.0; 126.8; 124.0; 106.3; 103.5; 84.0; 78.8;
77.8; 77.5; 77.3; 75.0; 74.9; 71.6; 66.9; 65.4; 64.7; 62.7; 51.7; 48.5; 47.7; 45.9; 44.1; 44.0; 43.3; 43.0; 42.8; 42.3;
41.6; 39.9; 37.9; 36.9; 35.1; 32.9; 31.0; 28.4; 27.9; 26.1; 24.1; 24.0; 23.3; 21.4; 21.0; 18.0; 17.6; 17.3; 14.5. ESI-
MS: 1001.56 ([M þ H]þ ), 1023.48 ([M þ Na]þ ), 999.54 ([MꢀH]ꢀ ).
Methyl (2b,3b)-3-{[4-O-(b-d-Glucopyranosyl)-b-d-xylopyranosyl]oxy}-2,23-dihydroxy-28-({3-[(4-
hydroxybenzoyl)amino]propyl}amino)-28-oxoolean-12-en-30-oate (5c). White powder (41.6 mg,
83.0%). M.p. 179.7–181.38. 1H-NMR (C5D5N): 7.92 (br. s, 1 H); 7.70–7.66 (m, 2 H); 6.85–6.81 (m,
2 H); 6.52 (br. s, 1 H); 5.38 (t, J ¼ 3.6, 1 H); 5.02 (d, J ¼ 7.8, 1 H); 4.94 (d, J ¼ 7.2, 1 H); 4.74 (d, J ¼ 1.2,
1 H); 4.67 (dd, J ¼ 2.4, 12.0, 1 H); 4.33–4.28 (m, 3 H); 4.24–4.20 (m, 2 H); 4.18 (d, J ¼ 9, 1 H); 4.15 (t,
J ¼ 9, 1 H); 4.08 (t, J ¼ 8.4, 1 H); 4.03 (t, J ¼ 8.4, 1 H); 4.01–3.96 (m, 1 H); 3.92 (t, J¼8.4, 1 H); 3.68 (d,
J ¼ 10.8, 1 H); 3.62 (s, 3 H); 3.58 (t, J ¼ 10.8, 1 H); 3.42–3.36 (m, 1 H); 3.26–3.23 (m, 4 H); 3.21–3.17
(m, 1 H); 3.09 (dd, J¼4.2, 13.2, 1 H); 2.65 (dd, J ¼ 2.4, 13.8, 1 H); 2.22 (d, J ¼ 13.2, 1 H); 2.16–1.76 (m,
12 H); 1.65–1.63 (m, 2 H); 1.61 (s, 3 H); 1.59–1.57 (m, 1 H); 1.52–1.50 (m, 2 H); 1.41 (td, J ¼ 4.2, 13.8,
1 H); 1.35 (s, 3 H); 1.33–1.30 (m, 2 H); 1.26 (s, 3 H); 1.19 (s, 3 H); 1.09 (d, J ¼ 13.2, 1 H); 0.86 (s, 3 H).
13C-NMR (C5D5N): 177.1; 175.8; 167.0; 160.8; 144.3; 129.0; 125.4; 124.0; 114.5; 106.3; 103.5; 84.0; 78.8;
77.8; 77.5; 77.3; 75.0; 74.9; 71.6; 66.9; 65.4; 64.7; 62.7; 51.7; 48.5; 47.7; 45.9; 44.1; 44.0; 43.3; 43.0; 42.8; 42.3;

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
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41.6; 39.9; 37.9; 36.9; 35.1; 32.9; 31.0; 28.4; 27.9; 26.1; 24.1; 24.0; 23.3; 21.4; 18.0; 17.6; 17.3; 14.5. ESI-MS:
1003.54 ([M þ H]þ ), 1025.51 ([M þ Na]þ ), 1001.52 ([MꢀH]ꢀ ).
Methyl (2b,3b)-3-{[4-O-(b-d-Glucopyranosyl)-b-d-xylopyranosyl]oxy}-2,23-dihydroxy-28-({3-[(4-
methoxybenzoyl)amino]propyl}amino)-28-oxoolean-12-en-30-oate (5d). White powder (40.2 mg,
79.1%). M.p. 241.9–243.28 1H-NMR (C5D5N): 7.95 (br. s, 1 H); 7.79–7.75 (m, 2 H); 6.89–6.85 (m,
2 H); 6.50 (br. s, 1 H); 5.39 (t, J ¼ 3.6, 1 H); 5.01 (d, J ¼ 7.8, 1 H); 4.93 (d, J ¼ 7.2, 1 H); 4.75 (d, J ¼ 1.2,
1 H); 4.68 (dd, J ¼ 2.4, 12.0, 1 H); 4.33–4.28 (m, 3 H); 4.24–4.20 (m, 2 H); 4.18 (d, J ¼ 9, 1 H); 4.15 (t,
J ¼ 9, 1 H); 4.08 (t, J ¼ 8.4, 1 H); 4.03 (t, J ¼ 8.4, 1 H); 4.01–3.96 (m, 1 H); 3.92 (t, J ¼ 8.4, 1 H); 3.68 (d,
J ¼ 10.8, 1 H); 3.79 (s, 3 H); 3.62 (s, 3 H); 3.58 (t, J ¼ 10.8, 1 H); 3.42–3.36 (m, 1 H); 3.26–3.22 (m, 4 H);
3.20–3.17 (m, 1 H); 3.09 (dd, J ¼ 4.2, 13.2, 1 H); 2.65 (dd, J ¼ 2.4, 13.8, 1 H); 2.22 (d, J ¼ 13.2, 1 H);
2.16–1.76 (m, 12 H); 1.65–1.63 (m, 2 H); 1.61 (s, 3 H); 1.59–1.57 (m, 1 H); 1.52–1.50 (m, 2 H); 1.41 (td,
J ¼ 4.2, 13.8, 1 H); 1.35 (s, 3 H); 1.33–1.30 (m, 2 H); 1.26 (s, 3 H); 1.19 (s, 3 H); 1.09 (d, J¼13.2, 1 H);
0.86 (s, 3 H). 13C-NMR (C5D5N): 177.1; 175.8; 166.9; 162.0; 144.3; 128.8; 126.6; 124.0; 113.5; 106.3; 103.5;
84.0; 78.8; 77.8; 77.5; 77.3; 75.0; 74.9; 71.6; 66.9; 65.4; 64.7; 62.7; 55.1; 51.7; 48.5; 47.7; 45.9; 44.1; 44.0; 43.3;
43.0; 42.8; 42.3; 41.6; 39.9; 37.9; 36.9; 35.1; 32.9; 31.0; 28.4; 27.9; 26.1; 24.1; 24.0; 23.3; 21.4; 18.0; 17.6; 17.3;
14.5. ESI-MS: 1017.55 ([M þ H]þ ), 1039.50 ([M þ Na]þ ), 1015.54 ([MꢀH]ꢀ ).
Methyl (2b,3b)-28-[(3-{[4-(Dimethylamino)benzoyl]amino}propyl)amino]-3-{[4-O-(b-d-glucopyra-
nosyl)-b-d-xylopyranosyl]oxy}-2,23-dihydroxy-28-oxoolean-12-en-30-oate (5e). White powder (43.8 mg,
85.1%). M.p. 173.7–176.88. 1H-NMR (C5D5N): 7.94 (br. s, 1 H); 7.76–7.72 (m, 2 H); 6.75–6.72 (m, 2 H);
6.51 (br. s, 1 H); 5.39 (t, J ¼ 3.6, 1 H); 5.01 (d, J ¼ 7.8, 1 H); 4.93 (d, J ¼ 7.2, 1 H); 4.75 (d, J ¼ 1.2, 1 H);
4.68 (dd, J ¼ 2.4, 12.0, 1 H); 4.33–4.28 (m, 3 H); 4.24–4.20 (m, 2 H); 4.18 (d, J ¼ 9, 1 H); 4.15 (t, J ¼ 9,
1 H); 4.08 (t, J ¼ 8.4, 1 H); 4.03 (t, J ¼ 8.4, 1 H); 4.00–3.96 (m, 1 H); 3.92 (t, J ¼ 8.4, 1 H); 3.68 (d, J ¼
10.8, 1 H); 3.62 (s, 3 H); 3.58 (t, J ¼ 10.8, 1 H); 3.41–3.38 (m, 1 H); 3.27–3.25 (m, 4 H); 3.21–3.18 (m,
1 H); 3.10 (dd, J ¼ 4.2, 13.2, 1 H); 3.08 (s, 6 H); 2.65 (dd, J ¼ 2.4, 13.8, 1 H); 2.22 (d, J ¼ 13.2, 1 H); 2.16–
1.76 (m, 12 H); 1.66–1.64 (m, 2 H); 1.61 (s, 3 H); 1.60–1.57 (m, 1 H); 1.53–1.49 (m, 2 H); 1.41 (td, J ¼
4.2, 13.8, 1 H); 1.35 (s, 3 H); 1.33–1.30 (m, 2 H); 1.26 (s, 3 H); 1.19 (s, 3 H); 1.09 (d, J¼13.2, 1 H); 0.86 (s,
3 H). 13C-NMR (C5D5N): 177.1; 175.8; 167.1,151.8; 144.3; 128.8; 121.1; 124.0; 110.7; 106.3; 103.5; 83.0;
78.8; 77.8; 77.5; 77.3; 75.0; 74.9; 71.6; 66.9; 65.4; 64.7; 62.7; 51.7; 48.5; 47.7; 45.9; 44.1; 44.0; 43.3; 43.0; 42.8;
42.3; 41.6; 39.9; 39.5; 37.9; 36.9; 35.1; 32.9; 31.0; 28.4; 27.9; 26.1; 24.1; 24.0; 23.3; 21.4; 18.0; 17.6; 17.3; 14.5.
ESI-MS: 1030.58 ([M þ H]þ ), 1052.47 ([M þ Na]þ ), 1028.57 ([MꢀH]ꢀ ).
Methyl (2b,3b)-3-{[4-O-(b-d-Glucopyranosyl)-b-d-xylopyranosyl]oxy}-2,23-dihydroxy-28-oxo-28-
({3-[(3-phenylpropanoyl)amino]propyl}amino)olean-12-en-30-oate (5f). White powder (43.5 mg,
1
86.1%). M.p. 177.5–180.18. H-NMR (C5D5N): 7.29 (m, 2 H); 7.26–7.23 (m, 2 H); 7.22–7.20 (m, 1 H);
6.97 (br. s, 1 H); 6.50 (br. s, 1 H); 5.40 (t, J ¼ 3.6, 1 H); 5.01 (d, J ¼ 7.8, 1 H); 4.96 (d, J ¼ 7.2, 1 H); 4.79 (d,
J ¼ 1.2, 1 H); 4.67 (dd, J ¼ 2.4, 12.0, 1 H); 4.33–4.28 (m, 3 H); 4.24–4.20 (m, 2 H); 4.18 (d, J ¼ 9, 1 H);
4.15 (t, J ¼ 9, 1 H); 4.08 (t, J ¼ 8.4, 1 H); 4.03 (t, J ¼ 8.4, 1 H); 4.01–3.96 (m, 1 H); 3.92 (t, J ¼ 8.4, 1 H);
3.68 (d, J ¼ 10.8, 1 H); 3.62 (s, 3 H); 3.58 (t, J ¼ 10.8, 1 H); 3.41–3.37 (m, 1 H); 3.27–3.23 (m, 4 H); 3.20–
3.17 (m, 1 H); 3.09 (dd, J ¼ 4.2, 13.2, 1 H); 2.98–2.95 (m, 2 H); 2.43–2.40 (m, 2 H); 2.35 (dd, J ¼ 2.4,
13.8, 1 H); 2.22 (d, J ¼ 13.2, 1 H); 2.16–1.76 (m, 12 H); 1.65–1.63 (m, 2 H); 1.61 (s, 3 H); 1.59–1.56 (m,
1 H); 1.52–1.48 (m, 2 H); 1.41 (td, J ¼ 4.2, 13.8, 1 H); 1.35 (s, 3 H); 1.33–1.30 (m, 2 H); 1.26 (s, 3 H); 1.20
(s, 3 H); 1.08 (d, J ¼ 13.2, 1 H); 0.86 (s, 3 H). 13C-NMR (C5D5N): 177.1; 174.1; 170.3; 142.3; 140.8; 128.7;
128.5; 126.2; 124.0; 106.5; 103.7; 83.0; 78.9; 77.8; 77.5; 77.3; 75.0; 74.9; 74.8; 71.8; 70.8; 65.4; 64.7; 62.7;
51.7; 48.5; 47.7; 45.9; 44.1; 44.0; 43.3; 43.0; 42.8; 42.3; 41.6; 39.9; 37.9; 37.8; 36.9; 35.1; 32.9; 31.0; 30.6; 28.4;
27.9; 26.1; 24.1; 24.0; 23.3; 18.0; 17.6; 17.3; 14.5. ESI-MS: 1015.57 ([M þ H]þ ), 1037.49 ([M þ Na]þ ),
1013.56 ([MꢀH]ꢀ ).
Methyl (2b,3b)-28-({3-[(N-Acetylglycyl)amino]propyl}amino)-3-{[4-O-(b-d-glucopyranosyl)-b-d-
xylopyranosyl]oxy}-2,23-dihydroxy-28-oxoolean-12-en-30-oate (5g). White powder (42.2 mg, 90.0%).
M.p. 115.2–117.18. 1H-NMR (C5D5N): 7.71 (br. s, 1 H); 7.21 (br. s,1 H); 6.50 (br. s, 1 H); 5.37 (t, J ¼ 3.5,
1 H); 5.02 (d, J ¼ 7.8, 1 H); 4.96 (d, J ¼ 7.2, 1 H); 4.79 (d, J ¼ 1.2, 1 H); 4.67 (dd, J ¼ 2.4, 12.0, 1 H); 4.35–
4.30 (m, 3 H); 4.24–4.20 (m, 2 H); 4.18 (d, J ¼ 9, 1 H); 4.15 (t, J ¼ 9, 1 H); 4.08 (t, J ¼ 8.4, 1 H); 4.03 (t,
J ¼ 8.4, 1 H); 4.01–3.96 (m, 1 H); 3.92 (t, J ¼ 8.4, 1 H); 3.73 (s, 2 H); 3.68 (d, J ¼ 10.8, 1 H); 3.62 (s, 3 H);
3.58 (t, J ¼ 10.8, 1 H); 3.42–3.36 (m, 1 H); 3.27–2.23 (m, 4 H); 3.20–3.18 (m, 1 H); 3.09 (dd, J ¼ 4.2,
13.2, 1 H); 2.35 (dd, J ¼ 2.4, 13.8, 1 H); 2.22 (d, J ¼ 13.2, 1 H); 2.16–1.76 (m, 12 H); 1.93 (s, 3 H); 1.65–

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1.63 (m, 2 H); 1.61 (s, 3 H); 1.59–1.56 (m, 1 H); 1.5 1(m, 2 H); 1.41 (td, J ¼ 4.2, 13.8, 1 H); 1.35 (s, 3 H);
1.33–1.30 (m, 2 H); 1.26 (s, 3 H); 1.19 (s, 3 H); 1.09 (d, J ¼ 13.2, 1 H); 0.86 (s, 3 H). 13C-NMR (C5D5N):
177.1; 174.1; 173.5; 171.37, 142.3; 123.5; 106.3; 103.5; 83.0; 78.8; 77.8; 77.5; 77.2; 75.0; 74.9; 71.6; 66.9; 64.3;
64.1; 63.3; 51.8; 47.7; 47.6; 45.9; 43.1; 42.7; 43.3; 43.0; 42.9; 42.3; 41.6; 39.9; 37.9; 36.9; 35.1; 32.9; 31.0; 28.4;
27.9; 26.1; 24.1; 24.0; 23.3; 18.1; 17.9. ESI-MS: 982.55 ([M þ H]þ ), 980.53([MꢀH]ꢀ ).
Methyl (2b,3b)-28-{[3-(Benzoylamino)propyl]amino}-2,3,23-trihydroxy-28-oxoolean-12-en-30-oate
1
(6a). White powder (31.2 mg, 90.0%). M.p. 191.4–193.18. H-NMR (C5D5N): 7.76 (d, 2 H); 7.47–7.42
(m, 3 H); 6.86 (br. s, 1 H); 6.52 (br. s, 1 H); 4.26–4.22 (m, 1 H); 4.14 (d, J ¼ 10.2, 1 H); 3.71 (d, J ¼ 10.2,
1 H); 3.67 (s, 3 H); 3.35–3.30 (m, 2 H); 3.14–3.09 (m, 2 H); 3.02 (d, J¼10.2, 1 H); 2.35 (d, J ¼ 14.4, 1 H);
2.20 (d, J ¼ 12.6, 1 H); 2.14–2.10 (m, 3 H); 2.07–2.01 (m, 4 H); 1.90–1.76 (m, 5 H); 1.73–1.71 (m, 1 H);
1.67–1.64 (m, 1 H); 1.63 (s, 3 H); 1.59–1.53 (m, 2 H); 1.47 (s, 3 H); 1.43–1.41 (m, 1 H); 1.38 (s, 3 H);
1.32–1.26 (m, 2 H); 1.23 (s, 3 H); 1.06 (d, J¼13.8, 1 H); 0.86 (s, 3 H). 13C-NMR (C5D5N): 177.1; 175.8;
167.0; 144.4; 134.9; 131.2; 128.3; 126.8; 123.3; 73.0; 71.5; 67.7; 51.6; 48.5; 48.1; 46.1; 44.8; 44.1; 43.3; 42.7;
42.4; 42.2; 39.8; 38.3; 37.9; 37.2; 34.5; 33.0; 30.8; 30.6; 28.4; 28.3; 26.2; 24.0; 23.9; 18.2; 17.4; 17.2; 14.5. ESI-
MS: 693.48 ([M þ H]þ ), 715.50 ([M þ Na]þ ), 691.43 ([MꢀH]ꢀ ).
Methyl (2b,3b)-2,3,23-Trihydroxy-28-({3-[(4-methylbenzoyl)amino]propyl}amino)-28-oxoolean-12-
en-30-oate (6b). White powder (29.9 mg, 84.6%). M.p. 181.2–183.78. 1H-NMR (C5D5N): 7.95 (br. s, 1 H);
7.68 (d, 2 H); 7.15 (d, 2 H); 6.52 (br. s, 1 H); 4.26–4.22 (m, 1 H); 4.14 (d, J ¼ 10.2, 1 H); 3.71 (d, J ¼ 10.2,
1 H); 3.67 (s, 3 H); 3.34–3.30 (m, 2 H); 3.13–3.09 (m, 2 H); 3.02 (d, J ¼ 10.2, 1 H); 2.35 (d, J ¼ 14.4,
1 H); 2.31 (s, 3 H); 2.20 (d, J ¼ 12.6, 1 H); 2.14–2.10 (m, 3 H); 2.07–2.01 (m, 4 H); 1.90–1.76 (m, 5 H);
1.74–1.70 (m, 1 H); 1.66–1.64 (m, 1 H); 1.63 (s, 3 H); 1.62–1.59 (m, 2 H); 1.47 (s, 3 H); 1.44–1.40 (m,
1 H); 1.38 (s, 3 H); 1.32–1.26 (m, 2 H); 1.23 (s, 3 H); 1.06 (d, J ¼ 13.8, 1 H); 0.86 (s, 3 H). 13C-NMR
(C5D5N): 177.1; 175.8; 166.8; 144.4; 141.7; 132.3; 129.1; 126.9; 123.3; 73.0; 71.5; 67.7; 51.6; 48.5; 48.1; 46.1;
44.8; 44.1; 43.3; 42.7; 42.4; 42.2; 39.8; 38.3; 36.6; 35.2; 34.5; 33.0; 30.8; 30.6; 29.7; 28.4; 28.3; 26.2; 24.0;
23.9; 18.2; 17.4; 17.2; 14.5. ESI-MS: 707.46 ([M þ H]þ ), 729.44 ([M þ Na]þ ), 705.45 ([MꢀH]ꢀ ).
Methyl (2b,3b)-2,3,23-Trihydroxy-28-({3-[(4-hydroxybenzoyl)amino]propyl}amino)-28-oxoolean-
1
12-en-30-oate (6c). White powder (27.0 mg, 76.0%). M.p. 143.4–146.18. H-NMR (C5D5N): 7.93 (br. s,
1 H); 7.72 (d, 2 H); 6.83 (d, 2 H); 6.51 (br. s, 1 H); 4.38 (br. s, 1 H); 4.26–4.22 (m, 1 H); 4.14 (d, J ¼ 10.2,
1 H); 3.71 (d, J ¼ 10.2, 1 H); 3.67 (s, 3 H); 3.33–3.29 (m, 2 H); 3.12–3.08 (m, 2 H); 3.02 (d, J ¼ 10.2,
1 H); , 2.35 (d, J ¼ 14.4, 1 H); 2.20 (d, J ¼ 12.6, 1 H); 2.14–2.10 (m, 3 H); 2.07–2.01 (m, 4 H); 1.90–1.76
(m, 5 H); 1.73- 1.70 (m, 1 H); 1.68–1.64 (m, 1 H); 1.63 (s, 3 H); 1.62–1.60 (m, 2 H); 1.47 (s, 3 H); 1.44–
1.40 (m, 1 H); 1.38 (s, 3 H); 1.32–1.26 (m, 2 H); 1.23 (s, 3 H); 1.06 (d, J ¼ 13.8, 1 H); 0.86 (s, 3 H).
13C-NMR (C5D5N): 177.1; 175.8; 166.9; 160.8; 144.4; 129.0; 125.5; 123.3; 73.0; 71.5; 67.7; 51.6; 48.5; 48.1;
46.1; 44.8; 44.1; 43.3; 42.7; 42.4; 42.2; 39.8; 38.3; 36.6; 35.2; 34.5; 33.0; 30.8; 30.6; 29.7; 28.4; 28.3; 26.2;
24.0; 23.9; 18.2; 17.4; 17.2; 14.5. ESI-MS: 709.44 ([M þ H]þ ), 707.45 ([MꢀH]ꢀ ).
Methyl (2b,3b)-2,3,23-Trihydroxy-28-({3-[(4-methoxybenzoyl)amino]propyl}amino)-28-oxoolean-
1
12-en-30-oate (6d). White powder (29.4 mg, 81.3%). M.p. 105.9–108.38. H-NMR (C5D5N): 7.92 (br. s,
1 H); 7.78 (d, 2 H); 6.69 (d, 2 H); 6.52 (br. s, 1 H); 4.26–4.22 (m, 1 H); 4.14 (d, J ¼ 10.2, 1 H); 3.79 (s,
3 H); 3.71 (d, J ¼ 10.2, 1 H); 3.67 (s, 3 H); 3.33–3.29 (m, 2 H); 3.14–3.10 (m, 2 H); 3.02 (d, J ¼ 10.2,
1 H); 2.35 (d, J ¼ 14.4, 1 H); 2.20 (d, J ¼ 12.6, 1 H); 2.14–2.10 (m, 3 H); 2.07–2.01 (m, 4 H); 1.90–1.76
(m, 5 H); 1.73–1.71 (m, 1 H); 1.66–1.64 (m, 1 H); 1.63 (s, 3 H); 1.62–1.59 (m, 2 H); 1.47 (s, 3 H); 1.44–
1.40 (m, 1 H); 1.38 (s, 3 H); 1.32–1.26 (m, 2 H); 1.23 (s, 3 H); 1.06 (d, J ¼ 13.8, 1 H); 0.86 (s, 3 H).
13C-NMR (C5D5N): 177.1; 175.8; 166.9; 162.0; 144.4; 128.7; 126.7; 123.3; 73.0; 71.5; 67.7; 55.1; 51.6; 48.5;
48.1; 46.1; 44.8; 44.1; 43.3; 42.7; 42.4; 42.2; 39.8; 38.3; 36.6; 35.2; 34.5; 33.0; 30.8; 30.6; 29.7; 28.4; 28.3;
26.2; 24.0; 23.9; 18.2; 17.4; 17.2; 14.5. ESI-MS: 723.46 ([M þ H]þ ), 745.44 ([M þ Na]þ ), 721.44 ([Mꢀ
H]ꢀ ).
Methyl (2b,3b)-28-[(3-{[4-(Dimethylamino)benzoyl]amino}propyl)amino]-2,3,23-trihydroxy-28-
1
oxoolean-12-en-30-oate (6e). White powder (30.3 mg, 82.4%). M.p. 100.8–102.38. H-NMR (C5D5N):
7.95 (br. s, 1 H); 7.73 (d, 2 H); 6.74 (d, 2 H); 6.52 (br. s, 1 H); 4.26–4.22 (m, 1 H); 4.14 (d, J ¼ 10.2, 1 H);
3.79 (s, 3 H); 3.71 (d, J ¼ 10.2, 1 H); 3.67 (s, 3 H); 3.33–3.30 (m, 2 H); 3.12–3.09 (m, 2 H); 3.08 (s, 6 H);
3.02 (d, J ¼ 10.2, 1 H); 2.35 (d, J ¼ 14.4, 1 H); 2.20 (d, J ¼ 12.6, 1 H); 2.14–2.10 (m, 3 H); 2.07–2.01 (m,
4 H); 1.90–1.76 (m, 5 H); 1.73–1.71 (m, 1 H); 1.67–1.64 (m, 1 H); 1.63 (s, 3 H); 1.62–1.60 (m, 2 H); 1.47
(s, 3 H); 1.44–1.40 (m, 1 H); 1.38 (s, 3 H); 1.32–1.26 (m, 2 H); 1.23 (s, 3 H); 1.06 (d, J ¼ 13.8, 1 H); 0.86

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1843
(s, 3 H). 13C-NMR (C5D5N): 177.1; 175.8; 167.1; 151.8; 144.4; 128.8; 123.3; 121.1; 110.7; 73.0; 71.5; 67.7;
51.6; 48.5; 48.1; 46.1; 44.8; 44.1; 43.3; 42.7; 42.4; 42.2; 39.8; 39.5; 39.5; 38.3; 36.6; 35.2; 34.5; 33.0; 30.8;
30.6; 29.8; 28.4; 28.3; 26.2; 24.0; 23.9; 18.2; 17.4; 17.2; 14.5. ESI-MS: 736.49 ([M þ H]þ ), 758.47 [M þ
Na]þ , 734.47 ([MꢀH]ꢀ ).
Methyl (2b,3b)-2,3,23-Trihydroxy-28-oxo-28-({3-[(3-phenylpropanoyl)amino]propyl}amino)olean-
12-en-30-oate (6f). White powder (30.3 mg, 84.1%). M.p. 93.4–95.38. 1H-NMR (C5D5N): 7.29–7.21
(m, 5 H); 6.98 (br. s, 1 H); 6.51 (br. s, 1 H); 4.26–4.22 (m, 1 H); 4.1 3(d, J ¼ 10.2, 1 H); 3.71 (d, J ¼ 10.2,
1 H); 3.67 (s, 3 H); 3.25–3.23 (m, 2 H); 3.22–3.20 (m, 2 H); 3.02 (d, J ¼ 10.2, 1 H); 2.97 (t, J ¼ 2.1, 7.8,
2 H); 2.41 (t, J ¼ 2.3, 8.1, 2 H); 2.34 (d, J ¼ 14.4, 1 H); 2.20 (d, J ¼ 12.6, 1 H); 2.14–2.10 (m, 3 H); 2.07–
2.01 (m, 4 H); 1.90–1.76 (m, 5 H); 1.73–1.71 (m, 1 H); 1.67–1.64 (m, 1 H); 1.63 (s, 3 H); 1.62–1.60 (m,
2 H); 1.47 (s, 3 H); 1.44–1.40 (m, 1 H); 1.38 (s, 3 H); 1.32–1.26 (m, 2 H); 1.23 (s, 3 H); 1.06 (d, J ¼ 13.8,
1 H); 0.86( s, 3 H). 13C-NMR (C5D5N): 177.1; 174.1; 170.3; 144.4; 140.8; 128.7; 128.5; 126.2; 123.3; 73.0;
71.5; 67.7; 51.6; 48.5; 48.1; 46.1; 44.8; 44.1; 43.3; 42.7; 42.4; 42.2; 39.8; 38.3; 37.9; 37.8; 37.2; 34.5; 33.0; 30.8;
30.7; 30.6; 28.4; 28.3; 26.2; 24.0; 23.9; 18.2; 17.4; 17.2; 14.5. ESI-MS: 721.48 ([M þ H]þ ), 743.45 ([M þ
Na]þ ), 719.46 ([MꢀH]ꢀ ).
Methyl (2b,3b)-28-({3-[(N-Acetylglycyl)amino]propyl}amino)-2,3,23-trihydroxy-28-oxoolean-12-en-
1
30-oate (6g). White powder (27.7 mg, 80.6%). M.p. 103.6–104.38. H-NMR (C5D5N): 7.70 (br. s, 1 H);
7.20 (br. s, 1 H); 6.52 (br. s, 1 H); 4.26–4.22 (m, 1 H); 4.14 (d, J ¼ 10.2, 1 H); 3.73 (s, 2 H); 3.71 (d, J ¼
10.2, 1 H); 3.67 (s, 3 H); 3.29–3.25 (m, 2 H); 3.24–3.20 (m, 2 H); 3.02 (d, J ¼ 10.2, 1 H); 2.35 (d, J ¼ 14.4,
1 H); 2.20 (d, J ¼ 12.6, 1 H); 2.14–2.10 (m, 3 H); 2.07–2.01 (m, 4 H); 1.90–1.76 (m, 5 H); 1.74–1.70 (m,
1 H); 1.68–1.66 (m, 1 H); 1.64–1.63 (m, 2 H);1.62 (s, 3 H); 1.47 (s, 3 H); 1.44–1.40 (m, 1 H); 1.38 (s,
3 H); 1.32–1.26 (m, 2 H); 1.23 (s, 3 H); 1.06 (d, J ¼ 13.8, 1 H); 0.86 (s, 3 H). 13C-NMR (C5D5N): 177.1;
174.1; 173.6; 171.4; 144.4; 123.3; 73.0; 71.5; 67.7; 51.6; 48.5; 48.1; 46.1; 44.8; 44.1; 43.3; 42.9; 42.7; 42.4;
42.2; 38.9; 38.8; 38.3; 37.9; 37.2; 34.5; 33.0; 30.8; 29.6; 28.4; 28.3; 26.2; 24.0; 23.9; 22.9; 18.2; 17.4; 17.2; 14.5.
ESI-MS: 688.45 ([M þ H]þ ), 686.46 ([MꢀH]ꢀ ).
General Procedure for Preparing Compounds 5h–5j or 6h–6j. Fmoc-Amino acid (0.06 mmol) was
dissolved in dry DMF/THF 1:3 (v/v). To this soln., DCC (18.6 mg, 0.09 mmol) and HOBt (12.2 mg,
0.09 mmol) were added. After stirring for 1 h at r. t., 3 (44.1 mg, 0.05 mmol) or 4 (29.4 mg, 0.05 mmol)
was added, and the mixture was stirred at 608 under N2 for 4–8 h. The precipitate was filtered, and the
filtrate was concentrated in vacuo. The residue was subjected to a FC (silica gel; MeOH/CH2Cl2) to
obtain the intermediate, and its structure was confirmed by MS and NMR. Then, the intermediate was
dissolved in Et2NH/DMF 1:4 (v/v). After stirring for 2 h, the mixture was concentrated in vacuo, and the
residue was subjected to a FC (silica gel; MeOH/CH2Cl2) and was repurified by RP-HPLC (MeOH/
H2O) to provide the compounds 5h–5j or 6h–6j.
Methyl (2b,3b)-3-{[4-O-(b-d-Glucopyranosyl)-b-d-xylopyranosyl]oxy}-2,23-dihydroxy-28-oxo-28-
{[3-(l-phenylalanylamino)propyl]amino}olean-12-en-30-oate (5h). White powder (36.6 mg, 71.0%).
1
M.p. >2508. H-NMR (C5D5N): 7.17 (br. s, 1 H); 7.11–7.15 (m, 5 H); 6.50 (br. s, 1 H); 5.39 (t, J ¼ 3.6,
1 H); 5.01 (d, J ¼ 7.8, 1 H); 4.96 (d, J ¼ 7.2, 1 H); 4.91 (br. s, 2 H); 4.79 (d, J ¼ 1.2, 1 H); 4.67 (dd, J ¼ 2.4,
12.0, 1 H); 4.33–4.28 (m, 3 H); 4.24–4.20 (m, 2 H); 4.18 (d, J ¼ 9, 1 H); 4.15 (t, J ¼ 9, 1 H); 4.08 (t, J ¼
8.4, 1 H); 4.03 (t, J ¼ 8.4, 1 H); 4.01–3.96 (m, 1 H); 3.92 (t, J ¼ 8.4, 1 H); 3.71 (t, 1 H); 3.68 (d, J ¼ 10.8,
1 H); 3.62 (s, 3 H); 3.58 (t, J ¼ 10.8, 1 H); 3.42–3.36 (m, 1 H); 3.25–3.21 (m, 2 H); 3.20–3.18 (m, 1 H);
3.09 (dd, J ¼ 4.2, 13.2, 1 H); 3.09–3.05 (m, 2 H); 3.01–2.97 (m, 2 H); 2.35 (dd, J ¼ 2.4, 13.8, 1 H); 2.22 (d,
J ¼ 13.2, 1 H); 2.16–1.76 (m, 12 H); 1.66–1.63 (m, 2 H); 1.61 (s, 3 H); 1.59–1.57 (m, 1 H); 1.53–1.50 (m,
2 H); 1.41 (td, J ¼ 4.2, 13.8, 1 H); 1.35 (s, 3 H); 1.33–1.30 (m, 2 H); 1.26 (s, 3 H); 1.19 (s, 3 H); 1.09 (d, J ¼
13.2, 1 H); 0.86 (s, 3 H). 13C-NMR (C5D5N): 177.1; 175.0; 174.8; 144.3; 138.2; 129.2; 128.4; 126.3; 123.5;
106.3; 103.5; 83.0; 78.8; 78.2; 77.5; 76.4; 75.0; 74.2; 71.8; 70.8; 65.4; 64.7; 62.7; 56.5; 51.6; 48.5; 47.7; 45.9;
44.1; 44.0; 43.3; 43.0; 42.8; 42.3; 41.6; 41.1; 39.9; 37.9; 36.9; 35.1; 32.9; 31.0; 28.4; 27.9; 26.1; 24.0; 24.0; 23.2;
18.0; 17.6; 17.3; 14.5. ESI-MS: 1030.58 ([M þ H]þ ), 1028.57 ([MꢀH]ꢀ ) .
Methyl (2b,3b)-3-{[4-O-(b-d-Glucopyranosyl)-b-d-xylopyranosyl]oxy}-28-{[3-(l-glutaminylamino)-
propyl]amino}-2,23-dihydroxy-28-oxoolean-12-en-30-oate (5i). White powder (40.9 mg, 81.1%). M.p.
175.5–177.68. 1H-NMR (C5D5N): 7.15 (br. s, 1 H); 6.83 (br. s, 2 H); 6.52 (br. s, 1 H); 5.39 (t, J ¼ 3.6, 1 H);
5.01 (d, J ¼ 7.8, 1 H); 4.96 (d, J ¼ 7.2, 1 H); 4.91 (br. s, 2 H); 4.79 (d, J ¼ 1.2, 1 H); 4.67 (dd, J ¼ 2.4, 12.0,
1 H); 4.34–4.32 (m, 3 H); 4.30 (br. s, 2 H); 4.24–4.20 (m, 2 H); 4.18 (d, J ¼ 9, 1 H); 4.15 (t, J ¼ 9, 1 H);

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
4.08 (t, J ¼ 8.4, 1 H); 4.03 (t, J ¼ 8.4, 1 H); 4.01–3.96 (m, 1 H); 3.92 (t, J ¼ 8.4, 1 H); , 3.68 (d, J ¼ 10.8,
1 H); 3.62 (s, 3 H); 3.59 (t, J ¼ 10.8, 1 H); 3.58–3.56 (m, 1 H); 3.39 (m, 1 H); 3.25–3.22 (m, 2 H); 3.20–
3.17 (m, 1 H); 3.09 (dd, J ¼ 4.2, 13.2, 1 H); 3.08–3.06 (m, 2 H); 2.39–3.35 (m, 2 H); 2.33 (dd, J ¼ 2.4,
13.8, 1 H); 2.22 (d, J ¼ 13.2, 1 H); 2.16–1.76 (m, 14 H); 1.66–1.62 (m, 2 H); 1.61 (s, 3 H); 1.59–1.57 (m,
1 H); 1.53–1.50 (m, 2 H); 1.41 (td, J ¼ 4.2, 13.8, 1 H); 1.35 (s, 3 H); 1.33–1.30 (m, 2 H); 1.26 (s, 3 H); 1.19
(s, 3 H); 1.09 (d, J ¼ 13.2, 1 H); 0.86 (s, 3 H). 13C-NMR (C5D5N): 177.1; 175.8; 174.6; 174.1; 144.3; 123.5;
106.3; 103.5; 83.0; 78.8; 78.2; 77.5; 76.4; 75.0; 74.2; 71.8; 70.8; 65.4; 64.7; 62.7; 54.0; 51.6; 48.5; 47.7; 45.9;
44.1; 44.0; 43.3; 43.0; 42.8; 42.3; 41.6; 39.9; 37.9; 36.9; 35.1; 32.9; 31.9; 31.0; 28.4; 27.9; 26.1; 25.4;
24.0; 24.0; 23.2; 18.0; 17.6; 17.3; 14.5. ESI-MS: 1011.57 ([M þ H]þ ), 1033.54([M þ Na]þ ), 1009.59
MꢀH]ꢀ ).
Methyl (2b,3b)-28-{[3-(l-Asparaginylamino)propyl]amino}-3-{[4-O-(b-d-glucopyranosyl)-b-d-xy-
lopyranosyl]oxy}-2,23-dihydroxy-28-oxoolean-12-en-30-oate (5j). White powder (42.5 mg, 85.3%).
1
M.p. 174.3–175.58 H-NMR (C5D5N): 7.18 (br. s, 1 H); 6.92 (br. s, 2 H); 6.52 (br. s, 1 H); 5.38 (t, J ¼
3.6, 1 H); 5.01 (d, J ¼ 7.8, 1 H); 4.96 (d, J ¼ 7.2, 1 H); 4.91 (br. s,2 H); 4.79 (d, J ¼ 1.2, 1 H); 4.67 (dd, J ¼
2.4, 12.0, 1 H); 4.34–4.32 (m, 3 H); 4.24–4.20 (m, 2 H); 4.18 (d, J ¼ 9, 1 H); 4.15 (t, J ¼ 9, 1 H); 4.08 (t,
J ¼ 8.4, 1 H); 4.09 (br. s, 2 H); 4.03 (t, J ¼ 8.4, 1 H); 4.01–3.96 (m, 1 H); 3.92 (t, J¼8.4, 1 H); , 3.69 (d,
J ¼ 10.8, 1 H); 3.68–3.66 (m, 1 H); 3.62( s, 3 H); 3.59 (t, J ¼ 10.8, 1 H); 3.40–3.36 (m, 1 H); 3.25–3.22
(m, 2 H); 3.20–3.18 (m, 1 H); 3.09 (dd, J ¼ 4.2, 13.2, 1 H); 3.08–3.06 (m, 2 H); 2.53–2.49 (m, 2 H); 2.33
(dd, J ¼ 2.4, 13.8, 1 H); 2.22 (d, J ¼ 13.2, 1 H); 2.16–1.76 (m, 12 H); 1.65–1.63 (m, 2 H); 1.61 (s, 3 H);
1.59–1.57 (m, 1 H); 1.53–1.49 (m, 2 H); 1.4 1(td, J ¼ 4.2, 13.8, 1 H); 1.35 (s, 3 H); 1.33–1.30 (m, 2 H);
1.26 (s, 3 H); 1.19 (s, 3 H); 1.09 (d, J ¼ 13.2, 1 H); 0.86 (s, 3 H). 13C-NMR (C5D5N): 177.1; 175.2; 175.1;
174.1; 144.3; 123.5; 106.3; 103.5; 83.0; 78.8; 78.2; 77.5; 76.4; 75.0; 74.2; 71.8; 70.8; 65.4; 64.7; 62.7; 54.4;
51.6; 48.5; 47.7; 45.9; 44.1; 44.0; 43.3; 43.0; 42.8; 42.3; 41.6; 39.9; 37.9; 36.9; 35.1; 34.2; 32.9; 31.0; 28.4; 27.9;
26.1; 24.0; 24.0; 23.2; 18.0; 17.6; 17.3; 14.5. ESI-MS: 997.56 ([M þ H]þ ), 1019.54 ([M þ Na]þ ), 995.54
([MꢀH]ꢀ ).
Methyl (2b,3b)-2,3,23-Trihydroxy-28-oxo-28-{[3-(l-phenylalanylamino)propyl]amino}olean-12-en-
30-oate (6h). White powder (32.4 mg, 88.0%). M.p. 107.5–109.88. 1H-NMR (C5D5N): 7.18–7.07 (m,
6 H); 6.52 (br. s, 1 H); 4.96 (br. s, 2 H); 4.26–4.22 (m, 1 H); 4.14 (d, J ¼ 10.2, 1 H); 3.73 (t, J ¼ 2.1, 7.1,
1 H); 3.71 (d, J ¼ 10.2, 1 H); 3.67 (s, 3 H); 3.25–3.21 (m, 2 H); 3.09–3.07 (m, 2 H); 3.02 (d, J ¼ 10.2,
1 H); 2.94 (dd, J ¼ 3.6. 4.2), 2.35 (d, J ¼ 14.4, 1 H); 2.20 (d, J ¼ 12.6, 1 H); 2.14–2.10 (m, 3 H); 2.07–2.01
(m, 4 H); 1.90–1.76 (m, 5 H); 1.74–1.70 (m, 1 H); 1.67–1.64 (m, 1 H); 1.63 (s, 3 H); 1.62–1.60 (m, 2 H);
1.47 (s, 3 H); 1.44–1.40 (m, 1 H); 1.38 (s, 3 H); 1.32–1.26 (m, 2 H); 1.23 (s, 3 H); 1.06 (d, J ¼ 13.8, 1 H);
0.86 (s, 3 H). 13C-NMR (C5D5N): 177.1; 175.0; 174.1; 144.4; 138.2; 129.2; 128.4; 126.3; 123.3; 73.0; 71.5;
67.7; 56.5; 51.7; 48.5; 48.1; 46.1; 44.8; 44.1; 43.3; 42.7; 42.4; 42.2; 41.1; 39.0; 38.9; 37.9; 37.2; 34.5; 33.0; 30.8;
30.6; 28.4; 28.3; 26.2; 24.0; 23.9; 18.2; 17.4; 17.2; 14.5. ESI-MS: 736.49 ([M þ H]þ ), 758.44 ([M þ Na]þ ),
734.47 ([MꢀH]ꢀ ).
Methyl (2b,3b)-28-{[3-(l-Glutaminylamino)propyl]amino}-2,3,23-trihydroxy-28-oxoolean-12-en-30-
oate (6i). White powder (30.1 mg, 84.1%). M.p. 198.1–199.98. 1H-NMR (C5D5N): 7.18 (br. s, 1 H); 6.83
(br. s, 2 H); 6.53 (br. s, 1 H); 4.96 (br. s, 2 H); 4.26–4.22 (m, 1 H); 4.14 (d, J ¼ 10.2, 1 H); 3.71 (d, J ¼
10.2, 1 H); 3.67 (s, 3 H); 3.57 (t, J ¼ 1.2, 1.8), 3.24 (m, 2 H); 3.11–3.09 (m, 2 H); 3.02 (d, J ¼ 10.2, 1 H);
2.94 (dd, J ¼ 3.6. 4.2), 2.36 (t, J ¼ 2.4, 2.4, 2 H); 2.35 (d, J ¼ 14.4, 1 H); 2.20 (d, J ¼ 12.6, 1 H); 2.14–2.12
(m, 3 H); 2.10–2.07 (m, 4 H); 2.05–1.93 (m, 2 H); 1.90–1.76 (m, 5 H); 1.73–1.71 (m, 1 H); 1.68–1.67 (m,
2 H); 1.66–1.64 (m, 1 H); 1.63 (s, 3 H); 1.47 (s, 3 H); 1.44–1.40 (m, 1 H); 1.38 (s, 3 H); 1.32–1.26 (m,
2 H); 1.23 (s, 3 H); 1.06 (d, J ¼ 13.8, 1 H); 0.86 (s, 3 H). 13C-NMR (C5D5N): 177.1; 175.9; 174.6; 174.0;
144.4; 123.3; 73.0; 71.5; 67.7; 54.0; 51.7; 48.5; 48.1; 46.1; 44.8; 44.1; 43.3; 42.7; 42.4; 42.2; 41.1; 39.0; 38.9;
37.9; 37.2; 34.5; 33.0; 31.9; 30.8; 30.6; 28.4; 28.3; 26.2; 25.4; 24.0; 23.9; 18.2; 17.4; 17.2; 14.5. ESI-MS: 717.47
([M þ H]þ ), 739.43 ([M þ Na]þ ), 715.46 ([MꢀH]ꢀ ).
Methyl (2b,3b)-28-{[3-(l-Asparaginylamino)propyl]amino}-2,3,23-trihydroxy-28-oxoolean-12-en-
1
30-oate (6j). White powder (28.7 mg, 81.6%). M.p. 176.6–177.88. H-NMR (C5D5N): 7.18 (br. s, 1 H);
6.91 (br. s, 2 H); 6.52 (br. s, 1 H); 4.26–4.22 (m, 1 H); 4.14 (d, J ¼ 10.2, 1 H); 3.71 (d, J ¼ 10.2, 1 H); 3.68
(s, 3 H); 3.66 (t, J ¼ 1.2, 1.8), ,3.23 (m, 2 H); 3.05–3.03 (m, 2 H); 3.02 (d, J ¼ 10.2, 1 H); 2.65–2.61 (m,
2 H); 2.36 (t, J ¼ 2.4, 4.8, 2 H); 2.35 (d, J ¼ 14.4, 1 H); 2.20 (d, J ¼ 12.6, 1 H); 2.14–2.12 (m, 3 H); 2.10–
2.07 (m, 4 H); 1.90–1.76 (m, 5 H); 1.73–1.71 (m, 1 H); 1.70–1.68 (m, 2 H); 1.66–1.64 (m, 1 H); 1.63 (s,

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1845
3 H); 1.47 (s, 3 H); 1.44–1.40 (m, 1 H); 1.38 (s, 3 H); 1.32–1.26 (m, 2 H); 1.23 (s, 3 H); 1.06 (d, J ¼ 13.8,
1 H); 0.86 (s, 3 H). 13C-NMR (C5D5N): 177.1; 175.2; 175.1; 174.1; 144.4; 123.3; 73.0; 71.5; 67.7; 51.7; 50.4;
48.5; 48.1; 46.1; 44.8; 44.1; 43.3; 42.7; 42.4; 42.2; 39.0; 38.9; 37.9; 37.2; 34.2; 33.0; 31.9; 30.8; 30.6; 28.4;
28.3; 26.2; 25.4; 24.0; 23.9; 18.2; 17.4; 17.2; 14.5. ESI-MS: 703.46 ([M þ H]þ ), 725.41 ([M þ Na]þ ),
701.44 ([MꢀH]ꢀ ).
Preparation of Compounds 5k or 6k. Fmoc-Phe-OH (11.6 mg, 0.03 mmol) was dissolved in dry DMF/
THF 1:3 (v/v). To this soln., DCC (9.3 mg, 0.045 mmol) and HOBt (6.1 mg, 0.045 mmol) were added.
After stirring for 1 h at r.t., 3h (25.8 mg, 0.025 mmol) or 4h (18.4 mg, 0.025 mmol) was added, and the
mixture was stirred at 608 under N2 for 8 h. The precipitate was filtered, and the filtrate was concentrated
in vacuo. The residue was subjected to a FC (silica gel; MeOH/CH2Cl2) to obtain the intermediate, and its
structure was confirmed by MS and NMR. Then, the intermediate was dissolved in Et2NH /DMF 1:4
(v/v), and after stirring for 2 h, the mixture was concentrated in vacuo, and the residue was subjected to
FC (silica gel; MeOH/CH2Cl2) and repurified by RP-HPLC (MeOH/H2O) to provide 5k or 6k.
l-Phenylalanyl-N-(3-{[(2b,3b)-3-{[4-O-(b-d-glucopyranosyl)-b-d-xylopyranosyl]oxy}-2,23-dihy-
droxy-30-methoxy-28,30-dioxoolean-12-en-28-yl]amino}propyl)-l-phenylalaninamide (5k). White pow-
1
der (22.4 mg, 76%), M.p. 156.2–158.18. H-NMR (C5D5N): 8.30 (br. s, 1 H); 7.92 (br. s,1 H); 7.43–7.07
(m,10 H); 6.51 (br. s, 1 H); 5.39 (t, J ¼ 3.6, 1 H); 5.01 (d, J ¼ 7.8, 1 H); 4.96 (d, J¼7.2 , 1 H); 4.93 (br. s,
2 H); 4.79 (d, J ¼ 1.2, 1 H); 4.67 (dd, J ¼ 2.4, 12.0, 1 H); 4.53–4.49 (m, 1 H); 4.33–4.28 (m, 3 H); 4.24 ꢀ
4.20 (m, 2 H); 4.18 (d, J ¼ 9, 1 H); 4.15 (t, J ¼ 9, 1 H); 4.08 (t, J ¼ 8.4, 1 H); 4.03 (t, J ¼ 8.4, 1 H); 4.01–
3.99 (m, 1 H); 3.92 (t, J ¼ 8.4, 1 H); 3.71 (t, 1 H); 3.68 (d, J ¼ 10.8, 1 H); 3.62 (s, 3 H); 3.58 (t, J ¼ 10.8,
1 H); 3.41–3.37 (m, 1 H); 3.25–3.22 (m, 2 H); 3.20–3.18 (m, 1 H); 3.15 (d, J ¼ 17, 2 H); 3.09 (dd, J ¼ 4.2,
13.2, 1 H); 3.08–3.06 (m, 2 H); 3.05–3.01 (m,2 H); 2.35 (dd, J¼2.4, 13.8, 1 H); 2.22 (d, J ¼ 13.2, 1 H);
2.16–1.76 (m, 12 H); 1.65–1.63 (m, 2 H); 1.61 (s, 3 H); 1.59–1.56 (m, 1 H); 1.53–1.47 (m, 2 H); 1.41 (td,
J ¼ 4.2, 13.8, 1 H); 1.35 (s, 3 H); 1.33–1.30 (m, 2 H); 1.26 (s, 3 H); 1.19 (s, 3 H); 1.09 (d, J ¼ 13.2, 1 H);
0.86 (s, 3 H). 13C-NMR (C5D5N): 177.1; 174.1; 173.7; 170.7; 144.3; 138.3; 137.2; 129.3; 129.2; 128.3; 128.0;
126.3; 126.1; 123.5; 106.3; 103.5; 83.0; 78.8; 78.2; 77.5; 76.4; 75.0; 74.2; 71.8; 70.8; 65.4; 64.7; 62.7; 56.1;
53.5; 51.6; 48.5; 47.7; 45.9; 44.1; 44.0; 43.3; 43.0; 42.8; 42.3; 41.6; 41.1; 40.9; 39.9; 38.3,36.9; 35.1; 32.9; 31.0;
28.4; 27.9; 26.1; 24.0; 24.0; 23.2; 18.0; 17.6; 17.3; 14.5. ESI-MS: 1177.65 ([M þ H]þ ), 1199.61 ([M þ Na]þ ),
1175.63 ([MꢀH]ꢀ ).
l-Phenylalanyl-N-(3-{[(2b,3b)-2,3,23-trihydroxy-30-methoxy-28,30-dioxoolean-12-en-28-yl]amino}-
propyl)-l-phenylalaninamide (6k). White powder (15.4 mg, 70.0%). M.p. 117.4–119.18. 1H-NMR
(C5D5N): 8.33 (br. s, 1 H); 7.91 (br. s, 1 H); 7.40–7.12 (m, 10 H); 6.52 (br. s, 1 H); 4.96 (br. s, 2 H);
4.51 (t, J ¼ 1.8, 4.8), 4.26–4.22 (m, 1 H); 4.14 (d, J ¼ 10.2, 1 H); 4.00 (t, J ¼ 1.8, 1 H); 3.71 (d, J ¼ 10.2,
1 H); 3.67 (s, 3 H); 3.36 (d, J ¼ 13.2, 2 H); 3.28–3.24 (m, 2 H); 3.16–3.12 (m, 2 H); 3.02 (d, J ¼ 10.2,
1 H); 2.94 (dd, J ¼ 3.6. 4.2), 2.35(d, J ¼ 14.4, 1 H); 2.20 (d, J ¼ 12.6, 1 H); 2.14–2.10 (m, 3 H); 2.07–2.01
(m, 4 H); 1.90–1.76 (m, 5 H); 1.73–1.71 (m, 1 H); 1.67–1.65 (m, 1 H); 1.63 (s, 3 H); 1.58–1.54 (m, 2 H);
1.47 (s, 3 H); 1.44–1.40 (m, 1 H); 1.38 (s, 3 H); 1.32–1.26 (m, 2 H); 1.23 (s, 3 H); 1.06 (d, J ¼ 13.8, 1 H);
0.86( s, 3 H). 13C-NMR (C5D5N): 177.1; 174.1; 173.6; 170.7; 144.4; 138.3; 137.2; 129.3; 129.2; 128.3; 127.9;
126.3; 126.1; 123.3; 73.0; 71.5; 67.7; 56.1; 53.5; 51.7; 48.5; 48.1; 46.1; 44.8; 44.1; 43.3; 42.7; 42.4; 42.2; 40.9;
39.0; 38.9; 38.6; 38.3; 37.2; 34.5; 33.0; 30.8; 30.6; 28.4; 28.3; 26.2; 24.0; 23.9; 18.2; 17.4; 17.2; 14.5. ESI-MS:
883.56 ([M þ H]þ ), 881.54 ([MꢀH]ꢀ ).
General Procedure for Preparing Compounds 7a–7k or 8a–8k. Compound 3 (44.1 mg, 0.05 mmol)
or 4 (35.3 mg, 0.06 mmol) was dissolved in dry MeOH. To this soln., aromatic aldehyde derivative
(0.06 mmol) was added, and the mixture was stirred for 4 h at r.t. under N2. Then, NaBH3CN (7.54 mg,
0.12 mmol) was added, and stiring was continued for 2–8 h at r.t. under N2. The soln. was concentrated in
vacuo, and the residue was subjected to FC (silica gel; (MeOH/CH2Cl2) and was repurified by RP-HPLC
(MeOH/H2O) to provide the compounds 7a–7k or 8a–8k.
Methyl (2b,3b)-28-{[3-(Benzylamino)propyl]amino}-3-{[4-O-(b-d-glucopyranosyl)-b-d-xylopyra-
nosyl]oxy}-2,23-dihydroxy-28-oxoolean-12-en-30-oate (7a). White powder (19.5 mg, 40.0%). M.p.
1
>2508. H-NMR (C5D5N): 7.31–7.28 (m, 5 H); 6.51 (br. s, 1 H); 5.39 (t, J ¼ 3.6, 1 H); 5.01 (d, J ¼ 7.8,
1 H); 4.96 (d, J ¼ 7.2, 1 H); 4.79 (d, J ¼ 1.2, 1 H); 4.67 (dd, J ¼ 2.4, 12.0, 1 H); 4.33–4.28 (m, 3 H); 4.24–
4.20 (m, 2 H); 4.18 (d, J ¼ 9, 1 H); 4.15 (t, J ¼ 9, 1 H); 4.08 (t, J ¼ 8.4, 1 H); 4.04 (br. s, 1 H); 4.01 (t, J ¼
8.4, 1 H); 4.01–3.96 (m, 1 H); 3.92 (t, J ¼ 8.4, 1 H); 3.73 (s, 2 H); 3.68 (d, J ¼ 10.8, 1 H); 3.67 (s, 3 H);

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
3.57 (t, J ¼ 10.8, 1 H); 3.41–3.37 (m, 1 H); 3.25 (t, J¼7 2.3, 7.1, 2 H);3.19 (m, 1 H); 3.09 (dd, J ¼ 4.2, 13.2,
1 H); 2.35 (dd, J ¼ 2.4, 13.8, 1 H); 2.22 (d, J¼13.2, 1 H); 2.16–1.76 (m, 12 H); 1.70–1.66 (m, 2 H); 1.61
(s, 3 H); 1.59–1.57 (m, 1 H); 1.53–1.47 (m, 2 H); 1.41 (td, J ¼ 4.2, 13.8, 1 H); 1.35 (s, 3 H); 1.33–1.30 (m,
2 H); 1.26 (s, 3 H); 1.19 (s, 3 H); 1.09 (d, J ¼ 13.2, 1 H); 0.86 (s, 3 H). 13C-NMR (C5D5N): 177.1; 174.4;
144.3; 140.5; 128.5; 128.2; 127.2; 123.5; 106.3; 103.5; 83.0; 78.8; 78.2; 77.5; 76.4; 75.0; 74.2; 71.8; 70.8; 65.4;
64.7; 62.7; 53.9; 51.7; 48.7; 47.7; 45.9; 44.1; 44.0; 43.3; 43.0; 42.8; 42.3; 41.6; 39.8; 37.9; 36.9; 35.1; 32.9; 31.0;
28.4; 27.9; 26.1; 24.1; 24.0; 23.3; 21.4; 21.0; 18.1; 17.6; 17.3; 14.5. ESI-MS: 973.56 ([M þ H]þ ), 995.49
([M þ Na]þ ), 971.53 ([MꢀH]ꢀ ).
Methyl (2b,3b)-3-{[4-O-(b-d-Glucopyranosyl)-b-d-xylopyranosyl]oxy}-2,23-dihydroxy-28-({3-[(4-
methylbenzyl)amino]propyl}amino)-28-oxoolean-12-en-30-oate (7b). White powder (25.2 mg, 51.1%).
M.p. 182.4–184.78. 1H-NMR (C5D5N): 7.16–7.12 (m, 4 H); 6.50 (br. s, 1 H); 5.39 (t, J ¼ 3.6, 1 H); 5.01 (d,
J ¼ 7.8, 1 H); 4.96 (d, J ¼ 7.2, 1 H); 4.79 (d, J ¼ 1.2, 1 H); 4.67 (dd, J ¼ 2.4, 12.0, 1 H); 4.33–4.28 (m,
3 H); 4.24–4.20 (m, 2 H); 4.18 (d, J ¼ 9, 1 H); 4.15 (t, J ¼ 9, 1 H); 4.08 (t, J ¼ 8.4, 1 H); 4.03 (br. s, 1 H);
4.01 (t, J ¼ 8.4, 1 H); 4.01–3.96 (m, 1 H); 3.92 (t, J ¼ 8.4, 1 H); 3.71 (s, 2 H); 3.68 (d, J ¼ 10.8, 1 H); 3.67
(s, 3 H); 3.57 (t, J ¼ 10.8, 1 H); 3.41–3.37 (m, 1 H); 3.25 (t, J ¼ 2.3, 7.1, 2 H); 3.21–3.17 (m, 1 H); 3.09
(dd, J ¼ 4.2, 13.2, 1 H); 2.35 (dd, J ¼ 2.4, 13.8, 1 H); 2.23 (s, 3 H); 2.20 (d, J ¼ 13.2, 1 H); 2.16 ꢀ 1.76 (m,
12 H); 1.68–1.66 (m, 2 H); 1.61 (s, 3 H); 1.59–1.57 (m, 1 H); 1.53–1.49 (m, 2 H); 1.41 (td, J ¼ 4.2, 13.8,
1 H); 1.35 (s, 3 H); 1.33–1.30 (m, 2 H); 1.26 (s, 3 H); 1.19 (s, 3 H); 1.09 (d, J ¼ 13.2, 1 H); 0.86 (s, 3 H).
13C-NMR (C5D5N): 177.1; 174.4; 144.3; 136.2; 135.8; 129.2; 128.6; 123.6; 106.3; 103.5; 83.0; 78.8; 78.2;
77.5; 76.4; 75.0; 74.2; 71.8; 70.8; 65.4; 64.7; 62.7; 53.7; 51.7; 48.7; 47.7; 45.9; 44.1; 44.0; 43.3; 43.0; 42.8; 42.3;
41.6; 39.8; 37.9; 36.9; 35.1; 32.9; 31.0; 28.4; 27.9; 26.1; 24.1; 24.0; 23.3; 21.4; 21.0; 20.7; 18.1; 17.6; 17.3; 14.5.
ESI-MS: 987.58 ([M þ H]þ ), 1009.51 ([M þ Na]þ ), 985.56 ([MꢀH]ꢀ ).
Methyl (2b,3b)-28-({3-[(4-Bromobenzyl)amino]propyl}amino)-3-{[4-O-(b-d-glucopyranosyl)-b-d-
xylopyranosyl]oxy}-2,23-dihydroxy-28-oxoolean-12-en-30-oate (7c). White powder (20.5 mg, 39.1%).
M.p. 218.5–221.18. 1H-NMR (C5D5N): 7.44–7.42 (m, 2 H); 7.12–7.10 (m, 2 H); 6.52 (br. s, 1 H); 5.39 (t,
J ¼ 3.6, 1 H); 5.01 (d, J ¼ 7.8, 1 H); 4.96 (d, J ¼ 7.2, 1 H); 4.79 (d, J ¼ 1.2, 1 H); 4.67 (dd, J ¼ 2.4, 12.0,
1 H); 4.33–4.28 (m, 3 H); 4.24–4.20 (m, 2 H); 4.18 (d, J ¼ 9, 1 H); 4.15 (t, J ¼ 9, 1 H); 4.08 (t, J ¼ 8.4,
1 H); 4.04 (br. s, 1 H); 4.01 (t, J ¼ 8.4, 1 H); 4.01–3.96 (m, 1 H); 3.92 (t, J ¼ 8.4, 1 H); 3.72 (s, 2 H); 3.68
(d, J ¼ 10.8, 1 H); 3.67 (s, 3 H); 3.57 (t, J ¼ 10.8, 1 H); 3.41–3.37 (m, 1 H); 3.26 (t, J ¼ 2.3, 7.1, 2 H); 3.21–
3.17 (m, 1 H); 3.09 (dd, J ¼ 4.2, 13.2, 1 H); 2.35 (dd, J ¼ 2.4, 13.8, 1 H); 2.21 (d, J ¼ 13.2, 1 H); 2.16–1.76
(m, 12 H); 1.68–1.66 (m, 2 H); 1.62 (s, 3 H); 1.59–1.57 (m, 1 H); 1.53–1.49 (m, 2 H); 1.41 (td, J ¼ 4.2,
13.8, 1 H); 1.35 (s, 3 H); 1.33–1.30 (m, 2 H); 1.26 (s, 3 H); 1.19 (s, 3 H); 1.09 (d, J ¼ 13.2, 1 H); 0.86 (s,
3 H). 13C-NMR (C5D5N): 177.1; 174.4; 144.3; 139.6; 130.7; 130.2; 128.6; 123.6; 120.9; 106.3; 103.5; 83.0;
78.8; 78.2; 77.5; 76.4; 75.0; 74.2; 71.8; 70.8; 65.4; 64.7; 62.7; 53.4; 51.6; 48.7; 47.6; 45.8; 44.1; 44.0; 43.3; 43.0;
42.8; 42.3; 41.6; 39.8; 37.9; 36.9; 35.1; 32.9; 31.0; 28.4; 27.9; 26.1; 24.1; 24.0; 23.3; 21.4; 21.0; 18.1; 17.6; 17.3;
14.5. ESI-MS: 1051.47 ([M þ H]þ ), 1049.46 ([MꢀH]ꢀ ).
Methyl (2b,3b)-28-({3-[(3-Bromobenzyl)amino]propyl}amino)-3-{[4-O-(b-d-glucopyranosyl)-b-d-
xylopyranosyl]oxy}-2,23-dihydroxy-28-oxoolean-12-en-30-oate (7d). White powder (27.2 mg, 51.8%).
M.p. >2508. 1H-NMR (C5D5N): 7.57 (s, 1 H); 7.27–7.24 (m, 2 H); 7.14–7.10 (m, 1 H); 6.52 (br. s, 1 H);
5.39 (t, J ¼ 3.6, 1 H); 5.01 (d, J ¼ 7.8, 1 H); 4.96 (d, J ¼ 7.2, 1 H); 4.79 (d, J ¼ 1.2, 1 H); 4.67 (dd, J ¼ 2.4,
12.0, 1 H); 4.33–4.28 (m, 3 H); 4.24–4.20 (m, 2 H); 4.18 (d, J ¼ 9, 1 H); 4.15 (t, J ¼ 9, 1 H); 4.08 (t, J ¼
8.4, 1 H); 4.04 (br. s, 1 H); 4.01 (t, J ¼ 8.4, 1 H); 4.01–3.96 (m, 1 H); 3.92 (t, J ¼ 8.4, 1 H); 3.72 (s, 2 H);
3.68 (d, J ¼ 10.8, 1 H); 3.67 (s, 3 H); 3.57 (t, J ¼ 10.8, 1 H); 3.41–3.37 (m, 1 H); 3.26 (t, J ¼ 2.3, 7.1, 2 H);
3.21–3.17 (m, 1 H); 3.09 (dd, J ¼ 4.2, 13.2, 1 H); 2.35 (dd, J ¼ 2.4, 13.8, 1 H); 2.21 (d, J ¼ 13.2, 1 H);
2.16–1.76 (m, 12 H); 1.68–1.66 (m, 2 H); 1.62 (s, 3 H); 1.59–1.57 (m, 1 H); 1.53–1.49 (m, 2 H); 1.41 (td,
J ¼ 4.2, 13.8, 1 H); 1.35 (s, 3 H); 1.33–1.30 (m, 2 H); 1.26 (s, 3 H); 1.19 (s, 3 H); 1.09 (d, J ¼ 13.2, 1 H);
0.86 (s, 3 H). 13C-NMR (C5D5N): 177.1; 174.4; 144.3; 142.1; 131.8; 129.7; 129.5; 126.8; 123.6; 121.3; 106.3;
103.5; 83.0; 78.8; 78.2; 77.5; 76.4; 75.0; 74.2; 71.8; 70.8; 65.4; 64.7; 62.7; 53.4; 51.6; 48.7; 47.6; 45.8; 44.1;
44.0; 43.3; 43.0; 42.8; 42.3; 41.6; 39.8; 37.9; 36.9; 35.1; 32.9; 31.0; 28.4; 27.9; 26.1; 24.1; 24.0; 23.3; 21.4; 21.0;
18.1; 17.6; 17.3; 14.5. ESI-MS: 1051.47 ([M þ H]þ ), 1049.46 ([MꢀH]ꢀ ).
Methyl (2b,3b)-3-{[4-O-(b-d-Glucopyranosyl)-b-d-xylopyranosyl]oxy}-2,23-dihydroxy-28-({3-[(3-
hydroxybenzyl)amino]propyl}amino)-28-oxoolean-12-en-30-oate (7e). White powder (14.0 mg,
28.3%). M.p. >2508. 1H-NMR (C5D5N): 7.16–7.14 (m, 1 H); 6.84–6.76 (m, 3 H); 6.51 (br. s, 1 H);

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1847
5.39 (t, J ¼ 3.6, 1 H); 5.01 (d, J ¼ 7.8, 1 H); 4.96 (d, J ¼ 7.2, 1 H); 4.79 (d, J ¼ 1.2, 1 H); 4.67 (dd, J ¼ 2.4,
12.0, 1 H); 4.33–4.28 (m, 3 H); 4.24–4.20 (m, 2 H); 4.18 (d, J ¼ 9, 1 H); 4.15 (t, J ¼ 9, 1 H); 4.08 (t, J ¼
8.4, 1 H); 4.04 (br. s, 1 H); 4.01 (t, J ¼ 8.4, 1 H); 4.01–3.96 (m, 1 H); 3.92 (t, J ¼ 8.4, 1 H); 3.68 (s, 2 H);
3.67 (s, 3 H); 3.65 (d, J ¼ 10.8, 1 H); 3.57 (t, J ¼ 10.8, 1 H); 3.41–3.37 (m, 1 H); 3.26 (t, J ¼ 2.3, 7.1, 2 H);
3.21–3.17 (m, 1 H); 3.09 (dd, J ¼ 4.2, 13.2, 1 H); 2.35 (dd, J ¼ 2.4, 13.8, 1 H); 2.21 (d, J ¼ 13.2, 1 H);
2.16–1.76 (m, 12 H); 1.68–1.66 (m, 2 H); 1.62 (s, 3 H); 1.59–1.57 (m, 1 H); 1.53–1.49 (m, 2 H); 1.41 (td,
J ¼ 4.2, 13.8, 1 H); 1.35 (s, 3 H); 1.33–1.30 (m, 2 H); 1.26 (s, 3 H); 1.19 (s, 3 H); 1.09 (d, J ¼ 13.2, 1 H);
0.86 (s, 3 H). 13C-NMR (C5D5N): 177.1; 174.4; 144.3; 140.4; 129.5; 123.6; 119.5; 115.1; 113.1; 106.3; 103.5;
83.0; 78.8; 78.2; 77.5; 76.4; 75.0; 74.2; 71.8; 70.8; 65.4; 64.7; 62.7; 54.3; 51.6; 48.9; 47.6; 45.8; 44.1; 44.0;
43.3; 43.0; 42.8; 42.3; 41.6; 39.8; 37.9; 36.9; 35.1; 32.9; 31.0; 28.4; 27.0; 26.1; 24.1; 24.0; 23.3; 21.4; 21.0; 18.1;
17.6; 17.3; 14.5. ESI-MS: 989.56 ([M þ H]þ ), 1011.51 ([M þ Na]þ ), 987.54 ([MꢀH]ꢀ ).
Methyl (2b,3b)-28-[(3-{[4-(Dimethylamino)benzyl]amino}propyl)amino]-3-{[4-O-(b-d-glucopyra-
nosyl)-b-d-xylopyranosyl]oxy}-2,23-dihydroxy-28-oxoolean-12-en-30-oate (7f). White powder (23.5 mg,
46.2%). M.p. 246.4–248.18. 1H-NMR (C5D5N): 6.90–6.88 (m, 2 H); 6.65–6.63 (m, 2 H); 6.51 (br. s, 1 H);
5.39 (t, J ¼ 3.6, 1 H); 5.01 (d, J ¼ 7.8, 1 H); 4.96 (d, J ¼ 7.2, 1 H); 4.79 (d, J ¼ 1.2, 1 H); 4.67 (dd, J ¼ 2.4,
12.0, 1 H); 4.33–4.28 (m, 3 H); 4.24–4.20 (m, 2 H); 4.18 (d, J ¼ 9, 1 H); 4.15 (t, J ¼ 9, 1 H); 4.08 (t, J ¼
8.4, 1 H); 4.03 (br. s, 1 H); 4.01 (t, J ¼ 8.4, 1 H); 4.01–3.96 (m, 1 H); 3.92 (t, J ¼ 8.4, 1 H); 3.72 (s, 2 H);
3.68 (d, J ¼ 10.8, 1 H); 3.67 (s, 3 H); 3.57 (t, J ¼ 10.8, 1 H); 3.41–3.37 (m, 1 H); 3.26 (t, J ¼ 2.3, 7.1, 2 H);
3.21–3.17 (m, 1 H); 3.09 (dd, J ¼ 4.2, 13.2, 1 H); 2.89 (s, 6 H); 2.35 (dd, J ¼ 2.4, 13.8, 1 H); 2.21 (d, J ¼
13.2, 1 H); 2.16–1.76 (m, 12 H); 1.68–1.66 (m, 2 H); 1.62 (s, 3 H); 1.59–1.57 (m, 1 H); 1.53–1.47 (m,
2 H); 1.41 (td, J ¼ 4.2, 13.8, 1 H); 1.35 (s, 3 H); 1.33–1.30 (m, 2 H); 1.26 (s, 3 H); 1.19 (s, 3 H); 1.09 (d, J ¼
13.2, 1 H); 0.86 (s, 3 H). 13C-NMR (C5D5N): 177.1; 174.4; 149.7; 144.3; 128.8; 128.2; 123.6; 112.6; 112.5;
106.3; 103.5; 83.0; 78.8; 78.2; 77.5; 76.4; 75.0; 74.2; 71.8; 70.8; 65.4; 64.7; 62.7; 52.2; 51.6; 48.7; 47.6; 45.8;
44.1; 44.0; 43.3; 43.0; 42.8; 42.3; 41.6; 40.5; 39.8; 37.9; 36.9; 35.1; 32.9; 31.0; 28.4; 27.9; 26.1; 24.1; 24.0; 23.3;
21.4; 21.0; 18.1; 17.6; 17.3; 14.5. ESI-MS: 1016.60 ([M þ H]þ ), 1038.56 ([M þ Na]þ ), 1014.59 ([MꢀH]ꢀ ).
Methyl (2b,3b)-3-{[4-O-(b-d-Glucopyranosyl)-b-d-xylopyranosyl]oxy}-2,23-dihydroxy-28-({3-[(4-
nitrobenzyl)amino]propyl}amino)-28-oxoolean-12-en-30-oate (7g). Pale yellow powder (20.3 mg,
40.0%). M.p. 192.7–193.88. 1H-NMR (C5D5N): 8.15–8.13 (m, 2 H); 7.47–7.45 (m, 2 H); 6.52 (br. s,
1 H); 5.39 (t, J ¼ 3.6, 1 H); 5.01 (d, J ¼ 7.8, 1 H); 4.96 (d, J ¼ 7.2, 1 H); 4.79 (d, J ¼ 1.2, 1 H); 4.67 (dd, J ¼
2.4, 12.0, 1 H); 4.33–4.28 (m, 3 H); 4.24–4.20 (m, 2 H); 4.18 (d, J ¼ 9, 1 H); 4.15 (t, J ¼ 9, 1 H); 4.08 (t,
J ¼ 8.4, 1 H); 4.03 (br. s, 1 H); 4.01 (t, J ¼ 8.4, 1 H); 4.01–3.96 (m, 1 H); 3.92 (t, J ¼ 8.4, 1 H); 3.82 (s,
2 H); 3.68 (d, J ¼ 10.8, 1 H); 3.67 (s, 3 H); 3.57 (t, J ¼ 10.8, 1 H); 3.41–3.37 (m, 1 H); 3.26 (t, J ¼ 2.3, 7.1,
2 H); 3.21–3.17 (m, 1 H); 3.09 (dd, J ¼ 4.2, 13.2, 1 H); 2.35 (dd, J ¼ 2.4, 13.8, 1 H); 2.21 (d, J ¼ 13.2,
1 H); 2.16–1.76 (m, 12 H); 1.68–1.66 (m, 2 H); 1.62 (s, 3 H); 1.59–1.57 (m, 1 H); 1.53–1.47 (m, 2 H);
1.41 (td, J ¼ 4.2, 13.8, 1 H); 1.35 (s, 3 H); 1.33–1.30 (m, 2 H); 1.26 (s, 3 H); 1.19 (s, 3 H); 1.09 (d, J ¼ 13.2,
1 H); 0.86 (s, 3 H). 13C-NMR (C5D5N): 177.1; 174.4; 149.0; 146.9; 144.3; 128.5; 123.6; 122.9; 106.3; 103.5;
83.0; 78.8; 78.2; 77.5; 76.4; 75.0; 74.2; 71.8; 70.8; 65.4; 64.7; 62.7; 52.5; 51.6; 48.7; 47.6; 45.8; 44.1; 44.0; 43.3;
43.0; 42.8; 42.3; 41.6; 39.8; 37.9; 36.9; 35.1; 32.9; 31.0; 28.4; 27.9; 26.1; 24.1; 24.0; 23.3; 21.4; 21.0; 18.1; 17.6;
17.3; 14.5. ESI-MS: 1018.55 ([M þ H]þ ), 1016.53 ([MꢀH]ꢀ ).
Methyl (2b,3b)-3-{[4-O-(b-d-Glucopyranosyl)-b-d-xylopyranosyl]oxy}-2,23-dihydroxy-28-({3-[(3-
nitrobenzyl)amino]propyl}amino)-28-oxoolean-12-en-30-oate (7h). Pale yellow powder (17.6 mg,
1
34.5%). M.p. 247.4–248.88. H-NMR (C5D5N): 8.11–8.09 (m, 1 H); 8.04 (s, 1 H); 7.66–7.64 (m, 1 H);
7.51–7.49 (m, 1 H); 6.52 (br. s, 1 H); 5.39 (t, J ¼ 3.6, 1 H); 5.01 (d, J ¼ 7.8, 1 H); 4.96 (d, J ¼ 7.2, 1 H); 4.79
(d, J ¼ 1.2, 1 H); 4.67 (dd, J ¼ 2.4, 12.0, 1 H); 4.33–4.28 (m, 3 H); 4.24–4.20 (m, 2 H); 4.18 (d, J ¼ 9,
1 H); 4.15 (t, J ¼ 9, 1 H); 4.08 (t, J ¼ 8.4, 1 H); 4.03 (br. s, 1 H); 4.01 (t, J ¼ 8.4, 1 H); 4.01–3.96 (m, 1 H);
3.92 (t, J ¼ 8.4, 1 H); 3.88 (s, 2 H); 3.68 (d, J ¼ 10.8, 1 H); 3.67 (s, 3 H); 3.57 (t, J ¼ 10.8, 1 H); 3.41–3.37
(m, 1 H); 3.26 (t, J ¼ 2.3, 7.1, 2 H); 3.21–3.17 (m, 1 H); 3.09 (dd, J ¼ 4.2, 13.2, 1 H); 2.35 (dd, J ¼ 2.4,
13.8, 1 H); 2.21 (d, J ¼ 13.2, 1 H); 2.16–1.76 (m, 12 H); 1.68–1.66 (m, 2 H); 1.62 (s, 3 H); 1.59–1.57 (m,
1 H); 1.53–1.47 (m,2 H); 1.41 (td, J ¼ 4.2, 13.8, 1 H); 1.35 (s, 3 H); 1.33–1.30 (m, 2 H); 1.26 (s, 3 H); 1.19
(s, 3 H); 1.09 (d, J ¼ 13.2, 1 H); 0.86 (s, 3 H). 13C-NMR (C5D5N): 177.1; 174.4; 148.6; 144.3; 141.7; 132.9;
129.5; 123.6; 122.0; 106.3; 103.5; 83.0; 78.8; 78.2; 77.5; 76.4; 75.0; 74.2; 71.8; 70.8; 65.4; 64.7; 62.7; 52.6;
51.6; 48.7; 47.6; 45.8; 44.1; 44.0; 43.3; 43.0; 42.8; 42.3; 41.6; 39.8; 37.9; 36.9; 35.1; 32.9; 31.0; 28.4; 27.9; 26.1;
24.1; 24.0; 23.3; 21.4; 21.0; 18.1; 17.6; 17.3; 14.5. ESI-MS: 1018.55 ([M þ H]þ ), 1016.53 ([MꢀH]ꢀ ).

1848
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Methyl (2b,3b)-3-{[4-O-(b-d-Glucopyranosyl)-b-d-xylopyranosyl]oxy}-2,23-dihydroxy-28-({3-[(2-
methoxybenzyl)amino]propyl}amino)-28-oxoolean-12-en-30-oate (7i). White powder (17.6 mg, 34.5%).
M.p. 143.8–145.48. 1H-NMR (C5D5N): 7.18–7.16 (m, 2 H); 6.90–6.86 (m, 2 H); 6.52 (br. s, 1 H); 5.39 (t,
J ¼ 3.6, 1 H); 5.01 (d, J ¼ 7.8, 1 H); 4.96 (d, J ¼ 7.2, 1 H); 4.79 (d, J ¼ 1.2, 1 H); 4.67 (dd, J ¼ 2.4, 12.0,
1 H); 4.33–4.28 (m, 3 H); 4.24–4.20 (m, 2 H); 4.18 (d, J ¼ 9, 1 H); 4.15 (t, J ¼ 9, 1 H); 4.08 (t, J ¼ 8.4,
1 H); 4.02 (t, J ¼ 8.4, 1 H); 4.01–3.99 (m, 1 H); 3.97 (br. s, 1 H); 3.92 (t, J ¼ 8.4, 1 H); 3.81 (s, 3 H); 3.80
(s, 2 H); 3.68 (d, J ¼ 10.8, 1 H); 3.67 (s, 3 H); 3.57 (t, J ¼ 10.8, 1 H); 3.41–3.37 (m, 1 H); 3.26 (t, J ¼ 2.3,
7.1, 2 H); 3.21–3.17 (m, 1 H); 3.09 (dd, J ¼ 4.2, 13.2, 1 H); 2.35 (dd, J ¼ 2.4, 13.8, 1 H); 2.21 (d, J ¼ 13.2,
1 H); 2.16–1.76 (m, 12 H); 1.68–1.66 (m, 2 H); 1.62 (s, 3 H); 1.59–1.57 (m, 1 H); 1.53–1.47 (m, 2 H);
1.41 (td, J ¼ 4.2, 13.8, 1 H); 1.35 (s, 3 H); 1.33–1.30 (m, 2 H); 1.26 (s, 3 H); 1.19 (s, 3 H); 1.09 (d, J ¼ 13.2,
1 H); 0.86 (s, 3 H). 13C-NMR (C5D5N): 177.1; 174.4; 157.8; 144.3; 130.2; 129.3; 127.4; 123.6; 119.1; 106.3;
103.5; 83.0; 78.8; 78.2; 77.5; 76.4; 75.0; 74.2; 71.8; 70.8; 65.4; 64.7; 62.7; 54.9; 51.6; 48.6; 48.5; 47.6; 45.8;
44.1; 44.0; 43.3; 43.0; 42.8; 42.3; 41.6; 39.9; 37.9; 36.9; 35.1; 32.9; 31.0; 28.4; 27.1; 26.1; 24.1; 24.0; 23.3; 21.4;
21.0; 18.1; 17.6; 17.3; 14.5. ESI-MS: 1003.57 ([M þ H]þ ), 1025.55 ([M þ Na]þ ), 1001.56 ([MꢀH]ꢀ ).
Methyl (2b,3b)-28-({3-[(2,4-Dichlorobenzyl)amino]propyl}amino)-3-{[4-O-(b-d-glucopyranosyl)-b-
d-xylopyranosyl]oxy}-2,23-dihydroxy-28-oxoolean-12-en-30-oate (7j). White powder (19.6 mg, 37.6 %).
M.p. 228.5–230.68. 1H-NMR (C5D5N): 7.46 (s, 1 H); 7.23–7.18 (m, 2 H); 6.51 (br. s, 1 H); 5.38 (t, J ¼ 3.6,
1 H); 5.01 (d, J ¼ 7.8, 1 H); 4.96 (d, J ¼ 7.2, 1 H); 4.79 (d, J ¼ 1.2, 1 H); 4.67 (dd, J ¼ 2.4, 12.0, 1 H); 4.33–
4.28 (m, 3 H); 4.24–4.20 (m, 2 H); 4.18 (d, J ¼ 9, 1 H); 4.15 (t, J ¼ 9, 1 H); 4.10 (s, 2 H); 4.08 (t, J ¼ 8.4,
1 H); 4.02 (t, J ¼ 8.4, 1 H); 4.01–3.96 (m, 1 H); 3.93 (br. s, 1 H); 3.92 (t, J ¼ 8.4, 1 H); 3.68 (d, J ¼ 10.8,
1 H); 3.67 (s, 3 H); 3.57 (t, J ¼ 10.8, 1 H); 3.41–3.37 (m, 1 H); 3.26 (t, J ¼ 2.3, 7.1, 2 H); 3.21–3.17 (m,
1 H); 3.09 (dd, J ¼ 4.2, 13.2, 1 H); 2.35 (dd, J ¼ 2.4, 13.8, 1 H); 2.21 (d, J ¼ 13.2, 1 H); 2.16 ꢀ 1.76 (m,
12 H); 1.68–1.66 (m, 2 H); 1.62 (s, 3 H); 1.59–1.57 (m, 1 H); 1.53–1.47 (m, 2 H); 1.41 (td, J ¼ 4.2, 13.8,
1 H); 1.35 (s, 3 H); 1.33–1.30 (m, 2 H); 1.26 (s, 3 H); 1.19 (s, 3 H); 1.09 (d, J ¼ 13.2, 1 H); 0.86 (s, 3 H).
13C-NMR (C5D5N): 177.1; 174.4; 144.3; 134.8; 132.6; 130.7; 128.7; 126.9; 123.6; 106.3; 103.5; 83.0; 78.8;
78.2; 77.5; 76.4; 75.0; 74.2; 71.8; 70.8; 65.4; 64.7; 62.7; 51.6; 50.5; 48.6; 47.6; 45.8; 44.1; 44.0; 43.3; 43.0;
42.8; 42.3; 41.6; 39.9; 37.9; 36.9; 35.1; 32.9; 31.0; 28.4; 26.8; 26.1; 24.1; 24.0; 23.3; 21.4; 21.0; 18.1; 17.6; 17.3;
14.5. ESI-MS: 1041.49 ([M þ H]þ ), 1039.47 ([MꢀH]ꢀ ).
Methyl (2b,3b)-3-{[4-O-(b-d-Glucopyranosyl)-b-d-xylopyranosyl]oxy}-2,23-dihydroxy-28-oxo-28-
({3-[(2,4,6-trihydroxybenzyl)amino]propyl}amino)olean-12-en-30-oate (7k). White powder (12.2 mg,
24.0%). M.p. 146.5–148.28. 1H-NMR (C5D5N): 6.52 (br. s, 1 H); 5.90 (s, 2 H); 5.39 (t, J ¼ 3.6, 1 H); 5.01
(d, J ¼ 7.8, 1 H); 4.96 (d, J ¼ 7.2, 1 H); 4.79 (d, J ¼ 1.2, 1 H); 4.67 (dd, J ¼ 2.4, 12.0, 1 H); 4.33–4.28 (m,
3 H); 4.24–4.20 (m, 2 H); 4.19 (s, 2 H); 4.17 (d, J ¼ 9, 1 H); 4.15 (t, J ¼ 9, 1 H); 4.08 (t, J ¼ 8.4, 1 H); 4.02
(t, J ¼ 8.4, 1 H); 4.01–3.96 (m, 1 H); 3.93 (br. s, 1 H); 3.92 (t, J ¼ 8.4, 1 H); 3.68 (d, J ¼ 10.8, 1 H); 3.67 (s,
3 H); 3.57 (t, J ¼ 10.8, 1 H); 3.41–3.37 (m, 1 H); 3.26 (t, J ¼ 2.3, 7.1, 2 H); 3.21–3.17 (m, 1 H); 3.09 (dd,
J ¼ 4.2, 13.2, 1 H); 2.35 (dd, J ¼ 2.4, 13.8, 1 H); 2.21 (d, J ¼ 13.2, 1 H); 2.16–1.76 (m, 12 H); 1.68–1.66
(m, 2 H); 1.62 (s, 3 H); 1.59–1.57 (m, 1 H); 1.53–1.47 (m,2 H); 1.41 (td, J ¼ 4.2, 13.8, 1 H); 1.35 (s, 3 H);
1.33–1.30 (m, 2 H); 1.26 (s, 3 H); 1.19 (s, 3 H); 1.09 (d, J¼13.2, 1 H); 0.86 (s, 3 H). 13C-NMR (C5D5N):
177.1; 174.4; 162.3; 158.7; 144.3; 123.6; 106.3; 103.5; 101.5; 83.0; 78.8; 78.2; 77.5; 76.4; 75.0; 74.2; 71.8;
70.8; 65.4; 64.7; 62.7; 51.6; 48.8; 47.6; 45.8; 44.1; 44.0; 43.3; 43.0; 42.8; 42.3; 41.6; 40.9; 39.9; 37.9; 36.9; 35.1;
32.9; 31.0; 28.4; 27.4; 26.1; 24.1; 24.0; 23.3; 21.4; 21.0; 18.1; 17.6; 17.3; 14.5. ESI-MS: 1021.55 ([M þ H]þ ),
1043.50 ([M þ Na]þ ), 1019.53 ([MꢀH]ꢀ ).
Methyl (2b,3b)-28-{[3-(Benzylamino)propyl]amino}-2,3,23-trihydroxy-28-oxoolean-12-en-30-oate
1
(8a). White powder (18.0 mg, 41.0%). M.p. 217.1–219.18. H-NMR (C5D5N): 7.34–7.28 (m, 5 H); 6.52
(br. s, 1 H); 4.26–4.22 (m, 1 H); 4.12 (d, J ¼ 10.2, 1 H); 3.98 (d, J ¼ 10.2, 1 H); 3.73 (s, 2 H); 3.67 (s, 3 H);
3.26–3.22 (m, 2 H); 3.02 (d, J ¼ 10.2, 1 H); 2.99 (br. s, 1 H); 2.75–2.71 (m, 2 H); 2.35 (d, J ¼ 14.4, 1 H);
2.20 (d, J ¼ 12.6, 1 H); 2.14–2.11 (m, 3 H); 2.05–2.01 (m, 4 H); 1.90–1.78 (m, 5 H); 1.74–1.70 (m, 1 H);
1.69–1.67 (m, 2 H); 1.66–1.64 (m, 1 H); 1.63 (s, 3 H); 1.47 (s, 3 H); 1.44–1.40 (m, 1 H); 1.38 (s, 3 H);
1.32–1.26 (m, 2 H); 1.23 (s, 3 H); 1.06 (d, J ¼ 13.8, 1 H); 0.86 (s, 3 H). 13C-NMR (C5D5N): 177.1; 174.4;
144.4; 140.5; 128.5; 128.2; 127.2; 123.3; 73.0; 71.5; 67.7; 53.9; 51.6; 48.7; 48.5; 48.1; 46.1; 44.8; 44.1; 43.3;
42.7; 42.4; 42.2; 39.8; 38.3; 37.2; 34.5; 33.0; 30.8; 30.6; 28.4; 28.3; 27.0; 26.2; 24.0; 23.9; 18.2; 17.4; 17.2; 14.5.
ESI-MS: 679.47 ([M þ H]þ ), 701.43 ([M þ Na]þ ), 677.45 ([MꢀH]ꢀ ).

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1849
Methyl (2b,3b)-2,3,23-Trihydroxy-28-({3-[(4-methylbenzyl)amino]propyl}amino)-28-oxoolean-12-
1
en-30-oate (8b). White powder (20.4 mg, 49.2%). M.p. 118.4–120.78. H-NMR (C5D5N): 7.17–7.14 (m,
4 H); 6.52 (br. s, 1 H); 4.26–4.22 (m, 1 H); 4.12 (d, J ¼ 10.2, 1 H); 3.98 (d, J ¼ 10.2, 1 H); 3.73 (s, 2 H);
3.67 (s, 3 H); 3.28–3.24 (m, 2 H); 3.02 (d, J ¼ 10.2, 1 H); 2.99 (br. s, 1 H); 2.75–2.71 (m, 2 H); 2.35 (d, J ¼
14.4, 1 H); 2.23 (s, 3 H); 2.20 (d, J ¼ 12.6, 1 H); 2.14–2.11 (m, 3 H); 2.05–2.01 (m, 4 H); 1.90–1.78 (m,
5 H); 1.74–1.70(m, 1 H); 1.69–1.67 (m, 2 H); 1.66–1.64 (m, 1 H); 1.63 (s, 3 H); 1.47 (s, 3 H); 1.44–1.40
(m, 1 H); 1.38 (s, 3 H); 1.32–1.26 (m, 2 H); 1.23 (s, 3 H); 1.06 (d, J ¼ 13.8, 1 H); 0.86 (s, 3 H); 13C-NMR
(C5D5N): 177.1; 174.4; 144.4; 136.3; 135.8; 129.2; 128.6; 123.3; 73.0; 71.5; 67.7; 53.7; 51.6; 48.7; 48.5; 48.1;
46.1; 44.8; 44.1; 43.3; 42.7; 42.4; 42.2; 39.8; 38.3; 37.2; 34.5; 33.0; 30.8; 30.6; 28.4; 28.3; 27.0; 26.2; 24.0;
23.9; 20.7; 18.2; 17.4; 17.2; 14.5. ESI-MS: 693.48 ([M þ H]þ ), 715.45 ([M þ Na]þ ), 691.47([MꢀH]ꢀ ).
Methyl (2b,3b)-28-({3-[(4-Bromobenzyl)amino]propyl}amino)-2,3,23-trihydroxy-28-oxoolean-12-
en-30-oate (8c). White powder (19.4 mg, 42.7%). M.p. 79.2–81.88. 1H-NMR (C5D5N): 7.44–7.42 (m,
2 H); 7.14–7.12 (m, 2 H); 6.52 (br. s, 1 H); 4.26–4.22 (m, 1 H); 4.12 (d, J ¼ 10.2, 1 H); 3.98 (d, J ¼ 10.2,
1 H); 3.72 (s, 2 H); 3.67 (s, 3 H); 3.28–3.24 (m, 2 H); 3.02 (d, J ¼ 10.2, 1 H); 2.99 (br. s, 1 H); 2.75–2.71
(m, 2 H); 2.35 (d, J ¼ 14.4, 1 H); 2.20 (d, J ¼ 12.6, 1 H); 2.14–2.11 (m, 3 H); 2.05–2.01 (m, 4 H); 1.90–
1.78 (m, 5 H); 1.74–1.70 (m, 1 H); 1.69–1.67 (m, 2 H); 1.66–1.64 (m, 1 H); 1.63 (s, 3 H); 1.47 (s, 3 H);
1.44–1.40 (m, 1 H); 1.38 (s, 3 H); 1.32–1.26 (m, 2 H); 1.23 (s, 3 H); 1.06 (d, J ¼ 13.8, 1 H); 0.86 (s, 3 H).
13C-NMR (C5D5N): 177.1; 174.4; 144.4; 139.6; 130.7; 130.2; 123.3; 120.9; 73.0; 71.5; 67.7; 53.4; 51.7; 48.7;
48.5; 48.1; 46.1; 44.8; 44.1; 43.3; 42.7; 42.4; 42.2; 39.8; 38.3; 37.2; 34.5; 33.0; 30.8; 30.6; 28.4; 28.3; 27.0;
26.2; 24.0; 23.9; 18.2; 17.4; 17.2; 14.5. ESI-MS: 757.38 ([M þ H]þ ), 755.36 ([MꢀH]ꢀ ).
Methyl (2b,3b)-28-({3-[(3-Bromobenzyl)amino]propyl}amino)-2,3,23-trihydroxy-28-oxoolean-12-
1
en-30-oate (8d). White powder (19.4 mg, 42.7%). M.p. >2508. H-NMR (C5D5N): 7.57 (s, 1 H); 7.29–
7.27 (m, 2 H); 7.16–7.12 (m, 1 H); 6.52 (br. s, 1 H); 4.26–4.22 (m, 1 H); 4.12 (d, J ¼ 10.2, 1 H); 3.98 (d,
J ¼ 10.2, 1 H); 3.78 (s, 2 H); 3.67 (s, 3 H); 3.28–3.24 (m, 2 H); 3.02 (d, J ¼ 10.2, 1 H); 2.99 (br. s, 1 H);
2.75–2.71 (m, 2 H); 2.35 (d, J ¼ 14.4, 1 H); 2.20 (d, J ¼ 12.6, 1 H); 2.14–2.11 (m, 3 H); 2.05–2.01 (m,
4 H); 1.90–1.78 (m, 5 H); 1.74–1.70 (m, 1 H); 1.69–1.67 (m, 2 H); 1.66–1.64 (m, 1 H); 1.63 (s, 3 H); 1.47
(s, 3 H); 1.44–1.40 (m, 1 H); 1.38 (s, 3 H); 1.32–1.26 (m, 2 H); 1.23 (s, 3 H); 1.06 (d, J ¼ 13.8, 1 H); 0.86
(s, 3 H). 13C-NMR (C5D5N): 177.1; 174.4; 144.4; 142.1; 131.8; 129.7; 129.5; 126.8; 123.3; 73.0; 71.5; 67.7;
53.4; 51.7; 48.7; 48.5; 48.1; 46.1; 44.8; 44.1; 43.3; 42.7; 42.4; 42.2; 39.8; 38.3; 37.2; 34.5; 33.0; 30.8; 30.6;
28.4; 28.3; 27.0; 26.2; 24.0; 23.9; 18.2; 17.4; 17.2; 14.5. ESI-MS: 757.36 ([M þ H]þ ), 755.33 ([MꢀH]ꢀ ).
Methyl (2b,3b)-2,3,23-Trihydroxy-28-({3-[(3-hydroxybenzyl)amino]propyl}amino)-28-oxoolean-12-
1
en-30-oate (8e). White powder (18.0 mg, 39.6%). M.p. 145.5–147.28. H-NMR (C5D5N): 7.16 (m, 1 H);
6.83 (s, 1 H); 6.79–6.76 (m, 2 H); 6.52 (br. s, 1 H); 4.33 (br. s, 1 H); 4.26–4.22 (m, 1 H); 4.12 (d, J ¼ 10.2,
1 H); 3.98 (d, J ¼ 10.2, 1 H); 3.78 (s, 2 H); 3.67 (s, 3 H); 3.28–3.24 (m, 2 H); 3.02 (d, J ¼ 10.2, 1 H); 2.99
(br. s, 1 H); 2.77–2.73 (m, 2 H); 2.35 (d, J ¼ 14.4, 1 H); 2.20 (d, J ¼ 12.6, 1 H); 2.14–2.11 (m, 3 H); 2.05–
2.01 (m, 4 H); 1.90–1.78 (m, 5 H); 1.74–1.70 (m, 1 H); 1.69–1.67 (m, 2 H); 1.66–1.64 (m, 1 H); 1.63 (s,
3 H); 1.47 (s, 3 H); 1.44–1.40 (m, 1 H); 1.38 (s, 3 H); 1.32–1.26 (m, 2 H); 1.23 (s, 3 H); 1.06 (d, J ¼ 13.8,
1 H); 0.86 (s, 3 H). 13C-NMR (C5D5N): 177.1; 174.4; 156.7; 144.4; 140.4; 129.5; 123.3; 119.5; 115.1; 113.1;
73.0; 71.5; 67.7; 54.3; 51.7; 48.9; 48.5; 48.1; 46.1; 44.8; 44.1; 43.3; 42.7; 42.4; 42.2; 39.8; 38.3; 37.2; 34.5; 33.0;
30.8; 30.6; 28.4; 28.3; 27.0; 26.2; 24.0; 23.9; 18.2; 17.4; 17.2; 14.5. ESI-MS: 695.96 ([M þ H]þ ), 717.93
([M þ Na]þ ) , 693.92 ([MꢀH]ꢀ ).
Methyl (2b,3b)-28-[(3-{[4-(Dimethylamino)benzyl]amino}propyl)amino]-2,3,23-trihydroxy-28-
oxoolean-12-en-30-oate (8f). White powder (14.7 mg, 33.6%). M.p. 113.2–114.38. 1H-NMR (C5D5N):
6.90--6.88 (m, 2 H); 7.67–7.65 (m, 2 H); 6.52 (br. s, 1 H); 4.26–4.22 (m, 1 H); 4.12 (d, J ¼ 10.2, 1 H); 3.98
(d, J ¼ 10.2, 1 H); 3.67 (s, 3 H); 3.60 (s, 2 H); 3.28–3.24 (m, 2 H); 3.02 (d, J ¼ 10.2, 1 H); 2.99 (br. s, 1 H);
2.89 (s, 6 H); 2.75–2.71 (m, 2 H); 2.35 (d, J ¼ 14.4, 1 H); 2.20 (d, J ¼ 12.6, 1 H); 2.14–2.11 (m, 3 H);
2.05–2.01 (m, 4 H); 1.90–1.78 (m, 5 H); 1.74–1.70 (m, 1 H); 1.69–1.67 (m, 2 H); 1.66–1.64 (m, 1 H);
1.63 (s, 3 H); 1.47 (s, 3 H); 1.44–1.40 (m, 1 H); 1.38 (s, 3 H); 1.32 ꢀ1.26 (m, 2 H); 1.23 (s, 3 H); 1.06 (d,
J ¼ 13.8, 1 H); 0.86 (s, 3 H). 13C-NMR (C5D5N): 177.1; 174.4; 149.7; 144.4; 128.8; 128.2; 123.3; 112.6;
73.0; 71.5; 67.7; 52.2; 51.7; 48.7; 48.5; 48.1; 46.1; 44.8; 44.1; 43.3; 42.7; 42.4; 42.2; 40.5; 39.8; 38.3; 37.2; 34.5;
33.0; 30.8; 30.6; 28.4; 28.3; 27.0; 26.2; 24.0; 23.9; 18.2; 17.4; 17.2; 14.5. ESI-MS: 722.51 ([M þ H]þ ), 742.47
([M þ Na]þ ), 720.49 ([MꢀH]ꢀ ).

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Methyl (2b,3b)-2,3,23-Trihydroxy-28-({3-[(4-nitrobenzyl)amino]propyl}amino)-28-oxoolean-12-en-
30-oate (8g). Pale yellow powder (13.8 mg, 32.3%). M.p. 145.1–147.28. 1H-NMR (C5D5N): 8.14–7.12 (m,
2 H); 7.47–7.44 (m, 2 H); 6.52 (br. s, 1 H); 4.26–4.22 (m, 1 H); 4.12 (d, J ¼ 10.2, 1 H); 3.98 (d, J ¼ 10.2,
1 H); 3.82 (s, 2 H); 3.67 (s, 3 H); 3.28–3.24 (m, 2 H); 3.02 (d, J ¼ 10.2, 1 H); 2.99 (br. s, 1 H); 2.75–2.71
(m, 2 H); 2.35 (d, J ¼ 14.4, 1 H); 2.20 (d, J ¼ 12.6, 1 H); 2.14–2.11 (m, 3 H); 2.05–2.01 (m, 4 H); 1.90–
1.78 (m, 5 H); 1.74–1.70 (m, 1 H); 1.69–1.67 (m, 2 H); 1.66–1.64 (m, 1 H); 1.63 (s, 3 H); 1.47 (s, 3 H);
1.44–1.40 (m, 1 H); 1.38 (s, 3 H); 1.32–1.26 (m, 2 H); 1.23 (s, 3 H); 1.06 (d, J ¼ 13.8, 1 H); 0.86 (s, 3 H).
13C-NMR (C5D5N): 177.1; 174.4; 149.0; 146.9; 144.4; 128.5; 123.3; 122.9; 73.0; 71.5; 67.7; 52.3; 51.7; 48.7;
48.5; 48.1; 46.1; 44.8; 44.1; 43.3; 42.7; 42.4; 42.2; 39.8; 38.3; 37.2; 34.5; 33.0; 30.8; 30.6; 28.4; 28.3; 27.0;
26.2; 24.0; 23.9; 18.2; 17.4; 17.2; 14.5. ESI-MS: 724.55 ([M þ H]þ ), 722.51 ([MꢀH]ꢀ ).
Methyl (2b,3b)-2,3,23-Trihydroxy-28-({3-[(3-nitrobenzyl)amino]propyl}amino)-28-oxoolean-12-en-
30-oate (8h). Pale yellow powder (15.3 mg, 35.2%). M.p. 149.4–151.38. 1H-NMR (C5D5N): 8.12–8.10 (m,
1 H); 8.04 (s, 1 H); 7.66–7.63 (m, 1 H); 7.51 (m, 1 H); 6.52 (br. s, 1 H); 4.26–4.22 (m, 1 H); 4.12 (d, J ¼
10.2, 1 H); 3.98 (d, J ¼ 10.2, 1 H); 3.88 (s, 2 H); 3.67 (s, 3 H); 3.28–3.24 (m, 2 H); 3.02 (d, J ¼ 10.2, 1 H);
2.99 (br. s, 1 H); 2.75–2.71 (m, 2 H); 2.35 (d, J ¼ 14.4, 1 H); 2.20 (d, J ¼ 12.6, 1 H); 2.14–2.11 (m, 3 H);
2.05–2.01 (m, 4 H); 1.90–1.78 (m, 5 H); 1.74–1.70 (m, 1 H); 1.69–1.67 (m, 2 H); 1.66–1.64 (m, 1 H);
1.63 (s, 3 H); 1.47 (s, 3 H); 1.44–1.40 (m, 1 H); 1.38 (s, 3 H); 1.32–1.26 (m, 2 H); 1.23 (s, 3 H); 1.06 (d, J ¼
13.8, 1 H); 0.86 (s, 3 H). 13C-NMR (C5D5N): 177.1; 174.4; 148.6; 144.4; 141.7; 132.9; 129.5; 123.3; 122.0;
73.0; 71.5; 67.7; 52.6; 51.7; 48.7; 48.2; 48.1; 46.1; 44.8; 44.1; 43.3; 42.7; 42.4; 42.2; 39.8; 38.3; 37.2; 34.5; 33.0;
30.8; 30.6; 28.4; 28.3; 27.0; 26.2; 24.0; 23.9; 18.2; 17.4; 17.2; 14.5. ESI-MS: 724.57 ([M þ H]þ ), 746.47
([M þ Na]þ ), 722.52 ([MꢀH]ꢀ ).
Methyl (2b,3b)-2,3,23-Trihydroxy-28-({3-[(2-methoxybenzyl)amino]propyl}amino)-28-oxoolean-12-
1
en-30-oate (8i). White powder (15.5 mg, 36.4%). M.p. 141.5–143.98. H-NMR (C5D5N): 7.20–7.17 (m,
2 H); 6.92–6.87 (m, 2 H); 6.52 (br. s, 1 H); 4.26–4.22 (m, 1 H); 4.12 (d, J ¼ 10.2, 1 H); 3.98 (d, J ¼ 10.2,
1 H); 3.80 (s, 2 H); 3.67 (s, 3 H); 3.28–3.24 (m, 2 H); 3.02 (d, J ¼ 10.2, 1 H); 2.83 (br. s, 1 H); 2.75–2.71
(m, 2 H); 2.35 (d, J ¼ 14.4, 1 H); 2.20 (d, J ¼ 12.6, 1 H); 2.14–2.11 (m, 3 H); 2.05–2.01 (m, 4 H); 1.90–
1.78 (m, 5 H); 1.74–1.70 (m, 1 H); 1.68–1.66 (m, 2 H); 1.66–1.64 (m, 1 H); 1.63 (s, 3 H); 1.47 (s, 3 H);
1.44–1.40 (m, 1 H); 1.38 (s, 3 H); 1.32–1.26 (m, 2 H); 1.23 (s, 3 H); 1.06 (d, J ¼ 13.8, 1 H); 0.86 (s, 3 H).
13C-NMR (C5D5N): 177.1; 174.4; 157.8; 144.4; 130.2; 129.3; 129.3; 127.4; 123.3; 119.1; 109.9; 73.0; 71.5;
67.7; 54.9; 51.7; 48.9; 48.7; 48.2; 48.1; 46.1; 44.8; 44.1; 43.3; 42.7; 42.4; 42.2; 39.8; 38.3; 37.2; 34.5; 33.0; 30.8;
30.6; 28.4; 28.3; 27.1; 26.2; 24.0; 23.9; 18.2; 17.4; 17.2; 14.5. ESI-MS: 709.48 ([M þ H]þ ), 731.45 ([M þ
Na]þ ), 707.44 ([MꢀH]ꢀ ).
Methyl (2b,3b)-28-({3-[(2,4-Dichlorobenzyl)amino]propyl}amino)-2,3,23-trihydroxy-28-oxoolean-
12-en-30-oate (8j). White powder (13.2 mg, 29.5%). M.p. 116.8–118.48. 1H-NMR (C5D5N): 7.47 (s,
1 H); 7.21–7.17 (m, 2 H); 6.52 (br. s, 1 H); 4.26–4.22 (m, 1 H); 4.13 (d, J ¼ 10.2, 1 H); 4.10 (s, 2 H); 3.97
(d, J ¼ 10.2, 1 H); 3.67 (s, 3 H); 3.27–3.23 (m, 2 H); 3.02 (d, J ¼ 10.2, 1 H); 2.83 (br. s, 1 H); 2.78–2.76
(m, 2 H); 2.35 (d, J ¼ 14.4, 1 H); 2.20 (d, J ¼ 12.6, 1 H); 2.14–2.11 (m, 3 H); 2.05–2.01 (m, 4 H); 1.90–
1.78 (m, 5 H); 1.74–1.72 (m, 1 H); 1.71–1.69 (m, 2 H); 1.66–1.64 (m, 1 H); 1.63 (s, 3 H); 1.47 (s, 3 H);
1.44–1.40 (m, 1 H); 1.38 (s, 3 H); 1.32–1.26 (m, 2 H); 1.23 (s, 3 H); 1.06 (d, J ¼ 13.8, 1 H); 0.86 (s, 3 H).
13C-NMR (C5D5N): 177.1; 174.4; 144.4; 134.8; 132.5; 130.6; 128.7; 126.9; 123.3; 73.0; 71.5; 67.7; 51.7; 50.5;
48.6; 48.2; 48.1; 46.1; 44.8; 44.1; 43.3; 42.7; 42.4; 42.2; 39.8; 38.3; 37.2; 34.5; 33.0; 30.8; 30.6; 28.4; 28.3; 27.1;
26.2; 24.0; 23.9; 18.2; 17.4; 17.2; 14.5. ESI-MS: 747.39 ([M þ H]þ ), 745.36 ([MꢀH]ꢀ ).
Methyl (2b,3b)-2,3,23-Trihydroxy-28-oxo-28-({3-[(2,4,6-trihydroxybenzyl)amino]propyl}amino)-
1
olean-12-en-30-oate (8k). White powder (10.5 mg, 24.0%). M.p. 213.6–225.18. H-NMR (C5D5N): 6.52
(br. s, 1 H); 5.90 (dd, J ¼ 1.8, 2 H); 5.16 (br. s, 7 H); 4.26–4.22 (m, 1 H); 4.19 (s, 2 H); 4.13 (d, J ¼ 10.2,
1 H); 3.96 (d, J ¼ 10.2, 1 H); 3.67 (s, 3 H); 3.28–3.24 (m, 2 H); 3.02 (d, J ¼ 10.2, 1 H); 2.83 (br. s, 1 H);
2.75–2.71 (m, 2 H); 2.35 (d, J ¼ 14.4, 1 H); 2.20 (d, J ¼ 12.6, 1 H); 2.14–2.11 (m, 3 H); 2.05–2.01 (m,
4 H); 1.90–1.78 (m, 5 H); 1.74–1.72 (m, 1 H); 1.71–1.69 (m, 2 H); 1.66–1.64 (m, 1 H); 1.63 (s, 3 H); 1.47
(s, 3 H); 1.44–1.40 (m, 1 H); 1.38 (s, 3 H); 1.32–1.26 (m, 2 H); 1.23 (s, 3 H); 1.06 (d, J ¼ 13.8, 1 H); 0.86
(s, 3 H). 13C-NMR (C5D5N): 177.1; 174.4; 162.3; 158.7; 144.4; 123.3; 101.5; 94.1; 73.0; 71.5; 67.7; 51.7; 48.8;
48.2; 48.1; 46.1; 44.8; 44.1; 43.3; 42.7; 42.4; 42.2; 40.9; 39.8; 38.3; 37.2; 34.5; 33.0; 30.8; 30.6; 28.4; 28.3;
27.4; 26.2; 24.0; 23.9; 18.2; 17.4; 17.2; 14.5. ESI-MS: 727.45 ([M þ H]þ ), 749.39 ([M þ Na]þ ), 725.44
([MꢀH]ꢀ ).

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1851
Cell Culture. The murine celiac macrophage cell line RAW 264.7 was purchased from the
Experimental Animal Center of Second Military Medical University (Shanghai, P. R. China). RAW 264.7
Cells were cultured in Dulbeccoꢂs Modified Eagle Medium (DMEM) supplemented with 1000 U/ml of
penicilin, 10 mg/ml of streptomycin, and 10% fetal serum. The cells were incubated in an atmosphere of
5% CO2 at 378 and were subcultured every 5 d.
Measurement of NO. RAW 264.7 Macrophage cells were seeded into 48-well cell culture plates (2ꢂ
105 cells per well). The cells were co-incubated with testing drugs (100, 50, 25, 10, and 5 mm) and LPS
(1 mg/ml) for 24 h. Aminoguanidine (1 mg/ml) was used as positive, and control, and DMSO was used as
the negative control. The amount of NO was assessed by determining the nitrite concentration in the
culture supernatants with Griess reagent. Aliquots of supernatants (100 ml) were incubated in sequence
with 50 ml of 1% sulfanilamide and 50 ml of 0.1% N-(naphthalen-1-yl)ethylenediamine in 2.5%
phosphoric acid soln. The mixture was incubated in dark at r.t. for 15 min. The absorbance at 570 nm was
read with a microplate reader. All experiments were performed in triplicate.
The inhibition was calculated with the formula:
Inhibition [%]¼(ODc – ODt)/ODc ꢂ100
where ODc is the absorbance of negative control, and ODt is the absorbance of tested drug. The
concentration inhibiting 50% of the maximum NO was the IC50
.
Haemolytic Assay. Non-heparinized blood of healthy New Zealand rabbit (Experimental Animal
Center of Second Military Medical University, P. R. China) was used. The erythrocytes were washed three
times in PBS (phosphate-buffered saline: NaCl, 8 g/l; KCl, 0.2 g/l; Na2HPO4, 1.44 g/l; and KH2PO4,
0.24 g/l; pH 7.4) and then diluted with PBS to obtain a 10% suspension. All the tested compounds were
dissolved in DMSO at a concentration of 10 mm, then PBS was added to dilute the soln. to the testing
concentrations ranging from 5 to 500 mm, The final volume of the derivative soln. was 1.0 ml. The
erythrocyte suspension (100 ml) was added to 1 ml of the suspension to be tested, and the samples were
rapidly stirred and incubated at 378 with periodic stirring during a 60-min incubation period. The soln.
was then centrifuged at 3000 rpm for 5 min. Absorbance of the supernatant was measured at 540 nm
using Thermo Multiskan MK3 (Labsystems Dragon, Finland, model: 353); and the haemolytic % was
calculated by comparison with the 100% haemolytic activity caused by dist. H2O as maximal haemolytic
control. The haemolytic % developed by the PBS control was subtracted from all groups. Each
experiment was performed in triplicate at each concentration.
The haemolytic % was calculated as with the formula:
Haemolytic %¼(ODt – ODnc)/(ODpc – ODnc)ꢂ100
where ODt is the absorbance of tested drug, ODpc is the absorbance of postive control, and ODnc is the
absorbance of negative control. The concentration inducing 50% of the maximum haemolytic activity is
the HD50
.
Cell Viability. Cell viability was determined using the mitochondrial respiration-dependent MTT (¼
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) reduction method. Briefly, after
transferring the required supernatant to another plate for the Griess assay, the remaining supernatant
was aspirated from the 96-well plates, and 100 ml of fresh medium containing 5 mg/ml of MTTwas added
to each well. The cells were incubated at 378 in a humidified atmosphere containing 5% CO2. After
incubating for 3–4 h, the medium was removed, and the violet crystals of formazan in viable cells were
dissolved in DMSO. Absorbance at 570 nm was measured using a microplate reader. DMSO was used as
the negative control.
The inhibition was calculated with the formula:
Inhibition [%]¼(ODc – ODt)/ODc ꢂ100
where ODc is the absorbance of negative control, and ODt is the absorbance of tested drug.

1852
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
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Received November 27, 2010