Dammaranes from Gynostemma pentaphyllum and synthesis of their derivatives as inhibitors of protein tyrosine phosphatase 1B

Article abstract of DOI:10.1016/j.bmc.2010.04.073

Protein tyrosine phosphatase 1B (PTP1B) is a key factor in the negative regulation of insulin pathway and a promising target for treatment of diabetes and obesity. Herein, the sapogenin 2b, prepared from the natural triterpene saponin 1b, was modified at 3-position to establish the dammarane derivatives library via esterification, oxidation and reductive amination reaction and evaluated as PTP1B inhibitors. 3-O-para-Carboxylphenyl substituted derivative 5b was found with the best in vitro inhibition activity to protein tyrosine phosphatase 1B (IC₅₀ = 0.27 μM), where 3-O-meta-carboxylphenyl substituted 5a exhibited the best selectivity (nearly fivefolds) between PTP1B and T-cell protein tyrosine phosphatase.

Full text of DOI:10.1016/j.bmc.2010.04.073

Bioorganic & Medicinal Chemistry 18 (2010) 3934–3939
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Dammaranes from Gynostemma pentaphyllum and synthesis of their
derivatives as inhibitors of protein tyrosine phosphatase 1B
a,
Ji-Qing Xu a, Qiang Shen b, Jia Li b, , Li-Hong Hu
*
*
a State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai 201203, China
b National Center for Drug Screening, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Protein tyrosine phosphatase 1B (PTP1B) is a key factor in the negative regulation of insulin pathway and
a promising target for treatment of diabetes and obesity. Herein, the sapogenin 2b, prepared from the
natural triterpene saponin 1b, was modified at 3-position to establish the dammarane derivatives library
via esterification, oxidation and reductive amination reaction and evaluated as PTP1B inhibitors.
3-O-para-Carboxylphenyl substituted derivative 5b was found with the best in vitro inhibition activity
Received 10 March 2010
Revised 11 April 2010
Accepted 13 April 2010
Available online 28 April 2010
to protein tyrosine phosphatase 1B (IC50 = 0.27 lM), where 3-O-meta-carboxylphenyl substituted 5a
exhibited the best selectivity (nearly fivefolds) between PTP1B and T-cell protein tyrosine phosphatase.
Keywords:
Dammarane triterpene
PTP1B inhibitor
Ó 2010 Elsevier Ltd. All rights reserved.
Gynostemma pentaphyllum
SAR (structure–activity relationship)
1. Introduction
the lack of sufficient cell permeability and oral bioavailability due
to the presence of highly negative charged polar residues in these
Protein tyrosine phosphorylation is the predominant posttrans-
lational modification that cells utilize to regulate signal transduc-
tion and to control a wide variety of cellular functions, such as
growth, differentiation, survival, apoptosis, metabolism and gene
transcription.1–4 Protein tyrosine phosphatase 1B (PTP1B) is an
important regulator of tyrosine kinase receptor-mediated re-
sponses, and influences negatively insulin sensitivity.5–7 Indeed,
PTP1B knockout mice display increased insulin sensitivity and
tyrosine phosphorylation of the insulin receptor via catalyzing
the dephosphorylation of specific phosphotyrosine (pTyr) residues
on insulin receptor, insulin receptor substrate proteins and on Ja-
nus kinase 2, a protein tyrosine kinase which is associated with
leptin receptors.8–12 These results attracted considerable interest
for the development of pharmacological PTP1B inhibitors for the
treatment of insulin resistance, in particular non-insulin-depen-
dent diabetes mellitus (type 2 diabetes), which is characterized
by a deficient insulin cascade and is therefore also termed insulin
resistance (IR).13,11 Inhibitors of PTP1B, mainly involve non-hydro-
lysable phosphotyrosine mimics (or affinity-based), such as dif-
luoromethylphosphonates, cinnamic acid, oxalylamino benzoic
acid, isoxazole, carboxylic acid, salicyclic acid, and a-ketocarboxy-
lic acid. However, a major drawback of most of these inhibitors is
inhibitors. Moreover, due to their high conserved catalytic domain,
the selectivity between T-cell protein tyrosine phosphatase
(TCPTP) and PTP1B14 had still not been improved remarkably. Thus,
these two drawbacks resulted in the restriction of their further
development to clinical trials.
In the history of traditional Chinese medicine (TCM), medicinal
plant Gynostemma pentaphyllum (Thunb.) Makino and their
extracts were used to treat various diseases, especially diabetes,
referred as ‘thirsty disease’ in ancient China. Recently, in the inves-
tigation of the bioactive chemical constituents of the herb, the
dammarane type triterpene sapogenins 2a and 2b were found as
a moderate PTP1B inhibitor (IC50 = 5.3 and 15.7 lM), respec-
tively,15 but hard to be studied further due to its low content in
the plant (Fig. 1). In our research of G. pentaphyllum, compound
1b, the corresponding saponin of 2b, was found in the plant with
much richer source than its 20R epimer 1a. And its corresponding
sapogenin 2b, which can be obtained easily by acid hydrolysis of
1b, was also found with moderate activity (IC50 = 11.3
biological assessment, which was just a little weaker than 2a
(IC50 = 9.6 M). This result suggested 2b but not 2a was more suit-
lM) in our
l
able for further modification to establish the dammarane deriva-
tives library and analysis their SAR. In the present paper, we
report the synthesis and in vitro PTP1B inhibitory of 3-O-substi-
tuted ester (3a–3l, 5a–5c) and amine (7a–7c) derivatives of 2b.
Several compounds such as 5a–5c showed potent inhibitory activ-
* Corresponding authors. Tel./fax: +86 21 50800721 (J.L.);+86 21 50272221
(L.-H.H.).
ity to PTP1B (IC50 = 0.2–0.5
lM). Furthermore, selected compounds
E-mail addresses: jli@mail.shcnc.ac.cn (J. Li), simmhulh@mail.shcnc.ac.cn
in the series were evaluated their selectivity between PTP1B and
(L.-H. Hu).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.073

J.-Q. Xu et al. / Bioorg. Med. Chem. 18 (2010) 3934–3939
3935
O
O
HO
H
O
HO
H
O
20
AcO
HO
XylO
23
10% HCl
60 oC, 4 h
O
H
H
3
4
O
HO
H
H
ORha
µ
µ
2a 20R,23R IC50 = 9.6
2b
M
M
1a 20R,23R
1b
20S,23S
20S,23S IC50 = 11.3
Figure 1. Structure–activity of dammarane triterpene saponin PTP1B inhibitors.
TCPTP to estimate whether they were suitable for further
development.
2.2. Biological evaluation
To examine the potential inhibition of the natural and semi-
synthesis dammarane derivatives, their PTP1B inhibitory activities
were assayed by the method of pNPP using {4-[4-(4-oxalyl-
phenoxymethyl)-benzyloxy]-phenyl}-oxo-acetic acid as reference
compound.16 Moreover, their TCPTP inhibitory activities were
examined simultaneously by the same method for further selectiv-
ity studying.
Primarily, 3-OH of 2b was oxidized to give 6 to test whether the
hydroxyl group was necessary for preserving the activity. And to
our surprise, the value of IC50 of 6 only increased to 13.1 lM com-
2. Results and discussion
2.1. Chemistry
2.1.1. Preparation of compound 2b
The air-dried and powdered total saponins of G. pentaphyllum
was hydrolyzed by 10% HCl and purified by using silica gel CC to
give the starting material 2b.
pared with 2b, which suggested the hydroxyl might not influence
obviously the activity probably because it can not enter deeply en-
ough to the catalysis domain. Then various fatty acyl groups were
introduced to C-3 to provide 3a–3c. In spite of not consistent with
the design principle of phosphate mimics, introduction of fatty
chain gradually increased the activity along with the increasing
length of the carbon chain without suitable explanation till the
number of carbon on the chain reached 6.
In further study, aroyl was coupled with 3-hydroxyl to examine
the impact of aryl group. Interestingly, the introduction of aroyl, no
matter electron-donating or withdrawing groups bearing on it,
significantly improved the activity. The comparison of 3f–3h with
3i–3k indicated that aroyl with electron-withdrawing groups
2.1.2. Modification of compound 2b
Esterification of 2b with corresponding acid in normal condition
furnished the 3a–3l. Due to the steric hinderence caused by two
methyl groups at C-4, reaction of 2b with some acid might not give
acceptable yield, which could be easily figured out by increasing
largely the equivalent of substrate acid. 5a–5c can not be directly
synthesized by DCC/DMAP method because of large amount of side
products formation. Condensation of 2b and corresponding aldehy-
dic acid, the intermediate 4a–4c can be obtained via normal meth-
od mentioned above. Then oxidation of 4a–4c by using of Jone’s
reagent readily afforded 5a–5c. Oxidation of 2b with PDC yielded
the ketone derivative 6, which was reacted with different amine
using reductive amination to provide 7a–7c (Scheme 1).
O
O
O
HO
H
O
HO
H
O
O
HO
H
O
b
a
H
H
H
O
N
H
HO
H
H
H
2b
6
7a
d
c
O
O
O
HO
H
O
O
H
O
HO
O
O
OH
O
HO
H
O
H
e
H
H
H
O
O
H
O
O
H
H
5a
3d
4a
Scheme 1. Synthesis of 3-substituted derivatives. Reagents and conditions: (a) PDC, CH2Cl2, 2 h; (b) benzylamine, NaBH3CN, MeOH, rt, 8 h; (c) benzoic acid, DCC, DMAP,
CH2Cl2, rt, 4 h; (d) 4-formylbenzoic acid, DCC, DMAP, CH2Cl2, rt, 4 h; (e) Jones reagent, acetone, rt, 4 h.

3936
J.-Q. Xu et al. / Bioorg. Med. Chem. 18 (2010) 3934–3939
improved the activity more than those with electron-donating
groups. Moreover, comparison of 3k with 3i showed the nitro
group improved the activity at para position more than at ortho
position, whereas the situation of methoxyl-substituted derivative
was opposite, maybe because the nitro group was similar with
phosphate group to some extent, and that the nitro group entered
the catalysis domain more deeply could increase the activity.
When carboxyl group was introduced to the aryl (5a–5c), activ-
ity was largely enhanced. This result was consistent with the prin-
ciple17 that phosphate mimics with an aryl group nearby entering
the catalytic domain could improve the activity in large scale.
However, substitution of benzylamino group on C-3 (7a–7c) de-
creased the activity dramatically. Notwithstanding benzylamino
group not proper for C-3 modification, the activity data still indi-
cated that electron-deficient aromatic ring was more beneficial.
Most of the compounds were also subjected to TCPTP inhibitory
assay (see Tables 1). However, most of them did not exhibited sig-
nificant distinction between these two homogeneous enzymes ex-
cept for 5a, the selectivity of which reached to 4.9-fold with
relatively more potent on PTP1B activity. The conclusion was sim-
ilar to most reported PTP1B inhibitors.18
come the best lead compound for further modification. Although
potent activity was observed, the selectivity between PTP1B and
TCPTP of them was still not improved significantly, which will be
the priority of the further investigation. Moreover, acquisition of
the cellular result of compounds with good activity in vitro will
be moved forward quickly to find whether they can be suitable
for animal studies in diabetes and obesity.
4. Experimental section
4.1. General experimental procedure
1H (300 MHz) and 13C (75 MHz) NMR spectra were recorded on
a Varian Mercury-VX300 Fourier transform spectrometer. The
chemical shifts were reported in d (ppm) using the d 7.26 signal
of CDCl3 (1H NMR) and the d 77.23 signal of CDCl3 13C NMR) as
(
internal standards. ESI-MS was run on a Bruker Esquire 3000 plus
spectrometer in MeOH and HR-ESI-MS was run on a Bruker Atex III
spectrometer in MeOH, respectively. All commercially available
reagents were used without further purification. The solvents used
were all AR grade and were redistilled under positive pressure of
dry nitrogen atmosphere in the presence of proper desiccant when
necessary. The progress of the reactions was monitored by analyt-
ical thin-layer chromatography (TLC) on HSGF254 precoated silica
gel plates. Details of analytical HPLC profile of compounds in two
diverse systems were described in the Supplementary data.
3. Conclusion
Dammarane saponin compound 1b and its sapogenin 2b, as far
as we know, were first developed as PTP1B inhibitor derived from
nature product with SAR research. In the present paper, through
varying the structure at C-3 positions, several compounds
(5a–5c), were found potent on PTP1B inhibitory activity, with 5b
the utmost potency, up to 42-fold more effective than lead com-
pound 2b, with their SAR was preliminary studied and discussed.
Meanwhile, 5a was found the best selectivity which made it be-
4.2. Preparation of compound 2b
The air-dried and powdered total saponins of G. pentaphyllum
(1.0 kg) was dissolved to a 10 L solution of 10% HCl and stirred at
60 °C for 4 h. The mixture was then cooled to room temperature
and extracted with CHCl3 (10 L ꢀ 3). The combined organic layers
were concentrated under reduced pressure to give the residue
(870.1 g), which were subjected to CC ((SiO2; CHCl3/MeOH 100:0,
99:1, 98:2, 95:5, 9:1, 8:2) to afford eight fractions (Fr.A–H). Fr.D
(103.9 g) was isolated on further CC (SiO2; CHCl3/MeOH 99:1,
98:2, 95:5, 9:1) to yield pure product 2b (15.0 g). 1H NMR (CDCl3,
300 MHz): d 5.27 (d, J = 7.4 Hz, 1H), 5.12 (m, 1H), 3.17 (m, 1H), 1.74
(s, 3H), 1.76 (s, 3H), 0.96 (s, 3H), 0.95 (s, 3H), 0.76 (s, 3H), 0.74 (s,
3H), 0.74 (s, 3H); 13C NMR (CDCl3, 75 MHz): d 179.1 (s), 140.1 (s),
123.5 (d), 79.1 (d), 78.5 (s), 74.4 (d), 56.1 (d), 50.9 (d), 50.7 (s),
45.8 (s), 42.5 (d), 40.7 (t), 38.9 (t), 38.1 (s), 37.3 (s), 35.4 (s), 31.5
(t), 28.2 (t), 27.5 (d), 26.6 (t), 26.0 (q), 25.3 (t), 21.6 (q), 18.6 (q),
18.4 (t), 16.6 (q), 16.4 (q), 15.6 (q), 15.5 (q); HRMS calcd for
C30H48O4: 472.3553. Found: 472.3545.
Table 1
Inhibitory activity of all compounds against PTP1B and TCPTP
O
HO
H
O
H
R
H
a
Compounds
R
IC50
(l
M)
PTP1B
TCPTP
2b
3a
3b
3c
3d
3e
3f
3g
3h
3i
OH
11.25 0.42 10.04 0.32
12.69 0.31 NTb
4.60 0.55 14.70 0.93
4.14 0.08 11.75 0.20
Acetoxyl
Propionyl
Pentanoyl
Benzoyl
20-Pyridinylcarbonyloxy
20-Methoxybenzoyloxy
30-Methoxybenzoyloxy
40-Methoxybenzoyloxy
20-Nitrobenzoyloxy
30-Nitrobenzoyloxy
40-Nitrobenzoyloxy
20,40-Dinitrobenzoyloxy
30-Carboxybenzoyloxy
40-Carboxybenzoyloxy
30-Nitro-40-carboxybenzoyloxy
@O
40-Methoxybenzylamino
30,50-Difluorobenzylamino
40-Nitrobenzylamino
4.3. Chemical synthesis
2.75 0.50
2.36 0.32
1.90 0.45
4.15 0.12
5.14 0.17
4.05 0.20
4.3.1. General procedure for synthesis of 3a–3l
To a solution of 2b (30 mg, 0.064 mM) in anhydrous DCM (5 ml)
was added corresponding acid (0.25 mM), DCC (0.25 mM), and
DMAP (0.025 mM) in room temperature under N2. The solution
was stirred at room temperature for 4 h. TLC monitored the reac-
tion. After starting material 2b was consumed out, the solution
was diluted to 30 mL, washed with 2 N HCl (30 mL ꢀ 3) and NaH-
CO3 aqueous solution (30 mL ꢀ 3) successively. Then the combined
organics were washed with 10 mL water and 10 mL brine succes-
sively, dried (anhydrous Na2SO4). The crude product after concen-
tration was subject to column chromatography and eluted with
petrol and acetone (9:1) to afford the pure corresponding com-
pounds 3a–3l.
2.08 0.29 11.98 0.98
2.61 0.08
1.96 0.24
1.70 0.09
1.42 0.22
1.62 0.23
0.33 0.02
0.27 0.05
0.50 0.06
3.90 0.18
3.38 0.13
2.26 0.26
5.10 0.98
2.87 0.39
1.62 0.07
0.97 0.08
1.22 0.07
3j
3k
3l
5a
5b
5c
6
13.12 0.71 34.13 0.591
24% NT
11.59 0.87 NT
6.58 0.27 NT
7ac
7b
7c
a
The pNPP assay. IC50 values were determined by regression analyses and
4.3.1.1.
(3S,20S,23S)-3-Acetoxyl-20,23-dihydroxydammar-24-
expressed as means SD of three replications.
en-21-oic acid-21,23-lactone (3a). 92.3% Yield; 1H NMR (CDCl3,
300 MHz): d 5.25 (d, J = 7.1 Hz, 1H), 5.12 (m, 1H), 4.47 (m, 1H),
b
Not tested.
c
Inhibition rate to PTP1B in 20 lM concentration.

J.-Q. Xu et al. / Bioorg. Med. Chem. 18 (2010) 3934–3939
3937
2.03 (s, 3H), 1.74 (s, 3H), 1.76 (s, 3H), 0.97 (s, 3H), 0.90 (s, 3H), 0.86
(s, 3H), 0.84 (s, 6H); 13C NMR (CDCl3, 75 MHz): d 179.0 (s), 171.3
(s), 140.2 (s), 123.4 (d), 81.0 (d), 78.4 (s), 74.3 (d), 56.2 (d), 50.9
(d), 50.7 (s), 45.7 (s), 42.4 (d), 40.8 (t), 40.7 (s), 38.9 (t), 38.1 (s),
37.3 (s), 35.4 (s), 31.5 (t), 29.9 (t), 28.1 (q), 26.6 (t), 26.0 (q), 25.3
(t), 23.9 (t), 21.6 (q), 18.6 (q), 18.3 (t), 16.7 (q), 16.6 (q), 16.5 (q),
15.6 (q); HRMS calcd for C32H50O5: 514.3658. Found: 514.3670.
123.4 (d), 120.8 (s), 120.2 (d), 112.1 (d), 81.5 (d), 78.4 (s), 74.3
(s), 56.1 (d), 55.9 (q), 50.8 (d), 50.7 (s), 45.6 (d), 42.4 (d), 40.7 (t),
40.6 (s), 38.9 (t), 38.4 (s), 37.2 (s), 35.3 (t), 31.5 (t), 28.2 (q), 26.5
(t), 26.0 (q), 25.3 (t), 24.0 (t), 21.5 (t), 18.6 (q), 18.3 (t), 16.8 (q),
16.6 (q), 16.5 (q), 15.6 (q); HRMS calcd for C38H54O6: 606.3920.
Found: 606.3949.
4.3.1.7.
(3S,20S,23S)-3-(30-Methoxybenzoyloxy)-20,23-dihydr-
4.3.1.2. (3S,20S,23S)-3- Propionyl-20,23-dihydroxydammar-24-
en-21-oic acid-21,23-lactone (3b). 89.3% Yield; 1H NMR (CDCl3,
300 MHz): d 1H NMR (CDCl3, 300 MHz): d 5.25 (d, J = 7.1 Hz, 1H),
5.12 (m, 1H), 4.47 (m, 1H), 2.34 (q, J = 6.7 Hz, 2H), 1.74 (s, 3H),
1.76 (s, 3H), 1.17 (t, J = 6.7 Hz, 3H), 0.97 (s, 3H), 0.93 (s, 3H), 0.86
(s, 3H), 0.84 (s, 6H); 13C NMR (CDCl3, 75 MHz): d 179.0 (s), 174.5
(s), 140.4 (s), 123.3 (d), 80.8 (d), 78.3 (s), 74.3 (d), 56.2 (d), 50.9
(d), 50.8 (s), 45.8 (d), 42.4 (d), 41.0 (t), 40.7 (s), 38.9 (t), 38.2 (s),
37.3 (s), 35.4 (t), 31.6 (t), 28.3 (t), 28.1 (q), 26.6 (t), 26.0 (q), 25.3
(t), 23.9 (t), 21.6 (t), 18.7 (q), 18.3 (t), 16.7 (q), 16.6 (q), 16.5 (q),
15.6 (q), 9.6 (q); HRMS calcd for C33H52O5: 528.3815. Found:
528.3804.
oxydammar-24-en-21-oic acid-21,23-lactone (3g). 72.7% Yield
28.0 mg; 1H NMR (CDCl3, 300 MHz): d 7.61 (d, J = 7.2 Hz, 1H),
7.32 (t, J = 7.2 Hz, 1H), 7.54 (s, 1H), 7.06 (d, J = 7.2 Hz, 1H), 5.26
(d, J = 7.1 Hz, 1H), 5.12 (m, 1H), 4.67 (m, 1H), 3.83 (s, 3H), 1.72 (s,
3H), 1.74 (s, 3H), 0.98 (s, 6H), 0.89 (s, 9H); 13C NMR (CDCl3,
75 MHz): d 179.0 (s), 166.3 (s), 159.6 (s), 140.1 (s), 132.4 (s),
129.5 (d), 123.4 (d), 122.0 (d), 119.2 (d), 114.3 (d), 81.8 (d), 78.4
(s), 74.3 (s), 56.1 (d), 55.5 (q), 50.8 (d), 50.6 (s), 45.6 (d), 42.4 (d),
40.6 (t), 38.9 (t), 38.5 (s), 37.3 (s), 35.3 (t), 31.5 (t), 28.3 (q), 26.5
(t), 25.9 (q), 25.2 (t), 23.8 (t), 21.5 (t), 18.6 (q), 18.3 (t), 16.9 (q),
16.5 (q), 16.5 (q), 15.6 (q); HRMS calcd for C38H54O6: 606.3920.
Found: 606.3901.
4.3.1.3. (3S,20S,23S)-3-(20-pentanoyl)-20,23-dihydroxydammar-
24-en-21-oic acid-21,23-lactone (3c). 72.3% Yield; 1H NMR
(CDCl3, 300 MHz): d 5.25 (d, J = 7.2 Hz, 1H), 5.11 (m, 1H), 4.47
(m, 1H), 1.75 (s, 3H), 1.73 (s, 3H), 0.98 (s, 3H), 0.88 (s, 3H), 0.86
(s, 3H), 0.82 (s, 3H), 0.76 (s, 3H); 13C NMR (CDCl3, 75 MHz): d
179.1 (s), 173.9 (s), 140.1 (s), 123.4 (d), 80.7 (d), 78.4 (s), 74.3
(d), 56.1 (d), 50.9 (d), 50.7 (s), 45.6 (d), 42.4 (d), 40.7 (t), 40.7 (s),
38.8 (t), 38.0 (s), 37.2 (s), 35.3 (t), 34.7 (t), 31.5 (t), 28.1 (q), 27.4
(t), 26.6 (t), 26.0 (t), 25.3 (q), 23.8 (t), 22.5 (t), 21.5 (t), 18.6 (q),
18.3 (t), 16.7 (q), 16.6 (q), 16.5 (q), 15.6 (q), 13.9 (q); HRMS calcd
for C35H56O5: 556.4128. Found: 556.4119.
4.3.1.8.
(3S,20S,23S)-3-(40-Methoxybenzoyloxy)-20,23-dihydr-
oxydammar-24-en-21-oic acid-21,23-lactone (3h). 75.3% Yield;
1H NMR (CDCl3, 300 MHz): d 7.98 (d, J = 7.3 Hz, 2H), 6.90 (d,
J = 7.3 Hz, 2H), 5.26 (d, J = 7.1 Hz, 1H), 5.12 (m, 1H), 4.67 (m, 1H),
3.85 (s, 3H), 1.74 (s, 3H), 1.76 (s, 3H), 0.98 (s, 6H), 0.89 (s, 9H);
13C NMR (CDCl3, 75 MHz): d 179.5 (s), 166.1 (s), 163.7 (s), 140.1
(s), 132.5 (s), 131.7 (d), 123.4 (d), 113.8 (d), 81.3 (d), 78.2 (s),
74.4 (s), 56.2 (d), 55.7 (q), 50.9 (d), 50.8 (s), 45.8 (d), 42.4 (d),
40.9 (t), 40.7 (s), 39.0 (t), 38.5 (s), 37.3 (s), 35.4 (t), 31.6 (t), 28.3
(q), 26.6 (t), 26.0 (q), 25.3 (t), 24.0 (t), 21.6 (t), 18.7 (q), 18.4 (t),
17.0 (q), 16.6 (q), 16.6 (q), 15.7 (q); HRMS calcd for C38H54O6:
606.3920. Found: 606.3892.
4.3.1.4. (3S,20S,23S)-3-Benzoyl-20,23-dihydroxydammar-24-en-
21-oic acid-21,23-lactone (3d). 87.3% Yield; 1H NMR (CDCl3,
300 MHz): d 8.03 (d, J = 7.5 Hz, 2H), 7.50 (m, 3H), 5.25 (d,
J = 7.1 Hz, 1H), 5.12 (m, 1H), 4.47 (m, 1H), 1.74 (s, 3H), 1.76 (s,
3H), 1.01 (s, 6H), 0.87 (s, 9H); 13C NMR (CDCl3, 75 MHz): d 179.0
(s), 166.5 (s), 140.4 (s), 132.9 (d), 131.2 (s), 129.7 (d), 128.5 (d),
123.4 (d), 81.7 (d), 78.3 (s), 74.3 (d), 56.2 (d), 50.9 (d), 50.8 (s),
45.8 (d), 42.4 (d), 41.0 (t), 40.7 (s), 38.9 (t), 38.5 (s), 37.3 (s), 35.4
(t), 31.6 (t), 31.1 (d), 29.9 (t), 28.3 (q), 26.6 (t), 26.0 (d), 25.3 (t),
23.9 (t), 21.6 (t), 18.6 (q), 18.3 (t), 17.0 (q), 16.6 (q), 16.5 (q), 15.7
(q); HRMS calcd for C37H52O5: 576.3815. Found: 576.3792.
4.3.1.9.
dammar-24-en-21-oic acid-21,23-lactone (3i). 83.4% Yield; 1H
NMR (CDCl3, 300 MHz): 7.84 (d, J = 7.8 Hz, 1H), 7.73 (d,
(3S,20S,23S)-3-(20-Nitrobenzoyloxy)-20,23-dihydroxy-
d
J = 7.8 Hz, 1H),7.62 (m, 2H), 5.26 (d, J = 7.1 Hz, 1H), 5.12 (m, 1H),
4.73 (m, 1H), 1.76 (s, 3H), 1.74 (s, 3H), 0.97 (s, 3H), 0.92 (s, 3H),
0.89 (s, 3H), 0.87 (s, 3H), 0.83 (s, 3H); 13C NMR (CDCl3, 75 MHz):
d 179.0 (s), 165.3 (s), 148.5 (s), 140.3 (s), 132.8 (s), 131.8 (d),
130.2 (d), 128.2 (d), 124.0 (d), 123.3 (d), 84.0 (d), 78.4 (s), 74.3
(s), 56.2 (d), 50.8 (d), 50.7 (s), 45.7 (d), 42.4 (d), 40.8 (t), 40.6 (s),
38.9 (t), 38.3 (s), 37.2 (s), 35.3 (t), 31.5 (t), 28.2 (q), 26.5 (t), 26.0
(q), 25.3 (t), 23.1 (t), 21.6 (t), 18.6 (q), 18.2 (t), 16.6 (q), 16.6 (q),
16.4 (q), 15.6 (q); HRMS calcd for C37H51NO7: 621.3666. Found:
621.3659.
4.3.1.5. (3S,20S,23S)-3-(20-pyridinylcarbonyloxy)-20,23-dihydr-
oxydammar-24-en-21-oic acid-21,23-lactone (3e). 75.9% Yield;
1H NMR (CDCl3, 300 MHz): d 8.76 (d, J = 5.8 Hz, 1H), 8.04 (d,
J = 7.5 Hz, 1H), 7.70 (t, J = 7.5 Hz, 1H), 7.43 (m, 1H), 5.25 (d,
J = 7.2 Hz, 1H), 5.11 (m, 1H), 4.81 (m, 1H), 1.75 (s, 3H), 1.73 (s,
3H), 0.98 (s, 3H), 0.96 (s, 3H), 0.90 (s, 3H), 0.89 (s, 3H), 0.87 (s,
3H); 13C NMR (CDCl3, 75 MHz): d 179.0 (s), 164.7 (s), 150.2 (d),
148.7 (s), 140.2 (s), 137.1 (d), 126.8 (d), 125.0 (d), 123.3 (d), 82.5
(d), 78.3 (s), 74.2 (s), 56.0 (d), 50.8 (d), 50.6 (s), 45.6 (d), 42.3 (d),
40.8 (t), 40.6 (s), 38.8 (t), 38.4 (s), 37.2 (s), 35.3 (t), 31.5 (t), 28.2
(q), 26.5 (t), 25.9 (q), 25.2 (t), 23.8 (t), 21.5 (t), 18.6 (q), 18.3 (t),
16.9 (q), 16.5 (q), 15.6 (q); HRMS calcd for C36H51O5N: 577.3767.
Found: 577.3760.
4.3.1.10. (3S,20S,23S)-3-(30-Nitrobenzoyloxy)-20,23-dihydroxy-
dammar-24-en-21-oic acid-21,23-lactone (3j). 81.0% Yield; 1H
NMR (CDCl3, 300 MHz): d 8.82 (s, 1H), 8.40 (d, J = 7.8 Hz, 1H),
8.35 (d, J = 7.8 Hz, 1H), 7.64 (t, J = 7.8 Hz, 1H), 5.26 (d, J = 7.1 Hz,
1H), 5.12 (m, 1H), 4.76 (m, 1H), 1.76 (s, 3H), 1.74 (s, 3H), 1.01 (s,
3H), 0.96 (s, 3H), 0.83 (s, 9H); 13C NMR (CDCl3, 75 MHz): d 179.1
(s), 164.3 (s), 148.4 (s), 140.2 (s), 135.4 (s), 132.8 (d), 129.8 (d),
127.4 (d), 124.6 (d), 123.4 (d), 83.0 (d), 78.4 (s), 74.4 (s), 56.1 (d),
50.8 (d), 50.6 (s), 45.6 (d), 42.4 (d), 40.8 (t), 40.6 (s), 38.8 (t), 38.6
(s), 37.2 (s), 35.3 (t), 31.5 (t), 28.3 (q), 26.5 (t), 26.0 (q), 25.7 (t),
23.1 (t), 21.5 (t), 18.6 (q), 18.3 (t), 16.9 (q), 16.5 (q), 16.5 (q), 15.6
(q); HRMS calcd for C37H51NO7: 621.3666. Found: 621.3632.
4.3.1.6.
(3S,20S,23S)-3-(20-Methoxybenzoyloxy)-20,23-dihydr-
oxydammar-24-en-21-oic acid-21,23-lactone (3f). 79.1% Yield;
1H NMR (CDCl3, 300 MHz): d 7.80 (d, J = 7.8 Hz, 1H), 7.44 (t,
J = 7.8 Hz, 1H), 6.95 (m, 2H), 5.26 (d, J = 7.1 Hz, 1H), 5.12 (m, 1H),
4.67 (m, 1H), 3.87 (s, 3H), 1.72 (s, 3H), 1.74 (s, 3H), 0.98 (s, 3H),
0.95 (s, 6H), 0.89 (s, 3H), 0.84 (s, 3H); 13C NMR (CDCl3, 75 MHz):
d 179.0 (s), 166.1 (s), 159.4 (s), 140.2 (s), 133.5 (d), 131.8 (d),
4.3.1.11. (3S,20S,23S)-3-(40-Nitrobenzoyloxy)-20,23-dihydroxy-
dammar-24-en-21-oic acid-21,23-lactone (3k). 85.4% Yield; 1H
NMR (CDCl3, 300 MHz):
d 8.29 (d, J = 7.4 Hz, 1H), 8.17 (d,
J = 7.4 Hz, 1H), 5.26 (d, J = 7.2 Hz, 1H), 5.12 (m, 1H), 4.76 (m, 1H),
1.76 (s, 3H), 1.74 (s, 3H), 1.05 (s, 3H), 1.00 (s, 3H), 0.91 (s, 9H);

3938
J.-Q. Xu et al. / Bioorg. Med. Chem. 18 (2010) 3934–3939
13C NMR (CDCl3, 75 MHz): d 179.0 (s), 164.5 (s), 150.6 (s), 140.3 (s),
136.5 (s), 130.8 (d), 123.7 (d), 123.3 (d), 83.0 (d), 78.3 (s), 74.3 (s),
56.1 (d), 50.8 (d), 50.7 (s), 45.6 (d), 42.3 (d), 40.8 (t), 40.6 (s), 38.8
(t), 38.5 (s), 37.3 (s), 35.3 (t), 31.5 (t), 28.3 (q), 26.5 (t), 26.0 (q), 25.3
(t), 23.8 (t), 21.6 (t), 18.6 (q), 18.3 (t), 16.9 (q), 16.6 (q), 16.5 (q),
15.6 (q); HRMS calcd for C37H51NO7: 621.3666. Found: 621.3684.
4.3.2.3. (3S,20S,23S)-3-(30-Nitro-40-carboxybenzoyloxy)-20,23-
dihydroxydammar-24-en-21-oic acid-21,23-lactone (5c). 48.4%
Yield; 1H NMR (CDCl3+CD3OD, 300 MHz): d 8.39 (s, 1H), 8.19 (d,
J = 7.8 Hz, 1H), 7.76 (d, J = 7.8 Hz, 1H), 5.25 (d, J = 7.2 Hz, 1H),
5.11 (m, 1H), 4.72 (m, 1H), 1.76 (s, 3H), 1.75 (s, 3H), 1.01 (s, 6H),
0.92 (s, 3H), 0.90 (s, 6H); 13C NMR (CDCl3+CD3OD, 75 MHz): d
180.3 (s), 171.6 (s), 164.8 (s), 148.2 (s), 140.3 (s), 137.8 (s), 134.1
(d), 133.0 (s), 130.3 (d), 125.1 (d), 124.3 (d), 83.8 (d), 79.1 (s),
75.1 (d), 56.7 (d), 51.5 (d), 51.1 (s), 45.9 (d), 43.2 (d), 41.2 (t),
40.9 (s), 39.4 (t), 39.0 (s), 37.8 (s), 35.9 (t), 32.0 (t), 28.6 (q), 27.2
(t), 26.0 (q), 25.8 (t), 24.4 (t), 22.1 (t), 18.8 (q), 18.6 (t), 17.3 (q),
16.8 (q), 15.9 (q); HRMS calcd for C38H51O9N: 665.3564. Found:
665.3543.
4.3.1.12. (3S,20S,23S)-3-(20,40-Dinitrobenzoyloxy)-20,23-dihydr-
oxydammar-24-en-21-oic acid-21,23-lactone (3l). 69.1% Yield;
1H NMR (CDCl3, 300 MHz): d 8.73 (s, 1H), 8.50 (d, J = 7.4 Hz, 1H),
7.93 (d, J = 7.4 Hz, 1H), 5.26 (d, J = 7.2 Hz, 1H), 5.12 (m, 1H), 4.76
(m, 1H), 1.76 (s, 3H), 1.74 (s, 3H), 0.97 (s, 3H), 0.92 (s, 3H), 0.90
(s, 3H), 0.87 (s, 3H), 0.81 (s, 3H); 13C NMR (CDCl3, 75 MHz): d
179.0 (s), 163.7 (s), 149.0 (s), 148.3 (s), 140.2 (s), 133.5 (s), 131.5
(d), 127.6 (d), 123.4 (s), 119.7 (s), 85.3 (d), 78.4 (s), 74.3 (s), 56.2
(d), 50.8 (d), 50.6 (s), 45.6 (d), 42.4 (d), 40.6 (t), 38.8 (s), 38.3 (t),
37.2 (s), 35.3 (s), 34.0 (t), 31.5 (t), 28.2 (q), 25.9 (t), 25.7 (q), 25.2
(t), 23.1 (t), 21.6 (t), 18.6 (q), 18.2 (t), 16.6 (q), 16.4 (q), 15.6 (q);
HRMS calcd for C37H50O9N2: 666.3516. Found: 666.3548.
4.3.3. Synthesis of 6
To a solution of 2b (5 g, 0.02 mM) in anhydrous DCM (50 mL)
was added PDC 4 g in 0 °C under N2. The solution was stirred in
room temperature for 2 h. After TLC showed the starting material
was consumed out, the solution was diluted by 300 mL ether and
filtrated. The filtrate was collected and evaporated to give crude
product 4.21 g, which was subjected to column chromatography
and eluted with petrol and acetone (5.5:1) to afford the pure cor-
responding compound 3.52 g (70.9% yield).
4.3.2. General procedure for synthesis of 5a–5c
To a solution of 2b (30 mg, 0.064 mM) in anhydrous DCM
(5 mL) was added corresponding aldehyde acid (0.25 mM), DCC
(0.25 mM), and DMAP (0.025 mM) in room temperature under
N2. The solution was stirred at room temperature for 4 h. TLC mon-
itored the reaction. After starting materia 2b was consumed out,
the solution was diluted to 30 mL, washed with 2 N HCl
(30 mL ꢀ 3), 10 mL water and 10 mL brine successively, dried
(anhydrous Na2SO4). The organic layer was concentrated to give
4a–4c, which was added directly to a solution of equal equivalent
Jones reagent in 10 mL acetone in 0 °C. The solution was stirred at
room temperature for 4 h. When TLC showed the material was
consumed out, the solution was concentrated and partitioned be-
tween EtOAc (30 mL ꢀ 3) and 2 N HCl (30 mL ꢀ 3), washed with
30 mL NaHCO3 aqueous, 10 mL of water and 10 mL of brine succes-
sively, and then dried (anhydrous Na2SO4). The crude product after
concentration was subject to column chromatography and eluted
with petrol and acetone (3:1) to afford the pure corresponding
compounds 5a–5l.
4.3.3.1. (20S,23S)-Dihydroxydammar-24-en-21-oic acid-3-oxo-
21,23-lactone (6). 70.7% Yield; 1H NMR (CDCl3, 300 MHz): d 5.25
(d, J = 7.8 Hz, 1H), 5.12 (m, 1H), 1.74 (s, 3H), 1.76 (s, 3H), 1.07 (s,
3H), 1.03 (s, 3H), 1.01 (s, 3H), 0.94 (s, 3H), 0.90 (s, 3H); HRMS calcd
for C30H46O4: 470.3396. Found: 470.3371.
4.3.4. General procedure for synthesis of 7a–7c
To a solution of 6 (70 mg, 0.15 mM), corresponding amine
0
(0.44 mM) and 4 ÅA molecular sieves (30 mg) in 10 mL MeOH was
added NaBH3CN (28 mg, 0.45 mM) in room temperature under
N2. The solution was stirred at room temperature for 8 h. The solu-
tion was evaporated to give crude product, which was subjected to
column chromatography and eluted with petrol and acetone (4:1)
to afford the pure corresponding product 7a–7c.
4.3.4.1. (3S,20S,23S)-3-(40-Methoxybenzylamino)-20,23-dihydr-
oxydammar-24-en-21-oic acid-21,23-lactone (7a). 25.6% Yield;
1H NMR (CDCl3, 300 MHz): d 7.26 (d, J = 7.2 Hz, 2H), 6.85 (d,
J = 7.2 Hz, 2H), 5.25 (d, J = 7.2 Hz, 1H), 5.11 (m, 1H), 1.77 (s, 3H),
1.75 (s, 3H), 3.92 (d, J = 14.1 Hz, 1H), 3.79 (s, 3H), 3.60 (d,
J = 14.1 Hz, 1H), 2.46 (m, 1H), 0.96 (s, 3H), 0.93 (s, 3H), 0.86 (s,
3H), 0.82 (s, 3H), 0.68 (s, 3H); HRMS calcd for C38H57O4N:
591.4288. Found: 591.4292.
4.3.2.1. (3S,20S,23S)-3-(30-Carboxybenzoyloxy)-20,23-dihydroxy-
dammar-24-en-21-oic acid-21,23-lactone (5a). 62.1% Yield; 1H
NMR (CDCl3, 300 MHz): d 8.73 (s, 1H), 8.27 (m, 1H), 7.55 (t,
J = 7.8 Hz, 1H), 5.25 (d, J = 7.2 Hz, 1H), 5.11 (m, 1H), 4.74 (m, 1H),
1.75 (s, 3H), 1.74 (s, 3H), 1.01 (s, 3H), 0.98 (s, 3H), 0.94 (s, 3H),
0.91 (s, 3H), 0.89 (s, 3H); 13C NMR (CDCl3, 75 MHz): d 179.7 (s),
170.6 (s), 165.5 (s), 140.3 (s), 134.7 (d), 134.3 (d), 131.7 (s), 131.4
(d), 130.0 (s), 128.8 (s), 123.3 (d), 82.3 (d), 78.6 (s), 74.6 (d), 56.2
(d), 50.8 (d), 50.7 (s), 45.6 (d), 42.4 (d), 40.8 (t), 40.7 (s), 38.8 (t),
38.5 (s), 37.2 (s), 35.3 (t), 31.5 (t), 28.3 (q), 26.5 (t), 25.9 (q), 25.3
(t), 23.8 (t), 21.5 (t), 18.6 (q), 18.3 (t), 16.9 (q), 16.5 (q), 16.4 (q),
15.6 (q); HRMS calcd for C38H52O7: 620.3713. Found: 620.3729.
4.3.4.2. (3S,20S,23S)-3-(30,50-Difluorobenzylamino)-20,23-dihy-
droxydammar-24-en-21-oic acid-21,23-lactone (7b). 19.2%
Yield; 1H NMR (CDCl3, 300 MHz): d 6.89 (m, 2H), 6.62 (m, 1H),
5.25 (d, J = 7.2 Hz, 1H), 5.11 (m, 1H), 1.77 (s, 3H), 1.75 (s, 3H),
3.95 (d, J = 13.2 Hz, 1H), 3.62 (d, J = 13.2 Hz, 1H), 2.44 (m, 1H),
0.96 (s, 3H), 0.95 (s, 3H), 0.86 (s, 3H), 0.82 (s, 3H), 0.73 (s, 3H); E
HRMS calcd for C37H53O3NF2: 597.3994. Found: 597.4017.
4.3.2.2. (3S,20S,23S)-3-(40-Carboxybenzoyloxy)-20,23-dihydroxy-
dammar-24-en-21-oic acid-21,23-lactone (5b). 52.9% Yield; 1H
NMR (CDCl3, 300 MHz):
d 8.15 (d, J = 8.1 Hz, 1H), 8.13 (d,
J = 8.1 Hz, 1H), 5.25 (d, J = 7.2 Hz, 1H), 5.11 (m, 1H), 4.74 (m, 1H),
1.76 (s, 3H), 1.75 (s, 3H), 1.01 (s, 3H), 0.98 (s, 3H), 0.92 (s, 3H),
0.90 (s, 6H); 13C NMR (CDCl3, 75 MHz): d 179.5 (s), 170.6 (s),
165.6 (s), 140.5 (s), 135.6 (d), 133.0 (d), 130.3 (d), 129.8 (d),
123.3 (d), 82.5 (d), 78.5 (s), 74.5 (s), 56.2 (d), 50.9 (d), 50.7 (s),
45.7 (d), 42.4 (d), 40.9 (t), 40.7 (s), 38.9 (t), 38.5 (s), 37.3 (s), 35.3
(t), 31.6 (t), 28.3 (q), 26.5 (t), 26.0 (q), 25.3 (t), 23.9 (t), 21.6 (t),
18.7 (q), 18.3 (t), 17.0 (q), 16.6 (q), 16.5 (q), 15.6 (q); HRMS calcd
for C38H52O7: 620.3713. Found: 620.3732.
4.3.4.3. (3S,20S,23S)-3-(40-Nitrobenzylamino)-20,23-dihydroxy-
dammar-24-en-21-oic acid-21,23-lactone (7c). 34.5% Yield; 1H
NMR (CDCl3, 300 MHz):
d 8.12 (d, J = 7.2 Hz, 2H), 7.51 (d,
J = 7.2 Hz, 2H), 5.25 (d, J = 7.2 Hz, 1H), 5.11 (m, 1H), 4.08 (d,
J = 14.1 Hz, 1H), 3.62 (d, J = 14.1 Hz, 1H), 2.50 (m, 1H), 1.77 (s,
3H), 1.75 (s, 3H), 1.01 (s, 3H), 0.99 (s, 3H), 0.86 (s, 3H), 0.82 (s,
3H), 0.73 (s, 3H); HRMS calcd for C37H54O5N2: 606.4033. Found:
606.4001.

J.-Q. Xu et al. / Bioorg. Med. Chem. 18 (2010) 3934–3939
3939
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This work was supported by the Chinese NationalScience & Tech-
nology Major Project ‘Key New Drug Creation and Manufacturing
Program’ (Grants 2009ZX09301-001, 2009ZX09102-022), the Chi-
nese National High-Tech R&D Program (Grants 2007AA02Z147),
the National Natural Science Foundation of China (Grants 9071
3046, 30772638, 30925040) and CAS Foundation (Grant KSCX2-
YW-R-179).
14. Iverson, L. F.; Moller, K. B.; Pedersen, A. K.; Peters, G. H.; Petersen, A. S.;
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Supplementary data
15. Hung, T. M.; Hoang, D. M.; Kim, J. C.; Jang, H. S.; Ahn, J. S.; Min, B. S. J.
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