Asymmetric synthesis and biological activities of natural product (+)-balasubramide and its derivatives

Article abstract of DOI:10.1080/14786419.2015.1071363

The natural product (+)-balasubramide (3j) and its derivatives (3a-3i) were synthesized using a two-step asymmetric synthesis, and the biological activities of 3a-3j were determined in vitro. Methyl (2S,3R)-(+)-3-phenyloxirane-2-carboxylate (1h), the asym

Full text of DOI:10.1080/14786419.2015.1071363

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Asymmetric synthesis and biological
activities of natural product (+)-
balasubramide and its derivatives
Jun Lia, Jianzu Lia, Yuan Xub, Yunjie Wangb, Luyong Zhangb, Li
Dinga, Yining Xuana, Tao Pangb & Hansen Lina
a College of Pharmacy, Guangdong Pharmaceutical University,
Guangzhou P.R. China
b State Key Laboratory of Natural Medicines, Jiangsu Key
Laboratory of Drug Screening, China Pharmaceutical University,
Nanjing, P.R. China
Published online: 06 Aug 2015.
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To cite this article: Jun Li, Jianzu Li, Yuan Xu, Yunjie Wang, Luyong Zhang, Li Ding, Yining Xuan,
Tao Pang & Hansen Lin (2015): Asymmetric synthesis and biological activities of natural product (+)-
balasubramide and its derivatives, Natural Product Research: Formerly Natural Product Letters,
DOI: 10.1080/14786419.2015.1071363
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Natural Product research, 2015
http://dx.doi.org/10.1080/14786419.2015.1071363
Asymmetric synthesis and biological activities of natural
product (+)-balasubramide and its derivatives
Jun Lia, Jianzu Lia, Yuan Xub, Yunjie Wangb, Luyong Zhangb, Li Dinga, Yining Xuana,
Tao Pangb and Hansen Lina
b
acollege of Pharmacy, Guangdong Pharmaceutical university, Guangzhou P.r. china; state Key laboratory
of Natural Medicines, Jiangsu Key laboratory of drug screening, china Pharmaceutical university, Nanjing,
P.r. china
ARTICLE HISTORY
received 21 april 2015
accepted 26 June 2015
ABSTRACT
The natural product (+)-balasubramide (3j) and its derivatives (3a–
3i) were synthesized using a two-step asymmetric synthesis, and
the biological activities of 3a–3j were determined in vitro. Methyl
(2S,3R)-(+)-3-phenyloxirane-2-carboxylate (1h), the asymmetric
synthesis of which was described in a previous paper, was selected
as the starting material. Compounds 3a–3j were evaluated for their
neuroprotective, antioxidative, and anti-neuroinflammatory effects.
(+)-Balasubramide and its derivatives with different electronegative
groups in the 6-phenyl ring produced little neuroprotection and
antioxidation, but induced potent anti-neuroinflammatory effects
in BV-2 microglial cells (with the exception of 3g). Compound 3c, with
a trifluoromethyl group in its 6-phenyl ring, was a particularly potent
anti-neuroinflammatory agent. These results demonstrated that the
electronegativity of the 6-phenyl ring of (+)-balasubramide is an
important determinant of its inhibitory effect on neuroinflammation.
More electronegative substituents result in more potent anti-
neuroinflammatory effects. Moreover, cytotoxicity assays indicated
no significant effects of the tested compounds.
KEYWORDS
Balasubramide; asymmetric
catalysis; synthesis;
biological screening; anti-
neuroinflammatory effect
CONTACT hansen lin
linhansenyd@163.com; tao Pang
tpang@cpu.edu.cn
the supplementary material for this paper is available online at http://dx.doi.org/10.1080/14786419.2015.1071363.
© 2015 taylor & Francis

2
J. LI ET AL.
1. Introduction
(+)-Balasubramide is an eight-membered lactam compound with an absolute configuration
of 5S,6R (Juárez-Calderón et al. 2013) that is extracted from the leaves of the Sri Lankan
plant Clausena indica (Riemer et al. 1997) along with its biosynthetic precursor, (+)-prebala-
mide (Figure 1). Studies have shown that five-membered lactam compounds extracted from
C. indica have extensive pharmacological uses. For example, clausenamide (Figure 1) pro-
duces hepatoprotective (Yang et al. 1987) and neuroprotective effects (Xue et al. 2008), inhib-
its apoptosis (Yao et al. 2001) and lipid peroxidation (Lin et al. 1992), and acts as an oxygen
free radical scavenger (Jiang & Zhang 1998). However, eight-membered lactam compounds
are rarely reported to have pharmacological activity. Total synthesis of (+)-balasubramide is
difficult because of its eight-membered lactam ring and two chiral centers. Thus, the biolog-
ical activities and preliminary structure-activity relationships (SARs) of related compounds
have not yet been reported.
At present, although there are three synthetic routes regarding balasubramide (Johansen
et al. 2007; Yang et al. 2007; Zheng et al. 2009), (+)-balasubramide, a chiral natural product,
only could be obtained by one method (Johansen et al. 2007) which was synthesized by
chiral resolution and the overall yield was only 17%.
We investigated an additional method of total synthesis of (+)-balasubramide to allow
us to study its bioactivity and to perform a preliminary analysis of the SARs of (+)-balasu-
bramide and derivative compounds. We previously reported a short asymmetric synthetic
route of methyl (2S,3R)-(+)-3-phenyloxirane-2-carboxylate (1h) (Xuan et al. 2013). In this
study, we used compound 1h as the starting material to synthesize a series of (+)-balasubr-
amide derivatives in order to explore their biological activities, including neuroprotection,
anti-neuroinflammation, cytotoxic effects.
In recent years, as a catalytic method with the same status of enzyme catalysis and
metal catalysis, asymmetric catalysis has been an important tool for building chiral molec-
ular scaffold owing to the high efficiency and selectivity (Shi et al. 2015; Wang et al. 2015;
Woźniak et al. 2015; Zhao et al. 2015). Our group initially reported a model reaction of
cinnamaldehyde with organocatalyst, a diphenyl prolinol TES ether, to synthesize methyl
(2S,3R)-(+)-3-phenyloxirane-2-carboxylate (1h) via a one-pot reaction with 73% yield
and 95% enantioselectivity (Xuan et al. 2013). In this report, we used compound 1h to
prepare the linear amide 2j by amine–ester interchange with N-methyltryptamine, followed
by intramolecular cyclization using ytterbium(III) triflate (Yb(CF3SO3)3) as a catalyst, resulting
in (+)-balasubramide (3j) (Figure 2).
In order to introduce substituents with varying electronegativity into the 6-phenyl ring,
cinnamaldehydes with different substitutions were employed to prepare (+)-balasubramide
CH3
OH
N
O
H
N
Ph
H
6R
5S
N
H
O
O
O
Ph
OH
N
H
N
OH
(+)-Balasubramide
(+)-prebalamide
- Clausenamide
Figure 1. structures of (+)-balasubramide, (+)-prebalamide and ( )-clausenamide.

NATuRAL PRODuCT RESEARCH
3
CH3
O
Ph
O
N
N-Methyltryptamine
CH3ONa, CH3OH, -18
COOCH3
Ph
O
2j
1h
N
H
CH3
O
N
Yb(CF3SO3)3
THF, rt
5S
6R
N
H
Ph
OH
3j
Figure 2. total asymmetric synthesis of natural product (+)-balasubramide.
Ph Ph
H
N
SiEt3
O
O
CHO
COOCH3
a) organocatalyst
b) NBS, Na2CO3
CH3OH, 3h, rt
R
R
30%H2O2, DCM, 3h, rt
1a-1i
R
NH
O
H
N
Yb(CF3SO3)3
THF, rt
tryptamine
CH3ONa, CH3OH, -18
N
H
O
O
OH
N
H
R
2a-2i
3a-3i
R = 3-F (3a); 4-F (3b); 4-CF3 (3c); 2-NO2 (3d); 4-Cl (3e); 4-Br (3f); 3-Cl (3g); H (3h); 4-NO2 (3i)
Figure 3. asymmetric synthesis of natural product (+)-balasubramide derivatives.
analogues using organocatalyst followed by asymmetric epoxidation, oxidative esterification,
an amine–ester interchange reaction, and intramolecular cyclization (Figure 3).
2. Results and discussion
2.1. Chemistry
In the conversion of methyl (2S,3R)-(+)-3-phenyloxirane-2-carboxylate into the linear amide 2j, an
amine–ester interchange reaction with N-methyltryptamine was carried out at room temperature
in CH3OH for 10 h, but resulted in only a 25% yield.Therefore, we investigated reaction conditions
using temperatures less than −18 °C and added catalytic basic additives such as NaHCO3, K2CO3,
Na2CO3, t-BuOK, and CH3ONa. When CH3ONa was used as the additive, the process resulted in a
good yield of compound 2j. Despite our best efforts, the resultant amide 2j could not be sep-
arated from at least one other unidentified compound, we speculated that the compound in
column chromatography may be decomposed due to its instability, but the mixture could once
again be subjected to next step without using purification.
The intramolecular cyclization of compound 2j was found to be greatly influenced by
the use of Lewis acid activities. When AlCl3, FeCl3, or CuCl were used, compound 3j was not

4
J. LI ET AL.
Table 1. Yield and enantiomeric excess of (+)-balasubramide derivatives.
Entrya
R
Product
Yield (%)
eeb (%)
1
2
3
4
5
6
7
8
9
3-F
4-F
3a
3b
3c
3d
3e
3f
3g
3h
3i
76
70
65
60
75
72
72
75
62
97
>99
96
>99
>99
98
98
>99
97
4-cF3
2-No
4-cl2
4-Br
3-cl
h
4-No2
aunless otherwise stated, all reactions were performed with 2 (2 mmol) and Yb(cF3so3)3 (0.4 mmol) in thF.
bthe enantiomeric excess was determined by high-performance liquid chromatography (daicel chiralcel ad-h column).
obtained, but when LaCl3, p-TSA, or Yb(CF3SO3)3 was used, compound 3j was obtained with
varying yields. Yb(CF3SO3)3 produced the best yield with >99% enantioselectivity. Although
the solvent had no effect on enantioselectivity in the last reaction, a polar solvent was
found to increase the yield, as evidenced by the improvement produced by the addition of
tetrahydrofuran.
Toward the synthesis of (+)-balasubramide analogues, compounds 1a–1i were asym-
metrically synthesized via a one-pot reaction with a variety of α,β-unsaturated aldehydes
in organocatalyst. When compounds 1a–1i were treated with tryptamine instead of
N-methyltryptamine, intermediates 2a–2i were obtained in good yields after purification.
In the intramolecular cyclization of 2a–2i, (+)-balasubramide analogues 3a–3i were success-
fully produced using Yb(CF3SO3)3 as the catalyst (Table 1).
2.2. In vitro biological evaluation
The (+)-balasubramide (3j) and its derivatives 3a–3i were evaluated for their in vitro biolog-
ical activities, including neuroprotective, antioxidative and anti-neuroinflammatory effects.
Our results in Tables S1–3 showed that compounds 3a–3j didn’t exhibit the significant
neuroprotective effects in primary neurons against glutamate or nutrient deprivation and
antioxidative effects in PC12 neuronal cells against H2O2 incubation.
All compounds were further subjected to bioassay to test their in vitro anti-
neuroinflammatory effects against LPS-induced pro-inflammatory cytokineTNFαexpression
in microglial cells. The results are shown in Figure 4 and Table S4. With the exception of
140
120
100
***
***
***
80
60
40
20
0
***
***
***
***
***
***
***
1
M (+LPS)
10 M (+LPS)
Figure 4. Inhibitory effects of target compounds on lPs-inducedtNFα production in BV-2 microglial cells.
***p < 0.001 compared to lPs group.

NATuRAL PRODuCT RESEARCH
5
compound 3g (3-Cl-substituted), the target compounds at 10 μM markedly inhibited
LPS-induced pro-inflammatory cytokine TNFα release in BV-2 microglial cells. Compound
3c, with a strongly electronegative substituent (4-CF3), at 1 and 10 μM, exhibited significant
inhibition of LPS-inducedTNFα release in a dose-dependent manner. In addition, compound
3c dose-dependently inhibited LPS-inducedTNFα gene expression (Figure S1). This indicated
that a strongly electronegative substituent in the 6-phenyl ring might be required for the
anti-neuroinflammatory effects of (+)-balasubramide derivatives. Meanwhile, in the MTT
assay for cytotoxicity of these compounds in BV-2 microglial cells (Figure S2), (+)-balasubr-
amide and its derivatives 3a–3j did not show cytotoxic effects.
3. Conclusions
In this report, we describe a convenient and efficient method for the synthesis of natu-
ral (+)-balasubramide with a 44% overall yield and excellent enantioselectivity (>99%).
Substituents with varying electronegativity were introduced into the 6-phenyl ring of
(+)-balasubramide and the resulting target compounds 3a–3j were evaluated for neuropro-
tective, antioxidative, and anti-neuroinflammatory effects. Our results indicated that natural
(+)-balasubramide and its derivatives had little neuroprotective and antioxidative effects, but
significantly inhibited neuroinflammation (with the exception of 3g). These results show that
electronegativity in the 6-phenyl ring of (+)-balasubramide and its derivatives is an important
determinant of their inhibitory effects on neuroinflammation; substituents with stronger
electronegativity produced more potent anti-neuroinflammatory effects. Our preliminary
SAR study provided information that could facilitate the design of novel anti-neuroinflam-
matory drug candidates or leads. These molecular structures may be worthy of future study
with the aim of developing new anti-neuroinflammatory agents. Further studies to improve
anti-neuroinflammatory activity and clarify the molecular mechanism of these compounds
are in progress.
Supplementary material
All experimental sections relating to this article are available online, alongside Tables S1–4
and Figures S1–43.
Disclosure statement
No potential conflict of interest were reported by the authors.
Funding
This work was supported by the National Natural Science Foundation of China [grant number
21402241]; the Natural Science Foundation of Jiangsu Province [grant number BK20130653]; the
Fundamental Research Funds for the Central universities [grant number JKZD2013006]; and the Open
Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical university [grant
number SKLNMKF201407].
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