Studies on the synthesis and biological evaluation of the metabolite of clausenamide CM2 and its stereoisomers

Article abstract of DOI:10.1016/j.ejmech.2013.04.048

The synthesis and biological evaluation of 5-hydroxy clausenamide (CM ₂), one of the major metabolites of clausenamide, and its stereoisomers have been carried out. The absolute configurations of (-)- and (+)-CM₂ were assigned as 3S,

Full text of DOI:10.1016/j.ejmech.2013.04.048

European Journal of Medicinal Chemistry 74 (2014) 736e741
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Laboratory note
Studies on the synthesis and biological evaluation of the metabolite of
clausenamide CM2 and its stereoisomers
,
b
,
a
b
b
b,
XingZhou Li a
, Kangying Lai , KeMei Wu , DaoFei Huang , Liang Huang
**
*
a Beijing Institute of Pharmacology and Toxicology, 27 Taiping Rd., Beijing 100850, China
b Institute of Materia Medica, Chinese Academy of Medical Sciences, 1 Xian Nong Tan St., Beijing 100050, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and biological evaluation of 5-hydroxy clausenamide (CM2), one of the major metabolites
of clausenamide, and its stereoisomers have been carried out. The absolute configurations of (ꢀ)- and
(þ)-CM2 were assigned as 3S,4S,5S,6S and 3R,4R,5R,6R respectively based on 1H NMR spectroscopic
investigation and their chemical correlation to (ꢀ)- and (þ)-clausenamidone (3). Electrophysiological
assay showed that compound (þ)-CM2 and its C6 epimer (þ)-8a had significant effects on synaptic
transmission and thus induced the long-term potentiation of the dentate gyrus.
Received 13 December 2012
Received in revised form
19 April 2013
Accepted 20 April 2013
Available online 2 May 2013
Ó 2014 Published by Elsevier Masson SAS.
Keywords:
Nootropic agents
Metabolite
Clausenamide
Long-term potentiation
1. Introduction
Thus CM2 may be either (3S,4S,5S,6S)-5-hydroxy clausenamide (2a)
or (3S,4S,5R,6S)-5-hydroxy clausenamide (2b) as shown in Fig. 1. To
Dementia is one of the age related mental problems and char-
acteristic symptom of various neurodegenerative diseases
including Alzheimer’s disease. Nootropic agents are clinically used
to address organic disorders in learning abilities and to improve
memory, mood and behavior [1]. (ꢁ)-Clausenamide (1, as shown in
Fig.1) is a naturally occurring nootropic agent first isolated from the
Chinese medicine wampee [2]. The synthesis and configuration
determination of (ꢁ)-1 and its two optical isomers (þ)-1 and (ꢀ)-1
(Fig. 1) has been accomplished by our group [3,4]. Compound (ꢀ)-1
has been found to possess potent nootropic activity in many
physiological or behavioral experiments, but (þ)-1 does not have
such properties [5]. Currently (ꢀ)-1 is being investigated clinically
for the treatment of senile dementia.
support the clinical studies of (ꢀ)-1 and to investigate whether CM2
is also a nootropic agent, the synthesis, characterization and bio-
logical evaluation of CM2 and its isomers have been conducted and
described herein.
2. Chemistry
CM2 is the C5 hydroxylation product of 1. Direct introduction of a
tertiary hydroxyl group at C5 of 1 by means of traditional chemistry
is difficult. Accordingly a de novo synthetic route via dihydrox-
ylation of D5,6 clausenamide (7) or its acetylation product (6)
was designed and used to prepare CM2 and its stereoisomers
(Scheme 1). (ꢁ)-Clausenamidone (3) or its optical active isomers
(ꢀ)-3 and (þ)-3 were chosen as the starting material, where the
relative or absolute configurations had already been determined by
our group [5] and thus were an aid to the determination of the
configuration of CM2.
(ꢁ)-Clausenamidone (3), prepared according to literature [3],
was acetylated with acetic anhydride in pyridine to give (ꢁ)-4 in
quantitative yield. The ketone group on the side chain of com-
pound (ꢁ)-4 was reduced with sodium borohydride to afford (ꢁ)-5
as the sole product in 95% yield. The relative configuration at C6
of (ꢁ)-5 is presumed to be S according to the literature precedent
that reported similar transformation of clausenamidone to
In vitro and in vivo metabolic studies have demonstrated that
CM2, (Fig. 1) (the C5 hydroxylation product of clausenamide) is one
of the major metabolites of clausenamide (1) [6]. The hydroxylation
could take place on either side of the pyrrolidone ring of 1, while
the configurations of C3, C4 and C6 remain unchanged in all cases.
* Corresponding author.
** Corresponding author. Beijing Institute of Pharmacology and Toxicology, 27
Taiping Rd., Beijing 100850, China.
E-mail addresses: xingzhouli@aliyun.com (X. Li), huangliang1920@yahoo.cn
(L. Huang).
0223-5234/$ e see front matter Ó 2014 Published by Elsevier Masson SAS.
http://dx.doi.org/10.1016/j.ejmech.2013.04.048

X. Li et al. / European Journal of Medicinal Chemistry 74 (2014) 736e741
737
Fig. 1. The structure of clausenamide and CM2.
clausenamide [3]. Dehydration of (ꢁ)-5 with phosphorus oxy-
chloride in pyridine provided the E-(ꢁ)-olefin-6 exclusively in 70%
yield. The E configuration was confirmed by NOE correlation be-
(d 6.00) detected) was obtained in 30% yield. This result showed
that the cis-dihydroxylation of E-(ꢁ)-olefin-7 proceeded in a
kinetically controlled manner and predominantly took place at the
less hindered side of the double bond (opposite the C4 phenyl). The
gross structure of (ꢁ)-8b is the same as (ꢁ)-2b, one possible
structure of (ꢁ)-CM2 (Fig. 1). But, the physical constants and
spectral data of (ꢁ)-8b were inconsistent with those reported for
CM2. Then, it is clear that (ꢁ)-CM2 must (ꢁ)-(3S,4S,5S,6S)-5-
hydroxy clausenamide (2a, Fig. 1).
tween the olefin signal (d 6.11) and N-methyl signal (d 3.10). The
E-(ꢁ)-olefin-6 was deacetylated with NaOH in ethanol to give
E-(ꢁ)-olefin-7 in 91% yield. Syn-dihydroxylation of E-(ꢁ)-olefin-7
with osmium tetroxide and N-methylmorpholine oxide (NMO)
gave a crude mixture. Purification by chromatography on silica gel
gave
(ꢁ)-(3S,4S,5S,6R)-8a according to the positive NOE between C4-H
3.41) and C5-OH ( 6.42). The minor product, assigned as
(ꢁ)-(3S,4S,5R,6S)-8b (no NOE between C4-H ( 3.19) and C5-OH
a
major product (yield 62%), determined to be
To obtain (ꢁ)-CM2, attempts were made to trans-dihydroxylate
E-(ꢁ)-olefin-6 using several methods, such as WO3/H2O2 [7], silver
acetate/I2 [8], but none gave a successful result. Efforts were
(d
d
d
Scheme 1. The preparation of CM2 and its isomers. Reagents and conditions: a: Ac2O/pyridine; b: NaBH4/MeOH; c: POCl3/pyridine; d: 10% NaOH in ethanol/CH2Cl2; e: OsO4/NMO/
THF/acetone; f: chromatography; g: DMSO/oxalyl chloride/THF/TEA; h: NaBH4/MeOH; i: recrystallization or preparative thin-layer chromatography; j: Sm/I2/methanol.

738
X. Li et al. / European Journal of Medicinal Chemistry 74 (2014) 736e741
Table 1
The absolute configurations, melting point and optical rotation data of the synthesized compounds.
Compound
Melting point (ꢂC)
Optical rotation data
Compound
Melting point (ꢂC)
Optical rotation data
12
12
(ꢀ)-(3S,4R,5R)-4
157e160
244e246
119e120
125e127
Oil
147e149
125e128
125e128
153e157
Oil
[
[
[
[
[
[
[
[
[
[
a
a
a
a
a
a
a
a
a
a
]
]
]
]
]
]
]
]
]
]
¼ ꢀ305 (c, 0.343, CHCl3)
¼ ꢀ167 (c, 0.106, CHCl3)
¼ þ330 (c, 0.870, CHCl3)
¼ þ17.1 (c, 0.700, CHCl3)
¼ ꢀ113 (c, 0.505, CH3OH)
¼ ꢀ202 (c, 0.510, CH3OH)
¼ ꢀ323 (c, 0.870, CH3OH)
¼ ꢀ310 (c, 0.360, CHCl3)
¼ ꢀ31.9 (c, 0.455, CH3OH)
¼ ꢀ53.6 (c, 0.470, CH3OH)
(þ)-(3R,4S,5S)-4
158e160
243e245
119e120
125e127
Oil
145e147
125e127
127e129
152e156
Oil
[a]
[a]
[a]
[a]
[a]
[a]
[a]
[a]
[a]
[a]
¼ þ306 (c, 0.370, CHCl3)
¼ þ173 (c, 0.120, CHCl3)
¼ þ326 (c, 0.870, CHCl3)
¼ þ16.7 (c, 0.738, CHCl3)
¼ þ117 (c, 0.200, CH3OH)
¼ þ208 (c, 0.620, CH3OH)
¼ þ327 (c, 0.470, CH3OH)
¼ þ312 (c, 0.442, CHCl3)
¼ þ31.7 (c, 0.480, CH3OH)
¼ þ54.1 (c, 0.480, CH3OH)
D
D
14
15
(ꢀ)-(3S,4R,5R,6S)-5
Z-(þ)-(3S,4R)-6
(þ)-(3R,4S,5S,6R)-5
Z-(ꢀ)-(3R,4S)-6
D
D
18
D
18
D
18
D
18
D
Z-(þ)-(3S,4R)-7
Z-(ꢀ)-(3R,4S)-7
25
D
25
D
(ꢀ)-(3S,4S,5S,6R)-8a
(ꢀ)-(3S,4S,5R,6S)-8b
(ꢀ)-(3S,4S,5S,6R)-9a
(ꢀ)-(3S,4S,5S)-10
(ꢀ)-(3S,4S,5S,6S)-11
(ꢀ)-(3S,4S,5S,6S)-CM2
(þ)-(3R,4R,5R,6S)-8a
(þ)-(3R,4R,5S,6R)-8b
(þ)-(3R,4R,5R,6S)-9a
(þ)-(3R,4R,5R)-10
(þ)-(3R,4R,5R,6R)-11
(þ)-(3R,4R,5R,6R)-CM2
25
25
D
D
15
D
15
D
15
18
D
D
15
D
18
D
18
D
18
D
also made to prepare the (ꢁ)-Z-olefin-6 through alternative dehy-
dration methods, such as direct dehydration of (ꢁ)-5 with AlO3 [9],
demesylation of the mesylate of compound 5 with DBU [10], with
the aim of preparing (ꢁ)-CM2 through syn-dihydroxylation of
(ꢁ)-Z-olefin-6 in the next step. However, these attempts were
unsuccessful.
Therefore, a circuitous route was adopted to access the correct
configuration of C6, i.e. oxidation of the C6-OH to a ketone and then
reduction of the ketone to hydroxyl group in order to invert the
configuration of C6 from R to S. The (ꢁ)-E-olefin-6 was syn-dihy-
droxylated with OsO4 and NMO to produce a mixture of products.
This mixture was recrystallized from ethyl acetate to give pure
3. Pharmacology
Repeated stimulation on hippocampal neurons can induce an
immediate and prolonged increase in synaptic strength that is
called long-term potentiation (LTP). It is believed that LTP in the
hippocampus is the key form of long-lasting synaptic plasticity and
it connects the behavior of learning and memory with the plasticity
of neurons. Therefore, LTP has been considered as an important
index of cognitive activities in cellular and synaptic levels [13].
The nootropic activity of (ꢀ)-CM2 and (þ)-CM2 and their ste-
reoisomers were evaluated herein with LTP assays, with the
nootropic parent (ꢀ)-clausenamide as the control. As shown in
Table 2, at an estimated final brain concentration of 1 mM,
(ꢀ)-clausenamide caused an increase of 32e58% of population
spike amplitude (PSA) above the basal level (P < 0.01), whereas, its
enantiomer (þ)-clausenamide is inactive in LTP-inducing activity.
Unexpectedly, (ꢀ)-CM2, which is the metabolite of active
(ꢀ)-clausenamide, exhibited little effect on the LTP-inducing ac-
tivity, but (þ)-CM2, the metabolite of inactive (þ)-clausenamide,
caused 61% (P < 0.01) and 88% (P < 0.001) increase relative to basal
PSA at 45 min and 60 min After Administration (A.A.) respectively.
The effect (PS amplitude) of (þ)-CM2 is greater than that of
(ꢀ)-clausenamide. These findings suggest that the introduction of a
hydroxyl group at C5 position of (ꢀ)- or (þ)-clausenamide exerts a
dramatic influence on the nootropic activities. Compound (þ)-8a,
the C6 epimer of (þ)-CM2, caused 94% (P < 0.05) and 133%
(P < 0.05) increase relative to basal PSA at 45 min and 60 min A.A.
respectively. Its effect (PS amplitude) was greater than that of
(þ)-CM2 and (ꢀ)-clausenamide, which indicates that (þ)-8a is a
more potent nootropic agent than (þ)-CM2 and (ꢀ)-clausenamide.
The other three CM2 stereoisomers (ꢀ)-8a, (ꢀ)-8b and (þ)-8b
exhibited little effect on LTP-inducing activity. The common
“3R,4R,5R” configuration in compounds (þ)-8a and (þ)-CM2
demonstrated that the “3R,4R,6R” configuration might be respon-
sible for the nootropic activities. While, the absolute configuration
of C6 may be less important to the nootropic activities, 6S may be
preferred over 6R consistent with the different activities between
(þ)-CM2 and (þ)-8a. Our findings suggest that the configuration of
5-hydroxy clausenamide has important implications for the
LTP-inducing activities. This may provide a better understanding of
the SAR of clausenamide derivatives and may benefit the study of
the biological mechanisms of LTP.
(ꢁ)-(3S,4S,5S,6R)-9a (NOE correlation observed between C4-H (
d
3.74) and C5-OH ( 5.65)) as the major product in 82% yield. Pre-
d
parative thin-layer chromatography of the mother liquors afforded
the minor product (ꢁ)-(3S,4S,5R,6S)-9b in 11% yield. Compound
(ꢁ)-9a was oxidized by Swern oxidation [11] to yield
a-hydrox-
yketone (ꢁ)-(3S,4S,5S)-10 in 70% yield. Reduction of the ketone
group of (ꢁ)-10 with NaBH4 gave (ꢁ)-11a and (ꢁ)-11b in 73% and
16% yields respectively. Comparison of the 1H NMR spectral data
and Rf value of (ꢁ)-11b and (ꢁ)-9a suggests that they are the same
compound. Accordingly, (ꢁ)-11a must be the C6 epimer of (ꢁ)-9a,
namely (ꢁ)-(3S,4S,5S,6S)-3-O-acetyl-5-hydroxy clausenamide.
Attempted deacetylation of (ꢁ)-11a under acidic or basic condi-
tions gave complex mixtures of products, whereas deacetylation
proceeded smoothly under the mild and neutral conditions of
Sm/I2/MeOH [12], to afford a white solid in 65% yield, which
showed identical spectral data to that of (ꢁ)-CM2. The deacetyla-
tion did not affect the configuration, so the relative configuration of
the (ꢁ)-CM2 must be 3S,4S,5S,6S (Scheme 1). The structures of all
new compounds were unambiguously characterized with 1H NMR,
13C NMR, and mass spectrometry or high-resolution mass
spectrometry.
(ꢀ)-CM2 and (þ)-CM2 were prepared from the corresponding
(ꢀ)-(3S,4R,5R)-clausenamidone (3) or (þ)-(3R,4S,5S)-clausenami-
done (3) by the same process used to synthesize (ꢁ)-CM2. The
absolute configurations, optical rotation data and melting points of
intermediates and final products are listed in Table 1. The absolute
configurations of (ꢀ)-CM2 and (þ)-CM2 were assigned as
3S,4S,5S,6S and 3R,4R,5R,6R respectively (Fig. 2) based on their
chemical correlation to (ꢀ)- and (þ)-3.
4. Conclusion
In summary, an efficient and economic synthesis of CM2 and its
stereoisomers has been described. The absolute configurations of
(ꢀ)- and (þ)-CM2 were assigned as 3S,4S,5S,6S and 3R,4R,5R,6R
respectively based on 1H NMR spectroscopic studies and chemical
correlations to (ꢀ)-(3S,4R,5R)-3 and (þ)-(3R,4S,5S)-3. The activities
Fig. 2. The structure of (ꢀ)- and (þ)-CM2.

X. Li et al. / European Journal of Medicinal Chemistry 74 (2014) 736e741
739
Table 2
LTP screening results of CM2 and its stereoisomers at a concentration of 1
mM.
Compound
Animal number
Relative PS (PSA %)
P.A.a
15 min A.A.b
30 min A.A.
45 min A.A.
60 min A.A.
DMSO
3
3
4
3
3
3
4
6
6
100
100
100
100
100
100
100
100
100
107.1 ꢁ 10.1
78.9 ꢁ 13.9
144.9 ꢁ 49.5
110.6 ꢁ 7.3
81.5 ꢁ 6.1
103.5 ꢁ 6.1
83.7 ꢁ 13.6
168.0 ꢁ 49.2
103.1 ꢁ 10.1
77.3 ꢁ 8.9
97.8 ꢁ 3.2
94.6 ꢁ 8.9
96.0 ꢁ 11.1
102.7 ꢁ 2.9
(ꢀ)-(3S,4S,5S,6R)-8a
(þ)-(3R,4R,5R,6S)-8a
(ꢀ)-(3S,4S,5R,6S)-8b
(þ)-(3R,4R,5S,6R)-8b
(ꢀ)-(3S,4S,5S,6S)-CM2
(þ)-(3R,4R,5R,6R)-CM2
(þ)-(3R,4S,5S,6R)-1
(ꢀ)-(3S,4R,5R,6S)-1
194.3 ꢁ 56.7*c
94.5 ꢁ 10.5
82.6 ꢁ 1.8
233.2 ꢁ 55.6*
88.4 ꢁ 14.4
99.9 ꢁ 6.3
92.8 ꢁ 17.5
91.7 ꢁ 17.2
90.6 ꢁ 0.3
87.1 ꢁ 30.5
117.8 ꢁ 14.9
110.1 ꢁ 13.1
138.5 ꢁ 8.9*
97.3 ꢁ 25.7
161.1 ꢁ 13.9**
98.3 ꢁ 28.6
188.0 ꢁ 5.3***
106.4 ꢁ 4.1
158.1 ꢁ 4.2*
131.8 ꢁ 0.4*
a
P.A.: Prior administration.
A.A.: After administration.
Test *p < 0.05, **p < 0.01, ***p < 0.001 vs. control.
b
c
of (ꢀ)-CM2, (þ)-CM2 and their optically active stereoisomers for
inducing long-term potentiation (LTP) of the dentate gyrus were
tested by electrophysiological assay. Preliminary results showed
that compounds (þ)-(3R,4R,5R,6S)-8a and (þ)-(3R,4R,5R,6R)-CM2
had significant effects on synaptic transmission and thus induced
the LTP of the dentate gyrus, and that the configuration of CM2 and
its stereoisomers has important implications for their LTP-inducing
activities. These findings may provide a better understanding of the
SAR of clausenamide derivatives and may assist the study on the
biological mechanisms of LTP.
5.1.3. (ꢁ)-(3S,4S)-3-O-acetyl-D5,6 clausenamide (6)
To a solution of (ꢁ)-(3S,4S,5R,6S)-3-O-acetyl-clausenamide (5)
(1.00 g, 2.94 mmol) in anhydrous pyridine (6 mL), a solution of
distilled phosphorous oxychloride (1.0 mL) in anhydrous pyridine
(4 mL) was added dropwise over half an hour at 0 ꢂC. The mixture
was stirred at 0 ꢂC for 24 h and then poured into ice water. The
precipitate was separated by filtration. The filter cake was dissolved
with methylene dichloride, washed with water and brine succes-
sively, dried over anhydrous sodium sulfate, and the solvent
evaporated off to give a crude solid. The solid was purified by col-
umn chromatography eluted with methylene dichloride and
methanol to give title compound as white solid (746 mg, yield:
5. Experiment
71%), mp: 148e150 ꢂC, 1H NMR (300 MHz, CDCl3):
d 2.13 (3H, s),
3.30 (3H, s), 4.33 (1H, s), 5.14 (1H, s), 6.11 (1H, s), 7.02e7.36 (10H,
m); HRMS (QFT-ESI): calculated for C20H20N1O3 (MHþ) 322.1438,
found 322.1440.
5.1. Chemistry
All the reagents were commercially available and used without
further purification. Melting points were determined using a
Yanaco apparatus and the thermometer is uncorrected. 1H NMR
and 13C NMR spectra were measured using a Bruker-400 or YS-300.
Mass spectra were obtained from VG300, ZAD-2F or API3000
instruments.
5.1.4. (ꢁ)-(3S,4S)-D5,6 clausenamide (7)
To a solution of (ꢁ)-(3S,4S)-3-O-acetyl-D5,6 clausenamide 6
(400 mg, 1.24 mmol) in methylene dichloride (15 mL), was added
10% sodium hydroxide alcohol solution (3 mL) and the mixture was
stirred for 3 min. The mixture was neutralized with 2 M HCl, diluted
with methylene dichloride (20 mL), washed with 1 M sodium bi-
carbonate solution and brine, dried over anhydrous sodium sulfate,
and then concentrated to give title compound as white solid
(330 mg, yield: 95%), mp: 162e163 ꢂC. 1H NMR (300 MHz,
5.1.1. (ꢁ)-(3S,4S,5R)-3-O-acetyl-clausenamidone (4)
Clausenamidone (3) (2.00 g, 6.78 mmol) was dissolved in a
mixture of acetic anhydride and pyridine. The mixture was stirred
overnight at 0 ꢂC, and then was poured into cold water. The
precipitated solid was collected by suction, washed with water
and air dried to give title compound as white solid 2.15 g (yield:
94%), which was pure enough to be used in next step without
further purification. mp: 163e165 ꢂC. 1H NMR (400 MHz, CDCl3):
CD3COCD3):
d
3.18 (3H, s), 4.08 (1H, dd, J ¼ 5.1, 1.8 Hz), 4.36 (1H,
d J ¼ 1.8 Hz), 5.32 (1H, d, J ¼ 5.1 Hz (D2O exchangeable)), 6.12 (1H,
s), 6.95e7.22 (10H, m). HRMS (QFT-ESI): calculated for C18H18N1O2
(MHþ) 280.1332, found 280.1338.
d
2.07 (3H, s), 2.89 (3H, s), 4.02 (1H, dd, J ¼ 6.0, 8.8 Hz), 5.42 (1H, d,
5.1.5. (ꢁ)-(3S,4S,5R,6S)-5-hydroxy clausenamide (8a) and
J ¼ 8.8 Hz), 6.13 (1H, d, J ¼ 6.0 Hz), 7.00e7.52 (10H, m). HRMS
(QFT-ESI): calculated for C20H20N1O4 (MHþ) 338.1387, found
338.1389.
(ꢁ)-(3S,4S,5S,6R)-5-hydroxy clausenamide (8b)
To a solution of (ꢁ)-(3S,4S)-D5,6 clausenamide (7) (260 mg,
0.931 mmol) in acetone (1 mL) and THF (5 mL), N-methylmorpho-
line N-oxide (545 mg, 4.66 mmol) and 4% OsO4 aqueous solution
(0.6 mL, 0.093 mmol) were added with a syringe. After stirring at
room temperature for 24 h, a mixture of anhydrous sodium sulfite
(650 mg) and water (3 mL) was added. The resulting mixture was
stirred for 1 h, diluted with methylene dichloride, washed with
water then brine and dried over anhydrous sodium sulfate. Evapo-
ration of the solvent afforded an oily product (280 mg), which was
purified by chromatography to give (ꢁ)-(3S,4S,5R,6S)-5-hydroxy
clausenamide (8a) (150 mg, yield: 50%) and (ꢁ)-(3S,4S,5S,6R)-5-
hydroxy clausenamide (8b) (70 mg, yield: 24%.).
5.1.2. (ꢁ)-(3S,4S,5R,6S)-3-O-acetyl-clausenamide (5)
To a solution of (ꢁ)-(3S,4S,5R)-3-O-acetyl-clausenamidone (4)
(1.20 g, 3.56 mmol) in 10 mL methanol, was added sodium boro-
hydride (480 mg, 12.70 mmol). The reaction mixture was stirred at
ambient temperature for 30 min. The precipitated solid was
collected by suction, washed with water and dried to give pure title
compound as white solid (1.15 g, yield: 95%), which was pure
enough to be used in the next step without further purification,
mp: 230e232 ꢂC. 1H NMR (400 MHz, CDCl3):
d 2.05 (3H, s), 2.89 (3H,
s), 3.99 (1H, dd, J ¼ 11.2, 8.0 Hz), 4.16 (1H, dd, J ¼ 8.0, 4.0 Hz), 4.82
(1H, d, J ¼ 4.0 Hz), 5.87 (1H, d, J ¼ 11.2 Hz), 6.86e7.30 (10H, m).
HRMS (QFT-ESI): calculated for C20H22N1O4 (MHþ) 340.1543, found
340.1560.
8a, White solid, mp: 134e136 ꢂC. 1H NMR (400 MHz, DMSO-d6):
d
2.11 (3H, s), 3.39 (1H, d, J ¼ 10.08 Hz), 4.06 (1H, d, J ¼ 4.2 Hz), 4.92
(1H, d, J ¼ 10.08 Hz), 5.40 (1H, d, J ¼ 4.2 Hz, (D2O exchangeable)),

740
X. Li et al. / European Journal of Medicinal Chemistry 74 (2014) 736e741
5.60 (1H, brs, (D2O exchangeable)), 6.42 (1H, s, (D2O exchange-
able)), 6.90e7.53 (10H, m); 13C NMR (DMSO-d6):
26.18, 61.53,
7.04e8.22 (10H, m). 13C NMR (DMSO-d6):
d 20.73, 25.88, 55.99,
d
70.62, 93.56, 127.98, 128.15, 128.40, 128.51, 128.55, 131.60, 133.30,
135.43, 169.66, 169.93, 198.57. FAB-MS (m/e, %) 354 (MHþ, 100), 276
(80), 376 (MNaþ, 20), 131 (82), 105 (PhCO, 53). HRMS (QFT-ESI):
calculated for C20H22N1O5 (MHþ) 354.1336, found 354.1339.
70.03, 71.98, 90.10, 126.67, 126.83, 127.81, 127.86, 128.88, 136.35,
141.44, 173.67. FAB-MS (m/e, %) 314 (MHþ, 60), 296 (7), 277 (11), 185
(90), 93 (100). HRMS (QFT-ESI): calculated for C18H20N1O4 (MHþ)
314.1387, found 314.1389.
8b, White solid, mp: 127e129 ꢂC, 1H NMR (400 MHz, DMSO-d6):
5.1.8. (3S,4S,5S,6S)-3-O-acetyl-5-hydroxy clausenamide (11a)
d
2.87 (3H, s), 3.18 (1H, d, J ¼ 9.9 Hz), 3.51 (1H, d, J ¼ 8.9 Hz), 4.19
(ꢁ)-(3S,4S,5S)-3-O-acetyl-5-hydroxy
clausenamidone
(10)
(1H, d, J ¼ 4.8 Hz), 4.69 (1H, d, J ¼ 4.2 Hz), 5.52 (1H, bs, (D2O
exchangeable)), 5.78 (1H, d, J ¼ 4.2 Hz, (D2O exchangeable)), 6.00
(1H, s, (D2O exchangeable)), 6.90e7.36 (10H, m); 13C NMR (DMSO-
(82 mg, 0.260 mmol) was dissolved in methanol (3 mL) and cooled
to 0 ꢂC. Sodium borohydride (30 mg, 0.78 mmol) was added and the
mixture stirred for 10 min. The mixture was neutralized carefully
with 1 M hydrochloric acid and the organic solvents were distilled
off under reduced pressure .The residue was extracted with ethyl
ether, washed with brine, dried over anhydrous sodium sulfate, and
the solvent distilled off to give a crude oil, which was purified by
column chromatography (eluted with CH2Cl2 and MeOH) to give
the title compound (60 mg, yield: 73%.) as white solid, mp: 144e
d6):
d 24.72, 50.90, 72.37, 75.12, 90.18, 125.84, 126.66, 126.74,
126.87, 127.77, 130.19, 138.16, 140.03, 173.67; FAB-MS (m/e, %): 314
(MHþ, 15), 312 (20), 176 (100), 120 (29), 93 (71). HRMS (QFT-ESI):
calculated for C18H20N1O4 (MHþ) 314.1387, found 314.1390.
5.1.6. (ꢁ)-(3S,4S,5S,6R)-3-O-acetyl-5-hydroxy clausenamide (9a)
and (ꢁ)-(3S,4S,5S,6R)-3-O-acetyl-5-hydroxy clausenamide (9b)
To a solution of (ꢁ)-(3S,4S)-3-O-acetyl-D5,6 clausenamide (6)
(240 mg, 0.748 mmol) in THF (4 mL) and acetone (1 mL), N-meth-
ylmorpholine N-oxide (581 mg, 3.738 mmol) and 10 mg/mL OsO4
aqueous solution (1.9 mL, 0.093 mol) were added. After stirring at
room temperature for 48 h, anhydrous sodium sulfite (500 mg) and
water (10 mL) was added. The resultant mixture was stirred for 1 h
more, diluted with methylene dichloride, washed with water then
brine and dried over anhydrous sodium sulfate. Evaporation of the
solvent afforded 300 mg of an oil, which was recrystallized from
ethyl acetate to give 150 mg of (ꢁ)-(3S,4S,5S,6R)-3-O-acetyl-5-
hydroxy clausenamide (9a). Separation of the mother liquors with
preparative thin-layer chromatography (eluted with ethyl acetate)
gave 70 mg of (ꢁ)-(3S,4S,5S,6R)-3-O-acetyl-5-hydroxy clausena-
mide (9b) and 67 mg of 9a.
147 ꢂC, 1H NMR (300 MHz, CD3COCD3):
3.77 (1H, d, J ¼ 11.1 Hz), 4.73 (1H, d, J ¼ 4.2 Hz), 5.05 (1H, d,
J ¼ 4.2 Hz, (D2O exchangeable)), 5.20 (1H, d, J ¼ 11.1 Hz), 5.96 (1H, s,
(D2O exchangeable)), 6.72e7.29 (10H, m). 13C NMR (DMSO-d6):
d 1.93 (3H, s), 3.04 (3H, s),
d
20.51, 25.14, 55.25, 69.80, 73.81, 90.78, 126.45, 126.66, 127.05,
127.64, 127.81, 128.31, 133.38, 139.14, 167.91, 169.59. HRMS (QFT-
ESI): calculated for C20H22N1O5 (MHþ) 356.1493, found 356.1503.
5.1.9. (ꢁ)-(3S,4S,5S,6S)-5-hydroxy clausenamide (CM2)
(ꢁ)-(3S,4S,5S,6S)-3-O-acetyl-5-hydroxy clausenamide (11a)
(50 mg, 0.142 mmol) was dissolved in anhydrous methanol (5 mL),
to the mixture was added iodine (38 mg, 0.149 mmol) and
samarium (23 mg, 0.149 mmol). The resultant mixture was stirred
in air at room temperature for 12 h and then partitioned between
ethyl acetate and water. The organic layer was washed with 25%
sodium hyposulfite solution, brine, dried over sodium sulfate and
concentrated in vacuo. The resulting light yellow oil was purified by
column chromatography (eluted with CH2Cl2 and MeOH) to give
the title compound, which was recrystallized from ethyl acetate/
petroleum ether to give a white solid (25 mg, yield: 56%), mp: 114e
9a, White solid, yield: 82%, mp: 134e136 ꢂC. 1H NMR (300 MHz,
CD3COCD3):
d
1.99 (3H, s), 2.23 (3H, s), 3.74 (1H, d, J ¼ 10.5 Hz), 4.31
(1H, d, J ¼ 4.5 Hz), 4.76 (1H, d, J ¼ 4.5 Hz (D2O exchangeable)), 5.66
(1H, s (D2O exchangeable)), 6.59 (1H, d, J ¼ 10.5 Hz), 7.20e7.63
(10H, m). 13C NMR (DMSO-d6):
d 20.60, 26.54, 58.53, 70.77, 71.88,
90.83, 127.08, 127.35, 127.79, 128.30, 128.50, 134.46, 140.80, 138.87,
169.86. HRMS (QFT-ESI): calculated for
356.1493, found 356.1501.
116 ꢂC. 1H NMR (300 MHz, CD3COCD3):
d 3.11 (3H, s), 3.58 (1H, d,
C
20H22N1O5 (MHþ)
J ¼ 8.4 Hz), 4.79 (1H, d, J ¼ 4.2 Hz), 3.45 (1H, dd, J ¼ 8.4, 4.5 Hz), 4.39
(1H, d, J ¼ 4.5 Hz, (D2O exchangeable)), 5.00 (1H, d, J ¼ 4.2 Hz, (D2O
exchangeable)), 5.88 (1H, s, (D2O, exchangeable)), 6.67e7.53 (10H,
9b, White solid, yield: 11%, mp: 146e148 ꢂC. 1H NMR (300 MHz,
CD3COCD3):
d
1.93 (3H, s), 3.00 (3H, s), 3.60 (1H, d, J ¼ 8.4 Hz), 4.98
m), 13C NMR (DMSO-d6):
d 25.89, 57.10, 68.73, 75.48, 89.84, 126.72,
(1H, d, J ¼ 4.5 Hz), 4.98 (1H, s (D2O exchangeable)), 5.07 (1H, d,
J ¼ 4.5 Hz (D2O exchangeable)), 5.75 (1H, d, J ¼ 8.4 Hz), 7.04e7.52
(10H, m). HRMS (QFT-ESI): calculated for C20H22N1O5 (MHþ)
356.1493, found 356.1503.
127.01, 127.30, 127.51, 127.68, 128.04, 128.68, 135.19, 139.29, 172.44.
FAB-MS (m/e, %) 314 (MHþ, 100), 296 (15), 289 (83). HRMS (QFT-
ESI): calculated for C18H20N1O4 (MHþ) 314.1387, found 314.1390.
Elemental Analysis: C18H19N1O4 found (calc.) C: 68.59, 68.85
(68.88), H: 5.98, 5.95 (6.11), N: 4.47, 4.48 (4.47).
5.1.7. (ꢁ)-(3S,4S,5S)-5-hydroxy clausenamidone (10)
To a solution of re-distilled oxalyl chloride (453
mL, 5 mmol) in
5.1.10. Preparation of optically active (ꢀ)-(3S,4S,5R,6S)-8b,
(þ)-(3R,4R,5S,6R)-8b, (ꢀ)-(3S,4S,5S,6R)-8a, (þ)-(3R,4R,5R,6S)-8a
and (ꢀ)-(3S,4S,5S,6S)-CM2 and (þ)-(3R,4R,5R,6R)-CM2
THF (5 mL) at ꢀ50e60 ꢂC, DMSO (767
mL, 10 mmol) was carefully
added under nitrogen atmosphere. After stirring for 5 min, a so-
lution of (ꢁ)-(3S,4S,5S,6R)-3-O-acetyl-5-hydroxy clausenamide
(9a) (150 mg, 0.423 mmol) in THF (10 mL) was added and stirred for
a further 2 h at the temperature, then Et3N (1 mL) was added. The
cooling bath was removed, and the reaction mixture was allowed to
warm to room temperature. The solvent was removed under
reduced pressure, and the residue was extracted with ethyl acetate.
The extract was washed with 1 M hydrochloric acid, aqueous so-
dium bicarbonate, saturated sodium chloride solution and dried
over anhydrous sodium sulfate. After removal of the solvent under
reduced pressure, the crude oil (250 mg) was purified by column
chromatography (eluted with CH2Cl2 and MeOH) to give the title
compound as white solid (95 mg, yield: 71%). mp: 103e106 ꢂC. 1H
These compounds were prepared from the corresponding
(ꢀ)-(3S,4R,5R) clausenamidone (1) or (þ)-(3R,4S,5S) clausenami-
done (1) by the same process as that described above for their
racemic counterpart. The absolute configurations, melting point and
optical rotation data of the synthesized compounds are listed Table 1.
5.2. Pharmacology
5.2.1. Effects of our compounds on LTP induced in rat hippocampus
Male adult SD rats (5 per group) were anesthetized with 20%
(w/v) urethane carbamate (1.0 g kgꢀ1, ip) and fixed in a stereotaxic
head holder. Recording electrodes (stainless steel needle of 0.2 mm
diameter, insulated except for 0.2 mm tip) were placed in the
dentate gyrus granule cell layer. Our compounds were injected into
NMR (300 MHz, CDCl3):
d 2.10 (3H, s), 2.81 (3H, s), 4.16 (1H, d,
J ¼ 10.2 Hz), 4.64 (1H, s (exchangeable)), 6.09 (1H, d, J ¼ 10.2 Hz),

X. Li et al. / European Journal of Medicinal Chemistry 74 (2014) 736e741
741
lateral ventricle at a concentration of 10ꢀ6 mol Lꢀ1. The stimulating
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.04.048.