Asymmetric total synthesis and identification of tetrahydroprotoberberine derivatives as new antipsychotic agents possessing a dopamine D1, D2 and serotonin 5-HT1A multi-action profile

Article abstract of DOI:10.1016/j.bmc.2012.12.016

An effective and rapid method for the microwave-assisted preparation of the key intermediate for the total synthesis of tetrahydroprotoberberines (THPBs) including l-stepholidine (l-SPD) was developed. Thirty-one THPB derivatives with diverse substituents on A and D ring were synthesized, and their binding affinity to dopamine D₁, D₂ and serotonin 5-HT _1A and 5-HT_2A receptors were determined. Compounds 18k and 18m were identified as partial agonists at the D₁ receptor with K_i values of 50 and 6.3 nM, while both compounds act as D₂ receptor antagonists (K_i = 305 and 145 nM, respectively) and 5-HT_1A receptor full agonists (K_i = 149 and 908 nM, respectively). These two THPBs compounds exerted antipsychotic actions in animal models. Further electrophysiological studies employing single-unit recording in intact animals demonstrated that 18k-excited dopaminergic (DA) neurons are associated with its 5-HT_1A receptor agonistic activity. These results suggest that these two compounds targeted to multiple neurotransmitter receptors may present novel lead drugs with new pharmacological profiles for the treatment of schizophrenia.

Full text of DOI:10.1016/j.bmc.2012.12.016

Bioorganic & Medicinal Chemistry 21 (2013) 856–868
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Asymmetric total synthesis and identification of tetrahydroproto-
berberine derivatives as new antipsychotic agents possessing
a dopamine D1, D2 and serotonin 5-HT1A multi-action profile
Haifeng Sun a, , Liyuan Zhu b, , Huicui Yang c, Wangke Qian a, Lin Guo b, Shengbin Zhou a, Bo Gao c, Zeng Li a,
Yu Zhou a, Hualiang Jiang a, Kaixian Chen a, Xuechu Zhen b,c, , Hong Liu a,
⇑
⇑
a State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, China
b Neuropharmacological Laboratory, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China
c Department of Pharmacology, Soochow University College of Pharmaceutical Sciences, Suzhou, China
a r t i c l e i n f o
a b s t r a c t
Article history:
An effective and rapid method for the microwave-assisted preparation of the key intermediate for the
total synthesis of tetrahydroprotoberberines (THPBs) including l-stepholidine (l-SPD) was developed.
Thirty-one THPB derivatives with diverse substituents on A and D ring were synthesized, and their bind-
ing affinity to dopamine D1, D2 and serotonin 5-HT1A and 5-HT2A receptors were determined. Compounds
18k and 18m were identified as partial agonists at the D1 receptor with Ki values of 50 and 6.3 nM, while
both compounds act as D2 receptor antagonists (Ki = 305 and 145 nM, respectively) and 5-HT1A receptor
full agonists (Ki = 149 and 908 nM, respectively). These two THPBs compounds exerted antipsychotic
actions in animal models. Further electrophysiological studies employing single-unit recording in intact
animals demonstrated that 18k-excited dopaminergic (DA) neurons are associated with its 5-HT1A recep-
tor agonistic activity. These results suggest that these two compounds targeted to multiple neurotrans-
mitter receptors may present novel lead drugs with new pharmacological profiles for the treatment of
schizophrenia.
Received 5 November 2012
Revised 12 December 2012
Accepted 14 December 2012
Available online 21 December 2012
Keywords:
Asymmetric synthesis
Tetrahydroprotoberberines
Dopamine receptors
Serotonin receptors
Multi-action
New pharmacological profile
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
symptoms and cognitive function remain the main challenge in
antipsychotic drug development.
Schizophrenia, a chronic and devastating psychotic disorder, is
clinically characterized by the presentation of positive symptoms
such as auditory hallucinations, disorganized thoughts, delusions,
and irrational fears, and negative symptoms including social with-
drawal, diminished affect, poverty of speech, lack of energy, and
the inability to experience pleasure, as well as cognitive impair-
ments.1–3 It is believed that abnormal neurotransmission, particu-
larly an imbalance of the dopamine system (mesolimbic
hyperactivity and mesocortical hypoactivity) is involved in the
pathophysiology of schizophrenia.3,4 Indeed, almost all currently
available antipsychotic drugs behave as dopamine D2 receptor
antagonists. However, dopamine D2 receptor-directed antipsy-
chotic drug therapy is dampened by the lack of efficacies in nega-
tive symptoms and cognitive function. Improvements in negative
Accumulating evidence suggests that dysfunction of dopamine
D1 receptors in the medial prefrontal cortex (mPFC) may be
responsible for the negative symptoms and cognitive deficits of
schizophrenia.5,6 Impaired D1 receptor function has been docu-
mented in schizophrenic patients and in experimental animal
models.7,8 Application of a D1 receptor agonist has been found to
improve the negative symptoms and working memory.9 Unfortu-
nately, currently available antipsychotic drugs do not bear dual
D1 agonistic and D2 antagonistic functions.
In addition to the dopamine (DA) system, the limbic forebrain
structures are also innervated by serotonin (5-HT) neurons. The
influence of the 5-HT system on midbrain DA neurons has been
demonstrated by
a
variety of experimental approaches.10–13
Abnormal serotonin transmission is an important tenet of the sero-
tonergic/dopaminergic hypothesis of schizophrenia which contrib-
utes to the pathology of the disease.14–22 Atypical antipsychotic
medications such as clozapine, risperidone solanzapine, ziprasi-
done, and aripiprazole (Fig. 1), have been shown to produce less se-
vere side-effects whilst effectively relieving positive symptoms.1,23
The relative advantage of these drugs is thought to be associated
with their modulation of 5-HT receptors, particularly the 5-HT2A
⇑
Corresponding authors. Tel.: +86 512 6588 1160; fax: +86 512 6588 0369 (X.Z.);
tel./fax: +86 21 5080 7042 (H.L.).
E-mail addresses: zhenxuechu@suda.edu.cn (X. Zhen), hliu@mail.shcnc.ac.cn (H.
Liu).
These two authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.12.016

H. Sun et al. / Bioorg. Med. Chem. 21 (2013) 856–868
857
OH
O
N
N
N
N
N
N
Cl
N
N
N
H
O
HN
S
Quetiapine
Bifeprunox
O
Clozapine
O
N
S
N
O
N
N
F
N
N
N
O
N
H
Cl
Ziprasidone
N
Risperidone
N
N
N
O
N
Cl
HO
N
N
Cl
S
H
O
O
N
O
H
Olanzapine
Aripiprazole
OH
l-SPD
Figure 1. Representative atypical antipsychotic agents.
receptor.24,25 In addition, the 5-HT1A receptor is believed to control
the output of cortical glutamatergic neurons in prefrontal cortex
(PFC) and regulate NMDA receptor channels through a microtu-
bule-dependent mechanism.26–28 Moreover, 5-HT1A stimulation
has been found to improve the acute extrapyramidal syndrome
(EPS) liability profile,29 while drugs such as aripiprazole with 5-
HT1A receptor agonistic activity have been shown to have a supe-
rior effect in the treatment of negative symptoms and cognition
impairments in schizophrenic patients.30–35
uents the on D ring (18g–18j and 24a–24e, Table 1); secondly, the
hydroxyl group at C10 was preserved and different alkoxy groups
were introduced on the A ring (18k–18r, Table 1); thirdly, C3 or
C9 was substituted with a hydroxyl group and C2 or C10 was
substituted with an alkoxy groups (18s–18z and 17a–17d, Table 1);
finally, we investigated compounds devoid of hydroxyl groups and
the effect of the C14-configuration.
2.2. Synthesis of target compounds
In this regard, development of drugs which are D1 receptor and
5-HT1A receptor agonists or partial agonists as well as D2 receptor
antagonists may provide a new approach for antipsychotic drug
discovery. l-Stepholidine (l-SPD) (Fig. 1), a tetrahydroberberine
(THPB) alkaloid isolated from the Chinese herb Stephania interme-
dia, exhibits a unique pharmacological profile; it elicits dual dopa-
mine receptor action (D1 agonistic and D2 antagonistic) while
Derivatives (17a–18z, Table 1) including l-SPD, which required
common intermediates 4 and 10, were synthesized efficiently
according to the procedures outlined in Schemes 1 and 2. First,
phenylethylamines 4 were prepared from aldehyde 1 using a pro-
cedure reported previously.39 To prepare the intermediates 10,
methyl 2-(4-hydroxyphenyl) acetate was selected as the starting
material, and 6 was easily accessible by the bromination of 5 with
bromine followed by protection with benzyl bromide or methyl io-
dide in acetone to give the intermediate 7. Although the synthesis
of phenols from halogen benzene has been previously reported,
some methods take a long time to reach completion and produce
low yields while others require iodobenzene with electron-with-
drawing substituent.40–42 Therefore, compound 8 was obtained un-
der microwave-assisted condition without ligand. Phenylacetic
acid 8 was subjected to hydroxymethylation in the presence of
phenylboric acid and para-formaldehyde, yielding borate, which
may be hydrolyzed without isolation of the lactone 9.43,44 Finally,
the lactone 9 was methylated with iodomethane to yield the re-
quired intermediates 10.
Condensation of lactone 10 with several phenylethylamines
proceeded in ethanol afforded 11 in high yields. The benzyl alcohol
group was converted to its acetate 12 using acetyl chloride and
pyridine. The imine 13 was obtained in excellent yields by promo-
tion with phosphoryl trichloride in acetonitrile according to the
Bischler–Napieralski reaction. Asymmetric transfer hydrogenation
of imines with formic acid/triethylamine catalyzed by a suitably
designed chiral Ru complex developed by Noyori and co-workers45
generated the amines 14 and their enantiomers asymmetrically in
dimethylformamide (DMF) at room temperature; the correspond-
ing amines were hydrolyzed to benzyl alcohol with sodium
acting as
a 5-HT1A receptor partial agonist in vitro and
in vivo.29,36–38 However, its poor physicochemical properties and
low oral bioavailability limits the application of l-SPD, in addition
to its relatively weak agonistic activity on the 5-HT1A receptor.
Although it is believed that l-SPD present a potential novel cate-
gory of antipsychotic drug acting as neurotransmitter stabilizer, a
detail structure–activity study of l-SPD has not been done. We de-
signed and synthesized a series of novel THPBs by introducing var-
ious substituents such as methyl, methyoxyl, hydroxymethyl and
methylenedioxy groups at different positions on the THPB pharma-
cophore as initial effort to find better drug candidate. In this paper,
we report the synthesis of these novel THPBs compounds and their
pharmacological evaluation at dopamine (D1 and D2), and seroto-
nin (5-HT1A and 5-HT2A) receptors.
2. Chemistry
2.1. Design of THPBs compounds
We presumed that the shortcoming of l-SPD most likely re-
sulted from the two phenolic hydroxyl groups on the A ring and
D ring. Therefore, further structural modification would be neces-
sary to explore natural products with new pharmacological pro-
files. We designed the THPBs compounds as follows. Firstly, we
preserved the hydroxyl group at C2 and introduced diverse substit-
hydroxide without purification. Closure of the
C ring was

858
H. Sun et al. / Bioorg. Med. Chem. 21 (2013) 856–868
Table 1
Binding affinity of THPBs for D1, D2, 5-HT1A and 5-HT2A receptors from HEK293 or CHO cellsa
R2
A
B
N
C
R1O
R5
R6
D
R8
R7
No.
R1
R2
R5
R6
R7
R8
Config
C-14b
D1
IRc (%) Ki (nM)
D2
5-HT1A
5-HT2A
IRc (%) Ki (nM)
IRc (%) Ki (nM)
IRc (%) Ki (nM)
17a
17b
17c
17d
18ed
18f
18g
18h
18i
18j
24a
24b
24c
24d
24e
18k
18l
18m
18n
18o
18p
18q
18r
18s
18t
18u
18v
18w
18x
18y
18z
SCH-23390
Spiperone
5-HT
CH3
CH3
CH2O
CH2O
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
CH2O
CH2O
CH2CH2O
CH2CH2
CH3
CH3
CH3
CH3
CH2O
CH2O
CH3
CH3
CH3
CH3
CH3O CH3O CH3O
CH3O CH3O CH3O
CH3O CH3O
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
S
R
S
R
S
R
S
R
S
R
S
S
S
S
S
S
R
S
R
S
S
S
R
S
R
S
R
S
R
S
R
96.9
39.8
98.3
75.5
99.8
94.4
99.2
99.5
99.1
84.5
97.5
99.8
99.0
104
99.6
98.8
54.4
99.7
95.8
22.6
99.3
98.3
67.1
87.7
35.9
98.7
78.9
84.1
88.8
95.2%
49.4
100
231 41
—
66 18
>5000
3.4 0.7
135 15
4.5 0.4
6.6 0.8
85.4
20.5
88.7
26.6
97.5
53.7
90.9
90.5
93.4
31.8
90.0
83.8
90.1
94.8
100
88.8
3.4
92.8
40.5
73.1
63.9
94.2
27.7
22.7
1.5
>5000
—
84.9
30.4
84.4
34.5
89.3
53.6
71.1
64.2
14.2
2.0
65.6
56.9
74.6
80.9
61.0
97.4
65.5
95.7
63.6
90.8
79.5
76.7
13.2
30.5
6.2
>5000
—
>5000
—
>5000
—
—
—
—
—
—
—
—
>5000
—
33.0
29.7
46.6
18.3
45.6
16.4
40.5
40.6
20.9
26.3
68.7
32.4
27.1
18.4
49.9
68.8
27.9
73.6
25.3
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
119
—
2
CH3O CH3O
CH3O CH3O OH
CH3O CH3O OH
CH3O CH3O CH3O
CH3O CH3O CH3O
11
—
1
91
105
214
—
9
3
2
CH3O OH
CH3O OH
CH3O
CH3O
H
H
H
22
4
529 64
35.8 2.6
2.5 0.2
28.9 2.7
17.3 0.5
4.2 0.1
50 10
—
6.3 0.4
565 12
—
7.5 1.0
200 25
—
CH3O
H
CH3O
CH3O
CH3
CH3O CH2OH
H
347
—
3
CH3O CH3O
CH3O CH3
CH3O CH3O
H
H
H
161
147
32
305
—
2
1
3
4
5
CH3O
H
OCH2O
CH3O CH3O OH
CH3O CH3O OH
CH3O OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
149
4
—
—
—
—
363
480
145
—
908 10
—
208 45 97.3
>5000
>5000
—
—
—
>5000
—
—
—
—
—
—
—
CH3O OH
CH3O OH
CH3O OH
—
—
4
6
91.7
84.0
13.9
20.0
19.3
49.1
10.9
38.0
40.4
59.1
36.0
—
OH
OH
CH3O OH
CH3O OH
113
—
2
>5000
—
—
—
CH3O OH
CH3O OH
OH
CH3O
CH3O
CH3O
CH3O
443 42
—
—
—
>5000
—
—
—
>5000
—
60 8.4
>5000
310 61
1189 23
530 74
—
1.2 0.4
—
—
78.0
20.4
63.2%
71.5
79.8
8.16
—
82.8
25.5
50.5
55.4
60.5
31.5
—
OH
OH
OH
OH
OH
CH3O CH3O
CH3O CH3O
—
—
—
—
OH
OH
CH3O
CH3O
—
—
—
—
100
—
1.2 0.3
—
—
100
100
—
2.9 0.1
—
0.9 0.1
a
The initial screening was carried out at a concentration of 10 lM for each compound and Kis were measured for compounds that inhibited binding by more than 80% for
DA receptors or 90% for the serotonin receptors.
b
Config C-14 represents the configuration of C-14.
IR represents inhibition ratio.
Compound 18e is l-SPD; dash line denote that no experiment was conducted.
c
d
accomplished in one pot in the presence of thionyl chloride and
aqueous NaHCO3 in good yields. The deprotection was carried
out by refluxing 17 in concentrated hydrochloric acid and ethanol,
when 18 was collected in moderate isolated yield and good enan-
tiomeric excess (ee). All compounds (17a–18z) prepared by these
general methods are listed in Table 1.
Compounds 24a–24e (Table 1) were synthesized from 3c and
19a–19c and 19e in five steps (Scheme 3). Condensation of inter-
mediates 3c with commercially available 2-phenylacetic acids 19
generated amides 20 in good yields. Using the Bischler–Napieralski
reaction, treatment of the amides 20 with phosphoryl trichloride
(POCl3) gave access to imines 21 in excellent yields. Asymmetric
hydrogenation of 21 catalyzed by a chiral Ru-(II) complex (Noyori’s
catalyst) afforded chiral amines 22. Cyclization of amines 22 via
the Pictect–Spengler reaction provided products 23a–c and 23e
in good isolated yields and excellent enantiomeric excess. Products
24a–e were obtained by deprotection of compounds 23.
3. Results and discussion
3.1. Chemistry
Although several racemic syntheses of tetrahydroberberines
(THPBs) with substituents at C9 and C10 have been reported along
with a few asymmetric syntheses, there remains a lack of general
methods to prepare THPBs in enantiopure form with the desired
ring substitution patterns.46–51 Although the racemic synthesis of
( )-SPD was reported by Chiang and Brochmann-Hassen in
1977,52 the total synthesis of l-SPD with good yield and high enan-
tiomeric excess was only recently reported.53 We developed an
effective and rapid microwave-assisted method to prepare the
key intermediate of THPBs, which was then applied in the total
synthesis of THPBs including l-SPD. The chemical structures of 31
designed compounds (17a–18z and 24a–24e) are shown in Table 1.
These compounds were synthesized through the routes outlined in

H. Sun et al. / Bioorg. Med. Chem. 21 (2013) 856–868
859
R2
CHO
R2
NH2
R2
NO2
a
b
R1O
R1O
R1O
4
2
3
a, R1, R2 = CH3, OCH3
b, R1, R2 = CH2O
c, R1, R2 = Bn, OCH3
d, R1, R2 = CH3, OBn
e, R1, R2 = CH2CH2
f, R1, R2 = CH2CH2O
O
O
O
O
O
d
R3O
c
e
HO
O
HO
Br
O
Br
5
7
6
O
O
O
OH
f
g
OR4
OR3
OH
O
R3O
OH
8
OR3
10
9
a, R3, R4 = OCH3, OCH3
b, R3, R4 = OBn, OCH3
c, R3, R4 = OCH3, OBn
Scheme 1. Preparation of the amine and isochroman-3-one. Reagents and conditions: (a) CH3NO2, CH3COOH, CH3COONH4, 100 °C, 70–85%; (b) THF, LiAlH4, reflux, 80–87%;
(c) Br2, CH3COOH, 90%; (d) BnBr, acetone, K2CO3, reflux, 95–99%; (e) w, 140 °C, 60 min, KOH, H2O, CuO/Cu, 56%; (f) C6H5B(OH)2, toluene, 110 °C, 1 h; (HCHO)n, 4 Å molecular
sieve, 46 h, then H2O, reflux, 2 h, 82–85%; (g) CH3I or BnBr, acetone, K2CO3, reflux, 92–95%.
l
OH
OR4
O
O
H
N
H
N
R2
O
R2
NH2
R2
h
OR4
OR3
i
+
R1O
O
R1O
R1O
OR3
4
11
R3O
12
10
a, R3, R4 = CH3, CH3
b, R3, R4 = Bn, CH3
c, R3, R4 = CH3, Bn
OR4 OAc
R2
R1O
R2
R2
R1O
N
OAc
OR4
NH OH
j
R1O
NH OAc
OR4
k
l
∗
∗
OR4
OR3
OR3
OR3
15
13
R2
R2
m
o
n
NH Cl
18a-18z
R1O
N
R1O
∗
∗
OR4
OR3
OR4
OR3
16
17
O
R2
R2
HO
R1O
N
N
HO
R1O
∗
N
∗
N
R1O
OR4
OR3
∗
∗
O
OR4
O
OH
O
18e-18j
18k-18r
OH
18w-18z
18k and 18l, R1, R2 = CH3, OCH3
18m and 18n, R1, R2 = CH2O
18o, R1, R2 = CH3CH2O
18s-18v
18e and 18f, R3, R4 = H, CH3
18g and 18h, R3, R4 = CH3, CH3
18i and 18j, R3, R4 = CH3, H
18w and 18x , R1, R4 = CH3, CH3
18y and 18z, R1, R4 = CH3, H
18s and 18t, R1, R2 = CH3, OCH3
18u and 18v, R1, R2 = OCH2O
18p, R1, R2 = CH3CH2
18q and 18r, R1, R2 = H, OH
Scheme 2. Synthesis of (ꢀ)-THPBs and (+)-THPBs from amines and isochroman-3-ones. Reagents and conditions: (h) C2H5OH, reflux, 84–92%; (i) CH3COCl, pyridine, CH2Cl2,
89–95%; (j) POCl3, CH3CN, reflux; (k) (Ru[(R,R)-Tsdpen](
6-p-cymene) or (Ru[(S,S)-Tsdpen]( 6-p-cymene), DMF, HCOOH/TEA, rt, 89–95%; (l) NaOH, EtOH/H2O, 90–98%; (m)
SOCl2, CH2Cl2; (n) saturated NaHCO3, 84–92%; (o) concentrated HCl, C2H5OH, reflux, 72–85%.
g
g

860
H. Sun et al. / Bioorg. Med. Chem. 21 (2013) 856–868
H
O
R5
R6
O
NH2
R7
R6
OH
O
N
p
q
+
N
O
O
BnO
BnO
BnO
R5
R6
R5
R7
20
3c
19
21
a, R5, R6, R7, R8 = H, H, OCH3, H
b, R5, R6, R7, R8 = OCH3, H, OCH3, H
c, R5, R6, R7, R8 = CH3, H, CH3, H
e, R5 = H, R6, R7 = OCH2O, R8 = H
R7
O
O
O
r
s
NH
t
N
N
BnO
HO
BnO
R5
R6
R5
R6
R5
R6
R8
23
R8
24
R7
R7
R7
22
a, R5, R6, R7, R8 = H, H, OCH3, H
b, R5, R6, R7, R8 = OCH3, H, OCH3, H
c, R5, R6, R7, R8 = CH3, H, CH3, H
d, R5, R6, R7, R8 = OCH3, H, OCH3, CH2OH
e, R5 = H, R6, R7 = OCH2O, R8 = H
Scheme 3. Synthesis of compounds 24a–24e. Reagents and conditions: (p) EDCI, HOBt, DCM, TEA, 87–93%; (q) POCl3, CH3CN, reflux; (r) (Ru[(R,R)-Tsdpen](
DMF, HCOOH/TEA, rt, 87–96%; (s) HCOOH, 40% HCHO, 25–90 °C, 2 h, 79–91%; (t) concentrated HCl, C2H5OH, reflux, 83–90%.
g
6-p-cymene),
Schemes 1–3, and the details of the synthetic procedures and
structural characterizations are described in the Section 5.
group at C11 exhibited good binding affinities at the D1 receptor
(35.8 and 2.5 nM), especially 24a were more potent than 18e (l-
SPD) at the D1 receptor. Compared to compound 24b, compound
24c with methyl group at C9 and C11 was found to be more potent
than 24b at the D2 receptor and lower binding affinity at the D1
receptor (28.9 nM for D1 and 161 nM for D2). Compound 24d, orig-
inated from 24b by introducing hydroxymethyl at the C12, also
displayed higher binding affinity than 24a–c at the D2 receptor
(147 nM) and good binding affinity at the D1 receptor (17.3 nM).
In addition, 24e which formed a ring between C10 and C11 with
methylenedioxy showed similar high binding affinity at the D1
and D2 receptors (4.2 nM for D1 receptor and 32.1 nM for D2 recep-
tor) with 18e. Therefore, compounds with good potency at the D1
and D2 receptors can be explored through retaining hydroxyl group
at the C2 and diverse substituents on the D ring.
Compounds 18k, 18m, 18o and 18p retained hydroxyl groups at
C10 but with an alkoxyl group at C2 showed an improved binding
affinity to 5-HT1A and 5-HT2A receptors except for 18p, and com-
pounds 18k and 18o displayed Ki values of 149 and 208 nM at
the 5-HT1A receptor (inhibition ratio 97.4% and 95.7% for 5-HT1A
receptor, respectively), which indicated a moderate improvement
compared to 18e (l-SPD, inhibition ratio 89.3% for 5-HT1A receptor).
In addition, 18o and 18p were found to be more potent than 18e (l-
SPD) at the 5-HT2A receptor (363 and 468 nM, respectively). Mean-
while, 18m and 18p showed a similar high binding affinity to the
D1 receptor with Ki values of 6.3 and 7.5 nM, respectively. How-
ever, only moderate affinity was observed for 18k and 18m at
the D2 receptor (305 and 145 nM, respectively), while 18o and
18p exhibited remarkably weakened binding to the D2 receptor.
Introducing a hydroxyl group to 18k at C3 generated compound
18q, which exhibited improved binding potency compared to
18k at the D2 receptor and a somewhat reduced affinity for D1
and 5-HT1A receptors. Compared to18e (l-SPD), a hydroxyl group
at C10 and alkyloxyl group at C2 improved binding affinity at the
5-HT1A receptor. In addition, the cyclization of C2 and C3 also
boosted binding potency at the 5-HT2A receptor. The reduced affin-
ity for D2 receptors following substitution with an alkyloxyl group
at C2 further confirmed that the alkyloxyl group at C2 hampered
the interaction of the ligand with D2 receptor, and the hydroxyl
group at C10 can improve the binding affinity to the D1 and 5-
HT1A receptors. As enantiomers 18f and 18e, compounds 18l, 18n
3.2. Binding assays
All THPBs compounds were subjected to the competitive bind-
ing assays for DA (D1 and D2) and serotonin (5-HT1A and 5-HT2A
)
receptors using a membrane preparation obtained from HEK293T
cells stably transfected with the respective receptors. The initial
screening was carried out at a concentration of 10 lM for each
compound to test its ability to inhibit the binding of a tritiated
radioligand to the corresponding receptor. Kis were measured for
compounds that inhibited binding by more than 80% for DA recep-
tors or 90% for the serotonin receptors. The procedures used
were similar to those reported previously by us.13,21,29 [3H]-
8-Chloro-3-methyl-5-phenyl-2,3,4,5-tetrahydro-1H-benzo-[d]aze-
pin-7-ol (SCH23390), [3H] spiperone, [3H]-8-OH-DPAT, and [3H]
Ketanserin were used as standard radioligands for DA D1 and D2
and serotonin 5-HT1A and 5-HT2A receptors, respectively.
The inhibition ratio and Ki values of the newly synthesized
THPBs (17a–18z and 24a–24e) are summarized in Table 1. Similar
to the natural product 18e (l-SPD), compared to the high binding
affinity of C14-S-configured THPBs, the R-configured compounds
showed remarkably weakened binding affinity to all the receptors
(D1, D2, 5-HT1A and 5-HT2A receptors) except compound 18h, indi-
cating that the C14-S configuration in THPBs is an important deter-
minant of receptor binding affinity.
Unlike 18e (l-SPD, Ki = 3.4 nM for D1 receptor), 18g bearing only
a hydroxyl group at C2 showed a similar high binding affinity for
the D1 receptor with a Ki value of 4.5 nM. However, it was nine
times less potent than 18e (l-SPD, Ki = 11 nM for D2 receptor) at
the D2 receptor with a Ki value of 91 nM. Unlike other enantiomers,
a similar binding affinity was also observed for the enantiomer of
18g and 18h with Ki values of 6.6 and 105 nM at the D1 and D2
receptors respectively. 18i, produced by introducing a hydroxyl
group at C9 in 18g, displayed a somewhat reduced binding affinity
compared to 18g at the D1 and D2 receptors. These results suggest
that the hydroxyl group at C2 may be essential to maintain potency
at the D1 and D2 receptors. Based on the early results achieved
from compounds 18g to 18j, further SAR was explored retaining
hydroxyl group at C2. Compounds 24a and 24b with methoxyl

H. Sun et al. / Bioorg. Med. Chem. 21 (2013) 856–868
861
and 18r exhibited reduced potency at D1, D2 and 5-HT1A receptors
compared to their corresponding enantiomers. Another important
difference between 18e (l-SPD) and compounds such as 18k, 18m,
18o and 18p was that the latter were found to be more soluble in
organic solvents.
0.19
l
M) had a 70-fold higher potency in activating the 5-HT1A
receptor than that of compound 18m (EC50, 13.5
lM). In agree-
ment with l-SPD, both 18k and 18m displayed antagonistic activity
at the D2 receptor, where 18m was 20 times more potent in antag-
onizing the D2 receptor than that of 18k. Thus, 18k and 18m ap-
pear to act as partial D1 receptor agonists and full 5-HT1A
agonists while exerting antagonistic activity at the D2 receptor.
We are searching for compounds that act as neurotransmitter
stabilizer that can target to multiple related neurotransmitter
receptors, presumably has advantage than that of single recep-
tor-targeted drug such as classical D2 receptor antipsychotic drug.
Since the pharmacological profile of 18k and18m meet such crite-
ria, they therefore were selected for efficacy study in vivo to prove
the concept.
Compounds 18s and 18u containing only a hydroxyl group at C9
showed reduced binding affinity for the D1 receptor with Ki values
of 334 and 60 nM, with weak binding affinity for 5-HT1A, 5-HT2A
and D2 receptors. Thus, a hydroxyl group at C9 can have a relatively
strong influence on the interaction between compounds and recep-
tors. Compounds 18w and 18y represented a subseries of C3 hy-
droxyl group THPBs. Compound 18w bearing a hydroxyl group at
C3 retained a moderate affinity for the D1 receptor, with a Ki value
of 310 nM. Not unexpectedly, 18y containing two hydroxyl groups
at C3 and C9 with a Ki value of 530 nM were less potent than 18w
at the D1 receptor. Compound 18q with Ki values of 200 and
113 nM at D1 and D2 receptors respectively, introducing hydroxyl
group to 18w at C10, was more potent than 18w at all tested recep-
tors, which indicated that hydroxyl group at C10 was beneficial to
improve binding affinity to dopamine (D1 and D2) and serotonin
(5-HT1A and 5-HT2A) receptors. Just as other enantiomers, 18t,
18v, 18x and 18z also showed lower potency than their corre-
sponding enantiomer, which once again demonstrated that the
R-configured compounds showed remarkably weakened binding
affinity to all the receptors.
3.4. Efficacy studies of compound 18k and 18m in vivo
Phencyclidine (PCP)-induced hyperlocomotion rat model is a
widely used in schizophrenia drug research. We therefore em-
ployed this model to study the potential antipsychotic efficacy of
compounds 18k and 18m.
3.4.1. Compounds 18k and 18m attenuated phencyclidine
(PCP)-induced hyperlocomotion
Rats received intraperitoneal injections of saline or the respec-
tive compounds 10 min prior to the administration of PCP. Animal
activity was monitored 5 min after PCP treatment for a period of
30 min. As shown in Figure 2, acute 5 mg/kg PCP treatment caused
hyperlocomotion in rats. Pretreatment with 18k or 18m signifi-
A direct comparison between 18e (l-SPD) and 17a and 17c indi-
cated that the hydrogen bonding donor (OH) at C2 and C10 is a
determining factor to D1 and D2 receptor binding. To our surprise,
non-hydroxyl THPBs retained a similar binding affinity to the 5-
HT1A and 5-HT2A receptors.
cantly attenuated PCP-induced hyperlocomotive activity in
a
In our studies, we found that the configuration of the C14 posi-
tion in the THPB skeleton is a critical factor determining receptor
binding affinity. Moreover, the hydrogen bonding donor (OH) at
C2 and C10 has a strong impact on both D1 and D2 receptor bind-
dose-dependent manner. A significant decrease in total travel dis-
tance was already observed with 2.5 mg/kg 18k and 10 mg/kg
18m.
ing, and also with much effect on binding to 5-HT1A and 5-HT2A
.
3.4.2. The effect of 18k and 18m on phencyclidine-induced PPI
disruption
We also demonstrated that the hydroxyl group at C2 is a key factor
in maintaining activity at D1 and D2 receptors, evidenced from the
data on 18g (D1, 4.5 nM; D2, 91 nM) and 24e (D1, 4.2 nM; D2,
32 nM). Compounds 18k, 18m, 18o with a hydroxyl group at C10
exhibited higher potency than l-SPD at 5-HT1A and 5-HT2A recep-
tors, indicating that the C10 hydroxyl group is crucial for improv-
ing 5-HT1A and 5-HT2A receptors binding. Most of the
corresponding THPB compounds with C10 hydroxyl group showed
appreciable binding to the D1 receptor and reduced potency at the
D2 receptor. Given that 18k and 18m bore the optimal receptor
binding profiles, and these were selected as the lead compounds
for further study.
Impairment of sensorimotor gating is a common feature in
schizophrenia. Rats were pretreated with either saline or the
respective compounds 10 min prior to the administration of PCP.
Prepulse inhibition (PPI) was measured 5 min after PCP treatment.
As shown in Figure 3, pretreatment with 18k (5 mg/kg, ip) or 18m
(5 mg/kg and 10 mg/kg, ip) or atypical antipsychotic risperidone
(0.5 mg/kg, ip) significantly attenuated PPI disruption induced by
PCP(5 mg/kg, ip), at least at one prepulse level. There was no signif-
icant effect of drug treatment (18k, 18m, risperidone and/or PCP)
on absolute acoustic startle responses produced by a 120 dB noise
stimulus (Fig. 3D–F), indicating that the drug treatment, at the dos-
age used in the present study, had no effect on baseline startle
reactivity. In addition, there was no significant effect on PPI when
18k was administered alone (data not shown).
3.3. [35S]GTP
cS binding assays for compounds 18k and 18m
To determine the agonistic or antagonistic activities of com-
pounds 18k and 18m, [34S]GTP
S binding assays were employed.
Stably transfected respective D1, D2 or 5-HT1A cell membrane frac-
c
3.5. Compound 18k excited tegmental area (VTA) DA neurons
in vivo via its 5-HT1A receptor agonistic effect
tions were prepared, and the [35S]GTP
cS binding assays were per-
formed as previously described.9,54 Compounds 18k and 18m were
diluted to various concentrations and added to the reaction tubes.
The D1 receptor agonist SKF38393 (1-phenyl-2,3,4,5-tetrahydro-
1H-3-benzazepine-7,8-diol) and antagonist SCH23390 (7-chloro-
To further determine the agonistic properties of 18k at the 5-
HT1A receptor, we studied the effect of this compound on the ven-
tral tegmental area (VTA) dopaminergic (DA) neuron firing using
single-unit recording system as reported previously.55–62 Because
the effect of 5-HT1A agonists on VTA DA neurons are known to be
masked by D2 receptor activation, the 5-HT1A-excited VTA DA neu-
rons can only be observed while the D2 action is blocked by a D2
receptor selective antagonist.62 Therefore, rats were administrated
with raclopride, a D2-like receptor antagonist, before treatment
with 18k. Consistent with literature reports,58,62 raclopride
(0.1 mg/kg) alone produced only a small effect on DA neuron activ-
ities (n = 5, data not shown). Subsequent injection of 18k (1 mg/kg)
3-methyl-1-phenyl-1,2,4,5-tetrahydro-3-benzazepin-8-ol),
D2
receptor agonist quinpirole [(4aR,8aR)-5-propyl-4,4a,5,6,7,8,8a,9-
octahydro-1H-pyrazolo[3,4-g]quinoline] and 5-HT1A receptor ago-
nist 5-HT (5-hydroxytryptamine) were used for reference. The re-
sults are summarized in Table 2, where 18k and 18m showed
partial agonistic activity at the D1 receptor. Both 18k and 18m
exhibited full agonistic activity at the 5-HT1A receptor with Emax
values of 156.4% and 80.7%, respectively. Compound 18k (EC50
,

862
H. Sun et al. / Bioorg. Med. Chem. 21 (2013) 856–868
Table 2
[
35S]GTP
cS binding assays of compounds 18k and 18m for D1, D2 and 5-HT1A receptors
Compounds
Agonist
D1 Receptor
Antagonist
IC50 M)
Antagonist
D2 Receptor
Agonist
5-HT1A Receptor
EC50
(l
M)
Emax
%
(
l
Imax
%
IC50
(lM)
Imax
%
EC50
(
lM)
Emax %
18k
1.35 0.09
31.3 4.5
21.27 1.30
60.1 4.2
18.73 2.72
82.3 2.4
0.19 0.02
156.4 44
18m
15.97 4.75
35.6 9.1
17.37 0.58
78.2 4.7
1.00 0.06
58.9 6.0
13.5 1.3
80.7 2.9
SKF38393
SCH23390
Haloperidol
5-HT
0.13 0.01
100
—
—
—
—
—
—
—
—
—
—
—
—
—
—
100
—
—
—
0.60 0.01
—
—
83.5 0.3
—
—
0.55 0.01
—
94.6 0.6
—
—
3.75 0.50(ꢁ10ꢀ3
)
Note, the dash line denote that no experiment was conducted.
Figure 2. Compound 18k and 18m dose-dependently inhibit acute PCP-induced hyperlocomotion in rats. The locomotor activity was measured for a 30 min duration after
PCP administration and the total traveled distance was expressed as mean SEM. Statistical analysis showed 2.5 mg/kg and 5 mg/kg 18k (A, one-way ANOVA followed by
Tamhane’s post hoc test: F(5,51) = 48.883, n = 7–12), as well as 10 mg/kg 18m (B, one-way ANOVA followed by Tamhane’s post hoc test: F(4,33) = 42.928, n = 6–10)
significantly attenuated hyperlocomotor activity induced by PCP (⁄⁄⁄P <0.001 vs saline, ##P <0.01 ###P <0.001 vs PCP). SAL: saline; PCP: phencyclidine; HAL: haloperidol.
Figure 3. Compounds 18k and 18m alleviated PCP-induced PPI deficit without affecting the baseline startle reactivity and normal PPI function. (A, B, C) 18k (5 mg/kg, ip) or
18m (5 and 10 mg/kg, ip) or atypical antipsychotic risperidone (0.5 mg/kg, ip) significantly attenuated PPI disruption induced by PCP (5 mg/kg, ip) at least at one prepulse
level. (Two-way ANOVA followed by Tamhane’s post hoc test: ⁄⁄⁄P <0.001 vs saline, #P <0.05 vs PCP, n = 9–15.); (D, E, F) There’s no significant effect of drug treatment on the
absolute startle amplitude (one way ANOVA, n = 9–15). SAL: saline; PCP: phencyclidine.

H. Sun et al. / Bioorg. Med. Chem. 21 (2013) 856–868
863
Figure 4. Compound 18k excited VTA DA neurons in vivo. (A) Typical rate scatter diagram showing the excitatory effect by 18k (1.0 mg/kg), suggesting that 18k can increase
the firing rate, which can be reversed by 5-HT1A receptor antagonist WAY100635. (B) Summary bar graph showing that firing rates were all increased after 18k injection
compared to the baseline. (C) Typical burst scatter diagram showing the excitatory effect by 18k (1.0 mg/kg), suggesting that 18k can increase the bursts, which also can be
reversed by WAY100635. (D) Summary bar graph showing that bursting level was increased after 18k injection compared to the baseline. All values were expressed as
percent of baseline (the firing rates or bursting levels after raclopride administration, respectively) in B and D (⁄P <0.05, ⁄⁄P <0.01 compared to baseline).
Figure 5. Compound 18k fails to excite VTA DA neurons in antagonist WAY100635-pretreated rats. (A) Typical rate scatter diagram showing that 5-HT1A antagonist
WAY100635 completely blocks the firing rate increase induced by 18k injection. (B) Summary bar graph showing that firing rate had no significantly change after 18k
injection compared to the baseline. (C) Typical rate scatter diagram showing that 5-HT1A antagonist WAY100635 also completely blocks the bursting increase induced by 18k
injection. (D) Summary bar graph showing that bursting level had no significantly change after 18k injection compared to the baseline. All values are expressed as percent of
baseline (the firing rates or bursting levels after raclopride administration, respectively) in B and D.
significantly increased the firing rate of DA neurons in all cells
tested (n = 5, Fig. 4). Compared to the results after raclopride
administration (paired t-test, p <0.05), the firing rate of DA neurons
significantly increased to 128.3 9.89% after treatment with 18k.
Compound 18k also increased the bursts/s (bursts per second) of
DA neurons compared to the basal control (paired t-test, p
<0.01). To confirm the role of 5-HT1A receptor activation in 18k-en-
hanced firing of VTA DA neurons, compound WAY100635 (1.0 mg/
kg), a selective 5-HT1A antagonist, was administered following
injection with 18k. Compound 18k-enhanced DA neuron firing rate
and bursting were blunted, suggesting that 5-HT1A agonistic activ-
ity plays an important role in 18k excitement of DA neurons.
To further confirm excitation by 18k via the 5-HT1A receptor,
rats were injected with 5-HT1A antagonist WAY100635 prior to
administration of 18k. WAY100635 alone had little effect on DA
neuron activities. However, pretreatment with WAY100635 abol-
ished the 18k-enhanced firing and bursting of DA neurons (n = 5,
Fig. 5). Taken together, these results demonstrate that 5-HT1A
receptor agonistic activity contributes to the excitation of VTA
DA neurons by 18k in vivo.
As can be seen from the above results, compounds 18k and 18m
displayed full agonistic response at the 5-HT1A receptor, partial
agonistic activity at the D1 receptor and antagonistic activity at
the D2 receptor. In the PCP-induced rat models of schizophrenia,

864
H. Sun et al. / Bioorg. Med. Chem. 21 (2013) 856–868
both 18k and 18m dose-dependently reduced PCP-induced spon-
taneous activity, indicating that 18k and 18m may effectively re-
lieve the positive symptoms of schizophrenia. Although 18k was
20 times less potent than 18m in antagonizing the D2 receptor in
enantiomeric excess (ee) were also indentified by HPLC (Supple-
mentary data, Table S2). The details for purity analyses of com-
pounds 17a–17d, 18e–18z and 24a–24e are described in the
Supplementary data.
the [34S]GTP
cS assays in vitro (Table 2), 18k was more efficacious
than 18m in the locomotion test in vivo (Fig. 2). We further found
5.1. General procedure for the synthesis of THPBs
that 18k and 18m significantly improved PCP-impaired PPI.
Improvement of PPI is
a
common feature of antipsychotic
5.1.1. 4-(Benzyloxy)-3-methoxybenzaldehyde (2c)
drugs.63–65 These results demonstrated that compounds with ago-
nistic activity at the 5-HT1A receptor could be more effective
in vivo. This is further supported by our data from the in vivo elec-
trophysiological studies (Fig. 5), in which we showed that, like
other antipsychotic drugs that excite DA neurons,66,67 18k-excited
DA neurons and this excitation effects are found to associate with
its 5-HT1A receptor agonistic activity. It is noted that 18k with the
weaker affinity for D1 and D2 shows a higher potency in attenuat-
ing PCP-induced locomotor activity than compound 18m with the
higher binding affinity to D1 and D2. The discrepancy may result
from the distinct in PK/PD property, penetration of brain blood-
barrier and receptor occupancy in vivo.
To a solution of 4-hydroxy-3-methoxybenzaldehyde (1c, 7.6 g,
50 mmol) in acetone (150 mL) was added K2CO3 (10.4 g, 75 mmol)
and BnBr (9.41 g, 55 mmol), and the mixture was heated at reflux
for 4 h before it was cooled to room temperature. The solid was fil-
tered off, and the filtrate was concentrated under reduced pressure
to get yellow oil, which was purified by flash chromatography
(ethyl acetate/petroleum ether = 1:6) to yield 4-(benzyloxy)-3-
methoxybenzaldehyde (2c, 11.5 g, 95%) as white solid.
1H NMR (CDCl3, 300 MHz): d 9.85 (s, 1H), 7.44–7.34 (m, 7H),
6.99 (d, J = 8.1 Hz, 1H), 5.26 (s, 2H), 3.96 (s, 3H).
5.1.2. 1-(Benzyloxy)-2-methoxy-4-(2-nitrovinyl) benzene (3c)
To the solution of 2c (11.5 g, 49.5 mmol) in acetic acid (50 mL)
was added ammonium acetate (3.82 g, 49.5 mmol) and nitro meth-
ane (9.2 g, 150 mmol), and the mixture was reflux overnight, then
the reaction mixture was cooled to room temperature and the
product was obtained by filtration (9.88 g; yellow solid, 70%).
1H NMR (CDCl3, 300 MHz): d 7.95 (d, J = 13.2 Hz, 1H), 7.51 (d,
J = 13.2 Hz, 1H), 7.42–7.32 (m, 5H), 7.10 (dd, J = 8.4 Hz, J = 1.8 Hz,
1H), 7.02 (d, J = 1.8 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 5.22 (s, 2H),
3.92 (s, 3H).
4. Conclusions
This study reported the development, characterization, and anti-
schizophrenic effects of a series of THPB compounds. The rationale is
based on the recent consensus that an ideal antipsychotic drug
should behave as a neurotransmitter stabilizer with multiple targets
to correct imbalances of neurotransmission in schizophrenia.68 In
addition to D2 receptor antagonistic activity, the beneficial effects
of D1 and 5-HT1A receptor agonistic activities have been proven in
human and experimental animals studies of schizophrenia, by
improving cognitive function or negative symptoms which are the
major challenge of schizophrenia drug therapy.69,70
In conclusion, we have developed a series of derivatives of
THPB, among them, pharmacological profile assays indicate that
18k and 18m act as partial D1 receptor agonists and full 5-HT1A
agonists while exerting antagonistic activity at the D2 receptor.
Further animal and electrophysiological studies in vivo indicate
that 18k and 18m are potential antipsychotic agents with new
pharmacological profiles.
5.1.3. 2-(4-(Benzyloxy)-3-methoxyphenyl)ethanamine (4c)
To a slurry of LiAlH4 (2.28 g, 60 mmol) in THF (40 mL) at room
temperature was added 3c (5.7 g, 20 mmol) as a solution in THF
(20 mL), and the reaction mixture was heated to reflux for 2 h.
The reaction was cooled to room temperature, and certain amount
of water was added slowly to the reaction mixture. Then, the solid
was filtered off, and the filtrate was dried with Na2SO4, concen-
trated to give yellow oil.
Yellow oil, yield 85%; 1H NMR (CDCl3, 300 MHz): d 8.20 (br, 2H),
7.46–7.33 (m, 5H), 6.99 (d, J = 8.4 Hz, 1H), 6.92 (d, J = 1.5 Hz, 1H),
6.75 (dd, J = 8.4 Hz, J = 1.5 Hz, 1H), 5.06 (s, 2H), 3.79 (s, 3H), 3.01
(t, J = 6.6 Hz, 2H), 2.85 (m, 2H).
5. Experimental section
5.1.4. Methyl 2-(3-bromo-4-hydroxyphenyl) acetate (6)
The reagents (chemicals) were purchased from Lancaster, Sig-
ma, Acros and Shanghai Chemical Reagent Company, and used
without further purification. Analytical thin-layer chromatography
To
a solution of methyl 4-hydroxyphenyl acetate (16.6 g,
100 mmol) in 200 mL acetic acid was added liquid bromine
(16.8 g, 105 mmol) in 10 mL acetic acid slowly. The mixture was
stirred for 2 h at room temperature, then saturation NaHSO3 was
added to quench the reaction. The solvent was evaporated and
the residue was purified by flash chromatography (ethyl acetate/
petroleum ether = 1:4) to give compound 6.
(TLC) was performed on HSGF 254 (150–200 lm thickness, Yantai
Huiyou Company, China). Yields were not optimized. Column chro-
matography was performed with CombiFlashÒ Companion system
(Teledyne Isco, Inc.). Nuclear magnetic resonance (NMR) spectra
were performed on a Brucker AMX-400 and AMX-300 NMR (TMS
as IS). Chemical shifts were reported in parts per million (ppm, d)
downfield from tetramethylsilane. Proton coupling patterns were
described as singlet (s), doublet (d), triplet (t), quartet (q), multi-
plet (m), and broad (br). Low- and high-resolution mass spectra
(LRMS and HRMS) were given with electric, electrospray and ma-
trix-assisted laser desorption ionization (EI, ESI and MALDI) pro-
duced by Finnigan MAT-95, LCQ-DECA spectrometer and IonSpec
4.7 Tesla. Optical rotations of compounds were obtained on PE-
431 polarimeter and the melting points were performed on SGW
X-4 (SHANGHAI PRECISION & SCIENTIFIC INSTRUMENT CO., LTD).
LC–MS analysis were conducted on Agilent 1100 Series HPLC with
Yield 90%; 1H NMR (CDCl3, 300 MHz): d 7.39 (d, J = 1.5 Hz, 1H),
7.11 (dd, J = 8.4 Hz, J = 1.5 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 3.70 (s,
3H), 3.54 (s, 2H).
5.1.5. Methyl 2-(4-(benzyloxy)-3-bromophenyl) acetate (7b)
To methyl 2-(4-(hydroxyl)-3-bromophenyl) (22.9 g) was added
potassium carbonate and acetone, benzyl bromide was added to
the stirring solution and the mixture was heated at reflux. The mix-
ture cooled after 3 h, and the solution was filtered. The acetone was
evaporated to give an oil crude product, which was purified by
flash chromatography (ethyl acetate/petroleum ether = 1:6) to give
7b (31.0 g).
Yield 99%; 1H NMR (CDCl3, 300 MHz): d 7.51–7.33 (m, 6H), 7.15
(dd, J = 8.4 Hz, J = 2.4 Hz, 1H), 6.89 (d, J = 8.4 Hz, 1H), 5.15 (s, 2H),
3.70 (s, 3H), 3.55 (s, 2H).
an Agilent Extend-C18 (4.6 ꢁ 50 mm, 5
lm) reversed phase col-
umn. Compounds 17a–17d, 18e–18z and 24a–24e were confirmed
P95% purity (Supplementary data, Table S1) and the compounds

H. Sun et al. / Bioorg. Med. Chem. 21 (2013) 856–868
865
5.1.6. 2-(4-(Benzyloxy)-3-hydroxyphenyl)acetic acid (8b)
5.1.12. N-(2-(Benzo[d][1,3]dioxol-5-yl)ethyl)-2-(2-
To a solution of 7b (2.0 g) and KOH (2.0 g) in water (8 mL) was
added 200 mg copper power and 200 mg copper oxide, and the
mixture was stirred 10 min at room temperature. The sealed tube
was filled with Ar gas, and the mixture was irradiated on micro-
wave under 140 °C for 60 min. The solution was filtered and ad-
justed to acid with hydrochloric acid, and then the precipitation
was collected and purified by column chromatography (MeOH/
CH2Cl2 = 1:100) to give 8b (white solid, yield 56%).
(hydroxymethyl)-3,4-dimethoxyphenyl)acetamide (11c)
White solid, yield 88%; 1H NMR (CDCl3, 300 MHz): d 6.92 (d,
J = 8.4 Hz, 1H), 6.82 (d, J = 8.4 Hz, 1H), 6.68 (d, J = 7.8 Hz, 1H),
6.57 (d, J = 1.8 Hz, 1H), 6.48 (dd, J = 7.8 Hz, J = 1.8 Hz, 1H), 5.44
(m, 1H), 4.69 (s, 2H), 3.88 (s, 3H), 3.86 (s, 3H), 3.52 (s, 2H), 3.40
(m, 2H), 2.60 (m, 2H).
5.1.13. 2-(4-(Benzyloxy)-2-(hydroxymethyl)-3-methoxyphenyl)-
N-(4-(benzyloxy)-3-methoxyphenethyl)acetamide (11e)
White solid, yield 84%; 1H NMR (CDCl3, 300 MHz): d 7.42–7.25
(m, 10H), 6.82 (m, 2H), 6.75 (d, J = 8.4 Hz, 1H), 6.64 (d, J = 1.9 Hz,
1H), 6.47 (dd, J = 8.4, 1.9 Hz, 1H), 6.32 (m, 1H), 5.08 (s, 2H), 5.06
(s, 2H), 4.65 (s, 2H), 3.91 (s, 3H), 3.79 (s, 3H), 3.47 (s, 2H), 3.39
(q, J = 6.8 Hz, 2H), 2.65 (t, J = 6.8 Hz, 2H).
1H NMR (CDCl3, 300 MHz): d 7.43–7.38 (m, 5H), 6.90 (s, 1H),
6.89 (dd, J = 8.1 Hz, J = 2.1 Hz, 1H), 6.74 (d, J = 8.1 Hz, 1H), 5.10 (s,
2H), 3.56 (s, 2H).
5.1.7. 7-Methoxy-8-hydroxyisochroman-3-one (9a)
To homoisovanillic acid (3.0 g, 16.5 mmol) was added phenylb-
oric acid (4.0 g, 33.0 mmol) and dry toluene (70 mL). The mixture
was heated at reflux for 1 h, and H2O was collected in a Dean–Stark
trap. The hot solution was poured over molecular sieves (4 Å,
2.07 g) in a sealed tube. Para formaldehyde (2.72 g) was added
along with enough toluene (10 mL). The tube was heated at
100 °C for 46 h. The sealed tube was opened and the hot solution
filtered. The toluene was evaporated, and 70 mL water was added
to the residue. After heating at reflux for 2 h, the mixture was
cooled to room temperature and extracted with CH2Cl2 (150 mL).
The solution was dried (MgSO4) and the solvent evaporated. The
residue was stirred in Et2O (60 mL) for 3 h and the solid lactone
9a (white solid; 2.2 g, 85%) was filtered.
5.1.14. 6-(2-(3,4-Dimethoxyphenethylamino)-2-oxoethyl)-2,3-
dimethoxyphenyl acetate (12a)
To a solution of compound 11a (1.0 g, 2.57 mmol) in CH2Cl2
(15 mL) was added pyridine (3.8 mL, 3.86 mmol). The mixture
was cooled to 0 °C, and acetyl chloride (1.6 mL, 3.86 mmol) was
added dropwise. After being stirred 1.5 h at room temperature,
the reaction mixture was diluted with CH2Cl2 (10 mL), washed
with 1 N HCl and brine, dried over Na2SO4, filtered, and
concentrated. The residue was purified by flash chromatogra-
phy (MeOH/CH2Cl2 = 1:100) to afford a white solid 12a (1.09 g,
90%).
White solid, yield 85%; 1H NMR (CDCl3, 300 MHz): 6.82 (d,
J = 8.1 Hz, 1H), 6.69 (d, J = 8.1 Hz, 1H), 5.82 (s, 1H), 5.41 (s, 1H),
3.89 (s, 3H), 3.63 (s, 2H).
1H NMR (CDCl3, 300 MHz): d 6.90 (d, J = 8.4, 1H), 6.86 (d,
J = 8.4 Hz, 1H), 6.72 (d, J = 8.1 Hz, 1H), 6.62 (d, J = 1.8 Hz, 1H),
6.55 (dd, J = 8.1 Hz, J = 1.8 Hz, 1H), 5.45 (m, 1H), 5.14 (s, 2H), 3.87
(s, 3H), 3.85 (s, 3H), 3.83 (s, 3H), 3.83 (s, 3H), 3.54 (s, 2H), 3.43
(m, 2H), 2.68 (t, J = 6.9 Hz, 2H), 2.00 (s, 3H).
5.1.8. 7-Benzyloxy-8-hydroxyisochroman-3-one (9c)
White solid, yield 82%; 1H NMR (CDCl3, 300 MHz): d 7.45–7.34
(m, 5H), 6.93 (d, J = 8.4 Hz, 1H), 6.86 (d, J = 8.4 Hz, 1H), 5.40 (s,
1H), 5.12 (s, 1H), 3.91 (s, 3H), 3.62 (s, 2H).
5.1.15. 6-(2-(2-(Benzo[d][1,3]dioxol-5-yl)ethylamino)-2-
oxoethyl)-2,3-dimethoxybenzyl acetate (12c)
White solid, yield 93%; 1H NMR (CDCl3, 300 MHz): d 6.92 (d,
J = 8.4 Hz, 1H), 6.87 (d, J = 8.4 Hz, 1H), 8.64 (d, J = 8.1 Hz, 1H),
8.52 (d, J = 2.1 Hz, 1H), 8.43 (dd, J = 8.1 Hz, J = 2.1 Hz, 1H), 5.91 (s,
2H), 5.13 (s, 2H), 3.87, (s, 3H), 3.86 (s, 3H), 3.53 (s, 2H), 3.38 (m,
2H), 2.82 (m, 2H), 1.99 (s, 3H).
5.1.9. 7,8-Dimethoxyisochroman-3-one (10a)
To a solution of 9a (1.94 g, 10 mmol) in acetone (50 mL) was
added K2CO3 (2.07 g, 15 mmol) and CH3I (0.77 mL, 15 mmol),
and the mixture was heated at reflux for 4 h before it was cooled
to room temperature. The solid was filtered off, and the filtrate
was concentrated under reduced pressure to yield a yellow oil,
which was purified by flash chromatography (ethyl acetate/petro-
leum ether = 1:4) to yield 10a (1.98 g, 92%) as white solid.
1H NMR (CDCl3, 300 MHz): 6.83 (d, J = 8.1 Hz, 1H), 6.69 (d,
J = 8.1 Hz, 1H), 5.41 (s, 1H), 3.92 (s, 3H), 3.89 (s, 3H), 3.62 (s, 2H).
5.1.16. 6-((4-(Benzyloxy-3-methoxyphenethyl)carbamoyl)
methyl)-3-(benzyloxy)-2-methoxybenzyl acetate (12e)
White solid, yield 92%; 1H NMR (CDCl3, 300 MHz): d 7.46–7.28
(m, 10H), 6.90 (d, J = 8.4 Hz, 1H), 6.84 (d, J = 8.4 Hz, 1H), 6.73 (d,
J = 8.1 Hz, 1H), 6.64 (d, J = 1.9 Hz, 1H), 6.46 (dd, J = 8.1 Hz, 1.9 Hz,
1H), 5.46 (m, 1H), 5.15 (s, 2H), 5.12 (s, 2H), 5.10 (s, 2H), 3.88 (s,
3H), 3.83 (s, 3H), 3.53 (s, 2H), 3.42 (q, J = 7.0 Hz, 2H), 2.65 (t,
J = 7.0 Hz, 2H), 2.00 (s, 3H).
5.1.10. 7-(Benzyloxy)-8-methoxyisochromen-3-one (10b)
White solid, yield 95%; 1H NMR (CDCl3, 300 MHz): d 7.45–7.34
(m, 5H), 6.92 (d, J = 8.1 Hz, 1H), 6.85 (d, J = 8.1 Hz, 1H), 5.40 (s,
2H), 5.12 (s, 2H), 3.91 (s, 3H), 3.62 (s, 2H).
5.1.17. 6-((-6,7-Dimethoxy-3,4-dihydroisoquinolin-1-
yl)methyl)-2,3-dimethyloxybenzyl acetate (13a)
5.1.11. N-[2-(3,4-Dimethoxyphenyl)ethyl]-2-(hydroxymethyl)-
3,4-dimethoxybenzeneacetamide (11a)
To a solution of the 12a (1.09 g, 2.52 mmol) in dry acetoni-
trile (25 mL) was added POCl3 (1.38 mL, 15.1 mmol) then the
mixture was refluxed for 40 min under nitrogen. The reaction
mixture was cooled to room temperature and concentrated
under vacuum. The residue was dissolved in CH2Cl2, washed
with saturated NaHCO3 and brine, dried over Na2SO4, and con-
3,4-Dimethoxyphenylethanamine (0.54 g, 3.6 mmol) was added
to a stirred solution of compound 10a (0.8 g, 3.0 mmol) in C2H5OH
(5 mL). The mixture was reflux for 14 h. Evaporation of the sol-
vent left a little yellow powder, which was purified by flash chro-
matography (MeOH/CH2Cl2 = 1:80) to yield a white solid 11a (1.0,
89%).
centrated to yield
purification.
a yellow solid (1.0 g) without further
1H NMR (CDCl3, 300 MHz): d 6.88 (d, J = 8.4 Hz, 1H), 6.82 (d,
J = 8.4 Hz, 1H), 6.74 (d, J = 8.0 Hz, 1H), 6.64 (d, J = 2.0 Hz, 1H),
6.57 (dd, J = 8.0 Hz, J = 2.0 Hz, 1H), 6.03 (m, 1H), 4.68 (d,
J = 4.5 Hz, 2H), 3.89 (s, 3H), 3.86 (s, 3H), 3.85 (s, 3H), 3.83 (s, 3H),
3.51 (s, 2H), 3.45 (m, 2H), 2.70 (t, 2H).
Yellow solid, yield 99%; 1H NMR (CDCl3, 300 MHz): d 6.90 (s,
1H), 6.88 (d, J = 8.7 Hz, 1H), 6.82 (d, J = 8.7 Hz, 1H), 6.69 (s, 1H),
5.28 (s, 2H), 5.28 (s, 2H), 4.09 (s, 2H), 3.91 (s, 3H), 3.85 (s,
3H), 3.83 (s, 3H), 3.74 (s, 3H), 3.70 (m, 2H), 2.68 (m, 2H), 2.04 (s,
2H).

866
H. Sun et al. / Bioorg. Med. Chem. 21 (2013) 856–868
5.1.18. 3-(Benzyloxy)-6-((7-(benzyloxy)-3,4-dihydro-6-
methoxyisoquinolin-1-yl)methyl)-2-methoxybenzyl acetate
(13e)
5.1.24. (3-(Benzyloxy)-6-(((S)-7-(benzyloxy)-1,2,3,4-tetrahydro-
6-methoxyisoquinolin-1-yl)-methyl)-2-
methoxyphenyl)methanol (15e)
Yellow solid, yield 98%; 1H NMR (CDCl3, 300 MHz): d 7.44–7.28
(m, 10H), 6.96 (s, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.72 (d, J = 8.4 Hz,
1H), 6.70 (s, 1H), 5.24 (s, 2H), 5.08 (s, 2H), 5.00 (s, 2H), 3.98 (s,
2H), 3.92 (s, 3H), 3.90 (s, 3H), 3.69 (t, J = 7.5 Hz, 2H), 2.66 (t,
J = 7.5 Hz, 2H), 2.03 (s, 3H).
A slightly green solid, yield 75%; 1H NMR (CDCl3, 300 MHz): d
7.56–7.25 (m, 10H), 7.04 (d, J = 8.4 Hz, 1H), 6.91 (d, J = 8.4 Hz,
1H), 6.83 (s, 1H), 6.64 (s, 1H), 5.17 (s, 2H), 5.13 (s, 2H), 4.89 (d,
J = 11.1 Hz, 1H), 4.47 (d, J = 11.1 Hz, 1H), 4.12–4.09 (m, 1H), 3.92
(s, 3H), 3.89 (s, 3H), 3.26–3.20 (m, 2H), 3.07–2.89 (m, 3H), 2.74–
2.72 (m, 1H), 2.56–2.54 (m, 1H).
5.1.19. (S)-6-((6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinolin-1-
yl)methyl)-2,3-dimethoxylbenzyl acetate (14a)
The imine 13a (0.42 g, 1.0 mmol) was dissolved in anhydrous
DMF (3 mL), and RuCl[(R,R)-TsDPEN(P-cymene)] (7.0 mg, 1 mol %)
was added followed by formic acid/triethylamine (v/v = 5/2,
5.1.25. (ꢀ)-Tetrahydropalmatine (17a)
A solution of 15a (300 mg, 0.8 mmol) in CH2Cl2 (10 mL) was
cooled to 0 °C, and thionyl chloride (350 lL, 4.8 mmol) was added
dropwise. The reaction mixture was stirred at room temperature
for 1.5 h before saturated NaHCO3 (20 mL) was added slowly. After
being stirred for another 1.5 h, the organic layer was washed with
CH2Cl2 (20 mL) and brine, dried over Na2SO4, filtered, and concen-
trated to yield a yellow solid, which was purified by flash chroma-
tography (ethyl acetate/petrol ether = 1:3) to give white solid 17a
(247 mg, 87%).
300 lL), and the reaction mixture was stirred at room temperature
overnight. The reaction mixture was adjusted pH to 8 with satu-
rated NaHCO3 and extracted with ethyl acetate. The combined or-
ganic layer was washed with brine, dried over Na2SO4, filtered, and
concentrated. The crude product was without further purified and
directly employed in the next step.
Green solid, yield 93%; 1H NMR (CDCl3, 300 MHz): d 7.01 (d,
J = 8.4 Hz, 1H), 6.88 (d, J = 8.4 Hz, 1H), 6.75 (s, 1H), 6.60 (s, 1H),
5.29 (s, 2H), 4.84 (d, J = 7.6 Hz, 1H), 4.48 (d, J = 7.6 Hz, 1H), 4.11–
4.08 (m, 1H), 3.91 (s, 3H), 3.85 (s, 3H), 3.83 (s, 3H), 3.74 (s, 3H),
3.24–3.18 (m, 2H), 2.76–2.67 (m, 2H), 2.61 (s, 3H).
Mp 140–141 °C; ½a D24
ꢂ
+238.7 (c 0.45, CHCl3). 1H NMR (CDCl3,
400 MHz): d 6.87 (d, J = 8 Hz, 1H), 6.77 (d, J = 8 Hz, 1H), 6.72 (s,
1H), 6.61 (s, 1H), 4.25–4.21 (m, 1H), 3.88 (s, 3H), 3.86 (s, 3H),
3.84 (s, 6H), 3.55–3.51 (m, 2H), 3.28–3.11 (m, 3H), 2.79–2.85 (m,
1H), 2.68–2.61 (m, 2H). 13C NMR (CDCl3, 100 MHz): 150.2, 147.4,
147.3, 144.9, 129.6, 128.6, 127.6, 126.7, 123.8, 111.2, 110.8,
108.5, 60.1, 59.2, 56.0, 55.8, 53.9, 51.4, 36.2, 29.0. MS (ESI) m/z:
355(M+). HRMS (ESI): Calcd for 356.1682, found 356.1684. The
optical purity of 17a was determined to be 99.9% by HPLC analyses
(Chiralcel AD-H (0.46 cm ꢁ 25 cm; hexanes/i-PrOH = 70/30 with
flow rate = 1.0 mL/min and a UV detector at 214 nm; retain
time = 17.35 min).
5.1.20. (S)-2,3-Dimethoxy-6-((5,6,7,8-tetrahydro-
[1,3]dioxolo[4,5-g]isoquinolin-5-yl)methyl)benzyl acetate (14c)
Yellow solid, yield 91%; 1H NMR (CDCl3, 300 MHz): d 7.01 (d,
J = 8.4 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 6.66 (s, 1H), 6.57 (s, 1H),
5.90 (s, 2H), 5.28 (s, 2H), 4.02–3.98 (m, 1H), 3.90 (s, 3H), 3.89 (s,
3H), 3.22–3.16 (m, 2H), 2.90–2.84 (m, 3H), 2.74–2.72 (m, 3H),
2.06 (s, 2H).
5.1.26. (+)-Tetrahydropalmatine (17b)
5.1.21. (S)-3-(Benzyloxy)-6-((7-(benzyloxy)-6-methoxy-1,2,3,4-
tetrahydroisoquinolin-1-yl)methyl)-2-methoxybenzyl acetate
(14e)
White solid, yield, 87% (for two steps); Mp 138–139 °C; ½a D24
ꢂ
ꢀ244.3 (c 0.42, CHCl3). 1H NMR (CDCl3, 400 MHz): d 6.87 (d,
J = 8.0 Hz, 1H), 6.78 (d, J = 8.0 Hz, 1H), 6.72 (s, 1H), 6.61 (s, 1H),
4.25–4.22 (m, 1H), 3.88 (s, 3H), 3.86 (s, 3H), 3.84 (s, 6H), 3.55–
3.51 (m, 2H), 3.29–3.14 (m, 3H), 2.86–2.79 (m, 1H), 2.69–2.61
(m, 2H). 13C NMR (CDCl3, 100 MHz): 150.2, 147.4, 147.3, 144.9,
129.6, 128.6, 127.6, 123.8, 111.2, 110.8, 108.5, 60.1, 59.2, 56.0,
55.8, 53.9, 51.4, 36.2, 29.0. MS (ESI) m/z: 355(M+). HRMS (ESI):
Calcd for 356.1682, found 356.1853. The optical purity of 17a
was determined to be 99.5% by HPLC analyses (Chiralcel AD-H
Green solid, yield 92%; 1H NMR (CDCl3, 300 MHz): d 7.48–7.30
(m, 10H), 6.96 (d, J = 8.4 Hz, 1H), 6.90 (d, J = 8.4 Hz, 1H), 6.79 (s,
1H), 6.66 (s, 1H), 5.35 (d, J = 11.1 Hz, 1H), 5.20 (d, J = 11.1 Hz,
1H), 5.12 (s, 2H), 5.08 (s, 2H), 3.97–3.93 (m, 1H), 3.95 (s, 3H),
3.89 (s, 3H), 3.24–3.16 (m, 1H), 3.12–2.87 (m, 3H), 2.82–2.71 (m,
2H), 2.06 (s, 3H).
5.1.22. (S)-(6-((6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinolin-1-
yl)methyl)-2,3-dimethoxyphenyl)methanol (15a)
(0.46 cm ꢁ 25 cm;
rate = 1.0 mL/min and
time = 9.10 min).
hexanes/i-PrOH = 70/30
with
flow
a
UV detector at 214 nm; retain
To the solution of 14a (370 mg) in 3 mL ethanol was added 1 mL
H2O and 120 mg NaOH, the mixture was stirred for 3 h at room
temperature. 10 mL water was added to the solution and the solid
was filtered and dried in vacuum. Slightly green solid was obtained
(300 mg; yield 80%, two steps).
5.1.27. (ꢀ)-Tetrahydroberberine (17c)
White solid, yield, 91% (for two steps); Mp 130–131 °C; ½a D24
+250.0 (c 0.40, CHCl3). 1H NMR (CDCl3, 300 MHz): d 6.85 (d,
J = 8.0 Hz, 1H), 6.77 (d, J = 8.0 Hz, 1H), 6.72 (s, 1H), 6.58 (s, 1H),
5.91 (s, 2H), 4.25–4.21 (m, 1H), 3.84 (s, 6H), 3.55–3.51 (m, 2H),
3.24–3.11 (m, 3H), 2.84–2.77 (m, 1H), 2.67–2.59 (m, 2H). 13C
NMR (CDCl3, 100 MHz): 150.2, 146.1, 145.9, 145.0, 130.8, 128.6,
127.8, 127.6, 123.9, 110.9, 108.4, 105.5, 100.7, 60.2, 59.6, 55.8,
53.9, 51.4, 36.4, 29.5. MS (ESI) m/z: 339(M+). HRMS (ESI): Calcd
for 340.1549, found 340.1542. The optical purity of 17a was deter-
mined to be 97.8% by HPLC analyses (Chiralcel OD-H
ꢂ
A slightly green solid, yield 80%; 1H NMR (CDCl3, 300 MHz): d
7.01 (d, J = 8.4 Hz, 1H), 6.88 (d, J = 8.4 Hz, 1H), 6.76 (s, 1H), 6.62
(s, 1H), 5.29 (m, 2H), 4.84 (d, J = 11.7 Hz, 1H), 4.49 (d, J = 11.7 Hz,
1H), 4.11–3.90 (m, 1H), 3.91 (s, 3H), 3.89 (s, 3H), 3.85 (s, 3H),
3.82 (s,3H), 3.24–3.18 (m, 1H), 3.01–2.85 (m, 3H), 2.76–267 (m,
2H).
5.1.23. (S)-(2,3-Dimethoxy-6-((5,6,7,8-tetrahydro-
[1,3]dioxolo[4,5-g]isoquinolin-5-yl)methyl)phenyl)methanol
(15c)
(0.46 cm ꢁ 25 cm;
rate = 1.0 mL/min and
time = 44 min).
hexanes/i-PrOH = 70/30
with
flow
a
UV detector at 214 nm; retain
A slightly green solid, yield 83%; 1H NMR (CDCl3, 300 MHz): d
7.01 (d, J = 8.4 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 6.66 (s, 1H), 6.59
(s, 1H), 5.90 (m, 2H), 5.33 (d, J = 11.2 Hz, 1H), 5.22 (d, J = 11.2 Hz,
1H), 4.02–3.90 (m, 1H), 3.89 (s, 3H), 3.87 (s, 3H), 3.22–3.16 (m,
2H), 2.90–2.83 (m, 2H), 2.74–2.71 (m, 2H).
5.1.28. (+)-Tetrahydroberberine (17d)
White solid, yield, 87% (for two steps); Mp 133–134 °C; ½a D24
ꢀ184.3 (c 0.46, CHCl3). 1H NMR (CDCl3, 300 MHz): d 6.85 (d,
ꢂ

H. Sun et al. / Bioorg. Med. Chem. 21 (2013) 856–868
867
J = 8 Hz, 1H), 6.77 (d, J = 8 Hz, 1H), 6.72 (s, 1H), 6.58 (s, 1H), 5.91 (s,
2H), 4.25–4.21 (m, 1H), 3.84 (s, 6H), 3.55–3.51 (m, 2H), 3.24–3.11
(m, 3H), 2.84–2.77 (m, 1H), 2.67–2.59 (m, 2H). 13C NMR (CDCl3,
100 MHz): 150.2, 146.1, 145.9, 145.0, 130.8, 128.6, 127.8, 127.6,
123.9, 110.9, 108.4, 105.5, 100.7, 60.2, 59.6, 55.8, 53.9, 51.4, 36.4,
29.5. MS (ESI) m/z: 339(M+). HRMS (ESI): Calcd for 340.1549, found
340.1541. The optical purity of 17a was determined to be 99.9% by
HPLC analyses (Chiralcel OD-H (0.46 cm ꢁ 25 cm; hexanes/i-
PrOH = 70/30 with flow rate = 1.0 mL/min and a UV detector at
214 nm; retain time = 21.84 min).
lation cocktail was added and the radioactivity was determined in
a MicroBeta liquid scintillation counter. The IC50 and Ki values were
calculated by nonlinear regression (PRISM, Graphpad, San Diego,
CA) using a sigmoidal function.
5.2.2. [35S]GTP
For detection of the agonism action of the compounds, the
35S]GTP
S binding assay was performed at 30 °C for 40 min (for
cS binding assays
[
c
D1 and D2) or 20 min (for 5-HT1A) in reaction buffer containing
50 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EDTA, 100 mM NaCl, and
1 mM DL-dithiothreitol (DTT). The assay mixture (200
tained 20 g (for D2) or 30 g (for D1 and 5-HT1A) of membrane
protein, 0.1 nM [35S]GTP
S, and 40 M guanosine triphosphate
(GDP) with various concentration of the compound. The antago-
nism effects of the compounds were tested in the existence of
lL) con-
5.1.29. (ꢀ)-Stepholidine (18e)
l
l
Compound 17e (254 mg, 0.5 mmol) was refluxed in a mixture of
concentrated hydrochloric acid (13 mL) and EtOH (50 mL) for 2 h.
The reaction mixture was cooled to 0 °C and basified with concen-
trated ammonia solution. The mixture was extracted with CH2Cl2.
The combined organic layer was washed with brine, dried over
Na2SO4, filtered, and concentrated to give the crude product, which
was purified by column chromatograph (MeOH/CH2Cl2 = 1:60) to
give 18e.
c
l
100
lM SKF38393 for D1 or quinpirole for D2 receptor. Nonspecific
binding was measured in the presence of 100
lM 50-guanylimid-
odiphosphate (Gpp(NH)p). The reaction was terminated by addi-
tion of 3 mL of ice-cold washing buffer (50 mM Tris, pH 7.5,
5 mM MgCl2, 1 mM EDTA, and 100 mM NaCl) and was rapidly fil-
tered with GF/C glass fiber filters (Whatman) and rinsed three
times. Filters were dried and radioactivity was determined by li-
quid scintillation counting.
White solid, yield, 75%; Mp 127–128 °C; ½a D24
ꢀ197.3 (c 0.52,
ꢂ
CHCl3). 1H NMR (CD3OD, 300 MHz): d 6.78–6.69 (m, 3H), 6.63 (d,
1H), 4.15 (d, J = 15.6 Hz, 1H), 3.79 (s, 3H), 3.78 (s, 3H), 3.49–3.41
(m, 2H), 3.30–3.06 (m, 3H), 2.73–2.57 (m, 3H). 13C NMR (CD3OD,
100 MHz): 149.2, 148.3, 146.4, 145.4, 131.2, 129.2, 127.7, 126.7,
125.9, 116.8, 113.6, 112.9, 61.1, 60.9, 56.8, 55.3, 53.3, 37.0, 29.7.
MS (ESI) m/z: 327(M+). HRMS (ESI): Calcd for 328.1549, found
328.1565. The optical purity of 17a was determined to be 99.9%
by HPLC analyses (Chiralcel AD-H (0.46 cm ꢁ 25 cm; hexanes/i-
PrOH = 70/30 with flow rate = 1.0 mL/min and a UV detector at
214 nm; retain time = 14.68 min).
5.3. Animals
Male Sprague–Dawley rats (230–280 g), were purchased from
Shanghai Slac Laboratory Animal Co. Ltd. (Shanghai, China). All ani-
mals were maintained under standard laboratory conditions and
kept in temperature and humidity controlled rooms (22 1 °C,
55 5%) on a 12:12 h of light:dark cycle. Food and filtered water
were available ad libitum. All procedures were in compliance with
the Guidelines for the Care and Use of Laboratory Animals (Na-
tional Research Council, People’s Republic of China, 1996). The ani-
mal protocols were approved by the Institutional Animal Care and
Use Committee of Shanghai Institute of Materia Medica (SIMM).
5.1.30. (+)-Stepholidine (18f)
White solid, yield, 80%; Mp 128–129 °C; ½a D24
ꢀ195.7 (c 0.53,
ꢂ
CHCl3). 1H NMR (CD3OD, 300 MHz): d 6.79–6.70 (m, 3H), 6.65 (d,
1H), 4.16 (d, J = 15.6 Hz, 1H), 3.80 (s, 3H), 3.79 (s, 3H), 3.50–3.45
(m, 2H), 3.26–3.02 (m, 3H), 2.74–2.57 (m, 3H). 13C NMR (CD3OD,
100 MHz): 149.3, 148.3, 146.5, 145.5, 131.2, 129.3, 127.7, 126.8,
125.9, 116.8, 113.6, 113.0, 61.2, 60.9, 56.8, 55.4, 53.4, 37.0, 29.8.
MS (ESI) m/z: 327(M+). HRMS (ESI): Calcd for 328.1549, found
328.1530. The optical purity of 17a was determined to be 99.9%
by HPLC analyses (Chiralcel AD-H (0.46 cm ꢁ 25 cm; hexanes/i-
PrOH = 70/30 with flow rate = 1.0 mL/min and a UV detector at
214 nm; retain time = 8.74 min).
5.3.1. Locomotion test
Rats were placed into
a
Plexiglas open field arena
(40 ꢁ 40 ꢁ 45 cm, Jiliang Co. Ltd., Shanghai, China) with a video
camera connected to a video recorder. Automated activity was re-
corded for 30 min. The total distance and the distance traveled per
5 min were calculated by Jiliang Vision software (Jiliang Co. Ltd.,
Shanghai, China).
5.2. Biology
5.3.2. Prepulse inhibition test
PPI of the acoustic startle response was measured as described
previously47,48 in three startle chambers from Coulbourn Instru-
ments (Whitehall, PA, USA). Briefly, following a 3-min acclimation
period, animals were exposed to four different types of trial: a
PULSEALONE trial in which a 40 ms, 120 dB white noise burst was
presented; three PREPULSE (79 dB, 83 dB, 87 dB) + PULSE trials in
which 20 ms, 3 kHz sounds were presented for 100 ms, respectively,
before the onset of a 120 dB pulse. All trial types were presented 12
times in random order. In addition, 3 PULSE ALONE trials, which
were not used in data analyses, were presented at the beginning
of the test session to achieve a relatively stable level of startle reac-
tivity for the remainder of the session. The inter-trial interval was
ranged from 15 to 25 s. The total duration of the session was approx-
imately 20 min. A ventilating fan built into the chamber provided a
background noise of 74 dB throughout the test.
5.2.1. Radioligand binding assays
The affinity of compounds to D1 and D2 dopamine receptors, 5-
HT1A and 5-HT2A receptors were determined by competition bind-
ing assays. Membrane homogenates of HEK293T cells stably trans-
fected with D1, D2, 5-HT1A or 5-HT2A receptors were prepared as
described previously.14,22 Duplicated tubes were incubated at
30 °C for 50 min(for D1, D2 and 5-HT1A) or 37 °C for 15 min (for
5-HT2A) with increasing concentrations of respective compound
and with [3H]SCH23390 (for D1 dopamine receptors), [3H]Spipe-
rone (for dopamine D2 receptor), [3H]8-OH-DPAT (for 5-HT1A
receptor), or [3H] Ketanserin (for 5-HT2A receptor) in a final volume
of 200
7.4. Nonspecific binding was determined by parallel incubations
with either 10 SCH23390 for D1, Spiperone for D2,
lL binding buffer containing 50 mM Tris, 4 mM MgCl2, pH
lM
WAY100635 for 5-HT1A, or spiperone for 5-HT2A receptors respec-
tively. The reaction was started by addition of membranes (15 ng/
tube) and stopped by rapid filtration through Whatman GF/B glass
fiber filter and subsequent washing with cold buffer (50 mM Tris,
5 mM EDTA, pH 7.4) using a Brandel 24-well cell harvester. Scintil-
PPI values were calculated as percentages using the following
formulation: %PPI = [1 ꢀ (startle response for PREPULSE + PULSE
trial)/(startle response for PULSE ALONE trial)] ꢁ 100%. The acous-
tic startle magnitude was calculated as the average response of the
PULSE ALONE trials (excluding the first 3 PULSE ALONE trials).

868
H. Sun et al. / Bioorg. Med. Chem. 21 (2013) 856–868
15. Schechter, L. E.; Ring, R. H.; Beyer, C. E.; Hughes, Z. A.; Khawaja, X.; Malberg, E.;
Rosenzweig-Lipson, S. NeuroRx 2005, 2, 590.
5.3.3. 5-HT1A agonism-involved DA neuron firing
Rats were anesthetized with chloral hydrate (400 mg/kg, ip
with supplemental doses administered via a lateral tail vein) and
mounted in a stereotaxic instrument. Their body temperatures
were maintained at 37 °C by means of a heating pad throughout
the experiment. The skull was exposed, and the wound margins
were infiltrated with a 0.3% solution of mepivacaine hydrochloride.
16. Newman, T. A.; Cussac, D.; Depoortere, R. Curr. Opin. Invest. Drugs 2007, 8, 539.
17. Wolf, W. A. Curr. Opin. Invest. Drugs 2003, 4, 878. SLV-308 (Solvay).
18. Bibbiani, F.; Oh, J. D.; Chase, T. N. Neurology 2001, 57, 1829.
19. Melamed, E.; Zoldan, J.; Friedberg, G.; Ziv, I.; Weizmann, A. Adv. Neurol. 1996,
69, 545.
20. Lucas, G.; Bonhomme, N.; Deurwaerdere, P.; Moal, M. L.; Spampinato, U.
Psychopharmacology 1997, 131, 57.
21. Liu, Z. Z.; Chen, X. T.; Sun, P. H.; Yu, L. P.; Zhen, X. C.; Zhang, A. Bioorg. Med.
Chem. 2008, 16, 8335.
A glass microelectrode (5–10 MX) filled with 2 M NaCl solution
containing 2% pontamine skyblue dye was lowered through a small
burr hole drilled above the VTA (3.0 mm anterior to the lambda,
0.5–0.9 mm lateral to the midline, 6.5–8.5 mm ventral to the corti-
cal surface). DA neurons were identified and recorded as de-
scribed:55–58 positive/negative action potentials of long duration
(2–5 ms), firing rate (1–10 Hz) with slow irregular or burst firing
pattern, low pitch sound produced on audio amplifier, duration
of 1.1 ms from the start of the action potential to the negative
trough.59 Interspike intervals (ISI) and firing rates were collected
online to a personal computer.
All data analyses were performed using programs written in Vi-
sual Basic for Applications in Microsoft Excel.60 Bursting was iden-
tified according to the ‘80/160 ms’ definition proposed by Grace
and Bunney.55,56 Thus, the onset of a burst was identified as the
concurrence of two spikes with an interspike interval less than
80 ms and the termination of a burst was defined as an interspike
interval greater than 160 ms.61 Data were analyzed off-line with a
software package from BSPS Mathlib (Fudan University, Shanghai,
China). The firing rate and bursting were analyzed every 10 s.62 Ef-
fects of drugs were determined by comparing measures before and
after drug injection using paired t test. All numerical data were ex-
pressed as mean ( SEM).
22. Mcintyre, J. A.; Castaner, J.; Bayes, M. Drugs Future 2006, 31, 314.
23. Assié, M. B.; Ravailhe, V.; Faucillon, V.; Newman-Tancredi, A. J. Pharmacol. Exp.
Ther. 2005, 315, 265.
24. Jordan, S.; Koprivica, V.; Chen, R.; Tottori, K.; Kikuchi, T.; Altar, C. A. Eur. J.
Pharmacol. 2002, 441, 137.
25. Stahl, S. M.; Shayegan, D. K. J. Clin. Psychiatry 2003, 64, 6.
26. Newman, T. A.; Assie, M. B.; Leduc, N.; Ormiere, A. M.; Danty, N.; Cosi, C. Int. J.
Neuropsychopharmacol. 2005, 8, 341.
27. Cayra, A.; Czepiel, K.; Mackowiak, M.; Chocyk, A.; Wedzony, K. Brain Res. 2003,
989, 42.
28. Yuen, E. Y.; Jiang, Q.; Chen, P.; Chen, P.; Gu, Z.; Feng, J.; Yan, Z. J. Neurosci. 2005,
25, 5488.
29. Mo, J.; Zhang, H.; Yu, L. P.; Sun, P. H.; Jin, G. Z.; Zhen, X. C. Neurobiol. Aging 2010,
31, 926.
30. Stark, A. D.; Jordan, S.; Allers, K. A.; Bertekap, R. L.; Chen, R. Y.; Kannan, T. M.;
Molski, T. F.; Yocca, F. D.; Sharp, T.; Kikuchi, T.; Burris, K. D. Psychopharmacology
2007, 190, 373.
31. Mamo, D.; Graff, A.; Mizrahi, R.; Shammi, C. M.; Romeyer, F.; Kapur, S. Am. J.
Psychiatry 2007, 164, 1411.
32. Lewicka, D. M.; Nichols, D. E. Psychopharmacology 2004, 172, 415.
33. Shapiro, D. A.; Renock, S.; Arrington, E.; Chiodo, L. A.; Liu, L. X.; Sibley, D.; Roth,
B. L.; Mailman, R. Neuropsychopharmacology 2003, 28, 1400.
34. Mamo, D.; Graff, A.; Mizrahi, R.; Shammi, C. M.; Romeyer, F.; Kapur, S. Am. J.
Psychiatry 2007, 164, 1411.
35. Stark, A. D.; Jordan, S.; Allers, K. A.; Bertekap, R. L.; Chen, R.; Mistry, K. T.;
Molski, T. F.; Frank, D. Y.; Sharp, T.; Kikuchi, T.; Burris, K. D. Psychopharmacology
2007, 190, 373.
36. Jin, G. Z.; Zhu, Z. T.; Fu, Y. Trends Pharmacol. Sci. 2002, 23, 4.
37. Gao, M.; Liu, C. L.; Yang, S.; Zhen, X. C.; Jin, G. Z. Acta Pharmacol. Sin. 2007, 28,
627.
38. Mo, J.; Guo, Y.; Yang, Y. S.; Shen, J. S.; Jin, G. Z.; Zhen, X. C. Curr. Med. Chem.
2007, 14, 2996.
Acknowledgments
39. Wiegand, S.; Schafer, H. J. Tetrahedron 1995, 51, 5341.
40. Kormos, C. M.; Leadbeater, N. E. Tetrahedron 2006, 62, 4728.
41. Tlili, A.; Xia, N.; Monnier, F.; Taillefer, M. Angew. Chem., Int. Ed. 2009, 48, 8725.
42. Zhao, D. B.; Wu, N. J.; Zhang, S.; Xi, P. H.; Su, X. Y.; Lan, J. B.; You, J. S. Angew.
Chem., Int. Ed. 2009, 48, 8729.
43. Spangler, R. J.; Beckmann, B. G.; Kim, J.; Ho J. Org. Chem. 1977, 42, 2989.
44. Cushman, M.; Dekow, F. W. J. Org. Chem. 1979, 44, 407.
45. Hashiguchi, S.; Fujii, A.; Takehara, J.; Ikariya, T.; Noyori, R. J. Am. Chem. Soc.
1995, 117, 7562.
46. Franklin, A. D.; Pradyumna, K. M. J. Org. Chem. 2002, 67, 1290.
47. Mark, A. M.; Meyers, A. I. J. Org. Chem. 1996, 61, 573.
48. Gema, R. G.; Luis, C.; Domingo, D.; Carlos, S. J. Org. Chem. 1999, 64, 877.
49. Satinder, V. K.; Paramjit, S.; Rahul, V.; Nachhattar, P. K.; Venugopal, D. J. Org.
Chem. 1992, 57, 6716.
We gratefully acknowledge financial support from National Ba-
sic Research Program of China (Grants 2009CB940903,
2009CB52201, 2011CB5C4403 and 2012CB518000), the National
Natural Science Foundation of China (Grants 21021063,
30825042, 81130023 and 81025017), and National S&T Major Pro-
jects (2012ZX09103-101-072). The support from The Priority Aca-
demic Program Development (PAPD) of Jiangsu Higher Education
Institutions is also appreciated.
Supplementary data
50. Glynn, D. W.; Charles, E. W.; Martin, W. Chem. Commun. 2005, 4735.
51. Manon, C.; Riccardo, P.; Olivier, B. Angew. Chem., Int. Ed. 2009, 48, 179.
52. Chiang, H. C.; Brochmann, H. E. J. Org. Chem. 1977, 42, 3190.
53. Cheng, J. J.; Yang, Y. S. J. Org. Chem. 2009, 74, 9225.
54. Liu, Z.; Zhang, H.; Ye, N.; Zhang, J.; Wu, Q.; Sun, P.; Li, L.; Zhen, X.; Zhang, A. J.
Med. Chem. 2010, 53, 1319.
55. Bunney, B. S.; Aghajanian, G. K.; Roth, R. H. Nat. Rev. Biol. 1973, 245, 123.
56. Grace, A. A.; Bunney, B. S. Science 1980, 210, 654.
57. Grace, A. A.; Bunney, B. S. Neuroscience 1983, 10, 301.
58. Zhang, D.; Yang, S.; Yang, C. H.; Jin, G. Z.; Zhen, X. Psychopharmacology 2008,
199, 625.
59. Zhang, D.; Zhang, H.; Jin, G. Z.; Zhang, K.; Zhen, X. Mol. Pain 2008, 4, 57.
60. Ungless, M. A.; Magill, P. J.; Bolam, J. P. Science 2004, 303, 2040.
61. Grace, A. A.; Bunney, B. S. J. Neurosci. 1984, 4, 2877.
62. Shi, W. X.; Zhang, X. Y.; Pun, C. L.; Bunney, B. S. Neuropsychopharmacology 2007,
32, 1922.
63. Bakshi, V. P.; Swerdlow, N. R.; Geyer, M. A. J. Pharmacol. Exp. Ther. 1994, 271, 787.
64. Johansson, C.; Jackson, D. M.; Svensson, L. Psychopharmacology 1994, 116, 437.
65. Kumari, V.; Soni, W.; Sharma, T. Schizophr. Res. 2002, 55, 139.
66. Gessa, G. L.; Devoto, P.; Diana, M.; Flore, G.; Melis, M.; Pistis, M.
Neuropsychopharmacology 2000, 22, 642.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.12.016.
References and notes
1. Freedman, R. N. Engl. J. Med. 2003, 349, 1738.
2. Laviolette, S. R. Schizophr. Bull. 2007, 33, 971.
3. Abi-Dargham, A. Int. J. Neuropsychopharmacol. 2004, 7, S1.
4. Davis, K. L.; Kahn, R. S.; Ko, G.; Davidson, M. Am. J. Psychiatry 1991, 148, 1474.
5. Brozoski, T. J.; Brown, R. M.; Rosvold, H. E., et al Science 1979, 205, 929.
6. Takahashi, H.; Kato, M.; Takano, H.; Arakawa, R.; Okumura, M.; Otsuka, T.;
Kodaka, F.; Hayashi, M.; Okubo, Y.; Ito, H.; Suhara, T. J. Neurosci. 2008, 28,
12032.
7. Castner, S. A.; Williams, G. V.; Goldman-Rakic, P. S. Science 2000, 289, 56.
8. BiDargham, A.; Mawlawi, O.; Lombardo, I.; Gil, R.; Martinez, D.; Huang, Y., et al
J. Neurosci. 2002, 22, 3708.
9. Guo, Y.; Zhang, H.; Chen, X.; Cai, W.; Cheng, J.; Yang, Y.; Jin, G.; Zhen, X.
Schizophr. Res. 2009, 115, 41.
10. Barone, P. Eur. J. Neurol. 2010, 17, 364.
11. Wang, W.; Zhou, Y. Q.; Sun, J.; Pan, L. F.; Kang, L.; Dai, Z. Z.; Yu, R.; Jin, G. Z.; Ma,
L. Neuropharmacology 2007, 52, 355.
12. Zhang, A.; Neumeyer, J. L.; Baldessarini, R. J. Chem. Rev. 2007, 107, 274.
13. Liu, Z. Z.; Chen, X. T.; Yu, L. P.; Zhen, X. C.; Zhang, A. Bioorg. Med. Chem. 2008, 16,
6675.
67. Diaz-Mataix, L.; Scorza, M. C.; Bortolozzi, A.; Toth, M.; Celada, P.; Artigas, F. J.
Neurosci. 2005, 25, 10831.
68. Ohlsen, R. I.; Pilowsky, L. S. J. Psychopharm. 2005, 19, 408.
69. Okubo, Y.; Suhara, T.; Suzuki, K.; Kobayashi, K.; Inoue, O.; Terasaki, O.; Someya,
Y.; Sassa, T.; Sudo, Y.; Matsushima, E.; Iyo, M.; Tateno, Y.; Toru, M. Nature 1997,
385, 634.
14. Broekkamp, C. L. E.; Leysen, D.; Peeters, B. W. M. M.; Pinder, R. M. J. Med. Chem.
1995, 38, 4615.
70. Williams, G. V.; Goldman-Rakic, P. S. Nature 1995, 376, 572.