Synthesis, antidepressant activity, and toxicity of the erythro/threo racemates and optical isomers of 2-(4-benzylpiperazin-1-yl)-1-(5-chloro-6-methoxynaphthalen-2-yl)hexan-1-ol

Article abstract of DOI:10.1111/cbdd.12438

The erythro/threo racemates and their four optical isomers of 2-(4-benzylpiperazin-1-yl)-1-(5-chloro-6-methoxynaphthalen-2-yl)hexan-1-ol were synthesized and evaluated for their antidepressant activity, toxicity, and pharmacokinetics as novel triple multi

Full text of DOI:10.1111/cbdd.12438

Received Date : 28-Jun-2014
Revised Date : 30-Aug-2014
Accepted Date : 09-Sep-2014
Article type
: Research Article
Synthesis, antidepressant activity and toxicity of the erythro/threo racemates and
optical isomers of 2-(4-benzylpiperazin-1-yl)-1-(5-chloro-6- methoxynaphthalen
-2-yl)hexan-1-ol
Running title: Synthesis, antidepressant activity and toxicity
Zhijie Weng,a,b ǂYongyong Zheng,a ǂ Jianqi Lia∗
a State Key Laboratory of New Drug and Pharmaceutical Process, Shanghai Institute
of Pharmaceutical Industry, 1111 North Zhongshan No.1 Road, Shanghai 200437, PR
China
∗Corresponding author. Tel.: +86 21 55514600; fax: +86 21 65312830. E-mail address: lijianqb@
126.com (J.Q. Li).
This article has been accepted for publication and undergone full peer review but has
not been through the copyediting, typesetting, pagination and proofreading process
which may lead to differences between this version and the Version of Record. Please
cite this article as an 'Accepted Article', doi: 10.1111/cbdd.12438
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b School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan RD, Shanghai
200240, PR China
ǂ These authors contributed equally to this work.
Abstract
The erythro/threo racemates and their four optical isomers of
2-(4-benzylpiperazin-1-yl)-1-(5-chloro-6-methoxynaphthalen-2-yl)hexan-1-ol were
synthesized and evaluated for their antidepressant activity, toxicity, and pharmacokinetics as
novel triple multiple reuptake inhibitors of monoamine transmitters. The racemates and
optical isomers were synthesized respectively through two different routes. Pharmacological
data indicate that the erythro racemate (SIPI5357) which has better inhibitory activity and
lower toxicity than the other racemate and optical isomers is worthy of further evaluation.
Keywords: Erythro/threo racemate; optical isomer; antidepressant; triple reuptake inhibitor
Introduction
Depression is a major health problem estimated to affect 17% of the population during their lifetime.
Economically, depression is responsible for tremendous health care costs, with a total of 53 billion
dollars per year spent in the US alone (1,2).
Selective serotonin (5-HT) reuptake inhibitors (SSRIs) are extensively used as first-line treatment for
major depression, but these drugs are not effective for 30% of depressed patients (3,4). Continuous
research for novel antidepressants with improved efficacies and acceptable tolerability prompted
discoveries of dual-reuptake inhibitors, such as venlafaxine, which blocks both the 5-HT transporter
(SERT) and norepinephrine transporter (NET) (5-7).
Recently, a promising strategy for new antidepressant development targeting monoaminergic circuits
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involves triple reuptake inhibitors, which simultaneously inhibit the reuptake of serotonin (5-HT),
norepinephrine (NE), and dopamine (DA) by blocking their transporters. Preclinical and clinical
studies indicate that a dopaminergic component may improve the speed of onset, efficacy or the side
effect profile (8-11), when added to a single- or dual-uptake inhibitor. These triple reuptake inhibitors
include DOV-21947, SEP-225289, and NS-2359.
Previously, we described the synthesis of 1-butyl-2-hydroxyl aryl alkanol piperazine derivatives and
biological evaluation of their antidepressant activities in our patents (12). We discovered that the
erythro
(SIPI5357)
and
threo
(SIPI5358)
racemates
of
2-(4-benzyl
piperazin-1-yl)-1-(5-chloro-6-methoxy- naphthalen-2-yl) hexan-1-ol (1) have triple reuptake inhibitory
activities for 5-HT, NE and DA. Furthermore, these compounds had potent antidepressant activities in
the mouse forced swimming test (FST).
Herein we reported our synthesis of the erythro/threo racemates and their four optical isomers of
2-(4-benzylpiperazin-1-yl)-1-(5-chloro-6-methoxynaphthalen-2-yl)hexan-1-ol, and investigations of
their antidepressant activity, toxicity, and pharmacokinetics as novel triple multiple reuptake inhibitors
of monoamine transmitters. The results indicate that the erythro racemate (SIPI5357) has better
inhibitory activity and lower toxicity than the other racemate and optical isomers is worthy of further
evaluation.
Chemistry
Structurally, compound 1 has two chiral carbon centers and therefore contains four optical isomers.
We studied the bioactivity and safety of each optical isomer and compared this with the erythro/threo
racemates to identify an optimal drug candidate with high efficacy and low toxicity for further
preclinical development.
The erythro racemate (SIPI5357) and the threo racemate (SIPI5358) were synthesized through 5
steps from1-chloro-2-methoxynaphthalene according to the methods described in Scheme 1. These two
racemates (SIPI5357 and SIPI5358) were separated by column chromatography on silica gel (200–300
mesh; dichloromethane/methanol, 20:1 v/v).
Four individual optical isomers shown in Table 1 were prepared through 11 steps from their
respective chiral norleucines as chiral educts, according to the methods described in Scheme 2.
Compound 9 was prepared from norleucine according to the procedure described in the literature (13).
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Compound 12 was synthesized via Grignand reaction of 9 with 11, then reduction, hydrolysis and
chloro-substitution to obtain the key intermediate 17, which was subsequently treated with 18 to form
the piperazine ring. Compound 19 was a mixture of two epimerides, which had different Rf values by
thin-layer chromatography and was separated by column chromatography on silica gel (200–300 mesh;
dichloromethane/methanol, 20:1 v/v). Therefore, (1R, 2S)/(1S, 2S) isomers were prepared from
L-norleucine and (1S, 2R)/(1R, 2R) isomers were prepared from D-norleucine through the
above-mentioned synthetic process.
The yields and the optical rotation values of target compounds are shown in Table 1. All of the
isomers were prepared with >99%ee, as determined by chiral HPLC analysis (Daicel 100 OJ-H chiral
column, 5 μm, 250×4.6 mm; Mobile phase: N-hexane/isopropanol/diethylamine, 80:20:0.1 v/v/v;
Temperature: 30 ℃; Detection: UV, 230 nm).
Compound 12 was reduced with NaBH4 to yield 13 as a mixture of two isomers. The erythro-isomer
was obtained as major product with formation of 5% of threo-isomer when NaBH4 or AIP (aluminum
isopropoxide) was used as the reductant. Meanwhile, compound 13 was obtained as a 1:1 isomer
mixture using Pd/C catalyzed hydrogenation.
The conformation inherited from its chiral norleucine starting material was confirmed by comparing
the coupling constants from their collective NMR spectra. The definitive configuration of SIPI5357
was established by X-ray diffraction (Fig. 1). Crystallographic data (including structure factors) for the
structure in this paper have been submitted with the Cambridge Crystallographic Data Centre as
supplementary publication numbers CCDC 837280. Copies of the data can be obtained, free of charge,
on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax: +44 (0)1223-336033 or
e-mail: deposit@ccdc.cam.ac. uk].
Pharmacology
According to the method described in the literature (14,15), rat synaptosomes were prepared from
3
cerebral cortices and used with H-labeled 5-HT, NE and DA to test reuptake inhibition of SIPI5357,
SIPI5358 and their four isomers in vitro. SIPI5357 was also tested in a commercial (CEREP,
www.CEREP.com) panel of 53 human targets (G-protein coupled receptors, ion channels and
transporters).
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Two depression-related behavioral models—the mouse forced tail suspension test (TST) and the rat
forced swim test (FST) (16, 17) were used to measure the antidepressant activity of the six compounds.
Acute and chronic (two-week) toxicities of these compounds were studied in the rat. Pharmacokinetics
of the six compounds was also tested in the rat.
Results and discussion
The synthesized compounds described above were test for their inhibition of functional uptake of
5-HT, NE, and DA. The detailed results were summarized in Table 1.
Studies on the erythro and threo compounds revealed that the erythro conformation was relatively
better for 5-HT and DA reuptake inhibitions (SIPI5357 and SIPI5358, Table 1). The erythro
compound SIPI5357 showed good selectivity for 5-HT and DA reuptake inhibitions (5-HT, IC50=0.87
μM; NE, IC50=1.40 μM; DA, IC50=0.96 μM), whereas the threo isomer SIPI5358 was less potent and
equal for all three transporters (5-HT, IC50=1.59 μM; NE, IC50=1.50 μM; DA, IC50=1.30 μM).
For their four optical isomers, the D-conformation compounds D-SIPI5357, and D-SIPI5358
exhibited higher potency for all three monoamine transporters than their L-conformation compounds.
On the basis of in vitro studies, the six target compounds were tested in the mouse TST and rat FST,
which have predictive value for antidepressant-like activity. For comparative purpose, venlafaxine was
also included in the experiment. The results of single administration and chronic treatment were
displayed in Table 2 and Table 3.
As show in Table 2 (the single administration), the six target compounds have antidepressant
efficacy in the mouse TST and rat FST. Compounds SIPI5357, L-SIPI5357, SIPI5358, and
D-SIPI5358 are dose dependently reduced the immobility time in the TST, which are statistically
significant compared to the vehicle at 20 and 40 mg/kg dose. In the FST model, compounds SIPI5357,
and L-SIPI5358 are dose dependently reduced the immobility time.
All six target compounds reduced the immobility time in the TST and FST model after the chronic
treatment (10 days, Table 3). Compounds D-SIPI5357, and SIPI5358 are dose dependently reduced
the immobility time in the TST, meanwhile, SIPI5358 is dose dependently reduced the immobility time
in the FST.
To explore the safety profile, the acute and chronic (two-week) toxicity of these six compounds were
measured in SD rat. In the acute toxicity test, compound SIPI5358 and its two threo-isomers
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(L-SIPI5358 and D-SIPI5358) took more safety risks than SIPI5358 and its two erythro-isomers
(L-SIPI5357 and D-SIPI5357) at the dose of 400, 800, and 1600 mg/kg. In chronic toxicity tests, all of
the six compounds were observed to induce apparent toxic reactions and animal death except SIPI5357.
The minimum effective dosage of SIPI5357 in the chronic toxicity test exceeded 400 mg/kg in rat
(Table 4). These data suggest that compound SIPI5357 have higher safety factor than the other five
compounds.
Compound SIPI5357 was further characterized in several CNS receptor binding assays to assess the
selectivity and specific interactions with monoamine transporters. Compound SIPI5357 was performed
against 53 receptors at Cerep, and the default concentration for primary binding experiments was 10
μM. Results indicating inhibition greater than 50% were considered to represent significant effects test
compound effects. Binding tests revealed that SIPI5357 was not only a triple reuptake inhibitor but
could inhibit 100% of 5-HT1A at 10 μM, the further studies shown compound SIPI5357 exhibit high
affinity for 5-HT1A receptor (IC50 = 21 nM, Ki = 13 nM). Thus, these results suggested that compound
SIPI5357 might be regarded as multi-target agent.
To further evaluate this series of compounds, the pharmacokinetics of the six compounds were
studied in the rat. After intravenous injection 5mg/kg or intragastric administration 25mg/kg of the six
compounds to Wister rats, there were no significant differences in pharmacokinetic behavior between
the racemate and it’s two optical isomers (Table 5). The absolute bioavailability of SIPI5358 was
slightly higher than that of SIPI5357. After a single oral dose (25 mg/kg) of SIPI5357 to rats, a Cmax
= 291.1 g/L was obtained at 1.88 h. The elimination half-life of SIPI5357 following oral administration
was 6.77 h in rats. This compound also have a moderately orally bioavailable (F = 21.1%) in rats
(Table 5).
Conclusion
The erythro/threo racemates and four optical isomers of
2-(4-benzylpiperazin-1-yl)-1-(5-chloro-6-methoxynaphthalen-2-yl) hexan-1-ol were synthesized and
evaluated. Their in vitro and in vivo properties were studied and compared. Data indicate that
SIPI5357 is worthy of further investigation due to its significant antidepressant activity and lower
toxicity compared with the other five compounds.
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Experimental protocols
Nuclear magnetic resonance (NMR) spectra were recorded on a Varian INOVA-400 spectrometer
with TMS as an internal standard and DMSO-d6 as solvent. Chemical shifts (d values) and coupling
constants (J values) are given in ppm and Hz, respectively. ESI mass spectra were performed on an
Agilent 6210 MS spectrometer. TLC analysis was carried out on silica gel plates GF254 (Qindao
Haiyang Chemical, China). Silica gel column chromatography was performed with Silica gel 200~300
m (Sinopharm Chemical Reagent, China). Commercial solvents were used without purification.
Analyses indicated by the symbols of the elements or functions were within 0.4 % of the theoretical
values.
Synthesis of 1-(5-chloro-6-methoxynaphthalen-2-yl) hexan-1-one (3)
AlCl3 (4.11 g, 0.031 mol, 1.3 equiv) was added to a solution of 1-chloro-2-methoxynaphthalene
(5.45 g, 0.028 mol, 1.2 equiv) in dichloromethane 30 mL. This reactant was stirred for 1 hour at room
temperature. Subsequently, hexanoyl chloride (3.19 g, 0.024 mol, 1 equiv) was added dropwise to the
mixture, with the temperature controlled below 10℃. Then the reactant was warmed naturally to room
temperature and stirred for another 1 h. The reaction solution was poured into a mixture of
hydrochloric acid 20 mL and crashed ice 50 g under stirring. The organic phase was separated, washed
with water till the aqueous phase being neutral, dried with Na2SO4 and concentrated under reduced
pressure to give 3 as yellow oil (5.55 g, 80.5% yield). The product was used in the next step without
further purification.
Synthesis of 2-bromo-1-(5-chloro-6-methoxynaphthalen-2-yl)hexan- 1-one (4)
CuBr2 (8.93 g, 0.040 mol, 2 equiv) was added to a solution of compound 3 (5.82 g, 0.020 mol, 1
equiv) in ethyl acetate 50 mL and chloroform 50 mL. The reaction mixture was refluxed for 3 h and
then filtered. The filtrate was washed with 100 mL water , dried with Na2SO4 and concentrated under
reduced pressure. Light yellow crystalline solid 4 was obtained by recrystallized with ethanol (6.28 g,
85.0% yield). MS (ESI) m/z: 370 (M+1).
Synthesis of 2-(4-benzylpiperazin-1-yl)-1-(5-chloro-6-methoxynaphthalen-2-yl)hexan-1-one (5)
Potassium iodide (0.17 g, 0.001 mol, 0.1 equiv) and K2CO3 ( 4.84 g, 0.035 mol, 3.5 equiv) were
added to the solution of 1-Benzylpiperazine (1.76 g, 0.01 mol, 1 equiv) and compound 4 (4.44 g, 0.012
mol, 1.2 equiv) in acetone 50 mL. The reaction was refluxed for 8 h. After filtered, the solvent was
evaporated under reduced pressure. The residue was diluted with water 50 mL and extracted with ethyl
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acetate (100 mL × 3). The organic phase was separated, dried with Na2SO4 and concentrated under
reduced pressure. Then the concentrate was dissolved in 30 mL ethanol, and adjusted to pH 2 with
HCl/ethanol (5N). The precipitated solid was filtered and recrystallized in methanol to obtain
compound 5 as white solid (3.80 g, 82.0% yield). MS (ESI) m/z: 466 (M+1)
Synthesis of 2-(4-benzylpiperazin-1-yl)-1-(5-chloro-6- methoxynaphthalen-2-yl)hexan-1-ol (1)
AlCl3 (4.67 g, 0.035 mol, 1 equiv) was added to a solution of aluminium isopropoxide (7.15 g, 0.035
mol, 1 equiv) in isopropanol 80 mL. The suspension was stirred for 30 min until it turned to clear at
45-50 ℃. A solution of compound 5 (16.28 g, 0.035 mol, 1 equiv) in isopropanol 160 mL was added to
it. The reaction mixture was heated to 60-65 ℃and stirred for 10h. Then the reactant was adjusted pH
to 7 with 15% of NaOH aqueous solution (w/w) and extracted with dichloromethane (100 mL × 3). The
organic phase was separated, dried with Na2SO4 and concentrated under reduced pressure to give 1 as
yellow oil (14.25 g, 87.2% yield).
Synthesis of SIPI5357 and SIPI5358
Compound 1 (14.25 g, 0.03 mol) was separated to the erythro and threo form by silica gel column
chromatography (dichloromethane/methanol, 20:1, v/v). The erythro form was dissolved in 30 mL
ethanol, and adjusted to pH 2 with HCl/ethanol (5 N). The precipitated solid was filtered and
recrystallized in methanol to obtain SIPI5357 as white solid (6.92g, 42.0% yield). Mp: 225.0-225.8 ℃.
1H NMR δ (ppm): 0.72-1.60 (m, 9H, CH2CH2CH2CH3), 2.27-2.70 (m, 9H, CH & piperazine-H), 3.40 (s,
2H, CH2Ph), 3.98 (s, 3H, OCH3), 5.000-5.011 (d, 1H, CH, J = 4.4 Hz), 5.10 (s, 1H, OH), 7.20-8.02 (m, 10H,
Ar-H); MS (ESI) m/z: 468 (M+1); Anal. Calcd for C28H35ClN2O2.2HCl: C, 62.28; H, 6.91; N, 5.19. Found: C, 62.20;
H, 6.78; N, 5.30.
SIPI5358 was prepared from the threo form through the above synthetic process as white solid
(6.87g, 41.7% yield). Mp: 223.1-224.3 ℃. 1H NMR δ (ppm): 0.66-1.50 (m, 9H, CH2CH2CH2CH3),
2.42-2.84 (m, 9H, CH & piperazine-H), 3.49 (s, 2H, CH2Ph), 4.02 (s, 3H, OCH3), 4.501-4.523 (d, 1H, CH, J
= 8.8 Hz), 5.11(s, 1H, OH), 7.26-8.08 (m, 10H, Ar-H); MS (ESI) m/z: 468 (M+1); Anal. Calcd for
C28H35ClN2O2.2HCl: C, 62.28; H, 6.91; N, 5.19. Found: C, 62.41; H, 6.94; N, 5.09.
Synthesis of methyl 1-(6-methoxynaphthalen-2-yl)- 1-oxohexan-2-ylcarbamate (12)
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Compound 9 and 11 was prepared according to the procedure described in the literature (13).
Compound 9 (23.2 g, 0.1 mol, 1 equiv) prepared from L-norleucine was dissolved in anhydrous
tetrahydrofuran 400 mL and the solution was stirred under N2 atmosphere at -20 ℃. Then a solution of
Compound 11 (52.28 g, 0.2 mol, 2 equiv) in tetrahydrofuran 350 mL was added dropwise to it. After
stirring at that temperature for 2 hours, the mixture was elevated gradually to room temperature. The
reactant was stirred for 10 h and concentrated under reduced pressure. Dichloromethane 500 mL and
1M phosphoric acid aqueous solution 200 mL was added to the residue and stirred for 30 min. The
aqueous phase was separated and extracted with dichloromethane (200 mL × 1). The combined extract
was dried with Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel
column chromatography (petroleum /ethyl acetate, 20:1, v/v) to obtain 12 as white solid (28.8 g, 87.4%
yield). MS (ESI) m/z: 330 (M+1).
Synthesis of methyl 1-hydroxy-1-(6-methoxynaphthalen- 2-yl)hexan-2-ylcarbamate (13)
10% Pd/C 0.7 g (10% w/w) was added to the solution of compound 12 (7 g, 0.021 mol, 1 equiv) in
methanol 70 mL. The mixture was stirred under H2 (2.0~2.5 MPa) atmosphere at 45 ℃ for 20 h. Pd/C
was filtered off and the filtrate was concentrated under reduced pressure to give 13 as white solid (7 g,
99.4% yield). MS (ESI) m/z: 332 (M+1).
Synthesis of 2-amino-1-(6-methoxynaphthalen-2-yl)hexan-1-ol (14)
30% of KOH aqueous solution 60 mL (w/w) was added to the solution of compound 13 (6 g, 0.018
mol, 1 equiv) in methanol 300 mL. The mixture was refluxed for 8 h and concentrated under reduced
pressure. The residue was diluted with water 100 mL and extracted with dichloromethane (50 mL × 3).
The extract was dried with Na2SO4 and concentrated under reduced pressure to obtain 14 as yellow oil
(5.3 g, 97.7% yield). The product was used in the next step without further purification.
Synthesis of 4-butyl-5-(6-methoxynaphthalen-2-yl)oxazolidin-2-one (15)
CDI (6.3 g, 0.039 mol, 2 equiv) and triethylamine（5.9 ml, 0.058 mol, 3 equiv） was added to the
solution of compound 14 (5.33 g, 0.0195 mol, 1 equiv) in dichloromethane 100 mL. The mixture was
stirred for 24 h at room temperature. Then water (25 mL) was added and the mixture was stirred for 30
min. The organic phase was separated and washed with water again, dried with Na2SO4 and
concentrated under reduced pressure to obtain 15 as yellow oil (5.75 g, 98.5% yield). The product was
used in the next step without further purification.
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Synthesis of 4-butyl-5-(5-chloro-6-methoxynaphthalen-2-yl) oxazolidin-2-one (16)
NaCl (2.3 g, 0.04 mol, 1.2 equiv) and 40% H2SO4 aqueous solution (10 ml, w/w) was added to the
solution of compound 15 (10 g, 0.033 mol, 1.0 equiv) in dichloromethane (100 ml). 11% NaClO
aqueous solution (50 ml, w/w, 0.074 mol, 2.2 equiv) was added dropwise to the mixture. After the
addition, the reactant was stirred for 15 min at room temperature. Then saturated NaCl aqueous
solution（100 mL）was added and the solution was extracted with dichloromethane (50 mL × 3). The
extract was dried with Na2SO4 and concentrated under reduced pressure to give yellow oil. The oil
was dissolved in the solution of hexane (50 mL) and ethyl acetate (25 mL) when heated to 60 ℃. The
residue was cooled to room temperature. The precipitation was filtrated to give 16 as yellow solid (6 g
, 54.5% yield). MS (ESI) m/z: 334 (M+1).
Synthesis of 2-amino-1-(5-chloro-6-methoxynaphthalen -2-yl)hexan-1-ol（17）
Compound 16 (5 g, 0.015 mol, 1 equiv) was added to the solution of NaOH aqueous solution (1 N,
27 mL) and methanol 50 mL. The mixture was refluxed for 20 h and concentrated under reduced
pressure to remove methanol. The precipitation from the solution was filtrated to give 17 as yellow
solid (4.2g, 91.3% yield). MS (ESI) m/z: 308 (M+1).
Synthesis of 2-(4-benzylpiperazin-1-yl)-1- (5-chloro-6-methoxynaphthalen-2-yl)hexan-1-ol (19)
Compound 18 was prepared according to the procedure described in the literature (18). Compound
17 (3.5 g, 0.011 mol, 1 equiv) and Compound 18 (5.3 g, 0.022 mol, 2 equiv）was added to the solution
of triethylamine (5.7 g, 0.055 mol, 5 equiv) and CH3CN (50 mL). The reactant was refluxed for 20 h and
concentrated under reduced pressure. The residue was diluted with water (100 mL) and extracted
with dichloromethane (50 mL × 3). The extract was dried with Na2SO4 and concentrated under
reduced pressure to give 19 as yellow oil (5g, 95.6% yield).
Synthesis of L-SIPI5357 and L-SIPI5358
Compound 19 was separated to the erythro and threo form by silica gel column chromatography
(dichloromethane/methanol, 20:1 v/v). The erythro form was dissolved in 30 mL ethanol, and
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adjusted to pH 2 with HCl/ ethanol (5N). The precipitated solid was filtered and recrystallized in
methanol to obtain L-SIPI5357 as white solid (2.34g, 40.5% yield). Mp: 225.1-225.8 ℃. 1H NMR δ
(ppm): 0.36-1.86 (m, 9H, CH2CH2CH2CH3), 3.54-3.80 (m, 9H, CH, piperazine-H), 3.96 (s, 2H, CH2Ph),
4.42 (s, 3H, OCH3), 5.519-5.530 (d, 1H, CH, J = 4.4 Hz), 5.77 (s, 1H, OH), 7.43-8.14 (m, 10H, Ar-H); EI-MS
m/z: 468 (M+1); Anal. Calcd for C28H35ClN2O2.2HCl: C, 62.28; H, 6.91; N, 5.19. Found: C, 62.30; H, 6.94; N, 5.17.
L-SIPI5358 was prepared from the threo form through the above synthetic process (2.2g, 38.2%
yield). Mp: 225.0-226.1 ℃. 1H NMR δ (ppm): 0.33-1.84 (m, 9H, CH2CH2CH2CH3), 3.57-3.82 (m, 9H, CH,
piperazine-H), 4.00 (s, 2H, CH2Ph), 4.42 (s, 3H, OCH3), 5.520-5.542 (d, 1H, CH, J = 8.8 Hz), 5.82 (s, 1H,
OH), 7.44-8.18 (m, 10H, Ar-H); MS(ESI) m/z: 468 (M+1); Anal. Calcd for C28H35ClN2O2.2HCl: C, 62.28; H,
6.91; N, 5.19. Found: C, 62.50; H, 7.01; N, 5.02.
Synthesis of D-SIPI5357 and D-SIPI5358:
D-SIPI5357 was prepared from D-norleucine through the above synthetic process (2.05 g, 35.5%
yield). Mp: 223.1-224.3 ℃. 1H NMR δ (ppm): 0.33-1.58 (m, 9H, CH2CH2CH2CH3), 3.62-3.79 (m, 9H, CH,
piperazine-H), 3.95 (s, 2H, CH2Ph), 4.42 (s, 3H, OCH3), 4.913-4.924 (d, 1H, CH, J = 4.4 Hz), 5.07 (s, 1H,
OH), 7.43-8.17 (m, 10H, Ar-H); MS(ESI) m/z: 468 (M+1); Anal. Calcd for C28H35ClN2O2.2HCl: C, 62.28; H,
6.91; N, 5.19. Found: C, 62.34; H, 6.88; N, 5.04.
D-SIPI5358: White solid (1.92 g, 33.2% yield). Mp: 223.0-224.2 ℃. 1H NMR δ (ppm): 0.37-1.58 (m,
9H, CH2CH2CH2CH3), 3.60-3.74 (m, 9H, CH, piperazine-H), 3.97 (s, 2H, CH2Ph), 4.39 (s, 3H, OCH3),
4.910-4.932 (d, 1H, CH, J = 8.8 Hz), 5.22 (s, 1H, OH), 7.43-8.19 (m, 10H, Ar-H); MS(ESI) m/z: 468 (M+1);
Anal. Calcd for C28H35ClN2O2.2HCl: C, 62.28; H, 6.91; N, 5.19. Found: C, 62.37; H, 6.88; N, 5.22.
Acknowledgement
This work is supported by National Natural Science Foundation of China (Grant No. 81302631),
Shanghai Rising-Star Program (Grant No. 13QB1403800), Key Technologies R&D Program of
Shanghai Municipal Science and Technology Commission (Grant No. 14431901500), and the National
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Science and Technology Major Project (Grant No. 2012ZX09102101-008; 2012ZX09103101-038). We
would like to gratefully acknowledge Dr. Linying Feng (Shanghai Institute of Materia Media, China)
for in vivo studies, Mr. Jialiang Zhong (Shanghai Institute of Pharmaceutical Industry, China) for X-ray
crystallography studies.
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Figure, Table and Scheme Legends
Figure 1 SIPI5357 is mixture of (1S,2R) and (1R,2S) isomer.
Table 1 In vitro reuptake inhibition of target compounds for 5-HT, NE, DA receptors.
Table 2 In vivo antidepressant activity of target compounds after a single administration (n=12, PO).
Table 3 In vivo antidepressant activity of target compounds after chronic treatment (10 days, n=12,
PO).
Table 4 Acute toxicity (n=10, female: male =1:1, PO) and chronic toxicity (two weeks, n=6, female:
male =1:1, PO) of target compounds.
Table 5 Pharmacokinetic characterizations of target compounds（n=4）
Scheme 1. Reagents and conditions: (a) Hexanoyl chloride, AlCl3, CH2Cl2, 0 ºC; (b) CuBr2, ethyl
acetate, CHCl3, reflux; (c) 1-Benzylpiperazine, K2CO3, KI, acetone, reflux; (d) Aluminum
isopropoxide, isopropyl alcohol, AlCl3, 45–50 ºC; (e) silica gel (200–300 mesh) column
chromatography, dichloromethane: methanol, 20:1 v/v.
Scheme 2. Reagents and conditions: (f) methyl chloroformate, 1N NaOH, rt; (g) SOCl2, CH2Cl2, -10ºC;
(h) N,O-dimethyl hydroxylamine hydrochloride, CH2Cl2, rt; (i)Mg, THF, 70ºC; (j) THF, -78 ºC; (k) H2,
10%Pd/C, 2-2.5atm, CH3OH, 45ºC; (i) 30% KOH/H2O, CH3OH, reflux; (m) CDI, Et3N, CH2Cl2, rt;(n)
NaClO/NaCl/40% H2SO4, CH2Cl2, rt; (o) 1N NaOH, MeOH, reflux; (p) TEA, CH3CN, reflux; (q) silica
gel (200–300 mesh) column chromatography, dichloromethane: methanol, 20:1 v/v.
This article is protected by copyright. All rights reserved.

Figure 1. SIPI5357 is mixture of (1S,2R) and (1R,2S) isomer
Table 1 In vitro reuptake inhibition of target compounds for 5-HT, NE, DA receptors
OH
H
1
N
N
2
H
1
O
Cl
IC50(μM)
5-HT NE
Yield
(%)
[α]D25(C 1, CH OH）
Compd.
Formula
Config.
3
DA
(1S,2R)/
(1R,2S)
SIPI5357
C28H35ClN2O2.2HCl
C28H35ClN2O2.2HCl
42.0%
0
0.87 1.40
0.96
L-SIPI5357
(1R,2S) 37.3%
(1S,2R) 35.5%
-2.2
2.9
1.89 2.10
0.83 1.40
1.01
0.67
D-SIPI5357 C28H35ClN2O2.2HCl
(1S,2S)/
41.7%
SIPI5358
C28H35ClN2O2.2HCl
C28H35ClN2O2.2HCl
0
1.59 1.50
1.30
(1R,2R)
L-SIPI5358
(1S,2S) 38.2%
32.9
3.10 1.40
1.19 1.21
1.11
1.10
D-SIPI5358 C28H35ClN2O2.2HCl (1R,2R) 33.2%
-32.7
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Table 2 In vivo antidepressant activity of target compounds after a single administration (n=12, PO)
Immobility (s) in mouse TST
Immobility (s) in rat FST
10mg/kg
Compd.
10mg/kg
20mg/kg
155.8±42.8
72.8±58.2a
40mg/kg
5mg/kg
20mg/kg
Blank
139.2±8.0
Venlafaxine
SIPI5357
NT
NT
NT
123.4±8.3
NT
127.8±27.9a 90.0±49.9a 63.2±38.7a
124.9±10.1
123.2±12.3
112.3±12.7
111.5±12.2
107.7±11.2a
122.4±12.3
106.0±14.1a
97.6±10.4b
98.6±11.0b
100.6±13.4b
96.6±13.2b
L-SIPI5357
D-SIPI5357
SIPI5358
93.0±46.1a 75.5±58.3a 71.0±34.2a 105.5±12.6a
66.8±47.4a 62.3±57.5a 73.8±37.5a 110.2±11.4a
125.9±47.0 82.0±35.7a 55.7±33.2a 106.3±11.5a
L-SIPI5358
105.9±53.8 113.7±62.1 75.9±50.2
101.42±
124.5±10.3
D-SIPI5358
78.83±63.1 66.42±57.3 117.4±12.5
93.2±8.1c
104.5±11.5a
64.0
a: p<0.05; b: p<0.01; c: p<0.001; NT: no test
Table 3 In vivo antidepressant activity of target compounds after chronic treatment (10 days, n=12,
PO)
Compd.
Immobility (s) in mouse TST
Immobility (s) in rat FST
10mg/kg
10mg/kg
20mg/kg
124.7±65.9
82.1±48.7a
94.6±61.4
65.8±56.4a
87.1±60.8
63.3±36.9a
84.7±56.7a
107.0±67.8
40mg/kg
NT
5mg/kg
NT
20mg/kg
Blank
154.2±11.4
Venlafaxine
SIPI5357
NT
124.5±7.3a
NT
80.2±51.9a
57.2±47.7a
102.7±53.9
71.3±57.1a
65.3±65.7a
67.8±59.2a
81.2±52.5a 107.2±10.4b
61.5±37.5a 113.6±11.4a
126.5±9.1a
148.5±7.9
121.3±12.2a
127.3±9.4
L-SIPI5357
D-SIPI5357
SIPI5358
85.6±75.2
48.3±55.9a
81.2±44.2a
64.8±51.1a
143.5±14.6
130.5±10.2
125.4±13.5
139.3±7.6
112.5±10.2b 140.4±12.5
119.4±10.5a 117.5±11.5a
L-SIPI5358
D-SIPI5358
131.6±9.1
131.9±11.2
137.2±9.4
131.3±11.4
a: p<0.05; b: p<0.01; NT: no test
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Table 4 Acute toxicity (n=10, female: male =1:1, PO) and chronic toxicity (two weeks, n=6, female:
male =1:1, PO) of target compounds
Acute toxicity/ mortality (%)
Chronic toxicity/ mortality (%)
Compd.
0mg/kg 400mg/kg 800mg/kg 1600mg/kg 0mg/kg 100mg/kg 200mg/kg 400mg/kg
SIPI5357
L-SIPI5357
D-SIPI5357
SIPI5358
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
NT
NT
100
0
0
0
0
0
0
0
0
0
0
0
0
0a
0
0
0a
0a
60
0
0a
100a
0a
100a
50a
100a
L-SIPI5358
D-SIPI5358
0a
30
80
16.7a
83.3a
a:toxic reactions were observed, including: ALT, AST, ALP ↑;TP, ALB ↓;weight loss; drooling;
adrenal glands tumefaction, et al. ; NT: no test
Table 5 Pharmacokinetic characterizations of target compounds（n=4）
Compd.
Dose
AUC
F
AUC
(0-t)
MRT
(0-t)
Cmax
t1/2
Tmax CLz
Vz
(mg/kg)
(%)
(0-∞)
ug/L
ug/L*h
ug/L*h
h
h
h
L/h/kg L/kg
SIPI5357(iv)
5
910.5 2530.0
291.1 2665.1
853.3 3194.0
408.3 4328.3
970.5 2822.3
399.5 2998.0
887.1 1547.2
2724.9 5.55
2909.0 7.37
3853.1 7.47
5285.1 8.23
2934.1 7.53
3172.4 6.41
1574.1 3.93
6.47
6.77
9.29
9.96
5.71
5.46
4.08
0.13
1.88
NT
7.46
6.89
5.92
4.17
5.83
6.31
12.9
69.5 21.1
67.5
SIPI5357(po)
L-SIPI5357(iv)
L-SIPI5357(po)
D-SIPI5357(iv)
25
5
70.5 33.1
60.1
25
5
2.75
NT
84.9 23.1
49.5
D-SIPI5357(po) 25
SIPI5358(iv)
2.00
NT
5
80.8 44.8
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SIPI5358(po)
L-SIPI5358(iv)
L-SIPI5358(po)
D-SIPI5358(iv)
25
5
379.3 3467.1
1137.2 2220.1
847.3 5577.0
738.5 1638.1
318.0 2674.0
3612.0 6.53
2253.0 4.32
5632.9 5.90
1823.0 5.51
3264.1 7.95
5.33
3.51
3.44
8.44
9.14
2.13
NT
5.68
9.09
3.91
11.0
7.94
43.9
44.9 50.2
20.3
25
5
1.75
NT
134.1 32.7
99.7
D-SIPI5358(po) 25
1.00
F: absolute bioavailability; NT: no test
O
O
a
b
c
Br
H3CO
H3CO
H3CO
N
H3CO
3
4
Cl
Cl
Cl
2
Cl
OH
SIPI5357
SIPI5358
O
N
N
N
d
C4H9
H3CO
C4H9
5
e
Cl
1
Scheme 1. Reagents and conditions: (a) Hexanoyl chloride, AlCl3, CH2Cl2, 0 ºC; (b) CuBr2, ethyl
acetate, CHCl3, reflux; (c) 1-Benzylpiperazine, K2CO3, KI, acetone, reflux; (d) Aluminum
isopropoxide, isopropyl alcohol, AlCl3, 45–50 ºC; (e) silica gel (200–300 mesh) column
chromatography, dichloromethane: methanol, 20:1 v/v.
O
O
O
O
g
f
h
C4H9
C4H9
*
C4H9
C4H9
*
*
*
HO
HO
Cl
N
O
O
NH2
NHCOOCH3
NHCOOCH3
NHCOOCH3
NHCOOCH3
j
*
8
9
6
7
C4H9
O
Br
MgBr
i
12
O
O
O
10
11
O
O
OH
OH
O
o
NH
NHCOOCH3
n
*
NH2
NH
C4H9
m
N
l
*
k
C4H9
C4H9
O
C4H9
O
O
O
16
N
Cl
15
14
13
N
Cl
Cl
OH
OH
OH
N
NH2
q
N
*
*
*
*
18
C4H9
C4H9
C4H9
O
O
O
p
17
Cl
Cl
Cl
19
TM
Scheme 2. Reagents and conditions: (f) methyl chloroformate, 1N NaOH, rt; (g) SOCl2, CH2Cl2, -10ºC;
(h) N,O-dimethyl hydroxylamine hydrochloride, CH2Cl2, rt; (i)Mg, THF, 70ºC; (j) THF, -78 ºC; (k) H2,
10%Pd/C, 2-2.5atm, CH3OH, 45ºC; (i) 30% KOH/H2O, CH3OH, reflux; (m) CDI, Et3N, CH2Cl2, rt;(n)
NaClO/NaCl/40% H2SO4, CH2Cl2, rt; (o) 1N NaOH, MeOH, reflux; (p) TEA, CH3CN, reflux; (q) silica gel
(200–300 mesh) column chromatography, dichloromethane: methanol, 20:1 v/v.
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