Discovery of 4-benzoylpiperidine and 3-(piperidin-4-yl)benzo[d]isoxazole derivatives as potential and selective GlyT1 inhibitors

Article abstract of DOI:10.1039/c5ra04714e

Regulation of glycine transporter 1 (GlyT1) activity is a currently investigated strategy in drug discovery for schizophrenia. This study developed a series of new 4-benzoylpiperidine derivatives as GlyT1 inhibitors by bioisosteric replacement and mimicking of the pyridine ring of RG1678. Among the 4-benzoylpiperidine derivatives, 23q showed an IC₅₀ of 30 nM. Preliminary optimization of the blood-brain barrier penetration led to the discovery of 3-(piperidin-4-yl)benzo[d]isoxazole derivatives. Both series showed good selectivity over GlyT2, D₁, D₂, D₃, 5-HT_1A and 5-HT_2A receptors. Moreover, behavioral testing showed 23q (40 mg kg^-1, intragastric) can inhibit the hyperlocomotion induced by acute treatment of phencyclidine, and improve the impaired negative and cognitive symptoms in chronic phencyclidine-induced C57BL/6J mice. An interesting finding showed that 3-(piperidin-4-yl)benzo[d]isoxazole was a privileged scaffold of atypical antipsychotic agents but exhibited high selectivity and potency as a GlyT1 inhibitor.

Full text of DOI:10.1039/c5ra04714e

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ARTICLE TYPE
Discovery of 4-benzoylpiperidine and 3-(piperidin-4-
yl)benzo[d]isoxazole derivatives as potential and selective GlyT1
inhibitors
Yang Liu,a Lin Guo,b Hongliang Duan,a Liming Zhang,a Neng Jiang,c Xuechu Zhen,*b and Jianhua
Shen*a
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
Regulation of glycine transporter 1 (GlyT1) activity is a currently investigated strategy in the drug
discovery for schizophrenia. This study developed a series of new 4-benzoylpiperidine derivatives as
10 GlyT1 inhibitors by bioisosteric replacement and mimic of the pyridine ring of RG1678. Among the 4-
benzoylpiperidine derivatives, 23q showed an IC50 of 30 nM. Preliminary optimization of the blood-brain
barrier penetration led to the discovery of 3-(piperidin-4-yl)benzo[d]isoxazole derivatives. Both series
showed good selectivity over GlyT2, D1, D2, D3, 5-HT1A and 5-HT2A receptors. Moreover, behavioral
testing showed 23q (40 mg/kg, intragastric) can inhibit the hyperlocomotion induced by acute treatment
15 of phencyclidine, and improve the impaired negative and cognitive symptoms in chronic phencyclidine-
induced C57BL/6J mice. Interesting finding showed that 3-(piperidin-4-yl)benzo[d]isoxazole was a
privileged scaffold of atypical antipsychotic agents but exhibited high selectivity and potency as GlyT1
inhibitors.
identified.7 One is the sarcosine-derived GlyT1 inhibitor, such as
18 and 2.9 However, some compounds of this series were
discontinued in clinical trials because of undesired adverse effects.
Another series is the non-sarcosine-based GlyT1 inhibitor, such
50 as 3,10 4,11 and 5.12 Among them, RG1678 demonstrated a
beneficial effect in patients with schizophrenia characterized with
predominant negative symptoms in a phase II proof-of-concept
study13 and it is currently in phase III clinical trial.
In our effort to find new GlyT1 inhibitors, we considered
55 RG1678 as a potential starting point for further structural
modification. A patent from Roche revealed some bioisosteric
replacements of piperazine ring, among them pyrrolidine series
was the most potent and well investigated (compound 6 with an
IC50 = 90 nM) while piperidine ring did not exhibit good
60 potency14. In this case, we considered that piperidine ring was an
appropriate bioisostere of piperazine and when the piperazine
ring was replaced with piperidine, some heteroatoms should be
incorporated to compensate for the loss of binding energy like
hydrogen bond. Based on this consideration, we first introduced a
65 carbonyl to 4-benzylpiperidine of compound 7 and a series of 4-
benzoylpiperidine derivatives were synthesized, and tested.
Introduction
20 Schizophrenia is a severe and chronic psychiatric illness,
characterized by positive, negative and cognitive symptoms.
Currently available drugs are effective in relief of the positive
symptoms, but elicit weak or no improvement on the negative
symptoms and cognitive impairments.1 It is generally believed
25 that the N-methyl-D-aspartate (NMDA) receptor hypofunction
plays a critical role in the pathophysiology of schizophrenia and
may associate with the development of negative symptoms and
cognitive deficits in the illness.2 Activating the NMDA receptor
and restoring function of glutamatergic neurons may be an
30 alternative therapeutic strategy for schizophrenia treatment.3
Because the NMDA receptor is widely distributed in the brain
and direct activation of the NMDA receptor would cause seizures
and serious neurotoxic side effects,4 efforts have turned to the
auxiliary glycine binding site. Glycine is an obligatory co-agonist
35 of the NMDA receptor, and elevated glycine levels in the
synaptic cleft can promote activation of the NMDA receptor.5
Glycine in the synaptic cleft can be uptaken into neurons or
surrounding glia by the glycine transporter (GlyT). There are two
types of glycine transporters in the brain: GlyT1, which is
40 localized to the NMDA receptor area, and GlyT2, which is
distributed around the glycinergic neurons.6 Therefore, selective
inhibition of GlyT1 could elevate glycine concentrations in the
glutamatergic synapse and enhance NMDA receptor activity,
thereby may be of potential therapeutic effects in schizophrenia.
Results and discussion
Chemistry
All described target compounds were prepared by condensation
70 of 2,5-disubstituted benzoic acids and corresponding piperidine
moieties. The 2,5-disubstituted benzoic acids were performed as
shown in Scheme 1 and 2. 8a–c were commercially available or
45
So far, at least two series of GlyT1 inhibitors have been
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DOI: 10.1039/C5RA04714E
Fig. 1. Structures of GlyT1 inhibitors.
Fig. 2. Design of new GlyT1 inhibitors.
5
synthesized as listed in references.10 The R2 group preferred an
electron-withdrawing groups (EWGs). So it was selected from
nitro, methylsulfonyl, and N-methylsulfamoyl. First, 2-alkoxy-5-
substituted benzoates 9a–h were prepared by substitution
coupled with 4-fluorophenylboronic acid or substituted by some
alkyl amines to yield 12a–b. Further hydrolysis was performed to
get 13a–b. Alternatively, considering the electron-withdrawing
groups of the phenyl ring, the alkoxy and alkyl amine group at
10 reactions with alkyl halides or Mitsunobu reactions with alkyl 20 position 2 was also introduced by 2-fluorobenzoic acid analogues
alcohols in good yields. Then the substituted esters were
hydrolyzed by sodium hydroxide to afford the corresponding
acids 10a–h. By reacting with triflic anhydride, the phenolic
following the synthetic route described in Scheme 2.
The synthesis of piperidine moieties is outlined in Scheme 3.
Substituted bromobenzene was converted to a Grignard reagent
and then reacted with Weinreb amide, resulting in 20a–b.
hydroxyl
group
of
8a–b
was
converted
into
15 trifluoromethylsulfonyloxy group in 72–79% yields and then 25 Following reflux in HCl (6N), 21a–b were prepared. 21c was
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Scheme 1. Reagents and conditions: (a) alkyl bromide, K2CO3, DMF, 40–60 °C, overnight; (b) alkyl alcohol, DIAD, PPh3, THF, rt, 24h; (c) NaOH,
MeOH/THF/H2O (1:1:1), 40 °C, 2 h; (d) triflic anhydride, Et3N, CH2Cl2, 0 °C-rt, 20 min; (e) alkylamine, NMP, microwave, 210 °C, 5 min; (f) NaOH,
MeOH/THF/H2O (1:1:1), 60 °C, 2 h.
5
Scheme 2. Reagents and conditions: (a) alkylamine, THF, 100 °C, microwave, 40 min; (b) NaOH (2N), THF, 60 °C, 2 h; (c) alkyl alcohol, t-BuOK, dry
THF, rt-60 °C, 2 h; (d) propane-2-thiol, Cs2CO3, DMA, 100 °C, 3 h.
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Scheme 3. Reagents and conditions: (a) Mg, I2, dry THF, 1-acetyl-N-methoxy-N-methylpiperidine-4-carboxamide, reflux to rt, overnight; (b) HCl (6N),
reflux, 4h; (c) HATU, Et3N, CH2Cl2, rt, overnight.
synthesized according to a previous study15 and 21d was
commercially available. Finally, the target compounds were
prepared in a single step by HATU-mediated condensation yields
ranging from 57% to 85%.
hydrogen bond donors, increasing rigidity and lowering polar
surface area (PSA).16 Herein we used two parameters, CLogP and
tPSA (topological PSA) calculated by ChembioDraw, to
represent the lipophilicity and PSA.
5
60
As shown in Table 4，23q had a similar tPSA value with
RG1678 while a slightly lower CLogP, which indicating that
higher lipophilicity may be helpful to improve the B/P ratio. At
the same time, we made a confirmation restriction of 4-
benzoylpiperidine moiety, leading to the 3-(piperidin-4-
In vitro biological evaluation and discussion
All final compounds were initially evaluated for their ability to
10 inhibit GlyT1, and the results are summarized in Table 1 and
Table 2. Considering the convenience of synthesis, we introduced
a nitro group to R2 position first and then explored the R3 and R4
group. The structure-activity relationship study (SAR) of RG1678
revealed that the aromatic ring of the left side preferred
15 hydrophobic substituents, so we incorporated trifluoromethyl and
fluoro to R3 and R4 positions. As shown in Table 1，for the R4
group, fluoro was more potent than hydrogen (23a vs. 23b, 23c
vs. 23d, and 23e vs. 23f), while on the R3 position,
trifluoromethyl was less suitable than fluoro (23a vs. 23c and 23b
20 vs. 23d). For the R1 group, introduction of some alkylaminos,
such as 23g and 23h, led to great loss in GlyT1 activity. Though
23e showed a slight increase of activity against 7, the others did
not meet our satisfaction. This result prompted us to optimize the
substituents of the benzoic moiety.
65 yl)benzo[d]isoxazole derivatives. By this transformation, we kept
the heteroatom in proper position, increased the rigidity and
lipophilicity of molecule, but caused a slight adverse effect to
tPSA. When 23q was transformed to 24a, a great loss in activity
was also observed (IC50 = 185 nM). A scale of optimization was
70 performed and the selected compounds summarized in Table 4
demonstrated that 3-(piperidin-4-yl)benzo[d]isoxazole derivatives
had moderate to acceptable GlyT1 inhibiting activities (24g with
an IC50 = 38 nM). But the initial optimization failed to reach a
balance among the activity, CLogP and tPSA. As we expected,
75 with a higher tPSA value, 24g did not have better performance
than 23q in vivo pharmacokinetic study (data not shown).
25
As shown in Table 2, nitro was first replaced with another
electron withdrawing N-methylsulfamoyl group while R3 and R4
were kept as the most potent fluoro. To our delight, the N-
methylsulfamoyl series exhibited well tolerance on the R1 group,
and all alkoxy and alkylamino groups on R1 position showed
30 moderate activities. However, none of the compounds had a
better performance with an IC50 value below 50 nM. Finally, we
tested methylsulfonyl on R2 position of benzoic moiety. The
result was consistent with nitro series and alkoxy groups were
more potent than alkylamino on R1 position. Surprisingly, 23q
35 gave the most satisfactory result (IC50 = 30 nM). The various
substituents at R1 position of benzoic that were tolerated
indicated that a lipophilic bulk pocket might exist at this position
for the GlyT1 receptor.
In vivo pharmacokinetic study
40 With the identification of 23q as the most potent GlyT1 inhibitor
in vitro, the pharmacokinetic parameters of 23q were evaluated in
ICR mice. After oral administration of 23q (10 mg/kg), blood
samples and brain tissues were collected and analyzed at different
time points. As shown in Table 3, 23q had a short half-life (T1/2
=
45 0.7 h), moderate plasma exposure (AUC0-∞ = 737 h*ng/mL) with
a low brain exposure (Cmax in brain = 26.3 ng/g), indicating a
poor B/P ratio. However, the concentration in the brain (Cmax in
brain = 26.3 ng/g) was just above the IC50 value. This result
promoted us to make structure modifications to improve the brain
50 exposure.
Initial attempts to improve brain exposure
The low B/P ratio of 23q indicated that it may be difficult to
penetrate the Blood-Brain Barrier (BBB). Strategies to improve
BBB permeability have been reviewed and the structure
55 optimization methods include increasing lipophilicity, reducing
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Table 1 In vitro biological evaluation of 23a-23h.
5 Table 2 In vitro biological evaluation of 23i-23t
GlyT1
GlyT1
R1
R2
R3
R4
Compd
R1
R2
IC50 (nM) a
Compd
IC50 (nM) a
23i
N-methylsulfamoyl
172±36
23a
23b
23c
23d
nitro
nitro
nitro
nitro
F
F
H
F
471±54
23j
N-methylsulfamoyl
N-methylsulfamoyl
143±7.25
117±5.37
F
2790±40
1855±45
23k
CF3
CF3
23l
N-methylsulfamoyl
N-methylsulfamoyl
184±14
H
77.87%@10μM
23m
80±10.47
23e
23f
nitro
nitro
F
F
F
310±94
23n
23o
N-methylsulfamoyl
methylsulfonyl
115±32.9
H
461±165
58.02%@10μM
23g
23h
nitro
nitro
F
F
F
F
78.53%@10μM
82.45%@10μM
23p
methylsulfonyl
501±84.9
23q
23r
methylsulfonyl
methylsulfonyl
30±5
a IC50 values were determined using [3H] glycine and rat glioma C6 cell
lines. Data are the mean ± SEM of at least three independent
determinations.
572±67
23s
23t
methylsulfonyl
methylsulfonyl
44.73%@10μM
955±101
a IC50 values were determined using [3H] glycine and rat glioma C6 cell
lines. Data are the mean ± SEM of at least three independent
determinations.
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Table 3 Pharmacokinetics parameters of 23q in ICR mice.
a
b
T1/2
(h)
Tmax
(h)
Cmax,p
AUC0-t
Cmax,b
AUC0-
MRT0-
(h)
∞
∞
Compd
B/P c
0.03
(ng/mL)
(h*ng/mL)
(ng/g)
(h*ng/mL)
23q
0.7
0.5
955
728
737
0.91
26.3
a Cmax, p = Cmax in plasma. b Cmax, b = Cmax in brain. c B/P = Cmax, b / Cmax, p. n = 3 mice/group; male ICR mice (18–20g); 10 mg/kg, intragastric administration,
formulated in 0.5% HMPC. PK parameters were calculated by non-compartmental model analysis on Phoenix WinNonlin 6.0 (Pharsight, Mountain View,
CA).
5
Table 4 In vitro biological evaluation of 3-(piperidin-4-yl)benzo[d]isoxazole derivatives.
Compd
R1
R2
GlyT1 IC50 (nM) a
CLogP b
tPSA c
RG1678
23q
-
-
-
-
30 d
30±5
2.99
2.67
79.28
80.75
24a
methylsulfonyl
185±56
3.28
85.27
24b
methylsulfonyl
159±43
2.93
85.27
24c
24d
methylsulfonyl
nitro
496±58.9
89±7.8
2.58
4.19
85.27
102.94
24e
24f
nitro
85±5.15
61±6.83
2.46
2.92
79.28
97.3
N-methylsulfamoyl
24g
N-methylsulfamoyl
38±7.63
3.54
97.3
a IC50 values were determined using [3H] glycine and rat glioma C6 cell lines. Data are the mean ± SEM of at least three independent determinations. b
Data were calculated by ChemBioDraw. c Data were calculated by ChemBioDraw. d Data was selected from published reference .
10
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Table 5 Selectivity evaluation of 23 and 24 series.
b
b
b
b
b
GlyT1
IC50 (nM)
172±36
117±5.37
80±10.47
30±5
159±43
89±7.8
61±6.83
38±7.63
GlyT2 a
@10μM
25.67%
11.94%
14.92%
14.62%
8.65%
18.95%
19.70%
17.61%
D1
D2
D3
5-HT1A
5-HT2A
hERG c
Compd
@10μM
19.73%
7.62%
14.87%
16.33%
22.25%
23.37%
6.21%
@10μM
5.87%
18.45%
6.20%
10.31%
24.24%
5.20%
@10μM
25.14%
14.43%
10.80%
15.29%
17.49%
21.51%
30.30%
25.81%
@10μM
2.90%
33.06%
5.80%
11.22%
3.91%
@10μM
0.66%
8.84%
4.86%
5.21%
4.05%
31.55%
6.39%
16.51%
IC50 (μM)
d
23i
23k
23m
23q
24b
24d
24f
-
-
-
8.1
-
-
-
1.75%
3.94%
22.48%
24.01%
35.67%
24g
8.02%
-
a GlyT2 inhibiting activity was examined with the cultured primary brain stem neurons. b D1, D2, D3, 5-HT1A, and 5-HT2A were obtained from HEK293
stable-transfecting cell lines. c hERG channels were obtained from HEK293 stable-transfecting cell lines. d Not tested.
5
Selectivity evaluation of 23 and 24 series
Though 24 series was unsuccessful to improve the B/P ratio, we
were
surprised
to
find
that
the
3-(piperidin-4-
yl)benzo[d]isoxazole moiety was a privileged scaffold that
10 appears frequently in atypical antipsychotic agents17 such as
risperidone18 and iloperidone,19 which were dopamine and
serotonin receptors dual antagonists. 4-Benzoylpiperidine moiety
was also incorporated to discover multitarget ligands of
aminergic receptors.20 With this in mind, we assessed the
15 selectivity of some selected compounds. Data were shown in Table 5.
Both 23 and 24 series exhibited high selectivity over GlyT2,
dopamine and serotonin receptors (IC50 > 10 μM), indicating that
23 and 24 series were selective GlyT1 inhibitors. On the other
hand,
the
result
demonstrated
that
3-(piperidin-4-
20 yl)benzo[d]isoxazole moiety was not only a privileged scaffold in
dopamine hypothesis, but also well tolerated in NMDA
hypofunction hypothesis.
35
Fig. 3. Effects of 23q on PCP-induced hyperlomotor activity in male
C57BL/6J mice. The locomotor activity was indicated by the total
travelling distance during 1 hour recording period after the injection of
PCP (5 mg/kg). Vehicle: treated with saline without PCP or 23q. PCP +
In vivo behavioral tests on C57BL/6J mice
With the identification of 23q as a potent and selective GlyT1
25 inhibitor, we assessed in vivo anti-psychotic activity using 40 vehicle: treated with PCP (5 mg/kg, i.p.). PCP + 23q: treated with 23q (20
mg/kg or 40 mg/kg, i.g.) for 60 min, then followed by the injection of
phencyclidine (PCP)-induced schizophrenia-like animal models.
PCP (5 mg/kg, i.p.). Data were expressed as mean ± SEM and analyzed
Considering the low B/P ratio, we assessed using higher doses
using one-way analysis of variance (ANOVA) and Dunnett’s post-hoc
(20, 40 mg/kg, intragastric).
Firstly, the effect of 23q on the acute PCP-induced
test. *: p < 0.05, vs. vehicle; #: p < 0.05, vs. PCP + vehicle. n = 10.
30 hyperlocomotor activity was examined as described previously.21,
23q alone did not change the mice’s locomotor activity.
Pretreatment of 23q (40 mg/kg) attenuated PCP (5 mg/kg) -
induced hyperlocomotion of mice, indicating that 23q is a
potential anti-psychotic drug in vivo (Figure 3).
45
We then examined whether 23q could improve the social
interaction and cognitive function. C57BL/6J mice received daily
injections of PCP (10 mg/kg) for 2 weeks prior to administration
of 23q for an additional 2 weeks. Then social interaction and
novel object recognition tests were performed. Methods are
50 summarized in the experimental section.21-23
As shown in Figure 4，mice treated with PCP exhibited a
social behavioral deficit in the social interaction test. Two weeks
of treatment with 23q (40 mg/kg) significantly restored the
impaired social interaction in PCP-treated mice. Similarly,
55 administration of 23q (40 mg/kg) significantly improved the
cognitive deficit in the chronic PCP-treated mice (Figure 5).
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with an IC50 value of 30 nM and showed high selectivity against
30 GlyT2 and dopamine and serotonin receptors. Further in vivo
study demonstrated that 23q was effective on the chronic PCP-
treated schizophrenia-like behavioral models with 40 mg/kg. The
relatively high dose of 23q was needed to achieve the anti-
psychotic efficacy may attribute to the poor pharmacokinetic
35 parameters and low B/P ratio. Preliminary optimization did not
improve the pharmacokinetic parameters but led to the discovery
of another potential and selective GlyT1 inhibitors: the 3-
(piperidin-4-yl)benzo[d]isoxazole derivatives. Next we will make
more structure modifications to improve the brain exposure. 4-
40 Benzoylpiperidine and
3-(piperidin-4-yl)benzo[d]isoxazole,
which were privileged scaffolds in atypical antipsychotic agents,
exhibited high potency in GlyT1 inhibition. We suspect whether
they may be potential templates to build multitarget ligands of
GlyT1, dopamine and serotonin receptors.
Fig. 4. Effects of 23q on PCP-induced C57BL/6J mice in the social
interaction test. Social interaction time was defined as sniffing at any part
of the partner's body, grooming, following, or crawling over/under.
Vehicle: treated with saline without PCP or 23q. PCP + vehicle: treated
with PCP (10 mg/kg, i.p.) once daily for 2 weeks and then saline for an
additional 2 weeks. PCP + 23q: treated with PCP (10 mg/kg, i.p.) once
daily for 2 weeks and then 23q (20 mg/kg or 40 mg/kg, i.g.) for another 2
weeks. Data were expressed as mean ± SEM and analyzed using one-way
45 Experimental Section
General
5
All reagents were purchased from commercial suppliers and used
without further purification unless otherwise stated. Yields were
not optimized. Microwave reactions were performed in a Biotage
50 Initiator. Column chromatography was performed using pre-
packed silica cartridges (from 4 to 40 g) from Bonna-Agela
10 analysis of variance (ANOVA) and Dunnett’s post-hoc test. *: p < 0.05,
vs. vehicle; #: p < 0.05, vs. PCP + vehicle. n = 10.
Technologies Inc. (Tianjin, China) and eluted with
a
CombiFlash® Rf 200 from Teledyne Isco. 1H NMR and 13C
NMR spectra was recorded on a Bruker AC300 or a Bruker
55 AC400 NMR spectrometer, using tetramethylsilane as an internal
reference. Low-resolution mass spectra were determined on
Agilent liquid-chromatography mass spectrometer system that
consisted of an Agilent 1260 infinity LC coupled to Agilent 6120
Quadrupole mass spectrometer (electrospray positive ionization;
60 ESI) using an Agilent ZORBAX 1.8 mm SB-C18 column
(2.1×50 mm) with aqueous CH3CN (30–90%) containing 0.05%
formic acid monitored at 240 nm. High-resolution mass spectra
were recorded on a Q-Tof Ultima Globe mass spectrometer
(Micromass, Manchester, UK). HPLC analysis for all compounds
65 tested in biological systems was performed on an Agilent 1200
series LC system (Agilent ChemStation, Agilent Eclipse XDB-
C18, 5 μm, 4.6×150 mm, 30 °C, UV240 nm, 1.0 ml/min) with
aqueous CH3CN (35–90%) containing formic acid (0.05%) for 25
min. Experiments in pharmacokinetics study and behavioral tests
70 with live animals were performed in compliance with the
Guidelines for the Care and Use of Laboratory Animals (National
Research Council, People's Republic of China, 1996). The animal
protocols were approved by the Institutional Animal Care and
Use Committees of Shanghai Institute of Materia Medica (SIMM)
75 and Soochow University.
Fig. 5. Effects of 23q on PCP-induced C57BL/6J mice in novel object
recognition test. Exploratory preference was a ratio of the amount of time
15 spent exploring the novel object over the total time spent exploring both
familiar and novel objects. Vehicle: treated with saline without PCP or
23q. PCP + vehicle: treated with PCP (10 mg/kg, i.p.) once daily for 2
weeks and then saline for an additional 2 weeks. PCP + 23q: treated with
PCP (10 mg/kg, i.p.) once daily for 2 weeks and then 23q (20 mg/kg or
20 40 mg/kg, i.g.) for another 2 weeks. Data were expressed as mean ± SEM
and analyzed using one-way analysis of variance (ANOVA) and
Dunnett’s post-hoc test. *: p < 0.05, vs. vehicle; #: p < 0.05, vs. PCP +
vehicle. n = 10.
General synthetic procedure for benzoic acids and piperidine
moieties.
Preparation of 10a-h, 13a-b, 16a-e, 18a-d and 21a-b was
summarized in Electronic Supplementary Information.
Conclusions
80 General synthetic procedure for 23a-23t, 24a-24g.
25 In summary, based on the bioisosteric replacement and mimic of
nitrogen atom of pyridine ring of RG1678, we synthesized and
evaluated a series of 4-benzoylpiperidine derivatives (23a-t).
Among them 23q was the most potent GlyT1 inhibitor in vitro
To a solution of benzoic acid moiety (1 equiv) in CH2Cl2 (15 mL)
was added HATU (1.3 equiv) and the mixture was stirred at room
temperature for 30 min. Then Et3N (3 equiv) and piperidine
moiety (1.2 equiv) were added subsequently and the reaction
8
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DOI: 10.1039/C5RA04714E
Page 9 of 14
RSC Advances
mixture was stirred at room temperature overnight. The mixture
126.78-122.09 (m), 125.95-125.82 (m, 2C), 125.46, 123.75,
was diluted with CH2Cl2 (10 mL) and washed with water. The 60 111.92, 71.78, 45.95, 42.92 (2C), 27.76 (2C), 21.37 (2C). HRMS
organic layer was dried over MgSO4 and concentrated. The
residue was purified by flash column chromatography to give the
final compounds.
(ESI): m/z [M+H]+ calcd for C23H24O5N2F3: 465.1637, found:
465.1625.
(1-(2-(Cyclopropylmethoxy)-5-nitrobenzoyl)piperidin-4-
yl)(2,4-difluorophenyl)methanone (23e): 23e was prepared
5
(4-(2,4-Difluorobenzoyl)piperidin-1-yl)(2-isopropoxy-5-
nitrophenyl)methanone (23a): 23a was prepared from 10a (0.1 65 from 10c (0.1 g, 0.42 mmol) and 21d (0.13 g, 0.51 mmol). Light
g, 0.44 mmol) and 21d (0.14 g, 0.53 mmol). Yellow solid (0.14 g,
76%). HPLC purity: 96.66%; H NMR (500 MHz, CDCl3): δ
10 8.28 – 8.08 (m, 2H), 7.95 – 7.81 (m, 1H), 7.04 – 6.94 (m, 2H),
6.93 – 6.83 (m, 1H), 4.79 – 4.62 (m, 2H), 3.51 (tt, J = 13.3, 4.1
yellow solid (0.15 g, 80%). HPLC purity: 98.09%; 1H NMR (400
MHz, CDCl3): δ 8.36 – 8.14 (m, 2H), 7.94 – 7.78 (m, 1H), 7.04 –
6.84 (m, 3H), 4.87 – 4.59 (m, 1H), 4.05 – 3.85 (m, 2H), 3.53 (t, J
= 13.7 Hz, 1H), 3.44 – 3.30 (m, 1H), 3.27 – 2.91 (m, 2H), 2.14 –
1
Hz, 1H), 3.44 – 3.30 (m, 1H), 3.23 – 2.91 (m, 2H), 2.13 – 2.02 (m, 70 1.91 (m, 2H), 1.79 – 1.19 (m, 3H), 0.78 – 0.61 (m, 2H), 0.46 –
1H), 1.94 – 1.53 (m, 3H), 1.48 – 1.35 ppm (m, 6H); 13C NMR
(126 MHz, CDCl3) 198.54, 166.83-164.88 (d, J = 245.7 Hz),
15 165.29, 163.05-161.02 (d, J = 255.8 Hz), 158.75, 140.92, 133.19-
133.00 (m), 127.60, 126.26, 124.22, 121.39-121.29 (d, J = 12.6
0.26 ppm (m, 2H); 13C NMR (101 MHz, CDCl3) 198.42, 167.13-
164.57 (d, J = 258.6 Hz), 165.02, 163.19-160.75 (d, J = 246 Hz),
159.64, 141.27, 132.97, 127.10, 126.30, 124.16, 121.48-121.36 (d,
J = 12.1 Hz), 112.66-112.45 (d, J = 21.2 Hz), 111.49 , 104.79
Hz), 112.70, 112.37-112.20 (d, J = 21.4 Hz), 105.07-104.64 (d, J 75 (m) , 74.34, 47.82, 46.78 (2C), 28.00 (2C), 9.86, 3.93 (2C).
= 27.1 Hz), 72.19, 47.66, 46.64 (2C), 28.14 (2C), 21.85 (2C).
HRMS (ESI): m/z [M+H]+ calcd for C22H23O5N2F2: 433.1575,
20 found: 433.1564.
HRMS (ESI): m/z [M+H]+ calcd for C23H23O5N2F2: 445.1575,
found: 445.1568.
(1-(2-(Cyclopropylmethoxy)-5-nitrobenzoyl)piperidin-4-yl)(4-
fluorophenyl)methanone (23f): 23f was prepared from 10c (0.1
(4-(4-Fluorobenzoyl)piperidin-1-yl)(2-isopropoxy-5-
nitrophenyl)methanone (23b): 23b was prepared from 10a (0.1 80 g, 0.42 mmol) and 21a (0.10 g, 0.51 mmol). Light yellow solid
g, 0.44 mmol) and 21a (0.11 g, 0.53 mmol). Light yellow solid
(0.14 g, 77%). HPLC purity: 99.86%; 1H NMR (600 MHz,
25 CDCl3): δ 8.28 – 8.13 (m, 2H), 8.06 – 7.93 (m, 2H), 7.22 – 7.12
(m, 2H), 6.99 (t, J = 8.7 Hz, 1H), 4.83 – 4.67 (m, 2H), 3.61 – 3.45
(0.13 g, 77%). HPLC purity: 99.71%; 1H NMR (600 MHz,
CDCl3): δ 8.28 – 8.14 (m, 2H), 8.03 – 7.94 (m, 2H), 7.20 – 7.13
(m, 2H), 6.96 (d, J = 8.0 Hz, 1H), 4.86 – 4.65 (m, 1H), 4.07 –
3.92 (m, 2H), 3.63 – 3.45 (m, 2H), 3.29 – 2.98 (m, 2H), 2.10 –
(m, 2H), 3.26 – 2.98 (m, 2H), 2.10 – 1.66 (m, 4H), 1.52 – 1.35 85 1.99 (m, 2H), 1.87 – 1.76 (m, 2H), 1.40 – 1.20 (m, 1H), 0.81 –
ppm (m, 6H); 13C NMR (151 MHz, CDCl3) 199.40, 166.20-
164.92 (d, J = 193.3 Hz), 164.76, 158.31, 140.43, 131.48, 130.45-
30 130.39 (d, J = 9.1 Hz, 2C), 127.14, 125.78, 123.75, 115.50,
111.90-111.72 (d, J = 27.2 Hz, 2C), 71.76, 46.04 (2C), 40.86,
0.60 (m, 2H), 0.46 – 0.31 ppm (m, 2H); 13C NMR (151 MHz,
CDCl3) 199.33, 166.16-164.47 (d, J = 255.2 Hz), 164.59, 159.21,
140.74, 131.51, 130.42-130.36 (d, J = 9.1 Hz, 2C), 126.54,
125.91, 123.74, 115.56-115.42 (d, J = 21.1 Hz, 2C), 111.02,
27.97 (2C), 21.36 (2C). HRMS (ESI): m/z [M+H]+ calcd for 90 73.94, 46.23, 42.82 (2C), 28.10 (2C), 9.40, 3.52 (2C). HRMS
C22H24O5N2F: 415.1669, found:415.1658.
(ESI): m/z [M+H]+ calcd for C23H24O5N2F: 427.1669, found:
427.1665.
(4-(2,4-Difluorobenzoyl)piperidin-1-yl)(2-(isopropylamino)-5-
nitrophenyl)methanone (23g): 23g was prepared from 13a (0.1
(4-(2-Fluoro-4-(trifluoromethyl)benzoyl)piperidin-1-yl)(2-
35 isopropoxy-5-nitrophenyl)methanone (23c): 23c was prepared
from 10a (0.1 g, 0.44 mmol) and 21c (0.14 g, 0.53 mmol).
Yellow solid (0.12 g, 57%). HPLC purity: 99.20%; 1H NMR (400 95 g, 0.45 mmol) and 21d (0.14 g, 0.54 mmol). Yellow solid (0.14 g,
1
MHz, CDCl3): δ 8.23 (dd, J = 9.2, 2.8 Hz, 1H), 8.15 (dd, J = 16.1,
2.8 Hz, 1H), 7.94 – 7.84 (m, 1H), 7.52 (d, J = 8.1 Hz, 1H), 7.43
40 (dd, J = 10.8, 6.4 Hz, 1H), 6.97 (dd, J = 9.2, 5.6 Hz, 1H), 4.80 –
4.61 (m, 2H), 3.57 – 3.45 (m, 1H), 3.45 – 3.33 (m, 1H), 3.22 –
73%). HPLC purity: 97.30%; H NMR (600 MHz, CDCl3): δ
8.14 (dd, J = 9.3, 2.6 Hz, 1H), 8.03 (d, J = 2.7 Hz, 1H), 7.89 (td, J
= 8.6, 6.5 Hz, 1H), 7.00 (ddd, J = 9.2, 7.6, 2.4 Hz, 1H), 6.90 (ddd,
J = 11.1, 8.6, 2.4 Hz, 1H), 6.66 (d, J = 9.3 Hz, 1H), 6.08 (d, J =
2.94 (m, 2H), 2.12 – 2.02 (m, 1H), 1.92 – 1.81 (m, 1H), 1.79 – 100 7.3 Hz, 1H), 4.25 (s, 1H), 3.79 – 3.69 (m, J = 6.5 Hz, 1H), 3.47 –
1.59 (m, 2H), 1.48 – 1.33 ppm (m, 6H); 13C NMR (101 MHz,
CDCl3) 199.29, 165.23, 161.82-159.29 (d, J = 255.5 Hz), 158.71,
45 140.99, 136.50-135.50 (m), 131.96-131.78 (m), 127.62, 126.21,
124.48-124.22 (d, J = 26.3 Hz), 123.95, 121.71, 128.10-121.23
3.37 (m, 1H), 3.18 (t, J = 12.9 Hz, 2H), 2.07 – 1.95 (m, 2H), 1.83
– 1.62 (m, 3H), 1.29 ppm (d, J = 6.4 Hz, 6H); 13C NMR (151
MHz, CDCl3) 197.87-197.83 (d, J = 6.04 Hz), 167.80, 166.28-
164.49 (dd, J = 12.08, 258.2 Hz), 162.40-160.63 (dd, J = 12.08,
(m), 114.50, 112.41-112.23 (m), 72.19, 47.92, 46.46 (2C), 27.88 105 255.2 Hz), 150.93, 135.29, 132.65 (m), 127.08, 124.77, 120.84-
(2C), 21.94 (2C). HRMS (ESI): m/z [M+H]+ calcd for
C23H23O5N2F4: 483.1543, found: 483.1533.
50 (1-(2-Isopropoxy-5-nitrobenzoyl)piperidin-4-yl)(4-
(trifluoromethyl)phenyl)methanone (23d): 23d was prepared
120.77 (d J = 10.57 Hz), 115.96, 112.25-112.09 (d, J = 21.14 Hz),
109.94 , 104.48-104.12 (t, J = 27.18 Hz), 47.13-47.08 (d, J = 7.55
Hz), 43.80 (3C), 27.74 (2C), 21.95 (2C). HRMS (ESI): m/z
[M+H]+ calcd for C22H24O4N3F2: 432.1735, found: 432.1722.
from 10a (0.1 g, 0.44 mmol) and 21b (0.14 g, 0.53 mmol). Off- 110 (4-(2,4-Difluorobenzoyl)piperidin-1-yl)(5-nitro-2-(piperidin-1-
1
white solid (0.14 g, 69%). HPLC purity: 98.61%; H NMR (600
MHz, CDCl3): δ 8.28 – 8.14 (m, 2H), 8.11 – 8.00 (m, 2H), 7.81 –
55 7.70 (m, 2H), 6.99 (t, J = 8.6 Hz, 1H), 4.82 – 4.67 (m, 2H), 3.62 –
3.51 (m, 2H), 3.27 – 3.01 (m, 2H), 2.12 – 1.69 (m, 4H), 1.52 –
yl)phenyl)methanone (23h): 23h was prepared from 13b (0.1 g,
0.40 mmol) and 21d (0.12 g, 0.48 mmol). Yellow solid (0.12 g,
66%). HPLC purity: 98.99%; H NMR (600 MHz, CDCl3): δ
1
8.20 – 8.05 (m, 2H), 7.95 – 7.83 (m, 1H), 7.05 – 6.85 (m, 3H),
1.36 ppm (m, 6H); 13C NMR (151 MHz, CDCl3) 200.08, 164.81, 115 4.85 – 4.56 (m, 1H), 3.56 – 3.46 (m, 1H), 3.44 – 3.28 (m, 3H),
158.29, 140.44, 137.83, 134.24-134.03 (m), 128.11 (2C), 127.03,
3.26 – 2.91 (m, 4H), 2.14 – 2.05 (m, 1H), 1.96 – 1.51 ppm (m,
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RSC Advances
DOI: 10.1039/C5RPA0a4g71e4E10 of 14
9H); 13C NMR (151 MHz, CDCl3) 198.22, 167.72, 166.23-164.61
(d, J = 244.6 Hz), 162.52-160.60 (d, J = 255.2 Hz), 154.24, 60 (piperidin-1-yl)benzenesulfonamide (23l): 23l was prepared
3-(4-(2,4-Difluorobenzoyl)piperidine-1-carbonyl)-N-methyl-4-
140.03, 132.78-132.52 (m), 127.54, 125.53, 125.01, 124.77 (m),
120.89, 116.58, 112.27-112.07 (d, J = 7.55 Hz), 104.58-104.17
(m), 51.85 (2C), 47.41, 46.03 (2C), 27.36 (2C), 25.27 (2C), 23.43.
HRMS (ESI): m/z [M+H]+ calcd for C24H26O4N3F2: 458.1891,
found: 458.1881.
4-(Cyclopropylmethoxy)-3-(4-(2,4-
difluorobenzoyl)piperidine-1-carbonyl)-N-
10 methylbenzenesulfonamide (23i): 23i was prepared from 10g
(0.1 g, 0.35 mmol) and 21.d (0.11 g, 0.42 mmol). White solid
(0.13 g, 80%). HPLC purity: 95.16%; 1H NMR (400 MHz, 70 104.62 (m), 52.76 (2C), 47.82, 46.49 (2C), 29.68 (2C), 27.82,
from 16d (0.1 g, 0.34 mmol) and 21d (0.10 g, 0.40 mmol). Off-
white solid (0.12 g, 69%). HPLC purity: 97.67%; H NMR (400
1
5
MHz, CDCl3): δ 7.98 – 7.82 (m, 1H), 7.81 – 7.63 (m, 2H), 7.09 –
6.96 (m, 2H), 6.89 (q, J = 9.6 Hz, 1H), 4.93 – 4.54 (m, 2H), 3.55
65 – 3.15 (m, 4H), 3.13 – 2.85 (m, 4H), 2.63 (d, J = 5.4 Hz, 3H),
2.18 – 1.65 (m, 8H), 1.29 ppm (d, J = 20.1 Hz, 2H); 13C NMR
(101 MHz, CDCl3) 198.84, 168.81, 167.02-164.5 (d, J = 254.5
Hz), 153.51, 133.16-132.95 (m), 130.71, 130.07, 129.62, 129.43,
127.98, 121.36, 117.77, 112.69-112.52 (d, J = 17.2 Hz), 104.88-
CDCl3): δ 7.94 – 7.79 (m, 2H), 7.78 – 7.69 (m, 1H), 7.07 – 6.75
(m, 3H), 5.05 – 4.88 (m, 1H), 4.85 – 4.52 (m, 1H), 4.05 – 3.77 (m,
15 2H), 3.55 (d, J = 13.2 Hz, 1H), 3.37 (tt, J = 10.7, 3.8 Hz, 1H),
3.27 – 2.87 (m, 2H), 2.61 (d, J = 5.3 Hz, 3H), 2.06 (d, J = 13.5
26.35 (2C), 24.00. HRMS (ESI): m/z [M+H]+ calcd for
C25H30O4N3F2S: 506.1925, found: 506.1911.
3-(4-(2,4-Difluorobenzoyl)piperidine-1-carbonyl)-N-methyl-4-
morpholinobenzenesulfonamide (23m): 23m was prepared
Hz, 2H), 1.75 (h, J = 9.8, 8.6 Hz, 1H), 1.39 – 1.19 (m, 2H), 0.76 – 75 from 16e (0.1 g, 0.33 mmol) and 21d (0.10 g, 0.40 mmol). White
1
0.58 (m, 2H), 0.48 – 0.23 ppm (m, 2H); 13C NMR (101 MHz,
CDCl3) 198.58, 167.12-164.44 (d, J = 257.5 Hz), 166.14, 163.20-
20 160.70 (d, J = 252.5 Hz), 157.94, 133.09 (m), 130.96, 130.08,
127.37, 127.05, 121.44, 112.66-112.45 (d, J = 21.2 Hz), 111.67,
solid (0.13 g, 77%). HPLC purity: 98.45%; H NMR (400 MHz,
CDCl3): δ 7.96 – 7.85 (m, 1H), 7.82 (dd, J = 8.6, 2.3 Hz, 1H),
7.73 (dd, J = 21.7, 2.3 Hz, 1H), 7.11 – 6.97 (m, 2H), 6.95 – 6.85
(m, 1H), 4.88 (p, J = 5.4 Hz, 1H), 4.80 – 4.57 (m, 1H), 3.94 –
105.10-104.57 (t, J = 26.8 Hz), 73.86, 47.94, 46.90 (2C), 29.32 80 3.73 (m, 4H), 3.50 – 3.18 (m, 4H), 3.13 – 2.91 (m, 4H), 2.63 (d, J
(2C), 28.21, 9.95, 3.14 (2C). HRMS (ESI): m/z [M+H]+ calcd for
C24H27O5N2F2S: 493.1609, found: 493.1595.
25 3-(4-(2,4-Difluorobenzoyl)piperidine-1-carbonyl)-N-methyl-4-
(2,2,3,3,3-pentafluoropropoxy)benzenesulfonamide (23j): 23j
= 5.3 Hz, 3H), 2.16 – 1.90 (m, 2H), 1.80 – 1.48 ppm (m, 2H); 13
C
NMR (101 MHz, CDCl3) 198.60-198.20 (d, J = 40.4 Hz), 168.38,
167.26-164.6 (d, J = 268.6 Hz), 163.45-160.78 (d, J = 268.7 Hz),
152.42, 133.24-133.02 (m), 132.41, 130.95, 129.51, 127.70,
was prepared from 18c (0.1 g, 0.27 mmol) and 21d (0.086 g, 0.33 85 121.38-121.25 (d, J = 13.1 Hz), 117.98, 112.78-112.57 (d, J =
1
mmol). Off-white solid (0.13 g, 71%). HPLC purity: 95.93%; H
NMR (600 MHz, CDCl3): δ 7.95 – 7.74 (m, 3H), 7.11 – 7.03 (m,
30 1H), 7.03 – 6.96 (m, 1H), 6.95 – 6.83 (m, 1H), 5.17 – 5.00 (m,
1H), 4.74 – 4.47 (m, 3H), 3.48 (dt, J = 13.9, 4.1 Hz, 1H), 3.37
21.2 Hz), 104.88-104.60 (m), 66.70 (2C), 51.88 (2C), 47.70,
46.55 (2C), 29.35 (2C), 28.04. HRMS (ESI): m/z [M+H]+ calcd
for C24H28O5N3F2S: 508.1718, found: 508.1704.
3-(4-(2,4-Difluorobenzoyl)piperidine-1-carbonyl)-4-
(dtd, J = 14.4, 10.5, 9.9, 4.7 Hz, 1H), 3.20 – 2.98 (m, 2H), 2.63 90 (isopropylthio)-N-methylbenzenesulfonamide (23n): 23n was
(dd, J = 5.4, 2.6 Hz, 3H), 2.07 (d, J = 13.7 Hz, 1H), 1.94 – 1.56
ppm (m, 3H); 13C NMR (151 MHz, CDCl3) 197.89-197.76 (d, J =
35 19.6 Hz), 166.25-164.54 (d, J = 258.2 Hz), 164.46, 162.35-
160.66 (d, J = 255.2 Hz), 155.35, 133.48, 132.67 (m), 129.68,
prepared from 18d (0.1 g, 0.35 mmol) and 21d (0.11 g, 0.42
mmol). Off-white solid (0.10 g, 58%). HPLC purity: 96.27%; H
NMR (400 MHz, CDCl3): δ 7.88 (q, J = 8.0 Hz, 1H), 7.81 – 7.74
(m, 1H), 7.66 (s, 1H), 7.47 (d, J = 8.4 Hz, 1H), 7.06 – 6.95 (m,
1
127.28, 127.00, 120.87, 116.98 (m), 112.20, 112.09 (m), 111.65, 95 1H), 6.95 – 6.80 (m, 1H), 5.11 – 4.95 (m, 1H), 4.67 (s, 1H), 3.62
104.43-104.07 (t, J = 27.2 Hz), 65.01-64.64 (m), 47.00, 45.60
(2C), 28.79 (2C), 27.35. HRMS (ESI): m/z [M+H]+ calcd for
40 C23H22O5N2F7S: 571.1138, found: 571.1121.
(hept, J = 6.8 Hz, 1H), 3.52 – 3.29 (m, 2H), 3.27 – 3.03 (m, 2H),
2.64 (d, J = 5.2 Hz, 3H), 2.16 – 1.57 (m, 4H), 1.37 ppm (d, J =
6.6 Hz, 6H); 13C NMR (101 MHz, CDCl3) 198.45, 167.16-164.61
(d, J = 257.5 Hz), 167.03, 163.18- 160.75 (d, J = 245.3 Hz),
3-(4-(2,4-Difluorobenzoyl)piperidine-1-carbonyl)-4-
(isopropylamino)-N-methylbenzenesulfonamide (23k): 23k 100 140.10, 137.76, 136.10, 133.11, 128.85, 127.64, 125.26, 121.43-
was prepared from 16c (0.1 g, 0.37 mmol) and 21d (0.11 g, 0.44
mmol). White solid (0.11 g, 63%). HPLC purity: 98.07%; 1H
45 NMR (600 MHz, CDCl3): δ 7.86 (td, J = 8.6, 6.5 Hz, 1H), 7.67
(dd, J = 8.8, 2.3 Hz, 1H), 7.55 (d, J = 2.3 Hz, 1H), 6.98 (ddd, J =
121.26 (d, J = 13.1 Hz), 112.72-112.51 (d, J = 21.2 Hz), 105.03-
104.49 (t, J = 27.3 Hz), 47.63, 46.75 (2C), 37.24, 29.34 (2C),
27.62, 22.69 (2C). HRMS (ESI): m/z [M+H]+ calcd for
C23H27O4N2F2S2: 497.1380, found: 497.1367.
9.5, 7.5, 2.4 Hz, 1H), 6.88 (ddd, J = 11.1, 8.5, 2.4 Hz, 1H), 6.70 105 (4-(2,4-Difluorobenzoyl)piperidin-1-yl)(2-isopropoxy-5-
(d, J = 8.9 Hz, 1H), 5.49 (d, J = 7.3 Hz, 1H), 4.83 (q, J = 5.4 Hz,
1H), 3.68 (h, J = 6.4 Hz, 1H), 3.43 – 3.33 (m, 1H), 3.13 (t, J =
50 12.7 Hz, 2H), 2.57 (d, J = 5.4 Hz, 3H), 2.07 – 1.89 (m, 3H), 1.77
– 1.60 (m, 2H), 1.24 ppm (d, J = 6.4 Hz, 6H); 13C NMR (151
(methylsulfonyl)phenyl)methanone (23o): 23o was prepared
from 10d (0.1 g, 0.38 mmol) and 21d (0.12 g, 0.46 mmol). White
solid (0.14 g, 83%). HPLC purity: 98.06%; H NMR (600 MHz,
1
CDCl3): δ 7.91 – 7.82 (m, 2H), 7.82 – 7.71 (m, 1H), 7.02 (t, J =
MHz, CDCl3) 198.02-197.99 (d, J = 4.53 Hz), 168.32, 166.21- 110 8.7 Hz, 1H), 6.98 (t, J = 8.3 Hz, 1H), 6.92 – 6.83 (m, 1H), 4.75 –
164.51 (d, J = 256.7 Hz), 162.37-160.67 (d, J = 256.7 Hz),
148.85, 132.64-132.55 (m), 129.96, 127.64, 123.00, 120.89,
55 117.13, 112.19-112.03 (d, J = 21.14 Hz), 110.58, 104.47-104.11
(t, J = 27.18 Hz), 47.11-47.06 (d, J = 7.55 Hz), 43.44 (2C), 38.10,
4.57 (m, 2H), 3.52 – 3.43 (m, 1H), 3.42 – 3.31 (m, 1H), 3.18 –
3.07 (m, 1H), 3.04 (d, J = 3.5 Hz, 3H), 3.00 – 2.91 (m, 1H), 2.08
– 1.99 (m, 1H), 1.91 – 1.52 (m, 3H), 1.44 – 1.31 ppm (m, 6H);
13C NMR (151 MHz, CDCl3) 198.21-197.97 (d, J = 36.2 Hz),
28.75 (2C), 27.71, 22.01 (2C). HRMS (ESI): m/z [M+H]+ calcd 115 166.25-164.54 (d, J = 258.2 Hz), 165.57, 162.38- 160.77 (d, J =
for C23H28O4N3F2S: 480.1769, found: 480.1757. 243.1 Hz), 157.35, 132.61-132.41 (m), 131.79-131.60 (m),
1
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DOI: 10.1039/C5RA04714E
Page 11 of 14
RSC Advances
129.77, 129.61, 127.47, 120.88-120.79 (d, J = 13.60 Hz), 112.50,
1H), 6.74 (d, J = 8.9 Hz, 1H), 5.65 (d, J = 7.3 Hz, 1H), 3.71 (h, J
112.23-112.01 (m), 104.53-104.18 (t, J = 26.43 Hz), 71.26, 47.11, 60 = 6.6 Hz, 1H), 3.46 – 3.34 (m, 1H), 3.15 (t, J = 12.6 Hz, 2H),
46.22, 44.17 (2C), 27.62 (2C), 21.11(2C). HRMS (ESI): m/z
[M+H]+ calcd for C23H26O5NF2S: 466.1500, found: 466.1494.
(4-(2,4-Difluorobenzoyl)piperidin-1-yl)(2-isobutoxy-5-
(methylsulfonyl)phenyl)methanone (23p): 23p was prepared
2.99 (d, J = 0.9 Hz, 3H), 2.00 (d, J = 13.9 Hz, 2H), 1.73 (q, J =
12.7, 12.1 Hz, 3H), 1.27 ppm (d, J = 6.3 Hz, 6H); 13C NMR (101
MHz, CDCl3) 198.34-198.30 (d, J = 4.04 Hz), 168.59, 167.16-
164.47 (d, J = 258.5 Hz), 163.29-160.62 (d, J = 256.5 Hz),
5
from 10e (0.1 g, 0.37 mmol) and 21d (0.11 g, 0.44 mmol). White 65 150.11, 133.11-133.04 (m), 130.47, 128.17, 125.42, 121.37-
1
solid (0.13 g, 73%). HPLC purity: 98.82%; H NMR (400 MHz,
CDCl3): δ 7.97 – 7.74 (m, 3H), 7.09 – 6.96 (m, 2H), 6.95 – 6.82
121.26 (d, J = 11.1 Hz), 117.64, 112.70-112.49 (d, J = 21.2 Hz),
111.14, 105.01-104.48 (t, J = 26.7 Hz), 47.59-47.52 (d, J = 7.07
Hz), 45.00, 43.97 (2C), 38.56 , 28.19 (2C), 22.44 (2C). HRMS
(ESI): m/z [M+H]+ calcd for C23H27O4N2F2S: 465.1660, found:
10 (m, 1H), 4.78 – 4.62 (m, 1H), 4.02 – 3.73 (m, 2H), 3.57 – 3.28 (m,
2H), 3.22 – 3.08 (m, 1H), 3.04 (d, J = 2.8 Hz, 3H), 2.25 – 2.11 (m,
1H), 2.10 – 1.66 (m, 5H), 1.04 ppm (d, J = 6.6 Hz, 6H); 13C NMR 70 465.1651.
(101 MHz, CDCl3) 198.52-198.27 (d, J = 25.3 Hz), 167.15-
164.47 (d, J = 270.6 Hz), 165.71, 163.21-160.67 (d, J = 256.5
15 Hz), 159.15, 133.23-132.93 (m), 132.79, 130.35, 130.20, 127.27,
121.48-121.33(d, J = 15.1 Hz), 112.77-112.47 (m), 112.26,
(4-(2,4-Difluorobenzoyl)piperidin-1-yl)(5-(methylsulfonyl)-2-
(piperidin-1-yl)phenyl)methanone (23t): 23t was prepared from
16b (0.1 g, 0.35 mmol) and 21d (0.11 g, 0.42 mmol). Off-white
1
solid (0.12 g, 72%). HPLC purity: 98.28%; H NMR (400 MHz,
105.09-104.56 (m), 75.52, 47.60 (m), 46.66, 44.76 (2C), 28.18 75 CDCl3): δ 7.92 – 7.66 (m, 3H), 7.09 – 6.93 (m, 2H), 6.87 (t, J =
(2C), 27.71, 19.09 (2C). HRMS (ESI): m/z [M+H]+ calcd for
C24H28O5NF2S: 480.1656, found: 480.1644.
20 (4-(2,4-Difluorobenzoyl)piperidin-1-yl)(5-(methylsulfonyl)-2-
(2,2,3,3,3-pentafluoropropoxy)phenyl)methanone (23q): 23q
10.0 Hz, 1H), 4.62 (d, J = 12.6 Hz, 1H), 3.44 – 2.87 (m, 8H), 3.01
(s, 3H) 2.10 – 1.99 (m, 1H), 1.96 – 1.55 ppm (m, 9H); 13C NMR
(101 MHz, CDCl3) 198.73, 168.48, 167.16-164.48 (d, J = 257.6
Hz), 163.15- 160.65 (d, J = 252.5 Hz), 154.10, 133.09, 132.11
was prepared from 18b (0.2 g, 0.57 mmol) and 21d (0.18 g, 0.69 80 (m), 129.54, 128.43, 121.43-121.27 (d, J = 13.1 Hz), 117.94 (2C),
mmol). White solid (0.26 g, 84%). HPLC purity: 98.78%; 1H
NMR (400 MHz, CDCl3): δ 7.98 (dt, J = 8.7, 2.6 Hz, 1H), 7.93 –
25 7.83 (m, 2H), 7.09 (t, J = 9.0 Hz, 1H), 7.04 – 6.96 (m, 1H), 6.94 –
6.84 (m, 1H), 4.74 – 4.45 (m, 3H), 3.52 – 3.41 (m, 1H), 3.42 –
112.73-112.52 (d, J = 21.2 Hz), 104.80 (m), 52.54 (2C), 47.81,
46.49 (2C), 38.61, 28.33 (2C), 26.21 (2C), 23.95. HRMS (ESI):
m/z [M+H]+ calcd for C25H29O4N2F2S: 491.1816, found:
491.1806.
3.29 (m, 1H), 3.21 – 2.96 (m, 2H), 3.06 (d, J = 3.2 Hz, 3H), 2.12 85 (4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)(5-
– 2.00 (m, 1H), 1.90 – 1.80 (m, 1H), 1.79 – 1.62 ppm (m, 2H);
13C NMR (101 MHz, CDCl3) 198.25, 167.10-164.65 (d, J = 247.5
30 Hz), 164.42, 163.27-160.75 (d, J = 254.5 Hz), 156.37, 135.49,
133.12 (m), 130.40, 128.23, 127.92, 121.42-121.29 (d, J = 13.1
(methylsulfonyl)-2-(2,2,3,3,3-
pentafluoropropoxy)phenyl)methanone (24a): 24a was
prepared from 18b (0.1 g; 0.29 mmol) and 6-fluoro-3-(4-
piperidinyl)-1,2-benzisoxazole hydrochloride (0.09 g, 0.34 mmol,
Hz), 121.42 (m), 112.77(m), 112.99, 112.43, 104.71-104.46 (t, J 90 commercial available). White solid (0.11 g, 71%). HPLC purity:
1
= 12.6 Hz), 65.38 (m), 47.50 (m), 46.63, 44.65 (2C), 26.65 (2C).
HRMS (ESI): m/z [M+H]+ calcd for C23H21O5NF7S: 556.1029,
35 found: 556.1013.
96.45%; H NMR (600 MHz, CDCl3): δ 8.03 – 7.86 (m, 2H),
7.70 – 7.61 (m, 1H), 7.27 (dq, J = 8.6, 2.1 Hz, 1H), 7.18 – 7.04
(m, 2H), 4.81 – 4.50 (m, 3H), 3.58 (td, J = 13.3, 3.7 Hz, 1H), 3.38
(td, J = 10.7, 5.0 Hz, 1H), 3.30 – 3.10 (m, 2H), 3.08 (dd, J = 4.5,
(4-(2,4-Difluorobenzoyl)piperidin-1-yl)(5-(methylsulfonyl)-2-
((1,1,1-trifluoropropan-2-yl)oxy)phenyl)methanone (23r): 23r 95 1.5 Hz, 3H), 2.29 – 1.97 ppm (m, 4H). 13C NMR (151 MHz,
was prepared from 18a (0.10 g, 0.32 mmol) and 21d (0.10 g, 0.38
mmol). White solid (0.13 g, 79%). HPLC purity: 98.73%; 1H
40 NMR (600 MHz, CDCl3): δ 7.99 – 7.94 (m, 1H), 7.94 – 7.83 (m,
2H), 7.18 – 7.07 (m, 1H), 7.00 (td, J = 9.2, 8.5, 2.5 Hz, 1H), 6.93
CDCl3) 164.55-162.89 (d, J = 250.6 Hz), 164.20, 163.46-163.37
(d, J = 13.6 Hz), 159.42 (m), 155.86, 134.82, 130.07, 127.82,
127.43, 121.73-121.67 (t, J = 4.53 Hz), 118.60 (m), 116.48,
112.60, 112.17 (m), 111.92, 97.15-96.98 (d, J = 25.67 Hz), 65.01
– 6.84 (m, 1H), 4.90 – 4.78 (m, 1H), 4.75 – 4.59 (m, 1H), 3.52 – 100 (m), 45.80 (2C), 44.07, 33.56, 29.81 (2C). HRMS (ESI): m/z
3.44 (m, 1H), 3.42 – 3.32 (m, 1H), 3.22 – 3.00 (m, 2H), 3.07 (d, J
= 2.8 Hz, 3H), 2.07 (dd, J = 13.7, 3.8 Hz, 1H), 1.91 – 1.82 (m,
45 1H), 1.80 – 1.49 ppm (m, 5H); 13C NMR (151 MHz, CDCl3)
197.82, 166.10-164.19 (d, J = 286.5 Hz), 164.37, 162.23-160.54
[M+H]+ calcd for C23H21O5N2F6S: 551.1075, found: 551.1060.
(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)(2-isobutoxy-
5-(methylsulfonyl)phenyl)methanone (24b): 24b was prepared
from 10f (0.1 g, 0.37 mmol) and 6-fluoro-3-(4-piperidinyl)-1,2-
(d, J = 255.2 Hz), 156.02-155.70 (m), 134.55, 132.71-132.61 (m), 105 benzisoxazole hydrochloride (0.11 g, 0.44 mmol). White solid
129.73, 129.57, 127.35, 124.47 (m), 120.88-120.80 (d, J = 12.08
Hz), 114.21, 112.21-112.06 (d, J = 22.65 Hz), 104.42-104.06 (t, J
50 = 27.18 Hz), 73.37-72.47 (m), 47.05, 46.31, 44.16 (2C), 27.31
(2C), 13.33-13.21 (m). HRMS (ESI): m/z [M+H]+ calcd for
C23H23O5NF5S: 520.1217, found: 520.1205.
(0.13 g, 76%). HPLC purity: 96.76%; 1H NMR (400 MHz,
CDCl3): δ 7.97 – 7.91 (m, 1H), 7.86 (dd, J = 32.0, 2.4 Hz, 1H),
7.70 – 7.59 (m, 1H), 7.28 – 7.24 (m, 1H), 7.13 – 7.02 (m, 2H),
4.80 (dd, J = 33.0, 13.5 Hz, 1H), 3.99 – 3.74 (m, 2H), 3.67 – 3.54
110 (m, 1H), 3.43 – 3.08 (m, 3H), 3.05 (d, J = 3.6 Hz, 3H), 2.32 –
1.94 (m, 5H), 1.11 – 0.96 ppm (m, 6H); 13C NMR (101 MHz,
CDCl3) 165.82, 165.42-162.92 (d, J = 252.5 Hz), 163.88, 159.99,
159.13, 132.85, 130.40, 127.62, 127.32, 122.05 (m), 117.14,
112.60 (m), 112.15, 97.68-97.41 (d, J = 27.3 Hz), 75.54, 46.82
(4-(2,4-Difluorobenzoyl)piperidin-1-yl)(2-(isopropylamino)-5-
(methylsulfonyl)phenyl)methanone (23s): 23s was prepared
55 from 16a (0.1 g, 0.39 mmol) and 21d (0.12 g, 0.47 mmol). Off-
1
white solid (0.11 g, 61%). HPLC purity: 98.63%; H NMR (400
MHz, CDCl3): δ 7.93 – 7.82 (m, 1H), 7.74 (dd, J = 8.9, 2.1 Hz, 115 (2C), 44.75, 34.35, 30.08 (2C), 28.18, 19.09 (2C). HRMS (ESI):
1H), 7.64 – 7.58 (m, 1H), 7.04 – 6.94 (m, 1H), 6.93 – 6.83 (m,
m/z [M+H]+ calcd for C24H28O5N2FS: 475.1703, found: 475.1690.
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RSC Advances
DOI: 10.1039/C5RPA0a4g71e4E12 of 14
(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)(5-
162.52 (d, J = 252.5 Hz), 163.40-163.27 (d, J = 13.1 Hz), 159.77,
(methylsulfonyl)-2-((1,1,1-trifluoropropan-2-
60 156.41, 130.38, 129.83, 129.61,127.25, 126.96, 126.53, 121.96-
121.68 (d, J = 28.3 Hz), 116.67-116.43 (d, J = 24.2 Hz), 112.19
(m), 71.11, 46.56 (2C), 33.77, 30.02, 28.78 (2C), 21.48 (2C).
HRMS (ESI): m/z [M+H]+ calcd for C23H27O5N3FS: 476.1655,
found: 476.1645.
yl)oxy)phenyl)methanone (24c): 24c was prepared from 18a
(0.1 g, 0.32 mmol) and 6-fluoro-3-(4-piperidinyl)-1,2-
benzisoxazole hydrochloride (0.10 g, 0.38 mmol). White solid
(0.13 g, 81%). HPLC purity: 98.52%; 1H NMR (400 MHz,
5
CDCl3): δ 7.98 (d, J = 8.4 Hz, 1H), 7.96 – 7.84 (m, 1H), 7.65 (td, 65 3-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidine-1-carbonyl)-4-
J = 8.2, 4.7 Hz, 1H), 7.28 – 7.24 (m, 1H), 7.22 – 7.04 (m, 2H),
4.95 – 4.69 (m, 2H), 3.63 – 3.53 (m, 1H), 3.44 – 3.12 (m, 3H),
10 3.08 (s, 3H), 2.25 (dd, J = 13.6, 4.0 Hz, 1H), 2.14 – 1.89 (m, 3H),
1.66 – 1.52 ppm (m, 3H); 13C NMR (101 MHz, CDCl3) 165.53-
isobutoxy-N-methylbenzenesulfonamide (24g): 24g was
prepared from 10f (0.1 g, 0.35 mmol) and 6-fluoro-3-(4-
piperidinyl)-1,2-benzisoxazole hydrochloride (0.11 g, 0.42 mmol).
1
White solid (0.13 g, 67%). HPLC purity: 96.83%; H NMR (500
163.03 (d, J = 252.5 Hz), 165.02, 164.08-163.94 (d, J = 14.1 Hz), 70 MHz, CDCl3): δ 7.90 – 7.71 (m, 2H), 7.71 – 7.60 (m, 1H), 7.27 –
160.02, 156.59, 135.09, 130.43, 128.52, 127.95, 122.34 (m),
122.32-122.21 (d, J = 11.1 Hz), 117.08, 113.64, 112.82 (m),
15 97.82-97.55 (d, J = 27.3 Hz), 73.64 (m), 47.12 (2C), 44.76, 34.25,
30.17 (2C), 13.89. HRMS (ESI): m/z [M+H]+ calcd for
C23H23O5N2F4S: 515.1264, found: 515.1251.
7.23 (m, 1H), 7.08 (tdd, J = 8.8, 3.9, 2.1 Hz, 1H), 7.01 (dd, J =
8.8, 5.2 Hz, 1H), 4.99 – 4.89 (m, 1H), 4.87 – 4.70 (m, 1H), 3.96 –
3.73 (m, 2H), 3.67 – 3.57 (m, 1H), 3.41 – 3.31 (m, 1H), 3.30 –
3.04 (m, 2H), 2.62 (d, J = 6.6 Hz, 3H), 2.31 – 1.88 (m, 5H), 1.07
75 – 1.00 ppm (m, 6H); 13C NMR (126 MHz, CDCl3) 166.25,
165.19-163.20 (d, J = 250.7 Hz), 163.87, 160.09, 158.27, 131.14,
130.35, 127.36, 126.92, 122.39-122.10 (d, J = 36.5 Hz), 117.18,
112.70 (m), 112.00-111.71 (d, J = 36.5 Hz), 97.68-97.46 (d, J =
27.7 Hz), 75.39, 46.90 (2C), 34.39, 30.36, 29.34 (2C), 28.25,
(2-(Cyclopentyloxy)-5-nitrophenyl)(4-(6-
fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)methanone (24d):
20 24d was prepared from 10b (0.1 g, 0.40 mmol) and 6-fluoro-3-(4-
piperidinyl)-1,2-benzisoxazole hydrochloride (0.12 g, 0.48 mmol).
Light yellow solid (0.12 g, 65%). HPLC purity: 97.55%; 1H 80 19.12 (2C). HRMS (ESI): m/z [M+H]+ calcd for C24H29O5N3FS:
NMR (400 MHz, CDCl3): δ 8.29 – 8.12 (m, 2H), 7.69 – 7.58 (m,
1H), 7.28 (d, J = 2.1 Hz, 1H), 7.09 (tt, J = 8.8, 2.1 Hz, 1H), 7.04
25 – 6.94 (m, 1H), 4.97 – 4.86 (m, 1H), 4.84 – 4.70 (m, 1H), 3.68 –
3.51 (m, 1H), 3.43 – 3.04 (m, 3H), 2.33 – 2.15 (m, 1H), 2.12 –
1.50 ppm (m, 11H); 13C NMR (101 MHz, CDCl3) 165.43-162.95
(d, J = 250.5 Hz), 165.26, 163.90, 160.02, 159.05, 141.03, 127.51,
126.18, 124.44, 124.01, 121.93 (m), 117.13, 112.80-112.49 (d, J
30 = 31.3 Hz), 97.72-97.45 (d, J = 27.3 Hz), 81.64, 46.84 (2C),
33.97, 33.01 (2C), 30.29 (2C), 23.96 (2C). HRMS (ESI): m/z
[M+H]+ calcd for C24H25O5N3F: 454.1778, found 454.1770.
490.1812, found: 490.1798.
[3H]Glycine uptake assay (GlyT1)
Rat glioma C6 cells that stably expressed GlyT1 were plated into
24-well culture plates (1×106 per well). After 18 h, the culture
85 medium was discarded and HEPES buffer solution (160 μL) was
3
added. Thereafter, tested compounds (20 μL) and H-glycine (20
μL; final concentration ranges: 10−10, 10−9, 10−8, 10−7, 10−6, and
10−5 M) were added to a total volume of 200 μL. In the total
uptake wells, the tested compounds were replaced with Hank’s
90 balanced salt solution (HBSS; 20 μL). NFPS24 (10 μM) was used
to determine nonspecific uptake. After a 30 min incubation,
HBSS was discarded and the plate was washed twice with
phosphate-buffered saline (500 μL). Then, NaOH (100 μL, 2M)
was added to lyse the cells. The lysate activity was examined, and
95 the inhibiting ratio and IC50 values were calculated.
(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)(5-nitro-2-
(piperidin-1-yl)phenyl)methanone (24e): 24e was prepared
35 from 13b (0.1 g, 0.40 mmol) and 6-fluoro-3-(4-piperidinyl)-1,2-
benzisoxazole hydrochloride (0.12 g, 0.48 mmol). Yellow solid
(0.12 g, 67%). HPLC purity: 97.32%; 1H NMR (600 MHz,
CDCl3): δ 8.24 – 8.08 (m, 2H), 7.70 – 7.60 (m, 1H), 7.31 – 7.24
(m, 1H), 7.10 (tdd, J = 8.8, 4.8, 2.1 Hz, 1H), 6.97 (dd, J = 11.2,
40 9.1 Hz, 1H), 4.95 – 4.59 (m, 1H), 3.67 – 3.53 (m, 1H), 3.43 –
3.25 (m, 4H), 3.23 – 3.02 (m, 3H), 2.35 – 2.21 (m, 1H), 2.19 –
1.82 (m, 3H), 1.80 – 1.59 ppm (m, 6H); 13C NMR (151 MHz,
CDCl3) 167.81, 164.60-162.94 (d, J = 250.7 Hz), 163.54-163.34
(d, J = 30.2 Hz), 159.63, 154.25, 140.06, 127.44, 125.62, 125.08,
45 121.72-121.45 (d, J = 40.7 Hz), 116.64, 116.74, 112.33-112.16 (d,
J = 25.6 Hz), 97.22-97.04 (d, J = 27.2 Hz), 51.86 (2C), 46.30
(2C), 33.98, 29.68 (2C), 25.71 (2C), 23.42. HRMS (ESI): m/z
[M+H]+ calcd for C24H26O4N4F: 453.1938, found: 453.1927.
3-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidine-1-carbonyl)-4-
50 isopropoxy-N-methylbenzenesulfonamide (24f): 24f was
prepared from 10h (0.1 g, 0.37 mmol) and 6-fluoro-3-(4-
piperidinyl)-1,2-benzisoxazole hydrochloride (0.11 g, 0.44 mmol).
[3H]Glycine uptake assay (GlyT2)
GlyT2 inhibiting activity was examined using cultured primary
brain stem neurons. In brief, brain stems were removed from
brains of rat pups. The tissues were minced with a sterile razor
100 blade and digested with 0.05% trypsin (Sigma) and 0.01% Dnase
I (Sigma) at 37 °C for 10 min. Then, the tissues were collected by
centrifugation for 5 min. After centrifugation, complete culture
media Dulbecco’s modified Eagle’s medium/F12 (3 mL; 1:1;
Gibco BRL, Gaithersburg, MD, USA) was added, which
105 contained 10% fetal bovine serum (Hyclone, Logan, UT, USA),
penicillin (100 U/mL), and streptomycin. Thereafter, this tissue
was mechanically dissociated into a single-cell suspension. The
dissociated cells were plated into 24 plates pre-coated with
polylysine (Sigma). At 48 h later, cytosine arabinoside (5 μM)
110 was added to kill the glial cells. Cultures were maintained at
37 °C in a humidified atmosphere of 5% CO2. On the 10th day of
culture, the inhibiting activity of GlyT2 was tested. Sarcosine
(10−3 M) was added to all wells to block GlyT1 activity, and
glycine (10−4 M) was used to determine nonspecific uptake action.
115 The remaining procedures were identical to those in the GlyT1
1
Off-white solid (0.10 g, 60%). HPLC purity: 97.72%; H NMR
(400 MHz, CDCl3): δ 7.85 – 7.68 (m, 2H), 7.68 – 7.59 (m, 1H),
55 7.25 – 7.18 (m, 1H), 7.06 (td, J = 8.8, 2.1 Hz, 1H), 6.98 (dd, J =
8.8, 2.9 Hz, 1H), 4.82 – 4.60 (m, 2H), 3.58 (d, J = 13.8 Hz, 1H),
3.42 – 3.01 (m, 3H), 2.58 (s, 3H), 2.27 – 1.82 (m, 5H), 1.42 –
1.31 ppm (m, 6H); 13C NMR (101 MHz, CDCl3) 166.07, 165.02-
1
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DOI: 10.1039/C5RA04714E
Page 13 of 14
RSC Advances
test.
were calculated by non-compartmental model analysis on
Phoenix WinNonlin 6.0 (Pharsight, Mountain View, CA).
Binding assay for dopamine and serotonin receptors
Locomotion test
Dopamine receptors 1, 2, and 3 were obtained from HEK293
stable-transfecting cell lines. Binding assays were conducted as 60 Male C57BL/6J mice were pre-treated with 23q (20, 40 mg/kg,
5
described before.25, 26 3H-SCH23390 (10 nM) was used for the
binding test of D1 receptor, and H-spiperone for the D2 or D3
receptor-binding assay. (+)-butaclamol (final concentration: 10
μM) was used to determine nonspecific binding activity. After
intragastric) 60 min before the injection of PCP (5 mg/kg,
intraperitoneal). The mice were placed in a Plexiglas open field
arena (40×40×45 cm, Jiliang Co. Ltd., Shanghai, China)
connected with a video-based recording system. Automated
3
incubating 1 h at 30 °C, Tris-HCl (pre-cooled, 3 ml) was added to 65 activity was recorded, and the total travelling distance was
10 stop the binding reaction. The receptor-bound isotope was
collected with a Whatman GF/B glass fiber filter. Radioactivity
on the fiber filter was examined, and the inhibiting ratio was
calculated.
Social interaction test
3
In this test, C57BL/6J mice received daily injections of either
saline or PCP (10 mg/kg, intraperitoneal) for 2 weeks prior to
70 administration of vehicle control (saline) or compound 23q (20,
40 mg/kg, intragastric) for an additional 2 weeks. After treatment,
the mice were individually placed into an unfamiliar arena
(40×40×45 cm, Jiliang Co., Ltd., Shanghai, China)
calculated. In the binding test of 5-HT receptor, H-8-OH-DPAT
(10 nM) was used to test binding of 5-HT1A, and nonspecific
15 binding activity was determined with 5-HT (10 μM). 3H-
ketanserin was used for the 5-HT2A test, and spiperone was used
for nonspecific binding activity. The remaining procedures were
identical to those in the dopamine receptor tests.
simultaneously with
a weight-matched male mouse. Their
hERG channel assay
75 behavior was video-recorded for 10 min. The time spent in
interaction was defined as sniffing at any part of the partner's
body (mainly the anogenital area), grooming, following, crawling
over/under, or boxing/wrestling. Interactions were manually
scored.
20 The K+ current by the hERG was recorded using an Axopatch
200 A amplifier at 24–25 °C. HEK293 cells stably expressing
hERG channels were held at a resting voltage of −80 mV.
Voltage protocols were controlled by pClamp 9.0 software via the
DigiData-1322A interface (Axon Instruments, USA). Electrodes
25 (a tip resistance of 3–5 mΩ) were pulled from borosilicate grass
pipettes (Sutter Instruments, USA) and filled with a pipette
solution consisted of the following (in mM): 140 KCl, 2 MgCl2, 1
CaCl2, 10 HEPES, 10 EGTA, pH 7.4 adjusted with KOH. The
test compound was diluted (1 nM – 0.1 mM) in extracellular
30 buffer (NaCl 150 mM, KCl 4 mM, CaCl2 1.2 mM, MgCl2 1 mM,
HEPES 10 mM, pH 7.4 with NaOH, 300–310 mOsm), and was
added directly using a RSC-100 rapid solution changer with a 9-
tube head (BioLogic Co, France). After the cells were stabilized
and the currents steady, the amplitude and kinetics of IKhERG were
35 recorded. Offline analysis of the peak tail current was performed
using pClamp software 9.0. The amplitude and kinetics of IKhERG
were recorded for each drug concentration. Concentration-
response curves were fitted by nonlinear regression analysis and
IC50 values were reported.
80 Novel object recognition test
Animal treatment was identical to that described in the social
interaction test. On the first day, mice were placed into an open
field box (40×40×45 cm) for 5 min and then immediately
returned to their home cages. This was repeated on the second
85 day. On the third day, two identical objects were placed into the
open-field box and animals were allowed to explore for 5 min.
After this training, the box and objects were cleaned with 75%
ethanol to avoid possible instinctive odorant cues. On the fourth
day, mice were placed back into the same box, a novel object
90 replaced one of the objects used during training, and mice were
allowed to explore freely for 5 min. The familiar object and the
novel one were different in shape and color, but similar in size.
An exploratory preference index, or the ratio of time spent
exploring novel object over total time spent exploring both
95 objects, was used to score recognition memory.
40 Pharmacokinetic study
Male ICR mice (body weight: 18-20 g) were purchased from the
Shanghai SLAC Laboratory Animal Co. Ltd (Shanghai, China).
Animals were fasted for 12h but free access to water before
administration of the compound. All animals were fed in 3h after
45 administration. Compound was formulated in 0.5% HMPC and
administered orally by gavage at a dose of 10 mg/kg. Blood
samples (0.1 mL) were drawn from retrobulbar venous plexus of
mice at 0.5, 1，2，4，8 and 24h postdose (n = 3 mice/group)
and stored in heparinized tubes. After centrifugation for 5 min,
50 plasma (50 μL) was added MeOH/ACN (1:1, 200 μL) and
centrifuged for 10 min. Supernatant fluid was stored at -20 °C for
analysis. Brain tissues were collected at the same time and stored
at -20 °C until analysis. Brain tissues were added 10 volumes of
MeOH/ACN (1:1). After vortex and centrifugation, supernatant
55 fluid was used to analysis. A LC-MS/MS method was used for
the quantification of the tested compound. The PK parameters
Abbreviations
THF: tetrahydrofuran; DMF: N,N-dimethylformamide; DMA:
N,N-dimethylacetamide; HATU: 2-(7-aza-1H-benzotriazole-1-
yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; DIAD:
100 diisopropyl azodicarboxylate; EWG: electron-withdrawing group;
NFPS:
N[3-(4’-fluorophenyl)-3-(4’-
phenylphenoxy)propyl]sarcosine; PCP: phencyclidine; SAR:
structure–activity relationship; HPLC: high performance liquid
chromatography; EGTA: ethylene glycol tetraacetic acid; ACN:
105 acetonitrile; HMPC: hydroxypropyl methyl cellulose; i.g.:
intragastric; i.p.: intraperitoneal.
Acknowledgments
This work was financially supported by grants from the National
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RSC Advances
DOI: 10.1039/C5RPA0a4g71e4E14 of 14
20. M. Barcelo, E. Ravina, M. J. Varela, J. Brea, M. I. Loza and C. F.
Nature Science Foundation of China (Grant 81402786,
81130023), National Basic Research Plan (973) of the Ministry of
Science and Technology of China (2011CB5C4403). Supports
from Priority Academic Program Development of Jiangsu Higher
Education Institutes (PAPD) and Grant from Jiangsu Science and
Technology commission (BY2011131) are also appreciated.
Masaguer, MedChemComm, 2011, 2, 1194-1200.
21. Y. Guo, H. Zhang, X. Chen, W. Cai, J. Cheng, Y. Yang, G. Jin and X.
Zhen, Schizophrenia Research, 2009, 115, 41-49.
22. P. R. Lee, D. L. Brady, R. A. Shapiro, D. M. Dorsa and J. I. Koenig,
Neuropsychopharmacology, 2005, 30, 1883-1894.
23. K. Hashimoto, Y. Fujita, E. Shimizu and M. Iyo, European Journal
of Pharmacology, 2005, 519, 114-117.
70
5
75 24. K. R. Aubrey and R. J. Vandenberg, British Journal of
Pharmacology, 2001, 134, 1429-1436.
Notes and references
25. J. Zhang, J. Huang, Z. Song, L. Guo, W. Cai, Y. Wang, X. Zhen and
A. Zhang, European Journal of Medicinal Chemistry, 2014, 85, 16-
26.
80 26. H. Zhang, N. Ye, S. Zhou, L. Guo, L. Zheng, Z. Liu, B. Gao, X. Zhen
and A. Zhang, Journal of Medicinal Chemistry, 2011, 54, 4324-4338.
a State Key Laboratory of Drug Research, Shanghai Institute of Materia
Medica, Chinese Academy of Sciences, Shanghai 201203, China
10 b Jiangsu Key Laboratory of Translational Research and Therapy for
Neuro-Psycho-Diseases and Department of Pharmacology, Soochow
University, Suzhou 215006, China
c China Pharmaceutical University, Nanjing 210009, China
Corresponding authors: jhshen@mail.shcnc.ac.cn
15 zhenxuechu@suda.edu.cn
† Electronic Supplementary Information (ESI) available: [Preparation of
10a-h, 13a-b, 16a-e, 18a-d and 21a-b was summarized in Electronic
Supplementary Information]. See DOI: 10.1039/b000000x/
1. J. T. Coyle, D. Balu, M. Benneyworth, A. Basu and A. Roseman,
20
Dialogues in Clinical Neuroscience, 2010, 12, 359.
2. W. Danysz and C. G. Parsons, Pharmacological Reviews, 1998, 50,
597-664.
3. J. Kantrowitz and D. C. Javitt, Current Opinion in Psychiatry, 2012,
25, 96-102.
25 4. M. J. Marino, L. J. Knutsen and M. Williams, Journal of Medicinal
Chemistry, 2008, 51, 1077-1107.
5. C. Sur and G. G. Kinney, Current Drug Targets, 2007, 8, 643-649.
6. L. Raiteri and M. Raiteri, Journal of Neurochemistry, 2010, 114,
647-653.
30 7. R. J. Harvey and B. K. Yee, Nat Rev Drug Discov, 2013, 12, 866-885.
8. B. Atkinson, S. Bell, M. De Vivo, L. Kowalski, S. Lechner, V.
Ognyanov, C.-S. Tham, C. Tsai, J. Jia and D. Ashton, Molecular
Pharmacology, 2001, 60, 1414-1420.
9. A. Molander, H. H. LIDÖ, E. Löf, M. Ericson and B. Söderpalm,
35
40
45
Alcohol and Alcoholism, 2007, 42, 11-18.
10. E. Pinard, A. Alanine, D. Alberati, M. Bender, E. Borroni, P.
Bourdeaux, V. Brom, S. Burner, H. Fischer, D. Hainzl, R. Halm, N.
Hauser, S. Jolidon, J. Lengyel, H.-P. Marty, T. Meyer, J.-L. Moreau,
R. Mory, R. Narquizian, M. Nettekoven, R. D. Norcross, B.
Puellmann, P. Schmid, S. Schmitt, H. Stalder, R. Wermuth, J. G.
Wettstein and D. Zimmerli, Journal of Medicinal Chemistry, 2010,
53, 4603-4614.
11. W. P. Blackaby, R. T. Lewis, J. L. Thomson, A. S. R. Jennings, S. C.
Goodacre, L. J. Street, A. M. MacLeod, A. Pike, S. Wood, S. Thomas,
T. A. Brown, A. Smith, G. Pillai, S. Almond, M. R. Guscott, H. D.
Burns, W. Eng, C. Ryan, J. Cook and T. G. Hamill, ACS Medicinal
Chemistry Letters, 2010, 1, 350-354.
12. D. Ouellet, S. Sutherland, T. Wang, P. Griffini and V. Murthy,
Clinical Pharmacology & Therapeutics, 2011, 90, 597-604.
50 13. D. Umbricht, D. Alberati, M. Martin-Facklam, E. Borroni, E. A.
Youssef, M. Ostland, T. L. Wallace, F. Knoflach, E. Dorflinger and J.
G. Wettstein, JAMA psychiatry, 2014.
14. S. Jolidon, R. Narquizian, R. Norcross and E. Pinard, PCT Int. Appl.
(2007), WO2007147770.
55 15. P. Angell, K. J. Bordeau, Y. Chiang, N. Collar, D. M. Fink, H.
Hemmerle, J. A. Hendrix, S. A. Jackson, J. G. Jurcak and T.
Nieduzak, PCT Int. Appl. (2002), WO2002066446.
16. A. K. Ghose, T. Herbertz, R. L. Hudkins, B. D. Dorsey and J. P.
Mallamo, ACS Chemical Neuroscience, 2011, 3, 50-68.
60 17. Y. Chen, Y. Lan, X. Cao, X. Xu, J. Zhang, M. Yu, X. Liu, B.-F. Liu
and G. Zhang, MedChemComm, 2015.
18. P. A. Janssen, C. J. Niemegeers, F. Awouters, K. H. Schellekens, A.
A. Megens and T. F. Meert, Journal of Pharmacology and
Experimental Therapeutics, 1988, 244, 685-693.
65 19. S. Caccia, L. Pasina and A. Nobili, Drug design, development and
therapy, 2010, 4, 33-48.
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