Isoquinolinone derivatives as potent CNS multi-receptor D2/5-HT1A/5-HT2A/5-HT6/5-HT7 agents: Synthesis and pharmacological evaluation

Article abstract of DOI:10.1016/j.ejmech.2020.112709

In this study, a series of novel Isoquinolinone derivatives were synthesized as potential multi-target antipsychotics. Among these, compound 13 showed high affinity for dopamine D₂ and serotonin 5-HT_1A, 5-HT_2A, 5-HT₆, and 5-HT₇ receptors, showed low affinity for off-target receptors (5-HT_2C, H₁, and α₁), and negligible effects on ether-a-gogo-related gene (hERG; i.e., reduced QT interval prolongation). An animal behavioral study revealed that compound 13 reversed APO-induced hyperlocomotion, MK-801-induced hyperactivity, and DOI-induced head twitch. Moreover, compound 13 exhibited a high threshold for acute toxicity, a lack of tendency to induce catalepsy, and did not cause prolactin secretion or weight gain when compared to risperidone. Furthermore, in the forced swim test, tail suspension test, and novel object recognition test, treatment with compound 13 resulted in improvements in depression and cognitive impairment. In addition, compound 13 had a favorable pharmacokinetic profile in rats. Thus, the antipsychotic drug-like effects of compound 13 indicate that it may be useful for developing a novel class of drugs for the treatment of schizophrenia.

Full text of DOI:10.1016/j.ejmech.2020.112709

European Journal of Medicinal Chemistry 207 (2020) 112709
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Isoquinolinone derivatives as potent CNS multi-receptor D /5-HT /5-
2
1A
HT /5-HT /5-HT agents: Synthesis and pharmacological evaluation
2A
6
7
Jian Jin a , Kunxiao Zhang , Fei Dou , Chao Hao , Yifang Zhang , Xudong Cao ,
,
1
a
,
1
a
,
1
b
b
c
b
b
b
b
a,
b,
*
, Yin Chen a
, **
Lanchang Gao , Jiaying Xiong , Xin Liu , Bi-Feng Liu , Guisen Zhang
Jiangsu Key Laboratory of Marine Biological Resources and Environment, Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, School
of Pharmacy, Jiangsu Ocean University, Lianyungang, 222005, China
Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, 430074, China
Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, 209 Tongshan Road, Xuzhou, 221004, Jiangsu, China
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 May 2020
Received in revised form
In this study, a series of novel Isoquinolinone derivatives were synthesized as potential multi-target
antipsychotics. Among these, compound 13 showed high affinity for dopamine D
HT1A, 5-HT2A, 5-HT , and 5-HT receptors, showed low affinity for off-target receptors (5-HT2C, H
), and negligible effects on ether-a-gogo-related gene (hERG; i.e., reduced QT interval prolongation). An
2
and serotonin 5-
6
7
1
, and
8
July 2020
a
1
Accepted 30 July 2020
animal behavioral study revealed that compound 13 reversed APO-induced hyperlocomotion, MK-801-
induced hyperactivity, and DOI-induced head twitch. Moreover, compound 13 exhibited a high
threshold for acute toxicity, a lack of tendency to induce catalepsy, and did not cause prolactin secretion
or weight gain when compared to risperidone. Furthermore, in the forced swim test, tail suspension test,
and novel object recognition test, treatment with compound 13 resulted in improvements in depression
and cognitive impairment. In addition, compound 13 had a favorable pharmacokinetic profile in rats.
Thus, the antipsychotic drug-like effects of compound 13 indicate that it may be useful for developing a
novel class of drugs for the treatment of schizophrenia.
Available online 20 August 2020
Keywords:
Isoquinolinone
Piperidine
Multi-receptor
Atypical antipsychotics
©
2020 Elsevier Masson SAS. All rights reserved.
1
. Introduction
cardiovascular risk factors, and abnormalities in prolactin
secretion.[2e6] Therefore, novel drug types that are effective
against not only positive symptoms but also negative symptoms
and cognitive impairment would represent a significant advance-
ment in schizophrenia treatment.
In our previous study, we found that compounds AeD exerted
obvious multi-target characteristics (Fig. 2).[7-10] As shown in
Table 1, Compared to compounds A-C, compound D exhibited high
Schizophrenia is characterized by positive, negative, and
cognitive symptoms. Approximately 20% of diagnosed individuals
have permanent and severe symptoms that cause disability, and
more than 50% have non-persistent symptoms that follow a long
course.[1] Current antipsychotics (Cozapine, Olanzapine, Risperi-
done, etc, Fig. 1) are marginally effective in treating positive
symptoms but are less effective against negative symptoms and
cognitive dysfunction. Furthermore, the treatment of schizophrenia
with antipsychotics is impaired by a number of side effects, such as
motor side effects, weight gain, metabolic disturbances,
affinity for D
affinity to the 5HT
2
, D
3
, 5-HT1A, and 5-HT2A, but also had strong binding
receptor. Moreover, compound D was not only
6
effective in the animal model of positive symptoms, but also
significantly displayed procognition properties in a novel object
recognition task in rats.[10] Compound D received China Food and
Drug Administration (CFDA) approval for clinical trials in 2018.
Aripiprazole was approved by the Food and Drug Administration
for treatment of schizophrenia in 2002. Aripiprazole exhibits high
*
Corresponding author. Jiangsu Key Laboratory of Marine Biological Resources
and Environment, Jiangsu Key Laboratory of Marine Pharmaceutical Compound
Screening, School of Pharmacy, Jiangsu Ocean University, Lianyungang, 222005,
China.
affinity for dopamine D
receptors, as well as moderate affinity for dopamine D
-HT2C and 5-HT , alpha- adrenergic, and histamine H
Aripiprazole functions as a partial agonist at dopamine D
2
and D
3
and serotonin 5-HT1A and 5-HT2A
, serotonin
receptors.
and
4
*
* Corresponding author.
E-mail addresses: gszhang@mail.hust.edu.cn (G. Zhang), 2019000015@jou.edu.
5
7
1
1
cn (Y. Chen).
2
1
Jian Jin, Kunxiao Zhang and Fei Dou contributed equally to this work.
https://doi.org/10.1016/j.ejmech.2020.112709
223-5234/© 2020 Elsevier Masson SAS. All rights reserved.
0

2
J. Jin et al. / European Journal of Medicinal Chemistry 207 (2020) 112709
Fig. 1. Representative current antipsychotics.
Fig. 2. Atypical antipsychotics of our previous study.
Table 1
(Tables 1e3), and structure-activity relationship studies and
Binding affinities of compounds A-D.
competitive receptor-binding assays were performed to evaluate
their pharmacological efficacy and relative affinity for the multi-
receptor, respectively. The target compounds were subjected to
preliminary pharmacological evaluation to determine their affinity
Compound
Ki(nM)
D
2
D
3
5-HT1A
5-HT2A
5-HT2C
H
1
5-HT
6
8
A
B
C
11.9
2.6
8.7
7.5
4.3
16.6
1.66
5.2
3.3
16.9
8.6
7.5
0.3
0.79
0.72
14.1
562.5
e
for D
13 exhibited higher affinity for D
HT receptors and low affinity for H
1
2
, 5-HT1A, 5-HT2A, 5-HT
6
, 5-HT
, 5-HT1A, 5-HT2A, 5-HT
and 5-HT2C receptors.
7
, and H
1
receptors. Compound
9
1700.7
998.2
616
1125.3
2845.2
630.3
e
10
6
, and 5-
e
2
1
1
D
2.9
5.55
7
Furthermore, compound 13 reduced apomorphine-induced
climbing in animal behavioral studies as well as MK-801-induced
hyperactivity, with a high threshold for inducing catalepsy. In
addition, compound 13 lead to negligible weight gain and did not
significantly influence serum prolactin levels compared with ris-
peridone. Moreover, compound 13 displayed procognitive proper-
ties in a novel object recognition task in rats and showed favorable
pharmacokinetic properties. Compared to compounds A-D, com-
pound 13 showed also antipsychotic-like effects in vivo models in
low doses, and it was also found effective in anti-depressant and
procognitive tests. Thus, compound 13 was used to validate our
novel approach to atypical antipsychotics based on its unique
polypharmacological antipsychotic profile.
serotonin 5-HT1A receptors, and as an antagonist at the serotonin 5-
HT2A receptor.[11] Therefore, it is considered to be a dopamine-
serotonin system stabilizer, which has not been observed in the
activity of any other antipsychotic drug.[12] Brexpiprazole is a
partial agonist of 5-HT1A and D
and 0.3 nM, respectively, as well as a 5-HT2A receptor antagonist
with a K of 0.47 nM.[13] RP5063, a novel multimodal dopamine
2 i
receptors with K values of 0.12 nM
i
and 5-HT modulator, shows high affinity for D2,3,4 and 5-HT2A,2B,7
receptors. As an adjunctive treatment, RP5063 has multifaceted
effects in improving some of the cognitive deficits associated with
schizophrenia.[14] These observations encouraged us to design
multi-target ligands that can precisely modulate the activity of
several monoaminergic receptors (dopamine D
2
and serotonin 5-
2. Results and discussion
HT1A, 5-HT2A, 5-HT , and 5-HT receptors), expecting that this
6
7
multifunctional profile will contribute to the therapeutic potential
for patients with schizophrenia.
2.1. Chemistry
Aripiprazole, brexpiprazole, and RP5063 contain lactam, with
high affinity to 5-HT1A, 5HT and D receptors. In this study, a new
7 2
The general strategy for the synthesis of the studied compounds
is summarized in Schemes 1e6. As shown in Scheme 1, interme-
diate 2 was obtained under triethylamine conditions with ethox-
compound was designed to form lactam by reversing an amide
bond (Fig. 3) and linking to an arylpiperazine (piperidine) group,
which is one of the most important classes for CNS activity.[15] A
series of new compounds were produced with an appropriate
linker between the lactam molecule and privilege structures
yphenylamine and ethyl chloroformate. Under P O
2
5
and
methanesulfonic acid conditions, intermediate 2 self-condensed to
obtain intermediate 3, and reacted with iodomethane to obtain
intermediate 4. Intermediate 4 was demethylated under HBr

J. Jin et al. / European Journal of Medicinal Chemistry 207 (2020) 112709
3
Fig. 3. Design of isoquinolinone derivatives.
conditions to obtain intermediate 5. Intermediate 5 reacted with
,2-dibromoethane, 1,3-dibromopropane, and 1,4-dibromobutane
to form intermediate 6. Compound 6 reacted with arylpiperazine
2.2. In vitro studies of new compounds
2.2.1. Structure-activity relationships
1
(
piperidine), yielding compounds 7e30.
As shown in Scheme 2, intermediate 5 reacted with 2-(chlor-
2.2.1.1. Effect of compounds with different amine moieties. In this
work, our initial focus was to investigate the affinities of different
amine moieties and four-carbon chain derivative compounds on
omethyl)oxirane to produce intermediate 31, and subsequently
reacted with 6-fluoro-3-(piperidin-4-yl)benzo[d]isoxazole hydro-
chloride to give compound 32. Compound 32 reacted with dieth-
2
the D , 5-HT1A, and 5-HT2A receptors (Table 2, compounds 7e12).
ꢁ
ylamino sulfur trivalent at ꢀ78 C to obtain compound 33. As
As shown in Table 2, compound 7 bearing the (6-fluorobenzo[d]
shown in Scheme 3, intermediate 3 reacted with iodoethane,
yielding intermediate 34. Intermediate 34 was demethylated under
HBr conditions to obtain intermediate 35. Intermediate 35 reacted
with 1,3-dibromopropane to form intermediate 36. Compound 36
then reacted with 6-fluoro-3-(piperidin-4-yl)benzo[d]isoxazole
hydrochloride to produce compound 37. As shown in Scheme 4,
carboxymethyl-5-methoxybenzoic acid [38] was condensed with
methylamine to obtain intermediate 39. Intermediate 39 was
placed under sodium borohydride conditions with dilute hydro-
chloric acid under the catalysis of the generated intermediate 40.
Intermediate 40 was demethylated under HBr conditions to obtain
intermediate 41. Intermediate 41 reacted with 1,3-dibromopropane
to form intermediate 42. Compound 42 reacted with 6-fluoro-3-
isoxazol-3-yl)piperidine moiety showed moderate affinity for D
5-HT1A, and 5-HT2A receptors (D
¼ 41.6 nM; 5-HT1A
¼ 30.6 nM; 5-HT2A, K
showed a higher affinity for 5-HT1A receptors than risperidone
(K
¼ 182 nM). When the amine moieties were arylpiperazines,
compounds 8e12 exhibited low affinity for the D , 5-HT1A, and 5-
2
,
2
, K
i
,
K
i
i
¼ 28.3 nM). Moreover, compound 7
i
2
HT2A receptors. These results indicate that compounds bearing
(6-fluorobenzo[d]isoxazol-3-yl)piperidine (compound 7) possess
higher affinity for all three receptors compared with those with
other amine moieties.
Next, we investigated the affinity of different amine moieties
2
and three-carbon chain derivative compounds for the D , 5-HT1A,
and 5-HT2A receptors (Table 3, compounds 13e29). Notably, com-
pound 13 bearing a three-carbon chain showed significantly higher
(
piperidin-4-yl)benzo[d]isoxazole hydrochloride to produce com-
pound 43.
As shown in Scheme 5, 2-bromo-5-methoxybenzoic acid [44]
reacted with SOCl to obtain acyl chloride 45, and then reacted with
affinity for the D
HT1A, K
¼ 9.1 nM; 5-HT2A, K
carbon chain compound 7 (D
5-HT2A, K
¼ 28.3 nM). Moreover, compound 13 showed higher
affinity for the 5-HT1A receptor compared with risperidone
(K
2
, 5-HT1A, and 5-HT2A receptors (D
¼ 0.65 nM) compared with the four-
, K ¼ 30.6 nM;
¼ 41.6 nM; 5-HT1A, K
2
, K
i
¼ 6.9 nM; 5-
i
i
2
2
i
i
methylamine to obtain intermediate 46, which was condensed into
intermediate 47 under cuprous bromide conditions. Intermediate
7 was demethylated under HBr conditions to obtain intermediate
i
4
4
i
¼ 182 nM). The 5-HT1A receptor is implicated in the therapeutic
8. Intermediate 48 reacted with 1,3-dibromopropane to form in-
efficacy of atypical antipsychotic drugs. Specifically, 5-HT1A recep-
tor activity reduces negative symptoms and the incidence of
extrapyramidal symptoms (EPS) in patients with schizophrenia
[32],[33]. Specifically, compound 13 also displayed higher affinity
termediate 49. Compound 49 reacted with 6-fluoro-3-(piperidin-4-
yl)benzo[d]isoxazole hydrochloride to produce compound 50.
As shown in Schemes 6 and 2-(3-methoxyphenyl)ethan-1-
amine [51] reacted with ethyl chloroformate, yielding intermedi-
2 2
for the D , 5-HT1A, and 5-HT2A receptors than aripiprazole (D ,
ate 52. Under P
2
O
5
conditions with methanesulfonic acid, inter-
K
i
¼ 9.7 nM; 5-HT1A, K
i
¼ 19.8 nM; 5-HT2A, K ¼ 12.1 nM). However,
i
mediate 52 self-condensed to obtain intermediate 53, and reacted
with iodomethane, yielding intermediate 54. Intermediate 54 was
demethylated under HBr conditions to obtain intermediate 55. In-
termediate 55 reacted with 1,3-dibromopropane to form interme-
diate 56. Compound 56 reacted with arylpiperazine (piperidine) to
give compound 57.
substitution of (6-fluorobenzo[d]isoxazol-3-yl)piperidine (com-
pound 13) with (2,4-difluorophenyl)(piperidin-4-yl)methanone
(compound 14) reduced the affinity for all three receptors. Com-
pound 15 bearing the 3-(trifluoromethyl)phenyl)piperazine moiety
displayed moderate affinity for the D
¼ 123.2 nM; 5-HT2A, K ¼ 101.5 nM). Compound 16 bearing 3-
piperazin-1-yl)benzo[d]isothiazole showed high affinity for the 5-
2 2
and 5-HT2A receptors (D ,
K
i
i
(

4
J. Jin et al. / European Journal of Medicinal Chemistry 207 (2020) 112709
Table 2
Binding Affinities for D
, 5-HT1A and 5-HT2A receptors of compounds 7e12 and reference antipsychoticsa.
2
Compound
7
Receptor affinity Ki ± SEM(nM)
D
2
5-HT1A
5-HT2A
41.6 ± 5.6
30.6 ± 3.1
28.3 ± 2.9
8
9
316.3 ± 42.1
119.2 ± 12.3
215.9 ± 33.9
261.4 ± 37.6
>10000b
1039 ± 133
>10000b
131.5 ± 15.3
>10000b
1
0
1
1
>10000b
1295.5 ± 139.6
890.2 ± 91.2
298 ± 32.1
12
161.9 ± 28.6
431 ± 54
risperidone
aripiprazole
e
3.7 ± 0.3
9.7 ± 1.1
182 ± 15
19.8 ± 2.6
0.19 ± 0.02
12.1 ± 1.6
a
Ki values are taken from three experiments, expressed as means ± SEM.
The Ki values were not calculated because the inhibition percentages at 10
b
m
M were too low.
HT1A and 5-HT2A receptors (5-HT1A, K
i
¼ 12.3 nM; 5-HT2A
,
,
moderate activity at all three receptors, while that substituted with
methoxy (compounds 28 and 29) showed lower activity at all three
receptors compared with compounds 26 and 27. These results
indicate that compounds bearing electron-withdrawing groups
possess higher affinity for all three receptors compared to those
bearing electron-donating groups. According to our results, com-
pounds bearing (6-fluorobenzo[d]isoxazol-3-yl)piperidine (com-
pound 13) possessed higher affinity for all three receptors
compared to those with other amine moieties.
K
K
i
¼ 19.2 nM), but displayed weak affinity for the D receptor (D
2
2
i
¼ 923 nM). 1-(Benzo[b]thiophen-4-yl)piperazine derivative 17
, K
¼ 29.3 nM;
¼ 75.9 nM). Compound 18 bearing
-(benzo[d][1,3]dioxol-5-ylmethyl)piperazine showed low affinity
receptor (K > 10,000 nM) and high affinity for 5-HT1A
¼ 18.3 nM) and 5-HT2A (K ¼ 25.9 nM). Compound 19 bearing the
-(piperazin-1-yl)quinoline moiety showed high affinity for 5-HT1A
and 5-HT2A receptors.
showed moderate affinity for all three receptors (D
2
i
5
1
-HT1A, K
i
¼ 65.2 nM; 5-HT2A, K
i
for the D
2
i
(K
i
i
2
(K
i
¼ 10.2 nM) and weak affinity for the D
2
Phenylpiperazine derivative 20 and compound 21 bearing 1-(pyr-
idin-2-yl)piperazine showed low affinity to the three receptors.
When the 2-(piperazin-1-yl)pyrimidine moiety was the amine
moiety, compound 22 exhibited moderate affinity for the 5-HT1A
2.3. Effect of the lactam and piperidine ring linker
According to the above results, the compound bearing a three-
carbon chain (compound 13,
¼ 9.1 nM; 5-HT2A, K ¼ 0.65 nM) possessed higher affinity for all
three receptors compared with the four-carbon chain compound 7
(D , K
D
2
,
K
i
¼
6.9 nM; 5-HT1A,
and 5-HT2A receptors and weak affinity for the D
stitution of phenylpiperazine with the 1-(2-chlorophenyl)pipera-
zine or 1-(4-chlorophenyl)piperazine groups (compounds 23 and
2
receptor. Sub-
K
i
i
2
i
¼ 41.6 nM; 5-HT1A, K
i
¼ 30.6 nM; 5-HT2A, K ¼ 28.3 nM).
i
2
4) improved the affinity for all three receptors. Moreover, com-
pound 25 bearing 1-(2,3-dichlorophenyl)piperazine showed higher
affinity for the three receptors (D , K
¼ 68.2 nM; 5-HT1A
¼ 20.1 nM; 5-HT2A, K ¼ 16.8 nM) than compounds 23 and 24. The
phenyl ring substituted with F (compounds 26 and 27) displayed
Therefore, we aimed to determine the effects of the length of the
linker between the lactam and the piperidine ring. As shown in
Table 4, a chain length of two carbon atoms (30) resulted in
2
i
,
K
i
i
2
significantly reduced D , 5-HT1A, and 5-HT2A receptor binding.
Introducing OH to the carbon chain of compound 32 resulted in

J. Jin et al. / European Journal of Medicinal Chemistry 207 (2020) 112709
5
Table 3
Binding Affinities for D
, 5-HT1A and 5-HT2A receptors of compounds 13e30 and reference antipsychoticsa.
2
Compound
Receptor affinity Ki ± SEM(nM)
D
2
5-HT1A
5-HT2A
1
3
4
6.9 ± 0.7
9.1 ± 0.8
0.65 ± 0.1
1
902 ± 110
>10000b
>10000b
1
5
6
123.2 ± 21.5
923 ± 108.2
1039 ± 133
12.3 ± 3.1
101.5 ± 12.3
19.2 ± 2.8
1
17
18
19
29.3 ± 4.2
>10000b
>10000b
65.2 ± 8.9
18.3 ± 3.2
10.2 ± 2.1
79.1 ± 112.5
25.9 ± 4.5
>10000b
2
2
0
1
>10000b
>10000b
>10000b
>10000b
>10000b
1156.9 ± 26.8
2
2
3
1368 ± 236
391.2 ± 40.1
59.3 ± 7.1
65.3 ± 8.2
2
89.2 ± 10.4
179.3 ± 21.2
2
2
4
5
268.3 ± 39.2
68.2 ± 8.3
356.2 ± 42.3
20.1 ± 2.9
589.4 ± 69.1
16.8 ± 2.7
26
117.2 ± 19.1
262.2 ± 30.3
305.7 ± 41.8
(
continued on next page)

6
J. Jin et al. / European Journal of Medicinal Chemistry 207 (2020) 112709
Table 3 (continued )
Compound
Receptor affinity Ki ± SEM(nM)
D
2
5-HT1A
5-HT2A
27
28
29
532.4 ± 63.5
>10000b
158.2 ± 17.3
246.2 ± 36.9
>10000b
>10000b
896 ± 101
1109 ± 153
992 ± 130
Risperidone
Aripiprazole
e
3.9 ± 0.3
9.7 ± 1.1
182 ± 15
19.8 ± 2.6
0.19 ± 0.02
12.1 ± 1.6
a
Ki values are taken from three experiments, expressed as means ± SEM.
The Ki values were not calculated because the inhibition percentages at 10
b
m
M were too low.
weak affinities of all three receptors. Moreover, compound 33
replacement of the OH with F) showed moderate affinity for 5-
HT1A (K ¼ 23.7 nM), and low affinity
¼ 68.2 nM) and 5-HT2A (K
for the D receptor. Taken together, these data indicate that a three-
2
this replacement resulted in reduced affinity for the D and 5-
(
HT2A receptors compared with compound 13. Based on the
above results, we observed that modifications of the alkyl group
in the 4- position (R) of the lactam provided analogues with the
i
i
2
carbon chain length (compound 13) resulted in the most active
following order of
D
2
,
5-HT1A and 5-HT2A receptors:
compound.
methyl > ethyl.
2.4. Effect of substitution on the 2-position (R)
2.5. Effect of the lactam double bond
We investigated the effects of replacing the methyl (R) group
As shown in Table 4, replacing the single bond with double bond
with an ethyl group (Table 4, compound 37). Compound 37
displayed moderate activity at all three receptors (D
¼ 249.2 nM; 5-HT1A, K ¼ 198.3 nM; 5-HT2A, K ¼ 92.1 nM), and
2
resulted in preserved high affinity for the D , 5-HT1A, and 5-HT2A
2
,
receptors (compound 43, D
HT2A, K
¼ 3.5 nM).
2
, K
i
¼ 9.2 nM; 5-HT1A, K ¼ 12.1 nM; 5-
i
K
i
i
i
i
ꢁ
ꢁ
ꢁ
ꢁ
Scheme 1. Reagents and conditions: (i)ethyl chloroformate, Et
TEBA, reflux; (vi)K CO ,CH CN, reflux.
3
N, CH
2
Cl
2
,0 C-rt; (ii), P
2
O
5
, MSA, 130 C, 10h; (iii) CH
3
I,NaH,DMF, 0 C-r.t.; (iv)HBr/H
2
O, 100 C; (v) K
2
CO
3
,acetone,
2
3
3
ꢁ
Scheme 2. Reagents and conditions: (i) 2-(cholomethyl)oxirane, K
2
CO
3
, KI, acetone, reflux; (ii)K
2
CO
3
, acetonitrile, reflux.; (iii) DAST, DCM, 0 C-rt.

J. Jin et al. / European Journal of Medicinal Chemistry 207 (2020) 112709
7
ꢁ
ꢁ
Scheme 3. Reagents and conditions: (i) CH
3
CH
2
I, NaH, DMF,0 C-r.t.; (ii) HBr/H
2
O, 100 C; (ii) K
2
CO
3
2 3 3
, acetone, reflux; (iv)K CO , CH CN,reflux.
ꢁ
ꢁ
Scheme 4. Reagents and conditions: (i) methylamine, MeOH, o-dichlorobenzene, reflux; (ii) NaBH
reflux.
4
, EtOH, 0 C; (iii) HBr/H
2
O, 100 C; (iv) K
2 3
CO , acetone, reflux; (v)K
2
CO
3
, CH
3
CN,
ꢁ
ꢁ
Scheme 5. Reagents and conditions: (i) SOCl
2
, DCM; (ii) methylamine, Et
3
N, DCM, 0 C-r.t.; (iii)CuBr, 1,10-Phenanthroline, CH
2
Cl
2
, DMSO, 100 C; (iv) BBr
3
, DCM; (v) BrCH
2
CH
2
CH
2
Br,
K
2
CO , acetone, reflux; (vi)K CO , CH CN, reflux.
3
2
3
3
ꢁ ꢁ ꢁ ꢁ
3 2 2 2 5 3 2 2 2 2
N, CH Cl ,0 C-r.t.; (ii) P O , MSA, 130 C, 10h; (iii) CH I, NaH, DMF,0 C-r.t.; (iv) HBr/H O, 100 C; (v) BrCH CH CH Br,
Scheme 6. Reagents and conditions: (i) ethyl chloroformate, Et
CO , acetone, reflux; (vi)K CO , CH CN,reflux.
K
2
3
2
3
3

8
J. Jin et al. / European Journal of Medicinal Chemistry 207 (2020) 112709
Table 4
Binding Affinities for D
, 5-HT1A, and 5-HT2A receptors of compounds 30, 32, 33, 37, 43, 50, 57 and reference antipsychotics a
.
2
Compound
Structure
Receptor affinity Ki ± SEM(nM)
D
2
5-HT1A
5-HT2A
30
568.2 ± 69.5
>10000b
>10000b
3
2
3
1237 ± 145
856 ± 112
768 ± 97
3
359.2 ± 46.1
68.2 ± 8.7
23.7 ± 4.1
3
7
3
249.2 ± 35.6
198.3 ± 24.5
92.1 ± 11.2
4
9.2 ± 1.1
12.1 ± 1.3
3.5 ± 0.5
5
0
7
698 ± 79
187 ± 25.8
598 ± 72
5
>10000b
>10000b
692 ± 78
risperidone
aripiprazole
e
3.9 ± 0.3
9.7 ± 1.1
182 ± 15
19.8 ± 2.6
0.19 ± 0.02
12.1 ± 1.6
a
Ki values are taken from three experiments, expressed as means ± SEM.
The Ki values were not calculated because the inhibition percentages at 10 mM were too low.
b
2
.6. Effect of replacing the oxygen atom with methylene
Overall, compounds 13 and 43 exhibited high affinity for the D
-HT1A, and 5-HT2A receptors. These two compounds showed
higher affinity for 5-HT1A receptors compared with aripiprazole
(K ¼ 182 nM). Furthermore, com-
¼ 19.8 nM) and risperidone (K
pounds 13 (K ¼ 3.5 nM) showed higher
¼ 0.65 nM) and 43 (K
affinity for the 5-HT2A receptor than aripiprazole (K
¼ 12.1 nM).
Therefore, compounds 13 and 43 were selected for additional
studies to determine their affinity for the H , 5-HT2C , 5-HT , and
-HT receptors due to their high affinity for the D , 5-HT1A, and 5-
2
,
5
We investigated the effects of replacing the oxygen atom in
compound 13 with a CH group (Table 4, compound 50). Compound
0 displayed a decreased affinity for all three receptors compared
with compound 13.
2
i
i
5
i
i
i
2.7. Effect of changing the phenyl linker position
1
,
a
1
6
5
7
2
Finally, we changed the linker 7 position (compound 13) to the 6
HT2A receptors.
1
Blockade of a adrenergic receptors results in orthostatic hy-
potension[16], which is an unwanted side effect associated with
position (compound 57), which caused a dramatic decrease in af-
finity for the D and 5-HT1A receptors.
2

J. Jin et al. / European Journal of Medicinal Chemistry 207 (2020) 112709
9
many antipsychotic agents. According to Table 4, compounds 13
¼ 991 nM) and 43 (K ¼ 718 nM) showed lower affinity for
receptors compared with risperdone (K
¼ 3.2 nM) and aripiprazole
2.9. Intrinsic activity of compound 13 at selected receptors
(
K
i
i
a
1
i
In an agonist assay including the D
5-HT
ficacy at the 10
inhibited four receptors, D
95%. Specifically, compound 13 showed antagonism against the
following five receptors: D
, IC50 ¼ 6.3 nM; 5-HT1A, IC50 ¼ 56.9 nM;
5-HT2A, IC50 ¼ 46.3 nM; 5-HT , IC50 ¼ 39.7 nM; and 5-HT
IC50 ¼ 68.5 nM. Aripiprazole exhibits functions as a partial agonist
at D and 5-HT1A receptors, and as an antagonist at the 5-HT2A
2
, 5-HT1A, 5-HT2A, 5-HT
6
, and
(K
i
¼ 8.9 nM), suggesting that compounds 13 and 43 may not cause
7
receptors, compound 13 displaying <10% of the agonist ef-
M (Table 6). In an antagonist assay, compound 13
, 5-HT1A, 5-HT2A, 5-HT , and 5-HT , by >
orthostatic hypotension.
m
Treatment of schizophrenia with atypical antipsychotic drugs
has been associated with weight gain. Several studies have
demonstrated that there is significant correlation between affinity
2
6
7
2
for H
pound 13, which had much lower affinity (K
receptors than risperidone (K
1
and 5-HT2C receptors and weight gain[17e19]. Thus, com-
¼ 1196 nM) for H
¼ 32.6 nM) and aripiprazole
¼ 48.9 nM) may have a lower propensity to induce weight gain
Table 4). Moreover, compound 13 had lower affinity for the 5-HT2C
¼ 1687 nM) compared to risperidone (K ¼ 20.9 nM)
and aripiprazole (K
¼ 18.2 nM). Therefore, compound 13 may
exhibit a low potential to elicit treatment-associated weight gain.
Some preclinical studies have found that the 5-HT receptor
plays an important role in preventing depressive-like behaviors
20],[21] and in improving cognitive function[22],[23]. As shown
in Table 4, compound 43, risperidone, and aripiprazole display
moderate affinity for the 5-HT receptor. Interestingly, compound
3 (K receptor
¼ 4.9 nM) exhibited a higher affinity for the 5-HT
than risperidone (K ¼ 89.4 nM).
¼ 40.7 nM) and aripiprazole (K
The 5-HT receptor has been proposed as a target for atypical
antipsychotic drugs, and various pharmacological studies have
suggested that 5-HT antagonism might improve cognitive
symptoms[24],[25]. As shown in Table 4, compounds 13
¼ 9.6 nM) and 43 (K ¼ 167.2 nM) exhibit moderate affinity for
the 5-HT receptor compared with risperidone (K
¼ 1329 nM)
and aripiprazole (K
6
7
,
i
1
i
2
(K
i
receptor,[11] however, compound 13 shows antagonism to D
HT1A, and 5-HT2A receptors.
2
, 5-
(
receptor (K
i
i
Overall, the binding profile of compound 13 showed high af-
finity for dopaminergic D and serotonergic (5-HT1A, 5-HT2A, 5-HT
and 5-HT ) receptors and low affinity for , H , and 5-HT2C re-
i
2
6
,
7
a
1
1
7
ceptors and hERG channels. Clozapine to reverse phencyclidine-
elicited cortical desynchronization-a model of neural activity un-
derlying negative symptoms-requires activation of 5-HT1A re-
ceptors[27]. Clozapine can attenuate negative symptoms and
cognitive decline through its actions as a 5-HT1A partial agonist
[28],[29]. Additionally, it has been argued that antagonism of se-
rotonin 5-HT2A receptors by second generation antipsychotics
(SGAs) contributes to their efficacy, reducing the need for relatively
extensive D receptor occupancy, consequentially reducing side
2
effects[30],[31]. Pimavanserin, a selective 5-HT2A antagonist,
effectively treats psychosis in Parkinson’s disease without causing
EPS[32],[33].5-HT antagonists enhance dopamine release in the
6
[
7
1
i
7
i
i
6
6
(K
i
i
6
i
prefrontal cortex and regulate interactions between the cholinergic
and glutamatergic systems, which is beneficial for the excitability
and conductibility of neurotransmission and results in improved
i
¼ 318 nM). According to these results, com-
pound 13 may improve cognitive symptoms and depressive-like
behaviors.
7
memory and cognitive functions[34].5-HT receptor antagonists
have been shown to produce a rapid-onset antidepressant effect in
rodent models, while much more complex actions have been
observed in cognitive assays.[35],[36] Therefore, combined with its
2.8. Ether-a-gogo-related gene KC channels and acute toxicity
6 7
affinity for 5-HT and 5-HT , the actions of compound 13 at the
Recent studies have shown that a wide range of medications can
desired receptors suggest a promising therapeutic potential in
terms of enhancing cognitive function in subjects with schizo-
phrenia as well as an adjunctive treatment for depression. Several
studies have demonstrated that 5-HT2C and H antagonists likely
1
lead to weight gain. The two antipsychotic drugs with the greatest
effects on body weight, olanzapine and clozapine, also have high
prolong the QT interval, defined as the length of time between the
start of the Q wave and the end of the T wave, on electrocardiogram.
Specific examples include sertindole and grepafloxacin[26]. Efforts
to predict increased risk for long QT syndrome have focused on
assays that test hERG channel activity. As shown in Table 5, the
affinity of compound 43 (IC50 ¼ 598 nM) for hERG was comparable
affinity for the 5-HT2C and H
1
receptors, which implicates these
to that of risperidone (IC50 ¼ 0.467
mM). Interestingly, the low af-
receptors in antipsychotic-induced weight gain[37]. Compound 13
showed low affinity for 5-HT2C and H , likely resulting in a lower
1
propensity to induce weight gain.
finity of compound 13 (IC50 ¼ 2312 nM) for hERG compared with
risperidone and aripiprazole may signify a decreased propensity for
eliciting treatment-induced QT interval prolongation. More
importantly, when tested in vivo in an anesthetized guinea pig
model, compound 13 showed no QT prolongation or cardiac lia-
bility, even at the highest dose (0.04, 0.2, and 0.4 mg/kg, intraper-
itoneal, Supporting Information). Next, we determined the acute
2.10. In vivo studies
Initial behavioral screening was performed on compound 13 on
the basis of its multiple receptor affinity profile. Atypical antipsy-
chotics have been used to relieve positive symptoms at doses that
do not result in EPS. In this study, the side effect profile of com-
pound 13 was evaluated using the horizontal bar test, which is
toxicity profiles of the compounds by determining their LD50
.
Compound 13 exhibited low affinity for the H and 5-HT2C re-
1
ceptors and hERG, and displayed a good safety profile, even at the
highest dose tested (LD50 > 2000 mg/kg).
Table 5
Binding affinities for D
3
, 5-HT2C and H
Receptors of compounds 13, 43 and reference antipsychoticsa.
1
Compound
Receptor affinity Ki ± SEM (nM)
(IC50, nM)
hERG
a
1
5-HT2C
H
1
5-HT
7
5-HT
6
1
4
3
3
991 ± 122
718 ± 80
3.2 ± 0.5
8.9 ± 1.2
1687 ± 159
129.3 ± 14.9
20.9 ± 2.2
18.2 ± 2.9
1196 ± 122
117.9 ± 11.9
32.6 ± 4.7
48.9 ± 5.8
4.9 ± 0.6
9.6 ± 1.1
2312 ± 298
598 ± 65
167 ± 23
896 ± 98
108.7 ± 16.8
40.7 ± 5.3
89.4 ± 9.7
167.2 ± 8.3
1329 ± 143
318 ± 49
risperidone
aripiprazole
a
Ki values are taken from three experiments, expressed as means ± SEM.

10
J. Jin et al. / European Journal of Medicinal Chemistry 207 (2020) 112709
Table 6
Activities of compound 13 and reference compounds to D
2
, 5-HT1A, 5-HT2A, 5-HT
6
and 5-HT
7
receptors.
EC50 (nM)
Receptor
Compd
Activation (10
m
M, %) (n ¼ 3)
Inhibition (10
n ¼ 3)
m
M, %)
IC50 (nM)
(
D
2
Dopamine
SCH 2 3390
99.2 ± 5.3
18.2
5.8
100.1 ± 2.4
99.9 ± 4.3
25.8
6.3
13
5.2 ± 0.5
98.3 ± 3.5
5
5
5
5
-HT1A
5-HT
WAY-100635
98.8 ± 1.8
98.7 ± 2.5
2.8
56.9
13
5.8 ± 0.3
101.6 ± 1.2
-HT2A
5-HT
ketanserin
1.1
99.4 ± 0.6
100.1 ± 1.4
19.6
46.3
13
22 ± 0.3
96.1 ± 3.1
-HT
-HT
6
7
5-HT
Clozapine
42.1
5.1
97.6 ± 5.3
99.5 ± 4.9
20.3
39.7
13
3.5 ± 0.3
99.1 ± 4.9
5-CT
Methiothepin
100.1 ± 4.2
99.1 ± 2.8
11.3
12.5
13
2.3 ± 0.3
sensitive for catalepsy induced by dopamine D
2
receptor blockade.
suggest that the therapeutic index of compound 13 as a function of
its efficacy, measured with apomorphine, MK-801, and DOI tests,
and its side effects, measured with the catalepsy test, was in the
range of 119e139, while the therapeutic indices of both risperidone
and aripiprazole were roughly 19e80. Thus, in contrast to risperi-
done and aripiprazole, compound 13 had a higher threshold for
inducing catalepsy, which may translate into lower clinical EPS
liability.
Overall, compound 13 significantly inhibited apomorphine-
induced climbing behavior, MK-801-induced hyperactivity, and
DOI-induced head twitch without causing catalepsy. These results
suggest a preferential ability of compound 13 to modulate meso-
limbic instead of nigrostriatal dopaminergic neurotransmission.
These findings highlight its atypicality and low propensity to
induce unwanted extrapyramidal motor disturbances at thera-
peutically useful doses.
The antipsychotic potential of compound 13 was assessed using the
apomorphine-induced climbing, dizocilpine (MK-801)-induced
hyperactivity, and 2,5-dimethoxy-4-iodoamphetamine (DOI)-
induced head twitch tests.
The apomorphine-induced climbing model has been classically
linked to motor agitation as one of the schizophrenia positive
symptoms[38]. Based on its binding profile (Table 6), compound 13
was selected for evaluation in a mouse model of the positive
symptoms of schizophrenia (Fig. 4). Compound 13 exhibited dose-
dependent antagonism of apomorphine-climbing behavior in mice,
with higher doses producing greater inhibition (ED50, 0.06 mg/kg).
In comparison, risperidone, aripiprazole, and haloperidol produced
reversal of apomorphine-induced climbing with ED50 values of
0
.02, 0.25, and 0.09 mg/kg, respectively (Table 7).
MK-801, a highly selective non-competitive NMDA receptor
antagonist, induces hyperlocomotion and other signs of disorga-
nized behavior[39]. Compound 13 showed significant dose-
dependent responses of MK-801-induced hyperactivity (Fig. 5),
with an ED50 value of 0.08 mg/kg (Table 7). In comparison, risper-
idone, aripiprazole, and haloperidol yielded ED50 values of 0.04,
0
.32, and 0.11 mg/kg, respectively. These results indicate that
compound 13 was more potent than aripiprazole in inhibiting MK-
01-induced hyperactivity.
,5-DOI is a 5-HT2A/2C agonist that can elicit head twitch
8
2
behavior mediated by activation of the 5-HT2A receptor in mice[40].
To further evaluate the inhibitory effect of compound 13 on the 5-
HT2A receptor in vivo, mice were treated with compound 13 and the
DOI-induced head twitch test was performed. As shown in Fig. 6,
DOI caused head twitch responses in mice, which were suppressed
by pretreatment with compound 13 in a dose-dependent manner
(
ED50, 0.07 mg/kg) (Table 7). In comparison, risperidone and ari-
piprazole yielded ED50 values of 0.04 and 0.58 mg/kg, respectively.
Based on the antagonistic effects of compound 13 on 5-HT2A re-
ceptors observed in vitro (i.e., antagonism of 5-HT2A receptor in
functional test) and in vivo (i.e., suppressing DOI-induced head
twitch behavior in mice) compared with aripiprazole, we speculate
that the therapeutic potential of compound 13 in treatment-
resistant depression largely may rely on its 5-HT2A receptor
antagonistic activity.
Catalepsy is often used as a measure to predict the incidence of
extrapyramidal motor disorders. Haloperidol had the highest pro-
pensity to induce catalepsy (ED50, 0.32 mg/kg), in agreement with
the high affinity of this drug to block D receptors (Table 7). By
2
contrast, compound 13 exhibited a low potential to induce cata-
Fig. 4. Effect of compound 13 administered po on APO (apomorphine)-induced
climbing in mice (1.0 mg/kg (sc) of the apomorphine,10/group). Results are expressed
as means SEM of score. statistical significances of drug effects were analyzed by the
nonparametric two-tailed ManneWhitney U test: #p < 0.05 versus vehicle treatment;
lepsy, with an ED50 of 8.35 mg/kg (Table 7). Moreover, these results
**p < 0.01, *p < 0.05 versus apomorphine treatment.

J. Jin et al. / European Journal of Medicinal Chemistry 207 (2020) 112709
11
Table 7
In vivo pharmacological profile of compound 13. Inhibition of different behavioral responses after oral administration of the test and reference Compounds.
Compound
ED50(mg/kg, po)
APOa
CAT/APO
CAT/MK-801
CAT/DOI
MK-801b
DOIc
CATd
1
3
0.06
0.02
0.25
0.09
0.08
0.04
0.32
0.11
0.07
0.04
0.58
e
8.35
1.62
11.23
0.32
139.2
81
44.9
3.5
104.3
40.5
35.1
2.9
119.3
40.5
19.4
e
risperidone
aripiprazole
haloperidol
a
APO:Apomorphine-induced climbing.
MK-801-induced Hyperactivity.
DOI: DOI-induced head twitch (mg/kg, po).
Catalepsy.
b
c
d
reduced immobility time in mice[43]. Next, the FST and TST were
performed in 5-HT receptor gene knockout mice, resulting in
significantly reduced immobilization time compared with wild-
type mice, indicating that the 5-HT receptor gene knockout
resulted in an antidepressant effect[44]. Several studies have
shown that 5-HT antagonists result in excellent antidepressant-
7
7
7
like effects during the FST[45],[46]. The antidepressant effects of
compound 13 were evaluated using the TST and FST in a mouse
model of acute depression with duloxetine as the positive control.
As shown in Figs. 7 and 8, oral administration of duloxetine (20 mg/
kg) decreased the duration of immobility. Compared to the control
group, compound 13 caused a dose-dependent reduction in
immobility time, with a lower MED （Minimum Effect Dose) of
0.06 mg/kg in both tests.
Fig. 5. Effect of compound 13 administered po on MK-801-induced hyperactivity in
mice (0.3 mg/kg (sc) of MK-801, 10/group). Results are expressed as means SEM of
distance traveled. Statistical evaluation was performed by two-way ANOVA followed
by Tukey test for multiple comparisons. #p < 0.05 versus vehicle treatment; **p < 0.01,
2
.12. Evaluations of learning, memory, and cognitive function
The present in vitro results indicated that compound 13 exerted
*p < 0.05 versus MK-801 treatment.
potential effects on 5-HT
6
(K
i
¼ 9.6 nM) and 5-HT
7
(K
i
¼ 4.9 nM),
which are both associated with cognitive ability[36],[38]. The ob-
ject recognition test, also known as the novel object recognition
test (NOR), is a relatively fast and efficient means for testing
different phases of learning and memory in mice[47], and can be
used to predict the potential of a novel drug for cognitive
enhancement[48].
2.11. Forced swim test and tail suspension test in mice
The antidepressant-like activity of the test compound was
screened using the forced swim test (FST)[41] and the tail sus-
pension test (TST)[42]. In the FST and TST models, the 5-HT re-
7
ceptor antagonist SB-269970 did not affect locomotor activity but
In this model, compound 13 was orally administered 1 h prior to
the acquisition trial, and the exploration times for the two identical
objects were recorded (Fig. 9A). After a 24 h acquisition trial, one of
the familiar objects was replaced with a novel object, the time spent
investigating each of the objects was recorded (Fig. 9B), and the
novelty discrimination index (NDI) was calculated as the percentage
of novel object interaction time relative to total interaction time
during the retention trial (Fig. 9C). Oral administration of compound
13 (0.02, 0.06, and 0.2 mg/kg) had no obvious influence on total
exploration time (Fig. 9A), similar to oral administration of risperi-
done and rivastigmine (0.2 mg/kg). As a positive control, riva-
stigmine (0.2 mg/kg) exerted obvious influence on total exploration
time (Fig. 9B). Furthermore, rats treated with 0.06 and 0.2 mg/kg of
compound 13 during the retention trial (Fig. 9B) explored the novel
object for a longer time period, indicating preserved memory for the
familiar object presented during the acquisition trial. By contrast,
rats treated with vehicle or dosed with 0.02 mg/kg of compound 13
did not exhibit differences in exploration time for the familiar and
novel objects, indicating deterioration or loss of memory for the
familiar object. Moreover, a 0.2-mg/kg oral dose of compound 13
and rivastigmine significantly increased the NDI (Fig. 9C). These
results suggest that compound 47 enhanced recognition memory
during the NOR task in rats. Taken together, these results demon-
strate that compound 13 potentially plays a role in the promotion of
recognition memory in this rat model, which suggests that this
novel compound may improve cognitive function.
Fig. 6. Effect of compound 13 administered po on DOI (2,5-dimethoxy-4-
iodoamphetamine)-induced head twitch in mice (1.0 mg/kg (ip) of DOI, 10/group).
Results are expressed as means SEM of head twitch. Statistical evaluation was per-
formed by two-way ANOVA followed by Tukey test for multiple comparisons. #p < 0.05
versus vehicle treatment; **p < 0.01, *p < 0.05 versus DOI treatment.

12
J. Jin et al. / European Journal of Medicinal Chemistry 207 (2020) 112709
Fig. 7. Effect of treatment of mice with compound 13 given po at graded doses on the
immobility time in the forced swim test. Results are represented as mean ± SEM. One-
way ANOVA followed by Dunnett’s test: **p < 0.01, *p < 0.05 compared with the
vehicle control group (n ¼ 10).
2.13. Locomotor activity test
The present study included a locomotor activity test to identify
false positive effects and to check for the possible occurrence of
drug-induced changes in locomotor activity that may have
contributed to altered behavior in the models. Previous studies
have shown that clonazepam can cause sedation[49], and thus, this
drug was used as a reference compound. Locomotor activity ana-
lyses indicated that compound 13 does not significantly alter total
travel distance at doses of 0.06, 0.2, and 0.6 mg/kg, which likely
excludes false positive antipsychotic and antidepressant effects
(Table 8).
2.14. Weight gain and serum prolactin
SGAs have many side effects, including weight gain and hyper-
prolactinemia[6]. As shown in Fig. 10, compound 13 resulted in
Fig. 9. Effects of compound 13 on NOR test in rats (10/group). Experimental results
acquired 60 min after rats oral administration of vehicle and 13 (0.02, 0.0.6, and
0.2 mg/kg), rivastigmine (0.2 mg/kg) and risperidone (0.2 mg/kg). (A)The time spent in
exploring two identical objects during acquisition trials; (B) The time spent in
exploring a familiar and novel object during acquisition trials. (C) Novelty discrimi-
nation index (NDI) are shown as the mean ± SEM (n ¼ 10). **, p < 0.01， *, p < 0.05 vs
familiar object by paired t-test; #, p < 0.05 vs vehicle by one-tailed Williams’ test.
Fig. 8. Effect of treatment of mice with compound 13 given po at graded doses on the
immobility time in the Tail suspension test. Results are represented as mean ± SEM.
One-way ANOVA followed by Dunnett’s test: **p < 0.01, *p < 0.05 compared with the
vehicle control group (n ¼ 10).

J. Jin et al. / European Journal of Medicinal Chemistry 207 (2020) 112709
13
Table 8
potential multi-target antipsychotics. Among the derivatives syn-
thesized, compound 13 showed high affinity for dopamine D and
serotonin 5-HT1A, 5-HT2A, 5-HT , and 5-HT receptors, and a
satisfactory selectivity profile for non-target receptors (5-HT2C, H
and ) that have been closely linked to the negative side effects of
Effects of the compounds 13, haloperidol and clonazepam on the spontaneous lo-
comotor activity in mice (mean ± SD, n¼8).
2
6
7
Compound
Dose (mg/kg)
Total distance (mm)
1
,
control
clonazepam
e
1
3
13579 ± 8864
12188 ± 5801
5784 ± 2302*
762 ± 253**
12697 ± 6818
10829 ± 5512
8876 ± 2095
a
1
other marketed antipsychotics. In vivo animal models showed that
compound 13 reversed APO-induced hyperlocomotion, MK-801-
induced hyperactivity, and DOI-induced head twitch. Moreover,
compound 13 exhibited low levels of inhibition at the hERG chan-
nel, which is associated with no QT prolongation in a guinea pig
model, a high threshold for acute toxicity, the lack of a tendency to
induce catalepsy, and no effect on prolactin secretion or weight
gain. Furthermore, the results of the FST, TST, and NOR tests
demonstrated that compound 13 showed improvements in
depression and cognitive impairment. Finally, pharmacokinetic
studies demonstrated that compound 13 had a favorable drug-like
pharmacokinetic profile. We anticipate that compound 13 might be
useful for developing a novel class of drugs for the treatment of
schizophrenia.
10
13
0.06
0
0
.2
.6
**p < 0.01, *p < 0.05 vs control.
negligible weight gain in mice receiving chronic dosing for 28 days.
Risperidone, used as a positive control, resulted in significantly
increased weight gain in mice that received medium and high
doses (Fig.10). This was also consistent with the estimated K
for the H (risperidone, 32.6 nM; compound 13, 1196 nM) and 5-
HT2C (risperidone, 20.9 nM; compound 13, 1687 nM) receptors.
Moreover, compound 13 did not significantly influence serum
prolactin levels compared with risperidone (Fig. 11).
i
values
1
4. Experimental section
2
.15. Selectivity profile of compound 13
Chemistry. All commercially available chemicals and reagents
were used without further purification. Reagents were all of
analytical grade or of chemical purity (>95%). Melting points were
The interactions between compound 13 and other receptors
1
related to CNS disorders were evaluated and a selectivity profile
was created using additional receptors, including the D , D , H
SERT, NET, DAT, sigma-1, and sigma-2 receptors. Compound 13
showed moderate affinity for the D receptor (K
¼ 226 nM), with
no significant affinity (K > 1000 nM) for any other putative target.
determined in open capillary tubes and are uncorrected. H NMR
1
3
,
a
2
3
,
spectra was recorded on a Bruker Avance III 600 spectrometer at
1
400 MHz ( H) using CDCl
3
as solvent. Chemical shifts were given in
3
i
d values (ppm), using tetramethylsilane (TMS) as the internal
standard; coupling constants (J) were given in Hz. Signal multi-
plicities were characterized as s (singlet), d (doublet), t(triplet), q
(quartet), m (multiplet), br (broad signal). Analytical thin layer
chromatography (TLC) was performed on silica gel GF254. Column
chromatographic purification was carried out using silica gel.
Compound purity is determined by high performance liquid chro-
matography (HPLC), and all final test compounds display purity
higher than 95%. HPLC methods used the following: Shimadzu LC-
i
2
.16. Pharmacokinetic studies of compound 13
The pharmacokinetic properties of compound 13 including its
in vitro and in vivo efficacy and its safety profile were explored in
rats (Table 9). Intravenous administration of compound 13 in rats
(
0.5 mg/kg, n ¼ 6) resulted in detectable plasma levels [half-life (t1/
) ¼ 0.94 h]. Oral administration of compound 13 in rats (5 mg/kg,
2
0AD
(4.6 mm ꢂ 250 mm, 5
KH PO (0.2%Et
0.01 mol/L KH PO
spectrometer;
column,
SHIMADZU
VP-ODS
2
n ¼ 6) resulted in a t1/2 of 3.25 h. The area under the curve value of
compound 13 was 2350 ng ꢂ h/mL after intravenous administra-
tion and 5194 ng ꢂ h/mL after oral administration. The Cmax value
after oral dosing was 1686 ng/mL, and the Tmax value was 0.54 h.
The bioavailability of compound 13 was 22.1%. These encouraging
preclinical data suggest that compound 13 possesses desirable
drug-like pharmacokinetic properties that may be applicable to
humans.
mm) C18-253; mobile phase A: 0.01 mol/L
2
4
3
N, pH ¼ 3.5) aq./CH OH ¼ 90/10; mobile phase B:
3
2
4
(0.2%Et
3
N, pH ¼ 3.5) aq./CH
3
OH ¼ 20/80; flow
ꢁ
rate, 1.0 mL/min; column temperature, 35 C. UV detection was
performed at 210 nm.
4.1. Ethyl 4-methoxyphenethylcarbamate [2]
To a solution of ethyl chloroformate (300 mmol) in 200 mL of
dry dichloromethane a solution of 1 (250mol) and triethylamine
3
. Conclusion
(375 mmol) in 600 mL of dichloromethane was added dropwise
In summary, we here described the synthesis and pharmaco-
logical evaluation of a series of Isoquinolinone derivatives as
under ice-cooling. A white precipitate (the hydrochloride of the
amine) formed immediately. After vigorous stirring for 1 h at r.t.,
Fig. 10. Influence of compound 13 and risperidone on weight gain. Each value is the mean ± SEM of 10 mice per group. Student’s t-test: **p < 0.01, *p < 0.05 versus vehicle group.

14
J. Jin et al. / European Journal of Medicinal Chemistry 207 (2020) 112709
Fig. 11. Serum prolactin (PRL) multiple (28 days) administration of compound 13 and risperidone in mice. Each value is the mean ± SEM of 10 mice per group. Student’s t-test:
*p < 0.01, *p < 0.05 versus vehicle group.
*
the reaction mixture was quenched with water (100 mL), washed
with 2 N HCl solution, brine and dried over Na SO . The organic
was purified with column chromatography (petroleum ether/
2
4
EtOAc ¼ 10: 1) to afford compound 4, as a yellow oil (yield,
1
phase was evaporated under reduced pressure to give crude oil. The
resulting oil solidified on standing. The crude mixture was purified
with column chromatography (petroleum ether/EtOAc ¼ 1: 20) to
90.1%). H NMR (400 MHz, CDCl
3
)
d
7.59 -7.57 (m, 1H), 7.10 - 6.78 (m,
2H), 3.80 (s, 3H), 3.52 - 3.47 (m, 2H), 3.11 (s, 3H), 2.91 - 2.87 (m,
13
3
2H). C NMR (100 MHz, CDCl ) d 164.66, 158.62, 130.15, 128.03,
1
afford compound 2, as a pale-yellow oil (yield, 91.8%). H NMR
118.79, 111.39, 55.44, 48.34, 35.17, 26.97, 21.00. MS (ESI) m/z: 192.0
þ
þ
(
(
400 MHz, CDCl
3
)
d
7.14 (d, J ¼ 8.5 Hz, 1H), 6.97 - 6.80 (m, 1H), 4.13
(calcd 192.1 for C10
H
12NO
2
[M þ H] ).
q, J ¼ 7.1 Hz, 1H), 3.81 (s, 1H), 3.41 (t, J ¼ 6.7 Hz, 1H), 2.78 (t,
13
J ¼ 7.1 Hz, 1H), 1.26 (t, J ¼ 7.1 Hz, 1H). C NMR (100 MHz, CDCl
3
)
4.4. Synthesis of 7-hydroxy-2-methyl-3,4-dihydroisoquinolin-
1(2H)-one [5]
d
3
H] ).
158.24, 156.67, 130.88, 129.76, 129.73, 114.02, 60.68, 55.25, 42.34,
þ
5.27, 14.69. MS (ESI) m/z: 224.1 (calcd 224.2 for C12
H17NO
3
[M þ
þ
To a stirred solution of compound 4 (41.9 mmol) was added
hydrobromic acid in water solution (48%) (32.0 mL) and reaction
ꢁ
4.2. 7-Methoxy-3,4-dihydroisoquinolin-1(2H)-one [3]
mixture was refluxed at 100 C overnight. The reaction mixture was
quenched with water (64.0 mL)and then extracted with DCM
To a solution of P (300 mmol) in 70 mL of methanesulfonic
2
O
5
(5 ꢂ 50 mL), The organic phase was dried over Na
2 4
SO and
acid, 2 (200 mmol) were added. The solution was heated for 2 h
concentrated under reduced pressure to colorless solid. The solid
was further purified with column chromatography (DCM/
MeOH ¼ 30:1) to afford compound 5, as a white solid. (yield, 83.8%),
ꢁ
under 130 C, excess methanesulfonic acid was removed in vacuo,
and the resulting residue was quenched with ice-water. The solu-
ꢁ
1
tion was neutralised with NaHCO
dichloromethane. The combined organic phases were washed
twice with water, dried with Na SO4.The organic phase was evap-
3
and extracted three times with
mp: 188e189 C. H NMR (400 MHz, CDCl ) d 8.47 (s, 1H), 8.08 (d,
3
J ¼ 2.6 Hz, 1H), 7.05 (d, J ¼ 8.2 Hz, 1H), 6.99 (dd, J ¼ 8.2, 2.6 Hz, 1H),
13
2
3.58 (dd, J ¼ 8.7, 4.8 Hz, 2H), 3.19 (s, 3H), 2.94 (t, J ¼ 6.7 Hz, 2H). C
orated under reduced pressure to give crude oil. The crude mixture
was purified with column chromatography (petroleum ether/
EtOAc ¼ 1: 1) to compound 3, as a white yellow solid (yield, 52.5%),
NMR (100 MHz, CDCl 165.44, 156.22, 129.54, 129.02, 128.11,
3
) d
119.52, 115.22,48.67, 35.39, 26.87. MS (ESI) m/z: 177.9 (calcd 178.1
þ
þ
for C10
H
11NO
2
[M þ H] ).
ꢁ
1
3
mp:117.5e118.5 C. H NMR (400 MHz, CDCl ) d 7.65 (s, 1H), 7.58 (d,
J ¼ 2.8 Hz, 1H), 7.11 (d, J ¼ 8.3 Hz, 1H), 6.99 (dd, J ¼ 8.3, 2.8 Hz, 1H),
4.5. 7-(3-Bromopropoxy)-2-methyl-3,4-dihydroisoquinolin-1(2H)-
one [6]
13
3
.83 (s, 4H), 3.54 (td, J ¼ 6.6, 2.8 Hz, 2H), 2.90 (t, J ¼ 6.6 Hz, 2H). C
NMR (100 MHz, CDCl 166.78, 158.69, 131.14, 129.95, 128.40,
3
) d
1
19.52, 111.07, 55.52, 40.29, 27.41. MS (ESI) m/z: 178.09 (calcd 178.1
The
dibromopropane(56.5 mmol), K2CO3 (84.6 mmol), TEBA
1.4 mmol) and acetone (50.0 mL), was heated under reflux for 6 h.
Water (300 ml) was added to the reaction solution, which was then
extracted with DCM, The organic phase was dried over Na SO
mixture
of
compound
5
(28.2
mmol),1,3-
þ
for C11
H13NO
2
þ[M þ H] )
(
4.3. 7-Methoxy-2-methyl-3,4-dihydroisoquinolin-1(2H)-one [4]
2
4
To a solution of sodium hydride (97.5 mmol) in 50 mL of dry
evaporated under reduced pressure to give crude oil. The residue
N,N-dimethylformamide, a solution of compound 3 (65.0 mmol) in
0 mL of N,N-dimethylformamide and iodomethane (71.5 mmol)
was purified with column chromatography (petroleum ether/
1
3
EtOAc ¼ 15: 1) to yield compound 6, as a yellow oil (yield, 87.3%). H
was added dropwise under ice-cooling. The reaction mixture was
stirred at room temperature for 1.5 h. The reaction was quenched
with 480 ml water, extracted with ethyl acetate (100 mL ꢂ 3),
3
NMR (400 MHz, CDCl ) d 7.63 - 7.60 (m, 1H), 7.16 - 7.04 (m, 1H), 6.99
- 6.95 (m, 1H), 4.18 - 4.12 (m, 2H), 3.63 - 3.51 (m, 4H), 3.26 - 3.09 (m,
13
2H), 3.02 - 2.84 (m, 2H), 2.35 - 2.29 (m, 2H). C NMR (100 MHz,
CDCl 164.63, 164.60, 157.75, 157.73, 130.44, 130.34, 130.33,
128.12, 119.21, 119.19, 119.15, 112.31, 65.54, 65.53, 48.39, 48.37,
washed with brine, and dried over Na
2
SO
4
. The organic phase was
3
) d
evaporated under reduced pressure to give crude oil. The residue
Table 9
Pharmacokinetic profile of compound 13 in rats (n ¼ 6/group).
Cmax (ng/mL)
T
1/2 (h)
T
max (h)
CLz (mL/min/kg)
Vz (L/kg)
AUC(0-t) (ng ꢂ h/mL)
AUC(0-∞)
(ng ꢂ h/mL)
F (%)
Dose
0
5
.5 mg (i.v)
mg (po)
2566
1686
0.94
3.25
0.08
0.54
14.7
7.68
1.16
30.71
2350
5194
2370
5288
e
22.1

J. Jin et al. / European Journal of Medicinal Chemistry 207 (2020) 112709
15
3
5.27, 35.25, 32.31, 32.29, 30.03, 27.06, 27.04. MS (ESI) m/z: 178.2
1H), 7.39 - 7.29 (m, 1H), 7.10 - 7.01 (m, 1H), 7.01 - 6.88 (m, 1H), 4.13 -
3.98 (m, 2H), 3.64 - 3.45 (m, 6H), 3.21 - 3.08 (m, 3H), 2.98 - 2.84 (m,
þ
(
calcd 178.1 for C10
H12NO
2
þ[M þ H] ).
2
H), 2.78 - 2.62 (m, 4H), 2.54 - 2.41 (m, 2H), 1.90 - 1.78 (m, 2H), 1.78
13
4.6. General procedures for the preparation of compounds 7-31
- 1.59 (m, 2H). C NMR (101 MHz, CDCl
3
) d 164.78, 163.97, 158.12,
152.71, 130.27, 130.07, 128.04, 127.54, 123.97, 123.90, 120.56, 119.37,
The mixture of compound 6 (16.8 mmol), arylpiperazine
112.15, 67.94, 58.31, 53.06, 50.10, 48.43, 35.28, 27.23, 27.07, 23.40.
þ
(
(
piperidine) (18.5 mmol), K
2
CO
3
(50.4 mmol) and acetonitrile
HRMS (ESI) m/z: 451.2159 (calcd 451.2168 for C25
H
31
N
4
O
2
S
[M þ
þ
50.0 mL), was heated under reflux for 8 h. Water (300 ml) was
H] ).
added to the reaction solution, which was then extracted with
DCM, The organic phase was dried over Na SO evaporated under
reduced pressure to give yellow solid, The residue was purified with
column chromatography (DCM: MeOH ¼ 20 : 1) to afford target
compounds.
7-(4-(4-(Benzo[b]thiophen-4-yl)piperazin-1-yl)butoxy)-2-
2
4
methyl-3,4-dihydroisoquinolin-1(2H)-one [12] as a white solid
ꢁ
1
(yield, 69.6%), mp: 118e120 C H NMR (400 MHz, CDCl ) d 7.61 (d,
3
J ¼ 2.8 Hz, 1H), 7.53 (d, J ¼ 8.0 Hz, 1H), 7.39 (dd, J ¼ 10.8, 5.2 Hz, 2H),
7.31 - 7.21 (m 1H), 7.05 (d, J ¼ 8.0 Hz, 1H), 6.96 (dd, J ¼ 6.8, 1.6 Hz,
1H), 6.88 (d, J ¼ 7.2 Hz, 1H), 4.05 (t, J ¼ 6 Hz, 2H), 3.50 (t, J ¼ 6.8 Hz,
2H), 3.25 - 3.09 (m, 7H), 2.90 (t, J ¼ 6.8 Hz, 2H), 2.70 (s, 4H), 2.54 -
7
-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)
butoxy)-2-methyl-3,4-dihydroisoquinolin-1(2H)-one [7] as a
ꢁ
1
13
white solid (yield, 86.0%), mp: 130e132 C. H NMR (400 MHz,
CDCl 7.76 - 7.68 (m, 1H), 7.66 - 7.56 (m, 1H), 7.27 - 7.16 (m, 1H),
.11 - 7.00 (m, 2H), 7.00 - 6.92 (m,1H), 4.10 - 3.99 (m, 2H), 3.58 - 3.48
m, 2H), 3.20 - 3.01 (m, 6H), 2.98 - 2.86 (m, 2H), 2.56 - 2.39 (m, 2H),
.23 - 2.00 (m, 6H), 1.92 - 1.79 (m, 2H), 1.73 (s, 2H). 13C NMR
101 MHz, CDCl 165.31, 164.76, 163.85, 162.82, 161.10, 158.11,
30.17, 128.03 (s, 8H), 122.75, 119.34, 117.28, 112.45, 112.17, 97.51,
2.46 (m, 2H), 1.90 - 1.78 (m, 2H), 1.78 - 1.67 (m, 2H). C NMR
3
)
d
3
(101 MHz, CDCl ) d 164.79, 158.16, 148.54, 141.10, 134.08, 130.28,
130.09, 128.05, 125.06, 124.94, 121.99, 119.41, 116.96, 112.19, 112.15,
67.98, 58.31, 53.60, 53.53, 52.15, 48.43, 35.29, 27.30, 27.07, 23.48.
7
(
2
HRMS (ESI) m/z: 450.2205 (calcd 450.2214 for C26
H] ).
32
H N
3
O
2
Sþ[M þ
þ
(
3
)
d
1
9
2
7-(3-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)pro-
poxy)-2-methyl-3,4-dihydroisoquinolin-1(2H)-one [13] as
7.25, 67.93, 58.46, 53.49, 48.41, 35.25, 34.57, 30.44, 27.27, 27.05,
a
þ
ꢁ
1
3.44. HRMS (ESI) m/z: 452.2339 (calcd 452.2349 for C26
H31FN
3
O
3
white solid (yield, 86.0%), mp: 150e152 C. H NMR (400 MHz,
CDCl
þ
[M þ H] ).
3
)
d
7.72 (dd, J ¼ 8.7, 5.1 Hz, 1H), 7.61 (d, J ¼ 2.5 Hz, 1H), 7.21 (d,
7-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butoxy)-2-
J ¼ 8.5 Hz, 1H), 7.10-7.00 (m, 2H), 6.96 (dd, J ¼ 8.2, 2.6 Hz, 1H), 4.08
(t, J ¼ 6.3 Hz, 2H), 3.52 (t, J ¼ 6.6 Hz, 2H), 3.14 (s, 3H), 3.06 (d,
J ¼ 10.0 Hz, 3H), 2.91 (t, J ¼ 6.6 Hz, 2H), 2.56 (t, J ¼ 7.2 Hz, 2H), 2.25
methyl-3,4-dihydroisoquinolin-1(2H)-one [8] as a white solid
ꢁ
1
(
yield, 82.5%), mp: 155e157 C. H NMR (400 MHz, CDCl ) d 7.60 (d,
3
13
J ¼ 2.8 Hz, 1H), 7.18 - 7.10 (m, 2H), 7.08 (s, 0.4H), 7.06 (s, 0.6H), 6.99 -
3
-1.88 (m, 8H). C NMR (100 MHz, CDCl ) d 165.27, 164.71, 163.90,
6
3
.93 (m, 2H), 4.04 (t, J ¼ 6.0 Hz, 2H), 3.53 (t, J ¼ 6.8 Hz, 2H), 3.15 (s,
H), 3.08 (s, 4H), 2.92 (t, J ¼ 6.8 Hz, 2H), 2.66 (s, 4H), 2.49 (t,
163.76, 162.78, 161.13, 158.12, 130.26, 130.08, 127.97, 122.80, 122.69,
119.31, 117.28, 117.27, 112.38, 112.24, 112.13, 97.46, 97.19, 66.58,
13
J ¼ 7.2 Hz, 2H), 1.91 - 1.78 (m, 2H), 1.78 - 1.66 (m, 2H). C NMR
55.34, 53.60, 48.39, 35.21, 34.68, 30.57, 27.04, 26.86. HRMS (ESI) m/
þ
(
1
1
100 MHz, CDCl
58.19, 131.12, 130.09, 127.46, 127.27, 122.91, 122.82, 122.71, 117.26,
12.41, 112.16, 108.22,105.83, 97.50, 97.24, 66.70, 55.38, 53.60, 37.10,
3
)
d
165.30, 163.95, 163.81, 162.81, 162.26, 161.11,
z: 438.2183 (calcd 438.2192 for C25
H
29FN
3
O
3
þ[M þ H] ).
7-(3-(4-(2,4-difluorobenzoyl)piperidin-1-yl)propoxy)-2-
methyl-3,4-dihydroisoquinolin-1(2H)-one [14] as a white solid
ꢁ
1
3
C
4.67, 30.52, 26.79. HRMS (ESI) m/z: 462.1707 (calcd 462.1715 for
(yield, 76.2%), mp: 141e142 C H NMR (400 MHz, CDCl ) d 7.88 -
3
þ
þ
24
H
30Cl
2
N
3
O
2
[M þ H] ).
7.82 (m, 1H), 7.59 (d, J ¼ 2.8 Hz, 1H), 7.08 (d, J ¼ 8.3 Hz, 1H), 7.00 -
6.95 (m, 2H), 6.90 - 6.85 (m, 1H), 4.07 (t, J ¼ 6.3 Hz, 2H), 3.54 (t,
J ¼ 6.7 Hz, 2H), 3.15 (s, 3H), 3.04 - 3.01 (m, 2H), 2.95 - 2.92 (m, 2H),
2.66 - 2.55 (m, 2H), 2.25 - 2.18 (m, 2H), 2.05 -1.05 (m, 4H), 1.85 - 1.76
2
-Methyl-7-(4-(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)
butoxy)-3,4-dihydroisoquinolin-1(2H)-one [9] as a white solid
ꢁ
1
(
yield, 72.2%), mp: 100e101 C. H NMR (400 MHz, CDCl ) d 7.61 (d,
3
13
J ¼ 2.8 Hz,1H), 7.33 (t, J ¼ 8.0 Hz,1H), 7.11 (s, 1H), 7.09 - 7.03 (m, 3H),
3
(m, 2H). C NMR (100 MHz, CDCl ) d 199.65, 199.60, 164.72, 158.00,
6
2
.97 (d, J ¼ 2.8 Hz, 0.6H), 6.95 (d, J ¼ 2.8 Hz, 0.4H), 4.05 (t, J ¼ 6.4 Hz,
133.00, 132.96, 132.90, 132.85, 130.24, 130.15, 128.03, 119.15, 112.48,
112.45, 112.35, 112.27, 112.24, 104.92, 104.67, 104.65, 104.39, 66.46,
H), 3.58 - 3.48 (m, 2H), 3.27 - 3.20 (m, 4H), 3.14 (s,3H), 2.92 (t,
J ¼ 6.8 Hz, 2H), 2.66 - 2.56 (m, 4H), 2.51 - 2.40 (m, 2H), 1.89 - 1.78
55.28, 53.10, 48.40, 35.26, 27.79, 27.04, 26.58. HRMS (ESI) m/z:
13
þ
(
m, 2H), 1.76 - 1.65 (m, 2H). C NMR (101 MHz, CDCl
58.13, 151.42, 131.84, 131.52, 131.21, 130.89, 130.29, 130.09, 129.54,
28.44, 128.03, 125.73, 123.02, 120.31, 119.41, 118.62, 115.70, 115.66,
12.11, 112.05, 112.01, 67.92, 58.15, 53.03, 48.64, 48.43, 35.27, 27.22,
3
)
d
164.78,
438.2183 (calcd 438.2191 for C25
H
29FN
3
O
3
þ[M þ H] ).
1
1
1
7-(3-(4-(3-(Trifluoromethyl)phenyl)piperazin-1-yl)propoxy)-
2-Methyl-3,4-dihydroisoquinolin-1(2H)-one [15] as a white solid
ꢁ
1
(yield, 76.2%), mp: 103e105 C H NMR (400 MHz, CDCl ) d 7.60 (d,
3
2
C
7.08, 23.40. HRMS (ESI) m/z: 462.2360 (calcd 462.2368 for
J ¼ 2.8 Hz,1H), 7.32 (t, J ¼ 8.0 Hz,1H), 7.10 (s,1H), 7.08 - 7.02 (m, 3H),
6.96 (d, J ¼ 2.8 Hz, 0.6H), 6.93 (d, J ¼ 2.8 Hz, 0.4H), 4.04 (t, J ¼ 6.4 Hz,
2H), 3.55 - 3.47 (m, 2H), 3.28 - 3.21 (m, 4H), 3.16 (s,3H), 2.93 (t,
þ
þ
H F
25 31 3
N
3
O
2
[M þ H] ).
7
-(4-(4-(2-Methoxyphenyl)piperazin-1-yl)butoxy)-2-methyl-
3
,4-dihydroisoquinolin-1(2H)-one [10] as a white solid (yield,
J ¼ 6.8 Hz, 2H), 2.67 - 2.58 (m, 4H), 2.52 - 2.42 (m, 2H), 1.91 - 1.86
ꢁ
1
13
6
5.8%), mp: 98e100 C. H NMR (400 MHz, CDCl
3
)
d
7.60 (d,
(m, 2H). C NMR (101 MHz, CDCl
3
)
d
164.72, 158.11, 151.41, 131.81,
J ¼ 2.8 Hz, 1H), 7.06 (s, 0.4H), 7.04 (s, 0.6H), 7.01 - 6.88 (m, 4H), 6.86
s, 0.6H), 6.84 (s, 0.4H), 4.04 (t, J ¼ 6.4 Hz, 2H), 3.85 (s, 3H), 3.51 (t,
J ¼ 6.4 Hz, 2H), 3.22e2.97 (m, 7H), 2.90 (t, J ¼ 6.8 Hz, 2H), 2.67 (s,
131.51, 131.22, 130.87, 130.26, 130.06, 129.51, 128.41, 128.01, 125.70,
123.01, 120.30, 119.31, 118.51, 115.73, 115.61, 112.12, 112.03, 112.04,
67.91, 58.12, 53.01, 48.61, 48.39, 27.21, 27.19, 23.44. HRMS (ESI) m/z:
(
13
þ
þ
4
H), 2.52 - 2.41 (m, 2H), 1.90 - 1.77 (m, 2H), 1.76 - 1.65 (m, 2H).
NMR (101 MHz, CDCl 164.76, 158.13, 152.25, 141.36, 130.24,
30.04, 128.02, 122.86, 120.97, 119.37, 118.19, 112.11, 111.13, 67.97,
C
448.2204 (calcd 448.2212 for C24
H
29
F
3
N
3
O
2
[M þ H] ).
3
)
d
7-(3-(4-(Benzo[d]isothiazol-3-yl)piperazin-1-yl)propoxy)-2-
methyl-3,4-dihydroisoquinolin-1(2H)-one [16] as a white solid
1
5
ꢁ
1
8.33, 55.36, 53.47, 50.65, 48.40, 35.26, 27.30, 27.05, 23.45. HRMS
(yield, 73.7%), mp: 122e124 C H NMR (400 MHz, CDCl ) d 7.92 (d,
3
þ
þ
(
ESI) m/z: 424.2593 (calcd 424.2600 for C25
H
34
N
3
O
3
[M þ H] ).
J ¼ 8.1 Hz, 1H), 7.81 (d, J ¼ 8.1 Hz, 1H), 7.63 (d, J ¼ 2.0 Hz, 1H), 7.47 (t,
J ¼ 7.5 Hz, 2H), 7.36 (t, J ¼ 7.6 Hz, 2H), 7.09 (d, J ¼ 8.3 Hz,2H), 6.99
(dd, J ¼ 8.2, 2.0 Hz, 1H), 4.11 (t, J ¼ 6.2 Hz, 2H), 3.75-3.39 (m, 6H),
3.16 (s, 3H), 2.94 (t, J ¼ 6.6 Hz, 2H), 2.81-2.43 (m, 6H), 2.15-1.90 (m,
7
-(4-(4-(Benzo[d]isothiazol-3-yl)piperazin-1-yl)butoxy)-2-
methyl-3,4-dihydroisoquinolin-1(2H)-one [11] as a white solid
ꢁ
1
(
yield, 64.9%), mp: 114e116 C. H NMR (400 MHz, CDCl
3
) d 7.95 -
13
7.87 (m, 1H), 7.84 - 7.74 (m, 1H), 7.66 - 7.55 (m, 1H), 7.49 - 7.40 (m,
3
2H). C NMR (100 MHz, CDCl ) d 164.74, 163.91, 158.08, 152.71,

16
J. Jin et al. / European Journal of Medicinal Chemistry 207 (2020) 112709
130.19, 128.02, 127.51, 123.90, 120.55, 119.29, 112.26, 66.47, 55.23,
157.73, 130.30, 130.18, 128.04, 119.29, 112.25, 109.97, 66.33, 55.25,
5
3.06, 50.04, 48.41, 35.26, 27.06, 26.69. HRMS (ESI) m/z: 437.2003
53.04, 48.42, 43.40, 35.28, 27.07, 26.45. HRMS (ESI) m/z: 382.2235
þ
þ
þ
(
calcd 437.2011 for C24
H
29
N
4
O
2
S
[M þ H] ).
(calcd 382.2243 for C22
H
28
N
5
O
2
þ[M þ H] ).
7
-(3-(4-(Benzo[b]thiophen-4-yl)piperazin-1-yl)propoxy)-2-
7-(3-(4-(2-Chlorophenyl)piperazin-1-yl)propoxy)-2-methyl-
methyl-3,4-dihydroisoquinolin-1(2H)-one [17] as a white solid
3,4-dihydroisoquinolin-1(2H)-one [23] as a white solid (yield,
ꢁ
1
ꢁ
1
(
(
1
4
2
yield, 68.5%), mp: 131e132 C H NMR (400 MHz, CDCl
3
)
d
7.73
71.9%), mp: 118e120 C H NMR (400 MHz, CDCl ) d 7.66 (d,
3
d,J ¼ 2.7 Hz,1H), 7.60 - 7.58 (m,1H), 7.47-7.41 (m, 2H), 7.34-7.29 (m,
J ¼ 2.7 Hz, 1H), 7.40 (dd, J ¼ 1.6, 7.9 Hz, 1H), 7.29-7.24 (m, 1H), 7.15 -
7.07 (m, 2H), 7.05 - 6.95 (m, 2H), 4.14 (t, J ¼ 6.3 Hz, 2H), 3.60 - 3.56
H), 7.13 - 7.10 (m, 1H), 7.04 - 7.01 (m, 1H), 6.96 - 6.9 (m, 1H), 4.17 -
.13 (m, 2H), 3.99 - 3.53 (m, 2H), 3.27-3.18 (m, 7H), 2.99-2.94 (m,
(m, 2H), 3.20 - 3.17 (s, 7H), 2.98 (t, J ¼ 6.7 Hz, 2H), 2.82 - 2.44 (m,
13
13
H), 2.79-2.67 (m, 6H), 2.13-2.06 (m, 2H). C NMR (100 MHz,
164.76, 158.11, 148.43, 141.11, 134.09, 130.32, 130.18, 121.94,
6H), 2.26 - 1.88 (m, 2H). C NMR (101 MHz, CDCl
3
)
d
164.76, 158.09,
CDCl
3
)
d
149.21, 130.63, 130.31, 130.16, 128.78, 128.03, 127.63, 123.73, 120.47,
119.39, 112.22, 66.47, 55.19, 53.39, 51.03, 48.44, 35.29, 27.10, 26.60.
119.35, 117.04, 112.27, 66.52, 55.23, 53.60, 52.01, 48.43, 35.29, 27.09,
2
6.70. HRMS (ESI) m/z: 436.2051 (calcd 436.2059 for C25
H
30
N
3
O
2
HRMS (ESI) m/z: 414.1939 (calcd 414.1948 for C23
H] ).
H
29ClN
3
O
2
þ[M þ
þ
þ
Sþ[M þ H] ).
7
-(3-(4-(Benzo[d][1,3]dioxol-5-ylmethyl)piperazin-1-yl)pro-
7-(3-(4-(4-Chlorophenyl)piperazin-1-yl)propoxy)-2-methyl-
poxy)-2-methyl-3,4-dihydroisoquinolin-1(2H)-one [18] as
a
3,4-dihydroisoquinolin-1(2H)-one [24] as a white solid (yield,
ꢁ
1
ꢁ
1
white solid (yield, 77.2%), mp: 125.5e126.5 C H NMR (400 MHz,
CDCl
7.60 (d, J ¼ 2.4 Hz, 1H), 7.09 (d, J ¼ 8.0 Hz, 1H), 6.97 (dd,
80.8%), mp: 146e147 C H NMR (400 MHz, CDCl ) d 7.66 (d,
3
3
)
d
J ¼ 2.7 Hz, 1H), 7.28 - 7.20 (m, 2H), 7.12 (d, J ¼ 8.3 Hz, 1H), 7.01 (dd,
J ¼ 2.7, 8.3 Hz, 1H), 6.93 - 6.83 (m, 2H), 4.13 (t, J ¼ 6.3 Hz, 2H), 3.58
J ¼ 5.6, 2.8 Hz, 1H), 6.91 (d, J ¼ 1.6 Hz, 1H), 6.84 - 6.75 (m, 2H), 5.97
(
(
(
s, 2H), 4.08 (t, J ¼ 6.0 Hz, 2H), 3.61 - 3.46 (m, 4H), 3.17 (s, 3H), 2.95
(dd, J ¼ 6.3, 7.1 Hz, 2H), 3.31 - 3.10 (m, 7H), 2.98 (t, J ¼ 6.7 Hz, 2H),
13
13
t, J ¼ 6.8 Hz, 2H), 2.84 - 2.65 (m, 9H), 2.15 - 2.05 (m, 2H). C NMR
100 MHz, CDCl 164.71, 157.86, 147.75, 147.05, 130.32, 130.26,
2.74 - 2.52 (m, 6H), 2.09 - 2.02 (m, 2H). C NMR (101 MHz, CDCl
3
)
3
)
d
d 164.76, 158.11, 149.97, 130.32, 130.14, 128.94, 128.03, 124.42,
1
6
30.18, 128.10, 122.84, 119.23, 112.31, 109.77, 108.04, 101.04, 66.08,
119.38, 117.21, 112.21, 66.49, 55.11, 53.14, 49.15, 48.44, 35.29, 27.09,
2.15, 54.91, 53.53, 52.47, 51.69, 48.41, 35.30, 27.05, 25.97. HRMS
26.75. HRMS (ESI) m/z: 414.1937 (calcd 414.1948 for
þ
þ
(
ESI) m/z: 438.2386 (calcd 438.2393 for C25
H
32
N
3
O
4
þ[M þ H] ).
C
23
H29ClN
3
O
2
þ[M þ H] ).
7
-(3-(4-(Quinolin-2-yl)piperazin-1-yl)propoxy)- 2-methyl-
7-(3-(4-(2,3-Dichlorophenyl)piperazin-1-yl)propoxy)-2-
methyl-3,4-dihydroisoquinolin-1(2H)-one [25] as a white solid
(yield, 79.7%), mp: 111e113 C H NMR (400 MHz, CDCl ) d7.66 (d,
3
,4-dihydroisoquinolin- 1(2H)-one [19] as a white solid (yield,
ꢁ
1
ꢁ
1
73.7%), mp: 121e123
C
H NMR (400 MHz, CDCl
3
)
d
7.88 (d,
3
J ¼ 8.8 Hz, 1H), 7.70 (d, J ¼ 8.4 Hz, 1H), 7.65 -7.56 (m, 2H), 7.56 - 7.48
J ¼ 2.9 Hz, 1H), 7.19 - 7.17 (m, 2H), 7.12 - 7.09 (m, 1H), 7.04 -6.91 (m,
(
m,1H), 4.10 (t, J ¼ 6.4 Hz, 2H), 3.83 - 3.69 (m, 4H), 3.53 (t, J ¼ 6.8 Hz,
2H), 4.33 - 3.96 (m, 2H), 3.71 - 3.45 (m, 2H), 3.24 - 3.04 (m, 7H), 2.97
1
3
2
H), 3.15 (s, 3H), 2.93 (t, J ¼ 6.8 Hz, 2H), 2.71 - 2.41 (m, 6H), 2.06 -
(t, J ¼ 6.6 Hz, 3H), 2.81 - 2.54 (m, 6H), 2.06 (t, J ¼ 6.7 Hz, 2H).
C
2
.00 (m, 2H). 13C NMR (101 MHz, CDCl
3
)
d
164.79, 158.11, 157.43,
NMR (100 MHz, CDCl 164.76, 158.09, 151.26, 133.99, 130.30,
3
) d
147.90, 137.45, 130.30, 130.15, 129.52, 128.05, 127.23, 126.64, 123.09,
130.15, 128.04, 127.49, 124.57, 119.40, 118.68, 112.18, 66.46, 55.10,
1
3
22.36, 119.38, 112.24, 109.57, 66.51, 55.25, 53.23, 48.44, 45.10,
5.30, 27.08, 26.72.
53.31, 51.23, 48.43, 35.28, 27.08, 26.66. HRMS (ESI) m/z: 448.1551
þ
(calcd 448.1559 for C23
H
28Cl
2
N
3
O
2
þ[M þ H] ).
HRMS (ESI) m/z: 431.2439 (calcd 431.2447 for C26
31
H N
4
O
2
þ[M þ
7-(3-(4-(2-Fluorophenyl)piperazin-1-yl)propoxy)-2-methyl-
þ
H] ).
3,4-dihydroisoquinolin-1(2H)-one [26] as a white solid (yield,
ꢁ
1
7
-(3-(4-Phenylpiperazin-1-yl)propoxy)-
2-methyl-3,4-
80.2%), mp: 119e121 C. H NMR (400 MHz, CDCl3) d 7.66 (t,
dihydroisoquinolin- 1(2H)-one [20] as a white solid (yield,
J ¼ 1.9 Hz,1H), 7.19 - 6.78 (m, 6H), 4.24 - 4.02 (m, 2H), 3.60 - 3.55 (m,
ꢁ
1
8
5.3%), mp: 98.5e99.5 C. H NMR (400 MHz, CDCl
3
)
d
7.67 (d,
2H), 3.20 -3.15 (m, 7H), 2.97 (m, 2H), 2.77 - 2.55 (m, 6H), 2.19 - 1.87
13
J ¼ 2.7 Hz, 1H), 7.37 - 7.26 (m, 2H), 7.12 (d, J ¼ 8.3 Hz, 1H), 7.05 - 6.95
3
(m, 2H). C NM (101 MHz, CDCl ) d 164.76, 158.12, 156.96, 154.51,
(
3
2
m, 3H), 6.90 (tt, J ¼ 1.1, 7.3 Hz, 1H), 4.14 (t, J ¼ 6.3 Hz, 2H), 3.60 -
140.22, 140.14, 130.30, 130.12, 128.02, 124.49, 124.46, 122.43, 122.35,
119.40, 118.96, 118.93, 116.18, 115.98, 112.20, 66.52, 55.17, 53.34,
.57 (m, 2H), 3.35 - 3.23 (m, 4H), 3.20 (s, 3H), 2.98 (t, J ¼ 6.7 Hz, 2H),
.77 - 2.44 (m, 6H). 13C NMR (101 MHz, CDCl
)
d
164.79, 158.13,
50.54, 50.51, 48.43, 35.28, 27.09, 26.72. HRMS (ESI) m/z: 398.2233
3
þ
151.36, 130.31, 130.13, 129.12, 128.02, 119.66, 119.39, 116.07, 112.24,
(calcd 398.2244 for C23
H
29FN
3
O
2
þ[M þ H] ).
6
6.55, 55.20, 53.32, 49.15, 48.45, 35.30, 27.10, 26.76. HRMS (ESI) m/
7-(3-(4-(4-Fluorophenyl)piperazin-1-yl)propoxy)-2-methyl-
þ
z: 430.2327 (calcd 380.2336 for C23
H
30
N
3
O
2
þ[M þ H] )
3,4-dihydroisoquinolin-1(2H)-one [27] as a white solid (yield,
ꢁ
1
7
-(3-(4-(Pyridin-2-yl)piperazin-1-yl)propoxy)- 2-methyl-3,4-
79.2%), mp: 114e115 C. H NMR (400 MHz, CDCl ) d 7.62 (d,
3
dihydroisoquinolin- 1(2H)-one [21] as a white solid (yield,
J ¼ 2.3 Hz, 1H), 7.07 (d, J ¼ 8.3 Hz, 1H), 6.96 (t, J ¼ 8.4 Hz, 3H), 6.91-
6.82 (m, 2H), 4.09 (t, J ¼ 6.3 Hz, 2H), 3.53 (t, J ¼ 6.7 Hz, 2H), 3.23-
3.03 (m, 7H), 2.92 (t, J ¼ 6.6 Hz, 2H), 2.78-2.47 (m, 6H), 2.21-1.80 (m,
ꢁ
1
5
8.9%), mp: 85e87 C. H NMR (400 MHz, CDCl ) d 8.21 (s, 1H), 7.64
3
(
d, J ¼ 2.5 Hz, 1H), 7.49 (dd, J ¼ 9.2, 4.2 Hz, 1H), 7.10 (dd, J ¼ 8.0,
13
2
2
2
d
.7 Hz, 1H), 7.04 - 6.90 (m, 1H), 6.80 - 6.50 (m, 2H), 4.33 - 3.96 (m,
3
2H). C NMR (100 MHz, CDCl ) d 164.72, 158.17, 155.89, 147.99,
H), 3.59 - 3.44 (m, 6H), 3.31 - 3.10 (m, 3H), 2.95 (d, J ¼ 3.4 Hz, 2H),
130.19, 128.01, 119.31, 117.75, 115.56, 115.34, 112.19, 66.46, 55.07,
1
3
.62 (s, 6H), 2.05 (d, J ¼ 6.2 Hz, 2H). C NMR (101 MHz, CDCl
3
)
53.24, 50.10, 48.39, 35.24, 27.04, 26.70. HRMS (ESI) m/z: 398.2235
þ
164.73, 159.50, 158.07, 147.93, 137.47, 130.28, 130.14, 128.03,
19.32, 113.29, 112.23, 107.09, 66.46, 55.22, 53.06, 48.41, 45.14,
(calcd 398.2244 for C23
H
29FN
3
O
2
þ[M þ H] ).
1
7-(3-(4-(4-Methoxyphenyl)piperazin-1-yl)propoxy)-2-
3
C
5.27, 27.06, 26.65. HRMS (ESI) m/z: 381.2282 (calcd 381.2291 for
methyl-3,4-dihydroisoquinolin-1(2H)-one [28] as a white solid
þ
ꢁ
1
22
H
29
N
4
O
2
þ[M þ H] )
(yield, 68.5%), mp: 96e97 C. H NMR (400 MHz, CDCl ) d 7.65 (d,
3
7
-(3-(4-(pyrimidin-2-yl)piperazin-1-yl)propoxy)-2-methyl-
J ¼ 2.7 Hz,1H), 7.11 (dd, J ¼ 3.4, 8.9 Hz,1H), 7.04 - 6.75 (m, 6H), 4.14 -
3
6
2
6
2
-
,4-dihydroisoquinolin-1(2H)-one [22] as a white solid (yield,
3.8%), mp: 105e107 C H NMR (400 MHz, CDCl
H), 7.64 - 7.63 (m, 1H), 7.11 - 7.08 (m, 1H), 7.01 - 6.97 (m, 1H), 6.53 -
.50 (m, 1H), 4.14 - 4.10 (m, 2H), 3.98 - 3.81 (m, 4H), 3.64 - 3.45 (m,
H), 3.25 - 3.07 (m, 3H), 2.98 - 2.93 (m, 2H), 2.74 - 2.53 (m, 6H), 2.12
4.10 (m, 2H), 3.89 - 3.71 (m, 3H), 3.59 - 3.54 (m, 2H), 3.26 - 3.11 (m,
ꢁ
1
13
3
)
d
8.52 - 8.13 (m,
7H), 2.98 - 2.93 (m, 2H), 2.78 - 2.55 (m, 6H), 2.16 - 1.82 (m, 2H).
NMR (100 MHz, CDCl
C
3
)
d
164.74, 158.07, 153.82, 145.66, 130.30,
130.16, 128.04, 119.30, 118.25, 114.43, 112.27, 66.48, 55.58, 55.18,
53.36, 50.50, 48.42, 35.28, 27.08, 26.64. HRMS (ESI) m/z: 410.2438
2.05 (m, 2H). 13C NMR (101 MHz, CDCl
)
d
164.73, 161.58, 158.01,
(calcd 410.2444 for C24
þ[M þ H] ).
þ
3
32
H N
3
O
3

J. Jin et al. / European Journal of Medicinal Chemistry 207 (2020) 112709
17
7
-(3-(4-(2-Methoxyphenyl)piperazin-1-yl)propoxy)-2-
(100 mL). The layers were separated, and the aqueous was re-
extracted into CH Cl . The organics were dried and concentrated.
The light brown residue was purified by means of chromatography
methyl-3,4-dihydroisoquinolin-1(2H)-one [29] as a white solid
2
2
ꢁ
1
(
yield, 71.8%), mp: 99e101 C. H NMR (400 MHz, CDCl
3
)
d
7.64 (m,
H), 7.12 - 7.09 (m, 1H), 7.07 - 6.91 (m, 4H), 6.91- 6.87 (m, 1H), 4.15 -
.10 (m, 2H), 3.96 - 3.74 (m, 3H), 3.58 - 3.54 (m, 2H), 3.31 - 3.10 (m,
1
4
7
(DCM/MeOH ¼ 40/1) to afford compound 33, as a yellow oil (yield,
1
33.8%). H NMR (400 MHz, CDCl
3
)
d
7.92 (d, J ¼ 8.1 Hz, 1H), 7.81 (d,
13
H), 3.03 - 2.67 (m, 8H), 2.18 - 2.11 (m, 2H). C NMR (100 MHz,
164.71,157.95,152.23,140.85,130.31,130.27,128.08,123.21,
21.04, 119.21, 118.38, 112.36, 111.19, 66.30, 55.41, 55.26, 53.32,
J ¼ 8.1 Hz, 1H), 7.63 (d, J ¼ 2.0 Hz, 1H), 7.47 (t, J ¼ 7.5 Hz, 2H), 7.36 (t,
J ¼ 7.6 Hz, 2H), 7.09 (d, J ¼ 8.3 Hz,2H), 6.99 (dd, J ¼ 8.2, 2.0 Hz, 1H),
4.11 (t, J ¼ 6.2 Hz, 2H), 3.75-3.39 (m, 6H), 3.16 (s, 3H), 2.94 (t,
3
CDCl ) d
1
4
4
13
9.95, 48.41, 35.28, 27.07, 26.18. HRMS (ESI) m/z: 410.2436 (calcd
10.2444 for C24
J ¼ 6.6 Hz, 2H), 2.81-2.43 (m, 6H), 2.15-1.90 (m, 2H). C NMR
þ
H
32
N
3
O
3
þ[M þ H] ).
3
(100 MHz, CDCl ) d 164.74, 163.91, 158.08, 152.71, 130.19, 128.02,
7
-(2-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)
127.51, 123.90, 120.55, 119.29, 112.26, 77.39, 77.08, 76.76, 66.47,
ethoxy)-2-methyl-3,4-dihydroisoquinolin-1(2H)-one [30] as a
55.23, 53.06, 50.04, 48.41, 35.26, 27.06, 26.69. HRMS (ESI) m/z:
ꢁ
1
þ
white solid (yield, 65.3%), mp: 151e153 C. H NMR (400 MHz,
CDCl
7.71 (dd, J ¼ 8.7, 5.1 Hz, 1H), 7.63 (d, J ¼ 2.4 Hz, 1H), 7.26-
.16 (m, 1H), 7.15-6.81 (m, 3H), 4.19 (t, J ¼ 5.7 Hz, 2H), 3.53 (t,
J ¼ 6.7 Hz, 2H), 3.30-3.00 (m, 6H), 2.94-2.86 (m, 4H), 2.36-2.30 (m,
456.2091 (calcd 456.2099 for C25
H
28
F
2
N
3
O
3
þ[M þ H] ).
3
) d
7
4.10. 2-Ethyl-7-methoxy-3,4-dihydroisoquinolin-1(2H)-one [34]
13
2
1
1
H), 2.19-1.88 (m, 4H). C NMR (100 MHz, CDCl
63.88, 163.75, 162.78, 161.05, 157.84, 130.31, 130.28, 128.04, 122.73,
22.62, 119.38, 117.28, 117.27, 112.41, 112.34, 112.16, 97.49, 97.22,
3
)
d
165.27, 164.65,
To a solution of sodium hydride (75.0 mmol) in 50 mL of dry N,
N-dimethylformamide, a solution of compound 3 (50.0 mmol) in
30 mL of N, N-dimethylformamide and iodoethane (60.0 mmol)
was added dropwise under ice-cooling. The reaction mixture was
stirred at room temperature for 1.5 h. The reaction was quenched
with 480 ml water, extracted with ethyl acetate (100 mL ꢂ 3),
6
6.20, 57.36, 53.87, 48.37, 35.23, 34.39, 30.48, 27.03. HRMS (ESI) m/
þ
z: 424.2029 (calcd 424.2036 for C24
H27FN
3
O
3
þ[M þ H] ).
4
1
.7. 2-Methyl-7-(oxiran-2-ylmethoxy)-3,4-dihydroisoquinolin-
(2H)-one [31]
2 4
washed with brine, and dried over Na SO . The organic phase was
evaporated under reduced pressure to give crude oil. The residue
was purified with column chromatography (petroleum ether/
EtOAc ¼ 1: 5) to yield compound 34, as a yellow oil (yield, 89.0%).
To a suspension of compound 5 (10 mmol) and 2-(chloromethyl)
oxirane (11 mmol) in acetone (100 mL), potassium carbonate
20 mmol) and a catalytic amount of potassium iodide (1% mol)
were added. The resulting mixture was heated and refluxed for
e6 h, and the progress of the reaction was monitored by TLC. After
(
4.11. 2-Ethyl-7-hydroxy-3,4-dihydroisoquinolin-1(2H)-one [35]
4
To a stirred solution of compound 34 (35.0 mmol) was added
hydrobromic acid in water solution (48%) (70.0 mL) and reaction
cooling to room temperature, the mixture was filtered and the
solvent was evaporated under reduced pressure. The crude product
was purified by means of chromatography (petroleum ether/
EtOAc ¼ 40/1) to yield compound 31, as a pale-yellow oil (yield,
ꢁ
mixture was refluxed at 100 C overnight.The reaction mixture was
quenched with water (210.0 mL)and then extracted with DCM
(5 ꢂ 50 mL), The organic phase was dried over Na
2 4
SO and
7
0.0%).
concentrated under reduced pressure to colorless solid. The solid
was further purified with column chromatography (DCM/
MeOH ¼ 30:1) to yield compound 35 as a white solid (yield, 80.8%),
4.8. 7-(3-(4-(6-Fluorobenzo[d]isoxazol-3-yl) piperidin-1-yl)-2-
ꢁ
hydroxypropoxy)-2-methyl-3,4- dihydroisoquinolin-1(2H)-one [32]
mp: 169-171 C.
A mixture of 31 (5 mmol), 6-fluoro-3-(piperidin-4-yl)benzo[d]
isoxazole hydrochloride (5.5 mmol) and potassium carbonate
4.12. 7-(3-Bromopropoxy)-2-ethyl-3,4-dihydroisoquinolin-1(2H)-
one [36]
(
10 mmol) in acetonitrile (50 ml) was stirred and heated to reflux
for 6e8 h. The solvent was evaporated under reduced pressure, and
the crude product was purified by means of chromatography (DCM/
MeOH ¼ 30/1) to yield compound 32, as a yellow oil (yield, 62.8%),
The
mixture
of
compound 35
(20
mmol),1,3-
dibromopropane(40.0 mmol),
(0.2 mmol) and acetone (50.0 mL), was heated under reflux for 6 h.
Water (300 ml) was added to the reaction solution, which was then
2 4
extracted with DCM, The organic phase was dried over Na SO
evaporated under reduced pressure to give crude oil. The residue
was purified with column chromatography (petroleum ether/
EtOAc ¼ 1: 10) to yield compound 36, as a yellow oil (5.36 g, 86.2%).
K
2
CO (60.0 mmol), TEBA
3
ꢁ
1
mp: 131e133 C. H NMR (400 MHz, CDCl
3
)
d
7.68 (dd, J ¼ 8.7,
5
6
3
2
.1 Hz, 1H), 7.61 (d, J ¼ 2.4 Hz, 1H), 7.23 (dd, J ¼ 8.4, 1.5 Hz, 1H), 7.17-
.82 (m, 3H), 4.17-4.12 (m, 1H), 4.09-4.00 (m, 2H), 3.53 (t, J ¼ 6.7 Hz,
H), 3.31-2.97 (m, 6H), 2.92 (t, J ¼ 6.7 Hz, 2H), 2.72-2.54 (m, 2H),
13
.53-2.46 (m, 1H), 2.27-2.21 (m, 1H), 2.17-1.90 (m, 4H). C NMR
(
100 MHz, CDCl
3
)
d
165.31, 164.62, 163.81, 162.82, 160.91, 157.84,
1
9
3
30.47, 130.26, 128.07, 122.53, 119.38, 117.24, 112.51, 112.23, 97.55,
7.28, 70.63, 65.73, 60.62, 54.94, 52.51, 48.35, 35.24, 34.25, 30.69,
4.13. 2-Ethyl-7-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-
yl)propoxy)-3,4-dihydroisoquinolin-1(2H)-one [37]
0.46, 27.02. HRMS (ESI) m/z: 454.2136 (calcd 454.2142 for
þ
C
25
H
29FN
3
O
4
þ[M þ H] ).
The mixture of compound 36 (10.0 mmol),6-fluoro-3-(piper-
idin-4-yl)benzo[d]isoxazole hydrochloride (12.0 mmol), K CO
2 3
4.9. 7-(2-Fluoro-3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-
(30.0 mmol) and acetonitrile (30.0 mL), was heated under reflux for
yl)propoxy)-2-methyl-3,4-dihydroisoquinolin-1(2H)-one [33]
8 h. Water (300 ml) was added to the reaction solution, which was
2 4
then extracted with DCM, The organic phase was dried over Na SO
ꢁ
At first, compound 32 (3.0 mmol) was added dropwise at ꢀ78 C
to a solution of diethylamino sulfur trifluoride (9.0 mmol) in CH Cl
50 mL) and the reaction was stirred for 2.5 h, warming to room
evaporated under reduced pressure to give yellow solid, The res-
idue was purified with column chromatography (DCM/MeOH ¼ 20
: 1) to yield compound 37, as a white solid (yield, 84.5%), mp:
2
2
(
ꢁ
1
temperature. The resulting solution was stirred at room tempera-
ture for 18 h. The reaction was quenched by slowly pouring the
135e137 C. H NMR (400 MHz, CDCl
3
)
d
7.73 (dd, J ¼ 8.7, 5.1 Hz,
1H), 7.63 (d, J ¼ 1.8 Hz, 1H), 7.23 (d, J ¼ 8.4 Hz, 1H), 7.06 (dd, J ¼ 12.1,
solution onto an ice-cold saturated aqueous NaHCO
3
solution
8.5 Hz, 2H), 6.97 (dd, J ¼ 8.0, 2.1 Hz,1H), 4.10 (t, J ¼ 6.3 Hz, 2H), 3.65-

18
J. Jin et al. / European Journal of Medicinal Chemistry 207 (2020) 112709
3
.52 (m, 4H), 3.08 (d, J ¼ 9.8 Hz, 3H), 2.92 (t, J ¼ 6.6 Hz, 2H), 2.57 (t,
evaporated under reduced pressure to give crude oil. The residue
was purified with column chromatography (petroleum ether/
EtOAc ¼ 1: 10) to yield compound 42, as a white oil (yield, 92.7%).1H
13
J ¼ 7.2 Hz, 2H), 2.23-1.90 (m, 8H), 1.22 (t, J ¼ 7.1 Hz, 3H). C NMR
(
100 MHz, CDCl 165.28, 164.12,163.68, 162.79, 161.16, 158.13,
3
) d
130.60, 130.09, 127.93, 122.76, 119.32, 117.27, 112.40, 112.17, 97.49,
NMR (400 MHz, CDCl3)
d
7.84 (d, J ¼ 2.6 Hz, 1H), 7.44 (dd, J ¼ 1.7,
97.23, 66.57, 55.37, 53.62, 45.79, 42.27, 34.71, 30.58, 27.34, 26.87,
8.7 Hz, 1H), 7.30 - 7.13 (m, 1H), 6.97 (dd, J ¼ 1.8, 7.2 Hz, 1H), 6.45 (dd,
J ¼ 1.9, 7.3 Hz, 1H), 4.36 - 4.14 (m, 2H), 3.77 (td, J ¼ 1.5, 6.4 Hz, 2H),
12.77. HRMS (ESI) m/z: 452.2342 (calcd 452.2349 for
þ
13
C
26
H31FN
3
O
3
þ[M þ H] ).
3.61 (d, J ¼ 1.8 Hz, 3H), 2.28 (td, J ¼ 1.7, 6.1 Hz, 2H). C NMR
3
(101 MHz, CDCl ) d 162.19, 157.79, 131.33, 130.29, 127.57, 127.21,
4
.14. 7-Methoxy-2-methylisoquinoline-1,3(2H,4H)-dione(39)
122.75, 108.25, 105.78, 64.63, 41.47, 37.14, 32.14. MS (ESI) m/z: 296.2
þ
þ
(calcd 296.0 for C13
H
15BrNO
2
[M þ H] ).
To compound 38 (30.0 mmol) was added a 40% aq solution of
7-(3-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)pro-
methylamine (15 mL) at r.t. and the reaction mixture was stirred for
min. It was then concentrated in vacuo and dried at the vacuum
poxy)-2-methylisoquinolin-1(2H)-one [43].
5
The mixture of compound 42 (5.0 mmol),6-fluoro-3-(piperidin-
pump. To the obtained salt was added o-dichlorobenzene (60 mL)
and the mixture was vigorously refluxed for 10 h. The reaction
mixture was allowed to cool to r.t. The silica gel column chro-
matographic purification of the resulting reaction mixture using
petroleum ether/EtOAc (1:3) as an eluent compound 39, as a white
solid (yield, 57.6%).
4-yl)benzo[d]isoxazole hydrochloride (6.0 mmol),
(15.0 mmol) and acetonitrile (20.0 mL), was heated under reflux for
8 h. Water (300 ml) was added to the reaction solution, which was
2 4
then extracted with DCM, The organic phase was dried over Na SO
evaporated under reduced pressure togive yellow solid, The residue
was purified with column chromatography (DCM: MeOH ¼ 20 : 1)
to yield compound 43, as a white solid (yield, 68.2%). mp:
2 3
K CO
ꢁ
1
4.15. 7-Methoxy-2-methylisoquinolin-1(2H)-one [40]
147e149 C. H NMR (400 MHz, CDCl
(
3
)
d
7.86 (d, J ¼ 2.6 Hz,1H), 7.74
dd, J ¼ 8.7, 5.1 Hz, 1H), 7.45 (d, J ¼ 8.7 Hz, 1H), 7.33-7.19 (m, 2H),
To
a
stirred solution of compound 39 (15 mmol) in
7.06 (td, J ¼ 8.9, 2.1 Hz, 1H), 6.98 (d, J ¼ 7.3 Hz, 1H), 6.46 (d,
ꢁ
EtOH(200 mL) was added NaBH
was stirred under argon atmosphere for 7 h at 0 C while 2 drops of
4
(150 mmol) at 0 C. The mixture
J ¼ 7.3 Hz,1H), 4.19 (t, J ¼ 6.4 Hz, 2H), 3.62 (s, 3H), 3.13-3.06 (m, 3H),
ꢁ
13
2.62 (t, J ¼ 7.3 Hz, 2H), 2.32-2.04 (m, 8H). C NMR (100 MHz, CDCl
3
)
a solution of aq 2 N HCl (20 mL) and EtOH (8 mL) were added at
d 165.30, 163.95, 163.81, 162.81, 162.26, 161.11, 158.19, 131.12, 130.09,
ꢁ
intervals of 15 min. The excess of NaBH
4
was quenched at 0 C by
127.46, 127.27, 122.91, 122.82, 122.71, 117.26, 112.41, 112.16, 108.22,
105.83, 97.50, 97.24, 66.70, 55.38, 53.60, 37.10, 34.67, 30.52, 26.78.
the addition of aq 2 N HCl in EtOH (100 mL) until the mixture was
acidic. The reaction mixture was concentrated in vacuo and the
obtained residue was dissolved in EtOAc (25 mL). The organic layer
HRMS (ESI) m/z: 436.2029 (calcd 436.2036 for C25
H] ).
H27FN
3
O
3
þ[M þ
þ
2 2 4
was washed with H O (150 mL), brine (150 mL), and dried (Na SO ).
Concentration of the dried organic layer in vacuum followed by
silica gel column chromatographic purification of the resulting
residue using EtOAc/PE ¼ 2:3 as a fluent gave the pure compound
4.18. 2-Bromo-5-methoxybenzoyl chloride [45]
To a solution of compound 44 (30.0 mmol) in 50 mL of dry
Cl , a solution of thionyl chloride (90.0 mmol) was added
4
0, as yellow oil (yield, 76.4%).1H NMR (400 MHz, CDCl3)
d
7.88 -
CH
2
2
7
1
.75 (m, 1H), 7.46 - 7.40 (m, 1H), 7.29 - 7.12 (m, 1H), 6.99 - 6.94 (m,
H), 6.47 - 6.42 (m, 1H), 4.00 - 3.86 (m, 3H), 3.70 - 3.51 (m, 3H). 13C
dropwise under ice-cooling. The resulting mixture was stirred at
room temperature for 4 h. The resulting mixture was concentrated
under reduced pressure to afford compound 45, as a pale-yellow
solid (6.34g, 85.6%).
NMR (101 MHz, CDCl3)
d
162.24, 158.74, 131.20, 130.15, 127.51,
127.21, 122.67, 107.33, 105.85, 55.64, 37.15. MS (ESI) m/z: 190.2
þ
þ
(
calcd 190.1 for C11
H12NO
2
[M þ H] ).
4.19. 2-Bromo-5-methoxy-N-methylbenzamide [46]
4.16. 7-Hydroxy-2-methylisoquinolin-1(2H)-one [41]
To a solution of methylamine hydrochloride (25.0 mmol) and
To a stirred solution of compound 40 (10 mmol) was added
2 2
triethylamine (40.0 mmol) in 50 mL of dry CH Cl , a solution of
hydrobromic acid in water solution (48%) (20.0 mL) and reaction
mixture was refluxed at 100 C overnight.The reaction mixture was
quenched with water (60.0 mL)and then extracted with DCM
compound 45 (20.0 mmol) was added dropwise under ice-cooling.
The resulting mixture was stirred at room temperature for 8 h. The
reaction mixture was quenched with water (100 mL), washed with
ꢁ
(
5 ꢂ 50 mL), The organic phase was dried over Na
2
SO
4
and
2 4
2 N HCl solution, brine and dried over Na SO . The organic phase
concentrated under reduced pressure to colorless solid. The solid
was further purified with column chromatography (DCM/
MeOH ¼ 30:1) to yield compound 41, as a white solid. (yield, 76.0%),
was evaporated under reduced pressure to give crude oil. The
resulting oil solidified on standing. The crude mixture was purified
with column chromatography (petroleum ether/EtOAc ¼ 1: 20) to
afford compound 46, as a yellow solid (yield, 77.8%). mp:
ꢁ
1
mp: 131e133 C. H NMR (400 MHz, DMSO)
d
7.56 (d, J ¼ 2.6 Hz,
ꢁ
1
1
2
H), 7.49 (d, J ¼ 8.6 Hz, 1H), 7.23 (d, J ¼ 7.3 Hz, 1H), 7.17 (dd, J ¼ 8.5,
137e139 C. H NMR (400 MHz, CDCl
3
) d 7.50 - 7.46 (m, 1H), 7.20 -
13
.6 Hz, 1H), 6.49 (d, J ¼ 7.3 Hz, 1H), 3.47 (s, 3H). C NMR (101 MHz,
7.03 (m, 1H), 6.88 - 6.84 (m, 1H), 3.88 - 3.78 (m, 3H), 3.07 - 3.04 (m,
13
DMSO)
1
d
161.42, 156.76, 130.84, 130.08, 128.26, 127.21, 122.35,
3
3H). C NMR (100 MHz, CDCl ) d168.02, 158.93, 138.41, 134.14,
þ
10.66,105.29, 36.74. MS (ESI) m/z: 176.3 (calcd 176.1 for C10
H10NO
2
117.87, 117.84, 117.82, 114.72, 114.70, 114.69, 109.37, 77.38, 77.07,
þ
[M þ H] ).
76.75, 55.66, 26.78. MS (ESI) m/z: 244.2 (calcd 244.0 for
þ
þ
C
9
H
11BrNO
2
[M þ H] ).
4
.17. 7-(3-Bromopropoxy)-2-methylisoquinolin-1(2H)-one [42]
4.20. 6-Methoxy-3-methyl-2,3-dihydro-4H-benzo[e][1,3]oxazin-4-
The
dibromopropane(14.0 mmol), K2CO3 (21.0 mmol), TEBA
0.7 mmol) and acetone (30.0 mL), was heated under reflux for 6 h.
mixture
of
compound
41
(7.0
mmol),1,3-
one [47]
(
In a 250 mL tube were added compound 46 (15.0 mmol), 15 mL
Water (300 ml) was added to the reaction solution, which was then
extracted with DCM, The organic phase was dried over Na2SO4
dichloromethane, LiOH (135.0 mmol), CuBr (0.8 mmol),1,10-
Phenanthroline (1.5 mmol) and DMSO (50 mL). The vessel was

J. Jin et al. / European Journal of Medicinal Chemistry 207 (2020) 112709
19
ꢁ
then sealed with a Teflon cap and heated up to100 C and stirred at
4.24. Ethyl (3-methoxyphenethyl)carbamate [52]
the same temperature for 12 h. After completion(TLC), the vessel
was cooled down to room temperature, and water (150 mL) was
added. The resulting suspension was extracted with ethyl acetate
To a solution of ethyl chloroformate (300 mmol) in 200 mL of
dry dichloromethane a solution of compound 51(250 mol) and
triethylamine (375 mmol) in 600 mL of dichloromethane was
added dropwise under ice-cooling. A white precipitate (the hy-
drochloride of the amine) formed immediately. After vigorous
stirring for 1 h at r.t., the reaction mixture was quenched with water
(100 mL), washed with 2 N HCl solution, brine and dried over
Na SO . The organic phase was evaporated under reduced pressure
2 4
to give crude oil. The resulting oil solidified on standing. The crude
mixture was purified with column chromatography (petroleum
(
10 mL ꢂ 3). The organic layer was combined and dried with
anhydrous Na SO . After filtration, the solvent in the acquired so-
2
4
lution was removed under reduced pressure. The residue obtained
therein was subjected to silica gel column chromatography(petro-
leum ether/EtOAc ¼ 1: 10)to afford compound 47, as a pale-yellow
1
oil (yield, 77.8%). H NMR (400 MHz, CDCl
7
3
)
d
7.43 (d, J ¼ 3.1 Hz, 1H),
.01 (dd, J ¼ 8.9, 3.1 Hz, 1H), 6.89 (d, J ¼ 8.9 Hz, 1H), 3.82 (s, 3H), 3.11
(
s, 3H). 13C NMR (100 MHz, CDCl
3
) d
162.47, 154.99, 151.82, 121.78,
1
19.04, 117.39, 110.14, 55.85, 31.42. MS (ESI) m/z: 194.1 (calcd 194.1
ether/EtOAc ¼ 1: 20) to afford compound 52, as a pale-yellow oil
þ
þ
1
for C10
H12NO
3
[M þ H] ).
(yield, 95.9%). H NMR (400 MHz, CDCl
3
)
d
7.26 (t, J ¼ 7.8 Hz, 1H),
6
.90 - 6.70 (m, 3H), 4.14 (q, J ¼ 7.1 Hz, 2H), 3.83 (s, 3H), 3.47 (q,
13
J ¼ 6.7 Hz, 2H), 2.83 (t, J ¼ 7.0 Hz, 2H), 1.27 (t, J ¼ 7.2 Hz, 3H).
C
4.21. 6-Hydroxy-3-methyl-2,3-dihydro-4H-benzo[e][1,3]oxazin-4-
NMR (100 MHz, CDCl 159.80, 156.64, 140.49, 129.62, 121.12,
3
) d
one [48]
1
14.49, 111.83, 60.73, 55.17, 42.04, 36.23, 14.68. MS (ESI) m/z: 224.4
þ
þ
(calcd 224.1 for C12
H18NO
3
[M þ H] ).
A solution of borontribromide in dichloromethane (1 M solu-
tion, 40 mmol) was added dropwise with stirring to an ice-cooled
solution of compound 47 (10 mmol) in dichloromethane
4
.25. 6-Methoxy-3,4-dihydroisoquinolin-1(2H)-one (53)
To a solution of P (300.0 mmol) in 70 mL of methanesulfonic
(
120 ml). After stirring at room temperature for 16 h the reaction
2 5
O
mixture was poured onto a mixture of crushed-ice and aqueous
ammonia (400 ml). The resulting mixture was extracted with
dichloromethane (3 ꢂ 200 ml).he combined organics were washed
acid, compound 52 (200.0 mmol) were added. The solution was
heated for 2 h under 130 C, excess methane sulfonic acid was
removed in vacuo, and the resulting residue was quenched with
ice-water. The solution was neutralised with NaHCO
three times with dichloromethane. The combined organic phases
were washed twice with water, dried with Na SO4. The organic
ꢁ
2 4
with brine (200 ml) then dried (Na SO ) and the solvent evaporated
3
and extracted
in vacuum to afford compound 48, as a pale yellow solid (yield,
ꢁ
1
7
8.8%), mp: 148e150 C. H NMR (400 MHz, CDCl
3
) d 7.73 (d,
2
J ¼ 3.0 Hz, 1H), 7.05 (dd, J ¼ 8.8, 3.0 Hz, 1H), 6.92 (d, J ¼ 8.8 Hz, 1H),
13
phase was evaporated under reduced pressure to give crude oil. The
crude mixture was purified with column chromatography (petro-
leum ether/EtOAc ¼ 1: 1) to afford compound 53, as pale-yellow
3
3
.18 (s, 3H). C NMR (100 MHz, CDCl ) d 151.91, 151.43, 122.06,
1
for C
18.87, 117.43, 113.72, 79.50, 31.60. MS (ESI) m/z: 180.3 (calcd 180.1
þ
þ
9
H10NO
3
[M þ H] ).
ꢁ
1
3
solid (yield, 54.4%), mp: 133-135 C. H NMR (400 MHz, CDCl )
d
1
8.14e7.89 (m, 1H), 6.89 (dd, J ¼ 8.7, 2.5 Hz, 1H), 6.74 (t, J ¼ 2.2 Hz,
4
.22. 6-(3-Bromopropoxy)-3-methyl-2,3-dihydro-4H-benzo[e][1,3]
13
H), 3.98 - 3.77 (m, 3H), 3.61 - 3.57 (m, 2H), 3.01 - 2.97 (m, 2H).
C
oxazin-4-one [49]
3
NMR (100 MHz,CDCl ) d
162.56, 141.07, 130.02, 121.85, 112.48,
1
C
12.33, 55.42, 40.23, 28.75. MS (ESI) m/z: 178.3 (calcd 178.1 for
The
dibromopropane(12.0 mmol),
0.6 mmol) and acetone (30.0 mL), was heated under reflux for 6 h.
Water (300 ml) was added to the reaction solution, which was then
extracted with DCM, The organic phase was dried over Na SO
evaporated under reduced pressure to give crude oil. The residue
was purified with column chromatography (petroleum ether/
EtOAc ¼ 1: 15) to yield compound 49, as white oil (1.61 g, 89.9%).
mixture
of
compound
48
(6.0
mmol),1,3-
þ
þ
10
H12NO
2
[M þ H] ).
K
2
CO
3
(18.0 mmol), TEBA
(
4.26. 6-Methoxy-2-methyl-3,4-dihydroisoquinolin-1(2H)-one (54)
2
4
To a solution of sodium hydride (75.0 mmol) in 50 mL of dry N,
N-dimethylformamide, a solution of compound 53 (50.0 mmol) in
0.0 mL of N, N-dimethylformamide and iodomethane (60.5 mmol)
3
was added dropwise under ice-cooling. The reaction mixture was
stirred at room temperature for 1.5 h. The reaction was quenched
with 480 ml water, extracted with ethyl acetate (100 mL ꢂ 3),
4.23. 6-(3-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)
propoxy)-3-methyl-2,3-dihydro-4H-benzo[e][1,3]oxazin-4-one [50]
2 4
washed with brine, and dried over Na SO . The organic phase was
evaporated under reduced pressure to give crude oil. The residue
The mixture of compound 49 (5.0 mmol),6-fluoro-3-(piperidin-
was purified with column chromatography (petroleum ether/
4
(
8
-yl)benzo[d]isoxazole hydrochloride (6.0 mmol),
15.0 mmol) and acetonitrile (20.0 mL), was heated under reflux for
h. Water (300 ml) was added to the reaction solution, which was
then extracted with DCM, The organic phase was dried over Na SO
K
2
CO
3
EtOAc ¼ 10: 1) to afford compound 54, as yellow oil (yield,
1
93.5%). H NMR (400 MHz, CDCl
3
)
d
8.15 - 7.77 (m, 1H), 6.82 (dd,
J ¼ 8.6, 2.5 Hz, 1H), 6.64 (d, J ¼ 2.6 Hz, 1H), 3.85- 3.78 (m, 3H), 3.53
13
2
4
(t, J ¼ 6.7 Hz, 2H), 3.11 (s, 3H), 2.95 (d, J ¼ 4.4 Hz, 2H). C NMR
(100 MHz, CDCl 164.85, 162.53, 162.08, 140.07, 130.09, 122.24,
evaporated under reduced pressure togive yellow solid, The residue
was purified with column chromatography (DCM/MeOH ¼ 20 : 1)
to yield compound 50, as a white solid (yield, 55.0%), mp:
3
) d
112.37, 111.86, 77.50, 77.18, 77.18, 76.86, 55.34, 48.11, 36.49, 34.99,
þ
31.40, 28.20. MS (ESI) m/z: 192.2 (calcd 192.1 for C11
H] ).
H
14NO
2
[M þ
ꢁ
1
þ
152e154 C. H NMR (400 MHz, CDCl
3
)
d
7.72 (dd, J ¼ 8.7, 5.1 Hz,
1
H), 7.44 (d, J ¼ 2.9 Hz, 1H), 7.22 (dd, J ¼ 8.5, 1.6 Hz, 1H), 7.07-6.99
(
3
m, 2H), 6.88 (d, J ¼ 8.9 Hz, 1H), 5.12 (s, 2H), 4.05 (t, J ¼ 6.3 Hz, 2H),
4.27. 6-Hydroxy-2-methyl-3,4-dihydroisoquinolin-1(2H)-one (55)
13
.28-2.85 (m, 6H), 2.57 (t, J ¼ 7.2 Hz, 2H), 2.27-1.76 (m, 8H). C NMR
165.27, 163.83, 162.78, 162.47, 161.11, 154.41,
51.75, 122.72, 122.19, 119.03, 117.29, 112.40, 112.15, 111.03, 97.49,
(
100 MHz, CDCl
3
)
d
To a stirred solution of compound 54 (40.0 mmol) was added
hydrobromic acid in water solution (48%) (30.0 mL) and reaction
1
9
ꢁ
7.23, 79.49, 66.98, 55.33, 53.59, 34.64, 31.41, 30.54, 26.83. HRMS
mixture was refluxed at 100 C overnight. The reaction mixture was
þ
(
ESI) m/z: 440.1981 (calcd 440.1986 for C24
H27FN
3
O
4
þ[M þ H] ).
quenched with water (60.0 mL)and then extracted with DCM

20
J. Jin et al. / European Journal of Medicinal Chemistry 207 (2020) 112709
3
(
5 ꢂ 50 mL), The organic phase was dried over Na
2
SO
4
and
receptor, [ H]prazosin, from rat cerebral cortex; For 5-HT1A recep-
tor binding assays, total binding (TB) was determined in the pres-
concentrated under reduced pressure to colorless solid. The solid
was further purified with column chromatography (DCM/
MeOH ¼ 30:1) to afford compound 55, as yellow solid (yield, 83.1%),
3
ence of the radioligand [ H]8-OH-DPAT. Nonspecific binding (NB)
was determined in the presence of the radioligand [ H]8-OHDPAT
and serotonin, whereas compound binding (CB) was determined
in the presence of the radioligand [ H]8-OH-DPAT and the com-
3
ꢁ
1
mp:124e126 C. H NMR (400 MHz, DMSO)
d
7.69 (d, J ¼ 8.5 Hz,
3
1
H), 6.69 (dd, J ¼ 8.4, 2.3 Hz, 1H), 6.61 (d, J ¼ 2.3 Hz, 1H), 3.46 (td,
13
J ¼ 6.7, 3.0 Hz, 2H), 2.96 (s, 3H), 2.86 (td, J ¼ 6.7, 2.4 Hz, 2H). C NMR
pound of interest. Each specific binding (SB) was calculated as the
total binding (TB) minus the nonspecific binding (NB) at a particular
concentration of radioligand. Each percentage of inhibition (%) was
calculated as follows: percentage of inhibition (%) ¼ [(TB - CB)/(TB -
NB)] ꢂ 100.
(
100 MHz, DMSO‑d 164.28, 160.69, 141.10, 129.88, 120.90, 114.19,
6
) d
1
C
13.73, 47.82, 34.81, 27.88. MS (ESI) m/z: 178.3 (calcd 178.1 for
þ
þ
10
H
12NO
2
[M þ H] ).
4
1
.28. 6-(3-Bromopropoxy)-2-methyl-3,4-dihydroisoquinolin-
(2H)-one (56)
Blank binding experiments contained 0.25% (v/v) DMSO were
performed; DMSO had no effect. All compounds were tested at least
ꢀ5
ꢀ10
three times over a 6-fold concentration range (10 M to 10 M).
IC values were determined by nonlinear regression analysis with
fitting to the Hill equation curve. Ki values were calculated using
5
0
The
dibromopropane(40.0 mmol),
2.0 mmol) and acetone (50.0 mL), was heated under reflux for 6 h.
Water (300 ml) was added to the reaction solution, which was then
extracted with DCM, The organic phase was dried over Na SO
mixture
of
compound
55
(20.0
mmol),1,3-
K
2
CO
3
(60.0 mmol), TEBA
(
the Cheng and Prussoff equation, Ki ¼ IC50/(1 þ C/K
d
), where C
represents the concentration of the hot ligand used and Kd the
receptor dissociation constant of each labeled ligand. The mean Ki
values and SEM were derived in at least three independent
experiments.
2
4
evaporated under reduced pressure to give crude oil. The residue
was purified with column chromatography (petroleum ether/
EtOAc ¼ 15: 1) to yield compound 56, as pale-yellow oil (3.89 g,
6
5.5%).
5.1.1. 5-HT1A receptor binding assay8,10
Rat cerebral cortex was homogenized in 20 vol of ice-cold
TriseHCl buffer (50 mM, pH 7.7) using an ULTRA TURAX homoge-
niser, and was then centrifuged at 32000 g for 10 min. The resulting
pellet was then resuspended in the same buffer, incubated for
4
.29. 6-(3-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)
propoxy)-2-methyl-3,4-dihydroisoquinolin-1(2H)-one (57)
ꢁ
The mixture of compound 56 (10.0 mmol),6-fluoro-3-(piper-
10 min at 37 C, and centrifuged at 32000 g for 10 min. The final
idin-4-yl)benzo[d]isoxazole hydrochloride (12.0 mmol), K
2
CO
3
pellet was resuspended in TriseHCl buffer containing 10
Pargyline, 4 mM CaCl and 0.1% ascorbic acid. Total binding each
assay tube was added 900 L of the tissue suspension, 50 L of
0.5 nM [ H]8-OH-DPAT (187.4 Ci/mmol, PerkinElmer Life Sciences,
Boston, MA, USA), 50 L TriseHCl buffer containing 10 M Pargy-
line, 4 mM CaCl and 0.1% ascorbic acid. Non-specific binding each
mM
(
30.0 mmol) and acetonitrile (40.0 mL), was heated under reflux for
2
8
h. Water (300 ml) was added to the reaction solution, which was
SO
4
m
m
3
then extracted with DCM, The organic phase was dried over Na
2
evaporated under reduced pressure to give yellow solid, The res-
m
m
idue was purified with column chromatography (DCM: MeOH ¼ 20
2
:
1
1) to afford compound 57, as white solid (yield, 52.2%), mp:
assay tube was added 900
mL of the tissue suspension, 50
mL of
ꢁ
1
3
16e118 C. H NMR (400 MHz, CDCl
3
)
d
7.82-7.62 (m, 2H), 7.24 (dd,
0.5 nM [ H]8-OH-DPAT, 50
m
L of 10
mM serotonin.Specific binding
J ¼ 8.5, 1.8 Hz, 1H), 7.08-7.03 (m, 1H), 6.95 (dd, J ¼ 8.3, 1.7 Hz, 1H),
each assay tube was added 900
m
L of the tissue suspension, 50 mL of
3
6
2
2
d
.84-6.79 (m, 1H), 4.11 (t, J ¼ 6.2 Hz, 2H), 3.11 (d, J ¼ 10.2 Hz, 3H),
.94 (s, 2H), 2.62 (t, J ¼ 7.2 Hz, 2H), 2.24-2.00 (m, 7H), 1.95-1.82 (m,
0.5 nM [ H]8-OH-DPAT, 50
mL of new compounds or reference
ꢁ
drug.The tubes were incubated at 37 C for 30 min. The incubation
was followed by a rapid vacuum filtration through Whatman GF/B
glass filters, and the filtrates were washed twice with 5 mL cold
buffer and transferred to scintillation vials. Scintillation fluid
(3.0 mL) was added and the radioactivity bound was measured
using a PE Microbeta 2450 liquid scintillation counter.
1
3
H), 1.43 (s, 2H), 0.38 (t, J ¼ 7.3 Hz, 3H). C NMR (101 MHz, CDCl
3
)
167.84, 165.31, 163.83, 162.82, 162.18, 161.05, 151.59, 124.74,
22.55, 117.28, 114.31, 112.46, 112.20, 106.53, 97.55, 97.28, 66.67,
4.99, 55.33, 53.59, 34.51, 30.49, 29.95, 26.84, 24.66, 23.78, 7.52.
1
6
HRMS (ESI) m/z: 438.2185 (calcd 438.2193 for C25
H] ).
H
29FN
3
O
3
þ[M þ
þ
5
.1.2. 5-HT2A receptor binding assay 8,10
5
. In vitro binding assays
Rat cerebral cortex was homogenized in 20 vol of ice-cold
TriseHCl buffer (50 mM, pH 7.7) using an ULTRA TURAX homoge-
niser, and centrifuged at 32000 g for 20 min. The resulting pellet
was resuspended in the same quantity of the buffer centrifuged for
20min. The final pellet was resuspended in 50 vol of the TriseHCl
buffer.
5
.1. General procedures for the binding assays
All of the new compounds were solved in 50% (v/v) DMSO, and
ꢀ3
the compound concentration was 2 ꢂ 10 M; dilution to the initial
ꢀ
4
concentration of the new compound, 2 ꢂ 10 M, contained 5%
DMSO. The following specific radioligands and tissue sources were
Total binding each assay tube was added 900
m
L of the tissue
L of 0.6 nM [ H]ketanserin (60.0 Ci/mmol, Perki-
nElmer Life Sciences, Boston, MA, USA), 50 L TriseHCl buffer.Non-
specific binding each assay tube was added 900 L of the tissue
L of 10 M methi-
sergide.Specific binding each assay tube was added 900 L of the
3
suspension, 50
m
used:
m
3
(
a) the serotonin 5-HT1A receptor, [ H]8-OH-DPAT, from rat
m
3
3
brain cortex; (b) the serotonin 5-HT2A receptor, [ H]ketanserin in
the present of 4-dione hydrochloride hydrate (35 nM), from rat
brain cortex; (c) the serotonin 5-HT2C receptor, [ H]mesulergine in
suspension, 50
m
L of 0.6 nM [ H]ketanserin, 50
m
m
m
3
3
tissue suspension, 50
m
L of 0.6 nM [ H]ketanserin, 150
m
L of new
ꢁ
the present of spiperone (40 nM), from rat brain cortex; (d) the
compounds or reference drug.The tubes were incubated at 37 C for
15 min. The incubation was followed by a rapid vacuum filtration
through Whatman GF/B glass filters, and the filtrates were washed
twice with 5 mL cold buffer and transferred to scintillation vials.
Scintillation fluid (3.0 mL) was added and the radioactivity bound
was measured using a PE Microbeta 2450 liquid scintillation counter.
3
serotonin 5-HT
HT
CT, from rat cerebral cortex. (f) the dopamine D
spiperone, from rat striatum; (g) the histamine H
6
receptor, [ H]lysergic acid diethylamide, from 5-
3
6
6
-C3 cells (CHO-K1); (e) the serotonin 5-HT receptor, [ H]-5-
3
2
receptor, [ H]
receptor, [ H]
3
1
mepyramine, from guinea pig cerebellum; (h) the adrenergic
a
1

J. Jin et al. / European Journal of Medicinal Chemistry 207 (2020) 112709
21
5
.1.3. 5-HT2C receptor binding assay8,10
Rat cerebral cortex was homogenized in 20 vol of ice-cold
Scintillation fluid(1.0 mL) was added, and the radioactivity bound
was measured using a PE Microbeta 2450 liquid scintillation
counter.
TriseHCl buffer (50 mM, pH7.7) using ULTRA TURAX homoge-
niser, and centrifuged at 32000 g for 20 min. The resulting pellet
was resuspended in the same quantity of the buffer centrifuged for
5.1.6. Dopaminergic D
2
receptor binding assay8,10
2
0min. The final pellet was resuspended in 50 vol of the TriseHCl
buffer. Total binding each assay tube was added 900 L of the tis-
L of 1 nM [ H]mesulergine (85.4 Ci/mmol;
PerkinElmer Life Sciences, Boston, MA, USA), 50 L TriseHCl
buffer.Non-specific binding each assay tube was added 900 L of
Rat striatum was homogenized in 20 vol of ice-cold 50 mM
TriseHCl buffer (pH 7.7) using an ULTRA TURAX homogeniser, and
centrifuged twice for 10 min at 48,000 g with resuspension of the
pellet in fresh buffer. The final pellet was resuspended in 50 mM
m
3
sue suspension, 50
m
m
m
2
ice-cold TriseHCl containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl ,
3
the tissue suspension, 50
mL of 1 nM [ H]mesulergine, 50
mL of
1 mM MgCl
2
, 0.1% ascorbic acid and 5
mM pargyline. Total binding
1
0
m
M mianserin.Specific binding each assay tube was added 900
m
L
each assay tube was added 900
m
L of the tissue suspension, 50 mL of
3
3
of the tissue suspension, 50
new compounds or reference drug.The tubes were incubated at
mL of 1 nM [ H]mesulergine, 50
mL of
0.5 nM [ H]spiperone (16.2 Ci/mmol; PerkinElmer Life Sciences,
Boston, MA, USA), 50 L TriseHCl buffer containing 120 mM NaCl,
5 mM KCl, 2 mM CaCl , 1 mM MgCl , 0.1% ascorbic acid and 5
pargyline.Non-specific binding each assay tube was added 900 mL
m
ꢁ
37 C for 15 min. The incubation was followed by a rapid vacuum
2
2
mM
filtration through Whatman GF/B glass filters, and the filtrates were
washed twice with 5 mL cold buffer and transferred to scintillation
vials. Scintillation fluid (3.0 mL) was added and the radioactivity
bound was measured using a PE Microbeta 2450 liquid scintillation
counter.
3
of the tissue suspension, 50
10
900
50
bated at 37 C for 15 min. The incubation was followed by a rapid
vacuum filtration through Whatman GF/B glass filters, and the fil-
trates were washed twice with 5 mL cold buffer and transferred to
scintillation vials. Scintillation fluid (3.0 mL) was added and the
radioactivity bound was measured using a PE Microbeta 2450
liquid scintillation counter.
m
L of 0.5 nM [ H]spiperone, 50 mL of
m
M (þ)-butaclamol.Specific binding each assay tube was added
3
m
L of the tissue suspension, 50 mL of 0.5 nM [ H]spiperone,
m
L of new compounds or reference drug.The tubes were incu-
ꢁ
5
.1.4. 5-HT
Membranes were prepared from CHO-5-HT
ected with the human serotonin 5-HT receptor cell. The harvested
receptor binding assay 8,10
6
6
cells stably trans-
6
cells are suspended in 1 volume of fresh physiological phosphate
buffered saline (PBS) solution and centrifugedat 1000 g. This ho-
mogenate was centrifuged at 100000 g for 60 min, the resulting
pellet was suspended in Tris-HCl (pH 7.4) to obtain a concentration
5.1.7. Histamine H
1
receptor binding assay8,10
7
corresponding to 4 ꢂ 10 cells/ml and aliquots were stored
Guinea pig cerebellum was homogenized in 20 vol of ice-cold
50 mM phosphate buffer (pH ¼ 7.4) using an ULTRA TURAX
homogeniser, and centrifuged twice for 10 min at 50,000 g with
resuspension of the pellet in fresh buffer. The final pellet was
resuspended in phosphate buffer.
ꢁ
at ꢀ80 C. Total binding each assay tube was added 900
mL of the
3
tissue suspension, 50
84.0 Ci/mmol, PerkinElmer Life Sciences, Boston, MA, USA), 50
Tris-HCl buffer. Non-specific binding each assay tube was added
mL of 2 nM [ H]lysergic acid diethylamide
(
mL
3
9
00
ylamide, 50
tube was added 900
lysergic acid diethylamide, 50
mL of the tissue suspension, 50
m
L of [ H]lysergic acid dieth-
Total binding each assay tube was added 900
m
L of membranes
L of 1 nM [ H] pyrilamine (20.0 Ci/mmol; PerkinElmer Life
Sciences, Boston, MA, USA), 50 L phosphate buffer.Non-specific
binding each assay tube was added 900 L of membranes, 50
M promethazine. Specific binding
3
m
L of 10 mM serotonin. Compound binding each assay
50 m
3
mL of the tissue suspension, 50
mL of [ H]
m
m
L of new compounds or reference
m
mL
ꢁ
3
drug. The tubes were incubated at 37 C for 30 min. The incubation
was followed by a rapid vacuum filtration through Whatman GF/B
glass filters, and the filtrates were washed twice with 5 mL cold
buffer and transferred to scintillation vials. Scintillation fluid
of [ H] pyrilamine, 50
mL of 1 m
3
each assay tube was added 900
m
L of Membranes, 50 mL of [ H]
pyrilamine, 50
m
L of new compounds or reference drug. The tubes
ꢁ
were incubated at 30 C for 60 min. The incubation was followed by
a rapid vacuum filtration through Whatman GF/B glass filters, and
the filtrates were washed twice with 5 mL cold buffer and trans-
ferred to scintillation vials. Scintillation fluid (3.0 mL) was added
and the radioactivity bound was measured using a PE Microbeta
2450 liquid scintillation counter.
(
1.0 mL) was added and the radioactivity bound was measured
using a PE Microbeta 2450 liquid scintillation counter.
5.1.5. 5-HT
7
receptor binding assay8,10
Rat cerebral cortex was homogenized in 20 vol of ice-cold
membrane buffer (50 mM Tris-HCl, pH 7.7 containing 4 mM
CaCl
2
, 0.1% ascorbic acid and 10
m
M pargyline) using an ULTRA
5.1.8. Adrenergic a
1
receptor binding assay 8,10
Rat cerebral cortex was homogenized in 20 vol of ice-cold Tris-
HCl buffer containing 5 mM EDTA (50 mM, pH 7.4) using an ULTRA
ꢁ
TURAX homogeniser, and centrifuged at 50,000g for 12 min at 4 C.
The resulting membrane pellets were washed by resuspension in
membrane buffer (50 mM Tris-HCl, pH 7.7 containing 4 mM CaCl
ꢁ
2
,
TURAX homogeniser and centrifuged at 44000 g for 20 min at 4 C.
0
2
.1% ascorbic acid and 10
0 min at 37 C. The final pellet was resuspended in 50 vol of the
m
M pargyline) and centrifuged for
The resulting pellet was resuspended in the same quantity of the
buffer centrifuged for 20 min. The final pellet was resuspended in
ꢁ
Tris-HCl buffer containing 4 mM CaCl
2
, 0.1% ascorbic acid and 10
m
M
50 vol of the Tris-HCl buffer. For total binding, to each assay tube
3
pargyline. For total binding, to each assay tube was added 900
m
m
L of
L of
membrane buffer. For nonspecific binding, to each assay tube was
was added 900
mL of the tissue suspension, 50
mL of 1 nM [ H]
3
the tissue suspension, 50
mL of 5 nM [ H]-5-CT, and 50
prazosin (85.4 Ci/mmol; PerkinElmer Life Sciences, Boston, MA,
USA), and 50 L of Tris-HCl buffer. For nonspecific binding, to each
assay tube was added 900 L of the tissue suspension, 50 L of 1 nM
L of 10 mMprazosin. Compound binding, to
each assay tube was added 900 L of the tissue suspension, 50 L of
m
3
added 900
L of 1
added 900
L of compound 47 solution in various concentrations (10 mol
mL of the tissue suspension, 50
m
L of 5 nM [ H]-5-CT, and
m
m
3
5
0
m
m
M5-HT. For specific binding, to each assay tube was
[ H]prazosin, and 50 m
3
mL of the tissue suspension, 50
m
L of 5 nM [ H]-5-CT,
m
m
ꢀ
5
3
5
0
m
[ H]prazosin, 50
m
L of new compounds, or reference drug. The
ꢀ10
ꢁ
ꢁ
to 10
mol). The tubes were incubated at 37 C for 30 min. The
tubes were incubated at 25 C for 60 min. The incubation was fol-
lowed by rapid vacuum filtration through Whatman GF/B glass
filters, and the filtrates were washed twice with 5 mL of cold buffer
and transferred to scintillation vials. Scintillation fluid (1.0 mL) was
incubation was followed by rapid vacuum filtration through
Whatman GF/B glass filters, and the filtrates were washed twice
with 5 mL of cold buffer and transferred to scintillation vials.

22
J. Jin et al. / European Journal of Medicinal Chemistry 207 (2020) 112709
added, and the radioactivity bound was measured using a PE
Microbeta 2450 liquid scintillation counter.
were controlled by pCLAMP software (version 10.1, Molecular De-
vices). The bath solution consisted of 137 mM NaCl, 5.4 mM KCl,
1
0 mM glucose, 10 mM HEPES, and 2 mM CaCl
adjusted to 7.5 by addition of NaOH. The pipet filling solution
consisted of 140 mM KCl, 1 mM MgCl , 5 mM EGTA, 10 mM HEPES,
and 5 mM Na ATP. The pH was adjusted to 7.2 by addition of KOH.
To study voltage dependence of steady-state block of hERG
channels on different drug concentrations (0.3, 1, 3, and 10 M) in
2
. The pH was
5
.1.9. Experimental in vitro pharmacology for intrinsic activity
10
Assessment
2
CHO-K1 cells expressing five receptors (HEK293/D
HT1A, HEK293/5-HT2A, HEK293/h5-HT and HEK293/h5-HT
seeded in a 384-well black-walled, clear bottom plate at a density of
2
, HEK293/5-
2
6
7
) were
m
2
0,000 cell per well in 20
m
L of growth medium, 18 h prior to the
HEK cells, the holding membrane potential was switched from ꢀ80
to þ50 mV for 2 s following return to ꢀ50 mV for 3 s (sampling rate
of 4 kHz, low-pass filtered at 1 kHz) in intervals of 30 s. Tail currents
were measured at ꢀ50 mV in control and in the presence of the
drug at concentrations determined empirically. All raw measure-
ments were performed using Clampfit (version 10.2), a part of
pCLAMP software (version 10.1). Results were transferred to the
program Statistical Package for the Social Sciences (SPSS) spread-
sheets for further analysis.
ꢁ
day of experiment, and maintained at 37 C/5% CO
2
.
For the agonist assay, 20
mL of dye-loading solution (20 mM
HEPES/HBSS with 2.5 mM Probenecid, pH 7.4) was added to the
well. Then, the plate was placed into a 37 C incubator for 60 min,
followed by 15 min at room temperature. Finally, 10 L of com-
pounds or control agonist were added into respective wells of the
assay plate.
ꢁ
m
For the antagonist assay, 20
mL of dye-loading solution (20 mM
HEPES/HBSS with 2.5 mM Probenecid, pH 7.4) and 10
mL of com-
pound or control antagonist solution were added into the well.
Then, the plate was placed into a 37 C incubator for 60 min, fol-
6. Behavioral tests
ꢁ
lowed by 15 min at room temperature. Finally, 12.5 mL of control
agonist was added into respective wells of the assay plate during
reading in FLIPR.
6
.1. Animals
Chinese Kun Ming (KM) Mice (20 ± 2.0 g) and Sprague-Dawley
For the agonist test, the compounds or agonist plate contained
compounds or control agonist at 5 ꢂ final test concentration and
were placed at Source 2. Compounds or control agonist was added
to the reading plate at 20 s and the change in cell fluorescence
signal was measured in a FLIPR (FLIPR Calcium 4 assay kit, Molec-
ular Devices) for an additional 100 s (21e120 s). For the antagonist
test, only compounds or control agonist solution at the Source 2
plate was replaced with control agonist solution at 5 ꢂ EC80
concentration.
Data were recorded by ScreenWorks (version 3.1) as FMD files
with FLIPR. Data acquisition and analyses were performed using the
ScreenWorks (version 3.1) program and exported to Excel. The
average value of 20 (1 se20 s) seconds of reading was calculated as
(
SD) rats (250 ± 5.0 g) were used as experimental animals in this
study. Animals were housed under standardized conditions for
light and temperature and received standard rat chow and tap
water and libitum. Animals were randomly assigned to different
experimental groups, each kept in a separate cage. All studies
involving animals in this research follow the guidelines of the
bylaw of experiments on animals and have been approved by the
Ethics and Experimental Animal Committee of Jiangsu Nhwa
Pharmaceutical Co., Ltd.
6.1.1. Acute toxicity study8,10
Mice (5 mice in each group) were orally dosed with increasing
doses of the compound 13 (100, 200, 500, 1000, 1500 and 2000 mg/
kg). The number of surviving animals was recorded after 24h of
drug administration, and the percent mortality in each group was
calculated. The LD50 values were calculated by using the program
SPSS (Statistical Package for the Social Science).
the baseline reading and the relative fluorescent unit (
tensity values were calculated with the maximal fluorescent units
DRFU) in-
(
21 se120 s) subtracting the average value of baseline reading.
The % activation of the compound was calculated from the
following equation:
%
D
activation ¼ {(
D
RFUCompound
-
D
RFUBackground)/(
D
RFUAgonist control
-
.1.2. MK-801-induced hyperactivity8,10
Mice (10 mice in each group) were orally dosed with vehicle or
6
RFUBackground)} ꢂ 100
increasing doses of the haloperidol (0.06, 0.2, 0.6, 2.0 and 6 mg/kg),
aripiprazole (0.3, 1.0, 3.0 and 10 mg/kg), risperidone (0.01, 0.03, 0.1,
The % activation was then plotted as a function of the log of the
cumulative doses of compounds.
The % inhibition of the test article was calculated from the
following equation:
0.3 and 1.0 mg/kg) and compound 13 (0.03, 0.1, 0.3 and 1.0 mg/kg).
Animals were placed in Plexiglas cages for evaluating locomotor
activity. After 30 min, the animals were challenged with 0.3 mg/kg
(
sc) of MK-801 and the locomotor activity of each animal was
%
inhibition ¼ {1 - (
D
RFUCompound
- DRFUBackground)/(DRFUAgonist
recorded for 90 min.
control
- D
RFUBackground)} ꢂ 100
6.1.3. Apomorphine-induced climbing8,10
The % inhibition was then plotted as a function of the log of the
cumulative doses of compounds.
Mice (10 mice in each group) were orally dosed with vehicle or
increasing doses of the haloperidol (0.1, 0.13, 0.17, 0.23 and 0.3 mg/
kg), aripiprazole (0.3, 1.0, 3.0 and 10 mg/kg), risperidone (0.01, 0.03,
0.1 and 0.3 mg/kg), compound 13 (0.03, 0.1, 0.3 and 1.0 mg/kg).
Animals were then challenged at 30 min post-injection with
5
.1.10. hERG Affinity10
Ability to block hERG potassium channels was determined using
the whole-cell patch clamp method and cloned hERG potassium
channels (expressed in HEK 293 cells) as biological material. For
this purpose, the patch clamp amplifier (Axopatch 200B, Molecular
Devices) and digital converter (Digidata 1440A, Molecular Devices)
were used. Recording electrodes were made from borosilicate glass
with filament (BF120-94-15, Sutter Instrument Company). Creation
of voltage-clamp command pulse protocols and data acquisition
þ
1.0 mg/kg of the apomorphine in 0.9% NaCl 0.1% ascorbic acid,
placed in cylindrical wire cages (12 cm in diameter, 14 cm in
height), and observed for climbing behavior at 10, 20 and 30 min
post dose. The climbing behavior was scored as follows: 3e4 paws
on the cage floor ¼ 0 score; 2 and 3 paws on the cage ¼ 1 score; 4
paws on the cage ¼ 2 score.

J. Jin et al. / European Journal of Medicinal Chemistry 207 (2020) 112709
23
6
.1.4. DOI-induced head twitch 50
6.3.1. Weight gain and serum prolactin 8,10
Mice (10 mice in each group) were orally dosed with increasing
Mice (10 mice in each group) were orally dosed with vehicle or
increasing doses as follows: risperidone (0.03, 0.1, and 0.3 mg/kg)
and 13 (0.08, 0.3 and 0.8 mg/kg). The animals received multiple
doses (28 day) of each compound. The weight of mice was tested
every day. The mice were killed by decapitation 180 min after the
last treatment. Blood samples (2 mL) were collected and centri-
fuged (300 g for 30 min), and the resulting serum samples were
doses of the aripiprazole (0.3, 1.0, 3.0 and 10 mg/kg), risperidone
0.01, 0.03, 0.1 and 0.3 mg/kg), compound 13 (0.03, 0.1, 0.3 and
.0 mg/kg). or vehicle 60 min before i.p. administration of DOI
1 mg/kg). Immediately after DOI treatment, animals were indi-
vidually placed into observation chambers; after a 5-min wait, the
number of head twitch responses that occurred during the next
(
1
(
ꢁ
1
5 min was counted. The mean (SEM) number of head twitches was
calculated for each group.
stored at ꢀ20 C until analyzed for prolactin (PRL). Serum PRL was
determined by an EIA-kit from Amersham. Data were analyzed by
Student’s t-test (##p < 0.01).
6
.1.5. Forced swim test (FST) 51
A total of 110 naive mice were randomly assigned to eleven
.3.2. Pharmacokinetics study in rats 8,10
The HPLC conditions were as follows: column, XSELECT CSH XP
6
groups consisting of vehicle control, duloxetine (20 mg/kg), and
compound 13 (0.02, 0.06 and 0.2 mg/kg) with each group con-
taining 10 mice. One hour after oral administration of test com-
pounds, each mouse was forced to swim in an open cylindrical
container (diameter of 10 cm, height of 25 cm, containing 15 cm of
C18 (2.1 mm ꢂ 50 mm, 2.5
mm); mobile phase, 0.025% FA and 1 mM
NH4OAc (ROE SCIENTIFIC INC, USA) in water/acetonitrile (Merck
Company, Germany) (v:v, 45:55); flow rate, 0.6 mL/min; column
ꢁ
temperature, 50 C. UV detection was performed at 210 nm. For
ꢁ
water with temperature maintained at 24 ± 1 C). The duration of
routine compound 13 screening, rats (n ¼ 6/group) were dosed via
immobility during the last 4 min of total 6 min was recorded, and
animals were judged to be immobile when they floated motionless,
making only necessary movements to keep their heads above the
water.
the lateral tail vein at the indicated dose for iv administration
(
0.5 mg/kg, 100% saline) or via oral gavage (5 mg/kg, suspension in
0.5% methylcellulose). At 30 min and 1, 2, 3, 4, 5, 6, 8 and 24 h after
administration, serial blood samples were collected from the lateral
tail vein into heparinized collection tubes (approximately 0.25 mL).
The plasma was separated by centrifugation, and the sample was
prepared for LC/MS analysis by protein precipitation with aceto-
nitrile. The plasma samples were analyzed for drug and internal
standard via the LCꢀMS/MS protocol.
6
.1.6. Tail suspension test (TST) 52
A total of 110 naive mice were randomly assigned to eleven
groups consisting of vehicle control, duloxetine (20 mg/kg), and
compound 13 (0.02, 0.06 and 0.2 mg/kg) with each group con-
taining 10 mice. After oral administration of compounds for 60 min,
each mouse was suspended on the top of apparatus using adhesive
tape placed approximately 1 cm from the tail tip. The immobility
duration of the last 4 min of total 6 min period was recorded. Mice
were considered to be immobile when hung passively without
moving.
6
.3.3. Novel object recognition training and testing 10
Rats were acclimated for 1 week prior to the experiment. The
rats were housed in groups of two/cage in a light-controlled room
12 h light/dark cycles with lights on at 07:00). Food and water
(
were provided ad libitum. Rats were habituated to handling and an
empty test arena (a gray-colored polyvinyl chloride box
3
6
.1.7. Catalepsy test 8,10
Mice (10 mice in each group) were orally dosed with vehicle or
(40 ꢂ 40 ꢂ 50 cm )) for 7 min on each of 3 consecutive days. On the
training day, the drug was administered p.o. in a vehicle of physi-
ological saline at a volume of 1 mL/kg 1h after dosing, each rat was
placed in the test arena, which now contained two identical objects
located centrally in the arena. Rats were given either 3 min explore
the arena and objects. Memory retention was tested 24 h after
training. Rats were placed back in the arena with one “familiar”
(previously trained) and one “novel” object and given 5 min to
explore. The spatial position of objects leftꢀright position) and
which object was novel (ball or cube) was counterbalanced across
subjects. Objects and arenas were cleaned with diluted 75% alcohol
solution between trials to remove rat feces and urine. To determine
memory performance, the novelty discrimination index (NDI) was
calculated using the following equation: novel object interaction
time/total interaction time ꢂ 100 (%). Rats were excluded from the
analysis if total exploration time in the acquisition trial was less
than 10 s. Numbers of rats treated with vehicle and those treated
with 0.02, 0.06, and 0.2 mg/kg of compound 13 were 14, 15, 15, and
increasing doses of the haloperidol (0.18, 0.35, 0.75, 1.5 and 3.0 mg/
kg), aripiprazole (5,15, 30, 100 and 150 mg/kg), risperidone (0.1, 0.6,
1.2, 2.5 and 5.0 mg/kg), compound 13 (0.5, 1.5, 3, 15 and 30 mg/kg).
Catalepsy was evaluated on a metal bar 0.6 cm in diameter posi-
tioned 4.5 cm above the tabletop. The test consisted in positioning
the animal with its forepaws on the bar and recording how long it
remained hanging onto the bar; the end-point was 60 s and an all-
or-none criterion was used.
6.2. Locomotor activity
Mice (8 mice in each group) were orally dosed with vehicle or
increasing doses of the clonazepam (1, 3 and 10 mg/kg) and com-
pound 13 (0.06, 0.2 and 0.6 mg/kg). Locomotor activity was assessed
in automated activity frames equipped with an automated video
tracking system. Test compounds were orally administered by
intragastric administration 1 h before test start. Mice were accli-
mated to the test chamber for 30 min before starting the experiment.
Locomotor activity was then measured for 10 min. The horizontal
distance traveled in centimeters by the animals was analyzed.
14, respectively. To estimate the potency of test and reference
compounds, the ED50 values and their 95% confidence limits were
calculated by using the program SPSS (Statistical Package for the
Social Science).
6.3. Statistics
Declaration of competing interest
To estimate the potency of test and reference compounds, the
ED50 values and their 95% confidence limits were calculated by
using the program SPSS (Statistical Package for the Social Science).
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.

2
4
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