Synthesis and pharmacological characterization of novel N-(trans-4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)ethyl)cyclohexyl)amides as potential multireceptor atypical antipsychotics

European Journal of Medicinal Chemistry 123 (2016) 332e353

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European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Research paper

Synthesis and pharmacological characterization of novel N-(trans-4-

(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)ethyl)cyclohexyl)

amides as potential multireceptor atypical antipsychotics

,

, **

Xiao-Wen Chen ^{a b}, Yuan-Yuan Sun ^a, Lei Fu ^{a *}, Jian-Qi Li ^b

^aSchool of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, PR China

^bNovel Technology Center of Pharmaceutical Chemistry, Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry,

285 Gebaini Road, Shanghai 201203, PR China

a r t i c l e i n f o

a b s t r a c t

Article history:

A series of novel benzisothiazolylpiperazine derivatives combining potent dopamine D₂and D₃, and

serotonin 5-HT_1Aand 5-HT_2Areceptor properties were synthesized and evaluated for their potential

antipsychotic properties. The most-promising derivative was 9j. The unique pharmacological features of

9j were a high afﬁnity for D₂, D₃, 5-HT_1A, and 5-HT_2Areceptors, together with a 20-fold selectivity for the

D₃versus D₂subtype, and a low afﬁnity for muscarinic M₁(reducing the risk of anticholinergic side

effects), and for hERG channels (reducing incidence of QT interval prolongation). In animal behavioral

models, 9j inhibited the locomotor-stimulating effects of phencyclidine, blocked conditioned avoidance

response, and improved the cognitive deﬁcit in the novel object recognition tests in rats. 9j exhibited a

low potential for catalepsy, consistent with results with risperidone. In addition, favorable brain pene-

tration of 9j in rats was detected. These studies have demonstrated that 9j is a potential atypical anti-

psychotic candidate.

Received 6 April 2016

Received in revised form

29 June 2016

Accepted 19 July 2016

Available online 22 July 2016

Keywords:

Dopamine

Serotonin

Multireceptor

X-ray crystallography

Atypical antipsychotic

1. Introduction

create an unmet clinical need for patients suffering from schizo-

phrenia. Therefore, innovative antipsychotic agents that provide

Schizophrenia is a chronic, severe, and often disabling mental

disorder that affects ~1% of the global population [1,2]. Regarded as

a spectrum disorder [3], schizophrenia is categorized by positive,

negative, and cognitive symptoms. First generation antipsychotic

agents (FGAs), such as dopamine D₂receptor antagonists, effec-

tively treat positive symptoms, but are ineffective in alleviating

negative symptoms or cognitive impairment. FGAs can also cause

serious side effects such as extrapyramidal symptoms (EPS) [4,5].

Second-generation agents (SGAs) (i.e., lurasidone, ziprasidone and

risperidone, Fig. 1) are characterized by multi-receptor afﬁnity and

offer a variety of therapeutic advantages, while causing fewer EPSs

than FGAs. However, SGAs cause adverse metabolic, anticholinergic

and cardiovascular effects such as weight gain, constipation and

prolonged heart rate-corrected QT intervals [6e8]. Inadequacies in

treating these negative side effects and cognitive impairments

superior therapeutic and decreased side effect proﬁles are urgently

needed.

Drugs that have multiple targets are generally accepted to be

better antipsychotic agents [9e12]. SGAs studies have conﬁrmed

that simultaneous action on both 5-HT_1Aand 5-HT_2Areceptors play

a major role in managing nonpsychotic schizophrenia symptoms

and reducing incidents of EPS [13,14]. Studies have also indicated

that the D₂receptor is an indispensable target for drug action, as all

current antipsychotics block the D₂receptor; aripiprazole, brexpi-

prazole and cariprazine (Fig.1) are D₂partial agonists. Dopamine D₃

receptors, which belong to a subfamily of D₂-like receptors [15], are

mainly expressed in the limbic system and are engaged in regu-

lating emotional and cognitive functions [16,17]. Selective D₃versus

D₂receptor (D₃vs D₂R) antagonists enhance frontocortical

cholinergic transmission and improve social cognition, which may

be beneﬁcial in managing of cognitive impairment in schizophrenia

[18]. Early studies have demonstrated that ligands with high pref-

erence for D₃receptors (>60-fold selectivity for D₃vs D₂R) were

inactive in animal models with potential antipsychotic activity,

such as those with conditioned avoidance behavior [19] or

* Corresponding author.

** Corresponding author.

E-mail addresses: leifu@sjtu.edu.cn (L. Fu), lijianqb@126.com (J.-Q. Li).

http://dx.doi.org/10.1016/j.ejmech.2016.07.038

X.-W. Chen et al. / European Journal of Medicinal Chemistry 123 (2016) 332e353

333

Fig. 1. Structures of reference compounds.

locomotor response to phencyclidine (PCP) in rats [20]. Recent ef-

forts developing selective D₃vs D₂R antagonists as potential anti-

psychotic agents have been focused on achieving compounds with

a minimum of 10-fold D₃/D₂selectivity, as well as a potent degree

(subnanomolar) of D₃antagonism coupled to a moderate degree

(nanomolar) of D₂antagonism to achieve simultaneous D₂and D₃

antagonism [21e25]. Cariprazine was recently released in America

as the ﬁrst orally active dopamine D₃-preferring D₃/D₂receptor

partial agonist that has ~10-fold selectivity for the D₃versus D₂

subtype and subnanomolar afﬁnity for D₂and D₃receptors, and this

drug exhibited good antipsychotic activity with favorable safety

and tolerability [26,27].

Previously, Meltzer et al. studied the biological proﬁles of a se-

ries of neuroleptic agents and postulated that mixed D₂/5-HT₂

antagonists (higher afﬁnity ratio for 5-HT_2Areceptors relative to D₂

receptors) should possess atypical antipsychotic properties [28].

Based on this 5-HT₂/D₂receptor antagonism concept, several

atypical antipsychotic agents have been developed including ris-

peridone [29], which has been widely used in clinical therapies.

Based on these ﬁndings, efforts were focused on obtaining new

atypical antipsychotic agents that possess the following multi-

receptor afﬁnity proﬁles: (a) high afﬁnity for D₂, D₃, 5-HT_1A, and 5-

HT_2Areceptors (K_i< 10 nM); (b) 10e60 fold selectivity for the D₃

versus D₂subtype as well as potent D₃antagonism coupled to

moderate D₂antagonism; (c) a higher receptor afﬁnity for 5-HT_2A

than D₂(K_iratio > 1) [28]; (d) low afﬁnity for muscarinic M₁re-

ceptors (to reduce the risk of anticholinergic side effects) and for

hERG channels (to reduce incidence of QT interval prolongation).

The main hurdle in discovering novel multireceptor antipsy-

chotics lies in balancing desired receptor afﬁnity with receptor

subtype selectivity. Subtype selectivity is further complicated due

to the highly similar nature of the D₂and D₃dopamine receptors

which share approximately 50% sequence homology [30]. Studies

have revealed that most D₃-speciﬁc ligands share a common

structure including an amide moiety, an arylpiperazine system, and

receptors share common arylpiperazine fragments [37e39]. 3-

Benzisothiazolyl-piperazine, a known serotonin pharmacophore,

was combined with dopamine activity-related pharmacophores to

achieve potential antipsychotic agents that incorporate high afﬁn-

ity and antagonism for both 5-HT_2Aand D₂receptors [40e43]. In

order to validate the proposed multireceptor afﬁnity proﬁle

approach to developing novel antipsychotics, while achieving an

optimum interaction with dopamine and serotonin receptors, a

new set of compounds with a cyclohexylamide system linked to the

benzisothiazolylpiperazine moiety was designed (Fig. 2).

Herein, we report the synthesis and pharmacological evaluation

of benzisothiazolylpiperazine derivatives as potential novel multi-

target antipsychotics. The structure-activity relationships of target

compounds for the D₂, D₃, 5-HT_1Aand 5-HT_2Areceptors were

investigated as a function of various substituents in the amide

moiety and spacer groups. The effect on receptor afﬁnity of various

substitutions at the 6-position of the benzo[d]isothiazole moiety

was further studied, which has been seldom reported [44]. The

selected compounds balanced for optimum dopamine to serotonin

receptor afﬁnity, were further subjected to behavioral study.

Among these compounds, compound 9j was chosen for further

pharmacological investigation including afﬁnity for crucial anti-

targets (muscarinic M₁receptor and hERG channels) and potential

atypicality (intrinsic activity at selected receptors and catalepto-

genic propensity). Moreover, brain and blood pharmacokinetics of

9j in rats were further explored as druggable properties.

2. Chemistry

The synthesis of target compounds 6e9,12,18, 35e37, 41 and 45

is depicted in Schemes 1e5. Scheme 1 outlines the synthesis of 6e9

and 12. The starting material N-(4-oxocyclohexyl)acetamide was

converted into the ethyl ester 1 via Horner Wittig reaction and

hydrogenation, which was then hydrolyzed and recrystallized in

DMF to give a trans carboxylic acid derivative (2) (57.6% yield). After

an esteriﬁcation reaction and NaBH₄/MeOH reduction, the alcohol

derivative 4 was prepared. Intermediate 4 was reacted with mesyl

chloride to give an activated-ester 5, which was coupled with 1-

(1,2-benzisothiazol-3-yl)piperazine to give compound 6. Then, 6

was reﬂuxed in 5% aqueous hydrochloric acid solution to yield the

a

ﬂexible/partially rigid-spacer (e.g., cyclohexyl, cyclopropyl,

alkenyl or alkynyl). Structural variations in amide moiety inﬂuence

both the D₃receptor afﬁnity and D₃/D₂receptor selectivity ratio

[31,32], and this ratio is also altered by the spacer group [33e36].

Ligands that preferentially bind to dopamine and serotonin

334

X.-W. Chen et al. / European Journal of Medicinal Chemistry 123 (2016) 332e353

Fig. 2. Design of benzisothiazolylpiperazine cyclohexylamides.

Scheme 1. Reagents and conditions: (i) Triethyl phosphonoacetate, tBuOK, THF, 0e5 ^ꢀC; (ii) H₂, 10%Pd/C, EA, rt; (iii) LiOH$H₂O, THF, H₂O, DMF, recrystallization; (iv) EtOH, conc.

H₂SO₄, reﬂux; (v) NaBH₄, MeOH, THF, reﬂux; (vi) Et₃N, MsCl, CH₂Cl₂, 0e5 ^ꢀC; (vii) Na₂CO₃, KI, Acetonitrile, reﬂux; (viii) 5%HCl, reﬂux; (ix) for 8: BTC, Et₃N, IPA, CH₂Cl₂; for 9a-q: Et₃N,

CH₂Cl₂, 0e5 ^ꢀC; for 9r: EDC.HCl, DMAP, CH₂Cl₂; for 10: DIBOC, Et₃N, CH₂Cl₂; (x) LiAlH₄, THF, reﬂux; (xi) Et₃N, CH₂Cl₂, 0e5 ^ꢀC.

key intermediate 7. Condensation of 7 with various amines afforded

compound 8. Compounds 9a-q were obtained via acylation of 7

with acyl chlorides. 7 reacted with indole-2-carboxylic acid to give

compound 9r. Reduction of intermediate 10 using LiAlH₄in THF

afforded compound 11, which reacted with 2-thiophenesulfonyl

chloride to give target compound 12.

Scheme 2 illustrates the synthesis of compound 18. Using a cis/

trans mixture 13 (obtained from the mother liquor of 2) as a starting

material, cis/trans mixture 15 was achieved by esteriﬁcation,

reduction, activation, and condensation with 1-(1,2-benzisothiazol-

3-yl)piperazine. The cis stereoisomer 16 was obtained via column

chromatography and recrystallization from 15. Finally, compound

18 was achieved by hydrolysis and acylation.

Scheme 3 depicts the synthesis of compounds 35e37. First, in-

termediate 23 was prepared as previously described [44]. Then, 23

was treated with sulfuryl chloride in chloroform and ammonia

hydrate to provide 25, which was chlorinated and condensed with

piperazine to afford 29 [44]. In synthesizing of 3-piperazinyl de-

rivative 30, ﬁrst benzyl mercaptan and 2-ﬂuoro-4-nitrobenzoic 20

were condensed in the presence of DIPEA in DMF to provide

compound 22. Then, 22 was converted into the corresponding acyl

chloride 24, which was then treated with sulfuryl chloride in

X.-W. Chen et al. / European Journal of Medicinal Chemistry 123 (2016) 332e353

335

Scheme 2. Reagents and conditions: (i) EtOH, conc. H₂SO₄, reﬂux; (ii) NaBH₄, MeOH, THF, reﬂux; (iii) Et₃N, MsCl, CH₂Cl₂, 0e5 ^ꢀC; (iv) Na₂CO₃, KI, Acetonitrile, reﬂux; (v) 5%HCl,

reﬂux; (vi) Et₃N, CH₂Cl₂, 0e5 ^ꢀC.

Scheme 3. Reagents and conditions: (i) for 21: Benzyl mercaptan, CuCl, K₂CO₃, DMF, 120 ^ꢀC, Nitrogen ambience; for 22: Benzyl mercaptan, DIPEA, DMF, 65 ^ꢀC; (ii) for 23: SOCl₂,

DMF, Toluene, reﬂux; for 24: (COCl)₂, DMF, CHCl₃, reﬂux; (iii) SO₂Cl₂, CHCl₃, NH₃$H₂O, rt; (iv): POCl₃, PCl₅, DIPEA, reﬂux; (v) for 29: Piperazine, Chlorobenzene, reﬂux; for 30:

Piperazine, Chlorobenzene, K₂CO₃, DMF, 80 ^ꢀC, Nitrogen ambience; (vi) Na₂CO₃, KI, Acetonitrile, reﬂux; (vii) 5%HCl, reﬂux; (viii) Et₃N, CH₂Cl₂, 0e5 ^ꢀC; (ix) ReNi, 85% Hydrazine

monohydrate, MeOH, rt.

chloroform and ammonia hydrate to provide 26. Compound 26 was

chlorinated and subsequently condensed with piperazine to afford

30. With 29 and 30 in hand, compounds 35e36 were achieved by

condensation of 29 and 30 with 5, and subsequent hydrolysis, and

acylation, respectively. Finally, nitro reduction of 36 by nickel Raney

afforded 37.

corresponding NaBH₄/MeOH reduction yielded trans-cyclohexane-

1,4-diyldimethanol 42. Subsequent treatment with mesyl chloride

and condensation with 3-(piperazin-1-yl)benzo[d]isothiazole

afforded 43. The key intermediate 44 was obtained using Gabriel

reaction from 43. Finally, 44 was condensed with 2-furoyl chloride

to furnish 45.

Scheme 4 describes the synthesis of compound 41. Intermediate

39 was obtained by Horner Wittig reaction, hydrogenation, and

subsequent reduction and activation with mesyl chloride. The key

intermediate 40 was obtained by deprotecting the tert-butox-

ycarbonyl group. Finally, acylation of 40 with acyl chlorides fur-

nished 41.

In order to assign the cis/trans stereochemistry, a single X-ray

crystal structure was obtained for compound 6. The single crystal

image for compound 6 was numbered by atom, demonstrating that

the cyclohexane ring was in the trans conformation (Fig. 3). It is

worth noting that the corresponding compounds 7e9, 12 and

35e37 were preferentially in the trans conformation of cyclohexane

ring as observed in the crystal structure of 6.

Scheme 5 depicts the synthesis of compound 45. The methyl

esteriﬁcation of trans-cyclohexane-1,4-dicarboxylic acid and

336

X.-W. Chen et al. / European Journal of Medicinal Chemistry 123 (2016) 332e353

Scheme 4. Reagents and conditions: (i) Triethyl phosphonoacetate, tBuOK, THF, 0e5 ^ꢀC; (ii) H₂, 10%Pd/C, EtOH, rt; (iii) NaBH₄, MeOH, reﬂux; (iv) Et₃N, MsCl, CH₂Cl₂, 0e5 ^ꢀC; (v)

Na₂CO₃, KI, Acetonitrile, reﬂux; (vi) TFA, CH₂Cl₂, rt; (vii) Et₃N, CH₂Cl₂, 0e5 ^ꢀC.

Scheme 5. Reagents and conditions: (i) SOCl₂, CH₃OH, reﬂux; (ii) NaBH₄, CH₃OH, THF; (iii) Et₃N, MsCl, CH₂Cl₂, 0e5 ^ꢀC; (iv) Na₂CO₃, KI, Acetonitrile, reﬂux; (v) Potassium

phthalimide, DMF, 100 ^ꢀC; (vi) 85% Hydrazine monohydrate, EtOH, reﬂux; (vii) 2-Furoyl chloride, Et₃N, CH₂Cl₂, 0e5 ^ꢀC.

Fig. 3. ORTEP diagram of 6 with 30% thermal ellipsoid plot for non-hydrogen atoms with atom labeling. Details about X-ray crystallographic data of 6 was reported in the Sup-

plementary Material (Table S1 and Fig. S1; CCDC 1414993).

3. Pharmacology

The IC₅₀values and Hill coefﬁcients (nH) were determined by

non-linear regression analysis of the competition curves generated

with mean replicate values using Hill equation curve ﬁtting. The

inhibition constants (K_i) were calculated using the Cheng Prusoff

equation: K_i¼ IC₅₀/(1 þ L/K_D), where L ¼ concentration of radio-

ligand in the assay, and K_D¼ afﬁnity of the radioligand for the re-

ceptor. A scatchard plot was used to determine the K_D.

The ability to block hERG potassium channels was determined

using an electrophysiological method and cloned hERG potassium

channels (expressed in CHO-K1 cells).

3.1. In vitro binding studies

The afﬁnities (K_ivalues) of the synthesized compounds to hu-

man D_2L, D₃, 5-HT_1A, 5-HT_2Aand M₁receptors were evaluated by

in vitro binding assays. The following speciﬁc radioligands and

tissue sources were used: (a) D_2Lreceptors, [³H]spiperone, human

recombinant (CHO cells); (b) D₃receptors, [³H]methyl-spiperone,

human recombinant (CHO cells); (c) 5-HT_1Areceptors, [³H]8-OH-

DPAT, human recombinant (HEK-293 cells); (d) 5-HT_2Areceptors,

[³H]ketanserin, human recombinant (HEK-293 cells); (e) M₁re-

ceptors, [³H]pirenzepine, human recombinant (CHO cells). Binding

studies were conducted by Euroﬁns Cerep SA, Celle l'Evescault,

France. Cariprazine was used as a reference.

3.2. In vivo studies

The selected compounds were further subjected to preliminary

pharmacological evaluation to determine their antipsychotic

X.-W. Chen et al. / European Journal of Medicinal Chemistry 123 (2016) 332e353

337

activity in vivo animal models, including PCP-induced locomotive

pharmacokinetic studies in rats.

activity, conditioned avoidance response (CAR), novel object

recognition (NOR) task, and catalepsy models.

4. Results and discussion

3.3. Intrinsic activity studies

4.1. In vitro studies of new compounds

The most-promising compound was subjected to intrinsic ac-

tivity studies at D₂, D₃, 5-HT_1A, and 5-HT_2Areceptors.

4.1.1. Effect of various substituents in amide moiety

4.1.1.1. Effect of different urea moieties. Initially, the afﬁnity proﬁle

of compound 7 bearing unsubstituted amine moiety at the cyclo-

hexyl side of the molecule was investigated (Table 1). Nanomolar

afﬁnities for D₂, D₃and 5-HT_1Areceptors (K_i¼ 4.60 nM, 9.60 nM

and 7.20 nM respectively), and subnanomolar afﬁnity for 5-HT_2A

3.4. In vivo pharmacokinetic studies

The selected compound was further subjected to

Table 1

Binding afﬁnities for recombinant human D_2L, D₃, 5-HT_1A, and 5-HT_2Areceptors with compounds 7, 8a-h, 9p, and reference antipsychotic.^a

Compound

Structure

Receptor afﬁnity K_i(nM)

hD_2L

4.60

hD₃

h5-HT_1A

7.20

h5-HT_2A

0.21

K_iratio D₂/D₃

7

9.60

0.48

8a

8b

0.53

0.72

0.40

1.40

1.20

2.30

0.60

0.38

1.33

0.51

8c

0.51

2.20

0.78

1.60

1.40

0.50

3.20

0.65

1.38

8d

15.00

8e

1.40

0.18

5.30

0.91

7.78

8f

0.63

0.51

2.40

0.043

0.055

1.20

1.40

12.00

5.20

0.21

0.14

0.35

14.65

9.27

2.00

8g

8h

9p

0.71

1.30

0.058

0.096

0.20

3.10

0.53

12.24

13.54

cariprazine

23.00

a

Shown are the mean K_ivalues from two independent experiments with eight concentrations in duplicates (Binding assays were conducted by Euroﬁns Cerep SA, Celle

l'Evescault, France).

338

X.-W. Chen et al. / European Journal of Medicinal Chemistry 123 (2016) 332e353

Table 2

Binding afﬁnities for recombinant human D_2L, D₃, 5-HT_1A, and 5-HT_2Areceptors with compounds 6, 9a-o, 9q-r, and 12.^a

Compound

Structure

Receptor afﬁnity K_i(nM)

hD_2L

9.60

hD₃

h5-HT_1A

3.0

h5-HT_2A

0.24

K_iratio D₂/D₃

9q

0.31

30.97

6

25.00

18.00

0.16

0.69

1.50

5.80

1.10

156.25

346.15

9a

0.052

9b

9c

2.80

1.70

0.14

0.27

2.10

1.10

0.87

0.63

20.00

6.30

9d

8.9

0.095

1.30

0.28

93.68

9e

9f

1.90

0.94

0.22

0.19

1.10

5.60

0.34

0.36

8.64

4.95

9g

9h

0.85

0.33

0.21

0.92

1.70

0.35

0.24

4.05

7.02

0.047

9i

9j

0.42

2.90

0.062

0.13

3.90

1.30

0.26

0.23

6.77

22.31

9k

9l

0.19

0.21

0.85

0.056

0.31

1.10

0.52

2.10

0.15

0.29

3.40

0.68

9.44

9m

0.090

X.-W. Chen et al. / European Journal of Medicinal Chemistry 123 (2016) 332e353

Receptor afﬁnity K_i(nM)

339

Table 2 (continued )

Compound

Structure

hD_2L

0.43

hD₃

h5-HT_1A

1.1

h5-HT_2A

0.30

K_iratio D₂/D₃

9n

12

9o

9r

0.043

10.00

0.30

0.35

0.43

0.046

0.058

0.029

2.9

1.2

6.52

11.00

7.60

8.10

0.64

6.03

14.83

a

Shown are the mean K_ivalues from two independent experiments with eight concentrations in duplicates (Binding assays were conducted by Euroﬁns Cerep SA, Celle

l'Evescault, France).

receptor (K_i¼ 0.21 nM) were observed. These encouraging results

prompted the introduction of urea moieties to further increase

afﬁnity for the D₃receptor duo to small amide-like functionalities

at the cyclohexyl side that are important structural elements of the

D₃receptor pharmacophore [21]. Afﬁnity for the D₃receptor, as

well as the D₂and 5-HT_1Areceptors was increased to ~1 nM after

introducing the alkyl urea; afﬁnity for 5-HT_2Areceptor remained

within subnanomolar range (8a-c). Analogues with increased af-

ﬁnity for the D₃and 5-HT_1Areceptors displayed the following order

of afﬁnity: methyl > ethyl > isopropyl substitution. Further work

was focused on improving afﬁnity at the D₃receptor to obtain

appropriate selectivity for the D₃versus D₂subtype. Aromatic or

heteroaromatic ureas, such as phenyl and pyridyl ureas, were

introduced and resulted in picomolar afﬁnities for the D₃receptor

(8f, K_i¼ 0.043 nM; 8g, Ki ¼ 0.055 nM). After addition of methyl or

introduction of benzyl, 8h and 8e reduced both D₂and D₃receptor

afﬁnity compared to 8f. Aryl substituents (except for 8f) reduced

the 5-HT_1Areceptor afﬁnity in contrast with alkyl substituents,

while the afﬁnity for 5-HT_2Areceptor stayed within the sub-

nanomolar range (8e-h). 8d bearing the morpholine ring in the

urea moiety displayed lower afﬁnities than other derivatives for

these four receptors, overall indicating that alkyl and aryl sub-

stituents had greater inﬂuence on afﬁnity to the D₃and 5-HT_1A

receptors than the D₂and 5-HT_2Areceptors. Among these de-

rivatives, 8f had both high afﬁnities for D₂, D₃, 5-HT_1A, and 5-HT_2A

receptors with approximately 15-fold selectivity for D₃vs D₂R (K_i

ratio D₂/D₃¼ 14.65).

was explored and the afﬁnities for D₂, D₃, and 5-HT_1Areceptors

were decreased in comparison to 9p. A butyryl moiety (9b) was

introduced to obtain an isostere of 9p causing a decrease in potency

for the D₂, D₃, and 5-HT_1Areceptors was observed in contrast with

9p. Afﬁnities for the D₂and 5-HT_2Areceptors were further reduced

by shortening the carbon length (afﬁnity order: 6 <9a <9b). An

inverse trend was observed in regard to the 5-HT_1Areceptor (af-

ﬁnity order: 6 >9a >9b). Moreover, introduction of sulfonamide

(9d-e) brought negative effects to afﬁnity with the D₂, D₃and 5-

HT_1Areceptors compared to 9p. The D₂and 5-HT_2Areceptors

maintained afﬁnity with either ﬂuoro-substitution (9c) or intro-

duction of a cycloalkyl (9f-g), but afﬁnities for the D₃and 5-HT_1A

receptors were moderately reduced. Based on these results, struc-

tural modiﬁcation with alkyl amides did not yield ideal ligands that

possessed both high afﬁnity for D₂, D₃, 5-HT_1A, 5-HT_2Areceptors

and good D₃receptor subtype preference.

4.1.1.4. Effect of different aryl amides. Considering the positive ef-

fect on afﬁnity when an aryl group was introduced (8f-g) in the

urea derivatives, phenyl and heteroaryl were introduced to the

cyclohexyl amide. Compounds 9h-o, 9r and 12 exhibited sub-

nanomolar and picomolar afﬁnities for the D₂and D₃receptors,

respectively (Table 2). All of the aryl carbamides (9h, 9j-m and 9r)

had subnanomolar afﬁnities for the 5-HT_2Areceptor and ~1 nM

afﬁnity for the 5-HT_1Areceptor (except 9m and 9r). In contrast, aryl

sulfonamides (9i, 9o and 12, excepting 9n) displayed lower afﬁn-

ities for the 5-HT_1Areceptor, and 9o (K_i¼ 8.10 nM) had the lowest

afﬁnity for the 5-HT_2Areceptor.

4.1.1.2. Effect of carbamate. The effect of replacing the alkyl urea

moiety at the cyclohexyl side of the molecule with carbamate

moiety was investigated (Table 1, compound 9p). Unlike alkyl urea

derivatives (8a-c), the ethyl carbamate 9p demonstrated sub-

nanomolar afﬁnities for D₂, 5-HT_1A, and 5-HT_2Areceptors, pico-

molar afﬁnity for D₃receptor, and 12-fold higher afﬁnity for D₃vs

D₂R (K_iratio D₂/D₃¼ 12.24). These results suggested that carbamate

modiﬁcations were important for designing ligands with both high

D₂, D₃, 5-HT_1A, 5-HT_2Areceptor afﬁnity and good selectivity for D₃

vs D₂R.

In regards to the selectivity of the D₃versus D₂subtype, 9j

demonstrated the highest K_iratio (D₂/D₃¼ 22.31). 9j analogues

displayed the following ascending order of D₃receptor preference:

1H-indole-2-yl (9r, K_iratio D₂/D₃¼ 14.83), pyridine-3-yl (9m, K_i

ratio D₂/D₃¼ 9.44), phenyl (9h, K_iratio D₂/D₃¼ 7.02), thiophene-2-

yl (9k, K_iratio D₂/D₃¼ 3.40), and pyrrole-2-yl (9l, K_iratio D₂/

D₃¼ 0.68) substitution. Aryl sulfonamides displayed the following

ascending order of D₃receptor preference: thiophene-2-yl (9n, K_i

ratio D₂/D₃

¼

10.00) > 4-methylphenyl (9i, K_iratio D₂/

D₃¼ 6.77) > pyridine-3-yl (9o, K_iratio D₂/D₃¼ 6.03).

These results suggested that structural modiﬁcation by intro-

ducing aryl carbamides was more favorable than introduction of

4.1.1.3. Effect of different alkyl amides. Considering the possible

instability of 9p, the 2-methoxyacetamide compound 9q (Table 2)

alkyl amides in designing multireceptor ligands for D₂, D₃, 5-HT_1A

,

340

X.-W. Chen et al. / European Journal of Medicinal Chemistry 123 (2016) 332e353

and 5-HT_2Areceptors. Introduction of the furyl (9j) was more likely

to improve D₃vs D₂R selectivity.

NH₂decreased the afﬁnity of the D₂, D₃, 5-HT_1A, and 5-HT_2Are-

ceptors (36 vs 9j, 37 vs 9j). Cl substitution (35) increased D₃re-

ceptor afﬁnity (K_i¼ 0.051 nM), maintained afﬁnities for the D₂and

5-HT_2Areceptors (K_i¼ 2.10 nM, 0.32 nM respectively), and reduced

afﬁnity for the 5-HT_1Areceptor (K_i¼ 15.00 nM). Therefore, the 5-

HT_1Areceptor afﬁnity is sensitive to substitutions at the 6-

position of benzo[d]isothiazole.

Through speciﬁc investigation of amide moiety substituents,

different spacer groups and substitutions at the 6-position of benzo

[d]isothiazole, the optimal dopamine/serotonin receptors' afﬁnity

balance was obtained for compounds 8f, 9j, 9n and 9p. The new

compounds were characterized by nanomolar or picomolar range

afﬁnities for the D₂, D₃, 5-HT_1A, and 5-HT_2Areceptors, a 10e20-fold

increase in selectivity for the D₃over the D₂receptor, and a higher

ratio of afﬁnity for 5-HT_2Areceptor relative to D₂receptor (K_iratio,

1.3e12.6). Speciﬁcally, 8f, 9j, 9n and 9p (K_i< 1.00 nM) demon-

strated subnanomolar afﬁnities for the 5-HT_2Areceptor, which was

higher in comparison to cariprazine (K_i¼ 23.00 nM). Therefore, 8f,

9j, and 9n were selected for further in vivo experiments concerning

the possibly unstable carbamate 9p.

4.1.1.5. Effect of conformational changes of the amide nitrogen.

Compounds 41a-b (Table 3) were prepared in order to investigate

the effect of conformational changes of the amide nitrogen.

Increasing the rigidity of the nitrogen via piperidine ring resulted in

a decrease of D₂and D₃receptor afﬁnity (41a vs 9j and 41b vs 9p).

Afﬁnities were only slightly affected for the 5-HT_1Aand 5-HT_2A

receptors.

4.1.2. Effect of various spacers

The effect of spacer identity on receptor afﬁnity was further

studied following speciﬁc structural modiﬁcations at the amide

moiety (Table 4, compounds 18a-b and 45). The cis isomers 18a-b

(D₂, K_i¼ 6.30, 4.00; D₃, K_i¼ 2.30, 2.00, respectively) displayed

lower afﬁnities for the D₂and D₃receptors compared with the trans

isomers (9j and 9p). The cis-conﬁguration had virtually no inﬂu-

ence on 5-HT_1Aand 5-HT_2Areceptor afﬁnity. Shifting the position of

the cyclohexyl by one carbon (45) diminished the afﬁnities for the

D_2,D₃, 5-HT_1A, and 5-HT_2Areceptors. These results indicated that

the trans conﬁguration of the cyclohexyl was important in obtain-

ing high afﬁnity for D₂and D₃receptors, while also suggesting that

the cyclohexylethyl spacer allowed for high afﬁnity towards the D₂,

D₃, 5-HT_1A, and 5-HT_2Areceptors.

4.2. Behavioral studies

Compounds 8f, 9j, and 9n were selected as potential atypical

antipsychotic agents and evaluated in animal models for behavioral

studies due to their prominent effect on serotonergic and dopa-

minergic receptors.

4.1.3. Effect of substitution on the 1,2-benzisothiazole moiety

The effect of substitution with Cl, NO₂, and NH₂at the 6-position

of benzo[d]isothiazole on receptor afﬁnity was investigated

(Table 5). Both electron-withdrawing NO₂and electron-donating

Since the 1990s, PCP has been used to simulate schizophrenia

symptoms in animals as a non-competitive N-methyl-

D-aspartate

receptor antagonist. While acute PCP can imitate positive

Table 3

Binding afﬁnities for recombinant human D_2L, D₃, 5-HT_1A, and 5-HT_2Areceptors with compounds 41a-b.^a

Compound

Structure

Receptor afﬁnity K_i(nM)

hD_2L

15

hD₃

7.3

h5-HT_1A

0.72

h5-HT_2A

2.2

K_iratio D₂/D₃

41a

2.2

41b

10

8.7

1.3

3.3

1.15

a

Shown are the mean K_ivalues from two independent experiments with eight concentrations in duplicates (Binding assays were conducted by Euroﬁns Cerep SA, Celle

l'Evescault, France).

Table 4

Binding afﬁnities for recombinant human D_2L, D₃, 5-HT_1A, and 5-HT_2Areceptors with compounds 18 and 45.^a

Compound

Structure

Receptor afﬁnity K_i(nM)

hD_2L

6.30

hD₃

h5-HT_1A

2.20

h5-HT_2A

0.19

K_iratio D₂/D₃

18a

2.30

2.74

18b

4.00

8.9

2.00

1.0

0.57

5.3

0.18

16

2.0

8.9

45

a

Shown are the mean K_ivalues from two independent experiments with eight concentrations in duplicates (Binding assays were conducted by Euroﬁns Cerep SA, Celle

l'Evescault, France).

X.-W. Chen et al. / European Journal of Medicinal Chemistry 123 (2016) 332e353

341

Table 5

Binding afﬁnities for recombinant human D_2L, D₃, 5-HT_1A, and 5-HT_2Areceptors with compounds 35e37.^a

Compound

Structure

Receptor afﬁnity K_i(nM)

hD_2L

2.10

hD₃

h5-HT_1A

15.00

h5-HT_2A

0.32

K_iratio D₂/D₃

35

0.051

41.18

36

37

26.00

14.00

0.24

1.10

39.00

33.00

1.80

4.30

108.33

12.73

a

Shown are the mean K_ivalues from two independent experiments with eight concentrations in duplicates (Binding assays were conducted by Euroﬁns Cerep SA, Celle

l'Evescault, France).

symptoms [45]. Moreover, PCP-induced hyperlocomotion can be

reversed through atypical antipsychotic administration [45]. 9j and

cariprazine produced signiﬁcant dose-dependent responses (0.1,

0.2, and 0.4 mg/kg, i.p.) in this model compared with PCP-treated

group (Fig. 4). In comparison, treatment with 8f (0.1, 0.2 and

0.4 mg/kg, i.p.) and 9n (0.1 and 0.2 mg/kg, i.p.) did not signiﬁcantly

cause inhibition of the PCP-induced locomotion.

the novel object in comparison to the familiar object (P < 0.05),

whereas rats treated with chronic PCP exhibited deﬁcits discrimi-

nating between novel and familiar objects (Fig. 6B). The chronic

PCP-induced impairment in NOR was reversed by 9j 0.1 mg/kg

(p.o.) (P < 0.05), 9j 0.2 mg/kg (p.o.) (P < 0.05), and cariprazine

0.1 mg/kg (p.o.) (P < 0.05) (Fig. 6B).

Previously, almost all antipsychotics have been demonstrated to

inhibit CAR in rats; CAR is used to assess the antipsychotic activity

for potential agents that display high afﬁnities for dopamine re-

ceptors [46,47]. In this model, the tested compound 9j and car-

iprazine effectively inhibited CAR in the shuttle-box in a dose range

of 1.0e2.0 mg/kg i.p. (Fig. 5). 8f and 9n displayed weak efﬁcacies at

the dose tested.

These results implied that 9j is more effective as a potential

antipsychotic agent than 8f and 9n, and chosen for further studies.

Improving cognitive impairment in schizophrenia is critically

important for efﬁciently treating schizophrenia. NOR is commonly

used in research to investigate memory performance. Repeated PCP

administration can effectively produce cognitive deﬁcits in the NOR

paradigm in rodents, and this PCP-based model has been widely

used to evaluate the efﬁcacy of atypical antipsychotics in attenu-

ating cognitive impairment [48,49]. In this model, there were no

signiﬁcant differences in the exploration time of two identical ob-

jects for any group in the training session (Fig. 6A). In the retention

session, saline treated rats spent signiﬁcantly more time exploring

4.3. Afﬁnity for M₁receptor and hERG channels

Afﬁnity with muscarinic M₁receptors is linked to anticholin-

ergic side effects such as dry mouth, constipation and blurred

vision [50]; atypical antipsychotics like ziprasidone, aripiprazole,

risperidone and cariprazine show low M₁receptor afﬁnity [51].

Interestingly, 9j exhibited weak afﬁnities for M₁receptors (Table 6).

Many antipsychotic drugs produce QT interval prolongation on

the electrocardiogram, and hERG channel blockade is an important

indicator of QT interval prolongation liability. 9j was subject to the

hERG channel binding assay, and 9j (IC₅₀¼ 2.11

mM) and cariprazine

(IC₅₀¼ 20.72

m

M) displayed micromolar afﬁnities for hERG channel

and over 700-fold selectivity for D₂, D₃, 5-HT_1Aand 5-HT_2Areceptor

binding compared to their hERG channel IC₅₀values (Table 6).

Based on these results, 9j showed high selectivity for the afﬁnity

proﬁle compared to M₁receptor binding and hERG channel IC₅₀

.

Fig. 4. Inhibition of PCP induced hyperactivity by tested compounds and reference antipsychotic drug cariprazine in male Sprague-Dawley rats. Compounds were administered

10 min before PCP injection. Locomotor activities were measured for a 1.5 h duration after PCP administration and the total travel distance was expressed as Mean

SEM

(n ¼ 8e10). þþþ, P < 0.001 versus vehicle treated control group (Veh). *, **, and ***, P < 0.05, P < 0.01, and P < 0.001 versus PCP treated group.

342

X.-W. Chen et al. / European Journal of Medicinal Chemistry 123 (2016) 332e353

Fig. 5. Inhibition of CAR by tested compounds and reference antipsychotic drug cariprazine in male Sprague-Dawley rats. Compounds were administered 1 h before CAR testing via single

intraperitoneal injection. Results were expressed as percentage of avoidance. Data was expressed as Mean SEM (n ¼ 6). ***, P < 0.001 versus vehicle treated control group (Basal).

Fig. 6. Mean exploration time SEM for (A) Two identical objects in the training phase and (B) One familiar and one novel object in the retention phase of the novel recognition test

following acute treatment with saline, 9j, or cariprazine in chronic PCP-treated rats (n ¼ 10). Data were analyzed by post-hoc Student's t-test; *P < 0.05 for time spent exploring the

familiar versus the novel object.

Table 6

Binding afﬁnities for the M₁receptor and hERG channels with 9j and reference antipsychotics.

Compound

M₁K_i, nM

hERG IC₅₀

,

m

M^a

IC₅₀/Ki

D₂

D₃

5-HT_1A

5-HT_2A

9j

50.6%^b

2.11

20.72

727.6

15938.5

16230.8

215833.3

1623.1

6683.9

9173.9

900.9

Cariprazine

<20%^b,c

a

Shown are the mean IC₅₀values from two independent experiments with six concentrations.

b

c

Percent inhibition measured at a concentration of 1

See ref [27].

mM.

Table 7

Activities of 9j and reference compounds to D₂, D₃, 5-HT_1Aand 5-HT_2Areceptors.^a

Receptor

D₂

Compound

Activation (10

103

m

M, %)

EC₅₀(nM)

0.69

Inhibition (10

m

M, %)

IC₅₀(nM)

Dopamine

Amisulpride

9j

Quinpirole

9j

102.17

99

3.04

61.45

ꢁ2.1

90.8

25.04

99.7

>10,000

186.30

>10,000

15.33

D₃

98.63

1.39

5-HT_1A

5-HT

Methiothepin mesylate

9j

5-HT

108

115

71

46.12

ꢁ5.0

>10,000

47

5-HT_2A

100

Methiothepin mesylate

9j

100

97

56

31.75

ꢁ1.4

>10,000

a

Shown are the mean EC₅₀or IC₅₀values from two independent experiments with eight concentrations in duplicates.

X.-W. Chen et al. / European Journal of Medicinal Chemistry 123 (2016) 332e353

343

Fig. 7. Cataleptogenic effects of compound 9j, risperidone, and cariprazine in rats. 9j, cariprazine and risperidone (1e10 mg/kg, p.o.) were individually evaluated for cataleptic

potential in SD rat (n ¼ 7). Catalepsy was measured at 0.5, 1, and 2 h after dosing, with the mean time S.E.M. spent in the catalepsy position for each dose at each time point

expressed as a percentage of maximal catalepsy possible (60 s). Statistically signiﬁcant effect at post-administration time: *p < 0.05, **p < 0.01, ***p < 0.001.

Table 8

Pharmacokinetic parameters for 9j in male rats following i.v. and p.o. administration.

Dose (mg/kg)

0.5 (iv)

Region

C_max(ng/mL)

t_1/2(h)

T_max(h)

AUC_0-t(ng/mL*h)

CL_Z(L/h/kg)

F (%)

Plasma

Brain

Plasma

Brain

135.0

259.4

18.4

0.8

15.8

1.7

105.8

1066.7

55.0

4.6

0.3

34.9

1.5

2.0 (op)

0.5

2.0

13.0

20.7

52.8

16.3

883.9

4.4. Intrinsic activity and cataleptogenic effect of 9j

4.4.1. Intrinsic activity of 9j at selected receptors

(IC₅₀

¼

61.45 nM), 5-HT_1A(IC₅₀

¼

46.12 nM), and 5-HT_2A

(IC₅₀¼ 31.75 nM) antagonism.

The intrinsic activity of 9j was selected for further investigation.

9j displayed no agonist activity against D₂, D₃, 5-HT_1A, and 5-HT_2A

receptors (Table 7). In antagonist assays, 9j had potent D₃

(IC₅₀¼ 1.39 nM) antagonism, which was coupled to moderate D₂

4.4.2. Cataleptogenic effect of 9j

Catalepsy is widely used to predict the incidence of EPS caused

by antipsychotic agents in humans [52]. In this model, 9j exhibited

a low potential for catalepsy, consistent with the marketed atypical

antipsychotic risperidone, and had a higher propensity to induce

catalepsy compared with cariprazine in rats (1.0, 3.0 and 10.0 mg/

kg, p.o.; Fig. 7).

Taken together, the antagonistic effect at four receptors (D₂, D₃,

5-HT_1Aand 5-HT_2A) and cataleptogenic effect of 9j indicated its

potential atypicality.

4.5. In vivo pharmacokinetics of 9j in rats

The pharmacokinetics of 9j, including plasma and brain, were

measured in rats (Table 8). The area under the curve (AUC) values of

9j were 105.8 ng/mL*h for plasma and 1066.7 ng/mL*h for the brain

after intravenous administration. After oral administration of 9j,

AUC values of 9j were 55.0 ng/mL*h for plasma and 883.9 ng/mL*h

for the brain. Furthermore, the half-life of the brain samples was

>15 h regardless of intravenous administration or oral adminis-

tration. 9j had high brain penetration with a brain-to-plasma AUC

ratio of 16.1:1 (Fig. 8) in spite of low orally bioavailable (F ¼ 13.0%)

in rats.

Fig. 8. Plasma concentrations (ng/mL) and brain concentrations (ng/g) of 9j in 2.0 mg/

kg po dosed SD male rats.

344

X.-W. Chen et al. / European Journal of Medicinal Chemistry 123 (2016) 332e353

5. Conclusion

6.1.2. trans-4-Acetamidocyclohexyl acetic acid (2)

A mixture of 1 (40.0 g, 176.0 mmol), Lithium hydroxide mono-

hydrate (14.8 g, 352.0 mmol), water (100 mL), and THF (400 mL)

was stirred at 45 ^ꢀC for 16 h. The resulting solution was evaporated

in vacuo to remove most of the THF and acidiﬁed by hydrochloric

acid (6.0 mol/L, 176 mL) in an ice bath and stirred for 1 h. The

resulting mixture was ﬁltered and washed with water (50 mL) to

give a colorless solid (31.6 g, 158.6 mmol). The solid was recrys-

tallized from DMF to yield 2 (18.2 g, 57.6%). Mp: 237e239 ^ꢀC. ¹H

In this work, a series of novel benzisothiazolylpiperazine de-

rivatives were designed and synthesized in order to discover novel

multitarget antipsychotics. A detailed structure-activity relation-

ship investigation of these derivatives revealed that multiple fac-

tors, such as substituents on the amide, the conﬁguration of the

spacer, and substitutions at the 6-position of benzo[d]isothiazole,

had inﬂuence on the afﬁnity for the D₂, D₃, 5-HT_1Aand 5-HT_2A

receptors both individually and collectively. Among these de-

rivatives, compound 9j showed unique afﬁnities for the D₂, D₃, 5-

HT_1Aand 5-HT_2Areceptors, along with a 20-fold selectivity for

the D₃vs D₂R and low afﬁnities for M₁receptors and hERG chan-

nels. Further in vivo animal model tests displayed that 9j was a

promising multireceptor antipsychotic. 9j exhibited a low potential

for catalepsy, consistent with the atypical antipsychotic risperi-

done. Moreover, 9j showed high brain penetration in rats.

NMR (CDCl₃):

d 1.14e1.30 (m, 5H), 1.64e1.71 (m, 4H), 1.99 (s, 3H),

2.32 (d, 2H, J ¼ 4.0 Hz), 4.02e4.03 (m, 1H), 5.42 (d, 1H, J ¼ 4.0 Hz).

6.1.3. Procedure B. trans-Ethyl-2-(4-acetamidocyclohexyl)acetate

(3)

A mixture of 2 (16.5 g, 82.8 mmol), concentrated H₂SO₄(1.0 mL)

and ethanol (300 mL) was stirred at reﬂux for 7 h. The resulting

solution was evaporated in vacuo to give a colorless solid (19.6 g).

The crude residue was extracted with dichloromethane (100 mL)

and water (50 mL). The organic layer was washed with brine

(50 mL) and dried over anhydrous magnesium sulfate. The solvent

was then removed in vacuo to afford 3 as a white solid (18.2 g,

This study sheds light on the development of novel benziso-

thiazolylpiperazine derivatives as potential multitarget atypical

antipsychotics for the treatment of schizophrenia.

6. Experimental

96.7%). Mp: 113e115 ^ꢀC. ¹H NMR (CDCl₃):

d 1.06e1.17 (m, 4H),

1.20e1.27 (m, 3H), 1.72e1.79 (m, 3H), 1.95e1.99 (m, 5H), 2.19 (d, 2H,

J ¼ 6.8 Hz), 3.67e3.72 (m, 1H), 4.11 (q, 2H, J ¼ 4.0 Hz), 5.51 (d, 1H,

J ¼ 7.2 Hz).

6.1. Chemistry

Melting points were determined on an electrothermal melting

point apparatus and are uncorrected. Nuclear magnetic resonance

(NMR) spectra were recorded on a Bruker AVANCE II 400 (400 MHz)

with TMS in DMSO or CDCl₃solution as an internal standard.

6.1.4. Procedure C. trans-4-(2-Hydroxyethyl)acetamide (4)

A mixture of 3 (18.2 g, 80.1 mmol), sodium borohydride (15.1 g,

400.4 mmol), and THF (182 mL) was stirred at reﬂux for 0.5 h.

Methanol (30 mL) was then added dropwise to this mixture at

room temperature. The resulting solution was stirred at reﬂux for

20 h and quenched with 30% hydrochloric acid (50 mL) in an ice

bath. The resulting mixture was stirred for 1 h and alkalized with

50% sodium hydroxide solution (60 mL). The resulting mixture was

extracted with dichloromethane (3 ꢂ 50 mL). The combined

organic extracts were dried (MgSO₄) and evaporated in vacuo to

give 4 (13.2 g, 89.0%) as a colorless solid. Mp: 120e122 ^ꢀC. ¹H NMR

Chemical shifts were given in

d values (ppm) and coupling con-

stants (J) were given in Hz. Signal multiplicities were characterized

as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and

br (broad). ESI mass spectra was performed on a Waters ZQ2000

spectrometer. Reagents were all of analytical grade or of chemical

purity and used without any pretreatment. TLC on silica gel GF254

was used to monitor the progress of all reactions. Silica gel column

chromatography was carried out with Silica gel 60G. Aluminum

oxide column chromatography was carried out with Aluminum

oxide 70G. All solvent evaporation was performed under vacuum.

Compound purity was determined by high performance liquid

chromatography (HPLC), and all ﬁnal test compounds were >95%

purity. The HPLC methods used a Waters Symmetry column C18

(CDCl₃):

d 1.06e1.14 (m, 4H), 1.29e1.41 (m, 2H), 1.47e1.50 (m, 2H),

1.78e1.80 (m, 2H), 1.95 (s, 3H), 1.98e2.04 (m, 2H), 3.67e3.73 (m,

3H), 5.29 (d, 1H, J ¼ 4.0 Hz).

6.1.5. Procedure D. trans-4-(Acetamidocyclohexyl)ethyl

(5

m

m, 250 mm ꢂ 4.6 mm); with a mobile phase A (CH₃CN) and B

methanesulfonate (5)

(0.05% phosphoric acid solution), 10:90e90:10 v/v; detection at

Methylsulfonyl chloride (8.9 g, 77.8 mmol) in dichloromethane

(10 mL) was added dropwise to a solution of 4 (12.0 g, 64.8 mmol)

and triethylamine (22.6 mL, 162.0 mmol) in dichloromethane

(30 mL) at 0e5 ^ꢀC. The resulting mixture was stirred for 3 h at

ambient temperature and quenched with a saturated sodium bi-

carbonate solution (50 mL). The resultant mixture was extracted

with dichloromethane (2 ꢂ 50 mL). The combined organic extracts

were washed with 5% HCl (2 ꢂ 20 mL), water (1 ꢂ 50 mL) and brine

(1 ꢂ 50 mL), dried over anhydrous sodium sulfate. The solvent was

then removed under reduced pressure to give 5 (13.9 g, 81.7%) as a

233 nm; ﬂow: 1.0 mL/min; temp 30 ^ꢀC.

6.1.1. Procedure A. trans/cis-Ethyl-2-(4-acetamidocyclohexyl)

acetate (1)

A solution of triethyl phosphonoacetate (52.0 g, 232.0 mmol) in

THF (200 mL) was added dropwise while stirring to a solution of

potassium tert-butanolate (43.4 g, 386.6 mmol) in THF (400 mL) at

0e5 ^ꢀC and continuously stirred for 1 h. Then a suspension of N-(4-

oxocyclohexy)acetamide (30.0 g, 193.3 mmol) in THF (150 mL) was

added dropwise to the solution at 0e5 ^ꢀC. The resulting solution

was stirred for another 2 h at room temperature and subsequently

quenched with water (100 mL). The resultant mixture was extrac-

ted with acetic ether (2 ꢂ 20 mL) after separation. Palladium 10% on

carbon (1.5 g) was then added to the organic layer and hydroge-

nation was conducted at ambient temperature and pressure con-

ditions. The resulting solution was stirred for 24 h and then ﬁltered.

The ﬁltrate evaporated in vacuo to give a colorless oil. The residue

was partitioned between water (100 mL) and dichloromethane

(2 ꢂ 100 mL), and the combined extracts were dried (MgSO₄) and

evaporated in vacuo to afford 1 (40.7 g, 92.6%) as a colorless semi-

solid. MS (ESI) m/z: 228.2 ([M þ H]^þ).

light yellow solid. Mp: 79e81 ^ꢀC. ¹H NMR (CDCl₃):

d 1.17e1.19 (m,

5H),1.46e1.48 (m, 2H),1.64e1.68 (m, 2H),1.79e1.82 (m, 2H),1.99 (s,

3H), 3.57e3.58 (m, 1H), 3.68 (s, 3H), 4.26 (t, 2H, J ¼ 4.0 Hz)6.16 (d,

1H, J ¼ 4.0 Hz).

6.1.6. Procedure E. N-(trans-4-(2-(4-(benzo[d]isothiazol-3-yl)

piperazin-1-yl)ethyl)cyclohexyl)acetamide (6)

A mixture of 5 (12.0 g, 45.6 mmol), 3-(piperazin-1-yl)benzo[d]

isothiazole (11.0 g, 50.2 mmol), sodium carbonate (14.5 g,

136.8 mmol), potassium iodide (0.01 g, 0.05 mmol), and acetonitrile

(180 mL) was stirred at reﬂux for 36 h. The resulting solution was

cooled to ambient temperature, ﬁltered and washed with water

X.-W. Chen et al. / European Journal of Medicinal Chemistry 123 (2016) 332e353

345

(100 mL) and further recrystallized from ethanol (120 mL) to afford

(d, 1H, J ¼ 8.0 Hz), 7.44 (t, 1H, J ¼ 8.0 Hz), 7.56 (t, 1H, J ¼ 8.0 Hz),

8.04e8.07 (m, 2H). MS (ESI) m/z: 430.1 ([MþH] ^þ).

6 (12.0 g, 68.3%) as a colorless solid. Mp: 222e223 ^ꢀC. HPLC

t_R¼ 8.40 min, 99.9% purity. ¹H NMR (CDCl₃):

d 1.06e1.12 (m, 3H),

1.21e1.29 (m, 2H), 1.46e1.53 (m, 2H), 1.81e1.85 (m, 2H), 2.06e2.10

(m, 2H), 2.27 (s, 3H), 2.46 (t, 2H, J ¼ 7.6 Hz), 2.64e2.67 (m, 4H),

3.50e3.53 (m, 4H), 4.07e4.09 (m, 1H), 5.52 (d, 1H, J ¼ 7.6 Hz), 7.32

(t, 1H, J ¼ 8.4 Hz), 7.44 (t, 1H, J ¼ 8.4 Hz), 7.79 (d, 1H, J ¼ 8.4 Hz), 7.93

(d, 1H, J ¼ 8.0 Hz). MS (ESI) m/z: 387.3 ([MþH] ^þ).

6.1.8.4. N-(trans-4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)

ethyl)cyclohexyl)morpholine-4-carboxamide (8d). Yield 79.8%; mp:

137e139 ^ꢀC. HPLC t_R¼ 6.90 min, 95.8% purity. ¹H NMR (DMSO-d₆):

d

1.11e1.25 (m, 3H), 1.30e1.32 (m, 2H), 1.48e1.54 (m, 2H), 1.85e1.86

(m, 2H), 2.09e2.11 (m, 2H), 2.51 (t, 2H, J ¼ 7.6 Hz), 2.69e2.73 (m,

4H), 3.44e3.46 (m, 4H), 3.61e3.65 (m, 4H), 4.01e4.02 (m, 4H),

4.17e4.19 (m,1H), 5.62 (d,1H, J ¼ 8.0 Hz), 7.38 (t,1H, J ¼ 8.0 Hz), 7.49

(t, 1H, J ¼ 8.0 Hz), 7.85e7.92 (m, 2H). MS (ESI) m/z: 458.3 ([MþH] ^þ).

6.1.7. Procedure F. trans-4-(2-(4-(Benzo[d]isothiazol-3-yl)

piperazin-1-yl)ethyl)cyclohexanamine (7)

A mixture of 6 (9.8 g, 25.4 mmol) and 5%HCl (130 mL) was

stirred at reﬂux for 30 h. The resulting solution was cooled to

ambient temperature and 20% sodium hydroxide solution (65 mL)

was added and stirred for another 1 h. The resulting solution was

ﬁltered and the ﬁltrate was extracted with dichloromethane

(3 ꢂ 50 mL). The combined organic extracts were washed with

water (1 ꢂ 50 mL) and brine (1 ꢂ 50 mL) and dried over anhydrous

sodium sulfate. The solvent was removed under reduced pressure

to yield 7 (7.5 g, 86.1%) as a colorless solid. Mp: 66e68 ^ꢀC. HPLC

6.1.8.5. 1-(trans-4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)

ethyl)cyclohexyl)-3-benzylurea (8e). Yield 85.4%; mp: 158e160 ^ꢀC.

HPLC t_R¼ 10.10 min, 97.3% purity. ¹H NMR (DMSO-d₆):

d 0.94e1.09

(m, 4H), 1.23e1.24 (m, 1H), 1.38e1.39 (m, 2H), 1.73e1.85 (m, 4H),

2.38 (t, 2H, J ¼ 7.6 Hz), 2.59e2.62 (m, 4H), 3.44e3.47 (m, 4H),

3.62e3.64 (m, 1H), 4.07 (d, 2H, J ¼ 8.0 Hz), 5.78 (d, 1H, J ¼ 8.0 Hz),

6.43e6.46 (m, 5H), 7.44 (t, 1H, J ¼ 8.0 Hz),7.57 (t, 1H, J ¼ 8.0 Hz),

8.04e8.07 (m, 2H). MS (ESI) m/z: 477.8 ([MþH] ^þ).

t_R¼ 6.23 min, 99.2% purity. ¹H NMR (CDCl₃):

d 0.97e1.12 (m, 4H),

1.23e1.26 (m, 1H), 1.43e1.47 (m, 2H), 1.76e1.87 (m, 6H), 2.45 (t, 2H,

J ¼ 4.0 Hz), 2.59e2.63 (m, 1H), 2.66e2.68 (m, 4H), 3.56e3.58 (m,

4H), 7.35 (t, 1H, J ¼ 8.0 Hz), 7.46 (t, 1H, J ¼ 8.0 Hz), 7.81 (d, 1H,

J ¼ 4.0 Hz), 7.91 (d, 1H, J ¼ 4.0 Hz). MS (ESI) m/z: 345.2 ([MþH]^þ).

6.1.8.6. 1-(trans-4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)

ethyl)cyclohexyl)-3-phenylurea (8f). Yield 88.6%; mp: 234e236 ^ꢀC.

HPLC t_R¼ 10.18 min, 98.6% purity. ¹H NMR (DMSO-d₆):

d 0.97e1.14

(m, 4H), 1.24e1.25 (m, 1H), 1.37e1.42 (m, 2H), 1.76e1.78 (m, 2H),

1.87e1.90 (m, 2H), 2.38 (t, 2H, J ¼ 7.2 Hz), 2.58e2.61 (m, 4H),

3.44e3.47 (m, 4H), 3.61e3.63 (m, 1H), 6.00 (d, 1H, J ¼ 7.6 Hz), 6.87

(t, 1H, J ¼ 7.6 Hz), 7.19e7.23 (m, 2H), 7.36 (d, 1H, J ¼ 8.0 Hz), 7.44 (t,

1H, J ¼ 7.6 Hz), 7.56 (t, 1H, J ¼ 7.6 Hz), 8.04e8.07 (m, 2H). MS (ESI)

m/z: 464.3 ([MþH] ^þ).

6.1.8. General procedure for the preparation of compounds 8a-h

Compound 7 (0.5 g, 1.5 mmol) in dichloromethane (2 mL) was

added dropwise to a solution of bis(trichloromethyl)carbonate

(0.5 g, 1.7 mmol) and triethylamine (0.4 mL, 3.0 mmol) in

dichloromethane (5 mL) at ꢁ5toꢁ10 ^ꢀC and stirred for 1 h. The

reaction mixture obtained was added to a solution of substituted

amines (13.5 mmol) in isopropyl alcohol (10 mL) and cooled

at ꢁ10~e0 ^ꢀC which kept the temperature of the reaction mixture

under 0 ^ꢀC. After stirring at ꢁ5~0 ^ꢀC for 6 h a saturated ammonium

chloride aqueous solution (10 mL) was added to the reaction

mixture. The resulting mixture was extracted with dichloro-

methane (2 ꢂ 10 mL). The combined organic extracts were dried

(Na₂SO₄) and evaporated in vacuo. The residue was puriﬁed by

recrystallization from ethanol to yield 8a-h.

6.1.8.7. 1-(trans-4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)

ethyl)cyclohexyl)-3-(pyridin-3-yl)urea (8g). Yield 80.5%; mp:

226e229 ^ꢀC. HPLC t_R¼ 7.15 min, 99.1% purity. ¹H NMR (DMSO-d₆):

d

0.97e1.06 (m, 2H), 1.11e1.19 (m, 2H), 1.24e1.25 (m, 1H), 1.37e1.42

(m, 2H), 1.76e1.79 (m, 2H), 1.87e1.90 (m, 2H), 2.39 (t, 2H,

J ¼ 7.2 Hz), 2.59e2.62 (m, 4H), 3.38e3.40 (m, 1H), 3.44e3.47 (m,

4H), 6.16 (d,1H, J ¼ 8.0 Hz), 7.23e7.26 (m,1H), 7.44 (t,1H, J ¼ 7.6 Hz),

7.56 (t, 1H, J ¼ 7.6 Hz), 7.86e7.88 (m, 1H), 8.04e8.11 (m, 3H),

8.50e8.51 (m, 1H). MS (ESI) m/z: 465.3 ([MþH] ^þ).

6.1.8.1. 3-(trans-4-(2-(4-(Benzo[d]isothiazol-3-yl)piperazin-1-yl)

6.1.8.8. 3-(trans-4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)

ethyl)cyclohexyl)-1-methyl-1-phenylurea (8h). Yield 91.7%; mp:

106e109 ^ꢀC. HPLC t_R¼ 10.31 min, 98.9% purity. ¹H NMR (DMSO-d₆):

ethyl)cyclohexyl)-1,1-dimethylurea

(8a). Yield

86.1%;

mp:

137e139 ^ꢀC. HPLC t_R¼ 8.90 min, 96.3% purity. ¹H NMR (DMSO-d₆):

d

1.08e1.24 (m, 3H),1.27e1.28 (m, 2H),1.44e1.50 (m, 2H),1.80e1.81

d 0.91e1.00 (m, 2H), 1.12e1.23 (m, 3H), 1.33e1.39 (m, 2H),1.71e1.77

(m, 2H), 2.05e2.06 (m, 2H), 2.46 (t, 2H, J ¼ 7.6 Hz), 2.66e2.69 (m,

4H), 2.89 (s, 6H), 3.57e3.59 (m, 4H), 4.11e4.13 (m, 1H), 5.54 (d, 1H,

J ¼ 8.0 Hz), 7.35 (t, 1H, J ¼ 8.4 Hz), 7.46 (t, 1H, J ¼ 8.4 Hz), 7.81 (d, 1H,

J ¼ 8.4 Hz), 7.91 (d, 1H, J ¼ 8.0 Hz). MS (ESI) m/z: 416.2 ([MþH] ^þ).

(m, 4H), 2.36 (t, 2H, J ¼ 7.2 Hz), 2.56e2.59 (m, 4H), 3.34 (s, 3H),

3.39e3.41 (m, 1H), 3.43e3.46 (m, 4H), 5.60 (d, 1H, J ¼ 8.0 Hz),

7.16e7.20 (m, 1H), 7.23e7.25 (m, 2H), 7.34e7.38 (m, 2H), 7.43 (t, 1H,

J ¼ 7.6 Hz), 7.56 (t, 1H, J ¼ 7.6 Hz), 8.03e8.06 (m, 2H). MS (ESI) m/z:

478.2 ([MþH] ^þ).

6.1.8.2. 3-(trans-4-(2-(4-(Benzo[d]isothiazol-3-yl)piperazin-1-yl)

ethyl)cyclohexyl)-1,1-diisopropylurea (8b). Yield 89.7%; mp:

142e143 ^ꢀC. HPLC t_R¼ 10.69 min, 97.3% purity. ¹H NMR (DMSO-d₆):

6.1.9. Procedure G. General procedure for the preparation of

compounds 9a-q

d

0.84e1.00 (m, 2H), 1.14e1.16 (m, 12H),1.19e1.24 (m, 3H), 1.35e1.41

Acyl chloride (1.7 mmol) in dichloromethane (2 mL) was added

dropwise to a solution of 7 (0.5 g, 1.5 mmol) and triethylamine

(0.4 mL, 3.0 mmol) in dichloromethane (5 mL) at 0e5 ^ꢀC. The

resulting mixture was stirred at room temperature for 7 h. Water

(10 mL) was added to the reaction mixture and extracted with

dichloromethane (2 ꢂ 10 mL). The combined organic extracts were

dried (Na₂SO₄) and evaporated in vacuo. The residue was puriﬁed

by recrystallization from ethanol to yield 9a-q as free base. The free

base of 9j, 9n and 9p were then dissolved in methanol (5 mL) and a

5% hydrogenchloride solution (0.5 mL) was added. The resulting

mixture was stirred for 1 h at room temperature. The resulting

mixture was evaporated to until dry under reduced pressure and

(m, 2H), 1.64e1.77 (m, 4H), 2.38 (t, 2H, J ¼ 8.0 Hz), 2.58e2.60 (m,

4H), 3.36e3.40 (m, 1H), 3.44e3.46 (m, 4H), 3.62e3.72 (m, 2H), 5.46

(d, 1H, J ¼ 8.0 Hz), 7.44 (t, 1H, J ¼ 8.0 Hz), 7.56 (t, 1H, J ¼ 8.0 Hz),

8.04e8.12 (m, 2H). MS (ESI) m/z: 472.2([MþH] ^þ).

6.1.8.3. 3-(trans-4-(2-(4-(Benzo[d]isothiazol-3-yl)piperazin-1-yl)

ethyl)cyclohexyl)-1-ethyl-1-methylurea (8c). Yield 90.3%; mp:

108e110 ^ꢀC. HPLC t_R¼ 9.20 min, 96.7% purity. ¹H NMR (DMSO-d₆):

d

0.96e1.05 (m, 4H), 1.19e1.24 (m, 4H), 1.38 (m, 2H), 1.74e1.76 (m,

4H), 2.38 (t, 2H, J ¼ 8.0 Hz), 2.51e2.53 (m, 4H), 2.73 (s, 3H),

3.32e3.36 (m, 1H), 3.19 (q, 2H, J ¼ 8.0 Hz), 3.44e3.46 (m, 4H), 5.81

346

X.-W. Chen et al. / European Journal of Medicinal Chemistry 123 (2016) 332e353

puriﬁed by recrystallization from ethanol/water to yield 9j, 9n and

9p hydrochloride.

6.1.9.8. N-(trans-4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)

ethyl)cyclohexyl)benzamide (9h). Yield 91.1%; mp: 189e191 ^ꢀC.

HPLC t_R¼ 9.95 min, 97.4% purity. ¹H NMR (DMSO-d₆):

d 1.08e1.12

(m, 3H), 1.21e1.29 (m, 2H), 1.46e1.53 (m, 2H), 1.81e1.85 (m, 2H),

2.02e2.08 (m, 2H), 2.49 (t, 2H, J ¼ 7.6 Hz), 2.64e2.67 (m, 4H),

3.50e3.53 (m, 4H), 4.17e4.19 (m, 1H), 7.30e7.42 (m, 4H), 7.48e7.53

(m, 2H), 7.79 (d, 1H, J ¼ 8.4 Hz), 7.90e7.96 (m, 2H), 8.02 (d, 1H,

J ¼ 5.6 Hz). MS (ESI) m/z: 449.2 ([MþH] ^þ).

6.1.9.1. N-(trans-4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)

ethyl)cyclohexyl)propionamide (9a). Yield 93.5%; mp: 259e261 ^ꢀC.

HPLC t_R¼ 8.74 min, 98.7% purity. ¹H NMR (DMSO-d₆):

d 0.81e1.06

(m, 7H), 1.11e1.19 (m, 2H), 1.45e1.46 (m, 1H),1.74e1.80 (m, 4H), 2.03

(q, 2H, J ¼ 8.0 Hz), 2.38 (t, 2H, J ¼ 7.2 Hz), 3.19e3.24 (m, 4H),

3.43e3.46 (m, 4H), 3.70e3.72 (m, 1H), 5.56 (d, 1H, J ¼ 7.6 Hz), 7.48

(t, 1H, J ¼ 8.0 Hz), 7.63 (t, 1H, J ¼ 8.0 Hz), 8.11e8.16 (m, 2H). MS (ESI)

m/z: 401.4 ([MþH] ^þ).

6.1.9.9. N-(trans-4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)

ethyl)cyclohexyl)-4-methylbenzenesulfonamide (9i). Yield 89.4%;

mp: 143e146 ^ꢀC. HPLC t_R¼ 10.83 min, 98.1% purity. ¹H NMR

(DMSO-d₆):

d 0.76e0.85 (m, 2H), 1.06e1.15 (m, 3H), 1.24e1.31 (m,

6.1.9.2. N-(trans-4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)

ethyl)cyclohexyl)butyramide (9b). Yield 92.8%; mp: 172e174 ^ꢀC.

2H), 1.56e1.63 (m, 4H), 2.29 (t, 2H, J ¼ 7.6 Hz), 2.51e2.53 (m, 4H),

2.79e2.85 (m, 1H), 3.40e3.42 (m, 4H), 7.36e7.44 (m, 3H), 7.54e7.58

(m, 2H), 7.67e7.69 (m, 2H), 8.00e8.03 (m, 2H). MS (ESI) m/z: 499.0

([MþH] ^þ).

HPLC t_R¼ 9.16 min, 99.1% purity. ¹H NMR (DMSO-d₆):

d 0.84 (t, 3H,

J ¼ 8.0 Hz), 1.03e1.10 (m, 2H), 1.13e1.19 (m, 2H), 1.36e1.38 (m, 2H),

1.45e1.54 (m, 3H), 1.62e1.64 (m, 2H), 1.74e1.79 (m, 4H), 2.00 (t, 2H,

J ¼ 8.0 Hz), 3.19e3.24 (m, 4H), 3.43e3.46 (m, 4H), 3.60e3.62 (m,

1H), 5.58 (d, 1H, J ¼ 7.6 Hz), 7.48 (t, 1H, J ¼ 8.0 Hz), 7.60 (t, 1H,

J ¼ 8.0 Hz), 8.10e8.15 (m, 2H). MS (ESI) m/z: 415.4 ([MþH] ^þ).

6.1.9.10. N-(trans-4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)

ethyl)cyclohexyl)furan-2-carboxamide (9j) hydrochloride. Yield

81.3%; mp: >270 ^ꢀC. HPLC t_R¼ 10.97 min, 99.0% purity. ¹H NMR

(DMSO-d₆):

d 1.03e1.09 (m, 2H), 1.30e1.40 (m, 3H), 1.65e1.68 (m,

6.1.9.3. N-(trans-4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)

ethyl)cyclohexyl)-2,2,2-triﬂuoroacetamide (9c). Yield 80.2%; mp:

170e172 ^ꢀC. HPLC t_R¼ 9.92 min, 98.4% purity. ¹H NMR (DMSO-d₆):

2H),1.77e1.82 (m, 4H), 3.18e3.25 (m, 4H), 3.48 (t, 2H, J ¼ 7.6 Hz),

3.59e3.61 (m, 2H), 3.68e3.73 (m, 1H), 4.07e4.08 (m, 4H),

6.60e6.61 (m, 1H), 7.09e7.10 (m, 1H), 7.47e7.49 (m, 1H), 7.59e7.62

(m, 1H), 7.80e7.81 (m, 1H), 8.11 (d, 1H, J ¼ 5.2 Hz), 8.14e8.16 (m,

2H). MS (ESI) m/z: 439.3 ([MþH] ^þ).

d

0.97e1.08 (m, 2H), 1.29e1.40 (m, 5H), 1.77e1.80 (m, 4H), 2.39 (t,

2H, J ¼ 8.0 Hz), 2.59e2.62 (m, 4H), 3.44e3.47 (m, 4H), 3.58e3.60

(m, 1H), 7.44 (t, 1H, J ¼ 8.0 Hz), 7.57 (t, 1H, J ¼ 8.0 Hz), 8.04e8.07 (m,

2H), 9.26 (d, 1H, J ¼ 8.0 Hz). MS (ESI) m/z: 440.8 ([MþH] ^þ).

6.1.9.11. N-(trans-4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)

ethyl)cyclohexyl)thiophene-2-carboxamide (9k). Yield 88.5%; mp:

178e181 ^ꢀC. HPLC t_R¼ 9.88 min, 98.2% purity. ¹H NMR (DMSO-d₆):

6.1.9.4. N-(trans-4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)

ethyl)cyclohexyl)methanesulfonamide (9d). Yield 81.9%; mp:

152e154 ^ꢀC. HPLC t_R¼ 8.88 min, 98.0% purity. ¹H NMR (CDCl₃):

d

0.99e1.07 (m, 2H), 1.23e1.43 (m, 5H), 1.76e1.86 (m, 4H), 2.39 (t,

2H, J ¼ 7.6 Hz), 2.59e2.61 (m, 4H), 3.44e3.46 (m, 4H), 3.67e3.70

(m, 1H), 7.11e7.14 (m, 1H), 7.42e7.45 (m, 1H), 7.71e7.72 (m, 1H),

7.77e7.78 (m, 1H), 8.04e8.07 (m, 2H), 8.21 (d, 1H, J ¼ 8.0 Hz). MS

(ESI) m/z: 455.0 ([MþH] ^þ).

d

1.02e1.19 (m, 2H), 1.20e1.29 (m, 3H), 1.42e1.58 (m, 2H), 1.81e1.84

(m, 2H), 2.05e2.08 (m, 2H), 2.64e2.66 (m, 4H), 2.68 (t, 2H,

J ¼ 7.8 Hz), 2.96 (s, 3H), 3.23e3.27 (m, 1H), 3.55e3.57 (m, 4H), 6.92

(d, 1H, J ¼ 7.6 Hz), 7.33 (t, 1H, J ¼ 8.0 Hz), 7.43 (t, 1H, J ¼ 8.0 Hz),

7.81e7.86 (m, 2H). MS (ESI) m/z: 423.2 ([MþH] ^þ).

6.1.9.12. N-(trans-4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)

ethyl)cyclohexyl)-1H-pyrrole-2-carboxamide (9l). Yield 80.0%; mp:

200e202 ^ꢀC. HPLC t_R¼ 9.54 min, 96.3% purity. ¹H NMR (DMSO-d₆):

6.1.9.5. N-(trans-4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)

ethyl)cyclohexyl)propane-1-sulfonamide (9e). Yield 87.0%; mp:

169e171 ^ꢀC. HPLC t_R¼ 9.71 min, 98.8% purity. ¹H NMR (DMSO-d₆):

d

1.00e1.06 (m, 2H), 1.19e1.32 (m, 3H), 1.43e1.47 (m, 2H),1.48e1.81

(m, 4H), 2.66 (t, 2H, J ¼ 7.6 Hz), 2.88e2.91 (m, 4H), 3.52e3.53 (m,

4H), 3.63e3.66 (m, 1H), 4.81 (s, 1H), 6.04e6.06 (m, 1H), 6.75e6.77

(m, 1H), 6.81e6.82 (m, 1H), 7.44e7.46 (m, 1H), 7.55e7.57 (m, 1H),

8.05e8.08 (m, 2H), 8.15 (d, 1H, J ¼ 7.6 Hz). MS (ESI) m/z: 438.0

([MþH] ^þ).

d

0.95e0.99 (m, 4H),1.16e1.26 (m, 4H),1.33e1.38 (m, 2H),1.63e1.67

(m, 2H), 1.72e1.75 (m, 2H), 1.84e1.87 (m, 2H), 2.36 (t, 2H,

J ¼ 7.6 Hz), 2.55e2.58 (m, 4H), 2.92e3.02 (m, 3H), 3.41e3.44 (m,

4H), 7.00 (d, 1H, J ¼ 7.6 Hz), 7.43 (t, 1H, J ¼ 7.6 Hz), 7.56 (t, 1H,

J ¼ 7.6 Hz), 8.03e8.07 (m, 2H). MS (ESI) m/z: 451.1 ([MþH] ^þ).

6.1.9.13. N-(trans-4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)

ethyl)cyclohexyl)nicotinamide (9m). Yield 78.7%; mp: 176e178 ^ꢀC.

6.1.9.6. N-(trans-4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)

ethyl)cyclohexyl)cyclohexanecarboxamide (9f). Yield 87.0%; mp:

221e223 ^ꢀC. HPLC t_R¼ 10.33 min, 97.5% purity. ¹H NMR (CDCl₃):

HPLC t_R¼ 8.05 min, 98.1% purity. ¹H NMR (CDCl₃):

d 1.08e1.14 (m,

3H), 1.20e1.28 (m, 2H), 1.44e1.50 (m, 2H), 1.79e1.81 (m, 2H),

2.01e2.05 (m, 2H), 2.45 (t, 2H, J ¼ 7.6 Hz), 2.66e2.68 (m, 4H),

3.56e3.59 (m, 4H), 4.12e4.14 (m, 1H), 7.41e7.43 (m, 1H), 7.50e7.52

(m, 1H), 7.65e7.66 (m,1H), 7.79e7.83 (m, 2H), 8.31 (d, 1H,

J ¼ 7.6 Hz), 8.47e8.48 (m, 1H), 8.90e8.91 (m, 1H), 9.24 (d, 1H,

J ¼ 2.0 Hz). MS (ESI) m/z: 450.0 ([MþH] ^þ).

d

1.06e1.15 (m, 4H), 1.19e1.31 (m, 4H), 1.37e1.48 (m, 4H), 1.65e1.67

(m, 1H), 1.78e1.85 (m, 6H), 1.97e2.04 (m, 3H), 2.42e2.56 (m, 2H),

2.64e2.67 (m, 4H), 3.55e3.57 (m, 4H), 3.70e3.72 (m, 1H), 7.34 (t,

1H, J ¼ 8.0 Hz), 7.45 (t,1H, J ¼ 8.0 Hz), 7.83e7.88 (m, 2H), 7.78 (d,1H,

J ¼ 7.2 Hz). MS (ESI) m/z: 455.2 ([MþH] ^þ).

6.1.9.7. N-(trans-4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)

ethyl)cyclohexyl)cyclopropanecarboxamide (9g). Yield 94.6%; mp:

213e215 ^ꢀC. HPLC t_R¼ 9.06 min, 99.2% purity. ¹H NMR (DMSO-d₆):

6.1.9.14. N-(trans-4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)

ethyl)cyclohexyl)thiophene-2-sulfonamide

(9n)

hydrochloride.

Yield 72.5%; mp: > 270 ^ꢀC. HPLC t_R¼ 12.15 min, 99.1% purity. ¹H

d

0.59e0.61 (m, 4H), 0.95e1.00 (m, 2H), 1.13e1.23 (m, 4H),

NMR (DMSO-d₆): d 1.08e1.14 (m, 3H), 1.20e1.28 (m, 2H), 1.44e1.50

1.37e1.49 (m, 2H), 1.74e1.78 (m, 4H), 2.37 (t, 2H, J ¼ 7.6 Hz),

2.57e2.59 (m, 4H),3.42e3.44 (m, 4H), 3.45e3.47 (m, 1H), 7.43e7.45

(m, 1H), 7.54e7.56 (m, 1H), 7.90 (d, 1H, J ¼ 6.8 Hz), 8.05e8.09 (m,

2H). MS (ESI) m/z: 413.1 ([MþH] ^þ).

(m, 2H), 1.79e1.81 (m, 2H), 2.01e2.05 (m, 2H), 2.45 (t, 2H,

J ¼ 7.6 Hz), 2.66e2.68 (m, 4H), 3.56e3.59 (m, 4H), 4.12e4.14 (m,

1H), 7.15e7.17 (m, 1H), 7.43e7.47 (m, 1H), 7.56e7.60 (m, 2H),

7.87e7.91 (m, 2H), 8.08e8.13 (m, 2H). MS (ESI) m/z: 490.5 ([MþH]

X.-W. Chen et al. / European Journal of Medicinal Chemistry 123 (2016) 332e353

347

^þ).

(m, 2H), 3.15e3.17 (m, 4H), 3.19e3.22 (m, 1H), 3.40 (t, 2H,

J ¼ 7.6 Hz), 3.56e3.58 (m, 2H), 4.05e4.07 (m, 2H), 6.52 (d, 1H,

J ¼ 7.6 Hz), 7.45e7.48 (m, 1H), 7.57e7.59 (m, 1H), 8.12e8.15 (m, 2H).

6.1.9.15. N-(trans-4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)

ethyl)cyclohexyl)pyridine-3-sulfonamide (9o). Yield 81.7%; mp:

137e140 ^ꢀC. HPLC t_R¼ 9.36 min, 98.8% purity. ¹H NMR (DMSO-d₆):

6.1.12. Ethyl trans-4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)

ethyl)-N-methylcyclohexanamine (11)

d

0.87e0.93 (m, 2H), 1.03e1.05 (m,1H), 1.10e1.23 (m, 3H),1.34e1.36

(m, 1H), 1.59e1.66 (m, 4H), 2.33 (t, 2H, J ¼ 7.6 Hz), 2.96e2.98 (m,

4H), 3.41e3.43 (m, 4H), 3.74e3.79 (m, 1H), 7.35e7.36 (m, 1H),

7.42e7.46 (m,1H), 7.63e7.66 (m,1H), 7.95 (d, 1H, J ¼ 8.0 Hz),

8.06e8.08 (m, 2H), 8.19e8.21 (m,1H), 8.81e8.82 (m,1H), 8.97e8.98

(m, 1H). MS (ESI) m/z: 485.5 ([MþH] ^þ).

10 (0.5 g, 1.1 mmol) in THF (3 mL) was added dropwise with

stirring to a solution of lithium aluminum hydride (0.1 g, 3.4 mmol)

in THF (5 mL) at 0e5 ^ꢀC. The resulting mixture was stirred at room

temperature for 10 h. The reaction was quenched with water

(0.5 mL). 20% sodium hydroxide solution (0.5 mL) and water

(1.5 mL) were then added and stirred for 0.5 h. The resulting so-

lution was ﬁltered. The ﬁltrate was evaporated in vacuo to give a

yellow oil (0.8 g) which was puriﬁed using Aluminium oxide, which

was eluted with 1e10% methanol/dichloromethane to 11 (0.2 g,

61.4%) as a colorless solid. Mp: 135e137 ^ꢀC. ¹H NMR (CDCl₃):

6.1.9.16. Ethyl (trans-4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-

yl)ethyl)cyclohexyl)carbamate (9p) hydrochloride. Yield 89.3%; mp:

218e219 ^ꢀC. HPLC t_R¼ 11.44 min, 98.5% purity. ¹H NMR (DMSO-d₆):

d

0.97e1.04 (m, 2H), 1.13e1.18 (m, 5H), 1.24e1.25 (m, 1H), 1.60e1.64

(m, 2H), 1.73e1.75 (m, 2H), 1.79e1.81 (m, 2H), 3.17e3.23 (m, 5H),

3.41 (t, 2H, J ¼ 7.6 Hz), 3.58e3.60 (m, 2H), 3.95 (q, 2H, J ¼ 4.8 Hz),

4.06e4.09 (m, 2H), 7.00 (d, 1H, J ¼ 5.2 Hz), 7.46e7.49 (m, 1H),

7.59e7.61 (m,1H), 8.11e8.15 (m, 2H). MS (ESI) m/z: 416.9 ([MþH] ^þ).

d

0.96e1.11 (m, 4H), 1.22e1.24 (m, 1H), 1.42e1.45 (m, 2H), 1.75e1.86

(m, 5H), 2.43 (t, 2H, J ¼ 4.0 Hz), 2.59e2.63 (m, 1H), 2.65e2.67 (m,

4H), 2.96 (d, 3H, J ¼ 3.6 Hz), 3.55e3.57 (m, 4H), 7.35 (t, 1H,

J ¼ 8.0 Hz), 7.45 (t, 1H, J ¼ 8.0 Hz), 7.88e7.93 (m, 2H).

6.1.9.17. N-(trans-4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)

ethyl)cyclohexyl)-2-methoxyacetamide (9q). Yield 86.3%; mp:

132e133 ^ꢀC. HPLC t_R¼ 8.81 min, 99.7% purity. ¹H NMR (DMSO-d₆):

6.1.13. N-(trans-4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)

ethyl)cyclohexy)-N-methylthiophene-2-sulfonamide (12)

Compound 11 (0.2 g, 0.6 mmol) in dichloromethane (5 mL) was

treated with thiophene-2-sulfonyl chloride (0.1 g, 0.7 mmol) in

dichloromethane (2 mL) using procedure G. The crude product was

puriﬁed by recrystallization from ethanol to yield 12 (0.2 g, 66.4%)

as a colorless solid. Mp: 159e161 ^ꢀC. HPLC t_R¼ 10.93 min, 97.1%

d

0.93e1.02 (m, 2H), 1.22e1.25 (m, 3H), 1.34e1.40 (m, 2H), 1.70e1.76

(m, 4H), 2.37 (t, 2H, J ¼ 7.6 Hz), 2.57e2.59 (m, 4H), 3.29 (s, 3H),

3.43e3.45 (m, 4H), 3.54e3.58 (m, 1H), 3.76 (s, 2H), 7.43 (t, 1H,

J ¼ 7.6 Hz), 7.52e7.58 (m, 2H), 8.03e8.07 (m, 2H). MS (ESI) m/z:

417.4 ([MþH] ^þ).

purity. ¹H NMR (DMSO-d₆):

d 0.93e1.02 (m, 2H),1.19e1.21 (m,

1H),1.28e1.42 (m, 6H), 1.71e1.74 (m, 2H), 2.33 (t, 2H, J ¼ 7.2 Hz),

2.54e2.56 (m, 4H), 2.70 (s, 3H), 3.40e3.42 (m, 4H), 3.60e3.66 (m,

1H), 7.22e7.24 (m, 1H), 7.43 (t, 1H, J ¼ 7.6 Hz), 7.55 (t, 1H, J ¼ 7.6 Hz),

7.65e7.66 (m, 1H), 7.97e7.98 (m, 1H), 8.02e8.06 (m, 2H). MS (ESI)

m/z: 505.1 ([MþH] ^þ).

6.1.10. N-(trans-4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)

ethyl)cyclohexyl)-1H-indole-2-carboxamide (9r) hydrochloride

A mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

hydrochloride (0.3 g, 1.8 mmol) and 4-dimethylaminopyridine

(0.1 g, 1.2 mmol) in dichloromethane (5 mL) was added at room

temperature with stirring to a solution of 7 (0.5 g, 1.5 mmol) and

indole-2-carboxylic acid (0.3 g, 1.7 mmol) in dichloromethane

(5 mL). The resulting mixture was stirred for 12 h. Water (10 mL)

was added to the reaction mixture and extracted with dichloro-

methane (2 ꢂ 10 mL). The combined organic extracts were dried

(Na₂SO₄) and evaporated in vacuo. The residue was dissolved in

methanol (5 mL) and 5% hydrogenchloride solution (0.5 mL) was

added. The resulting mixture was stirred for 1 h at room temper-

ature. After stirring, the mixture was evaporated until dry under

reduced pressure and puriﬁed by recrystallization from ethanol/

water to yield 9r (0.7 g, 90.1%) as a white solid. Mp: 269e270 ^ꢀC.

6.1.14. N-(cis-4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)

ethyl)cyclohexyl)acetamide (16)

The trans/cis mixture 15 (9.0 g, 34.2 mmol) was obtained via

esteriﬁcation (procedure B), reduction (procedure C), and activation

(procedure D) using tans/cis mixture 13 (11.8 g, 59.2 mmol) as

starting material. Then a mixture of 14 (9.0 g, 34.2 mmol), 3-

(piperazin-1-yl)benzo[d]isothiazole (8.7 g, 37.6 mmol), sodium

carbonate (10.9 g, 102.6 mmol), potassium iodide (0.01 g,

0.05 mmol), and acetonitrile (135 mL) was stirred at reﬂux for 36 h.

The resulting solution was cooled to ambient temperature and

ﬁltered. The ﬁltrate was evaporated in vacuo to give a brown oil

(5.9 g), which was puriﬁed using silica gel, eluting with 1e5%

methanol/dichloromethane to give a colorless solid (2.6 g). This

material was further recrystallized from ethanol (26 mL) to afford

16 (1.9 g, 14.4%) as a white solid. Mp: 147e148 ^ꢀC. ¹H NMR (CDCl₃):

HPLC t_R¼ 12.65 min, 99.7% purity. ¹H NMR (DMSO-d₆):

d 1.02e1.11

(m, 2H),1.32e1.45 (m, 5H),1.81e1.90 (m, 4H), 2.41 (t, 2H, J ¼ 7.6 Hz),

2.59e2.61 (m, 4H), 3.44e3.46 (m, 4H), 3.76e3.78 (m, 1H), 7.01e7.04

(m, 1H), 7.15e7.19 (m, 2H), 7.41e7.46 (m, 2H), 7.55e7.61 (m, 2H),

8.05e8.08 (m, 2H), 8.30 (d, 1H, J ¼ 5.2 Hz), 11.58 (d, 1H, J ¼ 0.8 Hz).

MS (ESI) m/z: 488.0 ([MþH] ^þ).

d

1.23e1.25 (m, 2H),1.50e1.54 (m, 3H), 1.62e1.67 (m, 4H),1.84e1.88

(m, 2H), 1.99 (s, 3H), 2.45 (t, 2H,J ¼ 4.0 Hz), 2.67e2.69 (m, 4H),

3.57e3.59 (m, 4H), 4.01e4.03 (m, 1H), 5.50 (d, 1H, J ¼ 2.0 Hz), 7.35

(t, 1H, J ¼ 8.0 Hz), 7.47 (t, 1H, J ¼ 8.0 Hz), 7.81 (d, 1H, J ¼ 4.0 Hz), 7.90

(d, 1H, J ¼ 4.0 Hz).

6.1.11. tert-Butyl(trans-4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-

1-yl)ethyl)cyclohexyl)carbamate (10)

A mixture of 7 (0.5 g, 1.5 mmol), triethylamine (0.4 mL,

3.0 mmol) and di(tert-butyl) carbonate (0.4 g, 1.7 mmol), and

dichloromethane (10 mL) was stirred at room temperature for 5 h.

Water (10 mL) was then added to the resulting mixture was added

water and extracted with dichloromethane (1 ꢂ 10 mL). The

combined organic extracts were dried (Na₂SO₄) and evaporated in

vacuo. The residue was puriﬁed by recrystallization from ethanol to

yield 10 (0.6 g, 96.7%) as a white solid. Mp: 175e178 ^ꢀC. ¹H NMR

6.1.15. cis-4-(2-(4-(Benzo[d]isothiazol-3-yl)piperazin-1-yl)ethyl)

cyclohexanamine (17)

Compound 16 (1.5 g, 3.9 mmol) was treated with 5%HCl (20 mL)

using procedure F to yield 17 (1.2 g, 89.2%) as a colorless semisolid.

¹H NMR (CDCl₃):

d 1.34e1.39 (m, 4H), 1.44e1.47 (m, 4H), 1.50e1.55

(m, 2H), 1.72e1.74 (m, 3H), 2.37 (t, 2H, J ¼ 4.0 Hz), 2.60e2.61 (m,

4H), 2.87e2.89 (m, 1H), 3.49e3.50 (m, 4H), 7.27 (t, 1H, J ¼ 8.0 Hz),

7.38 (t,1H, J ¼ 8.0 Hz), 7.72 (d, 1H, J ¼ 4.0 Hz), 7.82 (d,1H, J ¼ 4.0 Hz).

(DMSO-d₆):

d 0.96e1.03 (m, 2H), 1.08 (s, 9H), 1.12e1.17 (m, 2H),

1.22e1.23 (m, 1H), 1.58e1.61 (m, 2H), 1.71e1.73 (m, 2H), 1.78e1.80

348

X.-W. Chen et al. / European Journal of Medicinal Chemistry 123 (2016) 332e353

6.1.16. General procedure for the preparation of compounds 18a-b

Compound 17 (0.5 g, 1.5 mmol) was treated with acyl chloride

(0.2 g, 1.8 mmol) in dichloromethane (5 mL) using procedure G to

give 18a-b.

6.1.20. 3-Chloro-6-nitro-1,2-benzisothiazole (28)

A mixture of 26 (3.6 g, 18.4 mmol), PCl₅(4.6 g, 22.0 mmol) and

N-ethyl-N-isopropylpropan-2-amine (6.5 mL) in POCl₃(10 mL) was

heated to 120 ^ꢀC with stirring. After 3 h the mixture was cooled to

room temperature, poured into ice water, and extracted with ethyl

acetate. The organic phase was concentrated in vacuo. The residue

was puriﬁed using silica gel, eluting with 1e10% ethyl acetate/

hexane to give 28 (2.0 g, 49.5%) as a colorless solid. Mp: 146e147 ^ꢀC.

6.1.16.1. N-(cis-4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)

ethyl)cyclohexyl)furan-2-carboxamide

(18a)

hydrochloride.

Yield 79.3%; mp: 163e165 ^ꢀC. HPLC t_R¼ 9.40 min, 99.6% purity. ¹H

NMR (DMSO-d₆):

d

1.54e1.58 (m, 7H), 1.66e1.69 (m, 2H), 1.79e1.81

¹H NMR (CDCl₃):

d

8.17 (t, 1H, J ¼ 4.0 Hz), 8.61e8.65 (m, 2H).

(m, 2H), 3.17 (t, 2H, J ¼ 4.0 Hz), 3.23e3.31 (m, 2H), 3.52e3.55 (m,

2H), 3.59e3.62 (m, 2H), 3.86e3.88 (m, 1H), 4.00e4.09 (m, 2H),

6.61e6.62 (m, 1H), 7.14e7.15 (m, 1H), 7.48 (t, 1H, J ¼ 7.6 Hz), 7.60 (t,

1H, J ¼ 7.6 Hz), 7.82 (s, 1H), 7.97e7.99 (m, 1H), 8.11e8.15 (m, 2H). MS

(ESI) m/z: 439.1 ([MþH] ^þ).

6.1.21. 1-(6- nitro-1,2-benzisothiazol-3-yl)-3-piperazine (30)

A mixture of 28 (0.44 g, 2.1 mmol), and piperazine (1.8 g,

21.0 mmol) in chlorobenzene (20 mL) was heated to 80 ^ꢀC with

stirring for 16 h. Cooled to room temperature, the resulting solution

was partitioned between water (100 mL) and ethyl acetate

(3 ꢂ 50 mL). The combined extracts were dried (Na₂SO₄) and

evaporated in vacuo. The residue was puriﬁed using silica gel,

eluting with 0.1e1% methanol/dichloromethane to give 30 (2.0 g,

49.5%) as a colorless solid. Mp: 164e166 ^ꢀC. ¹H NMR (DMSO-d₆):

6.1.16.2. Ethyl (cis-4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)

ethyl)cyclohexyl)carbamate (18b) hydrochloride. Yield 81.5%; mp:

202e204 ^ꢀC. HPLC t_R¼ 9.74 min, 98.9% purity. ¹H NMR (DMSO-d₆):

d

1.04e1.07 (m, 1H), 1.15e1.19 (m, 4H), 1.68e1.70 (m, 2H), 1.98e1.99

(m, 1H), 2.50 (t, 3H, J ¼ 1.6 Hz), 3.17 (t, 2H, J ¼ 5.6 Hz), 3.24e3.27 (m,

3H), 3.43e3.90 (m, 6H), 3.95e4.09 (m, 5H), 7.08e7.10 (m, 1H), 7.48

(t, 1H, J ¼ 7.6 Hz), 7.60 (t, 1H, J ¼ 7.6 Hz), 8.10e8.15 (m, 2H). MS (ESI)

m/z: 417.3 ([MþH] ^þ).

d

1.22 (s, 1H), 2.80e2.81 (m, 4H), 3.00e3.01 (m, 4H), 8.11e8.15 (m,

2H), 8.32 (s, 1H).

6.1.22. General procedure for the preparation of compounds 31e32

Compound 29 [44] (5.0 g, 19.7 mmol) and 30 (1.5 g, 5.7 mmol)

were separately treated with sodium carbonate (2.5 equivalent)

and 5 (1.1 equivalent) in acetonitrile using procedure E. The crude

product was puriﬁed by recrystallization from ethanol to yield

31e32.

6.1.17. 3-Hydroxy-6-chloro-1,2-benzisothiazole (25)

Sulfuryl dichloride (13.6 g, 100.9 mmol) in chloroform (20 mL)

was added dropwise to a stirred solution of 2-(benzylthio)-4-

chlorobenzoic acid chloride 23 [44] (20.0 g, 67.3 mmol) in chloro-

form (200 mL). After stirring for 2 h, the resulting solution was

added to ammonia hydrate (200 mL) at room temperature and

stirred for 2 h. The reaction was quenched with 30% hydrochloric

acid (700 mL) in an ice bath and stirred for 20 min. The resulting

mixture was ﬁltered and washed with water (100 mL) to give 25

(6.5 g, 52.0%) as a yellow solid. Mp: 198e200 ^ꢀC. ¹H NMR (CDCl₃):

6.1.22.1. N-(trans-4-(2-(4-(6-Chlorobenzo[d]isothiazol-3-yl)piper-

azin-1-yl)ethyl)cyclohexyl)acetamide (31). Yield 69.3%; Mp:

251e252 ^ꢀC. ¹H NMR (CDCl₃):

d 1.08e1.14 (m, 3H), 1.23e1.31 (m,

2H), 1.47e1.54 (m, 2H), 1.83e1.87 (m, 2H), 2.09e2.13 (m, 2H), 2.31

(s, 3H), 2.50 (t, 2H, J ¼ 7.6 Hz), 2.68e2.70 (m, 4H), 3.54e3.57 (m,

4H), 4.10e4.13 (m, 1H), 7.23 (d, 1H, J ¼ 3.6 Hz), 7.50 (d, 1H),

J ¼ 8.0 Hz, 7.88e8.04 (m, 2H).

d

7.40 (d, 1H, J ¼ 7.6 Hz), 7.96e7.99 (m, 2H), 8.17 (s, 1H).

6.1.18. 2-(Benzylthio)-4-nitrobenzoic acid (22)

A mixture of 2-ﬂuoro-4-nitrobenzoic acid (25 g, 135.1 mmol),

phenylmethanethiol (18.5 g, 148.6 mmol), and N-ethyl-N-iso-

propylpropan-2-amine (118 mL) in N,N-dimethylformamide

(300 mL) was stirred at 65 ^ꢀC for 35 h. The resulting solution was

evaporated in vacuo to less than a certain amount volume to give a

brown oil, which was poured into water (600 mL). 30% hydrochloric

acid (90 mL) was added to the resulting mixture and stirred for

another 1 h. The solution was ﬁltered and washed with water

(100 mL). The crude product was puriﬁed by recrystallization from

ethanol to yield 22 (19.8 g, 55.5%) as a yellow solid. Mp: 130e131 ^ꢀC.

6.1.22.2. N-(trans-4-(2-(4-(6-nitrobenzo[d]isothiazol-3-yl)piperazin-

1-yl)ethyl)cyclohexyl)acetamide (32). Yield 71.1%; Mp: > 270 ^ꢀC. ¹H

NMR (CDCl₃): d 1.02e1.07 (m, 2H), 1.16e1.25 (m, 3H), 1.37e1.40 (m,

1H), 1.53e1.56 (m, 2H), 1.82e1.87 (m, 3H), 2.30 (s, 3H), 2.42 (t, 2H,

J ¼ 7.6 Hz), 2.56e2.59 (m, 4H), 3.48e3.50 (m, 4H), 3.58e3.63 (m,

1H), 8.08e8.10 (d, 1H, J ¼ 8.4 Hz), 8.16e8.18 (m, 1H), 8.29 (s, 1H),

9.10 (d, 1H, J ¼ 2.0 Hz).

6.1.23. General procedure for the preparation of compounds 33e34

Compound 31 (2.0 g, 4.8 mmol) or 32 (1.0 g, 2.3 mmol) was

treated with 5% HCl (100 mL) using procedure F to yield 33e34.

¹H NMR (DMSO-d₆):

d 4.37 (s, 2H), 7.27e7.31 (m, 1H), 7.34e7.38 (m,

2H), 7.47e7.48 (m, 2H), 8.00 (dd, 1H, J ¼ 8.4 Hz, J ¼ 2.0 Hz), 8.09 (d,

1H, J ¼ 8.4 Hz), 8.20 (d, 1H, J ¼ 2.0 Hz), 13.81 (br, 1H).

6.1.23.1. trans-4-(2-(4-(6-Chlorobenzo[d]isothiazol-3-yl)piperazin-1-

6.1.19. 3-Hydroxy-6-nitro-1,2-benzisothiazole (26)

yl)ethyl)cyclohexanamine (33). Yield 82.3%; Mp: 98e100 ^ꢀC. ¹H

Oxalyl dichloride (13.2 g, 103.7 mmol) and N,N-dime-

thylformamide (4 mL) were added dropwise to a stirred solution of

22 (15.0 g, 51.9 mmol) in chloroform (150 mL) at 0e5 ^ꢀC. The

resulting mixture was stirred at reﬂux for 3 h. The resulting solu-

tion was cooled to room temperature. Sulfuryl dichloride (7.7 g,

57.0 mmol) in chloroform (10 mL) was added dropwise. After stir-

ring for 2 h, the resulting solution was added to ammonia hydrate

(187 mL) at room temperature and stirred for 2 h. The reaction was

quenched with 30% hydrochloric acid (600 mL) in an ice bath, and

stirred for 20 min. The resulting mixture was ﬁltered and washed

with water (50 mL) to give 26 (3.2 g, 31.0%) as a yellow solid. Mp:

NMR (DMSO-d₆):

d 0.94e1.05 (m, 4H),1.13e1.20 (m, 3H), 1.34e1.39

(m, 2H), 1.68e1.76 (m, 2H), 1.74e1.87 (m, 2H), 2.34 (t, 2H,

J ¼ 7.6 Hz), 2.48e2.52 (m, 1H), 2.58e2.60 (m, 4H), 3.46e3.48 (m,

4H), 6.98 (d, 1H, J ¼ 8.0 Hz), 7.45e7.48 (m, 1H), 8.03 (d, 1H,

J ¼ 8.4 Hz), 8.22 (d, 1H, J ¼ 2.0 Hz).

6.1.23.2. trans-4-(2-(4-(6-Nitrobenzo[d]isothiazol-3-yl)piperazin-1-

yl)ethyl)cyclohexanamine (34). Yield 85.6%; Mp: 131e133 ^ꢀC. ¹H

NMR (CDCl₃): d 0.95e1.07 (m, 4H), 1.15e1.22 (m, 3H), 1.37e1.41 (m,

2H), 1.69e1.72 (m, 2H), 1.77e1.80 (m, 2H), 2.39 (t, 2H, J ¼ 8.0 Hz),

2.50e2.55 (m, 1H), 2.60e2.62 (m, 4H),3.49e3.52 (m, 4H), 7.94 (d,

1H, J ¼ 8.0 Hz), 8.10e8.13 (m, 1H), 8.63 (d, 1H, J ¼ 4.0 Hz).

266e268 ^ꢀC. ¹H NMR (CDCl₃):

(m, 2H), 8.59 (s, 1H).

d

8.19 (d, 1H, J ¼ 8.0 Hz), 8.25e8.27

X.-W. Chen et al. / European Journal of Medicinal Chemistry 123 (2016) 332e353

349

6.1.24. General procedure for the preparation of compounds 35e36

Compound 33 (0.5 g, 1.3 mmol) and 34 (0.5 g, 1.3 mmol) were

separately treated with acyl chloride (0.1 g, 1.5 mmol) in

dichloromethane (2 mL) using procedure G. The crude product was

puriﬁed by recrystallization from ethanol to yield 35e36.

was stirred at reﬂux for 32 h. The resulting solution was cooled to

ambient temperature and ﬁltered. The ﬁltrate was evaporated in

vacuo to give a brown oil (6.3 g). A mixture of this material and TFA

(7.5 mL) in dichloromethane (60 mL) was stirred at room temper-

ature for 20 h. The resulting solution was evaporated in vacuo to

remove most of the TFA and partitioned between aqueous water

(40 mL) and dichloromethane (100 mL). Aqueous 50% NaOH

(20 mL) was added to the solution in an ice bath and stirred for

20 min. The solution was extracted with dichloromethane (50 mL)

and the combined extracts were washed with brine (100 mL), dried

with Na₂SO₄, and evaporated in vacuo to give 40 (2.6 g, 66.8%) as a

6.1.24.1. N-(trans-4-(2-(4-(6-Chlorobenzo[d]isothiazol-3-yl)piper-

azin-1-yl)ethyl)cyclohexyl)furan-2-carboxamide (35). Yield 89.2%;

mp: 204e206 ^ꢀC. HPLC t_R¼ 10.26 min, 99.8% purity. ¹H NMR

(DMSO-d₆):

d 0.97e1.05 (m, 2H), 1.23e1.25 (m, 2H), 1.30e1.41 (m,

3H), 1.77e1.79 (m, 4H), 2.38 (t, 2H, J ¼ 7.6 Hz), 2.51e2.57 (m, 4H),

3.42e3.47 (m, 4H), 3.66e3.70 (m, 1H), 6.59e6.61 (m, 1H), 7.08 (d,

1H, J ¼ 3.2 Hz), 7.45 (dd, 1H, J ¼ 8.4 Hz, J ¼ 2.0 Hz), 7.80 (d, 1H,

J ¼ 0.8 Hz), 8.04e8.25 (m, 2H),8.25 (d, 1H, J ¼ 2.0 Hz). MS (ESI) m/z:

473.1 ([MþH] ^þ).

light yellow solid. Mp: 61e63 ^ꢀC. ¹H NMR (CDCl₃):

d 1.23e1.25 (m,

3H), 1.44e1.52 (m, 3H), 1.71e1.73 (m, 2H), 2.46 (t, 2H, J ¼ 4.0 Hz),

2.59e2.64 (m, 2H), 2.66e2.68 (m, 4H), 3.09e3.11 (m, 2H),

3.56e3.58 (m, 4H), 7.35 (t, 1H, J ¼ 4.0 Hz), 7.46 (t, 1H, J ¼ 4.0 Hz),

7.80 (d, 1H, J ¼ 4.0 Hz), 7.90 (d, 1H, J ¼ 4.0 Hz).

6.1.24.2. N-(trans-4-(2-(4-(6-nitrobenzo[d]isothiazol-3-yl)piperazin-

1-yl)ethyl)cyclohexyl)furan-2-carboxamide (36). Yield 90.5%; mp:

230e232 ^ꢀC. HPLC t_R¼ 9.70 min, 99.0% purity. ¹H NMR (DMSO-d₆):

6.1.29. General procedure for the preparation of compound 41

Compound 40 (2.5 g, 7.6 mmol) in dichloromethane (25 mL) was

treated with acyl chloride (9.1 mmol) in dichloromethane (10 mL)

using procedure G to obtained the free base of 41. Then, a mixture of

oxalic acid dihydrate and the corresponding free base in ethyl ac-

etate (10 mL) were added and stirred at room temperature for 2 h.

The reaction was ﬁltered; the ﬁlter cake was washed with acetone

(5 mL) to give 41 as a white solid.

d

1.00e1.03 (m, 2H),1.34e1.39 (m, 5H),1.77e1.80 (m, 4H), 1.71e1.74

(m, 2H), 2.40 (t, 2H, J ¼ 7.6 Hz), 2.59e2.61 (m, 4H), 3.45e3.47 (m,

4H), 3.64e3.69 (m, 1H), 6.59e6.61 (m, 1H), 7.07 (d, 1H, J ¼ 3.6 Hz),

7.80 (s, 1H), 8.10 (d, 1H, J ¼ 8.4 Hz), 8.17 (dd, 1H, J ¼ 8.4 Hz,

J ¼ 4.0 Hz), 9.14 (d, 1H, J ¼ 4.0 Hz). MS (ESI) m/z: 484.2 ([MþH] ^þ).

6.1.25. N-(trans-4-(2-(4-(6-Aminobenzo[d]isothiazol-3-yl)

piperazin-1-yl)ethyl)cyclohexyl)furan-2-carboxamide (37)

6.1.29.1. Ethyl 4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)ethyl)

Hydrazine hydrate (0.5 mL) was added with stirring to a solution

of 36 (0.1g, 0.3 mmol) and Raney Nickel (0.01 g) in methanol

(10 mL). The resulting solution was stirred at room temperature for

7 h and ﬁltered. The ﬁltrate was evaporated in vacuo. The residue

was puriﬁed using Aluminum oxide, eluting with 1e2% methanol/

dichloromethane to afford 37 (0.08 g, 85.3%) as a yellow solid. Mp:

182e184 ^ꢀC. HPLC t_R¼ 8.16 min, 97.2% purity. ¹H NMR (DMSO-d₆):

piperidine-1-carboxylate

oxalate

(41a). Yield

73.1%;

mp:

190e193 ^ꢀC. HPLC t_R¼ 9.52 min, 99.3% purity. ¹H NMR (DMSO-d₆):

d

1.14e1.16 (m, 2H), 1.62e1.65 (m, 3H), 1.75e1.78 (m, 2H), 2.82 (t,

2H, J ¼ 4.0 Hz), 3.05e3.08 (m, 2H), 3.26e3.29 (m, 4H), 3.68e3.71

(m, 4H), 4.30e4.34 (m, 2H), 6.62 (s, 1H), 6.94e6.95 (m, 1H), 7.47 (t,

1H, J ¼ 7.6 Hz), 7.59 (t,1H, J ¼ 7.6 Hz), 7.83 (s,1H), 8.09e8.14 (m, 2H).

MS (ESI) m/z: 425.0 ([MþH] ^þ).

d

0.99e1.02 (m, 2H),1.23e1.24 (m,1H),1.33e1.39 (m, 4H), 1.77e1.79

(m, 4H), 2.36 (t, 2H, J ¼ 7.6 Hz), 2.50e2.53 (m, 4H), 3.34e3.37 (m,

4H), 3.63e3.68 (m, 1H), 5.64 (s, 2H), 6.59e6.60 (m, 1H), 6.66e6.69

(m, 1H), 6.88 (d, 1H, J ¼ 2.0 Hz), 7.07e7.08 (m, 1H), 7.63 (d, 1H,

J ¼ 8.4 Hz), 7.79e7.80 (m, 1H), 8.10 (d, 1H, J ¼ 8.0 Hz). MS (ESI) m/z:

454.3 ([MþH] ^þ).

6.1.29.2. (4-(2-(4-(Benzo[d]isothiazol-3-yl)piperazin-1-yl)ethyl)

piperidin-1-yl)(furan-2-yl)methanone oxalate (41b). Yield 76.8%;

mp: 196e198 ^ꢀC. HPLC t_R¼ 9.16 min, 99.5% purity. ¹H NMR (DMSO-

d₆):

d

1.00e1.08 (m, 2H),1.17 (t, 3H, J ¼ 7.2 Hz),1.60e1.69 (m, 5H),

2.75 (t, 2H, J ¼ 4.0 Hz), 3.06e3.08 (m, 2H), 3.28e3.31 (m, 4H),

3.67e3.70 (m, 4H), 3.94e3.97 (m, 2H), 4.02 (q, 2H, J ¼ 7.2 Hz), 7.47

(t, 1H, J ¼ 7.6 Hz), 7.59 (t, 1H, J ¼ 7.6 Hz), 8.09e8.14 (m, 2H). MS (ESI)

m/z: 403.3 ([MþH] ^þ).

6.1.26. tert-Butyl 4-(2-ethoxy-2-oxoethyl)piperidine-1-carboxylate

(38)

tert-Butyl 4-oxopiperidine-1-carboxylate (20.0 g, 100.4 mmol)

was treated with triethyl phosphonoacetate (24.8 g, 110.4 mmol) in

THF (250 mL), and subsequent 10% Pd/C (1.0 g) using procedure A to

6.1.30. trans-cyclohexane-1,4-dimethanol (42)

Thionyl chloride (16.6 g, 139.5 mmol) was added dropwise to a

stirred solution of trans-cyclohexane-1,4-dicarboxylic acid (10.0 g,

58.1 mmol) in methanol (160 mL) at 0e20 ^ꢀC. The resulting mixture

was stirred at reﬂux for 4 h. The resulting solution was evaporated

in vacuo and the residue was partitioned between aqueous water

(50 mL) and dichloromethane (100 mL). The organic layer was

washed with aqueous NaHCO₃(40 mL), brine (50 mL) and dried

(Na₂SO₄), and evaporated in vacuo to give a colorless solid (9.8 g,

84.0%). Next, a mixture of this solid (9.8 g) and NaBH₄(18.5 g,

488.2 mmol) in THF (196 mL) was stirred at reﬂux for 0.5 h. Then

methanol (69 mL) was added to the resulting solution at room

temperature.

obtain 38. Yield 96.5%,¹H NMR (CDCl₃):

d 1.14e1.17 (m, 2H), 1.26 (t,

3H, J ¼ 4.0 Hz), 1.49 (s, 9H), 1.68e1.72 (m, 2H), 1.93e1.97 (m, 1H),

2.23 (d, 2H, J ¼ 8.0 Hz), 2.72e2.74 (m, 2H), 4.09e4.11 (m, 2H), 4.12

(q, 2H, J ¼ 4.0 Hz).

6.1.27. tert-Butyl 4-(2-((methylsulfonyl)oxy)ethyl)piperidine-1-

carboxylate (39)

Compound 38 (20.0 g, 73.7 mmol) was treated with sodium

borohydride (13.9 g, 368.5 mmol) in THF (200 mL) and subse-

quently mesyl chloride (73.7 mmol) using procedure C to achieve

39. Yield 86.9%, ¹H NMR (CDCl₃):

d 1.13e1.15 (m, 2H), 1.45 (s, 9H),

1.66e1.68 (m, 2H), 1.69e1.72 (m, 3H), 2.70e2.72 (m, 2H), 3.01 (s,

3H), 4.08e4.10 (m, 2H), 4.29 (t, 2H, J ¼ 4.0 Hz).

The resulting solution was stirred at reﬂux for 12 h and

quenched with 30% hydrochloric acid (20 mL) in and ice bath. The

resulting mixture was stirred for 1 h and alkalized with 50% sodium

hydroxide solution (30 mL). The resultant mixture was extracted

with dichloromethane (3 ꢂ 60 mL). The combined organic extracts

were dried (MgSO₄) and evaporated in vacuo to give 42 (2.4 g,

28.6%) as a colorless solid. Mp: 63e65 ^ꢀC. ¹H NMR (CDCl₃):

6.1.28. 3-(4-(2-(Piperidin-4-yl)ethyl)piperazin-1-yl)benzo[d]

isothiazole (40)

A mixture of 39 (4.0 g, 13.0 mmol), 3-(piperazin-1-yl)benzo[d]

isothiazole (2.6 g, 11.8 mmol), sodium carbonate (3.8 g, 35.5 mmol),

potassium iodide (0.01 g, 0.05 mmol), and acetonitrile (100 mL)

d

0.93e1.03 (m, 4H), 1.45e1.47 (m, 2H), 1.84e1.88 (m, 4H),

350

X.-W. Chen et al. / European Journal of Medicinal Chemistry 123 (2016) 332e353

3.40e3.46 (m, 4H).

matrix least-squares method using a SHELXL97. All the non-

hydrogen atoms were located in different Fourier maps. The

hydrogen atoms were ﬁxed geometrically and reﬁned isotropically.

Graphical representations were generated using ORTEP. Crystallo-

graphic data for 6 has been deposited with the Cambridge Crys-

tallographic Data Centre (CCDC 1414993).

6.1.31. (trans-4-((4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)

methyl)cyclohexyl)methyl methanesulfonate (43)

Methylsulfonyl chloride (4.2 g, 36.6 mmol) in ethyl acetate

(10 mL) was added dropwise with stirring to a solution of 42 (2.4 g,

16.6 mmol) and triethylamine (9.0 mL, 66.6 mmol) in ethyl acetate

(40 mL) at 0 ^ꢀC. The resulting mixture was stirred for 3 h at ambient

temperature. The resulting mixture was ﬁltered and washed with

water (2 ꢂ 50 mL) to give a colorless solid (3.93 g, 78.7 mmol). A

mixture of this solid (2.6 g, 11.9 mmol), 3-(piperazin-1-yl)benzo[d]

isothiazole (3.9 g, 13.1 mmol), sodium carbonate (2.5 g, 23.8 mmol),

potassium iodide (0.01 g, 0.05 mmol), and acetonitrile (100 mL)

was stirred at reﬂux for 22 h. The resulting solution was cooled to

ambient temperature and ﬁltered and washed with acetonitrile

(30 mL) to yield a colorless solid which was further puriﬁed using

silica gel, eluting with 2.5% methanol/dichloromethane to give 43

(2.2 g, 44.1%) as a white solid. Mp: 138e140 ^ꢀC. ¹H NMR (CDCl₃):

6.3. Biology evaluation

6.3.1. Animals

Male Sprague-Dawley (SD) rats (150e290 g) were used as

experimental animals in this study. Rats were housed under stan-

dardized conditions and had free access to standard rat chow and

water. Rats were randomly assigned to different experimental

groups, each separately kept in a cage. All research involving ani-

mals in this study follow the guidelines of the byelaw of experi-

ments on animals, and have been approved by the Ethics and

Experimental Animal Committee of Shanghai Institute of Pharma-

ceutical Industry.

d

0.94e0.98 (m, 2H), 1.02e1.10 (m, 2H), 1.52e1.53 (m, 1H), 1.72e1.74

(m, 2H), 1.85e1.94 (m, 4H), 2.23 (d, 2H, J ¼ 4.0 Hz), 2.61e2.63 (m,

4H), 3.00 (s, 3H), 3.54e3.55 (m, 4H), 4.04 (d, 2H, J ¼ 4.0 Hz),

7.34e7.36 (m, 1H), 7.45e7.47 (m, 1H), 7.34e7.36 (m, 1H), 7.80e7.81

(m, 1H), 7.90e7.92 (m, 1H).

6.3.2. In vitro binding assays

6.3.2.1. 5-HT_1Areceptor [53]. Cell membrane homogenates (36 mg

protein) were incubated for 60 min at 22 ^ꢀC with 0.3 nM [³H]8-OH-

DPAT in the absence or presence of the test compound in a buffer

containing 50 mM Tris-HCl (pH 7.4), 10 mM MgSO₄, 0.5 mM EDTA

6.1.32. (trans-4-((4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)

methyl)cyclohexyl)methanamine (44)

and 2

mg/ml aprotinine. Nonspeciﬁc binding was determined in the

A mixture of 43 (1.0 g, 2.4 mmol), potassium phthalimide (0.5 g,

2.6), and N,N-dimethylformamide (15 mL) was stirred at 100 ^ꢀC for

1.5 h. The resulting solution was poured into ice water (150.0 g) and

presence of 10

m

M 8-OH-DPAT. Following incubation, the samples

were ﬁltered rapidly under vacuum through glass ﬁber ﬁlters (GF/B,

Packard) presoaked with 0.3% PEI. The ﬁltered material was rinsed

several times with ice-cold 50 mM Tris-HCl using a 96-sample cell

harvester (Uniﬁlter, Packard). The ﬁlters were dried then counted

for radioactivity in a scintillation counter (Topcount, Packard) using

a scintillation cocktail (Microscint 0, Packard). The inhibition re-

sults were expressed as a percentage of sample binding compared

to the control radioligand speciﬁc binding. The standard reference

compound was 8-OH-DPAT and was tested in each experiment at

several concentrations to obtain a competition curve from which its

IC₅₀is calculated.

stirred for

1 h. The resultant mixture was extracted with

dichloromethane (2 ꢂ 75 mL). The combined organic extracts were

dried (Na₂SO₄) and evaporated in vacuo to give a semi-solid (1.1 g).

A mixture of this material (1.1 g), hydrazine hydrate (0.2 g,

2.6 mmol), and ethanol (40 mL) was stirred at reﬂux for 12 h. The

resulting mixture was cooled to ambient temperature, ﬁltered and

washed with ethanol (20 mL) to give 44 (0.5 g, 66.3%) as a white

solid. Mp: 198e200 ^ꢀC. ¹H NMR (CDCl₃):

d 0.91e0.94 (m, 4H),

1.26e1.28 (m, 2H), 1.54 (s, 2H), 1.82e1.83 (m, 2H), 1.89e1.90 (m,

2H), 2.23 (d, 2H, J ¼ 4.0 Hz), 2.54 (d, 2H, J ¼ 4.0 Hz), 2.61e2.63 (m,

4H), 3.54e3.56 (m, 4H), 7.34e7.36 (m, 1H), 7.45e7.47 (m, 1H),

7.34e7.36 (m, 1H), 7.80e7.81 (m, 1H), 7.90e7.92 (m, 1H).

6.3.2.2. 5-HT_2Areceptor [54]. Cell membrane homogenates (40 mg

protein) were incubated for 60 min at 22 ^ꢀC with 0.5 nM [³H]

ketanserin in the absence or presence of the test compound in a

buffer containing 50 mM Tris-HCl (pH 7.4). Nonspeciﬁc binding was

6.1.33. N-((trans-4-((4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)

methyl)cyclohexyl)methyl)furan-2-carboxamide (45)

determined in the presence of 1 mM ketanserin. Following incu-

Compound 44 (0.2 g, 0.6 mmol) in dichloromethane (5 mL) was

treated with pyromrcyl chloride (0.1 g, 0.7 mmol) in dichloro-

methane (1 mL) using procedure G. The crude product was puriﬁed

by recrystallization from ethanol to yield 45 (0.2 g, 75.8%). Mp:

159e160 ^ꢀC. HPLC t_R¼ 9.24 min, 97.1% purity. ¹H NMR (DMSO-d₆):

bation, the samples were ﬁltered rapidly under vacuum through

glass ﬁber ﬁlters (GF/B, Packard) presoaked with 0.3% PEI. The

ﬁltrate was rinsed several times with ice-cold 50 mM Tris-HCl using

a 96-sample cell harvester (Uniﬁlter, Packard). The ﬁlters were

dried then counted for radioactivity in a scintillation counter

(Topcount, Packard) using a scintillation cocktail (Microscint 0,

Packard). The inhibition results were expressed as a percentage of

sample binding compared to the control radioligand speciﬁc

binding. The ketanserin was used as a standard reference com-

pound and was tested in each experiment at several concentrations

to obtain a competition curve from which its IC₅₀is calculated.

d

0.81e0.95 (m, 4H), 1.47e1.50 (m, 2H), 1.72e1.74 (m, 2H),1.81e1.82

(m, 2H), 2.16 (d, 2H, J ¼ 4.8 Hz), 2.54e2.56 (m, 4H), 3.06 (t, 2H,

J ¼ 4.4 Hz), 3.43e3.45 (m, 4H), 6.60e6.61 (m, 1H), 7.07 (d, 1H,

J ¼ 2.0 Hz), 7.43 (t,1H, J ¼ 5.2 Hz), 7.55 (t,1H, J ¼ 5.2 Hz), 7.81 (s, 1H),

8.03e8.06 (m, 2H), 8.31e8.32 (m, 1H). MS (ESI) m/z: 439.2 ([MþH]

^þ).

6.2. Single crystal X-ray crystallographic study

6.3.2.3. Dopaminergic D_2Lreceptor [55]. Cell membrane homoge-

nates (16

m

g protein) were incubated for 60 min at 22 ^ꢀC with

The X-ray crystallographic analysis of 6 was carried out on a

colorless crystal, with dimensions 0.10 mm ꢂ 0.23 mm ꢂ 0.25 mm.

The crystal was grown from the slow evaporation of an acetone

solution of 6 at room temperature. A suitable crystal was selected

and mounted on a Bruker SMART APEX-II CCD diffractometer using

0.3 nM [³H]methyl-spiperone in the absence or presence of the test

compound in a buffer containing 50 mM Tris-HCl (pH 7.4), 120 mM

NaCl, 10 mM MgCl₂and 1 mM EDTA. Nonspeciﬁc binding was

determined in the presence of 10 mM butaclamol. Following incu-

bation, the samples were ﬁltered rapidly under vacuum through

glass ﬁber ﬁlters (GF/B, Packard) presoaked with 0.3% PEI. The

ﬁltrate was rinsed several times with ice-cold 50 mM Tris-HCl using

CuK_aradiation (

l

¼ 1.54178 Å). Data collection was carried out at

298 (2) K. The crystal structure solution was worked out by the full

X.-W. Chen et al. / European Journal of Medicinal Chemistry 123 (2016) 332e353

351

a 96-sample cell harvester (Uniﬁlter, Packard). The ﬁlters were

dried then counted for radioactivity in a scintillation counter

(Topcount, Packard) using a scintillation cocktail (Microscint 0,

Packard). The inhibition results were expressed as a percentage of

sample binding compared to the control radioligand speciﬁc

binding. The butaclamol was used as a standard reference com-

pound and was tested in each experiment at several concentrations

to obtain a competition curve from which its IC₅₀is calculated.

6.3.3. In vitro pharmacology for intrinsic activity assessment

6.3.3.1. Dopamine D₂, serotonin 5-HT_1Aand serotonin 5-HT_2Are-

ceptors [58,59]. CHO-K1 cells expressing three receptors (CHOK1/

D₂/G

seeded in a 384-well Poly-

sity of 20,000 cell per well in 20

the day of experiment, and maintained at 37 ^ꢀC/5% CO₂.

20 L of dye-loading solution (20 mM 2X (8 uM) Fluo-4 Direct™

a

15, CHO-K1/5-HT_1A/G

-Lysine protein coating plate at a den-

L of growth medium, 18 h prior to

a15, and CHO-K1/5-HT_2A/Ga15) were

D

m

Loading Buffer with 2.5 mM Probenecid) was added to the 384-well

cell culture plate using pipette. Then, the plate was placed into a

37 ^ꢀC/5% CO₂incubator for 50 min, followed by 10 min at room

temperature. Cell plate was removed from incubator and placed

into FLIPR. The compound plate and tip box were then placed into

FLIPR.

6.3.2.4. Dopaminergic D₃receptor [56]. Cell membrane homoge-

nates (8 m

g protein) were incubated for 60 min at 22 ^ꢀC with 0.3 nM

[³H] methyl-spiperone in the absence or presence of the test

compound in a buffer containing 50 mM Tris-HCl (pH 7.4), 120 mM

NaCl, 5 mM KCl, 5 mM MgCl₂and 5 mM EDTA. Nonspeciﬁc binding

was determined in the presence of 10

m

M (þ)butaclamol. Following

For the DRC plate, 10

384-well plate to the cell plate, followed by transfer of 10

m

L of assay buffer was transferred from

incubation, the samples were ﬁltered rapidly under vacuum

through glass ﬁber ﬁlters (GF/B, Packard) presoaked with 0.3% PEI.

The ﬁltrate was rinsed several times with ice-cold 50 mM Tris-HCl

using a 96-sample cell harvester (Uniﬁlter, Packard). The ﬁlters

were dried then counted for radioactivity in a scintillation counter

(Topcount, Packard) using a scintillation cocktail (Microscint 0,

Packard). The results were expressed as a percent inhibition of the

control radioligand speciﬁc binding. The standard reference com-

pound was (þ)butaclamol, which was tested in each experiment at

several concentrations to obtain a competition curve from which its

IC₅₀is calculated.

mL of the

compounds from the DRC plate to the cell plate. Then, the “Max-

Min” starting from Read 90 to Maximum allowed was calculated.

The EC₈₀values for each cell line were calculated using FLIPR.

Finally, 6 ꢂ EC₈₀concentrations of agonist reference compounds

were prepared.

For the compound plate (agonist test and antagonist test), 10

of references and compounds was transferred from the compound

plate to the cell plate, followed by transfer of 10

concentrations of agonist reference compounds to the cell plate. For

agonist test, the “Max-Min” starting from Read 1 to 90 was calcu-

lated. For antagonist test, the “Max-Min” starting from Read 90 to

Maximum allowed was calculated. Data were analyzed using Prism.

mL

mL of 6 ꢂ EC₈₀

6.3.2.5. Muscarinic M₁receptor [57]. Cell membrane homogenates

(45 m

g protein) are incubated for 60 min at 22 ^ꢀC with 2 nM [³H]

pirenzepine in the absence or presence of the test compound in a

buffer containing 50 mM Tris-HCl (pH 7.4), 120 mM NaCl, 5 mM KCl,

5 mM MgCl₂and 1 mM EDTA. Nonspeciﬁc binding is determined in

6.3.3.2. Dopamine D₃receptors. For detection of the agonism action

of the compounds at D₃receptor, the [³⁵S]GTP

gS binding assay was

performed at 30 ^ꢀC for 40 min in reaction buffer containing 50 mM

Tris, pH 7.4, 5 mM MgCl₂, 1 mM EDTA, 100 mM NaCl, and 1 mM DL-

the presence of 1 mM atropine. Following incubation, the samples

are ﬁltered rapidly under vacuum through glass ﬁber ﬁlters (GF/B,

Packard) presoaked with 0.3% PEI and rinsed several times with ice-

cold 50 mM Tris-HCl using a 96-sample cell harvester (Uniﬁlter,

Packard). The ﬁlters are dried then counted for radioactivity in a

scintillation counter (Topcount, Packard) using a scintillation

cocktail (Microscint 0, Packard). The results are expressed as a

percent inhibition of the control radioligand speciﬁc binding. The

standard reference compound is pirenzepine, which is tested in

each experiment at several concentrations to obtain a competition

curve from which its IC₅₀is calculated.

dithiothreitol (DTT). The assay mixture (200

m

L) contained 20

mg of

membrane protein, 0.1 nM [³⁵S]GTP

gS, and 40

mM guanosine

triphosphate (GDP) with various concentration of the compound.

The antagonism effects of the compounds were tested in the exis-

tence of 100

mM quinpirole. Nonspeciﬁc binding was measured in

the presence of 100

mM 50-guanylimidodiphosphate (Gpp(NH)p).

The reaction was terminated by addition of 3 mL of ice-cold

washing buffer (50 mM Tris, pH 7.4, 5 mM MgCl₂, 1 mM EDTA,

and 100 mM NaCl) and was rapidly ﬁltered with GF/C glass ﬁber

ﬁlters (Whatman) and rinsed three times. Filters were dried and

radioactivity was determined by liquid scintillation counting.

6.3.2.6. hERG afﬁnity evaluation. CHO-K1 (Chinese Hamster Ovary)

cells stably transfected with human hERG cDNA were used. The

cells were harvested by trypsinization and maintained in Serum

Free Medium at room temperature before recording. The test so-

lutions were prepared in the Extracellular Solution on the day of

patch clamp assay. The assay can tolerate up to 1% DMSO. After

whole cell conﬁguration was achieved, the cell was held at ꢁ80 mV.

A ꢁ50 mV pulse was delivered for 50 ms to measure the leaking

current, which was subtracted from the tail current on-line. Then

the cell was depolarized to þ20 mV for 5 s, followed by a 5 s pulse

to ꢁ50 mV to reveal the hERG tail current. This paradigm was

delivered once every 15 s to monitor the current amplitude. The

Extracellular Solution (control) was applied ﬁrst and the cell was

stabilized in the solution for 2 min. Then the test compound was

applied from low to high concentrations sequentially on the same

cell. The cells were incubated with each test concentration for

2 min. Cisapride was tested concurrently at multiple concentrations

to obtain an IC₅₀value. The percent inhibition of the hERG channel

is calculated by comparing the tail current amplitude before and

after application of the compound (the current difference is

normalized to control).

6.3.4. Behavioral tests

6.3.4.1. PCP-induced hyperlocomotion. Male SD rats (200e250 g,

8e10 rats in each group) were used. Animals were individually

placed into a Plexiglas open ﬁeld arena (40 ꢂ 40 ꢂ 45 cm, Jiliang Co.

Ltd., Shanghai, China) for 2 min 10 min after intraperitoneal in-

jection of the test compounds (0.1, 0.2 and 0.4 mg/kg) or cariprazine

(0.1, 0.2 and 0.4 mg/kg), animals were treated with PCP (5 mg/kg,

i.p.), and placed back into the experimental apparatus. Animals

were habituated for 10 min before the 1.5-h measurement period.

The results of the locomotor activity tests were expressed as the

mean SEM. The percent inhibition of activity was calculated for

each dose. Statistical evaluation was performed by two-way

ANOVA followed by the Tukey Test. þþþ, P < 0.001 versus

vehicle treated control group (Veh). *, **, and ***, P < 0.05, P < 0.01,

and P < 0.001 versus phencyclidine treated group (PCP).

6.3.4.2. Conditioned avoidance response (CAR) [60]. Male SD rats

(150e180 g, 6 rats in each group) were used. CAR behavior was

assessed using auto-mated shuttle-boxes placed in a sound-

attenuated chamber (73

ꢂ

43

ꢂ

37 cm) (LM100,

352

X.-W. Chen et al. / European Journal of Medicinal Chemistry 123 (2016) 332e353

HamiltoneKinder Scientiﬁc, USA). Daily sessions consisted of 30

trials with a 10-s continuous sound and light as conditioned

stimulus, paired with a 10-s electric foot shock (shock intensity:

0.8 mA) as an unconditioned stimulus. Rats who achieved greater

than 80% avoidance responses were chosen for drug treatments.

Test compounds (i.p. 1.0 and 2.0 mg/kg) and Cariprazine (i.p. 1.0 and

2.0 mg/kg) were administered 1 h before testing via single intra-

peritoneal injection. The test session consisted of 20 trials. Results

were expressed as percentage of avoidance. Data was expressed as

Mean SEM (n ¼ 6). ***, P < 0.001 versus vehicle treated control

group (Basal).

Pharmaceutical Industry, China), Dr. Yu Liu (Shanghai Institute of

Pharmaceutical Industry, China) and Dr. Jennifer Batara (Shanghai

Jiao Tong University, China) for helpful discussion. This work is also

sponsored by National Natural Science Foundation of China (Grant

No. 81302630), Key Technologies R&D Program of Shanghai

Municipal Science and Technology Commission (Grant No.

13431900201), Natural Science Foundation of Shanghai (Grant

No.14ZR1440300), and the National Science and Technology Major

Project (Grant No. 2012ZX09102101-014).

Appendix A. Supplementary data

6.3.4.3. Novel object recognition paradigm. Male SD rats

(160e180 g, N ¼ 50, 40 PCP- and 10 saline-treated) were used. Rats

were randomly assigned to receive either saline (once a day, i.p.) or

PCP (7.5 mg/kg once a day, i.p.) for 14 days. Subsequently, rats were

given a 6-day drug-free period prior to NOR testing. Rats were

habituated in groups to an empty test box for 5e10 min on day 1.

The test consisted of two 3-min trials separated by an inter-trial

interval (ITI) of 1 h. In the ﬁrst (training session) trial, animals

were placed in the test box and allowed to explore two identical

objects. In the second (retention session) trial, one of the objects

was replaced by a novel one. 9j 0.1, or 0.2 mg/kg (PO), cariprazine

0.1 mg/kg (PO), or saline was administered 60 min prior to testing.

The time spent in exploring any one of the two objects (training

session) or the novel one (retention session) which including the

animals licking, snifﬁng, or touching the object but not including

time spent leaning against, standing or sitting on the object was

recorded for used to score recognition memory. Data were analyzed

by post-hoc Student's t-test; *P < 0.05 for time spent exploring

familiar versus novel object.

Supplementary data related to this article can be found at http://

dx.doi.org/10.1016/j.ejmech.2016.07.038.

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Article Doi

Synthesis and pharmacological characterization of novel N-(trans-4-(2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)ethyl)cyclohexyl)amides as potential multireceptor atypical antipsychotics

DOI: 10.1016/j.ejmech.2016.07.038

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Full text of DOI:10.1016/j.ejmech.2016.07.038

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