Synthesis and pharmacological evaluation of piperidine (piperazine)-substituted benzoxazole derivatives as multi-target antipsychotics

Article abstract of DOI:10.1016/j.bmcl.2015.09.045

The present study describes the optimization of a series of novel benzoxazole-piperidine (piperazine) derivatives combining high dopamine D₂ and serotonin 5-HT_1A, 5-HT_2A receptor affinities. Of these derivatives, the pharm

Full text of DOI:10.1016/j.bmcl.2015.09.045

Accepted Manuscript
Synthesis and pharmacological evaluation of piperidine (piperazine)-substituted
benzoxazole derivatives as multi-target antipsychotics
Ling Huang, Wenjun Zhang, Xiaohua Zhang, Lei Yin, Bangyin Chen, Jinchun
Song
PII:
S0960-894X(15)30079-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.09.045
BMCL 23126
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
29 July 2015
5 September 2015
18 September 2015
Please cite this article as: Huang, L., Zhang, W., Zhang, X., Yin, L., Chen, B., Song, J., Synthesis and
pharmacological evaluation of piperidine (piperazine)-substituted benzoxazole derivatives as multi-target
antipsychotics, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.
2015.09.045
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Synthesis and pharmacological evaluation of piperidine (piperazine)-substituted
benzoxazole derivatives as multi-target antipsychotics
Ling Huang1,2, Wenjun Zhang1, Xiaohua Zhang1, Lei Yin1, Bangyin Chen2,
Jinchun Song3,*
1Wuhan Docan Pharmaceutical Co.,Ltd., Wuhan 430040, China
2Pharmacy School of Tongji Medical college, Huazhong University of Science and
Technology, Wuhan 430030, China
3Department of Pharmacy, Renmin Hospital of Wuhan University, Wuhan 430060,
China
* Author to whom correspondence should be addressed;
E-Mail: songjcwhu@163.com; Tel.: +86-27-88047471; Fax: +86-27-88047471.
Abstract
The present study describes the optimization of
a
series of novel
benzoxazole-piperidine (piperazine) derivatives combining high dopamine D2 and
serotonin 5-HT1A, 5-HT2A receptor affinities. Of these derivatives, the
pharmacological features of compound 29 exhibited high affinities for the DA D2,
5-HT1A and 5-HT2A receptors, but low affinities for the 5-HT2C and histamine H1
receptors and human ether-a-go-go-related gene (hERG) channels. Furthermore,
compound
29
reduced
apomorphine-induced
climbing
and
1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI)-induced head twitching
without observable catalepsy, even at the highest dose tested. Thus, compound 29 is a
promising candidate as a multi-target antipsychotic treatment.
Keywords: benzoxazole; piperazine; piperidine; antipsychotic
1

Schizophrenia is a psychiatric disorder that affects approximately 1% of the
general population. This disorder may be characterized by positive symptoms such as
delusions, hallucinations and agitation, negative symptoms such as anhedonia,
asociality and apathy, and/or cognitive disturbances.1 While the specific
pathophysiology of schizophrenia remains unclear, a large body of evidence suggests
that this disorder is associated with abnormalities in monoamine neurotransmission.
More specifically, pharmacological studies have indicated that alterations in
dopamine (DA) neurotransmission underlie the manifestation of schizophrenia; this is
known as the DA hypothesis of schizophrenia.2
The classic treatment options for schizophrenia consist of typical antipsychotics
such as chlorpromazine and haloperidol (Figure 1), that reduce DA
neurotransmission by blocking DA receptors, predominantly the D2 subtype.
Although these types of drugs effectively reduce positive symptoms, they do not treat
negative symptoms or ameliorate cognitive dysfunction and also commonly cause
detrimental side effects, including extrapyramidal syndrome (EPS).3 The introduction
of clozapine as the first atypical antipsychotic in 1989 represented a novel
development for the treatment of schizophrenia (Figure 1). It is now apparent that
atypical antipsychotics, including clozapine, olanzapine and risperidone, act as
antagonists at both the serotonin (5-HT) 5-HT2A and DA D2 receptors, and that the
antipsychotic efficacy of this class of drugs depends on the activity in these receptor
systems.4 Atypical antipsychotics are efficacious for the control of positive symptoms
of schizophrenia and, at the same time, are less likely to cause EPS.5,6 However, the
majority of patients treated with atypical antipsychotics report clinically significant
weight gain, hyperglycemia and dyslipidemia that are primarily caused by the high
affinity binding of these drugs to off-target receptors, such as the histamine H1 and
5-HT2C receptors.7-11 Thus, novel drugs that possess improved efficacy and favorable
side effect profiles are needed for the safe treatment of patients with schizophrenia.
Several recent studies have described the development of novel multi-target
atypical antipsychotic drugs, such as compounds 1-4 (Figure 2), that preferentially
bind to 5-HT and DA receptors.12-15 Additionally, benzimidazole-piperazinyl
2

derivative 5 has been shown to display high affinities for the D2 and D3 receptors (Ki
= 8.8 and 1.7 nM, respectively).16 This finding was from a study conducted by our
research group in which a new class of antipsychotic agents, compounds 10-33
(Figure 3), was developed by replacing benzimidazole with benzoxazole using the
principles of bioisosterism (Figure 3). The structure-activity relationships (SARs) of
these compounds at dopaminergic and serotonergic receptors are associated with
variations in the arylpiperazine (piperidine) moieties.17-19 Target compounds 10-33
have been subjected to preliminary pharmacological evaluations to determine their
affinities for the D2, 5-HT1A, 5-HT2A, H1, 5-HT2C, 5-HT7, and noradrenergic α1 and α2
receptors. Among the derivatives prepared, compound 29 showed high affinities for
the D2, 5-HT1A, 5-HT2A, 5-HT7, and α2 receptors, and low affinities for the 5-HT2C, H1
and α1 receptors and human ether-a-go-go-related gene (hERG) channels.
Behaviorally, compound 29 inhibited apomorphine-induced climbing and
1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane DOI-induced head twitching
without catalepsy, even at the highest dose tested.
The general strategy used to synthesize compounds 10-33 is summarized in
Scheme 1. Compound 8 was synthesized following the reaction of compound 6 with
aryl acid 7 and a catalytic amount of p-toluenesulfonic acid,17 while compound 9 was
created by
a
reaction between compound
8
and 1,3-dibromopropane
(1,4-dibromobutane) in acetone. Subsequently, compound 9 was reacted with an
arylpiperazine (piperidine) in acetonitrile in the presence of K2CO3 and a catalytic
amount of potassium iodide to produce compounds 10-33 with good yields (Tables
1-2).
The initial focus of the present study was to investigate the affinities of various
amine moieties of these derivative compounds for the D2, 5-HT1A and 5-HT2A
receptors (compounds 10-22; Table 1). When the amine moieties were
phenylpiperazine, 2,3-dichlorophenylpiperazine, 3-trifluoromethyl phenylpiperazine,
2,3-dimethylphenylpiperazine,
(pyridin-2-yl)piperazine,
compounds
and
((2,3-dihydrobenzo[b][1,4]dioxin-2-yl)methyl)piperazine,
10-15
demonstrated weak affinities for the D2, 5-HT1A and 5-HT2A receptors. Compounds
3

16 [2-(piperidin-4-yl)benzo[d]oxazole] and 17 [2-(piperidin-4-yl)benzo[d]thiazole]
showed moderate affinities for the 5-HT1A and 5-HT2A receptors, while the
2-methoxyphenylpiperazine and 2-(piperazin-1-yl)pyrimidine derivatives (compounds
18-19) displayed high affinities for the 5-HT1A and 5-HT2A receptors and moderate
affinities
(2,4-difluorophenyl)(piperidin-4-yl)methanone derivative 20 showed moderate
affinities for these three receptors. It should be noted that
for
the
D2
receptor.
Similarly,
(6-fluorobenzo[d]isoxazol-3-yl) piperidine derivative 21 also showed high affinities
for these three receptors (D2, Ki = 22 nM; 5-HT1A, Ki = 83 nM; 5-HT2A, Ki = 8.3 nM,
respectively), but the substitution of (6-fluorobenzo[d]isoxazol-3-yl) piperidine with a
(benzo[d]isothiazol-3-yl)piperazine group (compound 22) reduced all of these
affinities. These results suggest that the (6-fluorobenzo[d]isoxazol-3-yl) piperidine
group has greater affinities for the D2, 5-HT1A and 5-HT2A receptors than the other
amine moieties.
In order to evaluate the SARs of these compounds, further studies were
conducted by introducing either methyl (CH3) or chloro groups on the R1 of the
phenyl ring. Compound 23 with CH3 showed good affinity for the 5-HT1A receptor
but weak affinities for the D2 and 5-HT2A receptors, while chlorinated compound 24
had moderate affinities for the D2 and 5-HT2A receptors and a low affinity for the
5-HT1A receptor. The introduction of a CH3 group (compound 25) or methoxy (OCH3)
group (compound 26) on the R2 ofthe phenyl ring resulted in a dramatic decrease in
the affinities for all three receptors. Substituents with fluorine (F; compound 27) on
the R2 of the phenyl ring resulted in moderate affinities for the three receptors, while
a comparison of the 4-position (compound 21) and 3-position (compound 28)
formations of the R2 substituent resulted in a dramatic decrease in affinities for the
three receptors. It should be noted that the introduction of a methylene (CH2) group to
the phenyl ring of benzoxazole in compound 29 resulted in high affinities for the three
receptor types (D2, Ki = 10 nM; 5-HT1A, Ki = 21 nM; 5-HT2A, Ki = 2.7 nM,
respectively).
The present study also determined the effects of the length of the linker between
4

the phenyl ring and piperidine ring. Chain lengths of four carbon atoms (compound 30)
resulted in significant reductions in D2, 5-HT1A and 5-HT2A receptor binding. Thus, in
conjunction with the data presented above, the length of the alkyl chain appears to
have a direct impact on the affinity for these three receptors. When benzoxazole was
replaced with a benzothiazole group (compound 31), there was a dramatic decrease in
the affinities for the D2, 5-HT1A and 5-HT2A receptors. This study also investigated
the effects of the length of the linker with the phenyl ring of benzoxazole. Chain
lengths of one (compound 29) or two (compound 32) carbon atoms resulted in
reduced D2, 5-HT1A and 5-HT2A receptor binding (Table 2), indicating that the
binding affinities for the D2, 5-HT1A and 5-HT2A receptors are influenced by chain
length. Finally, the introduction of CH3 on the CH2 group (compound 33) resulted in
decreased affinities for all three receptors relative to compound 29.
In line with approaches that aim to identify drugs that target multiple receptor
types for the development of new antipsychotic agents, compounds 19, 21, 29, 32, and
33 were selected for further evaluation due to their high affinities for the D2, 5-HT1A
and 5-HT2A receptors. Additionally, these compounds were screened to assess their
binding affinities at the α2 and 5-HT7 receptors to determine their capacity to improve
cognitive function, but they showed only moderate binding affinities at both sites. The
5-HT7 receptor is abundant in the hippocampus, which is involved in learning and
memory,18,19 and it has been suggested that the α2 receptor is also potentially relevant
to cognitive function.20,21 Compound 29 exhibited a higher affinity for the α2 receptor
(Ki = 16 nM) than risperidone (Ki = 28 nM) and clozapine (Ki = 51 nM), as well as a
higher affinity (Ki = 18 nM) for the 5-HT7 receptor compared to clozapine (Ki = 62
nM; Table 3). These findings suggest that compound 29 has the potential to enhance
clinical efficacy.
Treatment of schizophrenia with atypical antipsychotic drugs is associated with
weight gain, and the H1 and 5-HT2C receptors have been implicated in the
development of this adverse side effect. Compound 29 had a much lower affinity
(Ki > 3000 nM) for the H1 receptor than risperidone (Ki = 38 nM) and clozapine (Ki =
12 nM), as well as a lower affinity for the 5-HT2C receptor (Ki > 1500 nM) relative to
5

risperidone (Ki = 30 nM) and clozapine (Ki = 41 nM; Table 3). Taken together, these
results indicate that compound 29 exhibits a low potential to produce
treatment-associated weight gain.
Blockade of the α1 receptor resulted in orthostatic hypotension, which is an
unwanted side effect associated with many antipsychotic agents.22 Compound 29
showed a lower affinity (Ki > 3000 nM) for the α1 receptor than risperidone (Ki = 11
nM) and clozapine (Ki = 38 nM; Table 3), indicating that compound 29 may not
cause orthostatic hypotension. Blockade of the hERG channel is an important
predictor of cardiac toxicity, and assessment of this capacity is an important step in
the drug discovery trajectory in the pharmaceutical industry. In the present study, the
patch-clamp technique was used in an in vitro assay to determine hERG inhibition
with a half maximal inhibitory concentration (IC50) cutoff value of > 3 μM.
Additionally, acute toxicities of the compounds were evaluated in terms of the lethal
dosage for 50% of the population (LD50; Table 4). Compounds 21 and 29 exhibited
good profiles, even at the highest dose tested (LD50 > 2,000 mg/kg)
The apomorphine-induced climbing model is based on the creation of a
hyperdopaminergic state by the administration of apomorphine,23 and has been
classically linked to motor agitation, a positive symptom of schizophrenia. Compound
29 (1 mg/kg) resulted in a significant reduction in apomorphine-induced climbing and
reaching in mice (p < 0.05; Figure 4). Furthermore, the effective dose (ED50) for
compound 29 to reduce apomorphine-induced climbing was 0.86 mg/kg, while the
ED50 values for risperidone and clozapine are 0.034 and 3.51 mg/kg, respectively
(Table 5). These findings indicate that compound 29 was slightly more potent at
blocking D2 receptors in vivo than clozapine, which is consistent with their estimated
Ki values at the D2 receptor. The present study also assessed the in vivo efficacies of
compound 29 and other selected analogs to antagonize 5-HT2A receptors using a
DOI-induced head twitch model in mice. DOI, a non-subtype-selective 5-HT2 agonist,
induces a stereotypic head twitch that is blocked by selective 5-HT2A receptor
antagonists.24 Compound 29 attenuated DOI-elicited head twitching in a
dose-dependent manner with significant effects at each dose; its ED50 value was 0.71
6

mg/kg (Figure 5 and Table 5).
Catalepsy is often used to predict the incidence of extrapyramidal motor disorders.
In order to evaluate their liabilities to produce striatal-mediated side effects and
cataleptogenic symptoms, selected compounds were tested in mice using the vertical
grid test and the elevated bar test.24 In these models, compound 29 exhibited a low
potential for the induction of catalepsy (ED50: 70.5 mg/kg, Table 5) and yielded a
greater than 80-fold therapeutic index relative to the apomorphine or DOI models in
terms of its inhibitory potency. Thus, in contrast to risperidone and clozapine, the
absence of catalepsy following the administration of compound 29 may predict a low
liability for EPS in clinical settings.
Additionally, the present study included a locomotor activity test to identify false
positive effects and to check for the possible occurrence of drug-induced changes in
locomotor activity that may have contributed to altered behavior in the apomorphine
and DOI models. Previous studies have shown that clonazepam and haloperidol can
cause sedation,25,26 and thus, these drugs were used as reference compounds.
Pretreatment with 1, 3 or 10 mg/kg compound 29 did not significantly reduce
spontaneous exploratory locomotor activity (Table 6), while pretreatment with
clonazepam and haloperidol did.
Thus, after oral administration, the effects of compound 29 in these animal
models may predict its antipsychotic effects in clinical settings due to the
compound-related reductions in apomorphine-induced climbing and DOI-induced
head twitching, as well as its marginal effects on spontaneous exploratory locomotor
activity. Furthermore, compound 29 also exhibited a low propensity to induce
unwanted extrapyramidal motor disturbances at therapeutically useful doses.
A detailed SAR analysis of the above Structures A and B revealed that several
factors influence the binding affinities of these compounds for the D2, 5-HT1A and
7

5-HT2A receptors. First, the presence of a 6-fluorobenzo[d]isoxazol-3-yl)-piperidine
moiety resulted in higher affinities for these three receptors than the other amine
moieties. Second, substitutions on R1, R2 and R3 appeared to more efficacious, and
hydrogen was the favored substituent. Third, the importance of the oxygen atom (X)
was preferred over the sulphur atom and, finally, a chain length (n1and n2) of one was
preferred over a length of two as the alkyl linker.
Findings from this SAR analysis revealed that compound 29, which has unique
affinities for the DA, 5-HT and adrenergic receptors, is a potential candidate for a
multi-receptor antipsychotic drug. The binding profile of compound 29 showed high
affinities for the D2, 5-HT1A, 5-HT2A, 5-HT7, and α2 receptors, and low affinities for the
5-HT2C, H1 and α1 receptors and hERG channels. The animal model studies revealed
that compound 29 significantly inhibited apomorphine-induced climbing behavior and
DOI-induced head twitching without catalepsy, even at the highest dose tested.
Moreover, following oral administration, compound 29 reversed behavior in the
apomorphine and DOI models with only minor effects on spontaneous locomotor
activity and no propensity to induce catalepsy. Additionally, compound 29 had a
higher threshold for catalepsy induction than two currently marketed atypical
antipsychotics, risperidone and clozapine. Thus, compound 29 may facilitate the
development of a novel class of drugs for the treatment of schizophrenia.
Conflict of Interest
There is no conflict of interests among the authors.
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11

Structures of a typical (haloperidol) and atypical antipsychotics
12

Structures of novel antipsychotic drugs 1-4
13

Design of benzoxazole derivatives
14

Figure 4. Effect of compound 29 administered po on APO (apomorphine)-induced
climbing in mice (10/group). Results are expressed as means ± SEM of score.
Statistical evaluation was performed by two-way ANOVA followed by Tukey test for
multiple comparisons: # p<0.05 versus vehicle treatment; ** p<0.01, * p<0.05 versus
apomorphine treatment.
15

Figure 5. Effect of compound 29 administered po on DOI-induced head twitch in
mice (10/group). Results are expressed as means ± SEM of distance traveled.
Statistical evaluation was performed by two-way ANOVA followed by Tukey test for
multiple comparisons. ## p<0.01 vs vehicle treatment; ** p<0.01, * p<0.05 vs DOI
treatment.
16

Reagents and conditions: (i) p-TsOH·H2O, xylenes, refluxing; (ii) Br(CH2)3 or 4Br,
K2CO3, Acetone, reflux; (iii) CH3CN, K2CO3, KI, reflux.
17

Table 1. Binding Affinities for D2, 5-HT1A and 5-HT2A receptors of compounds
10-22a
Receptor affinity Ki ± SEM (nM)
Compound
HNRR
D2
5-HT1A
>10000
5-HT2A
10
11
12
>10000
4681±569
>10000
>10000
>10000
>10000
2889±312
4479±509
13
>10000
4689.1±513.2
2135 ±299
14
15
16
17
>10000
>10000
>10000
>10000
1988.2±243.9
>10000
2341±401
1423±198
568.1 ±69.2
146.2 ±18.3
243±37
281±34
18
19
467±38
198±20
46.8 ±6.9
56.2 ±7.3
29.3±4.7
38.5±5.9
20
408±67
114 ±18
199±25
21
22
22.1±2.9
879.1±91
83.5 ±9.2
8.3± 0.9
16.9±2.2
969.7 ±110
-
5
16.3±3.5
155±17
258±36
161±18
109±17
clozapine
-
14.5±1.7
18

risperidone
-
3.6±0.3
211±24
0.31±0.03
a Ki values are taken from three experiments, expressed as means ±SEM.
b The Ki values were not calculated because the inhibition percentages at 10 μM were too low.
19

Table 2. Binding Affinities for D2, 5-HT1A and 5-HT2A receptors of compounds
23-33a
Receptor affinity Ki ± SEM (nM)
Compound
R
D2
5-HT1A
5-HT2A
23
24
>10000 b
76.3±2.9
>10000
660±72
>10000 b
135±14
25
26
27
1589±171
2142±248
41.1 ±6.8
1192 ±112
143±15
1556±229
232±39
72.2±8.9
23.7±3.4
28
>10000 b
>10000 b
>10000 b
29
30
31
10.6±1.6
2198±331
>10000 b
21.3 ±2.6
996±112
1756±182
2.7± 0.6
729±83
1268±132
32
33
53.1±6.8
60.3±5.7
31.1 ±4.2
89.6±9.1
38.1± 4.9
30.5± 4.2
a Ki values are taken from three experiments, expressed as means ±SEM.
b The Ki values were not calculated because the inhibition percentages at 10 μM were too low.
20

Table 3. Binding affinities for the 5-HT7 , H1, 5-HT2C, alpha 1, alpha 2, and M1
receptors (Ki nM ± SEM) of compounds 21, 29 and reference antipsychotics a.
Receptor affinity Ki ± SEM (nM)a
Compound
5-HT7
3.2±0.5
62.6±7.1
765±89
18.9±2.5
alpha 2
28.2±4.1
51.9±6.3
153±20
H1
5-HT2C
30.1±3.9
41.9±5.1
186±19
alpha 1
11.9±1.8
38.8±4.4
95±10
ripersidone
clozapine
21
38.3±4.8
12.2±1.3
54.2±6.2
3153±388
29
16.3±2.1
1987±212
3276±418
a Ki values are taken from three experiments, expressed as means ±SEM.
21

Table 4. hERG and acute toxicity LD50 of the compounds19, 21, 29, 32 and 33.
Compound hERG IC50 (μM)
LD50 (mg/kg)
20
21
29
32
33
0.85
3.76
5.98
0.63
1.01
369.1 (352.1 – 582.9)a
> 2000
> 2000
398.2 (281.9 – 478.0) a
614.4 (572.8 – 891.7) a
a 95% Confidence limits given in parentheses.
22

Table 5. In vivo pharmacological profile of compound 29. Inhibition of different
behavioral responses after oral administration of the test and reference compounds in
mice.
DOIb ED50
Compound
APOa ED50
CATc ED50
CAT/APO
CAT/DOI
risperidone
0.034
(0.026-0.046) d
3.51
0.045
(0.027-0.068)d
4.92
1.3
38.2
28.9
(1.07-1.84)d
clozapine
50.5
14.4
10.3
(0.668-5.44) d
(2.85-7.54) d
(38.68-73.67) d
70.5
29
0.86
(0.67-1.12) d
0.71
(0.21-0.85) d
81.9
99.3
(55.87-94.61) d
aAPO: apomorphine-induced climbing (mg/kg, po); DOI: DOI-induced head twitch
b
(mg/kg, po); c CAT: catalepsy (mg/kg, po); d 95% Confidence limits given in parentheses.
23

Table 6. Effects of the compounds 24, haloperidol and clonazepam on the
spontaneous locomotor activity in mice (mean±SD, n=8).
Compound
Dose (mg/kg)
Total distance (mm)
13881.4±8595.8
13188.4±8224.5
4784.1±6319.7*
655.7±662.3**
control
-
1
3
10
-
0.1
0.3
1
-
1
3
10
clonazepam
control
13415.6±9555.8
8980.9±6808.4
3580.6±1344.9*
744.5±712.9**
13263.8±8034.2
13046.7±6831.7
10447.5±4454.9
9492.4±4018.4
haloperidol
control
29
**p<0.01, *p<0.05 vs control
24

Graphical abstract
25