Discovery and SAR of N-(1-((substituted piperidin-4-yl)methyl)-3-methoxypiperidin-4-yl)-2-methoxybenzamide derivatives: 5-Hydroxytryptamine receptor 4 agonist as a potent prokinetic agent

Article abstract of DOI:10.1016/j.ejmech.2015.12.006

A series of novel benzamide derivatives, altering the 4-fluorophenylalkyl moiety in cisapride, were synthesized as 5-HT₄ receptor agonists, and SAR of these analogs was examined on in vitro and in vivo prokinetic activities. These compounds were synthesized for high 5-HT₄ receptor binding affinities and low hERG affinities. Several types of analogs were obtained and screened for 5-HT₄ binding, hERG blocking, agonism, and gastric emptying assessment. Among the analogues, compound 23g showed promising results compared with the other analogs with respect to gastric emptying rates in rats. Therefore, we suggest that it may be a clinical candidate for the development of a potent prokinetic agent to treat GI disorders.

Full text of DOI:10.1016/j.ejmech.2015.12.006

Accepted Manuscript
Discovery and SAR of N-(1-((substituted piperidin-4-yl)methyl)-3-methoxypiperidin-4-
yl)-2-methoxybenzamide derivatives; 5-hydroxytryptamine receptor 4 agonist as a
potent prokinetic agent
Jung Sang Park, Weonbin Im, Sunghak Choi, Sook Jin Park, Jun Min Jung, Ki seon
Baek, Han Pyo Son, Satyasheel Shrama, In Su Kim, Young Hoon Jung
PII:
S0223-5234(15)30393-7
10.1016/j.ejmech.2015.12.006
EJMECH 8246
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 September 2015
Revised Date: 17 September 2015
Accepted Date: 2 December 2015
Please cite this article as: J.S. Park, W. Im, S. Choi, S.J. Park, J.M. Jung, K.s. Baek, H.P. Son,
S. Shrama, I.S. Kim, Y.H. Jung, Discovery and SAR of N-(1-((substituted piperidin-4-yl)methyl)-3-
methoxypiperidin-4-yl)-2-methoxybenzamide derivatives; 5-hydroxytryptamine receptor 4 agonist
as a potent prokinetic agent, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2015.12.006.
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ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
Discovery and SAR of N-(1-((Substituted piperidin-4-yl)methyl)-3-methoxypiperidin-4-
yl)-2-methoxybenzamide Derivatives; 5-Hydroxytryptamine receptor 4 Agonist as a
Potent Prokinetic Agent
Jung Sang Parka,b,*, Weonbin Ima, Sunghak Choia, Sook Jin Parkb, Jun Min Jungb, Ki seon
Baekb, Han Pyo Sonb, Satyasheel Shramab, In Su Kimb, Young Hoon Jungb,*
a Dong-A ST Research Institute, Yongin 446-905, Republic of Korea
b School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea
*Corresponding Authors:
Tel: +82 31 260-5850, fax: +82 31 282-9604, email: pjs2002@donga.co.kr (J.S.P.)
Tel: +82 31 290-7711, fax: +82 31 290-7773, email: yhjung@skku.edu (Y.H.J.)

ACCEPTED MANUSCRIPT
Discovery and SAR of N-(1-((Substituted piperidin-4-yl)methyl)-3-methoxypiperidin-4-
yl)-2-methoxybenzamide Derivatives; 5-Hydroxytryptamine receptor 4 Agonist as a
Potent Prokinetic Agent
Jung Sang Parka,b,*, Weonbin Ima, Sunghak Choia, Sook Jin Parkb, Jun Min Jungb, Ki seon
Baekb, Han Pyo Sonb, Satyasheel Shramab, In Su Kimb, Young Hoon Jungb,*
a Dong-A ST Research Institute, Yongin 446-905, Republic of Korea
b School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea
Abstract
A series of novel benzamide derivatives, altering the 4-fluorophenylalkyl moiety in cisapride, were synthesized
as 5-HT4 receptor agonists, and SAR of these analogs was examined on in vitro and in vivo prokinetic activities.
These compounds were synthesized for high 5-HT4 receptor binding affinities and low hERG affinities. Several
types of analogs were obtained and screened for 5-HT4 binding, hERG blocking, agonism, and gastric emptying
assessment. Among the analogues, compound 23g showed promising results compared with the other analogs
with respect to gastric emptying rates in rats. Therefore, we suggest that it may be a clinical candidate for the
development of a potent prokinetic agent to treat GI disorders.
Keywords: 5-HT4, hERG inhibition, gastric emptying rate, prokinetic, GI disorder
1. Introduction
Serotonin (5-hyroxytryptamine, 5-HT) functions as both a hormone and a neurotransmitter, interacting
with different receptors [1-4]. There exist several members of the serotonin receptor family, which all play
important roles in various disorders. In particular, the role and distribution of 5-HT4 receptors within the
gastrointestinal (GI) tract have been well-established in different species including humans, guinea pigs [5],
mice [6], and rats [7]. Due to its potential roles in central and peripheral disorders, 5-HT4 has been an attractive
target for the treatment of GI disorders such as irritable bowel syndrome, chronic constipation, gastroparesis,
dyspepsia, and gastroesophageal reflux disease [8].
Dyspepsia is defined as pain or discomfort in the upper abdomen [9]. In particular, functional
dyspepsia is associated with abnormalities in gastric or duodenal physiology, including abnormal sensitivity to
gastric acid, delayed gastric emptying, impaired gastric accommodation, and visceral hypersensitivity [9-11].
Prokinetic agents such as 5-HT4 agonists are potential therapeutic agents to treat GI tract disorders including

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functional dyspepsia (Figure 1)[12, 13]. Of the representative prokinetic agents, cisapride (PropulsidTM) was
launched in 1988 and has shown potent effects in patients with GI disorders [14-16]. However, cisapride was
withdrawn from the US market in 2000 as a result of adverse cardiovascular effects associated with QT
prolongation due to the fact that it is a potent blocker of the hERG (human ether-a-go-go related gene) channel
[17, 18].
Figure 1. Examples of known prokinetic agents (5-HT4 agonists).
cis-4-Amino-5-chloro-2methoxy-N-(3-methoxypiperidin-4-yl)benzamide (cis-norcisapride) is
a
metabolite of cisapride [19], and a pharmacophore that is a potential 5-HT4 receptor agonist and a potential 5-
HT3 receptor antagonist [20]. Moreover, cis-norcisapride does not bind the hERG channel (IC50 > 10 µM). The
portion of cisapride that binds to the 5-HT4 receptor can be divided into parts A and B (Figure 2) [21]. Part A
contains cis-norcisapride and part B contains linkers with functional groups. We expected, as shown in figure 2
(c), that modifying part B into adequate structures may result in an enhanced binding affinity to the 5-HT4
receptor and a reduced inhibition of the hERG potassium ion channel.

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Figure 2. (a) The pharmacophore parts of cisapride, known as prokinetics, bind to the 5-HT4 receptor; (b) part A, cis-norcisapride is a
known 5-HT4 receptor agonist and a partial 5-HT3 receptor antagonist; (c) part B, three main structures of novel benzamide derivatives.
2. Results and discussion
2.1. Chemistry
Our approach was to examine various linkers and functional groups in cisapride part B that show
effective binding affinity and agonism to the 5-HT4 receptor, but are not hERG blockers. Herein we describe the
synthetic strategy and structure-activity relationships (SAR) of diverse benzamide derivatives as 5-HT4 receptor
agonists.
Scheme 1. Reagents and conditions: (a) PhI(OAc)2, KOH, MeOH, 0°C for 30 min, then r.t. 3 h, 71%; (b) NaH, MeI, DMF, 0°C for 30 min,
then r.t. 4 h, 75%; (c) 5% H2SO4, r.t., 78%; (d) NaBH3CN, NH4OAc, MeOH, 80°C, 3 h, 35%; (e) TEA, EDC, HOBt, DMF, r.t., 5 h, 83%; (f)
KOH, 2-propanol, 0°C for 15 min, then reflux for 6 h, 72%. Compound 1 and 6 are commercially available.
Initially, we synthesized compound 8, well-known as cis-norcisapride by following the route as
depicted in Scheme 1 [22, 23]. Further, we modified part B of the pharmacophore by incorporating three
different types of moieties such as alkyl heteroaryl, alkyl amide, and alkyl piperidinyl groups into compound 8
as shown in Scheme 2. Compounds 13a-e containing alkyl heteroaryl moieties were synthesized as shown in
Scheme 2A. 1-Bromo-3-chloropropane underwent SN2 reaction with heteroaryl compounds like pyrrole,
imidazole, triazole, and tetrazole to provide compounds 10a-e, which were coupled with compound 8 to give the
desired products 13a–e. Subsequently, we synthesized the alkyl amide derivatives 17a–d using the strategy
shown in Scheme 2B. Acryloyl chloride in the presence of triethylamine reacted with cyclic amines to provide
the corresponding amides 16a–d, which upon coupling with compound 8 in ethanol at room temperature
furnished the desired compounds 17a–d.
In addition, compounds 23a–o containing alkyl piperidinyl groups were synthesized using the
synthetic routes depicted in Scheme 2C. Substituted piperidinyl alcohols 18a–c were reacted with different acyl
chlorides to afford the corresponding amides 19a–o. Subsequently, the free hydroxyl group of the amides was
substituted with either mesylates 20a–i or halides 21j–o. Compounds 20a–i, 21j–k, and 21n–o were coupled
with compound 8 to afford the products 23a–i, 23j–k, and 23n–o, respectively. Additionally, compounds 21l–m
were converted to the sulfur analogues by Lawesson’s reagent to furnish the thioamides 22l–m, which on
reaction with compound 8 provide the thio-substituted compounds 23l–m. All compounds were purified by

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column chromatography and characterized by 1H and 13C NMR. Detailed experimental procedures and
characterization are provided in the experimental section.
Scheme 2 Reagents and conditions: (a) NaH, DMF, 0°C for 20 min, then r.t. 8 h; (b) K2CO3, KI, DMF, 90°C, 12 h; (c) Et3N, CH2Cl2, from
.
0°C to r.t., 2 h; (d) EtOH, r.t.; (e) DIPEA, MeOH, CH2Cl2, from 0°C to r.t., 3 h; (f) MsCl, Et3N, CH2Cl2, from 0°C to r.t.; (g) PPh3, NBS,
DCM, from 0°C to r.t., 12 h; (h) Lawesson’s reagent, THF, from 0°C to reflux, 20 h; (i) K2CO3, KI, DMF, 80 100°C.
–
Further, we also synthesized various salts of benzamide derivative 23g. As depicted in Scheme 3,
compound 23g was reacted with several acids like hydrochloric acid, hydrobromic acid, citric acid,
methanesulfonic acid, maleic acid, and L-tartaric acid in appropriate solvent to furnish the corresponding salts
24–29. These acid salts showed good solubility in distilled water. In addition, single enantiomers of 23g were
prepared using the diastereomeric salt resolution method as described in Scheme 4. cis
separated into the (+) form and (–) form using suitable optically active acids ((+) DBTA and (–)
and then SN2 reactions between 20g and a single enantiomer of cis-norcisapride were performed to obtain
compound 11 and 12, i.e. the (+) form and (–) form of 23g, respectively.
-Norcisapride (8) was
-
-
-
-DBTA) [24],
-
-

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Scheme 3. Diverse salt forms of compound 23g: (a) 2-Propanol, from r.t. to 0°C for 2 h; (b) 2-Propanol, from r.t. to 0°C for 2 h; (c)
Acetone, reflux for 1 h, cooled to 0°C for 12 h; (d) 2-Propanol, from r.t. to 0°C for 2 h; (e) 2-Propanol, from r.t. to 0°C for 3 h; (f) Acetone,
r.t. for 2 h.
Scheme 4. Preparation of each single enantiomer of compound 23g by diastereomeric salt resolution.

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2.2. Pharmacological evaluation
2.2.1. Structure-activity relationship (in vitro and in vivo efficacy, and hERG binding assay)
Structure-activity relationship (SAR) data based on 5-HT4 binding affinity (IC50), hERG binding
affinity (IC50), and functional potency (pEC50) for the compounds (13a–23d) are shown in Table 1. Compounds
containing alkyl heteroaryl functional groups (13a–e) showed relatively low binding affinities for the 5-HT4
receptor. Moreover, compounds 13b, 13c, and 13d weakly inhibited hERG. Compounds containing alkyl amide
functional groups (17a, 17b, and 17d) showed lower binding affinities for the 5-HT4 receptor than that of
cisapride. However, compound 17c, containing a benzyl group along with an alkylamide functional group,
showed dramatically higher binding affinity to the 5-HT4 receptor than that of cisapride, however, it also
strongly inhibited hERG.
Table 1. SAR for 13a–23d, based on 5-HT4 binding affinity (IC50), hERG binding (IC50), and functional potency (pEC50).
Functional potency
Compound
cisapride
L
5-HT4 (IC50, µM)
hERG (IC50, µM)
(pEC50, µM)
0.483
<1
6.99
13a
13b
13c
13d
13e
5.759
0.648
1.165
1.271
1.121
>10
6.835
7.261
7.711
>10
-
6.17
6.60
6.15
-
17a
17b
17c
0.920
0.560
0.066
>10
>10
-
5.98
5.22
3.701
17d
23a
0.954
0.329
>10
>10
-
6.42

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23b
0.493
>10
5.81
23c
23d
0.805
0.190
>10
10
-
-
In accordance with the SAR data for compounds 23a–d, consisting of amide derivatives of the alkyl
piperidinyl group in part B, they have higher binding affinities for the 5-HT4 receptor than that of cisapride and
lower affinities for hERG. The 4-piperidyl analog of compound 23a displayed a higher binding affinity and
functional potency value (pEC50) for the 5-HT4 receptor than that of 3-piperidyl derivative 23b. Furthermore, a
comparison between 23a and 23c illustrated that the –CH2– linker was better for 5-HT4 binding than –CH2CH2–
in these series. Interestingly, introducing carbamate protection to the piperidinyl nitrogen (23d) improved 5-HT4
receptor binding affinity and did not affect hERG binding. Thus, the initial SAR results for compounds 13a–23d
revealed that benzamide derivatives, which are composed of cis-norcisapride (part A) and piperidine analogs,
have potential as long as the nitrogen does not act as a tertiary amine (part B).
Table 2. Pharmacological evaluation of N-substituted piperidinyl methoxypiperidine benzamide derivatives.
Gastric empting rate %
Compound
hERG (IC50, µM)
5-HT4 IC50 (µM)
P
(rat iv 5mg/kg, normal model)
cisapride
23e
0.483
0.592
<1
57.1
39.5
-
>10
23a
23f
0.329
0.178
>10
>10
57.8
48.6
23g
23h
0.134
0.118
>10
>10
59.1
56.4
23i
23d
23j
23k
0.078
0.190
0.377
0.134
10
>10
4.580
<1
54.3
56.2
55.9
57.7

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23l
0.593
0.155
2.862
1.132
61.0
67.3
23m
23n
23o
0.052
0.073
>10
46.0
59.9
7.551
Next, various N-substituted analogs of compound 23a were tested for hERG and 5-HT4 receptor
binding affinities as well as for gastric emptying rate (Table 2). Biological assays of compounds 23a and 23e–i
indicated that the binding affinities for 5-HT4 receptor increased gradually by lengthening or branching of the
alkyl chain of the amide group. However, the gastric emptying results were not always relevant to homologation.
The gastric emptying rates of compounds 23a and 23g were comparatively equal or superior to that of cisapride,
whereas that of compound 23h was slightly less than that of cisapride. However, compounds 23g and 23h were
found to have good binding affinities for the 5-HT4 receptor and negligible inhibition of hERG (IC50 > 10 µM)
relative to cisapride.
Compounds containing piperidyl carboxylates (23d, 23j, and 23k) had similar binding affinities to those of the
compounds 23a and 23e–i, however, their hERG binding affinities were relatively higher and gastric emptying
rates were not improved from that of compound 23g. Pharmacological evaluation results of thio-derivatives 23l
and 23m were inferior to compound 23g in terms of hERG binding. Although 23n of the piperidyl carboxamide
derivatives showed the highest binding affinity (0.052 µM of 50% inhibition concentration) of all the
compounds, it showed a very low gastric emptying rate (46%). On the other hand, 23o showed a high binding
affinity (0.073 µM of 50% inhibition concentration) for the 5-HT4 receptor, however, also slightly inhibited
hERG (7.551 µM of 50% inhibition concentration).
Thus, after screening all compounds for 5-HT4 and hERG binding affinities, agonism, and gastric
emptying rates in rats, compound 23g was found to be the best pre-candidate for a novel prokinetic agent.
Figure 3 shows that a 0.1 mg/kg oral dose of compound 23g minimally enhanced gastric emptying,
whereas that of cisapride was 0.5 mg/kg. The mean gastric emptying rates were 46.2% and 46.9% in the rat
model at 0.1 and 0.5 mg/kg doses of compound 23g, respectively. However, the gastric emptying rate was
44.5% in the same model at a 0.5 mg/kg dose of cisapride [21]. Consequently, compound 23g produced more
potent gastric emptying stimulation in rats than that of cisapride.

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* P < 0.05
Figure 3. Gastric emptying rates of a semi-solid meal in rats.
In addition to the study as described above, the chiral (+)-form of 23g, 11 was more potent than the
racemic mixture, in binding affinity to the 5-HT4 receptor and in vivo gastric emptying rate as shown in Table 3.
The melting point and solubility in aqueous conditions of the different salts 24–29 are shown in Table 4. Some
salts of 23g, hydrochlorate 24, methansulfonate 27, and maleate 28, synthesized as shown in scheme 3, were
found to be stable and soluble in aqueous conditions.
Table 3. 5-HT4 binding affinities and GE (%) rates of 23g and each single enantiomer of compound 23g.
Gastric empting rate %
Compound
5-HT4 IC50 (µM)
(rat iv 5 mg/kg, normal model)
23g
0.134
0.068
59.1
11 ((+)-isomer of 23g)
.61.7
12 ((–
)-isomer of 23g)
>10
56.5
Table 4. Melting point and solubility of several salts of 23g.
24
25
26
27
28
29
Melting point (
̊
234.9
208.4
120.1
273
210.2
124.7
Solubility in D.W.
(mg/ml)
> 15
< 5
> 30
> 10
> 10
> 30
2.2.2. Pharmacokinetic properties of 23g and 23h
Compounds 23g and 23h are illustrated in Table 5 for in vivo efficacy, functional potency, and rat
pharmacokinetic (PK) tests. As shown in Table 5, compound 23g displayed gastric emptying results superior to

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those of compound 23h and cisapride in the rat model (normal model: 59.1% compared with 56.4% and 57.1%;
cisplatin model: 58.6% compared with 56.6% and 57.2%). Furthermore, with respect to pharmacokinetic
properties, 23g showed longer T1/2 and higher Cmax and AUCinf values than 23h in a rat PK study.
Table 5. Functional potency, hERG binding, in vivo efficacy and oral PK data of 23g and 23h in rats.
In vivo efficacy
Rat PK (oral, 5 mg/kg)
Functional
potency
pEC50
hERG
(IC50,
µM)
(rat gastric emptying rate (%))
Item / parameters
Cmax
AUCinf
normal
cisplatin
T1/2 (h)
Tmax (h)
(ng/ml)
(ng/h/ml)
cisapride
23g
6.99
7.34
7.00
<1
57.1
59.1
56.4
57.2
58.6
56.6
1.48
0.98
0.77
0.25
0.25
0.25
104
572
345
142
483
263
>10
>10
23h
2.2.3. Off-target screening study of 23g
We examined compound 23g (1–2 µM) for binding affinities to other pharmacologically relevant
receptors. Figure 4 shows that compound 23g selectively bound to the 5-HT4 receptor, whereas it did not show
even 50% inhibition of any other receptors. Therefore, it is expected that compound 23g is a potent prokinetic
agent without concern for side effects arising from non-specificity.

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Figure 4. Binding affinities of compound 23g (1–2µM) for other receptors.
3. Conclusion
In this study, we intended to find compounds that retained the prokinetic efficacies of cisapride but did
not block the hERG channel. We designed novel benzamide derivatives of 5-HT4 receptor agonists and tested
diverse compounds. We successfully found part B of a compound with negligible hERG binding and an
enhanced gastric emptying rate. We determined that compound 23g was better than cisapride in terms of high 5-
HT4 receptor binding, lower cardiac adverse effects due to lower hERG binding, and enhanced gastric emptying
in various models. In particular, the chiral (+)-form of 23g was more potent than the racemic mixture with
respect to binding affinity to the 5-HT4 receptor and in vivo gastric emptying rate. Thus, compound 23g or (+)-
form of 23g could be a good candidate to treat GI disorders, particularly functional dyspepsia. We plan to
investigate additional in vivo efficacy and toxicity for the single enantiomer of 23g.
4. Experimental Section
4.1. Chemistry

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Reagents and solvents were purchased from commercial suppliers (Sigma-Aldrich and TCI) used as provided,
unless indicated otherwise. All other solvents used for reactions were analytical grade and used as provided.
Column chromatography was carried out using Merck silica gel 60 (230-400 mesh). The melting points were
1
determined on Mettler Toledo FP900 thermosystem. H NMR and 13C NMR spectra were recorded on Varian
400MHz spectrophotometer by CDCl3 or DMSO-d6 and the chemical shifts are reported in δ (ppm) and are
relative to the central peak of these solvents. Mass spectra were obtained with JMS-700 Mstation mass
spectrometer. (JEOL Ltd.)
4.1.1. 1-(3-Chloropropyl)-1H-1,2,4-triazole (10d)
1,2,4-Triazole sodium derivative (1 g, 10.983 mmol) was dissolved in N,N-dimethylformamide (10mL) and
cooled to 0 ºC. Sodium hydride (60% dispersion in mineral oil, 570 mg, 14.25 mmol) was added to the reaction
mixture and stirred for 20 minutes at 0 ºC. 1-Bromo-3-chloropropane (2.08 g, 13.2mmol) was added dropwise to
the reaction mixture. The reaction mixture was stirred for 12h at room temperature and extracted with ethyl
acetate and water. The organic layer was dried over anhydrous MgSO4 and evaporated in vacuo. The residue
was purified by column chromatography (hexane:ethyl acetate = 4:1) to obtain the target compound (600 mg,
38% yield).
1H NMR (400 MHz, CDCl3): δ 8.09 (s, 1H), 7.91 (s, 1H), 4.33 (t, J = 6 Hz, 2H), 3.43(t, J = 6 Hz, 2H), 2.33–
2.26 (m, 2H). 13C NMR (400 MHz, CDCl3): δ 152.35, 143.59, 46.03, 41.09, 31.80.
4.1.2.
cis-4-Amino-5-chloro-N-[1-(3-(1H-pyrrol-1-yl)ethyl)-3-methoxypiperidin-4-yl]-2-methoxybenzamide
(13a)
1-(2-Bromoethyl)-1H-pyrrole (717 mg, 4.12 mmol), potassium carbonate (660 mg, 4.775 mmol) and potassium
iodide (106 mg, 0.64 mmol) were added to a stirred solution of cis-4-amino-5-chloro-2-methoxy-N-(3-
methoxypiperidin-4-yl)benzamide (1 g, 3.188 mmol) in N,N-dimethylformamide (20mL) in order. The reaction
mixture was heated to 110 ºC for 15 hours and then cooled to room temperature and extracted with ethyl acetate
and water. The organic layer was dried over anhydrous MgSO4 and evaporated in vacuo. The residue was
purified by column chromatography (chloroform:acetone = 4:1) to obtain the target compound (430 mg, 33%
yield).
1H NMR(CDCl3): δ 8.21 (d, J = 8Hz, 1H), 8.08 (s, 1H), 6.69 (s, 2H), 6.29 (s, 1H), 6.13 (s, 2H), 4.40 (s, 2H),
4.20–4.12 (m, 1H), 4.04 (t, J = 7.2Hz, 2H), 3.87 (s, 3H), 3.45–3.37 (m, 4H), 3.02–2.93 (m, 1H), 2.85–2.73 (m,
3H), 2.34–2.18 (m, 2H), 1.93–1.78 (m, 2H). 13C NMR (400 MHz, CDCl3): δ 163.75, 157.57, 146.69, 132.96,
120.76, 112.56, 111.50, 108.20, 97.89, 76.66, 59.20, 57.17, 55.97, 53.82, 52.02, 47.98, 47.55, 27.78. HRMS
(FAB): calcd for C20H27ClN4O3 [M + H]+ 407.1845, found 407.1845.
4.1.3.
cis-4-Amino-5-chloro-N-[1-(3-(1H-pyrrol-1-yl)propyl)-3-methoxypiperidin-4-yl]-2-methoxybenzamide
(13b)
1-(3-Bromopropyl)-1H- pyrrole (775 mg, 4.12 mmol), potassium carbonate (660 mg, 4.775 mmol) and
potassium iodide (106 mg, 0.64 mmol) were added to a stirred solution of cis-4-amino-5-chloro-2-methoxy-N-
(3-methoxypiperidin-4-yl)benzamide (1g, 3.188 mmol) in N,N-dimethylformamide (20mL) in order. The

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reaction mixture was heated to 90 ºC for 8 hours and then cooled to room temperature and extracted with ethyl
acetate and water. The organic layer was dried over anhydrous MgSO4 and evaporated in vacuo. The residue
was purified by column chromatography (chloroform:acetone = 4:1) to obtain the target compound (800 mg,
60% yield).
1H NMR(CDCl3): δ 8.21 (d, J = 8.0Hz, 1H), 8.07 (s, 1H), 6.65 (s, 2H), 6.28 (s, 1H), 6.13 (s, 2H), 4.56 (s, 2H),
4.21–4.10 (m, 1H), 3.97–3.87 (m, 2H), 3.85(s, 3H), 3.45–3.37 (m, 4H), 3.05–2.92 (m, 1H), 2.80–2.68 (m, 1H),
2.38–2.22 (m, 2H), 2.20–2.05 (m, 2H), 2.01–1.73 (m, 4H). 13C NMR (400 MHz, CDCl3): δ 163.75, 157.57,
146.71, 132.93, 120.61, 112.56, 111.46, 107.89, 97.89, 76.72, 57.02, 55.97, 55.03, 53.55, 51.77, 48.15, 47.38,
28.83, 27.83. HRMS (FAB): calcd for C21H29ClN4O3 [M + H]+ 421.2001, found 421.2010.
4.1.4. cis-4-Amino-5-chloro-N-[1-(3-(1H-imidazol-1-yl)propyl)-3-methoxypiperidin-4-yl]-2-methoxybenzamide
(13c)
1-(3-Chloropropyl)-1H-imidazole (596 mg, 4.12 mmol), potassium carbonate (660 mg, 4.775 mmol) and
potassium iodide (106 mg, 0.64 mmol) were added to a stirred solution of cis-4-amino-5-chloro-2-methoxy-N-
(3-methoxypiperidin-4-yl)benzamide (1g, 3.188 mmol) in N,N-dimethylformamide (20mL) in order. The
reaction mixture was heated to 90 ºC for 12 hours and then cooled to room temperature and extracted with ethyl
acetate and water. The organic layer was dried over anhydrous MgSO4 and evaporated in vacuo. The residue
was purified by column chromatography (chloroform:acetone = 4:1) to obtain the target compound (700 mg,
52% yield).
1H NMR (400 MHz, CDCl3): δ 8.19 (d, J = 8.4 Hz, 1H), 8.06 (s, 1H), 7.49 (s, 1H), 7.03 (s, 1H), 6.92(s, 1H),
6.28 (s, 1H), 4.49 (bs, 2H), 4.19–4.08 (m, 1H), 4.08–3.92 (m, 2H), 3.85(s, 3H), 3.44–3.38 (m, 4H), 2.99–2.83(m,
1H), 2.78–2.65 (m, 1H), 2.35–2.18(m, 2H), 2.18–2.03 (m, 2H), 1.99–1.67(m, 4H). 13C NMR (400 MHz,
CDCl3): δ 163.75, 157.56, 146.76, 137.41, 132.92, 129.28, 112.45, 111.44, 97.85, 57.14, 55.96, 54.19, 53.74,
51.44, 48.15, 44.53, 28.14, 27.73. HRMS (FAB): calcd for C20H28ClN5O3 [M + H]+ 422.1954, found
422.1955.
4.1.5.
cis-4-Amino-5-chloro-N-[1-(3-(1H-1,2,4-triazol-1-yl)propyl)-3-methoxypiperidin-4-yl]-2-
methoxybenzamide (13d)
1-(3-Chloropropyl)-1H-1,2,4-triazole (600 mg, 4.12 mmol), potassium carbonate (660 mg, 4.775 mmol) and
potassium iodide (106 mg, 0.64 mmol) were added to a stirred solution of cis-4-amino-5-chloro-2-methoxy-N-
(3-methoxypiperidin-4-yl)benzamide (1g, 3.188 mmol) in N,N-dimethylformamide (20mL) in order. The
reaction mixture was heated to 90 ºC for 12 hours and then cooled to room temperature and extracted with ethyl
acetate and water. The organic layer was dried over anhydrous MgSO4 and evaporated in vacuo. The residue
was purified by column chromatography (chloroform:acetone = 4:1) to obtain the target compound (610 mg,
45% yield).
1H NMR (400 MHz, CDCl3): δ 8.17 (d, J = 8 Hz, 1H), 8.11 (s, 1H), 8.0 (s, 1H), 7.91 (s, 1H), 6.27 (s, 1H), 4.48
(s, 2H), 4.28–4.06 (m, 3H), 3.84(s, 3H), 3.43–3.37 (m, 4H), 2.99–2.83(m, 1H), 2.77–2.64 (m, 1H), 2.34–1.97 (m,
6H), 1.90–1.65(m, 2H). 13C NMR (400 MHz, CDCl3): δ 163.75, 157.55, 151.92, 146.76, 143.65, 132.91, 112.44,

ACCEPTED MANUSCRIPT
111.43, 97.83, 57.16, 55.97, 53.99, 53.71, 51.26, 48.17, 47.11, 27.69, 26.56. HRMS (FAB): calcd for
C19H27ClN6O3 [M + H]+ 423.1906, found 423.1911.
4.1.6. cis-4-Amino-5-chloro-N-[1-(3-(1H-tetrazol-1-yl)propyl)-3-methoxypiperidin-4-yl]-2-methoxybenzamide
(13e)
1-(3-Chloropropyl)-1H-tetrazole (604 mg, 4.12 mmol), potassium carbonate (660 mg, 4.775 mmol) and
potassium iodide (106 mg, 0.64 mmol) were added to a stirred solution of cis-4-amino-5-chloro-2-methoxy-N-
(3-methoxypiperidin-4-yl)benzamide (1g, 3.188 mmol) was dissolved in N,N-dimethylformamide (20mL) in
order. The reaction mixture was heated to 100 ºC for 12 hours and then cooled to room temperature and
extracted with ethyl acetate and water. The organic layer was dried over anhydrous MgSO4 and evaporated in
vacuo. The residue was purified by column chromatography (chloroform:acetone = 4:1) to obtain the target
compound (795 mg, 59% yield).
1H NMR(DMSO-d6): δ 9.38 (s, 1H), 8.08 (d, J=7.6Hz, 1H), 7.72 (s, 1H), 6.49 (s, 1H), 5.99 (s, 2H), 4.47 (t, J =
6.8Hz, 2H), 4.00 (bs, 1H), 3.85 (s, 3H), 3.39–3.28 (m, 5H), 2.75 (bs, 1H), 2.25 (t, J = 6.6 Hz, 2H), 2.22–2.03
(m, 2H), 2.00 (t, J = 6.6 Hz, 2H), 1.75–1.58 (m, 2H). 13C NMR (400 MHz, CDCl3): δ 163.11, 157.85, 149.11,
144.57, 132.06, 110.38, 109.62, 98.09, 76.30, 56.54, 56.35, 54.25, 53.40, 50.73, 47.77, 46.17, 28.08, 26.81.
HRMS (FAB): calcd for C18H26ClN7O3 [M + H]+ 424.1859, found 424.1872.
4.1.7. 1-(Piperidin-1-yl)prop-2-en-1-one (16b)
Piperidine (2 g, 23.49 mmol) was dissolved in dichloromethane (20mL) and cooled to 0 ºC. Triethylamine
(6.6mL, 46.98 mmol) was added to the reaction mixture and stirred for 20 minutes at 0 ºC. Acryloyl chloride
(2.34 g, 25.84mmol) was added dropwise to the reaction mixture and stirred for 1h at 0 ºC. The reaction mixture
was extracted with ethyl acetate and water. The organic layer was dried over anhydrous MgSO4 and evaporated
in vacuo. The residue was purified by column chromatography (ethyl acetate:hexane = 4:1) to obtain the target
compound (1.32 g, 40% yield).
1H NMR (400 MHz, CDCl3): δ 6.58 – 6.47 (m, 1H), 6.18 (d, J = 16.8Hz, 1H), 5.59 (d, J = 10.4Hz, 1H), 3.64–
3.36 (m, 4H), 1.69–1.38 (m, 6H).
4.1.8.
cis-4-Amino-5-chloro-N-[1-(3-oxo-3-(pyrrolidin-1-yl)propyl)-3-methoxypiperidin-4-yl]-2-
methoxybenzamide (17a)
1-(Pyrrolidin-1-yl)prop-2-en-1-one (400 mg, 3.19 mmol) was slowly added to a stirred solution of cis-4-amino-
5-chloro-2-methoxy-N-(3-methoxypiperidin-4-yl)benzamide (500 mg, 1.59 mmol) in ethanol (5 mL). The
reaction mixture was stirred for 12 hours at room temperature and extracted with ethyl acetate and water. The
organic layer was dried over anhydrous MgSO4 and evaporated in vacuo. The residue was purified by column
chromatography (chloroform:methanol = 20:1) to obtain the target compound (370 mg, 53% yield).
1H NMR (400 MHz, CDCl3): δ 8.20 (d, J = 8.4Hz, 1H), 8.07 (s, 1H), 6.29 (s, 1H), 4.42 (s, 2H), 4.23–4.13 (m,
1H), 3.86(s, 3H), 3.51–3.38 (m, 8H), 3.10–2.97 (m, 1H), 2.87–2.71 (m, 3H), 2.58–2.46 (m, 2H), 2.34–2.18 (m,
2H), 1.99–1.78 (m, 6H). 13C NMR (400 MHz, CDCl3): δ 170.15, 163.70, 157.54, 146.61, 132.98, 112.66,

ACCEPTED MANUSCRIPT
111.52, 97.88, 57.11, 55.95, 53.83, 53.72, 51.55, 47.86, 46.64, 45.64, 32.31, 27.79, 26.07, 24.40. HRMS
(FAB): calcd for C21H31ClN4O4 [M + H]+ 439.2107, found 439.2108.
4.1.9.
cis-4-Amino-5-chloro-N-[1-(3-oxo-3-(piperidin-1-yl)propyl)-3-methoxypiperidin-4-yl]-2-
methoxybenzamide (17b)
1-(Piperidin-1-yl)prop-2-en-1-one (405 mg, 2.91 mmol) was slowly added to a stirred solution of cis-4-amino-5-
chloro-2-methoxy-N-(3-methoxypiperidin-4-yl)benzamide (500 mg, 1.59 mmol) in ethanol (5 mL). The reaction
mixture was stirred for 12 hours at room temperature and extracted with ethyl acetate and water. The organic
layer was dried over anhydrous MgSO4 and evaporated in vacuo. The residue was purified by column
chromatography (chloroform:methanol = 20:1) to obtain the target compound (360mg, 50% yield).
1H NMR (400 MHz, CDCl3): δ 8.20 (d, J = 8.4Hz, 1H), 8.07 (s, 1H), 6.28 (s, 1H), 4.43 (s, 2H), 4.23–4.11 (m,
1H), 3.86(s, 3H), 3.52(t, J = 5.2Hz, 2H), 3.46–3.37 (m, 4H), 3.09–2.98 (m, 1H), 2.85–2.69 (m, 3H), 2.61–2.50
(m, 2H), 2.34–2.18 (m, 2H), 1.91–1.77(m, 2H), 1.67–1.46 (m, 6H). 13C NMR (400 MHz, CDCl3): δ 169.75,
163.70, 157.53, 146.62, 132.97, 112.63, 111.51, 97.87, 57.13, 55.94, 54.15, 53.85, 51.60, 47.86, 46.63, 42.62,
30.94, 27.80, 26.50, 25.53, 24.53. HRMS (FAB): calcd for C22H33ClN4O4 [M + H]+ 453.2263, found
453.2265.
4.1.10.
cis-4-Amino-5-chloro-N-[1-(3-(4-benzylpiperidin-1-yl)-3-oxopropyl)-3-methoxypiperidin-4-yl]-2-
methoxybenzamide (17c)
1-(4-Benzylpiperidin-1-yl)prop-2-en-1-one (438 mg, 1.908 mmol) was slowly added to a stirred solution of cis-
4-amino-5-chloro-2-methoxy-N-(3-methoxypiperidin-4-yl)benzamide (500 mg, 1.59 mmol) in ethanol (5 mL).
The reaction mixture was stirred for 12 hours at room temperature and extracted with ethyl acetate and water.
The organic layer was dried over anhydrous MgSO4 and evaporated in vacuo. The residue was purified by
column chromatography (chloroform:methanol = 20:1) to obtain the target compound (318 mg, 37% yield).
1H NMR (400 MHz, CDCl3): δ 8.19 (d, J = 8.4Hz, 1H), 8.06 (s, 1H), 7.30–7.22 (m, 2H), 7.22–7.13 (m, 1H),
7.13–7.07 (m, 2H), 6.27 (s, 1H), 4.56 (d, J = 13.2Hz, 1H), 4.43 (s, 2H), 4.21–4.12 (m, 1H), 3.87–3.76 (m, 4H),
3.38 (m, 4H), 3.09–2.99 (m, 1H), 2.99–2.83 (m, 1H), 2.59–2.40 (m, 5H), 2.31–2.17 (m, 2H), 1.91–1.62 (m, 5H),
1.20–1.02 (m, 2H) . 13C NMR (400 MHz, CDCl3): δ 170.00, 163.72, 157.55, 146.67, 139.91, 132.97, 129.08,
128.30, 126.06, 124.96, 112.59, 111.50, 97.88, 57.14, 55.94, 54.15, 53.91, 53.86, 53.47, 51.60, 47.87, 45.86,
42.93, 41.94, 38.25, 32.55, 31.76, 27.80. HRMS (FAB): calcd for C29H39ClN4O4 [M + H]+ 543.2733, found
543.2739.
4.1.11.
cis-4-Amino-5-chloro-N-[1-(3-oxo-3-(morpholino-1-yl)propyl)-3-methoxypiperidin-4-yl]-2-
methoxybenzamide (17d)
1-Morpholinoprop-2-en-1-one (269 mg, 1.908 mmol) was slowly added to a stirred solution of cis-4-amino-5-
chloro-2-methoxy-N-(3-methoxypiperidin-4-yl)benzamide (500 mg, 1.59 mmol) in ethanol (5 mL). The reaction
mixture was stirred for 12 hours at room temperature and extracted with ethyl acetate and water. The organic
layer was dried over anhydrous MgSO4 and evaporated in vacuo. The residue was purified by column
chromatography (chloroform:methanol = 20:1) to obtain the target compound (292 mg, 40% yield).

ACCEPTED MANUSCRIPT
1H NMR (400 MHz, CDCl3): δ 8.19 (d, J = 8.4Hz, 1H), 8.06 (s, 1H), 6.28 (s, 1H), 4.44 (s, 2H), 4.20–4.12 (m,
1H), 3.85 (s, 3H), 3.68–3.56 (m, 6H), 3.47–3.39 (m, 6H), 3.09–2.97 (m, 1H), 2.84–2.67 (m, 3H), 2.54 (t, J =
7.6Hz, 2H), 2.28–2.17 (m, 2H), 1.86–1.77 (m, 2H). 13C NMR (400 MHz, CDCl3): δ 170.31, 163.71, 157.53,
146.67, 132.95, 112.54, 111.49, 97.85, 66.87, 66.61, 60.40, 57.17, 55.94, 53.99, 53.93, 51.67, 47.92, 45.94,
41.87, 30.78, 27.75. HRMS (FAB): calcd for C21H31ClN4O5 [M + H]+ 455.2056, found 455.2065.
4.1.12. 1-(4-(Hydroxymethyl)piperidin-1-yl)-2-methylpropan-1-one (19g)
Diisopropylethylamine(DIPEA) was slowly added to a stirred solution of piperidin-4-ylmethanol (1.3 g, 11.29
mmol) in methanol (20mL) and the reaction mixture was stirred for 30 minutes at 0ºC. Isobutyryl chloride (1.42
mL, 13.55 mmol) was added dropwise to the reaction mixture at the same temperature. The reaction mixture
stirred for more 2 h at 0ºC and 1 h at room temperature and extracted with ethyl acetate and water. The organic
layer was dried over anhydrous MgSO4 and evaporated in vacuo. The residue was purified by column
chromatography (chloroform:acetone = 2:1) to obtain the target compound (1.42 g, 7.68 mmol).
1H NMR (400 MHz, CDCl3): δ 4.68–4.47 (m, 1H), 4.03–3.79 (m, 1H), 3.44 (d, J = 5.2Hz, 2H), 2.83–2.64 (m,
2H), 2.62–2.37 (m, 1H), 1.90–1.62 (m, 3H), 1.19–0.97 (m, 8H).
4.1.13. (1-Isobutyrylpiperidin-4-yl)methyl methanesulfonate (20g)
1-(4-(Hydroxymethyl)piperidin-1-yl)-2-methylpropan-1-one (19g) (1 g, 5.398 mmol) was dissolved in
dichloromethane 20mL and cooled to 0 ºC. Triethylamine (1.51 mL , 10.796 mmol) was added to the reaction
mixture and stirred for 30 minutes at 0 ºC. Methanesulfonyl chloride (0.5 mL, 6.478 mmol) was added dropwise
for 30 minutes and stirred for 3 hours at the same temperature. The reaction mixture was stirred for 1 hour at
room temperature and extracted with dichloromethane and 1M citric acid aqueous solution. The organic layer
was dried with MgSO4 and evaporated in vacuo. The residue was purified by column chromatography (n-
hexane:EtOAc = 1:1) to obtain the target compound (1.21 g, 85% yield)
1H NMR (400 MHz, CDCl3): δ 4.79–4.49 (m, 1H), 4.08–3.81 (m, 1H), 3.67–3.40 (m, 2H), 3.07–2.73 (m, 1H),
2.65–2.71 (m, 2H), 2.64–2.38 (m, 1H), 1.99–1.74 (m, 4H), 1.27–1.03 (m, 9H).
4.1.14.
cis-4-Amino-5-chloro-N-(1-((1-propionylpiperidin-4-yl)methyl)-3-methoxypiperidin-4-yl)-2-
methoxybenzamide (23a)
(1-Propionylpiperidin-4-yl)methyl methanesulfonate (20a) (381 mg, 1.53 mmol), potassium carbonate (246 mg,
1.78 mmol) and potassium iodide (42 mg, 0.25 mmol) were added to a solution of cis-4-amino-5-chloro-2-
methoxy-N-(3-methoxypiperidin-4-yl)benzamide (400 mg, 1.275 mmol) in N,N-dimethylformamide (10mL) in
order. The reaction mixture was heated to 100 ºC for 12 hours and then cooled to room temperature and
extracted with ethyl acetate and water. The organic layer was dried over anhydrous MgSO4 and evaporated in
vacuo. The residue was purified by column chromatography (chloroform:acetone = 4:1) to obtain the target
compound (115 mg, 19% yield).
1H NMR (400 MHz, CDCl3): δ 8.18 (d, J = 8Hz, 1H), 8.08 (s, 1H), 6.29 (s, 1H), 4.60 (d, J = 13.2Hz, 1H), 4.41
(s, 2H), 4.25–4.16 (m, 1H), 3.96–3.78 (m, 4H), 3.42 (s, 4H), 3.04–2.83 (m, 2H), 2.78–2.61 (m, 1H), 2.54 (t, J =
13.2Hz, 1H), 2.39–2.07 (m, 6H), 1.96–1.64 (m, 6H), 1.20–0.99 (m, 5H). 13C NMR (400 MHz, CDCl3): δ

ACCEPTED MANUSCRIPT
172.17, 163.76, 157.55, 146.60, 133.01, 112.68, 111.53, 97.86, 64.29, 56.82, 56.00, 54.02, 52.22, 47.91, 45.57,
41.76, 34.01, 31.54, 31.41, 30.64, 30.52, 27.80, 9.63. HRMS (FAB): calcd for C23H35ClN4O4 [M + H]+
467.2420, found 467.2425.
4.1.15.
cis-4-Amino-5-chloro-N-(1-((1-propionylpiperidin-3-yl)methyl)-3-methoxypiperidin-4-yl)-2-
methoxybenzamide (23b)
(1-Propionylpiperidin-3-yl)methyl methanesulfonate (20b) (190 mg, 0.765 mmol), potassium carbonate (123
mg, 0.89 mmol) and potassium iodide (21 mg, 0.125 mmol) were added to a solution of cis-4-amino-5-chloro-2-
methoxy-N-(3-methoxypiperidin-4-yl)benzamide (200 mg, 0.638 mmol) in N,N-dimethylformamide (5mL) in
order. The reaction mixture was heated to 100 ºC for 12 hours and then cooled to room temperature and
extracted with ethyl acetate and water. The organic layer was dried over anhydrous MgSO4 and evaporated in
vacuo. The residue was purified by column chromatography (chloroform:acetone = 4:1) to obtain the target
compound (30 mg, 10% yield). Four stereoisomers were blended at the ratio of 0.24 : 0.26 : 0.26 : 0.24
(1R3S4R : 1S3S4R : 1R3R4S : 1S3R4S); 4-amino-5-chloro-2-methoxy-N-((3S,4R)-3-methoxy-1-(((R)-1-
propionylpiperidin-3-yl)methyl)piperidin-4-yl)benzamide : 4-amino-5-chloro-2-methoxy-N-((3R,4S)-3-
methoxy-1-(((R)-1-propionylpiperidin-3-yl)methyl)piperidin-4-yl)benzamide
:
4-amino-5-chloro-2-
methoxy-N-((3S,4R)-3-methoxy-1-(((S)-1-propionylpiperidin-3-yl)methyl)piperidin-4-yl)benzamide : 4-
amino-5-chloro-2-methoxy-N-((3R,4S)-3-methoxy-1-(((S)-1-propionylpiperidin-3-yl)methyl)piperidin-4-
yl)benzamide
4.1.16.
cis-4-Amino-5-chloro-N-(1-(2-(1-propionylpiperidin-4-yl)ethyl)-3-methoxypiperidin-4-yl)-2-
methoxybenzamide (23c)
2-(1-Propionylpiperidin-4-yl)ethyl methanesulfonate (20c) (202 mg, 0.765 mmol), potassium carbonate (123 mg,
0.89 mmol) and potassium iodide (21 mg, 0.125 mmol) were added to a solution of cis-4-amino-5-chloro-2-
methoxy-N-(3-methoxypiperidin-4-yl)benzamide (200 mg, 0.638 mmol) in N,N-dimethylformamide (50mL) in
order. The reaction mixture was heated to 90 ºC for 16 hours and then cooled room temperature and extracted
with ethyl acetate and water. The organic layer was dried over anhydrous MgSO4 and evaporated in vacuo. The
residue was purified by column chromatography (chloroform:acetone = 3:1) to obtain the target compound (83
mg, 27% yield).
1H NMR (400 MHz, CDCl3): δ 8.20 (d, J = 8Hz, 1H), 8.06 (s, 1H), 6.28 (s, 1H), 4.57 (d, J = 12.8Hz, 1H), 4.46
(s, 2H), 4.21–4.09 (m, 1H), 3.88–3.76 (m, 4H), 3.41 (s, 4H), 3.12–2.91 (m, 2H), 2.85–2.70 (m, 1H), 2.58–2.26
(m, 5H), 2.23–2.03 (m, 2H), 1.93–1.62(m, 4H), 1.58–1.37 (m, 3H), 1.21–1.00 (m, 5H). 13C NMR (400 MHz,
CDCl3): δ 172.09, 163.74, 157.57, 146.70, 132.94, 112.57, 111.47, 97.90, 76.64, 57.02, 56.03, 55.98, 55.95,
53.47, 53.41, 52.10, 48.09, 45.69, 41.90, 34.68, 33.42, 33.38, 32.91, 32.00, 27.78, 26.59, 9.64. HRMS (FAB):
calcd for C24H37ClN4O4 [M + H]+ 481.2576, found 481.2589.
4.1.17. Methyl 4-((cis-4-(4-amino-5-chloro-2-methoxybenzamido)-3-methoxypiperidin-1-yl)methyl)piperidine-1-
carboxylate (23d)
Methyl 4-(((methylsulfonyl)oxy)methyl)piperidine-1-carboxylate (20d) (437 mg, 1.74 mmol), potassium

ACCEPTED MANUSCRIPT
carbonate (281 mg, 2.03 mmol) and potassium iodide (48 mg, 0.29 mmol) were added to a solution of cis-4-
amino-5-chloro-2-methoxy-N-(3-methoxypiperidin-4-yl)benzamide (455 mg, 1.45 mmol) in N,N-
dimethylformamide (10mL) in order. The reaction mixture was heated to 100 ºC for 12 hours and then cooled to
room temperature and extracted with ethyl acetate and water. The organic layer was dried over anhydrous
MgSO4 and evaporated in vacuo. The residue was purified by column chromatography (chloroform:acetone =
4:1) to obtain the target compound (189 mg, 28% yield).
1H NMR (400 MHz, CDCl3): δ 8.18 (d, J = 7.6Hz, 1H), 8.04 (s, 1H), 6.27 (s, 1H), 4.50 (s, 2H), 4.23–3.08 (m,
3H), 3.83 (s, 3H), 3.65 (s, 3H), 3.38 (s, 4H), 2.98–2.83 (m, 1H), 2.79–2.58 (m, 3H), 2.29–2.07 (m, 5H), 1.90–
1.56 (m, 5H), 1.14–0.97 (m, 2H). 13C NMR (400 MHz, CDCl3): δ 163.80, 157.55, 155.98, 146.77, 132.85,
112.40, 111.37, 97.83, 76.67, 64.36, 56.72, 55.94, 54.00, 52.45, 52.19, 47.93, 43.94, 33.75, 30.72, 30.60, 27.79,
14.16. HRMS (FAB): calcd for C22H33ClN4O5 [M + H]+ 469.2212, found 469.2215.
4.1.18.
cis-4-Amino-5-chloro-N-(1-((1-acetylpiperidin-4-yl)methyl)-3-methoxypiperidin-4-yl)-2-
methoxybenzamide (23e)
(1-Acetylpiperidin-4-yl)methyl methanesulfonate (20e) (360 mg, 1.53 mmol), potassium carbonate (246 mg,
1.78 mmol) and potassium iodide (42 mg, 0.25 mmol) were added to a solution of cis-4-amino-5-chloro-2-
methoxy-N-(3-methoxypiperidin-4-yl)benzamide (400 mg, 1.275 mmol) in N,N-dimethylformamide (10mL) in
order. The reaction mixture was heated to 90 ºC for 12 hours and then cooled to room temperature and extracted
with ethyl acetate and water. The organic layer was dried over anhydrous MgSO4 and evaporated in vacuo. The
residue was purified by column chromatography (chloroform:acetone = 4:1) to obtain the target compound (27
mg, 5 % yield).
1H NMR (400 MHz, CDCl3): δ 8.18 (d, J = 8Hz, 1H), 8.07 (s, 1H), 6.29 (s, 1H), 4.57 (d, J = 13.2Hz, 1H), 4.44
(s, 2H), 4.23–4.14 (m, 1H), 3.86 (s, 3H), 3.78 (d, J = 13.2Hz, 1H), 3.41 (s, 4H), 3.07–2.82 (m, 2H), 2.75–2.59
(m, 1H), 2.53 (t, J = 13.2Hz, 1H), 2.26–2.09 (m, 4H), 2.07 (s, 3H), 1.94–1.65 (m, 5H), 1.18–0.98 (m, 2H). 13C
NMR (400 MHz, CDCl3): δ 168.83, 163.75, 157.55, 146.65, 132.97, 112.61, 111.48, 97.86, 64.27, 56.79, 55.98,
54.15, 52.20, 47.91, 46.55, 41.65, 41.61, 33.96, 31.44, 31.31, 30.56, 30.42, 27.83, 21.58. HRMS (FAB): calcd
for C22H33ClN4O4 [M + H]+ 453.2263, found 453.2267.
4.1.19.
cis-4-Amino-5-chloro-N-(1-((1-butyrylpiperidin-4-yl)methyl)-3-methoxypiperidin-4-yl)-2-
methoxybenzamide (23f)
(1-Butyrylpiperidin-4-yl)methyl methanesulfonate (20f) (381 mg, 1.53 mmol), potassium carbonate (246 mg,
1.78 mmol) and potassium iodide (42 mg, 0.25 mmol) were added to a solution of cis-4-amino-5-chloro-2-
methoxy-N-(3-methoxypiperidin-4-yl)benzamide (400 mg, 1.275 mmol) in N,N-dimethylformamide (10mL) in
order. The reaction mixture was heated to 100 ºC for 12 hours and then cooled to room temperature and
extracted with ethyl acetate and water. The organic layer was dried over anhydrous MgSO4 and evaporated in
vacuo. The residue was purified by column chromatography (chloroform:acetone = 4:1) to obtain the target
compound (115 mg, 19% yield).
1H NMR (400 MHz, CDCl3): δ 8.16 (d, J = 7.6Hz, 1H), 8.02 (s, 1H), 6.28 (s, 1H), 4.63–4.47 (m, 3H), 4.28–4.07
(m, 1H), 3.85–3.63 (m, 4H), 3.37 (s, 4H), 2.99–2.83 (m, 2H), 2.68–2.55 (m, 1H), 2.54–2.44 (m, 1H), 2.29–2.04

ACCEPTED MANUSCRIPT
(m, 6H), 1.88–1.53 (m, 7H), 1.12–0.96 (m, 3H), 0.91 (t, J = 7.6Hz, 3H). 13C NMR (400 MHz, CDCl3): δ 171.40,
163.82, 157.56, 146.91, 132.78, 112.25, 111.30, 97.83, 76.66, 64.23, 64.18, 56.73, 55.92, 54.06, 53.99, 52.12,
47.92, 45.77, 41.71, 41.68, 35.39, 33.96, 31.53, 31.42, 30.62, 30.50, 27.77, 18.84, 14.00. HRMS (FAB): calcd
for C24H37ClN4O4 [M + H]+ 481.2576, found 481.2584.
4.1.20.
cis-4-Amino-5-chloro-N-(1-((1-isobutyrylpiperidin-4-yl)methyl)-3-methoxypiperidin-4-yl)-2-
methoxybenzamide (23g)
(1-Isobutyrylpiperidin-4-yl)methyl methanesulfonate (20g) (403 mg, 1.53 mmol), potassium carbonate (246 mg,
1.78 mmol) and potassium iodide (42 mg, 0.25 mmol) were added to a solution of cis-4-amino-5-chloro-2-
methoxy-N-(3-methoxypiperidin-4-yl)benzamide (400 mg, 1.275 mmol) in N,N-dimethylformamide (10mL) in
order. The reaction mixture was heated to 100 ºC for 12 hours and then cooled to room temperature and
extracted with ethyl acetate and water. The organic layer was dried over anhydrous MgSO4 and evaporated in
vacuo. The residue was purified by column chromatography (chloroform:acetone = 4:1) to obtain the target
compound (428 mg, 70% yield).
1H NMR (400 MHz, CDCl3): δ 8.18 (d, J = 7.6Hz, 1H), 8.06 (s, 1H), 6.29 (s, 1H), 4.60 (d, J = 12.4Hz, 1H),
4.49(s, 2H), 4.24–4.13 (m, 1H), 3.99–3.80(m, 4H), 3.40(s, 4H), 3.05–2.44(m, 3H), 2.30–1.99(m, H), 1.97–1.62
(m, 5H), 1.24–0.96 (m, 8H). 13C NMR (400 MHz, CDCl3): δ 175.24, 163.77, 157.54, 146.73, 132.89, 112.47,
111.41, 97.84, 64.26, 56.77, 55.95, 54.11, 53.99, 52.25, 52.15, 47.92, 45.49, 41.90, 34.09, 31.78, 31.66, 30.69,
30.57, 30.06, 27.80, 19.57, 19.32. HRMS (FAB): calcd for C24H37ClN4O4 [M + H]+ 481.2576, found
481.2579.
4.1.21.
cis-4-Amino-5-chloro-N-(1-((1-pivaloylpiperidin-4-yl)methyl)-3-methoxypiperidin-4-yl)-2-
methoxybenzamide (23h)
(1-Pivaloylpiperidin-4-yl)methyl methanesulfonate (20h) (530 mg, 1.912 mmol), potassium carbonate (308 mg,
2.23 mmol) and potassium iodide (53 mg, 0.32 mmol) were added to a solution of cis-4-amino-5-chloro-2-
methoxy-N-(3-methoxypiperidin-4-yl)benzamide (500 mg, 1.593 mmol) in N,N-dimethylformamide (10mL) in
order. The reaction mixture was heated to 90 ºC for 12 hours and then cooled to room temperature and extracted
with ethyl acetate and water. The organic layer was dried over anhydrous MgSO4 and evaporated in vacuo. The
residue was purified by column chromatography (chloroform:acetone = 4:1) to obtain the target compound (228
mg, 29% yield).
1H NMR (400 MHz, CDCl3): δ 8.17 (d, J = 8.0Hz, 1H), 8.04 (s, 1H), 6.28 (s, 1H), 4.54 (s, 2H), 4.43–4.30 (m,
2H), 4.22–4.11 (m, 1H), 3.82 (s, 3H), 3.38 (m, 4H), 2.97–2.58 (m, 4H), 2.24–2.08 (m, 4H), 1.92–1.67 (m, 5H),
1.34–1.19 (m, 10H), 1.15–0.97 (m, 2H). 13C NMR (400 MHz, CDCl3): δ 176.13, 163.78, 157.54, 146.82, 132.82,
112.35, 111.34, 97.83, 76.67, 64.26, 60.36, 56.77, 55.93, 53.95, 52.20, 47.90, 45.31, 38.65, 34.04, 31.16, 31.10,
28.39, 27.80, 21.03, 14.16. HRMS (FAB): calcd for C25H39ClN4O4 [M + H]+ 495.2733, found 495.2739.
4.1.22.
cis-4-Amino-5-chloro-N-(1-((1-(3-methylbutanoyl)piperidin-4-yl)methyl)-3-methoxypiperidin-4-yl)-2-
methoxybenzamide (23i)
(1-(3-Methylbutanoyl)piperidin-4-yl)methyl methanesulfonate (20i) (424 mg, 1.53 mmol), potassium carbonate

ACCEPTED MANUSCRIPT
(246 mg, 1.78 mmol) and potassium iodide (42 mg, 0.25 mmol) were added to a solution of cis-4-amino-5-
chloro-2-methoxy-N-(3-methoxypiperidin-4-yl)benzamide (400 mg, 1.275 mmol) in N,N-dimethylformamide
(10mL) in order. The reaction mixture was heated to 90 ºC for 12 hours and then cooled to room temperature
and extracted with ethyl acetate and water. The organic layer was dried over anhydrous MgSO4 and evaporated
in vacuo. The residue was purified by column chromatography (chloroform:acetone = 4:1) to obtain the target
compound (148 mg, 24% yield).
1H NMR (400 MHz, CDCl3): δ 8.18 (d, J = 8.0Hz, 1H), 8.08 (s, 1H), 6.29 (s, 1H), 4.62 (d, J = 13.2Hz, 1H),
4.43 (s, 2H), 4.25–4.13 (m, 1H), 3.90–3.78 (m, 4H), 3.41 (s, 4H), 3.04–2.85 (m, 2H), 2.76–2.47 (m, 1H), 2.52 (t,
J = 13.2Hz, 1H), 2.47–2.01 (m, 7H), 1.97–1.66 (m, 6H), 1.15–0.98 (m, 2H), 0.95 (d, J = 6.0Hz, 6H). 13C NMR
(400 MHz, CDCl3): δ 170.89, 163.75, 157.54, 146.61, 132.97, 112.62, 111.49, 97.85, 64.25, 56.78, 55.97, 54.15,
54.00, 52.23, 47.89, 46.00, 42.23, 41.74, 41.71, 34.02, 31.65, 31.53, 30.62, 27.80, 25.84, 25.82, 22.80, 22.70.
HRMS (FAB): calcd for C25H39ClN4O4 [M + H]+ 495.2733, found 495.2737.
4.1.23. Ethyl 4-((cis-4-(4-amino-5-chloro-2-methoxybenzamido)-3-methoxypiperidin-1-yl)methyl)piperidine-1-
carboxylate (23j)
Ethyl 4-(bromomethyl)piperidine-1-carboxylate (410 mg, 1.64 mmol), potassium carbonate (281 mg, 2.03
mmol) and potassium iodide (48 mg, 0.29 mmol) were added to a stirred solution of cis-4-amino-5-chloro-2-
methoxy-N-(3-methoxypiperidin-4-yl)benzamide (455 mg, 1.45 mmol) in N,N-dimethylformamide (10mL) in
order. The reaction mixture was heated to 90 ºC for 12 hours and then cooled to room temperature and extracted
with ethyl acetate and water. The organic layer was dried over anhydrous MgSO4 and evaporated in vacuo. The
residue was purified by column chromatography (chloroform:acetone = 5:1) to obtain the target compound (222
mg, 32% yield).
1H NMR (400 MHz, CDCl3): δ 8.18 (d, J = 7.2Hz, 1H), 8.09 (s, 1H), 6.29 (s, 1H), 4.39 (s, 2H), 4.30–4.02 (m,
5H), 3.88 (s, 3H), 3.42 (s, 4H), 3.02–2.84 (m, 1H), 2.81–2.60 (m, 3H), 2.34–2.06 (m, 4H), 1.98–1.58 (m, 6H),
1.25 (t, J = 7.2Hz, 3H), 1.09–0.99 (m, 2H). 13C NMR (400 MHz, CDCl3): δ 163.73, 157.56, 155.61, 146.55,
133.04, 112.75, 111.56, 97.88, 64.43, 61.16, 56.78, 56.02, 54.05, 52.29, 47.88, 43.87, 33.85, 30.81, 30.69, 27.84,
14.72. HRMS (FAB): calcd for C23H35ClN4O5 [M + H]+ 483.2369, found 483.2379.
4.1.24. Propyl 4-((cis-4-(4-amino-5-chloro-2-methoxybenzamido)-3-methoxypiperidin-1-yl)methyl)piperidine-1-
carboxylate (23k)
Propyl 4-(bromomethyl)piperidine-1-carboxylate (434 mg, 1.64 mmol), potassium carbonate (281 mg, 2.03
mmol) and potassium iodide (48 mg, 0.29 mmol) were added to a stirred solution of cis-4-amino-5-chloro-2-
methoxy-N-(3-methoxypiperidin-4-yl)benzamide (455 mg, 1.45 mmol) in N,N-dimethylformamide (10mL) in
order. The reaction mixture was heated to 90 ºC for 12 hours and then cooled room temperature and extracted
with ethyl acetate and water. The organic layer was dried over anhydrous MgSO4 and evaporated in vacuo. The
residue was purified by column chromatography (chloroform:acetone = 4:1) to obtain the target compound (200
mg, 28% yield).
1H NMR (400 MHz, CDCl3): δ 8.18 (d, J = 8.0Hz, 1H), 8.06 (s, 1H), 6.28 (s, 1H), 4.45 (s, 2H), 4.24–4.03 (m,
3H), 4.00 (t, J = 6.8Hz, 2H), 3.85 (s, 3H), 3.40 (s, 4H), 2.99–2.84 (m, 1H), 2.80–2.59 (m, 3H), 2.28–1.57 (m,

ACCEPTED MANUSCRIPT
13H), 1.16–1.00 (m, 2H), 0.92 (t, J = 7.6Hz, 3H). 13C NMR (400 MHz, CDCl3): δ 163.79, 157.55, 155.68,
146.67, 132.91, 112.53, 111.44, 97.84, 76.66, 66.80, 64.38. 56.74, 55.96, 53.98, 52.20, 47.88, 43.88, 33.80,
30.75, 30.65, 27.74, 22.35, 10.43. HRMS (FAB): calcd for C24H37ClN4O5 [M + H]+ 497.2525, found
497.2529.
4.1.25.
4-((cis-4-(4-Amino-5-chloro-2-methoxybenzamido)-3-methoxypiperidin-1-yl)methyl)-N,N-
dimethylpiperidine-1-carboxamide (23n)
4-(Bromomethyl)-N,N-dimethylpiperidine-1-carboxamide (719 mg, 2.886 mmol), potassium carbonate (465 mg,
3.364 mmol) and potassium iodide (80 mg, 0.482 mmol) were added to a stirred solution of cis-4-amino-5-
chloro-2-methoxy-N-(3-methoxypiperidin-4-yl)benzamide (755 mg, 2.406 mmol) in N,N-dimethylformamide
(15mL) in order. The reaction mixture was heated to 90 ºC for 12 hours and then cooled to room temperature
and extracted with ethyl acetate and water. The organic layer was dried over anhydrous MgSO4 and evaporated
in vacuo. The residue was purified by column chromatography (chloroform:acetone = 3:1) to obtain the target
compound (310 mg, 27% yield).
1H NMR (400 MHz, CDCl3): δ 8.17 (d, J = 8.0Hz, 1H), 8.02 (s, 1H), 6.27 (s, 1H), 4.56 (s, 2H), 4.21–4.08 (m,
1H), 3.81 (s, 3H), 3.60 (d, J = 13.2Hz, 2H), 3.37 (s, 4H), 2.98–2.83 (m, 1H), 2.76 (s, 6H), 2.73–2.56 (m, 4H),
2.50–2.31 (m, 1H), 2.22–2.01 (m, 4H), 1.90–1.55 (m, 5H), 1.09–1.02 (m, 2H). 13C NMR (400 MHz, CDCl3): δ
165.17, 163.82, 157.55, 146.89, 132.79, 112.26, 111.30, 97.82, 76.66, 64.44, 56.73, 55.92, 53.89, 52.20, 47.93,
46.97, 38.51, 34.01, 30.81, 30.71, 27.78. HRMS (FAB): calcd for C23H36ClN5O4 [M + H]+ 482.2529, found
482.2531.
4.1.26.
4-((cis-4-(4-Amino-5-chloro-2-methoxybenzamido)-3-methoxypiperidin-1-yl)methyl)-N,N-
dimethylpiperidine-1-carboxamide (23o)
4-(Bromomethyl)-N-isopropylpiperidine-1-carboxamide (3.76 g, 14.287 mmol), potassium carbonate (2.18 g,
15.773 mmol) and potassium iodide (373 mg, 2.247 mmol) were added to a stirred solution of cis-4-amino-5-
chloro-2-methoxy-N-(3-methoxypiperidin-4-yl)benzamide (3.53 g, 11.25 mmol) in N,N-dimethylformamide
(70mL) in order. The reaction mixture was heated to 90 ºC for 12 hours and then cooled to room temperature
and extracted with ethyl acetate and water. The organic layer was dried over anhydrous MgSO4 and evaporated
in vacuo. The residue was purified by column chromatography (chloroform:acetone = 3:1) to obtain the target
compound (2.02 g, 36% yield).
1H NMR (400 MHz, CDCl3): δ 8.17 (d, J = 7.6Hz, 1H), 8.04 (s, 1H), 6.29 (s, 1H), 4.54 (s, 2H), 4.38–4.10 (m,
2H), 4.04–3.77 (m, 6H), 3.38 (s, 4H), 3.00–2.81 (m, 1H), 2.79–2.57 (s, 3H), 2.30–2.03 (m, 4H), 1.93–1.55 (m,
5H), 1.27–0.96 (m, 8H). 13C NMR (400 MHz, CDCl3): δ 163.78, 157.55, 157.15, 132.84, 146.89, 132.79,
112.37, 111.35, 97.84, 76.67, 64.35, 56.72, 55.94, 54.00, 43.99, 42.46, 33.80, 30.66, 30.53, 27.80, 23.48.
HRMS (FAB): calculated for C24H38ClN5O4 [M + H]+ 496.2685, found 496.2696.
4.1.27. 1-(4-(Bromomethyl)piperidin-1-yl)-2-methylpropane-1-thione (22m)
Lawesson’s reagent (724 mg, 1.79 mmol) was added to a stirred solution of 1-(4-(bromomethyl)piperidin-1-yl)-
2-methylpropane-1-one (740 mg, 2.982 mmol) in tetrahydrofuran (10mL) at 0 ºC. The reaction mixture was

ACCEPTED MANUSCRIPT
refluxed for 20 hours and then cooled to room temperature and extracted with ethyl acetate and water. The
organic layer was dried over anhydrous MgSO4 and evaporated in vacuo. The residue was purified by column
chromatography (chloroform:acetone = 8:1) to obtain the target compound (730 mg, 93 % yield).
1H NMR (400 MHz, CDCl3): δ 5.77 (d, J = 12.0Hz, 1H), 4.44 (d, J = 12.8Hz, 1H), 3.49–3.05 (m, 4H), 2.93 (t, J
= 12.0Hz, 1H), 2.13–1.92 (m, 3H), 1.47–1.15 (m, 8H).
4.1.28. cis-4-Amino-5-chloro-N-(1-((1-(2-methylpropanethioyl)piperidin-4-yl)methyl)-3-methoxypiperidin-4-yl)-
2-methoxybenzamide (23m)
1-(4-(Bromomethyl)piperidin-1-yl)-2-methylpropane-1-thione (730 mg, 2.763 mmol), potassium carbonate (480
mg, 3.473 mmol) and potassium iodide (74 mg, 0.446 mmol) were added to a stirred solution of cis-4-amino-5-
chloro-2-methoxy-N-(3-methoxypiperidin-4-yl)benzamide (722 mg, 2.3 mmol) in N,N-dimethylformamide
(15mL) in order. The reaction mixture was heated to 90 ºC for 12 hours and then cooled to room temperature
and extracted with ethyl acetate and water. The organic layer was dried over anhydrous MgSO4 and evaporated
in vacuo. The residue was purified by column chromatography (chloroform:acetone = 4:1) to obtain the target
compound (380 mg, 33 % yield).
1H NMR (400 MHz, CDCl3): δ 8.17 (d, J = 7.6Hz, 1H), 8.03 (s, 1H), 6.28 (s, 1H), 5.64 (d, J = 12.4Hz, 1H),
4.49 (s, 2H), 4.36 (d, J = 13.2Hz, 1H), 4.21–4.08 (m, 1H), 3.83 (s, 3H), 3.38 (s, 4H), 3.23–3.03 (m, 2H), 3.01–
2.78 (m, 2H), 2.72–2.57 (m, 1H), 2.26–1.68 (m, 9H), 1.34–1.03 (m, 8H). 13C NMR (400 MHz, CDCl3): δ
208.95, 208.90, 163.79, 157.56, 146.77, 132.83, 112.38, 111.36, 97.87, 76.64, 63.76, 63.73. 56.80, 56.76, 55.98,
54.28, 54.05, 52.26, 51.95, 50.71, 50.67, 49.06, 49.04, 47.96, 36.42, 33.89, 31.96, 31.86, 30.21, 30.10, 27.77,
23.12, 23.08, 23.03, 23.01. HRMS (FAB): calcd for C24H37ClN4O3S [M + H]+ 497.2348, found 497.2355.
4.1.29.
cis-4-Amino-5-chloro-N-(1-((1-propanethioylpiperidin-4-yl)methyl)-3-methoxypiperidin-4-yl)-2-
methoxybenzamide (23l)
1-(4-(Bromomethyl)piperidin-1-yl)propane-1-thione (658 mg, 2.763 mmol), potassium carbonate (480 mg,
3.473 mmol) and potassium iodide (74 mg, 0.446 mmol) were added to a stirred solution of cis-4-amino-5-
chloro-2-methoxy-N-(3-methoxypiperidin-4-yl)benzamide (722 mg, 2.3 mmol) in N,N-dimethylformamide
(15mL) in order. The reaction mixture was heated to 90 ºC for 12 hours and then cooled to room temperature
and extracted with ethyl acetate and water. The organic layer was dried over anhydrous MgSO4 and evaporated
in vacuo. The residue was purified by column chromatography (chloroform:acetone = 4:1) to obtain the target
compound (400 mg, 36 % yield).
1H NMR (400 MHz, CDCl3): δ 8.18 (d, J = 8.0Hz, 1H), 8.06 (s, 1H), 6.29 (s, 1H), 5.52 (d, J = 13.2Hz, 1H),
4.44 (s, 2H), 4.25–4.13 (m, 2H), 3.86 (s, 3H), 3.40 (s, 4H), 3.17 (t, J = 13.2Hz, 1H), 3.04–2.82 (m, 4H), 2.75–
2.63 (m, 1H), 2.31–2.10 (m, 4H), 2.04–1.73 (m, 6H), 1.35–1.11 (m, 5H). 13C NMR (400 MHz, CDCl3): δ
203.64, 203.60, 163.78, 157.54, 146.67, 132.91, 112.53, 111.45, 97.87, 76.73, 63.72. 56.83, 56.78, 55.99, 54.28,
54.06, 52.25, 51.98, 50.47, 50.44, 49.60, 49.58, 47.94, 37.27, 37.25, 33.57, 31.69, 31.57, 30.19, 30.06, 27.72,
13.47. HRMS (FAB): calcd for C23H35ClN4O3S [M + H]+ 483.2191, found 483.2202.
4.2. Biological evaluation

ACCEPTED MANUSCRIPT
4.2.1. 5-HT4 receptor binding assay
5-HT4 receptor binding assay were performed using membrane preparations form Cos-7 cells expressing human
5-HT4. The membrane protein preparation was conducted as described previously. [25] Briefly, cells were
washed with phosphate buffered saline (PBS) and centrifuged at 300 g for 5min. The resulting pellet was
suspended in ice-cold HEPES buffer (50mM, pH7.4), centrifuged at 40,000 g for 30 min at 4 ºC. The final pellet
was resuspended in HEPES buffer, and protein quantification assay. Competitive (30-4000 nM) of test
compounds and 30nM of [3H]-GR113808. Nonspecific binding was defined using 10 µM of 5-HT. The IC50
value (the concentration of test compound that inhibits the binding of the radioactive ligand by 50%) was
determined by linear regression of the displacement curve.
4.2.2. hERG channel assay
hERG binding affinities of test compounds were obtained using PredictorTM hERG fluorescence polarization
assay (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. The IC50 values of compounds (10 –
10,000 nM) were calculated by eliminating maximum polarization value of 30 µM E-4031, a specific hERG
blocker.
4.2.3. Functional potency evaluation; 5-HT4receptor agonistic activity on carbachol-induced contraction of rat
esophageal thoracic muscularis mucosae (TMM) preparations
Rats were sacrificed by a blow on the head, and the most distal 1.2 cm of the esophagus was isolated. The
esophageal segments were prepared as described previously. [26] Briefly, the external muscularis propria,
containing the outer longitudinal and circular muscle layers of the esophagus, was carefully removed in order to
isolate the smooth muscle of the tunica muscularis mucosae. The preparations were suspended longitudinally
under an initial tension of approximately 0.5 g in modified Krebs–Henseleit solution at 37 ºC and saturated with
95% O2 and 5% CO2. The ionic composition of the Krebs–Henseleit solution (mM) was NaCl 118, KCl 4.75,
CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25 and glucose 10. This solution routinely contained
indomethacin (3 ꢀM) to prevent the relaxation effects of prostanoids, methysergide(1 ꢀM) to block 5-HT1 and
5-HT2 receptor.
Tissues were left to equilibrate with Krebs–Henseleit solution for 60 min (with washing every 15 min) before
starting the experiment. Responses were recorded isometrically through a force displacement transducer (FT03,
GRASS technology, U.S.A.) coupled to a chart recorder (Labchart 5, AD Instruments, Australia).
The preparations were contracted by addition of a submaximal concentration of carbachol (3 ꢀM) into the
bathing solution. Upon establishing a stable contraction, accumulative concentration–effect curve for relaxation
to 5-HT was constructed. After construction of the control curve, the tissue was washed with fresh modified
Krebs–Henseleit solution and allowed to recover for 60 min before recontracting with carbachol. Potency
relative to 5-HT was calculated from experiments in which two concentration–effect curves were constructed in
the same preparation: the first to5-HT itself and the second to a test compound.

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4.2.4. Gastric emptying evaluation
Gastric emptying was measured according to the method [27] of with some modifications. Male Sprague-
Dawley rats (220—250 g) were fasted for 18 h with ad libitum access to water. (i) Normal rats were given 2 mL
of semisolid meals by gavages at 50 min after drug administration. Following 30 min animals were sacrificed,
and the weights of stomachs and contents in the stomachs were measured to determine gastric emptying. Gastric
emptying (%) = [1−weight of test stomach/weight of 0 time control stomach]×100. (ii) Animals were given 2
mL of semisolid meal at 30 min after drug administration, and simultaneously injected with cisplatin (i.v., 5
mg/kg). Following 40 min, gastric emptying was determined by the same method described above.
4.2.5. Other receptor binding assay
Other receptor binding assays were performed by MDS Pharma Services, Taiwan Ltd., using compound 23g at a
screening concentration of 1 or 2 uM. The radioligand binding affinities of 23g were determined at the
following receptors: 5-HT receptors (5-HT1A, 5-HT1B, 5-HT2A, 5-HT2B, 5-HT3, 5-HT4 receptors), non-5-HT
receptors (Adrenergic α1, Adrenergic α2, Adrenergic β1, Cholecystokininin 1, Dopamine 2L, Dopamine 2S,
Dopamine 3, Motilin, Muscarinic M2, Muscarinic M3, Opiate к (OP2), Opiate ꢀ (OP3), Somatostatin sst2,
Tachykinin NK1, Tachykinin NK2)
4.3. Chiral HPLC analyses
4.3.1. Instruments
Analytical HPLC apparatus consisted on a Agilent G1311A quaternary pump, a G1319A autosampler, a
G1316A column oven, a DAD G1315B UV detector, data were acquired and processed by a ChemStation
Datasystem. (Agilent)
4.3.2. HPLC operating condition
Analytical chromatographic separations were carried out on a Chiralpak IA column (250 mm × 4.6 mm I.D.)
with a mobile phase consisting of heptane : isopropanol : ethanol : diethylamine in the ratio 80 : 10 : 10 : 0.1
(v/v/v/v) at a flow rate of 1.0 mL/min and maintaining the column temperature at 30 ºC. The injection volume
was 10 ꢀL and the detection wavelength was set at 220 nm.
4.3.3. Preparation of sample
Sample was prepared by dissolving of accurate weight of 25 mg in 50 mL of methanol (0.5 mg/mL)

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Acknowledgements
The authors are grateful to Hyungkeun Lee for the tests of the biological activity and to A-yeon Park for the
analyses of chiral HPLC separation. We also appreciate the support and effort with the PK studies of Dong-A
ST Research Institute.
References and Notes
[1] D.E. Baker, Rationale for using serotonergic agents to treat irritable bowel syndrome,
American journal of health-system pharmacy : AJHP : official journal of the American
Society of Health-System Pharmacists, 62 (2005) 700-711; quiz 712-703.
[2] J.R. Grider, A.E. Foxx-Orenstein, J.G. Jin, 5-Hydroxytryptamine4 receptor agonists
initiate the peristaltic reflex in human, rat, and guinea pig intestine, Gastroenterology, 115
(1998) 370-380.
[3] M.B. Hansen, E. Skadhauge, Signal transduction pathways for serotonin as an intestinal
secretagogue, Comparative biochemistry and physiology. Part A, Physiology, 118 (1997)
283-290.
[4] J.G. Jin, A.E. Foxx-Orenstein, J.R. Grider, Propulsion in guinea pig colon induced by 5-
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Highlights
• 26 novel benzamide derivatives of cisapride were synthesized as potential prokinetic agents
• Amide derivatives of the alkyl piperidinyl group showed good binding affinities for the 5-
HT4 receptor and low affinities for hERG.
• Compound 23g could be a good candidate to treat GI disorders, particularly functional
dyspepsia.