Synthesis of quinoline derivatives as 5-HT4 receptor ligands

Article abstract of DOI:10.1071/CH08505

A general and convenient synthesis of 6-methoxyquinoline-3-carboxamides commencing with a cyclization step that involves -anisidine and diethyl (ethoxymethylene)malonate is described. An additional tetrahydroquinoline scaffold 19 is prepared from 6-methox

Full text of DOI:10.1071/CH08505

Full Paper
CSIRO PUBLISHING
Aust. J. Chem. 2009, 62, 150–156
www.publish.csiro.au/journals/ajc
Synthesis of Quinoline Derivatives as 5-HT4 Receptor Ligands
Amir Hanna-Elias,A David T. Manallack,A Isabelle Berque-Bestel,B,C
Helen R. Irving,A Ian M. Coupar,A and Magdy N. IskanderA,D
AMedicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences,
Monash University, 381 Royal Parade, Parkville, Vic. 3052, Australia.
BInserm U869, Bordeaux, 33076, France.
CUniversité Victor Segalen, Bordeaux 2, 33076, France.
DCorresponding author. Email: magdy.iskander@pharm.monash.edu.au
A general and convenient synthesis of 6-methoxyquinoline-3-carboxamides commencing with a cyclization step that
involves ρ-anisidine and diethyl (ethoxymethylene)malonate is described. An additional tetrahydroquinoline scaffold
19 is prepared from 6-methoxyquinoline-3-carboxamide and this represents a novel serotinergic lead structure. These
compounds show reasonable affinity at 1 × 10−6 M, and docking experiments suggest that they may bind in a similar
manner to serotonin.
Manuscript received: 18 November 2008.
Final version: 9 January 2009.
Introduction
agonist, 5-methoxytryptamine (2) (Fig. 1) as well as a novel
tetrahydroquinoline that contains a basic side chain.
Compounds that modulate serotonin (5-HT, 1) (Fig. 1) trans-
mission have many varied uses in clinical settings. In particular,
clinical agents that activate 5-HT4 receptors have use in gastro-
esophageal reflux disease[1] and irritable bowel syndrome.[2]
The endogenous ligand 1 acts on 5-HT4 receptors in the smooth
muscle of the gut and the bladder to regulate muscle tone.[3]
While compounds like cisapride and tegaserod also act as 5-HT4
receptor agonists, they have been restricted in their use because
of cardiovascular side effects.[4,5] The need for compounds that
have improved selectivity for the 5-HT4 receptor and reduced
toxicity is clear.
A range of molecular scaffolds have been used for 5-HT4
ligands including benzamides, indoles, and benzimidazoles.[6]
While various quinoline and quinolinone derivatives have been
explored,[7–9] the methods employed to generate these com-
pounds were not fully elucidated. Given this lack of detail
and the desire to explore the use of quinolines as potential
5-HT4 receptor ligands we have undertaken a more exten-
sive synthetic campaign. Initial molecular docking studies
using a 6-methoxyquinoline-3-carboxamide moiety showed that
favourable interactions could occur between this molecule and
the receptor. This result prompted our research and here we
report the design and synthesis of novel 5-HT4 compounds
based on a 6-methoxyquinoline ring system as analogues of the
Results and Discussion
Thesynthesisofthesimplestcarboxamidedescribedinthisstudy
was carried out in a four step reaction sequence (Scheme 1) that
involved cyclization of ρ-anisidine 3 and diethyl (ethoxymethy-
lene)malonate 4 to afford ethyl 4-hydroxy-6-methoxyquinoline-
3-carboxylate 5.[10] The difficulty in the cyclization of this
product was the requirement of high temperatures to achieve sig-
nificant yields.Various measures were taken to maintain the high
temperatures needed, without exceeding the maximum temper-
atures as this led to dimerization of the uncyclized intermediate.
Hydrolysis of the ester 5 was easily achieved by both base and
acid catalyzed hydrolysis; however, the efficiency of the latter
(98% of 6), and the ease of work up (where filtration was ade-
quate to achieve a pure product), were factors in choosing this
method over base catalysis. Acyl chloride formation and simul-
taneous chlorination were achieved by refluxing in phosphorus
oxychloride in a one pot synthesis. This reaction was quenched
in a solution of saturated ammonia gas in dichloromethane, to
afford the equivalent amide 7 in quantitative yield. The 4-chloro
adduct was treated with sodium borohydride in the presence of
palladium chloride catalyst to give the dechlorinated species 8.
Synthesis of further analogues employed an alternative route
found in Scheme 2. The cyclized compound 5 was treated with
phosphorus oxychloride to give the chlorinated species 9 to facil-
itate removal of this 4-position substituent in subsequent steps.
The dechlorination step was achieved with a palladium chloride
catalyst and sodium borohydride hydrogen donor in anhydrous
methanol to give product 10. As previously described for the
ester hydrolysis of compound 5, this was readily achieved in
acidic conditions to yield 11. The acid chloride intermediate
12 was synthesized using phosphorus oxychloride under reflux
NH2
NH2
HO
H3CO
N
H
N
H
1
2
Fig. 1. Structures of serotonin 1 and 5-methoxytryptamine 2.
© CSIRO 2009
10.1071/CH08505
0004-9425/09/020150

Synthesis of 5-HT4 Receptor Ligands
151
OH
N
CO2C2H5
H3CO
CO2C2H5
H3CO
O
O
O
(a)
(b)
C2H5O2C
O
O
NH2
H3CO
NH
3
4
5
(c)
OH
N
Cl
O
Cl
COCl
H3CO
H3CO
CO2H
H3CO
(e)
(d)
NH2
N
N
7
6
(f)
O
H3CO
NH2
N
8
Scheme 1. (a) 130◦C neat 4 h. (b) Dowtherm A, reflux, 1 day. (c) 20% HCl, reflux, 3 h. (d) POCl3, reflux, 3 h. (e) Sat. NH3 in DCM,
0◦C, 0.5 h→rt. (f) PdCl2, NaBH4, anhydrous MeOH, 30◦C, 3 h.
OH
Cl
H3CO
H3CO
H3CO
CO2C2H5
CO2C2H5
CO2C2H5
(a)
(b)
N
N
N
5
9
10
(c)
H3CO
H3CO
CONHR
COCl
H3CO
CO2H
(e)
(d)
N
N
N
I
12
11
N
R ؍
O
OH
S
NH2
OCH3
O
13
16
14
15
N
N
O
N
OH
17
18
Scheme 2. (a) POCl3, reflux, 3 h. (b) PdCl2, NaBH4, anhydrous MeOH, 30◦C, 3 h. (c) 20% HCl, reflux, 3 h. (d) POCl3, reflux, 3 h.
(e) RNH2, DCM, reflux, 4–24 h.
conditions. Analogues of I (Scheme 2) were synthesised in situ
with the acid chloride using an excess of a base.
(Fig. 2a).[11] For each analogue, five poses were evaluated for
both their docking score and a visual analysis of the binding
mode relative to both 1 and 2. Table 1 gives the G-score of the
best pose for each compound, which shows that there was no
relationship with their biological activity. This result was not
unexpected and it should be remembered that this exercise was
oriented to idea generation and to facilitate future design work.
One noticeable observation was that the analogues synthesized
in this study lacked a basic amine and as such they were unable to
form a strong charge-assisted hydrogen bond with residue D100.
Among the binding modes there were cases where the quinoline
ring behaved in a similar manner to the indole of 1 where the
amide nitrogen had an interaction with D100 and the quinoline
ring stacked with F201 (Fig. 2b). In other cases, however (e.g., 16
Docking
Docking studies were performed on analogues 8, 10, 11, and
13–19 using a homology model of the human 5-HT4 receptor.
This model was built based on the crystal structure of bovine
rhodopsin and was optimized in a lipid bilayer environment as
well as using information from docking experiments and site-
directed mutagenesis studies.[11–13] Using the Glide Package
(Schrodinger, NY, USA) and appropriate charge states for both
the active site residues and the ligands, 1 and 2 were successfully
docked into the protein in agreement with the previous model

152
A. Hanna-Elias et al.
(i) BH3·S(CH3)2/THF
(ii) HCl
(a) Ser 197
O
H3CO
H3CO
NH2
NH2
N
H
N
19
8
Scheme 3.
Asp 100
Phe 201
Ser 197
Ser 197
(b)
Phe 201
Asp 100
Fig. 3. Compound 19 (modelled as R isomer) docked into the homology
model of the 5-HT4 receptor.
Asp 100
Phe 201
Fig. 2. (a) Compound 1 docked into the homology model of the 5-HT4
receptor. (b) Compound 14 docked into the homology model of the 5-HT4
receptor.
displacements of 98 and 100% at 1 × 10−6 M, respectively,
which were not significantly different. The highest displace-
ment among the quinoline derivatives was observed with 14 (the
pyridine analogue), followed by 13 and 15.
Table 1. Radioligand displacement values and G-scores
Displacement of [3H]-GR113808 ( s.e.m.) at 1 × 10−6 M against the
5-HT4b receptor. G-score in Kcal mol−1
Tetrahydroquinoline
Given the affinity of the quinoline analogues at 1 × 10−6 M
together with their lack of an ionizable nitrogen in the side
chain that could interact with D100, an additional scaffold was
explored using the chemistry approaches described here. Com-
pound 19 was generated by the reduction of 8 to the amine in
anhydrous tetrahydrofuran, in the presence of borane–dimethyl
sulfide at room temperature (Scheme 3) in 50% yield. The
reduction of the pyridine ring formed an asymmetric centre at
the 3-position of the piperidine ring, which could lead to the
possibility of two enantiomers.
Molecular docking demonstrated that this molecule behaved
in a similar manner to 2 showing an interaction with D100
(Fig. 3). Encouragingly this compound displayed good potency
(Table 1) showing 75% displacement at 1 × 10−6 M. The com-
pound itself has reduced flexibility relative to 2 and the ring
nitrogen was estimated to be neutral at physiological pH (est.
pKa 4.7, ACD/Laboratories, Toronto, Canada). Molecular dock-
ing did not show any significant preference for the R or S isomers
and the results presented here are given for the R isomer. We
feel that compound 19 represents an important new scaffold for
5-HT4 receptors and potentially for other serotinergic targets.
As a lead compound it can be envisaged that substitution onto
the basic side-chain nitrogen atom would enable the optimiza-
tion of both potency and selectivity for the 5-HT4 receptor. This
compound will be the subject of future research in our laboratory.
Compound number
% Displacement
G-score
1
2
8
98
100
52
39
54
69
73
70
65
62
62
75
2
3
3
5
4
3
4
6
3
4
5
3
−7.3
−8.0
−7.8
10
11
13
14
15
16
17
18
19
−8.5
−8.2
−8.7
−3.4
−5.5
−10.0
−6.7
−2.8
−9.1 (R isomer)
and 18), the best pose showed that the side chain of the analogue
was placed adjacent to S197 and the amide made a hydrogen
bond with D100. This suggests that the presence of an ionizable
nitrogen is no doubt influential in orienting the compounds in
the binding site.
Radioligand Binding
A series of radioligand binding assays were conducted using
[3H]-GR113808, which is a high affinity and highly selec-
tive 5-HT4 receptor antagonist.[14] All molecules were screened
against the 5-HT4b receptor, the most commonly expressed
5-HT4 receptor splice variant.[5] Table 1 gives the displacement
values for compounds 1, 2, 8, 10, 11, 13–19, in this series
against the 5-HT4b receptor.The known agonists 1 and 2 showed
Conclusions
The results presented here demonstrate that the 6-methoxy-
quinoline-3-carboxamide scaffold is a useful structure to gener-
ate potential ligands for the 5-HT4 receptor. More importantly,

Synthesis of 5-HT4 Receptor Ligands
153
the 6-methoxy-tetrahydroquinoline compound (19) represents
a novel and useful lead structure in the design of 5-HT4 lig-
ands. Synthetically, the procedure we outline is convenient and
can be generally applied. The potency of several compounds
was of great interest and will form the basis of future medici-
nal chemistry efforts. The combination of docking experiments
and radioligand binding has given some insight into how these
compounds may interact with the receptor. Of course, further
screening is necessary at a range of concentrations to determine
IC50 values as well as exploring additional 5-HT4 splice vari-
ants for selectivity studies. Moreover, functional studies will be
undertaken to determine whether these compounds act as ago-
nists or antagonists. Although there was no correlation between
the binding affinities and their G-score, it was useful to visualize
each compound in the binding pocket to help influence future
design efforts.
Anhydrous sodium sulfate was used as the drying agent in
organic solutions. Concentration and evaporation of organic
solutions was performed using a Buchi rotary evaporator. All
reactions requiring reflux were conducted under inert nitro-
gen atmosphere using oven-dried glassware (120◦C). Analytical
TLC was performed using 0.2 mm, aluminium backed Silica Gel
60 F254 sheets. Preparative TLC was performed on 2 mm glass
backed Silica Gel 60 F254 sheets. Isolated compounds were ana-
lyzed byTLC to ensure only one spot was visible for high purity.
In all cases purity was determined to be over 90%.
Ethyl 4-Hydroxy-6-methoxyquinoline-3-carboxylate[10] (5)
A mixture of ρ-anisidine (3) (2.46 g, 20.0 mmol) and diethyl
(ethoxymethylene)malonate (4) (4.32 g, 20.0 mmol) was heated
without solvent at 130◦C for 4 h in a sand bath. The prod-
uct was cooled in an ethanol/dry ice slurry until a solid black
film developed on the glass flask. The solid was re-dissolved
in 15 mL of diethyl ether, and again cooled in a dry ice slurry.
White crystals formed upon cooling, and were filtered on a pre-
viously cooled Büchner funnel, and then quickly transferred
to a beaker where they rapidly melted. Dowtherm A (25 mL,
26.5% biphenyl and 73.5% diphenyl ether) was added to the
melted crystals and the dark coloured solution was heated at
reflux (290◦C) for 24 h. The reaction mixture was allowed to
cool to room temperature, and 25 mL of petroleum ether (100–
130◦C fraction) was added to precipitate the product. The light
brown coloured precipitate was filtered from the solution and
washed twice with petroleum ether (10 mL). Ethyl 4-hydroxy-
6-methoxyquinoline-3-carboxylate (5) (2.0 g, 41% yield) was
obtained as the product; mp 280–283◦C (lit.[10] 274–277◦C).
m/z 248 [M + H+]. δH ((D6)DMSO) 13.52 (1H, s, OH), 8.49
(1H, s, CH), 7.61 (1H, d, J 8.7, CH), 7.50 (1H, s, CH), 7.37 (1H,
dd, J 9.0, CH), 4.23 (2H, q, J 7.2, CH2), 3.87 (3H, s, OCH3),
1.30 (3H, t, J 7.2, CH3). δC ((D6)DMSO) 165.7, 163.3, 156.1,
150.7, 143.0, 127.2, 123.5, 119.7, 107.6, 102.9, 62.3, 56.4, 15.7.
(Found: [M + H+] 248.0922. C13H13NO4 requires [M + H+]
248.0923.)
Experimental
Molecular Modelling
A homology model of the 5-HT4 receptor developed by Mialet
and coworkers[11–13] was used for the docking experiments.
Details concerning the construction of this model can be found
in the references above. Molecules were constructed using Sybyl
(St Louis, MO, USA) employing Gasteiger Huckel charges. The
Glide Package was used for the docking experiments employing
the extra precision (XP) protocol without the specification of any
constraints. The top five poses were examined and the highest
scored pose was tabulated.
General
Diethyl (ethoxymethylene)malonate, diphenyl ether, and
biphenyl were all obtained from Fluka. Borane–dimethyl sul-
fide complex and ρ-anisidine were obtained from Aldrich.
Hydrochloric acid and phosphorus oxychloride as well as all
other solvents were obtained from Merck. Anhydrous com-
pressed ammonia gas was obtained from BOC.
All synthesized compounds∗ were assessed using electro-
1
spray ionization (ESI) mass spectrometry and H NMR spec-
4-Hydroxy-6-methoxyquinoline-3-carboxylic
troscopy. NMR spectra were recorded at room temperature on a
BrukerAvance300 MHzNMRspectrometer. Chemicalshiftsare
reported relative to tetramethylsilane at 0 ppm. Low-resolution
mass spectrometry analyses were performed using a Micromass
Platform II single quadropole mass spectrometer equipped with
an atmospheric pressure (ESI/APCI) ion source. Sample man-
agement was facilitated by an Agilent 1100 series HPLC system
and the instrument was controlled using MassLynx software
version 3.5. High-resolution mass spectrometry analyses were
collected on a Waters Micromass LCT Premier XE Time Of
Flight mass spectrometer fitted with either an electrospray (ESI)
or Ion Sabre (APCI) ion source and controlled with MassLynx
software version 4.1. All compounds were named using Chem-
Acid·HCl[10] (6)
Ethyl 4-hydroxy-6-methoxyquinoline-3-carboxylate (5) (2.0 g,
8.1 mmol) was heated to reflux in 20% hydrochloric acid for 3 h,
and upon cooling, beige needle-like crystals precipitated from
the solution. The product, 4-hydroxy-6-methoxyquinoline-3-
carboxylic acid (6), was isolated in 95% yield (1.96 g, 7.7 mmol);
mp 283–284◦C (lit.[15] 278–279◦C).
m/z (ESI) 220 [M + H+]. δH ((D6)DMSO) 17.75 (1H, s,
OH), 13.47 (1H, s, OH), 8.77 (1H, s, CH), 7.76 (1H, d, J 9.3,
CH), 7.61 (1H, s, CH), 7.51 (1H, dd, J 9.0, CH), 3.87 (3H,
s, OCH3). δC ((D6)DMSO) 169.7, 161.7, 158.4, 147.3, 144.1,
129.2, 124.9, 117.1, 106.5, 101.7, 56.9. (Found: [M + H+]
220.0609. C11H9NO4 requires [M + H+] 220.0610.)
1
draw Ultra 10.0 and predicted H NMR shifts were also used
for comparison with experimental data. It was observed that
in (D6)DMSO as NMR solvent the indole nitrogen proton was
always observed at the same chemical shift, whereas in CDCl3,
it was consistently absent. In reporting spectroscopic data the
following abbreviations have been used: DMSO, dimethyl sulf-
oxide; s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet;
dd, doublet of doublets; M, molecular ion.
4-Chloro-6-methoxyquinoline-3-carboxamide (7)
4-Hydroxy-6-methoxyquinoline-3-carboxylic acid (6) (1.96 g,
8.9 mmol) was heated at reflux in phosphorus oxychloride for 5 h
to afford the 4-chloro substituent of the quinoline and the for-
mation of the acyl chloride in a one pot reaction. Conversion
into the 3-amido derivative was achieved by addition of a
∗Compounds 5, 6, 8, and 11 have been reported previously but without detailed structure identification. As such, they have been described in the experimental
section.

154
A. Hanna-Elias et al.
saturated ammonia solution in dichloromethane (50 mL) at 0◦C
for 30 min, and then slowly allowing the mixture to return to
room temperature. The product, 4-chloro-6-methoxyquinoline-
3-carboxamide (7), was isolated by filtration of inorganic salts,
and washing with chloroform to obtain a 90% yield (1.9 g,
8.1 mmol); mp 261–263.5◦C. m/z (ESI) 237 [M + H+]. δH
((D6)DMSO) 9.24 (1H, s, CH), 7.86 (1H, d, J 9.3, CH), 7.85
(2H, s, NH2), 7.71 (1H, s, CH), 7.42 (1H, dd, J 9.0, CH), 3.83
(3H, s, OCH3). δC ((D6)DMSO) 168.7, 167.1, 158.1, 141.6,
139.3, 131.7, 129.7, 128.3, 119.4, 99.1, 57.3. (Found: [M + H+]
237.0421. C11H9ClN2O2 requires [M + H+] 237.0431.)
(0.58 g, 2.4 mmol); mp 210–211◦C (lit.[18] 273◦C for the free
base). m/z (ESI) 204 [M + H+]. δH ((D6)DMSO) 12.95 (1H, s,
OH), 9.51 (1H, s, CH), 8.75 (1H, s, CH), 7.96 (1H, d, J 9.3,
CH), 7.31 (1H, dd, J 9.0, CH), 7.15 (1H, s, CH), 3.81 (3H,
s, OCH3). δC ((D6)DMSO) 165.3, 157.6, 145.3, 144.0, 138.7,
130.2, 128.5, 124.3, 123.1, 108.5, 55.4. (Found: [M + H+]
204.0659. C11H9NO3 requires [M + H+] 204.0661.)
N-(4-Hydroxy-3-methoxybenzyl)-6-methoxyquinoline-
3-carboxamide (13)
The procedure for the preparation of compounds 13 to 18
employed compound 12 as an acyl chloride intermediate, which
was generated by reflux of 11 in POCl3 for 3 h followed by
removal of excess solvent under reduced pressure. The acid
chloride (12) was re-constituted in dichloromethane and reacted
immediately with the appropriate amine to generate compounds
13 to 18. During this procedure compound 12 was not isolated
or characterized.
4-Hydroxy-3-methoxy benzylamine (75.0 mg, 0.5 mmol)
was added to a stirred solution of 6-methoxyquinoline-3-
carbonyl chloride (12) (100 mg, 0.5 mmol) and TEA (250 µL)
in dichloromethane (5 mL) at room temperature. The solution
was heated to reflux for 24 h, and monitored by analytical TLC.
The reaction mixture was filtered through silica gel to facili-
tate removal of primary amines and inorganic salts. The filtered
product was identified as N-(4-hydroxy-3-methoxybenzyl)-6-
methoxyquinoline-3-carboxamide and was isolated as a yellow
powder in 43% yield (71.0 mg). m/z (ESI) 339 [M + H+]. δH
((D6)DMSO) 9.83 (1H, s, OH), 9.18 (1H, s, CH), 8.93 (1H, d, J
9.3, CH), 8.76 (1H, s, NH), 7.89 (1H, d, J 9.3, CH), 7.39 (1H,
dd, J 9.0, CH), 7.23 (1H, d, J 9.0, CH), 6.97 (1H, s, CH), 6.75
(1H, d, J 9.0, CH), 6.72 (1H, d, J 9.0, CH), 4.11 (2H, s, CH2),
3.73 (3H, s, OCH3). (Found: [M + H+] 339.1345. C19H18N2O4
requires [M + H+] 339.1345.)
6-Methoxyquinoline-3-carboxamide[16] (8)
Sodium borohydride (304 mg, 8.1 mmol) was added portion
wise to a stirred solution of 4-chloro-6-methoxyquinoline-
3-carboxamide (7) (1.9 g, 8.1 mmol) and palladium chloride
(1.41 g, 8.0 mmol) in dry methanol at room temperature over
a period of 1 h.[17] The inorganic salts were filtered off, the
methanol was removed under reduced pressure, and the reac-
tion mixture was reconstituted in chloroform. The organic phase
was washed with water and dried over anhydrous sodium sul-
fate. The dechlorinated product (8) was retained in 90% yield
(1.45 g, 7.1 mmol); mp 230–232◦C (lit.[16] 220◦C). m/z (ESI)
203 [M + H+]. δH ((D6)DMSO) 9.18 (1H, s, CH), 8.73 (1H, s,
CH), 7.71 (1H, d, J 9.3, CH), 7.91 (2H, s, NH2), 7.43 (1H, dd, J
9.0, CH), 7.25 (1H, s, CH), 3.78 (3H, s, OCH3). δC ((D6)DMSO)
167.2, 157.3, 148.0, 143.9, 134.1, 132.2, 130.7, 127.8, 125.7,
108.4, 56.5. (Found: [M + H+] 203.0819. C11H10N2O2 requires
[M + H+] 203.0821.)
Ethyl 6-Methoxyquinoline-3-carboxylate (10)
Ethyl 4-hydroxy-6-methoxyquinoline-3-carboxylate (5) (1.0 g,
4.1 mmol) was heated to reflux in phosphorus oxychloride
for 3 h. Upon completion, excess phosphorus oxychloride was
removed under reduced pressure. The reaction mixture was
reconstituted in chloroform and filtered through silica gel,
washed with water, and dried over anhydrous sodium sul-
fate. Ethyl 4-chloro-6-methoxyquinoline-3-carboxylate (9) was
recovered in 90% yield (0.95 g, 3.6 mmol).
Sodium borohydride (137 mg, 3.6 mmol) was added portion
wise to a stirred solution of ethyl 4-chloro-6-methoxyquinoline-
3-carboxylate (9) (0.95 g, 3.6 mmol) and palladium chloride
(634 mg, 3.6 mmol) in dry methanol at room temperature over a
period of 1 h. The inorganic salts were filtered off, the methanol
was removed under reduced pressure, and the reaction mix-
ture was reconstituted in chloroform. The organic phase was
washed with water and dried over anhydrous sodium sulfate.
The dechlorinated product (10) was retained in 90% yield
(0.74 g, 3.2 mmol); mp 203–205◦C. m/z (ESI) 232 [M + H+].
δH ((D6)DMSO) 9.46 (1H, s, CH), 9.10 (1H, s, CH), 7.89 (1H,
d, J 8.7, CH), 7.47 (1H, dd, J 9.0, CH), 7.35 (1H, s, CH), 4.33
(2H, q, J 7.2, CH2), 3.83 (3H, s, OCH3), 1.30 (3H, t, J 7.2,
CH3). δC ((D6)DMSO) 166.2, 157.5, 145.9, 145.2, 138.6, 130.7,
127.5, 124.9, 121.5, 108.6, 61.2, 56.9, 15.9. (Found: [M + H+]
232.0969. C13H13NO3 requires [M + H+] 232.0974.)
6-Methoxy-N-(pyridin-2-ylmethyl)quinoline-
3-carboxamide (14)
2-(Aminomethyl)pyridine (50 µL, 0.5 mmol) was added to a
stirred solution of 6-methoxyquinoline-3-carbonyl chloride (12)
(100 mg, 0.5 mmol) and TEA (250 µL) in dichloromethane
(5 mL) at room temperature. The solution was heated to reflux
for 4 h, and monitored by analytical TLC. The reaction mixture
was filtered through silica gel to facilitate removal of primary
amines and inorganic salts. The filtered product was identified
as 6-methoxy-N-(pyridin-2-ylmethyl)quinoline-3-carboxamide
and obtained as a white solid in 73% yield (105 mg); mp
113–114◦C. m/z (ESI) 294 [M + H+].
δH ((D6)DMSO) 9.28 (1H, s, CH), 8.84 (1H, d, J 9.3, CH),
8.76 (1H, s, NH), 8.46 (1H, d, J 9.3, CH), 7.79 (1H, d, J 9.3, CH),
7.73 (1H, d, J 9.3, CH), 7.31 (1H, dd, J 9.0, CH), 7.31 (1H, dd, J
9.0, CH), 7.17 (1H, d, J 9.0, CH), 4.37 (2H, s, CH2), 3.83 (3H,
s, OCH3). (Found: [M + H+] 294.1241. C17H15N3O2 requires
[M + H+] 294.1242.)
6-Methoxy-N-(4-sulfamoylphenethyl)quinoline-
3-carboxamide (15)
6-Methoxyquinoline-3-carboxylic Acid·HCl[18] (11)
4-(2-Aminoethyl)benzenesulfonamide (98.0 mg, 0.5 mmol) was
added to a stirred solution of 6-methoxyquinoline-3-carbonyl
chloride (12) (100 mg, 0.5 mmol) and TEA (250 µL) in
dichloromethane (5 mL) at room temperature. The solution
was heated to reflux for 24 h, and monitored by analyti-
cal TLC. The reaction mixture was filtered through silica
Ethyl6-methoxyquinoline-3-carboxylate(10)(0.74 g, 3.2 mmol)
was heated to reflux in a solution of 20% HCl (10 mL) for
2 h. Upon cooling, the product precipitated out of solution as
a black crystalline solid. This was filtered off and dried for use
in derivatization steps. The product was isolated in 75% yield

Synthesis of 5-HT4 Receptor Ligands
155
gel to facilitate removal of primary amines and inorganic
salts. The filtered product was identified as 6-methoxy-N-(4-
sulfamoylphenethyl)quinoline-3-carboxamide and obtained as a
beige powder in 29% yield (55 mg). m/z (ESI) 386 [M + H+].
δH ((D6)DMSO) 9.15 (1H, s, CH), 8.78 (1H, d, J 9.3, CH), 8.56
(1H, s, NH), 7.81 (2H, dd, J 9.3, CH, CH), 7.70 (1H, d, J 9.3,
CH), 7.45 (2H, dd, J 9.3, CH, CH), 7.39 (2H, s, NH2), 7.25 (1H,
dd, J 9.0, CH), 7.19 (1H, d, J 9.0, CH), 3.76 (3H, s, OCH3), 3.33
(2H, m, CH2), 2.53 (2H, m, CH2). (Found: [M + H+] 386.1175.
C19H19N3O4S requires [M + H+] 386.1172.)
m, CH2). (Found: [M + H+] 311.1510. C17H18N4O2 requires
[M + H+] 311.1508.)
( )(6-Methoxy-1,2,3,4-tetrahydroquinolin-
3-yl)methanamine (19)
Borane dimethyl sulfide (1.33 mL, 14 mmol) was added
dropwise to a stirred solution of 6-methoxyquinoline-3-
carboxamide (8) (1.45 g, 7.2 mmol) in dry THF (10 mL) at room
temperature over a period of 30 min.[19] Bubbling was observed
as sulfide gas and hydrogen were evolved. The solution was
stirred for a further hour upon completion of the addition and
then brought to reflux. Hydrochloric acid (6 M, 10 mL) was
slowly added to the refluxing solution to quench the remain-
ing hydride. The reaction was cooled to 0◦C in an ice bath
and neutralized with NaOH pellets. The amine product was
extracted with 3 × 15 mL quantities of diethyl ether. Analytical
HPLC produced a single clean peak, and the correspondingTLC
showed only 1 spot. The product was identified as (6-methoxy-
1,2,3,4-tetrahydroquinolin-3-yl)methanamine (19) in 50% yield
(0.670 g). m/z (ESI) 193 [M + H+]. δH ((D6)DMSO) 6.85 (1H,
s, CH), 6.59 (1H, s, CH), 6.44 (1H, d, J 9.0, CH), 4.25 (1H,
s, NH), 3.83 (1H, s, OCH3), 3.15–2.90 (2H, m, J 9.0, CH2),
2.73–2.34 (5H, m, CH2, CH2, CH), 2.30 (2H, s, NH2). δC
((D6)DMSO) 148.1, 138.3, 124.7, 113.6, 112.3, 111.9, 55.8,
55.6, 45.8, 42.7, 31.1. (Found: [M + H+] 193.1341. C11H16N2O
requires [M + H+] 193.1338.)
N-(4-Hydroxyphenethyl)-6-methoxyquinoline-
3-carboxamide (16)
Tyramine (67.0 mg, 0.5 mmol) was added to a stirred solu-
tion of 6-methoxyquinoline-3-carbonyl chloride (12) (100 mg,
0.5 mmol) and TEA (250 µL) in dichloromethane (5 mL) at
room temperature. The solution was heated to reflux for 24 h,
and monitored by analytical TLC. The reaction mixture was fil-
tered through silica gel to facilitate removal of primary amines
and inorganic salts. The filtered product was identified as N-
(4-hydroxyphenethyl)-6-methoxyquinoline-3-carboxamide and
isolated as a yellow powder in 24% yield (39 mg). m/z (ESI) 323
[M + H+]. δH ((D6)DMSO) 9.43 (1H, s, OH), 9.31 (1H, s, CH),
8.79 (1H, d, J 9.3, CH), 8.56 (1H, s, NH), 7.83 (1H, d, J 9.3, CH),
7.35(1H, dd, J9.0, CH), 7.27(1H, d, J9.0, CH), 7.16(2H, d, J9.0,
CH, CH), 6.76 (2H, d, J 9.0, CH, CH), 3.86 (3H, s, OCH3), 3.27
(2H, m, CH2), 2.72 (2H, m, CH2). (Found: [M + H+] 323.1390.
C19H18N2O3 requires [M + H+] 323.1396.)
Biology
The standard compounds 1 and 2 were screened along with
intermediates 8, 10, and 11, and derivatives 13–19 against the
5-HT4b receptor. Each compound was screened at 100 × 10−6 M
(data not shown) and 1 × 10−6 M in duplicate, which was
repeated three times to give n = 3. All assays were carried out
in 96-well plates using the Filtermate harvester (Packard, CT,
USA) to harvest the membrane-ligand complex onto a GF/B
Unifilter (Perkin–Elmer, Waltham, MA, USA). GF/B Unifilter
plates were prepared for the binding study by soaking in 0.5%
polyethyleneimine overnight. The potent and selective 5-HT4
receptor antagonist [3H]-GR113808 (GE Healthcare) was used
as the radioligand at a final concentration of 0.25 × 10−9 M
per well (300 µL final well volume). Test compounds, dis-
solved in phosphate buffered saline (PBS) and DMSO (max.
conc. 1% v/v) were then added, along with 20 µg of membrane
preparation (prepared from transient transfection of COS-7 cells
which expressed the 5-HT4b splice variant). The binding reac-
tion occurred at 25–30◦C for 60 min. The membrane complexes
were harvested onto the GF/B Unifilter plates using a vac-
uum pump and then washed three times with PBS. The filter
was then dried at 37◦C for 4–6 h before adding 50 µL of scin-
tillation fluid (Microscint 40). Binding was measured using
a TopCount Microplate Scintillation Counter (Packard). The
GraphPad Prism 5 package (La Jolla, CA, USA) was used for
data analysis. The mean displacement values of the compounds
in the screening exercise were determined. The standard error of
the mean was also calculated and the differences between means
were determined by one-way ANOVA followed by the Tukey–
Kramer post-hoc test. The level of significance was taken as
P < 0.01.
6-Methoxy-N-morpholinoquinoline-3-carboxamide (17)
4-Aminomorpholine (47 µL, 0.5 mmol) was added to a
stirred solution of 6-methoxyquinoline-3-carbonyl chloride (12)
(100 mg, 0.5 mmol) and TEA (250 µL) in dichloromethane
(5 mL) at room temperature. The solution was heated to reflux
for 4 h, and monitored by analytical TLC. The reaction mixture
was filtered through silica gel to facilitate removal of primary
amines and inorganic salts. The filtered product was identified
as 6-methoxy-N-morpholinoquinoline-3-carboxamide and iso-
lated as a brown solid in 31% yield (43 mg); mp 107–108◦C.
m/z (ESI) 288 [M + H+]. δH ((D6)DMSO) 9.19 (1H, s, CH),
8.71 (1H, d, J 9.3, CH), (1H, s, NH), 7.65 (1H, d, J 9.3, CH),
7.45 (1H, dd, J 9.0, CH), 7.28 (1H, d, J 9.0, CH), 3.75 (3H,
s, OCH3), 3.51 (4H, m, CH2, CH2), 2.90 (4H, m, CH2, CH2).
(Found: [M + H+] 288.1346. C15H17N3O3 requires [M + H+]
288.1348.)
N-(3-(1H-Imidazol-1-yl)propyl)-6-methoxyquinoline-
3-carboxamide (18)
1-(3-Aminopropyl)imidazole (58 µL, 0.5 mmol) was added to a
stirred solution of 6-methoxyquinoline-3-carbonyl chloride (12)
(100 mg, 0.5 mmol) and TEA (250 µL) in dichloromethane
(5 mL) at room temperature. The solution was heated to reflux
for 4 h, and monitored by analytical TLC. The reaction mixture
was filtered through silica gel to facilitate removal of primary
amines and inorganic salts. The filtered product was identi-
fied as N-(3-(1H-imidazol-1-yl)propyl)-6-methoxyquinoline-3-
carboxamide and obtained as a yellow oil in 34% yield (52 mg).
m/z (ESI) 311 [M + H+]. δH ((D6)DMSO) 9.21 (1H, s, CH),
8.79 (1H, d, J 9.3, CH), 8.47 (1H, s, NH), 7.98 (1H, s, CH), 7.72
(1H, d, J 9.3, CH), 7.36 (1H, dd, J 9.0, CH), 7.27 (1H, d, J 9.0,
CH), 7.23 (1H, d, J 9.0, CH), 6.69 (1H, d, J 9.0, CH), 4.07 (2H,
m, CH2), 3.71 (3H, s, OCH3), 3.12 (2H, m, CH2), 2.54 (2H,
Acknowledgement
The authors sincerely thank the Monash Institute of Pharmaceutical Sciences
(Monash University) for providing a Ph.D. scholarship, and Dr S. Egan

156
A. Hanna-Elias et al.
and Mr K. Chinkwo for their assistance. The mammalian expression vector
pcDNA3.1/5-HT4b was a gift from Dr F. O. Levy (University of Oslo).
5–HT4 receptor agonistic activity, WO Patent 2005049608 2005,
pp. 78.
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