New quinolinic derivatives as melatonergic ligands: Synthesis and pharmacological evaluation

Article abstract of DOI:10.1016/j.ejmech.2016.12.013

New series of melatonergic ligands issued from two methoxy-quinolinic scaffolds (2-MQ and 3-MQ), were designed and synthesized. Herein we report the synthetic scheme and pharmacological results of the new prepared compounds. Investigation of compound 11a, the strict 2-MQ analogue, revealed the promising potential of this series. Therefore, pharmacomodulation of the acetamide function of 11a has led to compounds with different pharmacological profiles and the emergence of an MT₂selectivity. Besides, sulphonamide 11b showed the most important MT₂selectivity of this series (167 folds) while methyl and ethyl-ureas 11f and 11g represented the most potent melatonergic ligands of this study.

Full text of DOI:10.1016/j.ejmech.2016.12.013

Accepted Manuscript
New quinolinic derivatives as melatonergic ligands: Synthesis and pharmacological
evaluation
Elodie Landagaray, Mohamed Ettaoussi, Marouan Rami, Jean A. Boutin, Daniel-Henri
Caignard, Philippe Delagrange, Patricia Melnyk, Pascal Berthelot, Saïd Yous
PII:
S0223-5234(16)31019-4
10.1016/j.ejmech.2016.12.013
EJMECH 9104
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 October 2016
Revised Date: 4 December 2016
Accepted Date: 5 December 2016
Please cite this article as: E. Landagaray, M. Ettaoussi, M. Rami, J.A. Boutin, D.-H. Caignard, P.
Delagrange, P. Melnyk, P. Berthelot, S. Yous, New quinolinic derivatives as melatonergic ligands:
Synthesis and pharmacological evaluation, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2016.12.013.
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ACCEPTED MANUSCRIPT
GRAPHICAL ABSTRACT

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New Quinolinic Derivatives as Melatonergic Ligands: Synthesis and
Pharmacological Evaluation
Elodie Landagaray,a,1 Mohamed Ettaoussi,a,1,* Marouan Rami,a Jean A. Boutin,b Daniel-Henri
Caignard,c Philippe Delagrange,c Patricia Melnyk,a,d Pascal Berthelot,a,d and Saïd Yousa,d,*
aUniversité de Lille, EA4481 – GRIIOT, F-59000 Lille, France
bBiotechnologies, Pharmacologie Moléculaire et Cellulaire, Institut de Recherches Servier, 78290
Croissy-sur-Seine, France.
cUnité de Recherche Chimie Neurosciences, Institut de Recherches Servier, 78290 Croissy-sur-Seine,
France.
d Univ. Lille, Inserm, CHU Lille, UMRS-1172 – JPArc – Centre de Recherches Jean-Pierre Aubert
Neurosciences et Cancer, F-59000 Lille, France
1Both investigators equally contributed to this work
Corresponding Author.
*Univ. Lille, Faculty of Pharmacy, UMRS-1172, F-59000 Lille, France
Dr. M. Ettaoussi, m.ettaoussi@yahoo.fr; Dr. S. Yous, said.yous@univ-lille2.fr.

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ABSTRACT
New series of melatonergic ligands issued from two methoxy-quinolinic scaffolds (2-MQ and
3-MQ), were designed and synthesized. Herein we report the synthetic scheme and
pharmacological results of the new prepared compounds. Investigation of compound 11a, the
strict 2-MQ analogue, revealed the promising potential of this series. Therefore,
pharmacomodulation of the acetamide function of 11a has led to compounds with different
pharmacological profiles and the emergence of an MT2 selectivity. Besides, sulphonamide
11b showed the most important MT2 selectivity of this series (167 folds) while methyl and
ethyl-ureas 11f and 11g represented the most potent melatonergic ligands of this study.
Key Words: Melatonin, quinoline derivatives, MT1, MT2-selectivity, agonist.

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1. Introduction
In light of the recent findings the question should we ask here is: may melatonin be the
promising ring in the search for efficient multitargets drug? In fact, recent advances in the
literature, permitted to highlight some physiological processes that might be great for
medicinal chemistry [1-5]. Melatonin (N-acetyl-5-methoxytryptamine) is a widely distributed
molecule in nature and has demonstrated functional roles in a wide range of organisms [6-7].
In humans, melatonin is primarily synthesized from L-tryptophan acid by the pinealocytes of
the pineal gland [8]. Once synthesized, this neurohormone is spread in the bloodstream where
it can hit peripheral organs or cross the blood-brain barrier and enter the central nervous
system (CNS) [9-10].
Within the pineal gland, melatonin secretion follows a circadian rhythm, with lower
concentration during the day and a maximal level in the night in both diurnal and nocturnal
species. This circadian rhythmicity of melatonin has been assigned to several biological
functions entrainment. In sleep, melatonin signal acts in the suprachiasmatic nucleus (SCN) to
regulate the sleep-wake cycle and other cyclic activities of the body [11-12]. In addition to its
implications in sleep and circadian rhythms, melatonin was shown to be involved in numerous
physiological functions including immune responses, core body temperature control [13-15],
insomnia [16-19], appetite and cardiac functions [20-21]. Much recent research demonstrated
the interest of melatonin in other physiological processes including, modulation of hormones
secretion [22], pain perception [23], cancer and inflammation [24-26], neurodegenerative
diseases [27-28], diabetes [29], mood regulation, depression and anxiety [31-32].
To date, most of these melatonin effects are mainly attributed to the activation of two major
receptors that belong to the family of high-affinity G protein-coupled receptors (GPCRs),
known as MT1 and MT2 [33]. Their activation induces downstream effects such as inhibition
of adenylate cyclase and c-AMP production [34-36] associated with effects responsible for

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the different physiological actions of melatonin [37]. In addition to MT1 and MT2 receptors,
another binding site of melatonin has been identified as a low-binding affinity site. Named
MT3, this binding site is a melatonin-sensitive form of the human enzyme quinone reductase
2, known for its detoxifying properties in humans [38-39].
However, despite the numerous above mentioned physiological functions of melatonin,
therapeutic potential is limited by its lack of selectivity, reduced bioavailability due to its high
first-pass metabolism and short half-life. To bypass these issues, during decades, several
efficient melatonergic ligands were prepared and biologically tested (Figure 1). Amongst a
huge number of them were non selective MT1/MT2 or MT2 selective ligands. In contrary, only
a limited number of MT1 selective ligands have been identified. Furthermore, comprehension
of intrinsic activity for MT1 also remains challenging. Hopelessly, all assays made by
molecular modelers to predict affinity and activity for MT1 in new designed ligands stayed
useless. This observed difficulty comes first from the lack of crystal structures for these two
receptor subtypes, then from the structural resemblance between MT1 and MT2 whom is
sharing about 60% of their amino acid identity. Further work is needed to clarify the exact
role of each of these two receptor subtypes in the above described physiological processes and
help to discover new potent ligands.
Within our Lab, our first objectives were first focused on the design and synthesis of selective
and non selective melatonergic ligands analogues of melatonin. By using pharmacological
concepts based first on bioisosteric replacement of melatonin indole ring to improve
pharmacokinetic properties of melatonin and establish structure-activity-relationships (SARs)
[40-42]. In this regard, our attention was first focused on nitrogen-based nucleus because of
their physicochemical and pharmacological properties! Consequently, our Lab has designed
and synthesized several nitrogen-based melatonergic analogues based on different scaffolds
such as isoquinolines, quinolines, aza-indoles, phthalazines and others. Obtained results

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permitted then the confirmation and the extension of our knowledge concerning the
melatonergic system. In this scoop, we report herein the synthesis and pharmacological
evaluation results of a new family of ligands issued from the replacement of the indole
nucleus by a quinolinic ring where the methoxy group is worn by the heterocycle (Figure 2).
The structure activity and biological results of new prepared compounds are shown hereafter.
<Insert Figure 1 here>
2. Results and discussion
2.1. Chemistry
In this part, we describe the synthetic routes leading to the designed ligands. The key
step to quinoline derivatives syntheses is the Heck cross-coupling reaction between
bromomethoxyquinoline derivatives (4-6) and vinyl phthalimide or methyl acrylate.
Compounds 4-6 were prepared from commercially available bromoquinolinic derivatives 1-3.
First, 2-chloroquinoline derivatives (1 and 2) were treated with sodium methoxide to give
compounds 4 and 5 in 97% and 94% yields, respectively. 5-Bromo-3-methoxyquinoline (6)
was prepared in two steps from compound 3 through methoxylation according to a variant of
Ullman process in the presence of sodium methoxide and copper iodide [43], and bromination
with NBS in an H2SO4 medium.
Heck cross-coupling reaction of bromoquinoline derivatives 4-6 with N-vinylphthalimide in
the presence of palladium acetate and tri(o-tolyl)phosphine [44] gave unsaturated compounds
7-8 and 13. Soft catalytic hydrogenation of the double bonds followed by cleavage of the
phthalimide with hydrazine gave primary amines 11-12 and 15 (Scheme 2). Similarly, acid 18
was prepared from 8-bromo-2-methoxyquinoline 4 through a treatment with ethyl acrylate in
the presence of bis(triphenylphosphine)palladium(II) dichloride and triethylamine. This

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unsaturated compound 16 was then hydrogenated over palladium on charcoal and saponified
in presence of sodium hydroxide leading to acid 18 after acidification (Scheme 2).
Finally, in Scheme 3 is illustrated the synthesis of targeted final compounds including,
amides, sulfonamides, carbamates, ureas and thioureas. First, amides 11a, 12a, and 15a-b
were synthesized from amines 11, 12 and 15 respectively according to a variant of the
Schotten-Baumann procedure [45], by reaction with the appropriate acid chloride. Retro-
amides 18a-c were prepared from acid 18 following two procedures: either, activation of acid
18 by a treatment with thionyl chloride followed by amidification with corresponding primary
amine (compound 18a), or a peptide coupling reaction by treatment with EDCI/HOBt and the
corresponding primary amine (compound 18b-c). Fluoroacetamide 15c was prepared by
refluxing amine 15 with ethyl fluoroacetate in 2,2,2-trifluoroethanol. Sulfonamides 11b-c and
carbamate 11d were obtained respectively by reaction of amine 11 with methanesulfonyl
chloride or methyl chloroformate in the presence of triethylamine. Ureas 11e and 15d were
prepared from the corresponding amines by reaction with potassium cyanate in an acidic
medium, while ethylureas (11g and 15e) and alkylthioureas (11h-i, 15f-g) were prepared from
corresponding amines by treatment with ethyl isocyanate or alkyl isothiocyanate. Finally,
methylurea 11f was prepared by treatment of primary amine 11 with methyl-N-
phenylcarbamate in dimethyl sulfoxide.
<Insert Scheme 1 here>
<Insert Scheme 2 here>
<Insert Scheme 3 here>

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2.2. Pharmacology
Herein we describe the synthesis and biological evaluation of new quinoline series
analogues of melatonin. Binding affinity and intrinsic activity of prepared compounds were
determined using competition binding studies against human MT1 and MT2 receptor subtypes
stably transfected in Human Embryonic Kidney (HEK 293) cells or Chinese Hamster Ovarian
(CHO) cells, using 2-[125I]iodomelatonin as radioligand. At each receptor, binding affinities
were verified for more than fifty selective and non-selective molecules, either using the
transfected HEK 293 or the CHO cell lines. The correlations between affinities in HEK 293
and CHO cells are highly significant (r = 0.98), for both MT1 and MT2 receptors. This high
correlation allowed the comparison of the affinities on the receptors expressed in two different
cell types and to draw structure–activity relationships (SARs).
<Insert Figure 2 here>
2.3. Biological results
Obtained results of binding affinity and intrinsic activity are depicted in Tables 1 and 2.
First, are reported in Table 1 the chemical structures and binding affinities of the new
synthesized compounds. Two series of new quinoline derivatives were designed and
synthesized (2-MQ and 3-MQ). Are reported hereafter the biological results obtained from the
pharmacological evaluation of the synthesized compounds. First, 2-MQ-acetamide derivative
11a was prepared as strict bioisostere of melatonin. Investigation of compound 11a showed a
pharmacological profile close to that of agomelatine, the strict naphthalenic analogue of
melatonin (Table 1). Encouraged by this result, we then extended our study to the second
developed series, the 3-methoxy-quinoline (3-MQ). Therefore and in contrary to 2-MQ, the
3-MQ has led to decrease of 2 logs and 1 log in binding affinities respectively at both MT1
and MT2 receptor subtypes (compound 15a). Modulation of the acetamide function of 15a

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through its replacement by different groups that previously showed beneficial effects on
melatonin binding affinity such as cyclopropanamide, fluoroacetamide, carbamate, ureas and
thioureas (15b-15g) has decreased the binding affinities at melatonin receptors MT1 and MT2.
In light of the interesting profile of compound 11a, this compound was chosen as a lead for
further pharmacomodulations. In this matter, preparation of retroamide compounds 18a-c,
analogues of 11a has shown the emergence of an MT2 selectivity by conserving a sub-
nanomolar affinity at MT2 and a slight decrease of 1 log of the MT1 binding affinity.
Cyclopropanamide 18b and fluoroacetamide 18c derivatives didn’t show any improvement in
the melatonergic binding affinities, while confirming an MT2 selectivity of this series (68 and
63 for 18b and 18c respectively). Similarly, the replacement of the lead 11a acetamide group
by sulphonamides, carbamates, ureas and thioureas (11b -11i) has resulted in the conservation
of the binding affinity at a sub-nanomolar level. The sulphonamide 11b showed the most
important MT2 selectivity of this series (167 folds) while ureas 11f and 11g represented the
most potent melatonergic ligands of this study (11f, Ki (nM): MT1 = 0.6, MT2= 0.09; 11g, Ki
(nM): MT1 = 0.8, MT2= 0.09). Another series derived from 2-MQ represented by compound
12a, isomer of acetamide 11a with the amide side chain in position 7, was also prepared.
However, evaluation of the compound 12a has shown noticeable decrease (2 logs) in binding
affinities at both MT1 and MT2 and drove to the abandonment of this series.
<Insert Table 1 here>
The most potent compounds 11a, 11b, 11d-11g and 18a from this series were further
evaluated for their functional activity using [35S]GTPγS binding assay (Table 2). Like
agomelatine, compounds 11a and 11g behaved as non-selective full agonists of the
melatonergic receptors MT1 and MT2. Most of the other tested compounds revealed a partial
agonist profile at both MT1 and MT2 except compound 18g. This latter compound exhibited

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an interesting pharmacological profile with an intrinsic activity of MT1 partial agonism and
MT2 full agonism accompanied with a light MT2 selectivity (27 folds), and can be used as a
pharmacological tool.
<Insert Table 2 here>
4. Conclusions
In conclusion, we report in this paper the synthesis and binding affinity results of new
quinoline derivatives issued from two series. Modulation of the amide group has led to new
melatonergic ligands with interesting profiles. The 2-MQ (2-methoxyquinoline) family (11a
and 18a) was revealed to be more convenient for the indole replacement than it was the 3-MQ
family (15a). Moreover, modulation of the acetamide function by its substitution by
cyclopropanamide, fluoroacetamide, sulfonamide, carbamate, urea and thiourea groups
produced decrease of binding affinity especially at MT1 leading to the appearance of an MT2
selectivity. Compounds 18b and 11b showed the most important MT2 selectivity of this series
(86 and 167 folds respectively). Substitution of acetamide group in 11a by methyl and
ethylurea (compounds 11f and 11g) produced the most interesting derivatives of this series
with binding affinities at melatonin receptors at the subnanomolar range (11f: Ki (nM), MT1=
0.6 and MT2= 0.09; 11f: Ki (nM), MT1= 0.8 and MT2= 0.09). Additional work is required in
order to study the behaviour of these derivatives in the binding pockets of MT1 and MT2.

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5. Experimental section
5.1. Chemistry
Chemicals and solvents were obtained from commercial sources, and used without
further purification unless otherwise noted. Reactions were monitored by TLC performed on
Macherey–Nagel Alugram® Sil 60/UV254 sheets (thickness 0.2 mm). Purification of products
was carried out by recrystallization or column chromatography. Column chromatography was
carried out using Macherey–Nagel silica gel (230–400 mesh). Melting points were determined
on a Büchi SMP-20 capillary apparatus and are uncorrected. FT-IR spectra were recorded on
a Thermo Nicolet Avatar 320 FT-IR spectrometer. NMR spectra were recorded on a Bruker
1
13
DRX 300 spectrometer (operating at 300 MHz for H and 75 MHz for C). Chemical shifts
are expressed in ppm relative to either tetramethylsilane (TMS). Chemical shifts are reported
as position (δ in ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, p = pentet,
dd = double doublet, br = broad and m = multiplet), coupling constant (J in Hz), relative
integral and assignment. Mass spectra were recorded on a Varian triple quadrupole 1200W
mass spectrometer equipped with a non-polar C18 TSK-gel Super ODS (4.6 × 50 mm)
column, using electrospray ionization and a UV detector (diode array). HRMS-ESI spectra
were recorded on a Thermo Scientific Exactive spectrometer.
5.1.1. 8-Bromo-2-methoxyquinoline (4).
To a solution of 8-bromo-2-chloroquinoline (1) (6.06 g, 25 mmol) in 50 mL of
methanol, was added sodium methanolate (8.10 g, 150 mmol). The mixture was refluxed for
16 h, concentrated and partitioned between ethyl acetate and water. The organic layer was
washed with water, dried over MgSO4 and concentrated under reduced pressure to afford
compound 4 (97% yield); Mp 87-89 °C; 1H NMR (300 MHz, CDCl3): δ 7.98 (d, 1H, 8.7 Hz),
7.96 (dd, 1H, 7.8 Hz and 1.2 Hz), 7.69 (dd, 1H, 7.8 Hz and 1.2 Hz), 7.24 (t, 1H, 7.8 Hz), 6.95
(d, 1H, 8.7 Hz), 4,16 (s, 3H); MS (APCI, pos. 30 V) m/z: [M+H]+, 239.08.

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5.1.2. 7-Bromo-2-methoxyquinoline (5).
This compound was prepared from 7-bromo-2-chloroquinoline (2) in 98% yield as
1
described for compound 1; Mp 68-69 °C; H NMR (300 MHz, CDCl3): δ 8.05 (d, 1H, 1.8
Hz), 7.93 (d, 1H, 8.7 Hz), 7.57 (d, 1H, 9.0 Hz), 7.47 (dd, 1H, 8.7 Hz and 1.8 Hz), 6.91 (d, 1H,
9.0 Hz), 4.00 (s, 3H); MS (APCI, pos. 30 V) m/z: [M+H]+, 239.06.
5.1.3. 5-Bromo-3-methoxyquinoline (6).
Step 1: To a solution of 3-bromoquinoline (3) (6.5 mL, 48 mmol) in dimethylformamide
(80 mL) were added sodium methanolate 30% (16 mL, 54 mmol) and copper iodide (458 mg,
2.4 mmol). The mixture was refluxed for 16 h, hydrolyzed and extracted with Et2O. The
organic layer was washed with water and brine, dried over MgSO4 and evaporated under
1
vacuum to afford 3-methoxyquinoline in 94% yield as a colorless oil ; H NMR (300 MHz,
CDCl3) δ : 8.45 (d, 1H, 2.9 Hz), 7,85 (d, 1H, 8.1 Hz and 1.2 Hz), 7.49 (d, 1H, 8.1 Hz and 1.2
Hz), 7.36-7.25 (m, 2H), 7.12 (d, 1H, 2.9 Hz), 3.67 (s, 3H) ; MS (APCI, pos. 30 V) m/z:
[M+H]+, 160.
Step 2: To a solution of 3-methoxyquinoline (7.96 g, 50 mmol) in sulfuric acid (30 mL)
was added dropwise NBS (8.90 g, 50 mmol) at 0 °C. After stirring at room temperature for 4
h, the mixture was hydrolyzed and alkalinized with 3M solution of NaOH. The formed solid
was filtrated and the filtrate was extracted with EtOAc. The organic layer was washed with
water and brine, dried over MgSO4 and evaporated under vacuum. The crude product was
purified by flash chromatography (cyclohexane/EtOAc : 6/4) to afford compound 6 (90%) as
1
a white solid ; mp 81-83°C ; H NMR (300 MHz, CDCl3) δ : 8.70 (d, 1H, 2.4 Hz), 8.00 (d,
1H, 8.1 Hz), 7.92 (d, 1H, 8.1 Hz), 7.67 (d, 1H, 2.4 Hz), 7.52 (t, 1H, 8.1 Hz), 3.99 (s, 3H) ; IR
(υ, cm-1, KBr) : 803 (CBr) ; MS (APCI, pos. 30 V) m/z: [M+H]+, 239, 241.
5.1.4. (E)-N-(2-(2-Methoxyquinolin-8-yl)vinyl)isoindoline-1,3-dione (7).

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In a sealed tube under argon atmosphere, were introduced compound 4 (4.74 g, 20
mmol), DMF (20 mL), triethylamine (11.16 mL, 80 mmol), palladium diacetate (225 mg, 1
mmol), tri(o-tolyl)-phosphine (609 mg, 2 mmol) and N-vinylphthalimide (5.20 g, 30 mmol).
The mixture was stirred at 110 °C for 3 h and hydrolyzed. The formed solid was solubilized in
CH2Cl2, washed with water and brine, dried over MgSO4 and evaporated under reduced
pressure. The crude product was recrystallized from acetonitrile to afford 7 as a white solid
1
(91% yield); Mp 199-200 °C; H NMR (300 MHz, CDCl3): δ 8.49 (d, 1H, 15 Hz), 8.25 (d,
1H, 15 Hz), 8.00 (d, 1H, 9.0 Hz), 7.92 (m, 2H), 7.81 (d, 1H, 7.8 Hz), 7.76 (m, 2H), 7.65 (d,
1H, 7.8 Hz), 7.38 (t, 1H, 7.8 Hz), 6.96 (d, 1H, 9.0 Hz), 4.23 (s, 3H); IR (υ, cm-1, KBr): 1715
(C=O); MS (APCI, pos. 30 V) m/z: [M+H]+, 331.34. HRMS (ESI+): m/z = calcd. for
C20H15N2O3 [M+H]+ 331.10772 found: 331.10606.
5.1.5. (E)-N-(2-(2-Methoxyquinolin-7-yl)vinyl)isoindoline-1,3-dione (8).
This compound was obtained in 80% yield from compound 5 as described for
1
compound 7; Mp 179-180 °C; H NMR (300 MHz, CDCl3): δ 7.97-7.92 (m, 4H), 7.86-7.77
(m, 3H), 7.70 (d, 1H, 8.4 Hz), 7.56-7.52 (m, 2H), 6.87 (d, 1H, 8.7 Hz), 4.11 (s, 3H); IR (υ,
cm-1, KBr): 1716 (C=O); MS (APCI, pos. 30 V) m/z: [M+H]+, 331.34. HRMS (ESI+):
m/z = calcd. for C20H15N2O3 [M+H]+ 331.10772 found: 331.10606.
5.1.6. (E)-N-(2-(3-Methoxyquinolin-5-yl)vinyl)isoindoline-1,3-dione (13).
This compound was obtained in 88% yield from compound 6 as described for
1
compound 7; Mp 205-206 °C; H NMR (300 MHz, CDCl3): δ 8.70 (d, 1H, 2.4 Hz), 8.13 (d,
1H, 14.7 Hz), 8.00-7.90 (m, 5H), 7.81 (d, 1H, 7.5 Hz), 7.73 (d, 1H, 2.4 Hz), 7.61 (t, 1H, 7.5
Hz), 7.28 (d, 1H, 14.7 Hz), 3.98 (s, 3H); IR (υ, cm-1, KBr): 1717 (C=O); MS (APCI, pos. 30
V) m/z: [M+H]+, 331.34. HRMS (ESI+): m/z = calcd. for C20H15N2O3 [M+H]+ 331.10772
found: 331.10696.
5.1.7. N-(2-(2-Methoxyquinolin-8-yl)ethyl)isoindoline-1,3-dione (9).

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To a solution of compound 7 (3.30 g, 10 mmol) in EtOH (30 mL) was added Pd/C 10%
(0.1 g). The mixture was stirred and placed under hydrogen atmosphere at room temperature
overnight. The catalyst was filtered on celite and the solvent was evaporated under reduced
pressure. The obtained compound was recrystallized from toluene to afford 9 as a white solid
1
(95% yield); Mp 150-152 °C; H NMR (300 MHz, CDCl3): δ 7.95 (d, 1H, 9.0 Hz), 7.80-7.69
(m, 4H), 7.60 (d, 1H, 7.8 Hz), 7.52 (d, 1H, 7.8 Hz), 7.27 (t, 1H, 7.8 Hz), 6.88 (d, 1H, 9.0 Hz),
4.19 (s, 3H), 4.15 (t, 2H, 7.5 Hz), 3.53 (t, 2H, 7.5 Hz); IR (υ, cm-1, KBr): 1702 (C=O); MS
(APCI, pos. 30 V) m/z: [M+H]+, 333.35. HRMS (ESI+): m/z = calcd. for C20H17N2O3 [M+H]+
333.12337 found: 333.12259.
5.1.8. N-(2-(2-Methoxyquinolin-7-yl)ethyl)isoindoline-1,3-dione (10).
This compound was prepared in 84% yield from 8 as described for compound 9; Mp
1
164-166 °C ; H NMR (300 MHz, CDCl3): δ 7.94 (d, 1H, 8.7 Hz), 7.87-7.81 (m, 2H), 7.77
(mt, 1H), 7.74-7.70 (m, 2H), 7.66 (d, 1H, 8.1 Hz), 7.33 (dd, 1H, 8.1 Hz and 1.8 Hz), 6.87 (d,
1H, 8.7 Hz), 4.06-4.01 (m, 5H), 3.17 (t, 2H, 7.8 Hz); IR (υ, cm-1, KBr): 1712 (C=O); MS
(APCI, pos. 30 V) m/z: [M+H]+, 333.35. HRMS (ESI+): m/z = calcd. for C20H17N2O3 [M+H]+
333.12337 found: 333.12259.
5.1.9. N-(2-(3-Methoxyquinolin-5-yl)ethyl)isoindoline-1,3-dione (14).
This compound was obtained in 95% yield from 13 as described previously for
compound 9; Mp 173-175 °C; 1H NMR (300 MHz, CDCl3): δ 8.70 (d, 1H, 2.7 Hz), 8.02-7.97
(m, 2H), 7.90-7.73 (m, 4H), 7.53-7.46 (m, 2H), 4.13 (s, 3H), 3.99 (mt, 2H), 3.37 (mt, 2H); IR
(υ, cm-1, KBr): 1704 (C=O); MS (APCI, pos. 30 V) m/z: [M+H]+, 333.35. HRMS (ESI+):
m/z = calcd. for C20H17N2O3 [M+H]+ 333.12337 found: 333.12229.
5.1.10. 2-(2-Methoxyquinolin-8-yl)ethanamine (11).
To a solution of compound 9 (3.32 g, 10 mmol) in 40 mL of ethanol, was added
hydrazine monohydrate (4.85 mL, 100 mmol). The mixture was refluxed overnight and

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filtered. The filtrate was concentrated under reduced pressure then solubilized in diethyl ether.
The organic layer was washed with water, dried over MgSO4 and evaporated under reduced
pressure. The residue was purified by column chromatography (CH2Cl2/CH3OH) : 8/2) to give
11 as a yellow oil in 83% yield; 1H NMR (300 MHz, CDCl3): δ 7.98 (d, 1H, 8.7 Hz), 7.61 (d,
1H, 7.8 Hz), 7.51 (d, 1H, 7.8 Hz), 7.31 (t, 1H, 7.8 Hz), 6.91 (d, 1H, 8.7 Hz), 4.07 (s, 3H), 3.36
(t, 2H, 6.6 Hz), 3.29 (mt, 2H), 3.21 (t, 2H, 6.6 Hz); IR (υ, cm-1, KBr): 3366 (NH2); MS
(APCI, pos. 30 V) m/z: [M+H]+, 203.25. HRMS (ESI+): m/z = calcd. for C12H15N2O [M+H]+
203.11789 found: 203.11718.
5.1.11. 2-(2-Methoxyquinolin-7-yl)ethanamine (12).
This compound was obtained from compound 10 as described for 11. The residue was
purified by column chromatography (CH2Cl2/CH3OH) : 8/2) to give 12 as a yellow oil in 86
1
% yield; H NMR (300 MHz, DMSO-d6): δ 8.16 (d, 1H, 9.0 Hz), 7.77 (d, 1H, 8.4 Hz), 7.58
(mt, 1H), 7.28 (dd, 1H, 8.4 Hz and 1.5 Hz), 7.93 (d, 1H, 9.0 Hz), 3.96 (s, 3H), 3.03 (br s, 2H),
2.88-2.80 (m, 4H); IR (υ, cm-1, KBr): 3365 (NH2); MS (APCI, pos. 30 V) m/z: [M+H]+,
203.25. HRMS (ESI+): m/z = calcd. for C12H15N2O [M+H]+ 203.11789 found: 203.11746.
5.1.12. 2-(3-Methoxyquinolin-5-yl)ethanamine (15).
This compound was obtained from compound 14 as described for 11. The residue was
purified by column chromatography (CH2Cl2/CH3OH) : 8/2) to give 15 as a yellow oil in 88%
yield; 1H NMR (300 MHz, CDCl3): δ 8.67 (d, 1H, 2.8 Hz), 7.93 (d, 1H, 8.4 Hz), 7.55 (d, 1H,
2.8 Hz), 7.47 (t, 1H, 8.4 Hz), 7.36 (d, 1H, 8.4 Hz), 4.82 (s, 2H), 3.96 (s, 3H), 3.19-3.06 (m,
4H); IR (υ, cm-1, KBr): 3022 (NH); MS (APCI, pos. 30 V) m/z: [M+H]+, 203.25. HRMS
(ESI+): m/z = calcd. for C12H15N2O [M+H]+ 203.11789 found: 203.11738.
5.1.13. (E)-Ethyl 3-(2-methoxyquinolin-8-yl)acrylate (16).
In a sealed tube under argon atmosphere, were introduced a solution of compound 3
(4.74 g, 20 mmol) in dry DMF (15 mL), triethylamine (3.35 mL, 24 mmol), PdCl2(PPh3)2

ACCEPTED MANUSCRIPT
(1.36 g, 2 mmol) and ethyl acrylate (2.61 mL, 24 mmol). The mixture was stirred at 120 °C
overnight and hydrolyzed then extracted twice with ethyl acetate. The organic layer was
washed, dried over MgSO4, filtered and evaporated under reduced pressure. The crude
product was purified by flash chromatography eluted with a mixture cyclohexane/ethyl
acetate (8:2) to afford compound 14 in 90% yield as a yellow solid; Mp 191-193 °C; 1H NMR
(300 MHz, CDCl3): δ 8.80 (d, 1H, 16.2 Hz), 8.00 (d, 1H, 9.0 Hz), 7.93 (dd, 1H, 8.1 Hz and
1.2 Hz), 7.77 (dd, 1H, 8.1 Hz and 1.2 Hz), 7.40 (t, 1H, 8.1 Hz), 6.97 (d, 1H, 9.0 Hz), 6.90 (d,
1H, 16.2 Hz), 4.32 (q, 2H, 7.2 Hz), 4.15 (s, 3H), 1.38 (t, 3H, 7.2 Hz); IR (υ, cm-1, KBr): 1720
(C=O); MS (APCI, pos. 30 V) m/z: [M+H]+, 258.28.
5.1.14. Ethyl 3-(2-methoxyquinolin-8-yl)propanoate (17).
This compound was prepared from compound 16 as described for compound 9. The
crude product was recrystallized from toluene to afford compound 17 in 88% yield as a white
solid; Mp 185-187 °C; 1H NMR (300 MHz, CDCl3): δ 7.97 (d, 1H, 9.0 Hz), 7.60 (dd, 1H, 8.1
Hz and 1.2 Hz), 7.52 (dd, 1H, 8.1 Hz and 1.2 Hz), 7.29 (t, 1H, 8.1 Hz), 6.91 (d, 1H, 9.0 Hz),
4.13 (q, 2H, 7.2 Hz), 4.08 (s, 3H), 3.48 (t, 2H, 7.8 Hz), 2.85 (t, 2H, 7.8 Hz), 1.23 (t, 3H, 7.2
Hz); IR (υ, cm-1, KBr): 1718 (C=O); MS (APCI, pos. 30 V) m/z: [M+H]+, 260.30.
5.1.15. 3-(2-Methoxyquinolin-8-yl)propanoic acid (18).
To a solution of compound 17 (7.77 g, 30 mmol) in 90 mL of ethanol, was added 15 mL
of an aqueous solution of 6 M NaOH. The mixture was stirred at room temperature overnight
and concentrated under reduced pressure. The mixture was then acidified with a solution of
2M HCl. The formed solid was solubilized in CH2Cl2 and the organic layer was washed, dried
over MgSO4 and evaporated under reduced pressure. The obtained compound was then
recrystallized from methanol to afford 18 as a white solid (88% yield); Mp 261-263 °C;
1H NMR (300 MHz, CDCl3): δ 7.99 (d, 1H, 9.0 Hz), 7.62 (dd, 1H, 8.1 Hz and 0.9 Hz), 7.53
(dd, 1H, 8.1 Hz and 0.9 Hz), 7.31 (t, 1H, 8.1 Hz), 6.92 (d, 1H, 9.0 Hz), 4.08 (s, 3H), 3.50 (t,

ACCEPTED MANUSCRIPT
2H, 7.8 Hz), 2.94 (t, 2H, 7.8 Hz); IR (υ, cm-1, KBr): 3033 (OH), 1719 (C=O); MS (APCI, pos.
30 V) m/z: [M+H]+, 232.25.
5.1.16. General procedure for preparation of amides 11a, 12a, and 15a-b.
Potassium carbonate (3 mmol) was added to a solution of the corresponding amine 11,
12 or 15 (1.0 mmol), in 30 mL of water and 50 mL of ethyl acetate. After stirring for 10 min
at 0°C, 1.5 mmol of the appropriate acid chloride was added dropwise at this temperature. The
reaction mixture was stirred at room temperature for 2 h. The organic phase was separated,
washed with a 1M HCl solution then with water, dried over MgSO4, filtered and concentrated
under reduced pressure. Recrystallization from the adequate solvent gave the desired amide:
N-(2-(2-Methoxyquinolin-8-yl)ethyl)acetamide (11a). Recrystallized from cyclohexane as a
1
white solid (84% yield); Mp 98-100 °C; H NMR (300 MHz, DMSO-d6): δ 8.19 (d, 1H, 8.7
Hz), 7.72-7.69 (m, 2H), 7.50 (d, 1H, 8.7 Hz), 7.32 (t, 1H, 8.7 Hz), 7.00 (d, 1H, 8.7 Hz), 4.00
(s, 3H), 3.41 (q, 2H, 6.7 Hz), 3.22 (t, 2H, 6.7 Hz), 1.89 (s, 3H); 13C NMR (75 MHz, DMSO-
d6): δ 170.5, 161.5, 155.3, 139.1, 133.7, 130.6, 126.9, 125.8, 124.6, 112.6, 55.9, 40.1, 31.9,
23.2; IR (υ, cm-1, KBr): 3251 (NH), 1662 (C=O); MS (APCI, pos. 30 V) m/z: [M+H]+,
245.12. HRMS (ESI+): m/z = calcd. for C14H17N2O2 [M+H]+ 245.12845 found: 245.12128
N-(2-(2-Methoxyquinolin-7-yl)ethyl)acetamide (12a). Recrystallized from cyclohexane as a
white solid (77% yield); Mp 104-106 °C; 1H NMR (300 MHz, CDCl3): δ 7.96 (d, 1H, 8.4 Hz),
7.67 (m, 2H), 7.23 (dd, 1H, 8.4 Hz and 1.8 Hz), 6.87 (d, 2H, 8.7 Hz), 5.50 (br s, 1H), 4.07 (s,
13
3H), 3.64 (m, 2H), 3.00 (t, 2H, 6.87 Hz), 1.94 (s, 3H); C NMR (75 MHz, CDCl3): δ 170.1,
162.7, 146.7, 140.5, 138.4, 127.7, 126.6, 125.1, 123.7, 112.7, 53.4, 40.4, 35.8, 23.3; IR (υ, cm-
1, KBr): 3254 (NH), 1656 (C=O); MS (APCI, pos. 30 V) m/z: [M+H]+, 245.19. HRMS
(ESI+): m/z = calcd. for C14H17N2O2 [M+H]+ 245.12845 found: 245.12128
N-(2-(3-Methoxyquinolin-5-yl)ethyl)acetamide (15a). Recrystallized from toluene as a
1
white solid (77% yield); Mp 120-122 °C; H NMR (300 MHz, DMSO-d6): δ 8.64 (d, 1H, 2.9

ACCEPTED MANUSCRIPT
Hz), 8.12 (br s, 1H), 8.03 (d, 1H, 2.9 Hz), 7.82 (d, 1H, 8.1 Hz), 7.48 (t, 1H, 8.1 Hz), 7.40 (d,
13
1H, 8.1 Hz), 3.99 (s, 3H), 3.31 (q, 2H, 7.1 Hz), 3.14 (t, 2H, 7.1 Hz), 1.82 (s, 3H); C NMR
(75 MHz, DMSO-d6): δ 170.1, 153.3, 144.0, 143.7, 135.6, 127.9 (2C), 127.8, 126.6, 109.9,
56.1, 40.3, 32.9, 23.1; IR (υ, cm-1, KBr): 3398 (NH), 1664 (C=O); MS (APCI, pos. 30 V) m/z:
[M+H]+, 245.12. HRMS (ESI+): m/z = calcd. for C14H17N2O2 [M+H]+ 245.12845 found:
245.12128
N-(2-(3-Methoxyquinolin-5-yl)ethyl)cyclopropanecarboxamide (15b). Recrystallized from
toluene as a white solid (56% yield); Mp 130-132 °C; 1H NMR (300 MHz, DMSO-d6): δ 8.64
(d, 1H, 2.9 Hz), 8.29 (br t, 1H, 5.5 Hz), 7.96 (d, 1H, 8.1 Hz), 7.83 (d, 1H, 2.9 Hz), 7.49 (t, 1H,
8.1 Hz), 7.40 (d, 1H, 8.1 Hz), 3.98 (s, 3H), 3.36 (q, 2H, 6.7 Hz), 3.16 (t, 2H, 6.7 Hz), 1.51
(mt, 1H), 0.71-0.60 (m, 4H); 13C NMR (75 MHz, DMSO-d6): δ 173.3, 153.3, 144.0, 143.7,
135.7, 127.9 (2C), 127.8, 126.6, 110.0, 56.1, 40.3, 32.9, 14.1, 6.6 (2C); IR (υ, cm-1, KBr):
3265 (NH), 1659 (C=O); MS (APCI, pos. 30 V) m/z: [M+H]+, 271.33. HRMS (ESI+):
m/z = calcd. for C16H19N2O2 [M+H]+ 271.14410 found: 271.14303.
5.1.17. 2-Fluoro-N-(2-(3-methoxyquinolin-5-yl)ethyl)acetamide (15c).
To a solution of amine 15 (0.4 g, 2 mmol) in 20 mL of 2,2,2-trifluoroethanol (TFE), was
added ethyl fluoroacetate (0.78 mL, 8 mmol) and the mixture was refluxed for 12 h. After
cooling, the mixture was hydrolyzed and extracted with diethyl ether. The organic phase was
washed with an aqueous solution of 1M HCl then water, dried over MgSO4, filtered and
concentrated under reduced pressure. Recrystallization from toluene gave 15c as a white solid
1
(40% yield); Mp 120-122 °C; H NMR (300 MHz, DMSO-d6): δ 8.64 (d, 1H, 2.9 Hz), 8.49
(br s, 1H), 7.99 (d, 1H, 2.9 Hz), 7.83 (d, 1H, 8.1 Hz), 7.48 (t, 1H, 8.1 Hz), 7.40 (d, 1H, 8.1
13
Hz), 4.92 (d, 2H, 47.0 Hz), 3.98 (s, 3H), 3.40 (q, 2H, 6.6 Hz), 3.19 (t, 2H, 6.6 Hz); C NMR
(75 MHz, DMSO-d6): δ 168.0 (d, JC-F= 75 Hz), 153.3, 144.0, 143.7, 135.3, 128.1, 127.9 (2C),
126.6, 109.8, 80.6 (d, JC-F= 718 Hz), 56.1, 39.7, 32.7; IR (υ, cm-1, KBr): 3328 (NH), 1661

ACCEPTED MANUSCRIPT
(C=O); MS (APCI, pos. 30 V) m/z: [M+H]+, 263.28. HRMS (ESI+): m/z = calcd. for
C14H16FN2O2 [M+H]+ 263.11903 found: 263.11788.
5.1.18. 3-(2-Methoxyquinolin-8-yl)-N-methylpropanamide (18a).
To a solution of acid 18 (0.5 g, 2.16 mmol) in 10 mL of CH2Cl2, was added thionyl
chloride (0.31 mL, 4.32 mmol). The mixture was stirred at room temperature for 2 h,
concentrated under reduced pressure. The obtained crude was then solubilized in 10 mL of
CH2Cl2, and methylamine (0.57 mL, 6.48 mmol) was added dropwise. The mixture was
stirred for 3 h, then hydrolyzed and extracted with CH2Cl2. The organic phase was washed
with an aqueous solution of 1 M NaOH and water, dried over MgSO4, filtered and
concentrated under reduced pressure. The crude product was recrystallized from toluene to
afford 18a as a white solid (57% yield); Mp 101-103 °C; 1H NMR (300 MHz, CDCl3): δ 7.98
(d, 1H, 9.0 Hz), 7.59 (dd, 1H, 8.1 Hz and 0.9 Hz), 7.51 (dd, 1H, 8.1 Hz and 0.9 Hz), 7.30 (t,
1H, 8.1 Hz), 6.91 (d, 1H, 9.0 Hz), 5.66 (br s, 1H), 4.08 (s, 3H), 3.49 (t, 2H, 7.2 Hz), 2.72 (t,
2H, 7.2 Hz), 2.72 (d, 3H, 4.8 Hz); 13C NMR (75 MHz, DMSO-d6): δ 172.6, 161.4, 144.5,
140.2, 138.1, 129.6, 126.4, 125.1, 124.2, 112.9, 53.4, 36.3, 27.7, 26.2; IR (υ, cm-1, KBr): 3327
(NH), 1649 (C=O); MS (APCI, pos. 30 V) m/z: [M+H]+, 245.29. HRMS (ESI+): m/z = calcd.
for C14H17N2O2 [M+H]+ 245.12845 found: 245.12775.
5.1.19. General procedure for synthesis of amides 18b and 18c.
To a solution of acid 18 (1 g, 4.32 mmol) in 100 mL of dry THF at 0 °C were added
HOBt (701 mg, 5.19 mmol), EDCI (806 mg, 5.19 mmol) and triethylamine (1.80 mL, 12.97
mmol). The mixture was stirred at for 30 min then the corresponding amine chloride (6.49
mmol) was added. The mixture was maintained under stir at room temperature for 2 h, then
hydrolyzed and extracted with CH2Cl2. The organic phase was washed with an aqueous
solution of 1 M NaOH and water, dried over MgSO4, filtered and concentrated under reduced
pressure.

ACCEPTED MANUSCRIPT
N-Cyclopropyl-3-(2-methoxyquinolin-8-yl)propanamide (18b). Recrystallized from
toluene as a white solid (72% yield); Mp 131-133 °C; 1H NMR (300 MHz, CDCl3): δ 7.98 (d,
1H, 8.7 Hz), 7.59 (d, 1H, 8.1 Hz), 7.50 (d, 1H, 8.1 Hz), 7.29 (t, 1H, 8.1 Hz), 6.91 (d, 1H, 8.7
Hz), 5.70 (br s, 1H), 4.07 (s, 3H), 3.47 (t, 2H, 7.2 Hz), 2.68 (t, 2H, 7.2 Hz), 2.60 (mt, 1H, 3.5
Hz), 0.65 (m, 2H), 0.22 (m, 2H); 13C NMR (75 MHz, DMSO-d6): δ 174.2, 161.6, 144.5,
139.3, 137.3, 129.7, 126.1, 125.0, 123.8, 112.6, 53.3, 37.3, 27.5, 22.4, 6.4 (2C); IR (υ, cm-1,
KBr): 3396 (NH), 1647 (C=O); MS (APCI, pos. 30 V) m/z: [M+H]+, 271.33. HRMS (ESI+):
m/z = calcd. for C16H19N2O2 [M+H]+ 271.14410 found: 271.14377.
N-(2-Fluoroethyl)-3-(2-methoxyquinolin-8-yl)propanamide (18c). Recrystallized from
toluene as a white solid (71% yield); Mp 111-113 °C; 1H NMR (300 MHz, DMSO-d6): δ 8.19
(d, 1H, 9.0 Hz), 8.04 (br t, 1H, 5.1 Hz), 7.71 (dd, 1H, 8.1 Hz and 1.2 Hz), 7.51 (dd, 1H, 8.1
Hz and 1.2 Hz), 7.32 (t, 1H, 8.1 Hz), 7.00 (d, 1H, 9.0 Hz), 4.37 (dt, 2H, 47.4 Hz and 4.8 Hz),
4.00 (s, 3H), 3.33 (m, 4H), 2.58 (t, 2H, 7.8 Hz); 13C NMR (75 MHz, DMSO-d6): δ 172.6,
161.5, 144.5, 140.2, 137.9, 129.7, 126.4, 125.1, 124.2, 112.9, 82.9 (d, JC-F= 656 Hz), 53.4,
39.6 (d, JC-F= 82 Hz), 36.2, 27.6; IR (υ, cm-1, KBr): 3248 (NH), 1647 (C=O); MS (APCI, pos.
30 V) m/z: [M+H]+, 277.31. HRMS (ESI+): m/z = calcd. for C15H18FN2O2 [M+H]+ 277.13468
found: 277.13000.
5.1.20. General procedure for sulfonamides 11b and 11c synthesis.
To a solution of amine 11 (404 mg, 2 mmol) in 50 mL of dry CH2Cl2, were added at 0
°C triethylamine (0.70 mL, 5 mmol) and the corresponding alkylsulfonyl chloride (2.6 mmol).
The mixture was then stirred at room temperature for 2 h and hydrolyzed. The organic phase
was washed with an aqueous solution of 1 M HCl and water, dried over MgSO4, filtered and
concentrated under reduced pressure.
N-(2-(2-Methoxyquinolin-8-yl)ethyl)methylsulfonamide (11b). Recrystallization from
1
toluene gave 11b as a white solid (78% yield); Mp 66-68 °C; H NMR (300 MHz, DMSO-

ACCEPTED MANUSCRIPT
d6): δ 8.20 (d, 1H, 9.0 Hz), 7.74 (dd, 1H, 8.1 Hz and 1.2 Hz), 7.56 (dd, 1H, 8.1 Hz and 1.2
Hz), 7.35 (t, 1H, 8.1 Hz), 7.17 (br t, 1H, 5.4 Hz), 7.01 (d, 1H, 9.0 Hz), 4.02 (s, 3H), 3.41-3.26
13
(m, 4H), 2.82 (s, 3H); C NMR (75 MHz, DMSO-d6): δ 161.6, 144.7, 140.2, 135.4, 130.6,
126.9, 125.2, 124.3, 53.5, 43.4, 39.8, 32.9; MS (APCI, pos. 30 V) m/z: [M+H]+, 281.34.
HRMS (ESI+): m/z = calcd. for C13H17N2O3S [M+H]+ 281.09544 found: 281.09475.
N-(2-(2-Methoxyquinolin-8-yl)ethyl)ethylsulfonamide (11c). Recrystallization from toluene
1
gave 11c as a white solid (84% yield); Mp 69-70°C; H NMR (300 MHz, DMSO-d6): δ 8.21
(d, 1H, 9.0 Hz), 7.75 (dd, 1H, 8.1 Hz and 1.5 Hz), 7.56 (dd, 1H, 8.1 Hz and 1.5 Hz), 7.35 (t,
1H, 8.1 Hz), 7.21 (br t, 1H, 5.4 Hz), 7.01 (d, 1H, 9.0 Hz), 4.02 (s, 3H), 3.38-3.25 (m, 4H),
2.92 (q, 2H, 7.5 Hz), 1.12 (t, 3H, 7.5 Hz); 13C NMR (75 MHz, DMSO-d6): δ 161.6, 144.7,
140.2, 135.4, 130.7, 126.9, 125.2, 124.3, 113.0, 53.5, 45.7, 43.3, 33.1, 8.5; MS (APCI, pos. 30
V) m/z: [M+H]+, 295.37. HRMS (ESI+): m/z = calcd. for C14H19N2O3S [M+H]+ 295.11014
found: 295.11002.
5.1.21. Methyl (2-(2-methoxyquinolin-8-yl)ethyl)carbamate (11d).
To a solution of amine 11 (404 mg, 2 mmol) in 20 mL of CH2Cl2, were added
triethylamine (0.70 mL, 5 mmol) and methyl chloroformate (0.23 mL, 3 mmol). The mixture
was stirred overnight at room temperature then hydrolyzed. The organic phase was washed
with an aqueous solution of 1 M HCl and water, dried over MgSO4, filtered and concentrated
under reduced pressure. Recrystallization from toluene 11d as a white solid (65% yield); Mp
1
67-68 °C; H NMR (300 MHz, DMSO-d6): δ 8.19 (d, 1H, 8.7 Hz), 7.72 (dd, 1H, 8.1 Hz and
1.5 Hz), 7.50 (dd, 1H, 8.1 Hz and 1.5 Hz), 7.33 (t, 1H, 8.1 Hz), 7.21 (br t, 1H, 5.1 Hz), 7.00
(d, 1H, 8.7 Hz), 4.02 (s, 3H), 3.50 (s, 3H), 3.39 (q, 2H, 7.8 Hz), 3.23 (t, 2H, 7.8 Hz);
13C NMR (75 MHz, DMSO-d6): δ 161.5, 157.1, 144.8, 140.1, 136.0, 130.4, 126.6, 125.2,
124.2, 112.9, 53.4, 51.6, 41.4, 32.4; MS (APCI, pos. 30 V) m/z: [M+H]+, 261.29. HRMS
(ESI+): m/z = calcd. for C14H17N2O3 [M+H]+ 261.12337 found: 261.12249.

ACCEPTED MANUSCRIPT
5.1.22. General procedure for ureas 11e and 15d synthesis.
To a solution of amine 11 or 15 (2.0 mmol) in 10 mL of water, were added 1 mL of 1 M
HCl and potassium cyanate (2.4 mmol). The mixture was stirred at room temperature for 2 h,
hydrolyzed and extracted with ethyl acetate. The organic phase was washed with water, dried
over MgSO4, filtered and concentrated under reduced pressure. The obtained product was
then purified by flash chromatography using a mixture of cyclohexane/ethyl acetate (8:2).
N-(2-(2-Methoxyquinolin-8-yl)ethyl)urea (11e). Recrystallized from acetonitrile as a white
1
solid (34% yield); Mp 163-165 °C; H NMR (300 MHz, DMSO-d6): δ 8.20 (d, 1H, 8.7 Hz),
7.73 (d, 1H, 7.8 Hz), 7.51 (d, 1H, 7.8 Hz), 7.35 (t, 1H, 7.8 Hz), 7.01 (d, 1H, 8.7 Hz), 5.93 (br
s, 1H), 5.37 (br s, 2H), 4.02 (s, 3H), 3.37 (m, 2H), 3.21 (m, 2H); 13C NMR (75 MHz, DMSO-
d6): δ 161.5, 159.1, 144.8, 140.1, 136.4, 130.3, 126.5, 125.2, 124.2, 112.9, 53.4, 41.3, 32.7; IR
(υ, cm-1, KBr): 3423 (NH), 3338 (NH), 1627 (C=O); MS (APCI, pos. 30 V) m/z: [M+H]+,
246.28. HRMS (ESI+): m/z = calcd. for C13H16N3O2 [M+H]+ 246.12370 found: 246.12271.
N-(2-(3-Methoxyquinolin-5-yl)ethyl)urea (15d). Recrystallized from acetonitrile as a white
1
solid (36% yield); Mp 213-215 °C; H NMR (300 MHz, DMSO-d6): δ 8.63 (d, 1H, 2.8 Hz),
8.10 (d, 1H, 2.8 Hz), 7.82 (d, 1H, 8.2 Hz), 7.49 (t, 1H, 8.2 Hz), 7.39 (d, 1H, 8.2 Hz), 6.13 (br
13
t, 1H, 5.3 Hz), 5.54 (br s, 2H), 3.99 (s, 3H), 3.29-3.14 (m, 4H); C NMR (75 MHz, DMSO-
d6): δ 159.5, 153.3, 144.0, 143.7, 136.1, 128.1, 127.8, 126.6, 110.2, 56.1, 41.0, 34.0; IR (υ,
cm-1, KBr): 3325 (NH), 3261 (NH), 1627 (C=O); MS (APCI, pos. 30 V) m/z: [M+H]+,
246.28. HRMS (ESI+): m/z = calcd. for C13H16N3O2 [M+H]+ 246.12370 found: 246.12269.
5.1.23. N-(2-(2-Methoxyquinolin-8-yl)ethyl)-N-methylurea (11f).
To a solution of amine 11 (0.8 g, 3.35 mmol) in 20 mL of DMSO, were added N-
methylphenylcarbamate (0.76 g, 5.03 mmol) and triethylamine (0.7 mL, 5.03 mmol). The
mixture was stirred and heated at 60 °C for 3 h, then hydrolyzed and extracted with CH2Cl2.
The organic layer was washed with an aqueous solution of 1 M HCl and water, dried over

ACCEPTED MANUSCRIPT
MgSO4, filtered and concentrated under reduced pressure. The obtained product was then
purified by flash chromatography using a mixture of cyclohexane/ethyl acetate (8:2).
Recrystallization from toluene gave 11f as a white solid (45% yield); Mp 142-144 °C;
1H NMR (300 MHz, CDCl3): δ 7.98 (d, 1H, 8.7 Hz), 7.62 (dd, 1H, 7.8 Hz and 1.2 Hz), 7.51
(dd, 1H, 7.8 Hz and 1.2 Hz), 7.32 (t, 1H, 7.8 Hz), 6.92 (d, 1H, 8.7 Hz), 4.80 (br s, 1H), 4.23
(br s, 1H), 4.09 (s, 3H), 3.60 (q, 2H, 6.6 Hz), 3.38 (t, 2H, 6.6 Hz), 2.67 (d, 3H, 4.8 Hz);
13C NMR (75 MHz, CDCl3): δ 161.7, 159.1, 145.0, 139.3, 136.1, 130.2, 126.3, 125.1, 123.9,
112.7, 53.4, 41.6, 32.0, 27.1; IR (υ, cm-1, KBr): 3368 (NH), 3291 (NH), 1653 (C=O); MS
(APCI, pos. 30 V) m/z: [M+H]+, 260.30. HRMS (ESI+): m/z = calcd. for C14H17N3O2 [M+H]+
260.13935 found: 260.13831.
5.1.24. General procedure for synthesis of alkyl ureas 11g, 15e and thioureas 11h, 11i, 15f,
15g.
To a solution of amine 11 or 15 (2.0 mmol) in 10 mL of CH2Cl2, were added at 0 °C
triethylamine (42 mmol), the corresponding alkyl isocyanate or isothiocyanate (2.2 mmol) and
the reaction mixture was stirred at room temperature for 2 h. The mixture was then
hydrolyzed and extracted twice with CH2Cl2. The organic phase was washed with an aqueous
solution of 1 M HCl then with water, dried over MgSO4, filtered and concentrated under
reduced pressure. The obtained product was then purified by flash chromatography using a
mixture of cyclohexane/ethyl acetate (8:2).
N-Ethyl-N-(2-(2-methoxyquinolin-8-yl)ethyl)urea (11g). Recrystallized from toluene as a
1
white solid in 86% yield; Mp 98-100 °C; H NMR (300 MHz, DMSO-d6): δ 8.20 (d, 1H, 8.7
Hz), 7.72 (d, 1H, 8.7 Hz), 7.50 (d, 1H, 8.7 Hz), 7.34 (t, 1H, 8.7 Hz), 7.00 (d, 1H, 8.7 Hz),
5.80-5.72 (m, 2H), 4.01 (s, 3H), 3.40 (q, 2H, 6.7 Hz), 3.20 (t, 2H, 6.7 Hz), 2.98 (mt, 2H), 0.95
13
(t, 3H, 7.2 Hz); C NMR (75 MHz, DMSO-d6): δ 161.5, 158.4, 144.8, 140.1, 136.5, 130.3,
126.5, 125.2, 124.2, 112.9, 53.4, 40.4, 34.5, 32.8, 16.2; IR (υ, cm-1, KBr): 3325 (NH), 3289

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(NH), 1613 (C=O); MS (APCI, pos. 30 V) m/z: [M+H]+, 274.33. HRMS (ESI+): m/z = calcd.
for C15H20N3O2 [M+H]+ 274.15500 found: 274.15421.
N-Ethyl-N-(2-(3-methoxyquinolin-5-yl)ethyl)urea (15e). Recrystallized from toluene as a
white solid in 68% yield; Mp 137-139 °C; 1H NMR (300 MHz, DMSO-d6): δ 8.63 (d, 1H, 2.1
Hz), 8.04 (d, 1H, 2.1 Hz), 7.82 (d, 1H, 8.1 Hz), 7.48 (t, 1H, 8.1 Hz), 7.38 (d, 1H, 8.1 Hz), 5.99
(br s, 1H), 5.89 (br s, 1H), 3.99 (s, 3H), 3.30-2.98 (m, 6H), 0.98 (t, 3H, 7.0 Hz); 13C NMR (75
MHz, DMSO-d6): δ 158.7, 153.3, 144.0, 143.7, 136.1, 128.1, 127.8 (2C), 126.6, 110.2, 56.0,
41.0, 34.6, 33.9, 16.1; IR (υ, cm-1, KBr): 3318 (NH), 3251 (NH), 1585 (C=O); MS (APCI,
pos. 30 V) m/z: [M+H]+, 274.33. HRMS (ESI+): m/z = calcd. for C15H20N3O2 [M+H]+
274.15500 found: 274.15431.
N-(2-(2-Methoxyquinolin-8-yl)ethyl)-N-methylthiourea (11h). Recrystallized from toluene
as a white solid (70% yield); Mp 100-102 °C; 1H NMR (300 MHz, CDCl3): δ 8.05 (d, 1H, 8.9
Hz), 7.65 (dd, 1H, 7.5 Hz and 1.2 Hz), 7.57 (dd, 1H, 7.5 Hz and 1.2 Hz), 7.36-7.32 (m, 3H),
6.96 (d, 1H, 8.9 Hz), 4.16 (s, 3H), 3.81 (q, 2H, 6.5 Hz), 3.48 (t, 2H, 6.5Hz), 2.84 (s, 3H);
13C NMR (75 MHz, DMSO-d6): δ 181.8, 161.0, 145.1, 140.3, 136.3, 130.3, 127.1, 125.2,
124.4, 113.2, 54.3, 43.9, 39.3, 35.7; IR (υ, cm-1, KBr): 3318 (NH), 1269 (C=S); MS (APCI,
pos. 30 V) m/z: [M+H]+, 276.37. HRMS (ESI+): m/z = calcd. for C14H18N3OS[M+H]+
276.10923 found: 276.10884.
N-Ethyl-N-(2-(2-methoxyquinolin-8-yl)ethyl)thiourea (11i). Recrystallized from toluene as
1
a white solid to afford 11i (76%); Mp 94-96 °C; H NMR (300 MHz, CDCl3): δ 8.21 (d, 1H,
9.0 Hz), 7.74 (d, 1H, 8.7 Hz), 7.55 (d, 1H, 8.7 Hz), 7.39-7.33 (m, 3H), 7.01 (d, 1H, 9.0 Hz),
4.04 (s, 3H), 3.74 (q, 2H, 6.7 Hz), 3.35-3.31 (m, 4H), 1.02 (t, 3H, 7.2 Hz); 13C NMR (75
MHz, DMSO-d6): δ 181.9, 161.6, 144.8, 140.2, 136.1, 130.4, 126.0, 125.2, 124.3, 113.0, 53.6,
44.6, 38.6, 31.2, 14.9; IR (υ, cm-1, KBr): 3239 (NH), 1269 (C=S); MS (APCI, pos. 30 V) m/z:

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[M+H]+, 290.40. HRMS (ESI+): m/z = calcd. for C15H20N3OS[M+H]+ 290.13216 found:
290.13112.
N-(2-(3-Methoxyquinolin-5-yl)ethyl)-N-methylthiourea (15f). Recrystallized from toluene
as a white solid (80% yield); Mp 127-129 °C; 1H NMR (300 MHz, CDCl3): δ 8.64 (d, 1H, 2.9
Hz), 8.04 (d, 1H, 2.9 Hz), 7.91 (d, 1H, 8.4 Hz), 7.46 (t, 1H, 8.4 Hz), 7.36 (d, 1H, 8.4 Hz), 6.20
(br s, 1H), 6,00 (br s, 1H), 4.03 (s, 3H), 3.92 (q, 2H, 7.9 Hz), 3.40 (t, 2H, 7.9 Hz), 2.87 (d, 3H,
13
4.1 Hz); C NMR (75 MHz, DMSO-d6): δ 182.4, 153.2, 144.4, 143.5, 136.0, 128.2, 128.1,
127.8, 126.5, 110.4, 56.2, 44.8, 38.6, 32.8; IR (υ, cm-1, KBr): 3213 (NH), 1258 (C=S); MS
(APCI, pos. 30 V) m/z: [M+H]+, 276.37. HRMS (ESI+): m/z = calcd. for C14H18N3OS[M+H]+
276.10923 found: 276.10864.
N-Ethyl-N-(2-(3-methoxyquinolin-5-yl)ethyl)thiourea (15g). Recrystallized from toluene as
a white solid (75% yield); Mp 135-137 °C; 1H NMR (300 MHz, CDCl3): δ 8.63 (m, 1H), 8.15
(m, 1H), 7.83 (d, 1H, 8.0 Hz), 7.52-7.42 (m, 4H), 3.98 (s, 3H), 3.67-3.28 (m, 6H), 1.04 (t, 3H,
13
6.8 Hz); C NMR (75 MHz, DMSO-d6): δ 182.4, 153.3, 144.1, 143.7, 135.8, 128.2, 128.1,
127.9, 126.6, 110.3, 56.3, 45.1, 38.6, 32.8, 14.8; IR (υ, cm-1, KBr): 3210 (NH), 1252 (C=S);
MS (APCI, pos. 30 V) m/z: [M+H]+, 290.40. HRMS (ESI+): m/z = calcd. for
C15H20N3OS[M+H]+ 290.13216 found: 290.13104.
5.2. Pharmacological methods
5.2.1. Reagents and Chemicals.
2-[125I]-Iodomelatonin (2200 Ci/mmol) was purchased from NEN (Boston, MA). Other
drugs and chemicals were purchased from Sigma-Aldrich (Saint Quentin, France).
5.2.2. Cell Culture.
HEK (provided by A.D. Strosberg, Paris, France) and CHO cell lines stably expressing
the human melatonin MT1 or MT2 receptors were grown in DMEM medium supplemented

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with 10% fetal calf serum, 2 mM glutamine, 100 IU/mL penicillin and 100 µg/ml
streptomycin. Grown at confluence at 37 °C (95%O2/5%CO2), they were harvested in PBS
containing EDTA 2 mM and centrifuged at 1000 x g for 5 min (4 °C). The resulting pellet
was suspended in TRIS 5 mM (pH 7.5), containing EDTA 2 mM and homogenized using a
Kinematica polytron. The homogenate was then centrifuged (95 000g, 30 min, 4 °C) and the
resulting pellet suspended in 75 mM TRIS (pH 7.5), 12.5 mM MgCl2 and 2 mM EDTA.
Aliquots of membrane preparations were stored at -80 °C until use.
5.2.3. Binding Assays.
2-[125I]iodomelatonin binding assay conditions were essentially as previously described
[46]. Briefly, binding was initiated by addition of membrane preparations from stable
transfected HEK or CHO cells diluted in binding buffer (50 mM Tris-HCl buffer, pH 7.4
containing 5 mM MgCl2) to 2-[125I]-iodomelatonin (25 or 200 pM for MT1 and MT2
receptors, respectively, expressed in HEK cells or 20 pM for MT1 and MT2 receptors
expressed in CHO cells) and the tested drug. Nonspecific binding was defined in the presence
of 1 µM melatonin. After 120 min incubation at 37 °C, reaction was stopped by rapid
filtration through GF/B filters presoaked in 0.5% (v/v) polyethylenimine. Filters were washed
three times with 1 ml of ice-cold 50 mM Tris-HCl buffer, pH 7.4.
Data from the dose-response curves (7 concentrations in duplicate) were analysed using
PRISM program (Graph Pad Software Inc., San Diego, CA) to yield IC50 (inhibitory
concentration 50). Results are expressed as Ki= IC50 / 1 + ([L]/KD), where [L] is the
concentration of radioligand used in the assay and KD, the dissociation constant of the
radioligand characterising the membrane preparation [47].
[35S] GTPγS binding assay was performed according to published methodology [46]. Briefly,
membranes from transfected CHO cells expressing MT1 or MT2 receptor subtype and
compounds were diluted in binding buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 3 µM

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GDP, 3 mM MgCl2, and 20 µg/mL saponin). Incubation was started by the addition of 0.2 nM
[35S]GTPγS to membranes (20 µg/ml) and drugs, and further followed for 1 h at room
temperature. For experiments with antagonists, membranes were pre-incubated with both the
melatonin (3 nM) and the antagonist for 30 min prior the addition of [35S]GTPγS. Non
specific binding was defined using cold GTPγS (10 µM). Reaction was stopped by rapid
filtration through GF/B filters followed by three successive washes with ice cold buffer.
Usual levels of [35S]GTPγs binding (expressed in dpm) were for CHO-MT1 or MT2
membranes: 2000 for basal activity, 8000 in the presence of melatonin 1 µM and 180 in the
presence of GTPγS 10 µM which defined the non specific binding. Data from the dose-
response curves (7 concentrations in duplicate) were analyzed by using the program PRISM
(Graph Pad Software Inc., San Diego, CA) to yield EC50 (Effective concentration 50 %) and
Emax (maximal effect) for agonists. Antagonist potencies are expressed as KB= IC50 / 1 +
([Ago]/EC50 ago), where IC50 is the inhibitory concentration of antagonist that gives 50%
inhibition of [35S] GTPγS binding in the presence of a fixed concentration of melatonin
([Ago]) and EC50 ago is the EC50 of the molecule when tested alone. Imax (maximal inhibitory
effect) was expressed as a percentage of that observed with melatonin at 3 nM for MT2
receptor.
Acknowledgement
Our acknowledgments go to the Region Hauts de France (France), the Ministère de la
Jeunesse, de l’Education Nationale et de la Recherche (MJENR) and the Fonds Européens de
Développement Régional (FEDER) for funds allowed for NMR facilities.

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