Quinazoline and phthalazine derivatives as novel melatonin receptor ligands analogues of agomelatine

Article abstract of DOI:10.1016/j.ejmech.2020.112078

For further development of successors of Agomelatine through modulation of its pharmacokinetic properties, we report herein the design, synthesis and pharmacological results of a new family of melatonin receptor ligands. Issued from the introduction of quinazoline and phthalazine scaffolds carrying an ethyl amide lateral chain and a methoxy group as bioisosteric ligands analogues of previously developed Agomelatine. The biological activity of the prepared analogues was compared with that of Agomelatine. Quinazoline and phthalazine rings proved to be a versatile scaffold for easy feasible MT₁ and MT₂ ligands. Potent agonists with sub-micromolar binding affinity were obtained. However, the presence of two nitrogen atoms resulted in compounds with lower affinity for both MT₁ and MT₂, in comparison with the parent compound, balanced by the exhibition of good pharmacokinetic properties.

Full text of DOI:10.1016/j.ejmech.2020.112078

Journal Pre-proof
Quinazoline and phthalazine derivatives as novel melatonin receptor ligands
analogues of agomelatine
Raphaël Bolteau, Florian Descamps, Mohamed Ettaoussi, Daniel H. Caignard,
Philippe Delagrange, Patricia Melnyk, Saïd Yous
PII:
S0223-5234(20)30045-3
DOI:
https://doi.org/10.1016/j.ejmech.2020.112078
EJMECH 112078
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 September 2019
Revised Date: 12 November 2019
Accepted Date: 16 January 2020
Please cite this article as: Raphaë. Bolteau, F. Descamps, M. Ettaoussi, D.H. Caignard, P. Delagrange,
P. Melnyk, Saï. Yous, Quinazoline and phthalazine derivatives as novel melatonin receptor ligands
analogues of agomelatine, European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/
j.ejmech.2020.112078.
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Graphical Abstract

Quinazoline and Phthalazine Derivatives as Novel Melatonin Receptor
Ligands Analogues of Agomelatine
Raphaël Bolteau,a┴ Florian Descamps,a┴ Mohamed Ettaoussi,a* Daniel H Caignard,b,c Philippe
Delagrange,b Patricia Melnyk,a and Saïd Yousa*
aUniv. Lille, Inserm, CHU Lille, UMR-S1172 - JPArc - Centre de Recherche Jean-Pierre AUBERT
Neurosciences et Cancer, F-59000 Lille, France
bPEX Biotechnologie Chimie & Biologie, Institut de Recherches Servier, 78290 Croissy sur Seine,
France
cInstitut de Recherches Internationales Servier, 92150 Suresnes, France
┴These authors contributed equally
Corresponding Authors:
*Corresponding author. aUniversity of Lille, Inserm, CHU Lille, UMR-S1172 - JPArc - Centre de
Recherche Jean-Pierre AUBERT Neurosciences et Cancer, F-59000 Lille, France. Emails:
m.ettaoussi@yahoo.fr; said.yous@univ-lille2.fr

Abstract
For further development of successors of Agomelatine through modulation of its
pharmacokinetic properties, we report herein the design, synthesis and pharmacological
results of a new family of melatonin receptor ligands. Issued from the introduction of
quinazoline and phthalazine scaffolds carrying an ethyl amide lateral chain and a methoxy
group as bioisosteric ligands analogs of previously developed Agomelatine. The biological
activity of the prepared analogs was compared with that of Agomelatine. Quinazoline and
phthalazine rings proved to be a versatile scaffold for easy feasible MT1 and MT2 ligands.
Potent agonists with sub-micromolar binding affinity were obtained. However, the presence
of two nitrogen atoms resulted in compounds with lower affinity for both MT1 and MT2, in
comparison with the parent compound, balanced by the exhibition of good pharmacokinetic
properties.
Key words
Agomelatine, agonist, melatonin receptor, MT1, MT2, quinazoline, phthalazine
Abbreviations used
AGM, Agomelatine; MLT, Melatonin; MT1, melatonin receptor subtype 1; MT2, melatonin
receptor subtype 2

1.
Introduction
Melatonin (MLT) is a neurohormone mainly synthesized by the pineal gland following
a circadian rhythm [1]. This rhythmicity is important for melatonin physiological functions
[2]. Once synthesized, melatonin is then released into the bloodstream and travels to different
organs and tissues of the body [1] to obtain desirable physiological responses mainly on the
central nervous system (CNS) [3]. MLT regulates a variety of physiological functions through
the activation of two membrane receptors MT1 and MT2 [4]. Both, MT1 and MT2 receptor
subtypes are classified as class A G-protein coupled receptors (GPCRs). Since its discovery,
several studies have demonstrated the involvement of MLT and its receptors in the regulation
of different physiological processes such as sleep and wakefulness [5], core body temperature
and blood pressure [6]. Some studies have demonstrated possible role of MT1 and/or MT2
receptors in several disorders including insomnia and mood disorders, depression, anxiety,
Alzheimer, Parkinson’s diseases [7]. Melatonin was also demonstrated to possess antioxidant
and anti-inflammatory effects [8].
Consequently, the melatonergic system represents an interesting target for the
development of new therapeutic agents. Accordingly, research for melatonin receptor ligands
has led to the design and synthesis of several ligands. Many of these ligands have been or are
in their way to be used as therapeutic agents. Besides, Ramelteon a high affinity nonselective
MT1/MT2 receptor agonist was developed and commercialized for the treatment of insomnia
and sleep disorders [9]. More interestingly, the discovery of Agomelatine (AGM) was
ingenious because of its unique pharmacological profile. AGM is a high-affinity nonselective
MT1/MT2 melatonin receptor agonist and 5-HT2C serotonin receptor selective antagonist. It is
marketed as an antidepressant with a high efficacy on sleep, circadian rhythm disorder, and
depression [10]. However, up to recently the absence of experimental 3D models of MT1 and
MT2 hindered the structure-based drug design. Indeed, melatonin receptor models were

constructed and refined using 3D structures of different templates including serotonin
receptors such as the human 5-HT1B and 5-HT2B [11]. Fortunately, very recently a group of
researchers described high-resolution XFEL (X-ray free electron laser) structures of the
human MT1 receptor in complex with ramelteon, 2-phenylmelatonin, 2-iodomelatonin, and
AGM; and the human MT2 receptor in complex with 2-phenylmelatonin (2-PMT) and
ramelteon [12]. Progress in understanding the role of melatonin receptors will be boosted by
these structural findings of the MT1 and MT2 receptors subtypes. Moreover, the determination
of the human MT2 melatonin receptor subtype selectivity reported in this work will lead
certainly to the design and synthesis of highly selective ligands and hence the comprehension
of this system.
Following our contribution to the understanding of the melatonin system, we reported
the design and synthesis of analogues of agomelatine issued from the replacement of the
naphthalene scaffold by different heterocycles (Figure 1) [13]. Herein we report the synthesis
and the pharmacological evaluation of two aza-cycles bioisosteric analogues of AGM.
Quinazoline and phthalazine are two heterocycles that attracted much interest among the
scientific community and have been under deep investigation due to their different biological
properties. Besides, phthalazine was extensively used in the preparation of a variety of
bioactive molecules with different pharmacological properties including antimicrobial,
antibacterial, cardio tonic, anticancer, antifungal, vasodilatation activity, antimalarial,
anticonvulsant, and anti-inflammatory activities [14]. On the other hand, quinazoline is a
pharmacologically promising heterocycle due to its different biomedical properties. This
heterocycle is present in different powerful antibacterial, antiviral, antifungal, anti-
inflammatory, antimalarial, antitubercular and anti-HIV agents [14]. It is also present in some
inhibitors of the epidermal growth factor (EGF) receptors of tyrosine kinase. Quinazolines
were also employed as ligands for benzodiazepine and GABA-receptors in the CNS system or

as DNA binders, and as anticancer with remarkable activity [15]. Our main objective was to
investigate the effect of the pharmacokinetic properties modulation via the introduction of two
nitrogen atoms into the naphthalene cycle.
2.
Results and discussion
2.1. Chemistry
Synthesis of amides 7a-d is depicted in Scheme 1. First, key intermediate 3 was
prepared following two synthetic routes. Using 5-methoxytryptamine, the primary amine was
protected with a phthalimide group to give intermediate 1. Oxidative cleavage of the indole
heterocycle into compound 2 was achieved using NaIO4 in methanol [16]. Quinazoline 3 was
then obtained by cyclization of 2 in the presence of ammonium formate in formamide. The
second method employed the commercially available 6-methoxyquinazolin-2-one as starting
material. Chlorination of 6-methoxyquinazolin-2-one by POCl3 gave 4-chloroquinazoline 4.
Heck cross-coupling reaction of 4 with N-vinylphthalimide in the presence of palladium
acetate and tri(o-tolyl)phosphine [17] led to the unsaturated compound 5. Hydrogenation of
this compound gave the desired quinazoline 3. Deprotection of the phthalimide group via
hydrazinolysis in ethanol furnished the primary amine 6. Finally, target amides 7a-d were
synthesized from 6 by reaction with the appropriate acid chloride [18].
<Insert Scheme 1 here>
Synthesis of 2,4,6-trisubstituted quinazolines 11a-c is described in Scheme 2. First,
kinurenamine 8 was obtained by oxidative cleavage of melatonin under the same conditions
previously described for compound 2 [16]. Compound 8 was then submitted to a selective
acidic hydrolysis of its formamide function leading to primary amine 9. N-acylation of
compound 9 by treatment with different acyl chlorides leads to amides 10a-c. Substituted
quinazolines 11a-c were obtained by cyclization of compounds 10a-c under the same

conditions as previously described for compound 3. Moreover, this method also allowed
access to the acetamide 7a previously described in Scheme 1 directly from kinurenamine 8.
Amino-quinazoline 12 and piperazino-quinazolines 13-14 were prepared in one step (Scheme
3). Reaction of commercially available N-acetylethylene-diamine, 1-(cyclopropanoyl)-
piperazine and N-ethylpiperazine-1-carboxamide with 4-chloro-6-methoxyquinazoline 4 at
room temperature in the presence of triethylamine yielded the desired quinazolines 12-14 in
good yields.
<Insert Scheme 2 here>
<Insert Scheme 3 here>
The phthalazine derivatives were prepared following the method described in Scheme 4.
Therefore, phthalazines 20a-d were prepared in six steps starting from commercially available
2-formyl-5-methoxybenzoic acid. First, condensation of this later compound with hydrazine
in EtOH led to 7-methoxyphthalazin-(2H)-one (15) [19]. Chlorination of 15 with phosphorous
oxychloride followed by treatment with ethyl cyanoacetate in the presence of sodium hydride
(NaH) produced the cyanoester 17. Refluxing of compound 17 in an acidic medium (5M HCl)
led, after decarboxylation, to cyanomethyl 18. Selective catalytic hydrogenation of the cyano
group of 18, with Raney-nickel, provided the aminoethyl phthalazine 19 which was then
transformed into the desired amides 20a-d according to a variant of the Schotten-Baumann
procedure, by reaction with the appropriate acid chloride in the presence of potassium
carbonate in a biphasic medium (ethyl acetate/water).
<Insert Scheme 4 here>
2.2. Pharmacology
<Insert Table 1 here>
First, all synthesized compounds were submitted to binding affinity experiments to the
serotonergic 5-HT2C receptor subtype using Chinese Hamster Ovarian (CHO) cell lines stably

expressing the human 5-HT2C receptors and revealed the absence of any binding affinity or
activity towards this receptor subtype. Second, binding assays for melatonin MT1 and MT2
receptor subtypes consisted of addition of membrane preparations from transfected CHO cells
stably expressing the human melatonin MT1 or MT2 diluted in binding buffer (50 mM Tris–
HCl buffer, pH 7.4, containing 5 mM MgCl2) to 2-[125I]iodomelatonin (20 pM for MT1 and
MT2 receptors expressed in CHO cells) and the tested compound. Non-specific binding was
defined in the presence of 1 µM melatonin, and data from the dose-response curves were
analyzed using the program PRISM (Graph Pad Software Inc., San Diego, CA, USA) to yield
IC50. Results are expressed as pKi (pKi = -Log10 (Ki)) with Ki = IC50/1 + ([L]/KD), where
[L] is the concentration of radioligand used in the assay and KD, the dissociation constant of
the radioligand characterizing the membrane preparation [21]. Evaluation of the prepared
quinazoline and phthalazine derivatives for their affinities for melatonin receptors MT1 and
MT2 receptors subtypes using transfected CHO cells stably expressing the human melatonin
MT1 or MT2, revealed that strict bioisosteric replacement of the naphthalene moiety of AGM
with quinazoline or phthalazine derivatives decreased the affinity for both melatonergic
receptors MT1 and MT2 and showed weak MT2-selectivity (7a, Table 1; 20, Table 2). To
further investigate the effect of this pharmacomodulation on the melatonergic affinity, we
then analysed the effect of the replacement of the acetamide function of 7a by an ethylamide
(7b) and a propyl amide (7c). The obtained results showed a conservation of the MT1 affinity
and an improvement of MT2 binding affinity in comparison with the acetamide 7a. However,
the introduction of a cyclopropylamide (7c) decreased the binding affinities at both MT1 and
the MT2 (Table 1).
Further modulations of 7a were performed specially the introduction of aromatic
substituents at position C-2 of the quinazoline ring the equivalent position to C-3 of

agomelatine, site to hydroxylation during its metabolism. First, the introduction of a phenyl
(11a) or a m-methoxyphenyl (11b) has led to an increase of the MT1 and a decrease of MT2
binding affinities and the improvement of the MT2-selectivity (57 and 11-folds respectively)
in comparison with the parent compound 7a (Table 1). In contrary, the introduction of a
methyl cyclopropyl group (11c) showed the most interesting results of this series, an increase
in both MT1 and MT2 binding affinities in comparison with the parent 7a, and in accordance
with the previously observed effects on the naphthalene derivatives [20]. The ethyl amide side
chain was also replaced by an aminoethyl amide chain (12) or piperazine derivatives (13-14).
These two modulations have led to a loss in the melatonergic affinity as shown in Table 1.
This loss may result from the addition of a H-bond donor/acceptor (compound 12) and a
molecular restriction (compounds 13-14) which hinder the free rotation of the lateral chain
and hence disturb its binding to the receptor.
<Insert Table 2 here>
The observed decrease of the melatonergic affinity by the replacement of the
naphthalene scaffold with quinazoline was confirmed with the second modulation. In fact, the
modification of the two nitrogen atoms pattern via the introduction of a phthalazine ring
proved to be unfavourable for the melatonergic affinity. Accordingly, compound 20a, the
strict bioisostere of the AGM, showed a significant decrease in affinity for both MT1 and MT2
receptors in comparison with agomelatine and quinazoline 7a. Further modulations of the
acetamide group had no positive effect on the affinity (Table 2). The replacement of the
acetamide with an ethyl or a propyl group (compounds 20b-d) showed a weak improvement
of both melatonin MT1 and MT2 affinities in comparison with the acetamide 20a, and the
conservation of a weak MT2 selectivity over MT1. Finally, the replacement of the acetamide
over a cyclopropyl amide 20d has led to the drop in both MT1 and MT2 affinities (Table 2).
<Insert Table 3 here>

In addition, the intrinsic activities of the synthesized compounds were determined. The
obtained results revealed that most compounds behaved as MT1/MT2 full agonists except 11b
(Table 3). Compound 11b bearing a methoxyphenyl in the C2-position exhibited an MT1 full
agonist and MT2 partial agonist activity. Such a pharmacological profile is very sought in
pharmacology as a tool to assess the role of each receptor subtype. Moreover, to investigate
the pharmacokinetics of the prepared compounds, the calculation of the logP (ClogP) was
realized using Data Warrior software. The obtained results showed that due to the presence of
more nitrogen atoms in the skeleton, quinazoline and phthalazine derivatives are more
hydrophilic than agomelatine. This high hydrophilicity may grant good solubility and
absorption to these compounds and hence improve their pharmacokinetic properties (Table 4).
On another important aspect, drug absorption and permeability test (Caco2 test) of the most
interesting prepared compounds is shown in Table 5. Obtained results showed a 100%
absorption for the tested compounds except for the phthalazine 20a which exhibit only 2%
absorption hence confirming the ClogP results above (Table 4). Bioavailability results (Table
5) showed in general a good bioavailability in human and rodents for the tested compounds
except compound 11a which exhibits a very poor bioavailability of only 2 to 7% due to the
presence of the sterically hindered phenyl in the C-2 position of the quinazoline ring.
<Insert Table 4 here>
<Insert Table 5 here>
3.
Conclusions
Herein we investigated the effect of bioisosteric modulation of agomelatine through
replacement of the naphthalene moiety by quinazoline and phthalazine nucleus. Quinazoline
derivatives (7a-14) showed a decrease in the affinity for both MT1 and MT2 and the
appearance of an MT2-selectivty. The modulation of the C2-position of quinazoline led to

some interesting compounds with pronounced MT2-selectivty for compounds 11a-11b.
Conversely to quinazoline, the introduction of phthalazine decreased dramatically the
melatonergic affinity for both receptors. In fact, the introduction of two nitrogen atoms in the
naphthalene led to the drop of the melatonergic affinity. The disposition of the two nitrogen
seems to play a role in this decrease in the affinity. More interestingly, in general most tested
compounds showed good absorption and bioavailability. Finally, with the recent exciting
news of the description of both binding sites of melatonin MT1 and MT2 receptors, our group
is planning to use this new data to construct new 3D models and probe the binding modes of
these melatonin ligands. This model is primordial to explain why the introduction of nitrogen
atoms to the aromatic cycle bearing the acetamide lateral chain led to the drop of the
melatonergic binding affinity.
4.
Experimental Section
4.1. 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 DRX 300 spectrometer (operating at 300 MHz for 1H and 75 MHz
13
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. The purity of final compounds was
determined by high pressure liquid chromatography (HPLC) using two columns: C18
Interchrom UPTISPHERE. The HPLC analysis was carried out on a Shimadzu LC-2010AHT
system equipped with a UV detector set at 254 and 215 nm. The compounds were dissolved in
100 mL of buffer B and 900 mL of buffer A. The eluent system used was: buffer A
(H2O/TFA, 100:0.1) and buffer B (ACN/H2O/TFA, 80:20:0.1). Retention times (tr) were
obtained at a flow rate of 0.2 mL/min for 37 min using a gradient form 100% of buffer A over
1 min, to 100% buffer B over the next 30 min, to 100% of buffer A over 1 min and 100% of
buffer A over 1 min. All tested compounds showed a purity of > 95%. The melting point
analyses were performed on Barnstead Electrothermal Melting Point Series IA9200 and were
not corrected.
4.1.1. 2-(2-(5-Methoxy-1H-indol-3-yl)ethyl)isoindoline-1,3-dione (1). To a solution of 5-
methoxytryptamine (800 mg, 4.2 mmol) in toluene (30 mL) was added phthalic anhydride
(686 mg, 4.62 mmol) and Et3N (0.64 mL, 4.62 mmol). The reaction mixture was heated at
reflux for 24 h with continuous removal of water with a Dean−Stark trap. The mixture was
cooled to room temperature and concentrated under vacuum. The residue was putted into 1M
NaOH (20 mL) and extracted with DCM (3 x 100 mL). The combined organic layers were
washed with 1M HCl and water. The organic phase was dried with MgSO4, filtered, and the
solvent was evaporated under reduced pressure. The residue was purified by flash
chromatography (PE/AcOEt); 10:0 to 7:3 (v/v)) to afford 1 as a beige powder (87% yield); mp
165 °C; 1H NMR (300 MHz, CD2Cl2): δ 8.08 (br s, 1H), 7.86 – 7.80 (m, 2H), 7.88 – 7.69 (m,
2H), 7.26 (d, J = 8.8 Hz, 1H), 7.15 (d, J = 2.5 Hz, 1H), 7.09 (d, J = 2.1 Hz, 1H), 6.82 (dd, J =
13
8.8, 2.5 Hz, 1H), 3.99 (m, 2H), 3.84 (s, 3H), 3.13 (m, 2H); C NMR (75 MHz, CD2Cl2): δ
168.2, 154.0, 133.9, 132.2, 131.3, 127.8, 122.9, 112.2, 111.8, 100.3, 55.6, 38.4, 24.3

4.1.2. N-(2-(3-(1,3-Dioxoisoindolin-2-yl)propanoyl)-4-methoxyphenyl)formamide (2). To
solution of 1 (437 mg, 1.36 mmol) in methanol (26 mL) was added dropwise a solution of
NaIO4 (1.17 g, 5.46 mmol) in water (30 mL) at 0 °C. The mixture was stirred at 80 °C for 12 h
and concentrated under vacuum. The residue was putted into water (25 mL) and extracted
with DCM (3 x 50 mL). The combined organic layers were dried over MgSO4 and
evaporated. The residue was purified by flash chromatography (PE/AcOEt); 10:0 to 7:3 (v/v))
to afford 2 as a brown powder (71% yield); mp 158 °C; 1H NMR (300 MHz, CD2Cl2): δ 11.09
(br s, 1H), 8.65 (d, J = 9.2 Hz, 1H), 8.42 (d, J = 1.7 Hz, 1H), 7.94 – 7.81 (m, 2H), 7.81 – 7.70
(m, 2H), 7.40 (d, J = 2.9 Hz, 1H), 7.15 (dd, J = 9.2, 2.9 Hz, 1H), 4.11 (t, J = 7.2 Hz, 2H), 3.84
13
(s, 3H), 3.49 (t, J = 7.2 Hz, 2H); C NMR (75 MHz, CD2Cl2): δ 201.7, 168.0, 159.4, 154.8,
134.1, 133.3, 132.1, 123.1, 122.9, 120.5, 115.4, 55.7, 38.1, 33.5, 29.7
4.1.3. 4-Chloro-6-methoxyquinazoline (4). A suspension of 6-methoxy-(3H)-quinazolin-4-
one (4.4 g, 25 mmol) in POCl3 (20 mL) was refluxed for 12h. The mixture was concentrated
in vacuo, carefully hydrolyzed in ice water and alkalinized with a 28% ammonia solution. The
solid was filtered off, dissolved in dichloromethane dried over MgSO4 and concentrated in
vacuo. Crude product was recrystallized from toluene to afford 4 (65% yield) as a yellow
solid; mp 78-79 °C; 1H (300 MHz, DMSO-d6): δ 8.13 (s, 1H), 7.41 (d, 1H, J = 2.9 Hz), 7.32
(d, 1H, J = 9.0 Hz), 7.25 (dd, J = 9.0, 2.9 Hz, 1H), 3.95 (s, 3H).
4.1.4. (E)-2-(2-(6-Methoxyquinazolin-4-yl)vinyl)isoindoline-1,3-dione (5). In a sealed tube
under argon atmosphere, were introduced compound 4 (0.39 g, 2.0 mmol), DMF (5 mL),
triethylamine (1.2 mL, 8 mmol), palladium diacetate (22.5 mg, 0.1 mmol), tri(o-tolyl)-
phosphine (61 mg, 0.2 mmol) and N-vinylphthalimide (0.52 g, 3 mmol). The mixture was
stirred at 110 °C for 5 h and then 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 toluene to afford 5 as a brown solid (50% yield); mp

236 °C; 1H NMR (300 MHz, DMSO-d6): δ 9.00 (s, 1H), 8.42 (d, J = 15 Hz, 1H), 7.92 (d, J =
9.0 Hz, 1H), 7.83 – 7.82 (m, 2H), 7.80 – 7.74 (m, 2H), 7.53 (dd, J = 9.0, 2.8 Hz, 1H), 7.46 (d,
J = 2.8 Hz, 1H), 6.25 (d, J = 15 Hz, 1H), 3.88 (s, 3H); 13C NMR (75 MHz, CD2Cl2): δ 165.2,
162.2, 159.5, 153.6, 145.1, 132.8, 132.1, 130.2, 126.5, 127.4, 123.7, 123.3, 100.6, 95.8, 55.8
4.1.5. 2-(2-(6-Methoxyquinazolin-4-yl)ethyl)isoindoline-1,3-dione (3).
Method 1. To a solution of 2 (200 mg, 0.57 mmol) in formamide (2 mL) was added
ammonium formate (302 mg, 4.79 mmol) in sealed tube. The reactional mixture was stirred at
135 °C for 24 h. The mixture was putted into water (20 mL) and extracted with DCM. The
combined organic layers were dried over MgSO4 and evaporated. The residue was washed
with methanol to give 3 as a brown powder (61% yield).
Method 2. To a solution of 5 (0.32 g, 1 mmol) in 30 mL of ethanol/THF (1/1) was added
Pd/C 10%. 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 solid was recrystallized from toluene to afford 3 as a
white solid (90% yield); mp 236 °C; 1H NMR (300 MHz, CD2Cl2): δ 9.07 (s, 1H), 7.94 (d, J =
9.2 Hz, 1H), 7.88 – 7.82 (m, 2H), 7.80 – 7.74 (m, 2H), 7.53 (dd, J = 9.2, 2.7 Hz, 1H), 7.46 (d,
13
J = 2.7 Hz, 1H), 4.34 – 4.14 (m, 2H), 4.00 (s, 3H), 3.69- 3.45 (m, 2H); C NMR (75 MHz,
CD2Cl2): δ 168.1, 165.5, 158.5, 152.6, 146.1, 134.0, 132.1, 130.5, 126.4, 124.9, 123.1, 101.6,
55.8, 36.3, 33.0
4.1.6. 2-(6-Methoxyquinazolin-4-yl)ethanamine (6). To a solution of 3 (200 mg, 0.34 mmol)
in EtOH was added hydrazine monohydrate (0.17 mL, 3.45 mmol). The mixture was stirred at
room temperature for 16 h. The mixture was concentrated under vacuum and purified by flash
chromatography (DCM/MeOH, NH3 sat; 10:0 to 9:1 (v/v)) to give 6 as brown powder (97%,
yield); mp 71 °C; 1H NMR (300 MHz, CD2Cl2): δ 9.08 (s, 1H), 7.94 (d, J = 9.2 Hz, 1H), 7.55
(dd, J = 9.2, 2.7 Hz, 1H), 7.35 (d, J = 2.7 Hz, 1H), 3.98 (s, 3H), 3.40- 3.34 (m, 2H), 3.33 –

3.26 (m, 2H); 13C NMR (75 MHz, CD2Cl2): δ 167.7, 158.2, 152.6, 145.9, 130.5, 125.9, 125.2,
102.0, 55.7, 40.2, 37.9
4.1.7. N-(2-(6-Methoxyquinazolin-4-yl)ethyl)acetamide (7a).
Method 1. To a solution of 6 (64 mg, 0.32 mmol), K2CO3 (133 mg, 0.96 mmol) in water (3
mL) and ethyl acetate (20 mL) was added acetyl chloride (34 µL, 0.48 mmol). The mixture
was stirred over 4 h. The biphasic mixture was separated, and the aqueous layer was extracted
with EtOAc (3 x 20 mL). The combined organic layers were dried over MgSO4, filtrated and
concentrated. The residue was dissolved in DCM and precipitated by petroleum ether. The
resulting precipitate was recovered and washed with PE to give 7a as a white powder (44%
yield).
Method 2. This compound was prepared from compound 8 according to the procedure
described for 3 (Method 1). Recrystallization from acetonitrile gave 7a (60% yield) as a white
solid; mp 78 °C; 1H NMR (300 MHz, CD2Cl2): δ 9.08 (s, 1H), 7.94 (d, J = 9.2 Hz, 1H), 7.52
(dd, J = 9.2 Hz, 2.8, 1H), 7.43 (d, J = 2.7 Hz, 1H), 6.47 (br s, 1H, NH), 3.99 (s, 3H), 3.89 –
13
3.80 (q, J = 6.3 Hz, 2H) 4.34 – 4.14 (t, J = 6.3 Hz, 2H), 1.92 (s, 3H); C NMR (75 MHz,
CD2Cl2): δ 169.9, 166.8, 158.5, 152.3, 145.8, 130.4, 126.4, 125.2, 101.8, 55.8, 36.7, 33.6,
23.1; HPLC: C18 column: tR = 19.799 min, purity > 99%.
4.1.8. N-(2-(6-Methoxyquinazolin-4-yl)ethyl)propionamide (7b). This compound was
prepared from compound 6 according to the procedure described for 7a (Method 1).
Recrystallized from acetonitrile as a yellow solid (85% yield); mp 89-91 °C; IR: 3268 cm-1
(νNH), 1668 cm-1 (νCO); 1H NMR (300 MHz, CDCl3): δ 9.08 (s, 1H), 7.94 (d, J = 9.2 Hz, 1H),
7.55 (dd, J = 9.2, 2.8 Hz, 1H), 7.40 (d, J = 2.8 Hz, 1H), 6.42 (br s, 1H), 3.99 (s, 3H), 3.86 (q,
J = 6.3 Hz, 2H), 3.46 (t, J = 6.3 Hz, 2H), 2.16 (q, J = 7.5 Hz, 3H), 1.09 (t, J = 7.5 Hz, 3H);
13C NMR (75 MHz, CD2Cl2): δ 173.5, 166.9, 158.5, 152.3, 145.8, 130.4, 126.3, 125.2, 101.8,
55.8, 36.6, 33.6, 29.6, 9.5; HPLC: C18 column: tR = 20.382 min, purity > 99%.

4.1.9. N-(2-(6-Methoxyquinazolin-4-yl)ethyl)butyramide (7c). This compound was prepared
from compound 6 according to the procedure described for 7a (Method 1). Recrystallized
from acetonitrile as a yellow solid (85% yield); mp 146-148 °C; IR: 3266 cm-1 (νNH), 1624
cm-1 (νCO); 1H NMR (300 MHz, CD2Cl2): δ 9.08 (s, 1H), 7.93 (d, J = 9.3 Hz, 1H), 7.55 (dd, J
= 9.3, 2.9 Hz, 1H), 7.40 (d, J = 2.9 Hz, 1H), 6.44 (br s, 1H), 3.99 (s, 3H), 3.86 (q, J = 6.1 Hz,
2H), 3.46 (t, J = 6.2 Hz, 2H), 2.11 (t, J = 7.2 Hz, 2H), 1.60 (p, J = 7.5 Hz, 2H), 0.89 (t, J =
13
7.4 Hz, 3H); C NMR (75 MHz, CD2Cl2): δ 174.7, 168.8, 160.4, 154.3, 147.7, 132.3, 128.3,
127.1, 103.8, 57.7, 40.5, 38.5, 35.6, 21.0, 15.4. HPLC: C18 column: tR = 21.437 min, purity >
95%.
4.1.10. N-(2-(6-Methoxyquinazolin-4-yl)ethyl)cyclopropanecarboxamide
(7d).
This
compound was prepared from compound 6 according to the procedure described for 7a
(Method 1). Recrystallized from acetonitrile as a white solid (82% yield); mp 115-117 °C. IR:
1
3304 cm-1 (νNH), 1663 cm-1 (νCO); H NMR (300 MHz, CD2Cl2): δ 9.09 (s, 1H), 7.93 (d, J =
9.2 Hz, 1H), 7.55 (dd, J = 9.2, 2.7 Hz, 1H), 7.38 (d, J = 2.7 Hz), 6.66 (br s, 1H), 3.98 (s, 3H),
3.88 (q, J = 6.2 Hz, 2H), 3.46 (t, J = 6.2 Hz, 2H), 1.41 – 1.31 (m, 1H), 0.92 – 0.83 (m, 2H),
0.74 – 0.65 (m, 2H); 13C NMR (75 MHz, CD2Cl2): δ 175.2, 168.9, 160.4, 154.3, 147.7, 132.3,
128.2, 127.1, 103.8, 57.7, 38.7, 35.7, 16.4, 8.5. HPLC: C18 column: tR = 20.382 min, purity >
99%.
4.1.11. N-(3-(2-Formamido-5-methoxyphenyl)-3-oxopropyl)acetamide (8). This compound
was prepared from melatonin according to the procedure described for 2. Crude product was
recrystallized from acetonitrile to afford 8 as a yellow solid (55% yield); mp 153-154 °C; IR:
3329 cm-1 (νNH), 1681 cm-1 (νCO). 1H NMR (300 MHz, CD2Cl2): δ 11.16 (br s, 1H, NH), 8.65
(d, J = 9.2 Hz, 1H), 8.43 (d, J = 1.9 Hz, 1H), 8.45 (br s, 1H), 7.43 (d, J = 2.9 Hz, 1H), 7.16
(dd, J = 9.2, 2.9 Hz, 1H), 6.09 (br s, 1H), 3.85 (s, 3H), 3.62 (q, J = 5.9 Hz, 2H), 3.28 (t, J =

13
5.9 Hz, 2H), 1.92 (s, 3H); C NMR (75 MHz, CDCl3): δ 203.4, 169.6, 161.9, 159.3, 154.9,
133.2, 122.9, 120.5, 115.5, 55.7, 39.8, 34.5, 23.0
4.1.12. N-(3-(2-Amino-5-methoxyphenyl)-3-oxopropyl)acetamide (9). To a solution of 8 (2.65
g, 10 mmol) in EtOH was added 4 M aqueous hydrochloric acid (60 ml). The solution was
refluxed for 2 h and EtOH was evaporated. The mixture was cooled to room temperature and
adjusted to pH = 8 with 10 M aqueous sodium hydroxide solution and extracted with ethyl
acetate. The organic layer was dried over MgSO4, filtered, and concentrated under reduced
pressure to afford 9 as yellow solid (65% yield); mp 89-92 °C. IR: 3407, 3274 cm-1 (νNH2),
1657, 1635 cm-1 (νCO); 1H (300 MHz, CDCl3): δ 8.75 (d, J = 9.3 Hz, 1H), 7.16 (d, J = 3.0 Hz,
1H), 7.02 (dd, J = 9.3, 3.0 Hz, 1H), 6.00 (br s, 2H), 3.78 (s, 3H), 3.69 (q, J = 4.9 Hz, 2H),
13
3.21 (t, 2H, J = 4.9 Hz), 1.96 (s, 3H); C NMR (75 MHz, CDCl3): δ 203.2, 161.5, 159.3,
153.4, 132.2, 122.7, 120.5, 117.5, 55.6, 39.8, 34.5, 23.2
4.1.13. N-(2-(3-Acetamidopropanoyl)-4-methoxyphenyl)benzamide (10a). To a cold solution
of 9 (0.4 g, 1.7 mmol) and TEA (0.35 mL, 2.5 mmol) in methylene chloride, was added
dropwise benzoyl chloride (0.24 mL, 2.0 mmol). The solution was stirred at room temperature
for 2h and water was added. The organic phase was wached with 1M NaOH and 1M HCl. The
organic layer was dried over MgSO4 and evaporated. The obtained solid was recrystallized
from acetonitrile to afford 10a (67% yield) as a yellow solid; mp 160-162 °C; IR: 3355, 3342
cm-1 (νNH), 1672, 1643, 1635 cm-1 (νCO); 1H NMR (300 MHz, CDCl3): δ 12.29 (br s, 1H), 8.89
(dd, J = 9.3, 1.5 Hz, 1H), 8.05-8.03 (m, 2H), 7.58-7.51 (m, 3H), 7.41 (m, 1H), 7.21-7.18 (m,
1H), 6.24 (m, 1H), 3.85 (m, 3H), 3.69 (q, J = 5.7 Hz, 2H), 3.32 (t, J = 5.1 Hz, 2H), 1.99 (s,
13
3H). C NMR (75 MHz, CDCl3): δ 203.7, 170.3, 165.7, 154.6, 134.9, 131.9, 128.8, 127.3,
122.5, 122.5, 121.2, 115.5, 55.7, 39.7, 34.5, 23.3
4.1.14. N-(2-(3-Acetamidopropanoyl)-4-methoxyphenyl)-3-methoxybenzamide (10b). This
compound was prepared from 9 according to the procedure described for 10a.

Recrystallization from acetonitrile gave 10b (70% yield) as a yellow solid; mp 148-153 °C;
IR: 3366, 3215 cm-1 (νNH), 1662, 1652, 1638 cm-1 (νCO); 1H NMR (300 MHz, CDCl3): δ 12.26
(br s, 1H), 8.86 (d, 1H, J = 9.2 Hz), 7.59-7.57 (m, 2H), 7.45-7.39 (m, 2H), 7.18 (dd, J = 9.2,
2.8 Hz, 1H), 7.10 (m, 1H), 6.28 (m, 1H), 3.90 (s, 3H), 3.85 (s, 3H), 3.68 (q, J = 5.8 Hz, 2H),
13
3.30 (t, J = 5.7 Hz, 2H), 1.98 (s, 3H). C NMR (75 MHz, CDCl3): δ 203.6, 170.4, 165.5,
159.9, 154.6, 136.4, 134.8, 129.8, 122.6, 122.4, 121.1, 119.1, 118.1, 115.5, 112.7, 55.7, 55.4,
39.7, 34.5, 23.3
4.1.15. N-(2-(3-Acetamidopropanoyl)-4-methoxyphenyl)-2-cyclopropylacetamide (10c). This
compound was prepared from 9 according to the procedure described for 10a.
Recrystallization from acetonitrile afford 10c (70% yield) as a yellow solid; mp 122-125 °C;
1
IR: 1669, 1658, 1648 cm-1; H NMR (300 MHz, CDCl3): δ 11.24 (br s, 1H), 8.67 (d, J = 9.5
Hz, 1H), 8.45 (br s, 1H), 7.38 (d, J = 2.9 Hz, 1H), 7.14 (dd, J = 9.2, 2.8 Hz, 1H), 3.84 (s, 3H),
3.67 (q, J = 5.8 Hz, 2H), 3.27 (t, J = 5.8 Hz, 2H), 1.35 (m, 2H), 1.12-0.70 (m, 5H). 13C NMR
(75 MHz, CDCl3): δ 203.5, 173.8, 159.5, 154.9, 133.2, 123.2, 122.7, 120.7, 115.6, 55.7, 39.8,
34.5, 29.7, 14.7, 7.25, 7.25
4.1.16. N-(2-(6-Methoxy-2-phenylquinazolin-4-yl)ethyl)acetamide (11a). This compound was
prepared from compound 10a according to the procedure described for 7a (Method 1).
1
Recrystallized from acetonitrile as a white solid (58% yield); mp 157-158 °C; H NMR (300
MHz, CD2Cl2): δ 8.61 (m, 2H), 8.00 (d, J = 9.2 Hz, 1H), 7.64-7.47 (m, 4H), 7.40 (d, J = 2.8
13
Hz, 1H), 6.43 (br s, 1H), 4.07-3.88 (m, 5H), 3.60-3.45 (t, J = 6.2 Hz, 2H), 1.92 (s, 3H); C
NMR (75 MHz, CD2Cl2): δ 169.9, 166.9, 158.3, 157.6, 146.5, 138.3, 130.6, 130.1, 128.5,
127.9, 126.4, 123.6, 102.0, 55.8, 36.6, 33.7, 23.1; HPLC: C18 column: tR = 26.753 min, purity
> 99%.
4.1.17. N-(2-(6-Methoxy-2-(3-methoxyphenyl)quinazolin-4-yl)ethyl)acetamide (11b). This
compound was prepared from compound 10b according to the procedure described for 7a

(Method 1). Recrystallized from acetonitrile as a white solid (54% yield); mp 151-152 °C; 1H
NMR (300 MHz, CD2Cl2): δ 8.24-8.15 (m, 2H), 7.99 (d, J = 9.2 Hz, 1H), 7.55 (dd, J = 9.2,
2.8 Hz, 1H), 7.46 (t, J = 8.0 Hz, 1H), 7.38 (d, J = 2.8 Hz, 1H), 7.06 (ddd, J = 8.0, 2.7, 0.9 Hz,
1H), 6.42 (br s, 1H), 4.00 (s, 3H), 3.98-3.92 (m, 5H), 3.51 (t, J = 6.3 Hz, 2H), 1.92 (s, 3H);
13C NMR (75 MHz, CD2Cl2): δ 169.9, 166.8, 160.1, 158.3, 157.4, 146.4, 139.9, 130.6, 129.5,
126.4, 123.7, 120.4, 116.0, 112.9, 102.0, 55.8, 55.3, 36.6, 33.7, 23.1; HPLC: C18 column: tR
= 27.341 min, purity > 99%.
4.1.18. N-(2-(2-(Cyclopropylmethyl)-6-methoxyquinazolin-4-yl)ethyl)acetamide (11c). This
compound was prepared from compound 10c according to the procedure described for 7a
(Method 1). Recrystallized from acetonitrile as a white solid (85% yield); mp 114-115 °C;
1
IR: 3318 cm-1 (νNH), 1638 cm-1 (νCO); H (300 MHz, DMSO-d6): δ 7.90 (d, J = 9.2 Hz, 1H),
7.52 (dd, J = 9.2, 2.8 Hz, 1H), 7.29 (d, J = 2.8 Hz, 1H), 6.66 (br s, 1H), 3.96 (s, 3H), 3.93 (m,
2H), 3.46 (t, J = 6.0 Hz, 2H), 2.98 (d, J = 7.1 Hz, 2H), 1.96 (s, 3H), 1.34 (m, 1H), 0.60 (m,
4H); 13C NMR (75 MHz, DMSO-d6): δ 170.29, 166.73, 163.80, 157.89, 146.07, 129.96,
126.41, 123.05, 101.57, 55.76, 44.07, 36.65, 33.39, 23.40, 10.46, 4.59, 4.59; HPLC: C18
column: tR = 20. 544 min, purity > 95%.
4.1.19. N-(2-((6-Methoxyquinazolin-4-yl)amino)ethyl)acetamide (12). To a solution of 4 (0.5
g, 2.5 mmol) in THF was added Et3N (0.52 mL, 3.76 mmol) and N-(2-aminoethyl)acetamide
(0.25 g, 2.5 mmol). The mixture was stirred at 30 °C for 1h, cooled to room temperature and
concentrated in vacuo. The residue was suspended in water and extracted with EtOAc. The
organic phase was dried over MgSO4 and concentrated by evaporation in vacuo. Solid was
recrystallized from toluene to afford 12. Recrystallized from acetonitrile as a white solid (55%
yield); mp 176-178 °C; IR: 3304 cm-1 (νNH), 3195 cm-1 (νNH), 1663 cm-1 (νCO); 1H NMR (300
MHz, CDCl3): δ 8.52 (s, 1H), 7.88 (br s, 1H), 7.74 (d, J = 9.1 Hz, 1H), 7.36 (dd, J = 9.1, 2.6
Hz, 1H), 7.30 (d, J = 2.6 Hz, 1H), 6.97 (br s, 1H), 4.56 (br s, 1H), 3.94 (s, 3H), 3.82-3.72 (m,

2H), 3.70-3.61 (m, 2H), 2.04 (s, 3H); 13C NMR (CDCl3): δ 172.9, 159.3, 157.8, 152.9, 143.9,
129.3, 124.2, 115.4, 100.6, 55.8, 43.7, 39.9, 23.3; HPLC: C18 column: tR = 20.382 min, purity
> 99%.
4.1.20. N-Ethyl-4-(6-methoxyquinazolin-4-yl)piperazine-1-carboxamide (13). This compound
was prepared from compound 4 according to the procedure described for 12. Recrystallized
from toluene as a white solid (75% yield); mp 145-147 °C; IR: 3255 cm-1 (νNH), 1643 cm-1
(νCO); 1H NMR (300 MHz, CD2Cl2): δ 8.66 (s, 1H), 7.84 (d, J = 9.1 Hz, 1H), 7.45 (dd, J = 9.1,
2.8 Hz, 1H), 7.20 (d, J = 2.8 Hz, 1H ) 4.60 (br s, 1H), 3.94 (s, 3H), 3.64 - 3.56 (m, 4H), 3.75-
13
3.70 (m, 4H), 3.33 - 3.23 (m, 2H), 1.17 (t, J = 7.2 Hz, 3H) ; C NMR (75 MHz, CD2Cl2): δ
164.3, 157.6, 157.0, 152.1, 147.3, 130.2, 124.2, 117.6, 103.4, 55.6, 49.3, 43.4, 35.7, 15.3;
HPLC: C18 column: tR = 20.382 min, purity > 99%.
4.1.21. Cyclopropyl-(4-(6-methoxyquinazolin-4-yl)piperazin-1-yl)methanone
(14).
This
compound was prepared from compound 4 according to the procedure described for 12.
Recrystallized from toluene as a white solid (75% yield); mp 145-147 °C; IR: 3255 cm-1
(νNH), 1643 cm-1 (νCO); 1H NMR (300 MHz, CD2Cl2): δ 8.68 (s, 1H), 7.86 (d, J = 9.2 Hz, 1H),
7.45 (dd, J = 9.2, 2.8 Hz, 1H), 7.21 (d, J = 2.8 Hz, 1H), 4.00-3.65 (m, 11H), 1.83 (m, 1H),
13
1.02-0.93 (m, 2H), 0.86 – 0.77 (m, 2H); C NMR (75 MHz, CD2Cl2): δ 172.1, 164.3, 157.1,
152.1, 147.4, 130.2, 124.2, 117.6, 103.4, 55.6, 49.8, 49.3, 45.0, 41.7, 29.7, 10.9, 7.1; HPLC:
C18 column: tR = 20.382 min, purity > 99%.
4.1.22. 1-Chloro-7-methoxyphthalazine (16). A suspension of 7-methoxyphthalazin-(2H)-one
(15) (1.0 g, 5.7 mmol) in POCl3 (3 mL) was refluxed for 1h. After cooling, the reaction
mixture was carefully hydrolyzed in crunched ice and alkalinized with a 15% NaOH solution.
The solid was filtered off, dissolved in dichloromethane dried over MgSO4 and concentrated
in vacuo. Crude product 16 (86% yield) was used without further purification; mp 139-141

1
°C; H NMR (300 MHz, CDCl3): δ 9.30 (s, 1H), 7.90 (d, J = 8.6 Hz, 1H), 7.60 (dd, J = 8.6,
2.5 Hz, 1H), 7.50 (d, J = 2.5 Hz, 1H), 4.05 (s, 3H).
4.1.23. Ethyl 2-cyano-2-(7-methoxyphthalazin-1-(2H)-ylidene)acetate (17). In anhydrous THF
(10 mL), a solution of ethyl cyanocetate (0.66 mL, 6.2 mmol) was added to a mixture of NaH
(60% in mineral oil) (250 mg, 6.2 mmol). The mixture was stirred for 30 min at r.t., then a
solution of 15 (0.8 g, 4.1 mmol) in anhydrous THF (10 mL) was added. The reaction mixture
was heated at reflux for 12 h, cooled and quenched with water. The solid was filtered, washed
with water, dried. Recrystallization from acetonitrile gave 17 as beige powder (80% yield);
1
mp 185-187 °C; IR 2250 cm-1 (νCN), 1650 cm-1 (νCO); H NMR (300 MHz, CDCl3): δ 13.55
(br s, 1H), 9.00 (d, J = 2.6 Hz, 1H), 8.30 (s, 1H), 7.70 (d, J = 8.4 Hz, 1H), 7.45 (dd J = 8.4,
13
2.6 Hz, 1H,), 4.25 (q, J = 7.0 Hz, 2H), 4.00 (s, 3H), 1.40 (t, J = 7.0 Hz, 2H); C NMR (75
MHz, CDCl3): δ 173.3, 165.2, 161.5, 139.1, 135.3, 130.0, 128.4, 116.2, 113.0, 105.9, 70.2,
61.4, 56.1, 13.5
4.1.24. 2-(7-Methoxyphthalazin-1-yl)acetonitrile (18). A mixture of 17 (0.46 g, 1.7 mmol) in
5M HCl (80 ml) was refluxed until complete dissolution. The solution was refluxed for an
additional 2h then ethanol was evaporated. The mixture was cooled to room temperature and
adjusted to pH 8 with an10 M NaOH solution and extracted with ethyl acetate. The organic
layer was dried over MgSO4, filtered, and concentrated under reduced pressure to afford after
recrystallization in toluene 18 as beige solid (60%, yield) ; mp 165-166 °C; IR 2250 cm-1
(νCN); 1H NMR (300 MHz, CDCl3): δ 9.40 (s, 1H), 7.96 (d, J = 8.9 Hz, 1H), 7.58 (dd, J = 8.9,
13
2.3 Hz, 1H), 7.29 (m, 1H), 4.45 (s, 2H), 4.05 (s, 3H); C NMR (75 MHz, CDCl3): δ 163.1,
129.3, 129.0, 126.9, 125.2, 121.0, 115.9, 105.4, 101.4, 56.1, 23.5
4.1.25. 2-(7-Methoxyphthalazin-1-yl)ethanamine (19). A NH3-saturated solution of 18 (2.7 g,
8.5 mmol) in methanol (50 mL) was hydrogenated over Raney nickel under pressure (3 bars)
at room temperature for 48 h. After filtration and evaporation, the residual oil was dissolved

in a mixture of toluene/methanol in the presence of 10% Pd/C. The mixture was refluxed for
5h, filtered and evaporated. The residue was purified by flash chromatography
(CH2Cl2/MeOH); 10:0 to 0:10 (v/v)) to afford 19 as a brown oil (70% yield); IR 3250 cm-1
1
(νNH2); H NMR (300 MHz, CDCl3): δ 9.30 (s, 1H), 7.85 (d, J = 8.8 Hz, 1H), 7.45 (dd, J =
8.8, 2.1 Hz, 1H), 7.30 (d, J = 2.1 Hz, 1H), 4.00 (s, 3H); 3.50 (m, 2H), 3.40 (t, J = 5.5 Hz,
2H), 3.00 (br s, 2H). 13C NMR (75 MHz, CDCl3): δ 170.8, 163.3, 156.2, 149.7, 128.6, 127.1,
125.6, 122.8, 101.2, 55.0, 40.3, 35.7
4.1.26. N-(2-(7-Methoxyphthalazin-1-yl)ethyl)acetamide (20a). To a solution of 19 (0.5 g, 2.5
mmol), K2CO3 (1.0 g, 14.7 mmol) in water (20 mL) and ethyl acetate (40 mL) was added
acetyl chloride (0.35 mL, 5 mmol). The mixture was stirred for 2h. The biphasic mixture was
separated, and the aqueous layer was extracted with EtOAc. The combined organic layers
were dried over MgSO4, filtrated and concentrated. The residue was first purified by flash
chromatography (CH2Cl2/MeOH; 9/1) and recrystallized from toluene to give 20a as a white
1
powder (40% yield); mp 160-163 °C; IR: 3250 cm-1 (νNH), 1665 cm-1 (νCO); H NMR (300
MHZ, CDCl3): δ 9.30 (s, 1H), 7.85 (d, J = 8.8 Hz, 1H), 7.50 (dd, J = 8.8, 2.4 Hz, 2H), 7.40
(d, J = 2.4 Hz, 1H), 6.70 (br s, 1H), 4.05 (s, 3H), 3.95 (q, J = 6.2 Hz, 2H), 3.50 (t, J = 6.2 Hz,
13
2H), 1.98 (s, 3H). C NMR (75 MHz, CDCl3): δ 170.8, 162.3, 157.2, 149.6, 128.8, 128.1,
124.6, 121.8, 102.2, 56.0, 37.3, 32.7, 23.3; HPLC: C18 column: tR = 17.266 min, purity >
99%.
4.1.27. N-(2-(7-Methoxyphthalazin-1-yl)ethyl)propionamide (20b). This compound was
prepared from compound 19 according to the procedure described for 20a. Recrystallized
from toluene as a white solid (55% yield); mp 137-139 °C; IR: 3250 cm-1 (νNH), 1660 cm-1
(νCO); 1H NMR (300 MHz, CDCl3): δ 9.30 (s, 1H), 7.85 (d, J = 8.8 Hz, 1H), 7.50 (dd, J = 8.8,
2.3 Hz, 1H), 7.40 (d, J = 2.3 Hz, 1H), 6.60 (br s, 1H), 4.05 (s, 3H), 3.95 (q, J = 6.2 Hz, 2H),
13
3.50 (t, J = 6.2 Hz, 2H), 2.20 (q, J = 7.5 Hz, 2H), 1.10 (t, J = 7.5 Hz, 3H). C NMR (75

MHz, CDCl3): δ 174.2, 162.7, 157.2, 149.6, 128.8, 128.1, 124.5, 121.8, 102.1, 56.0, 36.9,
32.6, 29.8, 9.9; HPLC: C18 column: tR = 17.813 min, purity > 99%.
4.1.28. N-(2-(7-methoxyphthalazin-1-yl)ethyl)butyramide (20c). This compound was prepared
from compound 19 according to the procedure described for 20a. Recrystallized from toluene
as a white solid (50% yield); mp 127-129 °C; IR: 3250 cm-1 (νNH), 1655 cm-1 (νCO); 1H NMR
(300 MHz, CDCl3): δ 9.40 (s, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.51 (dd, J = 8.8, 2.4 Hz, 1H),
7.43 (m, 1H), 6.66 (br s, 1H), 4.05 (s, 3H), 3.96 (q, J = 6.1 Hz, 2H), 3.53 (t, J = 6.3 Hz, 2H),
2.15 (t, J = 7.4 Hz, 2H), 1.64 (m, 2H), 0.90 (t, J = 7.5 Hz, 3H); 13C NMR (75 MHz, CDCl3):
δ 173.4, 162.7, 157.2, 149.6, 128.8, 128.1, 124.5, 121.8, 102.1, 56.0, 38.8, 36.7, 32.6, 19.2,
13.7; HPLC: C18 column: tR = 18.688 min, purity > 99%.
4.1.29. N-(2-(7-Methoxyphthalazin-1-yl)ethyl)cyclopropanecarboxamide
(20d).
This
compound was prepared from compound 19 according to the procedure described for 20a.
Recrystallized from toluene as a white solid (50% yield); mp 162-164 °C; IR: 3260 cm-1
(νNH), 1655 cm-1 (νCO)1H NMR (300 MHz, CDCl3): δ 9.26 (s, 1H), 7.86 (d, J = 8.8 Hz, 1H),
7.49 (dd, J = 8.8, 2.3 Hz, 1H), 7.41 (m, 1H), 7.03 (m, 1H), 4.02 (s, 3H), 3.96 (q, J = 6.0 Hz,
13
2H), 3.52 (t, J = 6.3 Hz, 2H), 1.43-1.35 (m, 1H), 0.95-0.90 (m, 2H), 0.72-0.66 (m, 2H). C
NMR (75 MHz, CDCl3): δ 173.9, 162.7, 157.3, 149.6, 128.8, 128.1, 124.4, 121.8, 102.2, 56.0,
37.1, 32.7, 14.7, 7.0; HPLC: C18 column: tR = 18.179 min, purity > 99%.
4.2. Binding studies
4.2.1. Reagents and chemicals
2-[125I]Iodomelatonin (2200 Ci/mmol) was purchased from NEN (Boston, MA). Other
drugs and chemicals were purchased from Sigma–Aldrich Sigma-Aldrich (Saint Quentin,
France) and used without further purification.
4.2.2. 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
with 10% fetal calf serum, 2 mM glutamine, 100 IU/mL penicillin and 100 mg/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 5mM (pH 7.5), containing EDTA 2mM and homogenized using a
Kinematica polytron. The homogenate was then centrifuged (95000 g, 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.
4.2.3. Binding assays for MT1 and MT2 receptors subtypes
2-[125I] Iodomelatonin competition binding assay conditions were essentially as
previously described [21]. Briefly, binding was initiated by addition of membrane
preparations from transfected CHO cells stably expressing the human melatonin MT1 or MT2
diluted in binding buffer (50 mM Tris–HCl buffer, pH 7.4, containing 5 mM MgCl2) to 2-
[125I]iodomelatonin (20 pM for MT1 and MT2 receptors expressed in CHO cells) and the tested
drug. Non-specific binding was defined in the presence of 1 µM of 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 (seven concentrations in duplicate) were analysed
using the program PRISM (Graph Pad Software Inc., San Diego, CA) to yield IC50 (inhibitory
concentration 50). Affinities are expressed as pKi (pKi = -Log10 (Ki)) with Ki = IC50/1 +
([L]/KD), where [L] is the concentration of radioligand used in the assay and KD, the
dissociation constant of the radioligand characterizing the membrane preparation [22].

[35S] GTPγS binding assay was performed according to published methodology [21].
Briefly, membranes from transfected CHO cells expressing MT1 or MT2 receptor subtype and
compounds were diluted in binding buffer (20 mM HEPES, pH 7.4,100mMNaCl, 3 mM
GDP, 3 mM MgCl2, and 20 mg/mL saponin). Incubation was started by the addition of 0.2
nM [35S]GTPγS to membranes (20 mg/mL) and drugs, and further followed for 1h 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 mM). 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 mM and 180 in the
presence of GTPγS 10 mM 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.
Serotonin 5-HT2C binding assay was determined according to reported tests [23].
4.2.4. cLogP and cytotoxicity
The n-octanol-water partition coefficients, which are well established measures of the
compounds’ hydrophilicity, were calculated for final prepared compounds by use of
theoretical procedures previously [24]. Numerical values of the partition coefficients were

obtained as follows using using Data Warrior software: the logP value, which is the logarithm
of the partition coefficient of a compound between n-octanol and water log(Coctanol/Cwater), is
calculated as increment system the contributions of every atom based on its atom type.
Cytotoxic activities towards standard cancer cell line Caco-2 was performed using the same
procedure as was previously described [25].
Acknowledgement
The authors would like to gratefully acknowledge 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 the 300 MHz
NMR facilities.

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