Discovery of coumarin-dihydroquinazolinone analogs as niacin receptor 1 agonist with in-vivo anti-obesity efficacy

Article abstract of DOI:10.1016/j.ejmech.2018.04.037

In this study, we presented rational designing and synthesis of coumarin-dihydroquinazolinone conjugates to evaluate their agonist activity at GPR109a receptor. Among the synthesized small molecule library, compound 10c displayed robust agonist action at

Full text of DOI:10.1016/j.ejmech.2018.04.037

Accepted Manuscript
Discovery of coumarin-dihydroquinazolinone analogs as niacin receptor 1 agonist
with in-vivo anti-obesity efficacy
L. Ravithej Singh, Ajeet Kumar, Akanksha Upadhyay, Sampa Gupta, Gopala Reddy
Palanati, Kamakshi Sikka, Mohammad Imran Siddiqi, Prem N. Yadav, Koneni V.
Sashidhara
PII:
S0223-5234(18)30371-4
10.1016/j.ejmech.2018.04.037
EJMECH 10386
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 February 2018
Revised Date: 5 April 2018
Accepted Date: 18 April 2018
Please cite this article as: L.R. Singh, A. Kumar, A. Upadhyay, S. Gupta, G.R. Palanati, K. Sikka, M.I.
Siddiqi, P.N. Yadav, K.V. Sashidhara, Discovery of coumarin-dihydroquinazolinone analogs as niacin
receptor 1 agonist with in-vivo anti-obesity efficacy, European Journal of Medicinal Chemistry (2018),
doi: 10.1016/j.ejmech.2018.04.037.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Discovery of coumarin-dihydroquinazolinone analogs as niacin
receptor 1 agonist with in-vivo anti-obesity efficacy
L. Ravithej Singh‡#, Ajeet Kumar§#, Akanksha Upadhyay‡, Sampa Gupta‡, Gopala Reddy
Palanati‡, Kamakshi Sikka†, Mohammad Imran Siddiqi†, Prem N. Yadav§,*, Koneni V.
Sashidhara‡,*
‡
§
Medicinal and Process Chemistry Division, Pharmacology Division, †Molecular and Structural Biology Division,
CSIR-Central Drug Research Institute, BS-10/1, Sector 10, Jankipuram extension, Sitapur Road, Lucknow 226031,
India.
# These authors contributed equally
Keywords:
Niacin, niacin receptor 1, GPR109a, pharmacophore hybridization, coumarin-
dihydroquinazolinone, Homology modeling
*Corresponding authors.
Tel.: +91 9919317940; Fax: +91-522-2628493.
Email: kv_sashidhara@cdri.res.in, sashidhar123@gmail.com (Dr. K. V. Sashidhara),
pn.yadav@cdri.res.in (Dr. P. N. Yadav)
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Graphical abstract
Abstract:
In this study, we presented rational designing and synthesis of coumarin-dihydroquinazolinone
conjugates to evaluate their agonist activity at GPR109a receptor. Among the synthesized small
molecule library, compound 10c displayed robust agonist action at GPR109a with EC50< 11nM.
Homology model of human GPR109a protein was generated to realize the binding interaction of
the active molecule with the active site of GPR109a. Further, the efficacy of active compound
10c was supported by in-vivo experiments which showed reduced body weight in diet induced
obese mice model. Interestingly, compound 10c reduced leptin in blood plasma and total serum
cholesterol. These results suggest that the coumarin-dihydroquinazolinone conjugate is a suitable
scaffold to further expand the chemical diversity and make them potential niacin receptor 1
agonist.
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1. Introduction
Nicotinic acid (NA), also known as niacin or vitamin B-3, is a well-known lipid modifying agent
in humans [1]. NA therapy has been shown to produce several beneficial effects, such as
reducing the major coronary events by 25%, stroke by 26%, and all cardiovascular events by
27% [2]. Moreover, NA has also been shown to quite effective for lowering triglycerides and
LDL cholesterol, raising HDL level in blood [3]. Multiple lines of evidence confirmed that the G
protein-coupled receptor GPR109a is a molecular target of NA [4-6]. GPR109a, also known as
hydroxy-carboxylic acid 2 (HCA2) receptor or niacin receptor 1(NIACR1), is highly expressed
in adipocytes, spleen and immune cells such as macrophage, keratinocytes and Langerhans cells
and mediates the lipid-lowering effect of NA [5]. Rahman M et al. reported that GPR109a is also
expressed in brain microglia and exhibited neuroprotective effect in mice [7]. However, NA
mediated activation of GPR109a in langerhans cells and keratinocytes induces expression of
prostaglandin D2 (PGD2) and ultimately cutaneous flushing [8, 9] by β-arrestin1 mediated
signaling [10]. Thus, a G-protein bias agonist of GPR109a that exhibits minimal or no β-
arrestin1 dependent signaling would be a better candidate to avoid the side effect of targeting
GPR109a. Therefore, there are renewed interests to synthesize G-protein biased agonist of
GPR109a.
Although NA has been shown to inhibit lipolysis through activation of the GPR109a in adipose
tissues [5], one study provided quite strong evidence that the NA effects on blood lipid profile
are independent of the GPR109a [11]. Besides, these studies also demonstrated the effects of NA
via GPR109a expressed in adipose tissue increased plasma level of adiponectin and anti-
inflammatory cytokine expression in obesity [12, 13]. Expressions of GPR109a have been shown
to increase during differentiation of adipocytes from preadipocytes as well as after activation of
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peroxisome proliferator-activated receptor-gamma (PPARγ) [14]. Thus, it is quite clear that
GPR109a modulates pleiotropic effects besides well known therapeutic effects on lipid
metabolism [15]. Therefore, search for new ligands that bind to GPR109a to accomplish desired
effects by subtracting or minimizing adverse effects has gained momentum. Medicinal chemistry
efforts from academia and industries towards the development of potent GPR109a agonist
resulted in the discovery of several novel small molecules. For example, pyridopyrimidinone and
aza-pyridopyrimidinone derivatives (1 and 2, Figure 1) were identified as GPR109a agonists
[16]. Similarly, Merck research laboratory discovered pyrano[2,3-d]pyrimidine (3, Figure 1)
class of compound processing nicotinic acid receptor agonist activity for dyslipidemia with in-
vivo efficacy [17].
Fig. 1. Chemical structures with potent GPR109a agonist activity and general structure of our synthesized prototype
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Interestingly, a distinct fused heterocyclic motif, pyranopyrimidine (4 and 5, Figure 1),
comprising barbituric acid and pyrone ring exhibited promising activity in a cell-based cAMP
assay, which is also a GPR109a agonist [18]. In the quest for new drug leads, the hybridization
approach has been a promising one, which involves the combination of pharmacophoric moieties
to produce a hybrid structure with improved efficacy and reduced toxicity. The derivatives of
coumarin are well known pharmacophores with potential benefits in the prevention of obesity
and liver dysfunction [19]. Also, we have reported the utility of coumarin analogs as good lipid-
lowering agents [20]. On the other hand, quinazolinone and its derivatives are the vital
constituents of number of natural products and modern drugs [21-23]. Quinazolinone and its
derivatives exhibit a wide range of biological activities including antitumor, antiviral, anti-
inflammatory, analgesic, antifungal, antimalarial, antidiabetic, anticonvulsant, antimicrobial, and
angiotensin II AT1 receptor antagonists [24-31]. Inspired by wide and interesting biological
activity of Quinazolinone scaffold, we synthesized the novel coumarin- dihydroquinazolinone
hybrids (6, Figure 1), and evaluated for their G-protein and arrestin biased agonist activity at
GPR109a receptors.
2. Results and Discussion:
2.1 Chemistry
The synthesis of target compounds (10a-10p) is shown in Scheme 1. The first step involved a
reaction of commercially available 2-alkyl phenols (7a-7g) with hexamethylenetetramine
(HMTA) in trifluoroacetic acid at 110°C [32]. The resulting dialdehyde compounds (8a-8g) were
then treated with substituted phenylacetic acids in the presence of cyanuric chloride, and N-
methyl morpholine (NMM) in DMF at 110°C conditions promoted the desired 3-aryl coumarins
(9a-9p) [33]. Further which were engaged in multicomponent reaction with methylamine and
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isatoic anhydride in acetic acid resulted in the formation of final target coumarin-
dihydroquinazolinone conjugates (10a-10p) in good yields.
To study the role of aryl coumarin, we made structural diversity on the 2, 3-
dihydroquinazolinone scaffold, the key 2-hydroxy-3-methyl-5-(3-methyl-4-oxo-1, 2, 3, 4-
tetrahydroquinazolin-2-yl) benzaldehyde (11) intermediate was obtained from the
multicomponent reaction of isatoic anhydride, methylamine and 5-methyl-4-
hydroxyisophthalaldehyde (8a) in acetic acid [34]. Access to the coumarin-dihydroquinazolinone
Scheme 1: Synthesis of coumarin-dihydroquinazolinone conjugates (10a-10p).
Reagents and conditions: (i) a. HMTA / TFA, 120 ºC, 3.0 h; b. 10% H2SO4, 90-100 ºC, 2.0 h; (ii) Cyanuric
chloride, NMM, DMF, 110 ºC, 30-90 min.; (iii) Methyl amine, acetic acid, 110 ºC, 1.0-2.0 h
ester derivatives (12a and 12b) were achieved upon treatment of intermediate 11, with malonic
esters in ethanol catalyzed by piperidine [35]. Benzofuran-dihydroquinazolinones (13a and 13b)
were obtained by treatment of 11 with phenacyl bromide derivatives in acetonitrile and K2CO3 as
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a base under reflux conditions [36]. Furthermore, enamines-dihydroquinazolinone (14a and 14b)
were obtained by the reaction of 11 and aniline derivatives in ethanol [37]. The complete
reaction conditions and reagents are illustrated in Scheme 2. The structures of all the synthesized
1
intermediates and targeted compounds were supported by the spectral H NMR and 13C NMR
data, the results of mass spectrometry which were in agreement with the proposed structures. The
purity of the tested compounds was found to be >95% by HPLC analysis.
Scheme 2: Structural diversity on the 2, 3 dihydroquinazolinone scaffolds (11)
Reagents and conditions: (i) AcOH, 110 ºC, 2.0h; (ii) Piperidine, ethanol, 80 ºC, 1.0 h; (iii) K2CO3, CH3CN,
reflux, 4.0 h; (iv) Ethanol, rt, 15 min.
2.2. Pharmacology
2.2.1 Cell-based high throughput screening for GPR109a biased agonist
First, we evaluated twenty-two coumarin-dihydroquinazolinone (10a-10p, 12a, 12b, 13a, 13b,
14a and 14b) analogs for agonist activity at GPR109a receptor using GloSensor assay that
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measures cAMP in live cells according to previously used method with some modification [38].
We observed that the compound 10b and 10c showed cAMP inhibition in a dose-dependent
manner (EC50 30.5 nM & 10.6 nM Fig-2A & Table 1), while NA, which was used as reference
agonist exhibited EC50 20.7 nM. Compound 10c showed slightly better affinity than NA with
EC50 < 11 nM vs 20.7 nM (Fig- 2A). However, compound 10g, 10j & 10k were modestly active
and inhibited cAMP formation at 10µM concentration (Table 1).
Fig. 2. Representative dose-response curve of GPR109a mediated cAMP inhibition and β-arrestin
recruitment: Concentration response curve of 10b, 10c with niacin (reference ligand) at GPR109a for agonist
activity in transiently transfected HEK293T cells by using cAMP GloSensor assay (A). Similarly, concentration
dependent curve of 10b, 10c and niacin (B) and 10g, 10j and 10k (C) for recruitment of β-arrestin was assessed in
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transiently transfected HTLA cells. Data are expressed as mean ± S.E.M. of normalized results of three individual
experiments.
To determine the β-arrestin dependent signaling of these compounds, we examined five active
compounds in the β-arrestin-Tango assay. Following 12 h of treatment with 10b, 10c, 10g, 10j,
10k and NA as a reference compound, we found that all the tested compounds show a dose-
dependent increase in β-arrestin recruitment (Fig 2b & 2c). we observed that all compounds have
micromolar range affinity and are close to NA in the β-arrestin-Tango assay (Table 1). In vitro
data suggests that 10c has slightly more potency than NA in Gi-protein dependent GPR109a
signaling (GloSensor assay), but have quite a similar effect in β-arrestin dependent GPR109a
signaling (arrestin-tango assay).
Table 1: Functional selectivity of Niacin, 10c and its derivatives at GPR109a receptor
EC50 in
EC50 in
Compounds
Niacin
10b
GloSensor Assay Tango Assay
20.7 nM
(pEC50=7.683±0.09)
2727 nM
(pEC50=5.56±0.07)
1
2
3
4
5
6
30.5 nM
232 nM
(pEC50=7.516±0.38) (pEC50=6.63±0.07)
10.6 nM
1686 nM
10c
(pEC50=7.975±0.18) (pEC50=5.77±0.06)
454 nM
≥10 µM
10g#
(pEC50=6.34±0.09)
1005 nM
≥10 µM
10j#
(pEC50=5.99±0.08)
276 nM
≥10 µM
10k#
(pEC50=6.56±0.10)
#
These compounds did not show dose dependent activity (active only at 10µM concentration) in GloSensor assay.
2.2.2 Structure activity relationship
A series of coumarin-dihydroquinazolinone analogs were synthesized and screened for agonist
activity on GPR109a. Our aim was to determine if coumarin-dihydroquinazolinone analogs
exhibit G-protein bias or β–arrestin bias agonist activity at GPR109a. Preliminary analysis
reveals that compound having mono substitution at para position of phenyl ring with strong
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electron donating group (EDG) such as methoxy (10c) showed greater affinity towards G-
protein dependent pathway (pEC50=7.98) compared to β-arrestin dependent pathway (pEC50=
5.77). Furthermore, compound with weak EDG such as methyl derivative (10b) displayed
slightly low affinity (pEC50=7.52) for the G-protein dependent pathway, while increased affinity
for β-arrestin dependent pathway (pEC50=6.6). The substitutions at both meta and para position
with methelendioxy (10g) or dimethoxy (10k) was not tolerated (pEC50≥ 5) in G- protein
dependent pathway but no change was observed in affinity (pEC50=6.0 to 6.6) in β-arrestin
dependent pathway. Interestingly, heteroaromatic substitution of phenyl ring for example
thiophenyl ring (10j), exhibited persistent β-arrestin dependent activity (pEC50=5.99), but
decrease in G-protein dependent activity (pEC50≥ 5).
2.2.3 Docking studies
Homology models were built due to the non-availability of the crystal structure of Homo sapiens
GPR109a and processed for validation study using PDBsum Generate tool [47]. Out of 10
models, a model was selected whose most of the residues (93.8%) lie in the favored region of
Ramachandran Plot created by PROCHECK, whereas 4.7%, 1.6%, 0.0% residues occur in the
additional allowed region, generously allowed region and disallowed region respectively (Fig
3a). The selected model shows a root mean square deviation (RMSD) of 0.43Aº on
superimposition with the template (Fig 3b). In order to get insight into the binding mode of (R)
and (S) isomers in the racemic mixture of the compound 10c, docking study was performed.
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Fig 3: (A) Ramachandran plot of GPR109a modeled protein. The different colored areas indicate “disallowed” (light
yellow), “generously allowed” (yellow), “additional allowed” (brown), and “most favored” (red) regions.
(B) Superimposed structures of GPR109a homology modeled (blue) and template of active µ-opioid receptor bound
to an agonist selected as a template (PDB identifier: 5C1M pink)
GPR109a has a well-characterised agonist binding pocket with residues surrounding mainly from
transmembrane (TM) and extracellular (EC) regions [48]. Based on the previous studies [49, 50],
Asn86 (EC1), Trp91 (EC1), Arg111 (TM3), Asn171 (EC2), Ser178 (EC2), Ser179 (EC2),
Ser247 (TM6), Arg251 (EC3), Ile254 (EC3), Phe255 (EC3), His259 (EC3), Try283 (TM7),
Tyr284 (TM7), residues were selected for docking study. Those docked conformations were
chosen which show interaction with key binding site residues with the suitable binding pose.
Both (R) and (S) isomers fit well into the binding cavity of the protein and showed relatively
similar molecular interaction with the surrounding residues. (R)-isomer of the compound 10c
docked into the trans-membrane binding pocket has formed a cluster of 15 poses with the lowest
binding energy of -8.87 kcal/mol and manifestation of strong molecular interaction is in the form
of hydrogen bonds with Arg111 at 2.039Aº and Tyr284 at 2.14Aº (Fig 4a). Further inspection of
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the docked structure has also revealed the presence of non-bonded T-shaped π-π interaction with
Phe276 and hydrophobic interactions with Leu34, Leu83, Leu107, and Leu280. In the case of
(S)-isomer, a cluster of 26 poses was selected based on the lowest binding energy of -9.49
kcal/mol. Apart from hydrogen bond interaction with Arg111 and Tyr284, (S)-isomer also
formed a hydrogen bond with the backbone of Cys177 (Fig 4b). Along with T-shaped π-π
interaction with Phe276, π-cation interaction was also observed with Arg111. Various
hydrophobic interactions with residues Leu83, Trp91, Leu107, Arg111, Leu176, Cys177,
Phe277, Leu280 were also observed. Consequently, we further examined the in-vivo anti-
dyslipidemic and antiobesity effect of compound 10c in high-fat diet induced obese mice.
Fig. 4a. Interaction of (R)-isomer (A) and (S)-isomer (B) of compound 10c with the GPR109a model protein.
2.2.4 Beneficial effect of compound 10c on obese mice
It has been previously reported that GPR109a have the benignant effect on obesity [12, 13, 51].
Therefore we examined the anti-obesity effect of chronic (six weeks) oral administration of
compound 10c (20 mg/kg body weight) in high-fat diet (HFD) induced obese mice. Interestingly,
compound 10c significantly reduced cumulative body weight gain starting at day 20 of treatment,
which was even better than reference anti-obesity drug lorcaserin (Figure 5a; n=4-5; *P<
0.05,.**P< 0.01,.***P< 0.001; Two-Way ANOVA). Finally, at the end of the experiment, we
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found a significant decrease in net body weight of compound 10c treated mice compared to HFD
vehicle-treated mice (Table 2; n=6-5; p<0.0356, t-test). Consistent with the reduced body weight,
mice treated with compound 10c also had modest but not significant reduced body fat mass as
compared to control mice (Fig. 5b and Table. 2; n=4-5, P< 0.0002; One-Way ANOVA). In
compound, 10c treated obese mice did not show any significant reduction in lean weight
compared to vehicle-treated mice (Table 2; n=4-5; p<0.0865, One-Way ANOVA).
Fig. 5. Anti-obesity effect, after six weeks chronic treatment of GPR109a agonist 10c and lorcaserin (LRC) in
high-fat diet (HFD) induced obese mice: HFD induced obese mice were treated with lorcaserin (10mg/kg; p.o.) or
10c (20mg/kg; p.o.) significantly decreased body weight gain compare to vehicle-treated HFD mice *P< 0.05,.**P<
0.01,.***P< 0.001 Two-Way ANOVA. (a). Lorcaserin treatment significantly decreased total body fat compared to
vehicle-treated HFD mice, but we did not observe the significant effect in total body fat of compound 10c treated
mice P< 0.001 One Way ANOVA followed by Newman-Keuls Multiple Comparison Test. (b). Compound 10c and
LRC treated obese mice significantly decreased the blood plasma level of leptin compared to vehicle-treated HFD
mice P< 0.001 One-Way ANOVA followed by Newman-Keuls Multiple Comparison Test. (c) Data represented as.
mean ± SEM of 4-5 mice/ group
.
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2.2.5. Chronic treatment of compound 10c decreased obesity-induced blood plasma leptin level
in obese mice
Since obesity is typically associated with high leptin levels due to the insensitivity of leptin [52],
we evaluated if compound 10c treatment modulates blood plasma level in obese mice.
Interestingly, chronic treatment with compound 10c significantly decreased blood plasma leptin
level in diet-induced obese (DIO) mice (Fig 5c & Table2; P< 0.001 One-Way ANOVA). These
observations suggest that treatment with active compound attenuated leptin resistance in DIO
mice. Although we found no difference in glucose tolerance (Fig. 6a), we noticed significant
difference in fasting blood glucose level in 10c treated mice (Table 2, n=4-6; p<0.0273 t-test).
2.2.6 Effect of compound 10c on lipid profile in DIO mice
Given the well established antilipolytic effect of NA [53], we measured the effect of compound
10c on the level of various types of lipids. We found that significant decrease in total serum
cholesterol in compound 10c treated mice as compared to vehicle-treated obese mice (Fig. 6b,
n=4-5; P< 0.05; t-test). However, we did not observe any significant effect of this compound on
triglycerides, high-density lipoprotein (HDL) cholesterol and low-density lipoprotein (LDL)
cholesterol levels in serum of DIO mice.
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Fig. 6. Chronic treatment of GPR109a agonist have no effect in glucose tolerance test but significantly
decreased total blood plasma cholesterol in obese mice: We did not observe any effect on the intraperitoneal
glucose tolerance test (IPGTT) in compound 10c treated mice. IPGTT was performed in 16 hours fasted mice, after
the glucose administration (2g/kg body weight, i.p.) blood glucose level was measured at different time points 15’
30’ 60’ 90’ and 120 minutes (a). Chronic treatment of compound 10c significantly decreased total blood cholesterol
level compared to vehicle-treated mice, but we did not observe any effects in triglycerides, HDLc and LDLc level of
blood serum of compound 10c treated obese mice P< 0.05;t-test (b). Data represented as. mean ± SEM of 4-5 mice/
group
.
Table 2: Summary of measured parameters in obese mice treated with 10c or Vehicle
S. No. Parameters
HFD Vehicle HFD+10c
3.867±1.108 -0.72±1.54
p-value n
5-6
0.0356
Net Body weight Change
1
2
3
4
5
6
7
8
9
Fat mass [g/mouse]
16.52 ± 1.154 13.01 ± 1.476 0.0551 4-5
25.58 ± 0.6789 23.16 ± 0.664 0.0865 5-6
Lean mass [g/mouse]
Fasting glucose [mg/dl]
93.17 ± 5.793 63.00 ± 10.80
4-6
4-5
0.0273
0.0360
Plasma leptin [pg/mg protein] 1751 ± 325.4
807.6 ± 199.2
TG [mg/dl]
40.96 ± 4.592 36.97 ± 8.558 0.7080 5-6
120.8 ± 7.206 95.60 ± 6.007 4-5
57.80 ± 3.385 45.92±5.727 0.0956 5-6
11.56±1.587 10.40±0.867 0.5178 5-6
Total cholesterol [mg/dl]
HDL-c [mg/dl]
LDL-c [mg/dl]
0.0302
3. Conclusion
In conclusion, coumarin-dihydroquinazolinone conjugates (10a-10p) have been synthesized and
tested for their agonist and antagonist activity towards GPR109a receptor. The compound 10c
found to have cAMP inhibition at 10nM concentration and modulates β-arrestin dependent
signaling. Subsequently, compound 10c exhibited beneficial effects such as reduction in
cumulative body weight gain and net body weight. It is evident from the blood plasma analysis
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that compound 10c decreased leptin level in plasma and reduced total serum cholesterol levels;
In order to explore the role of 3-aryl coumarin, we synthesized simple 2, 3-
dihydroquinazolinone, coumarin-dihydroquinazolinone ester (12a & 12b), furan (13a & 13b)
and enamines (14a & 14b), however, none of them were found to be active . Taken together,
these findings highlighted the coumarin-dihydroquinazolinone conjugate as a suitable scaffold to
further expand the chemical diversity and make them potential niacin receptor 1 agonist.
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Experimental section:
4. Chemistry
4.1.1. General synthetic procedure for preparation of 4-hydroxy-5-alkyl isophthalaldehydes
(8a-8g)
2-Alkyl phenol (1.0 mmol) and hexamethylenetetramine (1.2 mmol) were dissolved in TFA (25
mL), and the solution was heated at 120 ºC for 3.0 h. After cooling to room temperature 10 %
aq.H2SO4 (25 mL) was added and again the temperature maintained at 90-100 ºC for two more
hours. After completion, the solution was basified with Na2CO3 to pH 8 and extracted 3-fold with
25 mL of CHCl3. The combined organic layers were dried on Na2SO4, filtered, and concentrated
to dryness under reduced pressure. The crude product was purified on silica gel column (100-200
mesh) using 20% EtOAc/hexane as eluent to afford compounds 8a-8g in good yields.
4.1.1. 4-hydroxy-5-methylisophthalaldehyde (8a)
1
White solid; Yield: 60%; m.p.: 125 - 127 ºC; IR (neat): 3262, 2865, 1703, 1626, 1013 cm-1; H
NMR (CDCl3, 300 MHz): δ 11.82 (s, 1H), 9.97 (s, 1H), 9.90 (s, 1H), 7.97 (d, J = 1.8 Hz, 1H),
7.93 (brs, 1H), 2.33 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ 196.4, 189.9, 165.0, 137.2, 134.8,
128.7, 125.2, 119.7, 15.1; ESI-MS: (m/z): 164 [M+H]+.
4.1.2. 5-Ethyl-4-hydroxyisophthalaldehyde (8b)
1
White solid, yield: 69%; mp: 173-174 °C; IR (neat, cm-1): 3259, 2870, 1700, 1628, 1013; H
NMR (CDCl3, 400 MHz): δ 11.85 (s, 1H), 9.98 (s, 1H), 9.91 (s, 1H), 7.97-7.95 (m, 2H), 2.78-
2.72 (m, 2H), 1.26 (t, J = 7.5 Hz, 3H); 13C NMR (CDCl3, 75 MHz): δ 196.4, 189.7, 164.6, 135.4,
134.7, 134.3, 128.8, 119.7, 22.1, 13.2; ESI-MS (m/z): 179 (M+H)+.
4.1.3.. 4-Hydroxy-5-propylisophthalaldehyde (8c)
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Oily; yield: 62%; IR (neat, cm-1): 3259, 2872, 1710, 1620, 1010; 1H NMR (CDCl3, 400 MHz): δ
11.85 (s, 1H), 9.96 (s, 1H), 9.89 (s, 1H), 7.96 (d, J = 2.0 Hz, 1H), 7.91 (d, J = 2.0 Hz, 1H), 2.68
(t, J = 6.6 Hz, 2H), 1.71-1.61 (m, 2H), 0.96 (t, J = 7.3 Hz, 3H); 13C NMR (CDCl3, 100 MHz): δ
196.4, 189.7, 164.7, 136.4, 134.7, 132.8, 128.7, 119.8, 30.9, 22.1, 13.8; ESI-MS: (m/z): 193
(M+H)+.
4.1.4.. 4-hydroxy-5-isopropylisophthalaldehyde (8d)
White solid; yield; 89%; m.p.: 164-163 ºC; IR (KBr): 3028, 1677, 1665, 1615, 1229, 768 cm-1;
1H NMR (CDCl3, 300 MHz): δ 11.94 (s, 1H), 9.97 (s, 1H), 9.91 (s, 1H), 7.99 (brs, 1H), 7.96 (d, J
=1.47 Hz,1H), 3.43-3.33 (m, 1H), 1.27 (d, J =5.1 Hz, 6H); 13C NMR (CDCl3, 75 MHz): δ 196.6,
189.9, 164.3, 138.9, 134.8, 133.2, 129.0, 119.9, 26.5, 22.1 ; ESI-MS: (m/z) 265 [M+H]+.
4.1.5.. 5-sec-butyl-4-hydroxy-benzene-1,3-dicarbaldehyde (8e)
Oily; Yield: 64%; IR (neat): 3267, 2862, 1709, 1622, 1018 cm-1; 1H NMR (CDCl3, 200 MHz): δ
11.99 (s, 1H), 10.05 (s, 1H), 9.96 (s, 1H), 8.08 (brs, 1H), 8.00 (s, 1H), 3.27-3.10 (m, 1H), 1.74-
1.58 (m, 2H), 1.25 (d, J = 7.0 Hz, 3H), 0.87 (t, J = 7.3 Hz, 3H); 13C NMR (CDCl3, 75 MHz): δ
196.4, 189.4, 163.9, 137.1, 134.5, 133.4, 128.7, 119.6, 32.7, 28.8, 19.3, 11.5; ESI-MS (m/z): 207
[M+H]+.
4.1.6. 5-tert-butyl-4-hydroxyisophthalaldehyde (8f)
Oily; Yield: 65%; IR (neat): 3252, 2865, 1703, 1626, 1013 cm-1; 1H NMR (CDCl3, 300 MHz): δ
13
12.39 (s, 1H), 9.99 (s, 1H), 9.93 (s, 1H), 8.07 (brs, 1H), 7.99 (brs, 1H), 1.45 (s, 9H); C NMR
(CDCl3, 75 MHz): δ 196.9, 190.0, 166.1, 140.0, 135.4, 133.9, 128.6, 120.4, 35.2, 29.1; ESI-MS:
(m/z): 207 [M+H]+.
4.1.7. 4-hydroxy-5, 6, 7, 8-tetrahydronaphthalene-1, 3-dicarbaldehyde (8g)
18

ACCEPTED MANUSCRIPT
White solid; yield; 65%; m.p.: 145-147 ºC; IR (KBr): 3026, 1677, 1665, 1615, 1249, 768 cm-1;
1H NMR (CDCl3, 300 MHz): δ 11.86 (s, 1H), 10.15 (s, 1H), 9.92 (s, 1H), 7.92 (s, 1H), 3.24 (d, J
=5.2 Hz, 2H), 2.74 (d, J =5.1 Hz, 2H), 1.84-1.82 (m, 4H); 13C NMR (CDCl3, 100 MHz): δ 195.9,
190.3, 163.6, 149.3, 136.8, 127.7, 126.9, 117.3, 27.4, 22.6, 21.9, 21.1; ESI-MS: (m/z) 205
[M+H]+.
4.2.1. 8-methyl-2-oxo-3-phenyl-2H-chromene-6-carbaldehyde (9a)
Light yellow solid; yield: 86%; m.p.: 124-126 ºC; IR (KBr): 3041, 1716, 1709, 1652, 1024 cm-1;
1H NMR (CDCl3, 400 MHz): δ 10.03 (s, 1H), 7.92 (d, J = 4.0 Hz, 2H), 7.89 (s, 1H), 7.74 (d, J =
1.6 Hz, 1H), 7.72 (d, J = 1.3 Hz, 1H) 7.49-7.46 (m, 3H), 2.58 (s, 3H); 13C NMR (CDCl3, 100
MHz): δ 190.4, 159.7, 155.5, 139.4, 134.1, 132.4, 129.2, 129.1, 128.6, 128.5, 128.2, 127.3,
119.6, 15.5; ESI-MS (m/z): 265 [M+H]+.
4.2.2. 8-methyl-2-oxo-3-(p-tolyl)-2H-chromene-6-carbaldehyde (9b)
Light yellow solid; yield: 81%; m.p.: 128-130 ºC; IR (KBr): 3044, 1721, 1715, 1644, 1019 cm-1;
1H NMR (CDCl3, 300 MHz): δ 10.03 (s, 1H), 7.92 (d, J = 5.2 Hz, 2H), 7.86 (s, 1H), 7.64 (d, J =
7.4 Hz, 2H), 7.30 (d, J = 7.4 Hz, 2H), 2.58 (s, 3H), 2.43 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ
190.4, 159.8, 155.5, 139.4, 138.7, 132.4, 132.3, 131.2, 129.3, 129.1, 128.3, 128.1, 127.2, 119.7,
21.3, 15.5; ESI-MS (m/z): 279 [M+H]+.
4.2.3. 3-(4-Methoxyphenyl)-8-methyl-2-oxo-2H-chromene-6-carbaldehyde (9c)
Light yellow solid; yield: 96%; m.p.: 148-150 ºC; IR (KBr): 3033, 1725, 1705, 1639, 1021 cm-1;
1H NMR (CDCl3, 300 MHz): δ 9.98 (s, 1H), 7.86 (d, J = 4.5 Hz, 2H), 7.79 (s, 1H), 7.67 (d, J =
8.9 Hz, 2H), 6.96 (d, J = 8.5 Hz, 2H), 3.84 (s, 3H), 2.53 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ
190.5, 160.5, 159.9, 155.3, 137.9, 132.4, 132.3, 129.9, 128.6, 128.0, 127.2, 126.4, 119.8, 114.1,
55.4, 15.5; ESI-MS (m/z): 295 [M+H]+.
19

ACCEPTED MANUSCRIPT
4.2.4. 3-(3-Methoxyphenyl)-8-methyl-2-oxo-2H-chromene-6-carbaldehyde (9d)
Light yellow solid; yield: 95%; m.p.: 150-152 ºC; IR (KBr): 3025, 1716, 1707, 1645, 1016 cm-1;
1H NMR (CDCl3, 300 MHz): δ 10.01 (s, 1H), 7.88 (d, J = 10.4 Hz, 3H), 7.40-7.35 (m, 1H), 7.28-
7.26 (m, 2H), 6.99-6.95 (m, 1H), 3.86 (s, 3H), 2.56 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ 190.6,
159.7, 155.7, 139.7, 135.5, 132.8, 132.5, 129.8, 129.1, 128.4, 127.5, 121.0, 119.7, 114.9, 114.4,
55.5, 15.7; ESI-MS (m/z): 294 [M+H]+.
4.2.5. 3-(3,4-Dimethoxyphenyl)-8-methyl-2-oxo-2H-chromene-6-carbaldehyde (9e)
Light yellow solid; yield: 98%; m.p.: 178-180 ºC; IR (KBr): 3053, 1730, 1695, 1634, 1026 cm-1;
1H NMR (CDCl3, 300 MHz): δ 10.01 (s, 1H), 7.90 (d, J = 7.3 Hz, 2H), 7.84 (s, 1H), 7.32-7.29
(m, 2H), 6.96 (d, J = 9.0 Hz, 1H), 3.95 (s, 3H), 3.94 (s, 3H), 2.56 (s, 3H); 13C NMR (CDCl3, 75
MHz): δ 190.5, 159.9, 155.4, 150.2, 148.9, 138.2, 132.5, 132.4, 128.7, 128.0, 127.3, 126.8,
121.4, 119.8, 111.8, 111.2, 56.1, 56.0, 15.5; ESI-MS (m/z): 325 [M+H]+.
4.2.6. 8-Methyl-2-oxo-3-(3,4,5-trimethoxyphenyl)-2H-chromene-6-carbaldehyde (9f)
1
White solid; yield: 99%; m.p.: 196-198 ºC; IR (KBr): 3074, 1728, 1695, 1632, 1010 cm-1; H
NMR (CDCl3, 300 MHz): δ 10.02 (s, 1H), 7.91 (m, 2H), 7.86 (s, 1H), 6.95 (s, 2H), 3.93 (s, 6H),
13
3.91 (s, 3H), 2.57 (s, 3H); C NMR (CDCl3, 75 MHz): δ 190.5, 159.8, 155.5, 153.4, 139.4,
139.1, 132.8, 132.6, 129.6, 129.0, 128.2, 127.4, 119.7, 106.2, 61.0, 56.4, 15.6; ESI-MS (m/z):
355 [M+H]+.
4.2.7. 3-(benzo[d][1,3]dioxol-5-yl)-8-methyl-2-oxo-2H-chromene-6-carbaldehyde (9g)
1
Yellow solid; yield: 85%; m.p.: 166-168 ºC; IR (KBr): 3067, 1723, 1671, 1617, 1030 cm-1; H
NMR (CDCl3, 400 MHz): δ 10.03 (s, 1H), 7.91 (bs, 2H), 7.82 (s, 1H), 7.26 (d, J = 1.5 Hz 1H),
7.23 (d, J = 7.9 Hz, 1H), 6.92 (d, J = 7.9 Hz, 1H), 6.05 (s, 2H), 2.58 (s, 3H); 13C NMR (CDCl3,
20

ACCEPTED MANUSCRIPT
100 MHz): δ 190.4, 159.8, 155.3, 148.6, 147.8, 138.4, 132.4, 132.4, 128.7, 128.0, 127.3, 122.6,
119.6, 109.0, 108.4, 101.4, 15.5; ESI-MS (m/z): 309 [M+H]+.
4.2.8. 3-(2-chlorophenyl)-8-methyl-2-oxo-2H-chromene-6-carbaldehyde (9h)
1
Yellow solid; yield: 81%; m.p.: 203-205 ºC; IR (KBr): 3058, 1757, 1679, 1644, 1017 cm-1; H
NMR (CDCl3, 400 MHz): δ 10.04 (s, 1H), 7.96 (s, 1H), 7.93 (s, 1H), 7.83 (s, 1H), 7.54-7.52 (m,
1H), 7.45-7.42 (m, 1H), 7.41-7.38 (m, 2H), 2.59 (s, 3H); 13C NMR (CDCl3, 100 MHz): δ 190.3,
158.9, 156.0, 142.3, 133.5, 133.2, 133.0, 132.4, 131.2, 130.3, 130.0, 128.4, 127.9, 127.6, 126.9,
118.9, 15.5; ESI-MS (m/z): 299 [M+H]+.
4.2.9. 8-methyl-3-(2-nitrophenyl)-2-oxo-2H-chromene-6-carbaldehyde (9i)
1
White solid; yield: 94%; m.p.: 285-288 ºC; IR (KBr): 3065, 1718, 1697, 1607, 1022 cm-1; H
NMR (CDCl3, 400 MHz): δ 10.02 (s, 1H), 8.18 (d, J = 8.0 Hz, 1H), 7.94 (d, J = 4.0 Hz, 2H) 7.83
(s, 1H), 7.75 (t, J = 7.4 Hz, 1H), 7.66-7.62 (m, 1H), 7.50 (d, J = 7.4 Hz, 1H), 2.56 (s, 3H); ESI-
MS (m/z): 310 [M+H]+.
4.2.10. 8-methyl-2-oxo-3-(thiophen-2-yl)-2H-chromene-6-carbaldehyde (9j)
1
Brown solid; yield: 84%; m.p.: 117-119 ºC; IR (KBr): 3047, 1711, 1675, 1607, 1083 cm-1; H
NMR (CDCl3, 400 MHz): δ 10.02 (s, 1H), 8.06 (s, 1H), 7.93 (s, 1H), 7.89 (s, 1H), 7.85 (d, J =
13
3.0 Hz, 1H), 7.48 (d, J = 5.1 Hz, 1H), 7.16 (t, J = 4.1 Hz, 1H), 2.57 (s, 3H); C NMR (CDCl3,
100 MHz): δ 190.3, 160.0, 158.6, 154.6, 135.3, 134.9, 132.5, 132.3, 128.3, 128.0, 127.7, 127.3,
122.6, 119.3, 15.5; ESI-MS (m/z): 271 [M+H]+.
4.2.11. 3-(3, 4-dimethoxyphenyl)-8-ethyl-2-oxo-2H-chromene-6-carbaldehyde (9k)
Light yellow solid; yield: 95%; m.p.: 160-162 ºC; IR (KBr): 3065, 1728, 1697, 1607, 1032 cm-1;
1H NMR (CDCl3, 300 MHz): δ 10.04 (s, 1H), 7.92 (s, 2H), 7.85 (s, 1H), 7.33-7.28 (m, 2H), 6.97
21

ACCEPTED MANUSCRIPT
(d, J = 8.0 Hz, 1H), 3.95 (s, 6H), 3.00 (q, J = 7.4 Hz, 2H), 1.38 (t, J = 7.5 Hz, 3H); ESI-MS
(m/z): 339 [M+H]+.
4.2.12. 3-(3,4-dimethoxyphenyl)-2-oxo-8-propyl-2H-chromene-6-carbaldehyde (9l)
1
Yellow solid; yield: 94%; m.p.: 152-154 ºC; IR (KBr): 3061, 1731, 1688, 1612, 1017 cm-1; H
NMR (CDCl3, 400 MHz): δ 10.04 (s, 1H), 7.93 (s, 1H), 7.91 (s, 1H) 7.86 (s, 1H), 7.34 (s, 1H),
7.31 (d, J = 2.1 Hz, 1H), 6.98 (d, J = 8.1 Hz, 1H), 3.97 (s, 3H), 3.96 (s, 3H), 2.95 (t, J = 7.4 Hz,
13
2H), 1.85-1.76 (m, 2H), 1.05 (t, J = 7.0 Hz, 3H); C NMR (CDCl3, 100 MHz): δ 190.5, 159.8,
155.0, 150.0, 148.8, 138.2, 132.4, 131.6, 128.6, 128.0, 126.7, 121.3, 119.8, 111.7, 111.1, 56.0,
55.9, 31.2, 22.7, 13.8; ESI-MS (m/z): 353 [M+H]+.
4.2.13. 3-(3,4-dimethoxyphenyl)-8-isopropyl-2-oxo-2H-chromene-6-carbaldehyde (9m)
1
Yellow solid; yield: 89%; m.p.: 149-151 ºC; IR (KBr): 3067, 1727, 1681, 1611, 1027 cm-1; H
NMR (CDCl3, 300 MHz): δ 10.03 (s, 1H), 7.97 (s, 1H), 7.92 (s, 1H), 7.85 (s, 1H), 7.28 (d, J =
7.9 Hz, 2H), 6.98 (d, J = 8.0 Hz, 1H), 3.95 (s, 3H), 3.94 (s, 3H), 3.73-3.66 (m, 1H), 1.37 (d, J =
6.8 Hz, 6H); ESI-MS (m/z): 353 [M+H]+.
4.2.14. 8-tert-butyl-3-(3, 4-dimethoxyphenyl)-2-oxo-2H-chromene-6-carbaldehyde (9n)
Light yellow solid; yield: 91%; m.p.: 160-162 ºC; IR (KBr): 3055, 1728, 1687, 1607, 1022 cm-1;
1H NMR (CDCl3, 300 MHz): δ 10.05 (s, 1H), 8.04 (s, 1H), 7.95 (s, 1H), 7.87 (s, 1H), 7.38-7.32
(m, 2H), 6.98 (d, J = 8.3 Hz, 1H), 3.97 (s, 3H), 3.95 (s, 3H), 1.60 (s, 9H); ESI-MS (m/z): 367
[M+H]+.
4.2.15. 8-sec-Butyl-3-(3, 4-dimethoxyphenyl)-2-oxo-2H-chromene-6-carbaldehyde (9o)
Light yellow solid; yield: 98%; m.p.: 140-142 ºC; IR (KBr): 3065, 1728, 1697, 1607, 1032 cm-1;
1H NMR (CDCl3, 300 MHz): δ 10.04 (s, 1H), 7.93 (d, J = 1.6 Hz, 2H), 7.86 (s, 1H), 7.33-7.29
(m, 2H), 6.96 (d, J = 8.6 Hz, 1H), 3.96 (s, 3H), 3.95 (s, 3H), 3.54-3.44 (m, 1H), 1.81-1.72 (m,
22

ACCEPTED MANUSCRIPT
2H), 1.34 (d, J = 7.0 Hz, 3H), 0.91 (t, J = 7.4 Hz, 3H); 13C NMR (CDCl3, 75 MHz): δ 190.8,
160.0, 154.8, 150.2, 150.0, 138.6, 136.8, 132.8, 129.2, 128.8, 128.0, 126.9, 121.4, 120.1, 111.9,
111.3, 56.2, 56.1, 33.5, 29.8, 20.6, 12.1; ESI-MS (m/z): 367 [M+H]+.
4.2.16. 3-(3, 4-dimethoxyphenyl)-2-oxo-7, 8, 9, 10-tetrahydro-2H-benzo[h]chromene-6-
carbaldehyde (9p)
Light yellow solid; yield: 95%; m.p.: 185-187 ºC; IR (KBr): 3065, 1718, 1695, 1607, 1132 cm-1;
1H NMR (CDCl3, 300 MHz): δ 10.29 (s, 1H), 7.87 (s, 1H), 7.81 (s, 1H), 7.30-7.26 (m, 2H), 6.95
(d, J = 6.1 Hz, 1H), 3.95 (s, 3H), 3.93 (s, 3H), 3.29 (s, 2H), 2.99 (s, 2H), 1.89-1.87 (m, 4H); ESI-
MS (m/z): 365 [M+H]+.
4.3. Typical multicomponent synthesis of coumarin-quinoline conjugates (10a-10p)
The reaction of 9a-9p (1.0 mmol), isatoic anhydride (1.5 mmol) and methyl amine (excess) was
carried out in presence of sufficient quantity of acetic acid. The reaction mixture was subjected
to refluxing conditions at about 110 °C for 1.0-2.0 h. After completion of the reaction, the
reaction mixture was allowed to cool up to room temperature and then cold water was added to
it. A solid was obtained which was thoroughly washed with water to remove acetic acid. The
crude product thus obtained was purified by column chromatography using 5% MeOH/ DCM
solvent system.
4.3.1. 3-methyl-2-(8-methyl-2-oxo-3-phenyl-2H-chromen-6-yl)-2, 3-dihydroquinazolin-4(1H)-
one (10a)
1
White solid; Yield: 90%, m.p.: 156-158 ºC; IR (KBr): 3416, 3021, 2926, 1622, 1215 cm-1; H
NMR (DMSO-d6, 300 MHz): δ 8.25 (s, 1H), 7.73-7.66 (m, 3H), 7.52 (s, 2H), 7.44-7.42 (m, 3H),
13
7.32 (s, 1H), 7.23-7.18 (m, 1H) 6.69-6.64 (m, 2H), 5.91 (s, 1H), 2.89 (s, 3H), 2.39 (s, 3H); C
NMR (CDCl3, 75 MHz): δ 162.9, 160.0, 151.7, 146.5, 141.1, 136.9, 134.9, 133.6, 131.3, 129.0,
23

ACCEPTED MANUSCRIPT
128.9, 128.6, 127.9, 127.3, 125.7, 124.2, 119.4, 117.6, 114.8, 114.6, 71.9, 32.4, 15.6; HRMS
(ESI) calcd for C25H20N2O3 [M + H]+, 397.1547, found 397.1571.
4.3.2. 3-methyl-2-(8-methyl-2-oxo-3-p-tolyl-2H-chromen-6-yl)-2, 3-dihydroquinazolin-4(1H)-one
(10b)
White solid; yield: 85%; m.p.: 238-240 ºC; IR (KBr): 3415, 3015, 2946, 1670, 1210, 1052 cm-1;
1H NMR (DMSO-d6, 300 MHz): δ 8.23 (s, 1H), 7.68-7.61 (m, 3H), 7.50 (s, 2H), 7.36 (d, J = 1.0
Hz, 1H), 7.26-7.18 (m, 3H), 6.69-6.63 (m, 2H), 5.91 (d, J = 1.9 Hz, 1H), 2.88 (s, 3H), 2.38 (s,
13
3H), 2.33 (s, 3H); C NMR (DMSO-d6, 100 MHz): δ 162.9, 160.1, 151.5, 146.6, 140.4, 138.6,
136.9, 133.7, 132.0, 131.1, 129.2, 128.8, 127.9, 127.2, 125.7, 124.0, 119.5, 117.6, 114.8, 114.6,
71.8, 32.5, 21.3, 15.6; HRMS (ESI) calcd for C26H22N2O3 [M + H]+, 411.1703, found 411.1659.
4.3.3. 2- (3 -( 4- methoxyphenyl) -8- methyl -2-oxo-2H –chromen -6- yl) -3- methyl- 2, 3-
dihydroquinazolin -4(1H)-one (10c)
White solid; yield: 85%; m.p.: 228-230 ºC; IR (KBr):3409, 3025, 2944, 1660, 1220, 1042 cm-1;
1H NMR (CDCl3, 300 MHz): δ 7.98 (d, J = 7.6 Hz, 1H), 7.67-7.64 (m, 3H), 7.36 (d, J = 11.7 Hz,
2H), 7.30-7.25 (m, 1H), 6.98 (d, J = 8.6 Hz, 2H), 6.89-6.84 (m, 1H), 6.60 (d, J = 7.9 Hz, 1H),
5.74 (s, 1H), 4.76 (s, 1H), 3.86 (s, 3H), 2.92 (s, 3H), 2.42 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ
163.6, 160.6, 160.3, 151.8, 145.3, 138.3, 135.6, 133.7, 130.5, 129.8, 128.4, 127.8, 126.8, 126.7,
123.5, 119.5, 119.2, 115.3, 114.4, 114.0, 73.5, 55.4, 32.1, 15.6; HRMS (ESI) calcd for
C26H22N2O4 [M + H]+, 427.1652, found 427.1661.
4.3.4.
2-(3-(3-methoxyphenyl)-8-methyl-2-oxo-2H-chromen-6-yl)-3-methyl-2,
3-
dihydroquinazolin-4(1H)-one (10d)
White solid; Yield: 86%, m.p.: 160-162 ºC; IR (KBr): 3420, 3025, 2944, 1660, 1220, 1042 cm-1 ;
1H NMR (CDCl3, 300 MHz): δ 7.97 (d, J = 7.7 Hz, 1H), 7.74 (s, 1H), 7.41-7.35 (m, 3H), 7.28-
24

ACCEPTED MANUSCRIPT
7.24 (m, 3H), 6.97 (d, J = 7.5 Hz, 1H), 6.86 (s, 1H), 6.60 (d, J = 7.9 Hz, 1H), 5.74 (s, 1H), 4.81
(s, 1H), 3.86 (s, 3H), 2.93 (s, 3H), 2.42 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ 163.6, 160.6,
159.6, 152.1, 145.2, 139.9, 135.7, 135.6, 133.7, 130.9, 129.6, 128.4, 128.2, 126.9, 123.7, 120.8,
119.2, 115.3, 114.7, 114.4, 114.2, 73.5, 55.4, 32.2, 15.6; HRMS (ESI) calcd for C26H22N2O4 [M
+ H]+, 427.1652, found 427.1650.
4.3.5. 2- (3- (3, 4-dimethoxyphenyl) -8-methyl -2 -oxo- 2H- chromen- 6-yl) -3 -methyl- 2, 3-
dihydroquinazolin- 4(1H)-one (10e)
Light yellow solid; Yield: 87%; m.p.: 168-170 ºC; IR (KBr): 3415, 3013, 2921, 1635, 1252,
1
1026 cm-1; H NMR (CDCl3, 300 MHz): δ 7.98 (d, J = 7.4 Hz, 1H), 7.71 (s, 1H), 7.39 (s, 1H),
7.36 (s, 1H), 7.31-7.28 (m, 3H), 6.94 (d, J = 8.1 Hz, 1H), 6.89-6.84 (m, 1H), 6.60 (d, J = 7.8 Hz,
1H), 5.75 (s,1H), 4.77 (s, 1H), 3.95 (s, 3H), 3.93(s, 3H), 2.94 (s, 3H), 2.44 (s, 3H); 13C NMR
(CDCl3, 75 MHz): δ 163.7, 160.6, 151.9, 149.9, 148.8, 145.1, 138.6, 135.6, 133.7, 130.5, 128.5,
127.9, 127.0, 126.9, 123.4, 121.3, 119.5, 119.3, 115.4, 114.4, 111.6, 111.3, 75.3, 56.1, 32.2,
15.6; HRMS (ESI) calcd for C27H24N2O5 [M + H]+, 457.1758, found 457.1755.
4.3.6. 3- Methyl -2 - (8-methyl-2-oxo-3- (3, 4, 5-trimethoxyphenyl) -2H-chromen-6-yl) -2, 3-
dihydroquinazolin-4(1H)-one (10f)
Yellow solid; Yield: 85%; m.p.: 178-180 ºC; IR (KBr):3402, 3018, 2929, 1641, 1216, 1047 cm-1;
1H NMR (CDCl3, 300 MHz): δ 7.97 (d, J = 7.6 Hz, 1H), 7.74 (s, 1H), 7.40 (s, 1H), 7.36 (s, 1H),
7.29-7.25 (m, 1H), 6.94 (s, 2H), 6.88- 6.83 (m, 1H), 6.59 (d, J = 7.8 Hz, 1H), 5.75 (s, 1H), 4.85
13
(s, 1H), 3.92 (s, 3H), 3.89 (s, 6H), 2.95 (s, 3H), 2.45 (s, 3H); C NMR (DMSO-d6, 75 MHz): δ
159.0, 155.7, 148.5, 147.3, 140.5, 134.8, 134.3, 131.0, 129.1, 126.2, 125.2, 123.8, 123.4, 122.2,
118.8, 114.7, 114.5, 110.7, 109.8, 101.3, 68.7, 56.2, 51.7, 27.6, 10.8; HRMS (ESI) calcd for
C28H26N2O6 [M + H]+, 487.1864, found 487.1857.
25

ACCEPTED MANUSCRIPT
4.3.7. 2- (3-(benzo[d] [1, 3] dioxol-5-yl) -8-methyl-2-oxo-2H-chromen-6-yl) -3-methyl-2, 3-
dihydroquinazolin- 4(1H)-one (10g)
White solid; Yield: 84%; m.p.: 165-167 ºC; IR (KBr): 3412, 3011, 2921, 1634, 1243, 1039 cm-1;
1H NMR (CDCl3, 300 MHz): δ 7.95 (d, J = 7.6 Hz, 1H), 7.64 (s, 1H), 7.38 (s, 1H), 7.32 (s, 1H)
7.28-7.24 (m, 1H), 7.19-7.14 (m, 2H), 6.88-6.82 (m, 2H), 6.58 (d, J = 7.9 Hz, 1H), 6.00 (s, 2H),
5,73 (s, 1H), 4.77 (s, 1H), 2.91 (s, 3H), 2.40 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ 163.6, 160.5,
151.9, 148.4, 147.8, 145.2, 138.9, 135.7, 133.7, 130.6, 128.4, 128.2, 127.9, 126.8, 123.5, 122.5,
119.4, 119.2, 115.3, 114.4, 108.9, 108.4, 101.5, 73.5, 32.3, 15.6; HRMS (ESI) calcd for
C26H20N2O5 [M + H]+, 441.1445, found 441.1447.
4.3.8.
2-(3-(2-chlorophenyl)-8-methyl-2-oxo-2H-chromen-6-yl)-3-methyl-2,3-
dihydroquinazolin 4(1H)-one (10h)
White solid; yield: 83%; m.p.: 228-230 ºC; IR (KBr): 3408, 3025, 2946, 1670, 1220, 1052 cm-1;
1H NMR (DMSO-d6, 300 MHz): δ 8.13 (s, 1H), 7.66 (d, J = 7.2 Hz, 1H), 7.55-7.45 (m, 6H), 7.31
(s, 1H), 7.23-7.19 (m, 1H), 6.69-6.64 (m, 2H), 5.91 (s, 1H), 2.88 (s, 3H), 2.40 (s, 3H); 13C NMR
(DMSO-d6, 75 MHz): δ 162.9, 159.2, 152.1, 146.6, 143.6, 137.1, 134.4, 133.7, 133.2, 132.2,
131.7, 130.8, 129.7, 127.9, 127.7, 126.9, 126.1, 124.4, 118.8, 117.7, 114.8, 114.6, 71.8, 32.5,
15.6; HRMS (ESI) calcd for C25H19ClN2O3 [M + H]+, 431.1157, found 431.1149.
4.3.9. 3- methyl-2- (8-methyl-3-(2-nitrophenyl) -2-oxo-2H-chromen-6-yl) -2, 3-
dihydroquinazolin 4(1H)-one (10i)
Light yellow solid; Yield: 82%; m.p.: 188-190 ºC; IR (KBr): 3441, 3015, 2944, 1660, 1220,
1
1032 cm-1; H NMR (DMSO-d6, 300 MHz): δ 8.34 (s, 1H), 8.13 (s, 1H), 7.86 (s, 1H) 7.71 (s,
3H), 7.57 (d, J = 7.4 Hz, 2H), 7.39 (s, 1H), 7.22 (s, 1H), 6.67 (s, 2H), 5.95 (s, 1H), 2.92 (s, 3H),
2.40 (s, 3H); 13C NMR (DMSO-d6, 100 MHz): δ 163.0, 159.5, 151.7, 148.6, 146.5, 141.6, 137.4,
26

ACCEPTED MANUSCRIPT
134.6, 133.7, 132.9, 131.9, 130.8, 129.9, 127.9, 126.8, 126.2, 124.8, 124.2, 119.1, 117.7, 114.8,
114.6, 71.8, 32.5, 15.5; HRMS (ESI) calcd for C25H19N3O5 [M + H]+, 442.1397, found 442.1379.
4.3.10. 3- methyl- 2- (8-methyl-2-oxo-3- (thiophen-2-yl)-2H-chromen-6-yl)-2, 3-
dihydroquinazolin-4(1H)-one (10j)
1
Green solid; Yield: 84%; m.p.: 178-180 ºC; IR (KBr): 3410, 2921, 1611, 1213 cm-1; H NMR
(DMSO-d6, 300 MHz): δ 8.59 (s, 1H), 7.89-7.88 (m, 1H), 7.69-7.66 (m, 2H), 7.51 (s, 2H), 7.37-
7.36 (m, 1H), 7.24-7.16 (m, 2H), 6.70-6.63 (m, 2H), 5.92-5.91 (m, 1H), 2.91 (s, 3H), 2.38 (s,
13
3H); C NMR (DMSO-d6, 75 MHz): δ 163.0, 159.4, 150.9, 146.6, 137.2, 136.7, 133.7, 131.2,
129.4, 128.0, 127.8, 127.3, 125.9, 123.8, 121.1, 119.3, 117.7, 114.9, 114.7, 71.9, 32.6, 15.6;
HRMS (ESI) calcd for C23H18N2O3S [M + H]+, 403.1111, found 403.1104.
4.3.11. 2- (3- (3, 4-dimethoxyphenyl) -8-ethyl-2-oxo-2H-chromen-6-yl) -3-methyl-2, 3-dihydro
quinazolin-4(1H)-one (10k)
1
Yellow solid; Yield: 86%; m.p.: 95-97 ºC; IR (KBr): 3409, 2921, 1611, 1213 cm-1 ; H NMR
(CDCl3, 300 MHz): δ 7.99 (d, J = 7.7 Hz, 1H), 7.72 (s, 1H), 7.41 (s, 1H), 7.37 (s, 1H), 7.29-7.26
(m, 3H), 6.96-6.85 (m, 2H), 6.59 (d, J = 8.0 Hz, 1H), 5.77 (s, 1H), 4.69 (s, 1H), 3.95 (s, 3H),
3.93 (s, 3H), 2.94-2.85 (m, 5H), 1.31-1.27 (m, 3H); 13C NMR (DMSO-d6, 75 MHz): δ 163.6,
160.4, 151.4, 149.9, 148.7, 145.0, 138.6, 135.6, 133.6, 132.8, 128.9, 128.4, 127.9, 127.0, 123.3,
121.2, 119.6, 119.3, 115.5, 114.3, 111.6, 111.0, 73.5, 56.0, 55.9, 32.1, 22.5, 14.0; HRMS (ESI)
calcd for C28H25N2O5 [M + H]+, 471.1914, found 471.1911.
4.3.12. 2- (3- (3, 4-dimethoxyphenyl)-2-oxo-8-propyl-2H-chromen-6-yl)-3-methyl-2, 3 dihydro
quinazolin-4(1H)-one (10l)
1
White solid; Yield: 88%; m.p.: 98-100 ºC; IR (KBr): 3410, 2921, 1611, 1213 cm-1; H NMR
(CDCl3, 300 MHz): δ 8.00 (d, J = 7.7 Hz, 1H), 7.74 (s, 1H), 7.40-7.39 (m, 2H), 7.29 (d, J = 7.7
27

ACCEPTED MANUSCRIPT
Hz, 3H), 6.99-6.86 (m, 2H), 6.58 (d, J = 7.9 Hz, 1H), 5.77 (s, 1H), 4.57 (s, 1H), 3.95 (s, 3H),
3.94 (s, 3H), 2.95 (s, 3H), 2.88-2.83 (m, 2H), 1.78-1.68 (m, 2H), 1.01-0.97 (m, 3H); 13C NMR
(CDCl3, 75 MHz): δ 163.6, 160.4, 151.5, 149.8, 148.7, 144.9, 138.6, 135.4, 133.6, 131.3, 129.7,
128.4, 128.0, 127.0, 123.4, 121.2, 119.7, 119.3, 115.5, 114.3, 111.6, 111.0, 73.4, 56.0, 32.1, 22.8,
13.8; HRMS (ESI) calcd for C29H28N2O5 [M + H]+, 485.2071, found 485.2073.
4.3.13. 2-(3-(3, 4-dimethoxyphenyl)-8-isopropyl-2-oxo-2H-chromen-6-yl) -3-methyl-2, 3
dihydro quinazolin-4(1H)-one (10m)
Light yellow solid; Yield: 88%; m.p.: 110-112 ºC; IR (KBr): 3419, 3017, 2931, 1639, 1217,
1
1026 cm-1; H NMR (CDCl3, 300 MHz): δ 8.00-7.98 (m, 1H), 7.74 (s, 1H), 7.47 (d, J = 1.5 Hz,
1H), 7.38 (d, J = 1.7 Hz, 1H), 7.32-7.26 (m, 3H), 6.96-6.93 (m, 1H), 6.90-6.85 (m, 1H), 6.59 (s,
J = 7.9 Hz, 1H), 5.78 (s, 1H), 4.65 (s, 1H), 3.95 (s, 3H), 3.93 (s, 3H), 3.69-3.60 (m, 1H), 2.94 (s,
13
3H), 1.33-1.32 (m, 3H), 1.31-1.30 (m, 3H); C NMR (CDCl3, 75 MHz): δ 163.6, 160.4, 150.9,
149.8, 148.7, 145.0, 138.8, 137.3, 135.6, 133.6, 128.4, 127.9, 127.0, 126.4, 123.2, 121.2, 119.7,
119.3, 115.5, 114.3, 111.5, 111.0, 73.5, 55.9, 32.1, 26.7, 22.6; HRMS (ESI) calcd for
C29H28N2O5 [M + H]+, 485.2071, found 485.2056.
4.3.14. 2- (8-tert-butyl-3- (3, 4-dimethoxyphenyl) -2-oxo-2H-chromen-6-yl) -3-methyl-2, 3-
dihydro quinazolin-4(1H)-one (10n)
Light yellow solid; Yield: 86%; m.p.: 115-117 ºC; IR (KBr): 3410, 2921, 1611, 1213 cm-1 ;1H
NMR (CDCl3, 300 MHz): δ 8.01-7.99 (m, 1H), 7.75 (s, 1H), 7.53 (d, J = 1.6 Hz, 1H), 7.40 (d, J
= 1.6 Hz, 1H), 7.35-7.26 (m, 3H), 6.96-6.86 (m, 2H), 6.59 (d, J = 7.9 Hz, 1H), 5.77 (s, 1H), 4.61
(s, 1H), 3.95 (s, 3H), 3.94 (s, 3H), 2.95 (s, 3H), 1.52 (s, 9H); 13C NMR (CDCl3, 75 MHz): δ
162.9, 159.6, 151.6, 149.8, 148.8, 146.7, 140.3, 137.3, 136.7, 133.7, 127.4, 127.1, 126.2, 124.5,
28