Novel 2,3-disubstituted quinazoline-4(3H)-one molecules derived from amino acid linked sulphonamide as a potent malarial antifolates for DHFR inhibition

ACCEPTED MANUSCRIPT

Novel 2,3-disubstituted quinazoline-4(3H)-one molecules derived from amino acid

linked sulphonamide as a potent malarial antifolates for DHFR inhibition

a

b

c

Tarosh S. Patel , Satish F. Vanparia , Urmila H. Patel , Ritu B. Dixit , Chaitanya J.

d

e

a

Chudasama , Bhavesh D. Patel and Bharat C. Dixit *

a

Chemistry Department, V. P. & R. P. T. P Science College, Affiliated to Sardar Patel

University, Vallabh Vidyanagar – 388 120, Gujarat, India.

b

Department of Physics, Sardar Patel University, Vallabh Vidyanagar – 388 120, Gujarat,

India.

c

Ashok & Rita Patel Institute of Integrated Studies and Research in Biotechnology and Allied

Sciences, New Vallabh Vidyanagar – 388121, Gujarat, India.

d

Department of Biochemistry, Shree Alpesh N. Patel P. G. Institute, Affiliated to Sardar Patel

University, Anand – 388001, Gujarat, India.

e

Microbiology Department, V. P. & R. P. T. P Science College, Affiliated to Sardar Patel

University, Vallabh Vidyanagar – 388 120, Gujarat, India.

Abstract

An optimization of a modified Grimmel’s method for N-heterocyclization of Leucine linked

sulphonamide leading to 2,3-disustituted-4-quinazolin-(3H)-ones was accomplished. Further,

nineteen hybrid quinaloinone motifs (5a-5s) were synthesized by N-heterocyclization

reaction under microwave irradiation using TEAA (IL) as green solvent as well as catalyst.

The in vitro screening of the hybrid entities against the plasmodium species P. falciparum

yielded five antimalarial potent molecules 5g, 5l, 5m, 5n & 5p owing comparable activity to

the reference drugs. The active scaffolds were further evaluated for enzyme inhibition

efficacy against alleged receptor Pf-DHFR computationally as well as in vitro, proving their

candidature as lead dihydrofolate reductase inhibitors. The prediction of the ADMET

properties of the potent molecules also indicated their good oral bioavailability.

Keywords: Triethylammonium Acetate; p-LDH assay; MTT assay; Maestro; Glide;

Cytotoxicity

*

Corresponding

Author:

Tel:

+91-2692-230011#31;

Fax:

+91-2692-235207;

E-mail: tarosh_patel@yahoo.com (T.S. Patel), dixits20002003@yahoo.co.in (B.C. Dixit).

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1

. Introduction

Malaria is a life threatening disease caused by parasites that are transmitted to people through

the bites of infected female mosquitoes. In 2015 the World Health Organization has estimated

approximately 3.2 billion people; nearly half of the world's population was at risk of malaria.

Most malaria cases and deaths occur in sub-Saharan Africa. However, Asia, Latin America,

and, to a lesser extent, the Middle East, are also at risk. In 2015, 97 countries and territories

had ongoing malaria transmission [1]. Malaria is caused by different parasites of

Plasmodium, of which Plasmodium falciparum is the most vicious one [2]. The rapid

emergence of P. falciparum strains resistant to currently available antimalarial drugs [3-6];

and the inefficacy of malarial vaccines [7] have alarmed the emergence to stimulate new

efforts regarding medical and molecular studies about malaria. The resistance of the strain

against the available antimalarial drugs and an urge for development of newer class of

antimalarials has motivated the medicinal chemists towards design of inhibitors of various

key enzymes involved in the lifecycle of the parasite [8]. Among the various target enzymes,

Dihydrofolate reductase is the key enzyme that catalyses the NADPH-dependent reduction of

7

,8-dihydrofolate to 5,6,7,8-tetrahydrofolate, which is the precursor of the co-factors required

for the synthesis of purine nucleotides, thymidylate and several amino acids [9]. Thus,

inhibition of DHFR can lead to the disruption of DNA synthesis and the death of the rapidly

proliferating cells [9, 10]. Early studies have shown that the parasite DHFR and TS, as with

other protozoa, reside on the same polypeptide as a DHFR-TS bifunctional protein [11].

More than past decade, synthesis of the heterocyclic compounds has become an important

landmark of synthetic organic chemistry as a result of broad diversity of their application in

medicinal and pharmaceutical chemistry [12]. The important major area in medicinal

chemistry comprises of investigation of heterocycles as privileged structures in drug

discovery [13]. Among them, quinazoline ring systems from natural or synthetic origin are

known to give a wide variety of biological responses [14-20], which has stimulated the

synthesis and pharmacological evaluation of a great number of quinazolinone derivatives.

Accounting the excellent biological properties of 4-quinazolin-(3H)-one derivatives [21],

particularly 2,3-disubstituted-4(3H)-quinazolinone, numerous synthetic methods have been

investigated and studied by the researchers, which are found in the book chapters and in

recent literature reviews [22, 23].

Sulphonamides were the first effective agents against most Gram-positive and many Gram-

negative organisms, as well as employed systematically for the prevention and cure of

bacterial infections [24, 25]. The discovery and development of sulphonamides as an

antibacterial agent, was one of the most intriguing and enlightening breakthrough in the field

of medicinal chemistry demonstrating skillful planning and fate in drug research [24]. Since

discovery, the sulphonamide group has been found as a key structural motif shared by a large

number of bioactive compounds, spanning a wide variety of biological effects, such as,

specific enzyme inhibition, hormone regulation, and several others [26]. Apart from the

commercialized application as antibacterial/antibiotic agents, various sulphonamides such as

Celecoxib and Valdecoxib (COX-2 inhibitor), Darunavir, Tipranavir and Fosamprenavir

(Protease Inhibitors), Probenecid (PBN), Sulfasalazine (SSZ), Sumatriptan (SMT) among

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others are also known to inhibit several enzymes [27]. The proteinogenic α-amino acids serve

as precursors to a wide range of naturally occurring heteroaromatic substances including

alkaloids, antibiotics and peptides [28]. Further, special attention has been devoted to the

development of methodologies for the synthesis of optically active heterocyclic compounds,

due to their biological importance. In this context, proteinogenic α-amino acids are the most

extensively used precursors for the synthesis of enantiomerically pure heterocycles, for the

reason that they constitute a natural source of optical activity [29]. Foreseeing, the mutations

and resistance developing in the parasite against currently prescribed drug regime, the best

way to combat the diseased condition is by developing hybrid molecules owing

pharmacological activities [30]. Some of the potent hybrid molecules rendering antimalarial

potency are depicted in Figure 1, demonstrating analogical relationship with the targeted 2,3-

disubstituted quinazolinone-sulphonamide hybrids derived from amino acid.

Heating chemical reactions by microwave energy continues to be a popular theme in the

organic and medicinal chemistry community. It has been known since the first published

reports in 1986 by groups of Gedye and Giguere, on the use of microwave irradiation to carry

out organic chemical transformations, numerous articles have been published in this fast

moving and exciting field, generally referred to as microwave assisted organic synthesis [31].

This “non conventional” synthetic method has shown broad applications as a very efficient

way to accelerate the course of many organic reactions, producing high yields and higher

selectivity, lower quantities of side products and, consequently, easier work up and

purification of the products [8, 31, 32].

In continuation of our previous work [8, 33], with an aim to synthesize potent antimalarial

leads derived from 2,3-disubstituted quinazolinone motifs withholding leucine linked

sulphonamide side arm at position-2. We hereby report the synthesis of 2,3-disubstituted

quinazolinone molecules utilizing conventional as well as microwave assisted irradiation.

Further, to get an insight of the potency of the synthesized molecules, in vitro efficacy was

evaluated against P. falciparum employing MTT assay and LDH assay. Progressively the

active entities were scrutinized via docking experimentation against Pf-DHFR enzyme for the

comprehensive know how of the mechanism of action as well as their bioavailability

parameters were predicted. Finally, the potent molecules screened for their enzyme inhibitory

activity as well as toxicity against vero cells to establish their selectivity.

2

. Chemistry

A series of novel quinazolinone-sulphonamide hybrid derivatives derived from Leucine were

designed and synthesized as depicted in Scheme 1. The desired entities were synthesized by

N-hetero cyclization of previously synthesized N-acylanthranilic acid (3) with various

aromatic amines. The synthetic protocol was optimized utilizing the classical catalysts as well

as TEAA as green catalyst and reaction medium under conventional and microwave assisted

heating conditions. Finally, the targeted sulphonamide incorporated 2,3-disubstituted

quinazolinone hybrids 4a-s were synthesized using TEAA as green solvent as well as catalyst

under microwave irradiation as a source of energy at 350 watts power level. Further, the final

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entitled de-acetylated derivatives 5a-s were procured by acidic hydrolysis of 4a-s under

conventional as well as microwave assisted heating conditions.

3

. Pharmacology

3

.1 In vitro antimalarial activity

The newly synthesized Quinazolinone-Sulphonamide hybrids were evaluated against

P. falciparum for their antimalarial potency considering Chloroquine, Pyrimethamine and

Trimethoprime as standard reference drugs. The outcomes of the antimalarial screening are

expressed as the concentration of the molecules resulting in 50% inhibition (IC ) of parasite

5

0

growth and are as outlined in Table 5.

3

.2 The antiplasmodial assay (p-LDH and MTT)

The antiplasmodial efficacy of the synthesized Quinazolinone-Sulphonamide hybrid entities

was evaluated by in vitro quantitative enzyme-based method (Malstat® assay) involving

spectrophotometric measurement of the parasite lactate dehydrogenase activity

(p-LDH). The inhibition rendered by each drug concentration was calculated as compared to

the untreated control to obtain the IC values.

5

0

The in vitro antiplasmodial and cytotoxic effect of Quinazolinone-Sulphonamide hybrids on

parasite growth and normal uninfected RBCs was tested by the quantitative enzyme based

method (MTT assay) involving spectrophotometric measurement of the host RBC’s

Mitochondrial Succinate Dehydrogenase catalysed MTT reduction. The effective

concentration (EC ) and cytotoxic concentration (CC ) were ascertained as well as

5

0

50

selectivity was determined from the values obtained.

3

.3 The enzyme inhibition assay

The inhibitory efficacy of the synthesized Quinazolinone-Sulphonamide hybrids on Pf-DHFR

was determined against bovine DHFR owing resemblance to Pf-DHFR. Results are reported

as % inhibition of enzymatic activity.

3

.4 The cytotoxicity against Vero cells

The cytotoxic behaviour of the synthesized hybrid motifs was evaluated against Vero cells

using MTT assay. The toxicity of the compounds was evaluated and enumerated as IC₅₀

values indicating 50% inhibitory concentration of the molecules.

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4

. Computational and Docking study

To study and understand the functionality and mode of action of the in vitro active

Quinazolinone-Sulphonamide hybrids, in silico studies including prediction of

physicochemical properties, docking as well as prediction of the pharmacokinetic properties

was accomplished against mutant Pf-DHFR as well as human DHFR (1MVT) obtained from

RSC protein databank.

5

. Result and discussion

5

.1 Synthetic aspect

To establish a more general, efficient and convenient synthetic method, our efforts begins

with the hetero cyclization approach to derive desired quinazolinone-sulphonamide hybrids

(4a-s) under milder reaction conditions and to obtain high isolated yield. We recently have

been engaged for the reinvestigation of this heterocyclization approach, and the successful

modifications is reported in our article describing the synthesis of various N-(4-(N-((3-

substituted-4-oxo-3,4-dihydroquinazolin-2-yl)methyl)sulfamoyl)

phenyl)

acetamide

derivatives [33]. With the relative experience in hand and keeping in mind the literature

methods, synthesis of desired quinazolinone‐sulphonamide hybrid molecules from amino

acids as initial precursor would not be a difficult task. Hence, it was decided to explicit

various approaches which are feasible in common laboratories. Therefore, we chose to

explore some plausible synthetic approaches based on the intermediates to be opted in the

crucial reaction step, i.e. ring formation or ring closure step for the synthesis of diverse

quinazolinone-sulphonamide hybrid molecules from Leucine in our laboratory, and is

outlined in Scheme 1. Consequently, to probe the scope and limitations of newly developed

protocol for the synthesis of desired quinazolinone-sulphonamide hybrid 4a under classical

heating conditions, it was planned to employ optimized conditions for the hetero cyclization

of

N-acylanthranilic acid (3) with a aniline as an amine (Scheme 2). Therefore, it was decided to

reinvestigate again the solvent and catalyst effects on the efficiency of cyclization reactions

with the hope to identify an ideal combination that could improve reaction duration and

contribute eventually the higher conversion to quinazolinone-sulphonamide hybrids in the

ring formation step as well as proved itself to be environmentally benign. Hence, the effect of

various solvents and catalyst on the reaction time and yield were examined as shown in

Table 1. An excellent conversion of 3 to 4a was achieved (entry 7) within short reaction span

(14 min.) as indicated by isolated yield (90 %) utilizing an ionic liquid triethylammonium

acetate (TEAA) as reaction medium as well as catalyst.

In addition to this, to study the effect of microwave irradiation on the reaction time and yield

of the product it was decided further to employ microwave heating conditions for the

synthesis of the quinazolinone-sulphonamide hybrid (4a). The optimization of reaction

between 3 with appropriate aryl amine (Scheme 2) was examined under microwave

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irradiation conditions for the formation of 4a was achieved by considering the effect of

change of power levels, solvents and catalysts (Table 2). Amongst the solvents examined,

good results were observed again when the TEAA was employed as a solvent as well as

catalyst (Entry 9-10). Under the optimized condition (Entry 9), reaction under microwave

irradiation (350 W) has afforded desired 4a in excellent yield (96 %). Thus, the optimized

condition utilizing TEAA as reaction medium and catalyst as well under microwave

irradiation at 350 Watts power level for the conversion of N-acylanthranilic acid (3) to

quinazolinone-sulphonamide derivative (4a) was further employed for the synthesis of

quinazolinone-sulphonamide hybrids (4a-s) and the substitution pattern is as depicted in

Table 3.

Proceedingly to obtain our final entitled de-acetylated derivatives (Scheme 3) it was decided

to employ acidic hydrolysis conditions which utilized dilute HCl (40 % solution) in R-spirit

and H O mixture under conventional and microwave-assisted heating conditions to afford

2

entitled amino derivatives (5a-s) in quantitative yields as compared in Table 4.

5

.2 Analytical aspect

The structures of all the synthesized compounds were confirmed using various physico-

chemical methods and spectroscopic investigations, and the relevant analytical data have

been given in experimental section.

Mass spectra and elemental analysis of compounds reveals that they are consistent with the

proposed structures. Further, the results of elemental analysis of all compounds were in good

agreement (± 0.4 %) with their predicted molecular formula. All the ESI-MS (positive mode)

+

spectra of synthesized compounds showed molecular ion peak [M+H] corresponding to

either exact mass or molecular weight of respective compound [34, 35]. The mass spectrum

+

of compound 5a exhibited a parent peak at m/z 463.0 [M+H] , which confirms the proposed

formula. Compounds, 5k (chloro-derivative) and 5n (bromo-derivative) exhibited ionization

+

+2

peaks ([M+H] and [M+H] ) at m/z (497.0, 499) and at m/z (540.8, 542.8) respectively.

+

Further, ESI-MS spectra of some compounds appeared with [M+H+Na] peak along with

+

molecular ion peak [M+H] .

The FT-IR spectra represent an obvious tool for initial identification of the presence of

various functional groups in the moiety [34, 35]. In IR spectra of all the compounds, presence

of –SO NH– group was evidenced by the two strong bands appeared at 1318-1340 and 1144-

2

−

1

165 cm due to asymmetric and symmetric S═O stretching vibrations respectively, and

−

1

further absorption band at ~3250 cm assigned to –SO N–H stretching. For the quinazolinone

2

derivatives, strong intensity bands appeared in the IR spectra at 1690-1670, 1610-1650 and at

−

1

~

1495 cm attributed to C=O, C=N and C=C stretching vibrations respectively, provides a

strong evidence for the parent heterocyclic ring skeleton vibrations. In IR spectra of 5a-s

bands observed at ~2930 and ~2850 (methylene) due to the aliphatic C–H strechtching

vibrations, while C–H bending vibrations due to isobutyl group was observed at 1395-1385

−

1

cm .

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1

In H-NMR spectra of compounds 4a-s with acetamido group (Ar–NHCOCH ), protons of

3

–

COCH and –NHCO were appeared as a singlet at δ >10.0 ppm due to –NHCO and

3

between δ 2.0-2.3 ppm due to –COCH respectively. After the hydrolysis, disappearance of

3

these two signals (siglets) in 5a-s and appearance of signal (singlet at δ 5.6-6.3 ppm) due to

Ar–NH supports the formation of compounds, 5a-s. In addition to this, the aromatic protons

2

of benzenesulfonyl ring (Ar-NH in 5a-s) appeared as two separate doublets (J=8.8/8.4) and

2

with two integral proton to each peak (at δ 7.4-7.2 and 6.2-6.8 ppm), however, these four

protons (Ar–NHAc group in 4a-s) appeared as broad multiplet (at δ 7.5-7.8 ppm) due to the

symmetry might have been influenced by acetamido group. The proton at C of quinazolinone

5

ring appeared as a doublet around δ >8 ppm. All the other protons of aromatic and

1

3

heteroaromatic ring resonated in aromatic region (δ 6.2-8.6 ppm). All C-NMR spectra of

a-s were characterized by two signals (quarternary carbons) at low frequencies (δ 150-157

5

and 160-162 ppm) associated with a cyclic amide or lactam ring of quinazolinone (with a

cyclic –N–C═N carbon conjugated with C═O group) further, –CH CH(CH ) (secondary

2

3 2

carbon) appeared at δ ~42 ppm. All the other carbons (CH, C) of aryl and quinazolinone ring

were resonated in expected region (δ 112-150 ppm).

5

.3 In vitro antimalarial screening and assay

The antimalarial screening of the synthesized compounds was carried out against

P. falciparum strain which resulted in varied results as given in Table 5. The spectrum of

activity of entitled compounds showed improved potency IC <0.20 µg/mL, even better than

5

0

reference compound Pyrimethamine. The results of in vitro assay suggest that moderate to

good activity was achieved from the synthesized 2,3-disubstituted quinazolinones. Among

the screened scaffolds, compounds 5g, 5l, 5m, 5n and 5p were found to be most potent

entities having IC value of 0.068 µg/mL.

5

0

The core Quinazolinone motif consisting of sulphonamide linked amino acid at position-2

was found to be most promising structure owing comparatively better potency against

plasmodium species. Considering substitutional pattern at position-3, varied results were

noted which indicated the presence of electron withdrawing group greatly enhanced the

efficacy of the molecules. The compounds with 4-OMe (5g), 4-Cl (5m), 3-Cl (5l), 4-Br (5p)

and 2-Br (5n) substituted phenyl ring were found to have enhanced potency against

P. falciparum.

Progressively to cross validate the results of the above in vitro study and to determine the

selectivity of the potent molecules as true antimalarial drugs we executed p-LDH assay and

MTT assay as a pure indication of the efficacy as well as cytotoxicity of the synthesized

entities. The results of the p-LDH assay as enumerated in Table 6 indicated that the

compounds demonstrated lower to moderate potency as compared to standard drug

Chloroquine. Considering the results of lactate dehydrogenase assay, molecules 5l and 5m

noted their candidature to be most potent among the tested molecules.

Further, the results of MTT assay against P. falciparum indicated the potency of the

molecules as well as toxicity against RBC. The results in form of effective concentration and

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cytotoxic concentration revealed that the molecules were effective against the parasite but

had lower toxicity against blood cells. Based on the calculation of EC and CC values,

5

0

50

selectivity indices (SI) were calculated for P. falciparum. All the compounds tested have

exhibited the SI values of >1. Therefore, these compounds are specific against the malarial

parasites and not against the RBCs.

5

.4 Docking study

To enhance the knowhow of mechanism of the antimalarial potency of the in vitro active

scaffolds (5g, 5l, 5m, 5n and 5p), the molecules were further evaluated by docking them

against wild type mutant Pf-DHFR (4DPD) protein structure obtained from protein data bank

(RSC). For completion of docking study, 3D structures of the molecules were prepared and

were docked against the active site of the enzyme using glide 6.6.

A potential interaction was observed between the active molecules and the Pf-DHFR enzyme.

The molecules interacted with the protein active pocket by forming H-bonds as well as π-π

stacking interactions. As enumerated in Table 7, docking score as well as binding energies of

the molecules against the active site indicated higher affinity of the molecules to bind with

the protein leading to its inhibition, further more the RMSD values were found to be lower

than 2 which can be considered to be in acceptable range. The docking score of the molecules

(5g, 5l, 5m, 5n and 5p) was found to be ranging from -3.07 to -4.21 and binding energies

ranging from -34.56 to -43.08 which were found to be better as compared to standard drugs

Trimethoprime and Pyrimethamine. The compound 5g interacted with the active pocket of

the enzyme by forming hydrogen bonds with Phe295 at a distance of 2.732 Å and Asn294

with bond length 1.993 Å (Figure 2). The molecule 5l occupied the active pocket with great

ease as depicted in Figure 3. A hydrogen bond was observed between the nitrogen of

backbone of Quinazolinone hybrid and Lys97 with bond length of 1.938 Å, further side chain

amide group interacted with same amino acid Lys97 at distance of 1.99 Å and terminal amine

group if the hybrid interacted with Asp139 at a bond length of 1.791 Å. For molecule 5m

(Figure 4) similar mode of interaction was observed where a hydrogen bonding between

nitrogen and Lys97 was observed at a distance of 2.26 Å. The same amino acid Lys97 also

established hydrogen bond with amide group of side chain with bond length of 2.214 Å,

finally a hydrogen bond was observed between amine group and Asp139 at a distance of 1.75

Å. The compound 5n interacted in the similar fashion forming hydrogen bonding as well as

π-π stacking interactions with amino acids Leu98 and Lys97 with the bond length of 2.23 Å

and 3.927 Å respectively as elucidated in Figure 5. The active site invited 5p molecule to

occupy the pocket by forming hydrogen bond between nitrogen and Lys297 as well as

Asn294 at a distance of 2.43 Å and 2.071 Å respectively as visualized in Figure 6. The 2D

and 3D binding model of the standard inhibitors showing the interaction with the protein can

be observed in Figure S1 and Figure S2 in supplementary material.

Further, to understand the selectivity of the quinazolinone-sulphonamide hybrids they were

again docked against human DHFR (1MVT) procured from protein data bank (RSC). The

glide score and energies displayed in Table 7 as well as interactions of the ligand molecules

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against the active site indicated that the potent molecules poorly interacted with

h-DHFR as compared to Pf-DHFR. Moreover, the active site of the Pf-DHFR was found to

be flexible accommodating the ligand molecules. Thus the results of docking study also

indicated the selectivity of the potent ligand molecules.

5

.5 Pharmacokinetic properties prediction

The drug likeness properties of the synthesized molecules demonstrating superior

antimalarial potency was evaluated by predicting the pharmacokinetic properties in silico.

The potent molecules (5g, 5l, 5m, 5n and 5p) were preliminarily screened considering the

basic parameters of Lipinski’s rule of 5. The results of the Quickprop module with their

permissible rang are delineated in Table 8. For an orally active compound, 2 violations of the

Lipinski’s rule are acceptable. The molecules of the present study were found to follow the

rule with maximum violation of one rule and thus proving their drug likeness properties.

The oral bioavailability of the drug molecules are greatly influenced by the optimum values

of the descriptors, polar surface area and rotatable bonds. The important pharmacokinetic

parameters with their permissible ranges are delineated in Table 9. The active test

quinazolinone derivatives show results of the optimum value of rotatable bonds (<15) and

polar surface area (7-200 Å) to be in the prescribed range [36] thus owing good

bioavailability. The Caco-2 cell permeability (QPPCaco), used as model for gut-blood barrier

[

37] acts as the important factor to be studied in concern with the absorption of the drug

molecule across the intestine. Caco-2 cell permeability prediction of the test compounds

indicates excellent results predicting good intestinal absorption. Further, the compounds were

tested for human serum albumin binding indicated by the predicted values of QPlogkhsa

descriptor of Quickprop and all the test molecules were found to fall in the permissible range

(-1.5 to 1.5). Also, the Quickprop descriptor for blood/brain partition coefficient QPlogBB

showed reliable prediction for all the test compounds and reference drugs. The test entities

were assessed for aqueous solubility parameter (QPlogS) and the compounds were found to

be falling in the permissible range (-6.5-0.5). Finally, the active molecules were accessed for

IC₅₀value of HERG K+ channel blockage prediction, which indicated that the predicted

values fall in the acceptable range (<-5) as compared to the standard reference entities.

5

.6 DHFR inhibition assay and Cytotoxicity assay

The potent entities traced out of in vitro antimalarial screening were further evaluated for

inhibitory efficacy against bovine liver DHFR enzyme and cytotoxicity against vero cells. As

enumerated in Table 10, the test molecules rendered a good inhibitory potency against the

enzymes as compared to the standard inhibitors. The inhibitory concentrations of the

compounds were obtained as IC₅₀values which indicated that among the tested molecules,

compounds 5g, 5l and 5m were found to be most potent inhibitors. The cytotoxic

concentration of the test compounds against vero cells was found to be exceeding 15 µg/mL,

and thus showing up a non toxic behavior. The selective index calculated was found to be

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more than 100 which support the harmony towards utilization of the scaffolds as active

antimalarial compounds inhibiting dihydrofolate reductase enzyme.

6

. Conclusion

Withholding the aim of synthesizing Leucine linked sulphonamide incorporated hybrid

quinazolinone molecules via environmental friendly methodology; we have modified and

developed an efficient, convergent and facile methodology of N-heterocyclization leading

towards synthesis of 2,3-disustituted quinazolinone motifs in satisfactory yields. We hereby

report synthesis of nineteen qinazolinone-sulphonamide hybrids (5a-5s) under microwave

irradiation technique utilizing an ionic liquid TEAA as inexpensive, environmentally benign

and recyclable reaction medium as well as catalyst. Among the synthesized molecules, 5g, 5l

and 5m were found to be the most potent entities against Plasmodium falciparum species.

The in silico docking study directed towards their potentiality of interacting and inhibiting the

enzyme Plasmodium falciparum dihydrofolate reductase occupying the active binding pocket

with great ease. The antimalarial efficacy was further proved by the molecules exhibiting an

active inhibition of enzyme proving their potency as dihydrofolate reductase inhibitors and

thus interrupting malarial folate pathway. The calculated pharmacokinetic properties for the

test compounds predicted good oral bioavailability. Thus promising in vitro antimalarial

activity, inhibition as well as interactions with the enzyme Plasmodium falciparum

dihydrofolate reductase and the pharmacokinetic properties revealed in the present study,

indicates their potential for further development as antimalarial lead entities.

7

. Experimental section

.1 Experimental protocols and analytical data

.1.1 Synthesis of N-{4-[({1-[3-(aryl substituted)-4-oxo-3,4-dihydroquinazolin-2-yl]-2-

phenylethyl}amino)sulfonyl]phenyl}acetamide (4a-4s)

To a mixture of N-acylanthranilic acid (3) (0.783 g, 0.002 mol) and appropriate mono-

substituted aromatic amines (0.022 mol) {for instance, aniline and its o-, m-, p- substituted

derivatives of toludine, anisidine, nitroaniline, chloroaniline, bromoaniline, fluoroaniline and

phenylhydrazine respectively} in were taken in 5.0 mL of TEAA in round bottom flask and

heated at 100 °C for specific time interval as visualized by TLC. To carry out the reaction

under microwave irradiation, the reaction mass was irradiated under microwave for required

time period as indicated in Table 3 at 350 W power level. After completion of the reaction,

the system was cooled to room temperature and finally poured onto crushed ice. The solid

product thus separated was collected, washed with acetone and finally recrystallized from

ethanol to obtain pure product. TEAA was recovered from the aqueous medium by heating it

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under reduced pressure at 80 °C. The ionic liquid recovered can be reused again for the same

experimental protocol.

N-[4-({[1-(3-phenyl-4-oxo-3,4-dihydroquinazolin-2-yl)-3-methylbutyl]amino}sulfonyl)-

–

1

phenyl]acetamide (4a): White solid; mp: 164-169 °C; FT-IR (KBr, ν_m_a_x, cm ): 3358, 3253

N–H str.), 3065, 2956 (C–H str.), 1679 (C═O str.), 1602, 1592, 1496 (C═N, C═C str.),

(

1

8

1

3

326, 1152 (S═O str.); H-NMR (400 MHz, DMSO-d , δ , ppm): 10.01 (s, 1H, Ar–NHAc),

6 H

.20 (d, J = 8.4 Hz, 1H, SO NH), 8.04 (dd, J = 8, 1.2 Hz, 1H, ArH), 7.82 (dt, J = 8, 1.2 Hz,

H, ArH), 7.67-7.52 (m, 6H, ArH), 7.47-7.21 (m, 5H, ArH), 3.79 (m, 1H, NH–CH), 2.01 (s,

H, Ar–NHCOCH ), 1.55-1.30 (m, 3H, CH–CH ), 0.77, 0.55 (d, J = 6.4 Hz, 6H, CH–

2

3

2

13

(CH ) ); C-NMR (100 MHz, DMSO-d , δ , ppm): 168.7, 161.2, 156.7, 146.7, 142.5, 135.6,

3 2 6 C

1

2

5

34.5, 133.7, 129.7, 129.4, 128.5, 127.6, 127.1, 126.9, 126.2, 120.6, 118.3, 52.1, 41.8, 24.1,

+

3.5, 23.1, 19.2; MS (ESI) m/z: 504.5 [M+H] ; Anal. Calcd. for C H N O S: C, 64.27; H,

2

7

28

4

.59; N, 11.10. Found: C, 64.49; H, 5.67; N, 11.22 %.

7

.1.2 Synthesis of 4-amino-N-[(4-oxo-3-substituted aryl-3,4-dihydroquinazolin-2-

yl)alkyl]benzenesulfonamide derivatives (5a-5s)

The appropriate product obtained in ring-closure step (4a-4s) (0.001 mol) was taken in a two-

necked round bottomed flask containing dilute HCl solution (40 %) in R-spirit and H O (1:1)

2

mixture (15-20 mL). The reaction mixture was heated to reflux for 45-75 min till the clear

solution obtained. Under microwave irradiation method, the reaction mixture was heated

under microwave irradiation at 210 W for an appropriate time (Table 4). After cooling to

room temperature, the reaction mixture was filtered, diluted with distilled water (100 mL)

and neutralized with saturated NaHCO solution. The precipitated product was filtered and

3

recrystallized from R-spirit.

4

-amino-N-[1-(4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)-3-methylbutyl]benzene-

–

1

sulfonamide (5a): White solid; mp: 183-191 °C; FT-IR (KBr, ν_m_a_x, cm ): 3378, 3253 (N–H

str.), 3060, 2954 (C–H str.), 1656 (C═O str.), 1623, 1593, 1502 (C═N, C═C str.), 1314, 1152

1

(

S═O str.) ; H-NMR (400 MHz, DMSO-d , δ , ppm): 8.05 (d, J = 7.6 Hz, 1H, ArH), 7.85-

6

H

7

.59 (m, 4H, SO NH & ArH), 7.49-7.32 (m, 5H, ArH), 7.29 (d, J = 8.8 Hz, 2H, ArH), 6.34

2

(

d, J = 8.4 Hz, 2H, ArH), 5.91 (s, 2H, Ar–NH ), 3.72 (m, 1H, NH–CH), 1.54-1.31 (m, 3H,

2

13

CH–CH ), 0.75, 0.51 (d, J = 6.4 Hz, 6H, CH–(CH ) ); C-NMR (100 MHz, DMSO-d , δ ,

2

3 2

6

C

ppm): 161.1, 156.7, 152.5, 146.6, 135.6, 134.6, 130.0, 129.2, 128.6, 128.5, 127.2, 127.0,

+

1

[

26.2, 124.5, 120.5, 112.4, 51.9, 42.0, 23.5, 23.1, 19.1; MS (ESI) m/z: 463.0 [M+H] , 484.9

+

M+H+Na] ; Anal. Calcd. for C H N O S: C, 64.91; H, 5.67; N, 12.11. Found: C, 65.16; H,

25 26 4 3

5

.76; N, 12.23 %.

-amino-N-{1-[3-(2-methylphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]-3-methylbutyl}-

4

–

1

benzenesulfonamide (5b): White solid; mp: 204-213 °C; FT-IR (KBr, ν_m_a_x, cm ): 3361,

3

1

240 (N–H str.), 3055, 2955 (C–H str.), 1683 (C═O str.), 1613, 1592, 1503 (C═N, C═C str.),

1

312, 1151 (S═O str.), 1370 (C–H def.); H-NMR (400 MHz, DMSO-d , δ , ppm): 8.08 (dd,

6

H

ACCEPTED MANUSCRIPT

J = 8, 1.2 Hz, 1H, ArH), 7.88 (t, J = 8 Hz, 1H, ArH), 7.71-7.55 (m, 3H, SO NH & ArH),

2

7

.45-7.39 (m, 3H, ArH), 7.30-7.14 (m, 3H, ArH), 6.78 (d, J = 8.8 Hz, 2H, ArH), 5.86 (s, 2H,

Ar–NH ), 3.67 (m, 1H, NH–CH), 2.41 (s, 3H, Ar–CH ), 1.55-1.34 (m, 3H, CH–CH ), 0.75,

2

3

2

13

0

1

.53 (d, J = 6.4 Hz, 6H, CH–(CH ) ); C-NMR (100 MHz, DMSO-d , δ , ppm): 161.3,

3 2 6 C

55.6, 152.6, 146.4, 141.4, 134.6, 134.5, 131.7, 129.8, 128.9, 128.4, 127.2, 126.8, 126.4,

25.5, 125.1, 120.6, 112.4, 51.6, 42.3, 23.5, 23.1, 21.5, 19.1; MS (ESI) m/z: 477.0 [M+H] ;

+

Anal. Calcd. for C H N O S: C, 65.52; H, 5.92; N, 11.76. Found: C, 65.74; H, 5.99; N,

2

6

28

4

3

1

1.88 %.

4

-amino-N-{1-[3-(3-methylphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]-3-methylbutyl}-

–

1

benzenesulfonamide (5c): White solid; mp: 188-195 °C; FT-IR (KBr, ν_m_a_x, cm ): 3375, 3251

N–H str.), 3059, 2957 (C–H str.), 1689 (C═O str.), 1616, 1590, 1508 (C═N, C═C str.),

(

1

313, 1154 (S═O str.), 1371 (C–H def.); H-NMR (400 MHz, DMSO-d , δ , ppm): 8.08 (d, J

6 H

=

8 Hz, 1H, ArH), 7.79-7.64 (m, 3H, SO NH & ArH), 7.59 (d, J = 8 Hz, 1H, ArH), 7.40-7.33

2

(m, 4H, ArH), 7.31 (d, J = 8.8 Hz, 2H, ArH), 6.74 (d, J = 8.8 Hz, 2H, ArH), 5.87 (s, 2H, Ar–

NH ), 3.63 (m, 1H, NH–CH), 2.41 (s, 3H, Ar–CH ), 1.57-1.33 (m, 3H, CH–CH ), 0.74, 0.52

2

3

2

13

(

d, J = 6.4 Hz, 6H, CH–(CH ) ); C-NMR (100 MHz, DMSO-d , δ , ppm): 161.2, 155.8,

3 2 6 C

1

52.5, 146.5, 140.6, 135.7, 134.5, 130.6, 129.5, 128.8, 128.4, 127.1, 126.8, 126.3, 125.2,

23.5, 120.5, 112.6, 51.7, 42.3, 23.5, 23.1, 21.3, 19.1; MS (ESI) m/z: 476.9 [M+H] ; Anal.

+

Calcd. for C H N O S: C, 65.52; H, 5.92; N, 11.76. Found: C, 65.71; H, 5.97; N, 11.85 %.

2

6

28

4

3

4

-amino-N-{1-[3-(4-methylphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]-3-methylbutyl}-

–

1

benzenesulfonamide (5d): White solid; mp: 169-178 °C; FT-IR (KBr, ν_m_a_x, cm ): 3367,

3

255 (N–H str.), 3056, 2953 (C–H str.), 1685 (C═O str.), 1615, 1596, 1505 (C═N, C═C str.),

1

314, 1153 (S═O str.), 1370 (C–H def.); H-NMR (400 MHz, DMSO-d , δ , ppm): 8.04 (d, J

6

H

=

7.6, Hz, 1H, ArH), 7.82-7.78 (m, 2H, SO NH & ArH), 7.56-7.49 (m, 2H, ArH), 7.43 (d, J

8.8 Hz, 2H, ArH), 7.34-7.27 (m, 4H, ArH), 6.71 (d, J = 8.8 Hz, 2H, ArH), 5.87 (s, 2H, Ar–

2

NH ), 3.69 (m, 1H, NH–CH), 2.41 (s, 3H, Ar–CH ), 1.58-1.34 (m, 3H, CH–CH ), 0.73, 0.51

2

3

2

13

(

d, J = 6.4 Hz, 6H, CH–(CH ) ); C-NMR (100 MHz, DMSO-d , δ , ppm): 161.2, 155.9,

3 2 6 C

1

52.5, 146.4, 143.1, 137.8, 134.4, 129.9, 128.8, 128.3, 127.0, 126.9, 126.4, 125.2, 120.6,

12.6, 51.7, 41.3, 23.5, 23.1, 21.4, 19.1; MS (ESI) m/z: 477.1 [M+H] ; Anal. Calcd. for

+

C H N O S: C, 65.52; H, 5.92; N, 11.76. Found: C, 65.73; H, 5.98; N, 11.89 %.

26

28

4

3

4

-amino-N-{1-[3-(2-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]-3-methylbutyl}-

–

1

benzenesulfonamide (5e): White solid; mp: 173-178 °C; FT-IR (KBr, ν_m_a_x, cm ): 3377, 3250

N–H str.), 3054, 2959 (C–H str.), 1688 (C═O str.), 1619, 1591, 1507 (C═N, C═C str.),

(

1

317, 1150 (S═O str.), 1263 (C–O–C str.); H-NMR (400 MHz, DMSO-d , δ , ppm): 8.21

6 H

(

d, J = 8 Hz, 1H, ArH), 8.08 (d, J = 8 Hz, 1H, ArH), 7.81-7.56 (m, 3H, SO NH & ArH),

2

7

.44-7.34 (m, 3H, ArH), 7.19-6.87 (m, 3H, ArH), 6.72 (d, J = 8.4 Hz, 2H, ArH), 5.89 (s, 2H,

Ar–NH ), 3.93 (s, 3H, Ar–OCH ), 3.81 (m, 1H, NH–CH), 1.53-1.29 (m, 3H, CH–CH ), 0.75,

2

3

2

13

0

1

.54 (d, J = 6.4 Hz, 6H, CH–(CH ) ); C-NMR (100 MHz, DMSO-d , δ , ppm): 161.2,

3 2 6 C

57.6, 156.7, 152.6, 146.3, 134.7, 134.4, 132.9, 128.4, 127.3, 127.2, 126.9, 126.3, 124.8,

24.0, 120.5, 117.3, 112.5, 56.8, 51.8, 42.2, 23.5, 23.1, 19.1; MS (ESI) m/z: 493.1 [M+H] ;

+

Anal. Calcd. for C H N O S: C, 63.40; H, 5.73; N, 11.37. Found: C, 63.64; H, 5.81; N,

2

6

28

4

1

1.52 %.

ACCEPTED MANUSCRIPT

4

-amino-N-{1-[3-(3-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]-3-methylbutyl}-

–

1

benzenesulfonamide (5f): White solid; mp: 159-163 °C; FT-IR (KBr, ν_m_a_x, cm ): 3373, 3249

N–H str.), 3059, 2952 (C–H str.), 1686 (C═O str.), 1618, 1593, 1509 (C═N, C═C str.),

(

1

318, 1152 (S═O str.), 1260 (C–O–C str.); H-NMR (400 MHz, DMSO-d , δ , ppm): 8.08

6 H

(

d, J = 8 Hz, 1H, ArH), 7.88 (d, J = 8 Hz, 1H, ArH), 7.79-7.64 (m, 3H, SO NH & ArH),

2

7

8

1

.39-7.36 (m, 2H, ArH), 7.31 (d, J = 8.8 Hz, 2H, ArH), 7.18-7.08 (m, 2H, ArH), 6.75 (d, J =

.8 Hz, 2H, ArH), 5.79 (s, 2H, Ar–NH ), 3.86 (s, 3H, Ar–OCH ), 3.73 (m, 1H, NH–CH),

2

3

13

.55-1.30 (m, 3H, CH–CH ), 0.76, 0.53 (d, J = 6.4 Hz, 6H, CH–(CH ) ); C-NMR (100

2

3 2

MHz, DMSO-d , δ , ppm): 161.3, 157.5, 156.7, 152.5, 146.3, 134.5, 133.8, 132.6, 128.4,

6

C

1

27.2, 127.1, 126.2, 126.2, 124.7, 120.5, 116.5, 112.6, 110.2, 56.1, 51.8, 42.2, 23.5, 23.1,

+

9.1; MS (ESI) m/z: 493.0 [M+H] ; Anal. Calcd. for C H N O S: C, 63.40; H, 5.73; N,

2

6

28

4

1.37. Found: C, 63.62; H, 5.79; N, 11.49 %.

4

-amino-N-{1-[3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]-3-methylbutyl}-

benzenesulfonamide (5g): White solid; mp: 179-184 °C; α = + 11.8 (AcOH); FT-IR (KBr,

obs

–1

ν_m_a_x, cm ): 3368, 3251 (N–H str.), 3056, 2960 (C–H str.), 1689 (C═O str.), 1614, 1596, 1506

1

(

C═N, C═C str.), 1313, 1153 (S═O str.), 1262 (C–O–C str.); H-NMR (400 MHz, DMSO-

d , δ , ppm): 8.08 (d, J = 7.6 Hz, 1H, ArH), 7.83 (t, J = 7.6 Hz, 1H, ArH), 7.77-7.48 (m, 5H,

6

H

SO NH & ArH), 7.34 (d, J = 8.8 Hz, 2H, ArH), 7.15 (d, J = 8.8 Hz, 2H, ArH), 6.73 (d, J =

2

8

1

.4 Hz, 2H, ArH), 5.86 (s, 2H, Ar–NH ), 3.80 (m, 1H, NH–CH), 3.88 (s, 3H, Ar–OCH ),

2 3

13

.58-1.33 (m, 3H, CH–CH ), 0.75, 0.52 (d, J = 6 Hz, 6H, CH–(CH ) ); C-NMR (100 MHz,

2

3 2

DMSO-d , δ , ppm): 161.3, 159.0, 156.6, 152.6, 146.3, 134.4, 129.7, 128.3, 128.2, 127.1,

6

C

1

27.0, 126.2, 124.8, 120.4, 114.7, 112.6, 55.5, 51.8, 42.2, 23.5, 23.1, 19.1; MS (ESI) m/z:

+

4

92.9 [M+H] ; Anal. Calcd. for C H N O S: C, 63.40; H, 5.73; N, 11.37. Found: C, 63.65;

2

6

28

4

H, 5.81; N, 11.50 %.

4

-amino-N-{1-[3-(2-nitrophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]-3-methylbutyl}-

–

1

benzenesulfonamide (5h): White solid; mp: 208-212 °C; FT-IR (KBr, ν_m_a_x, cm ): 3379,

3

1

8

7

249 (N–H str.), 3063, 2953 (C–H str.), 1680 (C═O str.), 1616, 1589, 1495 (C═N, C═C str.),

1

315, 1148 (S═O str.), 1530, 1350 (N═O str.); H-NMR (400 MHz, DMSO-d , δ , ppm):

6

H

.64 (d, J = 8.4 Hz, 1H, ArH), 8.31 (d, J = 8.4 Hz, 1H, ArH), 8.14 (d, J = 8 Hz, 1H, ArH),

.94 (d, J = 6 Hz, 1H, SO NH), 7.86-7.63 (m, 5H, ArH), 7.36 (d, J = 8.8 Hz, 2H, ArH), 6.71

2

(

d, J = 8.8 Hz, 2H, ArH), 5.84 (s, 2H, Ar–NH ), 4.10 (m, 1H, NH–CH), 1.50-1.36 (m, 3H,

2

13

CH–CH ), 0.77, 0.49 (d, J = 5.6 Hz, 6H, CH–(CH ) ); C-NMR (100 MHz, DMSO-d , δ ,

2

3 2

6

C

ppm): 160.8, 155.3, 152.6, 146.6, 146.5, 139.0, 135.3, 129.9, 128.4, 127.5, 127.2, 126.4,

25.8, 125.3, 125.1, 122.0, 120.2, 112.6, 52.1, 41.7, 23.5, 23.1, 19.1; MS (ESI) m/z: 507.9

1

+

[

M+H] ; Anal. Calcd. for C H N O S: C, 59.16; H, 4.96; N, 13.80. Found: C, 59.39; H,

25 25 5 5

5

.05; N, 13.93 %.

-amino-N-{1-[3-(3-nitrophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]-3-methylbutyl}-

4

–

1

benzenesulfonamide (5i): White solid; mp: 176-185 °C; FT-IR (KBr, ν_m_a_x, cm ): 3376, 3258

N–H str.), 3061, 2950 (C–H str.), 1685 (C═O str.), 1617, 1591, 1498 (C═N, C═C str.),

(

1

8

311, 1146 (S═O str.), 1532, 1353 (N═O str.); H-NMR (400 MHz, DMSO-d , δ , ppm):

6 H

.54 (s, 1H, ArH), 8.16 (d, J = 8 Hz, 1H, ArH), 7.93-7.78 (m, 4H, SO NH & ArH), 7.71-7.48

2

(m, 3H, ArH), 7.36 (d, J = 8.4 Hz, 2H, ArH), 6.69 (d, J = 8.4 Hz, 2H, ArH), 5.88 (s, 2H, Ar–

NH ), 3.97 (m, 1H, NH–CH), 1.51-1.37 (m, 3H, CH–CH ), 0.78, 0.50 (d, J = 6 Hz, 6H, CH–

2

ACCEPTED MANUSCRIPT

1

3

(

CH ) ); C-NMR (100 MHz, DMSO-d , δ , ppm): 160.7, 155.5, 152.4, 148.5, 146.5, 137.9,

3 2 6 C

1

2

4

35.3, 132.2, 131.9, 128.3, 127.6, 127.2, 126.4, 124.9, 124.3, 122.0, 120.2, 112.6, 52.3, 41.8,

+

3.5, 23.1, 19.1; MS (ESI) m/z: 508.0 [M+H] ; Anal. Calcd. for C H N O S: C, 59.16; H,

2

5

25

5

.96; N, 13.80. Found: C, 59.34; H, 5.02; N, 13.91 %.

4

-amino-N-{1-[3-(4-nitrophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]-3-methylbutyl}-

–

1

benzenesulfonamide (5j): White solid; mp: 204-209 °C; FT-IR (KBr, ν_m_a_x, cm ): 3374, 3248

N–H str.), 3060, 2858 (C–H str.), 1689 (C═O str.), 1613, 1595, 1497 (C═N, C═C str.),

(

1

8

7

310, 1149 (S═O str.), 1535, 1354 (N═O str.); H-NMR (400 MHz, DMSO-d , δ , ppm):

6 H

.51 (d, J = 8.8 Hz, 2H, ArH), 8.36 (d, J = 8.8 Hz, 2H, ArH), 8.14 (d, J = 7.6 Hz, 1H, ArH),

.89-7.84 (m, 2H, SO NH & ArH), 7.74 (t, J = 7.6 Hz, 1H, ArH), 7.51 (d, J = 7.6 Hz, 1H,

2

ArH), 7.37 (d, J = 8.8 Hz, 2H, ArH), 6.73 (d, J = 8.8 Hz, 2H, ArH), 5.79 (s, 2H, Ar–NH₂),

.08 (m, 1H, NH–CH), 1.52-1.38 (m, 3H, CH–CH ), 0.79, 0.51 (d, J = 6 Hz, 6H, CH–

4

2

1

3

(CH ) ); C-NMR (100 MHz, DMSO-d , δ , ppm): 160.8, 155.5, 152.6, 147.7, 146.5, 141.2,

3 2 6 C

1

35.2, 130.9, 128.4, 127.6, 127.1, 126.5, 125.1, 124.8, 120.1, 112.8, 52.3, 41.8, 23.5, 23.1,

+

9.1; MS (ESI) m/z: 508.1 [M+H] ; Anal. Calcd. for C H N O S: C, 59.16; H, 4.96; N,

2

5

25

5

3.80. Found: C, 59.38; H, 5.04; N, 13.92 %.

4

-amino-N-{1-[3-(2-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]-3-methylbutyl}-

–

1

benzenesulfonamide (5k): White solid; mp: 196-201 °C; FT-IR (KBr, ν_m_a_x, cm ): 3367,

3

255 (N–H str.), 3051, 2855 (C–H str.), 1682 (C═O str.), 1620, 1596, 1496 (C═N, C═C str.),

1

319, 1147 (S═O str.), 750 (C–Cl str.); H-NMR (400 MHz, DMSO-d , δ , ppm): 8.08 (d, J

6

H

=

8 Hz, 1H, ArH), 7.91-7.81 (m, 3H, SO NH & ArH), 7.74-7.59 (m, 3H, ArH), 7.51-7.47 (m,

2

H, ArH), 7.34 (d, J = 8.8 Hz, 2H, ArH), 6.72 (d, J = 8.8 Hz, 2H, ArH), 5.87 (s, 2H, Ar–

NH ), 3.78 (m, 1H, NH–CH), 1.56-1.30 (m, 3H, CH–CH ), 0.73, 0.52 (d, J = 6.4 Hz, 6H,

2

13

CH–(CH ) ); C-NMR (100 MHz, DMSO-d , δ , ppm): 161.1, 157.0, 152.6, 146.6, 135.0,

3

2

6

C

1

5

34.9, 132.4, 131.4, 130.2, 129.8, 128.8, 128.6, 127.3, 127.0, 126.3, 125.1, 120.4, 112.6,

1.8, 42.2, 23.5, 23.1, 19.1; MS (ESI) m/z: 497.0 [M+H] , 499.0[(M+H)+2] ; Anal. Calcd.

+

for C H ClN O S: C, 60.41; H, 5.07; N, 11.27. Found: C, 60.71; H, 5.15; N, 11.41 %.

2

5

25

4

3

4

-amino-N-{1-[3-(3-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]-3-methylbutyl}-

benzenesulfonamide (5l): White solid; mp: 157-164 °C; α = -27.6 (AcOH); FT-IR (KBr,

obs

–1

ν_m_a_x, cm ): 3365, 3252 (N–H str.), 3050, 2861 (C–H str.), 1680 (C═O str.), 1625, 1594, 1499

1

(

C═N, C═C str.), 1312, 1144 (S═O str.), 746 (C–Cl str.); H-NMR (400 MHz, DMSO-d ,

6

δ , ppm): 8.09 (d, J = 8.8 Hz, 1H, ArH), 7.84-7.75 (m, 3H, SO NH & ArH), 7.62-7.48 (m,

H

2

3

H, ArH), 7.44-7.39 (m, 2H, ArH), 7.29 (d, J = 8.8 Hz, 2H, ArH), 6.71 (d, J = 8.8 Hz, 2H,

ArH), 5.68 (s, 2H, Ar–NH ), 3.74 (m, 1H, NH–CH), 1.57-1.32 (m, 3H, CH–CH ), 0.75, 0.51

2

1

3

(

d, J = 6.4 Hz, 6H, CH–(CH ) ); C-NMR (100 MHz, DMSO-d , δ , ppm): 161.1, 157.2,

3 2 6 C

1

52.5, 146.6, 135.7, 134.9, 132.8, 132.3, 131.3, 130.8, 128.5, 128.3, 127.2, 127.1, 126.2,

+

25.0, 120.3, 112.6, 51.8, 42.2, 23.5, 23.1, 19.1; MS (ESI) m/z: 496.9 [M+H] , 498.9

+

[

(M+H)+2] ; Anal. Calcd. for C H ClN O S: C, 60.41; H, 5.07; N, 11.27. Found: C, 60.67;

2

5

25

4

3

H, 5.13; N, 11.38 %.

4

-amino-N-{1-[3-(4-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]-3-methylbutyl}-

benzenesulfonamide (5m): White solid; mp: 183-188 °C; α = -41.9 (CHCl ); FT-IR (KBr,

obs

3

–1

ν_m_a_x, cm ): 3375, 3250 (N–H str.), 3055, 2958 (C–H str.), 1681 (C═O str.), 1628, 1597, 1504

1

(

C═N, C═C str.), 1310, 1148 (S═O str.), 744 (C–Cl str.); H-NMR (400 MHz, DMSO-d ,

6

ACCEPTED MANUSCRIPT

δ , ppm): 8.09 (d, J = 7.6 Hz, 1H, ArH), 7.91-7.84 (m, 2H, SO NH & ArH), 7.70 (d, J = 8.4

H

2

Hz, 1H, ArH), 7.63-7.52 (m, 3H, ArH), 7.29 (d, J = 8.8 Hz, 2H, ArH), 7.21 (dd, J = 8.4, 2.4

Hz, 2H, ArH), 6.41 (d, J = 8.4 Hz, 2H, ArH), 5.84 (s, 2H, Ar–NH ), 3.70 (m, 1H, NH–CH),

2

13

1

.57-1.23 (m, 3H, CH–CH ), 0.69, 0.51 (d, J = 6.4 Hz, 6H, CH–(CH ) ); C-NMR (100

2 3 2

MHz, DMSO-d , δ , ppm): 161.0, 157.4, 152.4, 146.9, 134.8, 134.8, 134.0, 131.3, 130.7,

6

C

1

4

1

28.6, 127.0, 126.9, 126.3, 125.0, 120.2, 112.4, 51.8, 42.2, 23.5, 23.1, 19.1; MS (ESI) m/z:

+

96.8 [M+H] , 498.8 [(M+H)+2] ; Anal. Calcd. for C H ClN O S: C, 60.41; H, 5.07; N,

2

5

25

4

3

1.27. Found: C, 60.69; H, 5.16; N, 11.40 %.

4

-amino-N-{1-[3-(2-bromophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]-3-methylbutyl}-

benzenesulfonamide (5n): White solid; mp: 167-171 °C; α = -18.3 (AcOH); FT-IR (KBr,

obs

–1

ν_m_a_x, cm ): 3368, 3254 (N–H str.), 3059, 2955 (C–H str.), 1684 (C═O str.), 1624, 1590, 1503

1

(

C═N, C═C str.), 1313, 1153 (S═O str.), 610 (C–Br str.); H-NMR (400 MHz, DMSO-d ,

6

δ , ppm): 8.12 (dd, J = 8, 1.2, 1H, ArH), 7.89-7.78 (m, 3H, SO NH & ArH), 7.71 (d, J = 8,

H

2

1

6

3

H, ArH), 7.64-7.58 (m, 2H, ArH), 7.51-7.48 (m, 2H, ArH), 7.33 (d, J = 8.4 Hz, 2H, ArH),

.51 (d, J = 8.4 Hz, 2H, ArH), 5.88 (s, 2H, Ar–NH ), 3.66 (m, 1H, NH–CH), 1.55-1.33 (m,

2

13

H, CH–CH ), 0.75, 0.53 (d, J = 6.4 Hz, 6H, CH–(CH ) ); C-NMR (100 MHz, DMSO-d ,

2

3 2

6

δ_C, ppm): 161.1, 157.0, 152.4, 146.4, 135.0, 134.6, 133.6, 131.3, 130.8, 129.5, 128.6, 127.3,

27.2, 126.4, 124.7, 122.2, 120.4, 112.4, 52.2, 42.0, 23.5, 23.1, 19.1; MS (ESI) m/z: 540.8

1

+

[

M+H] , 542.8 [(M+H)+2] ; Anal. Calcd. for C H BrN O S: C, 55.46; H, 4.65; N, 10.35.

25 25 4 3

Found: C, 55.71; H, 4.76; N, 10.48 %.

4

-amino-N-{1-[3-(3-bromophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]-3-methylbutyl}-

–1

benzenesulfonamide (5o): White solid; mp: 184-189 °C; FT-IR (KBr, ν_m_a_x, cm ): 3357,

3

250 (N–H str.), 3057, 2926 (C–H str.), 1685 (C═O str.), 1621, 1594, 1500 (C═N, C═C str.),

1

312, 1154 (S═O str.), 597 (C–Br str.); H-NMR (400 MHz, DMSO-d , δ , ppm): 8.11 (d, J

6

H

=

8 Hz, 1H, ArH), 7.88-7.79 (m, 2H, SO NH & ArH), 7.74-7.69 (m, 3H, ArH), 7.59-7.50 (m,

2

5

6

1

H, ArH), 7.45 (s, 1H, ArH), 7.32 (d, J = 8.8 Hz, 2H, ArH), 6.65 (d, J = 8.8 Hz, 2H, ArH),

.89 (s, 2H, Ar–NH ), 3.62 (m, 1H, NH–CH), 1.56-1.35 (m, 3H, CH–CH ), 0.77, 0.54 (d, J =

2

13

.4 Hz, 6H, CH–(CH ) ); C-NMR (100 MHz, DMSO-d , δ , ppm): 159.9, 157.2, 152.6,

3

2

6

C

46.3, 137.2, 134.8, 132.2, 131.5, 131.2, 128.5, 127.9, 127.2, 127.2, 126.3, 124.8, 122.0,

+

20.3, 112.5, 51.8, 42.1, 23.5, 23.1, 19.1; MS (ESI) m/z: 541.0 [M+H] , 543.0 [(M+H)+2] ;

Anal. Calcd. for C H BrN O S: C, 55.46; H, 4.65; N, 10.35. Found: C, 55.69; H, 4.71; N,

2

5

25

4

3

1

0.47 %.

4

-amino-N-{1-[3-(4-bromophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]-3-methylbutyl}-

benzenesulfonamide (5p): White solid; mp: 193-199 °C; α = -36.7 (AcOH); FT-IR (KBr,

obs

–1

ν_m_a_x, cm ): 3367, 3258 (N–H str.), 3053, 2924 (C–H str.), 1682 (C═O str.), 1617, 1598, 1501

1

(

C═N, C═C str.), 1317, 1151 (S═O str.), 619 (C–Br str.); H-NMR (400 MHz, DMSO-d ,

6

δ , ppm): 8.10 (d, J = 7.6 Hz, 1H, ArH), 7.82-7.78 (m, 2H, SO NH & ArH), 7.74 (d, J = 8.8

H

2

Hz, 2H, ArH), 7.68 (d, J = 7.6 Hz, 1H, ArH), 7.56 (t, J = 7.6 Hz, 1H, ArH), 7.38 (d, J = 8.8

Hz, 2H, ArH), 7.32 (d, J = 8.4 Hz, 2H, ArH), 6.26 (d, J = 8.4 Hz, 2H, ArH), 5.89 (s, 2H, Ar–

NH ), 3.65 (m, 1H, NH–CH), 1.58-1.36 (m, 3H, CH–CH ), 0.78, 0.57 (d, J = 6.4 Hz, 6H,

2

13

CH–(CH ) ); C-NMR (100 MHz, DMSO-d , δ , ppm): 160.0, 157.3, 152.4, 146.3, 137.7,

3

2

6

C

1

35.4, 134.8, 131.0, 127.2, 127.1, 126.5, 126.3, 124.7, 122.9, 120.4, 112.4, 51.8, 42.1, 23.5,

ACCEPTED MANUSCRIPT

+

2

3.1, 19.1; MS (ESI) m/z: 540.9 [M+H] , 542.9 [(M+H)+2] ; Anal. Calcd. for

C H BrN O S: C, 55.46; H, 4.65; N, 10.35. Found: C, 55.72; H, 4.74; N, 10.48 %.

25

4

3

4

-amino-N-{1-[3-(2-fluorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]-3-methylbutyl}-

–

1

benzenesulfonamide (5q): White solid; mp: 166-170 °C; FT-IR (KBr, ν_m_a_x, cm ): 3364,

3

259 (N–H str.), 3059, 2859 (C–H str.), 1683 (C═O str.), 1622, 1593, 1504 (C═N, C═C str.),

1

319, 1152 (S═O str.), 1213 (C–F str.); H-NMR (400 MHz, DMSO-d , δ , ppm): 8.09 (d, J

6

H

=

8 Hz, 1H, ArH), 7.83-7.72 (m, 3H, SO NH & ArH), 7.68-7.56 (m, 2H, ArH), 7.34 (d, J =

2

8

.4 Hz, 2H, ArH), 7.32-7.18 (m, 3H, ArH), 6.60 (d, J = 8.4 Hz, 2H, ArH), 5.84 (s, 2H, Ar–

NH ), 3.77 (m, 1H, NH–CH), 1.52-1.34 (m, 3H, CH–CH ), 0.77, 0.54 (d, J = 6 Hz, 6H, CH–

2

13

(

CH ) ); C-NMR (100 MHz, DMSO-d , δ , ppm): 161.4, 158.2, 156.5, 152.6, 146.6, 135.7,

3 2 6 C

1

2

5

34.9, 130.6, 129.3, 128.6, 128.3, 128.2, 127.1, 126.3, 125.2, 120.3, 114.7, 112.2, 52.0, 41.8,

+

3.5, 23.1, 19.1; MS (ESI) m/z: 480.9 [M+H] ; Anal. Calcd. for C H FN O S: C, 62.48; H,

2

5

25

4

3

.24; N, 11.66. Found: C, 62.76; H, 5.32; N, 11.79 %.

4

-amino-N-{1-[3-(4-fluorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]-3-methylbutyl}-

–

1

benzenesulfonamide (5r): White solid; mp: 201-208 °C; FT-IR (KBr, ν_m_a_x, cm ): 3375, 3256

N–H str.), 3059, 2862 (C–H str.), 1686 (C═O str.), 1625, 1596, 1502 (C═N, C═C str.),

(

1

316, 1150 (S═O str.), 1206 (C–F str.); H-NMR (400 MHz, DMSO-d , δ , ppm): 8.10 (d, J

6 H

=

7.6 Hz, 1H, ArH), 7.84-7.64 (m, 4H, SO NH & ArH), 7.42 (d, J = 8.8 Hz, 2H, ArH), 7.36

2

(

d, J = 8.4 Hz, 2H, ArH), 7.28 (d, J = 8.8 Hz, 2H, ArH), 6.73 (d, J = 8.4 Hz, 2H, ArH), 5.91

s, 2H, Ar–NH ), 3.76 (m, 1H, NH–CH), 1.55-1.34 (m, 3H, CH–CH ), 0.79, 0.57 (d, J = 6.4

2

1

3

Hz, 6H, CH–(CH ) ); C-NMR (100 MHz, DMSO-d , δ , ppm): 161.1, 160.9, 156.8, 152.5,

3

2

6

C

1

2

46.6, 134.7, 132.3, 128.6, 128.4, 127.0, 126.9, 126.2, 125.2, 120.3, 116.6, 112.2, 52.0, 41.9,

3.5, 23.1, 19.1; MS (ESI) m/z: 481.0 [M+H] , 502.9 [M+Na] ; Anal. Calcd. for

+

C H FN O S: C, 62.48; H, 5.24; N, 11.66. Found: C, 62.72; H, 5.31; N, 11.77 %.

25

4

3

4

-amino-N-[1-(3-anilino-4-oxo-3,4-dihydroquinazolin-2-yl)-3-methylbutyl]benzene-

–1

sulfonamide (5s): White solid; mp: 198-203 °C; FT-IR (KBr, ν_m_a_x, cm ): 3350, 3252 (N–H

str.), 3051, 2920 (C–H str.), 1692 (C═O str.), 1612, 1596, 1496 (C═N, C═C str.), 1314, 1152

1

(

S═O str.); H-NMR (400 MHz, DMSO-d , δ , ppm): 8.93 (s, 1H, NH–Ar), 8.09 (d, J = 8

6

H

Hz, 1H, ArH), 7.83-7.73 (m, 3H, SO NH & ArH), 7.55 (t, J = 8 Hz, 1H, ArH), 7.35 (d, J =

2

8

.8 Hz, 2H, ArH), 7.19 (d, J = 8 Hz, 2H, ArH), 6.84 (t, J = 8 Hz, 1H, ArH), 6.56 (d, J = 8

Hz, 2H, ArH), 6.43 (d, J = 8.8 Hz, 2H, ArH), 5.92 (s, 2H, Ar–NH ), 4.17 (m, 1H, NH–CH),

2

13

1

.57-1.39 (m, 3H, CH–CH ), 0.79, 0.56 (d, J = 6 Hz, 6H, CH–(CH ) ); C-NMR (100 MHz,

2 3 2

DMSO-d , δ , ppm): 161.1, 156.9, 152.5, 146.8, 146.5, 134.8, 129.4, 128.6, 127.9, 127.2,

6

C

1

26.3, 124.9, 120.3, 120.2, 112.6, 112.5, 52.1, 42.0, 23.5, 23.1, 19.1; MS (ESI) m/z: 478.0

+

[

M+H] ; Anal. Calcd. for C H N O S: C, 62.87; H, 5.70; N, 14.66. Found: C, 63.06; H,

25 27 5 3

5

.78; N, 14.79 %.

7

.2 In vitro antimalarial assay

A positive control with reference antimalarial drugs (Chloroquine, Pyrimethamine and

Trimethoprime) in standard concentrations was used in each experiment. The stock solutions

were further diluted in complete medium (RPMI 1640 plus 10% human serum) to each of the

ACCEPTED MANUSCRIPT

used concentrations (0.0001 up to 100 mg/mL in seven dilutions). The halfmaximal

inhibitory (IC ) responses as compared to the drug-free controls were estimated by

5

0

interpolation using Microcal Origin software. Each duplicate experiment was repeated three

times and blood smears were read blind [38]. Results from above study are summarized in

Table 5 as IC values.

5

0

7

.3 The Parasite Lactate Dehydrogenase assay (p-LDH)

The in vitro antiplasmodial effect of synthetic Quinazolinone-Sulphonamide hybrids on

parasite growth is tested by the quantitative enzyme-based method (Malstat® assay)

involving spectrophotometric measurement of the parasite lactate dehydrogenase activity

(p-LDH) as described by Makler and Hinrichs [39], at 650 nm using a micro plate reader (EL

8

00, Biotech Instruments Inc.). The simultaneous control parasite cultures devoid of drug

were referred to as having 100 % p-LDH activity. The inhibition of each drug concentration

was calculated as compared to the untreated control obtain the IC values.

5

0

7

.4 The Tetrazolium based colorimetric assay (MTT-assay)

The in vitro antiplasmodial and cytotoxic effect of Quinazolinone-Sulphonamide hybrids on

parasite growth and normal uninfected RBCs was tested by the quantitative enzyme based

method (MTT assay) involving spectrophotometric measurement of the host RBC’s

Mitochondrial Succinate Dehydrogenase catalysed MTT reduction as described by Ayisi,

N.K. et al. [40], at 565 & 690 nm using a micro plate reader (EL 800, Biotech Instruments

Inc.). The simultaneous control RBC cultures devoid of drug and parasite were referred to as

having maximum enzyme activity. The inhibition and cytotoxicity of each drug concentration

was calculated as compared to the untreated, uninfected and cell free controls to obtain the

EC and CC values. Finally, the ratio, CC /EC was calculated to obtain selective indices

50

(SI), showing that the drug is only selective against parasite.

7

.5 DHFR inhibitory activity

The inhibitory activity was carried out using bovine liver DHFR as model enzymes

resembling to Pf-DHFR [41]. The utilized assay mixture contained 50 mM Tris-HCl buffer

(

pH 7.4), 50 µM NADPH, 20 µL DMSO or the same volume of DMSO solution containing

-11

-5

the test compounds to a final concentration of 10 -10 M, and 0.02 units of bovine liver

DHFR, in a final volume of 2.0 ml. After addition of the enzyme, the mixture was incubated

at room temperature for 2.0 min, and the reaction was initiated by adding 25 µM FH2, the

change in absorbance (∆A/min) was measured at 340 nm. The activity under these conditions

was linear for 10 min [41]. Results are reported as % inhibition of enzymatic activity

calculated using the following formula:

∆

ꢁ/ꢂꢃꢄ_ꢅ_ꢆ_ꢇ_ꢅ

%

Inhibition = ꢀ1 − _∆

ꢌ × 100

ꢁ/ꢂꢃꢄ_ꢈ_ꢉ_ꢊ_ꢋ

ACCEPTED MANUSCRIPT

The % inhibition values were plotted versus drug concentration (log scale). The 50%

inhibitory concentration (IC ) of each compound was obtained using the Graph Pad Prism

5

0

program, version 3 (San Diego, CA).

7

.6 Cytotoxicity study by MTT assay

MTT assay was used to determine the inhibition of vero cell proliferation by the synthesized

entities using Promega CellTiter 96 Non-radioactive Cell Proliferation Assay (Promega,

Madison, WI, USA). The viability was assessed on the basis of cellular conversion of MTT

into a formazan by the vero cells after 72 hours of incubation at 37 °C and the activity was

assessed by measuring the absorbance at 540 nm followed by the calculation of percentage

cell viability.

ꢎ ꢐꢎ_ꢑ

ꢏ

Percentage cell viability = ꢍ100 − ꢀ

ꢌ × ꢒ100ꢓ

ꢎ_ꢏ

Where, A = Absorbance of cells treated with 0.1% DMSO medium, A = Absorbance of cells

o

t

treated with various concentration of the samples.

Each treatment was performed in triplicate and the 50% inhibitory concentration (IC ) of

5

0

each compound was obtained using the Graph Pad Prism program, version 3 (San Diego,

CA).

7

.7 Computational studies

The molecular structures of all the compounds were drawn using ChemBioDraw Ultra 14.0

www.cambridgesoft.com). These structures were then imported into Maestro implemented

(

in Schrödinger, further energy of the 3D structures was minimized using Ligprep 3.3 module.

The possible Lewis structure, tautomers and ionization states (pH 7.0 ± 2.0) for each of these

compounds were generated and optimized with default settings (Ligprep 3.3, Schrödinger,

LLC, New York, NY, 2015). The crystal structure of mutant Pf-DHFR (PDB ID: 4DPD) and

h-DHFR (PDB ID: 1MVT) was extracted from protein data bank (www.rcsb.org). The

protein structure was further refined and hydrogen atoms were added to the structure utilizing

Protein Preparation Wizard (Maestro 10.1 Schrödinger, LLC, New York, NY, 2015). The

binding site in the protein molecules was predicted using Sitemap 3.4 wizard and further the

centre of the grid was defined, which was generated using Glide 6.6 (Schrödinger, LLC, New

York, NY, 2015) with default settings for all parameters. The grid size was kept sufficiently

high so as to include the atoms participating in the interaction and then all the compounds

were docked against the grid of prepared receptor (mutant Pf-DHFR & h-DHFR) using Glide

in XP (Extra Precession) mode [42] with other default setting for scoring function.

7

.8 ADMET prediction method

ACCEPTED MANUSCRIPT

The pharmacokinetic profile of the test compounds showing good antimalarial activity were

predicted by using programs Qikprop v4.3 (Schrödinger, Inc., New York, NY, 2015). The

compounds prepared by LigPrep 3.3 were utilized for the calculation of pharmacokinetic

parameters by QikProp v4.3. The program QikProp v4.3, utilizes the method of Jorgensen

[

43] to compute pharmacokinetic properties and descriptors such as octanol/water

partitioning coefficient, aqueous solubility, brain/blood partition coefficient, intestinal wall

permeability, plasma protein binding and others.

Acknowledgement

Authors are gratefully acknowledged to the University Grants Commission [UGC F. No. 34-

3

01/2008 (SR)], New Delhi-India for the financial support of this research work. We are

thankful to management (C.V.M) of V. P. & R. P. T. P. Science College, Vallabh Vidyanagar

for providing all the necessary research facilities and encouragement. The support of Dr.

Raghu R. and the team Schrodinger (Bangalore) for Maestro-2015-1 is greatly lauded. The

authors would like to thank The Director, SICART, Vallabh Vidyanagar for NMR, FTIR

analyses facility. We are grateful to Oxygen Healthcare Research Pvt. Ltd., Ahmedabad for

Mass analyses.

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List of Legends:

Table 1 Optimization of reaction condition under conventional heating for synthesis of

compound 4a

Table 2 Optimization of microwave promoted reaction condition for synthesis of compound

4

a

Table 3 Synthesis of quinazolinone-sulphonamide hybrids using TEAA 4a-4s

Table 4 Synthesis of quinazolinone-sulphonamide hybrids 5a-s

Table 5 In vitro antimalarial assay (IC ) values

5

0

Table 6 Antiplasmodial activity and cytotoxicity of the tested hybrids

Table 7 Glide docking score and docking energies of the active entries and reference

compound

Table 8 Prediction of Lipinski RO5 for active test entities

Table 9 Prediction of ADME parameters* of the active test compounds

Table 10 Enzyme inhibition assay and Cytotoxicity of 2,3-disubstituted quinazolinone*

Scheme 1 Schematic outline of reaction path leading to 2,3-disubstituted quinazolin-4-(3H)-

one hybrids

Scheme 2 Optimization of the reaction conditions leading to hybrid 2,3-disubstituted

quinazolin-4-(3H)-one (4a). Reagents & Conditions: (i) Method-I: Conventional method for

synthesis of 4a, PCl /Toluene, ∆. (i) Method-II: Conventional method for synthesis of 4a,

3

PCl /Xylene, ∆. (i) Method-III: Conventional OR Microwave Irradiation method for

3

synthesis of 4a, PCl /CH Cl , ∆. (i) Method-IV: Conventional OR Microwave Irradiation

3

2

method for synthesis of 4a, PCl /CH CN, ∆. (i) Method-V: Conventional OR Microwave

3

Irradiation method for synthesis of 4a, PCl /THF, ∆. (i) Method-VI: Conventional OR

3

Microwave Irradiation method for synthesis of 4a, PCl /Pyridine, ∆. (i) Method-VII:

3

Conventional OR Microwave Irradiation method for synthesis of 4a, TEAA, ∆.

Scheme 3 Synthesis of 5a-5s Quinazolinone-Sulphonamide hybrid molecules. Reagents &

Conditions: Conventional OR Microwave Irradiation method for synthesis of 5a-5s, dil. HCl

(

40%), R-spirit/H O.

2

Fig. 1 Potent antimalarial scaffolds owing analogical relationship with target molecules

Fig. 2 The binding model of docked compound 5g with Pf-DHFR enzyme. (a) 2D model of