Discovery of a new class of natural product-inspired quinazolinone hybrid as potent antileishmanial agents

Article

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Discovery of a New Class of Natural Product-Inspired Quinazolinone

Hybrid as Potent Antileishmanial agents

Moni Sharma,^†,‡Kuldeep Chauhan,^†Rahul Shivahare,^§Preeti Vishwakarma,^§Manish K. Suthar,^∥

Abhisheak Sharma,^⊥Suman Gupta,^§Jitendra K. Saxena,^∥Jawahar Lal,^⊥Preeti Chandra,^#Brijesh Kumar,^#

and Prem M. S. Chauhan*^,†

^†Medicinal and Process Chemistry Division, CSIRCentral Drug Research Institute, Lucknow-226 001, U.P., India

^‡Department of Chemistry, Bhagwant University, Ajmer, India

^§Division of Parasitology, CSIRCentral Drug Research Institute, Lucknow-226 001, U.P., India

^∥Division of Biochemistry, CSIRCentral Drug Research Institute, Lucknow-226 001, U.P., India

^⊥Pharmacokinetics & Metabolism Division, CSIRCentral Drug Research Institute, Lucknow-226 001, U.P., India

^#Sophisticated Analytical Instrumentation Facility, CSIRCentral Drug Research Institute, Lucknow 226001, India

S

* Supporting Information

ABSTRACT: The high potential of quinazolinone containing natural products and their derivatives in medicinal chemistry led

us to discover four novel series of 53 compounds of quinazolinone based on the concept of molecular hybridization. Most of the

synthesized analogues exhibited potent leishmanicidal activity against intracellular amastigotes (IC₅₀from 0.65 0.2 to 7.76

2.1 μM) as compared to miltefosine (IC₅₀= 8.4 2.1 μM) and nontoxic toward the J-774A.1 cell line and Vero cells. Moreover,

activation of Th1 type and suppression of Th2 type immune responses and induction in nitric oxide generation proved that 8a

and 8g induce murine macrophages to prevent survival of parasites. Compounds 8a and 8g exhibited signiﬁcant in vivo inhibition

of parasite 73.15

12.69% and 80.93

10.50% against Leishmania donovani/hamster model. Our results indicate that

compounds 8a, 8g, and 9f represent a new structural lead for this serious and neglected disease.

nisms to cause dysfunction in their host macrophages and the

INTRODUCTION

■

outcome of infection depends on the production and/or

secretion of immunosuppressive molecules that includes trans-

forming growth factor (TGF)-β, interleukin (IL)-10, and

prostaglandin E2 (PGE2).^5aThese molecules distort the normal

immune response by suppression of host-protective microbicidal

molecules, nitric oxide (NO), and reactive oxygen species (ROS)

and cytokines interferon (IFN)-γ, IL-1, IL-12, and tumor

necrosis factor-α (TNF-α).^5bAntileishmanial therapy relies on

handful number of drugs such as antimony-based drugs:

meglumine antimoniate (Glucantime), sodium stibogluconate

(Pentostam), pentamidine, amphotericin B, and miltefosine.⁶

Leishmaniasis is a debilitating disease caused by the protozoan

parasites of the genus Leishmania that pose serious public health

challenges for prevention, diagnosis, and treatment.¹Leishmania

donovani, the etiological agent of visceral leishmaniasis (VL),

aﬀects about 12 million people in nearly 90 countries and an

estimated 51000 annual deaths for leishmaniasis, representing a

signiﬁcant health problem in tropical and subtropical regions of

the world.²Leishmania parasites are transmitted to humans

almost exclusively by biting of infected phlebotomine sand ﬂies

as proliferative promastigotes. Inside the mammalian body, the

parasite multiplies within phagolysosomes of macrophages as

intracellular amastigotes.³Active VL is almost fatal if not treated

after the onset of clinicopathological abnormalities.⁴To survive

within macrophages, Leishmania has developed several mecha-

Received: January 11, 2013

Published: April 23, 2013

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Figure 1. Designing of quinazolinone-based hybrids showing antileishmanial activity.

Pentavalent antimony, a prodrug which was developed before

1959, is characterized by high toxicity and limited eﬃcacy.

Conventional amphotericin B as the ﬁrst-line treatment for VL

has replaced antimonials in some areas of the Bihar State of India

where more than 60% of newly diagnosed VL cases in this area do

not respond to antimonials.⁷Pentamidine (an aromatic

diamidine), the second line treatment options for VL, is also

far from satisfactory due to several side eﬀects and not active

orally. WHO/TDR is currently developing a research program

with miltefosine (a teratogenic drug), represents a fundamental

advance in antileishmanial chemotherapy because it is the ﬁrst

oral agent. Despite its great eﬃcacy, this drug has a long half-life

(more than 150 h) and parasite resistance is easily induced in

vitro, therefore its use is strictly outlawed in pregnant women.⁸

Because most of the drugs used in treatment have a list of

problems like unaﬀordable cost, diﬃculty in administration, high

toxicity, and more importantly the resistance problem.⁹In view

of the foregoing facts, the search for innovative drugs based on

new molecular scaﬀolds that target the speciﬁc metabolic

pathway of the parasite should be highly prioritized, which in

turn requires new medicinal chemistry approaches to discover

novel lead compounds that might populate a pipeline of new

therapeutics. Unfortunately, our limited discerning of leishmania

biology makes diﬃcult the rational designing of antileishmanial

agents.

An attractive concept of hybridization for neglected tropical

disease is becoming popular as an emerging structural

modiﬁcation tool to design new molecules with improved

bioactivity when compared to the parent compounds.¹⁰⁻¹²In

this context, biology oriented synthesis (BIOS) of natural

product-inspired scaﬀolds and their hybrids may show promising

results in ﬁnding new lead structures for chemical biology and

medicinal chemistry research.¹³⁻¹⁵Furthermore, bioactive small

molecule natural products possess all of the potency, selectivity,

and pharmacokinetic persona, an important paradigm for drug-

like molecules.¹⁶However, the number of natural products are

limited, hence incorporating either diﬀerent natural products or

drug fragments may provide millions of combinations which will

be enriched in biological activity and less toxicity.

Quinazolinone is a building block of naturally occurring

alkaloids and is utilized as a druglike scaﬀold in several natural

products¹⁷⁻²¹(trypanthrin, rutaecarpine, and luotonin A, Figure

1), as these possess a wide range of biological activities including

antitumor, anticonvulsant,²²antiviral,²³antiinﬂammatory, an-

algesic,²⁴antimicrobial,²⁵antifungal,²⁶antimalarial,²⁷antidia-

betic,²⁸cytotoxicity,²⁹and angiotensin II AT1 receptor

antagonists.³⁰Recently, we have developed an eﬃcient and

facile methodology for the preparation of this privileged

scaﬀold.³¹

On the other hand, among the diﬀerent heterocyclic structures

that have been disclosed, the nitrogen containing class of

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Figure 2. Examples of some pyrimidine-, triazine-, and tetrazole-based bioactive agents.

Figure 3. SAR of the synthesized quinazolinone−pyrimidine hybrids.

compounds such as pyrimidine, triazine, peptide, and tetrazole

that are found in many natural products (annomontine,

meridianin G, and almiramide A−C)³²⁻³⁴are the important

constituents of number of modern drugs³⁵⁻³⁷(Figures 1 and 2).

Interestingly, some of these from natural sources have been

screened as potent antileishmanial agents and a number of them

exhibited multifarious pharmacological proﬁles (Figure 1).³⁸⁻⁴⁰

Moreover, previously we have reported the pyrimidine and

triazine class of analogues as good antileishmanial agents (Figure

2).⁴¹

On the basis of this reasoning and as a part of our ongoing

interest toward the design and synthesis of novel heterocycles as

anti-infective agents,⁴²we have synthesized the quinazolinone

hybrids via introducing heterocyclic systems as discussed above

with diﬀerent possible functionalities. To the best of our

knowledge, the hybridization of these bioactive pharmacophores

and their antileishmanial activity has never been reported to date.

Through these processes, we have discovered novel scaﬀolds

with potent antileishmanial activity with a high selective index

which was comparable or superior to other current antileishma-

nials, miltefosine and SSG (sodium stibogluconate). The

compounds which have a combination of high eﬃcacy and no

cytotoxicity with the high value of selective index were tested for

in vivo study in a chronic hamster model. We also investigated

the interaction of the most potent compounds with bovine serum

albumin (BSA). Serum albumin, major soluble blood protein, has

many physiological functions such as maintenance of colloidal

osmotic pressure and blood pH. Another characteristic property

of albumins is their reversible interactions with various

endogenous and exogenous compounds which facilitate the

transportation and disposition of several compounds including

drugs, proteins, and fatty acids to speciﬁc targets.⁴³⁻⁴⁵BSA a 66

kDa monomer having domains and subdomains stabilized by 17

pairs of disulﬁde bridges shows 80% structural homology with

human serum albumin.⁴⁶⁻⁴⁸

RESULTS AND DISCUSSION

■

Chemistry. To realize a facile access toward quinazolinone

hybrids with various bioactive heterocycles from readily available

substrates, a set of compounds were synthesized for the

structure−activity relationship (SAR).

Synthesis of Quinazolinone−Pyrimidines (5a−i, Figure

3). 4-(4-Oxo-1,2,3,4-tetrahydroquinazolin-2-yl)benzaldehyde

(3)³¹and various substituted acetophenones were subjected to

Claisen−Schmidt condensation⁴⁹using KOH in ethanol to

aﬀord the corresponding quinazolinone−chalcones (4a−i).

Intermediate (4a−i) were reacted with the guanidine hydro-

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a

Scheme 1. Synthesis of Quinazolinone−Pyrimidine Derivatives (5a−i)

a

Reagents and conditions: (i) cyanuric chloride, CH₃CN, 15 min; (ii) substituted acetophenone, KOH, EtOH, 5h rt; (iii) guanidine hydrochloride,

NaH, DMF, 70 °C, 5 h.

Figure 4. SAR of the synthesized quinazolinone−triazine hybrids.

a

Scheme 2. Synthesis of Quinazolinone−Triazine Derivatives (8a−o)

a

Reagents and conditions: (i) acidic SiO₂, dry MeOH, 3 h, 60 °C; (ii) (a) cyanuric chloride, THF, 1 h rt; (b) various amines, K₂CO₃, DMF, reﬂux, 4

h.

Figure 5. SAR of the synthesized quinazolinone−peptide hybrids.

chloride in the presence of NaH in DMF to aﬀord the target

quinazolinone−pyrimidines (5a−i, Scheme 1).

Synthesis of Quinazolinone−Triazine (8a−r, Figure 4).

For the scope of the quinazolinone hybrid, introduction of 1,3,5-

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Scheme 3. Synthesis of Quinazolinone−Peptide Derivatives (9a−i)

Figure 6. SAR of the synthesized quinazolinone−tetrazole hybrids.

Scheme 4. Synthesis of Quinazolinone−Tetrazole Derivatives (10a−o)

Scheme 5. Synthesis of Quinazolinone−Tetrazole Derivatives (11a−e)

triazine at the quinazolinone ring was envisaged. In this respect, a

straightforward synthetic approach was followed for the

synthesis of compounds (8a−r, Scheme 2). The synthesis of

2,4,6-trisubstituted-[1,3,5]triazines were achieved by consec-

utive nucleophilic substitution of cyanuric chloride. The whole

sequence is performed in the same pot with the change of

solvent. Quinazolinone−triazine analogues (8a−r) were formed

by nucleophilic substitution of cyanuric chloride and 2-(4-

aminophenyl)-2-methyl-2,3-dihydroquinazolin-4(1H)-one (7)

in THF at room temperature followed by treatment with various

amines using K₂CO₃in dry DMF at 70 °C. Compound 7 was

prepared by the acidic silica-catalyzed condensation of

commercially available anthralinamide (1) and p-aminoaceto-

phenone (6) in MeOH at 60 °C.

Synthesis of Quinazolinone−Peptide (9a−i, Figure 5).

Because the isocyanide-based multicomponent reactions

(IMCRs) are considered as powerful tools in modern organic

synthesis and drug discovery,⁵⁰therefore, to further optimize the

SAR, the preparation of new chemical entities (NCEs), based on

multicomponent Ugi reaction, was designed for the creation of

quinazolinone−peptides. The 2-(4-aminophenyl)-2-methyl-2,3-

dihydroquinazolin-4(1H)-one (7), m-chlorobenzoic acid, vari-

ous commercially available aldehydes, and diﬀerent isocyanides

were subjected to IMCRs in methanol to aﬀord the

corresponding quinazolinone−peptides (9a−i, Scheme 3) with

good to excellent yield at room temperature.

Synthesis of Quinazolinone−Tetrazole (10a−o, 11a−

e). To further optimize the quinazolinone analogues, we have

synthesized a series of quinazolinone−tetrazoles (10a−o) for

generating the collections of molecules with high molecular

diversity via modiﬁed TMSN₃−Ugi MCR (Figure 6).^50bIn this

context, compound 7 was allowed to react at room temperature

with diﬀerent commercially available aldehydes, isocyanides, and

azidotrimethylsilane (TMSN₃) in anhydrous methanol to form

the desire quinazolinone−tetrazoles (10a−o, Scheme 4) in good

to excellent yield at room temperature. To introduce further

diversity in the quinazolinone−tetrazole hybrids for the SAR , we

have designed and synthesized another series of quinazolinone−

tetrazole analogue (11a−e) as shown in Scheme 5.

1

The structures of the compounds were substantiated by H

NMR, ¹³C NMR, mass spectrometry and IR spectroscopy.

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Table 1. In Vitro Antileishmanial Activity of Quinazolinone−Pyrimidine (5a−i)

a

b

c

IC₅₀values are the average of two independent assays expressed as average standard error. CC₅₀on J-774A.1 cells. The selectivity index (SI) is

b

a

d

e

deﬁned as the ratio of CC₅₀on J-774A.1 cells from ( ) to IC₅₀on L. donovani intracellar amastigotes ( ). ND: not determined. NA: not available.

Biological Activity. In an attempt to identify novel

quinazolinone hybrids, a novel series of 53 compounds was

subjected to the screening test against L. donovani. Each

candidate was evaluated for its in vitro activity against WHO

reference strain (MHOM/IN/80/Dd8) of extracellular promas-

tigotes⁴and intramacrophagic amastigotes (expressing luciferase

ﬁreﬂy reporter gene) of L. donovani.⁵¹The in vitro cytotoxicity

assay was performed using murine macrophage cells (J-774A.1

cell line) and mammalian kidney ﬁbroblast cells (Vero cell

line).⁵²The selectivity of a compound was deﬁned as the ratio of

the cytotoxicity in macrophage cells and the inhibitory activity in

intracellular amastigotes. Compounds which showed in vitro

potency with high selectivity were further tested for their in vivo

eﬃcacy in golden hamsters at a dosage of 50 mg/kg for ﬁve days

through the intraperitoneal (IP) route.⁵³Standard antileishma-

nials, miltefosine and SSG, were included in the study as control

drugs. SARs of the quinazolinone hybrids have been extensively

studied with the diﬀerent counterparts and wide variation of

substituents around it to optimize potency/selectivity. The

intracellular amastigote form is the clinically relevant parasitic

stage for biological evaluation and interestingly, we were very

pleased to ﬁnd that among the synthesized analogues most of the

analogues were more potent against intracellular amastigote form

than promastigotes (Tables 1−4).

In Vitro Antileishmanial Activity of Quinazolinone−

Pyrimidine. Initially, a series of quinazolinone−pyrimidines

(5a−i) was synthesized (Scheme 1) and evaluated for their in

vitro antileishmanial activity, and the results are shown in Table

1. All the synthesized hybrids in this series showed signiﬁcant to

moderate activity against promastigote with IC₅₀ranging from

1.75 0.9 to 4.23 1.2 μM. Compounds 5b−e, 5g, and 5i

displayed the signiﬁcant antiamastigote activity in the micro-

molar range better than that of miltefosine. Miltefosine and SSG

have been evaluated under the same conditions, miltefosine

displayed signiﬁcant antipromastigote (IC₅₀= 1.05 0.2 μM),

moderate antiamastigote activity (IC₅₀= 8.4 2.1 μM), and

toxic to the J-774A.1 cell line (CC₅₀values of 12.42 3.2 μM)

and less cytotoxicity toward Vero cells (CC₅₀values of 52.47

2.4 μM). SSG displayed a poor antiamastigote activity (IC₅₀

=

46.70 2.8 μM) although it is nontoxic to J-774A.1 (CC₅₀

297.38 10.2 μM) and Vero cells (CC₅₀> 400 μM). Compound

5a, which contained unsubstituted phenyl ring, was inactive

against amastigotes. A progressive enhancement in antiamasti-

gote potency was observed in the case of electron releasing

substituents such as; 3,4,5-trimethoxy substituted hybrid (5b),

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Table 2. In Vitro Antileishmanial Activity of Quinazolinone−Triazine (8a−o)

a

b

c

IC₅₀values are the average of two independent assays expressed as average standard error. CC₅₀on J-774A.1 cells. The selectivity index (SI) is

b

a

d

e

deﬁned as the ratio of CC₅₀on J-774A.1 cells ( ) to IC₅₀on L. donovani intracellular amastigotes ( ). ND: not determined. NA: not available.

displaying the promising activity (IC₅₀= 3.83

1.1 μM),

high to both J-774A.1 and Vero cells (CC₅₀value of 10.15 2.5

and 8.32 2.2 μM, respectively). Interestingly, most of the

followed by dimethoxy (5c, IC₅₀= 4.43

1.5 μM), and

monomethoxy (5d, IC₅₀= 10.08

1.4 μM). Moreover,

synthesized analogues exhibited better potency than the natural

product (annomontine).

compounds 5b−d was nontoxic to the J-774A.1 cell line (CC₅₀

from 103.32 8.5 to >400 μM) and Vero cells (85.86 8.8 -

>400 μM). Whereas compound 5c exhibited highest selectivity

(SI = 90.29). The presence of the p-chloro substituent enhanced

the antiamastigote activity of compound 5f (IC₅₀= 1.89 0.9

μM). Importantly, compound 5f was nontoxic to J-774A.1 and

In Vitro Antileishmanial Activity of Quinazolinone−

Triazine. To ﬁnd out the role of the eﬀect of triazine counterpart

at the quinazolinone skeleton for SAR, quinazolinone−triazine

hybrids (8a−o, Scheme 2) were prepared by varying the

substituents around the triazine and screened for their in vitro

antileishmanial activity (Table 2). Analogues 8b, 8k, 8m, and 8n

displayed promising antipromastigote activity having IC₅₀values

in the range of 1.02 0.4 to 6.38 1.5 μM. Compounds 8a, 8b,

8g−i, 8k, and 8n exhibited more potent antiamastigote activity,

having IC₅₀values in the range of 2.42 0.9 to 6.72 1.1 μM

than miltefosine. Compounds 8a−e, which have p-hydroxy

piperidine, morpholine and aminopropyl morpholine were

appeared to be inactive (IC₅₀> 40 μM). Compound 8a, having

piperidine substituents, exhibited signiﬁcant activity (IC₅₀= 3.95

0.8 μM). Homopiperidine containing analogue 8b exhibited

Vero cells (CC₅₀= 82.15

5.5 and 322.72

15.6 μM,

respectively). Having realized the importance of electron

withdrawing groups on an aryl ring, we synthesized o-chloro

substituted analogue 5h; unfortunately, antiamastigote activity

was lost in 5h. With replacement of p-Cl with p-Br on the aryl

ring (compound 5g), signiﬁcant reduction in the antileishmanial

activity was observed (IC₅₀= 7.02 1.5 μM), although it was

nontoxic to J-774A.1 and Vero cells (CC₅₀125.12 6.8 and

>400 μM, respectively). However, in the p-ﬂuoro substituted

compound (5e), the antiamastigote activity was completely lost.

Compound 5i, having p-phenylpiperazine substituent on the aryl

ring of the R₁group, was found to be the most active candidate

with an IC₅₀value of 0.65 0.2 μM, 13-fold more potent than

miltefosine (IC₅₀= 8.4 2.1 μM), however, its cytotoxicity was

better activity (IC₅₀= 2.59

1.2 μM) than 8a. Both the

analogues were nontoxic although 8a showed higher selectivity

(SI > 101.26) than 8b (SI = 91.64). Furthermore, compound 8a

proved to be more potent than compound 8d, carrying a less

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Table 3. In Vitro Antileishmanial Activity of Quinazolinone−Peptide (9a−i)

a

b

c

IC₅₀values are the average of two independent assays expressed as average standard error. CC50 on J-774A.1 cells. The selectivity index (SI) is

b

a

d

e

deﬁned as the ratio of ( ) to IC₅₀on L. donovani intracellular amastigotes ( ). ND: not determined. NA: not available.

basic morpholine. In addition, tetrahydroisoquinoline containing

analogue 8g exhibited potent antiamastigote activity (IC₅₀= 4.39

1.4 μM) and high selectivity (SI > 91.11), whereas the

tetrahydroquinoline containing analogue 8f was inactive,

indicating that the position of nitrogen atom on the tetrahydro

ring is critical in antiamastigote activity. Compound 8h, having

an amyl amine substituent, demonstrated the best potency

among all the synthesized quinazolinone−triazine analogues,

with IC₅₀value of 2.42 0.9 μM without any cytotoxicity toward

both J-774A.1 and Vero cells (CC₅₀> 400 μM). Moreover,

compound 8h has a very high selective index (SI > 165.29).

Considering the importance of alkyl amine, we have synthesized

n-butyl and n-propyl substituted derivatives 8i and 8j for SAR,

which were found to be less potent than 8h with IC₅₀= 4.80 1.1

μM and IC₅₀= 8.87 2.1 μM respectively. On the other side,

tert-butyl substituted derivative 8k exhibited comparable activity

(IC₅₀= 6.72 1.1 μM) without any toxic eﬀect to Vero cells

(CC₅₀value of 359.25 12.5 μM) and J-774A.1 cells (CC₅₀

400 μM) and has a signiﬁcant selectivity index (SI > 59.52).

However, polar methoxy group carrying benzyl functionality lost

the antiamastigote activity of compound 8o.

>

In Vitro Antileishmanial Activity of Quinazolinone−

Peptide. The encouraging results of the above series prompted

us to carry out further structural optimization with the

quinazolinone skeleton. Therefore, quinazolinone−peptides

(9a−i, Scheme 3) via Ugi multicomponent reaction, oﬀer

advantages and ﬂexibility to access the desired hybrid for further

SAR. The tert-butyl isocyanide and cyclohexyl isocyanide were

chosen as a model input for this reaction. Synthesized hybrids

that were found to be inactive against promastigote nevertheless

showed poor to excellent antiamastigote activity, which was

demonstrated in Table 3. Compounds 9a and 9c exhibited poor

antiamastigote activity (p-isopropylarylidine, IC₅₀12.03 1.4

μM; p-chloroarylidine, IC₅₀13.48 1.8 μM) but did not show

any toxicity. Compounds 9b and 9d, which contained the p-

methoxy and p-bromo arylidinesubstituents with tert-butyliso-

cyanide functionality, were inactive. Considering the importance

of ferrocene-containing heterocycles in organometallic synthesis

and in medicinal chemistry,⁵⁴we have introduced the ferrocene

functionality in the quinazolinone skeleton via Ugi multi-

(IC₅₀= 5.48

1.7 μM) and selectivity with the n-butyl

containing analogue 8i. Surprisingly, all the aminoalkyl

substituted analogues did not cause toxic eﬀects to Vero cells

(CC₅₀from 141.59

7.4 to >400 μM). Our SAR studies

indicated that aminoalkyl substituent is required for good

antileishmanial activity of the quinazolinone−triazine hybrid.

Introduction of a benzyl group into the quinazolinone−triazine

generated a compound (8n) with good antiamastigote activity

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Table 4. In Vitro Antileishmanial Activity of Quinazolinone−Tetrazole (10a−o)

a

b

c

IC₅₀values are the average of two independent assays expressed as average standard error. CC₅₀on J-774A.1 cells. The selectivity index (SI) is

b

a

d

e

deﬁned as the ratio of CC₅₀on J-774A.1 cells ( ) to IC₅₀on L. donovani intracellular amastigotes ( ). ND: not determined. NA: not available. NT:

not tested SSG: sodium stibogluconate.

f

component reaction (9e; tert-butyl as R₁) showed moderate

activity (IC₅₀= 14.18 1.0 μM). To evaluate the eﬀect of

cyclohexyl over tert-butyl, we synthesized the quinazolinone−

peptide 9f. To our surprise, hybrid 9f showed an excellent

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antileishmanial activity with IC₅₀value of 0.73 0.2 μM, which is

12-fold more potent than miltefosine. However, both the

ferrocene analogues (9e and 9f) were found to be nontoxic to

both J-774A.1 and Vero cells (CC₅₀> 400 μM). Compound 9f

showed the best selectivity, as indicated by the high value of

selective index (SI > 547.93, Table 3) and was found to be the

most promising in vitro among all the synthesized analogues.

SAR was achieved through the combination of a quinazolinone

with a metal (Fe) containing fragment; such a hybrid can

translate into an enhanced activity of 9f with high selectivity

compared to the other analogues. Introducing the isopropyl in

the p-position of aryl part and tert-butyl as the R₁group

to be inactive. Interestingly, compounds 10a, 10b, 10e, 10k,

10m, and 10n was nontoxic to J-774A.1 and Vero cells (CC₅₀

>

400 μM) with good selectivity (SI ranging from >30.65 to 56.65).

On the other side, another series of quinazolinone−tetrazole

hybrids (11a−e) was found to be inactive against the amastigote

stages of the parasite. However, compounds 11a−d demon-

strated moderate antipromastigote activity with IC₅₀value in the

range of 1.27 0.6 to 5.59 2.05 μM.

The compounds that combine with high leishmanicidal

activity and low or no cytotoxicity were 5b, 5c, 5f, 5g, 8a, 8b,

8g−k, 8n, 9a, 9c, 9f, 9g, 10a, 10b, 10e, 10f, 10k, 10m, and 10n.

Moreover, these analogues exhibited high selectivity as indicated

by the high value of selective index (SI ranging from 22.41 to

>547.94), which was several-fold better than miltefosine (SI =

1.48) and SSG (SI > 8.57).

The higher potency of quinazolinone analogues against the

amastigote stage of the parasite may be due to the activation of

murine macrophages to produce microbicidal molecules and

represent a new lead in the development of antileishmanial drugs.

Calculated physicochemical properties of the most potent

antileishmanial compounds were done (Table 5) using

(compound 9a) exhibited the moderate activity with IC₅₀

=

12.03 1.4 μM. While the replacement of the tert-butyl with

cyclohexyl as the R₁group with the same aromatic ring, i.e., p-

isopropyl arylidene (compound 9g; IC₅₀= 4.74 0.9 μM), led to

3-fold enhancement of the leishmanicidal activity as compared to

9a, it displayed no toxicity toward J-774A.1 and Vero cells (CC₅₀

> 400 μM). Interestingly, compounds 9f and 9g were more

potent than the natural product (Almiramide A−C). On the

contrary, p-methoxy and p-chloro arylidene as the R₂and

cyclohexyl as the R₁group substituted analogues 9h and 9i did

not show any antileishmanial activity.

In Vitro Antileishmanial Activity of Quinazolinone−

Tetrazole. To ﬁnd out the role of the eﬀect of Ugi products in

the form of tetrazole scaﬀolds, we have synthesized the

quinazolinone−tetrazole hybrid (10a−o, 11a−e, Schemes 3

and 4) and screened for their in vitro antileishmanial activity

(Table 4). Compounds 10a, 10e, 10f, 10h, 10i, and 10k

exhibited signiﬁcant antipromastigote activity with IC₅₀value in

the range of 1.21 0.2 to 7.00 2.5 μM, whereas compounds

10a, 10e, 10f, and 10k exhibited good antiamastigote activity

against the pathogen L. donovanii with IC₅₀values in the range of

7.06 1.4 to 9.07 1.2 μM (Table 4). The quinazolinone−

tetrazole hybrid 10a, having p-isopropyl substituent on the aryl

ring, displayed good antileishmanial activity (IC₅₀= 7.76 2.1

μM), while compound 10k with cyclohexyl instead of tert-butyl

displayed weak activity (IC₅₀= 9.07 1.2 μM). Interestingly,

both the derivatives (10a and 10k) were nontoxic to J-774A.1

and Vero cells (CC₅₀> 400 μM). Compounds 10e and 10f,

which contain halo substituents such as chloro and bromo on aryl

ring, displayed comparable activity (IC₅₀= 7.06 1.4 μM and

Table 5. Calculated Physical Properties of Best Active

Antileishmanial Compounds

entry acceptable

range

MW (g/mol)

<500

log

no. Lipinski

violations

P < 5 nrotb

5f

427.8

498.6

594.7

502.6

542.6

715.0

649.2

509.2

407.56

4.49

6.23

7.19

7.57

6.49

5.77

8.53

6.03

−0.21

3

5

0

1

2

0

8a

8g

5

8h

13

9

8n

9f

9

9g

8

10a

7

miltefosine

20

moleinspiration to assess if they were compliant with the

Lipinski “rule of ﬁve” criteria (Table 5); the data showed that all

molecules have (a) molecular weight in the range of 427−715 g/

mol, which is lower and higher than the accepted 500 g/mol, and

(b) log P close or higher to 5.0, which may indicate that the

compounds are too much lipophilic. The compounds 5f and 8a

did not show violation of the above criteria (Table 5), which

exhibited the good in vitro activity (Table 1 and 2). Although the

other most active compounds 8g, 8h, 8n, 9f, 9g, and 10a did not

meet the compliance to the Lipinski rule of ﬁve. Therefore,

obtained results of this informative analysis do not counsel that

any of the ligands should be discarded from this computational

assessment and they should be taken in account for further lead

optimization.

Production of Host Protective Cytokines in Murine

Macrophages Induced by Compounds 8a and 8g.

Leishmania prevents the activation of an eﬀective immune

response by repression of a number of pro-inﬂammatory

cytokines that included IL-1, IL-12, and TNF-α. For the eﬀective

clearance of parasite burden, it is necessary to augment host

immune responses by the production of these protective

cytokines. Th1 type cytokines, IL-12, and TNF-α play a critical

role in the repression of immunosuppressive cytokines IL-10 and

TGF-β. Our results indicate that quinazolinone−triazine

analogues, 8a and 8g, induce the activation of IL-12 and TNF-

α in treated murine macrophage cells and mediate eﬀective

IC₅₀= 8.46

1.7 μM, respectively) without any toxicity.

Modiﬁcation of R₁from tert-butyl to cyclohexyl in the analogues

10m and 10n, having p-chloro and p-bromo arylidine

substituents, exhibited less potency with IC₅₀value of 11.33

1.2 μM and 9.42 0.8 μM, respectively, than the compounds

10e and 10f. However, p-ﬂuoro aryl substituted analogue 10d

was inactive. tert-Butyl as the R₁group and a moderate electron

releasing group such as mono methoxyarylidene containing

analogue 10b showed poor leishmanicidal activity (IC₅₀= 13.05

1.6 μM), whereas trimethoxy arylidene substituted analogue

10c displayed no inhibition. These results indicated that tert-

butyl as R₁functionality containing hybrids have a pivotal role in

antileishmanial activity than the cyclohexyl carrying one.

Introduction of heterocyclic moiety like pyridine as a part of

R₂in the compound 10g resulted in a complete loss of activity.

For the sterical demand of the aryl portion, we introduced the

naphthyl and 4-methoxynaphthyl as the R₂and tert-butyl as the

R₁group (compounds 10h and 10i), did not show

antiamastigote activity, which is likely to be due to its steric

properties. To our surprise, compounds 10j and 10o bearing the

ferrocene fragment as a part of R₂and tert-butyl as R₁were found

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Figure 7. Compounds 8a and 8g mediated antileishmanial activity via up-regulation of Th1 cytokines (IL-12 and TNF-α) and down-regulation of Th2

cytokines (IL-10 and TGF-β) in infected murine macrophage (J774-A.1) cell line. The levels of cytokine release (pg/mL) in the culture supernatant

were evaluated by sandwich ELISA after 24 h post treatment. Results were expressed as mean SD from two independent experiments. The asterisks

indicate statistically signiﬁcant increase and decrease (*, p < 0.05; **, p < 0.01; ***, p < 0.001) in cytokine production compared between infected

macrophages versus 8a treated and infected versus 8g treated macrophages. Miltefosine was used as a reference drug.

suppression of parasite multiplication by abrogation of L.

donovani induced anti-inﬂammatory cytokines, IL-10 and TGF-

β. A comparative cytokine proﬁle of compound 8g treated

leishmania-infected cells showed >8- and 11-fold (p < 0.01)

increase in IL-12 and TNF-α production, respectively. On the

other hand, 8g treated cells showed ∼5- (p < 0.01) and >3-fold (p

< 0.05) decrease in IL-10 and TGF-β production, respectively, in

comparison with untreated infected cells. Moreover, 8a treated

cells also showed 6- and >9-fold (p < 0.01) increase in IL-12 and

TNF-α and >3- (p < 0.01) and 2.8-fold (p < 0.05) decrease in IL-

10 and TGF-β production, respectively (Figure 7).

Induction of Nitric Oxide Generation in Murine

Macrophages in Vitro by Compounds 8a and 8g. Along

Figure 8. Nitric oxide production (μM) by infected murine macrophage

J-774 cells in response to 8a, 8g, and miltefosine at IC₅₀dose. The

absorbance of the reaction product was measured at 540 nm using

Griess reagent. The asterisks indicate statistically signiﬁcant induction

(*, p < 0.05; **, p < 0.01; ***, p < 0.001) of NO production compared

between infected versus 8a treated and infected versus 8g treated J774

cells. Miltefosine was used as a reference drug, and lipopolysaccharide

(LPS 10 μg/mL) was used as a mitogen.

with pro-inﬂammatory cytokines, production of deadly micro-

bicidal agent nitric oxide (NO) by macrophages play a crucial

role in intracellular parasite killing by oxidative stress. IL-10 and

TGF-β inhibit the expression of inducible nitric oxide synthase

(iNOS), responsible for NO generation. Compounds 8a and 8g

treated leishmania-infected macrophages stimulated murine

macrophages and produced signiﬁcant amount of nitrite, 15.72

and 22.58 μM, respectively, which was >3- (p < 0.01) and ∼5-

fold (p < 0.05) higher than infected untreated cells (4.65 μM)

(Figure 8). Thus, signiﬁcant generation of nitrite by compounds

8a and 8g treated cells promotes elimination of L. donovani

parasites (Figure 8).

In Vivo Activity against L. donovani/Hamster Model.

Because of the highly promising results obtained for this novel

family of quinazolinone hybrids (5e, 8a, 8b, 8g, 8h, 8n, 9f, 9i, and

10a) in our in vitro studies, they appeared as excellent candidates

for further drug development; to that end, in vivo tests were

performed in the golden hamster model. The aqueous

suspensions of tested compounds were administered for ﬁve

consecutive days at 50 mg/kg/day by IP route. The post-

treatment splenic biopsies were done on day 7 of the last dose

administration, and amastigote counts were assessed by Giemsa

staining (Table 5). Compound 5e showed poor percentage

inhibition of 35.83 10.6. Compounds 8a and 8g showed the

best in vivo eﬃcacy among the all tested analogues, on average

73.15 12.69 and 80.93 10.50% inhibitions, respectively, in

parasite multiplication. In contrast, compounds 8h and 8n have

shown moderate inhibition of 43.01

10.30 and 44.83

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Figure 9. Fluorescence analysis of BSA−compound interaction. (a) Intrinsic ﬂuorescence emission spectra of BSA (1.0 × 10⁻⁵M) excited at 280 nm in

the presence of various concentrations (A−L represent 0, 1, 2, 3, 4, 9, 14, 19, 24, 29, 34, and 38 μM compound, respectively) of compound 8a, and M

represents emission spectra of 10 μM 8a alone excited at 280 nm. (b) Intrinsic ﬂuorescence of BSA in the presence of various concentrations of

compound 8g (A−K represents 0, 1, 6, 11, 16, 21, 26, 31, 36, 47, and 57 μM compound respectively), and L represents emission ﬂuorescence of 10 μM

8g alone). (c) Intrinsic ﬂuorescence spectra of BSA in the presence of various concentrations of compound 9f (A−K represents 0, 1, 6, 11, 16, 21, 26, 31,

36, 41, and 46 μM of 9f, respectively), and L represents emission spectra of 10 μM 9f alone excited at 280 nm. (d) Stern−Volmer plots for quenching of

BSA by compounds. ( e) Modiﬁed Stern−Volmer plots for quenching of BSA by compounds . (f) Plots of log [(F_o− F)/F] versus log [Q].

a

Table 7. Binding Parameters and Site of Compound−BSA Interaction at pH 7.4

Entry

K_sv(L mol⁻¹

)

K_q(L mol⁻¹s⁻¹

)

R^a

K_a(L mol⁻¹

)

R^b

N

R^c

8a

8g

9f

10.0 × 10⁴

6.0 × 10⁴

7.0 × 10⁴

10 × 10¹²

6.0 × 10¹²

7.0 × 10¹²

0.9983

0.9970

0.9962

7.9 × 10⁴

5.8 × 10⁴

4.6 × 10⁴

0.9988

0.9992

0.9998

1.08

1.05

1.2

0.9980

0.9946

a

R^a, R^b, and R^care correlation coeﬃcients for Stern−Volmer plots, modiﬁed Stern−Volmer plots, and plots of log analysis of ﬂuorescence,

respectively.

12.26%, respectively. Quinazolinone−peptide (9f) displayed

better in vivo inhibition of 51.42 15.67% than 9i (48.05

13.60), whereas quinazolinone−tetrazole hybrid 10a exhibited

above moderate inhibition of 54.01 10.65%.

where F_oand F are the ﬂuorescence intensities before and after

the addition of the compound or quencher, respectively, K_SVis

the dynamic quenching constant; K_qis the quenching rate

constant; [Q] is the concentration of compound added; τ₀is the

average lifetime of the molecule without quencher and its value is

considered to be 10⁻⁸s.⁵⁶

Stern−Volmer plots for compounds showed good linear

relationship with the increasing concentrations (Figure 9d). The

results showed that higher values of K_q(Table 7) than the

maximum value of the scattering collision quenching constant

(2.0 × 10¹⁰L mol⁻¹s⁻¹) for biomolecules,⁵⁷indicating the

probable quenching mechanism of BSA−compound interactions

was initiated by complex formation.

To conﬁrm that quenching of BSA ﬂuorescence by

compounds is due to static complex formation, the diﬀerence

absorption spectroscopy was employed. The UV−vis absorption

spectrum of BSA and the diﬀerence absorption spectrum

between BSA−compound complex and compound alone at the

same concentration could not be superposed (Figure 10),

demonstrating that BSA formed ground state complex with

compounds. BSA absorption spectra showed two peaks and

change in peak near 210 nm is due to change in conformation in

Intrinsic Fluorescence Quenching. To ﬁnd out the

binding parameters, mechanism of quenching and mode of

interaction of most active compounds 8a, 8g, and 9f with BSA,

ﬂuorescence spectroscopy, and UV−vis absorption spectroscopy

were performed. Fluorescence quenching is useful in ﬁnding the

binding behavior of small molecules with proteins. BSA showed

characteristic emission peak at 343 nm upon excitation at 280

nm. Fluorescence intensity was decreased gradually with

increasing concentration of compounds as shown in Figure

9a−c. All three compounds showed ﬂuorescence quenching and

shift in ﬂuorescence (345 to 359 nm with 8a, 345 to 336 nm with

8g, and 345 to 356 nm with 9f) of BSA. All the compounds

showed strong protein binding, and extent of protein binding was

similar for all compounds studied. The decrease in ﬂuorescence

intensity is usually described by the Stern−Volmer equation:⁵⁵

F /F = 1 + K_SV[Q] = 1 + K_qτ₀[Q]

o

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Binding Constant and Site. For static quenching of BSA

ﬂuorescence data were analyzed by Modiﬁed Stern−Volmer⁵⁹

equation:

Table 6. In Vivo Evaluation of Compounds against L.

donovani/Hamster Model

entry

% inhibition SD (dose − 50 mg/kg × 5 days, IP route)

F /ΔF = {1/(f K_a[Q])} + 1/f

5f

35.83 10.66

73.15 12.69

37.43 08.59

80.93 10.50

43.01 10.30

44.83 12.26

51.42 15.67

48.05 13.60

54.01 10.65

98.1 1.2

o

a

8a

8b

8g

8h

8n

9f

ΔF is the diﬀerence of ﬂuorescence in the absence and presence

of compounds at concentration [Q], f_ais the fraction of accessible

ﬂuorescence, and K_ais the eﬀective quenching constant for the

accessible ﬂuorophores, which is similar to the binding constant

for the quencher−acceptor systems. The dependence of F_o/ΔF

on the reciprocal value of concentration [Q]⁻¹is linear, with the

slope equaling to the value of (f_aK_a)⁻¹. The modiﬁed Stern−

Volmer plots were demonstrated in Figure 9e.

9g

10a

a

Miltefosine

b

In static quenching, the equilibrium between free and bound

SSG

86.89 5.45

molecules can be given by the equation:⁶⁰

a

b

Miltefosine (30 mg/kg × 5 days, oral). SSG, sodium stibogluconate

(40 mg/kg × 5 days, IP) used as reference drugs; SD, standard

deviation; IP, intraperitoneal.

log(F − F)/F = log K_A+ nlog[Q]

o

K_Aand n are binding constant and number of binding sites,

respectively.

The double logarithm plot has been shown in Figure 9f, and

the calculated binding sites (n) are shown in Table 7. The results

showed that the values of the binding sites n were approximately

1, indicating the formation of ground state complex is due to

single high aﬃnity binding site on protein.

Pharmacokinetics Studies. The pharmacokinetic studies

revealed that the animals tolerated the treatment as no

peculiarities in the animals’ behavior were observed. Calculations

of the pharmacokinetic data were based on the mean serum

concentrations and were performed by use of the compartmental

and noncompartmental approaches using WinNonlin (version

5.1) software. In a comparative pharmacokinetic study, both of

the compounds were rapidly absorbed with C_maxbetween 1 and 2

after oral dose (Figure 11). A one-compartment open model best

described serum concentration−time data of 8a, with elimination

from central compartment and a large volume of distribution

(3.22 L/kg). Oral serum concentrations were signiﬁcantly lower

than those observed by the intravenous administration as the

extent of absolute bioavailability of 8a following oral

administration was 5.75% (Table 8). The serum concentration

of 8a after intravenous administration decreased biexponentially

with the terminal half-life (t_1/2β) of 0.28 h, but its elimination

from the central compartment was higher (t_1/2k10, 0.84 h).

Figure 10. UV−vis absorption spectra of 1.0 × 10⁻⁵M BSA in the

presence of compounds at the 1:1 concentration ratio.

peptide backbone associated with helix coil transformation. The

change in microenvironment of tryptophan and tyrosine residues

is shown by change in 280 nm peak.⁵⁸Absorption spectra of BSA

(Figure 10) showed that ground state complex formation with

compounds is associated with alteration in the conformation of

BSA.

Figure 11. Concentration−time proﬁle of compounds 8a and 8g after oral and intravenous administration in male Sprague−Dawley rats. Bar represents

SEM.

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a

Table 8. Pharmacokinetic Parameters of Compounds (8a and 8g) after Oral and Intravenous Dose in Male Sprague−Dawley Rats

8a

8g

parameters

oral

intravenous

oral

intravenous

C

_max(ng/mL)

1

2

1

2

107.83 1.39

1829.48 172.93

44.72 1.87

63276.38 3152.55

81.66 4.22

t

_max(h)

2

0.08

1

0.08

6

AUC_0−t(ng h/mL)

_1/2(h)

MRT (h)

_ss(L/kg)

327

2843

0.84

3.07

5.44

1.77

716

24341

12.79

5.88

t

1.26

3.01

5.39

1.79

5.75

8.44

V

1.95

clearance (L/h/kg)

bioavailability (%)

0.36

2.94

0.33

a

Values of C_maxare mean SEM, and the value represents the average of three rats post dose. AUC_0−t: area under the serum concentration−time

curve up to last sampling time. C_max: serum peak concentration. MRT: mean residence time. t_max: time to C_max. t : elimination half-life. V_ss: volume

1/2

of distribution at steady-state.

Following oral administration, 8g gave two C_maxand, therefore,

model-independent pharmacokinetic parameters were calcu-

lated. The AUC_0−tand MRT were 2.2- and 2.8-fold higher and

clearance was 5-fold lower than those of 8a after a single oral dose

of 10 mg/kg in rats (Table 8). However, a three-compartmental

open model ﬁtted the raw data of 8g better after intravenous

administration than a two-compartmental model based on the

observed ﬁt, the residual sum of squared error, the correlation,

and the Akaike’s Information Criterion.⁶¹Using an open three-

compartment model, the terminal half-life (t_1/2γ) of 8g was 12.79

h. The AUC_0−twas 4-fold higher whereas the serum clearance

was 5-fold lower than that of 8a, although 8a showed

comparatively higher bioavailability but the extent of absorption

(AUC_0−t), indicating systemic availability and elimination half-

life were lower than that of 8g. However, the systemic clearances

of both the compounds were smaller than the hepatic blood ﬂow

of the rat (2.9 L/h/kg),⁶²suggesting an insigniﬁcant amount of

extrahepatic elimination.

EXPERIMENTAL SECTION

■

General. All reagents were commercial and were used without

further puriﬁcation. Chromatography was carried on silica gel (60−120

and 100−200 mesh). All reactions were monitored by thin-layer

chromatography (TLC), and silica gel plates with ﬂuorescence F254

were used. Melting points were taken in open capillaries on a Complab

melting point apparatus and are presented uncorrected. Infrared spectra

were recorded on a Perkin-Elmer AC-1 spectrometer. ¹H NMR and ¹³

C

NMR spectra were recorded using a BrukerSupercon Magnet DRX-300

1

spectrometer (operating at 300 MHz for H and 50 and 75 MHz for

¹³C) using CDCl₃as solvent and tetramethylsilane (TMS) as internal

standard. Chemical shifts are reported in parts per million. Multiplicities

are reported as follows: singlet (s), doublet (d), triplet (t), multiplet

(m), and broad singlet (brs). Some signals for NH proton are not

present in the few ¹H NMR data. Electrospray ionization mass spectra

(ESIMS) were recorded on Thermo Lcq Advantage Max-IT. High

resolution mass spectra (HRMS) were recorded on 6520 Agilent Q

TOF LC MS/MS (accurate mass). Purity of ﬁnal compounds was

determined by analytical HPLC, which was carried out on a Waters

HPLC system (model pump, 515; detector, PDA-2998). HPLC analysis

conditions: Waters Sunﬁre C18 (5.0 μM), 4.6 mm × 250 mm column,

ﬂow rate 0.5 mL/min, UV detection at 254 nm. All biologically evaluated

compounds are >95% pure.

Representative Procedure for the Synthesis of 2-(4-(3-Oxo-3-

phenylprop-1-enyl)phenyl)-2,3-dihydroquinazolin-4(1H)-one

(4a). To a stirred solution of 3 (500 mg, 1.98 mmol) and acetophenone

(0.23 mL, 1.92 mmol) in ethanol (5 mL) was added KOH as a catalyst.

The whole reaction mixture was stirred for 5 h at rt. The obtained

precipitate was ﬁltered and washed with water and recrystallized with

CONCLUSION

■

Four novel series of quinazolinone hybrids bearing interesting

bioactive scaﬀolds (pyrimidine, triazine, tetrazole, and peptide)

were synthesized. A number of such compounds exhibited better

potency against the intracellular amastigote of L. donovani than

the standard drugs and were not found to be cytotoxic. The SAR

analysis revealed that among the synthesized quinazolinone

hybrids, quinazolinone−pyrimidine, triazine, and ferrocene-

containing quinazolinone−peptide displayed potent antileish-

manial activity. Compounds 8a, 8g, and 9f showed very

consistent and promising leishmanicidal activity against intra-

cellular amastigotes and in vivo eﬃcacy in the golden hamster

model and displayed no toxicity for macrophages and Vero cells.

Furthermore, our data suggest that interaction of most potent

compounds 8a, 8g, and 9f with blood proteins showed static and

dynamic quenching of intrinsic ﬂuorescence. The data obtained

for albumin interaction and pharmacokinetic studies provide

information about mode of transport in blood, absorption,

distribution, and bioavailability of quinazolinone derivatives.

Therefore, these analogues are good candidates for a lead

optimization for identifying new analogues to curb the

unnecessary loss of life globally.

1

ethanol to obtain 4a as a cream solid (550 mg, yield 78%). H NMR

(300 MHz, DMSO-d₆) δ 8.43 (brs, 1H), 8.21 (d, J = 7.26 Hz, 2H), 7.97

(d, J = 7.26 Hz, 3H), 7.76 (s, 1H), 7.73−7.61 (m, 1H), 7.65 (t, J = 7.77

Hz, 4H), 7.33−7.28 (m, 1H), 7.25 (s, 1H), 7.18 (s, 1H), 6.83 (d, J = 8.07

Hz, 1H), 6.76 (t, J = 7.53 Hz, 1H) 5.86 (s, 1H). IR (KBr): 3436, 2943,

1634, 1425, 1216 cm⁻¹. ESI-MS C₂₃H₁₈N₂O₂(m/z): 355.0 (M⁺+ H).

Synthesis of Other Quinazolinone−Chalcone Hybrid 4b−i.

The above procedure was followed for 4b−i.

2-(4-(3-(3,4,5-Trimethoxyphenyl)acryloyl)phenyl)-2,3-dihydroqui-

nazolin-4(1H)-one (4b). Light-yellow solids (yield 72%). H NMR

1

(300 MHz, DMSO-d₆) δ 8.38 (brs, 1H), 7.87 (d, J = 7.87 Hz, 2H), 7.69

(s, 1H), 7.69−7.51 (m, 4H), 7.27−7.19 (m, 2H), 7.09 (d, J = 8.38 Hz,

1H), 6.70 (d, J = 8.12 Hz, 1H), 6.65 (t, J = 7.39 Hz, 1H), 5.80 (s, 1H),

3.87(s, 6H), 3.71 (s, 3H). IR (KBr): 3439, 2926, 1652, 1427, 1218, 780

cm⁻¹. ESI-MS C₂₆H₂₄N₂O₅(m/z): 445 (M⁺+ H).

2-(4-(2,3-Dimethoxystyryl)phenyl)-2,3-dihydroquinazolin-4(1H)-

one (4c). Light-yellow solids (yield 75%). ¹H NMR (300 MHz, DMSO-

d₆) δ 8.41 (brs, 1H), 7.92 (d, J = 7.92 Hz, 2H), 7.72 (s, 1H), 7.63−7.54

(m, 5H), 7.32−7.22 (m, 2H), 7.12 (d, J = 8.40 Hz, 1H), 6.78 (d, J = 8.04

Hz, 1H), 6.71 (t, J = 7.41 Hz, 1H), 5.80 (s, 1H), 3.87(s, 6H). IR (KBr):

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3424, 2928, 1648, 1418, 1219, 785 cm⁻¹. ESI-MS C₂₄H₂₂N₂O₃(m/z):

387 (M⁺+ H).

8.51 (brs, 1H), 7.49 (d, J = 7.62 Hz, 1H), 7.37 (brs, 1H), 7.20−7.10 (m,

3H), 6.72 (d, J = 8.07 Hz, 1H), 6.58 (t, J = 7.35 Hz, 1H), 6.44 (d, J = 8.04

Hz, 2H), 4.95 (brs, 2H). IR (KBr): 3423, 3314, 2932, 1624, 1410, 1224

cm⁻¹. ESI-MS C₁₅H₁₅N₃O (m/z): 254.0 (M⁺+ H).

2-(4-(4-Methoxystyryl)phenyl)-2,3-dihydroquinazolin-4(1H)-one

(4d). Cream solid (yield 72%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.34

(brs, 1H), 8.17 (d, J = 8.64 Hz, 2H), 7.96−7.87 (m, 3H), 7.72−7.54 (m,

4H), 7.28 (t, J = 7.80 Hz, 1H), 7.17 (s, 1H), 7.10 (d, J = 8.64 Hz, 2H),

6.78 (d, J = 8.13 Hz, 1H), 6.71 (t, J = 7.35 Hz, 1H), 5.80(s, 1H), 3.87(s,

3H). IR (KBr): 3426, 2923, 1620, 1416, 1218, 770 cm⁻¹. ESI-MS

C₂₃H₂₀N₂O₂(m/z): 357 (M⁺+ H).

2-(4-(4-Fluorostyryl)phenyl)-2,3-dihydroquinazolin-4(1H)-one

(4e). Cream solid (yield 72%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.07

(brs, 1H), 7.99 (s, 1H), 7.89 (s, 2H), 7.66−7.56 (m, 8H), 7.16 (s, 2H),

6.75−6.63 (m, 2H), 5.76(s, 1H). IR (KBr): 3435, 2927, 1634, 1423,

1218, 772 cm⁻¹. ESI-MS C₂₂H₁₇FN₂O (m/z): 345 (M⁺+ H).

2-(4-(4-Chlorostyryl)phenyl)-2,3-dihydroquinazolin-4(1H)-one

(4f). Light-yellow solid (yield 70%). ¹H NMR (300 MHz, DMSO-d₆) δ

8.35 (brs, 1H), 8.18 (d, J = 8.37 Hz, 2H), 7.92−7.89 (m, 3H), 7.77 (s,

1H), 7.65 (t, J = 8.10 Hz, 3H), 7.58 (d, J = 7.89 Hz, 2H), 7.28 (t, J = 7.05

Hz, 1H), 7.18 (s, 1H), 6.78 (d, J = 7.95 Hz, 1H), 6.71 (d, J = 7.41 Hz,

1H), 5.81(s, 1H). IR (KBr): 3436, 2943, 1634, 1425, 1216, 779 cm⁻¹.

ESI-MS C₂₂H₁₇ClN₂O (m/z): 361 (M⁺+ H).

Representative Procedure for the Synthesis of 2-(4-(4,6-

Di(piperidin-1-yl)-1,3,5-triazin-2-ylamino)phenyl)-2-methyl-

2,3-dihydroquinazolin-4(1H)-one (8a). The solution of compound

7 (500 mg, 1.98 mmol) in dry THF was added dropwise to an ice-cold

mixture of cyanuric chloride (401 mg, 2.17 mmol) in dry THF. The

reaction mixture was stirred at room temperature for 1 h, and then the

solvent was removed in vacuo and formed crude product was mixed with

K₂CO₃(550 mg, 3.95 mmol) and piperidine (0.39 mL, 4.58 mmol) in

dry DMF. The whole reaction mixture was heated at 70 °C until

completion of reaction. On completion of the reaction (checked by TLC

analysis), the reaction mixture was poured into ice−water, and obtained

precipitate was ﬁltered. The crude product was subjected to silica gel

column chromatography using chloroform/methanol as mobile phase

(9:1) to aﬀord the desired compound 8a as white solid (700 mg, yield

71%); mp 160−162 °C. HPLC-DAD: t_r= 9.6 min (% area = 97.3%). ¹H

NMR (300 MHz, CDCl₃+ DMSO-d₆) δ 7.72 (brs, 1H), 7.56−7.50 (m,

2H), 7.35 (s, 2H), 7.10 (s, 1H), 6.66 (d, J = 5.4 Hz, 1H), 6.53 (s, 2H),

3.63 (brs, 8H), 1.69 (s, 3H), 1.55−1.46 (m, 12H). ¹³C NMR (50 MHz,

CDCl₃+ DMSO-d₆) δ 169.51, 152.35, 145.02, 144.74, 138.26, 132.37,

130.43, 123.58, 121.83, 120.05, 119.47, 75.16, 48.84, 35.62, 35.76, 30.60,

29.65. IR (KBr): 3423, 2934, 2849, 1639, 1574, 1494, 1443, 1370, 1237,

1026 cm⁻¹. ESI-MS C₂₈H₃₄N₈O (m/z): 499 (M⁺+ H). HRMS: calcd,

499.2928 (MH⁺); found, 499.2929 (MH⁺).

2-(4-(4-Bromostyryl) phenyl)-2, 3-dihydroquinazolin-4(1H)-one

1

(4g). Cream solid (yield 69%). H NMR (300 MHz, DMSO-d₆) δ

8.36 (brs, 1H), 8.16 (d, J = 8.07 Hz, 2H), 7.94−7.83 (m, 2H), 7.76 (s,

1H), 7.68 (t, J = 8.10 Hz, 3H), 7.59 (d, J = 7.90 Hz, 2H), 7.25 (t, J = 7.15

Hz, 1H), 7.17 (s, 1H), 6.74 (d, J = 7.92 Hz, 1H), 6.70 (d, J = 7.31 Hz,

1H) 5.83 (s, 1H). IR (KBr): 3436, 2926, 1649, 1411, 1219, 680 cm⁻¹.

ESI-MS C₂₂H₁₇BrN₂O (m/z): 405 (M⁺+ H).

Representative Procedure for the Synthesis of N-(2-(tert-

Butylamino)-1-(4 isopropylphenyl)-2-oxoethyl)-3-chloro-N-(4-

(2-methyl-4-oxo-1,2,3,4 tetrahydroquinazolin-2 yl)phenyl)-

benzamide (9a). To a stirred solution of 7 (300 mg, 1.18 mmol) in

methanol, m-chlorobenzoic acid (218 mg, 1.18 mmol), isopropyl

benzaldehyde (0.17 mL, 1.37 mmol), and tert-butyl isocyanide (0.13

mL, 1.61 mmol) was successively added, and the reaction mixture was

stirred at rt for 6 h. After completion of reaction (checked by TLC

analysis), the methanol was removed and the residue was puriﬁed by

silica gel column chromatography (eluting with 2% methanol in

chloroform) to aﬀord the target product 9a as white solid (450 mg, yield

61%); mp162−164 °C. HPLC-DAD: t_r= 7.6 min (% area = 97.3%). ¹H

NMR (300 MHz, DMSO-d₆) δ 8.53 (brs, 1H), 7.69 (d, J = 4.1, 1H), 7.42

(d, J = 7.5 Hz, 1H), 7.36 (s, 1H), 7.18−7.13 (m, 2H), 7.09−7.03 (m,

4H), 6.93 (t, J = 7.7 Hz, 5H), 6.67−6.64 (m, 1H), 6.58 (t, J = 6.9 Hz,

1H), 6.09 (s, 1H), 2.73−2.64 (m, 1H), 1.42 (s, 3H), 1.18 (s, 9H), 1.08

(d, J = 5.2 Hz, 6H). IR (KBr): 3418, 3325, 2967, 1654, 1563, 1490, 1377,

1218, 767 cm⁻¹. ESI-MS C₃₇H₃₉ClN₄O₃(m/z): 623.0 (M⁺+ H).

HRMS: calcd, 623.2783 (MH⁺); found, 623.2783 (MH⁺); [M + H]⁺.

Representative Procedure for the Synthesis of 2-(4-((1-tert-

Butyl-1H-tetrazol-5-yl)(4-isopropylphenyl)methylamino)-

phenyl)-2-methyl-2,3-dihydroquinazolin-4(1H)-one (10a). Sol-

ution of 7 (300 mg, 1.18 mmol), isopropylbenzaldehyde (1.0 equiv, 1.65

mmol), and tert-butyl isocyanide (0.13 mL, 1.61 mmol) was stirred in

anhydrous methanol (5 mL) at rt for 10 min. Thereafter, trimethylsilyl

azide (0.23 mL, 2.03 mmol) was added and the resulting mixture was

further stirred for 7 h. On completion of the reaction (checked by TLC

analysis), the methanol was removed in vacuo and the crude product was

puriﬁed by chromatography on silica gel (eluting with 2% methanol in

chloroform) to aﬀord the target product 10a as white solid (390 mg,

yield 71%); mp 145−147 °C. HPLC-DAD: t_r= 8.6 min (% area =

97.9%). ¹H NMR (300 MHz, CDCl₃) δ 7.88 (d, J = 7.29 Hz, 1H), 7.35−

722 (m, 8H), 6.83 (t, J = 7.17 Hz, 1H), 6.66−6.60 (m, 2H), 6.31 (d, J =

7.29 Hz, 1H), 6.09 (s, 1H), 4.56 (bs, 1H), 2.91−2.87 (m, 1H), 1.82 (s,

3H), 1.68 (s, 9H), 1.24 (d, J = 6.81, 6H). ¹³C NMR (50 MHz, CDCl₃) δ

162.4, 155.2, 149.56, 145.84, 135.10, 133.95, 128.34, 127.76, 126.77,

118.69, 114.72, 113.59, 70.72, 61.80, 54.06, 33.74, 30.07, 29.68, 23.82.

IR (KBr): 3425, 3321, 2935, 1613, 1517, 1380, 1219, 772 cm⁻¹. ESI-MS

C₃₀H₃₅N₇O (m/z): 510.0 (M⁺+ H). HRMS: calcd, 510.2976 (MH⁺);

found, 510.2976 (MH⁺).

2-(4-(3-(2-Chlorophenyl)acryloyl)phenyl)-2,3-dihydroquinazolin-

4(1H)-one (4h). Yellow solid (yield 70%). ¹H NMR (300 MHz, DMSO-

d₆) δ 8.42 (brs, 1H), 7.85 (d, J = 8.19 Hz, 2H), 7.67−7.57 (m, 7H), 7.43

(s, 1H), 7.36 (s, 1H), 7.31−7.27 (m, 1H), 7.24 (s, 1H), 6.82 (d, J = 8.04

Hz, 1H), 6.75 (t, J = 7.53 Hz, 1H), 5.84 (s, 1H). IR (KBr): 3436, 2943,

1634, 1425, 1216, 776 cm⁻¹. ESI-MS C₂₂H₁₇ClN₂O (m/z): 361 (M⁺+

H).

2-(4-(4-(4-Methylpiperazin-1-yl)styryl)phenyl)-2,3-dihydroquina-

1

zolin-4(1H)-one (4i). Yellow solid (yield 70%). H NMR (300 MHz,

DMSO-d₆) δ 8.34 (brs, 1H), 8.05 (d, J = 8.64 Hz, 2H), 7.93 (s, 1H), 7.88

(d, J = 7.2 Hz, 2H), 7.71−7.61 (m, 2H), 7.56 (d, J = 8.04 Hz, 2H), 7.28

(t, J = 7.62 Hz, 1H), 7.16 (s, 1H), 7.03 (d, J = 8.67 Hz, 2H), 6.78 (d, J =

7.95 Hz, 1H), 6.71 (t, J = 7.47 Hz, 1H), 5.79 (s, 1H), 3.33 (brs, 4H), 2.44

(brs, 4H), 2.23 (s, 3H). IR (KBr): 3406, 2932, 1650, 1417, 1220, 770

cm⁻¹. ESI-MS C₂₇H₂₈N₄O (m/z): 425 (M⁺+ H).

Representative Procedure for the Synthesis of 2-(4-(2-

Amino-6-phenylpyrimidin-4-yl)phenyl)-2,3-dihydroquinazo-

lin-4(1H)-one (5a). To a stirred solution of NaH (108 mg, 4.5 mmol)

and guanidine hydrochloride (214 mg, 2.25 mmol) in DMF (3 mL) was

added gradually 4a (400 mg, 1.12 mmol), and the reaction mixture was

stirred for 10 min at rt and then heated at 60 °C for 5 h. After the

completion of reaction, the reaction mixture was poured into water and

the obtained precipitate was ﬁltered and puriﬁed by column

chromatography (eluting with 2% methanol in chloroform) to aﬀord

the quinazolinone-pyrimidine 5a as light-yellow solid (310 mg, yield

70%); mp 175−177 °C. HPLC-DAD: t_r= 6.5 min (% area = 97.2%). ¹H

NMR (300 MHz, DMSO-d₆) δ 8.38 (brs, 1H), 8.25−8.23 (m, 4H),

7.72−7.63 (m, 4H), 7.52 (s, 3H), 7.29 (t, J = 7.02 Hz, 1H), 7.20 (s, 1H),

6.79−6.76 (m, 3H), 6.72 (t, J = 7.47 Hz, 1H), 5.85(s, 1H). ¹³C NMR (75

MHz, DMSO-d₆) δ 165.39, 164.85, 164.47, 164.05, 148.28, 144.25,

137.96, 133.83, 130.94, 129.08, 127.84, 117.71, 115.48, 114.94, 102.37,

66.72. IR (KBr): 3411, 3019, 1654, 1569, 1451, 1218, 1063 cm⁻¹. ESI-

MS C₂₄H₁₉N₅O (m/z): 394 (M⁺+ H). HRMS: calcd, 394.1662 (MH⁺);

found, 394.1666 (MH⁺).

Representative Procedure for the Synthesis of 2-(4-Amino-

phenyl)-2-methyl-2,3-dihydroquinazolin-4(1H)-one (7). To a

stirred solution of anthralinamide (1.0 g, 7.39 mmol) and 4-

aminoacetophenone (1.0 g, 7.35 mmol) in methanol (10 mL) were

added acidic silica as a catalyst. The reaction mixture was heated at 60 °C

for 3h, and the conversion was monitored by TLC. The reaction mixture

was evaporated and formed residue was mixed with water and

chloroform and puriﬁed by crystallization with acetonitrile to aﬀord 7

as a cream solid (1.6 g, yield 85%). ¹H NMR (300 MHz, DMSO-d₆) δ

Representative Procedure for the Synthesis of 2-(4-

((Benzylamino)(1-tert-Butyl-1H-tetrazol-5-yl)methyl)phenyl)-

2,3-dihydroquinazolin-4(1H)-one (11a). A solution of compound 3

(300 mg, 1.19 mmol), benzylamine (0.13 mL equiv, 1.19 mmol), and

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tert-butyl isocyanide (0.13 mL, 1.61 mmol) was stirred in anhydrous

methanol (5 mL) at rt for 10 min. Thereafter, trimethylsilyl azide (0.23

mL, 2.03 mmol) was added and the resulting mixture was further stirred

for 6 h. On completion of the reaction (checked by TLC analysis), the

methanol was removed in vacuo. The crude product was subjected to

silica gel column chromatography using chloroform/methanol as

mobile phase to aﬀord the desired compound 11a as white solid (410

mg, yield 74%); mp 120−122 °C. HPLC-DAD: t_r= 8.3 min (% area =

98.1%). ¹H NMR (300 MHz, CDCl₃) δ 7.85 (d, J = 6.78 Hz, 1H), 7.50

(d, J = 6.51 Hz, 2H), 7.29−7.19 (m, 8H), 6.84 (t, J = 7.44 Hz, 1H),

6.61(d, J = 7.98 Hz, 1H), 5.88 (s, 1H), 5.81 (s, 1H), 5.21 (s, 1H), 4.38 (s,

1H), 3.78−3.73 (m, 1H), 3.64−3.60 (m, 1H), 1.45 (s, 9H). ¹³C NMR

(50 MHz, CDCl₃) δ 164.86, 155.22, 147.22, 140.37, 139.26, 138.65,

134.05, 128.74, 127.90, 127.4, 119.39, 115.45, 114.74, 68.23, 61.46,

56.64, 51.31, 29.92. IR (KBr): 3426, 3319, 2925, 1642, 1525, 1432, 1272,

771 cm⁻¹. ESI-MS C₂₇H₂₉N₇O (m/z): 468.0 (M⁺+ H). HRMS: calcd,

468.2506 (MH⁺); found, 468.2518 (MH⁺).

Experimental Section for Biology. Promastigote Viability Assay.

The in vitro eﬀects of the compounds on the growth of extracellular

promastigotes were assessed as described previously.^42aThe late log

phase of L. donovani promastigotes (MHOM/IN/60/Dd₈; originally

obtained from Imperial college, London) transfected with the ﬁreﬂy

luciferase gene^62,63was seeded with complete M-199 medium at 5 ×

10⁴/100 μL/well in 96-well plates and incubated with tested compounds

for 96 h. Miltefosine and SSG were used as reference controls. After

incubation, an aliquot (50 μL) of promastigote suspension was aspirated

from each well of a 96-well plate and mixed with an equal volume of

Steady Glo reagent (Promega) and luminescence was measured by a

luminometer. The values were expressed as relative luminescence unit

(RLU). The inhibition of parasitic growth is determined by comparison

of the luciferase activity of compound treated parasites with that of

untreated controls.

In Vitro Antiamastigote Assay. For assessing the activity of

compounds against the amastigote stage of the parasite, murine

macrophage cell line (J-774A.1) infected with promastigotes expressing

luciferase ﬁreﬂy reporter gene was used. Cells were seeded in a 96-well

plate (4 × 10⁴/100 μL/well) in RPMI-1640 containing 10% fetal calf

serum and the plates were incubated at 37 °C in a CO₂incubator. After

24 h, the medium was replaced with fresh medium containing stationary

phase promastigotes (4 × 10⁵/100 μL/well) at the ratio of 1:10

(cell:promastigote). Promastigotes were phagocytized by the macro-

phages, and inside the phagolysosomes, they were transformed into

amastigotes (nonmotile form). Each well of the plate was washed with

plain RPMI medium after 24 h of incubation to remove the

uninternalized promastigotes. The test compounds were added at 2-

fold dilutions up to 7 points in complete medium starting from 40 μM

concentration, and the plates were incubated at 37 °C in a CO₂

incubator for 72 h. After incubation, the drug containing medium was

aspirated and 50 μL of PBS was added in each well and mixed with an

equal volume of Steady Glo reagent. After gentle shaking for 1−2 min,

the reading was taken in a luminometer.⁵¹The values are expressed as

relative luminescence units (RLU). Data were transformed into a

graphic program (Excel). IC₅₀of antileishmanial activity was calculated

by nonlinear regression analysis of the concentration response curve

using the four parameter Hill equations.

In Vivo Assay in L. donovani/Hamster Model. The in vivo

antileishmanial activity was determined in golden hamsters (Mesocricetus

auratus) infected with MHOM/IN/80/Dd₈strain of L. donovani. The

method as described by Gupta et al.⁵⁴was used for in vivo evaluation.

Golden hamsters (inbred strain) of either sex weighing 40−45 g were

infected intracardiacally with 1 × 10⁷amastigotes per animal. After

establishment of infection in 15−20 days, pretreatment spleen biopsy in

all the animals was carried out to assess the degree of infection. The

animals with +1 grade infection (5−10 amastigotes/100 spleen cell

nuclei) were included in the chemotherapeutic trials. Five to six infected

animals were randomized into several groups used for each test sample.

Drug treatment (50 mg/kg) by intraperitoneal (IP) route was initiated

after 2 days pretreatment of biopsy and continued for 5 consecutive

days. Miltefosine and SSG were used as reference drugs. Post-treatment

biopsies were done on day 7 of the last dose administration, and

amastigote counts were assessed by Giemsa staining. Intensity of

infection in both treated and untreated animals, and also the initial count

in treated animals, was compared and the eﬃcacy was expressed in terms

of percentage inhibition (PI) using the following formula:

PI = 100 − [(ANAT × 100)/(INAT × TIUC)]

Where PI is the percent inhibition of amastigotes multiplication, ANAT

is the actual number of amastigotes in treated animals, INAT is the initial

number of amastigotes in treated animals, and TIUC is time increase of

parasites in untreated control animals.

Cytokines Assessment by ELISA. The level of various cytokines in

the murine macrophages were measured using an OptEIA set ELISA kit

(BD Biosciences, California, USA) according to manufacturer’s

instructions. Brieﬂy, 1 × 10⁶murine macrophages (J-774A.1) infected

with Leishmania amastigote (1:10 ratio) were incubated with tested

compounds at IC₅₀concentration in 6-well plates. Supernatants were

collected after 24 h for cytokines (IL-12, TNF-α, IL-10, and TGF-β)

determination. The absorbance of the test samples was measured at 450

nm in an ELISA reader.

Nitric Oxide Production Assay. NO was quantiﬁed by the

accumulation of nitrite in macrophage culture supernatant, and nitrite

was detected by the Griess reaction as described by Kar et al.⁶⁵Brieﬂy,

leishmania infected macrophage cells (1 × 10⁶/mL) were incubated

with tested compounds for 24 h before nitrite assay. LPS (10 μg/mL)

was used as a mitogen. Supernatant (500 μL) were collected after 24 h,

mixed with an equal volume of Griess reagent (Sigma, USA) and left for

10 min at room temperature. The absorbance of the test samples was

measured at 540 nm in an ELISA reader.

Statistical Analysis. Results were expressed as mean SD from two

independent experiments. The results were analyzed by one-way

ANOVA followed by student’s t test using GraphPad Prism (version

5.0) software.

ASSOCIATED CONTENT

* Supporting Information

■

S

Final compounds characterization data, pharmacokinetics study,

and material and methods of BSA interaction associated with this

article. This material is available free of charge via the Internet at

http://pubs.acs.org.

Cytotoxicity Assay. The cell viability was determined using the MTT

assay.⁵²As described previously, macrophage cells (J-774A.1 cell line)

and mammalian kidney ﬁbroblast cells, (Vero cell line) (1 × 10⁵cells/

100 μL/well) were incubated with test compounds at seven

concentrations starting from 400 μM. After 72 h of incubation, 25 μL

of MTT reagent (5 mg/mL) in PBS medium was added to each well and

incubated at 37 °C for 2 h. At the end of the incubation period, the

supernatant were removed and 150 μL of pure DMSO was added to

each well. After 15 min of shaking, the readings were recorded as

absorbance at 544 nm on a microplate reader. The 50% cytotoxic

concentration (CC₅₀) values were estimated as described by Huber &

Koella.⁶⁴The selectivity index (SI) for each compound was calculated as

the ratio between cytotoxicity (CC₅₀) and activity (IC₅₀) against

Leishmania amastigotes.

AUTHOR INFORMATION

Corresponding Author

*Phone: 91 522 2612411. Fax: +91 522 2623405. E-mail: prem_

chauhan_2000@yahoo.com; premsc58@hotmail.com.

Notes

■

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We are grateful to Indian Council of Medical Research (M.S.)

and Council of Scientiﬁc and Industrial Research (K.C.) for the

ﬁnancial support in the form of fellowship. We are also thankful

to R. K. Purshottam and Tofan Kumar Rout for providing the

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Journal of Medicinal Chemistry

Article

(12) Congreve, M.; Chessari, G.; Tisi, D.; Woodhead, A. J. Recent

developments in fragment-based drug discovery. J. Med. Chem. 2008, 51,

3661−3680.

HPLC data and the SAIF Division of CDRI, Lucknow, for

providing the spectroscopic data. CDRI communication no.

8453.

(13) Newman, D. J. Natural Products as Leads to Potential Drugs: An

Old Process or the New Hope for Drug Discovery? J. Med. Chem. 2008,

51, 2589−2599.

ABBREVIATIONS USED

■

(14) (a) Breinbauer, R.; Vetter, I. R.; Waldmann, H. From protein

domains to drug candidatesnatural products as guiding principles in

the design and synthesis of compound libraries. Angew. Chem., Int. Ed.

2002, 41, 2878−2890. (b) Basu, S.; Ellinger, B.; Rizzo, S.; Deraeve, C.;

BIOS, biology oriented synthesis; BSA, bovine serum albumin;

IC₅₀, half maximal inhibitory concentration; IMCRs, isocyanides

based multicomponent reactions; iNOS, inducible nitric oxide

synthase; IP, intraperitoneally; PK, pharmacokinetics; SAR,

structure−activity relationship; SSG, sodium stibogluconate; VL,

visceral leishmaniasis

Schurmann, M.; Preut, H.; Arndt, H.-D.; Waldmann, H. Biology-

̈

oriented synthesis of a natural-product inspired oxepane collection

yields a small molecule activator of the Wnt-pathway. Proc. Natl. Acad.

Sci. U. S. A. 2011, 108, 6805−6810.

(15) Tietze, L. F.; Bell, H. P.; Chandrasekhar, S. Natural Product

Hybrids as New Leads for Drug Discovery. Angew. Chem., Int. Ed. 2003,

42, 3996−4028.

REFERENCES

■

(1) Akopyants, N. S.; Kimblin, N.; Secundino, N.; Patrick, R.; Peters,

N.; Lawyer, P.; Dobson, D. E.; Beverley, S. M.; Sacks, D. L.

Demonstration of Genetic Exchange During Cyclical Development of

Leishmania in the Sand Fly Vector. Science 2009, 324, 265−268.

(2) WHO. Working to Overcome the Global Impact of Neglected Tropical

Diseases; Stylys: Geneva, 2010. (b) Majumder, H. K. Drug Targets in

Kinetoplastid Parasites; Springer Science+Business Media: New York,

2011.

(16) Wilson, R. M.; Danishefsky, S. J. Small molecule natural products

in the discovery of therapeutic agents: the synthesis connection. J. Org.

Chem. 2006, 71, 8329−8351.

(17) Mhaske, S. B.; Argade, N. P. The chemistry of recently isolated

naturally occurring quinazolinone alkaloids. Tetrahedron 2006, 62,

9787−9826.

(18) Michael, J. P. Quinoline, quinazoline and acridone alkaloids. Nat.

Prod. Rep. 2003, 20, 476−493.

(3) Pimenta, P. F.; Turco, S. J.; McConville, M. J.; Lawyer, P. G.;

Perkins, P. V.; Sacks, D. L. Stage-specific adhesion of Leishmania

promastigotes to the sandfly midgut. Science 1992, 256, 1812−1815.

(4) (a) Chappuis, F.; Sundar, S.; Hailu, A.; Ghalib, H.; Alvar, J.;

Boelaer, M. Visceral leishmaniasis: what are the needs for diagnosis,

treatment and control? Nature Rev. Microbiol. 2007, 5, 873−885.

(b) Alvar, J.; Canavate, C.; Gutierrez- Solar, B.; Jimenez, M.; Lagnon, F.;

Lopez-Velet, R.; Molina, R.; Moreno, J. Leishmania and human

immunodeficiency virus coinfection: the first 10 years. Clin. Microbiol.

Rev. 1997, 10, 298−319.

(5) (a) Matte, C.; Maion, G.; Mourad, W.; Olivier, M. Leishmania

donovani-induced macrophages cyclooxygenase-2 and prostaglandin E₂

synthesis. Parasite Immunol. 2001, 23, 177−184. (b) Bogdon, C.;

Gessner, A.; Solbach, W.; Rollinghoff, M. Invasion, control and

persistence of Leishmania parasites. Curr. Opin. Immunol. 1996, 8,

517−525.

(6) (a) Sundar, S.; Rai, M. Advances in the treatment of leishmaniasis.

Curr. Opin. Infect. Dis. 2002, 15, 593−598. (b) Pink, R.; Hudson, A.;

Mouries, M. A.; Bendig, M. Opportunities and challenges in antiparasitic

̀

drug discovery. Nature Rev. Drug Discovery 2005, 4, 727−740.

(7) (a) Berman, J. ABLE: A New and Improved Amphotericin B for

Visceral Leishmaniasis? Am. J. Trop. Med. Hyg. 2009, 8, 689−690.

(b) Guerin, P. J.; Olliaro, P.; Sundar, S.; Boelaert, M.; Croft, S. L.;

Desjeux, P.; Wasunna, K.; Bryceson, A. D. M. Visceral leishmaniasis:

current status of control, diagnosis, and treatment, and a proposed

research and development agenda. Lancet Infect. Dis. 2002, 2, 494−501.

(8) (a) Poola, N. R.; Kalis, M.; Plakogiannis, F. M.; Taft, D. R.

Characterization of pentamidine excretion in the isolated perfused rat

kidney. J. Antimicrob. Chemother. 2003, 52, 397−404. (b) Dorlo, T. P.

C.; Van Thiel, P. A. M.; Huitema, A. D. R.; Keizer, R. J.; De Vries, H. J.

C.; Beijnen, J. H.; De Vries, P. J. Pharmacokinetics of Miltefosine in Old

World Cutaneous Leishmaniasis Patients. Antimicrob. Agents Chemother.

2008, 52, 2855−2860. (c) Sindermann, H.; Engel, J. Development of

miltefosine as an oral treatment for leishmaniasis. Trans. R. Soc. Trop.

Med. Hyg. 2006, 100 (Suppl 1), S17−S20.

(9) (a) Croft, S. L.; Sundar, S.; Fairlam, A. H. Drug Resistance in

Leishmaniasis. Clin. Microbiol. Rev. 2006, 111−126. (b) Sundar, S. Drug

resistance in Indian visceral leishmaniasis. Trop. Med. Int. Health 2001, 6,

849−854.

(10) Cavalli, A.; Bolognesi, M. L. Neglected tropical diseases:

multitarget-directed ligands in the search for novel lead candidates

against Trypanosomia and Leishmania. J. Med. Chem. 2009, 52, 7339−

7359.

(19) (a) Bandekar, P. P.; Roopnarine, K. A.; Parekh, V. J.; Mitchell, T.

R.; Novak, M. J.; Sinden, R. R. Antimicrobial Activity of Tryptanthrins in

Escherichia coli. J. Med. Chem. 2010, 53, 3558−3565. (b) Bhattacharjee,

A. K.; Skanchy, D. J.; Jennings, B.; Hudson, T. H.; Brendle, J. J.;

Werbovetz, K. A. Analysis of stereoelectronic properties, mechanism of

action and pharmacophore of synthetic indolo[2,1-b]quinazoline-6,12-

dione derivatives in relation to antileishmanial activity using quantum

chemical, cyclic voltammetry and 3-D-QSAR CATALYST procedures.

Bioorg. Med. Chem. 2002, 10, 1979−1989.

(20) Chiou, W.; Liao, J.; Chen, C. Comparative Study on the

Vasodilatory Effects of Three Quinazoline Alkaloids Isolated from

Evodia rutaecarpa. J. Nat. Prod. 1996, 59, 374−378.

(21) Liang, J. L.; Cha, H. C.; Jahng, Y. Recent Advances in the Studies

on Luotonins. Molecules 2011, 16, 4861−4883.

(22) (a) Chinigo, G. M.; Paige, M.; Grindrod, S.; Hamel, E.;

Dakshanamurthy, S.; Chruszcz, M.; Minor, W.; Brown, M. L. Brown

Asymmetric Synthesis of 2,3-Dihydro-2-arylquinazolin-4-ones: Meth-

odology. Application to a Potent Fluorescent Tubulin Inhibitor with

Anticancer Activity. J. Med. Chem. 2008, 51, 4620−4631. (b) El-Azab, A.

S.; ElTahir, K. E. H. Design and synthesis of novel 7-aminoquinazoline

derivatives: antitumor and anticonvulsant activities. Bioorg. Med. Chem.

Lett. 2012, 22, 1879−1885. (c) Saravanan, G.; Alagarsamy, V.; Prakash,

C. R. Design, synthesis and anticonvulsant activities of novel 1-

(substituted/unsubstituted benzylidene)-4-(4-(6,8-dibromo-2-(meth-

yl/phenyl)-4-oxoquinazolin-3(4H)-yl)phenyl) semicarbazide deriva-

tives. Bioorg. Med. Chem. Lett. 2012, 22, 3072−3078.

(23) Wang, Z.; Wang, M.; Yao, X.; Li, Y.; Tan, J.; Wang, L.; Qiao, W.;

Geng, Y.; Liu, Y.; Wang, Q. Design, synthesis and antiviral activity of

novel quinazolinones. Eur. J. Med. Chem. 2012, 53, 275−282.

(24) (a) Abbas, S. E.; Awadallah, F. M.; Ibrahin, N. A.; Said, E. G.;

Kamel, G. M. New quinazolinone−pyrimidine hybrids: synthesis, anti-

inflammatory, and ulcerogenicity studies. Eur. J. Med. Chem. 2012, 53,

141−149. (b) Amin, K. M.; Kamel, M. M.; Anwar, M. M.; Khedr, M.;

Syam, Y. M. Synthesis, biological evaluation and molecular docking of

novel series of spiro[(2H,3H)quinazoline-2,10-cyclohexan]-4(1H)- one

derivatives as anti-inflammatory and analgesic agents. Eur. J. Med. Chem.

2010, 45, 2117−2131.

(25) (a) Al-Omary, F. A. M.; Abou-zeid, L. A.; Nagi, M. N.; Habib, E.

E.; Abdel-Aziz, A. M.; El-Azab, A. S.; Abdel-Hamide, S. G.; Al-Omar, M.

A.; Al-Obaid, A. M.; El-Subbagh, H. I. Non-classical antifolates. Part 2:

Synthesis, biological evaluation, and molecular modeling study of some

new 2,6-substituted-quinazolin-4-ones. Bioorg. Med. Chem. Lett. 2010,

18, 2849−2863. (b) Suresha, G. P.; Suhas, R.; Kapfo, W.; Gowda, D. C.

Urea/thiourea derivatives of quinazolinone lysine conjugates: synthesis

(11) Meunier, B. Hybrid molecules with a dual mode of action: dream

or reality? Acc. Chem. Res. 2008, 41, 69−77.

4390

dx.doi.org/10.1021/jm400053v | J. Med. Chem. 2013, 56, 4374−4392

Journal of Medicinal Chemistry

Article

and structure−activity relationships of a new series of antimicrobials.

Eur. J. Med. Chem. 2011, 46, 2530−2540.

(26) Mohameda, M. S.; Kamel, M. M.; Kassem, E. M. M.; Abotaleb, N.;

AbdEl-moez, S. I.; Ahmeda, M. F. Novel 6,8-dibromo-4(3H)-

quinazolinone derivatives of anti-bacterial and anti-fungal activities.

Eur. J. Med. Chem. 2010, 45, 3311−3319.

(38) (a) Torrence, P. F.; Fan, X.; Zhanga, X.; Loiseau, P. M.

Structurally diverse 5-substituted pyrimidine nucleosides as inhibitors of

Leishmania donovani promastigotes in vitro. Bioorg. Med. Chem. Lett.

2006, 16, 5047−5051. (b) Musonda, C. C.; Whitlock, G. A.; Witty, M. J.;

Brun, R.; Kaiser, M. Synthesis and evaluation of 2-pyridyl pyrimidines

with in vitro antiplasmodial and antileishmanial activity. Bioorg. Med.

Chem. Lett. 2009, 19, 401−405. (c) Determann, R.; Dreher, J.;

Baumann, K.; Preu, L.; Jones, P. G.; Totzke, F.; Schachtele, C.;

Kubbutat, M. H. G.; Kunick, C. 2-Anilino-4-(benzimidazol-2-yl)-

pyrimidines A multikinase inhibitor scaffold with antiproliferative

activity toward cancer cell lines. Eur. J. Med. Chem. 2012, 53, 254−263.

(39) (a) Smith, A. L.; D’Angelo, N. D.; Bo, Y. Y.; Booker, S. K.; Cee, V.

J.; Herberich, B.; Hong, F.; Jackson, C. L. M.; Lanman, B. A.; Liu, L.;

Nishimura, N.; Pettus, L. H.; Reed, A. B.; Tadesse, S.; Tamayo, N. A.;

Wurz, R. P.; Yang, K.; Andrews, K. L.; Whittington, D. A.; McCarter, J.

D.; Miguel, T. S.; Zalameda, L.; Jiang, J.; Subramanian, R.; Mullady, E.

L.; Caenepeel, S.; Freeman, D. J.; Wang, L.; Zhang, N.; Wu, T.; Hughes,

P. E.; Norman, M. H. Structure-Based Design of a Novel Series of

Potent, Selective Inhibitors of the Class I Phosphatidylinositol 3-

Kinases. J. Med. Chem. 2012, 55, 5188−5219. (b) Menicagli, R.;

Samaritani, S.; Signore, G.; Vaglini, F.; Via, L. D. In Vitro Cytotoxic

Activities of 2-Alkyl-4,6-diheteroalkyl-1,3,5-triazines: New Molecules in

Anticancer Research. J. Med. Chem. 2004, 47, 4649−4652. (c) Grave-

stock, D.; Rousseau, A. L.; Lourens, A. C. U.; Moleele, S. S.; Zyl, R. L. V.;

Steenkamp, P. A. Expeditious synthesis and biological evaluation of

novel 2,N⁶-disubstituted 1,2-dihydro-1,3,5-triazine-4,6-diamines as

potential antimalarials. Eur. J. Med. Chem. 2011, 46, 2022−2030.

(d) Vidal-Mosquera, M.; Fernandez-Carvajal, A.; Moure, A.; Valente, P.;

Planells-Cases, R.; Gonzalez-Ros, J. M.; Bujons, J.; Ferrer-Montiel, A.;

Messeguer, A. Triazine-Based Vanilloid 1 Receptor Open Channel

Blockers: Design, Synthesis, Evaluation, and SAR Analysis. J. Med. Chem.

2011, 54, 7441−7452.

(40) (a) Okandeji, B. O.; Greenwald, D. M.; Wroten, J.; Sello, J. K.

Synthesis and evaluation of inhibitors of bacterial drug efflux pumps of

the major facilitator superfamily. Bioorg. Med. Chem. 2011, 19, 7679−

7689. (b) Noren-Muller, A.; Reis-Correa, I., Jr.; Prinz, H.; Rosenbaum,

C.; Saxena, K.; Schwalbe, H. J.; Vestweber, D.; Cagna, G.; Schunk, S.;

Schwarz, O.; Schiewe, H.; Waldmann, H. Discovery of protein

phosphatase inhibitor classes by biology-oriented synthesis. Proc. Natl.

Acad. Sci. U. S. A. 2006, 103, 10606−10611. (c) Sutherland, H. S.; Blaser,

A.; Kmentova, I.; Franzblau, S. G.; Wan, B.; Wang, Y.; Ma, Z.; Palmer, B.

D.; Denny, W. A.; Thompson, A. M. Synthesis and structure−activity

relationships of antitubercular 2-nitroimidazooxazines bearing hetero-

cyclic side chains. J. Med. Chem. 2010, 53, 855−866. (d) Semple, G.;

Skinner, P. J.; Gharbaoui, T.; Shin, Y.; Jung, J.; Cherrier, M. C.; Webb, P.

J.; Tamura, S. Y.; Boatman, P. D.; Sage, C. R.; Schrader, T. O.; Chen, R.;

Colletti, S. L.; Tata, J. R.; Waters, M.; Cheng, K.; Taggart, A. K.; Cai, T.;

Carballo-Jane, E.; Behan, D. P.; Connolly, D. T.; Richman, J. G. 3-(1H-

Tetrazol-5-yl)-1,4,5,6-tetrahydro-cyclopentapyrazole (MK-0354): A

Partial Agonist of the Nicotinic Acid Receptor, G-Protein Coupled

Receptor 109a, with Antilipolytic but No Vasodilatory Activity in Mice.

J. Med. Chem. 2008, 51, 5101−5108. (e) Biot, C.; Bauer, H.; Schirmer, R.

H.; Davioud-Charvet, E. 5-Substituted Tetrazoles as Bioisosteres of

Carboxylic Acids. Bioisosterism and Mechanistic Studies on Glutathione

Reductase Inhibitors as Antimalarials. J. Med. Chem. 2004, 47, 5972−

5983. (f) Fan, X.; Zhang, X.; Bories, C.; Loiseau, P. M.; Torrence, P. F.

The Ugi reaction in the generation of new nucleosides as potential

antiviral and antileishmanial agents. Bioorg. Chem. 2007, 35, 121−136.

(41) (a) Sunduru, N.; Agarwal, A.; Katiyar, S. B.; Shakya, N.; Goyal, N.;

Gupta, S.; Chauhan, P. M. S. Synthesis of 2,4,6 trisubstituted pyrimidine

and triazine heterocycles as antileishmanial agents. Bioorg. Med. Chem.

2006, 14, 7706−7715. (b) Kumar, A.; Katiyar, S. B.; Gupta, S.; Chauhan,

P. M. S. Syntheses of new substituted triazino tetrahydroisoquinolines

and β-carbolines as novel antileishmanial agents. Eur. J. Med. Chem.

2006, 41, 106−113. (c) Sunduru, N.; Palne, S.; Chauhan, P. M. S.;

Gupta, S. Synthesis and antileishmanial activity of novel 2,4,6-

trisubstituted pyrimidines 1,3,5-triazines. Eur. J. Med. Chem. 2009, 44,

2473−2481. (d) Gupta, L.; Sunduru, N.; Verma, A.; Srivastava, S.;

Gupta, S.; Goyal, N.; Chauhan, P. M. S. Synthesis and biological

(27) Gemma, S.; Camodeca, C.; Brindisi, M.; Brogi, S.; Kukreja, G.;

Kunjir, S.; Gabellieri, E.; Lucantoni, L.; Habluetzel, A.; Taramelli, D.;

Basilico, N.; Gualdani, R.; Tadini-Buoninsegni, F.; Bartolommei, G.;

Moncelli, M. R.; Martin, R. E.; Summers, R. L.; Lamponi, S.; Savini, L.;

Fiorini, I.; Valoti, M.; Novellino, E.; Campiani, G.; Butini, S. Mimicking

the Intramolecular Hydrogen Bond: Synthesis, Biological Evaluation,

and Molecular Modeling of Benzoxazines and Quinazolines as Potential

Antimalarial Agents. J. Med. Chem. 2012, 55, 10387−10404.

(28) Rudolph, J.; Esler, W. P.; O’Connor, S.; Coish, P. D. G.; Wickens,

P. L.; Brands, M.; Bierer, D. E.; Bloomquist, B. T.; Bondar, G.; Chen, L.;

Chuang, C.; Claus, T. H.; Fathi, Z.; Fu, W.; Khire, U. R.; Kristie, J. A.;

Liu, X.; Lowe, D. B.; McClure, A. C.; Michels, M.; Ortiz, A.; Ramsden, P.

D.; Schoenleber, R. W.; Shelekhin, T. E.; Vakalopoulos, A.; Wang, L.; Yi,

L.; Gardell, S. J.; Livingston, J. N.; Sweet, L. J.; Bullock, W.;

Quinazolinone, H. Derivatives as Orally Available Ghrelin Receptor

Antagonists for the Treatment of Diabetes and Obesity. J. Med. Chem.

2007, 50, 5202−5216.

(29) Rhee, H.; Yoo, J. H.; Lee, E.; Kwon, Y. J.; Seo, H.; Lee, Y.; Choo,

H. P. Synthesis and cytotoxicity of 2-phenylquinazolin-4(3H)-one

derivatives. Eur. J. Med. Chem. 2011, 46, 3900−3908.

(30) Ismail, M. A. H.; Barker, S.; Abou El Ella, D. A.; Abouzid, K. A. M.;

Toubar, R. A.; Todd, M. H. Design and Synthesis of New Tetrazolyl-

and Carboxy-biphenylylmethyl-quinazolin-4-one Derivatives as Angio-

tensin II AT1 Receptor Antagonists. J. Med. Chem. 2006, 49, 1526−

1535.

(31) Sharma, M.; Pandey, S.; Chauhan, K.; Sharma, D.; Kumar, B.;

Chauhan, P. M. S. Cyanuric chloride catalyzed mild protocol for

synthesis of biologically active dihydro/spiro quinazolinones and

quinazolinone-glycoconjugates. J. Org. Chem. 2012, 77, 929−937.

(32) Costa, E. V.; Pinheiro, M. L. B.; Xavier, C. M.; Silva, J. R. A.;

Amaral, A. F.; Souza, A. D. L.; Barison, A.; Campos, F. R.; Ferreira, A. G.;

Machado, G. M. C.; Leon, L. P. L. A Pyrimidine-β-carboline and Other

Alkaloids from Annona foetida with Antileishmanial Activity. J. Nat. Prod.

2006, 69, 292−294.

(33) Akue-Gedu, R.; Debiton, E.; Ferandin, Y.; Meijer, L.;

Prudhomme, M.; Anizon, F.; Moreau, P. Synthesis and biological

activities of aminopyrimidyl-indoles structurally related to meridianins.

Bioorg. Med. Chem. 2009, 17, 4420−4424.

(34) Sanchez, L. M.; Lopez, D.; Vesely, B. A.; Togna, G. D.; Gerwick,

W. H.; Kyle, D. E.; Linington, R. G.; Almiramides, A-C Discovery and

Development of a New Class of Leishmaniasis Lead Compounds. J. Med.

Chem. 2010, 53, 4187−4197.

(35) Balasegaram, M.; Young, H.; Chappuis, F.; Priotto, G.;

Raguenaud, M. E.; Checchi, F. Effectiveness of melarsoprol and

eflornithine as first-line regimens for gambiense sleeping sickness in nine

Med

́ ̀

ecins Sans Frontieres programmes. Trans R. Soc. Trop. Med. Hyg.

2009, 103, 280−290.

(36) (a) Muh, U.; Schuster, M.; Heim, R.; Singh, A.; Olson, E. R.;

Greenberg, E. P. Novel Pseudomonas aeruginosa Quorum-Sensing

Inhibitors Identified in an Ultra-High-Throughput Screen. Antimicrob.

Agents Chemother. 2006, 50, 3674−3679. (b) Vitale, C.; Mercuro, G.;

Castiglioni, C.; Cornoldi, A.; Tulli, A.; Fini, M.; Volterrani, M.; Rosano,

G. M. C. Metabolic effect of telmisartan and losartan in hypertensive

patients with metabolic syndrome. Cardiovasc. Diabetol. 2005, 4, 6−13.

(37) Sivaprakasam, P.; Tosso, P. N.; Doerksen, R. J. Structure−Activity

Relationship and Comparative Docking Studies for Cycloguanil Analogs

as Pf DHFR-TS Inhibitors. J. Chem. Inf. Model. 2009, 49, 1787−1796.

(b) Rastelli, G.; Sirawaraporn, W.; Sompornpisut, P.; Vilaivan, T.;

Kamchonwongpaisan, S.; Quarrell, R.; Lowe, G.; Thebtaranonth, Y.;

Yuthavong, Y. Interactions of pyrimethamine, cycloguanil and

WR99210 and their analogues with Plasmodium falciparum dihydrofo-

late reductase: structural basis for antifolate resistance. Bioorg. Med.

Chem. 2000, 8, 1117−1128.

4391

dx.doi.org/10.1021/jm400053v | J. Med. Chem. 2013, 56, 4374−4392

Journal of Medicinal Chemistry

Article

evaluation of new [1,2,4]triazino[5,6-b]indol-3-ylthio-1,3,5-triazines

and [1,2,4]triazino[5,6-b]indol-3-ylthio-pyrimidines against Leishmania

donovani. Eur. J. Med. Chem. 2010, 45, 2359−2365.

(53) Gupta, S.; Ramesh; Sharma, S. C.; Srivastava, V. M. L. Efficacy of

picroliv combination with miltefosine, an orally effective antileishmanial

drug against experimental visceral leishmaniasis. Acta Trop. 2005, 94,

41−47.

(42) (a) Porwal, S.; Chauhan, S. S.; Chauhan, P. M. S.; Shakya, N.;

Verma, A.; Gupta, S. Discovery of Novel Antileishmanial Agents in an

Attempt to Synthesize Pentamidine−Aplysinopsin Hybrid Molecule. J.

Med. Chem. 2009, 19, 5793−5802. (b) Sharma, M.; Chauhan, K.;

Chauhan, S. S.; Kumar, A.; Singh, S. V.; Saxena, J. K.; Agarwal, P.;

Srivastava, K.; Kumar, S. R.; Puri, S. K.; Shah, P.; Siddiqi, M. I.; Chauhan,

P. M. S. Synthesis of hybrid 4-anilinoquinoline triazines as potent

antimalarial agents, their in silico modeling and bioevaluation as

Plasmodium falciparum transketolase and β-hematin inhibitors. Med.

Chem. Commun. 2012, 3, 71−79. (c) Sharma, M.; Chaturvedi, V.;

Manju, Y. K.; Bhatnagar, S.; Srivastava, K.; Puri, S. K.; Chauhan, P. M. S.

Substituted quinolinyl chalcones and quinolinyl pyrimidines as a new

class of anti-infective agents. Eur. J. Med. Chem. 2009, 44, 2081−2091.

(d) Sunduru, N.; Sharma, M.; Srivastava, K.; Kumar, S. R.; Puri, S. K.;

Saxena, J. K.; Chauhan, P. M. S. Synthesis of oxalamide and triazine

derivatives as a novel class of hybrid 4-aminoquinoline with potent

antiplasmodial activity. Bioorg. Med. Chem. 2009, 17, 6451−6462.

(e) Katiyar, S. B.; Srivastava, K.; Puri, S. K.; Chauhan, P. M. S. Synthesis

of 2-[3,5-substituted pyrazol-1-yl]-4,6-trisubstituted triazine derivatives

as antimalarial agents. Bioorg. Med. Chem. Lett. 2005, 15, 4957−4960.

(f) Kumar, A.; Srivastava, K.; Kumar, S. R.; Puri, S. K.; Chauhan, P. M. S.

Synthesis and bioevaluation of hybrid 4-aminoquinoline triazines as a

new class of antimalarial agents. Bioorg. Med. Chem. Lett. 2008, 18,

6530−6533. (g) Tyagi, V.; Khan, S.; Shivahare, R.; Srivastava, K.; Gupta,

S.; Kidwai, S.; Srivastava, K.; Puri, S. K.; Chauhan, P. M. S. A natural

product inspired hybrid approach towards the synthesis of novel

pentamidine based scaffolds as potential anti-parasitic agents. Bioorg.

Med. Chem. Lett. 2013, 23, 291−296.

(54) (a) Talancon, D.; Bosque, R.; Lopez, C. Study of the Effect

Induced by the Substituents on the Ring−Chain Tautomerism of Schiff

Bases Derived from Norephedrine. J. Org. Chem. 2010, 75, 3294−3300.

(b) Dubar, F.; Anquetin, G.; Pradines, B.; Dive, D.; Khalife, J.; Biot, C.

Enhancement of the Antimalarial Activity of Ciprofloxacin Using a

Double Prodrug/Bioorganometallic Approach. J. Med. Chem. 2009, 52,

7954−7957. (c) Bellot, F.; Cosledan, F.; Vendier, L.; Brocard, J.;

Meunier, B.; Robert, A. Trioxaferroquines as New Hybrid Antimalarial

Drugs. J. Med. Chem. 2010, 53, 4103−4109.

(55) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.;

Springer: New York, 2006; pp 11−12.

(56) Lakowicz, J. R.; Weber, G. Quenching of fluorescence by oxygen, a

probe for structural fluctuations in macromolecules. Biochemistry 1973,

12, 4161−4170.

(57) Maurice, R. E.; Camillo, A. G. Fluorescence quenching studies

with proteins. Anal. Biochem. 1981, 114, 199−212.

(58) Jayabharathi, J.; Thanikachalam, V.; Sathishkumar, R.;

Jayamoorthy, K. Fluorescence investigation of the interaction of 2-(4-

fluorophenyl)-1-phenyl-1H-phenanthro[9,10-d]imidazole with bovine

serum albumin. J. Photochem. Photobiol., B: Biol. 2012, 117, 222−228.

(59) Lehrer, S. S. Solute perturbation of protein fluorescence. The

quenching of the tryptophyl fluorescence of model compounds and of

lysozyme by iodide ion. Biochemistry 1971, 10, 3254−3263.

(60) Gong, A.; Zhu, X.; Hu, Y.; Yu, S. A fluorescence spectroscopic

study of the interaction between epristeride and bovin serum albumine

and its analytical application. Talanta 2007, 73, 668−673.

(61) Yamaoka, K.; Nakagawa, T.; Uno, T. Application of a Akaike’s

information criterion (AIC) in the evaluation of linear pharmacokinetic

equations. J. Pharmacokinet. Biopharm. 1978, 6, 65−175.

(43) Dockal, M.; Carter, D. C.; Ruker, F. Five recombinant fragments

of human serum albumintools for the characterization of the warfarin

binding site. J. Biol. Chem. 2000, 275, 3042−3050.

(62) Davies, B.; Morris, T. Physiological parameters in laboratory

animals and humans. Pharm. Res. 1993, 10, 1093−1095.

(44) Tian, J. N.; Liu, J. Q.; Zhang, J. Y.; Hu, Z. D.; Chen, X. G.

Fluorescence Studies on the Interactions of Barbaloin with Bovine

Serum Albumin. Chem. Pharm. Bull. 2003, 51, 579−582.

(63) Gupta, A. S. R.; Sundar, S.; Goyal, N. Use of Leishmania donovani

Field Isolates Expressing the Luciferase Reporter Gene in in Vitro Drug

Screening. Antimicrob. Agents Chemother. 2005, 49, 3776−3783.

(64) (a) Huber, W.; Koella, J. C. A. comparison of three methods of

estimating EC₅₀in studies of drug resistance of malaria parasites. Acta

Trop. 1993, 55, 257−261.

(45) Ran, D. H.; Wu, X.; Zheng, J. H.; Yang, J. H.; Zhou, H. P.; Zhang,

M. F.; Tang, Y. J. Study on the interaction between florasulam and

bovine serum albumin. J. Fluoresc. 2007, 17, 721−726.

(46) Kun, R.; Kis, L.; Dekany, I. Hydrophobization of bovine serum

albumin with cationic surfactants with different hydrophobic chain

length. Colloids Surf., B 2010, 79, 61−68.

(65) Kar, S.; Sharma, G.; Das, P. K. Fucoidan cures infection with both

antimony-susceptible and -resistant strains of Leishmania donovani

through Th1 response and macrophage-derived oxidants. J. Antimicrob.

Chemother. 2011, 66, 618−625.

(47) Liu, B.; Guo, Y.; Wang, J.; Xu, R.; Wang, X.; Wang, D.; Zhang, L.

Q.; Xu, Y. N. Spectroscopic studies on the interaction and sonodynamic

damage of neutral red (NR) to bovine serum albumin (BSA). J. Lumin.

2010, 130, 1036−1043.

(48) Xiang, G. H.; Tong, C. L.; Lin, H. Z. Nitroaniline isomers

interaction with bovine serum albumin and toxicological implications. J.

Fluoresc. 2007, 17, 512−521.

(49) Wattanasin, S.; Murphy, W. S. An improved procedure for the

preparation of chalcones and related enones. Synthesis 1980, 8, 647−

650.

NOTE ADDED AFTER ASAP PUBLICATION

■

This paper was published on the Web on May 7, 2013, with an

error to the Abstract graphic. The corrected version was reposted

May 16, 2013.

(50) (a) Domling, A.; Wang, W.; Wang, K. Chemistry and Biology of

Multicomponent Reactions. Chem. Rev. 2012, 112, 3083−3135.

(b) Domling, A.; Beck, B.; Magnin-Lachaux, M. 1-Isocyanomethylben-

zotriazole and 2,2,4,4-tetramethylbutylisocyanide-cleavable isocyanides

useful for the preparation of α-aminomethyl tetrazoles. Tetrahedron Lett.

2006, 47, 4289−4291.

(51) Gupta, L.; Talwar, A.; Shakya, N.; Palne, S.; Gupta, S.; Chauhan,

P. M. S. Synthesis of marine alkaloid: 8,9-dihydrocoscinamide B and its

analogues as novel class of antileishmanial agents. Bioorg. Med. Chem.

Lett. 2007, 17, 4075−4079.

(52) (a) Mosmann, T. Rapid colorimetric assay for cellular growth and

survival: application to proliferation and cytotoxicity assays. J. Immunol.

Methods 1983, 65, 55−63. (b) Carmichael, J.; DeGraff, W. G.; Gazdar, A.

F.; Minna, J. D.; Mitchel, J. B. Evaluation of a tetrazolium-based

semiautomated colorimetrie assay: assessment of radiosensitivity.

Cancer Res. 1987, 47, 943−946.

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Discovery of a new class of natural product-inspired quinazolinone hybrid as potent antileishmanial agents

DOI: 10.1021/jm400053v

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