Novel series of 1,2,4-trioxane derivatives as antimalarial agents

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2017

VOL. 32, NO. 1, 1159–1173

https://doi.org/10.1080/14756366.2017.1363742

RESEARCH PAPER

Novel series of 1,2,4-trioxane derivatives as antimalarial agents

a

b

Mithun Rudrapal , Dipak Chetia and Vineeta Singh

a

b

Department of Pharmaceutical Sciences, Dibrugarh University, Dibrugarh, India; Parasite Bank, National Institute of Malaria Research (ICMR),

Sector 8, Dwarka, New Delhi, India

ABSTRACT

ARTICLE HISTORY

Among three series of 1,2,4-trioxane derivatives, five compounds showed good in vitro antimalarial activity,

three compounds of which exhibited better activity against P. falciparum resistant (RKL9) strain than the

sensitive (3D7) one. Two best compounds were one from aryl series and the other from heteroaryl series

with IC50 values of 1.24 mM and 1.24 mM and 1.06 mM and 1.17 mM, against sensitive and resistant strains,

respectively. Further, trioxane derivatives exhibited good binding affinity for the P. falciparum cysteine pro-

tease falcipain 2 receptor (PDB id: 3BPF) with well defined drug-like and pharmacokinetic properties based

on Lipinski’s rule of five with additional physicochemical and ADMET parameters. In view of having anti-

malarial potential, 1,2,4-trioxane derivative(s) reported herein may be useful as novel antimalarial lead(s) in

the discovery and development of future antimalarial drug candidates as P. falciparum falcipain 2 inhibitors

against resistant malaria.

Received 12 June 2017

Revised 21 July 2017

Accepted 1 August 2017

KEYWORDS

P. falciparum; resistance;

trioxane derivatives;

falcipain 2 inhibitors;

antimalarial

Introduction

resistance. In view of this, the development of new antimalarial

drugs with novel mode(s) of action could be an attractive strategy

to address the above challenging issue.

Malaria continues to be a major lethal infectious disease of human

beings, affecting around 200–300 million people and causing

approximately 430,000 deaths every year globally. The most

affected populations are children and infants as well as pregnant

Though ART and related endoperoxides are the mainstay of

current antimalarial therapy, high treatment cost (relative to quin-

oline-based drugs), unsatisfactory physicochemical/pharmacoki-

netic properties, toxicities and limited availability of drugs are

some other notable pitfalls besides having potential resistance

1

women . The disease is caused by five species of the parasite

Plasmodium, namely, Plasmodium falciparum, Plasmodium vivax,

Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi.

Of these, P. falciparum is the most prevalent and deadly species

that causes severe malaria such as cerebral malaria, and it is also

7

,8

problem . All above issues are adversely affecting clinical out-

comes of ART-based drug regimens used in the treatment of mal-

aria. The clinical significance of ART-based drugs is attributed to

be due to the excellent antiparasitic action of the endoperoxide

component. Pharmacodynamic study also suggests that the key

pharmacophoric 1,2,4-trioxane ring system is responsible for the

biological activity of ART (natural lead molecule), its semi-synthetic

2

–4

responsible for most of the malaria-related deaths in humans

.

Every year over 50% of all malarial infections are caused by P. fal-

ciparum with about 20–50% of total mortality cases commonly

occur in children younger than five years old and pregnant

5

women . During the past two decades, the emergence of drug

6

,9,10

analogues and related endoperoxides

research into several synthetic endoperoxide scaffolds such as

,2,4-trioxane, 1,2,4-trioxolane and 1,2,4,5-teraoxane derivatives

. In recent years,

resistant strains of P. falciparum has become an increasingly ser-

ious concern in malaria control and prevention worldwide.

Reduced clinical efficacy of currently available drugs, especially

those in the aminoquinoline group (chloroquine, amodiaquine,

mefloquine, and piperaquine) including non-quinolines like antifo-

lates (sulfadoxine, proguanil, pyrimethamine), lumefantrine, atova-

quone, and antibiotics, and the novel artemisinin (ART) derivatives

1

and their hybrid analogues have gained significant interest in dis-

covery and development of potent antimalarial drugs. Some endo-

2

peroxide-based antimalarial compounds that are currently under

clinical phase of development are represented in Figure 1.

Endoperoxide molecules bearing 1,2,4-trioxane core have been

(dihydroartemisinin, artemether, and artesunate), to multi-drug

resistant P. falciparum strains has limited their clinical usefulness in identified to exhibit a broad spectrum of biological effects such as

2

11

the treatment of resistant malaria . Artemisinin-based combination anticancer, antimicrobial, and anti-parasitic effects . Several

therapies (ACTs) such as artemether/lumefantrine/amodiaquine design strategies have been initiated by MMV (Medicines for

and artesunate/mefloquine/piperaquine recommended by WHO Malaria Venture) program for the development of effective and

for the treatment of multi-drug resistant P. falciparum malaria affordable synthetic peroxides as alternative therapy to existing

2

,12

have also been reported to develop resistance in South-East ART-based drugs . However, considering all above facts, devel-

2

,6

Asia . This increasing burden of resistant malaria has stimulated opment of peroxide-based synthetic compounds is expected to

drug discovery scientists to search for new antimalarial drugs or provide effective antimalarials agents with limited toxicity issues

alternative therapeutic options to combat the problem of drug and ready to be available at affordable cost. It would offer a better

CONTACT Mithun Rudrapal

rsmrpal@gmail.com

Department of Pharmaceutical Sciences, Dibrugarh University, Dibrugarh 786 004, Assam, India

Supplemental data for this article can be accessed here.

ß 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distri-

bution, and reproduction in any medium, provided the original work is properly cited.

1

160

M. RUDRAPAL ET AL.

H

O O

O

O O

O

NH

NH₂

H

O

N

S

O

OZ 277 (Arterolane)

OZ 439 (Artefenomel)

BAY 44 9585 (Artemisone)

O

N

O O

O

HN

N

H

O O

N

O O

N

Fe

O

Cl

F

SS R97193

TDD E209

RKA 182

Figure 1. Structures of some new endoperoxide antimalarials.

therapeutic option for the treatment of malaria with good clinical properties) of molecules to exhibit good drug-likeness behaviour

1

4

outcomes (desired potency with optimal pharmacokinetics) and in human body . In fact, molecular properties are the fundamen-

resistant preventing action. It may therefore represent a new class tal structural parameters which determine the physicochemical

of synthetic, new-generation and orally active peroxides, with opti- (solubility and permeability) and biochemical (metabolic stability,

mal antimalarial potency against resistant P. falciparum malaria.

transport property, and protein/tissue affinity) properties, which

As a part of our research program towards developing potent ultimately determine molecule’s in vivo pharmacokinetics (bioavail-

antimalarial drugs that would be active against resistant P. falcip- ability and half life), toxicity, and pharmacodynamics (receptor

1

5

arum malaria, novel molecular manipulation approach was affinity and efficacy) . ADMET (absorption, distribution, metabol-

ism, excretion, and toxicity) properties have a predictable influence

on pharmacokinetic and pharmacodynamic effects of drug mole-

cules. The calculation of ADMET properties is therefore intended

as the first step towards optimising the new drug molecules,

whether to check the failure of lead molecules which may cause

toxicity and one unable to cross the intestinal membranes or

adopted to design and develop newer series of 1,2,4-trioxane

derivatives as single drug conjugates (single molecular entity

rather than hybrid drugs) based on the basic structural scaffold of

1

,2,4-trioxane ring system. After the synthesis of compounds, in

vitro antimalarial screening, molecular docking and drug-likeness

studies including ADMET prediction using in silico tools were also

carried out. Since, ART and its semi-synthetic analogues or ART

derived peroxides possess unfavourable pharmacokinetic issues

and certain tissue toxicities because of their high structural com-

1

6

metabolised by the body in to an inactive form .

Experimental

7

,8

plexities and some physicochemical insufficiencies , it is expected

that designed compounds would possess modified pharmacoki- Synthesis and analysis

netic properties and toxicities due to less complexities in the

All of the chemicals were procured commercially from Sigma-

Aldrich Corporation (St. Louis, MO), Merck Specialists Pvt. Ltd.

Darmstadt, Germany), HiMedia Lab. Pvt. Ltd. (Einhausen,

molecular structure. Moreover, single drug conjugate-based triox-

ane molecule can be given as single drug therapy that would also

be advantageous over hybrid antimalarial drugs and combination

therapies.

Molecular docking study was performed to validate the anti-

malarial efficacy of synthesised 1,2,4-trioxane derivatives by inves-

tigating binding modes as well as orientation of ligands in the

receptor pocket of target falcipain 2 protein (P. falciparum cysteine

protease enzyme). In our study, three-dimensional (3D) structure

of falcipain 2 protein molecule was used as possible antimalarial

drug target for 1,2,4-trioxane derivatives. Docking rationalises find-

ing new antimalarial lead molecules and also gives an insight into

structure–activity relationships (SARs) and mode(s) of antimalarial

(

Germany), or Spectrochem Pvt. Ltd. (Mumbai, India) and were

used without further purification. All the reactions were performed

in oven dried glass ware using synthetic grade chemicals. Melting

points (MP) were measured in open capillaries on an electrically

heated MP apparatus. Ultraviolet (UV)–visible spectra were

recorded on Shimadzu UV-1700 UV–visible spectrophotometer and

the wave lengths of maximum absorption (kmax, nm) are reported.

Infrared (IR) spectra were obtained on a Bruker Alpha Fourier

Transform (FT-IR) spectrophotometer (Billerica, MA) using KBR disc

ꢀ1

1

and are reported in terms of frequency of absorption (t, cm ). H

1

3

and C nuclear magnetic resonance (NMR) spectra were recorded

action based on scoring function and further binding modes ana- on Bruker Avance II 400 FT-NMR spectrometer (Billerica, MA) at 400

1

3

lysis from ligand–protein binding/interaction studies . Drug-like- and 100 MHz, respectively, using tetramethylsilane (TMS) as an

ness study includes calculation of fundamental molecular internal standard (d 0.00 ppm) and CDCl₃as a solvent. Chemical

(

physicochemical) properties and Lipinski’s parameters, which shift (d) values were expressed in parts per million (ppm) relative

1

assesses the acceptability of compounds as drug molecules. They to TMS (d 0.00 ppm). HNMR data are assigned in order: peak

represent the combined physicochemical, pharmacokinetic and multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet), number

pharmacodynamic properties (collectively termed as drug-like of protons (numerical integral value), coupling constants (J value)

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1161

1

3

in hertz. C NMR data represents different types of structural car- 2H, CO–CH

2

–CH), 4.23 (d, J ¼ 3.0 Hz, 1H, CO–CH

2

–CH), 6.06 (t,

bons with respect to their corresponding chemical shift values. J ¼ 6.0 Hz, 1H, CH–CH –CH ), 6.09 (d, J ¼ 10.0, 1H, CO–CH¼CH),

2

3

1

3

Mass spectra were obtained on a LC–MS Water 4000 ZQ instru- 6.34 (d, J ¼ 9.0, 1H, CO–CH¼CH); C NMR (100 MHz, CDCl ) d:

3

þ

ment using electrospray ionisation (ES ). The m/z values were 15.53, 18.18 (CH ), 22.43, 28.10, 32.62 (CH ), 36.36, 46.21 (CH),

3

2

þ

recorded in the range of m/z between 100 and 500 and the m/z 86.54, 93.26 (CH¼CH), 187.60 (C¼O); MS (ES ), m/z (%): 226.27

þ

values of the most intense molecular ion [M] peak, with relative (100), [M] , 182.12 (52), 176.12 (28), 152.38 (6); CHN anal. for

intensities in parentheses, are given followed by peaks corre- C H O (198.22), calc. (%): C, 63.70, H, 8.02, O, 28.28, found (%):

1

2 18 4

sponding to major fragment ions. Elemental analysis was per- C, 64.42, H, 8.71, O, 27.78.

formed on a ThermoFinnigan CHNS Analyzer (Somerset, NJ).

3

-(2-Hydroxyphenyl)-8a-methyl-4a,5-dihydrobenzo-1,2,4-trioxin-

1

1,17

0

00

0

6(8aH)-one (3 b)

General procedure of synthesis

A mixture of p-cresol, 1 (1 mmol, 0.1045 ml), distilled water UV spectrum (MeOH), k_m_a_x, nm: 284.2; IR (KBr, cm ) t: 3174.10

16.0 ml) and acetonitrile (CH CN, 4.0 ml) was vigorously stirred

until complete solution was achieved. A mixture of oxone (5 mmol,

.537 g) and NaHCO (15 mmol, 1.261 g), previously ground into

, 3a–e, 3 a–n, 3 a–e

ꢀ

1

(

3

(

O–H, bonded), 2970.21, 2879.23 (C–H, CH ), 1667.13 (C¼O, conj.),

3

1

595.12, 1514.23, 1423.14 (C¼C, aryl), 1161.15 (C–O), 832.19 (O–O);

1

3

H

3 3

NMR (400 MHz, CDCl ) d: 1.25 (s, 3H, CH ), 7.01 (1H,

powder, was slowly added in portion, and a septum with an

empty balloon was immediately placed into the flask in order to

avoid overpressure and loss of generated singlet oxygen. The mix-

ture was vigorously stirred at room temperature until total dis-

appearance of the phenol. After about 1 h of stirring, the reaction

was quenched with water and extracted several times (3–4 times)

with ethyl acetate (EtOAc). Combined EtOAc extracts were dried in

CO–CH¼CH), 7.02 (1H, CO–CH¼CH), 7.82 (Ar–H), 7.84 (Ar–H), 9.83

1

3

(

Ar–OH); C NMR (100 MHz, CDCl ) d: 15.42 (CH ), 29.72 (CH),

3 3

þ

1

(

16.02, 129.69, 132.55, 161.79 (Ar–C), 191.34 (C¼O); MS (ES ), m/z

þ

%): 262.26 (100), [M] , 230.38 (59), 192.37 (34), 176.03 (21); CHN

anal. for C14

14 5

H O (262.26), calc. (%): C, 64.12, H, 5.38, O, 30.50,

found (%): C, 64.62, H, 4.78, O, 30.00.

2 4

Na SO and the solvent was removed under reduced pressure to

yield the product of p-peroxyquinol, 2. The crude compound was 3-(3-Methoxyphenyl)-8a-methyl-4a,5-dihydrobenzo-1,2,4-trioxin-

0

recrystallised from EtOAc/ethanol mixture (3:1) to obtain a pure 6(8aH)-one (3 c)

product of white crystalline solid.

ꢀ

1

UV spectrum (MeOH), k_m_a_x, nm: 292.3; IR (KBr, cm ) t: 2956.33,

The intermediate compound, p-peroxyquinol (4-methyl-4-hydro-

peroxycyclohexa-2,5-dienone) 2 (1 mmol) was added slowly into a

mixture of aldehyde (1.5 mmol) and dichloromethane (4.0 ml), fol-

lowed by the addition of an acid catalyst, p-toluenesulfonic

2

1

880.16 (C–H, CH

424.10 (C¼C, aryl), 1152.82 (C–O), 878.41 (O–O);

) d: 1.19 (s, 3H, CH

–CH), 4.02 (s, 3H, OCH

), 4.23 (d, J ¼ 4.0 Hz, 1H,

–CH), 6.04 (d, J ¼ 10.0, 1H, CO–CH¼CH), 6.37 (d, J ¼ 10.0,

3

), 1674.20 (C¼O, conjugated), 1642.13, 1582.30,

1

H

NMR

(400 MHz, CDCl

3

), 2.43 (d, J ¼ 4.0 Hz, 2H,

CO–CH

2

3

acid (PTSA, 0.05 mmol). The reaction mixture was stirred at

ꢁ

2

4

5–5 C till the completion of reaction (8–12 h) and then concen-

1

H, CO–CH¼CH), 7.03 (d, 1H, J ¼ 7.0 Hz, Ar–H), 7.28 (d, J ¼ 7.0 Hz,

trated in vacuum. Colum chromatography (silica gel 60,

30–400 mesh) of the resulting residue was carried out using a

solvent system of hexane:EtOAc (80:20) to obtain the desired pure

13

Ar–H), 7.44 (d, J ¼ 8.0 Hz, Ar–H), 7.82 (d, 1H, J ¼ 8.0 Hz, Ar–H);

C

2

NMR (100 MHz, CDCl ) d: 15.42 (CH ), 29.12 (OCH ), 38.40 (CH),

3

0

00

84.65, 92.28 (CH¼CH), 118.18, 126.12, 132.17, 140.72, 152.32,

product of target compounds, 3a–e, 3 a–n, 3 a–e as crystalline

solids or semi-solids.

þ

1

62.25 (Ar–C), 190.20 (C¼O); MS (ES ), m/z (%): 276.48 (100), [M] ,

2

23.01 (64), 182.98 (32), 142.04 (18); CHN anal. for C15H O

16 5

(276.28), calc. (%): C, 65.21, H, 5.84, O, 28.95, found (%): C, 65.72,

3

-Ethyl-8a-methyl-4a,5-dihydrobenzo-1,2,4-trioxin-6(8aH)-one

H, 6.24, O, 28.55.

(

3a)

ꢀ

1

UV spectrum (MeOH), kmax, nm: 212.8; IR (KBr, cm ) t: 2940.31,

883.27 (C–H, CH

), 1656.48 (C¼O, conj.), 1145.20 (C–O), 892.28

O–O); H NMR (400 MHz, CDCl ),

) d: 0.89 (t, J ¼ 6.0 Hz, 3H, CH

.21 (s, 3H, CH –CH ), 2.46 (d,

), 1.43 (q, J ¼ 6.0 Hz, 3H, CH

J ¼ 3.0 Hz, 2H, CO–CH –CH), 4.12 (d, J ¼ 3.0 Hz, 1H, CO–CH –CH),

.24 (t, J ¼ 6.0 Hz, 1H, CH–CH –CH ), 6.04 (d, J ¼ 9.0, 1H,

CO–CH¼CH), 6.32 (d, J ¼ 9.0, 1H, CO–CH¼CH); C NMR (100 MHz,

CDCl ) d: 14.10, 18.24 (CH ), 22.90, 26.23 (CH ), 36.81, 42.37 (CH),

4.77, 92.40 (CH¼CH), 188.27 (C¼O); MS (ES ), m/z (%): 198.78 1H, CO–CH¼CH), 7.12 (d, 1H, J ¼ 8.0 Hz, Ar–H), 7.46 (d, J ¼ 8.0 Hz,

3

6

-(4-Chlorophenyl)-8a-methyl-4a,5-dihydrobenzo-1,2,4-trioxin-

(8aH)-one (3 d)

2

(

1

3

0

1

3

ꢀ

1

^U^V^s^p^e^c^t^r^u^m⁽^M^e^O^H⁾^,^kmax, nm: 255.0; IR (KBr, cm ) t: 2912.56,

3

2

3

2

898.20 (C–H, CH ), 1672.17 (C¼O, conj.), 1142.10 (C–O), 1054.36

3

2

1

(Ar. C–Cl), 878.12 (O–O); H NMR (400 MHz, CDCl

3

) d: 1.16 (s, 3H,

5

2

3

1

3

CH

CO–CH

3

), 2.41 (d, J ¼ 4.0 Hz, 2H, CO–CH

2

–CH), 4.22 (d, J ¼ 4.0 Hz, 1H,

2

–CH), 6.06 (d, J ¼ 10.0, 1H, CO–CH¼CH), 6.34 (d, J ¼ 10.0,

3

2

þ

8

(

þ

13

100), [M] , 182.12 (52), 176.12 (28), 152.38 (6); CHN anal. for Ar–H); C NMR (100 MHz, CDCl

3 3

) d: 15.47 (CH ), 36.38 (CH), 89.42,

10 14 4

C H O

C, 61.09, H, 6.72, O, 31.69.

(198.22), calc. (%): C, 60.59, H, 7.12, O, 32.29, found (%): 94.17 (CH¼CH), 117.20, 128.13, 134.60, 140.44, 152.02, 160.12

þ

(Ar–C), 188.00 (C¼O); MS (ES ), m/z (%): 262.71 (100), [M] , 242.12

62), 222.13 (18), 154.72 (6); CHN anal. for C H O Cl (280.70),

(

1

4 13 4

calc. (%): C, 59.90, H, 4.67, O, 22.80, found (%): C, 60.50, H, 4.07, O,

0.41.

3

-Butyl-8a-methyl-4a,5-dihydrobenzo-1,2,4-trioxin-6(8aH)-one

2

(

3c)

ꢀ

1

UV spectrum (MeOH), kmax, nm: 218.6; IR (KBr, cm ) t: 2923.67,

888.36 (C–H, CH

), 1668.59 (C¼O, conj.), 1177.34 (C–O), 886.56

O–O); H NMR (400 MHz, CDCl ),

) d: 0.92 (t, J ¼ 6.0 Hz, 3H, CH

.18 (s, 3H, CH ), 1.46 (m, 2H, CH –CH –CH

CH –CH –CH ), 1.63 (m, 2H, CH –CH –CH –CH

), 2.52 (d, J ¼ 3.0 Hz, 2887.12 (C–H, CH

3

6

-(4-Nitrorophenyl)-8a-methyl-4a,5-dihydrobenzo-1,2,4-trioxin-

(8aH)-one (3 e)

2

(

1

3

0

1

3

ꢀ

1

), 1.52 (m, 2H, UV spectrum (MeOH), kmax, nm: 265.0; IR (KBr, cm ) t: 2932.21,

), 1664.20 (C¼O, conj.), 1543.69 (N–O, NO ),

3

2

3

2

3

2

3

2

1

162

M. RUDRAPAL ET AL.

1

346 (N–O, NO

2

), 1142.78 (C–O), 873.11 (O–O);

H NMR 3-(1H-pyrrol-2-yl)-8a-methyl-4a,5-dihydrobenzo-1,2,4-trioxin-

0

(400 MHz, CDCl

3

) d: 1.20 (s, 3H, CH

3

), 2.44 (d, J ¼ 3.0 Hz, 2H, 6(8aH)-one (3 c)

CO–CH –CH), 4.62 (d, J ¼ 3.0 Hz, 1H, CO–CH –CH), 6.13 (d,

2

ꢀ1

UV spectrum (MeOH), k_m_a_x, nm: 242.6, 259.2; IR (KBr, cm ) t:

3247.01 (NH, 1H-pyrrol-3-yl), 2920.12, 2889.12 (C–H, CH ), 1657.26

J ¼ 10.0, 1H, CO–CH¼CH), 6.38 (d, J ¼ 10.0, 1H, CO–CH¼CH), 7.24

1

3

(

d, 1H, J ¼ 8.0 Hz, Ar–H), 7.74 (d, J ¼ 8.0 Hz, Ar–H);

C NMR

1

(

C¼O, conj.), 1123.05 (C–O), 874.11 (O–O); H NMR (400 MHz,

100 MHz, CDCl d: 14.13 (CH ), 39.11 (CH), 86.70, 90.12

3

)

3

CDCl ) d: 1.32 (s, 3H, CH ), 2.17 (d, J ¼ 3.0 Hz, 2H, CO–CH –CH),

3

2

(

1

2

(

CH¼CH), 116.12, 124.34, 132.26, 138.25, 147.10, 158.33 (Ar–C),

þ

4.26 (d, J ¼ 3.0 Hz, 1H, CO–CH

2

–CH), 6.17 (d, J ¼ 10.0, 1H,

92.12 (C¼O); MS (ES ), m/z (%): 291.45 (100), [M] , 272.11 (52),

CO–CH¼CH), 6.38 (d, J ¼ 10.0, 1H, CO–CH¼CH), 7.01 (d, J ¼ 8.0 Hz,

37.18 (27), 192.16 (12); CHN anal. for C14 (291.26), calc.

H13NO

6

1

H, 1H-pyrrole-H ), 7.12 (t, 1H, J ¼ 8.0 Hz, 1H-pyrrole-H ), 7.42 (t,

3

4

%): C, 57.73, H, 4.50, N, 4.81, O, 32.96, found (%): C, 58.32, H,

1

3

H, J ¼ 8.0 Hz, pyrrole-H

5

), 7.67 (s, 1H, NH, 1H-pyrrole-H

1

);

C

4

.10, N, 4.21, O, 32.16.

NMR (100 MHz, CDCl

1

(

3

) d: 15.32 (CH

3

), 86.52, 88.46 (CH¼CH),

07.23, 114.26, 118.40, 122.35 (Ar–C, 1H-pyrrole-2-yl), 186.38

þ

C¼O); MS (ES ), m/z (%): 235.44 (100), [M] , 182.48 (39), 136.59

3

-(4-(Dimethylamino)phenyl)-8a-methyl-4a,5-dihydrobenzo-1,2,4-

0

(24), 84.72 (7); CHN anal. for C H NO (235.24), calc. (%): C,

trioxin-6(8aH)-one (3 j)

12 13

4

6

5

1.27; H, 5.57; N, 5.95; O, 27.21, found (%): C, 62.06, H, 5.22, N,

.22, O, 26.68.

ꢀ1

UV spectrum (MeOH), kmax, nm: 242.0, 344.0; IR (KBr, cm ) t:

2

928.15, 2876.42 (C–H, CH

3

), 1664.74 (C¼O, conj.), 1623.16,

1

590.12, 1482.49 (C¼C, aryl), 1278.32 (C–N), 1164.23 (C–O), 846.45

1

(O–O); H NMR (400 MHz, CDCl

3

) d: 1.22 (s, 3H, CH

3

), 2.18 (d, 3-(1H-indol-3-yl)-8a-methyl-4a,5-dihydrobenzo-1,2,4-trioxin-

00

J ¼ 3.0 Hz, 2H, CO–CH

2

–CH), 2.82 (d, J ¼ 12.0 Hz, 3H, N–(CH

3 2

) ), 2.96 6(8aH)-one (3 d)

(

d, J ¼ 12.0 Hz, 3H, N–(CH ) ), 4.24 (d, J ¼ 3.0 Hz, 1H, CO–CH –CH),

3

2

ꢀ1

UV spectrum (MeOH), kmax, nm: 288.4, 295.8; IR (KBr, cm ) t:

358.34 (NH, 1H-indol-3-yl), 3066.28, 3040.28 (C–H, aryl), 2872.17

(C–H, CH

), 1694.10 (C¼O, conj.), 1481.36, 1450.25 (C¼C, conj.,

aryl), 1144.17 (C–O), 865.45 (O–O); H NMR (400 MHz, CDCl

.24 (s, 3H, CH

), 7.23 (1H, CO–CH¼CH), 7.21 (1H, CO–CH¼CH),

4

.88 (t, J ¼ 7.0 Hz, 1H, CH–CH

2

–CH

3

), 6.23 (d, J ¼ 9.0, 1H,

3

CO–CH¼CH), 6.36 (d, J ¼ 9.0, 1H, CO–CH¼CH), 7.22 (d, 1H,

1

3

J ¼ 8.0 Hz, Ar–H), 7.68 (d, J ¼ 8.0 Hz, Ar–H); C NMR (100 MHz,

1

3

) d:

CDCl

(

1

2

(

3

) d: 14.25, 15.60, 18.13 (CH

3

), 38.03 (CH), 86.40, 89.34

1

3

CH¼CH), 116.45, 128.02, 132.57, 136.44, 150.13, 160.11 (Ar–C),

þ

7.46 (d, J ¼ 7.0 Hz, 1H, 1H-indole-H

4

), 7.48 (1H, J ¼ 7.0 Hz, 1H-

90.34 (C¼O); MS (ES ), m/z (%): 289.56 (100), [M] , 262.22 (60),

indole-H ), 8.08 (1H, J ¼ 8.0 Hz, 1H-indole-H ), 8.15 (s, J ¼ 9.0 Hz,

5

6

4

32.17 (28), 182.03 (11); CHN anal. for C16H19NO (289.33), calc.

1

H, 1H-indole-H

7

), 8.17 (s, 1H, 1H-indole-H

2

), 9.87 (s, 1H, NH, 1H-

%): C, 66.42, H, 6.62, N, 4.84, O, 22.12, found (%): C, 66.82, H, 6.12,

1

3

indole-H

1

); C NMR (100 MHz, CDCl

3

) d: 113.15, 120.08, 122.39,

N, 4.98, O, 21.52.

1

23.63, 125.02, 125.68, 139.77, (Ar–C, 1H-indole-3-yl), 187.44

þ

(

C¼O); MS (ES ), m/z (%): 285.47 (100), [M] , 253.12 (43), 226.10

4

25), 187.19 (14); CHN anal. for C16H15NO (285.29), calc. (%): C,

3

1

-(4-Hydroxy-3-methoxyphenyl)-8a-methyl-4a,5-dihydrobenzo-

0

67.36, H, 5.30, N, 4.91, O, 22.43, found (%): C, 67.96, H, 4.70, N,

.41, O, 17.95.

,2,4-trioxin-6(8aH)-one (3 l)

5

ꢀ1

UV spectrum (MeOH), k_m_a_x, nm: 277.6, 305.4; IR (KBr, cm ) t:

3

269.19 (O–H, bonded), 3089.21 (C–H, aryl), 2972.24, 2944.25 (C–H,

CH

3

), 1679.11 (C¼O, conj.), 1642.14, 1593.10, 1510.14 (C¼C, aryl), 3-(Pyridin-4-yl)-8a-methyl-4a,5-dihydrobenzo-1,2,4-trioxin-

1

00

043.31 (C–O), 820.17 (O–O); H NMR (400 MHz, CDCl ) d: 1.18 (s, 6(8aH)-one (3 e)

3

1

3

H, CH

3

), 3.94 (s, 3H, OCH

3

), 7.03 (1H, CO–CH¼CH), 7.05 (1H,

ꢀ

1

13

UV spectrum (MeOH), kmax, nm: 296.8; IR (KBr, cm ) t: 3023.22,

010.23 (C–H, aryl), 2932, 2822.11 (C–H, CH

), 1667.34 (C¼O,

conj.), 1481.36, 1450.25 (C¼C, conj., aryl), 1154.20 (C–O), 872.13

CO–CH¼CH), 7.42 (Ar–H), 7.43 (Ar–H), 9.81 (Ar–OH); C NMR

3

(100 MHz, CDCl ) d: 56.08 (OCH ), 108.84, 114.47, 127.61, 129.73,

3 3

þ

1

47.23, 151.84 (Ar–C), 191.11 (C¼O); MS (ES ), m/z (%): 292.80

1

þ

(O–O); H NMR (400 MHz, CDCl

3

) d: 1.26 (s, 3H, CH

–CH), 4.32 (d, J ¼ 4.0 Hz, 1H, CO–CH

.21 (d, J ¼ 8.0, 1H, CO–CH¼CH), 6.40 (d, J ¼ 8.0, 1H, CO–CH¼CH),

.48 (d, J ¼ 8.0 Hz, 1H, 4-pyridyl-H2/6), 7.68 (1H, J ¼ 8.0 Hz, 4-pyr-

3

), 2.63 (d,

(100), [M] , 266.34 (62), 218.15 (36), 178.36 (12); CHN anal. for

J ¼ 4.0 Hz, 2H, CO–CH

2

–CH),

C

15

H

16

O

6

(292.28), calc. (%): C, 61.64, H, 5.52, O, 32.84, found (%):

6

7

C, 62.04, H, 5.93, O, 32.26.

1

3

idyl-H3/5); C NMR (100 MHz, CDCl

3

) d: 15.65 (CH

3

), 84.72, 92.68

(

CH¼CH), 116.20, 122.58, 128.34, 140.67 (Ar. C, 4-pyridyl), 188.42

3

6

-(3-Formylphenyl)-8a-methyl-4a,5-dihydrobenzo-1,2,4-trioxin-

(8aH)-one (3 n)

þ

C¼O); MS (ES ), m/z (%): 247.47 (100), [M] , 204.78 (67), 172.09

38), 156.20 (24); CHN anal. for C13 (247.25), calc. (%): C,

0

H13NO

4

ꢀ1

UV spectrum (MeOH), kmax, nm: 226.4, 244.6; IR (KBr, cm ) t: 63.15; H, 5.30; N, 5.67; O, 25.88, found (%):C, 63.87; H, 5.06; N,

932.12, 2882.00 (C–H, CH ), 2812.65, 2773.26 (C–H, CHO), 1672.45 6.12; O, 25.06.

2

3

(

C¼O, conjugated), 1632.12, 1568.23, 1534.00, 1482.34 (C¼C, aryl),

1

3

1

163.67 (C–O), 879.34 (O–O); H NMR (400 MHz, CDCl

H, CH ), 2.19 (d, J ¼ 3.0 Hz, 2H, CO–CH

–CH), 4.24 (d, J ¼ 3.0 Hz, Evaluation of antimalarial activity

H, CO–CH –CH), 6.13 (d, J ¼ 10.0, 1H, CO–CH¼CH), 6.32 (d,

3

) d: 1.34 (s,

3

2

Chemicals and parasite strains

J ¼ 10.0, 1H, CO–CH¼CH), 7.10 (t, J ¼ 8.0 Hz, 1H, Ar–H), 7.23 (d,

J ¼ 7.0 Hz, 1H, Ar–H), 7.44 (d, J ¼ 8.0 Hz, 1H, Ar–H), 7.53 (s, 1H, All the synthesised compounds, 3a–e, 3 a–n, 3 a–e were eval-

0

00

1

3

Ar–H), 9.71 (CHO); C NMR (100 MHz, CDCl ) d: 14.87 (CH ), 84.60, uated for in vitro antimalarial activity at multiple doses against

3

9

2.30 (CH¼CH), 112.43, 116.29, 122.70, 132.67, 148.70, 156.13 CQ-sensitive (3D7) and CQ-resistant (RKL9) strains of P. falciparum

þ þ

(

Ar–C), 189.76 (C¼O); MS (ES ), m/z (%): 274.27 (100), [M] , 243.37 using blood (erythrocytic) stage of parasite. The solvents and

(54), 210.00 (32), 189.56 (14); CHN anal. for C15

(274.27), calc. reagents used in the antimalarial study were of analytical grade

(

%): C, 65.69, H, 5.15, O, 29.17, found (%): C, 66.49, H, 5.34, O, and were procured from Sigma-Aldrich Corporation (St. Louis, MO)

8.56.

and HiMedia Lab. Pvt. Ltd. (Einhausen, Germany).

14 5

H O

2

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1163

Assay procedure

and Marvin Sketch (ChemAxon LLC, Cambridge, MA, 2015) soft-

ware. The 2D structures were later transformed into 3D structures

using the converter module of Biovia Discovery Studio (DS) v 4.5

The in vitro antimalarial activity screening was carried out by

microculture Giemsa Stained slide method based on light micros-

copy. Schizont Maturation Inhibition assay technique was used for

the quantitative assessment of parasitaemia and evaluation of

drug sensitivity. The assay was conducted using the blood parasite

of in vitro P. falciparum culture according to the method described

(2015) software (San Diego, CA). The compounds were prepared

using the prepare ligand module of the DS which includes assign-

ing bond orders, generating various tautomers, ring conformations

and stereochemistries. All the conformations generated were then

energetically minimised by a single step Steepest Descent method

using CHARMM (Chemistry at HARvard Macromolecular Mechanics,

Cambridge, MA) Force Field with 5000 iterations and a minimum

1

8,19

by Trager and Jensen

arum strain was maintained in vitro in A human red blood cells

. Briefly, a continuous culture of P. falcip-

þ

(RBCs) diluted to 5% haematocrit (HCT) in RPMI (Roswell Park

23

root mean square (RMS) gradient of 0.01 kcal/mol/Å .

Memorial Institute)-1640 medium supplemented with 25 mM

HEPES [(N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid)] buf-

fer, 1% D-glucose, 5.0% sodium bicarbonate, gentamycin (40 lg/

The X-ray crystal structure of falcipain 2-E-64 (PDB id: 3BPF)

was retrieved from the RCSB Protein Data Bank (http://www.rcsb.

org/pdb/) and Chain A of the protein determined at a resolution

þ

ꢁ

ml), and 10% human AB serum. Incubations were done at 37 C

and 5% CO level in a modular incubator. The culture was pas-

24

of 2.9 Å was used in the study . Prior to docking, protein was

2

prepared using protein preparation wizard tool of DS. Polar hydro-

gen atoms were added to the proteins and charges were assigned.

All bound water molecules, other heteroatoms and ligands were

excluded from the crystal structure as they were not significant for

the proteins’ function. Subsequently, the 3D structure of protein

was optimised by energy minimisation using CHARMM Force Field.

It was done in two steps to remove the bad steric clashes using

Steepest Descent and Conjugate Gradient methods for 5000 steps

saged with fresh mixture of erythrocytes and complete RPMI

medium for every day to maintain cell growth. D-Sorbitol (5%)

synchronised 1% ring stage parasitaemia in 5% HCT was used for

the antimalarial assay using 96 well microtitre plate. A stock solu-

tion of 1.0 mg/ml of the test compound was prepared in DMSO

and subsequent dilutions were made with incomplete RPMI to

obtain different concentrations (50, 25, 12.5, 6.25, 3.13, 1.56, 0.78,

2

3

0

.39, and 0.19 mg/ml) of samples (test/standard) in duplicate. In

at RMS gradients of 0.01 and 0.05 kcal/mol/Å, respectively .

Molecular docking studies were carried out using Biovia

Discovery Studio (DS) v 4.5 (2015) software (San Diego, CA). After

addition, drug free negative control to assess the parasite growth

and chloroquine diphosphate as positive control to assess the

integrity of the assay were also maintained in duplicate in the energy minimisation, the Chain A of falcipain 2 protein was defined

microtitre plate. After 40 h of incubation at 37 C, the smears were as a receptor and the binding site sphere was selected based on

ꢁ

prepared from each well (depending on the maturation of schiz- the ligand binding location of E-64. A receptor grid was thereby

onts in negative control, ꢂ10%), stained with 3% Giemsa and generated around the binding cavity (active sites) of protein by

examined under light microscope to ascertain drug sensitivity by specifying the key amino acid residues (Cys 42, Gly 83, and His

2

4

assessing the level of parasitaemia, i.e. the inhibition of parasite 174) . In DS, binding site sphere was set with a radius of 20 Å and

2

0,21

growth in terms of percent inhibition of schizonts’ maturation

.

x, y, z dimensions of –52.25, –4.46, –19.25, respectively. Flexible

molecular docking was performed where the protein was held rigid

while the ligands were allowed to be flexible during refinement.

During docking, the falcipain 2-E-64 complex was imported and

E-64 molecule (co-crystal ligand) was removed, and ligands were

placed in the predicted binding site (grid box) and docking was

performed using the Dock Ligands module of LibDock genetic

algorithm program of DS. The docking parameters were as fol-

lows: No. of hotspots 100, docking tolerance 0.25, No. of runs 100,

No. of iterations 1000, conformation method best, RMS gradient

Observation and assessment of activity

Each test compound was assayed in duplicate and number of

schizonts was counted against 200 asexual parasites per replica

under light microscope. The number of schizonts count was also

assessed in negative control maintained in duplicate in the micro-

titre plate. Mean number of schizonts for duplicate observations

was calculated at each concentration of test/standard samples

compounds. Test values were compared with the control values.

The percentage inhibition of parasite growth for each concentra-

tion was calculated as inhibition ¼100 – A, where A is the percent-

age inhibition of schizonts in test wells, which was determined

using the following formula:

2

5

0.01. All other docking and consequent scoring parameters used

were kept at their default settings. The LibDock scores of the

docked ligands were calculated. Different dock poses were studied

to know the best binding mode of receptor–ligand complex in

terms of scoring function. All docked poses were scored, ranked

and the best pose of each compound having the highest score was

selected and it was later used for the receptor–ligand interaction

analysis. Interaction of ligands with receptor was studied to know

Number of schizonts in the test well

Number of schizonts in the control well

A ¼

ꢃ 100

Further, the IC50 (concentration at which the inhibition of para- the best binding orientation of receptor–ligand complex having

maximum LibDock score. Binding modes of the best pose for each

compound was also analysed with the help of 3D receptor–ligand

complex. Different non-bonding interactions (hydrogen bonding

and hydrophobic) were also analysed with the help of 2D diagram

of docked receptor–ligand complexes. Receptor–ligand interaction

gives better understanding of the molecular interactions between

the binding site residues of receptor molecule and complimentary

groups/atoms of ligands involved in interactions.

site growth represents 50%) values in mg/ml were also calculated

using the NonLin v1.1 software . Test results were compared with

the standard results of CQ.

2

In silico studies

Molecular docking study

Molecular modelling studies were carried out using Dell Precision

work station T3400 running Intel Core2 Duo Processor, 4 GB RAM,

Molecular properties calculation and drug-likeness studies

2

50 GB hard disk and NVidia Quodro FX 4500 graphics card. Two-

dimensional (2D) structures of all compounds were built on In silico calculations of the molecular properties and drug-likeness

0

00

Chemdraw Ultra 10.0 (Cambridge Soft Co., Cambridge, MA, 2010) parameters for all compounds, 3a–e, 3 a–n, 3 a–e were performed

1

164

M. RUDRAPAL ET AL.

based on theoretical approaches to identify the compounds which all structure and property parameters that are relevant to bio-

violate the optimum requirements for drug-likeness. Molecular logical activity. The 1,2,4-trioxane ring system was considered as

properties (molecular weight, LogP value, number of hydrogen the basic requirement for the antimalarial activity and substitu-

bond acceptor(s) (HBA), number of hydrogen bond donor(s) (HBD), tions with groups/moieties having electronic property of (s) was

2

5

total polar surface area) incorporated in Lipinski’s rule of five

and other physicochemical parameters like aqueous solubility

LogS), molar refractivity (MR), and molar volume (MV) were calcu-

lated using Calculation of Molecular Properties module of Biovia

the main motif behind the design task.

(

Chemistry

DS v 4.5 software (San Diego, CA). The number of rotatable bonds In our study, three new series of 1,2,4-trioxane derivatives were

was predicted using Molsoft Online software (http://www.molsoft. synthesised and evaluated in vitro for their antimalarial activity.

com/, 2016) (San Diego, CA), and non-violation of drug-likeness Substitutions with different aliphatic, aromatic, and heteroaromatic

and drug-likeness score was calculated using Molinspiration online groups at the C-3 position of 1,2,4-trioxane ring system afforded

software (http://www.molindpiration.com/, 2016) (Slovensky Grob, target trioxane derivatives obtained as single drug conjugates. The

1

Slovakia).

standard reaction procedure involving a facile synthetic route

depicted in Figure 3 was employed for the preparation of 1,2,4-tri-

oxane derivatives. The oxidative dearomatisation of p-cresol, 1

with oxone first yielded the intermediate compound, p-peroxyqui-

nol, 2 which was later allowed to react with different aldehydes

ADMET prediction

ADME-Toxicity (ADMET) for all the target compounds was calcu-

lated in silico using ADMET descriptor module of Biovia DS v 4.5

software (San Diego, CA). Six mathematical models (aqueous solu-

bility, blood–brain barrier (BBB) penetration, cytochrome P450 2D6

inhibition, hepatotoxicity, human intestinal absorption, and plasma

protein binding) were used to quantitatively predict properties

related to ADMET characteristics or pharmacokinetics of mole-

(

aliphatic/aromatic/heteroaromatic) in the presence of an acid

catalyst called PTSA, to obtain desired trioxane derivatives, 3a–e,

0

00

3

a–n, 3 a–e. The first step of reaction was a [4 þ 2] cycloaddition

2

between electron rich p-alkyl phenol and 1O (singlet oxygen)

generated in situ in the reaction system from oxone, which gave

p-peroxyquinol intermediate through a 1,4-endoperoxide, i.e. per-

oxyhemiacetal. Subsequent desymmetrisation of p-peroxyquinol

after reaction with aldehydes with the help of PTSA catalyst

yielded different trioxane derivatives (Figure 4) . The progress of

reactions was monitored by the silica gel-G thin-layer chromatog-

raphy (TLC) and the spots were visualised by iodine vapours. The

purity of the synthesised compounds was ascertained by MP

determinations and silica gel G TLC. All the compounds were

2

6

cules . These properties influence oral bioavailability, cell perme-

ation, and metabolism of drug molecules.

1

7

Results and discussion

Design of compounds

Rationale behind the design strategy involved selection of 1,2,4-tri- obtained in good yields with high purity. The intermediate com-

oxane ring system as the parent peroxide scaffold considering the pound, 4-hydroperoxy-4-methyl-2,5-cyclohexadien-1one was obtained

ꢁ

pharmacodynamic importance of 1,2,4-trioxane component for as white solid in 89% yield with melting range of 102–104 C and

antimalarial activity of ART and related endoperoxides. Our study R value of 7.8 (cyclohexane:EtOAc:acetic acid ¼ 1:2:0.5). The physi-

f

was aimed at developing 1,2,4-trioxane derivatives as newer syn- cochemical details of synthesised compounds are summarised in

1

13

thetic peroxide-based antimalarial agents having comparatively Table 1. The spectral (UV, IR, HNMR, CNMR, Mass) and analytical

simpler molecular framework than conventional antimalarial drug data (of representative compounds) depicted in the Experimental

molecules with activity against resistant malaria parasites. Three section are in close agreement with the structures of synthesised

series of target compounds (Figure 2) were designed with diverse compounds. In UV spectra (in methanol), k_m_a_xvalues were

substitution patterns (alkyl/aryl/heteroaryl groups) at C-3 position observed at the range of 212.8–344.0 which was due to the pres-

of the 1,2,4-trioxane structural scaffold, based on the molecular ence of chromophoric trioxane ring system along with alkyl/aryl/

manipulation approach of drug design with due consideration to heteroaryl substituents at the C-3 position of the parent scaffold.

O

alkyl

O

^C^H3

Peroxide bond

CH₃

H

1

O

6

H C

3

2

O

3

H

O

5

O

R

aryl

O

8

7

O

4

3

O

H

O

H C

3

CH₃

Trioxane

O

heteroaryl

Lactone

O

ART:

Trioxane derivatives:

alkyl series, aryl series & heteroaryl series

1

,2,4-Trioxane scaffold:

Bicyclic peroxide

Tetracyclic endoperoxide

Figure 2. Design of 1,2,4-trioxane derivatives.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1165

O

OH

H

O

R

a

b

O

^C^H3

3a-e: alkyl series

3'a-n: aryl series

H C

OOH

3

CH₃

3

''a-e: heteroaryl series

1

2

3

0

00

ꢁ

2

CN/H O, rt; (b) RCHO, 40–50 C, 8–12 h,

Figure 3. Scheme of synthesis of target compounds, 3a–e, 3 a–n, 3 a–e. Reagents and conditions: (a) Oxone, NaHCO

3

/CH

3

2 2

CH Cl , PTSA.

O

OH

O

H

O

R

¹O₂

CH CN/H O

H

R

H O

2

O

3

2

CH Cl /H O

2 2 2

4

O

rt

°

0-50 C

CH₃

^C^H3

H C OOH

CH₃

1,4-Endoperoxide

3

p-Cresol

p-Peroxyquinol

Trioxane derivative(s)

Figure 4. Reaction mechanism of synthesis of 1,2,4-trioxane derivative(s).

Table 1. Physicochemical data of synthesised compounds.

Comp.

ꢁ

a

R

Colour (state)

% Yield

MP ( C)

R

f

3

a

b

c

Ethyl

Propyl

Butyl

Pentyl

1-Formyl-but-4-yl

Phenyl

2-Hydroxyphenyl

3-Methoxyphenyl

4-Chlorophenyl

4-Nitrophenyl

4-Tolyl

4-Bromophenyl

4-Fluorophenyl

1-Naphthyl

4-Dimethylaminophenyl

3-Cinnamyl

4-Hydroxy-3-methoxyphenyl

3,4-Dimethoxyphenyl

Isophthalyl

Colourless (solid)

Brown (semi-solid)

Colourless (solid)

Off-white (solid)

Pale yellow (solid)

Colourless (solid)

Yellow (solid)

Colourless (solid)

Brown (semi-solid)

Pale yellow (solid)

Yellow (solid)

Colourless (solid)

Dark brown (semi-solid)

Light brown (semi-solid)

Light yellow (solid)

Colourless (solid)

Brown (semi-solid)

78

74

69

66

65

72

83

74

84

80

69

68

66

70

78

65

82

76

69

68

66

64

75

68

112–114

118–122

–

122–124

128–130

145–147

136–140

156–158

142–144

–

0.70

0.72

0.74

0.67

0.72

0.76

0.72

0.74

0.72

0.78

0.68

0.70

0.72

0.66

0.76

0.74

0.79

0.65

0.66

0.67

0.74

0.68

d

e

0

a

0

b

c

d

e

f

g

h

i

–

j

k

l

168–170

–

148–150

128–130

134–136

–

m

n

0

a

b

c

d

e

Furan-2-yl

Thiophen-2-yl

Pyrrole-2-yl

Indole-3-yl

Pyridin-4-yl

0

–

145–147

157–160

–

Cyclohexane:EtOAc:acetic acid (1:2:0.5).

ꢀ

1

IR spectral data showed absorption frequencies characteristic to at 1054.36 cm was due to the aromatic C–Cl stretch for com-

0

specific functional groups and/or bonds present in the structure of pound 3 d.

A

weak absorption band observed at

ꢀ

1

synthesised compounds. The appearance of a broad peak at 892.28–820.17 cm was due to O–O stretching. The presence of

ꢀ

1

3

174.10 and 3269.19 cm

confirmed the presence of phenolic aliphatic C–H (CH ), aromatic C–H (CH¼C), and aromatic C¼C

3

0

–

OH group in the structure of 3 b and 3 l, respectively. N–H groups in the structure of synthesised compounds was also con-

stretching band of 1H-pyrrol-2-yl and 1H-indol-3-yl systems was firmed by characteristic peaks as depicted in the Experimental

ꢀ1

observed at 3247.01 and 3358.34 cm , respectively. The carbonyl section.

1

(

C¼O) group was assigned with a strong and sharp absorption

HNMR spectra exhibited resonance signals with chemical shift

ꢀ1

band in the region of 1694.10–1656.48 cm for all the synthesised (d, ppm) values characteristic to various structural protons which

compounds. The characteristic peak due to N–O stretching (NO

2

)

concorded the anticipated structure of synthesised compounds

group

strong intensity at 1543.69 cm . A weak band at frequency of of the trioxane ring system was observed in the range of 1.16–1.

was explicated due to the C–N stretch for com- 34 ppm. For olefinic protons (CH5CH) peaks at 2.17–2.63 and 4.

pound 3 j. Distinguished C–O stretching band appeared in the 02–4.32 ppm were assigned, while for other skeletal protons like

0

was also appeared for the compound 3 e by a sharp peak of (Supplementary material). A singlet for three protons of CH

3

ꢀ

1

ꢀ

1

278.32 cm

0

ꢀ

1

2

range of 1043.24–1177.34 cm . A peak of weak intensity observed CO–CH CH were assigned at 6.04–6.23 and 6.32–6.40 ppm.

1

166

M. RUDRAPAL ET AL.

Depending on the nature of the substituent at C-3 position of the both CQ-sensitive (3D7) and CQ-resistant (RKL9) strains of P. falcip-

trioxane ring, distinct d values were also observed with expected arum in a dose dependant manner at screening doses. The IC50

splitting patterns (doublets, triplets, or multiplets) and coupling values of tested compounds range from 1.24 mM to >1000 mM

constant (J) values. A broad singlet at 9.71 and 9.81 ppm was and 1.06 mM to >1000 mM against CQ-sensitive and CQ-resistant

0

observed due to aromatic OH proton for compounds, 3 b and 3 l, strains of P. falciparum, respectively. Out of 24 tested compounds,

0

00

respectively. The CHO proton in compound 3 h was appeared at five compounds (3d, 3e, 3 k, 3 a, and 3 b) showed poor activity

9

.71 ppm. Distinct singlet due to three protons of the OCH group with IC50 values of >1000 mM against both the strains.

3

0

00

0

Compounds 3a–3c, 3 a, 3 d–3 j, 3 m, 3 n, and 3 c exhibited mod-

erate activity with IC50 values ranging from 18.37 to 220.20 mM

and 14.72 to 220.68 mM against sensitive and resistant strains,

respectively. Compounds, 3b, 3c, 3 a, 3 e, 3 h, and 3 j showed bet-

ter activity against sensitive strain than the resistant one.

appeared at 4.02 and 3.94 ppm for compounds 3 c and 3 l,

respectively. Aromatic protons including heteroaryl substituents

appeared in the range of 7.01–8.15 ppm. A broad singlet was also

assigned to NH proton at 7.67 ppm and 9.87 ppm for compounds

0

00

3

c and 3 d, respectively. Aliphatic methyl and methylene protons

0

Among three series of compounds, five compounds (3 b, 3 c, 3 l,

were observed in multiplet pattern (doublet or triplet) at d values

in the downfield region for compounds 3a and 3c, as depicted in

0

3

d, and 3 e) showed good activity, three compounds (3 b, 3 l,

0

2

8

and 3 d) of which exhibited better activity against resistant strain

than the sensitive one. These five compounds were found com-

paratively more potent than rest of the synthesised analogues.

the Experimental section .

1

3

In C NMR spectra, distinguished resonance signal due to the

carbonyl (>C¼O) carbon appeared in the range of

0

00

0

The IC50 values of two best compounds (3 l and 3 d), one (3 l)

1

88.27–192.12 ppm. Aromatic/heteroaromatic carbons appeared at

07.23–162.25 ppm. Characteristic downfield signals for aliphatic

0

from aryl series and the other (3 d) from heteroaryl series were

found to be 1.24 mM and 1.24 mM, respectively, against sensitive

strain of P. falciparum. The IC50 values of the two compounds

against resistant strain were found to be 1.06 mM and 1.17 mM,

respectively. Results were comparable with that of the standard

drug, CQ (IC50¼1.23 mM and 47.16 mM against sensitive and resist-

ant strain of P. falciparum, respectively). Figure 5 shows photo-

carbons were observed for CH

22.43–32.62 ppm), OCH₃(56.08 ppm, 3 l; 29.12 ppm, 3 c), and CH

3

(14.10–15.65 ppm), CH

2

0

(

36.36–92.68 ppm) groups. Olefinic (CH¼CH) carbons were also

assigned and depicted in the Experimental section. The mass spec-

0

00

tra of 3a–e, 3 a–n, 3 a–e exhibited prominent molecular ion

þ

peaks, [M] which finally confirmed the structure of compounds

0

graphs of the effect of the most active compound, 3 l against

with the anticipated mass corresponding to their respective

molecular formula. The results of elemental analyses were within

the acceptable limits (±0.5%) of the calculated values for all the

synthesised compounds.

both CQ-sensitive (3D7) and CQ-resistant (RKL9) strains of

P. falciparum.

Results reveal that the degree of antimalarial (inhibitory) activ-

ity of 1,2,4-trioxane derivatives is dependent on the nature and

degree of substitution pattern. Different structural substitutions

such as non-bulky alkyl and bulky aryl/heteroaryl groups at C-3

position of the ring system modulate the antimalarial efficacy of

prepared 1,2,4-trioxane derivatives. Some degree of variations in

Antimalarial activity

The in vitro antimalarial activity data are summarised in Table 2.

Results of in vitro antimalarial activity revealed that all the synthes- activity among synthesised analogues might be due to diverse

0

00

ised compounds (3a–e, 3 a–n, 3 a–e) showed activity against structural substitutions. A brief SAR study can be depicted as fol-

lows: small alkyl substituents like ethyl, propyl, or butyl moieties

Table 2. In vitro antimalarial activity data.

are of considerable importance for the activity, while substitution

with larger alkyl like pentyl and 1-formyl-but-4-yl moieties is

comparatively less important for the activity. Substitution with

bulky aryl moieties except 3-cinnamyl group also contributes

good antimalarial potency to same extent or little less or even

more compared to alkyl substituted analogues. Compounds with

a

IC50 in mg/ml (mM)

Comp.

Pf 3D7

Pf RKL9

3

a

b

c

0.026 (26.20)

0.020 (20.44)

0.16 (160.47)

3.48 (>1000)

3.46 (>1000)

0.068 (68.46)

0.0054 (5.43)

0.0074 (7.62)

0.032 (32.49)

0.040 (40.78)

0.052 (52.06)

0.058 (58.20)

0.040 (40.21)

0.074 (74.22)

0.018 (18.37)

1.20 (>1000)

0.0012 (1.24)

0.056 (56.09)

0.045 (45.29)

2.07 (>1000)

3.50 (>1000)

0.22 (220.20)

0.0012 (1.24)

0.0072 (8.27)

0.0012 (1.23)

0.028 (28.46)

0.022 (22.26)

0.12 (120.13)

d

4.09 (>1000) phenyl, 4-chlorophenyl, 4-nitrophenyl, 4-tolyl, 3,4-dimethoxy-

4.07 (>1000) phenyl, and isophthalyl substitutions sufficiently retain the

e

0

a

0.066 (66.19)

0.0056 (5.68)

0.0078 (7.83)

0.032 (32.54)

0.036 (36.42)

0.054 (54.17)

0.058 (58.34)

0.036 (36.67)

0.078 (78.28)

0.014 (14.72)

activity, whereas, in case of 2-hydroxyphenyl, 3-methoxyphenyl,

4-hydroxy3-methoxyphenyl substituted analogues the activity sig-

nificantly increases. With bulky heteroaryl moieties like furan-2-yl

and thiophen-2-yl substitutions, the activity declines, while, pyr-

role-2-yl and pyridine-4-yl improve activity, and the indole-3-yl

substitution still potentiates the activity. It is also clear that com-

pounds with five membered heterocyclic rings are less important

for the activity than six membered or fused heteroaryl systems.

0

b

c

d

e

f

g

h

i

j

k

l

1.48 (>1000) Upon critical analysis of SAR, the most notable point found is

0.0010 (1.06)

0.064 (64.53)

0.048 (48.20)

5.02 (>1000)

that substitutions with electron withdrawing moieties are likely

less important for the activity, while electron releasing groups

substituents are considerably more important for activity. More

m

n

0

a

b

c

d

e

0

3.61 (>1000) illustratively, phenyl substituent with electron releasing (OH,

0.22 (220.68)

0.0011 (1.17)

0.0065 (6.52)

0.147 (47.16)

OCH , etc.) groups significantly increases the activity, whereas,

3

2

electron-withdrawing group (Cl, NO , etc.) substituted phenyl ring

reduces the activity (Figure 6). Substitutions of electron releasing

groups at ortho and para or both have greater contributing

effects than meta substitutions. Di-substitution (4-hydroxy-3-

CQ

a

Calculated using NonLin v1.1 software, mean of two replicate observations

counted against 200 cells per replicate), values in parentheses indicate activity

in micromolar (mM) concentration.

(

0

methoxyphenyl, compound 3 l) is more important for the activity

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1167

(

a)

(b)

Ring

Trophozoite

Schizont

0

Figure 5. Photographs showing in vitro antimalarial activity of the most active compound, 3 l against (a) CQ-sensitive 3D7 and (b) CQ-resistant RKL9 strains of P.

falciparum.

H

4

5

Substitution

O

R

(

modulates activity)

6

7

3

Key Pharmacophore

antimalarial specificty)

(

O

-

R: e releasing gr. increases activity

- R: e withdrawing gr. decreases activity

2

8

CH₃

1

Peroxide bond

bioactivation through cleavage)

(

Figure 6. Structural requirements and effect of substitutions on antimalarial activity of 1,2,4-trioxane derivatives.

Hb

Haem-Fe(II)

Pf HDE

H

O-O

cleavage

H

O

R

O

R

(

1)

(2)

Inhibitory

activity

Oxidative damage

to parasite proteins/lipids

O

Enzyme

inhibition

Fe(III)

LPO

O

CH₃

^C^H3

Trioxane peroxide

Oxyl radical

Figure 7. Hypothetical mode(s) of antimalarial action of 1,2,4-trioxane derivatives. (1) Inhibition of haemoglobin degrading enzyme in food vacuole of parasite,

(

2) direct lethal action to parasite by LPO process. Pf HDE: P. falciparum haemoglobin degrading enzyme; LPO: lipid peroxidation.

0

than mono-substitution (2-hydroxyphenyl, compound 3 b). The action (Figure 7) of 1,2,4-trioxane derivatives can therefore be

findings of our present study are also consistent with earlier explained as: target compounds may act either as direct inhibitor

2

8

report

on trioxane-based compounds which show excellent of enzyme (receptor antagonist) or as prodrug by interfering the

antiparasitic activity upon incorporation of an aryl group. It is haemoglobin degradation mechanism during erythrocytic stages

seen that bulkiness of substituents is important for the activity. of P. falciparum. Electronic property of the substituent may play a

However, aryl substitution seems to be more important than significant role for the bio-activation (through O–O cleavage) of

alkyl or heteroaryl substitution. It is important to note here that trioxane ring system to generate highly reactive oxyl radical which

besides bulkiness of substituents, lipophilicity (hydrophobicity) causes death of parasite by lipid peroxidation of parasitic cell

and ionisation potential (basicity) of the molecule are also con- membrane/protein.

sidered to play a crucial role for the activity. The lipophilicity

(

LogP) is inevitable for permeation of parasitic membrane and

Docking simulations

basicity (pKa) is required for accumulation of trioxane molecule

in the acidic food vacuole of parasites.

Plasmodium cysteine protease falcipain 2 enzyme plays an essen-

Literature report that 1,2,4-trioxane structure lacking the lac- tial role in the degradation of host haemoglobin into smaller pep-

tone ring plays an important role for the antimalarial activity, but tides which takes place in the acidic food vacuole of P. falciparum.

3

1

,2,4-trioxane ring alone is insufficient for optimal antimalarial Recent literature claims that endoperoxide antimalarials have

2

9

potency as compared to ART and its semi-synthetic analogues . potential to inhibit falcipain 2 enzyme and therefore, drug target-

The reductive cleavage of the peroxide bond in vivo by heme ing of this particular enzyme would be an attractive avenue in the

[Fe(II)] (released during haemoglobin degradation process in design and development of new antimalarial drugs. Accordingly, a

parasitised RBC) and subsequent production of cytotoxic carbon- molecular docking study was performed for all newly designed

0

00

centred radicals target cell proteins/enzymes and destroy para- compounds 3a–e, 3 a–n, 3 a–e using the falcipain 2 enzyme as

3

0–32

sites

. From the SAR study, possible mode(s) of antimalarial receptor molecule.

1

168

M. RUDRAPAL ET AL.

Figure 8. (a) Optimised co-crystal structure of falcipain 2 (chain A)-E-64 and (b) receptor grid for docking.

The protein model used for docking study was validated as fol- poses of bound ligands were visualised which revealed that the

lows: the 3D crystal structure of falcipain 2 co-crystallised with the best orientation of the ligand relative to the receptor as well as

inhibitor trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane the conformation of the ligand and receptor (best fit of ligand in

(E-64) with active site (receptor grid model) as defined by Cys 42, the receptor molecule). Analysis of 2D diagram indicated that vari-

Gln 36, and His 174 residues was optimised and used for the ous non-bonded interactions mainly polar hydrogen bonding

study. The co-crystal structure of falcipain 2-E-64 (PDB: 3BPF) and interactions were involved between binding site residues (active

the receptor grid model used for docking study are represented in site amino acids) and ligand moieties/atoms. Table 3 also depicts

Figure 8. Co-crystallised ligand, E-64 was re-docked using flexible the number of hydrogen bonds (H-bonds) and H-bond energies

docking simulations (LibDock program of DS) into the original for all docked compounds. Higher the number of hydrogen bonds,

structure of the target protein, falcipain-2 by non-covalent docking higher is the binding affinity. Apart from hydrogen bonding inter-

procedure. For this study, docking parameters were set to the actions, other non-bonded interactions like hydrophobic bonding

software’s default values. E-64 was successfully re-docked to the were also observed.

predicted active sites of falcipain 2 with an acceptable RMSD value

Table 4 reveals the hydrogen bonding interactions of five

of 1.124 Å. Further, in order to reproduce an experimentally most potent compounds (3 b, 3 c, 3 l, 3 d, and 3 e) with active

0

00

observed ligand-binding mode, the co-crystallised ligand, E-64 (a site residues such as Asp 154, Arg 12, Gln 36, Glu 14, Glu 15,

0

00

selective falcipain 2 inhibitor) was used as reference ligand. Glu 138, Gly 13, Val 152, and His 19. Compounds 3 l and 3 d

Results confirmed experimental binding conformations of E-64 in which showed the highest antimalarial activity among three ser-

the binding pocket of receptor molecule. The binding modes and ies, could bind the active sites of falcipain-2 mainly by hydrogen

ligand–protein interaction diagrams obtained in re-docking of E-64 bonding interactions. The 3D binding modes and 2D interaction

0 00

are shown in Figure 9(a).

diagrams of two most potent compounds, 3 l and 3 d are shown

Docking results revealed that LibDock program successfully in Figure 9(b,c), respectively. The first compound formed three

docked all 1,2,4-trioxane derivatives into the binding pocket of fal- strong hydrogen bonds with residues like Gln 36 (OꢄꢄHꢄꢄO), Asp

cipain 2 enzyme. All compounds which were active in in vitro test 154 (OꢄꢄHꢄꢄN), and Arg 12 (OꢄꢄHꢄꢄO) with bonding distances of

could bind with the active site of falcipain-2 with high docking 2.737, 2.985, and 1.834 Å, respectively. In later case, five bonds

score (LibDock) and binding affinities in the range of were observed with residues like Arg 12 (OꢄꢄHꢄꢄO), His 19

9

6.077–126.390. The LibDock scores are summarised in Table 3. All (OꢄꢄHꢄꢄH), Gly 13 (OꢄꢄHꢄꢄO), Glu 14 (OꢄꢄHꢄꢄO), and Glu 15 (OꢄꢄHꢄꢄO)

trioxane derivatives except 3a and 3b exhibited more binding with bonding distances of 1.868, 2.938, 2.308, 2.795, and 2.489 Å,

0

00

affinity (higher dock scores) than the co-crystal ligand, E-64 respectively. Analysis of best docking poses of 3 l and 3 d

(

LibDock score ¼100.364). Five compounds which showed most revealed that the trioxane scaffold was oriented in the binding

potency in in vitro study also ranked top in docking study with cavity (active site residues) of falcipain 2 receptor molecule. In

LibDock scores of 116.533, 126.390, 122.609, 110.646, and 124.737 2D diagram, the trioxane moiety could occupy the binding sites

0

for 3 a, 3 b, 3 l, 3 d, and 3 e, respectively. It is important to note of falcipain-2 through strong H-bonding interactions along with

here that docking results are generally analysed by a statistical hydrophobic interactions. Such interactions afforded good stabil-

scoring function which converts interacting energy (binding ity between receptor molecule and ligand. Trioxane moiety inter-

energy) into numerical values called as the docking score, and acted with multiple amino acid residues such as Arg 12, Asp

therefore docking score is nothing but an expression of binding 154, Val 12 and Gly 13, and Gln 36 and Glu 14 residues

3

4

energy . Binding energy (DG) includes the sum of all non-bonded interacted with the 4-hydroxy-3-methoxyphenyl and idole-3-yl

0

00

interactions including hydrogen bonding, hydrophobic and Vander substituents in compounds 3 l and 3 d, respectively. Strong

3

5

Walls between protein residues and bound ligand . LibDock score H-bonding interactions between C¼O group of the trioxane and

is useful to assess the antimalarial activity of ligands as P. falcip- amino acid residues were also observed. The OH and NH groups

arum falcipain 2 inhibitors. Docking scores of compounds sup- were involved for H-bonding interactions from the substituent

ported the results of in vitro antimalarial activity of synthesised moieties for compounds 3 l and 3 d, respectively. On further

,2,4-trioxane derivatives.

analysis of docking interactions, it was found that trioxane ring

Protein–ligand docking was performed to generate the bio- played a crucial role in protein–ligand binding. Substituents

0

00

1

active binding poses of designed inhibitors in the active site of fal- increased binding strength by forming additional H-bonds that

cipain 2 enzyme. LibDock is a high throughput docking algorithm facilitated much stronger interaction of ligands with the receptor

that finds various conformations of the ligands in the protein (falcipain-2 protein) molecule with optimum binding affinity to

3

6

active site based on polar interaction sites (hotspots) . The 3D achieve desired antimalarial activity.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1169

Figure 9. (a) Redocked conformer (pose) of E-64 in the active site of the protein falcipain 2 (left) and 2D representation of the binding interaction (right); (b, c) binding

0

00

mode (left) and 2D receptor–ligand interaction diagram (right) of compounds, 3 l and 3 d at binding pocket of falcipain 2 (left), respectively.

Docking is generally used to find the best binding orientation conformations of the molecule in the binding site. Scoring func-

of small molecules bound to their target protein molecules in tion is used to identify the most likely pose for an individual lig-

order to predict the binding affinity and thereby biological activity and to assign a priority order to a set of diverse ligands docked to

of the small molecules. Hence, docking plays an important role in the same protein and hence estimate binding affinity. Using scor-

the rational drug design. To evaluate the ligand–receptor interac- ing function, one can predict the binding conformation of a ligand

tions, the best pose of receptor–ligand complex should possess in its receptor and the affinity between the ligand and the recep-

the lowest free energy (binding energy) which differs from experi- tor with the correct poses of ligands in the binding pocket of a

3

4–38

mentally observed other structure and estimate the binding affin- protein

ity. Search algorithm is used to generate different poses of the

Furthermore, a correlation study between docking scores and

ligand within the active site of molecules, orientations of particular logIC50 (pIC50) values of top five compounds (3 b, 3 c, 3 l, 3 d,

.

0

00

1

170

M. RUDRAPAL ET AL.

Table 3. LibDock scores, no. of H-bonds and H-bond energies.

1

.8

.6

.4

Comp.

LibDock score

No. of H-bond (s)

H-Bond energy

0

3

a

b

c

93.077

96.153

101.69

3

5

4

6

4

3

4

5

2

3

4

1

3

1

4

3

5

6

5

6

7

ꢀ2.056

ꢀ1.648

ꢀ2.174

ꢀ2.056

ꢀ5.457

0.804

ꢀ3.727

ꢀ2.265

0.179

_R2 ₌₀_.₇₄₅

d

111.343

108.143

107.807

116.533

126.390

110.908

127.665

118.391

117.436

110.983

120.229

118.985

120.370

122.609

100.364

122.864

101.911

105.097

110.952

110.646

124.747

100.364

e

0

a

0

0.2

0

b

c

d

e

f

g

h

i

105

110

115

120

125

130

ꢀ2.246

0.173

LibDock score

0.182

0.174

0

j

k

l

ꢀ2.5

1

ꢀ2.526

0.976

0

.8

m

n

ꢀ2.526

0.780

.6

.4

0

a

b

c

d

e

ꢀ2.5

R²= 0.782

0

ꢀ2.5

ꢀ2.466

ꢀ2.478

0.031

0.2

E-64

0.086

0

1

05

110

115

LibDock score

Figure 10. Graphs showing correlation between in vitro antimalarial activity

120

125

130

(pIC50) and LibDock scores for five most potent compounds.

Table 4. Details of hydrogen bonding between five most active ligands and

receptor molecule.

Molecular properties and drug-likeness

H-binding

ligand

H-binding receptor

The results of predicted Lipinski’s parameters and other drug-like-

0

00

ness properties of the synthesised compounds, 3a–e, 3 a–n, 3 a–e

are depicted in Table 5. Results revealed that all the compounds

possessed good drug-like properties based on Lipinski’s rule of

five with additional parameters such as molar solubility (MS), MV,

MR, and number of rotatable bonds (nRotB). All compounds

obeyed Lipinski’s rule of five and Veber rule. Lipinski rule of five is

a rule to evaluate drug likeness to determine if a chemical com-

pound has a certain pharmacological or biological activity to make

H-bond

Comp. H-bond (s) Element Type Residue Element Type distance (Å)

0

3

b

6

O

H

O

H

O

H

O

H

O

H

A

D

A

D

A

D

A

D

A

D

Asp 154

His 19

N

H

O

N

H

O

H

N

H

O

H

O

H

O

D

A

D

A

D

A

D

A

D

A

2.749

2.811

1.720

2.393

2.488

2.262

2.234

2.534

2.874

3.016

2.737

2.985

1.834

1.868

2.938

2.308

2.795

2.489

2.976

2.107

2.804

2.515

2.247

2.735

Arg 12

Gly 13

Val 152

Asp 154

His 19

0

3

c

l

4

39

it an orally active drug . According to Lipinski’s rule, compounds

Asp 154

Glu 138

Gln 36

Asp 154

Arg 12

His 19

are more likely to be drug-like and orally bioavailable if they obey

the following criteria: LogP_o_/_w(octanol/water partition coef-

ficient) ꢅ 5, MW (molecular weight) ꢅ 500, HBA ꢅ 10, and HBD

0

3

5

15

ꢅ5 . To further substantiate, Veber et al. stated that compounds

0

3

d

with ꢅ10 rotatable bonds and TPSA (total polar surface area) of

2

Gly 13

Glu 15

Glu 14

Arg 12

Glu 14

Asp 154

Gly 13

Glu 14

ꢅ140 A are more likely to show membrane permeability and

3

9,40

good bioavailability

. Lipinski rule of five is considered predict-

ive for oral bioavailability; however, 16% of oral drugs violate at

00

3

e

6

3

9

least one of the criteria and 6% fail in two or more . In our study,

compounds did not violate Lipinski rule of five parameters. Poor

absorption or permeation of a ligand is more likely if a drug-like

molecule have more than one of five rule violations. Values of

LogP, MW, and TPSA indicated that compounds possessed good

membrane permeability and oral bioavailability, whereas, nRotb

bonds suggested that compounds had good intestinal availability.

MS data indicated good bioavailability of compounds if given by

00

and 3 e) was carried out. Graphs presented in Figure 10 revealed oral route. MV and MR values were also found in permissible

that a good correlation existed between in vitro antimalarial activ- range which indicated good oral bioavailability for all the com-

ity (against both CQ-sensitive and CQ-resistant P. falciparum pounds. Hydrophobicity, membrane permeability, and bioavailabil-

strains) of compounds and their LibDock scores. The in vitro activ- ity are dependent on molecule’s MW, LogP, MS, HBA, and HBD.

2

ity was found consistent (R ¼ 0.745 or 0.782) with the binding Molecules violating more than one of these rules fail to exhibit

affinity of compounds for the P. falciparum cysteine protease falci- optimum bioavailability. Sufficient water solubility is also import-

pain 2 receptor.

ant for optimal bioavailability of drugs. Number of rotatable bonds

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1171

Table 5. Calculated molecular properties and drug-likeness parameters.

Lipinski’s parameters

2

3

Comp.

MW

LogP

nHBA

nHBD

TPSA (A )

nViolations

MS

MR

MV (A )

nRotB

DL score

3

a

b

c

198.78

212.25

226.27

240.30

240.26

246.26

262.26

276.29

280.71

291.26

260.29

325.16

264.25

296.32

289.33

272.30

292.29

306.32

274.27

236.23

252.28

235.24

285.30

247.25

1.02

1.50

1.99

1.64

0.69

2.17

1.79

2.26

2.88

1.84

2.57

3.02

2.44

3.50

2.29

3.01

1.88

2.23

1.96

1.32

1.76

0.99

1.12

2.39

4

5

4

5

4

6

4

5

4

6

5

4

5

4

0

1

0

1

0

1

0

1

44.76

61.83

44.76

64.99

53.99

44.76

90.58

44.76

48.00

44.76

74.22

63.22

61.83

57.90

73.00

60.55

57.65

60.55

0

ꢀ1.33

ꢀ1.74

ꢀ2.21

ꢀ2.23

ꢀ1.81

ꢀ2.68

ꢀ2.21

ꢀ2.81

ꢀ3.48

ꢀ3.19

ꢀ3.85

ꢀ2.98

ꢀ4.39

ꢀ2.64

ꢀ3.66

ꢀ2.32

ꢀ2.89

ꢀ2.21

ꢀ2.08

ꢀ2.68

ꢀ1.63

ꢀ3.25

ꢀ2.21

50.01

54.65

59.25

59.68

59.99

65.27

66.97

71.74

70.08

72.60

70.32

72.90

65.49

81.73

79.70

75.59

73.43

78.21

62.76

57.68

64.13

59.93

76.36

71.87

220.52

238.37

256.28

258.69

264.63

255.79

268.63

287.64

272.98

280.74

276.73

277.64

261.70

306.92

305.35

296.66

299.06

319.92

284.05

241.85

251.15

243.60

251.09

296.39

1

2

3

4

1

2

1

2

1

2

3

2

1

ꢀ0.43

ꢀ0.41

ꢀ0.60

ꢀ0.68

ꢀ0.42

ꢀ0.82

ꢀ0.17

ꢀ0.54

ꢀ0.26

ꢀ1.06

ꢀ0.78

ꢀ0.65

ꢀ0.45

ꢀ0.76

ꢀ0.79

ꢀ0.70

ꢀ0.18

ꢀ0.10

ꢀ0.72

ꢀ0.70

ꢀ0.51

ꢀ1.22

ꢀ0.39

ꢀ1.24

d

e

0

a

0

b

c

d

e

f

g

h

i

j

k

l

m

n

0

a

b

c

d

e

0

MW: molecular weight; LogP: log of octanol/water partition coefficient; nHBA: no. of hydrogen bond acceptor(s); nHBD: no. of hydrogen bond donor(s); TPSA: total

polar surface area; nViolations: no. of rule of five violations; MS: molar aqueous solubility; MR: molar refractivity; MV: molar volume; nRotB: no. of rotatable bonds;

DL: drug-likeness.

is important for molecular conformational studies (i.e. stereo- and bioactivity could suitably transform a ligand to a good drug

4

1–44

selectivity of drug molecules) for optimal binding with the lead

. Better antimalarial activity of the top five compounds

0

00

receptor molecule. Reduced molecular flexibility, as measured by 3 b, 3 c, 3 l, 3 d, and 3 e might be probably due to their sufficient

the number of rotatable bonds, and polar surface area or total lipophilicity along with balanced polar properties likes HBD, HBA

hydrogen bond count (sum of donors and acceptors) are some groups, rotatable bonds, and PSA. They collectively determine

important predictors of good oral bioavailability, independent of membrane permeability as well as transport property of drug mol-

molecular weight . Further, TPSA, MV, and MR are also useful ecule. Due to optimal lipophilicity, compound readily penetrated

parameters for drug’s transport and biodistribution . The drug parasite’s cell membrane that led to attain an intracellular concen-

1

5

1

5

score combines drug-likeness, lipophilicity, solubility, molecular tration for desired antimalarial activity.

weight, and the risk of toxicity into a single numerical value

that can be used to predict a global value for each compound

as a potential new drug candidate . Drug-likeness score was

2

ADMET prediction

obtained in the range of ꢀ0.18 and ꢀ0.79. Even though com- The ADMET values of newly designed 1,2,4-trioxane derivatives

pounds exhibited negative drug-likeness values, scores were in presented in Table 6 were found in acceptable range with favour-

acceptable range and indicated that compounds possessed able ADMET properties. All the compounds were predicted to

potential as new drug candidates. The overall analysis of drug- have good intestinal absorption and non-inhibitors of cytochrome

likeness studies strongly suggested that newly designed 1,2,4-tri- P450 2D6 (CYP2D6) with medium to moderate BBB penetration.

oxane derivatives possessed good drug-likeness behaviour BBB penetration is mandatory for the drug to be used in the treat-

favourable for optimal membrane permeability, transport and ment of cerebral malaria. The CYP2D6 enzyme is one of the

bioavailability and eventual interaction with the receptor important enzymes involved in drug metabolism. The aqueous

ꢁ

molecule.

solubility prediction (defined in water at 25 C) indicated that

Since all newly designed compounds showed favourable drug- most of the compounds were soluble in water. The predictive hep-

like properties, a reasonable correlation can be drawn between atotoxicity was observed for a few compounds among three ser-

their calculated drug-like properties and in vitro antimalarial activ- ies. Some of the compounds were found to be highly bound with

ity profile. LogPo/w is a direct indicator of lipophilicity of drug sub- plasma protein, while some were poorly bound with plasma

stances. Higher the value of LogP, better the biological membrane protein.

permeability. It is inevitable for a molecule to have sufficient lipo-

philicity which is required for its optimal bioavailability and bio-

logical action. Hydrogen bond acceptor and donor groups are of

Conclusions

paramount importance required to achieve optimal drug action. Newer series of 1,2,4-trioxane derivatives reported herein have

PSA is also closely related to the hydrogen bonding potential of a potential in vitro antimalarial effectiveness and therefore, further

drug molecule. Practically, a compound with all drug-like proper- studies are required for the evaluation of their toxicity, antimalarial

ties in the desired range appears to exhibit high levels of thera- efficacy, and pharmacokinetics in animal models. Our work is in

peutic potency. Good drug-like properties and activity are progress to carry out in vivo toxicity and antimalarial activity of

complementary, and hence balanced attention to both properties potent compounds. Structure–activity relationship study helped us

1

172

M. RUDRAPAL ET AL.

Table 6. Theoretical ADMET parameters.

Funding

Aqueous

Comp. solubility penetration

BBB

CYP P450 2D6

inhibition

Intestinal

PP

One of the authors is grateful to UGC, New Delhi, India, for the

financial support in the form of Senior Research Fellowship (F/

Hepatotoxicity absorption binding

3

a

b

c

3

2

3

2

3

2

3

2

3

2

1

3

1

2

1

3

1

2

FALSE

TRUE

FALSE

TRUE

FALSE

TRUE

0

FALSE

TRUE

FALSE

TRUE

FALSE

2015-16/NFO-17-OBC-TRI-33908).

d

e

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Article Doi

Novel series of 1,2,4-trioxane derivatives as antimalarial agents

DOI: 10.1080/14756366.2017.1363742

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Article abstract of DOI:10.1080/14756366.2017.1363742

Full text of DOI:10.1080/14756366.2017.1363742

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