Design, synthesis and antimalarial activity of some new 2-hydroxy-1,4-naphthoquinone-4-hydroxyaniline hybrid mannich bases

Article abstract of DOI:10.14233/ajchem.2016.19478

In this study, some novel 2-hydroxy-1,4-naphthoquinone-4-hydroxyaniline hybrid Mannich bases were designed, synthesized and evaluated for in vitro antimalarial activity. The design strategy of novel hybrid molecules involves fusion between the pharmacopho

Full text of DOI:10.14233/ajchem.2016.19478

Asian Journal of Chemistry; Vol. 28, No. 4 (2016), 782-788
A
SIAN
J
OURNAL OF HEMISTRY
C
http://dx.doi.org/10.14233/ajchem.2016.19478
Design, Synthesis and Antimalarial Activity of Some New
2-Hydroxy-1,4-naphthoquinone-4-hydroxyaniline Hybrid Mannich Bases
*
DIPSHIKHA SHARMA, DIPAK CHETIA and MITHUN RUDRAPAL
Department of Pharmaceutical Sciences, Dibrugarh University, Dibrugarh-786 004, India
*Corresponding author: E-mail: rudrapal.m03@gmail.com
Received: 15 July 2015;
Accepted: 31 August 2015;
Published online: 30 December 2015;
AJC-17691
In this study, some novel 2-hydroxy-1,4-naphthoquinone-4-hydroxyaniline hybrid Mannich bases were designed, synthesized and evaluated
for in vitro antimalarial activity. The design strategy of novel hybrid molecules involves fusion between the pharmacophoric moieties of
lawsone (2-hydroxy-1,4-naphthoquinone, a residue from atovaquone) and Mannich substituted 4-hydroxyaniline (4-aminophenol, a
residue from amodiaquine) on the basis of molecular hybridization strategy. Newly designed compounds, 5a-f were also studied for drug-
likeness assessment based on Lipinski’s rule of five. All the synthesized compounds exhibited some degree of in vitro antimalarial activity
against the chloroquine-sensitive strain (RKL-2) of P. falciparum at the tested dose (1 mg/mL), which was considerably less than that of
the standard drug, chloroquine (0.1 mg/mL). However, compounds with propyl, 5a (IC50 0.453 µg/mL) and morpholinyl, 5f (IC50 0.391
µg/mL) substitutions showed comparatively better activity than rest of the synthesized analogues. Compound 5f (IC50 0.993 µg/mL) was
found to possess higher antimalarial effectiveness than compound 5a (IC50 2.92 µg/mL) against resistant strain (RKL-9) of P. falciparum.
The activity of these compounds against the resistant strain was also less than that of chloroquine (IC50 0.299 µg/mL). From results, it is
clear that compounds having substitutions like smaller alkyl groups (n-propyl, 5a; isopropyl, 5b) or saturated heterocyclic moiety
(morpholinyl, 5f) possess superior antimalarial activity in comparison to other compounds substituted with bulky alkyl (diisopropyl, 5c;
n-butyl, 5d) or aryl (phenyl, 5e) moieties. Further, since all the compounds exhibited favourable drug-like properties a reasonable correlation
therefore appears to exist between their drug-likeness and antimalarial activities.
Keywords: 2-Hydroxy-1,4-naphthoquinone, 4-Hydroxyaniline, Mannich base, Hybrid, Drug-likeness, Antimalarial.
To overcome the problem of drug resistance in P. falciparum
malaria, multiple traditional and modern approaches such as
development of new analogues of the existing drugs, use of
combination therapy, design of hybrid molecules are currently
being adopted for the discovery and development of potent
antimalarial agents [5]. The design of hybrid molecules by
the covalent fusion of two or more pharmacophoric units
having different mechanisms of action remains a very attractive
strategy for the development of new antimalarial drugs. This
novel rational drug design approach is based upon the concept
of molecular hybridization. A single hybrid molecule with
different or dual modes of action may be beneficial for the
treatment of malaria in terms of overcoming drug resistance
for showing action at multiple therapeutic targets with improved
biological affinities and reduced undesired side effects. This
strategy therefore not only remains therapeutically efficacious
but also cost-effective, which ultimately reduces the overall
drug pressure of the treatment. Some hybrid molecules comp-
rising two distinct pharmacophoric moieties like 4-amino-
quinoline-trioxane, 4-aminoquinoline-triazine, chloroquine-
INTRODUCTION
Malaria is a life-threatening parasitic disease affecting
around 300-500 million people, which causes over 1 million
deaths per year globally [1]. The development of resistance of
malaria parasites to most of the currently available antimalarial
drugs is mainly attributed to be responsible for the global rise
of malaria in recent times. Moreover, the emergence of multi-
drug resistant strains of Plasmodium falciparum has further
complicated the issues related to the chemotherapy of the
disease in malaria endemic regions of the world [2]. Chloroquine
(CQ, Fig. 1), a 4-aminoquinoline antimalarial was once used
as a first line drug for the treatment of malaria because of its
excellent clinical efficacy, limited host toxicity and cost-effec-
tiveness. Resistance to chloroquine in P. falciparum malaria
has seriously impaired the use of quinoline antimalarials in
the treatment of malaria [3]. Novel artemisinin-based antimala-
rials and their combination regimens such as mefloquine-
artemisinin derivatives have also currently become clinically
less effective in multi-drug resistant P. falciparum malaria [4].

Vol. 28, No. 4 (2016)
Synthesis of Some New 2-Hydroxy-1,4-naphthoquinone-4-hydroxyaniline Hybrid Mannich Bases 783
ATQ
AQ
ferrocene and 4- aminoquinoline-pyrimidine hybrids are under
clinical trials as hybrid antimalarial agents [6-9].
Lawsone is chemically known as 2-hydroxy-1,4-naphtho-
quinone. Lawsone scaffold based synthetic drugs have potential
for antimalarial effectiveness. Atovaquone (ATQ, Fig. 1), a
3-substituted derivative of lawsone is a clinically useful anti-
malarial agent. Literature reveal the antiplasmodial potential
of natural and/or synthetic 2-amino substituted 1,4-naphtho-
quinones and 1,2-naphthoquinones. The antiparasitic activity
of the quinones is exhibited by several mechanisms such as
the competitive inhibition of the cytochrome bc1 complex, gene-
ration of reactive oxygen species (ROS), enzymatic inhibition
(e.g., glutathione reductase, dihydroorotate dehydrogenase and
glyceraldehyde-3-phosphate dehydrogenase), alkylation of
biomolecules etc. [10]. On the other hand, paracetamol (4-
hydroxyacetanilide) contains a 4-hydroxyanilino moiety,
which is believed to undergo Cyt. P-450 catalyzed oxidation
in vivo to a reactive quinoneimine [11]. Amodiaquine (AQ,
Fig. 1) is a 4-aminoquinoline antimalarial drug having Mannich
substitution at the 4-hydroxyaniline residue in the side chain.
Amodiaquine was effective against chloroquine-resistant
strains of P. falciparum, but the clinical use of amodiaquine
has been restricted because of its severe side effects like hepato-
toxicity and agranulocytosis [12]. The toxicity of amodiaquine
is due to the fact that 4-hydroxyanilino moiety undergoes enzy-
matic oxidative bioactivation to form reactive toxic quinone-
imine metabolites such as amodiaquine quinoneimine (AQQI)
or semiquinoneimine (AQSQI). However, its regioisomeric
analogue, isoquine (ISQ, Fig. 1) showed excellent oral in vivo
ED50 activity with less severe side effects as it did not undergo
bioactivation [11,13,14].
O
H
N
R1
O
N
2
H
4
R2
OH
O
Mannich base of
moiety
2-Hydroxy-1,4-naphthoquinone 4-hydroxyanilino
moiety
Molecular
hybridization
O
H
N
R1
Target compounds, 5a-f
N
5a:R1=H, R2=
5b:R1=H, R2=
n
-propyl
R2
OH
OH
i
-pr
O
5c:R1=H, R2= n-butyl
5d:R1, R2= -pr
5e:R1, R2= phenyl
5f:R1, R2=-(CH2)2O(CH2)2
i
2-Hydroxy-1,4-naphthoquinone-
4-hydroxyaniline
hybrid
Mannich bases, 5a-f
Fig. 2. Design of novel hybrid compounds
In the present study, some new 2-hydroxy-1,4-naphtho-
quinone-4-hydroxyaniline hybrid Mannich Bases were
designed, synthesized and screened for in vitro antimalarial
activity. The synthesized compounds were also evaluated in
silico for molecular properties assessment and drug-likeness
prediction with an aim to study their drug-like characteristics
based on Lipinski’s rule of five with additional parameters.
Molecular properties are fundamental structural properties
which determine the physicochemical (solubility, permeability)
and biochemical (metabolic stability, transport property,
protein/tissue affinity) properties, which in turn ultimately
determine molecule’s pharmacokinetics (bioavailability, half
life), toxicity and pharmacodynamics (receptor affinity and
efficacy) in biological systems [15]. Molecular properties are
therefore required to be assessed in order to evaluate the drug-
like characteristics of a particular molecule based on Lipinski’s
rule of five.
OH
N
N
HN
HN
Cl
N
Cl
N
Amodiaquine
Chloroquine
EXPERIMENTAL
Cl
N
All of the chemicals were procured commercially from
Sigma-Aldrich Corporation (USA), Merck Specialists Pvt. Ltd.
(Germany), HiMedia Lab. Pvt. Ltd. (Germany) or Spectrochem
Pvt. Ltd. (India) and were used without further purification,
unless otherwise stated. The solvents and reagents used in the
antimalarial study were of analytical grade. The progress
of reactions was monitored by the silica gel-G thin-layer
chromatography (TLC) and the spots were visualized by iodine
vapours. Melting points (m.p.) were measured in open
capillaries on an electrically heated melting point apparatus.
UV-visible spectra were recorded on Shimadzu UV-1700 UV-
visible spectrophotometer and the wavelength of maximum
absorption (λmax, nm) is reported. Infrared (IR) spectra were
obtained on a Bruker Alpha FT-IR spectrometer using KBR
disc and are reported in terms of frequency of absorption (ν,
cm-1). 1H NMR and 13C NMR spectra were recorded on Bruker
AD II 400 FT-NMR spectrometer at 400 and 100 MHz, respec-
O
HN
OH
OH
Cl
N
O
Isoquine
Atovaquone
Fig. 1. Chemical structures of some potent antimalarial drugs
Keeping in view the above facts, it was thought to design
some novel antimalarial derivatives through coupling between
the structural scaffolds of 2-hydroxy-1,4-naphthoquinone (a
residue from atovaquone) and Mannich substituted 4-hydroxy-
aniline (a residue from amodiaquine) based on the concept of
molecular hybridization strategy (Fig. 2). Newer hybrid anti-
malarial agents would be potent (improved efficacy and affinity)
and also active against resistant strains of P. falciparum malaria.

784 Sharma et al.
Asian J. Chem.
tively with tetramethylsilane (TMS) as an internal standard (δ
0.00 ppm) and CDCl3 as a solvent. Chemical shift (δ) values
were expressed in parts per million (ppm) relative to TMS (δ
reduced pressure and the residue was dissolved in dichloro-
methane. The organic solution was treated with dilute hydro-
chloric acid, basified to pH 9-10 and then again extracted with
dichloromethane. The combined extract was washed with water
and the separated product was collected.
1
0.00 ppm). HNMR data are assigned in order: peak multi-
plicity (b, broad; s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet), number of protons (numerical integral value), coupling
constants (J value) in hertz. Mass spectra were obtained with
the LC-MS Water 4000 ZQ instrument using atmospheric
pressure ionization (API-ES) in the range of m/z 150-1400
both in the negative and positive ion modes. The m/z values of
the most intense quasimolecular ion [M+H]+ peak, with relative
intensities in parentheses are stated.
General method of synthesis: The target compounds
were synthesized as Mannich bases of 2-hydroxy-1,4-naphtho-
quinone (lawsone)-4-hydroxyaniline conjugate. 2-hydroxy-
1,4-naphthoquinone (1) obtained commercially was first
chlorinated by thionyl chloride to give 3-chloro-2-hydroxy-
1,4-naphthoquinone (2) which was later fused with diffe-
rent Mannich products of 4-aminophenol to afford hybrid
compounds 5a-f. The Mannich reaction of commercially
available 4-acetamidophenol (3) and subsequent hydrolysis
of the amide function led to the formation of substituted
Mannich bases, 4a-f of 4-aminophenol/4-hydroxyaniline. The
general procedure [11] of synthesis of target compounds is
depicted in Scheme-I.
A solution of N-(4-hydroxy-3-[(substituted amino)-
methyl]phenyl)acetamide (17 mM) in hydrochloric acid (20
%, 27.3 mL) was heated under reflux for 6 h. The solvent was
then removed under reduced pressure and the resulting residue
was co-evaporated with ethanol. The residue was dissolved in
ethanol (20 mL), 3-chloro-2-hydroxy-1,4-naphthoquinone (3.8
g, 18 mM) was added into it and the mixture was heated under
reflux for 12 h. The solution was concentrated under reduced
pressure to give a viscous mass which was subsequently poured
into ice-cold ammonium hydroxide. The sticky solid thus
separated was dissolved in dichloromethane and was extracted
with alkaline solution. The organic solution was washed with
water, dried with anhydrous sodium sulphate and then
evaporated to dryness under reduced pressure to give the crude
product. The product was recrystallized from dichloromethane.
In vitro Antimalarial activity screening [3,12,16,17]:
All the synthesized compounds, 5a-f were screened for in
vitro antimalarial activity against a chloroquine-sensitive strain
of P. falciparum (RKL-2) and additionally, two compounds,
5a and 5f were also screened for activity against chloroquine-
resistant strain of P. falciparum (RKL-9). The antimalarial
activity screening was carried out by Giemsa stained slide method
in the Department of Pharmaceutical Sciences, Dibrugarh
University, Dibrugarh, Assam, India.
Continuous culture of P. falciparum strain was maintained
in vitro in O+ human red blood cells diluted to 6 % haematocrit
in RPMI 1640 medium supplemented with 25 mM HEPES,
1 % D-glucose, 0.23 % sodium bicarbonate, gentamycin (40
mg/mL), amphotericin-B (0.25 mg/mL) and 10 % humanAB+
serum. Incubations were done at 37 °C and 5 % CO2 level in a
modular incubator. D-sorbitol synchronized 1 % ring stage
parasitaemia in 3 % haematocrit was used for antimalarial
assays using 96 well microtitre plate. A stock solution of 5
mg/mL of the test compound was prepared in DMSO and
subsequent dilutions were made with incomplete RPMI in
duplicate.All the test compounds were assayed at a fixed dose
of 1 mg/mL. Each test well of the microtitre plate contained
20 µL of the compound and 180 µL of 1 % ring stage parasitaemia
in 3 % haematocrit. In addition, drug free negative control to
assess the parasite growth and chloroquine diphosphate (at
0.1 mg/mL dose) as positive control to assess the integrity of
the assay were also maintained in duplicate in the microtitre
plate. After 40 h of incubation the smears were prepared from
each well, stained with 3 % Giemsa and scanned under light
microscope to ascertain percentage dead rings and trophozoites
by examining a minimum of 400 asexual parasites.
O
O
Cl
a
OH
OH
OH
O
1
O
2
H3COCHN
H3COCHN
R1
b
N
R2
OH
3
4a-f
c
O
H
N
R1
N
R2
OH
OH
O
5a-f
Scheme-I: Synthesis of target compounds (5a-f). Reagents and conditions:
(a) SOCl2, pyridine, 70 °C, 1 h; (b) primary/secondary amine,
Aq. CH2O, EtOH, reflux, 24 h; (c) (i) 2O % HCl, EtOH, reflux,
6 h; (ii) 2, EtOH, reflux 12 h
To a mixture of 2-hydroxy-1,4-naphthoquinone (7 g, 40
mM) and pyridine (3.2 g, 3.2 mL, 40 mM) thionyl chloride (6
mL) was added drop wise and the resulting solution was heated
under reflux at 70 °C for about 1 h. The solvent was removed
under vacuum and the product thus separated was collected.
A mixture of 4-acetamidophenol (6 g, 39.69 mM),
appropriate primary/secondary amine (39.69 mM) and aqueous
formaldehyde (2.46 mL) in ethanol (28.29 mL) was heated
under reflux for 24 h. The solvent was then removed under
Test results were compared with the standard result of
chloroquine. Each test compound was assayed in two replicates
and counted against 400 asexual parasites (% dead rings +
trophozoites) per replicate. The percentage of inhibition of
parasite growth was obtained as mean of duplicate studies.
The minimum inhibitory concentration (MIC) and IC50 values
(in µg/mL) were calculated using the NonLin V1.1 software.

Vol. 28, No. 4 (2016)
Synthesis of Some New 2-Hydroxy-1,4-naphthoquinone-4-hydroxyaniline Hybrid Mannich Bases 785
Molecular properties and drug-likeness prediction:
Drug-like properties of the synthesized compounds, 5a-f was
studied in silico using web based Molsoft (molsoft.com/molprop/)
and Molinispiration Cheminformatics (www.molinspiration.com)
academic softwares. The following molecular descriptors were
calculated for drug-likeness prediction: Molecular weight, water
solubility (log S), octanol-water partition coefficient (log P),
hydrogen bond acceptor and donor count, polar surface area
(PSA), rotable bonds. The drug-likeness score were also calcu-
lated for all the synthesized compounds.
49.27, 32.65, 12.21 (aliphatic CH2 & CH3); MS (API), m/z
(%): 353.18 (100), [M+H]+.
2-Hydroxy-3-(4-hydroxy-3-[(isopropylamino)methyl]-
phenylamino)-2,3-dihydronaphthalene-1,4-dione (5b): UV
spectrum (dichloromethane), λmax (nm): 243.5; IR (KBr, νmax
,
cm-1): 3520, 3452 (2 O-H), 3344, 3329 (2 N-H), 3015, 3010
(C-H aryl), 2962, 2922, 2868, 2826 (C-H, CH3 & CH2), 1677,
1635 (2 C=O), 1620, 1590, 1544, 1488 (C=C aryl), 1364, 1352
(C-N), 1211, 1178 (C-O); 1H NMR (400 MHz, DMSO-d6), δ
(ppm): 9.90 (bs, 1H, alcoholic OH), 8.06-7.98 (m, 4H, Ar-H),
6.62 (d, 1H, J = 7.88 Hz, Ar-H), 6.56 (d, 1H, J = 7.58 Hz, Ar-
H), 6.43 (s, 1H, Ar-H), 6.37 (bs, 1H, phenolic OH), 5.64 (s,
1H, NH-aryl), 3.66 (s, 2H, CH2-NH), 3.07 (s, 1H, NH-alkyl),
2.29 (m, 1H, NH-CH(CH3)2), 1.08 (d, 6H, J = 7.88 Hz,
CH(CH3)2);13C NMR (100 MHz, DMSO-d6), δ (ppm): 184.86,
178.00 (2 C=O), 154.67, 128.02 (2 C-OH), 138.72, 133.93,
130.25, 128.20, 124.52, 118.00 (aromatic CH), 115.39 (olefinic
C=C, CO-C=C-CO), 48.66, 46.28, 12.80 (aliphatic CH2, CH
& CH3); MS (API), m/z (%): 353.70 (100), [M+H]+.
RESULTS AND DISCUSSION
In the present study, a new series of 2-hydroxy-1,4-
naphthoquinone-4-hydroxyaniline hybrid Mannich bases were
synthesized and evaluated for in vitro antimalarial activity.
Several acyclic and cyclic primary/secondary amines were used
for different Mannich substitutions on a conjugated framework
of 2-hydroxy-1,4-naphthoquinone (lawsone) and 4-hydroxy-
aniline scaffolds. The target compounds are thus the Mannich
bases of hybrid skeleton made up of two distinct pharmacophoric
moieties from two potent antimalarial drugs, atovaquone and
amodiaquine, respectively. The physicochemical details of the
synthesized compounds are given in Table-1. The purity of the
synthesized compounds was ascertained by melting point
determinations and silica gel G TLC. The Rf values of the
synthesized compounds are reported herein.All the compounds
were obtained in good yields with high purity. The structural
assignments of the synthesized compounds were made on the
2-Hydroxy-3-(3-hydroxy-4-[(butylamino)methyl]-
phenylamino)-2,3-dihydronaphthalene-1,4-dione (5c): UV
spectrum (dichloromethane), λmax (nm): 247.5; IR (KBr, νmax
,
cm-1): 3530, 3460 (2 O-H), 3355, 3328 (2 N-H), 3012, 3002
(C-H aryl), 2966, 2927, 2865, 2830 (C-H, CH3 & CH2), 1674,
1632 (2 C=O), 1612, 1583, 1556, 1488 (C=C aryl), 1376, 1358
(C-N), 1212, 1180 (C-O); 1H NMR (400 MHz, DMSO-d6), δ
(ppm): 9.90 (bs, 1H, alcoholic OH), 8.04-7.98 (m, 4H, Ar-H),
6.63 (d, 1H, J = 7.68 Hz, Ar-H), 6.54 (d, 1H, J = 7.82 Hz, Ar-
H), 6.42 (s, 1H, Ar-H), 6.38 (bs, 1H, phenolic OH), 5.64 (s,
1H, NH-aryl), 3.63 (s, 2H, CH2-NH), 3.08 (s, 1H, NH-alkyl),
2.32 (t, 2H, J = 7.88 Hz, NH-CH2CH2), 2.02 (m, 2H, CH2CH2CH2),
1.48 (m, 2H, CH2CH2CH3), 1.09 (t, 3H, J = 7.64 Hz, CH2CH3);
13C NMR (100 MHz, DMSO-d6), δ (ppm): 186.40, 178.46 (2
C=O), 155.00, 127.20 (2 C-OH), 138.52, 132.00, 131.58,
128.36, 124.22, 118.36 (aromatic CH), 115.30 (olefinic C=C,
CO-C=C-CO), 52.68, 46.20, 38.29, 33.62, 30.27, 12.10 (aliphatic
CH2 & CH3); MS (API), m/z (%): 367.61 (100), [M+H]+.
2-(4-[(Diisopropylamino)methyl]-3-hydroxyphenyl-
amino)-3-hydroxy-2,3-dihydronaphthalene-1,4-dione (5d):
UV spectrum (dichloromethane), λmax (nm): 250.0; IR (KBr,
1
basis of UV, FT-IR, H NMR, 13C NMR and Mass spectral
studies. The spectral data of 5a-f are depicted below:
TABLE-1
PHYSICO-CHEMICAL DATA OF
SYNTHESIZED COMPOUNDS, 5a-f
Comp.
5a
5b
5c
5d
m.f.
m.w.
Yield (%) m.p. (°C) Rf value*
C20H20N2O4 352.14
C20H20N2O4 352.14
C21H22N2O4 366.16
C23H26N2O4 394.19
C29H22N2O4 462.16
C21H20N2O5 380.14
57.54
82.54
70.76
89.86
58.56
68.67
57-59
71-74
81-84
149-152
47-49
141-143
0.67
0.44
0.49
0.97
0.87
0.21
5e
5f
ν
max, cm-1): 3540, 3452 (2 O-H), 3350, 3335 (2 N-H), 3018,
*Solvent system: Dichloromethane/MeOH-5:5
3000 (C-H aryl), 2964, 2922, 2860, 2826 (C-H, CH3 & CH2),
1677, 1640 (2 C=O), 1632, 1586, 1563, 1498 (C=C aryl), 1364,
1352 (C-N), 1212, 1165 (C-O);1H NMR (400 MHz, DMSO-
d6), δ (ppm): 9.87 (bs, 1H, alcoholic OH), 8.02-7.96 (m, 4H,
Ar-H), 6.66 (d, 1H, J = 7.52 Hz, Ar-H), 6.54 (d, 1H, J = 7.48
Hz, Ar-H), 6.43 (s, 1H, Ar-H), 6.37 (bs, 1H, phenolic OH),
5.65 (s, 1H, NH-aryl), 3.66 (s, 2H, CH2-N), 2.28 (m, 1H, NH-
CH(CH3)2), 1.06 (d, 6H, J = 7.45 Hz, CH(CH3)2); 13C NMR
(100 MHz, DMSO-d6), δ (ppm): 186.04, 178.54 (2 C=O),
155.54, 128.32 (2 C-OH), 138.26, 132.39, 131.60, 128.00,
124.42, 118.00 (aromatic CH), 115.26 (olefinic C=C, CO-
C=C-CO), 48.40, 46.72, 12.20 (aliphatic CH, CH2 & CH3);
MS (API), m/z (%): 395.26 (100), [M+H]+.
2-Hydroxy-3-(4-hydroxy-3[(propylamino)methyl]-
phenylamino)-2,3-dihydronaphthalene-1,4-dione (5a): UV
spectrum (dichloromethane), λmax (nm): 250.0; IR (KBr, νmax
,
cm-1): 3526, 3454 (2 O-H), 3345, 3327 (2 N-H), 3015, 3008
(C-H aryl), 2962, 2920, 2866, 2824 (C-H, CH3 & CH2), 1678,
1630 (2 C=O), 1620, 1592, 1543, 1490 (C=C aryl), 1363, 1350
(C-N), 1210, 1175 (C-O); 1H NMR (400 MHz, DMSO-d6), δ
(ppm): 9.93 (bs, 1H, alcoholic OH), 8.02-7.96 (m, 4H, Ar-H),
6.64 (d, 1H, J = 7.86 Hz, Ar-H), 6.57 (d, 1H, J = 7.68 Hz, Ar-
H), 6.42 (s, 1H, Ar-H); 6.36 (bs, 1H, phenolic OH), 5.63 (s,
1H, NH-aryl), 3.67 (s, 2H, CH2-NH), 3.05 (s, 1H, NH-Alkyl),
2.22 (t, 2H, J = 7.62 Hz, NH-CH2CH2), 1.46 (m, 2H, CH2CH2CH3),
1.13 (t, 3H, J = 7.67 Hz, CH2CH3); 13C NMR (100 MHz,
DMSO-d6), δ (ppm): 184.46, 178.32 (2 C=O), 152.32, 126.26
(2 C-OH), 138.50, 132.23, 130.24, 128.26, 123.26, 118.10
(aromatic CH), 115.39 (olefinic C=C, CO-C=C-CO), 52.20,
2-(4-[(Diphenylamino)methyl]-3-hydroxyphenyl-
amino)-3-hydroxy-2,3-dihydronaphthalene-1,4-dione (5e):
UV spectrum (dichloromethane), λmax (nm): 242.0; IR (KBr,
ν
max, cm-1): 3530, 3450 (2 O-H), 3376, 3330 (2 N-H), 3016,

786 Sharma et al.
Asian J. Chem.
3000 (C-H aryl), 2966, 2928, 2867, 2825 (C-H, CH3 & CH2),
1680, 1634 (2 C=O), 1610, 1595, 1544, 1492 (C=C aryl), 138,
1352 (C-N), 1212, 1180 (C-O);1H NMR (400 MHz, DMSO-
d6), δ (ppm): 9.95 (bs, 1H, alcoholic OH), 8.05-7.98 (m, 4H,
Ar-H), 7.18-7.09 (m, 5H, Ar-H),, 6.92-6.85 (m, 5H, Ar-H),
6.63 (d, 1H, J = 7.54 Hz, Ar-H), 6.58 (d, 1H, J = 7.64 Hz, Ar-
H), 6.52 (s, 1H, Ar-H), 6.47 (bs, 1H, phenolic OH), 5.52 (s,
1H, NH-Aryl), 3.58 (s, 2H, CH2-N); 13C NMR (100 MHz,
DMSO-d6), δ (ppm): 186.24, 176.53 (2 C=O), 156.68, 125.20
(2 C-OH), 146.23, 138.26, 136.34, 132.53, 130.02, 128.61,
129.02, 124.26, 119.26, 118.10 (aromatic CH), 116.32 (olefinic
C=C, CO-C=C-CO), 48.63 (CH2); MS (API), m/z (%): 3467.20
(100), [M+H]+.
methyl Mannich side chain. The mass spectra of 5a-f exhibited
prominent molecular ion peaks, [M+H]+ which are in accor-
dance with the anticipated mass corresponding to their respective
molecular formula..
Antimalarial activity: The in vitro antimalarial activity
data is depicted in Table-2. The IC50 (in µg/mL) represents the
concentration of compound that inhibits the growth of malaria
parasite by 50 %. Results clearly reveal that all the synthesized
compounds, 5a-f showed activity against chloroquine-sensitive
P. falciparum (RKL-2) strain at the tested dose which, however,
was considerably less than that of the standard reference drug,
chloroquine. Among the synthesized compounds, compounds
substituted with n-propyl (5a, IC50 0.453 µg/mL) and morpho-
linyl (5f, IC50 0.391 µg/mL) moieties showed comparatively
better activity against the sensitive strain of P. falciparum than
rest of the compounds having substitutions like isopropyl (5b,
IC50 0.639 µg/mL), n-butyl (5c, IC50 1.263 µg/mL), diisopropyl
(5d, IC50 0.613 µg/mL) and biphenyl (5e, IC50 0.911 µg/mL)
moieties. The IC50 value for chloroquine in sensitive strain of
P. falciparum was found 0.0391 µg/mL. It is apparent that
compounds with smaller alkyl substitutions such as n-propyl
and isopropyl groups and with saturated heterocycle such as
morpholinyl moiety possess superior antimalarial effectiveness
in comparison to other compounds having bulky alkyl or aryl
substitutions such as n-butyl group and phenyl rings.
2-Hydroxy-3[4-hydroxy-3-(morpholinomethyl)cyclo-
hexylamino]-2,3-dihydronaphthalene-1,4-dione (5f): UV
spectrum (dichloromethane), λmax (nm): 248.5; IR (KBr, νmax
,
cm-1): 3522, 3455 (2 O-H), 3365, 3338 (2 N-H), 3018, 3000
(C-H aryl), 2968, 2936, 2864, 2837 (C-H, CH3 & CH2), 1677,
1645 (2 C=O), 1622, 1590, 1544, 1460 (C=C aryl), 1364, 1362
(C-N), 1226, 1180 (C-O); 1H NMR (400 MHz, DMSO-d6), δ
(ppm): 9.89 (bs, 1H, alcoholic OH), 8.04-7.96 (m, 4H, Ar-H),
6.96 (d, 1H, J = 7.40 Hz, Ar-H), 6.72 (d, 1H, J = 7.38 Hz, Ar-
H), 6.55 (s, 1H, Ar-H); 6.34 (bs, 1H, phenolic OH), 5.60 (s,
1H, NH-aryl), 3.53 (s, 2H, CH2-N), 3.46 (t, 2H, J = 7.32 Hz,
morpholinyl CH2), 2.39 (t, 2H, J = 7.46 Hz, morpholinyl CH2);
13C NMR (100 MHz, DMSO-d6), δ (ppm): 185.02, 178.36 (2
C=O), 158.46, 128.28 (2 C-OH), 138.20, 134.24, 130.60,
128.35, 124.40, 118.00 (aromatic CH), 115.04 (olefinic C=C),
65.32 (morpholinyl CH2), 52.06 (morpholinyl CH2), 49.44
(CH2); MS (API), m/z (%): 381.29 (100), [M+H]+.
TABLE-2
In vitro ANTIMALARIAL ACTIVITY DATA#
Comp. code$
MIC (µg/mL)*
3.12 (> 25**)
12.5
IC50 (µg/mL)*
0.453 (2.921**)
0.639
5a
5b
The spectral data depicted above are in close agreement
with the structures of the synthesized compounds, 5a-f. All
the compounds in dichloromethane exhibited characteristic
absorption maxima (λmax in the range of 240-250 nm) indicating
the presence of chromophoric 2-hydroxy-1,4-naphthoquinone
system embedded aminoaryl moiety containing auxochromes
like phenolic hydroxyl group and alkyl/hetero ring substituted
amino methyl chain. The infrared spectral data showed charac-
teristic absorption bands for hydroxyl groups (O-H str., broad
peaks in the frequency range of 3540-3520 cm-1 and 3460-
3450 cm-1 for phenolic and alcoholic, respectively), 2° amino
moieties (Aryl N-H str. and alkyl N-H str., peaks of medium
intensity in the range of 3376-3328 cm-1), two cyclic carbonyl
functions (C=O str., sharp peaks around 1670 cm-1 and 1640-
1630 cm-1) and methylene group (C-H str., about 2900 cm-1
and about 2800 cm-1) [18], which ascertains the anticipated
25
1.263
5c
12.5
0.613
5d
> 25
0.911
5e
5f
1.56 (> 25**)
3.125 (> 25**)
0.391 (0.993**)
0.0391 (0.299**)
Chloroquine$$
#Data are presented as mean of duplicate observations.
*CQ-sensitive P. falciparum (RKL-2) strain.
**CQ-resistant P. falciparum (RKL-9) strain.
$Test dose: 1 mg/mL.
$$Standard dose: 0.1 mg/mL.
Compound 5a and compound 5f were further tested for
antimalarial activity against the resistant strain of P. falciparum
(RKL-9) and were found less active (IC50 2.921 and 0.993
µg/mL respectively) than that of the standard chloroquine
(IC50 0.299 µg/mL). However, compound 5f possesses higher
antimalarial effectiveness than compound 5a in resistant
P. falciparum strain. It has been reported [11] that replacement
of the diethyl functional moiety at the Mannich side chain of
4-aminoquinoline phenol conjugates with the metabolically
stable alkyl grouping and heterocyclic ring as in morpholinyl
modifications lead to a substantial increase in the antimalarial
activity of the compound.
Drug-likeness assessment: Drug-like properties are very
important in assessing that whether a particular molecule
satisfies all desirable properties of the known drugs or not. In
general, drug-like properties include both physicochemically
significant descriptors and pharmacokinetically relevant
1
structure of the synthesized compounds. H NMR spectra
displayed signals (chemical shift values, δ) for different structural
protons of aryl components of conjugated naphthoquinone-
aniline framework along with OH (broad singlet(s) at δ 9.9
and δ 6.3-6.4 for alcoholic and phenolic, respectively), NH
(sharp singlet(s) around δ 5.6 for 2° aryl amino and δ 3.05,
3.07, 3.08 for 2° alkyl amino, 5a-c) and methylene protons
(-CH2NH/CH2N, approximate δ values between 3.5-3.6) [20]
of the synthesized compounds. The 13C resonance data revealed
the presence of different carbon atoms such as aryl-C, C=O,
CH2, CH3, etc. in the structural scaffold of 1,4-napththoquinoe
nucleus and aminoaryl residue along with substituted amino

Vol. 28, No. 4 (2016)
Synthesis of Some New 2-Hydroxy-1,4-naphthoquinone-4-hydroxyaniline Hybrid Mannich Bases 787
properties. Drug-likeness prediction evaluates the acceptability
of derivatives as drug molecules based on Lipinski’s rule of
five [19].
the calculated drug-like properties and in vitro antimalarial
activity profile of the compounds 5a-5f. log Po/w is a direct
indicator of lipophilicity of drug substances. Higher the value
of log P, better the membrane permeability. It is essential for a
molecule to have sufficient lipophilicity for optimal bioavail-
ability and drug action. Hydrogen bond acceptor and donor
groups are of paramount importance in this regard. Polar
surface area is closely related to the hydrogen bonding potential
required for the bio-efficacy of a drug molecule. Practically, a
compound with all drug-like properties in the desired range
appears to exhibit high levels of therapeutic potency. Good
drug-like properties and activity are complementary and hence
balanced attention to both properties and bioactivity could
suitably transform a ligand to a good drug lead [15].
Higher antimalarial activity of the compound 5f might
be because of increased lipophilicity of the molecule. Polar
properties such as hydrogen bond donor (HBD) and hydrogen
bond acceptor (HBA) groups, polar surface area, etc. which
contribute transport property to the drug molecule are respon-
sible as well. Having desired lipophilicity compound 5f (log
P 2.01) readily penetrated parasite’s cell membrane that led to
attain an intracellular concentration which was favourable for
antimalarial action. A good correlation also exists between
other drug-like properties (HBA/HBD groups, polar surface
area, rotable bonds) and antimalarial activity of 5f. Drug-
likeness score of the compound (5f, 0.91) is in support of the
above consideration.
Finally, it can be concluded that 4-hydroxy-1,4-naphtho-
quinone (lawsone) coupled with 4-hydroxyaniline moiety is
attributed to have potential as a new hybrid antimalarial
scaffold. Different Mannich substitutions have contributing
effects towards improving the antimalarial efficacy of the basic
nuclear system. Some degree of variations in activity among
synthesized hybrid compounds might be because of different
alkyl/heterocyclic Mannich substitution pattern. From earlier
studies [11], it is apparent that the presence of a 4-arylamino
moiety and an aryl hydroxyl function appear to be important
for activity both in terms of resistant preventing activity and
antiparasitic activity. It is noteworthy to mention here that the
position of amino and hydroxyl functions in the aryl residue
(as in amodiaquine) would render molecule toxic (as it forms
toxic quinoneimine metabolites) [13,14]. However, because
of having antimalarial effectiveness newer hybrid Mannich
bases can be used as such as lead molecules for further struc-
tural modification/optimization to achieve an array of similar
compounds/analogous with better activity profile. From another
point of view, rational design of some different structural
The results of predicted drug-like properties and drug-
likeness score of the synthesized compounds, 5a-f is presented
in Table-3.All the compounds possess good drug-like properties
based on Lipinski’s rule of five with additional parameters
such as log S and polar surface area. Poor absorption or perme-
ation of a ligand is more likely when there are molecular weight
> 500, log Po/w > 5, number of HBA > 10, number of HBD > 5
and number of Rot B > 5 [15]. Hydrophobicity, membrane
permeability and bioavailability are always associated with
molecular weight, log P, log S and hydrogen bond acceptor
and donor count, etc. Molecules violating more than one of
these rules may have problems with bioavailability. Sufficient
water solubility is important for optimal bioavailability of
drugs. Number of rotable bonds is also important for molecular
conformational studies (i.e., stereoselectivity of drug
molecules) required for optimal binding with the receptor.
Molecular polar surface area is a useful parameter for drug
transport properties [20].
The values of the calculated drug-like properties are in
acceptable range for all the compounds except compound 5e
which violated one rule because of having log P value more
than 5 (5.69). Compounds 5a-e possess less than or equal to 4
hydrogen bond donors and have no more than 6 hydrogen
bond acceptors. The octanol-water partition coefficient (log P)
is less than 5 for all these compounds. None of their molecular
weights exceeds 500 daltons. The number of rotable bonds
and polar surface area are in permissible range for all of the
compounds. Furthermore, the drug-likeness score of the com-
pounds, 5a-5f ranges from 0.18-0.95. Higher drug-likeness
scores were found 0.95 and 0.91 for compounds 5b and 5f
with molecular weights of 352.14 and 380.14, log P values of
2.48 and 2.01, log S values of -5.44 and -3.90, polar surface
area values of 77.98 and 78.82, 5 and 6 hydrogen bond
acceptors (HBA), 4 and 3 hydrogen bond donors (HBD) and
5 and 4 rotable bonds, respectively. The results of drug-likeness
studies strongly suggest that newly synthesized compounds
have drug-likeness behaviour favourable for membrane
permeability, transport and bioavailability and also interaction
with receptor. Assessment of drug-likeness score implies that
the suitability of derivatives as drug-like molecules. Com-
pounds having zero or negative value should not be considered
as drug-like molecule.
Since all the studied compounds showed favourable drug-
like properties a reasonable correlation can be drawn between
TABLE-3
RESULTS OF PREDICTED DRUG-LIKENESS PROPERTIES
Violation of
rule of 5
Drug-likeness
score
Comp.
m.w.
log Pa
log Sb
HBAc
HBDd
Rot Be
PSAf (A2)
352.14
352.14
366.16
394.19
462.16
380.14
2.65
2.48
3.13
3.76
5.69
2.01
-5.29
-5.44
-5.54
-5.68
-7.62
-3.90
5
5
5
5
4
6
4
4
4
3
3
3
6
5
7
6
6
4
79.06
77.98
79.06
70.55
69.34
78.82
0
0
0
0
1
0
0.85
0.95
0.65
0.79
0.18
0.91
5a
5b
5c
5d
5e
5f
aOctanol/water partition coefficient, calculated lipophilicity; bWater solubility in log (mol/L); cNumber of hydrogen bond acceptor (HBA); dNumber
of hydrogen bond donor (HBD); fPolar surface area.

788 Sharma et al.
Asian J. Chem.
derivatives as potent antimalarial agents can be made on
the basis of conjugated 2-hydroxy-1,4-naphthoquinone-4-
hydroxyaniline hybrid scaffold. Metabolic and toxicity studies
are important to be carried out for optimizing those structural
congeners. To overcome the problem of toxicity, the 3-Mannich
side chain can be interchanged with the 4-OH function which
does not generate toxic quinoneimine metabolites (as in
isoquine) [11].
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Conclusion
The present work investigates the synthesis, in vitro
antimalarial activity and drug-likeness prediction of some
newer Mannich bases of 2-hydroxy-1,4-naphthoquinone-4-
hydroxyaniline hybrid. The synthesis and antimalarial activity
of these hybrid molecules have not been reported earlier. The
structural assignments of the new compounds were made on
the basis of IR spectroscopy, NMR spectroscopy and Mass
spectrometric analyses. All the synthesized compounds exhi-
bited some degree of in vitro antimalarial activity against the
chloroquine-sensitive strain (RKL-2) of P. falciparum at the
tested dose, which, however, was considerably less than that
of standard drug, chloroquine. Two of the synthesized com-
pounds with propyl and morpholinyl substitutions showed
good activity, whereas other compounds showed moderate
activity. Since all the compounds exhibited favourable drug-
likeness behaviour a reasonable correlation therefore appears
to exist between their drug-like properties and antimalarial
activity profile. On the basis of our present findings, future
work can be directed towards the rational design of such more
hybrid analogous in structurally diverse series based on the
nuclear scaffold of lawsone (4-hydroxy-1,4-naphthoquinone)
by replacement and/or modification of existing moieties/
groups with more metabolically stable moieties at the Mannich
side chain. This strategy would possibly lead to the develop-
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ACKNOWLEDGEMENTS
The authors are thankful to Sophisticated Analytical
Instrument Facility (SAIF), North-Eastern Hill University, Shillong
(India) for carrying out the spectral analysis of synthesized
compounds.