Synthesis, Polymorphism, and Insecticidal Activity of Methyl 4-(4-chlorophenyl)-8-iodo-2-methyl-6-oxo-1,6-dihydro-4H-pyrimido[2,1-b]quinazoline-3-Carboxylate Against Anopheles arabiensis Mosquito

Article abstract of DOI:10.1111/cbdd.12736

Mosquitoes are the major vectors of pathogens and parasites including those causing malaria, the most deadly vector-borne disease. The negative environmental effects of most synthetic compounds combined with widespread development of insecticide resistance encourage an interest in finding and developing alternative products against mosquitoes. In this study, pyrimido[2,1-b]quinazoline derivative DHPM3 has been synthesized by three-step chemical reaction and screened for larvicide, adulticide, and repellent properties against Anopheles arabiensis, one of the dominant vectors of malaria in Africa. The title compound emerged as potential larvicide agent for further research and development, because it exerted 100% mortality, while adulticide activity was considered moderate.

Full text of DOI:10.1111/cbdd.12736

Chem Biol Drug Des 2016; 88: 88–96
Research Article
Synthesis, Polymorphism, and Insecticidal Activity of
Methyl 4-(4-chlorophenyl)-8-iodo-2-methyl-6-oxo-1,6-
dihydro-4H-pyrimido[2,1-b]quinazoline-3-Carboxylate
Against Anopheles arabiensis Mosquito
Katharigatta N. Venugopala1,*, Susanta K.
fever, Japanese encephalitis, and other diseases (1).
Although malaria control programs have seen a tremendous
expansion over the last decade, according to the 2012
World Malaria Report 219 million cases (range 154–289 mil-
Nayak2, Raquel M. Gleiser3,4, Mariela E.
Sanchez-Borzone5, Daniel A. Garcia5 and
Bharti Odhav1,*
lion) and 660 000 deaths (range 490 000–836 000) still
1Department of Biotechnology and Food Technology,
occur every year (2). Vector control programs mainly use
four classes of chemical insecticides: organochlorines,
organophosphates, carbamates, and pyrethroids. Bacterial
insecticides and insect growth regulators have also become
more widely used in recent years. However, the use of
chemicals on a vast and increasing scale has led to the
widespread development of resistance as a result of selec-
tion for certain genes (3), and some species have even
become resistant to multiple insecticides (4). Mosquito
resistance to at least one insecticide used for malaria control
has been identified in 64 countries (4). Besides, many syn-
thetic organic insecticides, such as those currently used to
control mosquitoes, affect non-target organisms and result
in negative environmental effects (5). Thus, there is an inter-
est in the finding and development of alternative anti-
mosquito products. In continuation of our recent findings on
selection of South African indigenous plants (6) and synthe-
sis of heterocyclic compounds for antimosquito properties
against Anopheles arabiensis (7–9), in the present investiga-
tion, we decide to work on pyrimido[2,1-b]quinazoline sys-
tem. The synthesis and polymorphism property of methyl
4-(4-chlorophenyl)-8-iodo-2-methyl-6-oxo-1,6-dihydro-4H-
pyrimido[2,1-b]quinazoline-3-carboxylate has been the
focus of our continuing interest because the presence of
pharmacophore pyrimido[2,1-b]quinazoline in the title com-
pound is responsible for diverse pharmacological activities.
For example, the substituted dihydropyrimidine ring system
exhibited numerous pharmacological activities such as
antibacterial (10,11), anticancer (12,13), antifungal (13), anti-
hypertensive (14), antiinflammatory (15), and antitubercular
properties (16). Some of the substituted dihydropyrimidine
derivatives have been used as dipeptidyl peptidase-IV
(DPP-4) inhibitors (17) and non-nucleoside HIV-1 reverse
transcriptase inhibitors (18). Among the dihydropyrimidine
pharmacophores, wide spectrum of biological activities
include larvicide actions against mosquitoes (9,19).
Faculty of Applied Sciences, Durban University of
Technology, Durban 4001, South Africa
2Department of Chemistry, Visvesvaraya National Institute
of Technology, Nagpur, Maharashtra 440010, India
3CREAN-IMBIV (CONICET-UNC), Universidad Nacional de
ꢀ
Cordoba, Av. Valparaıso s.n., Cordoba 5000, Argentina
FCEFyN, Universidad Nacional de Cordoba, Av. Velez
ꢀ
ꢀ
4
ꢀ
ꢀ
ꢀ
Sarsfield 299, Cordoba 5000, Argentina
Instituto de Investigaciones Biologicas y Tecnologicas
ꢀ
5
ꢀ
ꢀ
ꢀ
(IIBYT), CONICET-Universidad Nacional de Cordoba, Av.
Velez Sarsfield 1611, Cordoba 5016, Argentina
ꢀ
ꢀ
*Corresponding authors: Katharigatta N. Venugopala,
katharigattav@dut.ac.za; Bharti Odhav, odhavb@dut.ac.za
Mosquitoes are the major vectors of pathogens and
parasites including those causing malaria, the most
deadly vector-borne disease. The negative environ-
mental effects of most synthetic compounds combined
with widespread development of insecticide resistance
encourage an interest in finding and developing alter-
native products against mosquitoes. In this study,
pyrimido[2,1-b]quinazoline derivative DHPM3 has been
synthesized by three-step chemical reaction and
screened for larvicide, adulticide, and repellent proper-
ties against Anopheles arabiensis, one of the dominant
vectors of malaria in Africa. The title compound
emerged as potential larvicide agent for further
research and development, because it exerted 100%
mortality, while adulticide activity was considered
moderate.
Key words: antimosquito properties, crystallography, poly-
morphism, pyrimido[2,1-b]quinazoline, synthesis
Received 3 November 2015, revised 12 January 2016 and
accepted for publication 26 January 2016
Mosquitoes are the major vectors of parasites and patho-
gens that cause malaria, filariasis, dengue fever, yellow
On the other hand, some of the quinazolinone derivatives
have been found to show anticancer (20), anticonvulsant
88
ª 2016 John Wiley & Sons A/S. doi: 10.1111/cbdd.12736

Insecticidal Activity of Pyrimido[2,1-b]Quinazoline Analogue
(21), antifungal (22), antiinflammatory (23), antimicrobial
properties (24). In addition, quinazolinone ring has been
the core structural skeleton in various alkaloids and a vari-
ety of natural products, such as luotonin A (25), oxo-
glyantrypine (26), and schizocommunin (27). Imidazo- and
pyrimido[2,1-b]quinazolines are reported for antihyperten-
sive and antidepressant activities (28). Most importantly,
quinoline moieties incorporated into the compounds have
previously shown activity against insects (29), and quino-
line compounds gave good inhibitory activity for acetyl-
were recorded using LC–MS-Agilent 1100 series with
MSD (Ion trap) using 0.1% aqueous trifluoroacetic acid in
acetonitrile system on C18-BDS column. Elemental analy-
sis was performed on Thermo Finnigan FLASH EA 1112
CHN analyzer. Single crystal X-ray diffraction study was
accomplished using Bruker KAPPA APEX II DUO diffrac-
tometer using graphite monochromator, Mo-Ka radiation
ꢀ
(k = 0.71073 A.
cholinesterase (AChE) (30). The introduction of
a
Synthesis of methyl 4-(4-chlorophenyl)-6-methyl-2-
oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate
(DHPM1)
pyrimidine system to quinazolinone ring is predicted to
influence the significant antimosquito properties. The title
compound methyl 4-(4-chlorophenyl)-8-iodo-2-methyl-6-
oxo-1,6-dihydro-4H-pyrimido[2,1-b]quinazoline-3-carboxy-
late has been achieved by three-step chemical reactions
starting from Biginelli reaction (31) and characterized by
IR, NMR, LC-MS, elemental analysis, and single crystal X-
ray studies and evaluated for antimosquito properties
against A. arabiensis for larvicide, adulticide, and repel-
lency properties. For adulticide properties, the test com-
pounds were sprayed onto ceramic tiles and screened
using the cone bioassay method. The larvicide activity was
tested by monitoring larval mortality daily and up to 3 days
of exposure. Repellency properties were tested in a feed-
ing-probe assay using unfed female A. arabiensis. Previ-
ously, we reported synthesis and polymorphic forms of
heterocyclic compounds (32,33). Polymorphism is known
to lead to changes in various physical and chemical prop-
erties of the compound such as melting point, particle
size, stability, tableting, dissolution rates, bioavailability,
and pharmacological activity (29). In some cases, it is also
observed that the polymorphic forms exhibit nearly similar
properties; for example, the polymorphic forms ato-
vaquone, antimalarial drug results in a similar binding fea-
ture with cytochrome bc1 (30). It is interesting to note that
the structures in solution state have been also observed to
be similar to those determined by X-ray crystallography or
other spectroscopic methods in solution state (31).
A solution of methyl acetoacetate (0.12 mol), 4-chlorobenzal-
dehyde (0.1 mol), and urea (0.1 mol) was refluxed in the pres-
ence of concentrated hydrochloric acid (0.05 mol) for 8 h in
50 mL of ethanol (Scheme 1). Completion of reaction was
monitored on TLC. The reaction mixture was then cooled to
room temperature, and the pure precipitate was collected by
filtration. The solid obtained was washed with cold Et-OH,
dried, and recrystallized using Et-OH solvent. Appearance:
yellow solid; yield 66%. m.p. 205–206 °C. IR (KBr) m cmÀ1
3240, 3110, 1724, 1704, 1649, 1490, 1461, 781; 1H-NMR
(400 MHz, DMSO-d6) d 2.30 (s, 3H), 3.92 (s, 3H), 5.43 (s,
1H), 7.13 (d, 2H, J = 9.05 Hz), 7.51 (s, 1H), 7.86 (d, 2H,
J = 9.05 Hz), 9.01 (s, 1H). 13C-NMR (400 MHz, DMSO-d6) d
18.66, 52.05, 54.35, 109.58, 113.20, 128.23, 136.26,
148.25, 153.40, 159.18, 167.76. MS: m⁄ z 281 [M + H]+.
Synthesis of methyl 2-chloro-4-(4-chlorophenyl)-6-
methyl-1,4-dihydropyrimidine-5-carboxylate
(DHPM2)
A solution of compound DHPM1 (1 mol) in POCl3 (5 mol)
was refluxed for 16 h (Scheme 1). The reaction was moni-
tored by TLC. Unreacted POCl3 was evaporated com-
pletely at room temperature, and the remaining residue
was taken in ethyl acetate and washed with 10% sodium
bicarbonate solution followed by water and finally brine
solution. The ethyl acetate layer was dried over sodium
sulfate and evaporated to obtain a solid which was recrys-
tallized using ethanol solvent. Appearance: brown solid;
yield 72%. m.p. 218–219 °C. IR (KBr) m cmÀ1 3244, 3108,
Methods and Materials
1
General chemistry
1708, 1641, 1491, 1466, 786. H-NMR (400 MHz, DMSO-
All the chemicals were obtained from Aldrich and Merck
chemical company. Reactions were monitored by thin-
layer chromatography (TLC). TLC was performed on Mer-
ck (Sandton, South Africa) 60 F-254 silica gel plates with
ethyl acetate and n-hexane (6:4) as solvent system and
visualization with UV-light. Melting points were determined
d6) d 2.32 (s, 3H), 3.91 (s, 3H), 5.44 (s, 1H), 7.13–7.86
(m, 4H), 9.02 (s, 1H). 13C-NMR (400 MHz, DMSO d6) d
18.72, 53.67, 54.35, 105.41, 113.20, 128.54, 135.29,
141.20, 145.67, 158.39, 167.76. MS m⁄ z 299 [M + H]+.
€
on a Buchi melting point B-545 apparatus. The IR spectra
Synthesis of methyl 4-(4-chlorophenyl)-8-iodo-2-
methyl-6-oxo-1,6-dihydro-4H-pyrimido[2,1-b]
quinazoline-3-carboxylate (DHPM3)
A mixture of methyl-2-chloro-4-(4-chlorophenyl)-6-methyl-
1,4- dihydropyrimidine-5-carboxylate DHPM2 (1 mmol), 2-
amino-5-iodobenzoic acid (1 mmol), and methanamine
(1 mmol) in 2-propanol (15 mL) was refluxed for 12 h. The
reaction completion was monitored by TLC. The reaction
were recorded on a Nicolet 6700 FT-IR spectrometry. 1H
and 13C NMR spectra were recorded on Bruker AVANCE
III 400 MHz instruments in DMSO as a solvent. Chemical
shifts (d) were indicated in parts per million downfield from
tetramethylsilane, and the coupling constants (J) are
recorded in Hertz. Splitting pattern is abbreviated as fol-
lows: s, singlet; d, doublet; m, multiplet. Mass spectra
Chem Biol Drug Des 2016; 88: 88–96
89

Venugopala et al.
Cl
Cl
O
O
NH2
(b)
(a)
+
+
O
O
NH
O
H2N
O
N
H
O
H
O
DHPM1
Cl
Cl
O
OH
O
O
O
H2N
(c)
+
I
N
O
N
O
I
N
H
N
N
H
Cl
DHPM2
DHPM3
Scheme 1: Synthetic routes to dihydropyrimidine analogue DHPM3: Reagents and conditions; (a) Ethanol, HCl, 8 h, reflux; (b) POCl3,
16 h, reflux; (c) Methanamine, 2-propanol, 12 h, reflux.
medium was cooled to room temperature, and the pro-
duct was filtered, washed with cold 2-propanol, and dried
to obtain the crude product. The product was purified by
recrystallization using ethanol. Appearance: yellow crys-
talline compound; yield 69%. m.p. 194 °C. IR (KBr):
m cmÀ1 3356, 3193, 1674, 1588, 1527, 1492, 838. 1H
NMR (400 MHz, DMSO-d6): d 2.38 (s, CH3, 3H), 3.60 (s,
CH3, 3H), 5.39 (s, CH, 1H), 7.32–7.83 (m, Ar-H, 7H),
10.77 (s, NH, 1H), 13C NMR (400 MHz, DMSO-d6): d
18.14, 51.82, 55.82, 98.97, 102.01, 104.77, 116.39,
128.92, 129.17, 130.16, 133.20, 141.01, 145.78, 150.20,
158.54, 161.16, 165.27. LCMS (m/z): 507 (M+). Anal.
Calcd for C20H15ClIN3O3: C, 47.31; H, 2.98; N, 8.28%.
Found C, 47.39; H, 3.01; N, 8.31%.
However, polymorphic form DHPM3b was obtained by
similar way employing a mixture of acetonitrile and tetrahy-
drofuran at 1:1 ratio as plates by slow evaporation method
at room temperature. Structural confirmation of poly-
morphs DHPM3a and DHPM3b was achieved by single
crystals X-ray diffraction study using Bruker KAPPA APEX
II DUO diffractometer using graphite monochromator, Mo-
ꢀ
Ka radiation (k = 0.71073 A; Table 1). Data collections
were carried out at 173 (2) K temperature using liquid
Nitrogen (N2) cryo-system attached with Oxford cryostat.
Cell refinement and data reduction were performed using
the program SAINT. The crystal structure solution was
worked out by full matrix least-squares method using
SHELXL97 and absorption correction performed using
SADABS (34). All the non-hydrogen atoms and some of
hydrogen atoms were located in difference Fourier maps
and were refined anisotropically, and the remaining
hydrogen atoms were fixed geometrically and refined
isotropically. Graphical presentations were drawn using
Ortep-3 and Mercury (35,36). The crystal structure solution
Crystallography
Crystals of polymorphic form DHPM3a was obtained from
mixture of methanol and tetrahydrofuran at 1:1 ratio as
plates by slow evaporation method at room temperature.
90
Chem Biol Drug Des 2016; 88: 88–96

Insecticidal Activity of Pyrimido[2,1-b]Quinazoline Analogue
Table 1: Crystal data and measurement details for polymorphs
DHPM3a and DHPM3b
monitored for larval mortality at 24-h intervals for a period
of 2 days, and the percentage mortality was calculated rel-
ative to the initial number of exposed larvae. Throughout
the experiment, larvae were fed specially made cat food
with reduced oil/fat content at regular intervals.
Crystal data
DHPM3a
DHPM3b
Formula
C20H15ClIN3O3
920295
506.98
C20H15ClIN3O3
1415679
506.98
CCDC Number
Formula weight
Crystal morphology
Crystal size(mm)
Temperature/K
Radiation
Plate
Plate
Adulticide activity
0.25 9 0.14 9 0.12 0.16 9 0.10 9 0.04
173 (2)
Mo Ka
Adulticide activity was assessed by exposing susceptible
adult mosquitoes to a treated surface, in accordance with
WHO protocol (1975) (1), and determining the knockdown
rate, which was based on temporary paralysis of the mos-
quitoes during a 60-min exposure period, and mortality
24-h postexposure. One mL of test compound solution (1,
1.5, or 2 mg/mL in acetone) was sprayed onto a clean,
dry, non-porous ceramic tile using a precalibrated Potter’s
Tower apparatus (38) that was then air-dried. The assay
began within 24 h of spraying, by fixing a cone over the
sprayed tile and introducing thirty non-blood-fed, 2 to 5-
day-old susceptible adult A. arabiensis mosquitoes into
the cone. Acetone and water were used as a negative
controls and deltamethrin (15 g/L; K-Othrineꢀ) as a posi-
tive control. All bioassays were triplicate. Lethal adulticide
concentration was estimated based on the following con-
centration gradient: 0 (control), 0.5, 1.0, and 2.0 lg/mL of
the test compound in acetone.
173 (2)
Mo Ka
ꢀ
Wavelength (A)
0.71073
Triclinic
P -1
0.71073
Triclinic
P -1
Crystal system
Space group
ꢀ
a (A)
7.3443 (15)
10.847 (2)
12.474 (3)
106.657 (3)
103.527 (3)
92.791 (3)
918.47 (1)
2
7.2435 (4)
9.8117 (5)
14.3351 (7)
72.132 (1)
81.315 (1)
83.968 (9)
956.68 (9)
2
ꢀ
b (A)
ꢀ
c (A)
a (°)
b (°)
c (°)
ꢀ3
Volume (A )
Z
Density (gm/cm3)
l (1/mm)
F (000)
1.917
1.91
500
1.76
1.84
500
ϑ (min, max)
2.9, 26.0
1.5, 26.0
13 403
3744
Total number of refln 7109
No. Unique refln
No. of parameters
R_ obs, wR2_obs
3605
255
0.024, 0.059
À0.518, 0.888
259
0.019, 0.050
À0.267, 0.447
ꢀÀ3
Dqmin(eA ),
ꢀÀ3
Dqmax(eA
)
Repellence assays
GooF
1.085
1.073
Repellent activity was assessed by topical application of
the compound to skin of a rodent and subsequent expo-
sure of the treated skin to unfed female mosquitoes, as
described in Venugopala et al. (8). Ethical approval for the
use of Mastomys coucha in these trials was approved
from the Medical Research Council’s Ethics Committee for
Research on Animals. Adult Mastomys were anesthetized
by injecting intraperitoneally with sodium pentobarbital
(concentration in accordance with the weight of the ani-
mal). The anesthetized rodents were then shaved on the
ventral surface, and a test compound in acetone (1 or
1.5 mL) was applied to each rodent’s abdomens. Acetone
was used as a solvent for the preparation of stock solution
(1 mg/mL). Laboratory grade DEET (IUPAC: N,N-Diethyl-3-
methylbenzamide) was used as the positive control, and
plane acetone was used as negative control. Thirty unfed
4-day-old A. arabiensis females were introduced into
paper cups (500 mL) whose base was replaced with mos-
quito netting and held in contact with the treated ventral
surface of each rodent. Mosquito activity was observed
through transparent plastic film closing the mouth of the
cup. After a period of 2 min, the numbers of mosquitoes
probing were recorded. The cups holding the mosquitoes
were removed, and mosquitoes were further observed for
24 h. All tests were triplicate. The rodent was then
returned to the animal facility and allowed to recover from
anesthetic. No adverse reactions to the applied compo-
nents were observed on any of the Mastomys rodents
during the 3 days they were monitored. Repellence of the
was worked out by full matrix least-squares method using
SHELXL97 and absorption correction performed using
SADABS. All the non-hydrogen atoms and some hydrogen
atoms were located in difference Fourier maps and were
refined anisotropically, and other hydrogen atoms were
fixed geometrically and refined isotropically.
Antimosquito properties
Larvicide activity
The A. arabiensis used were from a colonized strain from
Zimbabwe reared according to the WHO (1975) guidelines
(37) in an insectary reproducing the temperature (27.5 °C),
humidity (70%), and lighting (12/12) of a malaria endemic
environment. One milliliter of test compound (0.125–1 mg/
mL in acetone) was added to distilled water (249 mL) pro-
ducing a final concentration of 0.5–4 lg/mL. Groups of
thirty 3rd instar larvae were exposed to test concentrations
of 0 (control), 0.5, 1.0, 2.0, and 4.0 lg/mL of the test
compound in distilled water. The negative controls (0 lg/
mL) were set up using a solvent (acetone) and distilled
water, and a positive control included temephos (Mostop;
Agrivo), an effective emulsifiable organo-phosphate larvi-
cide used by the malarial control program. Bioassays were
triplicate to ensure validity of results. Each container was
Chem Biol Drug Des 2016; 88: 88–96
91

Venugopala et al.
test compounds was calculated as the percentage of
mosquitoes not biting from the total exposed to the
rodent.
DHPM3 is illustrated in Scheme 1. Intermediates DHPM1
and DHPM2 are achieved at 66% and 72% yield, respec-
tively. Title compound DHPM3 exhibited –NH- and -CO-
stretching at 3356 and 1674 cmÀ1, respectively. Molecular
ion peak M + 1 was in agreement with calculated molecu-
lar mass of the compound 507 (m/z). Carbonyl carbon in
DHPM3 compound was found at d 165.27 in 13C NMR
spectra and d 5.39 for –CH- in 1H NMR spectra. Elemen-
tal analysis result was according to the calculated values
of the proposed title compound DHPM3. The IR, NMR (1H
Statistical analysis
General linear mixed modelsa were used to determine dif-
ferences between treatments registered in larval mortality
(larvicide assays) and adulticide effects. LSD Fisher test
was used for post hoc analyses. In all cases, a value of
p < 0.05 was considered statistically significant. Through-
out the text, the results are presented as the mean plus/
minus the standard error. Dependent variables were
A. arabiensis knockdown or mortality. Fixed effects were
test compound (quinazoline, water, acetone, temephos, or
K-Othrine) and observation period (24 and 48 h for larvi-
cide assays; 30, 60 min, and 24 h for adulticide assays).
Random effects were mosquito groups (i.e. container in
larvicide tests; cup in adulticide). For repellent effects, gen-
eral linear models were used to determine differences
between treatments. LSD Fisher test was used for post
hoc analyses. Dependent variables were A. arabiensis
repellence and knockdown. Fixed effects were test com-
pound (quinazoline, water, acetone, DEET). Data gathered
from the larvicide and adulticide assays were used to
determine the concentration of each of the compounds
needed for 50% mortality. The concentration of compound
inducing 50% mortality (LC50) was calculated using
GraphPad Prism version 6.00 for Windowsb.
&
13C), and elemental analysis data are discussed in detail
under experimental section.
Crystallography
From X-ray crystallography, the DHPM3 shows polymor-
phism properties with differences in their preference of
hydrogen and/or halogen bonding intermolecular interac-
tions. The crystallographic details (Table 1) of DHPM3a
and DHPM3b prove clearly the existence of two different
crystalline form of DHPM3. The structural details of
DHPM3a form is only reported previously by us (39); how-
ever, for the comparison, we report both DHPM3a and
DHPM3b form. In DHPM3a form, molecule is stabilized by
two intermolecular C—HÁÁÁO type interactions (methyl and
aromatic hydrogen atoms), whereas in DHPM3b, the
molecule is stabilized by methyl hydrogen C—HÁÁÁO inter-
action. It is interesting to note that DHPM3a and DHPM3b
are structural isomers obtained from crystallization method
using combination of different solvents. In solution state,
the structural preferences of DHPM3 could be either one
of them due to their preferred non-covalent interactions.
Results and Discussion
Chemistry
The dihedral angle between the planes of the 4-chlorophe-
nyl and iodophenyl groups is 80.3 (2)° and 89.9 (2)° for
DHPM3a and DHPM3b, respectively (Figure 1). The crys-
tal structure of both forms is stabilized by N—HÁÁÁO infinite
Design of the molecule is achieved based on the pharma-
cophore available in dihydropyrimidine larvicide com-
pounds (9). The synthetic strategy employed to produce
Figure 1: The molecular structure with ellipsoids for non-H atoms drawn at the 50% probability level for DHPM3a and DHPM3b forms
with intramolecular C—HÁÁÁO interactions with dashed lines.
92
Chem Biol Drug Des 2016; 88: 88–96

Insecticidal Activity of Pyrimido[2,1-b]Quinazoline Analogue
hydrogen bond chains. It is worthy to mention that the
negative controls, and increased with time. The LC50 was
estimated to be 1.33 lg/mL (95% confidence intervals are
1.21–1.46 lg/mL).
ꢀ
halogen-involving short contacts IÁÁÁCl [3.427 (2) A,
h1 = 166.1 (2)°; h2 = 90.5 (2)°, Type II (40) are present in
DHPM3a form and absent in DHPM3b form which con-
firm the existence of packing polymorphs of the com-
pound DHPM3 (Figure 2).
Repellence assays
Treatment had a significant effect on mosquito repellence
(p < 0.0001). The results of repellent and knockdown
activities of the quinazoline compared to controls are
shown in Table 4. Although DHPM3 exerted a statistically
higher repellent activity than the negative controls, it was
only 3.3% repellent (only at 1 lg/mL) and significantly
lower than the positive control DEET (100%) (p < 0.05).
On the other hand, mosquito knockdown (96.7% and
98.9% for 1 and 1.5 lg/mL, respectively) was significantly
higher than the negative controls (p < 0.05), and equal to
or slightly lower than the positive control (100%).
Antimosquito properties
Larvicide activity
There were significant effects of treatment (p < 0.0001),
exposure time (p = 0.002), and treatment exposure inter-
action (p = 0.02) on larval mortality (Table 2). The interac-
tion was due to a mortality increase in larvae exposed to
the positive control temephos. The higher concentrations
of DHPM3 (2 and 4 lg/mL) exerted significantly higher
mortality (both 100%) than the positive controls (71.4%
and 73.9% at 24 and 48 h, respectively). At the lowest
concentration tested (0.5 lg/mL), mortalities were not sig-
nificantly higher than the negative controls. The LC50 was
estimated to be 1.08 lg/mL (95% confidence intervals are
0.99–1.19).
Table 2: Mortality of Anopheles arabiensis larvae exposed to
methyl 4-(4-chlorophenyl)-8-iodo-2-methyl-6-oxo-1,6-dihydro-4H-
pyrimido[2,1-b]quinazoline-3-carboxylate (DHPM3) at 0.5, 1, 2,
and 4 lg/mL, and their negative (acetone, water) and positive
(Temephos) controls
Mortality
Adulticide activity
On adulticide assays, there were also significant effects of
the compound (p < 0.0001), of exposure time
(p < 0.0001), and their interaction (p < 0.0001) on mos-
quito knockdown/mortality. The results of the adulticide
assays are shown in Table 3. The positive control K-Othr-
ine showed 100% knockdown/mortality from the first
30 min of exposure, while quinazoline killed 70% of the
mosquitoes after 24 h of exposure to the highest concen-
tration (2 lg/mL). Still, mortality from exposure to quinazo-
line at all concentrations was significantly higher than the
Compound
24 h
48 h
DHPM3 (4 lg/mL)
DHPM3 (2 lg/mL)
DHPM3 (1 lg/mL)
DHPM3 (0.5 lg/mL)
Temephos
100.0 Æ 8.8A
100.0 Æ 8.8A
33.3 Æ 8.8B
15.6 Æ 8.8BC
71.4 Æ 4.4D
0.0 Æ 4.4C
100.0 Æ 8.8A
100.0 Æ 8.8A
37.8 Æ 8.8B
16.7 Æ 8.8bc
73.9 Æ 4.4E
0.0 Æ 4.4C
Acetone
Water
0.0 Æ 4.4C
0.0 Æ 4.4C
Compounds not sharing a letter differ significantly (p < 0.05).
Figure 2: Intermolecular N—HÁÁÁO hydrogen bonds for both forms (DHPM3a and DHPM3b) and DHPM3a form shows additional IÁÁÁCl
short contacts.
Chem Biol Drug Des 2016; 88: 88–96
93

Venugopala et al.
Table 3: Mortality of Anopheles arabiensis adults exposed to
methyl 4-(4-chlorophenyl)-8-iodo-2-methyl-6-oxo-1,6-dihydro-4H-
pyrimido[2,1-b]quinazoline-3-carboxylate (DHPM3) at 1, 1.5, 2 lg/
mL, and their negative (acetone, water) and positive (K-Othrine)
controls
On the other hand, adulticide activity was moderate, which
could be due to stage-related differences in susceptibility or
to differences in actual exposure of the insect to the com-
pounds (larvicide is delivered to the water where the larvae
dwells; exposure of adult to compounds could be due to
either through surface contact or through vapors). Although
significantly higher than controls, it only achieved 70% mor-
tality after 48 h with the highest concentration tested (2 lg/
mL). Repellent activity was negligible; however, exposure to
rodent skin treated with the compounds knocked down a
high proportion of mosquitoes (close to 99%).
Compound
30 min
60 min
24 h
DHPM3 (2 lg/mL)
DHPM3
53.3 Æ 1.4A
63.3 Æ 1.4B
70.0 Æ 1.4C
45.6 Æ 1.4D
51.1 Æ 1.4A
52.2 Æ 1.4A
38.9 Æ 1.4F
(1.5 lg/mL)
DHPM3 (1 lg/mL)
K-Othrine
Acetone
Water
35.6 Æ 1.4E
38.9 Æ 1.4F
100.0 Æ 0.8G 100.0 Æ 0.8G 100.0 Æ 0.8G
0.0 Æ 0.8H
0.0 Æ 0.8H
0.0 Æ 0.8H
0.0 Æ 0.8H
0.0 Æ 0.8H
0.0 Æ 0.8H
Conclusion
Compounds not sharing a letter differ significantly (p < 0.05).
We designed and synthesized the title compound DHPM3
in good yield and developed two polymorphs such as
DHPM3a and DHPM3b. Confirmation and characterization
of the polymorphs were carried out by single crystal X-ray
studies, and antimosquito results indicate that the pyrim-
ido[2,1-b]quinazoline derivative DHPM3 shows larvicide
potential against the malaria vector A. arabiensis that mer-
its further research and development. Our future work will
further examine to understand the antimosquito activities
of both forms of DHPM3.
Table 4: Adult Anopheles arabiensis repellent and knockdown
activity after 24 h of a 2 min exposure to methyl 4-(4-chlorophe-
nyl)-8-iodo-2-methyl-6-oxo-1,6-dihydro-4H-pyrimido[2,1-b]quina-
zoline-3-carboxylate (DHPM3) at 1, 1.5 lg/mL, and their negative
(acetone, water) and positive (DEET) controls during repellence
assays
Repellent assay
Compound
Repellence
Knockdown
DHPM3 (1.5 lg/mL)
DHPM3 (1 lg/mL)
DEET
Acetone
Water
0.0 Æ 0.6A
3.33 Æ 0.6B
100.0 Æ 0.4C
0.0 Æ 0.4A
98.9 Æ 0.7A
96.7 Æ 0.7B
100.0 Æ 0.5A
0.0 Æ 0.5C
Acknowledgments
0.0 Æ 0.4A
0.0 Æ 0.5C
The authors are grateful to National Research Foundation
(Grant number 91995), South Africa, Ministerio de Ciencia,
Within each column, compounds not sharing a letter differ signifi-
cantly (p < 0.05).
ꢁ
ꢁ
e Innovacion Productiva de la Republica
ꢁ
Tecnologıa
Argentina South Africa/Argentina Joint Science and Tech-
nology Research, and Durban University of Technology for
support and facilities. RMG, MSB, and DAG are CONICET
Career Members. The funding institutions had no involve-
ment in the study design; in the collection, analysis, and
interpretation of data; in the writing of the manuscript; and
in the decision to submit the manuscript for publication.
No adverse reactions to the treatments applied were
observed on any of the Mastomys rodents during the
3 days they were monitored.
Quinazoline derivatives have been widely studied for their
antibacterial, antifungal, and anti-HIV among other bioac-
tivities (41). Fenazaquin (4-[[4-(1, 1-dimethylethyl)phenyl)
ethoxy]quinazoline), a quinazoline, has shown miticidal
activity, controlling all stages of mites including eggs. It is
a metabolic inhibitor that interrupts mitochondrial electron
transport at Site 1c. To our best knowledge, very few other
quinazolines have been researched for their insecticidal
activity. A series of 3-[(2- chloroquinolin-3-yl)methyl]quina-
zolin-4(3H)-ones evidenced larvicide activity against an
aquatic midge (Chironomus tentans Fabricius, Chironomi-
dae) (42). Consistently, the pyrimido[2,1-b]quinazoline
derivative DHPM3 showed larvicide activity against the
mosquito A. arabiensis, killing 100% larvae after 24-h
exposure to 2 lg/mL of this product. The estimated LC50
of 1.08 lg/mL was much lower than those for 3-[(2-chlor-
oquinolin-3-yl)methyl]quinazolin-4(3H)-ones against aquatic
Conflict of Interest
The authors declare no conflicts of interest.
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