Discovery and structure activity relationships of 2-pyrazolines derived from chalcones from a pest management perspective

Article abstract of DOI:10.1007/s00044-015-1415-8

Synthesis of chalcones and 2-pyrazoline derivatives has been an active field of research due to the established pharmacological effects of these compounds. In this study, a series of chalcone (1a-i), 2-pyrazoline-1-carbothioamides (2a-i), and 2-pyrazoline

Full text of DOI:10.1007/s00044-015-1415-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-015-1415-8
ORIGINAL RESEARCH
Discovery and structure activity relationships of 2-pyrazolines
derived from chalcones from a pest management perspective
1
Bedia Koc¸yigit-Kaymakc¸ıoglu Nagihan Beyhan1
•
•
˘
˘
Nurhayat Tabanca2 Abbas Ali2 David E. Wedge3
Stephen O. Duke3 Ulrich R. Bernier4 Ikhlas A. Khan2,5
•
•
•
•
•
Received: 2 April 2015 / Accepted: 11 July 2015
Ó Springer Science+Business Media New York 2015
Abstract Synthesis of chalcones and 2-pyrazoline deriva-
tives has been an active field of research due to the estab-
lished pharmacological effects of these compounds. In this
study, a series of chalcone (1a–i), 2-pyrazoline-1-carboth-
ioamides (2a–i), and 2-pyrazoline-1-carboxamide deriva-
tives (3a–g) were synthesized and screened for their potential
pesticide activities. The proposed structures of all the syn-
thesized compounds were confirmed using the elemental
analysis, UV, IR, 1H-NMR, and mass spectroscopy. Among
the total of 25 tested compounds, compounds 1g and 2a and
2e with Biting Deterrence Index (BDI) values of 0.85, 0.83,
and 0.8, respectively, at 25 nmol/cm2 showed the highest
biting deterrent activity against Aedes aegypti, which was
comparable to N,N-diethyl-3-methylbenzamide (DEET).
Compounds 1g, 2a and 2e were subsequently tested in
human-based repellent bioassays, and they showed MED
(minimum effective dose) values of 0.375, 0.094, and
0.375 mg/cm2, respectively. Compound 1e was the most
toxic compound (LC50 = 2.58 ppm), followed by 1f
(LC50 = 5.69 ppm) and 2g (LC50 = 15.14 ppm), against
1-day-old Ae. aegypti larvae. Compounds 1f and 2h 1f and
2h showed the greatest growth inhibition against Col-
letotrichum gloeosporioides (97.6 and 98.5 %, respectively)
at the lowest dose (0.3 lM), which was greater antifungal
activity than with standard commercial fungicides captan and
azoxystrobin. Compounds 2d, 2g and 2h produced 79.5,
98.3, and 82.3 % growth inhibition, respectively, at 30.0 lM
against Botrytis cinerea, which was similar to captan in the
antifungal activity. The active fungicidal compounds (2d, 2g,
and 2h) were weaky phytotoxic, with little or no phytotoxi-
city at concentrations that were fungitoxic. Compound 2h
stimulated the growth of Lemma paucicostata at concentra-
tions that are fungitoxic to several plant pathogens.
Keywords Chalcones Á 2-Pyrazolines Á Carbothioamide Á
Carboxamide Á Aedes aegypti Á
Colletotrichum gloeosporioides Á Botrytis cinerea Á
Lactuca sativa Á Agrostis stolonifera Á Lemna paucicostata
Introduction
˘
˘
& Bedia Koc¸yigit-Kaymakc¸ıoglu
bkaymakcioglu@marmara.edu.tr
The pyrazole moiety is present in several pharmaceuticals and
agrochemicals. Changes in pyrazole structure offer a high
degree of diversity which is useful for the development of
new therapeutic agents (Meegalla et al., 2004; Rueger et al.,
2012; Desai et al., 2013). Many pyrazole derivatives are
known to possess a wide range of bioactivities and are
employed as anti-inflammatory (Keche et al., 2012; Bansal
et al., 2011), antidepressant (Kaplancıkli et al., 2010;
Karuppasamy et al., 2010), antimicrobial (Pathak et al., 2012;
Mamolo et al., 2001; Bondock et al., 2011), antiviral (El-
Sabbagh et al., 2009), and anticancer (Peng-Cheng et al.,
2010; Banday et al., 2010) agents. Furthermore,
1
Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, Marmara University, Haydarpasa, 34668 Istanbul,
Turkey
2
National Center for Natural Products Research, The
University of Mississippi, University, MS 38677, USA
3
Natural Products Utilization Research Unit, USDA-ARS, The
University of Mississippi, University, MS 38677, USA
4
Center for Medical, Agricultural and Veterinary Entomology,
USDA-ARS, Gainesville, FL 32608, USA
5
Division of Pharmacognosy, Department of BioMolecular
Sciences, School of Pharmacy, The University of Mississippi,
University, MS 38677, USA
123

Med Chem Res
1-phenylpyrazoles with alkyl, acyl, thioalkyl, or cyano sub-
stituents at the position 4 exhibit potent insecticidal activity
(Zhong-Zheng and Guang-Fu, 2006; Meegalla et al., 2004;
Jiang et al., 2009). Moreover, 5-amino-1-(2,6-dichloro-4-
trifluoromethylphenyl)-4-trifluoro-methanesulfinyl-1H-pyra-
zole-3-carbonitrile (fipronil) is one of the most commercially
successful insecticides (Ozoe et al., 2000; Davari et al., 2007).
Pyrazoles block the c-aminobutyric acid (GABA)-regulated
chloride channel in insect neurons (Singh and Bhati, 2011).
Fipronil shows excellent insecticidal activity against Plutella
xylostella and has low toxicity to mammals (Nash and
Hoffmann, 2012). In recent years, a large number of active
compounds have been obtained by modifying fipronil at its
1-(3-chloropyridyl)pyrazole moiety, substituted phenyl moi-
ety, aliphatic amide moiety, and amide bridge (Hainzl and
Casida, 1996). Furthermore, amide bonds are a prevalent
motif in natural products as well as other biologically active
compounds. Sedaxane is the mixture of two cis-isomers 20-
[(1RS, 2RS)-1,10-bicycloprop-2-yl]-3-(difluoromethyl)-1-
methylpyrazole-4-carboxanilide and two trans-isomers 20-
[(1RS,2SR)-1,10-bicycloprop-2-yl]-3-(difluoromethyl)-1-
methylpyrazole-4-carboxanilide which has insecticidal
activity (Kang et al., 2013), but sedaxane is sold as a fungicide
on the market (Lamberth et al., 2013). Chalcones are the
versatile intermediates for the synthesis of various hetero-
cyclic systems such as oxazoline, thiazine, oxazine as well as
pyrazoline. The formation of these nucleuses is mainly con-
stituted with the reaction of unsaturated carbonyl unit of
chalcone (Singh and Bhati, 2011). Chalcone is a unique
template that is associated with several promising biological
activities such as antimicrobial (Ahmed et al., 2015), anti-
inflammatory (Sashidhara et al., 2011), antimalarial (Pingaew
prepared by reacting equimolar aldehyde and ketone in the
presence of a base by conventional Claisen–Schmidt con-
densation. 3,5-Disubstituted-4,5-dihydro-1H-pyrazole-1-
carbothioamides (2a–i) were synthesized by refluxing
compounds 1a–e and thiosemicarbazide in the presence of
alkaline medium. Physicochemical and spectroscopic
characterization of the 2-pyrazoline derivatives 1a–i and
2a–i has been previously described (Beyhan et al., 2015).
The purities of the synthesized compounds were checked
by reversed-phase HPLC (Chromasil C18 3.6 9 150 mm
column using acetonitrile and water (50:50 v/v) as the
eluent) and elemental analysis. All compounds showed a
single and sharp peak with
a retention time of
4.129–5.690 min, and also, elemental analysis results were
calculated within 0.3 %. N,3,5-trisubstituted-4,5-dihydro-
1H-pyrazole-1-carboxamides (3a–g) were synthesized by
refluxing selected chalcones with substituted semicar-
bazides in alkaline medium. All the compounds were iso-
lated in satisfactory yields (30–65 %) and purified by
recrystallization from ethanol. Structures of the synthesized
compounds were confirmed by elemental analysis and
spectral (UV, IR, 1H-NMR, 13C-NMR, and mass) data,
which were in line with the proposed structures. The IR
spectra of the compounds 3a–g afforded pyrazoline C=N
stretching (1589–1573 cm-1), carboxamide group N–H
stretching (3462–3232 cm-1), and C=O stretching
(1373–1348 cm-1) bands. The CH2 protons of the pyra-
zoline ring resonated as a pair of doublets of doublets at
3.10–3.22 ppm (HA) and 3.90–4.00 ppm (HB). The CH
(HX) proton appeared as a doublet of doublets at
5.50–6.32 ppm due to vicinal coupling with the two mag-
netically non-equivalent protons of the methylene group at
¨
¨ ¨
et al., 2014), anticancer (Gorgulu et al., 2015; Shankaraiah
et al., 2015), antileishmanial (Boeck et al., 2006), and anti-
malarial (Tadigoppula et al., 2013). Moreover, chalcones find
several applications in agriculture; for example, insect anti-
feedant (Thirunarayanan and Vanangamudi, 2014, Kumar
et al., 2012) and larvicidal (Pasquale et al., 2012; Satyavani
et al., 2015) activities have also been shown by chalcone
derivatives.
position 4 of the pyrazoline ring (JAB: 17.72–18.40, JAX:
5.10–5.50 Hz, JBX: 12.01–12.95 Hz). N–H protons of the
carboxamide group for compounds 3a–g were seen at
9.10–9.37 ppm as singlet. The protons belonging to the
aromatic ring and the other aliphatic groups are observed
with the expected chemical shift and integral values. Mass
spectra (ESI–MS) of compounds showed a [MH]? peak, in
line with their molecular formula.
On the basis of historical backgrounds of the pesticide
activity of chalcone and pyrazole derivatives, we synthe-
sized different substituted pyrazoles derived from chal-
cones and evaluated their activity as insect repellents,
insecticides, fungicides, and herbicides.
Biological activities
Mosquito activity
The biting deterrent activity of 25 tested compounds against
Ae. aegypti is given in Table 1. The chalcone derivative 1g
carrying 4-methylsulfonylphenyl and 4-metoxyphenyl ring
and the 2-pyrazoline-1-carbothioamide derivatives 2a hav-
ing 4-trifluoromethylphenyl with 4-methoxyphenyl and 2e
having thiophen-2-yl with 2,6-dichlorophenyl rings in
2-pyrazoline moiety showed the highest deterrent activity
Results and discussion
Chemistry
The synthetic route to the target compounds is outlined in
Scheme 1. The intermediate chalcones (1a–i) were
123

Med Chem Res
Ar1
O
O
O
a
N
b
+
N
Ar2
Ar1
CH3
Ar2
H
Ar1
Ar2
NH2
S
1a-1i
c
2a-i
Ar1
N
Ar2
N
NH
R
O
3a-g
1a, 2a: Ar1=4-Trifluoromethylphenyl, Ar2=4-Methoxyphenyl
1b, 2b: Ar1=5-Chlorothiophen-2-yl, Ar2=2,6-Dichlorophenyl
1c, 2c: Ar1 =5-Bromothiophen-2-yl, Ar2=2,6-Dichlorophenyl
1d, 2d: Ar1=4-Methylsulfonylphenyl, Ar2=2,6-Dichlorophenyl
1e, 2e: Ar1=Thiophen-2-yl, Ar2=2,6-Dichlorophenyl
1f, 2f : Ar1=Phenyl, Ar2=2,6-Dichlorophenyl
1g, 2g: Ar1=4-Methylsulfonylphenyl, Ar2=4-Metoxyphenyl
1h, 2h: Ar1=Thiophen-2-yl, Ar2=3,4-Dimetoxyphenyl
1i, 2i: Ar1= Thiophen-2-yl, Ar2=4-Dimethylaminophenyl
3a: Ar1=4-Trifluoromethylphenyl, Ar2=4-Methoxyphenyl (R:4-Cl)
3b: Ar1=4-Trifluoromethylphenyl, Ar2=4-Methoxyphenyl (R:4-CH3S)
3c: Ar1=4-Trifluoromethylphenyl, Ar2=4-Methoxyphenyl (R:4-CF3)
3d: Ar1=4-Trifluoromethylphenyl, Ar2=4-Methoxyphenyl (R:3-Cl)
3e: Ar1=4-Methylsulfonylphenyl, Ar2=4-Methoxyphenyl (R:4-Cl)
3f: Ar1=4-Methylsulfonylphenyl, Ar2=4-Methoxyphenyl (4-CH3S)
3g: Ar1=4-Methylsulfonylphenyl, Ar2=4-Methoxyphenyl (R:4-CF3)
Scheme 1 Synthetic pathway for compounds 1a–i, 2a–i, and 3a–g. Reagents and conditions: a NaOH, MeOH; b NH2CSNHNH2, C2H5OH,
NaOH c 4-substituted phenyl semicarbazide (S1–S4), NaOH
with BDI (Biting Deterrent Index) values of 0.85, 0.83, and
0.80 nmol/cm2 at 25 nmol/cm2 which was closed to posi-
tive control, N,N-diethyl-3-methylbenzamide (DEET)
(BDI = 1 nmol/cm2). Compounds 1g, 2a and 2e were
evaluated using human-based cloth-based repellent assay to
determine minimum effective dosage (MED) for repellency.
123

Med Chem Res
Antifungal activity
However, these compounds did not repel Ae. aegypti at
0.375, 0.094, and 0.375 mg/cm2, respectively, compared
with the reference standard, DEET (0.004 0.002 mg/
cm2). In larvicidal bioassays, only chalcone derivatives 1e,
1f and 2-pyrazoline-1-carbothioamide derivative 2g showed
larvicidal activity, whereas all the other compounds did not
show any larvicidal activity at the highest screening dose of
100 ppm. Based on the calculated LC50 values (Table 2),
chalcone derivative 1e carrying thiophene and 2,6-dichlor-
ophenyl was the most toxic compound (LC50 = 2.58 ppm)
against 1-day-old Ae. aegypti larvae, followed by 1f car-
rying phenyl and 2,6-dichlorophenyl (LC50 = 5.69 ppm)
and 2g having 4-methylsulfonylphenyl and 4-methox-
A total of 25 compounds were evaluated against seven
important plant pathogens, and the active compounds are
given in Fig. 1. At the lowest dose, 0.3 lM, chalcone
derivative 1f and 2-pyrazoline-1-carbothioamide derivative
2h having thiophen and 3,4-dimethoxyphenyl rings showed
97.6 and 98.5 % inhibition, respectively, against C.
gloeosporioides after 48 h. Compound 2e possessed good
antifungal activity with 64.6 % growth inhibition of C.
gloeosporioides at 30 lM, although it showed weak anti-
fungal activity against C. acutatum and C. fragariae at the
highest concentration. Compound 2h showed a decrease in
activity against C. gloeosporioides at 30 lM; this lower
antifungal activity at higher concentration was due to
precipitation in the aqueous growth medium. Out of the 25
compounds, only 1f, 2e, and 2h demonstrated weak anti-
fungal activity at 30 lM with 59.0, 41.9, and 73.3 %
inhibition, respectively, against F. oxysporum. Compounds
2e and 2h exhibited the most antifungal activity against
Phomopsis species with 83.5 and 72.3 %, respectively,
growth inhibition of P. obscurans at 30 lM, respectively.
At 30 lM, compounds 1f and 2a and 2g showed greater
fungal growth inhibition than positive control captan
against B. cinerea. The other compounds, 1c, 1g, 1i, 2d, 2e,
and 2 h exhibited fungal growth inhibition that was similar
or slightly higher than positive control azoxystrobin
(64.1 %) at the 30 lM (Fig. 1). 2-Pyrazoline-1-carbox-
amide derivatives (3a–g) were found to be less active than
chalcones (no data were shown).
yphenyl rings on the 2-pyrazoline moiety (LC50
15.1 ppm).
=
Table 1 Mosquito biting deterrent effects of synthesized compounds
against seven- to 13-day-old mated females of Ae. aegypti
Compound
Concentration (nmol/cm2)
BDI value ( SE) (A)
1a
1b
1c
1d
1e
1f
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
0.57 0.093
0.48 0.114
0.53 0.058
0.64 0.094
0.64 0.048
0.64 0.048
0.85 0.145
0.7 0.099
0.75 0.097
0.83 0.044
0.49 0.071
0.63 0.070
0.79 0.064
0.8 0.089
0.73 0.086
0.71 0.078
0.57 0.086
0.62 0.101
0.61 0.070
0.58 0.107
0.61 0.070
0.56 0.063
0.64 0.048
0.58 0.071
0.69 0.087
1g
1h
1i
2a
2b
2c
2d
2e
2f
Phytotoxicity activity
The most active antifungal compounds (2d, 2g, 2h) were
evaluated for their phytotoxicity because, to be used as
fungicides for plant protection, they should have little or no
phytotoxicity. A dose–response experiment was per-
formed using 33, 100, 333, and 1000 lM of each compound
against lettuce and agrostis (Fig. 2). None of the compounds
were significantly phytotoxic to lettuce at the highest con-
centration. Compounds 2g and 2h inhibited the growth of
bentgrass at concentrations of 333 lM and higher. Com-
pounds 2g and 2h were phytotoxic to L. paucicostata
(Fig. 3). 2g completely inhibited growth at 100 lM and
higher, whereas 2h was only effective at 333 lM and
higher. Both compounds stimulated growth at subtoxic
concentrations (hormesis), with remarkable hormesis using
2h at 33 lM. Hormesis caused by natural phytotoxins is a
common phenomenon (Belz et al., 2007). At concentrations
needed for fungicidal activity, these compounds were not
phytotoxic and may have growth stimulatory effects.
2g
2h
2i
3a
3b
3c
3d
3e
3f
3g
SE Standard error of the mean
Values within a column not followed by the same letter are signifi-
cantly different (P \ 0.05, DMRT). A Biting Deterrence Index (BDI)
value of 1 is similar to DEET, whereas a value of 0 is similar to
solvent control at 25 nmol/cm2
123

Med Chem Res
Table 2 Toxicity of the compounds 1e, 1f, and 2g against 1-day-old Ae. aegypti 24 h after treatment
Compound
LC50
(95 % CI)a
LC90
(95 % CI)a
v2
DFb
1e
1f
2g
a
2.58
5.69
(2.27–2.90)
(5.0–6.46)
4.63
11.14
51.12
(3.97–5.81)
59.9
81.0
37.7
48
48
48
(9.44–14.04)
(36.43–90.64)
15.14
(10.93–19.78)
LC50 and LC90 values are given in ppm (95 % confidence interval)
b
DF refers to degree of freedom
molecules can be utilized for the design of new molecules
to find new derivatives for controlling pest and pathogenic
fungi.
Conclusion
A series of chalcones (1a–i), 2-pyrazoline-1-carboth-
ioamides (2a–i), and 2-pyrazoline-1-carboxamide deriva-
tives (3a–g) have been synthesized and screened for the
first time for their pesticidal activities. 2-Pyrazoline-1-
carboxamide derivatives (3a–g) are new compounds and
synthesized for the first time. The chalcone derivative 1g
carrying 4-methylsulfonylphenyl and 4-metoxyphenyl ring
and the 2-pyrazoline-1-carbothioamide derivatives 2a
having 4-trifluoromethylphenyl with 4-methoxyphenyl and
2e having thiophen-2-yl with 2,6-dichlorophenyl rings in
2-pyrazoline moiety showed the highest deterrent activity
with Biting Deterrent Index (BDI) values of 0.85, 0.83, and
0.80 nmol/cm2 at 25 nmol/cm2 which was closed to posi-
tive control, N,N-diethyl-3-methylbenzamide (DEET,
BDI = 1 nmol/cm2). Based on the calculated LC50 values,
chalcone derivative 1e carrying thiophene and 2,6-
dichlorophenyl was the most toxic compound against
1-day-old Ae. aegypti larvae. The chalcone derivative 1f
and 2-pyrazoline-1-carbothioamide derivative 2h showed
the highest antifungal activity, each with similar or greater
activity than commercial fungicides. The most active
fungicidal compounds (2d, 2g, and 2h) were weakly phy-
totoxic, with little or no phytotoxicity at concentrations that
were highly fungitoxic. Compound 2h stimulated the
growth of L. paucicostata at concentrations that are fun-
gitoxic to several plant pathogens.
Experimental
All the reagents were obtained commercially and used by
further purification using standard procedures. Melting
points were determined by an open capillary method and are
uncorrected. The IR spectra were recorded on a PerkinElmer
1
(Spectrum ONE) FT-IR Spectrometer. The H NMR and
13C NMR spectra were recorded (in DMSO-d6) with a
Bruker NMR 400 and 100-MHz spectrometer, respectively.
The chemical shift values are expressed in ppm (d scale)
using tetramethylsilane as an internal standard. The mass
spectral measurements were taken by electrospray method
on LC–MS-Agilent 1100. Elemental analysis was performed
on Leco 215 CHNS-932 analyzer. All the compounds gave
C, H, and N analyses within 0.4 %.
The intermediated chalcones (1a–i) were prepared by
reacting equimolar aldehyde and ketone in the presence of
a sodium hydroxide and methanol through Claisen–Sch-
midt condensation. 3,5-disubstituted-4,5-dihydro-1H-pyra-
zole-1-carbothioamides (2a–i) were synthesized by
refluxing compounds 1a–i and thiosemicarbazide in the
presence of alkaline medium. Physicochemical and spec-
troscopic characterization of the 2-pyrazoline derivatives
1a–i and 2a–i has been previously described (Beyhan et al.,
2015).
The results of bioassays indicated that chalcones (1a–
i) and 2-pyrazoline-1-carbothioamides (2a–i) derivatives
possessed weak-to-excellent activity against mosquito and
plant pathogens. However, 2-pyrazoline-1-carboxamide
derivatives (3a–g) showed weak biting deterrent activity at
25 nmol/cm2 against adult female Ae. aegypti and showed
no mortality at 100 pm against 1-day-old Ae. aegypti lar-
vae and 3a–g compounds also demonstrated insignificant
antifungal activity at a highest concentration of 30 mM
against Colletotrichum acutatum, C. fragariae, C.
gloeosporioides, Botrytis cinerea, Phomopsis viticola, P.
obscurans, and Fusarium oxysporum. Based on these
results, we conclude that the position of the substituent
present on the ring affects the activity, and active
General synthesis methods of N,3,5-trisubstituted-
4,5-dihydro-1H-pyrazole-1-carboxamides (3a–g)
The 5 mmol 4-substituted phenylisothiocyanates were
dissolved in diethyl ether. In order to stir the mixture,
5 mmol hydrazine hydrate was added dropwise and stirred
for 15 min. The obtained 4-substituted phenyl semicar-
bazides (S1–S4) were filtered, dried, and washed with
petrol ether. 2 mmol chalcone (1a–1i) was dissolved in
ethanol (10 ml), and then, semicarbazides (S1–S4) were
added. Finally, solution of 5 mmol NaOH in 1 ml water
123

Med Chem Res
Colletotrichum acutatum Growth Responseto Samples at 48h
Colletotrichum fragariae Growth Response to Samples at 72h
100.0%
80.0%
100.0%
80.0%
0.3 μM
3.0 μM
30 μM
0.3 μM
3.0 μM
30 μM
60.0%
60.0%
40.0%
40.0%
20.0%
20.0%
0.0%
0.0%
(20.0%)
(40.0%)
(60.0%)
(80.0%)
(100.0%)
(20.0%)
(40.0%)
(60.0%)
(80.0%)
(100.0%)
1f
2e
2h
Azoxystrobin
Captan
1f
2e
2h
Azoxystrobin
Captan
Colletotrichum gloeosporoides Growth Response to Samples at 48h
Fusarium oxysporum Growth Responseto Samples at 48h
100.0%
80.0%
100.0%
80.0%
0.3 μM
3.0 μM
30 μM
0.3 μM
3.0 μM
30 μM
60.0%
60.0%
40.0%
40.0%
20.0%
20.0%
0.0%
0.0%
(20.0%)
(40.0%)
(60.0%)
(80.0%)
(100.0%)
(20.0%)
(40.0%)
(60.0%)
(80.0%)
(100.0%)
1f
2e
2h
Azoxystrobin
Captan
1f
2e
2h
Azoxystrobin
Captan
Phomopsis obscurans Growth Responseto Samples at 144h
100.0%
80.0%
0.3 μM
3.0 μM
30 μM
60.0%
40.0%
20.0%
0.0%
(20.0%)
(40.0%)
(60.0%)
(80.0%)
(100.0%)
1f
2e
2h
Azoxystrobin
Captan
Botryꢀs cinerea Growth Response to Samples at 48h
100.0%
80.0%
0.3 μM
3.0 μM
30 μM
60.0%
40.0%
20.0%
0.0%
(20.0%)
(40.0%)
(60.0%)
(80.0%)
(100.0%)
1c
1f
1g
1i
2a
2d
2e
2g
2h AzoxystrobinCaptan
123

Med Chem Res
500
400
300
200
100
bFig. 1 Growth inhibition of Colletotrichum, Fusarium, Phomopsis,
and Botrytis species using 96-well microdilution broth assays in a
dose–response experiment to tested compounds with the commercial
fungicide standard azoxystrobin and captan. Only active compounds
are presented here
2 g
0.14
Control
Lettuce
33
M
100
333
1000
M
M
M
0.12
0.10
0.08
0.06
0.04
0.02
2 h
400
300
200
100
0
Agrostis
12
10
8
6
1
10
100
1000
4
Concentration( M)
2
Fig. 3 Dose–growth response curve of the effects of the compounds
2g and 2h on Lemna pauciscostata growth 7 days after treatment. The
lowest concentration is zero
0
2d
2g
2h
Fig. 2 Growth effects of 2d, 2g, 2h on Lactuca sativa and Agrostis
stolonifera
N-(3-Chlorophenyl)semicarbazide (S4)
Yield 90 %, mp 260–262 °C (EtOH), UV (EtOH), kmax
(EtOH) (nm): 204, 247. IR mmax (cm-1): 3330, 3290, 3220,
3100, 2980, 2924, 1665, 1590, 1530 1490, 1455, 1400,
1093.
was added. After the completion of the reaction by TLC,
the reaction mixture was poured into ice, and the precipi-
tate was filtered, dried, and crystallized from ethanol.
N-(4-Chlorophenyl)semicarbazide (S1)
N-(4-Chlorophenyl)-5-(4-methoxyphenyl)-3-[4-
(trifluoromethylphenyl)]-4,5-dihydro-1H-pyrazole-
1-carboxamide (3a)
Yield 98 %, mp 282–284 °C (EtOH), UV (EtOH), kmax
(EtOH) (nm): 204, 247. IR mmax (cm-1): 3330, 3292, 3218,
3098, 2980, 2924, 1667, 1592, 1530 1491, 1455, 1405, 1093.
Yield 30 %, mp 183–185 °C (EtOH), UV (EtOH), kmax
(loge): 204 (4.00), 225 (4.12), 269 (4.22), 323 (4.25). IR
mmax (cm-1): 3385(NH), 1676 (C=O), 1585 (C=N). 1H-
NMR (400 MHz, DMSO-d6/TMS), d, ppm (J, Hz): 3.21
(1H, dd, HA, JAB: 17.72 Hz, JAX: 5.58 Hz); 3.72 (3H, s,
OCH3); 3.90 (1H, dd, HB, JBA: 17.72 Hz, JBX: 12.01 Hz);
5.53 (1H, dd, HX, JXA: 5.35 Hz, JXB: 12.01 Hz); 6.90 (2H,
d, J: 8.67 Hz, 4-methoxyphenyl H3, H5); 7.18 (2H, d, J:
8.64 Hz, 4-methoxyphenyl H2, H6); 7.31 (2H, d, J:
8.86 Hz, 4-chlorophenyl H3, H5); 7.66 (2H, d, J: 8.91 Hz,
4-chlorophenyl H2, H6); 7.84 (2H, d, J: 8.42 Hz,
N-[4-(Methylsulphanyl)phenyl]semicarbazide (S2)
Yield 92 %, mp 251–252 °C (EtOH), kmax (EtOH) (nm):
206, 240, 269. IR mmax (cm-1): 3248, 3209, 3095, 2976,
2914, 1664, 1587, 1525, 1491, 1402.
N-(4-Trifluoromethlphenyl)semicarbazide (S3)
Yield 90 %, mp 240–242 °C (EtOH), kmax (EtOH) (nm):
204, 245. IR mmax (cm-1): 3335, 3280, 3220, 3100, 2980,
2925, 1665, 1590, 1540 1495, 1450, 1409.
123

Med Chem Res
4-trifluoromethylphenyl H2, H6); 8.14 (2H, d, J: 8.27 Hz,
4-trifluoromethylphenyl H3, H5); 9.29 (1H, s, NH). 13C-
NMR (100 MHz), (DMSO-d6/TMS), d (ppm): 42.55
(pyrazole C4); 60.49 (pyrazole C5); 116.37, 129.15,
129.91, 130.59, 131.89, 132.16, 133.31, 135.59, 136.06,
137.02 (aromatic C and CF3); 149.98 (pyrazole C3);
164.18 (C=O). Anal. Calcd. for C24H19ClF3N3O2 (%); C,
59.67; H 3.89; N 8.86. Found: C, 59.83; H, 3.90; N, 8.84,
ESI–MS: m/z 474 (MH?, 32 %), 347 (15 %), 319 (100 %),
305 (40 %), 212 (10 %).
135.61, 137.14 (aromatic C and CF3); 150.92 (pyrazole
C3); 166.10 (C=O): Anal. Calcd. for C25H19F6N3O2 (%);
C, 59.17; H, 3.77; N, 8.28. Found: C, 59.52; H, 3.78; N,
8.30. ESI–MS: m/z 508 (MH?, 30 %), 347 (25 %) 319
(100 %), 305 (32 %), 212 (10 %), 160 (25 %).
N-(3-Chlorophenyl)-5-(4-methoxyphenyl)-3-[4-
(trifluoromethylphenyl)]-4,5-dihydro-1H-pyrazole-
1-carboxamide (3d)
Yield 30 %, mp 183–185 °C (EtOH), UV (EtOH), kmax
(loge): 204 (4.00), 225 (4.12), 269 (4.22), 323 (4.25), IR
mmax (cm-1): 3385(NH), 1676 (C=O), 1585 (C=N). 1H-
NMR (400 MHz, DMSO-d6/TMS), d, ppm (J, Hz): 3.21
(1H, dd, HA, JAB: 17.76 Hz, JAX: 5.35 Hz); 3.72 (3H, s,
OCH3); 3.90 (1H, dd, HB, JBA: 17.76 Hz, JBX: 12.01 Hz);
5.53 (1H, dd, HX, JXA: 5.35 Hz, JXB: 12.01 Hz); 6.90 (2H,
d, J: 8.67 Hz, 4-methoxyphenyl H3, H5); 7.18 (2H, d, J:
8.64 Hz, 4-methoxyphenyl H2, H6); 7.31 (1H, s, 3-chlor-
ophenyl H2); 7.66 (3H, m, 3-chlorophenyl H4, H5, H6);
7.84 (2H, d, J: 8.42 Hz, 4-trifluoromethylphenyl H2, H6);
8.14 (2H, d, J: 8.27 Hz, 4-trifluoromethylphenyl H3, H5);
9.29 (1H, s, NH). 13C-NMR (100 MHz), (DMSO-d6/TMS),
d (ppm): 42.55 (pyrazole C4); 60.49 (pyrazole C5); 116.37,
129.15, 129.91, 130.59, 131.89, 132.16, 133.31, 135.59,
136.06, 137.02 (aromatic C and CF3); 149.98 (pyrazole
C3); 164.18 (C=O). Anal. Calcd. for C24H19ClF3N3O2 (%);
C, 59.67; H, 3.89; N, 8.86, Found: C, 59.65; H, 3.90; N,
8.84. ESI–MS: m/z 474 (MH?, 20 %), 347 (27 %), 305
(100 %), 212 (10 %), 127 (25 %).
N-(4-(Methylsulfanyl)phenyl)-5-(4-methoxyphenyl)-
3-[4-(trifluoromethylphenyl)]-4,5-dihydro-1H-
pyrazole-1-carboxamide (3b)
Yield 46 %, mp 166–167 °C (EtOH), UV (EtOH), kmax
(loge): 204 (4.70), 225 (4.47), 323 (4.45), IR mmax (cm-1):
3383, 3325 (NH), 1678 (C=O), 1579 (C=N). 1H-NMR
(400 MHz, DMSO-d6/TMS), d, ppm (J, Hz): 2.44 (3H, s,
SCH3); 3.22 (1H, dd, HA, JAB: 18.10 Hz, JAX: 5.50 Hz);
3.72 (3H, s, OCH3); 3.90 (1H, dd, HB, JBA: 18.10 Hz, JBX
:
:
12.04 Hz); 5.50 (1H, dd, HX, JXA: 5.47 Hz, JXB
12.04 Hz); 6.90 (2H, d, J: 8.60 Hz, 4-methoxyphenyl H3,
H5); 7.19 (4H, dd, J1: 6.44 Hz, J2: 6.37 Hz, 4-methox-
yphenyl H2, H6 and 4-methylsulfanylphenyl H2, H6); 7.58
(2H, d, J: 8.66 Hz, 4-methylsulfanylphenyl H3, H5); 7.84
(2H, d, J: 8.40 Hz, 4-trifluoromethylphenyl H2, H6); 8.15
(2H, d, J: 8.27 Hz, 4-trifluoromethylphenyl H3, H5); 9.19
(1H, s, NH). 13C-NMR (100 MHz), (DMSO-d6/TMS), d
(ppm): 16.41 (SCH3); 42.55 (pyrazole C4); 55.76 (OCH3);
60.73 (pyrazole C5); 130.11, 130.43, 131.32, 135.53,
135.97, 137.57, 151.14 (aromatic C and CF3); 151.83
(pyrazole C3); 159.16 (C=O). Anal. Calcd. for C25H22F3
N3O2S (%); C, 61.84; H, 4.57; N, 8.65; S 6.60. Found: C,
61.86; H, 4.53; N, 8.62; S, 6.61. ESI–MS: m/z 486 (MH?,
20 %), 319 (100 %), 305 (40 %), 212 (10 %), 166 (25 %).
N-(4-Chlorophenyl)-5-(4-methoxyphenyl)-3-[4-
(methylsulfonylphenyl)]-4,5-dihydro-1H-pyrazole-1-
carboxamide (3e)
Yield 45 %, mp 256–257 °C (EtOH), UV (EtOH), kmax
(loge): 203 (4.05), 248 (4.15), 347 (4.20), IR mmax (cm-1):
3440, 3220 (NH), 1690 (C=O), 1580 (C=N). 1H-NMR
(400 MHz, DMSO-d6/TMS), d, ppm (J, Hz): 3.17 (1H, dd,
HA, JAB: 18.07 Hz, JAX: 5.10 Hz); 3.20 (3H, s, –SO2CH3);
3.96 (1H, dd, HB, JBA: 18.06 Hz, JBX: 12.95 Hz); 6.31 (1H,
dd, HX, JXA: 5.10 Hz, JXB: 12.95 Hz); 6.90 (2H, d, J:
8.67 Hz, 4-methoxyphenyl H3, H5); 7.16 (2H, d, J:
8.66 Hz, 4-methoxyphenyl H2, H6); 7.30 (2H, d, J:
8.90 Hz, 4-chlorophenyl H3, H5); 7.64 (2H, d, J: 8.90 Hz,
4-chlorophenyl H2, H6); 7.97 (2H, d, J: 8.60 Hz,
4-methylsulfonylphenyl H2, H6); 8.12 (2H, d, J: 8.20 Hz,
4-methylsulfonylphenyl H3, H5); 9.10 (1H, s, NH). 13C-
NMR (100 MHz), (DMSO-d6/TMS), d (ppm): 40.34
(pyrazole C4); 44.00 (SO2CH3); 60.0 (pyrazole C5);
129.28, 129.73, 131.17, 131.68, 133.09, 133.91, 134.01,
136.21, 137.62 (aromatic C); 151.20 (pyrazole C3); 164.20
(C=O). Anal. Calcd. for C24H12ClN3O4S (%); C, 60.07; H,
N-(4-Trifluoromethylphenyl)-5-(4-methoxyphenyl)-
3-[4-(trifluoromethylphenyl)]-4,5-dihydro-1H-
pyrazole-1-carboxamide (3c)
Yield 55 %, mp 252–254 °C (EtOH), UV (EtOH), kmax
(loge): 206 (4.10), 224 (4.12), 338 (4.20), IR mmax (cm-1):
3360, 3248 (NH), 1670 (C=O), 1590 (C=N). 1H-NMR
(400 MHz, DMSO-d6/TMS), d, ppm (J, Hz): 3.20 (1H, dd,
HA, JAB: 18.40 Hz, JAX: 5.20 Hz); 4.00 (1H, dd, HB, JBA
18.40 Hz, JBX: 12.80 Hz); 6.30 (1H, dd, HX, JXA: 5.20 Hz,
XB: 12.80 Hz); 6.90 (2H, d, J: 8.60 Hz, 4-methoxyphenyl
:
J
H3, H5); 7.19 (4H, dd, J1: 6.44 Hz, J2: 6.37 Hz, 4-meth-
oxyphenyl H2, H6); 7.58–8.18 (8H, m aromatic protons);
9.25 (1H, s, NH), 13C-NMR (100 MHz), (DMSO-d6/TMS),
d (ppm): 40.93 (pyrazole C4); 60.34 (pyrazole C5); 128.76,
129.14, 129.85, 130.58, 130.75, 131.35, 133.30, 134.26,
123

Med Chem Res
4.53; N, 8.66; S 6.60. Found: C, 60.02; H 4.54; N 8.68; S
6.61. ESI–MS: m/z 484 (MH?, 18 %), 358 (30 %), 329
(100 %), 224 (10 %), 127 (20 %).
8.20; S 6.21. ESI–MS: m/z 518 (MH?, 22 %), 358 (25 %),
329 (60 %), 305 (100 %), 160 (21 %).
N-(4-(Methylsulfanyl)phenyl)-5-(4-methoxyphenyl)-
3-[4-(methylsulfonylphenyl)]-4,5-dihydro-1H-
pyrazole-1-carboxamide (3f)
Biological activities
Insects
Yield 53 %, mp 158–160 °C (EtOH), UV (EtOH), kmax
(loge): 202 (4.14), 221 (4.10), 250 (4.20), 348 (4.10), IR
mmax (cm-1): 3400, 3250 (NH), 1686 (C=O), 1580 (C=N).
1H-NMR (400 MHz, DMSO-d6/TMS), d, ppm (J, Hz): 3.22
(1H, dd, HA, JAB: 17.43 Hz; JAX: 5.50 Hz); 3.25 (3H, s,
The Ae. aegypti (L.) mosquitoes used in these studies were
from a laboratory colony maintained since 1952, originally
from Orlando, FL, and now at the Mosquito and Fly
Research Unit at the Center for Medical, Agricultural and
Veterinary Entomology, USDA-ARS, in Gainesville,
Florida, USA. For biting deterrence bioassays, eggs were
hatched, and the insects were reared to the adult stage in a
laboratory maintained at 27 2 °C and 60 10 % RH
with a photoperiod regimen of 12:12 h (L–D), and eight- to
18-day-old adult females were used. For larval bioassays,
the eggs were hatched, and the larvae were maintained at
the above temperature.
–SO2CH3); 3.98 (1H, dd, HB, JBA: 17.43 Hz, JBX
:
:
12.90 Hz); 6.32 (1H, dd, HX, JXA: 5.50 Hz, JXB
12.90 Hz); 6.91 (2H, d, J: 8.67 Hz, 4-methoxyphenyl H3,
H5); 7.18 (2H, d, J: 8.64 Hz, 4-methoxyphenyl H2, H6);
7.19 (2H, d, J: 8.64 Hz, 4-methylsulfanylphenyl H2, H6);
7.58 (2H, d, J: 8.66 Hz, 4-methylsulfanylphenyl H3, H5);
7.97 (2H, d, J: 8.60 Hz, 4-methylsulfonylphenyl H2, H6);
8.12 (2H, d, J: 8.20 Hz, 4-methylsulfonylphenyl H3, H5);
9.28 (1H, s, NH). 13C-NMR (100 MHz), (DMSO-d6/TMS),
d (ppm): 16.40 (SCH3); 40.34 (pyrazole C4); 44.02 (SO2
CH3); 60.49 (pyrazole C5); 116.37, 129.15, 129.91, 130.59,
131.89, 132.16, 133.31, 135.59, 136.06, 137.02 (aromatic
C); 149.98 (pyrazole C3); 164.18 (C=O). Anal. Calcd. for
C25H25N3O4S2 (%); C, 61.14; H, 5.10; N, 8.91; S, 12.36.
Found: C, 61.12; H, 5.08; N, 8.88; S, 12.34. ESI–MS: m/z
496 (MH?, 18 %), 358 (25 %), 329 (100 %), 166 (25 %).
Mosquito biting bioassay
Experiments were conducted by using a six-celled in vitro
Klun & Debboun (K&D) module bioassay system devel-
oped by Klun et al. (2005) for quantitative evaluation of
biting deterrent properties of candidate compounds.
Briefly, the assay system briefly consists of a six-well
reservoir with each of the 3 9 4 cm wells containing 6 ml
of blood. As described by Ali et al. (2012), a feeding
solution consisting of CPDA-1 and ATP was used instead
of blood. Green fluorescent tracer dye (www.
blacklightword.com) was used to determine feeding by
the females. Chalcone and pyrazole derivatives were tested
in this study. Samples were applied at concentrations of
25 nmol/cm2, and DEET (97 %, N,N-diethyl-3-methyl-
benzamide) (Sigma-Aldrich, St. Louis, MO) at 25 nmol/
cm2 was used as positive control. All treatments were
freshly prepared in molecular biology grade 100 % ethanol
(Fisher Scientific Chemical Co. Fair Lawn, NJ) at the time
of bioassay. Temperature of the solution in the reservoirs
was maintained at 37 °C by continuously passing warm
water through the reservoir using a circulatory bath.
Reservoirs were covered with a layer of collagen mem-
brane (Devro, Sandy Run, SC). Test compounds were
randomly applied to six 4 9 5 cm areas of organdy cloth
and positioned over the membrane-covered CPDA-
1 ? ATP solution with a Teflon separator placed between
the treated cloth and the six-celled module to prevent the
contamination of the module. A six-celled K&D module
containing five female mosquitoes per cell was positioned
over the cloth treatments covering the six CPDA-1 ? ATP
solution membrane wells, and trap doors were opened to
N-(4-Trifluoromethylphenyl)-5-(4-methoxyphenyl)-
3-[4-(methylsulfonylphenyl)]-4,5-dihydro-1H-
pyrazole-1-carboxamide (3g)
Yield 65 %, mp 281–283 °C (EtOH), UV (EtOH), kmax
(loge): 204 (4.04), 250 (4.17), 342 (4.22), IR mmax (cm-1):
3430, 3225 (NH), 1695 (C=O), 1580 (C=N). 1H-NMR
(400 MHz, DMSO-d6/TMS), d, ppm (J, Hz): 3.12 (1H, dd,
HA, JAB: 18.07 Hz, JAX: 5.50 Hz); 3.24 (3H, s, –SO2CH3);
3.94 (1H, dd, HB, JBA: 18.06 Hz, JBX: 12.90 Hz); 6.30 (1H,
dd, HX, JXA: 5.50 Hz, JXB: 12.90 Hz); 6.92 (2H, d, J:
8.66 Hz, 4-methoxyphenyl H3, H5); 7.18 (2H, d, J:
8.66 Hz, 4-methoxyphenyl H2, H6); 7.82 (2H, d, J:
8.42 Hz, 4-trifluoromethylphenyl H2, H6); 8.10 (2H, d, J:
8.27 Hz, 4-trifluoromethylphenyl H3, H5); 7.97 (2H, d, J:
8.60 Hz, 4-methylsulfonylphenyl H2, H6); 8.12 (2H, d, J:
8.20 Hz, 4-methylsulfonylphenyl H3, H5); 9.12 (1H, s,
NH). 13C-NMR (100 MHz), (DMSO-d6/TMS), d (ppm):
40.30 (pyrazole C4); 44.01 (SO2CH3); 60.05 (pyrazole
C5); 129.30, 129.74, 131.15, 131.56, 133.02, 133.84,
134.00, 136.19, 137.02 (aromatic C); 151.10 (pyrazole C3);
164.40 (C=O). Anal. Calcd. for C25H22F3N3O4S (%); C,
58.42; H, 4.27; N, 8.22; S 6.22. Found: C 58.46; H 4.28; N
123

Med Chem Res
expose the treatments to these females. The number of
mosquitoes biting through cloth treatments in each cell was
recorded after a 3-min exposure, and mosquitoes were
prodded back into the cells to check the actual feeding.
Mosquitoes were squashed, and the presence or absence of
green fluorescent tracer dye in the gut was used as an
indicator of feeding. A replicate consisted of six treat-
ments: four test materials, DEET (commercial biting
deterrent standard), and ethanol-treated organdy as solvent
control applied randomly. One set of five replications each
with five females per treatment was conducted using a
newly treated organdy and a new batch of females in each
replication. Treatments were replicated five times.
droplet of water to each well of 24-well plates (BD Lab-
ware, Franklin Lakes, NJ) by use of a disposable 22.5-cm
Pasteur pipette. Fifty microliters of larval diet (2 % slurry
of 3:2 beef liver powder (Now Foods, Bloomingdale, Illi-
nois) and Brewer’s yeast (Lewis Laboratories Ltd., West-
port, CT) was added to each well by using a Finnpipette
stepper (Thermo Fisher, Vantaa, Finland). All chemicals
tested were diluted in ethanol. Eleven microliters of the test
chemical was added to the labeled wells, and 11 lL of
ethanol was added to control treatments. After the treat-
ment application, the plates were swirled in clockwise and
counterclockwise motions and front to back and side to
side five times to ensure even mixing of the chemicals.
Larval mortality was recorded 24 h post-treatment. Larvae
that showed no movement in the well after manual dis-
turbance of the water were recorded as dead. Compounds
that showed larvicidal activity in screening bioassays were
followed by dose–response bioassays. A series of five
concentrations ranging between 100 and 6.25 ppm were
used in each treatment to obtain a range of mortality.
Treatments were replicated 10 times for each compound.
Permethrin (46.1 % cis–53.2 % trans, Chemical Service,
West Chester, PA) at 0.025 ppm was used as positive
control. Larval mortality was recorded 24 h post-treatment.
Mosquito repellent bioassay
Repellency was determined as the minimum effective
dosage (MED), which is the minimum threshold surface
concentration necessary to prevent mosquitoes from biting
through the treated surface (US Department of Agriculture,
1977). Approximately 500 ( 10 %) mosquitoes were col-
lected and loaded into
45 cm 9 37.5 cm 9 5 cm) and held in the cage for 25
2.5) min before initiating repellency assays. Serial
a
test cage (size of
(
dilutions were then made such that the concentrations on
the cloth for the remaining 1 mL solution were 0.375 and
0.094 mg/cm2. Each concentration was tested to determine
the point where the repellent failed for each of the volun-
teers in the study; this concentration was averaged and
reported. Each test was conducted by having a volunteer
affix the treated cloth onto a plastic sleeve to cover a
32-cm2 window previously cut into the sleeve. Each of the
volunteers wore this sleeve/cloth assembly above a nylon
stocking covering their arm, with their hands protected by a
glove (Katritzky et al., 2010). The arm with the sleeve/-
cloth assembly was inserted into a cage in which approx-
imately 500 female Ae. aegypti mosquitoes (aged
6–10 days) had been preselected as host-seeking using a
draw box (Posey and Schreck 1981). Failure of the repel-
lent treatment was 1 % bite through, i.e., the volunteer
received five bites through the cloth over the sleeve win-
dow during the 1-min assay. There were three human
volunteers in this study, and all three provided written
informed consent to participate in this study as part of a
protocol (636-2005) approved by the University of Florida
Human Use Institutional Review Board (IRB-01).
Statistical analyses for mosquito biting
and larvicidal bioassays
As the K&D module bioassay system can handle only four
treatments along with negative and positive controls, in
order to make direct comparisons among more than four
test compounds and to compensate for variation in overall
response among replicates, biting deterrent activity was
quantified as Biting Deterrence Index (BDI) (Ali et al.,
2012). The BDIs were calculated using the following
formula:
ꢀ
ꢁ
Â
Ã
PNBi;j;k À PNBc;j;k
PNBd;j;k À PNBc;j;k
BDIi;j;k
¼
where PNBi,j,k denotes the proportion of females not biting
when exposed to test compound i for replication j and
day k (i = 1–4, j = 1–5, k = 1–1), PNBc,j,k denotes the
proportion of females not biting the solvent control ‘‘c’’ for
replication j and day k (j = 1–5, k = 1–1), and PNBd,j,k
denotes the proportion of females not biting in response to
DEET ‘‘d’’(positive control) for replication
j and
day k (j = 1–5, k = 1–1). This formula makes an adjust-
ment for inter-day variation in response and incorporates
information from the solvent control as well as the positive
control.
Larvicidal bioassay
Bioassays were conducted to test chalcone and pyrazole
compounds for their larvicidal activity against Ae. aegypti
by using the bioassay system described earlier (Pridgeon
et al., 2009). Five Ae. aegypti larvae were added in a
A BDI value of 0 indicates an effect similar to ethanol,
whereas a value significantly greater than 0 indicates biting
deterrent effect relative to ethanol. BDI values not
123

Med Chem Res
significantly different from 1 are statistically similar to
DEET. BDI values were analyzed using the SAS Proc
ANOVA procedure of SAS [single factor: test compound
(fixed)] (SAS Institute, 2007), and means were separated
using the Ryan–Einot–Gabriel–Welsch multiple range test.
To determine whether confidence intervals included the
values of 0 or 1 for treatments, Scheffe’s multiple com-
parison procedure with the option of CLM was used. LC50
values for larvicidal data were calculated by using the
PROBIT procedure of SAS. Control mortality was cor-
rected by using Abbott’s formula (Abbott 1925). Toxicity
was compared among treatments based on non-overlapping
95 % CIs (Savin et al., 1977).
placed in each well of the 24-well plates (Costar 3524,
Corning Incorporated). Test fractions were dissolved in
MeOH with the final concentration being 1 mg/ml. To each
test well was added 180 lL distilled deionized (DDI) H2O
and 20 lL appropriate dilution of the sample. Only 20 lL
MeOH and 180 lL DDI H2O were added to each control
well as control 1, while 200 lL DDI H2O was added to
each control well as control 2. The solvent was evaporated
in a fume hood. Five lettuce or 10 mg of bentgrass seeds
was placed into each well of the 24-well plates. The plates
were covered, sealed with Parafilm, and incubated in a
Percival Scientific CV-36L5 incubator under light condi-
tions at 26 °C and 120.1 lmol/s/m2 average light intensity
for at least 7 days. Phytotoxicity data were quantitatively
evaluated on lettuce by blotting and weighing the plants
after 7 days and on bentgrass by measuring average shoot
height after 7 days. Treatments were replicated so that
standard errors of the means could be calculated.
Antifungal activity against plant pathogens
A standardized 96-well microdilution broth assay was used
to evaluate antifungal activity of compounds toward B.
cinerea, C. acutatum, C. fragariae, C. gloeosporioides, P.
viticola, P. obscurans, and F. oxysporum (Wang et al.,
2011; Sobolev et al., 2011). The fungicides azoxystrobin
([98 %, Syngenta, Greensboro, NC) and captan ([ 98 %,
Chem Service, Inc., West Chester, PA) were used as
internal fungicide standards in all assays. Each fungus was
grown in 0.3, 3.0, and 30.0 lM solutions of each test
compound. Microtiter plates (Nunc MicroWell, untreated;
Roskilde, Denmark) were covered with a plastic lid and
incubated in a growth chamber as described previously
(Wang et al., 2013; Radwan et al., 2014). Fungal growth
was then evaluated by measuring the absorbance of each
well at 620 nm using a microplate photometer (BioTek
Instruments, Winooski, VT, USA). Each chemical was
evaluated in duplicate at doses 0.3, 3.0, and 30.0 lM.
Sixteen wells containing broth and inoculum served as
positive growth controls. Eight wells containing solvent at
the appropriate concentration and broth without inoculum
were used as negative growth controls. The experiments
were repeated three times over a period of time. Mean
absorbance values and standard errors were used to eval-
uate fungal growth at 48 and 72 h except for P. obscurans
and P. viticola where the data were recorded at 144 h.
Means for percent inhibition of each fungus at each dose of
test compound relative to the untreated positive growth
controls were used to evaluate fungal growth. The SAS,
Proc ANOVA procedure of SAS, was used to identify
significant factors, and Fisher’s protected LSD was used to
separate means (Steel and Torrie, 1980).
Bioassay against Lemna paucicostata
The bioassay used was almost identical to that of Michel
et al. (2004). This analysis determines the growth of L.
paucicostata by total frond area. L. paucicostata stocks
were grown from a single colony consisting of a mother
and two daughter fronds in a beaker in Hoagland’s No. 2
Basal Salt Mixture (Sigma H2395) (1.6 g/L) with added
iron (1 mL of 1000X FeEDTA solution to 1 L of Hoag-
land media). The 1000X iron solution contained 18.36 g/
L of Fe-EDTA. The pH of the media was adjusted to 5.5
with 1 N NaOH. The media was filter-sterilized using a
0.2-lm filter and stored in sterile 1-L bottles. The L.
paucicostata stocks were grown in approximately 100 mL
of media in sterile jars with vented lids in a Percival
Scientific CU-36L5 incubator under continuous light
conditions at 26 °C and 120 lmol/s/m-2 average PAR.
Media were changed every 2–3 days, or new stocks were
prepared in fresh media. Plant doubling time was
approximately 24–36 h. Both screening and replicate
series tests were conducted using non-pyrogenic poly-
styrene sterile six-well plates (CoStar 3506, Corning
Incorporated). Each well contained 4950 lL of the
Hoagland medium plus 50 lL of water, or the solvent, or
the compound dissolved in the appropriate solvent (at a
concentration of 1009). Final concentration of the solvent
was therefore approximately 1 % by volume. A graphic
template of the six-well plates was used for LemnaTec
image analysis software (LemnaTec, Wu¨rselen, Ger-
many). Each well was inoculated with two 3-frond plants
of the same age (four- to five-day olds) and approximate
size. All six-well plates were incubated in the Percival
incubator as described above at 26 °C and 120 lmol/s/m2
average PAR.
Phytotoxicity activity in Lactuca sativa (lettuce)
and Agrostis stolonifera (bentgrass) bioassays
Bioassays were performed in a similar manner to Dayan
et al. (2000). A filter paper (Whatman grade 1, 1.5 cm) was
123

Med Chem Res
Acknowledgments This study was supported by Marmara Univer-
sity Scientific Research Projects Commission (BAPKO, Grant No.
SAG-A-040609-0140) for the financial support and in part by USDA/
ARS Grant No. 56-6402-1-612, Deployed War-Fighter Protection
Research Program Grant funded by the US Department of Defense
through the Armed Forces Pest Management Board. We thank Jessie
Linda Robertson, Ramona Pace, Robert Johnson, Dr. Maia Tsikolia,
Natasha M. Agramonte, Nathan Newlon, and Greg Allen for per-
forming bioassays for this study and Dr. James J. Becnel, USDA-
ARS, Gainesville, FL, for supplying mosquito eggs.
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