The larvicidal activity of natural inspired piperine-based dienehydrazides against Culex pipiens

Article abstract of DOI:10.1016/j.bioorg.2019.103464

A series of piperine-based dienehydrazide derivatives were designed and synthesized to be used as insecticides against Culex pipiens. The chemical structure of compound 5n was confirmed by single-crystal x-ray diffraction. Their insecticidal activities of

Full text of DOI:10.1016/j.bioorg.2019.103464

Journal Pre-proofs
The larvicidal activity of natural inspired piperine-based dienehydrazides
against Culex pipiens
Ahmed H. Tantawy, Shaimaa M. Farag, Lamees Hegazy, Hong Jiang, Man-
Qun Wang
PII:
S0045-2068(19)31589-5
DOI:
https://doi.org/10.1016/j.bioorg.2019.103464
YBIOO 103464
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
23 September 2019
18 November 2019
21 November 2019
Please cite this article as: A.H. Tantawy, S.M. Farag, L. Hegazy, H. Jiang, M-Q. Wang, The larvicidal activity of
natural inspired piperine-based dienehydrazides against Culex pipiens, Bioorganic Chemistry (2019), doi: https://
doi.org/10.1016/j.bioorg.2019.103464
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The larvicidal activity of natural inspired piperine-based
dienehydrazides against Culex pipiens
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Ahmed H. Tantawya,b,c, Shaimaa M. Faragd, Lamees Hegazye, Hong Jiangb*, Man-
Qun Wanga*
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aHubei Insect Resources Utilization and Sustainable Pest Management Key
Laboratory, College of Plant Science and Technology, Huazhong Agricultural
University, Wuhan 430070, People's Republic of China
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bDepartment of Chemistry, College of Science, Huazhong Agricultural University,
Wuhan, 430070, China
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cDepartment of Chemistry, College of Science, Benha University, Benha 13518, Egypt
dDepartment of Entomology, Faculty of Science-Ain Shams University, Egypt
eCenter for Clinical Pharmacology, Washington University School of Medicine and
St. Louis College of Pharmacy, St. Louis, MO 63110, USA
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* Corresponding authors: mqwang@mail.hzau.edu.cn (Man-Qun Wang),
jianghong0066@126.com (Hong Jiang)
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Abstract
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A series of piperine-based dienehydrazide derivatives were designed and
synthesized to be used as insecticides against Culex pipiens. The chemical structure of
compound 5n was confirmed by single-crystal x-ray diffraction. Their insecticidal
activities of synthesized compounds were tested against third-instar larval of Cx.
pipiens at concentrations ranging from 0.1-1.2 mg/mL. Among all derivatives,
compounds 5a, 5b, 5f, 5g, 5m, 5n, 5o, 5p, and 5u displayed good activities. The final
mortality rates at the concentration of 0.75 mg/mL after 48 h treatment, were found to
be in the range from 80.00 to 83.33% and with LC50 values ranging from 0.221 to 0.094
mg/mL. These compounds demonstrated higher insecticidal activities than piperine and
Deltamethrin (a commercial positive control). Molecular modelling reveals several
molecular interactions between synthesized compounds and the substrate binding sits
of acetylcholinesterase (AChE) that are predicted to be responsible for its binding and
inhibition activity.
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Keywords: Piperine; Natural product; Molecular docking; Structural modification;
Culex pipens; Larvicidal activity
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1. Introduction
Although synthetic chemical pesticides have a significant role in modern
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agricultural pest management such as crops protection, repeat application of those
agrochemicals over the last years has led to increase in the insect pest populations
resistance and environmental problems[1–4]. Instead, plant secondary metabolites
result from the interaction between plants and surrounding environment (life and non-
life) during the long period of evolution, indicating that pesticides originated from plant
secondary metabolites may lead to less or slower resistance development and lower
pollution. Consequently, discovery of selective, low mammalian toxicity, effective and
ecofriendly insecticides is of great interest to many research groups[5–7]. Natural
products have been used for thousands of years as food preservatives, drugs and
pesticidal agents[8,9].
Black pepper extracts have drawn attention for their insecticidal constituents
toward Cx. Pipien, Aedesaegypti larvae[10], rice weevils, cowpea weevils[11] and
larvae of various mosquito vectors[12]. Piperine, an alkaloid from pepper (Piper
nigrum) exhibited a wide range of biological activities including inhibition of liver
CYP3A4 enzyme[13], and being used as antiadipogenic[14], anti-inflammatory[15]
and antimicrobial functions[16]. In addition, modified structures of piperine also
exhibited interesting insecticidal activity[17,18].
Cx. pipiens, the common house mosquito, is a worldwide vector. It has been
incriminated as a vector of serious diseases such as filarial worms, west Nile virus, St.
Louis encephalitis virus and also avian malaria[10]. The unwisely usage of chemical
insecticides led to several environmental problems[19]. The toxicity to non-target
population[19], development of resistance phenomenon and the residual effects of these
insecticides are main concerns of scientists[20]. With the emergence of Cx. pipiens
resistance to many insecticides, control is becoming more difficult and that led to
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increasing rates of diseases transmission[21]. There is a dire need to develop
environmental friendly insecticides to protect the environment from hazards of
chemical insecticides[22].
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Several researchers investigated the potential of natural compounds as a model
for the development of new insecticides. Many amides and hydrazones of piperine have
been synthesized and been investigated against insects [23–25]. Inspired by these, a
series of piperine-based dienehydrazide derivatives were designed and synthesized as
insecticidal agents against 3rd instar larvae of Cx. pipens.
2. Experimental and methods
2.1. Chemicals and Instruments.
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All utilized chemical reagents were obtained and used without further
purification. Piperine and hydrazine (≥99.0%) were obtained from Aladdin Industrial
Corporation (Shanghai, China). Oxalyl chloride (≥99.0%) was obtained from TCI
(Tokyo Chemical Industry Co. Ltd). Other chemicals were purchased from Sinopharm
Chemical Reagent Co., Ltd.
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Proton (1H NMR) and carbon nuclear magnetic resonance spectra (13C NMR)
were obtained in DMSO-d6 or CDCl3 on a Bruker Avance 600 MHz instrument using
tetramethylsilane (TMS) as the internal standard. Coupling constants (J) were reported
in Hz. Mass spectra were recorded on an Agilent 6530 accurate-mass Q-TOF
spectrometer. X-Ray data were collected using a BRUKER SMART APEX-CCD
diffractometer with Cu-Kα radiation (λ = 0.71073 Å). Gas sorption experiment was
carried out with a Micrometrics ASAP 2020 instrument. The infrared spectra of the
generated compounds were done on FT-IR spectra recorded in KBr on a thermo nicolet
iS10 FTIR spectrophotometer. Measurements of melting points (mp) were done on a
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XT-4 digital melting point apparatus (Beijing Tech Instrument Co., Ltd., Beijing,
China) and were incorrected.
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2.2. Maintenance of mosquito colony, Cx. pipiens L. (Diptera: Culicidae)
The egg rafts of common house mosquito, Cx. pipiens were obtained from the
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Research and Training Center on Vectors of Diseases (RTC), Faculty of Science, Ain
Shams University, Egypt. The mosquitoes were reared for at least eight generations in
insectary rooms, under controlled laboratory conditions at temperature 27±2 ºC, and
relative humidity RH 70±10%, for photoperiods 14:10 (light:dark) hours. Egg rafts
were placed in white enamel dishes 35-40 cm in diameter and 10 cm in depth filled with
1500 ml of distilled water. Newly hatched larvae were fed on fish food (Tetra-/Min,
Germany) as a diet sprinkled twice daily over the water surface of the breeding
pans[26]. Distilled water in each dish was stirred daily and changed every two days to
avoid scum formation on the water surface or on the walls and bottoms of pans. Small
air pump was used to aerate the breeding water gently every day for about 5 minutes.
Pupae were collected routinely and separated in plastic containers filled with distilled
water then introduced into screened wooden cages until emergence. Adults were reared
in (24 x 24 x 24 cm) wooden cages. Adults were provided daily with cotton pads soaked
in 10% sucrose solution for a period of four days. After this period the females were
allowed to take a blood meal from a pigeon host. To obtain best blood feeding, sucrose
was removed 24 hours prior to blood meal. Oviposition containers filled with distilled
water were placed in adult cages 48 hours after the females had been provided with
blood meals. Egg rafts were collected routinely and placed in white enamel dishes.
When mosquito larvae developed to the 2nd instar, they were poured into clean pans (25
x 30 x 15 cm) containing three liters of tap water left for 24 h and observed daily[26,27].
Larvicidal toxicity of experimental compounds was tested toward early third larval
instars of Cx. pipiens.
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2.3. Statistical analysis
The results of the insecticidal activities were analyzed using one-way analysis
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of variance (ANOVA) by SPSS 19.0 program. Moreover, the LC50 values were
calculated using probit analysis.
2.4. The Synthesis of target molecule
2.4.1. Piperic Acid (Compound 2)
The piperic acid was produced from the hydrolysis of piperine according
literature[28]. The piperic acid was recrystallized from methyl alcohol to afford piperic
acid as a pale-yellow solid (93.4% yield).
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2.4.2. General Procedure for Preparation of Piperic Acid hydrazide (Compound
4)
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According to literature[29] with some modification, oxalyl chloride (1.24 g, 10
mmol) in DCM (5 mL) and three drops of DMF as a catalyst were added to piperic acid
(217 mg, 1.01 mmol) and stirred at room temperature for 4 h. After completing the
reaction, the oxalyl chloride excess and solvent were then evaporated. The obtained
acid chloride was dissolved in DCM (5 mL) and was added dropwise to hydrazine (40
mg, 1.2 mmol) in DCM (5 mL) at room temperature. The reaction mixture was then
stirred for 7 h, and the solvent was evaporated under reduced pressure. Water was added
to the mixture, which was subsequently extracted with DCM. The organic layer was
separated and dried over anhydrous Na2SO4 and the solvent was evaporated. The crude
product was purified by silica gel column chromatography (DCM:MeOH=20:1) to give
the desired piperic acid hydrazide (210 mg, 87% yield).
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2.4.3. Synthesis of piperine-based dienehydrazide derivatives (Compounds 5a-u)
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A mixture of 4 (100 mg, 0.43 mmol), the corresponding carbonyl compounds (0.45
mmol), and four drops of glacial acetic acid in MeOH (10 mL) was heated to reflux.
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The reaction was completed in 2-6 h as shown by TLC. The obtaining reaction mixture
was cooled to precipitate the desired product. The crude solid product was collected via
filtration and washed with cold methanol to give compounds 5a-u in 55-85% yield.
(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)penta-2,4-dienoic acid (2): Pale yellow solid,
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m.p. 218oC; H NMR (600 MHz, DMSO-d6) δ 12.20 (s, 1H, -CO2H), 7.28-7.33 (m,
1H), 7.24 (d, J = 1.4 Hz, 1H), 6.92-7.02 (m, 4H), 6.05 (s, 2H, -OCH2O-), 5.92 (d, J =
15.44 Hz, 1H).
(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)penta-2,4-dienehydrazide (4): Pale yellow
solid, m.p. 180oC; 1H NMR (600 MHz, DMSO-d6) δ 9.28 (s, 1H, NH), 7.25 (d, J= 1.29
Hz, 1H), 7.15-7.19 (m, 1H), 6.85(d, , J= 15.55 Hz, 1H), 6.92-7.02 (m, 4H), 6.04 (s, 2H,
-OCH2O-), 6.02 (d, J=15.01Hz, 1H), 4.41 (s, 2H, NH).
(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N'-((Z)-2-hydroxybenzylidene)penta-2,4-
dienehydrazide (5a): Pale brown solid, m.p. 99-101oC; IR: 3587, 3197, 3037, 2887,
1652, 1617, 1567, 1488, 1312, 1245. 1H NMR (600 MHz, DMSO-d6) δ 11.84 (s, 1H,
NH), 11.20 (s, 1H, OH), 8.84 (s, 1H, CH=N), 7.52 (d, J = 7.8 Hz, 1H), 7.15-7.19 (m,
1H), 6.85(d, J = 15.55 Hz, 1H), 6.92-7.02 (m, 4H), 6.04 (s, 2H, -OCH2O-), 6.02 (d, J
= 15.01Hz, 1H), 4.41 (s, 2H, NH). 13C NMR (151 MHz, DMSO-d6) δ 166.51, 161.98,
159.81, 159.56, 148.45, 148.43, 148.41, 148.37, 147.18, 143.48, 142.80, 141.51,
139.57, 139.33, 131.29, 131.23, 129.30, 128.88, 126.26, 125.82, 125.67, 123.45,
123.41, 122.97, 120.39, 116.14, 108.95, 106.16, 106.11, 101.78.
(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N'-((Z)-2-chlorobenzylidene)penta-2,4-
dienehydrazide (5b): Yellow solid, m.p. 130-133oC; IR: 3283, 3189, 3027, 2903,
1642, 1596, 1569, 1497, 1443, 1374, 1259. 1H NMR (600 MHz, DMSO-d6) δ 11.30 (d,
J = 131.9 Hz, 1H), 8.02 (d, J = 112.6 Hz, 1H), 7.54 (d, J = 8.6 Hz, 2H), 7.35 (ddd, J =
22.4, 15.0, 10.7 Hz, 1H), 7.29 (d, J = 1.4 Hz, 1H), 7.18 – 7.07 (m, 1H), 7.02 (dd, J =
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18.3, 9.1 Hz, 1H), 6.97 (d, J = 11.8 Hz, 1H), 6.92 (dd, J = 8.0, 4.6 Hz, 1H), 6.83 (d, J
= 8.5 Hz, 2H), 6.16 (d, J = 14.9 Hz, 1H), 6.06 (d, J = 3.0 Hz, 2H). 13C NMR (151 MHz,
DMSO-d6) δ 166.51, 161.98, 159.81, 159.56, 148.45, 148.43, 148.41, 148.37, 147.18,
143.48, 142.80, 141.51, 139.57, 139.33, 131.29, 131.23, 129.30, 128.88, 126.26,
125.82, 125.67, 123.45, 123.41, 122.97, 120.39, 116.14, 108.95, 106.16, 106.11,
101.78.
(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N'-((Z)-1-(benzo[d][1,3]dioxol-5-
yl)ethylidene)penta-2,4-dienehydrazide (5c): Pale yellow solid, m.p. 150-160oC; IR:
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3212, 3153, 3015, 2885, 1642, 1616, 1547, 1444, 1285, 1244. H NMR (600 MHz,
DMSO-d6) δ 10.46 (d, J = 29.3 Hz, 1H), 7.46–7.26 (m, 4H), 7.23–7.10 (m, 1H), 7.01
(d, J = 15.3 Hz, 1H), 7.00–6.90 (m, 3H), 6.46 (d, J = 14.9 Hz, 1H), 6.07 (d, J = 9.5 Hz,
4H), 2.24 (d, J = 16.7 Hz, 3H). 13C NMR (151 MHz, DMSO-d6) δ: 167.38, 162.53,
148.42, 147.38, 138.44, 141.46, 139.76, 133.05, 131.25, 126.30, 125.58, 123.34,
123.20, 121.47, 121.14, 120.54, 109.17, 107.90, 106.35, 106.14, 101.38.
4-((Z)-(2-((2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)penta-2,4-dienoyl)hydrazono)
methyl)phenyl 1,1,2,2,3,3,4,4,5,5,6,6,6-tridecafluorohexane-1-sulfonate (5d): Pale
yellow solid, m.p. 185-190oC; IR: 3204, 3041, 2904, 1652, 1601, 1488, 1426, 1303,
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1211. H NMR (600 MHz, DMSO-d6) δ 11.64 (s, 1H), 8.17 (d, J = 128.5 Hz, 1H), 7.86
(s, 2H), 7.59 (s, 2H), 7.38 (s, 1H), 7.27 (s, 1H), 7.11 (s, 1H), 6.99 (s, 2H), 6.92 (s, 1H),
6.17 (s, 1H), 6.06 (s, 2H). 13C NMR (151 MHz, DMSO-d6) δ 148.47, 144.85, 143.09,
141.90, 141.07, 139.88, 131.20, 129.56, 129.23, 126.10, 125.54, 123.55, 122.44,
119.63, 108.96, 106.19, 101.82.
(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N'-((Z)-4-methoxybenzylidene)penta-2,4-
dienehydrazide (5e): Pale yellow solid, m.p. 216-219oC; IR: 3200, 3052, 2895, 1642,
1602, 1505, 1365, 1253. 1H NMR (600 MHz, DMSO-d6) δ 11.37 (d, J = 116.6 Hz, 1H),
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8.08 (d, J = 118.3 Hz, 1H), 7.65 (d, J = 8.8 Hz, 2H), 7.42 – 7.26 (m, 2H), 7.20 – 7.07
(m, 1H), 6.99 (ddd, J = 40.2, 24.5, 18.2 Hz, 5H), 6.16 (d, J = 14.9 Hz, 1H), 6.06 (s, 2H),
3.81 (s, 3H).13C NMR (151 MHz, DMSO-d6) δ 166.63, 162.03, 161.10, 148.40, 146.63,
142.95, 141.51, 139.47, 131.21, 129.09, 128.73, 127.37, 126.20, 125.68, 123.56,
122.96, 120.19, 114.93, 108.74, 106.14, 101.72, 55.63.
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(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N'-((Z)-4-nitrobenzylidene)penta-2,4-
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dienehydrazide (5f): Yellow solid, m.p. 230-233oC; H NMR (600 MHz, DMSO-d6)
δ 11.80 (d, J = 163.5 Hz, 1H), 8.37 – 8.20 (m, 2H), 8.13 (s, 1H), 7.98 (d, J = 8.8 Hz,
2H), 7.43 (d, J = 11.1 Hz, 1H), 7.28 (s, 1H), 7.14 (t, J = 14.0 Hz, 1H), 7.06 – 6.95 (m,
2H), 6.93 (s, 1H), 6.20 (d, J = 14.7 Hz, 1H), 6.06 (s, 2H). 13C NMR (151 MHz, DMSO-
d6) δ 166.22, 151.95, 150.75, 148.64, 148.49, 148.34, 147.13, 140.52, 131.36, 131.21,
128.93, 128.79, 128.54, 128.35, 123.43, 122.93, 122.24, 111.79, 111.53, 109.22,
108.27, 106.11, 106.16, 101.72.
(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N'-((Z)-furan-2-ylmethylene)penta-2,4-
dienehydrazide (5g): Pale yellow solid, m.p. 203-206oC; IR: 3423, 3130, 2990, 2900,
1661, 1600, 1496, 1406, 1259. 1H NMR (600 MHz, DMSO-d6) δ 11.45 (d, J = 119.8
Hz, 1H), 8.03 (d, J = 129.5 Hz, 1H), 7.84 (s, 1H), 7.37 (ddd, J = 20.7, 15.1, 10.8 Hz,
1H), 7.31 (d, J = 11.3 Hz, 1H), 7.15 (dd, J = 15.4, 11.1 Hz, 1H), 7.00 (ddd, J = 15.7,
13.1, 6.1 Hz, 3H), 6.96 – 6.85 (m, 2H), 6.63 (dd, J = 3.3, 1.7 Hz, 1H), 6.14 (d, J = 14.9
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Hz, 1H), 6.06 (s, 2H). C NMR (151 MHz, DMSO-d6) δ 166.61, 162.18, 150.00,
149.78, 148.45, 145.56, 145.28, 143.37, 142.04, 141.88, 139.99, 139.63, 136.64,
131.24, 131.20, 126.08, 125.64, 123.55, 122.93, 122.30, 119.70, 113.91, 113.73,
113.54, 109.02, 108.88, 106.25, 106.10, 101.80.
4-((Z)-(2-((2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)penta-2,4-dienoyl)hydrazono)
methyl)benzoic acid (5h): Pale yellow solid, m.p. 209-211oC; IR: 3209, 3062, 2893,
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2671, 2548, 1651, 1613, 1558, 1497, 1370, 1254. 1H NMR (600 MHz, DMSO-d6) δ
13.10 (s, 1H), 11.66 (d, J = 125.3 Hz, 1H), 8.07 (d, J = 23.2 Hz, 1H), 7.97 (s, 2H), 7.82
(s, 2H), 7.39 (s, 1H), 7.30 (s, 1H), 7.15 (s, 1H), 7.02 (s, 2H), 6.98 (d, J = 10.2 Hz, 1H),
6.93 (s, 1H), 6.18 (s, 1H), 6.07 (s, 2H). 13C NMR (151 MHz, DMSO-d6) δ 167.41,
166.94, 148.47, 142.28, 142.07, 138.94, 138.83, 132.03, 131.87, 131.23, 131.17,
130.21, 127.50, 127.18, 126.18, 125.61, 123.50, 108.92, 106.27, 106.11, 101.82.
(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N'-((Z)-naphthalen-1-ylmethylene)penta-
2,4-dienehydrazide (5i): Pale yellow solid, m.p. 215-218oC; IR: 3211, 3185, 3038,
1645, 1612, 1546, 1503, 1337, 1255. 1H NMR (600 MHz, DMSO-d6) δ 11.59 (d, J =
156.6 Hz, 1H), 8.93 – 8.72 (m, 1H), 8.55 (d, J = 8.0 Hz, 1H), 8.11 – 7.84 (m, 3H), 7.81
– 7.51 (m, 3H), 7.42 (d, J = 11.8 Hz, 1H), 7.32 (s, 1H), 7.18 (dd, J = 27.1, 15.0 Hz, 1H),
7.04 (dd, J = 18.1, 12.7 Hz, 2H), 6.94 (d, J = 7.6 Hz, 1H), 6.23 (d, J = 14.8 Hz, 1H),
6.07 (s, 2H).13C NMR (151 MHz, DMSO-d6) δ 165.78, 161.21, 148.47, 146.83, 143.27,
143.16, 142.05, 141.92, 139.72, 139.61, 134.82, 134.81, 131.22, 130.29, 129.36,
129.12, 127.51, 127.18, 126.24, 125.65, 123.54, 122.41, 120.12, 109.02, 108.91,
106.25, 106.20, 106.10, 101.82, 101.11.
(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N'-((Z)-pentylidene)penta-2,4-
dienehydrazide (5j): Pale yellow solid, m.p. 170-175oC; IR: 3206, 3066, 2929, 1648,
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1611, 1557, 1495, 1388, 1254. H NMR (600 MHz, DMSO-d6) δ 11.04 (d, J = 125.9
Hz, 1H), 7.41 (ddd, J = 51.8, 10.0, 4.6 Hz, 1H), 7.33 – 7.24 (m, 2H), 7.07 (dt, J = 28.1,
14.1 Hz, 1H), 7.03–6.97 (m, 1H), 6.98 – 6.87 (m, 3H), 6.09–6.06 (m, 1H), 6.05 (s, 2H),
2.30 – 2.13 (m, 2H), 1.52 – 1.40 (m, 2H), 1.39 – 1.27 (m, 2H), 0.96 – 0.86 (m, 3H). 13C
NMR (151 MHz, DMSO-d6) δ 166.24, 161.78, 151.27, 148.47, 147.65, 142.73, 141.34,
139.52, 139.16, 131.17, 126.12, 125.65, 123.36, 123.07, 120.27, 109.10, 106.21,
101.77, 32.17, 28.31, 22.22, 13.97.
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(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N'-((Z)-2,3-dihydro-1H-inden-1-ylidene)
penta-2,4-dienehydrazide (5k): Pale yellow solid, m.p. 212-215oC; IR: 3184, 3035,
2889, 1655, 1603, 1555, 1501, 1252. 1H NMR (600 MHz, DMSO-d6) δ 10.40 (d, J =
14.4 Hz, 1H), 7.70 (dd, J = 27.4, 7.6 Hz, 1H), 7.44 – 7.27 (m, 5H), 7.23 – 7.12 (m, 1H),
6.98 (ddd, J = 33.0, 20.3, 7.6 Hz, 3H), 6.46 (d, J = 15.0 Hz, 1H), 6.06 (s, 2H), 3.13 –
3.04 (m, 2H), 2.88–2.80 (m, 2H). 13C NMR (151 MHz, DMSO-d6) δ 167.19, 162.38,
161.14, 157.17, 149.14, 148.72, 148.36, 142.86, 141.38, 139.55, 139.27, 138.42,
131.31, 130.95, 130.73, 127.49, 127.36, 125.67, 123.48, 123.32, 121.88, 121.32,
120.65, 108.96, 106.23, 106.02, 101.87, 28.64, 28.60, 27.91, 27.41.
(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N'-((1Z,2E)-3-phenylallylidene)penta-2,4-
dienehydrazide (5l): Yellow solid, m.p. 179-184oC; IR: 3300, 3146, 3017, 2884, 1646,
1635, 1547, 1491, 1335, 1242. 1H NMR (600 MHz, DMSO-d6) δ 11.41 (d, J = 112.6
Hz, 1H), 8.10–7.76 (m, 1H), 7.61 (d, J = 7.6 Hz, 2H), 7.42–7.24 (m, 5H), 7.17 – 6.87
(m, 6H), 6.15 (d, J = 14.8 Hz, 1H), 6.06 (s, 2H). 13C NMR (151 MHz, DMSO-d6) δ
166.53, 162.07, 148.78, 148.45, 141.97, 141.82, 139.56, 139.17, 138.84, 136.44,
131.20, 129.40, 129.23, 127.56, 127.50, 126.20, 125.67, 123.51, 123.05, 122.44,
109.03, 108.91, 106.26, 106.11, 101.80.
(2E,4E)-N'-((Z)-anthracen-9-ylmethylene)-5-(benzo[d][1,3]dioxol-5-yl)penta-2,4-
dienehydrazide (5m): Yellow solid, m.p. 225-230oC; IR: 3443, 3184, 3048, 2900,
1
1651, 1607, 1558, 1496, 1335, 1248. H NMR (600 MHz, DMSO-d6) δ 11.76 (d, J =
169.7 Hz, 1H), 9.33 (s, 1H), 8.63 (s, 2H), 8.18 (s, 1H), 7.63 (d, J = 42.0 Hz, 3H), 7.44
(s, 1H), 7.31 (s, 1H), 7.00 (s, 3H), 6.27 (s, 1H), 6.06 (s, 2H). ESI-HRMS: Calcd for
C27H20N2O3 ([M+]), 420.27; Found, 420.26.
(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N'-((Z)-4-(diethylamino)-2-hydroxy
benzylidene)penta-2,4-dienehydrazide (5n): Yellow solid, m.p. 105-107oC; IR:
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3208, 3034, 2972, 2884, 1629, 1547, 1518, 1443, 1371, 1246. H NMR (600 MHz,
DMSO-d6) δ 11.52 (s, 1H), 11.34 (s, 1H), 8.20 (s, 1H), 7.36 – 7.28 (m, 2H), 7.20 (d, J
= 11.6 Hz, 1H), 7.02 (d, J = 14.7 Hz, 2H), 6.98 – 6.90 (m, 2H), 6.13 (d, J = 14.9 Hz,
1H), 6.11 (d, J = 2.3 Hz, 1H), 6.06 (s, 2H), 3.35 (s, 4H), 1.11 (s, 6H). 13C NMR (151
MHz, DMSO-d6) δ 161.26, 160.10, 150.52, 148.91, 148.34, 141.39, 139.36, 131.92,
131.69, 125.64, 123.41, 122.84, 122.23, 108.95, 107.01, 106.21, 106.21, 103.80,
101.78, 44.36, 12.86.
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Crystallographic description of 5-(2H-1,3-benzodioxol-5-yl)-N'-((4-(diethylamino)-
2-hydroxyphenyl)methylidene)penta-2,4-dienehydrazide methanol solvate (5n):
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C23H25N3O4.CH3OH, crystal dimensions 0.15 x 0.15 x 0.1 mm , Mr = 439.50,
monoclinic, space group P 21/c (14), Cell: a = 11.298(3), b = 13.398(3), c = 15.678(4)
Å, α = 90°, β = 107.247(4)°, γ = 90°, V = 2266.5(10) Å3, Z = 4, Density (calculated) =
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-1
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1.288 Mg/m , μ = 0.091 mm , F(000)= 936, reflection collected / unique = 17545 /
4630, refinement method = full-matrix least-squares on F2, Final R indices
[I>2sigma(I)]: R1 = 0.0475, wR2 = 0.1263, R indices (all data): R1 = 0.0948, wR2 =
2
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0.1535, goodness of fit on F = 1.037. CCDC 1934954 for 5-(2H-1,3-benzodioxol-5-
yl)-N'-((4-(diethylamino)-2-hydroxyphenyl)methylidene)penta-2,4-dienehydrazide
methanol solvate (5n): contains the supplementary crystallographic data for this paper.
These data can be obtained free of charge from The Cambridge Crystallographic Data
Centre via www.ccdc.cam.ac.uk/data_request/cif.
(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N'-((Z)-1-(4-nitrophenyl)ethylidene)penta-
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2,4-dienehydrazide (5o): Deep yellow solid, m.p. 201-205oC; IR: 3169, 3067, 2999,
1
1665, 1596, 1441, 1345, 1248. H NMR (600 MHz, DMSO-d6) δ 11.33-11.26 (d, J =
114.6 Hz, 1H), 7.66 (d, J= 8.77 Hz, 2H), 7.31-7.39 (m, 2H), 7.11-7.18 (m, 1H), 6.93-
7.06 (m, 4H), 6.92 (m, 1H), 6.95(s, 1H), 6.48 (d, J = 15.90 Hz, 1H), 6.05(s, 2H), 2.32(s,
3H). ESI-HRMS: Calcd for C20H17N3O5 ([M+]), 379.37; Found, 379.32.
(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N'-((Z)-1-(benzo[d][1,3]dioxol-5-yl)propan-
2-ylidene)penta-2,4-dienehydrazide (5p): Yellow solid, m.p. 107-110oC; IR: 3146,
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3017, 2884, 1649, 1635, 1547, 1491, 1335, 1242. H NMR (600 MHz, DMSO-d6) δ
10.13 (d, J = 43.0 Hz, 1H), 7.29 (s, 2H), 7.02 (s, 1H), 6.92 (d, J = 8.0 Hz, 3H), 6.86 (s,
1H), 6.80 (d, J = 6.8 Hz, 1H), 6.71 (s, 1H), 6.35 (d, J = 15.0 Hz, 1H), 6.05 (s, 2H), 5.95
(d, J = 4.8 Hz, 2H), 2.81 – 2.69 (m, 2H), 1.89 (d, J = 14.7 Hz, 3H). 13C NMR (151 MHz,
DMSO-d6) δ 166.70, 162.35, 148.30, 147.17, 145.42, 140.78, 139.23, 135.72, 135.09,
130.73, 126.10, 123.22, 121.67, 109.67, 109.38, 108.74, 108.11, 106.14, 101.30,
100.59, 31.68, 16.78.
(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N'-((Z)-2-hydroxy-3-methoxybenzylidene)
penta-2,4-dienehydrazide (5q): Pale yellow solid, m.p. 178-180oC; IR: 3449, 3229,
1
3016, 2992, 1673, 1609, 1502, 1490, 1340, 1254. H NMR (600 MHz, DMSO-d6) δ
11.82 (s, 1H), 10.92 (s, 1H), 8.42 (s, 1H), 7.37 (dd, J = 24.1, 10.8 Hz, 1H), 7.30 (s, 1H),
7.22 – 6.95 (m, 5H), 6.93 (d, J = 7.7 Hz, 1H), 6.85 (dd, J = 13.9, 7.0 Hz, 1H), 6.15 (d,
J = 14.9 Hz, 1H), 6.06 (s, 2H), 3.82 (d, J = 8.9 Hz, 3H). 13C NMR (150 MHz, DMSO-
d6) δ 166.46, 161.95, 149.39, 149.11, 148.48, 148.44, 148.37, 147.38, 143.74, 142.86,
141.50, 139.63, 139.35, 131.25, 126.26, 126.22, 125.68, 123.41, 123.01, 122.51,
121.41, 120.34, 116.06, 115.89, 110.25, 109.47, 108.94, 106.16, 101.79, 56.18.
4-((Z)-(2-((2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)penta-2,4-dienoyl)hydrazono)
methyl)phenyl 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-heptadecafluorooctane-1-sulfonate
(5r): Pale yellow solid, m.p. 227-231oC;IR: 3448, 3060, 2939, 1661, 1598, 1577, 1339,
1252. 1H NMR (600 MHz, DMSO-d6) δ 11.68 (s, 1H), 8.17 (s, 1H), 7.89 (s, 2H), 7.60
(s, 2H), 7.41 (s, 1H), 7.33 (s, 1H), 7.09 (s, 1H), 7.01 (s, 3H), 6.23 (s, 1H), 6.01 (s, 2H).
13C NMR (151 MHz, DMSO-d6) δ 167.41, 150.96, 148.57, 144.86, 143.93, 141.89,
139.97, 135.56, 131.30, 130.13, 128.75, 126.10, 125.26, 123.77, 122.53, 109.45,
105.93, 101.53.
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(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N'-((Z)-4-(trifluoromethyl)benzylidene)
penta-2,4-dienehydrazide (5s): Deep yellow solid, m.p. 198-201oC; IR: 3443, 3164,
3012, 2899, 1663, 1601, 1500, 1448, 1403, 1261. 1H NMR (600 MHz, DMSO-d6) δ
11.84 (s, 1H), 8.77 (d, J = 128.5 Hz, 1H), 7.56 (s, 2H), 7.49 (s, 2H), 7.46 (s, 1H), 7.37
(s, 1H), 7.18 (s, 1H), 6.92 (s, 2H), 6.82 (s, 1H), 6.17 (s, 1H), 6.06 (s, 2H). 13C NMR
(151 MHz, DMSO-d6) δ 168.47, 164.85, 150.09, 148.90, 145.07, 140.88, 137.20,
133.56, 130.23, 128.10, 126.54, 124.55, 108.96, 106.19, 103.82. 19F NMR (565 MHz,
CDCl3) δ -63.08.
(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N'-((Z)-benzylidene)penta-2,4-
dienehydrazide (5t): Pale yellow solid, m.p. 243-248 oC; IR: 3184, 3035, 2889, 1655,
1501, 1314, 1252. 1H NMR (600 MHz, DMSO-d6) δ 11.52 (d, J = 120.1 Hz, 1H), 8.14
(d, J = 121.7 Hz, 1H), 7.72 (d, J = 7.5 Hz, 2H), 7.51 – 7.34 (m, 4H), 7.30 (d, J = 1.4
Hz, 1H), 7.23 – 7.09 (m, 1H), 7.08 – 6.88 (m, 3H), 6.19 (d, J = 15.0 Hz, 1H), 6.07 (s,
2H).13C NMR (151 MHz, DMSO-d6) δ 166.78, 162.22, 148.47, 146.81, 143.27, 143.12,
142.05, 141.90, 139.79, 139.63, 134.88, 134.80, 131.21, 130.29, 129.36, 127.51,
127.18, 126.24, 125.65, 123.55, 120.02, 109.03, 108.90, 106.25, 106.20, 106.05,
101.81, 101.11.
(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N'-((Z)-(3,5a1-dihydropyren-1-yl)methylene)
penta-2,4-dienehydrazide (5u): Deep yellow solid, m.p. 250-256oC, IR: 3184, 3035,
2889, 1655, 1603, 1555, 1501, 1445, 1374, 1252. 1H NMR (600 MHz, DMSO-d6) δ
11.70 (d, J = 179.5 Hz, 1H), 9.21 (d, J = 65.8 Hz, 1H), 8.84 (d, J = 9.3 Hz, 1H), 8.59
(dd, J = 45.4, 7.9 Hz, 1H), 8.43 – 8.31 (m, 3H), 8.31 – 8.20 (m, 2H), 8.14 (t, J = 7.6 Hz,
1H), 7.54 – 6.90 (m, 6H), 6.27 (d, J = 14.6 Hz, 1H), 6.08 (s, 2H). ESI-HRMS: Calcd
for C29H20N2O3([M+]), 444.29; Found, 444.20.
2.5. Biological assays
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The larvicidal activity of the tested compounds against the third larval instar of Cx.
pipiens was evaluated by immersion methods at concentrations ranging from 0.1-1.2
mg/mL for two days. For each compound at least 75 larvae (25 newly moulted larvae /
group) were used. All compounds were dissolved in acetone solutions and deltamethrin
was used as a positive control. The 3rd instar larvae of Cx. pipiens were immersed into the
corresponding concentration for 24 and 48 hours. The untreated larvae were considered as
blank control group. The experiment procedure was done at 25±2 oC and relative humidity
(RH) 65–80%, and on 12 h/12 h (light/dark) photoperiod.
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2.6. Molecular modelling:
We performed molecular modeling of compounds 5a,5o, 5g and 5n in a
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homology model of acetyl cholinesterase of the organism Cx. Pipien. The homology
model was generated using I-TASSER [30,31]. The reference structure used for protein
structure prediction is the crystal structure of Acetylcholinesterase Catalytic Subunit of
the Malaria Vector Anopheles Gambiae ( PDB:5X61).[32] Compounds were modeled
based on the binding pose of the highly similar scaffold MR28926 in the X-ray structure
of acetyl cholinesterase (PDB:6ezh). Predicting the ligand binding poses based on
existing similar scaffolds is the recommended approach by the D3R Grand challenge
and pose prediction [33]. Refinement of the protein-ligand complex was performed
using Macromodel [34].
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3. Results and discussion
3.1. Chemistry
Piperine, the main ingredients of Piper nigrum, and its amide derivatives have
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a variety of biological activities[35]. Piperine is consisted of three parts: benzodioxol
group (A); a pentadienoic acid (B); the amine moiety (C) as shown in Figure 1.
Modulation of part A of piperine via introduction of an alkyl or halogen at phenyl ring,
or replacing the whole benzodioxol substructure with other phenyl groups has been
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achieved to give a new series of compounds[36]. Several of these molecules exhibited
a good bioevaluation indices[37].
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<<< Figure 1>>>>
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In this work, the modifications of part C was carried out via substitution of the
piperidine group (amine moiety) by aliphatic and aromatic hydrazones. We studied the
impact of these substituents on the overall insecticidal activities of synthesized
compounds against Cx. pipens. The synthesis of compounds 5a–u is outlined in Figure
2. The piperic acid (2) was obtained by alkaline hydrolysis of piperine. Then, piperic
acid hydrazide (4) was easily prepared via the reaction of 2 with oxalyl chloride in the
DCM as solvent and DMF as a catalyst, followed by reaction with hydrazine in DCM.
The obtained hydrazide derivative was reacted with different corresponding carbonyl
compounds in methanol in the presence acetic acid as a catalyst to afford piperine-based
hydrazo-amide derivatives (5a–u) in 70-90% yield. All compounds were characterized
by IR, 1H, 13C, 19F NMR, ESI-MS and mp (See supplementary data). Moreover, the
configuration of 5u was elucidated by single-crystal X-ray diffraction analysis (Figure
3)[38].
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<<< Figure 2>>>>
<<< Figure 3>>>>
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3.2. Biological activity
The evaluation of larvicidal activity of 5a–u was carried out against the 3rd instar
larvae of Cx. pipiens by immersion method at concentrations ranging from 0.1-1.2
mg/mL for two days[39]. Deltamethrin, a conventional insecticide was utilized as the
positive control at same concentrations. In this study, the work aimed to evaluate the
toxicity of each synthesized compound and find out if the structural modification
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carried out on piperine would result in a synthetic product more toxic than the natural
product. The results of the bioassays are shown in Table 1, Figures 4 and S22. The
corresponding mortality after 48 hours was far higher than those after 24 hours. This
phenomena indicated that the mechanism was different from neurotoxic insecticides
such as pyrethroids, organophosphates, and carbamates, which exhibit delayed
insecticidal activity[40,41]. Also, as noted in Table 1, where the amide group of 1 was
replaced by -COOH or hydrazide moiety, derivatives such as 2 and 4 did not improve
the insecticidal activity than piperine. Interestingly, when 4 was reacted with different
aldehydes and ketones to give piperine based dienehydrazide compounds 5a–u, the all
synthesized dienehydrazide derivatives gave more toxicity toward the investigated
insect than deltamethrin as conventional insecticide. Eleven compounds (5a-c, 5f, 5g,
5m, 5n, 5o, 5p, 5u, and 5q) gave more insecticidal activity against 3rd instar larvae of
Cx. pipiens than piperine as natural product insecticide. Their insecticidal activity was
ranged from 70.66 to 83.33% after 48 h. Compounds 5a, 5b, 5p, 5g, 5u and 5n exhibited
highest insecticidal activities compared to piperine and deltamethrin, and the mortality
percentages were 81.33%, 81.33%, 81.33%, 82.66%, 82.66% and 83.33%, respectively
compared to 26.25% as the final mortality rate of deltamethrin. The data shows that the
presence of substituents groups at the C-2 position on the phenyl ring of the
dienehydrazide derivatives was important to improve their insecticidal activity. For
example, the final mortality rate of 5t (not containing substituent group on the phenyl
ring) was 52.50 %, whereas the final mortality rate of 5q (containing 2-dihydroxy and
3-methoxy on the phenyl ring), 5b (containing 4-chloro on the phenyl ring), and 5a
(containing 2-hydroxy on the phenyl ring) were 70.66, 81.33 and 81.33% respectively.
It appears that there is a relationship between the chemical structure of phenolic
moieties and its insecticidal activity.
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<<< Table 1>>>>
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<<< Figure 4>>>>
These compounds contain aromatic nucleus attached to a polar functional group
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(OH). It is well known that phenolic hydroxyl group is quiet reactive and easily forms
hydrogen bonds with active sites of enzymes[42]. Similarly, the final mortality rate of
5f and 5o (containing 4-Nitro on the phenyl ring) were 80.00%; whereas the final
mortality rate of 5n (containing 2-OH and 4-N,N-diethylamino on the phenyl ring) was
83.33%. However, introduction of the substituent R as methoxy or carboxyl at 4-postion
(e.g., 5e and 5h) did not exhibit more effective compounds compared to their precursor
piperine. The introduction of OH group at position 2 with methoxy group at position 3
improved the final mortality to reach 70.66%. Interestingly, for compounds 5d, 5s and
5r (final mortality was 61.33, 65.33 and 68.00%, respectively), it was found that the
final mortality increased gradually as the number fluorinated carbons increased. Also,
the results showed that the final mortalities of 5t, 5i, 5m and 5u were 52.50, 54.66,
80.00 and 82.66%, respectively, where the final mortality increased gradually with
increasing the number of fused phenyl rings. This is may be due to the formation of
more π-π stacking interactions with the active site of the enzyme, which make it more
potent compound.
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<<< Figures 5>>>>
As shown in Figures 5, S23 and S24, the 3rd instar larvae of Cx. pipiens treated
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with the synthesized compounds exhibited various morphological aberrations including
the appearance of larval-pupal intermediate, malformed pupa and incomplete eclosions
of adults. Similar results were described by Bosly who found that distinct
malformations of larvae and pupae of the house fly were induced after treatment of the
third larval instar with LC50 and LC75 of Mentha piperita and Lavandula
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angustifoli[43]. These abnormalities could be attributed to hormonal disturbance that
caused by 5n and affect the insect metamorphosis. Mansour showed that Various ten
morphological abnormalities as abnormal pupal size in addition to pupal adult
intermediate were induced by using essential oils against Cx. Pipiens and Musca
domestica, respectively[44].
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3.3. Molecular docking studies
We performed molecular modelling followed by energy minimization of
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compounds 5a, 5f, 5n, 5g in AChE of Cx. Pipiens to gain insights into the possible
interactions between them and the binding site of AChE [32]. AChE is of interest
because it is a critical nervous system enzyme responsible for synaptic transmission and
is the target site for organophosphoate and carbamate insecticides [45,46]. Molecular
modeling shows that pentadienoic acid is predicted to make π-π stacking interactions
with Trp408. The phenol aromatic ring makes also π-π stacking interactions with
Trp212. Molecular modeling of 5a in the binding site of AchE shows that the hydroxyl
group of the phenol group can form hydrogen bonding with His567. This explains why
presence of substituents groups at the C-2 position on the phenyl ring of the
dienehydrazide derivatives was important to improve their insecticidal activity (Figure
6).
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<<< Figure 6>>>>
Adding a nitro group as in compound 5f results in formation of electrostatic interaction
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as a salt bridge between the nitrogen of the nitro group and the carboxyl group of
Glu326 hence this modification is predicted to strengthen the binding (Figure 7).
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<<< Figure 7>>>>
Alternatively, replacing the phenol group with furan as in 5g results in formation of π-
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π stacking interactions with His567 (Figure 8).
<<< Figure 8>>>>
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Adding 4-N,N-diethylamino to the phenol group in 5n resulted in similar interactions
as in 5a and possibly enhanced fitting of the compound in the binding site (Figure 9).
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<<< Figure 9>>>>
The molecular docking analysis suggests that these compounds are predicted to be tight
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binders of AChE and most probably producing their insecticidal activities through
inhibition of AChE enzyme.
4. Conclusion
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A series of piperine-based dienehydrazide derivatives were designed and
synthesized from piperine and their structures were elucidated by melting point, IR, 1H,
13C, 19F NMR and ESI-HRMS. An x-ray single crystal of compound 5n was obtained
to further illustrate the structural indices of this series of synthesized compounds. The
bioassays results showed that all these synthesized compounds showed better activities
against pre-third-instar larval of Cx. Pipiens than commercial insecticide deltamethrin.
And most of them exhibited stronger insecticidal activities than piperine. These kinds
of compounds deserve further exploration to discover green pesticides.
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Acknowledgments
The research was funded by the National Key Research and Development
Program of China (2017YFE0113900) and Huazhong Agriculture University, Talent
Young Scientist Program (Grant No. 42000481-7).
Supporting Information
Supporting information may be found in the online version of this article.
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5. References:
[1] C. Lamberth, S. Jeanmart, T. Luksch, A. Plant, Current challenges and trends in
the discovery of agrochemicals, Science (80-. ). 341 (2013) 742–746.
doi:10.1126/science.1237227.
[2] R. Pavela, Possibilities of botanical insecticide exploitation in plant protection.,
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Pest Technol. 1 (2007) 47–52.
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25

667
668
LIST OF FIGURES
No.
Caption
Figure 1
Figure 2
Chemical structure of piperine.
General scheme for synthetic route for the synthesis of 5a–u. Reagents and conditions: (a)
KOH, 95% methanol, reflux, 48h, 93.4%; (b) (COCl)2, DCM, three drops DMF, room
temperature, 4 h; (c) NH2NH2/DCM, room temperature, 7 h, 87%; (d) corresponding carbonyl
compounds, methanol, reflux, 2-6 h, 55-86%.
Figure 3
Figure 4
The X-ray single crystal structure of 5n.
The corrected mortality rates of Cx. pipens caused by more active compounds with the increase
of concentration after 48 h.
A- Normal 3rd instar larvae; B- 4th instar larva showing symptoms of dwarfism and body
shrinkage; C- Malformed larvae with the appearance of neck between head and 1st thoracic
segment; D- Larva has a swollen thorax, which finally lead to rupture of the larva; E- 4th instar
larva with old exuvium of the 3rd instar larva on the posterior part of the body.
Figure 5
A. two- dimensional and B. three-dimensional figures demonstrating molecular interactions of
compound 5a in the binding site of AchE. π-π stacking interactions are colored green lines while
hydrogen bonding interactions are shown as purple lines.
Figure 6
Figure 7
A. two- dimensional and B. three-dimensional figures demonstrating molecular interactions of
compound 5f in the binding site of AchE. π-π stacking interactions are colored green lines while
hydrogen bonding interactions are shown as purple lines
A. two- dimensional and B. three-dimensional figures demonstrating molecular interactions of
compound 5g in the binding site of AchE. π-π stacking interactions are colored green lines while
hydrogen bonding interactions are shown as purple lines.
Figure 8
Figure 9
A. two- dimensional and B. three-dimensional figures demonstrating molecular interactions of
compound 5n in the binding site of AchE. π-π stacking interactions are colored green lines while
26

hydrogen bonding interactions are shown as purple lines
669
670
671
672
673
674
675
676
677
678
679
680
Figure 1
O
O
N
Compartment B
O
Compartment C
Compartment A
681
682
683
684
685
686
687
688
689
690
691
692
27

693
Figure 2
O
O
a
O
O
O
O
O
H
N
[2]
[1]
b
O
O
O
O
O
O
c
Cl
N
NH2
H
[3]
[4]
d
O
N
O
O
R1
N
H
R2
[5a-u]
694
695
696
R1
R2
R1
H
R2
R1
R2
Cl
OH
O
O
5a:
5d:
H
H
5b:
5e:
5c:
5f:
CH3
80%
78%
80%
H
H
H
OSO2(CF2
)
5CF3
NO2
83%
O
69%
65%
O
5g:
5j:
H
H
5h:
5k:
5i:
5l:
H
H
65%COOH
70%
77%
55%
77%
81%
28

OH
5m:
5p:
H
5n:
5q:
H
H
5o:
5r:
CH3
N
No2
79%
85%
79%
OH
O
OCH3
O
CH3
H
OSO2(CF2)7CF3
66%
79%
75%
5s:
H
5t:
H
5u:
H
CF3
72%
69%
71%
697
698
699
Figure 3
700
701
702
703
704
Figure 4
29