Synthesis and Photoactivated Toxicity of 2-Thiophenylfuranocoumarin Induce Midgut Damage and Apoptosis in Aedes aegypti Larvae

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Synthesis and Photoactivated Toxicity of

2‑Thiophenylfuranocoumarin Induce Midgut Damage and

Apoptosis in Aedes aegypti Larvae

Jiecheng Wu,^§Lanying Wang,^§Yunfei Zhang, Shujing Zhang, Shakil Ahmad, and Yanping Luo*

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ABSTRACT: Furanocoumarins are photoactive compounds derived from secondary plant metabolites. They possess many

bioactivities, including antioxidative, anticancer, insecticidal, and bactericidal activities. Here, we designed a new scheme for

synthesizing 2-arylfuranocoumarin derivatives by condensation, esteriﬁcation, bromination, and Wittig reaction. We found that 2-

thiophenylfuranocoumarin (Iy) had excellent photosensitive activity. Three Iy concentrations (LC₂₅, LC₅₀, and LC₇₅) were used to

treat the fourth instar larvae of Aedes aegypti (A. aegypti). The photoactivated toxicity, sublethal dose, mitochondrial dysfunction,

oxidative stress level, intestinal barrier dysfunction, and apoptosis were studied. The results showed that Iy induced reactive oxygen

species (ROS) production in midgut cells under ultraviolet light. Ultrastructural analysis demonstrated that mitochondria were

damaged, and the activities of related enzymes were inhibited. Ultimately, Iy exposure led to excessive ROS production followed by

the inhibition of antioxidant enzymes, including SOD, CAT, GPx, and GR, which diminished ROS elimination and escalated

oxidative stress in midgut cells, aggravating the degree of oxidative damage in these cells. Histopathological changes were observed in

the midgut, which led to intestinal barrier dysfunction. When the elimination of ROS was blocked and it accumulated in cells,

apoptosis-related genes, including AeDronc, AeCaspase7, and AeCaspase8, were induced and activated. In addition, Iy aﬀected the

growth and development of A. aegypti at sublethal concentrations, and there was an obvious post-lethal eﬀect. Thus, we found that Iy

caused midgut damage and apoptosis in A. aegypti larvae under ultraviolet light, which preliminarily revealed the mode of action of Iy

in A. aegypti.

KEYWORDS: 2-thiophenylfuranocoumarin, Aedes aegypti, midgut, oxidative stress, apoptosis

antioxidant defense systems, superoxide dismutase (SOD) is

1. INTRODUCTION

the ﬁrst line of defense.¹⁹SOD converts O₂into hydrogen

−

Furanocoumarins are photosensitive compounds produced as

secondary metabolites by plants to resist the attack of fungi,

bacteria, and insects.¹⁻³In humans, furanocoumarins have

broad application prospects as anti-inﬂammatory and anti-

oxidative agents, along with their therapeutic potential in the

treatment of skin diseases and cancer.⁴⁻⁶Due to their

antibacterial and insecticidal properties, furanocoumarins are

regarded as natural pesticides.⁷Irradiation of furanocoumarins

by ultraviolet light causes electron transition and change to the

triplet state. The triplet status is persistent in vivo and involved

in energy transfer and phototoxicity.^8,9Furanocoumarins can

produce reactive oxygen species (ROS) and damage cells

through lipid peroxidation, enzyme inactivation, guanine

oxidation, and nucleic acid fragmentation.^3,10−12In eukaryotic

cells, mitochondria are critically involved in metabolite

biosynthesis and the production of the energy carrier molecule

ATP.¹³Typically, ROS originates from the electron transport

chain (ETC).¹⁴Speciﬁcally, nicotinamide adenine dinucleotide

(NADH) and reduced ﬂavin adenine dinucleotide (FADH₂)

produced by the tricarboxylic acid (TCA) cycle provide

electrons to the ETC complexes I and II, respectively.¹⁵When

the mitochondria are damaged, ETC electrons leak, and ROS

are produced in the form of superoxide anions.¹⁶Excessive

ROS levels can lead to oxidative stress.^17,18Among biological

peroxide (H₂O₂), which is decomposed into water by the

catalase (CAT) reaction. Concurrently, glutathione peroxidase

(GPx) catalyzes the reaction of glutathione (GSH) with H₂O₂

to form water,²⁰which reduces the ROS level in vivo.

Glutathione reductase (GR) can reduce oxidized glutathione

(GSSG) to GSH, which regenerates the antioxidant capacity of

the GSH antioxidant system. The ratio of GSH to GSSG

reﬂects the redox state of the organism,²¹while the levels of

malondialdehyde (MDA) and protein carbonyl reﬂect the

degree of lipid peroxidation and protein oxidation, respec-

tively.^22,23

Aedes aegypti (A. aegypti) is an eﬀective vector of dengue

fever, yellow fever, chikungunya fever, and other important

viral diseases. It is a global problem endangering public

health.²⁴⁻²⁶Photosensitizers with insecticidal activity are

Received: November 16, 2020

Revised: December 31, 2020

Accepted: December 31, 2020

Published: January 12, 2021

https://dx.doi.org/10.1021/acs.jafc.0c07237

J. Agric. Food Chem. 2021, 69, 1091−1106

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Journal of Agricultural and Food Chemistry

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Article

bromosuccinimide (NBS) were mixed with a catalytic amount of

benzoyl peroxide and 10 mL of anhydrous benzene then heated and

reﬂuxed. The reaction was monitored by TLC for approximately 6−

10 h. The resulting reaction mixture was concentrated into a solid

under reduced pressure, washed with hot water, ﬁltered, and dried.

The product was recrystallized with glacial acetic acid to obtain 6-

bromomethyl-7-ester coumarin 4, which was used in the next step

without further puriﬁcation.

Using a 25 mL round-bottom ﬂask, 6-bromomethyl-7-ester

coumarin 4 (1.0 mmol), triphenylphosphine (1.0 mmol), and 10

mL of anhydrous CH₃Cl were mixed. The reaction was monitored by

TLC after heating and reﬂuxing. The reaction mixture was

concentrated until a solid was formed, which was washed with

toluene and ﬁltered to obtain 6-bromotriphenylphosphine methyl-7-

ester coumarin 5. Without further puriﬁcation, the resulting material

was used in the next step.

The six-fold amount of anhydrous K₂CO₃and 10 mL of anhydrous

tetrahydrofuran (THF) were added to the product described above.

The reaction, which was performed under stirring at room

temperature for approximately 4−10 h, was monitored by TLC.

The products were separated by silica gel (200−300 mesh) column

chromatography using petroleum ether-ethyl acetate (10/1, v/v) as a

mobile phase. White or yellow solid I was produced.

2.2.4. Test Method for Insecticidal Activity of Target Compound

I. In the study on the activity of target compound I, compound I was

dissolved in acetone and prepared into a solution of 100 mg/L with

dechlorinated water, and the mosquito species tested was Culex

pipiens pallens. The fourth instar larvae of Culex pipiens pallens were

exposed to 100 mg/L compound I solution for 3 h in the dark. Then,

the larvae were exposed under a 365 nm black light lamp (Hitachi 6

W, 17 cm away from the culture dish) for 3 h and placed again in the

dark for 42 h. Finally, the mortality of larvae was calculated.

2.3. Toxicity Test. Thirty A. aegypti larvae at the fourth instar

were randomly selected and placed into culture dishes containing 9.8

mL of dechlorinated water, to which six diﬀerent concentrations

(2000, 2500, 3000, 3500, 4000, and 4500 mg/L) of 0.2 mL of Iy

solution were added. Therefore, the ﬁnal concentration of the solution

was 40, 50, 60, 70, 80, and 90 mg/L, respectively. The fourth instar

larvae were placed in the dark for 3 h to fully absorb the solution.

Then, the larvae were exposed under a 365 nm black light lamp

(Hitachi 6 W, 17 cm away from the culture dish) for 3 h and placed

again in the dark for 42 h. The solvent control was created by adding

0.2 mL of acetone to 9.8 mL of dechlorinated water, and the blank

control consisted of 10 mL of dechlorinated water. Five replicates of

each concentration were used, and each experiment was repeated

three times. The mortality and toxicity caused by Iy were measured by

enumerating dead and surviving larvae.

environmentally friendly and have a broad application prospect

in mosquito control.²⁷In A. aegypti, the core apoptosis

pathway depends on the initiator caspase AeDronc. Upon

induction by an apoptotic signal, the initiator caspase AeDronc

binds to its adaptor molecule AeArk and activates the

downstream caspases AeCaspase7 and AeCaspase8, leading

to apoptosis.^28,29This process is blocked by the A. aegypti

inhibitor of apoptosis protein 1 (AeIAP1), which inhibits

apoptosis through direct interaction with AeDronc, AeCas-

pase7, and AeCaspase8. AeMichelob_x is an antagonist

molecule of AeIAP1 that can neutralize the inhibitory eﬀect

of AeIAP1 on caspases and promote apoptosis.³⁰

In this study, we designed a new scheme for synthesizing 2-

arylfuranocoumarin derivatives from resorcinol by condensa-

tion, esteriﬁcation, bromination, and Wittig reaction. We

assessed the ﬂuorescence properties of the newly synthesized

derivatives. An in vivo assessment of target compound 2-

thiophenylfuranocoumarin (Iy) found that it exerted excellent

photoactivated toxicity in A. aegypti larvae. Therefore, the

eﬀects of Iy on the growth and development of A. aegypti larvae

at sublethal concentrations were evaluated. Furthermore, the

mechanism of midgut damage was elucidated by measuring the

oxidative stress level, performing a histological and ultra-

structural analysis of midgut paraﬃn sections using a

transmission electron microscope, and assessing the perme-

ability of the peritrophic membrane. Finally, a series of

physiological conditions related to apoptosis were applied to

study cell apoptosis.

2. MATERIALS AND METHODS

2.1. Test Materials and Equipment. The susceptible strain of A.

aegypti was provided by the Agricultural and Pharmaceutical

Laboratory at Hainan University, Haikou, China. A. aegypti was

reared, as described previously.³¹

The ROS assay kit, mitochondrial membrane potential (MMP)

assay kit, and oxidative stress and mitochondria-related indicator assay

kit were purchased from Solarbio Technology Company (Solarbio,

Beijing, China). The protein concentration assay kit and apoptosis

staining kit were purchased from Beyotime Biotechnology Company

(Beyotime Biotechnology, Shanghai, China). All other commercially

available chemical reagents were analytically pure or chemically pure.

Equipment included the micro melting point apparatus (Yanagi-

moto, Kyoto, Japan), an AV 400 NMR spectrometer (Bruker,

Watertown, MA, USA; CDCl₃was used as a solvent), a Trace MS

2000 GS-MS (Thermo Finnigan, San Jose, CA, USA), a Vario EL III

element analyzer (Elementar, Langenselbold, Germany), and a mixer

mill MM400 frozen (Retsch, Haan, Germany).

2.2. Synthesis of Furan [3,2-g] Coumarin I. 2.2.1. Synthesis of

7-Hydroxy-4,6-dimethylcoumarin 2. In a 250 mL round-bottom

ﬂask, 12.4 g (0.1 mol) of 4-methyl-1,3-resorcinol and 50 mL of

concentrated sulfuric acid were mixed in an ice bath for 1 h, during

which 0.12 mol of ethyl acetoacetate (or ethyl 2-chloroacetoacetate)

was added by slowly dripping into the mixture. Then, the ice bath was

removed, and the solution was stirred overnight at 25 °C. The

resulting reaction solution was poured into an ice-water mixture. A

large amount of a light-yellow precipitate formed, which was

recovered as white solid 2 by vacuum ﬁltration.

2.4. Eﬀect of Sublethal Dose on Larval Development

Parameters. Based on the calculated toxicity of Iy, the larvae were

treated at diﬀerent sublethal concentrations, and the eﬀects of Iy on

larval growth and development were evaluated, along with the post-

lethal eﬀect. Three hundred fourth instar larvae were randomly

selected and exposed to LC₁₀, LC₂₀, and LC₃₀(45, 51, and 56 mg/L)

of Iy solution, using the same procedure as in the toxicity test. The

larval death numbers of the treatment and control groups were

examined after 48 h, and the larval mortality was calculated.

The surviving larvae were washed 3 times with dechlorinated water

and raised normally. The pupae were collected and placed in adult

cages. The numbers of pupae and eclosion mosquitoes were regularly

checked every day. The cumulative pupation rate and emergence rate

were calculated, and the average pupation time and average

emergence time were documented. Furthermore, the numbers of

dead pupae and abnormal eclosion mosquitoes were regularly

recorded every day. The collected data were used to calculate the

dead pupa rate (dead pupa number/total pupa number) and the

abnormal emergence rate (abnormal eclosion mosquito number/total

eclosion mosquito number).

2.2.2. Synthesis of 7-Ester-4,6-dimethylcoumarin 3. One mmol of

7-hydroxy-4,6-dimethylcoumarin 2 and 0.17 g of anhydrous K₂CO₃

were placed in a grinding container, and 1.1 mmol of substituted aryl

chloride was quickly added. The mix was ground for 15 min at 30 Hz.

Then, the reaction mix was checked by thin-layer chromatography

(TLC), washed, and ﬁltered. The product, 7-ester-4,6-dimethylcou-

marin 3, was obtained by recrystallization with anhydrous ethanol.

2.2.3. Synthesis of Target Compound I. In a 25 mL round-bottom

ﬂask, 1.0 mmol of 7-ester coumarin 3 and 1.1 mmol of N-

2.5. Detection of Singlet Oxygen (¹O₂). The previously

published method for measuring ¹O₂was slightly modiﬁed in the

present study.³²Samples containing 100 mg/L Iy and 10 × 10⁻³mol/

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L 2,2,6,6-tetramethylpiperidine (Macklin, Shanghai, China) were

irradiated with 365 nm black light for 30 min. The O₂production

MO, USA), for 1 h (unbound FITC was removed by a Sephadex gel

G10 column). The stained larvae were washed three times with

dechlorinated water and placed in an ice bath for 10 min to slow

down their activity for microscopic analysis using the SZX16

ﬂuorescence microscope (Olympus). Green ﬂuorescence of FITC in

the larvae was measured using the GFP channel, and spontaneous

ﬂuorescence was captured using the RFP channel for subsequent

colocation analysis.

1

was detected by electron spin resonance (ESR) spectroscopy using

the AV 400 NMR spectrometer (Bruker). The ESR parameter settings

1

for measuring O₂in this experiment were as follows: center ﬁeld,

3565G; power, 20.02 mW; modulation frequency, 100 kHz; time

constant, 2.56 ms; receiver gain, 1 × 10³).

2.6. Detection of ROS. We used a previously described ROS

detection method with some modiﬁcations.³³The larvae were treated

with 65 mg/L Iy. The whole digestive organ system was recovered by

dissection at 0, 1, 2, 3, and 4 h after light treatment followed by

incubation in 2 μmol/L 2,7-dichloro-hydroﬂuorescein diacetate

(DCFH-DA) for 30 min. ROS production was analyzed qualitatively

and quantitatively. To perform a qualitative analysis, samples loaded

with the probe were washed three times with phosphate-buﬀered

saline (PBS), and green ﬂuorescence signals were observed with an

Olympus BX53 ﬂuorescence microscope (Olympus Corporation,

Tokyo, Japan) using 488 and 525 nm for excitation and emission,

respectively. For quantitative analysis, 30 digestive organs were

submerged in 5 mL of ultrapure water and completely ground with a

glass bead homogenizer. The tissue homogenates were centrifuged at

11,000g for 10 min at 4 °C, and the supernatants were collected for

ﬂuorescence measurements with a multifunction enzyme labeling

instrument (PerkinElmer, Waltham, MA, USA) using 488 and 525

nm for excitation and emission, respectively. The results were

expressed as fold changes in ﬂuorescence intensity at 1 h intervals

over the value measured at 0 h.

2.7. Observation by Transmission Electron Microscopy

(TEM). The dissected midgut was cleaned with PBS and ﬁxed

overnight in 2.5% glutaraldehyde solution. The samples were

processed as previously described and analyzed using a TEM

instrument (Hitachi, Tokyo, Japan).³⁴

2.8. Detection of Mitochondrial and Oxidative Stress

Indicators. The 60 midguts were dissected, washed with PBS, and

weighed before being ground with a glass bead homogenizer. The

tissue homogenates were used to detect mitochondria and oxidative

stress indicators. The protein concentrations of the samples were

normalized based on the results obtained with the Bradford protein

concentration assay kit (Solarbio).

The indicators of oxidative stress, including SOD, CAT, GR, GPx,

GSH, GSSG, MDA, and protein carbon, were determined by a kit

(cat. nos. BC0175, BC0200, BC1165, BC1190, BC1175, BC1185,

BC0025, and BC1270, Solarbio). Mitochondria-related indicators,

including complex I, complex II, complex III, complex IV, citric acid

synthase (CS), isocitrate dehydrogenase (ICDHm), α-ketoglutarate

dehydrogenase (α-KGDH), and ATP, were evaluated according to

the manufacturer’s instructions. (cat. nos. BC0515, BC3235, BC3245,

BC0945, BC1065, BC2165, BC0715, and BC0305, Solarbio).

2.9. Detection of Mitochondrial Membrane Potential

(MMP). As previously described,³⁵the dissected midgut samples

were incubated with 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimida-

zolyl carbocyanine iodide (JC-1) staining solution for 20 min at 37 °C

then washed three times with JC-1 staining buﬀer. Green ﬂuorescence

of JC-1 monomers was detected at 490/530 nm (excitation/

emission), and red ﬂuorescence of JC-1 aggregates was observed at

525/590 nm (excitation/emission). The images were captured with

the Olympus BX53 ﬂuorescence microscope. ImageJ software was

used to choose the images with the same exposure time for statistical

analysis of MMP, and the ratio of green to red ﬂuorescence was

calculated. Each calculation was based on ten images selected for the

statistical analysis.

2.10. Histopathological Evaluation of the Midgut. Iy-treated

larvae were ﬁxed in 4% paraformaldehyde solution for 48 h. The

samples were processed as described previously,³⁶observed under a

light microscope (Carl Zeiss AG, Oberkochen, Germany), and the

pictures were taken.

2.11. Detection of Peritrophic Membrane Permeability. As

previously described,³⁷the surviving larvae were washed three times

with chlorinated water and submerged in 0.5 mg/mL ﬂuorescein

isothiocyanate (FITC)-dextran, 40 kDa (Sigma-Aldrich, St. Louis,

2.12. Detection of Chitin Content. Thirty midguts were

dissected and washed with PBS before determining the wet weight.

The midgut samples were ground in 1 mL of ultrapure water using a

glass bead homogenizer. The samples were processed, as described

previously.³⁸The absorbance was measured at 650 nm using an

ultraviolet−visible spectrophotometer (MAPADA, Shanghai, China).

A stock solution containing 100 mg/L glucosamine (Sigma-Aldrich)

was used to construct a standard curve for normalizing the chitin

content values, which were expressed based on the amount of

glucosamine.

2.13. Terminal Deoxynucleotidyl Transferase dUTP Nick

End Labeling (TUNEL) Assay. TUNEL staining was performed as

previously described, with some modiﬁcations.³⁹The one-step

TUNEL cell apoptosis assay kit was used to assess the apoptosis

status. The midgut samples were incubated in 100 μL of TUNEL

solution at 37 °C for 1 h and then washed three times with PBS.

Apoptosis was observed using the Olympus BX53 ﬂuorescence

microscope. Appropriate ﬁlters were selected for the excitation

wavelength range of 450−500 nm and the emission wavelength range

of 515−565 nm.

2.14. Hoechst 33342 Staining. According to the manufacturer’s

instructions, the midgut samples were incubated in 1 mL of Hoechst

33342 working solution at 4 °C for 30 min followed by three times

washing with PBS. Apoptosis was observed using the Olympus BX53

ﬂuorescence microscope at 350/461 nm (excitation/emission).

2.15. Quantitative Reverse Transcription PCR (RT-qPCR).

For the RT-qPCR analysis, we used primer sequences as published

previously.³¹Total RNA was extracted from 15 midgut samples with

the TRIzol RNA reagent kit (Thermo Scientiﬁc, Waltham, MA,

USA), and the ﬁrst-strand cDNA was synthesized using the RevertAid

First Strand cDNA Synthesis Kit (Thermo Scientiﬁc). The qPCR step

was performed using the SYBR Select Master Mix (2X) kit (ABI,

Waltham, MA, USA) with a total volume of 20 μL. The ampliﬁcation

conditions were as follows: predenaturation at 95 °C for 10 min was

followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s. Dissolution

curve analysis was performed as follows: at 95 °C for 15 s, at 60 °C for

60 s, and at 95 °C for 15 s. The expression levels of AeDronc,

AeCaspase7, AeCaspase8, AeIAP1, AeMichelob_x, and the house-

keeping gene RP7S were analyzed. Using the RP7S gene as the

reference gene, the relative gene expression was calculated by the

2^−ΔΔCtmethod.

2.16. Statistical Analysis. All experiments were carried out in

triplicate and repeated three times, at least. Representative data values

are expressed as the mean standard deviation (SD). The analysis of

variance and Duncan’s test (SPSS 23.0) were used to evaluate the

diﬀerence between the average values of each treatment. The results

were considered statistically signiﬁcant when the probability (p) value

was <0.05.

3. RESULTS

3.1. Chemistry. Coumarin 2 was synthesized from O-

methylresorcinol and 1-dicarbonyl compound 1 in the

presence of concentrated sulfuric acid, and ester compound

3 was obtained by the reaction of aryl chloride with compound

2 in the presence of triethylamine; the physicochemical

properties of compound 3 derivatives are shown in Table 1.

Compound 4 was brominated by NBS at the methyl group on

position 6 of the coumarin core. Resulting compound 4 was

reacted with triphenylphosphine (PPh₃) to form Wittig reagent

5. In the presence of K₂CO₃, compound 5 underwent an

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Table 1. Physicochemical Properties of Compound 3

Derivatives

Table 2. Physicochemical Properties of Target Compound I

Derivatives

no.

R¹

R²

yield (%) m. p. (°C) physical state

no.

R¹

R²

yield (%) m. p. (°C) physical state

3a

3b

3c

3e

3f

3g

3i

C₆H₅

H

Cl

H

100

97

95

90

93

91

96

100

92

94

95

94

96

90

87

96

94

85

179−181

219−221

210−212

213−214

221−222

185−187

223−225

202−204

233−234

240−242

206−207

188−189

222−224

191−192

222−224

216−218

235−237

128−130

white crystal

white solid

white crystal

white solid

white crystal

white solid

white crystal

Ia

Ib

Ic

Ie

If

Ig

Ii

Ij

Im

Ip

It

Iu

Iw

Ix

C₆H₅

H

Cl

H

89

84

86

80

83

74

90

88

76

88

69

75

87

69

74

84

89

79

216−218

227−229

249−250

257−259

260−262

254−256

239−241

216−218

218−220

228−230

208−210

215−217

204−206

187−189

190−192

235−237

246−248

123−125

white solid

yellow solid

white solid

yellow solid

white solid

2-F-C₆H₄

3-F-C₆H₄

2-F-C₆H₄

3-F-C₆H₄

4-F-C₆H₄

3-Cl-C₆H₄

4-Cl-C₆H₄

3,4-2F-C₆H₃

2,4-2Cl-C₆H₃

3-CF₃-C₆H₃

4-CF₃-C₆H₃

4-OCH₃-C₆H₄

C₄H₃O

2-F-C₆H₄

3-F-C₆H₄

2-F-C₆H₄

3-F-C₆H₄

4-F-C₆H₄

3-Cl-C₆H₄

4-Cl-C₆H₄

3,4-2F-C₆H₃

2,4-2Cl-C₆H₃

3-CF₃-C₆H₃

4-CF₃-C₆H₃

4-OCH₃-C₆H₄

C₄H₃O

3j

3m

3p

3t

3u

3w

3x

3y

3aa

3bb

3cc

C₄H₃S

1-C₁₀H₇

2-C₁₀H₇

C₃H₅

Iy

Iaa

Ibb

Icc

C₄H₃S

1-C₁₀H₇

2-C₁₀H₇

C₃H₅

intramolecular Wittig reaction to generate target compound I

(Scheme 1); the physicochemical properties of target

compound I derivatives are shown in Table 2.

the 3-position hydrogen was replaced by chlorine, this peak

will disappear. There was a single peak between 2.4 and 2.6

ppm for the 4-methyl group. If there was chlorine substitution

in the adjacent position, the chemical shift will move to the

high ﬁeld. There was a single peak near 2.32 ppm for the

chemical shift of 6-methyl. The mass spectra showed that the

molecular ion peaks were unstable and the abundance was low.

3.1.2.1. 4,6-Dimethyl-2-oxo-2H-chromen-7-yl-benzoate

The synthesis of the target compound requires bromination,

along with Wittig reagent preparation and reaction, which

depend on humidity-sensitive substances. To minimize the

eﬀect of environmental humidity and other factors, our newly

designed synthesis scheme combined the three synthesis steps

without purifying the intermediates, which were directly used

as substrates in the next reaction to obtain target compound I.

3.1.1. Spectral Analysis of Coumarin 2. 7-Hydroxy-4,6-

dimethylcoumarin (2a): The yield is 96%, m. p. 258−260 °C

(literature value 268 °C). ¹H NMR (400 MHz, CDCl₃): δ 7.33

(s, 1H), 6.87 (s, 1H), 6.13 (s, 1H), 6.03 (s, 1H), 2.40 (s, 3H),

2.31 (s, 3H).

1

(3a). H NMR (400 MHz, CDCl₃): δ 8.30−8.13 (m, 2H),

7.68 (dd, J = 10.6, 4.4 Hz, 1H), 7.54 (dd, J = 18.5, 11.0 Hz,

3H), 7.20 (s, 1H), 6.28 (s, 1H), 2.45 (s, 3H), 2.31 (s, 3H). MS

(EI): m/z (%) 294 ([M]⁺, 4.58), 105 (100), 77 (41.08). Anal.

calcd for C₁₈H₁₄O₄: C, 73.46; H, 4.79. Found: C, 73.48; H,

4.81.

3.1.2.2. 4,6-Dimethyl-2-oxo-2H-chromen-7-yl-2-ﬂuoro-

1

3-Chloro-7-hydroxy-4,6-dimethylcoumarin (2b): The yield

benzoate (3b). H NMR (400 MHz, CDCl₃): δ 8.13 (t, J =

1

is 83%, m. p. 242−244 °C. H NMR (400 MHz, CDCl₃): δ

7.6, 1H), 7.73−7.56 (m, 1H), 7.50 (s, 1H), 7.31 (t, J = 7.6 Hz,

1H), 7.28−7.23 (m, 1H), 7.21 (s, 1H), 6.27 (s, 1H), 2.45 (s,

3H), 2.33 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 163.31,

162.01, 160.67, 152.43, 151.76, 135.74, 132.69, 126.94, 126.40,

124.37, 124.33, 118.18, 117.49, 117.27, 114.78, 110.94, 77.34,

77.18, 77.02, 76.82, 76.70, 18.73, 16.18. MS (EI): m/z (%)

7.14 (s, 1H), 6.98 (s, 1H), 5.12 (s, 1H), 2.53 (s, 3H), 2.35 (s,

3H).

3.1.2. Spectral Analysis of Compound 3. Nuclear magnetic

resonance (NMR) results show that there was a single peak

near 6.28 ppm for the 3-position hydrogen of the coumarins. If

Scheme 1. Synthesis of 2-Substituted Aryl-7H-furo [3,2-g] Coumarin I. (a) H₂SO₄and Ice; (b) ArCOCl and K₂CO₃; (c) NBS,

Benzene, and Reﬂux; (d) PPh₃, CHCl₃, and Reﬂux; (e) K₂CO₃and THF. R¹= H and Cl. Ar = Aryl Substitution.

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312 ([M]⁺, 7), 161 (1), 123 (100), 95 (17), 75 (3). Anal. calcd

for C₁₈H₁₃FO₄: C, 69.23; H, 4.20. Found: C, 69.22; H, 4.23.

3.1.2.3. 4,6-Dimethyl-2-oxo-2H-chromen-7-yl-3-ﬂuoro-

3.1.2.12. 4,6-Dimethyl-2-oxo-2H-chromen-7-yl-4-(triﬂuor-

omethyl) Benzoate (3u). ¹H NMR (400 MHz, CDCl₃): δ 8.36

(d, J = 8.2 Hz, 2H), 7.83 (d, J = 8.3 Hz, 2H), 7.55 (d, J = 8.7

Hz, 1H), 7.17 (d, J = 8.7 Hz, 1H), 6.32 (s, 1H), 2.47 (s, 3H),

2.34 (s, 3H). MS (EI): m/z (%) 362 ([M]⁺, 5), 173 (100),

145 (26), 95 (2), 44 (2). Anal. calcd for C₁₉H₁₃F₃O₄: C, 62.99;

H, 3.62. Found: C, 62.95; H, 3.64.

1

benzoate (3c). H NMR (400 MHz, CDCl₃): δ 8.02 (d, J =

7.8 Hz, 1H), 7.96−7.85 (m, 1H), 7.61−7.45 (m, 2H), 7.39

(dd, J = 8.3, 2.6 Hz, 1H), 7.19 (s, 1H), 6.28 (s, 1H), 2.45 (s,

3H), 2.30 (s, 3H). MS (EI): m/z (%) 312 ([M]⁺, 9), 161 (1),

123 (100), 95 (28). Anal. calcd for C₁₈H₁₃FO₄: C, 69.23; H,

4.20. Found: C, 69.25; H, 4.24.

3.1.2.13. 4,6-Dimethyl-2-oxo-2H-chromen-7-yl-4-methox-

1

ybenzoate (3w). H NMR (400 MHz, CDCl₃): δ 8.24−8.13

(m, 2H), 7.48 (s, 1H), 7.18 (s, 1H), 7.05−6.99 (m, 2H), 6.27

(s, 1H), 3.92 (s, 3H), 2.44 (s, 3H), 2.30 (s, 3H). MS (EI): m/z

(%) 324 ([M]⁺, 2), 135 (100), 107 (4), 77 (6), 44 (1). Anal.

calcd for C₁₉H₁₆O₅: C, 70.36; H, 4.97. Found: C, 70.33; H,

4.98.

3.1.2.4. 3-Chloro-4,6-dimethyl-2-oxo-2H-chromen-7-yl-2-

ﬂuorobenzoate (3e). H NMR (400 MHz, CDCl₃): δ 8.13

1

(m, J = 7.6, 1.8 Hz, 1H), 7.78−7.61 (m, 1H), 7.53 (s, 1H),

7.36−7.26 (m, 2H), 2.60 (s, 3H), 2.35 (s, 3H). MS (EI): m/z

(%) 346 ([M]⁺, 4), 123 (100), 95 (15), 75 (2). Anal. calcd for

C₁₈H₁₂ClFO₄: C, 62.35; H, 3.49. Found: C, 62.38; H, 3.46.

3.1.2.5. 3-Chloro-4,6-dimethyl-2-oxo-2H-chromen-7-yl-3-

ﬂuorobenzoate (3f). ¹H NMR (400 MHz, CDCl₃): δ 8.02 (d,

J = 7.8 Hz, 1H), 7.90 (d, J = 8.9 Hz, 1H), 7.54 (d, J = 5.2 Hz,

2H), 7.39 (d, J = 2.4 Hz, 1H), 7.23 (s, 1H), 2.61 (s, 3H), 2.32

(s, 3H). MS (EI): m/z (%) 346 ([M]⁺, 7), 139 (5), 123 (100),

95 (27), 75 (4). Anal. calcd for C₁₈H₁₂ClFO₄: C, 62.35; H,

3.49. Found: C, 62.36; H, 3.50.

3.1.2.14. 4,6-Dimethyl-2-oxo-2H-chromen-7-yl-furan-2-

1

carboxylate (3x). H NMR (400 MHz, CDCl₃): δ 7.72 (m,

1H), 7.49 (s, 1H), 7.45 (d, J = 3.5 Hz, 1H), 7.19 (s, 1H),

6.69−6.58 (m, 1H), 6.28 (s, 1H), 2.44 (s, 3H), 2.31 (s, 3H).

MS (EI): m/z (%) 284 ([M]⁺, 17), 96 (6), 95 (100), 67 (2).

Anal. calcd for C₁₆H₁₂O₅: C, 67.60; H, 4.25. Found: C, 67.64;

H, 4.22.

3.1.2.15. 4,6-Dimethyl-2-oxo-2H-chromen-7-yl-thio-

1

phene-2-carboxylate (3y). H NMR (400 MHz, CDCl₃): δ

3.1.2.6. 3-Chloro-4,6-dimethyl-2-oxo-2H-chromen-7-yl-4-

8.03 (dd, J = 3.8, 1.1 Hz, 1H), 7.72 (dd, J = 5.0, 1.1 Hz, 1H),

7.48 (s, 1H), 7.22 (t, J = 4.4 Hz, 2H), 6.28 (s, 1H), 2.45 (s,

3H), 2.32 (s, 3H). MS (EI): m/z (%) 300 ([M]⁺, 8), 161 (1),

111 (100), 83 (4), 77 (1). Anal. calcd for C₁₆H₁₂O₄S: C,

63.99; H, 4.03; S, 10.68. Found: C, 63.95; H, 4.04; S, 10.71.

3.1.2.16. 4,6-Dimethyl-2-oxo-2H-chromen-7-yl-1-naph-

1

ﬂuorobenzoate (3g). H NMR (400 MHz, CDCl₃): δ 8.25

(dd, J = 8.9, 5.4 Hz, 2H), 7.27 (s, 1H), 7.22 (dd, J = 9.9, 7.3

Hz, 3H), 2.61 (s, 3H), 2.32 (s, 3H). MS (EI): m/z (%) 346

([M]⁺, 5), 123 (100), 95 (22), 75 (3). Anal. calcd for

C₁₈H₁₂ClFO₄: C, 62.35; H, 3.49. Found: C, 62.36; H, 3.44.

3.1.2.7. 4,6-Dimethyl-2-oxo-2H-chromen-7-yl-3-chloro-

1

thoate (3aa). H NMR (400 MHz, CDCl₃): δ 9.04 (d, J =

1

benzoate (3i). H NMR (400 MHz, CDCl₃): δ 8.08 (d, J =

8.6 Hz, 1H), 8.55 (dd, J = 7.3, 1.2 Hz, 1H), 8.16 (d, J = 8.2 Hz,

1H), 7.96 (d, J = 8.1 Hz, 1H), 7.67 (ddd, J = 18.0, 9.7, 6.3 Hz,

2H), 7.63−7.56 (m, 2H), 7.53 (s, 1H), 6.30 (s, 1H), 2.47 (s,

3H), 2.37 (s, 3H). MS (EI): m/z (%) 334 ([M]⁺, 3), 155

(100), 127 (40), 65 (2), 44 (7). Anal. calcd for C₂₂H₁₆O₄: C,

76.73; H, 4.68. Found: C, 76.77; H, 4.65.

8.4 Hz, 2H), 7.85−7.62 (m, 2H), 7.49 (s, 1H), 7.18 (s, 1H),

6.28 (s, 1H), 2.45 (s, 3H), 2.29 (s, 3H). MS (EI): m/z (%)

374 ([M]⁺, 7), 183 (100), 157 (15), 104 (3), 76 (8). Anal.

calcd for C₁₈H₁₃ClO₄: C, 65.76; H, 3.99. Found: C, 65.73; H,

3.41.

3.1.2.8. 4,6-Dimethyl-2-oxo-2H-chromen-7-yl-4-chloro-

3.1.2.17. 4,6-Dimethyl-2-oxo-2H-chromen-7-yl-2-naph-

1

benzoate (3j). H NMR (400 MHz, CDCl₃): δ 8.23−8.11

1

thoate (3bb). H NMR (400 MHz, CDCl₃): δ 8.82 (s, 1H),

(m, 2H), 7.60−7.51 (m, 2H), 7.50 (s, 1H), 7.19 (s, 1H), 6.29

(s, 1H), 2.45 (s, 3H), 2.30 (s, 3H). MS (EI): m/z (%) 328

([M]⁺, 7), 139 (100), 111 (15), 75 (3),44 (2). Anal. calcd for

C₁₈H₁₃ClO₄: C, 65.76; H, 3.99. Found: C, 65.75; H, 3.43.

3.1.2.9. 4,6-Dimethyl-2-oxo-2H-chromen-7-yl-3,4-diﬂuor-

8.21 (dd, J = 8.6, 1.7 Hz, 1H), 8.05−7.91 (m, 3H), 7.72−7.58

(m, 2H), 7.52 (s, 1H), 7.25 (s, 1H), 6.29 (s, 1H), 2.46 (s, 3H),

2.35 (s, 3H). MS (EI): m/z (%) 344 ([M]⁺, 6), 155 (100),

127 (45), 77 (2), 44 (5). Anal. calcd for C₂₂H₁₆O₄: C, 76.73;

H, 4.68. Found: C, 76.71; H, 4.64.

3.1.2.18. 4,6-Dimethyl-2-oxo-2H-chromen-7-yl-cyclopro-

panecarboxylate (3cc). ¹H NMR (400 MHz, CDCl₃): δ

7.42 (s, 1H), 7.05 (s, 1H), 6.23 (s, 1H), 2.41 (s, 3H), 2.24 (s,

3H), 1.91−1.88 (m, 1H), 1.22−1.21 (m, 2H), 1.09−1.08 (m,

2H). MS (EI): m/z (%) 258 ([M]⁺, 16), 190 (7), 162 (5),69

(100), 41 (13). Anal. calcd for C₁₅H₁₄O₄: C, 69.76; H, 5.46.

Found: C, 69.77; H, 5.45.

1

obenzoate (3m). H NMR (400 MHz, CDCl₃): δ 8.13−7.94

(m, 2H), 7.54 (d, J = 8.7 Hz, 1H), 7.34 (dd, J = 17.2, 8.9 Hz,

1H), 7.14 (d, J = 8.7 Hz, 1H), 6.31 (s, 1H), 2.47 (s, 3H), 2.33

(s, 3H). MS (EI): m/z (%) 330 ([M]⁺, 7), 141 (100), 113

(16), 77 (3), 44 (4). Anal. calcd for C₁₈H₁₂F₂O₄: C, 65.46; H,

3.66. Found: C, 65.42; H, 3.64.

3.1.2.10. 4,6-Dimethyl-2-oxo-2H-chromen-7-yl-2,4-di-

1

chlorobenzoate (3p). H NMR (400 MHz, CDCl₃): δ 8.07

3.1.3. Spectral Analysis of Target Compound I. 3.1.3.1. 5-

Methyl-2-phenyl-7H-furo [3,2-g] Chromen-7-one (Ia). ¹H

NMR (400 MHz, CDCl₃): δ 7.85 (d, J = 7.3 Hz, 2H), 7.75 (d,

J = 1.7 Hz, 1H), 7.47 (t, J = 7.4 Hz, 3H), 7.40 (d, J = 7.4 Hz,

1H), 7.04 (s, 1H), 6.25 (s, 1H), 2.49 (s, 3H). ¹³C NMR (100

MHz, CDCl₃): δ 160.95, 157.90, 152.20, 151.68, 150.19,

132.82, 132.19, 126.06, 126.02, 125.52, 117.91, 114.56, 108.24,

106.12, 103.21, 19.01. MS (EI): m/z (%) 276 ([M]⁺, 100),

248 (36), 219 (7), 189 (9), 124 (12), 105 (29). Anal. calcd for

C₁₈H₁₂O₃: C, 78.25; H, 4.38. Found: C, 78.22; H, 4.35.

3.1.3.2. 2-(2-Fluorophenyl)-5-methyl-7H-furo [3,2-g]

(d, J = 8.5 Hz, 1H), 7.59 (d, J = 2.0 Hz, 1H), 7.50 (s, 1H),

7.42 (dd, J = 8.5, 2.0 Hz, 1H), 7.19 (s, 1H), 6.29 (s, 1H), 2.45

(d, J = 1.0 Hz, 3H), 2.33 (s, 3H). MS (EI): m/z (%) 362

([M]⁺, 3), 173 (100), 145 (11),108 (2), 77 (2). Anal. calcd for

C₁₈H₁₂Cl₂O₄: C, 59.53; H, 3.33. Found: C, 59.51; H, 3.37.

3.1.2.11. 4,6-Dimethyl-2-oxo-2H-chromen-7-yl-3-(triﬂuor-

omethyl) Benzoate (3t). ¹H NMR (400 MHz, CDCl₃): δ 8.49

(s, 1H), 8.42 (d, J = 7.8 Hz, 1H), 7.95 (d, J = 7.8 Hz, 1H),

7.71 (t, J = 7.8 Hz, 1H), 7.52 (s, 1H), 7.20 (s, 1H), 6.30 (s,

1H), 2.46 (s, 3H), 2.31 (s, 3H). MS (EI): m/z (%) 362 ([M]⁺,

10), 173 (100), 145 (26), 95 (22), 77 (1). Anal. calcd for

C₁₉H₁₃F₃O₄: C, 62.99; H, 3.62. Found: C, 62.96; H, 3.59.

1

Chromen-7-one (Ib). H NMR (400 MHz, CDCl₃): δ 8.04

(dd, J = 8.4, 6.9 Hz, 1H), 7.82 (s, 1H), 7.50 (s, 1H), 7.37 (d, J

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= 7.3 Hz, 1H), 7.33−7.27 (m, 2H), 7.20 (dd, J = 11.4, 8.2 Hz,

1H), 6.29 (s, 1H), 2.52 (s, 3H). ¹³C NMR (100 MHz,

CDCl₃): δ 161.05, 158.36, 155.64, 152.65, 151.97, 130.19,

127.06, 126.62, 124.65, 118.11, 116.87, 116.52, 116.26, 116.05,

113.50, 106.07, 99.57, 19.18. MS (EI): m/z (%) 295 ([M +

1]⁺, 14.25), 294 ([M]⁺, 100), 266 (58.72), 265 (26.93), 237

(3.85), 236 (1.66), 209 (3.10), 207 (2.54), 189 (1.98), 133

(8.76), 123 (2.42). Anal. calcd for C₁₈H₁₁FO₃: C, 73.47; H,

3.77. Found: C, 73.48; H, 3.75.

219 (8), 189 (18), 141 (18). Anal. calcd for C₁₈H₁₁ClO₃: C,

69.58; H, 3.57. Found: C, 69.55; H, 3.56.

3.1.3.8. 2-(4-Chlorophenyl)-5-methyl-7H-furo [3,2-g]

1

Chromen-7-one (Ij). H NMR (400 MHz, CDCl₃): δ 7.81

(s, 1H), 7.79 (s, 2H), 7.49 (s, 1H), 7.45 (d, J = 8.6 Hz, 2H),

7.06 (s, 1H), 6.29 (s, 1H), 2.52 (s, 3H). ¹³C NMR (100 MHz,

CDCl₃): δ 161.00, 156.92, 156.35, 152.55, 151.87, 135.11,

129.25, 128.15, 126.34, 126.27, 116.93, 116.16, 113.60, 101.19,

99.75, 19.18. MS (EI): m/z (%) 312 ([M + 1H]⁺, 31.34), 311

([M]⁺, 17.47), 310 ([M − 1]⁺, 100), 284 (16.71), 283 (19.22),

282 (51.21), 281 (34.76), 254 (1.40), 219 (2.05), 189 (13.05),

176 (3.33), 141 (13.25), 109 (4.15), 94(15.02). Anal. calcd for

C₁₈H₁₁ClO₃: C, 69.58; H, 3.57. Found: C, 69.57; H, 3.55.

3.1.3.9. 2-(3,4-Diﬂuorophenyl)-5-methyl-7H-furo [3,2-g]

3.1.3.3. 2-(3-Fluorophenyl)-5-methyl-7H-furo [3,2-g]

1

Chromen-7-one (Ic). H NMR (400 MHz, CDCl₃): δ 7.80

(s, 1H), 7.65 (d, J = 7.9 Hz, 1H), 7.58−7.53 (m, 1H), 7.50 (s,

1H), 7.45 (dd, J = 8.1, 5.8 Hz, 2H), 7.09 (s, 1H), 6.29 (s, 1H),

2.52 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 160.96, 156.35,

152.53, 151.97, 132.08, 130.58, 128.46, 126.20, 120.71, 116.98,

116.33, 116.17, 115.95, 113.64, 112.11, 111.88, 101.81, 99.80,

19.17. MS (EI): m/z (%) 294 ([M]⁺, 100), 266 (62), 237 (8),

207 (9), 133 (29), 104 (7), 95 (10). Anal. calcd for

C₁₈H₁₁FO₃: C, 73.47; H, 3.77. Found: C, 73.46; H, 3.76.

3.1.3.4. 6-Chloro-2-(2-ﬂuorophenyl)-5-methyl-7H-furo

1

Chromen-7-one (Im). H NMR (400 MHz, CDCl₃): δ 7.69

(dd, J = 6.1, 5.0 Hz, 1H), 7.59 (s, 1H), 7.50 (dd, J = 29.9, 8.7

Hz, 2H), 7.33 (s, 1H), 7.30 (d, J = 8.3 Hz, 1H), 6.30 (s, 1H),

2.51 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 160.69, 156.95,

154.87, 153.47, 147.72, 121.41, 121.13, 120.85, 118.58, 118.22,

118.04, 114.99, 114.35, 114.16, 113.11, 108.14, 99.35, 19.39.

MS (EI): m/z (%) 312 ([M]⁺, 100), 294 (1.11), 284 (97.00),

227 (2.92), 207 (3.67), 142 (10.52). Anal. calcd for

C₁₈H₁₀F₂O₃: C, 69.23; H, 3.23. Found: C, 69.25; H, 3.20.

3.1.3.10. 2-(2,4-Dichlorophenyl)-5-methyl-7H-furo [3,2-g]

Chromen-7-one (Ip). ¹H NMR (400 MHz, CDCl₃): δ 8.02 (d,

J = 8.6 Hz, 1H), 7.86 (s, 1H), 7.60 (s, 1H), 7.55 (s, 1H), 7.50

(s, 1H), 7.41 (d, J = 8.4 Hz, 1H), 6.30 (s, 1H), 2.53 (s, 3H).

¹³C NMR (100 MHz, CDCl₃): δ 160.92, 155.52, 153.02,

152.53, 152.25, 134.96, 131.92, 130.83, 129.66, 127.62, 126.76,

126.21, 117.04, 116.90, 113.66, 107.16, 99.57, 19.18. MS (EI):

m/z (%) 346 ([M + 1]⁺,66.15), 344 ([M − 1]⁺, 100), 316

(48.88), 315 (21.20), 189 (15.02), 173 (4.81), 157 (10.57),

122 (7.26), 99 (2.27), 94 (15.76). Anal. calcd for

C₁₈H₁₀Cl₂O₃: C, 62.63; H, 2.92. Found: C, 62.61; H, 2.94.

3.1.3.11. 5-Methyl-2-(3-(triﬂuoromethyl) phenyl)-7H-furo

1

[3,2-g] Chromen-7-one (Ie). H NMR (400 MHz, CDCl₃):

δ 8.04 (td, J = 7.7, 1.8 Hz, 1H), 7.86 (s, 1H), 7.53 (s, 1H),

7.43−7.35 (m, 1H), 7.33−7.27 (m, 2H), 7.21 (ddd, J = 11.4,

8.2, 1.1 Hz, 1H), 2.69 (s, 3H). ¹³C NMR (100 MHz, CDCl₃):

δ 160.92, 157.21, 155.36, 152.35, 149.83, 148.01, 130.39,

127.13, 124.69, 119.35, 117.96, 116.79, 116.66, 116.31, 116.09,

106.01, 99.59, 16.68. MS (EI): m/z (%) 330 ([M + 1]⁺,

25.78), 329 ([M]⁺, 8.35), 328 ([M − 1]⁺, 100), 300 (4.30),

265 (51.14), 207 (7.47), 181 (1.34), 163 (1.50), 150 (3.66),

133 (6.38), 122 (1.42), 118 (2.39), 108 (1.51), 104 (3.36), 94

(6.79). Anal. calcd for C₁₈H₁₀ClFO₃: C, 65.77; H, 3.07.

Found: C, 65.78; H, 3.04.

3.1.3.5. 6-Chloro-2-(3-ﬂuorophenyl)-5-methyl-7H-furo

1

[3,2-g] Chromen-7-one (If). H NMR (400 MHz, CDCl₃): δ

7.84 (s, 1H), 7.65 (d, J = 7.8 Hz, 1H), 7.56 (d, J = 9.7 Hz,

1H), 7.53 (s, 1H), 7.45 (d, J = 5.8 Hz, 1H), 7.11 (s, 2H), 2.69

(s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 164.39, 161.93,

157.13, 156.05, 149.80, 147.90, 130.72, 126.76, 120.81, 119.48,

116.59, 116.35, 116.14, 112.19, 111.95, 101.76, 99.81, 16.68.

MS (EI): m/z (%) 330 ([M + 1]⁺, 33.58), 329 ([M]⁺, 11.09),

328 ([M − 1]⁺, 100), 300 (4.84), 265 (64.93), 237 (4.34), 207

(15.73), 183 (1.40), 164 (1.51), 150 (3.75), 132 (17.78). Anal.

calcd for C₁₈H₁₀ClFO₃: C, 65.77; H, 3.07. Found: C, 65.75; H,

3.06.

1

[3,2-g] Chromen-7-one (It). H NMR (400 MHz, CDCl₃): δ

8.12 (s, 1H), 8.04 (d, J = 7.5 Hz, 1H), 7.82 (s, 1H), 7.67−7.58

(m, 2H), 7.53 (s, 1H), 7.17 (s, 1H), 6.30 (s, 1H), 2.53 (s, 3H).

¹³C NMR (100 MHz, CDCl₃): δ 160.90, 156.43, 156.32,

152.48, 152.10, 131.47, 130.47, 129.55, 128.05, 126.10, 125.63,

125.24, 121.86, 117.11, 116.48, 113.74, 102.16, 99.88, 19.17.

3.1.3.12. 5-Methyl-2-(4-(triﬂuoromethyl) phenyl)-7H-furo

1

[3,2-g] Chromen-7-one (Iu). H NMR (400 MHz, CDCl₃): δ

3.1.3.6. 6-Chloro-2-(4-ﬂuorophenyl)-5-methyl-7H-furo

7.98 (d, J = 8.2 Hz, 2H), 7.74 (d, J = 8.3 Hz, 2H), 7.56 (d, J =

8.6 Hz, 1H), 7.54−7.42 (m, 2H), 6.30 (s, 1H), 2.52 (s, 3H).

¹³C NMR (100 MHz, CDCl₃): δ 160.64, 157.14, 155.36,

153.45, 147.86, 132.84, 130.68, 126.03, 125.99, 125.21, 121.19,

118.51, 114.99, 113.15, 108.24, 100.55, 19.38. MS (EI): m/z

(%) 344 ([M]⁺, 93.74), 316 (100), 288 (4.16), 259 (2.79),

233(2.54), 189 (4.01), 173 (4.24), 158 (5.51), 148 (4.12), 132

(1.90), 115 (1.77), 91 (2.04). Anal. calcd for C₁₉H₁₁F₃O₃: C,

66.28; H, 3.22. Found: C, 66.30; H, 3.23.

1

[3,2-g] Chromen-7-one (Ig). H NMR (400 MHz, CDCl₃):

δ 7.89−7.83 (m, 2H), 7.82 (s, 1H), 7.51 (s, 1H), 7.18 (t, J =

8.7 Hz, 2H), 7.02 (s, 1H), 2.69 (s, 3H). ¹³C NMR (100 MHz,

CDCl₃): δ 157.53, 157.22, 156.03, 149.57, 147.96, 140.19,

127.12, 127.04, 119.37, 116.63, 116.30, 116.26, 116.08, 100.41,

99.71, 16.68. MS (EI): m/z (%) 330 ([M + 1]⁺, 17.18), 329

([M]⁺, 8.04), 328 ([M-1]⁺, 100), 300 (2.65), 272 (1.34), 265

(51.21), 237 (5.26), 207 (8.14), 183 (1.41), 170 (1.00), 150

(3.22), 132 (19.02). Anal. calcd for C₁₈H₁₀ClFO₃: C, 65.77; H,

3.07. Found: C, 65.73; H, 3.10.

3.1.3.13. 2-(4-Methoxyphenyl)-5-methyl-7H-furo [3,2-g]

1

Chromen-7-one (Iw). H NMR (400 MHz, CDCl₃): δ 7.81

3.1.3.7. 2-(3-Chlorophenyl)-5-methyl-7H-furo [3,2-g]

1

(d, J = 8.8 Hz, 2H), 7.75 (s, 1H), 7.48 (s, 1H), 7.00 (d, J = 8.8

Hz, 2H), 6.93 (s, 1H), 6.28 (s, 1H), 3.88 (s, 3H), 2.52 (s, 3H).

¹³C NMR (100 MHz, CDCl₃): δ 156.27, 154.05, 152.74,

151.44, 146.46, 126.86, 126.62, 122.45, 120.47, 116.63, 115.51,

114.44, 113.32, 99.57, 99.00, 55.43, 19.20. MS (EI): m/z (%)

278 ([M − 28]⁺,37.16), 277 (100), 201 (13.37), 199 (15.85),

183 (15.75), 171 (2.53), 152 (11.53), 128 (2.21), 95 (1.96),

Chromen-7-one (Ii). H NMR (400 MHz, CDCl₃): δ 7.78

(s, 1H), 7.73 (d, J = 8.5 Hz, 2H), 7.60 (d, J = 8.5 Hz, 2H),

7.49 (s, 1H), 7.07 (s, 1H), 6.29 (s, 1H), 2.52 (s, 3H). ¹³C

NMR (100 MHz, CDCl₃): δ 160.96, 156.95, 156.36, 152.52,

151.91, 132.19, 131.18, 129.88, 128.58, 126.49, 126.32, 123.32,

116.96, 116.18, 113.62, 101.30, 99.76, 19.16. MS (EI): m/z

(%) 312 ([M]⁺, 38), 310 (100), 282 (76), 276 (9), 254 (6),

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77 (21.53), Anal. calcd for C₁₉H₁₄O₄: C, 74.50; H, 4.61.

Found: C, 74.48; H, 4.65.

hydrogen atom was replaced by chlorine, the peak near 6.30

ppm disappeared, corresponding 4-methyl was aﬀected by 3-

position chlorine, and the chemical shift moved to the high

ﬁeld, at 2.63 ppm. The mass spectra show that the molecular

ion peaks of these compounds were abundant, and some of

them were basic peaks, indicating that the molecular ion peaks

of these substances are stable.

3.1.3.14. 2-(Furan-2-yl)-5-methyl-7H-furo [3,2-g] Chro-

1

men-7-one (Ix). H NMR (400 MHz, CDCl₃): δ 7.78 (s,

1H), 7.55 (s, 1H), 7.47 (s, 1H), 6.95 (s, 1H), 6.86 (d, J = 3.4

Hz, 1H), 6.56 (s, 1H), 6.29 (s, 1H), 2.52 (s, 3H). ¹³C NMR

(100 MHz, CDCl₃): δ 161.04, 158.11, 152.60, 151.78, 143.62,

126.07, 119.24, 116.07, 113.54, 112.78, 112.18, 111.92, 108.72,

100.35, 99.71, 19.19. MS (EI): m/z (%) 266([M]⁺, 100), 238

(44.49), 237 (29.10), 209 (9.47), 181 (5.65), 152 (11.79), 139

(2.37), 119 (11.82), 104 (2.28), 91 (6.12), 76 (10.17). Anal.

calcd for C₁₆H₁₀O₄: C, 72.18; H, 3.79. Found: C, 72.14; H,

3.76.

3.1.4. Insecticidal Activity of Target Compound I. The data

in Table 3 indicate that under light exposure, the activities of

Table 3. Photochemical Insecticidal Eﬀect of Target

a

Compound I

corrected mortality of mosquito larvae (%)

3.1.3.15. 5-Methyl-2-(thiophen-2-yl)-7H-furo [3,2-g] Chro-

men-7-one (Iy). ¹H NMR (400 MHz, CDCl₃): δ 7.74 (s, 1H),

7.53 (d, J = 3.6 Hz, 1H), 7.47 (s, 1H), 7.40 (d, J = 5.0 Hz,

1H), 7.18−7.08 (m, 1H), 6.90 (s, 1H), 6.28 (s, 1H), 2.51 (s,

3H). ¹³C NMR (100 MHz, CDCl₃): δ 161.06, 156.07, 153.37,

152.58, 151.73, 132.33, 128.09, 126.65, 126.44, 125.42, 116.89,

115.76, 113.52, 100.38, 99.66, 19.18. MS (EI): m/z (%) 282

([M]⁺, 100), 254 (47.83), 253 (21.80), 223 (2.31), 197 (4.13),

171 (1.41), 158 (1.80), 152 (1.37), 127 (12.93), 111 (9.69).

Anal. calcd for C₁₆H₁₀O₃S: C, 68.07; H, 3.57; S, 11.36. Found:

C, 68.04; H, 3.60; S, 11.33.

no.

light group

dark group

Ia

Ib

Ic

Ie

If

90.00 3.60 e

18.33 1.52 f

98.33 3.06 b

98.33 3.05 b

6.67 1.23 g

91.67 3.19 d

100.00 a

1.67 1.62 j

93.33 0.12 c

5.00 1.79 h

100.00 a

98.33 2.62 b

100.00 a

3.33 0.05 i

93.33 1.26 c

100.00 a

21.67 1.84 i

23.33 2.77 h

98.33 1.63 b

90.00 2.17 d

0.00 l

85.00 2.80 e

100.00 a

0.00 l

91.67 1.98 c

3.33 0.09 j

90.00 2.15 d

81.67 2.74 f

80.00 2.56 g

3.33 0.06 j

80.00 3.79 g

1.67 1.62 k

Ig

Ij

Im

Ip

Iu

Iw

Ix

3.1.3.16. 5-Methyl-2-(naphthalen-1-yl)-7H-furo [3,2-g]

1

Chromen-7-one (Iaa). H NMR (400 MHz, CDCl₃): δ 8.44

(d, J = 8.0 Hz, 1H), 8.01−7.91 (m, 3H), 7.88 (s, 1H), 7.65−

7.53 (m, 4H), 7.15 (d, J = 0.9 Hz, 1H), 6.31 (s, 1H), 2.55 (s,

3H). ¹³C NMR (100 MHz, CDCl₃): δ 161.13, 157.77, 156.41,

152.66, 151.77, 133.99, 130.56, 130.15, 128.81, 127.54, 127.35,

127.17, 126.37, 126.32, 125.28, 125.17, 116.78, 116.17, 113.51,

105.41, 99.77, 19.20. MS (EI): m/z (%) 326 ([M]⁺, 100), 314

(1.52), 298 (21.28), 297 (11.38), 239 (5.33), 226 (1.24), 200

(1.19), 155 (4.65), 149 (10.89), 127 (3.53), 120 (2.70), 113

(2.25), 91 (9.42). Anal. calcd for C₂₂H₁₄O₃: C, 80.97; H, 4.32.

Found: C, 80.96; H, 4.34.

Iy

Iaa

Icc

α-T

a

Note: Data are presented as the mean

independent experiments. Diﬀerent lowercase letters indicate a 0.05

level diﬀerence in p.

SD of at least three

compounds Ia, Ic, Ie, Ig, Ij, Ip, Iw, Ix, Iy, and Icc against Culex

pipiens pallens were more than 90%; especially, the activities of

Ij, Iw, and Iy were 100%, which were equivalent to the activity

of standard control alpha-terthienyl (α-T). In the dark, the

activities of Ic, Ie, Ig, Ij, Ip, Iw, Ix, Iy, and Icc against Culex

pipiens pallens were more than 80%. Among them, the activity

of Ij was 100%, but the activity of Ia was only 21.67%, while

the activity of the standard control α-T was 1.67%. It showed

that these compounds had photosensitive activity, but the

photosensitive activity was lower than that of α-T.

3.2. Toxicity of Iy to A. aegypti Larvae. The insecticidal

activity test identiﬁed Iy as a highly active compound. Because

its activity was 100% in light and 80% in the dark (Table 3), Iy

was selected for further experiments with A. aegypti. After a 48

h treatment with Iy, the LC₂₅, LC₅₀, and LC₇₅of the

compound in A. aegypti larvae were 53.96, 64.99, and 78.27

mg/L, respectively (Table 4). In the follow-up test, 0, 54, 65,

and 78 mg/L were used as control, low, medium, and high

concentrations, respectively, to further assess the eﬀects of Iy

3.1.3.17. 5-Methyl-2-(naphthalen-2-yl)-7H-furo [3,2-g]

1

Chromen-7-one (Ibb). H NMR (400 MHz, CDCl₃): δ 7.53

(d, J = 3.6 Hz, 1H), 7.51 (s, 1H), 7.49 (s, 1H), 7.45 (s, 1H),

7.43 (s, 1H), 7.40 (dd, J = 5.0, 1.0 Hz, 1H), 7.20 (s, 1H), 7.15

(s, 1H), 7.14 (d, J = 1.3 Hz, 1H), 7.13 (s, 1H), 6.28 (s, 1H),

2.50 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 160.79, 156.58,

153.49, 152.45, 147.46, 132.25, 128.08, 126.69, 125.48, 120.32,

118.71, 118.62, 114.95, 114.83, 113.00, 108.00, 98.27, 19.38.

MS (EI): m/z (%) 283 ([M − 43]⁺,14.09), 282 (100), 254

(66.68), 253 (19.41), 225 (4.10), 197 (4.43), 171 (4.23), 165

(1.86), 141 (1.05), 126 (10.38), 115 (1.74). Anal. calcd for

C₂₂H₁₄O₃: C, 80.97; H, 4.32. Found: C, 80.95; H, 4.29.

3.1.3.18. 2-Cyclopropyl-5-methyl-7H-furo [3,2-g] Chro-

1

men-7-one (Icc). H NMR (400 MHz, CDCl₃): δ 7.63 (s,

1H), 7.34 (s, 1H), 6.41 (s, 1H), 6.25 (s, 1H), 2.49 (s, 3H),

2.09−1.96 (m, 1H), 1.14−0.89 (m, 4H). ¹³C NMR (101 MHz,

CDCl₃): δ 162.77, 161.37, 155.83, 152.85, 150.98, 126.51,

116.20, 114.75, 113.11, 99.98, 99.25, 19.18, 9.44, 7.53. MS

(EI): m/z (%) 240 ([M]⁺, 100), 239 (7.03), 234 (1.25), 212

(29.34), 211 (26.01), 197 (1.34), 185 (12.60), 169 (1.09), 153

(1.86), 139 (1.89), 127 (2.26), 115 (8.64), 105 (8.64), 92

(2.41). Anal. calcd for C₁₅H₁₂O₃: C, 74.99; H, 5.03. Found: C,

75.02; H, 5.06.

NMR results showed that the chemical shift of the 3-

position hydrogen atom of coumarin in target compound I was

6.29 ppm nearby, that of 4-methyl was 2.50 ppm nearby, and

that of a furan ring was 7.09 ppm nearby. If the 3-position

a

Table 4. Toxic Eﬀects of Iy on the Larvae of A. aegypti

toxicity

Iy (mg/L)

LC₂₅

LC₅₀

LC₇₅

53.96 0.58*

64.99 0.15*

78.27 1.02*

a

LC, lethal concentration. *p < 0.05 compared with the control.

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Figure 1. Eﬀect of Iy on developmental parameters of A. aegypti. (a) Observation of poisoning symptoms in A. aegypti larvae (magniﬁcation, 2.5×;

scale, 4 μm). (b) Cumulative pupation rate. (c) Cumulative emergence rate. (d) Average pupation time. (e) Average emergence time. (f) Dead

pupa rate. (g) Abnormal emergence rate. (h) Mortality of A. aegypti. Data are presented as the mean

SD of at least three independent

experiments. Diﬀerent lowercase letters indicate a 0.05 level diﬀerence in p.

on A. aegypti larvae, including the poisoning symptoms (Figure

1a). A. aegypti larvae were exposed to diﬀerent Iy

concentrations in the dark. After the light treatment, the Iy-

exposed groups displayed diﬀerent degrees of toxic reactions,

and the activity of the larvae was increased during the initial

period, especially at the medium and high concentration of Iy.

After 48 h observation, the exposed larvae were weakened, and

they swam up and down spasmodically after being touched by

the needle. Most of the dead larvae were in a rigid state with a

black abdomen, and their color was deepened at the higher Iy

concentrations. Neither the blank control group nor the

solvent control group exhibited toxic symptoms.

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1

Figure 2. Eﬀect of Iy on ROS production. (a) Detection of O₂. (b) Qualitative observation of ROS (magniﬁcation, 10×; scale, 1 mm). (c)

Quantitative determination of ROS. Data are presented as the mean SD of at least three independent experiments. Diﬀerent lowercase letters

indicate a 0.05 level diﬀerence in p.

3.3. Iy Has an Obvious Post-Lethal Eﬀect on A.

aegypti. After treatment with diﬀerent sublethal Iy concen-

trations for 48 h, the cumulative pupation rate and emergence

rate were signiﬁcantly reduced (Figure 1b,c, P < 0.05), and the

pupation time and emergence time were signiﬁcantly

prolonged (Figure 1d,e, P < 0.05), compared with those in

the control group. Thus, the survival and development of A.

aegypti larvae were aﬀected by Iy exposure. Furthermore,

throughout the development from the pupa to the eclosion

stage, the dead pupa rate and abnormal emergence rate were

also increased (Figure 1f,g). The cumulative mortality of the

whole development period was 8.5, 13.9, and 17.8 times higher

in the low, medium, and high Iy groups than in the control

group (Figure 1h, P < 0.05). These results indicated that Iy

had an obvious post-lethal eﬀect on A. aegypti at diﬀerent

developmental stages.

3.4. Iy Induces Cellular ROS Production. When the

mixed solution was exposed to 365 nm black light for 30 min,

an ESR spectrum was recorded (Figure 2a) that was consistent

with the ESR signal, which showed that the presence of Iy in

the system triggered the light-dependent ¹O₂increase,

indicating the induction of ROS production.⁴⁰The analysis

of the qualitative observations identiﬁed a weak level of

ﬂuorescence at 0 h, which was gradually increased after 1 and 2

h of light treatment and signiﬁcantly enhanced after 3 h. At the

latter time point, obvious green ﬂuorescence was observed in

the digestive organs, including the gastric caeca, midgut, and

Malpighian tubules (Figure 2b). However, there was a decline

in ﬂuorescence after 4 h of light treatment. The quantitative

analysis detected a statistically signiﬁcant ROS burst after 3 h

of light treatment, which was 1.41 times higher than the 0 h

ROS level (Figure 2c, P < 0.05), which was consistent with the

qualitative observation. Combined with the observation of

poisoning symptoms, these results suggested that the midgut

may be a potential site for Iy activity.

3.5. Mitochondrial Dysfunction of Midgut Cells

Induced by Iy. The origin of ROS is related to mitochondria.

Therefore, to evaluate the eﬀect of Iy on mitochondria, TEM

was used to analyze their morphology (Figure 3a). The

structure of the control group mitochondria was intact, and

their membrane and cristae were clearly visible. In contrast, the

membrane and cristae of the Iy treatment group mitochondria

were fuzzy, and there were vacuoles, suggesting that the

structural integrity of the mitochondria was compromised.

Damage of mitochondria will inevitably aﬀect their function.

Therefore, we measured the enzymatic activities of respiratory

complex and TCA cycle components in midgut cell

mitochondria. All examined enzyme activities were increased

in the low Iy group but signiﬁcantly inhibited in the high Iy

group (Figure 3). At the main ROS production sites

speciﬁcally,⁴¹the activities of the respiratory complex I and

III enzymes were decreased by 35.19 and 59.57%, respectively,

compared with those in the control group (Figure 3b,d, P <

0.05). The ETC and TCA cycle enzymes were inhibited by Iy,

which inevitably aﬀected energy production. The ATP content

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Figure 3. Eﬀect of Iy on mitochondrial indicators of midgut cells. (a) Observation of the structure of mitochondria (magniﬁcation, 10000×; scale,

10 μm). (b) Activity of complex I. (c) Activity of complex II. (d) Activity of complex III. (e) Activity of complex IV. (f) Activity of CS. (g) Activity

of ICDHm. (h) Activity of α-KGDH. (i) Content of ATP. Data are presented as the mean SD of at least three independent experiments.

Diﬀerent lowercase letters indicate a 0.05 level diﬀerence in p.

was signiﬁcantly lower in the high Iy group than in the control

group (Figure 3i, P < 0.05).

synthesis, and lowered MMP, resulting in mitochondrial

dysfunction.

MMP changes were detected by JC-1 staining. JC-1

aggregates with strong red ﬂuorescence were observed in the

midgut cells of the control group (Figure 4a). The super-

position of ﬂuorescence images showed that the increase in the

Iy concentration was associated with a signiﬁcant increase in

the green-to-red ﬂuorescence ratio (Figure 4b, P < 0.05). Thus,

the increase in the Iy concentration damaged the mitochon-

dria, inhibited mitochondrial enzyme activities, blocked energy

3.6. The Oxidative Stress in Midgut Cells Was

Aggravated by Iy. The damaged mitochondria leaked ETC

electrons and continuously produced ROS, which is known to

cause oxidative stress. We found that the activities of SOD and

CAT were increased in the low Iy group but inhibited in the

medium and high Iy groups (Figure 5a,b, P < 0.05). High Iy

concentration inhibited the activities of GR and GPx (Figure

5c,d, P < 0.05). By increasing the activity of antioxidant

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Figure 4. Eﬀect of Iy on mitochondrial membrane potential of midgut cells. (a) Changes of mitochondrial membrane potential observed by a

ﬂuorescence microscope (magniﬁcation, 25×; scale, 400 μm). (b) Quantitative analysis of the mean green (JC-1 monomers)/red (JC-1 aggregates)

ﬂuorescence intensity ratio. Data are presented as the mean SD of at least three independent experiments. Diﬀerent lowercase letters indicate a

0.05 level diﬀerence in p.

Figure 5. Eﬀect of Iy on oxidative stress indicators of midgut cells. (a) Activity of SOD. (b) Activity of CAT. (c) Activity of GR. (d) Activity of

GPx. (e) Content of GSH and GSSG and the GSH/GSSG ratio. (f) Content of MDA. (g) Content of protein carbonyls. Data are presented as the

mean SD of at least three independent experiments. Diﬀerent lowercase letters indicate a 0.05 level diﬀerence in p.

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enzymes, the balance of the antioxidant system was maintained

in midgut cells exposed to a low ROS level. However, at higher

Iy concentrations, the antioxidant system was out of balance,

and the antioxidant enzyme activity was inhibited. In the

presence of Iy, GSH was continuously consumed, the GSH/

GSSG ratio was decreased (Figure 5e), the redox balance in

midgut cells was diminished, the MDA and protein carbonyl

levels were signiﬁcantly increased (Figure 5f,g, P < 0.05), and

the degree of oxidative damage was gradually increased.

Oxidative damage was easily detected by histopathological

evaluation (Figure 6a,b). The midgut tissue, going from inside

structure appeared to aﬀect the intestinal shielding function.

Based on this assumption, the permeability of the peritrophic

membrane was tested. When the peritrophic membrane was

destroyed, (FITC)-dextran will exude from the intestine. Iy

treatment was associated with increased ﬂuorescence intensity

and intestinal permeability, compared with those in the control

group (Figure 7a). We also measured the level of chitin as the

main component of the peritrophic membrane. Chitin can be

aﬀected when the peritrophic membrane is damaged.⁴³The

chitin level was signiﬁcantly lower in each Iy group than in the

control group (Figure 7b, P < 0.05). The increase in Iy

concentration decreased the chitin level and damaged the

peritrophic membrane.

3.8. Midgut Cell Apoptosis Induced by Iy. Because

ROS accumulation can induce apoptosis, we measured the

expression of apoptosis-related genes to assess the apoptosis

status of midgut cells. Iy treatment increased the expression

levels of AeDronc, AeCaspase7, and AeCaspase8, which were

signiﬁcantly upregulated in the high Iy group (Figure 8a−c, P

< 0.05). Although the expression of the negative regulatory

gene AeIAP1 was increased, the elevated expression of the

antagonist AeMichelob_x diminished the inhibition by AeIAP1

and induced cell apoptosis (Figure 8d,e).

Apoptosis-speciﬁc staining was applied. The Hoechst 33342

stain densely labeled the nuclei of midgut cells treated with Iy

(Figure 8f). The TUNEL staining detected broken genomic

DNA in the midgut cells. The terminal deoxynucleotidyl

transferase (TDT) added FITC-conjugated dUTP to exposed

3′-OH groups, which clearly labeled apoptotic spots (Figure

8g). The TEM analysis indicated that Iy treatment was

associated with karyopyknosis (Figure 8h). Overall, these

results suggested that Iy induced apoptosis in midgut cells.

Figure 6. Histological and ultrastructural analysis of the midgut. (a)

Structure of the midgut observed by transection (magniﬁcation, 40×;

scale, 400 μm; EP, epithelial cell; PM, peritrophic membrane; D, cell

disintegration; V, cell vacuolization). (b) Structure of the midgut

observed by a longitudinal section (magniﬁcation, 40×; scale, 400

μm; SB, striated border). (c) Observation of microvilli (magniﬁcation,

10000×; scale, 10 μm; MV, microvilli).

to outside, consists of the endo-peritrophic space, peritrophic

membrane, and ecto-peritrophic space, and the epithelial cells

were covered with microvilli.^37,42In the control group, the

midgut epithelial cells were intact and orderly arranged, the

nucleocytoplasmic staining generated strong signals, and the

striated border and peritrophic membrane were not broken. In

contrast, under Iy treatment, the midgut tissues were damaged

at varying degrees, the striated border was diminished, the cells

were swollen, and there were instances of vacuolation and cell

disintegration, associated with the appearance of intercellular

space and midgut disruption. Orderly arranged microvilli were

not detectable by TEM (Figure 6c).

4. DISCUSSION

The viral diseases transmitted by A. aegypti pose a threat to

human health on a global scale. Furanocoumarin possesses

photosensitive activity and has various pharmacological

properties. In this study, we newly designed and synthesized

2-arylfuranocoumarin derivatives, including Iy, which we

investigated for photoactivated toxicity in A. aegypti.

Speciﬁcally, Iy was activated by ultraviolet light to produce

singlet oxygen, which induced ROS production in body cells.

Our combined qualitative and quantitative ROS analysis

identiﬁed the gastric caeca, midgut, and Malpighian tubules

as sites with high ROS production. By monitoring the

3.7. Intestinal Shielding Dysfunction in A. aegypti

Larvae Caused by Iy. The destruction of the intestinal

Figure 7. Eﬀect of Iy on intestinal barrier dysfunction. (a) Detection of peritrophic membrane permeability (magniﬁcation, 2.5×; scale, 4 mm). (b)

Detection of chitin content. Data are presented as the mean SD of at least three independent experiments. Diﬀerent lowercase letters indicate a

0.05 level diﬀerence in p.

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Figure 8. Eﬀect of Iy on apoptosis of midgut cells. (a) Relative expression of AeDronc mRNA. (b) Relative expression of AeCaspase7 mRNA. (c)

Relative expression of AeCaspase8 mRNA. (d) Relative expression of AeIAP1 mRNA. (e) Relative expression of AeMichelob_x mRNA. (f) Hoechst

33342 staining. (g) TUNEL staining (magniﬁcation, 25×; scale, 400 μm). (h) Observation of the nucleus structure (magniﬁcation, 1500×; scale,

50 μm; N, nucleolus). Data are presented as the mean SD of at least three independent experiments. Diﬀerent lowercase letters indicate a 0.05

level diﬀerence in p.

poisoning symptoms, we found that the abdomen of the A.

aegypti larvae was blackened, suggesting that the site of Iy

action in the larvae could be the midgut. In general, ROS is

mainly generated by the ETC, and mitochondria not only

produce ROS, but they are also targeted by these

antioxidants.⁴⁴The mitochondria were damaged by the Iy

treatment, and the enzyme activities of ETC complexes were

inhibited at diﬀerent degrees, which especially applied to the

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J. Agric. Food Chem. 2021, 69, 1091−1106

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Article

respiratory complex III enzymes. Some earlier studies showed

that a respiratory complex II key enzyme, which connects the

ETC to the TCA, can also be a site of ROS production.^15,45,46

The activities of key enzymes in the TCA cycle are also

inhibited by Iy in varying degrees. Interestingly, some studies

have shown that α-KGDH can also participate in the

production of ROS.^47,48Furthermore, the function of

mitochondria is highly dependent on MMP as a critical

parameter for the real-time state of mitochondria, which plays

an important role in maintaining biosynthesis and apopto-

sis.⁴⁹⁻⁵¹Therefore, it was speculated that the decrease in

MMP might play a key role in inducing apoptosis. The

decrease in MMP leads to the dysfunction of mitochondria and

promotes the continuous production of ROS in midgut cells.

The cells have an eﬃcient antioxidant system, which can

rapidly remove ROS. At a low level of ROS stimulation, the

cells start the repair function, preventing ROS from reacting

with lipids and proteins,^52,53which causes oxidative damage.

Thus, the increase in antioxidant enzyme activity at low ROS

concentrations blocks further damage. However, mitochondrial

dysfunction leads to continuous ROS production. When it

exceeds the tolerance range of the intracellular antioxidant

system, it will cause oxidative damage to the cells, which is

associated with a gradual decrease in the GSH/GSSG ratio,

indicating that the intracellular antioxidant system is out of

balance. Histopathological changes in Iy-treated larvae

indicated substantial midgut damage, increased intestinal

permeability, and a decreased chitin content, which provided

evidence that the intestinal shielding function was impaired.

However, the excessive ROS production caused by Iy-

induced damage of the mitochondria cannot be rapidly

eliminated in time due to the impairment of the intracellular

antioxidant system by Iy. Thus, Iy treatment promoted the

accumulation of ROS, which led to the induction of apoptosis.

AeDronc was induced as an apoptosis initiator gene, which

signiﬁcantly upregulated and activated the downstream eﬀect

caspases, indicating that apoptosis was induced. Apoptosis

staining conﬁrmed the existence of apoptotic cells, and the

typical characteristics of apoptotic karyopyknosis were

observed.⁵⁴

out of balance, it causes oxidative damage to the midgut,

leading to histopathological changes. ROS accumulates

continuously, and apoptosis is induced, which leads to

apoptosis of midgut cells, eventually to the death of the A.

aegypti larva. However, although the control of A. aegypti at

sublethal Iy concentrations appeared to be eﬀective under

laboratory conditions, the toxic eﬀect of Iy on A. aegypti in a

natural environment needs to be further studied. Hence, there

is promising potential for the application of Iy in mosquito

prevention and control.

AUTHOR INFORMATION

Corresponding Author

■

Yanping Luo − Key Laboratory of Green Prevention and

Control of Tropical Plant Diseases and Pests, Ministry of

Education, College of Plant Protection, Hainan University,

Haikou, Hainan 570228, P.R. China; orcid.org/0000-

0002-2184-4291; Phone: +86-13648634489;

Email: yanpluo2012@hainanu.edu.cn

Authors

Jiecheng Wu − Key Laboratory of Green Prevention and

Control of Tropical Plant Diseases and Pests, Ministry of

Education, College of Plant Protection, Hainan University,

Haikou, Hainan 570228, P.R. China

Lanying Wang − Key Laboratory of Green Prevention and

Control of Tropical Plant Diseases and Pests, Ministry of

Education, College of Plant Protection, Hainan University,

Haikou, Hainan 570228, P.R. China

Yunfei Zhang − Key Laboratory of Green Prevention and

Control of Tropical Plant Diseases and Pests, Ministry of

Education, College of Plant Protection, Hainan University,

Haikou, Hainan 570228, P.R. China

Shujing Zhang − Key Laboratory of Green Prevention and

Control of Tropical Plant Diseases and Pests, Ministry of

Education, College of Plant Protection, Hainan University,

Haikou, Hainan 570228, P.R. China

Shakil Ahmad − Key Laboratory of Green Prevention and

Control of Tropical Plant Diseases and Pests, Ministry of

Education, College of Plant Protection, Hainan University,

Haikou, Hainan 570228, P.R. China

Thus, our analysis captures the complexity of the lethal eﬀect

of Iy on A. aegypti larvae. The peritrophic membrane is the ﬁrst

barrier of the midgut defense system, which can protect

epithelial cells from mechanical damage, promote digestion,

and facilitate the absorption of nutrients in the insects.^55,56

Furthermore, the microvilli provide an enlarged surface area

for nutrient absorption.⁵⁷When they are damaged, it will

inevitably lead to poor nutrient absorption, insuﬃcient energy

supply, and damage to mitochondrial functions, which aﬀect

energy production. A. aegypti needs suﬃcient energy to

complete its life cycle. When the nutrient intake is insuﬃcient,

along with reduced energy storage, it can lead to the death of

A. aegypti.⁵⁸Therefore, the larval stage continues to die, and

even if it can enter the pupa stage, there are dead pupae and

abnormal eclosion. Furthermore, some studies demonstrated

that when the integrity of epithelial cells is destroyed, the

proliferation and diﬀerentiation of epithelial cells in the process

of development are aﬀected, leading to the emergence of

malformed adult mosquitoes.^59,60Therefore, even at a

sublethal concentration of Iy, it can have a negative eﬀect on

the growth, development, and metamorphosis of A. aegypti.

In conclusion, the mechanism of the lethal eﬀect of Iy in A.

aegypti larvae can be proposed. When the antioxidant system is

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jafc.0c07237

Author Contributions

^§J.W. and L.W. contributed equally to this work.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This work was supported by the National Natural Science

Foundation of China (31860513) and the Hainan Provincial

Natural Science Foundation of China (2018CXTD335).

ABBREVIATIONS

■

Iy, 2-thiophenylfuranocoumarin; ROS, reactive oxygen species;

ETC, electron transport chain; NADH, nicotinamide adenine

dinucleotide; FADH₂, ﬂavin adenine dinucleotide; TCA,

tricarboxylic acid; SOD, superoxide dismutase; H₂O₂, hydro-

gen peroxide; CAT, catalase; GPx, glutathione peroxidase;

GSH, glutathione; GR, glutathione reductase; GSSG, oxidized

glutathione; MDA, malondialdehyde; AeIAP1, Aedes aegypti

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(14) Willems, P. H. G. M.; Rossignol, R.; Dieteren, C. E. J.; Murphy,

M. P.; Koopman, W. J. H. Redox homeostasis and mitochondrial

dynamics. Cell Metab. 2015, 22, 207−218.

(15) Nolfi-Donegan, D.; Braganza, A.; Shiva, S. Mitochondrial

Electron Transport Chain: Oxidative Phosphorylation, Mitochondrial

Oxidant Production, and Methods of Measurement. Redox. Biol.

2020, 37, 101674.

(16) Angelova, P. R.; Abramov, A. Y. Functional role of

mitochondrial reactive oxygen species in physiology. Free Radical

Biol. Med. 2016, 100, 81−85.

(17) Zhuang, S.; Yu, R.; Zhong, J.; Liu, P.; Liu, Z. Rhein from

Rheum rhabarbarum Inhibits Hydrogen-Peroxide-Induced Oxidative

Stress in Intestinal Epithelial Cells Partly through PI3K/Akt-Mediated

Nrf2/HO-1 Pathways. J. Agric. Food Chem. 2019, 67, 2519−2529.

(18) Hutnick, M. A.; Ahsanuddin, S.; Guan, L.; Lam, M.; Baron, E.

D.; Pokorski, J. K. PEGylated Dendrimers as Drug Delivery Vehicles

for the Photosensitizer Silicon Phthalocyanine Pc 4 for Candidal

Infections. Biomacromolecules 2017, 18, 379−385.

(19) Shah, D.; Sah, S.; Nath, S. K. Interaction between glutathione

and apoptosis in systemic lupus erythematosus. Autoimmun Rev. 2013,

12, 741−751.

(20) Nasimian, A.; Farzaneh, P.; Tamanoi, F.; Bathaie, S. Z.

Cytosolic and mitochondrial ROS production resulted in apoptosis

induction in breast cancer cells treated with Crocin: The role of

FOXO3a, PTEN and AKT signaling. Biochem. Pharmacol. 2020, 177,

113999.

inhibitor of apoptosis protein 1; MMP, mitochondrial

membrane potential; TLC, thin-layer chromatography; NBS,

N-bromosuccinimide; THF, tetrahydrofuran; ¹O₂, singlet

oxygen; ESR, electron spin resonance; DCFH-DA, 2,7-

dichloro-hydroﬂuorescein diacetate; PBS, phosphate-buﬀered

saline; TEM, transmission electron microscopy; CS, citric acid

synthase; ICDHm, isocitrate dehydrogenase; α-KGDH, α-

ketoglutarate dehydrogenase; JC-1, 5,5,6,6-tetrachloro-1,1,3,3-

tetraethylbenzimidazolyl carbocyanine iodide; FITC, ﬂuores-

cein isothiocyanate; TUNEL, transferase dUTP nick end

labeling; RT-qPCR, quantitative reverse transcription PCR;

RP7S, ribosomal protein 7S; PPh₃, triphenylphosphine; NMR,

nuclear magnetic resonance; α-T, alpha-terthienyl; LC, lethal

concentration; TDT, terminal deoxynucleotidyl transferase

REFERENCES

■

(1) Raquet, N.; Schrenk, D. Application of the equivalency factor

concept to the phototoxicity and −genotoxicity of furocoumarin

mixtures. Food Chem. Toxicol. 2014, 68, 257−266.

(2) Roselli, S.; Olry, A.; Vautrin, S.; Coriton, O.; Ritchie, D.; Galati,

́

G.; Navrot, N.; Krieger, C.; Vialart, G.; Berges, H.; Bourgaud, F.;

Hehn, A. A bacterial artificial chromosome (BAC) genomic approach

reveals partial clustering of the furanocoumarin pathway genes in

parsnip. Plant J. 2017, 89, 1119−1132.

(3) Melough, M. M.; Lee, S. G.; Cho, E.; Kim, K.; Provatas, A. A.;

Perkins, C.; Park, M. K.; Qureshi, A.; Chun, O. K. Identification and

Quantitation of Furocoumarins in Popularly Consumed Foods in the

U.S. Using QuEChERS Extraction Coupled with UPLC-MS/MS

Analysis. J. Agric. Food Chem. 2017, 65, 5049−5055.

(21) Rossbach, L. M.; Oughton, D. H.; Maremonti, E.; Coutris, C.;

Brede, D. A. In vivo assessment of silver nanoparticle induced reactive

oxygen species reveals tissue specific effects on cellular redox status in

the nematode Caenorhabditis elegans . Sci. Total Environ. 2020, 721,

137665.

(22) Jahedsani, A.; Khezri, S.; Ahangari, M.; Bakhshii, S.; Salimi, A.

Apigenin attenuates Aluminum phosphide-induced cytotoxicity via

reducing mitochondrial/Lysosomal damages and oxidative stress in

rat Cardiomyocytes. Pestic. Biochem. Physiol. 2020, 167, 104585.

(23) Kuzmic, M.; Galas, S.; Lecomte-Pradines, C.; Dubois, C.;

Dubourg, N.; Frelon, S. Interplay between ionizing radiation effects

and aging in C. elegans . Free Radical Biol. Med. 2019, 134, 657−665.

(24) Batool, K.; Alam, I.; Jin, L.; Xu, J.; Wang, J.; Huang, E.; Guan,

X.; Yu, X.-Q.; Zhang, L. CTLGA9 interacts with ALP1 and APN

receptors to modulate Cry11Aa toxicity in Aedes aegypti . J. Agric. Food

Chem. 2019, 67, 8896−8904.

(25) Tok, F.; Kocyigit-Kaymakcioglu, B.; Tabanca, N.; Estep, A. S.;

Gross, A. D.; Geldenhuys, W. J.; Becnel, J. J.; Bloomquist, J. R.

Synthesis and structure −activity relationships of carbohydrazides and

1, 3, 4-oxadiazole derivatives bearing an imidazolidine moiety against

the yellow fever and dengue vector, Aedes aegypti. Pest Manag. Sci.

2018, 74, 413−421.

(26) Wan, Y.; Wu, S.; Zheng, S.; Liang, E.; Liu, S.; Yao, X.; Zhu, Q.

A series of octahydroquinazoline-5-ones as novel inhibitors against

dengue virus. Eur. J. Med. Chem. 2020, 200, 112318.

(4) Ahmed, S.; Khan, H.; Aschner, M.; Mirzae, H.; Kupeli Akkol, E.;

̈

Capasso, R. Anticancer Potential of Furanocoumarins: Mechanistic

and Therapeutic Aspects. Int. J. Mol. Sci. 2020, 21, 5622.

(5) Pynam, H.; Dharmesh, S. M. Antioxidant and anti-inflammatory

properties of marmelosin from Bael (Aegle marmelos L.); Inhibition of

TNF-α mediated inflammatory/tumor markers. Biomed. Pharmac-

other. 2018, 106, 98−108.

(6) Stern, R. S.; PUVA Follow-up Study. The risk of squamous cell

and basal cell cancer associated with psoralen and ultraviolet A

therapy: A 30-year prospective study. J. Am. Acad. Dermatol. 2012, 66,

553−562.

(7) Melough, M. M.; Cho, E.; Chun, O. K. Furocoumarins: A review

of biochemical activities, dietary sources and intake, and potential

health risks. Food Chem. Toxicol. 2018, 113, 99−107.

̀

(8) Carbone, A.; Montalbano, A.; Spano, V.; Musante, I.; Galietta, L.

J. V.; Barraja, P. Furocoumarins as multi-target agents in the treatment

of cystic fibrosis. Eur. J. Med. Chem. 2019, 180, 283−290.

(9) Scott, B. R.; Pathak, M. A.; Mohn, G. R. Molecular and genetic

basis of furocoumarin reactions. Mutat. Res. 1976, 39, 29−74.

(10) Ahmad, S. I.; Yokoi, M.; Hanaoka, F. Identification of new

scavengers for hydroxyl radicals and superoxide dismutase by utilising

ultraviolet A photoreaction of 8-methoxypsoralen and a variety of

mutants of Escherichia coli: implications on certain diseases of DNA

repair deficiency. J. Photochem. Photobiol., B 2012, 116, 30−36.

(11) Melough, M. M.; Chun, O. K. Dietary furocoumarins and skin

cancer: A review of current biological evidence. Food Chem. Toxicol.

2018, 122, 163−171.

(27) Chang, K. P.; Kolli, B. K.; The New Light Group. New ″ light ″

for one-world approach toward safe and effective control of animal

diseases and insect vectors from leishmaniac perspectives. Parasites

Vectors 2016, 9, 396.

(28) Cooper, D. M.; Granville, D. J.; Lowenberger, C. The insect

caspases. Apoptosis. 2009, 14, 247−256.

(29) Liu, Q.; Clem, R. J. Defining the core apoptosis pathway in the

mosquito disease vector Aedes aegypti : the roles of iap1, ark, dronc,

and effector caspases. Apoptosis. 2011, 16, 105−113.

(12) Miolo, G.; Sturaro, G.; Cigolini, G.; Menilli, L.; Tasso, A.;

Zago, I.; Conconi, M. T. 4,6,4 ′-trimethylangelicin shows high anti-

proliferative activity on DU145 cells under both UVA and blue light.

Cell Proliferation 2018, 51, No. e12430.

(13) Martínez-Reyes, I.; Diebold, L. P.; Kong, H.; Schieber, M.;

Huang, H.; Hensley, C. T.; Mehta, M. M.; Wang, T.; Santos, J. H.;

Woychik, R.; Dufour, E.; Spelbrink, J. N.; Weinberg, S. E.; Zhao, Y.;

DeBerardinis, R. J.; Chandel, N. S. TCA Cycle and Mitochondrial

Membrane Potential Are Necessary for Diverse Biological Functions.

Mol. Cell 2016, 61, 199−209.

(30) Wang, H.; Clem, R. J. The role of IAP antagonist proteins in

the core apoptosis pathway of the mosquito disease vector Aedes

aegypti . Apoptosis. 2011, 16, 235−248.

(31) Zhang, J.; Ahmad, S.; Wang, L. Y.; Han, Q.; Zhang, J. C.; Luo,

Y. P. Cell death induced by α -terthienyl via reactive oxygen species-

mediated mitochondrial dysfunction and oxidative stress in the

midgut of Aedes aegypti larvae. Free Radical Biol. Med. 2019, 137, 87−

98.

1105

https://dx.doi.org/10.1021/acs.jafc.0c07237

J. Agric. Food Chem. 2021, 69, 1091−1106

Journal of Agricultural and Food Chemistry

pubs.acs.org/JAFC

Article

(32) Timoshnikov, V. A.; Kobzeva, T. V.; Polyakov, N. E.;

Kontoghiorghes, G. J. Inhibition of Fe(2+)- and Fe(3+)- induced

hydroxyl radical production by the iron-chelating drug deferiprone.

Free Radical Biol. Med. 2015, 78, 118−122.

(33) Xia, Q.; Wei, L.; Zhang, Y.; Kong, H.; Shi, Y.; Wang, X.; Chen,

X.; Han, L.; Liu, K. Psoralen Induces Developmental Toxicity in

Zebrafish Embryos/Larvae Through Oxidative Stress, Apoptosis, and

Energy Metabolism Disorder. Front. Pharmacol. 2018, 9, 1457.

(34) Vaidyanathan, R.; Scott, T. W. Apoptosis in mosquito midgut

epithelia associated with West Nile virus infection. Apoptosis. 2006,

11, 1643−1651.

(49) Bian, M.; Fan, R.; Jiang, G.; Wang, Y.; Lu, Y.; Liu, W. Halo and

Pseudohalo Gold(I)−NHC Complexes Derived from 4,5-Diary-

limidazoles with ExcellentIn VitroandIn VivoAnticancer Activities

Against HCC. J. Med. Chem. 2020, 63, 9197−9211.

(50) Marie, M.; Bigot, K.; Angebault, C.; Barrau, C.; Gondouin, P.;

Pagan, D.; Fouquet, S.; Villette, T.; Sahel, J.-A.; Lenaers, G.; Picaud, S.

Light action spectrum on oxidative stress and mitochondrial damage

in A2E-loaded retinal pigment epithelium cells. Cell Death Dis. 2018,

9, 287.

(51) Xiao, J.; Wu, C.; He, Y.; Guo, M.; Peng, Z.; Liu, Y.; Liu, L.;

Dong, L.; Guo, Z.; Zhang, R.; Zhang, M. Rice Bran Phenolic Extract

Confers Protective Effects against Alcoholic Liver Disease in Mice by

Alleviating Mitochondrial Dysfunction via the PGC-1 α -TFAM

Pathway Mediated by microRNA-494-3p. J. Agric. Food Chem.

2020, 68, 12284−12294.

(35) Dubey, S. K.; Shrinet, J.; Sunil, S. Aedes aegypti microRNA,

miR-2944b-5p interacts with 3 ’UTR of chikungunya virus and cellular

target vps-13 to regulate viral replication. PLoS Negl Trop Dis. 2019,

13, No. e0007429.

(52) Napolitano, G.; Fasciolo, G.; Di Meo, S.; Venditti, P. Vitamin E

Supplementation and Mitochondria in Experimental and Functional

Hyperthyroidism: A Mini-Review. Nutrients 2019, 11, 2900.

(53) Liu, S.-Y.; Song, J.-Y.; Fan, B.; Pan, Y.-R.; Che, L.; Sun, Y.-J.; Li,

G.-Y. Resveratrol protects photoreceptors by blocking caspase- and

PARP-dependent cell death pathways. Free Radical Biol. Med. 2018,

129, 569−581.

(54) Chen, G.; Zhang, P.; Huang, T.; Yu, W.; Lin, J.; Li, P.; Chen, K.

Polysaccharides from Rhizopus nigricans mycelia induced apoptosis

and G2/M arrest in BGC-823 cells. Carbohydr. Polym. 2013, 97, 800−

808.

(55) Liu, X.; Cooper, A. M. W.; Zhang, J.; Zhu, K. Y. Biosynthesis,

modifications and degradation of chitin in the formation and turnover

of peritrophic matrix in insects. J. Insect Physiol. 2019, 114, 109−115.

(56) Toprak, U.; Erlandson, M.; Baldwin, D.; Karcz, S.; Wan, L.;

Coutu, C.; Gillott, C.; Hegedus, D. D. Identification of the Mamestra

con f i gurata (Lepidoptera: Noctuidae) peritrophic matrix proteins and

enzymes involved in peritrophic matrix chitin metabolism. Insect Sci.

2016, 23, 656−674.

(57) Li, H.; Zhang, J.; Ma, T.; Li, C.; Ma, Z.; Zhang, X. Acting target

of toosendanin locates in the midgut epithelium cells of Mythimna

separate Walker larvae (lepidoptera: Noctuidae). Ecotoxicol. Environ.

Saf. 2020, 201, 110828.

(36) Ganesan, P.; Stalin, A.; Paulraj, M. G.; Balakrishna, K.;

Ignacimuthu, S.; Al-Dhabi, N. A. Biocontrol and non-target effect of

fractions and compound isolated from Streptomyces rimosus on the

immature stages of filarial vector Culex quinquefasciatus Say (Diptera:

Culicidae) and the compound interaction with Acetylcholinesterase

(AChE1). Ecotoxicol. Environ. Saf. 2018, 161, 120−128.

(37) Edwards, M. J.; Jacobs-Lorena, M. Permeability and disruption

of the peritrophic matrix and caecal membrane from Aedes aegypti and

Anopheles gambiae mosquito larvae. J. Insect Physiol. 2000, 46, 1313−

1320.

(38) Zhang, J.; Zhu, K. Y. Characterization of a chitin synthase

cDNA and its increased mRNA level associated with decreased chitin

synthesis in Anopheles quadrimaculatus exposed to diflubenzuron.

Insect Biochem. Mol. Biol. 2006, 36, 712−725.

(39) Eng, M. W.; Van Zuylen, M. N.; Severson, D. W. Apoptosis-

the mosquito vector, Aedes aegypti . Insect Biochem. Mol. Biol. 2016, 76,

70−83.

̀

(40) Dichiara, M.; Prezzavento, O.; Marrazzo, A.; Pittala, V.;

Salerno, L.; Rescifina, A.; Amata, E. Recent advances in drug

discovery of phototherapeutic non-porphyrinic anticancer agents. Eur.

J. Med. Chem. 2017, 142, 459−485.

(41) Zamani, E.; Shaki, F.; Abediankenari, S.; Shokrzadeh, M.

Acrylamide induces immunotoxicity through reactive oxygen species

production and caspase-dependent apoptosis in mice splenocytes via

the mitochondria-dependent signaling pathways. Biomed. Pharmac-

other. 2017, 94, 523−530.

(58) Sasmita, H. I.; Tu, W.-C.; Bong, L.-J.; Neoh, K.-B. Effects of

larval diets and temperature regimes on life history traits, energy

reserves and temperature tolerance of male Aedes aegypti (Diptera:

Culicidae): optimizing rearing techniques for the sterile insect

programmes. Parasites. Vectors. 2019, 12, 578.

(59) Bibi, R.; Tariq, R. M.; Rasheed, M. Toxic assessment, growth

disrupting and neurotoxic effects of red seaweeds ’ botanicals against

the dengue vector mosquito Aedes aegypti L. Ecotoxicol. Environ. Saf.

2020, 195, 110451.

́

(42) Fernandes, K. M.; Tome, H. V. V.; Miranda, F. R.; Conca

̧

lves,

W. G.; Pascini, T. V.; Serrao, J. E.; Martins, G. F. Aedes aegypti larvae

̃

treated with spinosad produce adults with damaged midgut and

reduced fecundity. Chemosphere 2019, 221, 464−470.

(43) Kelkenberg, M.; Odman-Naresh, J.; Muthukrishnan, S.;

Merzendorfer, H. Chitin is a necessary component to maintain the

barrier function of the peritrophic matrix in the insect midgut. Insect

Biochem. Mol. Biol. 2015, 56, 21−28.

(60) de Santa Silva, L. L.; Fernandes, K. M.; Miranda, F. R.; Silva, S.

C. C.; Coelho, L. C. B. B.; do Amaral Ferraz Navarro, D. M.;

Napoleao, T. H.; Martins, G. F.; Paiva, P. M. G. Exposure of mosquito

̃

(Aedes aegypti ) larvae to the water extract and lectin- rich fraction of

Moringa oleifera seeds impairs their development and future

fecundity. Ecotoxicol. Environ. Saf. 2019, 183, 109583.

(44) Chowdhury, A. R.; Zielonka, J.; Kalyanaraman, B.; Hartley, R.

C.; Murphy, M. P.; Avadhani, N. G. Mitochondria-Targeted Paraquat

and Metformin Mediate ROS Production to Induce Multiple

Pathways of Retrograde Signaling: A dose-dependent phenomenon.

Redox. Biol. 2020, 36, 101606.

(45) Markevich, N. I.; Galimova, M. H.; Markevich, L. N. Hysteresis

and bistability in the succinate-CoQ reductase activity and reactive

oxygen species production in the mitochondrial respiratory Complex

II. Redox Biol. 2020, 101630.

(46) Zorov, D. B.; Juhaszova, M.; Sollott, S. J. Mitochondrial reactive

oxygen species (ROS) and ROS-induced ROS release. Physiol. Rev.

2014, 94, 909−950.

(47) Meng, D.; Zhang, P.; Zhang, L.; Wang, H.; Ho, C. T.; Li, S.;

Shahidi, F.; Zhao, H. Detection of cellular redox reactions and

antioxidant activity assays. J. Funct. Foods. 2017, 37, 467−479.

(48) Quijano, C.; Trujillo, M.; Castro, L.; Trostchansky, A. Interplay

between oxidant species and energy metabolism. Redox. Biol. 2016, 8,

28−42.

1106

https://dx.doi.org/10.1021/acs.jafc.0c07237

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Article Doi

Synthesis and Photoactivated Toxicity of 2-Thiophenylfuranocoumarin Induce Midgut Damage and Apoptosis in Aedes aegypti Larvae

DOI: 10.1021/acs.jafc.0c07237

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