Journal of Photochemistry and Photobiology B: Biology 96 (2009) 170–177

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Journal of Photochemistry and Photobiology B: Biology

journal homepage: www.elsevier .com/locate / jphotobiol

The synthesis and photoactivated cytotoxicity of 2-methyl-4-oxo-3-prop-2-yn-1-ylcyclopent-2-en-1-yl-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylateconjugated with a-terthienyl derivatives

Na Li a, Han-Hong Xu a,*, Zheng-Yong Liu b, Zhuo-Hong Yang b

a Laboratory of Insect Toxicology and Key Laboratory of Natural Pesticide and Chemical Biology, Ministry of Education, South China Agricultural University, Guangzhou, Chinab College of Science, South China Agricultural University, Guangzhou, Guangdong 510642, China

a r t i c l e i n f o a b s t r a c t

Article history:Received 16 February 2009Received in revised form 2 June 2009Accepted 9 June 2009Available online 13 June 2009

Keywords:Pyrethroid insecticidea-terthienylPhotoactivated cytotoxicitySpodoptera litura cellROSMMP

1011-1344/$ - see front matter Crown Copyright � 2doi:10.1016/j.jphotobiol.2009.06.002

* Corresponding author. Tel.: +86 20 85285127; faxE-mail address: hhxu@scau.edu.cn (H.-H. Xu).

The synthesis of one pyrethroid insecticide [2-methyl-4-oxo-3-prop-2-yn-1-ylcyclopent-2-en-1-yl-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate (Abbrev. JZ) (Fig. 1)] conjugated with a seriesof a-terthienyl derivatives (2–8) (Fig. 1) by palladium/copper-catalyzed cross-coupling reaction is pre-sented here for evaluating the photoactivated cytotoxicity. The photoactivated cytotoxicity on Spodopteralitura (SL) cell line was detected by MTT assay. The inhibitory activity of all the conjugates was enhancedin the irradiation condition, compared with that of JZ. The IC50 values of the most effective compound 9(Fig. 1) treated with irradiation were 11.60 lg mL�1 at 24 h and 8.93 lg mL�1 at 48 h, respectively. Gen-eration of intracellular reactive oxygen species (ROS) and change of mitochondrial transmembranepotential (MMP) on SL cells treated with compound 9 were used for the further photoactivated study.A summary of these experiments on compound 9 demonstrated the notable ROS generation and dramaticMMP decrease when irradiated with UVA light. The results also represented statistically significant dif-ference between dark and irradiation treatment of compound 9. However, in control and JZ groups,the effects were not statistically different. It was proved that our prepared compounds were ideal candi-dates for new photoactivated pyrethroid insecticides.

Crown Copyright � 2009 Published by Elsevier B.V. All rights reserved.

1. Introduction

Pyrethroid insecticides are structural derivatives of naturallyoccurring pyrethrins extracting from Chrysanthemum cinerariaefoli-um [1]. The development of synthetic pyrethroid insecticides hasinvolved an iterative process of structural modification and biolog-ical evaluation. Early synthetic pyrethroid was a type of effectiveand safe insecticide; however, it may degrade quickly in the envi-ronment and could be limited in the crop protection. Then the fol-lowing synthetic pyrethroid with improved photostability andhigh insecticidal potency has gained extensive applications in thefield [1,2]. However, modifications of pyrethroid structure mayalso result in changes in pyrethroid toxicity on non-target species[3–5]. Evaluations of the cumulative risk of neurotoxicity are cur-rently underway in accordance with the mandate of the Food Qual-ity Protection Act [2,6]. Likewise, the ecosystem risk and residueproblem of pyrethroid were mainly generated by agricultural useand industrial effluent [7].

In the last few years, thiophene derivatives such as a-terthienyl(a-T, 1) (Fig. 1) have stimulated much interest because of their

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phototoxic activity against a great number of organisms includingbacteria, fungi, viruses, nematodes, eggs and larvae of insects [8–10]. Further study has revealed that a-T is acted as a powerful pho-tosensitizer, which efficiently generates singlet oxygen for biolog-ical activity [8]. a-T has desirable environmental characteristics asit will degrade in the presence of sunlight with a half-life ofapproximately 6 h [11]. The field trials have demonstrated thata-T was a commercially useful and environmentally non-threaten-ing biocidal agent [12,13].

In the current study, the hypothesis of novel photoactivatedpyrethroid insecticides to attain the expected purpose on improv-ing bioactivity and eliminating residue in the environment wasdiscussed. We therefore chose 2-methyl-4-oxo-3-prop-2-yn-1-ylcyclopent-2-en-1-yl-3-(2,2-dichlorovinyl)-2,2-dimethylcyclo-propanecarboxylate (Abbrev. JZ) (Fig. 1), a kind of pyrethroidinsecticide, as starting material to conjugate with 5,50-dibromo-2,20:50,50 0-terthienyl (2) (Fig. 1) by palladium/copper–catalyzedcross-coupling reaction of 1-alkynes with arylhalides, as the termi-nal alkyne of the pyrethroid is a suitable site to link with other sixinteresting derivatives (3–8) (Fig. 1). Compound 9 in our experi-ments showed excellent photoactivated cytotoxicity on Spodopteralitura (SL) cells. In addition, attempting to provide a better under-standing of photoactivated action mechanism of these conjugates,

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Fig. 1. General synthetic routes: (1) Mg I2, diethyl ether, rt, (2) Ni(dppp)Cl2, diethyt ether, rt, (3) N-bromosuccinimide, N,N-dimethylformamide, (4) and (5) Pb(CH3COO)2, CuI,PPh3, triethylamine.

N. Li et al. / Journal of Photochemistry and Photobiology B: Biology 96 (2009) 170–177 171

compound 9 (Fig. 1), as an example, was investigated further interms of ROS generation and MMP change. The results representedstatistically significant difference between dark and irradiationtreatment of compound 9. However, in control and JZ groups, theeffects were not statistically different. This finding has revealedthe hypothesis of new photoactivated pyrethroid insecticides.

2. Experimental

2.1. Reagents and Instrumental analysis

Melting points were measured with a WRR digital melting pointapparatus. 1H and 13C NMR spectra were recorded on a Bruker AV-600 instrument, chemical shifts are reported in ppm high fre-quency from tetramethylsilane as secondary reference standardand coupling constant in Hz. EIMS (electron impact ionizationmass spectra) were obtained on Thermo DSQ GC/MS instrumentby direct inlet. Enzyme labeled instrument (Bio-Rad) and flowcytometer (FACS Calibur, Becton Dickinson, NJ) were prepared forcytotoxicity experiments.

95% JZ was obtained from Zhongshan Aestar Fine Chemical Inc.Ltd., China. 2-bromothiophene (93%) and 90% 2, 5-dibromothio-phene were purchased from Shanghai Haiqu Chemical Co., Ltd.The terminal arylacetylenes (R )(Fig. 1), Palladium(II) acetate,cuprous(I) iodide, triphenylphosphine, 1,3-bis(diphenylphos-phino)-propanechloride [Ni(dppp)Cl2], 3-(4,5-dimethylthiazol-2-yl)- 2,5- diphenyltetrazolium bromide (MTT), 20,70-dichloro-fluorescein diacetate (DCFH-DA) and rhodamine 123 werepurchased from Sigma–Aldrich Chemical Co., USA.

2.2. Synthesis

The synthetic pathways are shown in Fig. 1. The Grignard re-agent 2-thienylmagnesium bromide was prepared from 2-bromo-thiophene by a procedure according to literature [14]. Thesynthesis of a-T was based on Ni(dppp)Cl2 catalyzed cross-cou-

pling reaction between 2,5-dibromothiophene and 2-thienylmag-nesium bromide in diethyl ether [15]. N-bromosuccinimideadded portion wise to a solution of a-T in N,N-dimethylformamidehas been successfully used in the preparation of 5,50-Dibromo-2,20:5020 0-terthienyl [16]. The intermediate products 3–8 and endproducts 9–15 were synthesized by palladium/copper–catalyzedcross-coupling reaction of 1-alkynes with arylhalides [17]. Theterthienyl bromide and terminal acetylenes (1:1 equiv) were dis-solved in triethylamine under nitrogen. After stirring for 5 min,Palladium(II) acetate, cuprous(I) iodide and triphenylphosphine(1:2:3) were added. The reaction mixture was stirred 2–3 h atroom temperature. The product was purified by column chroma-tography on silica gel.

2.2.1. 5,5”-Dibromo-2,20:5020 0-terthienyl (2)Gold yellow crystals, 84% yield, mp 156 �C (lit mp 156 and

157 �C). 1H NMR (CDCl3, 600 MHz): d 7.00 (s, 2H, thiophene-H),6.98 (d, J = 4.2 Hz, 2H, thiophene-H), 6.91 (d, J = 4.2 Hz, 2H, thio-phene-H).

2.2.2. 5-bromo-50 0-(phenylethynyl)-2,20:50,20 0-terthiophene (3)Yellow powder, 72% yield, mp 169–170 �C. 1H NMR (CDCl3,

600 MHz): d 7.51–7.53 (m, 2H, Ph-H), 7.35–7.36 (m, 3H, Ph-H),7.18 (d, J = 3.8 Hz, 1H, thiophene-H), 7.08 (d, J = 3.8 Hz, 1H, thio-phene-H), 7.06 (d, J = 3.8 Hz, 1H, thiophene-H), 7.02 (d, J = 3.8 Hz,1H, thiophene-H), 6.98 (d, J = 3.8 Hz, 1H, thiophene-H), 6.92 (d,J = 3.8 Hz, 1H, thiophene-H). 13C NMR (CDCl3, 150 MHz): d 82.6,94.6, 111.4, 122.6, 122.9, 123.7, 124.0, 124.7, 124.8, 128.4, 128.6,130.8, 131.5, 132.8, 135.8, 136.1, 138.3, 138.5. EIMS m/z (%): 428([M+2]+, 100.0), 426 (M+, 93.3), 347 (12.3), 303 (63.0), 214 (56.5),152 (77.8).

2.2.3. 5-bromo-50 0-[(3-methylphenyl)ethynyl]-2,20:50,20 0-terthiophene(4)

Yellow powder, 68% yield, mp 169–171 �C. 1H NMR (CDCl3,600 MHz): d 7.34 (s, 1H, Ph-H), 7.32 (d, J = 7.8 Hz, 1H, Ph-H), 7.24

172 N. Li et al. / Journal of Photochemistry and Photobiology B: Biology 96 (2009) 170–177

(t, J = 7.8 Hz, 1H, Ph-H), 7.16 (d, J = 3.8 Hz, 1H, thiophene-H), 7.15–7.16 (m, 1H, Ph-H), 7.08 (d, J = 3.8 Hz, 1H, thiophene-H), 7.05 (d,J = 3.8 Hz, 1H, thiophene-H), 7.01 (d, J = 3.8 Hz, 1H, thiophene-H),6.98 (d, J = 3.8 Hz, 1H, thiophene-H), 6.92 (d, J = 3.8 Hz, 1H, thio-phene-H), 2.36 (s, 3H, CH3�). 13C NMR (CDCl3, 150 MHz): d 21.2,82.2, 94.7, 111.3, 122.5, 123.7, 123.9, 124.7, 124.8, 128.3, 128.5,129.5, 130.7, 131.9, 132.7, 135.7, 136.0, 138.1, 138.4. EIMS m/z(%):442 ([M+2]+, 100.0), 440 (M+, 91.2), 361 (10.5), 317 (62.7),304 (6.8), 221 (60.5), 158 (69.3).

2.2.4. 5-bromo-50 0-[(4-methylphenyl)ethynyl]-2,20:50,20 0-terthiophene(5)

Yellow powder, 70% yield, mp 171–172 �C. 1H NMR (CDCl3,600 MHz): d 7.40 (d, J = 7.8 Hz, 2H, Ph-H), 7.16 (d, J = 7.8 Hz, 2H,Ph-H), 7.15 (d, J = 3.6 Hz, 1H, thiophene-H), 7.07 (d, J = 3.8 Hz, 1H,thiophene-H), 7.05 (d, J = 3.8 Hz, 1H, thiophene-H), 7.01 (d,J = 3.8 Hz, 1H, thiophene-H), 6.98 (d, J = 3.8 Hz, 1H, thiophene-H),6.92 (d, J = 3.8 Hz, 1H, thiophene-H), 2.37 (s, 3H, CH3�). 13C NMR(CDCl3, 150 MHz): d 21.5, 81.9, 94.7, 111.3, 119.7, 122.6, 123.6,123.9, 124.7 (124.66, 124.71), 129.2, 130.7, 131.3, 132.6, 135.6,136.0, 137.9, 138.4, 138.8. EIMS m/z (%): 442 ([M+2]+, 100.0), 440(M+, 88.7), 361 (10.2), 317 (57.8), 304 (6.3), 221 (56.2), 158 (76.2).

2.2.5. 5-bromo-50 0-[(4-ethylphenyl)ethynyl]-2,20:50,20 0-terthiophene(6)

Yellow powder, 64% yield, mp 181–182 �C. 1H NMR (CDCl3,600 MHz): d 7.43 (d, J = 8.4 Hz, 2H, Ph-H), 7.18 (d, J = 8.4 Hz, 2H,Ph-H), 7.15 (d, J = 3.8 Hz, 1H, thiophene-H), 7.07 (d, J = 3.8 Hz, 1H,thiophene-H), 7.05 (d, J = 3.8 Hz, 1H, thiophene-H), 7.01 (d,J = 3.8 Hz, 1H, thiophene-H), 6.98 (d, J = 3.8 Hz, 1H, thiophene-H),6.92 (d, J = 3.8 Hz, 1H, thiophene-H), 2.67 (q, J = 7.8 Hz, 2H,CH3CH2�), 1.24 (t, J = 7.8 Hz, 3H, CH3�). 13C NMR (CDCl3,150 MHz): d 15.3, 28.9, 81.9, 94.8, 111.3, 119.9, 122.7, 123.7,123.9, 124.7 (124.67, 124.71), 128.0, 130.8, 131.4, 132.6, 135.6,136.0, 137.9, 138.4, 145.1. EIMS m/z (%): 456 ([M+2]+, 84.9), 454(M+, 73.2), 439 (28.4), 375 (2.3), 331 (12.0), 316 (20.0), 303(12.2), 220 (54.7), 158 (100.0).

2.2.6. 5-bromo-50 0-[(4-propylphenyl)ethynyl]-2,20:50,20 0-terthiophene(7)

Yellow powder, 59% yield, mp 165–166 �C. 1H NMR (CDCl3,600 MHz): d 7.42 (d, J = 8.4 Hz, 2H, Ph-H), 7.16 (d, J = 8.4 Hz, 2H,Ph-H), 7.15 (d, J = 3.8 Hz, 1H, thiophene-H), 7.07 (d, J = 3.8 Hz, 1H,thiophene-H), 7.05 (d, J = 3.8 Hz, 1H, thiophene-H), 7.01 (d,J = 3.8 Hz, 1H, thiophene-H), 6.98 (d, J = 3.8 Hz, 1H, thiophene-H),6.92 (d, J = 3.8 Hz, 1H, thiophene-H), 2.60 (t, J = 7.8 Hz, 2H,CH3CH2CH2�), 1.629-1.667 (m, 2H, CH3CH2�), 0.94 (t, J = 7.8 Hz,3H, CH3�). 13C NMR (CDCl3, 150 MHz): d 13.8, 24.3, 38.0, 81.9,94.8, 111.3, 119.9, 122.7, 123.7, 123.9, 124.7 (124.68, 124.72),128.6, 130.8, 131.3, 132.6, 135.6, 136.0, 137.9, 138.4, 143.6. EIMSm/z (%): 470 ([M+2]+, 83.1), 468 (M+,75.5), 439 (36.6), 389 (1.7),345 (7.6), 316 (20.0), 303 (11.6), 220 (78.0), 158 (100.0).

2.2.7. 5-bromo-50 0-[(4-butylphenyl)ethynyl]-2,20:50,20 0-terthiophene(8)

Yellow powder, 63% yield, mp 168–170 �C. 1H NMR (CDCl3,600 MHz): d 7.42 (d, J = 8.4 Hz, 2H, Ph-H), 7.16 (d, J = 8.4 Hz, 2H,Ph-H), 7.15 (d, J = 3.8 Hz, 1H, thiophene-H), 7.07 (d, J = 3.8 Hz, 1H,thiophene-H), 7.05 (d, J = 3.8 Hz, 1H, thiophene-H), 7.01 (d,J = 3.8 Hz, 1H, thiophene-H), 6.98 (d, J = 3.8 Hz, 1H, thiophene-H),6.92 (d, J = 3.8 Hz, 1H, thiophene-H), 2.62 (t, J = 7.8 Hz, 2H,CH3CH2CH2CH2�), 1.59–1.62 (m, 2H, CH3CH2CH2�), 1.34–1.38 (m,2H, CH3CH2�), 0.93 (t, J = 7.8 Hz, 3H, CH3�). 13C NMR (CDCl3,150 MHz): d 13.9, 22.3, 33.4, 35.6, 81.9, 94.8, 111.3, 119.9, 122.7,123.7, 123.9, 124.7 (124.68, 124.72), 128.6, 130.8, 131.3, 132.6,135.6, 136.0, 137.9, 138.4, 143.8. EIMS m/z (%): 484 ([M+2]+,

100.0), 482 (M+, 89.9), 439 (58.6), 403 (1.6), 359 (12.5), 316(30.8), 303 (19.5), 220 (68.3), 158 (97.0).

2.2.8. 2-methyl-4-oxo-3-[3-(500-bromo-2,20:50,200-terthien-5-yl)prop-2-yn-1-yl]cyclopent-2-en-1-yl-3-(2,2-dichlorovinyl)-2,2-dimethycyclopropanecarboxylate (9)

Yellow powder, 39% yield, mp 78–80 �C. 1H NMR (Acetone-d6,600 MHz): d 7.27 (d, J = 3.8 Hz, 1H, thiophene-H), 7.24 (d,J = 3.8 Hz, 1H, thiophene-H), 7.22 (d, J = 3.8 Hz, 1H, thiophene-H),7.17 (d, J = 3.8 Hz, 1H, thiophene-H), 7.15 (d, J = 3.8 Hz, 2H, thio-phene-H), 5.97 (d, J = 8.6 Hz, 1H, CCl2@CHA), 5.78 (m, 1H, cyclo-pentene CH–), 3.46 (s, 2H, alkyne-CH2�), 2.89 (dd, J = 6.0 Hz,18.0 Hz, 1H, cyclopentene CH–), 2.24 (s, 3H, cyclopentene-CH3�),2.23 (dd, J = 5.0 Hz, 9.0 Hz, 1H, cyclopropane CH–), 2.23 (dd,J = 2.4 Hz, 18.0 Hz, 1H, cyclopentene CH–), 1.96 (d, J = 5.0 Hz, 1H,cyclopropane CH–), 1.30 (s, 3H, cyclopropane-CH3�), 1.25 (s, 3H,cyclopropane-CH3�). 13C NMR (Acetone-d6, 150 MHz): d 13.9,14.3, 20.3, 22.3, 33.8, 34.9, 42.2, 74.1, 74.3, 92.3, 111.6, 121.4,123.4, 124.9, 125.5, 126.2, 128.9, 132.4, 133.7, 136.1, 136.5,138.0, 138.8, 139.1, 167.3, 171.4, 202.2. EIMS m/z (%): 664 (M+,4.2), 456 (30.1), 428 (5.5), 208 (13.4), 173 (71.2), 163 (56.9), 27(75.1), 91 (100.0), 77 (37.8).

2.2.9. 2-methyl-4-oxo-3-{3-[500-(phenylethynyl)-2,20:50,200-terthien-5-yl]prop-2-yn-1-yl}cyclopent-2-en-1-yl-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate (10)

Yellow powder, 32% yield, mp 90–92 �C. 1H NMR (Acetone-d6,600 MHz): d 7.55-7.57 (m, 2H, Ph-H), 7.43–7.45 (m, 3H, Ph-H),7.33 (d, J = 3.8 Hz, 2H, thiophene-H), 7.30 (d, J = 3.8 Hz, 1H, thio-phene-H), 7.29 (d, J = 3.8 Hz, 1H, thiophene-H), 7.23 (d, J = 3.8 Hz,1H, thiophene-H), 7.16 (d, J = 3.8 Hz, 1H, thiophene-H), 5.96 (d,J = 8.6 Hz, 1H, CCl2@CH–), 5.78–5.79 (m, 1H, cyclopentene CH–),3.46 (s, 2H, alkyne-CH2�), 2.89 (dd, J = 6.0 Hz, 18.0 Hz, 1H, cyclo-pentene CH–), 2.25 (s, 3H, cyclopentene-CH3�), 2.23 (dd,J = 5.0 Hz, 9.0 Hz, 1H, cyclopropane CH–), 2.23 (dd, J = 2.4 Hz,18.0 Hz, 1H, cyclopentene CH–), 1.96 (d, J = 5.0 Hz, 1H, cyclopro-pane CH–), 1.31 (s, 3H, cyclopropane-CH3�), 1.25 (s, 3H, cyclopro-pane-CH3�). 13C NMR (Acetone-d6, 150 MHz): d 14.0, 14.3, 20.3,22.3, 33.8, 35.0, 42.3, 74.1, 74.3, 82.9, 92.3, 95.2, 121.4, 122.8,123.4, 125.0, 125.0, 125.2, 126.3, 126.5, 128.9, 129.6, 129.8,132.1, 133.7, 134.4, 136.5, 136.8, 138.0, 138.8, 139.0, 167.4,171.4, 202.3. EIMS m/z (%): 686 (M+, 2.7), 478 (30.6), 450 (6.0),435 (2.5), 208 (12.4), 173 (97.6), 163 (63.5), 127 (71.8), 91(100.0), 77 (38.4).

2.2.10. 2-methyl-4-oxo-3-(3-{500-[(3-methylphenyl)ethynyl]-2,20:50,200-terthien-5-yl}prop-2-yn-1-yl)cyclopent-2-en-1-yl-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate (11)

Yellow powder, 26% yield, mp 106–108 �C. 1H NMR (Acetone-d6,600 MHz): d 7.37 (s, 1H, Ph-H), 7.24–7.35 (m, 7H, Ph-3H, thio-phene-4H), 7.22 (d, J = 3.8 Hz, 1H, thiophene-H), 7.15 (d,J = 3.8 Hz, 1H, thiophene-H), 5.95 (d, J = 8.6 Hz, 1H, CCl2@CH–),5.78–5.79 (m, 1H, cyclopentene CH–), 3.46 (s, 2H, alkyne-CH2�),2.89 (dd, J = 6.0 Hz, 18.0 Hz, 1H, cyclopentene CH–), 2.36 (s, 3H,Ph-CH3�), 2.24 (s, 3H, cyclopentene-CH3�), 2.23 (dd, J = 5.0 Hz,9.0 Hz, 1H, cyclopropane CH–), 2.22 (dd, J = 2.0 Hz, 18.0 Hz, 1H,cyclopentene CH–), 1.95 (d, J = 5.0 Hz, 1H, cyclopropane CH–),1.30 (s, 3H, cyclopropane-CH3�), 1.25 (s, 3H, cyclopropane-CH3�).13C NMR (Acetone-d6, 150 MHz): d 13.9, 14.3, 20.3, 21.1, 22.3,33.8, 34.9, 42.2, 74.1, 74.3, 82.5, 92.3, 95.4, 121.4, 122.9, 123.2,123.4, 124.9, 125.1, 126.3, 126.4, 128.9, 129.2, 129.4, 130.6,132.6, 133.7, 134.2, 136.4, 136.7, 137.9, 138.8, 138.9, 139.3,167.3, 171.4, 202.2. EIMS m/z (%): 700 (M+, 5.48), 492 (25.37),464 (5.41), 449 (2.89), 208 (10.56), 173 (85.72), 163 (60.56), 127(74.50), 91 (100.00), 77 (38.14).

N. Li et al. / Journal of Photochemistry and Photobiology B: Biology 96 (2009) 170–177 173

2.2.11. 2-methyl-4-oxo-3-(3-{500-[(4-methylphenyl)ethynyl]-2,20:50,200-terthien-5-yl}prop-2-yn-1-yl)cyclopent-2-en-1-yl-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate (12)

Yellow powder, 30% yield, mp 107–109 �C. 1H NMR (Acetone-d6,600 MHz): d 7.43 (d, J = 7.2 Hz, 2H, Ph-H), 7.24–7.29 (m, 6H, Ph-2H,thiophene-4H), 7.21 (d, J = 3.8 Hz, 1H, thiophene-H), 7.14 (d,J = 3.8 Hz, 1H, thiophene-H), 5.95 (d, J = 8.6 Hz, 1H, CCl2@CH–),5.78–5.79 (m, 1H, cyclopentene CH–), 3.45 (s, 2H, alkyne-CH2�),2.89 (dd, J = 6.0 Hz, 18.0 Hz,1H, cyclopentene CH–), 2.37 (s, 3H,Ph-CH3�), 2.24 (s, 3H, cyclopentene-CH3�), 2.23 (dd, J = 5.0 Hz,9.0 Hz, 1H, cyclopropane CH–), 2.22 (dd, J = 2.0 Hz, 18.0 Hz, 1H,cyclopentene CH–), 1.95 (d, J = 5.0 Hz, 1H, cyclopropane CH–),1.30 (s, 3H, cyclopropane-CH3�), 1.25 (s, 3H, cyclopropane-CH3�).13C NMR (Acetone-d6, 150 MHz): d 13.9, 14.3, 20.3, 21.4, 22.3,33.8, 34.9, 42.2, 74.1, 74.3, 82.3, 92.3, 95.4, 120.3, 121.4, 123.1,123.3, 124.9, 125.1, 126.2, 126.3, 128.9, 130.2, 132.1, 133.7,134.0, 136.5, 136.7, 138.0, 138.7, 138.8, 140.0, 167.3, 171.4,202.2. EIMS m/z (%): 700 (M+, 0.3), 492 (15.2), 464 (3.6), 449(1.1), 208 (6.7), 173 (100.0), 163 (62.1), 127 (72.9), 91 (98.0), 77(36.1).

2.2.12. 2-methyl-4-oxo-3-(3-{500-[(4-ethylphenyl)ethynyl]-2,20:50,200-terthien-5-yl}prop-2-yn-1-yl)cyclopent-2-en-1-yl-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate (13)

Yellow powder, 33% yield, mp 116–117 �C. 1H NMR (Acetone-d6, 600 MHz): d 7.47 (d, J = 8.4 Hz, 2H, Ph-H), 7.30 (d, J = 3.8 Hz,1H, thiophene-H), 7.29 (d, J = 8.4 Hz, 2H, Ph-H), 7.28 (d, J = 3.8 Hz,2H, thiophene-H), 7.27 (d, J = 3.8 Hz, 1H, thiophene-H), 7.22 (d,J = 3.8 Hz, 1H, thiophene-H), 7.15 (d, J = 3.8 Hz, 1H, thiophene-H),5.96 (d, J = 8.6 Hz, 1H, CCl2@CH–), 5.78-5.79 (m, 1H, cyclopenteneCH–), 3.46 (s, 2H, alkyne-CH2�), 2.89 (dd, J = 6.0 Hz, 18.0 Hz, 1H,cyclopentene CH–), 2.68 (q, J = 7.8 Hz, 2H, Ph-CH2�), 2.24 (s, 3H,cyclopentene-CH3�), 2.23 (dd, J = 5.0 Hz, 9.0 Hz, 1H, cyclopropaneCH–), 2.22 (dd, J = 2.0 Hz, 18.0 Hz, 1H, cyclopentene CH–), 1.96(d, J = 5.0 Hz, 1H, cyclopropane CH–), 1.30 (s, 3H, cyclopropane-CH3�), 1.25 (s, 3H, cyclopropane-CH3�), 1.23 (t, J = 7.8 Hz, 3H, Ph-CH2CH3�). 13C NMR (Acetone-d6, 150 MHz): d 13.9, 14.3, 15.7,20.3, 29.3, 22.3, 33.8, 34.9, 42.2, 74.1, 74.3, 82.2, 92.3, 95.4,120.6, 121.4, 123.0, 123.4, 124.9, 125.1, 126.2, 126.4, 128.9,129.1, 132.2, 133.7, 134.0, 136.5, 136.7, 138.0, 138.7, 138.8,146.4, 167.3, 171.4, 202.2. EIMS m/z (%): 714 (M+, 2.6), 506(21.9), 478 (4.4), 463 (2.4), 208 (8.3), 173 (87.7), 163 (58.3), 127(67.5), 91 (100.0), 77 (38.4).

2.2.13. 2-methyl-4-oxo-3-(3-{500-[(4-propylphenyl)ethynyl]-2,20:50,200-terthien-5-yl}prop-2-yn-1-yl)cyclopent-2-en-1-yl-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate (14)

Yellow powder, 27% yield, mp 110–112 �C. 1H NMR (Acetone-d6, 600 MHz): d 7.47 (d, J = 7.8 Hz, 2H, Ph-H), 7.31 (d, J = 3.8 Hz,1H, thiophene-H), 7.27–7.29 (m, 5H, Ph-2H, thiophene-3H), 7.23(d, J = 3.8 Hz, 1H, thiophene-H), 7.15 (d, J = 3.8 Hz, 1H, thiophene-H), 5.96 (d, J = 8.6 Hz, 1H, CCl2@CH–) , 5.78–5.79 (m, 1H, cyclopen-tene CH–), 3.46 (s, 2H, alkyne-CH2�), 2.89 (dd, J = 6.0, 18.0 Hz, 1H,cyclopentene CH–), 2.63 (t, 2H, Ph-CH2�), 2.24 (s, 3H, cyclopen-tene-CH3�), 2.23 (dd, J = 5.0 Hz, 9.0 Hz, 1H, cyclopropane CH–),2.22 (dd, J = 2.0 Hz, 18.0 Hz, 1H, cyclopentene CH–), 1.96 (d,J = 5.0 Hz, 1H, cyclopropane CH–), 1.62–1.68 (m, 2H, Ph-CH2CH2�),1.30 (s, 3H, cyclopropane-CH3�), 1.25 (s, 3H, cyclopropane-CH3�),0.93 (t, J = 7.8 Hz, 3H,Ph-CH2CH2CH3�).

13C NMR (Acetone-d6, 150 MHz): d 13.9, 14.3, 20.3, 22.3, 25.0,33.8, 34.9, 38.4, 42.2, 74.1, 74.3, 82.3, 92.3, 95.5, 120.6, 121.4,123.0, 123.3, 124.9, 125.1, 126.2, 126.3, 128.9, 129.7, 132.0,133.7, 134.0, 136.5, 136.7, 138.0, 138.7, 138.8, 144.7, 167.3,171.4, 202.2. EIMS m/z (%): 728 (M+, 6.4), 520 (30.8), 492 (6.7),477 (1.5), 208 (10.5), 173 (93.4), 163 (63.9), 127 (76.3), 91(100.0), 77 (36.1).

2.2.14. 2-methyl-4-oxo-3-(3-{500-[(4-butylphenyl)ethynyl]-2,20:50,002-terthien-5-yl}prop-2-yn-1-yl)cyclopent-2-en-1-yl-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate (15)

Yellow powder, 31% yield, mp 111–113 �C. 1H NMR (Acetone-d6, 600 MHz): d 7.46 (d, J = 7.8 Hz, 2H, Ph-H), 7.29 (d, J = 3.8 Hz,1H, thiophene-H), 7.26-7.28 (m, 5H, Ph-2H, thiophene-3H), 7.21(d, J = 3.8 Hz, 1H, thiophene-H), 7.14 (d, J = 3.8 Hz, 1H, thiophene-H), 5.96 (d, J = 8.6 Hz, 1H, CCl2@CH–), 5.78–5.79 (m, 1H, cyclopen-tene CH–), 3.46 (s, 2H, CCl2@CH–), 2.89 (dd, J = 6.0 Hz, 18.0 Hz, 1H,cyclopentene CH–), 2.65 (t, 2H, Ph-CH2�), 2.25 (s, 3H, cyclopen-tene-CH3�), 2.23 (dd, J = 5.0, 9.0 Hz, 1H, cyclopropane CH–), 2.22(dd, J = 2.0 Hz, 18.0 Hz, 1H, cyclopentene CH–), 1.95 (d, J = 5.0 Hz,1H, cyclopropane CH–), 1.58–1.63 (m, 2H, Ph-CH2CH2�), 1.33–1.38 (m, 2H, PhCH2CH2CH2�), 1.30 (s, 3H, cyclopropane-CH3�),1.25 (s, 3H, cyclopropane-CH3�), 0.93 (t, J = 7.8 Hz, 3H,PhCH2CH2CH2CH3�). 13C NMR (Acetone-d6, 150 MHz): d 13.9,14.1, 14.3, 20.3, 22.3, 22.9, 33.8, 34.2, 34.9, 36.1, 42.2, 74.1, 74.3,82.3, 92.3, 95.5, 120.6, 121.4, 123.1, 123.3, 124.9, 125.1, 126.2,126.3, 128.9, 129.6, 132.1, 133.7, 134.0, 136.5, 136.7, 138.0,138.7, 138.8, 145.0, 167.3, 171.4, 202.2. EIMS m/z (%): 742 (M+,6.5), 534 (14.7), 506 (2.6), 491 (2.5), 208 (8.5), 173 (71.7), 163(52.6), 127 (67.7), 91 (100.0), 77 (35.2).

2.3. Biological studies

2.3.1. Cell cultureSpodoptera litura (SL) cell was obtained from Key Laboratory of

Pesticide and Chemical Biology of Ministry of Education, CentralChina Normal University, China, and cultured with Grace’s insectcell culture medium (Gibco, America) containing 9% new born calfserum at 27.5 �C. Cells in logarithmic phase of growth were used inall experiments. The compounds were stock solutions in dimethyl-sulfoxide (DMSO), which diluted with culture medium to the de-sired concentrations on the day of the experiments. Theconcentration of DMSO was kept 0.5% in treated groups. Controlcultures were performed in the presence of DMSO under the sameculture conditions.

2.3.2. Cytotoxicity of the compounds on SL cellCytotoxicity of the compounds (9–15) on SL cell was identified

by MTT assay, as previously reported [18]. Cells were incubated in96-well cell culture plates in absence (control) or presence of thecompounds. After 30 min of incubation, the cultures were eitherirradiated with light (3 min, 40 W, UV-A) or not, two Philips’ ultra-violet light were used as light source, giving a light intensity of2600 lW cm�2 at the spectrum peak 365 nm as measured with aHuandi UV-A radiometer (Photoelectric Instrument Factory of Bei-jing, Normal University, China), then cultured in the incubator for24 h [19]. The medium was replaced by a solution of MTT(0.5 mg mL�1), and the cells were incubated 4 h. Finally, the absor-bance was measured on a Bio-Rad ELISA reader at 570 nm. Theinhibition rate was calculated by the absorbance, and medianInhibitory concentration (IC50) values of the active compound 9and JZ were analyzed with up to the inhibition rates of five desiredconcentrations.

2.3.3. Measurement of intracellular ROSThe fluorescence probe DCFH-DA was used to detect the levels

of intracellular ROS. DCFH-DA diffuses through the cell membranereadily and is hydrolyzed by esterases to DCFH, which is then oxi-dized to highly fluorescent 20,70-dichlorofluorescein (DCF) in thepresence of ROS [20]. SL Cells were incubated onto cell culture dishwith compound 9 (10 lg mL�1) and JZ (40 lg mL�1) for 24 h and48 h, treated as described above for MTT assay. Cells were respec-tively harvested at 24 h or 48 h and stained with 10 lM ofDCFH-DA at 37 �C for 30 min in dark, washed, re-suspended in

Table 1Comparison of median Inhibitory concentration (IC50) between compound 9 and JZ onSL cells exposed to 24 h, determined by the MTT assay.

Compounds Treatment method IC50 (lg mL�1) 95%CI

9 Dark 108.52 85.96–137.00Irradiation 11.60 8.37–16.06

JZ Dark 40.80 27.40–60.76Irradiation 46.89 31.09–70.71

Table 2Comparison of median Inhibitory concentration (IC50) between compound 9 and JZ onSL cells exposed to 48 h, determined by the MTT assay.

Compounds Treatment method IC50 (lg mL�1) 95%CI

9 Dark 72.64 54.70–96.47Irradiation 8.93 6.98–11.43

JZ Dark 23.57 17.27–32.17Irradiation 28.44 20.86–38.76

174 N. Li et al. / Journal of Photochemistry and Photobiology B: Biology 96 (2009) 170–177

phosphate-buffered saline (PBS), and immediately analyzed byflow cytometry (the excitation wavelength sets at 485 nm andthe emission wavelength at 530 nm). ROS generation wasestimated by the fluorescence intensity of 20,000 cells.

2.3.4. Monitoring of MMPMitochondrial membrane was monitored using the fluorescent

probe rhodamine 123, a cell permeable cationic dye, which prefer-entially partitions into mitochondria based on the highly negativeMMP [21]. SL Cells were incubated onto cell culture dish with com-pound 9 (10 lg mL�1) and JZ (40 lg mL�1) for 24 h and 48 h, trea-ted as described above for MTT assay. Cells were respectivelyharvested at 24 h or 48 h and stained with 10 lM rhodamine 123at 37 �C for 30 min in dark, washed, re-suspended in PBS, andimmediately analyzed by flow cytometry (the excitation wave-length at 490 nm and the emission wavelength at 535 nm). MMPwas estimated by the fluorescence intensity of 20,000 cells.

2.4. Statistical analysis

All the experiments were repeated at least three times and val-ues were expressed as the mean ± SE. To evaluate the significancedifferences between the data of two chosen groups, the statisticalanalysis was obtained by StatView 5.0.1 software (SAS Institute,Mountain View, CA, USA). The level of significance was set atP < 0.05.

3. Results and discussion

3.1. Cytotoxicity of the compounds on SL cell

The MTT assay was performed to explore the photoactivatedcytotoxicity on SL cell exposed to the conjugates 9–15 and JZ.When the conjugates were irradiated with UV-A light, they gener-ally became more cytotoxic than in the dark. By comparison to JZ,the inhibition rate exhibited enhancement of all the conjugatestreated with irradiation, and compound 9 was the most activeone (Fig. 2). The values represented statistically significant differ-ences between compound 9–15 and JZ (P < 0.05). It implied that,to some extent, our conjugates portrayed a stronger activity andthe three thiophene ring played an important role to the photoac-tivation of the compounds. The photoactivated cytotoxicity was in-creased slightly by the shift of a donor group on the benzene ringfrom m- to p- in compounds 11–15. The statistically significant dif-ferences were showed between dark and irradiation treatment ofall the compounds (P < 0.05). The inhibition rate values exhibitednotable enhancement for compound 9–15 under irradiation treat-ment compared with the dark treatment, but for JZ, the inhibition

Fig. 2. Cytotoxicity of the compounds on Spodoptera litura (SL) cell was identified by Mcompound 9–15 and JZ at the concentration of l0 lg/mL or l00 lg/mL in absence (Dark)the mean ± SE (n = 3), � indicates statistically significant differences between compoundtreatment and irradiation-treatment of all compounds (P < 0.05).

rate value was slightly lower under the irradiation treatment thanthe dark treatment.

In the subsequent experiment, the IC50 values could furthershow the marked photoactivated effect (Tables 1 and 2). The IC50

values of SL cells exposed to compound 9 under irradiation were11.60 lg mL�1at 24 h and 8.93 lg mL�1 at 48 h respectively. Thevalues also indicated a distinct improvement in cytotoxicity effectscompared with JZ, and showed a time-dependent cytotoxic effect.

3.2. Measurement of intracellular ROS

Generally, absorption of the light triggers excitation of the pho-tosensitizer, which then either kills cells directly through forma-tion of highly reactive free radicals, or reacts with molecularoxygen to create singlet oxygen that disrupts cell function [22].Photodynamic therapy (PDT) is a novel and promising cancer treat-ment that involves the administration of photosensitizers coupledwith a specific wavelength of laser irradiation. And the basis of thephotodynamic damaging effect on cells consisting in the produc-tion of intracellular ROS was confirmed [22–25]. a-T is a potentphotosensitizing agent, and the activation occurs upon irradiationwith near-ultraviolet wavelengths (300–400 nm) [26]. Since ourconjugates were started from the corresponding a-T derivatives,we focused our study on whether the conjugates can increaseROS production within SL cells.

TT assay, as described m Section 2. The inhibition rate of SL cell after treating withor presence af irradiation (3 min, 2600 lW cm2, 365 nm) for 24 h. Results represent9–15 and JZ treatment (P < 0.05). # indicates significant differences between dark-

N. Li et al. / Journal of Photochemistry and Photobiology B: Biology 96 (2009) 170–177 175

In the current study, the photoactivated effect of the syntheticconjugates had been preliminarily corroborated by MTT assay.Attempting to elucidate the mechanism of the phototoxic action,we chose the active compound 9 as a typical example to measureintracellular ROS for further study. It is well known that the DCFH-DA fluorescence intensity is proportional to the amount of ROSformed intracellularly. Our results clearly demonstrated that SLcells treated with compound 9 (10 lg mL�1) or JZ (40 lg mL�1)for 24 h and 48 h generated substantial amount of ROS (Fig. 3),and represented significant differences between compound treat-ment and respective control cell cultures (P < 0.05) either underdark or irradiation treatment. In particular, the mean fluorescenceintensity values of SL cells exposed to compound 9 with irradiationexceeded three times of control values for 24 h and four times for48 h respectively. However, the values exhibited more remarkableenhancement in the ROS levels of SL cell exposed to compound 9under irradiation treatment compared with dark treatment. Butthe mean fluorescence intensity value of irradiation treatment ofJZ was slightly less than in the dark. The statistical results showedsignificant difference between dark and irradiation treatment ofcompound 9 (P < 0.05). However, in control and JZ groups(P > 0.05), the difference was not significant. This remarkable dif-ference of intracellular ROS levels further illustrated the photoacti-vated effect of our conjugates.

The photodynamic therapy (PDT) is a recently developed anti-cancer modality utilizing the generation of singlet oxygen andother reactive oxygen species. Multiple signaling cascades are

Fig. 3. ROS production alterations of SL cells after treatment for 24 h (A) and 48 h (B). RO2. Control: SL cells were incubated with culture medium containing 0.5% DMSO in the coincubated with 10 lg/mL compound 9 in the same condition; JZ: SL cells were incubatedmeasured by flow cytometric analysis, and the results represent mean ± SE of threecompounds treatment and respective control in dark or irradiation condition (P < 0.0compound 9 (P < 0.05), but there was no significant differences of control or JZ group (P

Fig. 4. MMP changes of SL cell after treatment for 24 h (A) and 48 h (B). MMP ware detercells were incubated with culture medium containing 0.5% DMSO in the condition of dawith l0 lg/mL compound 9 in the same condition: JZ: SL cells were incubated with 40 lg/flow cytometric analysis, and the results represent mean ± SE of three independenttreatment and respective control in dark or irradiation condition (P < 0.05); # indicat(P < 0.05), but there was no significant differences of control or JZ group (P > 0.05).

concomitant, and the direct cytotoxic effect of PDT is the resultof the incorporation of the sensitizer mainly into cellular mem-branes and the subsequent light-induced generation of ROS caus-ing irreparable damage [27,28]. Presumably, our conjugateswould generate singlet oxygen after UV-A irradiation. And theircytotoxicity would be related to the oxidative stress throughgeneration of ROS. The concentrations of the compounds wechose were more or less commensurate to their respective IC50

values. Future studies also should investigate the effect of otherdoses to different cell types.

3.3. Monitoring of MMP

Mitochondria are sometimes referred to as the powerhouses ofthe cell. They also have a significant role on regulating ROS levels,apoptosis and cellular signaling [29]. Cytotoxicity of the com-pounds on SL cell had been identified by MTT assay for measuringmitochondrial dehydrogenase activity. The central mitochondrialmechanism involved is the opening of the mitochondrial perme-ability transition pore, which results in mitochondrial membranepotential depolarization [23]. To further demonstrate the involve-ment of mitochondria function, we designed an experiment esti-mating the effects of the compounds on MMP. Depolarization ofMMP results in the loss of rhodamine 123 from the mitochondriaand the decrease in intracellular fluorescence.

As shown in Fig. 4, addition of compound 9 (10 lg mL�1) and JZ(40 lg mL�1) caused a loss of MMP and showed significant changes

S levels were determined by the fluorescent probe DCFH-DA as described in Sectionndition of dark or irradiation (3 min, 2600 lW cm�2 365 nm); Compound 9: SL cellswith 40 lg/mL JZ in the same condition. The fluorescence intensity of the cells was

independent experiments. � indicates statistically significant differences between5); # indicates significant difference between dark and irradiation treatment of> 0.05).

mined by the fluorescent probe rhodaminel23 as described in Section 2. Control: SLrk or irradiation (3 min, 2600 lW cm�2, 365 nm); Compound 9: SL cells incubatedmL JZ in the same condition The fluorescence intensity of the cells was measured byexperiments. � indicates statistically significant differences between compoundses significant difference between dark and irradiation treatment of compound 9

176 N. Li et al. / Journal of Photochemistry and Photobiology B: Biology 96 (2009) 170–177

of rhodamine 123 fluorescence between compound treatment andrespective control cell cultures (P < 0.05) either under dark or irra-diation treatment. These values further demonstrated that mito-chondrial function was severely impaired. Especially, the meanfluorescence intensity values of compound 9 with irradiation treat-ment were approximately 36% (24 h) and 27% (48 h) of the valuesof the control group, respectively. The values also showed the sig-nificant difference between dark and irradiation treatment of com-pound 9 (P < 0.05). However, in control and JZ groups, the effectswere not statistically different. Clearly, the statistical results ofMMP accorded with that of intracellular ROS levels.

The changes by ROS and MMP can damage the ability of mito-chondria to maintain several functions crucial to cellular viability.The mitochondria are perhaps a prominent site of ROS formation.In addition to producing ROS, mitochondria are also a target forROS which reduces mitochondrial potency and leads to more ROSgeneration in a vicious self-destructive cycle [23]. In conclusion,the results suggest that one possible mechanism of our conjugatesinducing cell death could be through mitochondria as its initial tar-get. The subsequent increase of ROS then inflicts cell death and fur-ther worsens mitochondria function.

The aim of this study was to obtain novel photoactivatedpyrethroid insecticides by conjugating pyrethroid insecticidewith a series of a-T derivatives. Seven a-T derivatives (2–8) weresynthesized successfully for our conjugation. It is presumed thatthe a-T derivatives may be valuable intermediate products toconjugate with the most effective but high residue pesticides,such as organochlorines pesticides by other structure modifica-tion. Over the past few decades, the occurrence of organochlo-rines pesticides in the environment has been of great concerndue to their persistent and long-range transportable nature aswell as toxic biological effects [30,31]. Some virulent kinds oforganophosphorus and carbamate pesticides may still becomethe transformation target.

4. Conclusions

In conclusion, we have synthesized seven a-T derivatives (2–8)and their corresponding end products (9–15), and compound 9had high-level photoactivated cytotoxicity on SL cell. A summaryof the three biological studies demonstrated the interesting photo-activated effect of our conjugates with the involvement of cytotox-icity, notable generation of ROS and dramatic decrease of MMP. Theresults strongly suggest that the bioactivity of our conjugates exci-tated by near-ultraviolet light is a promising strategy for photoacti-vated pesticide.

5. Abbreviations

a-T a-terthienyl

JZ 2-methyl-4-oxo-3-(prop-2-ynyl)cyclopent-2-enyl 3-

(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate

NMR

nuclear magnetic resonance EIMS electron impact ionization mass spectra Ni(dppp)Cl2 1,3-bis(diphenylphosphino)-propane]chloride MTT 3-(4,5-dimethylthiazol-2-yl)- 2,5-

diphenyltetrazolium bromide

DCFH-DA 20, 70-dichlorofluorescein diacetate IC50 median Inhibitory concentration 95% CI 95% confidence interval ROS intracellular reactive oxygen species MMP mitochondrial transmembrane potential

Acknowledgements

The authors thank Dr. Xiao-Yi Wei (South China Botanical Gar-den, Chinese Academy of Science, Xingke Road 723, Tianhe District,Guangzhou, 510650, China) for helping to review the manuscript.This study was partly supported by the National Science Founda-tion of China (Grant No. 30671386 and No. 30840058) and Guang-dong Natural Science Foundation (Grant No. 036837 and No.7006672).

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