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Industrial Crops and Products 55 (2014) 11–18

Contents lists available at ScienceDirect

Industrial Crops and Products

jo ur nal home p age: www.elsev ier .com/ locate / indcrop

esign, synthesis and bioactivity evaluation of novel acylthioureaerivatives of cantharidin

ei-Juan Wanga,1, Xiang Nana,1, Gang Fengc, Hai-Tao Yub,uan-Fang Hub, Ying-Qian Liua,∗

School of Pharmacy, Lanzhou University, Lanzhou 730000, ChinaInstitute of Plant Protection, Gansu Academy of Agricultural Sciences, Lanzhou 730070, ChinaEnvironment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Science, Danzhou 571737, China

r t i c l e i n f o

rticle history:eceived 31 October 2013eceived in revised form 7 January 2014ccepted 9 February 2014vailable online 2 March 2014

a b s t r a c t

In an attempt to find the biorational pesticides, a series of novel acylthiourea derivatives of cantharidinwere designed, synthesized and preliminarily evaluated for their bioactivities against Tetranychuscinnabarinus and Brevicoryne brassicae. All the new compounds showed significant acaricidal activitiesagainst T. cinnabarinus with LC50 values ranging from 0.695 to 2.934 mmol/L. In particular, compounds

eywords:antharidincaricidal activity

nsecticidal activity

6i and 6g exhibited the greatest acaricidal activity against T. cinnabarinus in all derivatives we pre-pared. Moreover, all of these compounds displayed better acaricidal activities against T. cinnabarinusthan insecticidal activities against B. brassicae. Based on the observed bioactivities, the structure–activityrelationship (SAR) of these analogs was also discussed. The present work indicated that some cantharidinderivatives can be used as potential lead compounds for developing novel insecticides and acaricides.

tructure–activity relationship

. Introduction

Significant increase in agricultural productivity over recentecades stems from the successful control of insect pests withynthetic chemical pesticides. However, the rising resistance tovailable synthetic pesticides, combined with their adverse sideffects to humans and to the environment, is driving the search forew alternative pesticides from natural products. Investigation ofatural products from plant, animal and microbial sources, not onlynhances diversity in the search for new prototype insecticides,ut may lead to new market products as the prominent examplesf spinosyns, pyrethroids and nereistoxins underline (Dayan et al.,009; Harmatha and Dinan, 2003; Walter, 2002; Crombie, 1999;osell et al., 2008; Petroski and Stanley, 2009; Pillmoor et al., 1993).articularly, as modern pesticide discovery efforts move forward,he insect-derived substances as an integral part of their chemicalefenses may constitute an important alternative for pest man-gement and very useful models for identifying new pesticides

Dossey, 2010; Meinwald, 2011).

Cantharidin is one of the most famous insect defensive com-ounds secreted by the male blister beetles of Meloidae and

∗ Corresponding author. Tel.: +86 0931 8618795; fax: +86 0931 8915686.E-mail address: yqliu@lzu.edu.cn (Y.-Q. Liu).

1 These authors contributed equally to this work.

ttp://dx.doi.org/10.1016/j.indcrop.2014.02.007926-6690/© 2014 Elsevier B.V. All rights reserved.

© 2014 Elsevier B.V. All rights reserved.

Oedemeridae families (Fig. 1), which has long been used in Chi-nese traditional medicine and is still being used as a folk medicine(Nicholls and Teare, 1954; Wang, 1989). It is a potent and selectiveinhibitor of protein phosphatase 2A (PP2A) that plays an importantrole in control of cell cycle, apoptosis, and cell-fate determina-tion (Sakoff et al., 2002). Recent pharmacologic studies provedthat cantharidin can interfere with the metabolism of nucleic acidsand of proteins in cancer cells, significantly inhibit the growthof various implanted tumor on animal models. It has inhibitoryeffect on primary hepatoma and other carcinomas, such as uter-ine cervix cancer, nasopharyngeal carcinoma, cutaneous cancer,leucemia, etc. (Li et al., 2010; McCluskey et al., 2003; Sagawa et al.,2008). In clinic, cantharidin has been demonstrated particular ther-apeutic efficacy in the treatment of cancer and some refractorydiseases.

Meanwhile, in agrochemical applications, cantharidin wasreported to display potent insecticidal/antifeedant activitiesagainst some economically important insect pests (Carrel andEisner, 1974; Carrel et al., 1986). In China, cantharidin has beenused traditionally to control pests, and it was reported that can-tharidin showed significant biological activities against Nilaparvatalugens, Plutella xylosella, Sogatella furcifera, Pryeria sinica, Spodoptera

frugiperda and T. cinnabarinus, etc. which suggested its potential useas a pesticide in the field (Li et al., 2008; Liu et al., 2010a,b). Previ-ous studies by Zhang et al. demonstrated that cantharidin stronglyinhibited the growth, development and reproduction of P. xylosella

12 M.-J. Wang et al. / Industrial Crops a

Fig. 1. A blister beetle (Epicauda sp., family Meloidae) deploying its typical defensivesecretion of blood (hemolymph) enriched in the blistering agent cantharidin (1).

P

ltlticallfospmpbotdka2

mp2batifici

2

2

aaTdcgu6

13C NMR (100 MHz, CDCl3) ı: 181.4, 180.8, 165.3, 165.8, 130.2 (2C),

hotograph by Aaron T. Dossey. Genus identification by Michael C. Thomas.8

arvae (Zhang, 1998). Subsequently Zhang et al. showed that can-haridin display potent cytotoxic effects to the tested insect cellines in a time- and dose-dependent manner. The DNA fragmenta-ion, activation of caspases and cytochrome c release were observedn both IOZCAS-Spex-II and Sf9 treated with cantharidin. The resultsonfirm the existence of the mitochondrial-dependent pathway ofpoptosis induced by cantharidin in Sf9 and IOZCAS-Spex-II cellines (Zhang et al., 2011; Chen et al., 2008). Furthermore, apoptosis-ike symptoms in insect tissues (midgut, Malpighian tubules, testes,at body and epidermis) due to cantharidin poisoning have beenbserved (Zhang et al., 2003). It also exerted in vivo and in vitroignificant inhibitory effects on protein serine/threonine phos-hatases (PSPs) of P. xylostella, suggesting that its mode of actionay be related to impeding insect protein serine/threonine phos-

hatases activity (Chen et al., 2014). Such findings have providedasic knowledge needed to understand the mechanisms of actionf cantharidin and produced definitive evidence to develop can-haridin as a lead compound and/or insect control agent in theevelopment of biorational pesticides. However, to the best of ournowledge, not much attention has been paid to cantharidin as

starting material for further transformations so far (Sun et al.,013).

As part of an ongoing effort to identify potential pesticideolecules derived from natural products, we have successfully

repared a number of pesticides (Liu et al., 2007, 2008, 2010a,b,011, 2012; Di et al., 2007). Meanwhile, introduction of theioactive acylthiourea groups into the molecule would usu-lly potentiate the biochemical or pharmacological properties ofhe original molecule. Therefore, as an extension to our stud-es among natural products-derived pesticides, in this paper werstly introduced the acylthiourea group to cantharidin via aoupling reaction for their insecticidal/acaricidal activity stud-es.

. Materials and methods

.1. General

Melting points were taken on a Kofler melting point apparatusnd are uncorrected; 1H NMR and 13C NMR spectra were recordedt 400 MHz and 100 MHz on a Bruker AM-400 spectrometer usingMS as reference (Bruker Company, USA). Elemental analyses wereetermined on a Vario El Gmbh elemental analyzer. The syntheticompounds were purified by flash chromatography on Merck silica

el (70–230 mesh). Thin-layer chromatography (TLC) involved these of silica gel plates with a fluorescent indicator (Merck Silica Gel0 F254 0.25 mm thick).

nd Products 55 (2014) 11–18

2.2. Synthesis of cantharidinimide 2

Cantharidin 1 (500 mg, 2.55 mmol) was added to a high pres-sure tube containing 10 mL of dried toluene and ethylenediamine(137.9 mg, 2.3 mmol). Then triethylamine (145 mg, 2.55 mmol) wasadded. The solution was stirred for 2 h at 200 ◦C, then cooled. Thetube cap was opened and the solvent was evaporated, the driedmass was separated by column Chromatography and recrystallizedfrom methanol to give cantharidinmide 2 (450 mg, yield 90%).

2.3. General procedure for cantharidinimide substitutedacylthiourea derivatives 6a–t

Various acids 3a–t (0.63 mmol), and thionyl chloride (10 mL)were placed in a dried CH2Cl2 and refluxed with stirring for 4 h.Then the excessive thionyl chloride was removed under reducedpressure to give a clear solution of corresponding carbonyl chlo-ride 4a–t. Without further purification, the active intermediateacyl chlorides 4a–t was dissolved in acetonitrile (10 mL) and themixture was added dropwise to a suspension of potassium thio-cyanate (0.9 mmol) in acetonitrile (10 mL). After refluxed for 1 h,the reaction mixture was filtered off to give corresponding carbonylisothiocyanate 5a–t, then equimolar quantity of cantharidinmide 2was added, the reaction mixture was stirring for 2 h at room tem-perature and then was concentrated, the residue was purified bychromatography on silica gel using ethyl acetate-petroleum etherto give 6a–t.

2.3.1. N-[(Benzoyl-thioureido)ethyl]cantharidinimide (6a)90% yield, white solid, mp: 157–178 ◦C; 1H NMR (400 MHz,

CDCl3) ı: 1.16 (s, 6H, 2CH3), 1.67–1.81 (m, 4H, 2CH2), 3.88 (dd, 2H,J = 3.8, 5.7 Hz, NCH2), 3.98 (dd, 2H, J = 5.3, 10.7 Hz, CCH2), 4.57 (s,2H, OCH), 7.49 (t, 2H, J = 7.8 Hz, 3′,5′-H), 7.61 (t, 1H, J = 7.4 Hz, 4′-H),7.82 (d, 2H, J = 7.7 Hz, 2′,6′-H), 9.01 (s, 1H, C ONH), 10.77 (s, 1H,C SNH); 13C NMR (100 MHz, CDCl3) ı: 181.4, 180.9, 166.4, 133.4,131.7, 129.0, 127.5, 83.6, 54.0, 43.5, 37.6, 23.6, 12.4. Anal. Calcd.for C20H23N3O4S: C 59.83%, H 5.77%, N 10.47%. Found: C 59.86%, H5.74%, N 10.47%.

2.3.2. N-[(4-Methylbenzoyl-thioureido)ethyl]cantharidinimide(6b)

98% yield, white solid, mp: 154–155 ◦C; 1H NMR (400 MHz,CDCl3) ı: 1.16 (s, 6H, 2CH3), 1.67–1.82 (m, 4H, 2CH2), 2.42 (s, 3H,4′-CH3), 3.87 (t, 2H, J = 5.4 Hz, NCH2), 3.98 (dd, 2H, J = 5.2, 10.6 Hz,CCH2), 4.57(s, 2H, OCH), 7.28 (d, 2H, J = 8.7 Hz, 3′,5′-H), 7.72 (d,2H, J = 8.2 Hz, 2′,6′-H), 8.97 (s, 1H, C ONH), 10.80 (s, 1H, C SNH);13C NMR (100 MHz, CDCl3) ı: 181.4, 181.0, 166.3, 144.4, 129.7,128.9, 127.5, 83.6, 54.0, 43.5, 37.6, 23.6, 21.6, 12.4. Anal. Calcd.for C21H25N3O4S: C 60.70%, H 6.06%, N 10.11%. Found: C 60.73%,H 6.02%, N 10.12%.

2.3.3. N-[(4-Fluorobenzoyl-thioureido)ethyl]cantharidinimide(6c)

93% yield, white solid, mp: 165–167 ◦C; 1H NMR (CDCl3,400 MHz) ı: 1.17 (s, 6H, 2CH3), 1.68–1.81 (m, 4H, 2CH2), 3.89 (dd,2H, J = 3.4, 5.9 Hz, NCH2), 3.99 (dd, 2H, J = 5.2, 10.7 Hz, CCH2), 4.58(t, 2H, J = 2.2 Hz, OCH), 7.19 (t, 2H, J = 8.4 Hz, 3′,5′-H), 7.87 (dd, 2H,J = 5.1, 3.7 Hz, 2′,6′-H), 8.94 (s, 1H, C ONH), 10.72 (s, 1H, C SNH);

127.9 (2C), 116.2 (2C), 83.6, 54.0, 43.6, 37.6, 23.6, 12.4. Anal. Calcdfor C20H22FN3O4S: C 57.27%, H 5.29%, N 10.02%, F 4.53%. Found: C57.29%, H 5.24%, N 10.02%, F 4.51%.

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.3.4. N-[(3-Fluorobenzoyl-thioureido)ethyl]cantharidinimide6d)

82% yield, white solid, mp: 167–169 ◦C; 1H NMR (400 MHz,DCl3) ı: 1.16 (s, 6H, 2CH3), 1.68–1.84 (m, 4H, 2CH2), 3.88 (dd, 2H,

= 3.7, 5.7 Hz, NCH2), 3.98 (dd, 2H, J = 5.2, 10.6 Hz, CCH2), 4.58 (s, 2H,CH), 7.31 (dd, 1H, J = 2.4, 8.2 Hz, 4′-H), 7.48 (dd, 1H, J = 8.2, 13.6 Hz,′-H), 7.58 (t, 2H, J = 6.5 Hz, 2′,6′-H), 8.99 (s, 1H, C ONH), 10.67s, 1H, C SNH); 13C NMR (100 MHz, CDCl3) ı: 181.4, 180.7, 165.1,62.8, 134.0, 130.8, 122.8, 120.6, 115.1, 83.7, 54.0, 43.7, 37.6, 23.6,2.4. Anal. Calcd. for C20H22FN3O4S: C 57.27%, H 5.29%, N 10.02%, F.53%. Found: C 57.26%, H 5.28%, N 10.02%, F 4.52%.

.3.5. N-[(2-Fluorobenzoyl-thioureido)ethyl]cantharidinimide6e)

98% yield, white solid, mp: 143–145 ◦C; 1H NMR (400 MHz,DCl3) ı: 1.17 (s, 6H, 2CH3), 1.68–1.83 (m, 4H, 2CH2), 3.88 (dd,H, J = 3.6, 10.8 Hz, NCH2), 4.00 (dd, 2H, J = 5.3, 10.8 Hz, CCH2), 4.58t, 2H, J = 2.4 Hz, OCH), 7.19 (dd, 1H, J = 8.4, 12.1 Hz, 5′-H), 7.31 (t,H, J = 7.3 Hz, 3′-H), 7.60 (dd, 1H, J = 1.8, 7.3 Hz, 4′-H), 8.06 (dd, 1H,

= 1.7, 7.8 Hz, 6′-H), 9.51 (d, 1H, J = 14.6 Hz, C ONH), 10.72 (s, 1H, SNH); 13C NMR (100 MHz, CDC13) ı: 181.4, 180.7, 162.5, 160.4,35.5, 132.2, 125.2, 119.1, 116.6, 83.7, 54.0, 43.6, 37.7, 23.6, 12.4.nal. Calcd. for C20H22FN3O4S: C 57.27%, H 5.29%, N 10.02%, F 4.53%.ound: C 57.28%, H 5.26%, N 10.02%, F 4.55%.

.3.6. N-[(4-Chlorobenzoyl-thioureido)ethyl]cantharidinimide6f)

97% yield, white solid, mp: 174–176 ◦C; 1H NMR (400 MHz,DCl3) ı: 1.16 (s, 6H, 2CH3), 1.68–1.82 (m, 4H, 2CH2), 3.87 (dd, 2H,

= 3.4, 5.8 Hz, NCH2), 3.98 (dd, 2H, J = 5.1, 10.5 Hz, CCH2), 4.57 (t, 2H, = 2.1 Hz, OCH), 7.47 (d, 2H, J = 8.6 Hz, 2′,6′-H), 7.78 (d, 2H, J = 8.6 Hz,′,5′-H), 8.98 (s, 1H, C ONH), 10.70 (s, 1H, C SNH); 13C NMR100 MHz, CDCl3) ı: 181.4, 180.7, 165.4, 140.0, 130.1, 129.3, 128.9,3.7, 54.0, 43.7, 37.6, 23.6, 12.4. Anal. Calcd. for C20H22ClN3O4S:

55.10%, H 5.09%, N 9.64%, Cl 8.13%. Found: C 55.09%, H 5.09%, N.72%, Cl 8.12%.

.3.7. N-[(3-Chlorobenzoyl-thioureido)ethyl]cantharidinimide6g)

6% yield, Yellow solid, mp: 160–161 ◦C; 1H NMR (400 MHz,DCl3) ı: 1.16 (s, 6H, 2CH3), 1.68–1.82 (m, 4H, 2CH2), 3.88 (dd,H, J = 3.4, 5.9 Hz, NCH2), 3.98 (dd, 2H, J = 5.1, 10.6 Hz, CCH2), 4.58t, 2H, J = 2.2 Hz, OCH), 7.42 (t, 1H, J = 7.9 Hz, 5′-H), 7.57 (d, 1H,

= 8.0 Hz, 4′-H), 7.68 (dd, 1H, J = 7.8, 0.96 Hz, 6′-H), 7.84 (t, 1H, = 1.8 Hz, 2′-H), 8.98 (s, 1H, C ONH), 10.66 (s, 1H, C SNH); 13CMR (100 MHz, CDCl3) ı: 181.4, 180.6, 165.1, 135.4, 133.6, 133.4,30.3, 128.0, 125.3, 83.7, 54.0, 43.7, 37.5, 23.6, 12.4. Anal. Calcd.or C20H22ClN3O4S: C 55.10%, H 5.09%, N 9.64%, Cl 8.13%. Found: C5.11%, H 5.09%, N 9.64%, Cl 8.14%.

.3.8. N-[(4-Bromobenzoyl-thioureido)ethyl]cantharidinimide6h)

48% yield, white solid, mp: 179–180 ◦C; 1H NMR (400 MHz,DCl3) ı: 1.16 (s, 6H, 2CH3), 1.68–1.82 (m, 4H, 2CH2), 3.88 (dd, 2H,

= 3.4, 5.9 Hz, NCH2), 3.98 (dd, 2H, J = 5.1, 10.6 Hz, CCH2), 4.57 (t, 2H, = 2.3 Hz, OCH), 7.64 (d, 2H, J = 8.6 Hz, 2′,6′-H), 7.70 (d, 2H, J = 8.6 Hz,′,5′-H), 8.98 (s, 1H, C ONH), 10.69 (s, 1H, C SNH); 13C NMR100 MHz, CDCl3) ı: 181.4, 180.7, 165.5, 132.3, 130.6, 129.0, 128.6,3.7, 54.0, 43.7, 37.6, 23.6, 12.4. Anal. Calcd. for C20H22BrN3O4S: C0.01%, H 4.62%, N 8.75%, Br 16.63%. Found: C 50.01%, H 4.64%, N.74%, Br 16.62%.

.3.9. N-[(3-Bromobenzoyl-thioureido)ethyl]cantharidinimide6i)

78% yield, white solid, mp: 145–147 ◦C; 1H NMR (400 MHz,DCl3) ı: 1.16 (s, 6H, 2CH3), 1.68–1.83 (m, 4H, 2CH2), 3.88 (dd,

nd Products 55 (2014) 11–18 13

2H, J = 3.2, 6.0 Hz, NCH2), 3.98 (dd, 2H, J = 5.2, 10.6 Hz, CCH2), 4.58(t, 2H, J = 2.6 Hz, OCH), 7.37 (t, 1H, J = 7.9 Hz, 5′-H), 7.73 (d, 2H,J = 7.9 Hz, 4′,6′-H), 7.99 (s, 1H, 2′-H), 8.99 (s, 1H, C ONH), 10.65(s, 1H, C SNH); 13C NMR (100 MHz, CDCl3) ı: 181.4, 180.6, 165.0,136.3, 133.7, 130.9, 130.5, 125.8, 123.2, 83.7, 54.0, 43.7, 37.5, 23.6,12.4. Anal. Calcd. for C20H22BrN3O4S: C 50.01%, H 4.62%, N 8.75%,Br 16.63%. Found: C 50.02%, H 4.67%, N 8.73%, Br 16.60%.

2.3.10. N-[(4-Acetylbenzoyl-thioureido)ethyl]cantharidinimide(6j)

71% yield, yellow solid, mp: 71–72 ◦C; 1H NMR (400 MHz, CDCl3)ı: 1.15 (s, 6H, 2CH3), 1.67–1.80 (m, 4H, 2CH2), 2.64 (s, 3H, C OCH3),3.87 (dd, 2H, J = 3.5, 5.8 Hz, NCH2), 3.97 (dd, 2H, J = 5.0, 10.5 Hz,CCH2), 4.56 (t, 2H, J = 2.2 Hz, OCH), 7.92 (d, 2H, J = 8.4 Hz, 3′,5′-H),8.03 (d, 2H, J = 8.4 Hz, 2′,6′-H), 9.17 (s, 1H, C ONH), 10.69 (s, 1H,C SNH); 13C NMR (100 MHz, CDCl3) ı: 196.9, 181.4, 180.6, 165.6,140.5, 135.5, 128.7, 127.9, 83.6, 54.0, 43.6, 37.5, 26.8, 23.6, 12.3.Anal. Calcd. for C22H25N3O5S: C 59.58%, H 5.68%, N 9.47%. Found: C59.56%, H 5.67%, N 9.47%.

2.3.11. N-[(3-Acetylbenzoyl-thioureido)ethyl]cantharidinimide(6k)

85% yield, yellow solid, mp: 59–60 ◦C; 1H NMR (400 MHz, CDCl3)ı: 1.16 (s, 6H, 2CH3), 1.68–1.82 (m, 4H, 2CH2), 2.66 (s, 3H, C OCH3),3.88 (dd, 2H, J = 3.5, 5.9 Hz, NCH2), 3.99 (dd, 2H, J = 5.2, 10.6 Hz,CCH2), 4.57 (t, 2H, J = 2.3 Hz, OCH), 7.62 (t, 1H, J = 7.8 Hz, 5′-H), 8.04(d, 1H, J = 7.8 Hz, 4′-H), 8.17 (d, 1H, J = 7.8 Hz, 6′-H), 8.40 (s, 1H, 2′-H), 9.17 (s, 1H, C ONH), 10.72 (s, 1H, C SNH);13C NMR (100 MHz,CDCl3) ı: 196.6, 181.4, 180.7, 165.7, 137.7, 132.8, 132.4, 131.7,129.4, 127.3, 83.7, 54.0, 43.7, 37.6, 26.7, 23.6, 12.4. Anal. Calcd. forC22H25N3O5S: C 59.58%, H 5.68%, N 9.47%. Found: C 59.58%, H 5.68%,N 9.47%.

2.3.12. N-[(4-Nitrobenzoyl-thioureido)ethyl]cantharidinimide(6l)

76% yield, yellow solid, mp: 161–163 ◦C; 1H NMR (400 MHz,CDCl3) ı: 1.16 (s, 1H, 2CH3), 1.68–1.83 (m, 4H, 2CH2), 3.88 (dd, 2H,J = 3.2, 5.8 Hz, NCH2), 3.98 (dd, 2H, J = 5.0 Hz, 10.4 Hz, CCH2), 4.58(t, 2H, J = 2.2 Hz, OCH), 8.03 (d, 2H, J = 8.8 Hz, 3′,5′-H), 8.34 (d, 2H,J = 8.8 Hz, 2′,6′-H), 9.18 (s, 1H, C ONH), 10.59 (s, 1H, C SNH); 13CNMR (100 MHz, CDCl3) ı: 181.4, 180.3, 164.6, 150.6, 137.3, 128.9,124.1, 83.7, 54.0, 43.8, 37.5, 23.6, 12.4. Anal. Calcd. for C20H22N4O6S:C 53.80%, H 4.97%, N 12.55%. Found: C 53.84%, H 4.96%, N 12.54%.

2.3.13. N-[(3-Nitrobenzoyl-thioureido)ethyl]cantharidinimide(6m)

50% yield, yellow solid, mp: 88–89 ◦C; 1H NMR (400 MHz, CDCl3)ı: 1.17 (s, 6H, 2CH3), 1.69–1.83 (m, 4H, 2CH2), 3.88 (dd, 2H, J = 5.7,4.6 Hz, NCH2), 3.98 (dd, 2H, J = 5.0, 10.3 Hz, CCH2), 4.58 (s, 2H, OCH),7.73 (t, 1H, J = 8.0 Hz, 5′-H), 8.18 (d, 1H, J = 7.8 Hz, 6′-H), 8.46 (d, 1H,J = 8.1 Hz, 4′-H), 8.71 (s, 1H, 2′-H), 9.26 (s, 1H, C ONH), 10.62 (s, 1H,C SNH); 13C NMR (100 MHz, CDCl3) ı: 181.5, 180.4, 164.3, 148.5,133.6, 133.1, 130.3, 127.7, 123.0, 83.7, 54.0, 43.8, 37.5, 23.6, 12.4.Anal. Calcd. for C20H22N4O6S: C 53.80%, H 4.97%, N 12.55%. Found:C 53.81%, H 4.96%, N 12.57%.

2.3.14. N-[Nicotinoyl-thioureido)ethyl]cantharidinimide (6n)40% yield, yellow solid, mp: 163–165 ◦C; 1H NMR (400 MHz,

DMSO-d6) ı: 1.09 (s, 6H, 2CH3), 1.50 (d, 2H, J = 8.2 Hz, CH2), 1.78(d, 2H, J = 7.6 Hz, CH2), 3.70 (t, 2H, J = 5.7 Hz, NCH2), 3.88 (dd, 2H,J = 5.8, 10.9 Hz, CCH2), 4.38 (s, 2H, OCH), 7.53 (dd, 1H, J = 4.9, 7.8 Hz,

5′-H), 8.21 (d, 1H, J = 8.0 Hz, 6′-H), 8.77 (d, 1H, J = 4.1 Hz, 4′-H), 8.97(s, 1H, 2′-H), 10.75 (t, 1H, J = 5.5 Hz, C ONH), 11.59 (s, 1H, C SNH);13C NMR (100 MHz, DMSO-d6) ı: 181.5, 181.3, 166.9, 153.6, 149.6,136.7, 128.8, 123.8, 83.5, 54.0, 42.9, 37.8, 23.8, 12.3. Anal. Calcd.

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or C19H22N4O4S: C 56.70%, H 5.51%, N 13.92%. Found: C 56.68%, H.50%, N 13.96%.

.3.15. N-[2-Chloronicotinoyl-thioureido)ethyl]cantharidinimide6o)

62% yield, yellow solid, mp: 252–254 ◦C; 1H NMR (400 MHz,CD3)2S O) ı: 1.10 (s, 6H, 2CH3), 1.52 (m, 2H, CH2), 1.80 (d, 2H,

= 7.6 Hz, CH2), 3.72 (dd, 2H, J = 4.0, 6.0 Hz, NCH2), 3.88 (dd, 2H, = 5.2, 10.0 Hz, CCH2), 4.41 (t, 2H, J = 2.4 Hz, OCH), 7.52 (dd, 1H, = 4.8, 7.6 Hz, 5′-H), 7.95 (dd, 1H, J = 2.0, 7.6 Hz, 6′-H), 8.52 (dd, 1H, = 1.6, 4.8 Hz, 4′-H), 10.50 (s, 1H, C ONH), 11.77 (s, 1H, C SNH);3C NMR (100 MHz, CDCl3) ı: 181.4, 167.9, 158.2, 155.0, 150.8,39.1, 124.8, 120.5, 83.7, 54.4, 42.0, 36.1, 23.6, 12.5. Anal. Calcd.or C19H21ClN4O4S: C 52.23%, H 4.84%, N 12.82%, Cl 8.11%. Found:

52.26%, H 4.86%, N 12.84%, Cl 8.12%.

.3.16. N-[4-Chloronicotinoyl-thioureido)ethyl]cantharidinimide6p)

92% yield, yellow solid, mp: 154–156 ◦C; 1H NMR (400 MHz,DCl3) ı: 1.15 (s, 6H, 2CH3), 1.67–1.82 (m, 4H, 2CH2), 3.87 (dd, 2H,

= 3.2, 5.6 Hz, NCH2), 3.96 (dd, 2H, J = 5.0, 10.3 Hz, CCH2), 4.56 (t,H, J = 2.2 Hz, OCH), 7.46 (d, 1H, J = 8.4 Hz, 5′-H), 8.11 (dd, 1H, J = 2.4,.4 Hz, 6′-H), 8.87 (d, 1H, J = 2.4 Hz, 2′-H), 9.21 (s, 1H, C ONH), 10.58s, 1H, C SNH);13C NMR (100 MHz, CDCl3) ı:181.5, 180.4, 164.0,56.1, 149.1, 137.9, 126.7, 124.5, 83.7, 54.0, 43.8, 37.5, 23.6, 12.4.nal. Calcd. for C19H21ClN4O4S: C 52.23%, H 4.84%, N 12.82%, Cl.11%. Found: C 52.24%, H 4.84%, N 12.82%, Cl 8.11%.

.3.17. N-[5-Bromonicotinoyl-thioureido)ethyl]cantharidinimide6q)

84% yield, yellow solid, mp: 88–89 ◦C; 1H NMR (400 MHz, CDCl3): 1.16 (s, 6H, 2CH3), 1.68–1.83 (m, 4H, 2CH2), 3.88 (dd, 2H, J = 3.0,0.1 Hz, NCH2), 3.97 (m, 2H, CCH2), 4.58 (t, 2H, J = 2.2 Hz, OCH),.31 (t, 1H, J = 2.0 Hz, 6′-H), 8.87 (d, 1H, J = 2.1 Hz, 4′-H), 8.98 (d,H, J = 1.8 Hz, 2′-H), 9.28 (s, 1H, C ONH), 10.55 (s, 1H, C SNH);3C NMR (100 MHz, CDCl3) ı: 181.5, 180.3, 163.6, 154.9, 146.5,38.0, 129.2, 121.0, 83.7, 54.0, 43.8, 37.5, 23.6, 12.4. Anal. Calcd.or C19H21BrN4O4S: C 47.71%, H 4.40%, N 11.64%, Br 16.60%. Found:

47.72%, H 4.44%, N 11.62%, Br 16.60%.

.3.18. N-[Furan-2-carbonyl-thioureido)ethyl]cantharidinimide6r)

96% yield, yellow solid, mp: 191–193 ◦C; 1H NMR (400 MHz,DCl3) ı: 1.15 (s, 6H, 2CH3), 1.67–1.80 (m, 4H, 2CH2), 3.86 (dd,H, J = 3.3, 5.9 Hz, NCH2), 3.96 (dd, 2H, J = 5.2, 10.6 Hz, CCH2), 4.57t, 2H, J = 2.4 Hz, OCH), 6.58 (dd, 1H, J = 1.6, 3.5 Hz, 4′-H), 7.32 (d, 1H,

= 3.6 Hz, 3′-H), 7.57 (d, 1H, J = 0.64 Hz, 5′-H), 9.07 (s, 1H, C ONH),0.50 (s, 1H, C SNH); 13C NMR (100 MHz, CDCl3) ı: 181.4, 180.5,56.3, 146.0, 145.0, 118.6, 113.2, 83.6, 54.0, 43.6, 37.6, 23.6, 12.3.nal. Calcd. for C18H21N3O5S: C 55.23%, H 5.41%, N 10.73%. Found:

55.26%, H 5.40%, N 10.72%.

.3.19.-[Thiophene-2carbonyl-thioureido)ethyl]cantharidinimide (6s)

70% yield, yellow solid, mp: 184–186 ◦C; 1H NMR (400 MHz,DCl3) ı: 1.16 (s, 6H, 2CH3), 1.68–1.82 (m, 4H, 2CH2), 3.87 (dd,H, J = 3.5, 5.9 Hz, NCH2), 3.97 (dd, 2H, J = 5.1, 10.6 Hz, CCH2), 4.57t, 2H, J = 2.3 Hz, OCH), 7.14 (t, 1H, J = 4.3 Hz, 4′-H), 7.67 (m, 2H, 3′,5′-), 8.86 (s, 1H, C ONH), 10.58 (s, 1H, C SNH); 13C NMR (100 MHz,DCl3) ı: 181.4, 180.6, 160.6, 136.1, 133.8, 130.6, 128.3, 83.7, 54.0,3.6, 37.6, 23.6, 12.4. Anal. Calcd. for C18H21N3O4S2: C 53.05%, H.19%, N 10.31%. Found: C 53.04%, H 5.19%, N 10.33%.

.3.20. N-[2-Naphthoyl-thioureido)ethyl]cantharidinimide (6t)98% yield, white solid, mp: 152–154 ◦C; 1H NMR (400 MHz,

DCl3) ı: 1.18 (s, 6H, 2CH3), 1.68–1.83 (m, 4H, 2CH2), 3.91 (dd, 2H,

nd Products 55 (2014) 11–18

J = 3.5, 6.0 Hz, NCH2), 4.02 (dd, 2H, J = 5.2, 10.8 Hz, CCH2), 4.60 (t, 2H,J = 2.5 Hz, OCH), 7.61 (m, 2H, 5′,6′-H), 7.83–7.96 (m, 4H, 3′,4′,7′,8′-H), 8.36 (s, 1H, 2′-H), 9.16 (s, 1H, C ONH), 10.84 (s, 1H, C SNH); 13CNMR (100 MHz, CDCl3) ı: 181.4, 181.0, 166.5, 135.5, 132.3, 129.2,129.1, 128.8, 127.8, 127.3, 123.1, 83.7, 54.0, 43.6, 37.6, 23.6, 12.4.Anal. Calcd. for C24H25N3O4S: C 63.84%, H 5.58%, N 9.31%. Found: C63.84%, H 5.56%, N 9.33%.

2.4. Biological activity

All bioassays were performed on representative test organismsreared in the laboratory. The bioassay was repeated at 25 ± 2 ◦Caccording to statistical requirements. Assessments were made on adead/alive basis, and mortality rates were corrected using Abbott’sformula (Abbott, 1925). Evaluations are based on a percentage scaleof 0–100, in which 0, no activity and 100, total kill. The deviationof values was ±5%. Probit analysis was used to determine lethalconcentrations of 50% (LC50) by using the SPSS program, version13.0. All bioassays result is summarized in Table 1.

2.4.1. Acaricidal assay against T. cinnabarinusThe acaricidal activities of all of these compounds 2, 6a–t and

cantharidin (1) were evaluated using the slide immersion methodrecommended by FAO (Wang et al., 2006). Each test sample wasprepared in acetone at a concentration of 2 g/L and diluted tothe required concentration with distilled water containing TW-80.Thirty adult spider mites were fixed dorsally to a strip of double-sided tape attached to the slide by using a small brush. The slidewas immersed and shaken for 3 s in the diluted solution of the testcompound. After the excessive solution was removed, the treatedslides with the mites were kept at 25 ± 2 ◦C in a Petri dish with moistfilter paper. The number of the dead mites was recorded 24 h aftertreatment. Each treatment was replicated with triplicate experi-ments and each replicate involved 30 adult mites. Control groupswere tested with acetone only.

2.4.2. Insecticidal assay against B. brassicaeThe insecticidal activities of all of these compounds 2, 6a–t

and cantharidin (1) against B. brassicae were evaluated accordingto the reported procedure (Zhao et al., 2010). Each test samplewas prepared in acetone at a concentration of 2 g/L and diluted tothe required concentration with distilled water containing TW-80.Thirty healthy adult aphid were dipped into the diluted solutionsof these compounds for 5 s, superfluous fluid was removed, and theaphid were placed in an air-conditioned room. Mortality was cal-culated 24 h after treatment. Each treatment was performed withtriplicate experiments. Control groups were tested with acetoneonly.

2.5. X-ray crystallography

X-ray quality crystal of 6c was obtained from the ethyl acetatesolution after 2 d. The crystal structure of compound 6c (Fig. 2)was determined, and X-ray intensity data were recorded on aBruker APEX II diffractometer using graphite monochromatedMo-Ka radiation (k = 0.71073 A). A total of 6141 reflections weremeasured, of which 3714 were unique (Rint = 0.0208) in the rangeof 3.10 < � < 25.68′ (h, −11 to 10; k, −5 to 8; l, −20 to 36),and 2586 observed reflections with I > 2�(I) were used in therefinement on F2. The structure was solved by direct methodwith the SHELXTL-97 program. All of the non-H atoms wererefined anisotropically by full-matrix least-squares to give the final

R = 0.0495 and WR = 0.1379. All hydrogen atoms were computedand refined using a riding model. The atomic coordinates for 6c havebeen deposited at the Cambridge Crystallographic Data Center.CCDC-910119 contains the supplementary crystallographic data

M.-J. Wang et al. / Industrial Crops and Products 55 (2014) 11–18 15

Table 1Biological activity of compounds 6a–t against Tetranychus cinnabarinus and Brevicoryne brassicae.

Compd Tetranychus cinnabarinus Brevicoryne brassicae

Mortality (%) in vivo at1day concn (g/L)

LC50 (mmol/L) (95% CI) Mortality (%) in vivo at1day concn (g/L)

LC50 (mmol/L) (95% CI)

0.5 1 0.5 1

6a 66.7 80.0 1.087 (0.718–1.499) 16.7 30.0 3.691 (2.798–4.799)6b 36.7 80.0 1.624 (1.324–1.813) 30.0 53.3 3.982 (2.703–5.065)6c 50.0 56.7 1.369 (1.082–1.519) 10.0 36.7 3.742 (2.817–4.674)6d 26.7 66.7 1.718 (1.475–2.032) 6.7 16.7 3.339 (2.708–4.175)6e 70.0 76.7 0.839 (0.582–1.281) 10.0 16.7 5.012 (3.142–7.210)6f 70.0 76.7 0.945 (0.448–1.511) 10.0 40.0 4.487 (3.013–6.336)6g 66.7 76.7 0.757 (0.354–1.180) 10.0 26.7 3.125 (2.521–3.969)6h 63.3 73.3 0.814 (0.429–1.278) 10.0 26.7 4.356 (2.624–6.134)6i 80.0 86.7 0.703 (0.362–1.142) 16.7 30.0 3.692 (2.465–5.013)6j 56.7 70.0 1.482 (1.118–1.234) 46.7 63.3 2.878 (2.017–3.613)6k 43.3 80.0 1.722 (1.350–2.273) 23.3 33.3 4.273 (2.591–5.747)6l 43.3 80.0 1.552 (1.185–1.977) 23.3 50.0 2.823 (1.960–3.991)6m 60.0 66.7 1.144 (0.745–1.580) 13.3 20.0 3.364 (2.657–3.873)6n 13.3 36.7 2.934 (2.448–3.447) 16.7 30.0 4.936 (3.160–5.289)6o 13.3 40.0 2.267 (1.879–2.646) 36.7 43.3 3.346 (2.461–4.315)6p 50.0 66.7 1.572 (1.284–1.802) 30.0 33.3 3.213 (2.448–4.060)6q 40.0 76.7 1.406 (1.070–1.789) 26.7 40.0 6.587 (2.270–9.520)6r 60.0 80.0 1.071 (0.542–1.631) 13.3 26.7 4.772 (3.196–6.296)6s 43.3 46.7 2.059 (1.653–2.466) 23.3 30.0 8.851 (2.124–13.877)

22)

41)

01)

fCR

3

3

dcma

6t 43.3 66.7 1.368 (0.940–1.92 50.0 80.0 2.896 (2.366–3.61 83.3 100 0.695 (0.091–1.1

or this paper. These data can be obtained free of charge from Theambridge Crystallographic Data Center via CCDC CIF Depositoryequest Form for data published from 1994.

. Results and discussion

.1. Chemistry

The synthetic chemical routes to the intermediates canthari-

inimide 2 and various acyl isothiocyanates 5a–t and the targetompounds 6a–t are depicted in Scheme 1. Firstly, the key startingaterial cantharidinimide 2 was synthesized in excellent yield by

modified Gabriel synthesis commencing from isolated available

Fig. 2. Molecular structur

30.0 36.7 3.087 (2.207–4.186)26.7 40.0 5.593 (4.305–6.978)53.3 83.3 2.324 (1.411–3.559)

cantharidin 1. The syntheses of key intermediates 5a–t wereconducted according to the following procedures. Briefly, commer-cially available acids 3a–t were firstly reacted with thionyl chloridein dry CH2Cl2, which afforded the active intermediate acyl chlorides4a–t. Subsequent treatment of 4a–t with potassium thiocyanate inCH3CN to provide the corresponding thioisocyanates 5a–t, whichwere used for further reactions in situ to prevent their decom-position. Nextly, the desired compounds 6a–t were obtained inthe yields of 40–98% by coupling corresponding thioisocyanates

5a–t with 2 in dry acetonitrile. All synthesized target compounds6a–t were purified by column chromatography and their struc-tures were characterized by mp, 1H NMR, 13C NMR and elementalanalyses.

e of compound 6c.

16 M.-J. Wang et al. / Industrial Crops and Products 55 (2014) 11–18

urea

3

6Tsm

cT0lFdpc6mtFcatLcr4cT

Scheme 1. Synthesis of acylthio

.2. Biological activity

Based on the methodology in Scheme 1, with twenty derivativesa–6t in hand, we next examined their biological activities against. cinnabarinus and B. brassicae, cantharidin was tested under theame conditions as a comparison compound. The results were sum-arized in Table 1.As illustrated in Table 1, all of these compounds 2, 6a–t and

antharidin (1) exhibited significant biological activity against. cinnabarinus and B. brassicae, with LC50 values ranging from.388 to 8.851 mmol/L, indicating that they may be a potential

ead compound for developing novel insecticides and acaricides.or example, when pests were fed with the diet containing 1 g/Liet, the corrected mortality rates of most of compounds showedromising biological activities against T. cinnabarinus and B. brassi-ae after 1 day. Even at 0.5 g/L diet, compounds 6a, 6e, 6f, 6g, 6h, andi still remain significant acaricidal activities against showed >60%ortality against T. cinnabarinus, as opposed to <20% mortality of

hese compounds against B. brassicae at the same concentration.rom the above results, we assumed that mite pests are sus-eptible to cantharidin-type derivatives. The biological selectivitygainst T. cinnabarinus may be the most important characteris-ic property of cantharidin-related derivatives. For example, TheC50 values of compounds 6a, 6e, 6f, 6g, 6h, and 6i against T.innabarinus were 1.087, 0.839, 0.945, 0.757, 0.814, 0.703 mmol/L,

espectively, whereas the LC50 values B. brassicae are 3.691, 5.012,.487, 3.125, 4.356, 3.692 mmol/L, respectively. As seen in Table 1,antharidin itself exhibited a remarkable biological effect against. cinnabarinus and B. brassicae. However, the introduction of the

derivatives of cantharidin 6a–t.

acyl isothiocyanate groups into cantharidin skeleton substantiallyreduced the biological potency. A possible explanation for loweractivity of these derivatives is that acyl isothiocyanate groupswould affect the conjugated system of cantharidin and either ofthe evens would influence their binding manners with enzyme andcause the reduction of activity, and the reason is not clear to ourcurrent knowledge and has to be subject of further investigations.

3.2.1. Acaricidal activity against T. cinnabarinusAs indicated in Table 1, all new compounds exhibited potent

acaricidal activity against T. cinnabarinus, with LC50 values rang-ing from 0.695 to 2.934 mmol/L. Most of these derivatives showedsignificant acaricidal activities against T. cinnabarinus, althoughsomewhat lower than that of cantharidin. In particular, analogs6i and 6g exhibited the greatest acaricidal activity against T.cinnabarinus in all derivatives we prepared. The activities of thesecompounds in Table 1 varied drastically, depending upon the typesand patterns of substitution on cantharidin skeleton. For the effectof substituents at phenyl ring, it was observed that compoundswith electron-withdrawing is favorable for high activities fromdata analysis present, the introduction of electron-withdrawinggroups (6c–6j) as R resulted in higher acaricidal potency thanthe corresponding analog with electron-donating group (6b). Forinstance, introducing methyl substituent at the 4-position of ben-zene generated analog 6b with decreased activity as compound

6b without any substituent on benzene. The introduction of flu-oro (6c–6e), chloro (6f, 6g), bromo (6h, 6i), acetyl (6j, 6k) groupsinto the 4-position of benzene significantly enhanced the insec-ticidal activities of these compounds compared with compound

rops a

6nwptepiogspctccds

3

csaccogfi

ait2taaaftsgft9TadMCtYbepsacctTbtsht

M.-J. Wang et al. / Industrial C

a. Unexpectedly, the activity was found to decrease when aitro group was introduced at the 4-position of benzene (6l, 6m),hereas it is still higher than 6a. For the effect of substitutedosition (compounds 6c–6e), it was observed that the introduc-ion of a fluro group at 2-position was beneficial for activities. Forxample, compounds 6e displayed higher activity than that of com-ounds 6c and 6d. These data showed that efficacy is strongly

nfluenced by the nature of the substitutents and their positionn the phenyl ring. Disappointingly, introduction of heterocyclicroups into cantharidin skeleton to generated analogues (6n–6s)howed decreasing tendency in acaricidal activity. For instance, theydrine-containing compounds (6n, 6o and 6p), furan or thiopheneontaining compounds (6r, 6s) gave obviously lower activities thanhe corresponding benzene-containing compounds. These resultslearly underlined the acaricidal difference could be ascribed to aombination of factors, like nature of the substitutes (which mayepend on the size of substitutes, electronic characteristics of sub-titutes, and other factors) or by a different interaction at the site.

.2.2. Insecticidal activity against B. brassicaeAs to the insecticidal activities, from Table 1, all of the target

ompounds showed moderate insecticidal activities against B. bras-icae. For example, all of these compounds displayed <50% activitygainst B. brassicae at 500 mg L−1, they exhibited lower insecti-idal activities against B. brassicae than those of the prototypicalompound cantharidin 1. The different insecticidal activity rangesf compounds 6a–t indicated that variation of acyl isothiocyanateroups at cantharidin skeleton markedly affected the activity pro-les of this compound class.

Overall, although the insecticidal activity of natural cantharidingainst some important insect pests has been well determined,ts in vivo insecticidal effects and mode of action has also inves-igated (Li et al., 2008; Liu et al., 2010a,b; Zhang, 1998; Zhang et al.,003; Chen et al., 2014), however, few attempts have been madeo explore cantharidin-modified derivatives and their insecticidal-ctivity relationships, more recently, two series of cantharidinnhydride-modified derivatives containing alkyl and aryl groupst 9-position have been synthesized, and most of compounds wereound to display similar or less potent activity against P. xylostellahan cantharidin (Sun et al., 2013). In contrast to these results, ourtudy herein showed that most of the target compounds possessedood-to-excellent activities against two insect species, but gave dif-erent structure–activity relationships for each species, suggestinghe possibility of further optimizing cantharidin through rational at-position modifications. Particularly, significant activity toward. cinnabarinus indicated a promising future for these derivativess acaricidal leads. Additionally, cantharidin, in the form of theried body of the Chinese blister beetles: Mylabris phalerata orylabris cichorii has been used by the Chinese as a traditional

hinese medicine for malignancy, steroidogenesis and antivirusreatments for the past 2000 years (Wang, 1989; Wang et al., 2000;u et al., 2001). Commercial preparations of cantharidin also haveeen commonly used for years. More recently, investigation by Cuit al. demonstrated that cantharidin emulsifiable concentrate (EC)esticide displayed low toxicity against quail, ladybird beetles andoil microorganisms according to the results of safety evaluationsgainst non-target organisms (Li et al., 2009), which indicated thatantharidin as insecticide led is safe to environment and health andould be used as an attractive precursor compound for the syn-hesis of new analogs to replace traditional chemical pesticides.hough, of course, environmental safety evaluation must be doneefore cantharidin-derived insecticide will be applied for registra-

ion. As we envisioned, comparing with these findings, althoughafety evaluation of cantharidin against some nontarget organismsad not been determined at the moment, in this study, some deriva-ives showed significant inhibitory activity against B. brassicae and

nd Products 55 (2014) 11–18 17

T. cinnabarinus, suggesting the development of cantharidin-derivedanalogs which could be used in plant protection is conceivable.

4. Conclusion

In conclusion, a series of novel acylthiourea derivatives of can-tharidin were designed, synthesized and their structures werecharacterized by mp, 1H NMR, 13C NMR and elemental analyses.The bioactivities were evaluated against T. cinnabarinus and B.brassicae. The results of bioassays indicated that most of the tar-get compounds possessed excellent acaricidal activities against T.cinnabarinus and exhibited moderate insecticidal activities againstB. brassicae. In particular, analogs 6i and 6g exhibited the greatestacaricidal activity against T. cinnabarinus in all derivatives we pre-pared. SARs indicated that variations of acyl isothiocyanate groupsin the cantharidin skeleton markedly affected the activity profilesof this compound class, and some important SAR information hasbeen revealed from it. The present work demonstrates that thecantharidin derivatives can be used as possible lead compoundsfor developing novel insecticides and acaricides. Further structuraloptimization and detailed bioassays of these novel compounds arewell under way.

Acknowledgments

This work was financially supported by the National Natural Sci-ence Foundation of China (30800720, 31371975); the Post-DoctorResearch Foundation (20090450142); the Fundamental ResearchFunds for the Central Universities(lzujbky-2013-69), as well as theFoundation of Priority forestry Disciplines in Zhejiang A&F Univer-sity (KF201325).

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