Synthesis, Characterization and Insecticidal Activity against Cotton Leaf Worm of New Heterocyclics Which Scaffold on Hydrazide-Hydrazone Derivative

Abstract A new hydrazide-hydrazone has been designed and synthesized by combining p-aminoacetophenone with cyanoacetohydrazide followed by acetylation. Afterwards, it was utilized as scaffold to synthesize different arylidenes, heteroarylidenes and heterocycles. The structures of these compounds were proved clearly based on their spectral and analytical data. The insecticidal activity for the newly synthesized compounds was tested against cotton leaf worm (Spodoptera littoralis). The obtained data indicated that, 2,4-dichlorobenzylidenecyanoacetohydrazide derivative 14c is the most potent one after methoxyfenozide (LC50 = 51.75 μg/ml) where its LC50 was (79.73 μg/ml). Interestingly, we recommended to use arylidene derivative 14c in the field of pest control after its formulation. Graphical Abstract


Introduction
Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae), the cotton leaf worm, is the most frequent, severe, and devastative pest, it attacks a wide range of ornamentals and commercial crops such as cotton, clover, maize, peanut, and soybean as well as other vegetable crops all over the world. [1][2][3][4][5][6] The insect causes significant damage by feeding on leaves, fruiting points, flower buds, and, on rare occasions, bolls. While feeding these, they also feed on other sections of the plant, producing varying degrees of harm depending on plant diversity. 7 Infestation usually results in total defoliation and leaf eating. Plant development is hampered by the larvae's destruction of growth points and blooms, as well as the hollowing out of seed bolls, which causes them to wilt and drop. 8,9 There are many tools to overcome the dangerous effect of this pest such as hand-picking of egg patches by children, 10 and physical means (Ultraviolet light 11 and Gamma irradiation 12 ). Moreover, cultivating some resistant plant varieties and increasing agricultural culture and phytosanitary measures are also consider as modern tools for controlling cotton leaf worm (S. littoralis). [13][14][15] In addition to uses of entomopathogen-based insecticides such as Bacillus thuringiensis or baculoviruses have been registered as biopesticides and used in IPM programs of cotton leaf worm (S. littoralis). [16][17][18] Although, the existence of different controlling tools for this pest insecticides application demonstrate a main tactic to tolerate the pest and safeguard crop yield. So, several insecticides were used to control (S. littoralis) in Egypt. 19 However, excessive usage of insecticides has demonstrated adverse impacts starting from evolution of pest impedance strain to major insecticide classes, [20][21][22] toxicity to non-target organism and natural enemy and environmental pollution as well as demise of pest caused by high application rate of insecticides. 23,24 So the harmful effect of chemical insecticides on the environment and human health has promoted the search for new chemical compound can be used as an active substance for controlling this pest.
Recently, hydrazide-hydrazones have gained great importance due to their diverse biological properties including antibacterial, antifungal, anticonvulsant, anti-inflammatory, antimalarial and antituberculosis activities, [25][26][27][28][29][30] and they were also reported to be valuable in medicinal chemistry as protein synthesis as peptide mimetic, [31][32][33] drug isoniazid, and in organic chemistry as candidates for pharmaceuticals 34,35 and insecticides. 36 In continuation of our previous work, [37][38][39][40][41][42][43][44][45][46] and as a part of our program intensified on the synthesis of some valuable heterocyclic compounds and with the aim of obtaining novel hydrazide-hydrazones possess a wide spectrum of biological applications, we report herein, synthesis of a new hydrazide-hydrazone as scaffold to synthesize a different arylidenes and heterocycles such as iminochromene, indolinone, thiazole, pyrazole and pyridinone derivatives and evaluation of their insecticidal activity against cotton leaf worm (Spodoptera littoralis).
The chemical structures of compounds 3 and 4 was ascertained by spectroscopic data. The IR spectra of compounds 3 and 4 displayed characteristic bands corresponding to NH, C ¼ O, C ¼ N and CN functionalities. Moreover, the 1 H NMR spectrum of 3 revealed two doublet peaks at 7.50 and 6.54 ppm with J ¼ 8.7 Hz corresponding to four aromatic protons, broad singlet peak at 5.44 ppm exchangeable with D 2 O corresponding to NH 2 protons, and six singlet peaks; three singlet peaks at 10.73, 4.14 and 2.12 ppm (for anti-isomer) and the other three singlet peaks at 10.43, 3.82 and 2.15 ppm (for syn-isomer) corresponding to NH, CH 2 and CH 3 protons, respectively as shown in Figure 1.
Meanwhile, the 1 H NMR spectrum of 4 exhibited the presence of two doublet peaks at 7.74 and 7.60 ppm with J ¼ 8.7 Hz corresponding to four aromatic protons, two singlet peaks at 10.07 (exchangeable with D 2 O) and 2.05 ppm corresponding to NH and CH 3 protons of acetamide moiety, and six singlet peaks; two singlet peaks at 10.96 and 10.61 ppm exchangeable with D 2 O corresponding to NH proton (anti-and syn-isomers, respectively), two singlet peaks at 4.22 and 3.88 ppm corresponding to CH 2 protons (anti-and syn-isomers, respectively) and two singlet peaks at 2.21 and 2.23 ppm corresponding to CH 3 protons (syn-and anti-isomers, respectively) as shown in Figure 2.
From the aforementioned facts, we were rational to explain that cyanoacetamides 3 and 4 existed as a diastereomeric mixture in the ratio 40:60 and 20:80 (syn/anti), respectively as depicted in Figure 3.
From this point, the cyanoacetamide derivative 4 was exploited as scaffold to prepare novel compounds mostly containing heterocycles to assay the insecticidal activity for them against  cotton leaf worm (Spodoptera littoralis). The reactivity of both the nucleophilicity of the active methylene group in basic medium and the electrophilicity of the cyano functionality of cyanoacetamide derivative 4 was examined as depicted in Scheme 2. Reaction of cyanoacetamide derivative 4 with elemental sulfur and phenyl isothiocyanate in the presence of triethyl amine as a base afforded 4-amino-3-phenylthiazole-2(3H)-thione derivative 6, through intracyclization of the intermediate 5 via 1,5-exo-dig cyclization (Scheme 2).
Proving the chemical structure of compound 6 was first based on the disappearance of cyano group from IR spectrum then the appearance of a singlet peak at 7.24 ppm exchangeable with D 2 O compatible with NH 2 protons beside displaying extra five protons in aromatic region corresponding to phenyl ring. Moreover, the 13 C NMR spectrum revealed a peak at 189.9 ppm corresponding to C ¼ S moiety.
Meanwhile, reaction of cyanoacetamide derivative 4 with o-salicylaldehyde and/or 2-hydroxynaphthaldehyde furnished 2-iminochromene and 3-iminobenzochromene derivatives 8 and 11, respectively. Also, the both formed via 1,6-exo-dig cyclization of the intermediate 7 and 10, respectively (Scheme 2). The 1 H NMR spectrum of compound 8 exhibited two singlet peaks one at 9.30 ppm which exchangeable with D 2 O corresponding to imino proton and the other at 8.60 ppm corresponding to C 4 -H (2-iminochromene) . Meanwhile, the 1 H NMR spectrum of compound 11 revealed three singlet peaks exchangeable with D 2 O at 13.53, 10.08 and 9.26 ppm corresponding to 3NH protons, three singlet peaks at 9.19, 2.30 and 2.06 ppm corresponding to  C 1 -H (3-iminobenzochromene) and two methyl protons, and six doublets and two triplet peaks in aromatic region corresponding to ten aromatic protons. Furthermore, all peaks which appeared in 13 C NMR spectrum are keeping with their foreseeable product.
Afterwards, the nucleophilicity of the imino group of iminochromene and iminobenzochromene derivatives 8 and 11 was tested by reaction of compounds 8 and/or 11 individually with benzoyl chloride in dry pyridine, which afforded N-benzoyl derivatives 9 and/or 12, respectively (Scheme 2). The benzoylation of the imino group was elucidated by helping variant spectroscopic techniques, The 1 H NMR spectra of compounds 8 and 11 revealed the presence of extra five protons in aromatic zone corresponding to benzoyl protons and disappearance of the imino proton at the same time. Moreover, the 13 C NMR of compound 8 is matching with the proposed structure.
On the other hand, reaction of cyanoacetamide derivative 4 with various aryl/heteroaryl aldehyde derivatives 13a-f under basic conditions (drops of piperidine) achieved the condensed products 14a-f via Knoevenagel condensation reaction (Scheme 3).
The chemical structures of arylidene/Heteroarylidene derivatives 14a-f were supported by spectral data and elemental analyses. The IR spectra were displayed a characteristic band corresponding to the conjugated cyano group at 2201-2017 cm À1 . Also, the 1 H NMR spectra of compounds 14a-f unambiguously ascertained the desired products. For example, the 1 H NMR spectrum of compound 14d showed three singlet peaks (exchangeable with D 2 O) at 10.63, 10.06 and 9.69 ppm corresponding to two NH and one OH protons and five singlet peaks at 8.11, 7.47, 3.83, 2.31 and 2.06 ppm corresponding to C-H (olefinic) , two Ar-H, two OCH 3 and two CH 3 protons, respectively. Furthermore, the 13 C NMR spectrum of 14d displayed a peak at 56.1 compatible with two carbons of OCH 3 group and also the other peaks were fit with this foreseeable structure.
In fact, treatment of pyrazolylidene derivative 14e with hydrazine monohydrate in absolute ethanol afforded the expected 3-aminopyrazole derivatives 15. Meanwhile, when repeating the same procedure with 4-methoxybenzylidene derivative 14a, compound 16 was afforded instead of the expected product (Scheme 3). Mechanistically, the formation of compounds 15 and 16 was occurred by attacking the lone pair of nitrogen atom of hydrazine on b-carbon of a,b-unsaturated nitrile system via Michael-addition reaction followed by 1,5-exo-dig cyclization, but the formation of compound 16 which undergoes hydrazinolysis at acetamide moiety as shown below in Scheme 4. the spectroscopic data confirmed the predictable structures for compounds 15 and 16 as recorded in experimental section.
The insertion of indolin-2-one moiety with compound 4 to give compound 17 was implemented by refluxing compound 4 with isatin in DMF containing drops of piperidine (Scheme 5). The IR spectrum of compound 17 exhibited characteristic bands at 2208, 1706 and 1667 cm À1 characteristic for CN, C ¼ O (indolinone) and C ¼ O (amide) functionalities, respectively.
Reaction of compound 4 with doubly electrophilic centers, namely acetylacetone and dimethyl acetylenedicarboxylate (DMAD) were studied. In case of, reaction of compound 4 with acetylacetone in DMF containing drops of piperidine the pyridinone moiety 19 was afforded, through condensation of compound 4 with one carbonyl of acetylacetone followed by ring closure on the other carbonyl of the intermediate 18 via 1,6-exo-trig cyclization (Scheme 5). The 1 H NMR spectrum of compound 19 clearly underscores the foreseeable structure for it, where it showed six singlet peaks at 10.27, 6.31, 2.41, 2.31, 2.20 and 2.08 ppm corresponding to NH proton (exchangeable with D 2 O), C 5 -H (pyridinone) and four CH 3 protons, respectively.
Under the same conditions, reaction of compound 4 with dimethyl acetylenedicarboxylate (DMAD) afforded pyridinedione derivative 21. The formation of 21 was done firstly through Michael-addition reaction to form the intermediate 20 followed by 1,6-exo-trig cyclization (Scheme 5). The chemical structure of compounds 21 is in keeping with their spectroscopic and elemental analyses.

Insecticidal activity
The results represented by Figure 4 and exhibited in Table 1 showed mortality data of the synthesized arylidene derivatives 14a-f and 17 against 4 th instar larvae of cotton leaf worm (Spodoptera littoralis) in terms of LC 50 and toxicity index (TI) compared with mortality data of methoxyfenozide as a reference registered insecticide which presented in Table 2. From these results we showed that, 2,4-dichlorobenzylidenecyanoacetohydrazide derivative 14c appeared as the most potent one after methoxyfenozide LC 50 (51.75 lg/ml) where its LC 50 was (79.73 lg/ml) followed by 4-nitrobenzylidene derivative 14b and its LC 50 (177.09 lg/ml) while arylidene derivative of isatin 17 showed the lowest insecticidal activity where its LC 50 was (1418.88 lg/ml). The insecticidal activity of other tested arylidene derivatives were in the following arrangement (14f, 14a, 14d and 14e) since their LC 50 recorded (361.51, 449.19, 522.16 and 723.15 lg/ml), respectively.
When concerning the tested cotton leaf worm (spodoptera littoralis), the toxicity line's slopes showed the extent of tested strain response homogeneity toward treated compounds and reference standard. From data in Table 1 we showed that, the homogeneous response of treated strain for tested arylidenes and methoxyfenozide were in order methoxyfenozide > 14a > 14d > 14e > 17 > 14c > 14f > 14 b where slope of their toxicity lines were 3.779, 2.400, 2.272, 2.021, 1.837, 1.147, 0.945 and 0.808, respectively.
Depending on data listed in Tables 2 and 3 and exhibited by Letha Dose Probit lines in Figure 5 which declare the insecticidal activity of methoxyfnozide and newly synthesized chromenes (8 and 9), and benzochromenes (11 and 12) against 4 th instar larvae of cotton leaf worm (Spodoptera littoralis).
These results showed that, methoxyfenozide has got the most potent insecticidal activity its LC 50 (51.75 lg/ml) while the other synthesized chromene and benzochromene derivatives have got convergent insecticidal activity where their recorded LC 50 were in the range (556.16 À 672.57 lg/ml) this is may be attributed to the high similarity in their structural skeleton. Despite the pervious reality benzochromene derivative 12 exhibited the highest insecticidal activity since its LC 50 was (556.16 lg/ml) while chromene derivative 8 had the lowest insecticidal activity as its LC 50 was (672.57 lg/ml). On the other hand, the insecticidal potency of other tested derivatives was as follow (11 and 9) as it was clear from the recorded LC 50 (605.36 and 622.68 lg/ml), respectively. This may be due to the presence of benzoyl group in (9 and 12) as a substituent on the nitrogen atom of imino moiety of (8 and 11).
Toxicity index (TI) is statistical parameter describing the insecticidal potency of tested chromene and benzochromene derivatives. From the shown data we concluded that, methoxyfenozide reference possess the highest insecticidal potency. On the other hand, benzochromene derivative 12 was 1.21, 1.12 and 1.08 fold more potent than chromenes (8 and 9) and benzochromene derivative 11.  On concerning the homologous response of tested strain we study the toxicity line 0 s slope as another statistical parameter to demonstrate the strain response. From Tables (2 and 3) and Figure 5 we showed that, strain response homogeneity of cotton leaf worm (Spodoptera littoralis) to chromene and benzochromene derivatives (8, 9, 11 and 12) found to be in the order 12 > 11 > 8 > 9 as a given from their toxicity line slopes which were 1.824, 1.590, 1.467 and 1.046 on sequence.
Tables 2, 4, and Figure 6 represented the insecticidal activity of reference insecticide and some hydrazide-hydrazone derivatives 4, 6, 15 and 16 against 4 th instar larvae of cotton leaf worm (Spodoptera littoralis). From this data we showed that, methoxfenozide recorded the most potent insecticidal activity as it shown from its LC 50 value also, 3-aminopyrazole derivative 16 its LC 50 was (209.78 lg/ml) has approximately double fold insecticidal activity more than the starting compound 4 and its LC 50 was (400.83 lg/ml) and triple fold insecticidal activity more than 2-thioxothiazole derivative 6 and bipyrazole derivative 15 where their recorded LC 50 were (691.56 and 678.15 lg/ml), respectively.
With the exception of methoxyfenozide, toxicity index (TI) has reflected the great difference in the insecticidal activity of tested compounds where it showed that, compound 16 was 1.91 and 3.30 and 3.23 fold more potent than compounds 4, 6 and 15 on sequence.
On the other hand, toxicity line 0 s slope of the newly synthesized heteocyles ranged from 1.245 to 1.558 this narrow range declared that the treated cotton leaf worm strain response to those derivatives was symmetric to high extent. Despite the mentioned fact, cotton leaf worm (Spodoptera littoralis) strain had high homologous response to 6 followed by 4, then 15 and finally 16 where their toxicity line 0 s slopes were 1.558, 1.433, 1.301 and 1.245, respectively.
On concerning Table 5 and Figure 7 which exhibited the insecticidal activity of pyridinone derivatives 19 and 21 expressed as mortality percentage against 4 th instar larvae of cotton leaf worm (Spodoptera littoralis).
It is clear that, these two pyridinone dervatives revealed the lowest insecticidal activity among all tested synthesized compounds.
Anyhow, from this data we deduce that, pyridinone derivative 19 with LC 50 (739.98 lg/ml) exhibited more insecticidal activity against treated larvae than compound 21 which has LC 50 (952.47 lg/ml).
The toxicity index of these two pyrdinones declared that, their insecticidal activity is so close to each other where its values were (10.833 and 8.371) for compounds 19 and 21, respectively. The homologous response of the treated starin also showed that, the strain has high response to pyridinone derivative 19 where its line 0 s slope was 1.127 as compared to compound 21 since its toxicity line 0 s slope was 1.044.
The above-mentioned finding of insecticidal activity experiment could be explained knowing that, the presence of groups (-Cl, -NO 2 ) are electron withdrawing groups are conjugate with benzene ring and thus the electron density of benzene ring which found to have positive influence on the insecticidal activity of tested compounds, while the introduction of (-OCH 3 , -OH and heterocyclic ring such as pyridine ring) could decrease the insecticidal activity. 47

Conclusion
In conclusion, a novel hydrazide-hydrazone, namely N-(4-(1-(2-(2-cyanoacetyl)hydrazono)ethyl)phenyl)acetamide (4) was utilized as a key starting material to synthesize different arylidenes, heteroarylidenes and heterocycles. The synthesized compounds were screened for their insecticidal efficacy against cotton leaf worm (Spodoptera littoralis). The obtained results indicated that, the most potent one is 2,4-dichlorobenzylidenecyanoacetohydrazide derivative 14c after methoxyfenozide (LC 50 ¼ 51.75 lg/ml) where its LC 50 was (79.73 lg/ml). So that, we recommended to use it in the field of pest control after formulating it in proper formulation.

Method of application
Thin film technique was used as a method of application in this study 54 in which the tested concentrations were applied through acetone to the surface of 9 cm in petri-dish. Where one ml of each concentration of the tested compound was spread on the inner surface of a petri-dish and moving the dish gently in circles to get series of concentration gradient from 250 to 1250 lg/ml. Petri-dish used as control was treated with 1 ml of acetone only. The solvent was evaporated under room conditions leaving a thin film of oil on the surface of petri-dish. Ten newly molted 4 th larval instar of S. littoralis were exposed to the tested chemicals in each petri-dish, with the presence of castor leaves for feeding larvae. Five replicates of each concentration and the control were made. The mortality percentages were recorded after 24 hr from treatment and corrected as compared to control larvae according to Abbott formula. 55 The LC 50 values for each compound were calculated according to Finney. 56 To evaluate the toxicity index (TI) of the tested derivative, the following equation (Sun equation) 57 was applied: Toxicity index ¼ LC 50 of most effective compound LC 50 of the compound used Â 100

Chemical used
The newly synthesized hydrazide-hydrazone derivatives.

Disclosure statement
No potential conflict of interest was reported by the authors.