Versatile Applications of Cyanoacetic Acid in Organic Chemistry: Active Methylene Compound for the Knoevenagel Condensation and Organocatalyst for the Biginelli Reaction

The application of cyanoacetic acid as a catalyst for the Biginelli reaction and as an active methylene compound for the Knoevenagel condensation reaction was evaluated. Using cyanoacetic acid as a Bronsted acid catalyst, after a synthetic optimization process, it was possible to synthesize eight dihydropyrimidinones with good yields (80-99%) using ethanol as solvent. It is the first time, to our knowledge, that the use of cyanoacetic acid is reported in the synthesis of this class of compounds, which have a wide bioactive potential. Also, cyanoacetic acid was used as a reagent in the Knoevenagel condensation, through which polyfunctionalized olefins were obtained and can be used as building blocks for structurally complex molecules. By using KOH as catalyst, eleven Knoevenagel adducts were synthesized with good yields (65-97%), under microwave irradiation as heating source, in water. Moreover, Knoevenagel adducts containing halogenated substituents (F, Cl) showed potential larvicidal activity with lethal concentrations (LC 50 ) of 19.63, 33.84 µg mL -1 and LC 90 of 27.46 and 48.16 µg mL -1 . This study showed the versatility of cyanoacetic acid as a catalyst for the synthesis of dihydropirimidinones, aldol compounds and presented the first study showing their larvicidal activity against Aedes aegypti.


Introduction
The Knoevenagel and Biginelli reactions are very important protocols for organic chemistry.The Knoevenagel condensation is a type of aldol reaction widely used in the construction of new C−C bonds.Also, their products are excellent building blocks for structurally more complex molecules with relevant applications. 1,2he Biginelli synthesis is a three-component reaction with aldehydes, β-ketoesters or dicarbonyl compounds, and urea derivatives.Its products are known as dihydropyrimidinones (DHPMs) and are highly polyfunctionalized heterocyclic compounds that have important applications included in the field of medicinal chemistry with the discovery of the antitumoral properties This is an open-access article distributed under the terms of the Creative Commons Attribution License.
2][13] Polymers based on DHPM structures are polyfunctionalized, which makes the material susceptible to structural modifications after polymer formation through derivatization reactions.These processes can induce or increase properties of interest, such as light absorption or solubility.Mao et al. 14 synthesized a highly conjugated polymer based on the Biginelli reaction.The capacity of these materials was tested against ultra-violet (UV) light absorption, and they proved to be very effective, being able to insert these compounds, for example, in the field of development of novel types of sunscreens.
There are several synthetic methodologies for Knoevenagel condensation, which are employed with acidic and basic catalysts.Gilanizadeh and Zeynizadeh 15 developed a heterogeneous and reusable nanostructure catalyst, based on immobilization of Cu and Fe on silica as catalyst Lewis acid, aromatic aldehydes and malononitrile, to synthesize thirteen Knoevenagel adducts with excellent yields (90-97%) in times of 4-50 min, with the advantage of using water as a solvent under reflux conditions.
Likewise, the Biginelli reaction is a versatile protocol for the synthesis of organic compounds with complex structures.However, based on literature data, the vast majority of studies use Bronsted or Lewis acids as catalysts.Zanin and Porto 16 developed a heterogeneous catalyst for the Biginelli reaction.Using HClO 4 -Al 2 O 3 , sixteen 3,4-dihydropyrimidin-2(1H)-ones/thiones were synthesized with good yields (80-94%) using aromatic aldehydes, ethyl acetoacetate, urea/thiourea and ethanol as solvent at 80 ºC for 1-3 h.
Another interesting reaction was studied by Jimenez et al. 17 using triethylamine in the synthesis of twenty-four Knoevenagel adducts with good yields (70-99%).The reactions were carried out in a microwave reactor, for 35 min, at 85 ºC and the solvents used were an aqueous solution of NaCl and EtOH (according to the activated methylene compound used malononitrile or methyl cyanoacetate).
Due to the various applications of DHPMs and Knoevenagel condensation, the research for new catalysts for these reactions remains relevant.Therefore, in this study, the versatility of cyanoacetic acid (CA) as an organocatalyst for the Biginelli reaction and a methylene active compound for the Knoevenagel reaction was evaluated.
Nuclear magnetic resonance (NMR) spectra were recorded on an Agilent  NMR spectra, the number of scans was 120 and for 13 C NMR was 881.
The reactions performed in a microwave (MW) reactor were performed using a Discover System from CEM Corporation at a 2.45 GHz frequency with maximal power output of about 200 W with internal magnetic stirring.For the reactions, the power of the microwave reactor used was 55 W (CEM Corporation, São Carlos, São Paulo, Brazil).
Melting points were measured in a Fisatom model 431 melting point apparatus in temperature interval of 25 to 300 ºC (Fisatom, São Carlos, São Paulo, Brazil).
The Biginelli reactions were monitored by a gas chromatograph-mass spectrometer (GC-MS-QP2010 Ultra) equipped with an auto-sampler injection AOC-20i (Shimadzu Corporation, São Carlos, São Paulo, Brazil).The detector used was of the electronic impact type (70 eV), scanning speed of 1.666, scan interval of 0.30 and fragments detected from 40 to 500 Da.Separations were performed on a fused silica capillary column (DB-5MS 5% phenyl-95%-dimethylpolysiloxane 30 m × 0.25 mm internal diameter, 0.25 mm film thickness) in a stream of helium of 0.95 mL min -1 .The injector temperature was 250 °C, the ion source was 200 °C, the interface was 270 °C and the split ratio was 5.The oven temperature program started at 70 °C, remaining for 2 min, with an increase of 20 ºC min -1 to 280 ºC, remaining for 8.54 min, resulting in a total time of 21 min.
The chromatographic yield of the optimization step of Biginelli reaction was performed by a Shimadzu liquid chromatograph system (HPLC-PDA) equipped with LC-20AT pump, DGU-20A5 degasser, SIL-20AHT autosampler, SPD-M20A PDA detector, CTO-20A column oven, and CBM-20A controller, using a C18-phenomenex chromatographic column (250 mm × 4.6 mm, particle size of 5 μm) as the stationary phase and a mixture of deionized water (eluent A) and acetonitrile (eluent B) as the mobile phase, with linear gradient elution protocol: 15 to 100% of the eluent B in 40 min.The flow rate was 1.0 mL min −1 (Shimadzu Corporation, São Carlos, São Paulo, Brazil).
The solvents (hexane, ethyl acetate, acetone, methanol and ethanol) were purchased from Aldrich, Synth, Merck and Vetec (São Paulo, SP, Brazil) and were used without further purification.

General procedure for the synthesis of DHPMs 4a-4h
In a typical procedure, a mixture of aldehyde (0.5 mmol), urea (0.5 mmol), ethyl acetoacetate (1 mmol) and cyanoacetic acid (20 mol%) was placed in a roundbottom flask.The suspension was stirred at 80 ºC for 2 h.In the sequence, 200 µL of EtOH were added and the reaction proceeded for more 4 h.The reaction was monitored by TLC in a mixture of EtOAc-hexane 6:4.After completion of the reaction, the products were directly purified by flash column chromatography (silica gel 230-400 mesh) using the same mixture used in TLC.
All products were characterized by 1 H NMR, 13 C NMR, FTIR, GC-MS and melting point.The spectral and analytical data of the compounds 4a-4h can be viewed in the Supplementary Information (SI) section.
General procedure for the synthesis of Knoevenagel adducts 6a-6e, 6h-6m using conventional heating In a typical procedure, a mixture of aldehyde (1.0 mmol), cyanoacetic acid (1 mmol), KOH (20 mol%, 0.7 M) and water (5 mL) was placed in a round-bottom flask.The suspension was stirred at 75 ºC for 20 min.The reaction was monitored by TLC in a mixture of EtOAc-hexane 8:2.After completion of the reaction, it was added 1 mL of HCl (3 M) and the solution was stirred for 30 min.At the end of the reaction, extraction with ethyl acetate (3 × 25 mL) was carried out.The combined organic phases were concentrated under reduced pressure until total evaporation of the solvent.The obtained product was purified by column chromatography (silica gel 230-400 mesh) using the same mixture used in TLC.
All products were characterized by 1 H NMR, 13 C NMR, FTIR, GC-MS and melting point.The spectral and analytical data of the compounds 6a-6e, 6h-6m can be viewed in the SI section.
General procedure for the synthesis of Knoevenagel adducts 6a-6e, 6h-6m under microwave irradiation A mixture of aldehyde (1 mmol), cyanoacetic acid (1 mmol), KOH (20 mol%, 0.7 M) and water (5 mL) was put in the MW reactor (50 W) for 20 min and stirred at 75 ºC.The reaction progress was monitored by TLC in a mixture of EtOAc-hexane 8:2.After completion of the reaction, it was added 1 mL of HCl (3 M) and the solution was stirred for 30 min.At the end of the reaction, extraction with ethyl acetate (3 × 25 mL) was carried out.The combined organic phases were concentrated under reduced pressure until total evaporation of the solvent.The obtained product was purified by column chromatography (silica gel 230 400 mesh) using the same mixture used in TLC.
All products were characterized by 1 H NMR, 13 C NMR, FTIR and melting point.The spectral and analytical data of the compounds 6a-6m can be viewed in the SI section.

Crystallization of Knoevenagel adducts 6a, 6h, 6l and 6m
All the crystallization of the Knoevenagel adducts followed the same protocols.First, 20 mg of each compound were put in a vial of 10 mL and were dissolved in 10 mL of acetone and methanol (1:1) and stirred at soft heating conditions (50 °C) for 5 min or until complete solubilization.Then, the crystallization batches were allowed to cool down slowly and covered the vial with Parafilm ® for slow evaporation of the solvent.All these systems were maintained at room temperature until the appearance of suitable single-crystals, which occurred within 1-2 days.
Single crystal structure determination of Knoevenagel adducts 6a, 6h, 6l and 6m The single crystal X-ray diffraction measurements for 6a, 6h, 6l and 6m were performed at 293 K on a Rigaku XtaLAB mini (ROW) diffractometer with graphite monochromated Mo Kα radiation (0.71073 Å).Cell refinements were performed using the CrysAlisPro software. 18Using Olex2, 19 the structures were solved by intrinsic phasing method SHELXT-14, 20 and refined by full-matrix least squares on F2 using the SHELXL. 21The non-hydrogen atoms were refined anisotropically.Then, all hydrogen atoms were located from electron-density difference maps and were positioned geometrically and refined using the riding model [Uiso(H) = 1.2Ueq or 1.5Ueq].The Olex2 was also used for analysis and visualization of the structures, and for graphic material preparation.The X-ray diffraction data and refinement parameters obtained for the elucidated crystal structures are summarized in Table 1.The Crystallographic Information File (CIF) of Knoevenagel adduct derivatives 6a, 6h, 6l and 6m were deposited in the Cambridge Structural Data Base 22 under the Cambridge Crystallographic Data Centre (CCDC) numbers 2298597, 2298598, 2298599 and 2298600, respectively.

Larvicidal bioassay
The larvae of Aedes aegypti and Rokeffeller colony were from the Arthropod Laboratory of the Federal University of Amapá (ARTHROLAB); all larvae used were in the 3 rd stage.They were kept in standard weather conditions with a temperature of 25 ± 2 °C and relative humidity of 75 ± 5% and photoperiod of 12 h according to the World Health Organization (WHO). 23ompounds 6a-6e, 6h-6m were prepared in different concentrations (75, 50, 25, 15 and 7.5 μg mL -1 ) solubilized in DMSO (1%) from stock solution of 900 μg mL -1 .In each bioassay was used 10 larvae in controlled conditions (25 ± 2 °C).The distilled water and DMSO (1%) were used with negative controls.All assays were performed in quintuplicate. 24or larvicidal activity the lethal concentrations (LC 50 and LC 90 ) were determined after 24 and 48 h of incubation and calculated using Probit analysis with Minitab 19 statistical software. 25

Design and optimization of Knoevenagel adducts
[28]

Molecular docking simulation
After obtaining bioactive poses, the compounds 6a-6e and 6h-6m were selected for molecular docking simulations, in order to evaluate the score of the binding free energy function (ΔG), as well as the analysis of conformations, interaction mode and binding affinity with the selected receptor.

Molecular docking study
Ligands and protein structure used in molecular docking were prepared using Discovery Studio 5.0 software. 29alidation of docking protocols in the molecular target (PDB ID 5V13) was performed by overlay the ligand crystal structure on the best docking pose for root mean square deviation (RMSD) calculation.In the juvenile hormone (Aedes aegypti organism) docking study, specific complexed ligands were used in AutoDock 4.2 30 / Vina 1.1.2 31via graphical interface PyRx 0.8.30. 32he x, y and z coordinates were determined according to the binding site on the receptor.The coordinates used for the grid center and dimensions can be seen in Table 2.

Cyanoacetic acid as a catalyst for Biginelli reaction
The first investigation of this study consisted of evaluating the ability of cyanoacetic acid to be a Bronsted acid-type organocatalyst for the Biginelli reaction.For this, the reaction using benzaldehyde 1a, ethyl acetoacetate 2, and urea 3 was evaluated as a protocol with different reactional conditions and the yields were calculated by HPLC-DAD (Table 3).
The first experiment was performed in the absence of the catalyst and, with the reagents in the proportion of 0.5 mmol of aldehyde, 1 mmol of ethyl acetoacetate and, 0.75 mmol of urea.It was observed that in approximately 2 h of reaction, the agitation of the system was stopped due to precipitation of DHPM 4a.Thus, 200 µL of EtOH were added to the reaction system, which regained its normal agitation.After 6 h of reaction, the DHPM 4a was obtained in 55% yield (Table 3, entry 1).
In the following experiments, using this strategy of starting the reactions without solvent and adding the ethanol after 2 h, the catalytic effect of cyanoacetic acid on the Biginelli reaction was evaluated (Table 3, entries 2-7).In the first attempt, using 10 mol% of CA, and at 60 ºC, the DHPM 4a was obtained in 66% yield with 4 h of reaction (Table 3, entry 2).Increasing the reaction time to 6 h, no significant changes were noted in the yield of 4a (Table 3, entry 3).Varying the amount of CA to 20 mol%, it was observed that the yield increased to 80% for 4a (Table 3, entry 4).Moreover, it was observed that when the temperature was changed to 80 ºC, the yield of DHPM 4a was increased to 88% (Table 3, entry 5).
It was possible to realize that CA was acting as an organocatalyst in the reaction, since the reaction yields in its presence were higher than in the control experiment in the absence of catalyst.Also, it was observed that temperature was an important factor, and the best yield values were obtained at 80 ºC.Subsequently, a new test was performed in which the proportion of urea was decreased (0.5 mmol aldehyde, 1 mmol ethyl acetoacetate and 0.5 mmol urea).
The result showed the same 88% yield for DHPM 4a compared to the Table 3, entry 5 experiment, using a small amount of urea (Table 3, entry 6).
Finally, the optimized condition was tested in a microwave reactor (Table 3, entry 7); however, the yield under microwave irradiation was practically the same as that obtained through conventional heating conditions.Therefore, the conventional heating was then selected for the development of a scope of compounds.
For the development of the synthetic optimized protocol, several aromatic aldehydes were used, both with acceptor and electron donor groups linked in the aromatic ring.At the end of the process, DHPMs 4a-4h were obtained with excellent yields 80-99% (Table 4) and characterized by NMR ( 1 H and 13 C), IR and melting point.It is noteworthy that the electronic effect of the aromatic ring substituents of the respective aldehydes did not influence the reactions and the yields of products, as well, although a significant amount of the CA was used, a gain of more than 30% in the reaction yield was observed, turning the catalytic role of cyanoacetic acid for the Biginelli reaction evident.
Figure 2 contains a proposed mechanistic route for the Biginelli reaction catalyzed by cyanoacetic acid (iminium route promoted by a Bronsted acid).The first step of the reaction includes the nucleophilic attack of the nitrogen from urea on the aldehyde, which was preliminary protonated by CA.In the sequence, after a dehydration, the iminium ion is formed.Following this route, the β-ketoester enolate attacked the iminium intermediate and CA was regenerated through a proton abstraction.In the final steps, The reactions were performed initially for 2 h under free solvent.Then, 200 μL of EtOH were added and the reactions were performed in the appropriate time; reagents proportion: aldehyde (0.5 mmol), ethyl acetoacetate (1 mmol) and urea (0.75 mmol).a Proportion: aldehyde (0.5 mmol), ethyl acetoacetate (1 mmol) and urea (0.50 mmol); b Performed in MW reactor.Conversion determined by HPLC-DAD.
an intramolecular cyclisation and another dehydration occurs, resulting in the DHPM structure. 33anoacetic acid as a reagent for Knoevenagel condensation reaction The reaction using furfural 1l and CA 5 to produce the Knoevenagel adduct 6l was selected for the optimization process involving different reaction conditions, using a MW reactor as heating source (Table 5).Initially, the experiment in absence of catalyst, for 20 min at 75 ºC did not produce compound 6l (Table 5, entry 1).The use of a basic catalyst KOH (5 mol%, 0.7 M) was the first attempt to improve the reaction.In 20 min at 75 ºC, adduct 6l was obtained with 51% yield (Table 5, entry 2).This experiment showed that the basic catalyst was essential for the formation of 6l.
In the sequence, the temperature was kept constant at 75 ºC, the amount of KOH was increased to 10 mol% and Table 4. Synthetic scope of DHPMs 4a-4h using cyanoacetic acid as catalyst and conventional heating in the Biginelli reaction The reactions were performed initially for 2 h under free solvent.Then, 200 μL of EtOH were added and the reactions were performed for more 4 h.Reagents proportion: aldehyde (0.5 mmol), ethyl acetoacetate (1 mmol) and urea (0.50 mmol).the yield of 6l was increased to 71% (Table 5, entry 3).
Increasing even more, using 20 mol%, adduct 6l was obtained in 93% yield; however, no improvement in reaction yield was observed when 30 mol% of KOH was used (Table 5, entry 5).The best condition achieved (Table 5, entry 4) was evaluated under conventional heating, to compare the effect of the heating source.The result was unsatisfactory and, adduct 6l was obtained with only a 31% yield, showing the efficiency of the MW irradiation as a heating source for this protocol (Table 5, entry 6).
Furthermore, the possibility of a decrease in reaction time was investigated; however, when the reaction was performed in just 10 min, the adduct 6l was obtained with 61% yield (Table 5, entry 8).In the last experiments, the effect of temperature was monitored.Through the results, it was possible to conclude that the temperature of 75 ºC had a direct influence on the yield of 6l, since at lower temperatures the yield decreased significantly (Table 5, entries 9 and 10).
After the synthetic optimization process, a scope of molecules was obtained using CA as an activated methylene compound for the Knoevenagel condensation reaction with several aromatic aldehydes.As shown in Table 6, the Knoevenagel adducts 6a-6e and 6h-6m were obtained with good yields (65-97%), independently of the electronic characteristic of the substituent group attached to the aromatic ring.
Single crystals of 6a, 6h, 6l, and 6m suitable for X-ray diffraction analyses were obtained with the slow evaporation of solutions prepared for each compound using a mixture of acetone and methanol (1:1).The Oak Ridge Thermal Ellipsoid Plot (ORTEP) type representation of the crystal structures of these compounds are shown in Figure 3. Selected bond lengths and angles are available in the SI section.
The Knoevenagel adducts 6a, 6h, and 6l crystallize in the triclinic space group P-1, while 6m crystallizes in the monoclinic space group P2 1 /c.As shown in Figure 3, the aromatic rings of adducts show high planarity, except 6h, which possesses the carbon from the p-methoxy group positioned out of the plane of the aromatic ring.Furthermore, through these results, it was possible to observe the tendency of the formation of diastereoisomeric adducts with (E)-configuration.
The structures are stabilized by O-H•••O hydrogen bonds, with these interactions giving rise to formation of dimeric arrangements.The non-classical hydrogen bonds such as C-H•••O and C-H•••N are also observed in the crystal packing of the adducts 6a, 6h and 6m, and the combination of the non-covalent interactions present on these structures is helpful for the organization of the molecules of 6a and 6h in a one-dimensional chain, and 6m in a two-dimensional network (see SI section).
According to the study of Bianco et al., 34 the stereoelectronic properties of compounds containing halogen substituents attached to the sesquiterpene showed  larvicidal activities (> 91% at 50 ppm).When investigating larvicidal activity, the authors observed that molecules with electronegative substituent groups, e.g., chlorine and fluorine, influenced the solubility of the substance, the steric volume, and the interaction at the binding site.
The larvicidal results of some halogenated Knoevenagel adducts were tested for A. aegypti in the study by Carvalho et al. 35 Benzylidenemalononitriles, e.g., the (2-(4-chlorobenzylidene) malononitrile showed an effect against 3 rd instar larvae showing LC 50 values of 9.42 and 9.44 μg mL -1 at 24 and 48 h, respectively.
Aryl and phenoxymethyl-(thio)semicarbazones were also evaluated against A. aegypti.It was possible to observe the high activity of thiosemicarbazines, and the authors highlighted the importance of halogen substituent groups in the para position for this biological property.Atoms can interact through a halogen bond at the binding site of biological targets, and the presence of electron-donating groups may not present this desired activity. 36inally, A. aegypti is an important vector for public health, and current strains show resistance to commercial insecticides.According to these results, the Knoevenagel compounds tested may be a promising alternative in the control of A. aegypti.

Larvicidal activity for the synthesized Biginelli compounds
The Biginelli compounds 4a-4h were tested for the 3 rd instar larvae of Aedes aegypti (A.aegypti) at different concentrations (75, 50, 25, 15 and 7.5 μg mL -1 ), These compounds did not show any potential larvicidal activity with a value of 0% mortality in 24 and 48 h.

Validation of molecular docking protocols
Recovering the inhibitor pose (JHIII) was possible to validate the molecular docking protocols by superimposing the crystallographic pose over the best docking pose, obtaining a root-mean-square deviation (RMSD) value of 1.15 Å.According to Ramos et al. 37,38 the predicted binding mode in molecular docking indicates that when the RMSD ≤ 2.0 Å in relation to the crystallographic pose of a respective ligand, the validation is considered satisfactory (Figure 4).

Evaluation of receptor-ligand interactions
Eleven compounds (Table 9) were subjected to molecular docking at the juvenile hormone (Aedes aegypti) binding site.In this study, only three adducts showed binding affinity values similar to or higher than the control used.Compound 6e had a binding affinity of −8.0 kcal mol -1 , followed by 6j with −8.0 kcal mol -1 and 6k with −8.1 kcal mol -1 compared to the control (JHIII).
Individual interactions observed in the docking of compounds 6e, 6j, and 6k were similar to those observed for JHIII in the juvenile hormone-binding site, around the α-helix between the amino acid residues Trp33-Val-65 and in the β-sheet with Val-51, Trp-53, and Pro-55, as shown in Figure 5.
A single well-ordered molecule of JHIII (refined occupations 0.91-0.96) is present in the binding pocket of the N-terminal domain of mJHB.According to Kim et al., 39 three molecules of the complex are present in the asymmetric unit of the crystal, and the ligand conformation is essentially identical in all three.The JHIII epoxy is located in the center of the domain, while the methyl ester group of the hormone is oriented towards its surface.The epoxy group forms a hydrogen bond with the phenolic hydroxyl of Tyr-129, and the remainder of the isoprenoid chain is surrounded by hydrophobic side chains including those of Phe-144, Tyr-64, Trp-53, Val-65, Val -68, Leu-72, Leu-74, Val-51 and Tyr-33.
Compound 6k presented significant contributions to the binding affinities (−8.1 kcal mol -1 ), as the study complies with the pocket binding site, in which they are represented by amino acids surrounded by hydrophobic side chains including Try-33, and Trp-53, and a hydrogen bond with Val-51.

Conclusions
The use and application of CA were evaluated in two distinct roles, as a catalyst and as a reagent for the synthesis of organic compounds.According to experimental results, CA had catalytic activity, improving the Biginelli reaction.Through its use as an organocatalyst, it was possible to synthesize eight DHPMs 4a-4h in good yields of 80-99% using ethanol as a green solvent.Furthermore, the use of CA as a reagent for the Knoevenagel condensation reaction was explored.Using KOH as a base catalyst, it was possible to obtain eleven examples of adducts in the (E)-configuration with good yields of 65-97%, using water as a green solvent under microwave irradiation.
The larvicide studies showed that Knoevenagel adducts, containing halogenated substituent groups  Therefore, CA is a promising stable compound with low cost that can be used as a catalyst and a reagent in organic chemistry.Moreover, the products obtained by its use are easily purified and can be used as larvicide control against Aedes aegypti.

Figure 2 .
Figure 2. Proposed mechanism for the synthesis of DHPMs using cyanoacetic acid as catalyst.

Table 9 .
Results of binding affinity of the compounds 6a-6m with the juvenile hormone proteinComplexBinding affinity / (kcal mol -aromatic ring have potential larvicidal activity with 100% mortality in 24 h with LC 50 and LC 90 for values of 19.63 and 33.84 μg mL -1 and, 27.46 and 48.16 μg mL -1 , respectively. Technologies 500/54 Premium Shielded or Agilent Technologies 400/54 Premium Shielded spectrometer, with dimethyl sulfoxide (DMSO-d 6 ) or acetone-d 6 as solvent and tetramethylsilane (TMS) as the internal standard (Agilent Technologies, São Carlos, São Paulo, Brazil).The chemical shifts (d) were expressed in parts per million (ppm) and referenced to the tetramethylsilane internal standard (TMS) signal and the deuterated solvent used DMSO-d 6 (d H 2.50, d C 39.52) and acetone-d 6 (d H 2.84 and 2.05, d C 206.26 and 29.8).For 1

Table 3 .
Synthetic optimization process of DHPM 4a using cyanoacetic acid as catalyst

Table 5 .
Synthetic optimization process of Knoevenagel adduct 6I using cyanoacetic acid and furfural under MW irradiation

Table 7 .
Lethal concentration values (LC 50 and LC 90 ) and confidence interval (CI) of compounds 6b-6d with halogenated substituents attached in the aromatic ring

Table 8 .
Lethal concentration values (LC 50 and LC 90 ) and confidence interval (CI) of compounds