Efficient Green Synthesis of Hydrazide Derivatives Using L-Proline: Structural Characterization, Anticancer Activity, and Molecular Docking Studies

: Green synthesis using L-proline as an organocatalyst is crucial due to its reusability, mild conditions, clean reactions, easy workup, high purity, short reaction times, and high yields. However, existing methods often involve harsh conditions and longer reaction times. In this study, 2-cyano-N ’-(2-cyanoacetyl)acetohydrazide ( 3 ) was prepared and condensed with various benzaldehyde derivatives to yield 2-cyano-N ’-(2-cyano-3-phenylacryloyl)-3-phenylacrylohydrazide derivatives ( 5a – e , 7a,b ) using a grinding technique with moist L-proline. Additionally, three 2-cyano-N ’-(2-cyano-3-heterylbut-2-enoyl)-3-heterylbut-2-enehydrazides ( 9 , 11 , 13 ) were synthesized by condensing compound 3 with respective (heteraryl)ketones ( 8 , 10 , 12 ) following the same method. The synthesized compounds were characterized using IR, NMR, and MS spectroscopy. L-proline’s reusability was confirmed for up to four cycles without significant yield loss, showcasing the protocol’s efficiency and sustainability. The new compounds were screened for anticancer activities against the HCT-116 colon carcinoma cell line using the MTT assay. Molecular docking studies revealed the binding conformations of the most potent compounds to the target protein (PDB ID 6MTU), correlating well with in vitro results. In silico ADMET analysis indicated favorable pharmacokinetic properties, highlighting these novel compounds as promising targeted anti-colon cancer agents.


Introduction
Green chemistry aims to design products and processes that reduce or eliminate hazardous substances [1].This field emphasizes the importance of developing safer chemicals, reducing waste, and improving energy efficiency.An overview of the twelve principles of green chemistry highlights key aspects such as the design of safer chemicals, waste reduction, and energy efficiency improvements.The focus is on creating environmentally sustainable chemical procedures that are safe for biological activity and industry-leading molecules [1][2][3][4].It emphasizes alternatives such as reduced solvent use, high atom economy and selectivity, elimination of hazardous wastes, simple product separation and purification methods, and alternative energy sources for organic transformations [5][6][7][8][9][10].Mechanosynthesis, using "ball milling" or simple grinding methods [11,12], fits this sustainable and "greener" definition.Manual grinding with a mortar and pestle is especially useful at the laboratory scale, offering an energy-efficient, economical, and ecologically advantageous process without external heating [13].Toda and colleagues demonstrated that many organic exothermic reactions could be performed using mortar and pestle grinding with good yields [14], including condensation reactions like Schiff base and oxime formation [15].Recently, this method has also proven efficient for constructing biologically important heteroaromatic compounds [16][17][18][19].
Organocatalysis has gained significant interest due to its ease of use, stability, nontoxicity, and cost-effectiveness.It does not require an inert atmosphere or anhydrous conditions, making it suitable for mild conditions [20,21].The field has grown exponentially, with new catalysts and reactions producing asymmetric products in classical reactions such as Diels-Alder, Mannich, and Michael reactions [22,23].L-proline, a naturally occurring amino acid, has gained significant attention as a catalyst in green chemistry due to its effectiveness and environmental friendliness [24][25][26][27].As an organocatalyst, L-proline is used in various organic transformations, such as aldol reactions, Michael additions, and Mannich reactions.It offers advantages, including biodegradability, non-toxicity, high selectivity, and mild reaction conditions, making it a valuable tool for synthesizing complex molecules with high enantioselectivity and yield [24][25][26][27].Proline's biological relevance extends to both natural and synthetic compounds.In nature, proline is a significant component of proteins, particularly collagen, contributing to structural stability.It also plays a role in neurotransmission and enzyme catalysis.In synthetic compounds, proline and its derivatives are used to design pharmaceuticals with enhanced stability, bioavailability, and therapeutic efficacy, including antiviral, anticancer, and anti-inflammatory agents [28][29][30].
Cancer continues to be a significant global health challenge, with its incidence rising in both developed and developing countries.Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the fourth leading cause of cancer-related deaths worldwide [31].This disease affects both men and women similarly, being the third most common cancer in men and the second most common in women [32].Despite its prevalence, CRC is considered one of the most preventable cancers through lifestyle modifications [17,33,34].Current CRC chemotherapy mainly involves 5-fluorouracil, often combined with oxaliplatin, irinotecan, leucovorin, or folic acid in various treatment protocols.While these treatments are effective, they come with significant side effects, including neurological and gastrointestinal disorders, myelosuppression, anemia, and neutropenia [35][36][37][38].Given the drawbacks of conventional chemotherapy, there is an urgent need to develop new treatment strategies for colorectal cancer.Chemoprevention, which involves using natural, synthetic, or biological agents to reverse, suppress, or prevent carcinogenesis at any stage (initiation, promotion, or progression), has emerged as a promising approach [39,40].
The pharmacological activities of hydrazides (-CO-NH-NH-) and hydrazones (-CO-NH-N=) have been extensively evaluated, making them a significant class of compounds [41][42][43].Hydrazones, important compounds with diverse pharmacological activities, are currently in clinical trials, such as pyridoxal isonicotinoyl hydrazone (I) [44].For instance, compounds (II) and (III) demonstrated antiproliferative activity with IC 50 values of 0.29 and 3.1 µM against HCT 116 cells, outperforming the drug 5-fluorouracil (IC 50 = 5 µM) [45].Hydrazone (IV) induced a cytostatic effect in p53-competent HCT116 cells, leading to cell cycle arrest at the S/G2 phase [46].Acylhydrazone (V) exhibited toxicity in colon cancer cells without harming nonmalignant cells, triggering apoptosis and cell cycle arrest in the G2/M phase [47].Compound (VII) showed greater antiproliferative activity than compound (I) due to its high chelation efficacy [48], while compound (VI) displayed strong antiproliferative activity on esophageal carcinoma cells with IC 50 values of 1.09 and 2.79 µM, inducing apoptosis and cell cycle arrest at the G0/G1 phase [49] (Figure 1).This makes hydrazides and their derivatives promising candidates for developing effective cancer therapies.compound (I) due to its high chelation efficacy [48], while compound (VI) display strong antiproliferative activity on esophageal carcinoma cells with IC50 values of 1.09 a 2.79 µM, inducing apoptosis and cell cycle arrest at the G0/G1 phase [49] (Figure 1).T makes hydrazides and their derivatives promising candidates for developing effect cancer therapies.Furthermore, the biological profile of the new hydrazides was evaluated, and the sults were followed by an in silico docking study.Recently, in silico computational me ods have become important in the drug discovery area [50].Using molecular docking, important computational technique, to understand the theoretical interactions of co pounds with a macromolecular structure and provide assistance to the results of in vi assays.In this research, we targeted this protein with PDB ID (6MTU) and docked it w novel synthesized compounds to present its effect as anti-colon cancer.For the major of therapeutic drugs, one of these investigations combined the methods of Absorpti Distribution, Metabolism, and Toxicity (ADMET).The procedure by which a medicat enters the systemic circulation after administration is known as absorption.Distribut is the movement of medication from systemic circulation to extravascular sites.Metab lism involves the enzymatic breakdown of a drug into metabolites that are cleared fro the body.Excretion is the passive or active transport of intact drug molecules into urine or bile [51].Toxicology is described in this article as the negative effects that dr substances have on humanity.Early-stage medication development is drawing attent to ADMET characteristics due to their increased significance [52,53].
Building on our research into bioactive heterocyclic frameworks [5,9,17,19,[54][55][56][57][58][59][60], synthesized novel hydrazide derivatives via solvent-free mechanical grinding of 2-cyan N'-(2-cyanoacetyl)acetohydrazide with various aldehydes and ketones, using L-proline an efficient organocatalyst.This method achieved high yields, short reaction times, a low costs.The synthesis was conducted using both thermal and grinding techniques.then assessed the in vitro and in silico cytotoxicity of these derivatives against the HC 116 colon carcinoma cell line.This comprehensive approach, integrating synthetic che istry, biological evaluation, and computational modeling, highlights the potential of h drazide derivatives as anticancer agents.The promising results from both experimen and in silico analyses support further development and optimization of these compoun for cancer therapy.

Results and Discussion
The main precursor for synthesizing new compounds, 2-cyano-N'-(2-cyanoa tyl)acetohydrazide (3), is formed by reacting 3-(3,5-dimethyl-1H-pyrazol-1-yl)-3-oxop panenitrile with 2-cyanoacetohydrazide under reflux in dioxane [61].Carbonyl co pounds, including aldehydes and ketones, can react with compound 3 to produce hyd zide derivatives through either conventional reflux or green grinding processes, usi Furthermore, the biological profile of the new hydrazides was evaluated, and the results were followed by an in silico docking study.Recently, in silico computational methods have become important in the drug discovery area [50].Using molecular docking, an important computational technique, to understand the theoretical interactions of compounds with a macromolecular structure and provide assistance to the results of in vitro assays.In this research, we targeted this protein with PDB ID (6MTU) and docked it with novel synthesized compounds to present its effect as anti-colon cancer.For the majority of therapeutic drugs, one of these investigations combined the methods of Absorption, Distribution, Metabolism, and Toxicity (ADMET).The procedure by which a medication enters the systemic circulation after administration is known as absorption.Distribution is the movement of medication from systemic circulation to extravascular sites.Metabolism involves the enzymatic breakdown of a drug into metabolites that are cleared from the body.Excretion is the passive or active transport of intact drug molecules into the urine or bile [51].Toxicology is described in this article as the negative effects that drug substances have on humanity.Early-stage medication development is drawing attention to ADMET characteristics due to their increased significance [52,53].
Building on our research into bioactive heterocyclic frameworks [5,9,17,19,[54][55][56][57][58][59][60], we synthesized novel hydrazide derivatives via solvent-free mechanical grinding of 2-cyano-N'-(2-cyanoacetyl)acetohydrazide with various aldehydes and ketones, using L-proline as an efficient organocatalyst.This method achieved high yields, short reaction times, and low costs.The synthesis was conducted using both thermal and grinding techniques.We then assessed the in vitro and in silico cytotoxicity of these derivatives against the HCT-116 colon carcinoma cell line.This comprehensive approach, integrating synthetic chemistry, biological evaluation, and computational modeling, highlights the potential of hydrazide derivatives as anticancer agents.The promising results from both experimental and in silico analyses support further development and optimization of these compounds for cancer therapy.
Table 1.Comparison of the synthesis of products 5a-e, 7a,b, 9, 11, and 13 using thermal and gre methods with L-proline organocatalyst.

Compounds
Thermal  1 compares the synthesis of products 5a-f, 7a,b, 9, 11, and 13 using both trad tional reflux and the eco-friendly grinding method with moist L-proline as the basic cat lyst.The green method demonstrated significantly shorter reaction times and general higher yields than the conventional reflux method.For example, compound 5a achieve a yield of 75% in 5 h under reflux, whereas the grinding method yielded 90% in just min.This trend was consistent for other compounds, highlighting that the grindin Additionally, a comparative study of the conventional (thermal) method and the green (grinding) method using moist L-proline as the basic catalyst is presented in Table 1.Table 1 compares the synthesis of products 5a-f, 7a,b, 9, 11, and 13 using both traditional reflux and the eco-friendly grinding method with moist L-proline as the basic catalyst.The green method demonstrated significantly shorter reaction times and generally higher yields than the conventional reflux method.For example, compound 5a achieved a yield of 75% in 5 h under reflux, whereas the grinding method yielded 90% in just 25 min.This trend was consistent for other compounds, highlighting that the grinding method is not only more time-efficient but also produces comparable or improved yields, making it a promising greener alternative for synthesizing these compounds.
Additionally, Table 2 presents the optimization of catalyst loading for the synthesis of compound 5a using the grinding method with moist L-proline as the basic catalyst.a The best reaction condition for the synthesis of compound 5a.
Table 2 presents the optimization process for catalyst loading in the synthesis of compound 5a.Each entry reflects a different catalyst percentage, along with corresponding reaction times, temperatures, and yields.Notably, entry 3, with a catalyst loading of 5 mol%, achieved the highest yield of 90%, indicating the optimal conditions for synthesizing compound 5a in this experimental setup.
Additionally, we explored the catalyst's regeneration and reusability.The reaction between compound 3 and p-anisaldehyde 4a was conducted under optimized conditions.Upon completion of the reaction, the mixture was diluted with 10 mL of water, and the crude solid product was filtered and washed with 2-5 mL of water.The L-proline catalyst was removed by washing with water, as it is water-soluble.The catalyst was then recovered by evaporating the water, washed with diethyl ether, and reused for subsequent catalytic runs, as detailed in Table 3.

State of Catalyst
New Catalyst Recycled Catalyst Yield of product 5a 90 87 81 78 74 49 Table 3 showcases the recyclability data for the L-proline organocatalyst, comparing its performance as a fresh catalyst (1) and after multiple recycling cycles (2 to 5).The yield of compound 5a serves as a benchmark for assessing the catalyst's efficiency.Initially, the new catalyst achieves a 90% yield.However, with each recycling cycle, a gradual decrease in yield is observed: 87% after the first cycle, 81% after the second, 78% after the third, and 74% after the fourth cycle.A notable decline in performance is evident after the third cycle, with the yield dropping to 49% by the fifth cycle.This trend indicates a reduction in the catalyst's recyclability over successive uses, although it remains effective for up to four cycles with minimal loss in activity.The loss in yield after the fifth cycle of recycling L-proline as a catalyst is primarily due to cumulative structural degradation, loss of catalyst quantity, contamination, and changes in physical properties affecting its catalytic performance.These cumulative effects lead to a significant reduction in yield, dropping from 90% with a new catalyst to 49% after the fifth recycle.

Antitumor Study
The in vitro cytotoxic activity of ten newly synthesized target compounds was evaluated against the HCT-116 colon carcinoma cell line.The bioactivity of these compounds was assessed using the MTT assay, which measures cell viability and proliferation.Cisplatin, a well-known chemotherapeutic agent, was used as a reference standard to compare the calculated IC 50 values of the new compounds.The results of this comparative study are presented in Table 4, providing insights into the potential efficacy of the synthesized compounds relative to the established drug.The outline data in Table 4 showed that the in vitro cytotoxic effect of synthesized compounds against the HCT-116 cell line reveals a range of efficacies, as indicated by their IC 50 values (µM).Among the synthesized compounds, compound 11 (IC 50 = 2.5 ± 0.81 µM) exhibits the highest cytotoxicity, closely followed by compound 5b (IC 50 = 3.2 ± 1.1 µM) and compound 13 (IC 50 = 3.7 ± 1.0 µM).These compounds show comparable efficacy to Cisplatin, a known chemotherapeutic agent, which has an IC 50 value of 2. 1 µM) underscores their potential as anticancer agents.These findings indicate that the nature and position of substituents, the core structure, and the overall size and flexibility of the compounds play significant roles in their cytotoxicity, providing a basis for optimizing structures to improve anticancer properties.

Molecular Docking Study
After identifying the potent compounds against the HCT-116 colon carcinoma cell line, docking studies were performed to understand their binding interactions with the prepared protein (PDB ID: 6MTU).Molecular docking was used to examine how the ligands interact with the protein's active site [62].The results, compared to the co-crystallized ligand SEP, revealed significant interactions with the protein residues, as shown in Figure 2. Key findings include (i) more negative binding energies for our ligands than the CCL (co-crystallized ligand), indicating greater stability; (ii) various interactions, including hydrogen donors, hydrogen acceptors, π-H, and π-π interactions; (iii) key binding residues were glycine, isoleucine, arginine, and leucine; (iv) primary interaction sites were oxygen, nitrogen, and six-membered rings; and (v) compounds 5a and 5b are predicted to exhibit the strongest inhibition against the 6MTU protein in cancer.As shown in Table 5, the test compound 5a formed four H-bond acceptors: two between oxygen atoms of the carbonyl moiety with the active site residues Gly739 and Ile740, and another two H-bond acceptors between nitrogen atoms of the cyano group with Arg733 and Gly737.Additionally, in test compound 5b, four H-bond acceptors were formed: three between the oxygen atoms of carbonyl groups with the active site residues Ile740, Gly737, and Gly739, and another one between the nitrogen atoms of the cyano moiety with Gly737.The 3D model of compound 5c showed one H-bond donor between its N and Ile740.On the contrary, the docking of 5d gave one H-bond acceptor between the oxygen atom and the residue of Gly739.Finally, the docking of 11 gave one H-bond donor between the N of the hydrazinylidene moiety and the residue of Ile740; additionally, there were three H-bond acceptors: two between the nitrogen atom of the cyano group with the Arg733 and Arg801, and another one between the oxygen atom of the carbonyl group with the Leu738.

In Silico Pharmacokinetic Profile (ADMET)
The tested compounds showed intestinal absorption rates between 65.21% and 92.60%, with a volume of distribution (log L/kg) ranging from −0.51 to 0.30.The derivative 5a inhibited CYP3A4, while 5b and 5c were metabolized by CYP1A2.Additionally, derivatives 5c and 5d inhibited CYP3A4, CYP2C19, and CYP2C9.The compound 11 was metabolized by all of the cytochrome substrates and inhibitors.The evaluated substances' excretion was determined using a log value of ml/min/kg, with values ranging from −0.61 to 1.24.Table 6 displays all the previous detailed data.Subsequently, the toxicity prediction findings showed that none of the investigated substances had AMES toxicity.

In Silico Pharmacokinetic Profile (ADMET)
The tested compounds showed intestinal absorption rates between 65.21% and 92.60%, with a volume of distribution (log L/kg) ranging from −0.51 to 0.30.The derivative 5a inhibited CYP3A4, while 5b and 5c were metabolized by CYP1A2.Additionally, derivatives 5c and 5d inhibited CYP3A4, CYP2C19, and CYP2C9.The compound 11 was metabolized by all of the cytochrome substrates and inhibitors.The evaluated substances' excretion was determined using a log value of ml/min/kg, with values ranging from −0.61 to 1.24.Table 6 displays all the previous detailed data.Subsequently, the toxicity prediction findings showed that none of the investigated substances had AMES toxicity.

Experimental
The melting points of the newly synthesized compounds were determined using an electrothermal Gallenkamp apparatus, Cambridge, UK.IR spectra were obtained with a Pye-Unicam SP300 instrument (Pye, Cambridge, UK) using potassium bromide discs.Mass spectra were acquired using GCMS-Q1000-EX Shimadzu (Kyoto, Japan) and GCMS 5988-A HP spectrometers (HP Inc., Palo Alto, CA, USA), operating at an ionizing voltage of 70 eV.The 1 H-NMR and 13 C-NMR spectra were recorded using a Jeol-500 spectrometer (JEOL, Tokyo, Japan), operating at 500 MHz for 1 H-NMR and 125 MHz for 13 C-NMR.Elemental analyses were conducted at the Microanalytical Center of Cairo University, Giza, Egypt.

Cytotoxicity Assay
In the cytotoxicity and antitumor studies, cells were planted in 96-well plates at 5 × 10 4 cells/well and incubated for 24 h.Subsequently, different concentrations of the compounds were applied in six replicates per concentration.Control wells contained only medium or 0.5% DMSO.After a 24 h treatment period, the MTT assay was employed to evaluate cell viability [17,56].

Docking Study
Ligand preparation: Compounds with the lowest IC 50 values in the MTT assay were selected for docking with the target protein.Using CheDraw Professional 16.0, the potent molecules were sketched, and molecular modeling was performed with MOE software (http://structure.bioc.cam.ac.uk/pkcsm, accessed 20 May 2024).Minimizations were performed until a root mean square deviation (RMSD) gradient of 0.5 kcal•mol −1 Å −1 was achieved using MMFF 94× (Merck Molecular Force Field 94×) [63].The oriented compounds were then saved in MDB format for docking.
Protein preparation: Protein preparation involved downloading the enzyme's X-ray crystal structure (PDB ID: 6MTU, resolution: 2.14 Å) from the Protein Data Bank [64].The enzyme was prepared for docking as follows [65]: (1) Retaining the SEP co-crystalline ligand and removing crystallographic water molecules.(2) Adding hydrogen atoms with standard geometry, reconnecting broken bonds, and fixing potentials.(3) Using Alpha Site Finder to identify large sites with dummy atoms.(4) Marking the protein's binding pocket.
(5) Saving the identified pocket in Moe format for modeling ligand-enzyme interactions.Figure 3 shows the prepared protein.(6) Analyzing interactions between the ligand and active site amino acids.Following the docking process, both 2D and 3D interactions with amino acid residues were visualized.As per established protocols, all docking procedures and scoring were meticulously documented [66].
interactions with amino acid residues were visualized.As per established protocols, all docking procedures and scoring were meticulously documented [66].

In silico pharmacokinetic profile (ADMET)
Using pkCSM, the pharmacokinetic profile of Cisplatin and the most significant synthesized compounds were predicted [67].A web server that is publicly available (http://structure.bioc.cam.ac.uk/pkcsm, accessed 27 July 2024) offers a comprehensive platform for quickly assessing pharmacokinetic [68] and toxicity properties [69].Predicting the features of new compounds related to ADMET is an effort that requires linking their pharmacokinetic and toxicological characteristics [70].

Conclusions
This study demonstrates the effectiveness of green synthesis using L-proline as an organocatalyst, highlighting its reusability, mild conditions, clean reactions, easy workup, high purity, short reaction times, and high yields.The synthesized compounds, including various 2-cyano-N'-(2-cyanoacetyl)acetohydrazide derivatives, were structurally characterized and screened for anticancer activities against the HCT-116 colon carcinoma cell line.The reusability of L-proline was confirmed, making the synthesis protocol efficient and sustainable.Molecular docking studies and in silico ADMET investigations revealed that the most potent compounds have favorable binding properties and pharmacokinetic profiles.The docking study showed that compound 11 was the most effective agent against the colon cancer cell line.Additionally, studies using the ADMET tool showed that hydrazide compounds have drug-like properties.These findings suggest that the novel compounds have significant potential as targeted anti-colon cancer agents.The information supports the conclusion that, in 3OCH3, the use of advanced technology may result in the development and production of novel, potent colon anticancer medications based on these molecules.

Figure 2 .
Figure 2. Three-dimensional and two-dimensional ligand interactions within the binding site of 6MTU for potent synthesized compounds 5a-d, 11, and CCL.

Table 1 .
Comparison of the synthesis of products

Table 2 .
Optimization of the catalyst loading for the synthesis of compound 5a using the grinding method.

Table 4 .
In vitro cytotoxic effect of synthesized compounds against the HCT-116 cell line.

Table 5 .
Docking score (kcal/mol), No. of H-bonding, and No. of arene interaction of potent synthesized compounds 5a-d and 11 with the 6MTU receptor relative to the CCL.

Table 6 .
Results of ADMET properties of potent synthetic compounds 5a-d, 11, and Cisplatin.

Table 6 .
Results of ADMET properties of potent synthetic compounds 5a-d, 11, and Cisplatin.