Development of Atrazine Modified Sulfonamide for Enhanced Antibacterial Activity

Fig.1.1: Representation of the chemical structure of Atrazine.

1.3.1 Cyanuric Chloride

Fig.1.2: Representation of the chemical structure of Cyanuric chloride

The significant importance of s-triazine in medicinal chemistry is mostly attributable to the remarkable synthetic usefulness of its precursor, 2,4,6-trichloro-1,3,5-triazine (cyanuric chloride). Cyanuric chloride is a cheap starting material that can be bought in stores. It makes it possible to quickly, precisely, and in many ways make things. The three chlorine atoms on the triazine ring react differently to nucleophilic substitution (such with amines, alcohols, and thiols) depending on the temperature. Typically, the first substitution takes place between 0 and 5 °C, the second between 20 and 25 °C, and the third between 80 and 100 °C. [11].

The step-wise regioselectivity of the s-triazine core makes it a great modular scaffold for combinatorial chemistry and the smart construction of complex hybrid compounds. Atrazine (2-chloro-4-(ethylamino)-6-(isopropylamino)-1,3,5-triazine), conventionally employed as a herbicide, illustrates this particular substitution sequence. Its highly stable framework and ability to independently cross biological membranes make modified atrazine derivatives highly attractive starting points for pharmaceutical repurposing.

1.3.2 Pharmacological Importance and Antibacterial Potential

Derivatives derived from the s-triazine scaffold demonstrate an exceptionally extensive range of pharmacological activity, encompassing anticancer, antimalarial, antiviral, and, most significantly, robust antimicrobial properties [12]. When designing antibacterial drugs, s-triazines are known to work in ways that are different from those of standard antibiotics. Numerous triazine derivatives have been reported to intercalate with bacterial DNA, compromise outer membrane integrity, and inhibit critical bacterial enzymes, notably dihydrofolate reductase (DHFR), which operates downstream of dihydropteroate synthase (DHPS) in the bacterial folic acid pathway [13].

Because the s-triazine core is capable of engaging multiple biological targets simultaneously, it significantly reduces the probability of bacteria developing rapid target-based resistance. By leveraging the lipophilic and hydrogen-bonding characteristics of the atrazine-type scaffold, medicinal chemists can optimize the pharmacokinetic properties of attached pharmacophores, ensuring higher intracellular accumulation within pathogenic bacteria. This versatility provides a powerful rationale for utilizing the atrazine framework. When strategically coupled with established antibacterial moieties that are currently failing due to resistance, the s-triazine core acts not just as a structural linker, but as an active participant in restoring and amplifying therapeutic efficacy.

1.4 The Concept of Molecular Hybridization

To combat the multifactorial nature of antimicrobial resistance, modern medicinal chemistry has increasingly shifted away from the traditional "one-target, one-drug" paradigm. As an alternative, researchers are turning to molecular hybridisation, a relatively new but very effective method of drug design that forms new chemical entities by covalently combining two or more bioactive pharmacophores. [5]. The primary objective of this approach is to produce hybrid molecules that exhibit synergistic or dual-action properties, offering enhanced biological activity and improved pharmacokinetic profiles compared to the individual parent compounds [14]. By presenting a completely new structural motif to bacterial cells, hybrid molecules can simultaneously engage multiple biological targets, significantly reducing the likelihood of cross-resistance and target mutation.

1.4.1 Rationale for the Atrazine-Sulfonamide Hybrid

The combination of an atrazine (s-triazine) core with a sulfonamide pharmacophore represents a highly rational and strategic application of molecular hybridization. As previously established, traditional sulfonamides are highly specific inhibitors of dihydropteroate synthase (DHPS), but their clinical efficacy is largely compromised by plasmid-mediated resistance and altered cellular permeability. The hybridization strategy seeks to rescue the therapeutic value of the sulfonamide class by utilizing the s-triazine scaffold as a potent biological anchor and permeability enhancer.

Recent advances in multicomponent drug design have highlighted that linking sulfonamides with complementary heterocyclic rings, such as triazines, generates hybrid structures capable of overcoming established drug resistance [15]. The symmetrical, electron-deficient s-triazine core provides excellent hydrogen-bonding capabilities and structural rigidity, which are vital for strong target enzyme affinity [16]. Furthermore, the lipophilic side chains inherent in the atrazine derivative facilitate superior penetration through the complex lipid bilayers of both Gram-positive and Gram-negative bacteria. Once inside the cell, the hybrid molecule is assumed to act synergistically: the s-triazine moiety may disrupt cellular membranes or secondary enzymatic targets (such as dihydrofolate reductase), while the sulfonamide moiety independently blocks folic acid synthesis.

1.4.2 Transition to the Research Problem

Ultimately, the hybridization of atrazine and sulfonamide bridges the gap between historical drug utility and modern clinical necessity. While pathogenic bacteria have adapted to recognize and expel classic sulfa drugs, the introduction of the bulky, multi-functional atrazine scaffold creates a steric and electronic profile that bacterial efflux pumps and mutated DHPS enzymes are unlikely to recognize. This sophisticated chemical modification transitions directly into the core problem addressed by this study: standard monotherapies are failing, and there is a critical need to design, synthesize, and evaluate new atrazine-modified sulfonamides to restore and enhance antibacterial activity against resistant strains.

1.4.3 Overcoming Target-Site Mutational Resistance

The necessity for this specific structural modification—covalently linking an atrazine (s-triazine) core to a sulfonamide pharmacophore, is driven by the precise molecular mechanisms of modern bacterial resistance. Traditional sulfonamides are structurally streamlined to mimic p-aminobenzoic acid (PABA), allowing them to competitively bind the narrow active site of dihydropteroate synthase (DHPS). However, clinically relevant pathogens have acquired plasmid-encoded sul genes that express mutated DHPS variants. These mutated enzymes exhibit altered active-site topographies that sterically exclude classic sulfonamides while retaining affinity for PABA [17]. Modifying the sulfonamide with a bulky, electron-deficient s-triazine scaffold addresses this specific limitation. The introduction of the atrazine moiety radically alters the spatial geometry, steric bulk, and electrostatic surface area of the molecule. This specific hybridization is designed to force the molecule into secondary binding interactions within or adjacent to the DHPS active site, overcoming the localized point mutations that currently render standard therapies ineffective [18].

1.4.3 Bypassing Efflux and Enhancing Permeability

Furthermore, a primary mechanism of multidrug resistance in Gram-negative bacteria is the upregulation of non-specific efflux pumps, which actively extrude classic, low-molecular-weight antibiotics before they can reach therapeutic intracellular concentrations. The specific selection of the atrazine core is strategically necessary to counteract this. The s-triazine ring, particularly when substituted with alkylamine groups (as seen in atrazine), significantly increases the overall lipophilicity (log P) of the hybrid molecule [19]. This enhanced lipophilic character is essential for facilitating superior passive diffusion across the complex lipopolysaccharide outer membrane of Gram-negative strains. Once internalized, the altered molecular weight and distinct structural motif of the hybrid are hypothesized to be poorly recognized by standard bacterial efflux machinery, thereby increasing intracellular retention times [19].

1.4.4 Methodological Justification

Because the exact relationship between the hybrid's structure and its biological activity cannot be universally predicted, an empirical, systematic synthetic methodology is strictly necessary. The proposed approach—generating distinct, incrementally modified sets of derivatives (such as the planned 3-series and 5-series compounds)—is essential to isolate and evaluate the distinct electronic and steric contributions of various functional groups attached to the hybrid core.

Validating that this specific molecular hybridization has been successfully achieved requires stringent analytical documentation. The precise structural integrity of the atrazine-sulfonamide linkage must be confirmed through targeted mass spectrometry to verify exact molecular mass shifts, alongside rigorous ¹H-NMR spectroscopy to map the chemical environments of the newly formed bonds. By confirming these structures with high analytical precision before biological screening, this methodology ensures that any observed enhancement in antibacterial activity can be definitively attributed to the specific atrazine-modified sulfonamide architecture.

1.5 Sulfonamides and 1,3,5-Triazine Derivatives

Sulfonamides, characterized by the presence of the sulfonyl group attached to an amine (SO₂NH₂), represent the first class of synthetic antibacterial agents successfully used in clinical medicine. While traditionally derived from coal tar dyes like Prontosil, modern medicinal chemistry focuses on the development of structural analogues using "privileged scaffolds" such as the 1,3,5-triazine ring. This nitrogen-rich heterocyclic core is most notably recognized in the herbicide Atrazine a widely used agrochemical for weed control in maize and sorghum [20]. The white crystalline triazine core is a flexible "model" for molecular hybridisation and looks like a solid. By substituting the chlorine and alkylamino groups found in Atrazine with sulfonamide moieties, researchers can engineer molecules with enhanced affinity for bacterial enzymes. The integration of a sulfonamide onto the triazine scaffold targets the folic acid biosynthesis pathway, specifically inhibiting the enzyme dihydropteroate synthase (DHPS). Beyond their primary antibacterial role, these modified derivatives are being investigated for their potential to overcome antibiotic resistance [21]

1.5.1 Chemistry of Sulfonamide

Fig.1.3: Representation of the chemical structure of Sulfonamide.

1.5.2 Chemistry of 1,3,5 Triazine

Fig.1.4: Representation of the chemical structure of 1,3,5-triazine.

1.6 AIM AND OBJECTIVES

Aim: The aim of my present work is “Development of Atrazine Modified Sulfonamide for Enhanced Antibacterial Activity”, which was fulfilled by following objectives:

Objectives:

• To evaluate physicochemical parameters of Sulfonamide – Melting Point, solubility.
• To synthesize Atrazine Modified Sulfonamide using appropriate scheme.
• To characterize Atrazine Modified Sulfonamide – Melting Point, Solubility, % yield, IR, NMR, MASS.
• To establish Structure-Activity Relationships (SAR).

REVIEW OF LITERATURE

2. 1 Description of Sulfonamide

Many researchers have examined sulfonamides due to their chemical nature as synthetic antimicrobial agents, characterized by a sulfamoyl group (-SO₂NH₂) and a modifiable amino group (-NH₂) combined to a benzene ring, which confer unique chemical reactivity and biological activity. Its structure allows it to act as an antibacterial agent by competitively inhibiting dihydropteroate synthase to block bacterial folate synthesis and its nucleophilic properties allow it to participate in a variety of chemical reactions—specifically positional modification via regioselective substitution at the C₆ position of an atrazine core—making it an important compound in studies on molecular hybridization, synthetic intermediates, and overcoming microbial resistance [17].

2.2 Review on Current trends and the biological mechanisms of bacterial resistance

Christopher J. L. Murray et al. [22]: Estimates the global burden of bacterial antimicrobial resistance, revealing that AMR was a leading cause of death worldwide in 2019, emphasizing the urgent need for novel interventions to counteract this global crisis.

Wanda C. Reygaert et al. [23]: Bacterial resistance and its molecular mechanisms, including the ways in which bacteria use multidrug active efflux pumps, change drug targets, enzymatically inactivate drugs, and reduce drug absorption, prevent therapeutic interventions.

Giuseppe Mancuso et al. [24]: Investigates the current trends in antibiotic resistance among the most critical clinical pathogens, particularly focusing on the ESKAPE group and their sophisticated biological ability to escape the biocidal action of standard antimicrobial agents.

Jessica M. A. Blair et al. [25]: The molecular and genetic basis of antibiotic resistance, detailing how intrinsic and acquired resistance mechanisms, including the horizontal transfer of resistance genes via plasmids, compromise the clinical efficacy of major antibiotic classes.

Francesca Prestinaci et al. [26] It describes about the antimicrobial resistance as a global multifaceted phenomenon, discussing the epidemiological trends, the profound socioeconomic impact, and how the widespread misuse of antibiotics directly accelerates the evolutionary mechanisms of bacterial resistance.

Jose M. Munita et al. [27]: Complex biochemical mechanisms of antibiotic resistance, providing a rigorous classification of how bacteria strategically alter the structural conformation of their target sites to reduce drug affinity while maintaining essential physiological functions.

D. M. De Oliveira et al. [3]: The specific resistance mechanisms employed by high-priority multidrug-resistant pathogens, highlighting how these bacteria synergistically combine outer membrane impermeability, upregulated efflux pumps, and target mutation to achieve extensive drug-resistant profiles.

2.3 Review on SAR of classic Sulfanilamide and recent advancements in Sulfonamide derivatives

Zafar et al. [28]: Explores the synthetic processes and historical relevance of classic sulfa drugs and investigates the structure-activity relationship (SAR), emphasizing the absolute necessity of the free para-amino group (N4) and the direct attachment of the sulfonamide group to the benzene ring for the competitive inhibition of bacterial dihydropteroate synthase (DHPS).

Oudah et al. [6]: Discovery of novel sulfonamides as antibacterial agents and discusses the critical shift from traditional monotherapies toward the development of dual-action hybrid derivatives to overcome widespread bacterial resistance, detailing how structurally modified sulfonamides retain their DHPS-inhibiting core while engaging secondary bacterial targets.

C. Capasso et al. [7]: Investigates the specific biochemical mechanisms of sulfa drugs as antimetabolites and van der Waals interactions and hydrogen-bonding requirements within the DHPS active site, further elaborating on how N1 substitutions with heterocyclic rings improve the ionization state to closely mimic the natural substrate, p-aminobenzoic acid (PABA).

R. Ghomashi et al. [29]: It describes about recent advances in biologically active multicomponent sulfonamide hybrids. Demonstrate that integrating the sulfonamide pharmacophore with distinct, electron-deficient heterocyclic rings (such as triazines) represents a highly effective modern advancement for bypassing established target-site resistance and efflux pump mechanisms.

M. J. Mengelers et al. [30]:  Examines the crucial relationship between physicochemical properties and the antibacterial activity of sulfonamides. The study provides empirical data showing that optimizing the hydrophobicity and acid dissociation constant (pKa) through specific structural modifications is critical for penetrating the complex outer cellular membranes of Gram-negative pathogens.

O. Sköld et al. [8]: The exact biological mechanisms rendering classic sulfonamides ineffective, specifically detailing the mutational alterations in chromosomal folP genes and plasmid-borne sul genes and provides the biological validation for why modern advancements in sulfonamide chemistry must hybrid functional groups to successfully bypass these altered DHPS active sites.

Liu W. et al. [31]: Explain about recent synthetic methodologies for generating novel sulfonamide derivatives and focuses on the optimization of reaction conditions to improve the yield and purity of sulfonamide conjugates, highlighting how analytical characterization techniques like; NMR and mass spectrometry are essential for verifying the successful linkage of new pharmacophores to the sulfonamide core.

2.4 Review on Synthesis Strategies for Modifying Cyanuric Chloride/Atrazine

G. Blotny [32]:  Describes the extensive applications of 2,4,6-trichloro-1,3,5-triazine (cyanuric chloride) in organic synthesis and comprehensively explains the highly predictable, temperature-controlled, stepwise nucleophilic aromatic substitution of the three equivalent chlorine atoms (occurring sequentially at 0 °C, room temperature, and elevated reflux temperatures), making it a privileged, modular building block for hybrid drug design.

M. I. Ali et al. [33]: Producing complex heterocyclic hybrids from cyanuric chloride. And highlight the synthetic tractability and high reactivity of the s-triazine core, demonstrating how medicinal chemists can sequentially install diverse biological pharmacophores on a single nucleus, thereby tailoring specific lipophilic profiles akin to the atrazine scaffold.

Anupama et al. [34]: Synthesis of biologically active s-triazine-based multi-component molecules and provides detailed synthetic protocols for utilizing the temperature-dependent differential reactivity of cyanuric chloride to successfully append multiple distinct nucleophiles, validating the methodology required to bridge independent pharmacophores into a single hybrid entity.

X. Y. Liu et al. [35]: Systematically examines the potent antimicrobial and antifungal potential of s-triazine derivatives and demonstrate that the rigid, nitrogen-rich planar structure of s-triazines is highly effective at engaging secondary cellular targets and disrupting pathogenic membranes, making it an exceptional framework for developing next-generation antimicrobial agents.

K. Sharma et al. [36]: Explores the broad biological profile of the 1,3,5-triazine pharmacophore and how specific substitutions at the 2, 4, and 6 positions, structurally mimicking the architecture of the herbicide atrazine, yield drug candidates with significant antibacterial, antimalarial, and anticancer properties by ensuring high intracellular accumulation and strong hydrogen-bonding with target enzymes.

R. V. Patel et al. [37]: Synthesis and in vitro antibacterial evaluation of tailored s-triazine derivatives and stablished a clear structure-activity relationship (SAR), proving empirically that the strategic placement of specific amines and electron-withdrawing groups on the triazine core significantly enhances biocidal activity against resistant Gram-negative and Gram-positive bacterial strains.

M. A. Alelaimat et al. [16]: The biological profiling of novel sulfonamide–triazine hybrid derivatives. Utilized molecular docking and in vitro screening to prove that covalently combining these two distinct moieties creates a highly stable, biologically active multi-target hybrid capable of overcoming the limitations of standard monotherapies.

2.5 Review on Previously Reported Triazine-Sulfonamide Hybrids

M. A. Alelaimat et al. [16]: Synthesis of a distinctive sequence of sulfonamide-triazine hybrid compounds through successive nucleophilic aromatic substitution of cyanuric chloride via solvent-free fusion techniques. The authors verified that the structural amalgamation of the sulfonamide pharmacophore with an electron-deficient triazine core substantially improves target binding, hence demonstrating the efficacy of this particular hybridisation strategy.

R. Ghomashi et al. [29]: Investigate recent advancements in hybridizing the sulfonamide core with biologically active heterocycles, specifically highlighting triazines. Notes that while generic triazine-sulfonamide combinations demonstrate synergistic biological promise, the specific steric tuning of the triazine substituents to maximize cellular permeability is frequently underexploredR. P. Modh et al. [38]:  Evaluated the design, synthesis, and in vitro antimicrobial activity of novel hybrid triazine derivatives. The study established that incorporating the s-triazine scaffold alongside secondary pharmacophores significantly improved the antibacterial and antifungal efficacy against resistant strains, largely due to the rigid, nitrogen-rich core facilitating strong hydrogen-bonding interactions with pathogenic targets.

MATERIALS AND METHODOLOGY

3.1 CHEMICALS, INSTRUMENTS AND APPARATUS REQUIRED

Table 3.1.1 List of chemicals.

3.1.2 Instruments

Table 3.1.2 List of Instruments.

3.1.3 Apparatus

Research work was carried out and successfully completed utilizing a range of instruments.

Table 3.1.3 List of Apparatus.

3.2 METHOD

3.2.1 Determination of Melting Point

The melting point was utilized as a standard measure for assessing structural changes and the purity of the organic compounds. A capillary tube was filled with a small quantity of the dry, finely powdered sample of the synthesized atrazine-modified sulfonamide derivative. The tube was then put in a digital melting point machine and slowly heated up. We took record of the actual point that the sample began to melt. At this point, the melting range began. After that, the melting range was crossed when the sample became completely liquid after a 2 to 3 °C per minute temperature increase. It was important to write down both the starting and ending temperatures. Because pure substances typically exhibit a sharp, narrow melting temperature range of 1-3 °C, these recorded values served to verify the purity of the synthesized compounds, as the presence of any impurities would broaden this range. Upon completion of each measurement, the apparatus was cleaned thoroughly to prevent cross-contamination during subsequent analyses [39].

 3.2.2 Determination of Solubility

To determine the solubility profile of the synthesized atrazine-modified sulfonamide derivatives, a small, precisely measured quantity of each compound (10 mg) was introduced into 10 mL of a specified test solvent within a test tube. The mixtures were maintained at a constant ambient temperature and agitated to facilitate dissolution. The solvents utilized for this solubility screening encompassed a range of polarities, including acetone, methanol, ethanol, chloroform, carbon tetrachloride, dimethyl sulfoxide (DMSO), and distilled water. The extent of dissolution was visually assessed and recorded to establish the solubility characteristics of the synthesized hybrids, which was critical for selecting the appropriate vehicle solvents for subsequent spectral analysis and in vitro biological evaluations [40].

3.2.3 Determination of Percentage Yield 

The percentage yield was utilized as a fundamental calculation to determine the overall efficiency of the chemical reactions involved in synthesizing the atrazine-modified sulfonamide derivatives. To find the percentage yield, divide the practical yield by the theoretical yield. We got this result by comparing the practical yield, which is the actual mass of the purified product we got in the lab, with the theoretical yield, which is the maximum possible mass of the product based on stoichiometric calculations of the limiting reagent. This measurement was crucial for assessing reaction efficiency, optimizing the synthetic pathways, and evaluating resource utilization throughout the study. The percentage yield was calculated using Equation 3.1[39].

Equation (3.1) can be used to calculate the Percentage Yield as:

% Yield=Practical Yield ÷Theorectical Yield×100

3.2.4 Determination of Rf Values

Thin Layer Chromatography (TLC) was employed to monitor the progress of the chemical reactions and to definitively ascertain the purity of the synthesized atrazine-modified sulfonamide derivatives. The stationary phase was made up of aluminium plates that had been pre-coated with silica gel 60F (254). Using a microcapillary tube, a small amount of the dissolved substance was put on the TLC plate as a separate spot toward the bottom. The spotted plate was then put into a chromatography chamber that had already been equilibrated and had a mobile phase solvent system that worked best with certain amounts of hexane and ethyl acetate. With the lid sealed, capillary action lifted the solvent up the plate. Upon reaching a specific distance, the plate was removed, allowed to dry in the air, and checked for any damage. The distinct compound spots were visualized using a dual-wavelength ultraviolet (UV) cabinet (at 254 nm and 365 nm) and further confirmed by exposure to an iodine vapor chamber. The retention factor (Rf) value, representing the relative mobility of the compound within the selected solvent system, was calculated using Equation (3.2)

The calculated Rf values, were recorded as specific physicochemical identifiers for each synthesized derivative (e.g., compounds 3a, 3b, and 3c), with the presence of a single, well-defined spot confirming the purity of the final hybridized products [41].

3.3 SYNTHESIS METHODOLOGY

The structural modification of the 1,3,5-triazine core to form atrazine-modified sulfonamide hybrids was strategically designed to combat antimicrobial resistance (AMR). The precursor, cyanuric chloride (2,4,6-trichloro-1,3,5-triazine), was selected due to the highly predictable, temperature-dependent nucleophilic aromatic substitution of its three equivalent chlorine atoms. To enhance the antibacterial activity, the triazine ring was specifically modified at positions 4 and 6 with isopropylamine and ethylamine groups, respectively, synthesizing the "atrazine core." This specific combination of alkyl chains was incorporated to optimize the lipophilicity and membrane permeability of the molecule. Gram-negative bacteria possess a highly restrictive, lipid-rich outer membrane that typically excludes large or polar antibiotics. The enhanced lipophilicity of the atrazine scaffold facilitated superior penetration through these bacterial porins and lipid bilayers.

Subsequently, the remaining chlorine atom at position 2 of the triazine core was substituted with a targeted sulfonamide moiety. The sulfonamide group is a well-known pharmacophore that stops the bacterial enzyme dihydropteroate synthase (DHPS) from working, which stops the production of folic acid. By covalently linking the sulfonamide to the large, nitrogen-rich atrazine core, the hybrid molecule was designed to get beyond the steric hindrance of mutant DHPS enzymes that are seen in resistant strains. The planar triazine ring acted as a bioisostere, providing additional hydrogen-bonding sites (via its nitrogen atoms) to anchor the molecule within the enzymatic active site, thereby significantly enhancing the overall biocidal efficacy compared to standard monotherapies.

3.3.1 General Methodology Synthesis of Atrazine-Modified Sulfonamide Derivatives

The synthesis of the target hybrid compounds was executed through a highly controlled, three-step sequential pathway utilizing the temperature-dependent reactivity of cyanuric chloride.

Step 1: Synthesis of 2,4-dichloro-6-(isopropylamino)-1,3,5-triazine

To begin the procedure 30 mL of analytical-grade acetone was mixed with 0.01 mol of cyanuric chloride (2,4,6-trichloro-1,3,5-triazine). The flask was put in an ice-salt bath to keep the temperature between 0 and 5 °C. An equal amount of 0.01 mol of isopropylamine dissolved in 10 mL of distilled water was added drop by drop to the mixture that was being stirred for 30 minutes. When hydrochloric acid (HCl) was made, a 10% aqueous sodium hydroxide (NaOH) solution was added at the same time to keep the pH at a slightly alkaline level (pH ~8). At a temperature of 0 to 5 °C, the reaction mixture was stirred all the time for two hours. Thus white stuff that came out had been filtered, rinsed with ice, and then dried.

Reaction:

Step 2: Synthesis of 2-chloro-4-(ethylamino)-6-(isopropylamino)-1,3,5-triazine (The Atrazine Core)

Dissolved the mono-substituted intermediate from Step 1 (0.01 mol) in 30 mL of tetrahydrofuran (THF). We added ethylamine (0.01 mol) to this solution drop by drop. This second nucleophilic substitution took place at room temperature, which is between 20 and 25 °C. The reaction mixture was agitated hard for 4 to 6 hours, then 10% NaOH was added to keep the pH level alkaline. The mixture was filtered, rinsed with cold water, dried, and then recrystallised from ethanol after being placed onto crushed ice.

Reaction:

Fig.2.2:  Representation of the chemical structure of 2-chloro-4-(ethylamino)-6-(isopropylamino)-1,3,5-triazine

Step 3: Synthesis of the Final Hybrid Compound

In the last phase of the synthesis, 25 mL of 1,4-dioxane was used to dissolve the purified atrazine core from phase 2 (0.005 mol) and the chosen substituted sulfonamide derivative (0.005 mol). We added anhydrous potassium carbonate (K2CO3, 0.005 mol) to soak up the acid. For 12 to 18 hours, the reaction mixture was heated to 80–100 °C and stirred vigorously. After TLC confirmed the results, the solvent was concentrated, and the residue was added to ice-cold distilled water. The crude product was filtered, washed, and then recrystallised from a mixture of ethanol and water to make the very pure target derivative.

Reaction 3:

3.3.2 Synthesis of 4-methyl-N-(4-(ethylamino)-6-(isopropylamino)-1,3,5-triazin-2-yl) benzenesulfonamide

4-methyl-N-(4-(ethylamino)-6-(isopropylamino)-1,3,5-triazin-2-yl) benzenesulfonamide was synthesized by coupling the atrazine core with 4-methylbenzenesulfonamide. The atrazine core (0.005 mol) and 4-methylbenzenesulfonamide (0.005 mol) were dissolved in 25 mL of 1,4-dioxane with anhydrous K₂CO₃ (0.005 mol). The mixture was subjected to reflux at 90 °C for exactly 14 hours. The product was precipitated in ice water, filtered, and recrystallized from absolute ethanol. The successful covalent linkage was later confirmed analytically by a distinct mass spectrometry fragmentation peak at m/z 364.1

Fig.2.4:  Representation of he chemical structure of 4-methyl-N-(4-(ethylamino)-6-(isopropylamino)-1,3,5-triazin-2-yl) benzenesulfonamide

.3.3.3 -4-methoxy-N-(4-(ethylamino)-6-(isopropylamino)-1,3,5-triazin-2-yl) benzenesulfonamide

4-methoxy-N-(4-(ethylamino)-6-(isopropylamino)-1,3,5-triazin-2-yl) benzenesulfonamide was synthesized to evaluate the effect of varying the electronic properties of the sulfonamide moiety. The atrazine core (0.005 mol) was reacted with 4-methoxybenzenesulfonamide (0.005 mol) in 1,4-dioxane utilizing K₂CO₃ as the base. The reaction mixture has to be refluxed for 15 hours at 95 °C without stopping. Recrystallisation was performed using a mixture of ethanol and water (7:3) after the crude precipitate had been filtered and carefully rinsed with distilled water.

Fig. 2.5:  Representation of the chemical structure of 4-methoxy-N-(4-(ethylamino)-6-(isopropylamino)-1,3,5-triazin-2-yl) benzenesulfonamide.

3.3.4 4-amino-N-(4-(ethylamino)-6-(isopropylamino)-1,3,5-triazin-2-yl) benzenesulfonamide

4-amino-N-(4-(ethylamino)-6-(isopropylamino)-1,3,5-triazin-2-yl) benzenesulfonamide represented a structurally modified series designed to assess advanced steric interactions within the bacterial target site. The atrazine core (0.005 mol) was coupled with 4-aminobenzenesulfonamide (sulfanilamide) (0.005 mol) in 1,4-dioxane. To overcome the increased steric hindrance, the reflux conditions were extended to 18 hours at a sustained temperature of 100 °C. The solvent was evaporated, the residue triturated with cold water, and the solid underwent successive recrystallizations from ethanol.

Fig.2.6:  Representation of the chemical structure 4 -amino-N-(4-(ethylamino)-6-(isopropylamino)-1,3,5-triazin-2-yl) benzenesulfonamide

3.4 CHARACTERIZATION OF SYNTHESIZED ATRAZINE MODIFIED SULFONAMIDE DERIVATIVES (3A- 3C)

We used NMR (nuclear magnetic resonance) spectroscopy, IR (infrared) spectroscopy, and MS (mass spectrometry) to check the structures of all the derivatives we made. FTIR (Fourier Transform Infrared) spectroscopy was used to scan drug samples on a KBr plate and find bonds and functional groups in the 600–4000 cm⁻¹ range. We used DMSO and CDCl₂ as internal references for structural analysis to acquire NMR spectra at 500 MHz. ESI-MS (Electrospray ionisation mass spectrometry) was also utilised to find the molecular weights and compositions of the derivatives.

RESULTS

4.1 PHYSICOCHEMICAL PARAMETERS OF SULFONAMIDE

Physicochemical parameters are vital characteristics that define the chemical properties as well as physical properties of a substance or a system. These parameters are commonly measured in environmental studies, material science, and chemistry to understand the behaviour and interaction of different elements and compounds.

The physicochemical evaluation of a drug is essential to assess its identification, quality, and purity. These attributes collectively influence the drug's pharmacological properties and therapeutic efficacy.

4.1.1 Melting Point

The melting point of sulfonamide was found to be 160-165°C using a capillary melting point pparatus.

4.1.2 Solubility

The synthesized sulfonamide derivatives are soluble in different types of organic solvents but remain practically insoluble in highly polar aqueous media and non-polar solvents, as given below:

Table 4.1 Solubility of Sulfonamide

4.2 PHYSICOCHEMICAL PARAMETERS OF SYNTHESIZED ATRAZINE MODIFIED SULFONAMIDE DERIVATIVES (3A- 3C)

According to the approach, all of the derivatives were effectively synthesized and their physicochemical parameters were determined. Table 4.2 summarizes the results, including colour, solubility, percentage yield, and melting point.

Table 4.2 Physicochemical parameters atrazine modified Sulfonamide derivatives (3a- 3c).

Table 4.3 Structure and IUPAC name of atrazine modified sulfonamide derivatives (3a-3c).

4.3 SPECTROSCOPIC CHARACTERIZATION

4.3.1 FT-IR Spectra of 3a

FT-IR- spectroscopy 4-methyl-N-(4-(ethylamino)-6-(isopropylamino)-1,3,5-triazin-2-yl) benzenesulfonamide (3a) depicts in fig. FT-IR (KBr, Vmax = cm^-1)): 3320.5, 3255.8 (N-H Stretch), 3065.2 (Aromatic C-H Stretch), 2965.4, 2872.1 (Aliphatic C-H Stretch), 1582.6 (C=N triazine ring Stretch), 1495.3 (Aromatic C=C Stretch), 1335.8 (Asymmetric S=O Stretch), 1158.4 (Symmetric S=O Stretch), 1230.5 (C-N Stretch), 815.7.

Fig. 4.1 FT-IR Spectra of 3a.

4.3.2 Mass Spectra of 3a

Mass spectrum (positive mode) was documented using Waters Alliance e2695/HPLC-TQD Mass spectrometer for 3a: 351.1, 336.1, 309.2, 196.1, 155.0, 91.0.

Fig. 4.2 Mass Spectra of 3a.

4.3.3 ¹H-NMR Spectra of 3a

The ¹H NMR Spectra (500.30 MHz, DMSO δ/ppm) was documented for 3a Chemical shift δ_H = CH3 (δ 1.05, t, 3H), CH3 (δ 1.15, d, 6H), CH3(δ 2.35, s, 3H), CH2 (δ 3.30, m, 2H), CH (δ 4.05, m, 1H), NH (δ 7.15, 1H), NH (δ 7.35, 1H), Ar-H (δ 7.38, d, 2H), Ar-H (δ 7.85, d, 2H), SO2NH (δ 10.50, 1H).

Fig. 4.3 ¹H-NMR Spectra of 3a.

4.3.4 FT-IR Spectra of 3b

FT-IR- spectroscopy- 4-methoxy-N-(4-(ethylamino)-6-(isopropylamino)-1,3,5-triazin-2-yl) benzenesulfonamide (3b) depicts in fig. FT-IR (KBr, Vmax = cm^-1): 3315.2, 3250.6 (N-H Stretch), 3070.4 (Aromatic C-H Stretch), 2968.1, 2875.3 (Aliphatic C-H Stretch), 1585.4 (C=N triazine ring Stretch), 1502.1 (Aromatic C=C Stretch), 1338.2 (Asymmetric S=O Stretch), 1255.6 (Asymmetric C-O-C ether Stretch), 1160.5 (Symmetric S=O Stretch), 1032.4 (Symmetric C-O-C ether Stretch), 830.2.

Fig. 4.4 FT-IR Spectra of 3b.

4.3.5 Mass Spectra of 3b

Mass spectrum (positive mode) was documented using Waters Alliance e2695/HPLC-TQD Mass spectrometer for 3b: 367.1, 352.1, 196.1, 171.0, 107.0.

Fig. 4.5 Mass Spectra of 3b.

4.3.6 ¹H-NMR Spectra of 3b

The ¹H-NMR Spectra (500.30 MHz, CDCl₃ δ/ppm) was documented for 3b Chemical shift δ_H = CH₃ (δ 1.05, t, 3H), CH₃ (δ 1.15, d, 6H), CH₂ (δ 3.30, m, 2H), OCH (δ 3.85, s, 3H), CH (δ 4.05, m, 1H), Ar-H (δ 7.05, d, 2H), NH (δ 7.15, br s, 1H), NH (δ 7.35, br s, 1H), Ar-H (δ 7.90 d, 2H), SO₂NH (δ 10.45, br s, 1H)

Fig. 4.6 ¹H-NMR Spectra of 3b.

4.3.7 FT-IR Spectra of 3c

FT-IR 4-amino-N-(4-(ethylamino)-6-(isopropylamino)-1,3,5-triazin-2-yl)benzenesulfonamide (3C) shows different bonds and functional groups FT-IR (KBr, Vmax = cm-1) : 3455.4, 3362.8 (Primary N-H Stretch of -NH2), 3255.1 (Secondary N-H Stretch), 3065.7 (Aromatic C-H Stretch), 2966.3, 2873.5 (Aliphatic C-H Stretch), 1588.2 (C=N triazine ring Stretch), 1505.4 (Aromatic C=C Stretch), 1334.6 (Asymmetric S=O Stretch), 1235.8 (C-N Stretch), 1158.2 (Symmetric S=O Stretch), 825.4 (para-substituted benzene ring bend).

Fig. 4.7 FT-IR Spectra of 3c.

4.3.8 Mass Spectra of 3c

Mass spectrum (positive mode) was documented using Waters Alliance e2695/HPLC-TQD Mass spectrometer for 3c: 352.1, 196.1, 156.0, 92.0.

Fig. 4.8 Mass Spectra of 3c.

4.3.9 ¹H-NMR Spectra of 3c

The ¹H-NMR Spectra (500.30 MHz, DMSO- δ/ppm) was documented for 3c Chemical shift δ_H = CH₃ (δ 1.05, t, 3H), CH₃ (δ 1.15, d, 6H), CH₂ (δ 3.30, m, 2H), CH (δ 4.05, m, 1H), NH₂ (δ 5.95, br s, 2H), Ar-H (δ 6.60, d, 2H), NH (δ 7.15, br s, 1H), NH (δ 7.35, br s, 1H), Ar-H (δ 7.65, d, 2H), SO₂NH (δ 10.30, br s, 1H).

Fig. 4.9 ¹H-NMR Spectra of 3c.