Innovative Thiophene Schiff Bases: Synthesis and Evaluation as Antitubercular Agents

Tuberculosis (TB) is a highly contagious disease caused by Mycobacterium tuberculosis, primarily affecting the lungs but also spreading to other parts of the body (extra-pulmonary TB). It spreads through the air via droplets from infected individuals. The death rate from TB has increased in recent years, particularly among those with compromised immune systems such as HIV/AIDS patients, who are 18 times more likely to develop TB. COVID-19 has also contributed significantly to TB-related mortality due to overlapping infection sites.^1,2 In 2022, approximately 1.3 million people died from TB, including 167,000 with HIV, while 10.6 million people globally fell ill with TB.

Despite the availability of antitubercular drugs and ongoing vaccine trials, tuberculosis remains a significant challenge due to antimicrobial resistance. Resistance arises from both intrinsic factors, which occur naturally at high levels and extrinsic factors, including acquired mutations due to sub-optimal drug exposure. Factors such as bacterial load, mutation rates and virulence contribute to this resistance. This has led to the emergence of resistant forms like MDR-TB, XDR-TB and TDR-TB, prompting global concern.^3–5 Identifying essential metabolic pathways and components vital for the organism’s survival is critical for developing effective anti-TB drugs.

The cell wall of Mycobacterium tuberculosis is rich in mycolic acids, which are crucial for the bacterium’s survival and resistance. Among the key enzymes in mycolic acid synthesis, Pks13, a type I polyketide synthase, plays a pivotal role by condensing fatty acid intermediates into mycolic acids.^6–10 Pks13 is less sensitive to resistance and helps form a mycolic acid layer that reduces drug permeability, making it an attractive target for tuberculosis treatment.^11–13 This has led to a focus on discovering new drugs with novel mechanisms, either as independent treatments or in combination.

Heterocyclic rings have shown potent antitubercular activity. This study involves designing thiophene derivatives through a Schiff base reaction. Schiff bases, derived from carbonyl compounds and primary amines, are biologically active and found in many natural and synthetic compounds. Although thiophene-containing heterocyclic derivatives have shown promise as antitubercular agents, none are currently on the market, justifying further research and development.^14–17

Schiff bases have gained importance in medicinal chemistry for their diverse biological activities, including anti-inflammatory, analgesic,¹⁸ anticonvulsant, anticancer, antioxidant, anthelmintic, antimicrobial¹⁹ and antitubercular properties.²⁰ Those derived from aromatic aldehydes are particularly noted for their antibacterial and antifungal effects. With the rise of multi-drug-resistant and extensively drug-resistant tuberculosis, there is a crucial need for new, effective agents. Research indicates that Schiff bases with aromatic and heterocyclic rings show promising activity against Mycobacterium tuberculosis and other microorganisms, making them potential candidates to combat drug resistance in tuberculosis treatment.^15–17

Computer-Aided Drug Design (CADD) uses structural data from target proteins (structure-based) or known bioactive compounds (ligand-based) to identify potential drug candidates. Virtual screening, a key in silico technique, aids in discovering new compounds. Rational drug design within CADD leverages knowledge-driven approaches to elucidate protein-ligand interactions and binding affinities.^21,22 This investigation focuses on evaluating Schiff bases and thiophene heterocyclic amino ester derivatives for their potential antibacterial and anti-mycobacterial activity.

Schiff base thiophene derivatives were synthesized by reacting cyclohexanone and substituted aliphatic cyanoacetate in the presence of diethyl-λ⁴-sulfanimine and ethanol which act as catalysts to produce an intermediate compound, further intermediate compound reacts with various types of aromatic aldehyde. The reactants like cyclohexanone and aliphatic cyanoacetate and various aldehydes like bromobenzaldehyde, 1,3-dinitro benzaldehyde, 1-chloro-4-vinyl benzaldehyde, benzaldehyde, 4-chloro benzaldehyde were purchased from Southern India Scientific Corporation Limited Chennai.

Using conventional techniques, Schiff base thiophene derivatives were synthesized and their melting points determined by the open capillary tube method. Chemical purity was confirmed using thin-layer chromatography. The designed compounds were sketched with Chem3D Pro 12.0 and docked using AutoDock 4.2. Protein preprocessing involved UCSF Chimera, assigning Kollmann charges, generating grid points and performing molecular docking to analyze binding affinities and interactions.²³ Docked complexes were visualized with Biovia Molecular Discovery Studio. The pdb 5v3W protein, validated by resolution and outliers, was selected for docking. The best-ranked compounds underwent in silico analyses, including drug-likeness (Molsoft), bioactivity (Molinspiration) and ADMET parameters (pkCSM).²⁴

Molecules were designed based on literature surveys and SAR studies, leading to a library of Schiff base compounds created from amino ester derivatives and various aromatic aldehydes. The molecules were then evaluated using in silico methods such as docking, ADMET, molecular properties and bioactivity. Molecular docking involved five steps: protein preparation, ligand preparation, grid generation, docking and result analysis. The protein, downloaded from the protein databank in .pdb format, was validated using the Ramachandran plot. Preprocessing included selecting the suitable chain, extracting the co-crystallized ligand, analyzing the active site, generating grid points and minimizing the protein using the OPLS2005 force field. Molecular structures were drawn with ChemDraw Ultra 12.0,²⁵ minimized with Chem3D Pro 12.0 and scrutinized using the MMFF94 force field. The grid was generated to enclose the active site fully and docking was performed to form macromolecular complexes ranked by energy. Interactions were visualized using Biovia Discovery Studio Visualizer.²⁶

Molecular properties were analyzed using Molinspiration cheminformatics by sketching the compounds and extracting their SMILES codes. These were then subjected to analysis. The Lipinski rule of five was used, focusing on molecular weight <500 Da, log P≤5, hydrogen bond donors ≤5 and hydrogen bond acceptors ≤10, which correlate with approximately 90% of orally bioavailable drugs reaching phase II clinical trials. Next, the pharmacokinetic profile, including ADMET^27,28 properties (Absorption, Distribution, Metabolism, Excretion and Toxicity), was analyzed using the pkCSM server, considering factors like intestinal permeability and aqueous solubility.

Various aromatic ketones are treated with the active methylene groups like Ethyl and Methyl cyanoacetate to produce the amino esters in the presence of sulfur and ethanol and further, the amino esters are treated with various aromatic aldehydes to form the corresponding Schiff base product^29–31 and it shown in Figure 1.

Figure 1:
Scheme of the designed schiff base derivatives.

The melting points and R_f values of the synthesized compounds were preliminarily checked for purity and homogeneity. The final compounds were found to be soluble in organic solvent Ethanol/ methanol; compounds were also subjected to FTIR spectral studies, Nuclear Magnetic Resonance (NMR) Spectroscopy, Mass Spectroscopy and Elemental Data Analytical studies for structural elucidation, showed good results indicating successful completion of the reaction and absence of impurities.

The anti-mycobacterial activity of compounds against M. tuberculosis was evaluated using the Microplate Alamar Blue Assay (MABA). This method uses a thermally stable reagent and correlates well with proportional and BACTEC radiometric methods. To prevent medium evaporation, 200 μL of sterile deionized water was added to the outer perimeter wells of a 96-well plate. Each well received 100 μL of Middlebrook 7H9 broth and serial dilutions of the compounds were prepared on the plate, with final concentrations ranging from 100 to 0.2 μg/ mL. The plates were sealed with parafilm and incubated at 37°C for 5 days. After incubation, 25 μL of a 1:1 mixture of Alamar Blue reagent and 10% Tween 80 were added to each well and incubated for another 24 hr. Blue wells indicated no bacterial growth, while pink indicated growth. The MIC was the lowest concentration preventing the color change from blue to pink. The standard strain used was M. tuberculosis (H37 RV strain) ATCC No., with Pyrazinamide, Ciprofloxacin and Streptomycin serving as controls at 3.125 μg/mL, 3.125 μg/mL and 6.21 μg/mL, respectively.³²

Molecular Docking Studies: In silico docking simulations were used to identify ligands with high predicted binding affinity for protein PDB 5V3W. The top-ranked compounds were selected for synthesis and docking results, ranked by lowest energy, are presented in Table 1. Figures 2 and 3 shows the 2D and 3D images of the docked complexes, highlighting ligand-protein interactions.

Figure 2:
3D Interactions of Ca1 -Ca 10.

Figure 3:
2D Interactions of Ca1 -Ca 10.

ADME Properties: The pharmacokinetic properties of 10 designed molecules were evaluated using the pkCSM server for ADMET prediction, focusing on drug-likeness. These properties are detailed in Table 2.

Molecular Properties of Synthesized Compounds: Key molecular properties, such as log P, TPSA, volume, molecular weight, total number of atoms, number of rotatable bonds, hydrogen bond acceptors and hydrogen bond donors, are shown in Table 3.

Bioactivity: The selected compounds were evaluated for bioactivity against various receptors, including nuclear ligand-receptors, G-protein coupled receptors, tyrosine kinase-linked receptors, ion channels and protease and enzyme inhibitors. The results are shown in Table 4.

The antitubercular activity for the synthesized compounds was performed by microplate alamar blue assay method and the results were tabulated in Table 5.

Yield 76%, Melting point: 145°C; IR (Cyclohexane cm^-1): 3310 (N-H stretching), 760 (Thiophene, C-H bending), 1710 (C=O stretching), 1180 (C-O stretching), 2920 (C-H stretching), 1590 (C=N stretching), 1480 (C=C stretching), 630 (C-Br stretching); 1H NMR(400 MHz, DMSO): 7.65 (d, 2H, Ar-H, J≈8 Hz), 7.50 (d, 2H, Ar-H, J≈8 Hz,), 8.40 (s, 1H), 1.90 (m, 4H, CH₂), 2.60 (m, 4H, CH₂), 4.20 (q, 2H, OCH₂, J≈7 Hz), 1.30 (t, 3H, CH₃, J≈7 Hz). Compound -1 Molecular formula: C₁₇H₁₆BrNO₂S, Molecular weight: 392.31, Elemental Analysis: C (53.97), H (4.26), Br (21.12), N (3.70), O (8.46), S (8.48).

Yield 75%, Melting point: 163°C, IR (Cyclohexane cm^-1): 1730-1750 (C=O stretching), 1600-1620 (C=C stretching), 1650-1690 (C-N stretching), 500-600 (C-Br stretching), 690-740 (C-S stretching), 2850-2950 (C-H stretching), 3000-3100 (Aromatic C-H stretching); 1H NMR(400 MHz, DMSO): 7.2-7.8 (m, 4H), 8.0-8.5 (s, 1H, CH=N), 3.5-4.0 (s, 3H,OCH₃), 1.2-2.5 (m, 8H). Compound-2 Molecular formula: C₁₈H₁₈BrNO₂S, Molecular weight: 364.26, Elemental Analysis: C (55.11), H (4.62), Br (20.37), N (3.57), O (8.16), S (8.17).

Yield 85%, Melting point: 136°C, IR (Cyclohexane cm^-1): 1730-1750 (C=O stretching), 1600-1620 (C=C Aromatic stretching), 1630-1680 (C-N stretching), 1510-1560, 1320-1350 (NO₂ stretching), 690-740 (C-S stretching), 2850-2950 (Alkyl C-H stretching), 3000-3100 (Aromatic C-H stretching); 1H NMR(400 MHz, DMSO): 7.4-8.0 (m, 4H), 8.0-8.5 (s, 1H, CH=N), 1.2-1.4 (d, 3H, CH₂CH₃, 1.5-2.5 (m, 8H, CH₂ groups); Compound-3 Molecular formula: C₁₈H₁₇N₃O₆S, Molecular weight: 403.41, Elemental Analysis: C (53.59), H (4.25), N (10.42), O (23.80), S (7.95).

Yield 72%, Melting point: 141°C, IR (Cyclohexane cm^-1): 1730-1750 (C=O stretching), 1600-1620 (C=C Aromatic stretching), 1630-1680 (C-N stretching), 1510-1560, 1320-1350 (NO₂ stretching), 690-740 (C-S stretching), 2800-2950 (Methyl stretching), 3000-3100 (Aromatic C-H stretching); ¹H NMR (400 MHz, DMSO): 7.4-8.0 (m, 4H), 8.0-8.5 (s,1H, CH=N), 2.0-2.5 (s, 3H, CH₃), 1.5-2.5 (m, 8H, CH₂); Compound-4 Molecular formula: C₁₇H₁₅N₃O₆S, Molecular weight: 389.38, Elemental Analysis: C (52.44), H (3.88), N (10.79), O (24.65), S (8.23).

Yield 84%, Melting point: 185°C, IR (Cyclohexane cm^-1): 1730-1750 (C=O stretching), 1600-1620 (C=C Aromatic stretching), 1630-1680 (C-N stretching), 1600-1650 (C=C stretching,), 700-800 (C-Cl stretching), 690-740 (C-S stretching), 2800-2950 (C-H stretching), 3000-3100 (Aromatic C-H stretching); ¹H NMR (400 MHz, DMSO): 7.0-8.0 (m, 5H, Aromatic protons), 5.5-6.5 (m, 2H, CH=CH₂), 8.0-8.5 (s, 1H, CH=N), 2.0-2.5 (s, 3H,CH₃), 1.5-2.5 (m, 8H, CH₂ groups); Compound-5 Molecular Formula: C₁₉H₁₈ClNO₂S Molecular weight: 359.87, Elemental Analysis: C (63.41), H (5.04), N (3.89), O (8.89), S (8.91), Cl (9.85).

Ten novel compounds were designed and subjected to docking studies. Compounds Ca3 and Ca5 exhibited the lowest binding energies and highest binding affinities. Ca3 had a binding energy of -8.6 kcal/mol, interacting with amino acids TYR B:1582, VAL B:1614, LEU B:1615, ARG B:1634, VAL B:1611, PHE B:1670 and MET B:1669 through pi-pi stacking, T-shaped interactions, alkyl, pi-alkyl, conjugated hydrogen bonding and van der Waals forces. Ca5 had a binding energy of -8.4 kcal/mol, interacting with TYR B:1582, TRP B:1579, ALA B:1583, VAL B:1614, ALA B:1586 and PRO B:1595 through Pi-Sulfur, Van der Waals, alkyl and pi-alkyl interactions. The docking results, ranked by lowest energy, are shown in Table 1 and Figures 2 and 3 present the 2D and 3D images of the docked complexes, highlighting the ligand-protein interactions.

Using pkCSM server results, predicted water solubility (log P) ranged from -6.48 to -4.89, indicating moderate to low water solubility. Intestinal absorption was high (91.79 to 98.77) and the Volume of distribution (Vd) ranged from 0.18 to 0.61, suggesting wider distribution for compounds with lower Vd values. Compounds showed varying interactions with metabolic enzymes Cytochrome P2D6 and P3A4. Total renal clearance ranged from -0.08 to 0.34, suggesting moderate clearance, with oral rat acute toxicity between 2.23 to 2.75 indicating moderate oral clearance rates in rats. Compounds with desirable pharmacokinetic properties and good molecular docking results were selected for further studies.

The compounds’ LogP values ranged from 4.04 to 5.62, indicating moderate to low water solubility. TPSA values ranged from 38.67 to 130.2 Ų, suggesting moderate polarity; compounds with lower TPSA may have better passive membrane diffusion and higher oral absorption, while higher TPSA compounds may have lower diffusion due to greater polarity. The molecular weights ranged from 359.88 to 403.42 Da and the hydrogen bond acceptors, donors and numbers of rotatable bonds were within the acceptable range for orally administered drugs according to the Lipinski Rule of Five.

Compounds with values ranging from -0.34 to -0.64 indicate weak to moderate binding affinity with GPCR, with lower values suggesting weaker binding. Ion channel interaction showed stronger inhibition at -0.47 and weaker inhibition at -0.95. For IC₅₀ values, a lower value (closer to -0.58) indicates a stronger inhibitor, requiring a lower concentration for 50% inhibition, while a higher value (closer to -0.68) indicates a weaker inhibitor. Inhibition of protease enzymes ranged from -0.50 to -0.91, indicating weak inhibition. Similarly, enzyme inhibition values ranged from -0.26 to -0.85, also indicating weak inhibition.

Molecules with favorable docking, ADME predictions and biological activity were synthesized. Cyclohexanone (0.1 moles) reacted with ethyl cyanoacetate, methyl cyanoacetates, sulfur and diethyl amine (0.125 moles) in 20 mL of ethanol. The mixture was stirred for 3 hr at room temperature and refrigerated overnight. After adding 20 mL of ice-cold water, an amino ester was obtained. In the second step, the amino ester reacted with various aromatic aldehydes in 10 mL of ethanol to yield an imine base. Melting points ranged from 136-185°C, with yields between 72% and 85%. Reaction completion was monitored by TLC using n-hexane and ethyl acetate. The R_f values varied and ethanol was used for recrystallization.

Figure 4:
Anti-tubercular activity by MABA test.

It shows that the values range between the concentration ranging from 0.8-100 μg/mL. The compounds Ca3 and Ca5 were found to be active at 25 μg/mL and 12 μg/mL respectively whereas Ca1, Ca2 and Ca4 were active only at 50 μg/mL concentration. The color change from blue to pink in the microtitre plate is scored as growth, whereas blue indicates no growth and it is shown in Figure 4.

In this study, a series of new thiophene derivatives were synthesized using Schiff base reaction and screened for in silico antitubercular activity. All compounds satisfied Lipinski’s Rule of Five, indicating good oral bioactivity. Molecular docking revealed that compounds Ca3 and Ca5 had the best interactions with the target, showing the lowest binding energy. These compounds were optimized for future development. This investigation suggests that these thiophene derivatives can be used as leads for novel antitubercular agents.

We thank the Research Council of SRMIST and the Dean of SRM College of Pharmacy for their valuable support.