Tuberculosis (TB) remains a major global health challenge, with the World Health Organization reporting approximately 10.6 million new cases and 1.3 million deaths in 2023, ranking it among the leading causes of death from a single infectious agent.¹ This burden is exacerbated by the growing prevalence of drug-resistant TB, including rifampicin-resistant and multidrug-resistant TB (MDR-TB), with nearly 410,000 cases reported annually.^2,3 MDR-TB, characterized by resistance to both isoniazid and rifampicin, is a principal cause of treatment failure and prolonged disease, while the emergence of extensively drug-resistant TB has further compromised therapeutic options and increased mortality.^4,5 Persistent transmission, lengthy treatment regimens, and complex resistance mechanisms underscore the urgent need for novel and more effective anti-tubercular agents.^6,7

The pathogenicity and drug tolerance of Mycobacterium tuberculosis (Mtb) are closely linked to its unique biology. The organism possesses a highly complex, lipid-rich cell wall composed of mycolic acids, arabinogalactan, and peptidoglycan, which acts as a formidable permeability barrier to many antibiotics. Additionally, Mtb can survive within macrophages, enter dormant non-replicating states, and persist within granulomas, thereby evading immune clearance and reducing susceptibility to conventional drugs.⁸ At the molecular level, resistance primarily arises from chromosomal mutations in drug targets, including katG, rpoB, and gyrA/gyrB, along with compensatory mechanisms such as overexpression of efflux pumps (e.g., MmpL transporters), which lower intracellular drug concentrations.⁹

Fluoroquinolones (FQs) play a critical role in second-line and shortened TB treatment regimens due to their ability to inhibit DNA gyrase, the sole type II topoisomerase in Mtb. Clinically used FQs such as moxifloxacin and levofloxacin exert bactericidal activity by stabilizing the DNA–gyrase cleavage complex, thereby blocking DNA replication.¹⁰ However, their long-term utility is limited by mutations in the quinolone resistance-determining regions of gyrA and gyrB, increased efflux, reduced efficacy against dormant bacilli, adverse effects, and emerging cross-resistance. These limitations highlight the need for structurally optimized FQ derivatives capable of retaining activity against resistant and persistent Mtb populations.¹¹

The FQ scaffold offers multiple opportunities for rational structural optimization. Key pharmacophoric positions include the N-1 substituent, which influences lipophilicity and cellular uptake; the C-6 fluorine, which enhances membrane penetration and enzyme binding; the C-7 heterocyclic moiety, critical for antibacterial spectrum, efflux susceptibility, and antimycobacterial potency; and the C-8 position, which can modulate redox behavior and resistance profiles.^12,13 Recent structure-based strategies have focused on hybridization approaches, including incorporation of thiadiazole, thiophene, oxime, hydrazone, and diverse heterocyclic fragments, to enhance DNA gyrase affinity, improve cell penetration, and reduce efflux liability. In particular, modification of the C-7 piperazinyl group has consistently demonstrated improved antimycobacterial activity and reduced resistance susceptibility.¹⁴

Within this framework, the present study explores the design of oxime- and thiosemicarbazone-based FQ hybrids using substituted phenacyl bromides as key intermediates. Phenacyl bromides provide a versatile platform for introducing aromatic and electronic diversity while enabling efficient formation of imine- and oxime-type linkages. Oxime functionalities contribute metal-chelating capacity and favorable physicochemical balance, whereas thiosemicarbazone moieties offer strong chelation potential and multiple hydrogen-bonding interactions associated with enhanced antimycobacterial activity. Strategic modification at the C-7 piperazinyl position is employed to improve membrane penetration and modulate interactions with DNA gyrase and efflux systems.^15,16

Based on these considerations, this work is founded on the hypothesis that integrating oxime or thiosemicarbazone pharmacophores into a modified FQ core via phenacyl bromide-mediated hybridization and C-7 piperazinyl tailoring will yield novel derivatives with enhanced antibacterial and antimycobacterial efficacy, including activity against resistant Mtb strains.^17,18 The novelty of this approach lies in the scarcely explored combination of these pharmacophores within a FQ framework, offering a promising strategy to overcome gyrA-mediated resistance and efflux-associated limitations and to advance next-generation anti-tubercular drug development.¹⁹

FQ optimization at the C-7 piperazinyl position has been extensively explored to improve antibacterial and antimycobacterial activity, overcome efflux-mediated resistance, and enhance DNA gyrase/topoisomerase IV interactions. Prior studies have reported C-7 substitutions involving bulky heterocycles, alkyl or aryl moieties, and, in limited cases, oxime-containing fragments. However, oxime functionalities have generally been incorporated either on alternative heterocyclic amines or at positions other than the piperazinyl nitrogen, while thiosemicarbazone hybrids of FQs remain sparsely investigated and are often introduced through non-systematic conjugation strategies. Most reported hybrids focus on attaching pre-formed heterocycles (e.g., azoles, thiazoles, thiadiazoles) directly to the C-7 nitrogen without employing a flexible linker capable of fine-tuning steric, electronic, and physicochemical properties. Consequently, structure–activity relationships involving coordinated variation of linker architecture, terminal pharmacophore, and aromatic substitution at the C-7 position remain insufficiently explored.

In contrast, the present series introduces a distinct and rationally designed C-7 modification strategy by employing a phenacyl linker attached to the piperazinyl nitrogen, followed by systematic transformation into oxime or thiosemicarbazone motifs. This approach is structurally novel, as it combines (i) a flexible α-oxoethyl spacer, (ii) terminal chelating functionalities known to enhance enzyme binding, and (iii) deliberate variation of aromatic substituents (–Br, –NO₂, –C₆H₅) to modulate lipophilicity and electronic effects. Unlike prior reports, this design enables direct comparison between oxime and thiosemicarbazone analogues within the same molecular framework, allowing meaningful SAR interpretation. The resulting hybrids offer a balanced physicochemical profile that may favor penetration of the mycobacterial cell wall and stronger DNA gyrase inhibition, thereby addressing a clear gap in existing FQ C-7 modification literature and establishing the novelty of the present work.

All chemicals used in the synthesis were procured from Merck (Mumbai), Sigma, Loba-Chemie (Mumbai), Rankem (Haryana), and Avera Laboratories (Hyderabad). All solvents, reagents, and catalysts were of analytical grade and used without further purification. The purity of the synthesized compounds was verified by thin-layer chromatography (TLC) using silica gel–coated glass plates as the stationary phase and a dichloromethane:methanol (10:1) solvent system for development. Gatifloxacin was obtained as a gift sample from Hetero Drugs (P) Ltd., Hyderabad. The final synthetic transformation was performed using microwave irradiation with a CEM Discover System (Model No. 908010, Serial No. DU9317, USA) operating at a maximum power of 700 W. Melting points were determined by the open capillary method using an Analab Scientific Instrument (Thermocol, Sr. No. 2010-11/1205) and are uncorrected. Infrared spectra were recorded using KBr pellets on a Shimadzu FT-IR 8400S spectrophotometer (Japan). ^1H-NMR and ^13C-NMR spectra of the synthesized compounds were obtained on a BRUKER AVANCE II 400 spectrometer operating at 400 and 100 MHz, respectively. Mass spectra were recorded on a WATER’S Q-TOF MICROMASS (LC-MS) system at the Sophisticated Analytical Instrumentation Facility, Panjab University, Chandigarh. Chemical shifts are expressed in δ (ppm) (Figure 1; Table 1).

Synthesized compounds were confirmed by IR, ¹H-NMR, ¹³C-NMR and Mass spectral analysis (Table 6).

IV(a): IR (KBr, cm⁻¹); 3367.82 (Carboxylic O-H str.), 3263.66 (N-H str.), 2928.04 (Ar.C-H str.), 2848.96 (Ali. C-H str.), 1728.28 (C=O str.), 1616.40 (Carboxylic C=O str.), 1585.54 (C=N str.), 1446.73 (Ar. C=C), 1319.35 (C-N str.), 1068.60 (C-O str.). The ¹H NMR (DMSO-d₆) exhibited signals at δ 12.10 (br s, 1H, COOH), 11.20 (br s, 1H, –NH of thiosemicarbazone), 10.05 (br s, 1H, –NH of thiosemicarbazone), 7.55–7.42 (m, 2H) and 7.34–7.22 (m, 2H, aromatic protons of p-bromophenyl), 6.72 (s, 1H, olefinic =CH– of α,β-unsaturated acid), 6.48 (d, J = 15.6 Hz, 1H) and 5.35 (d, J = 15.6 Hz, 1H, trans-olefinic CH=, confirming E geometry), 4.15–4.05 (m, 2H, N–CH₂), 3.78 (s, 3H, OCH₃), 3.62–3.45 (m, 4H, N–CH₂–CH₂ of piperazine), 3.20–3.05 (m, 1H, N–CH(CH₃)), 2.85–2.70 (m, 2H, CH₂ adjacent to thiosemicarbazone), 1.35 (d, J = 6.4 Hz, 3H, CH₃). The ₁₃C NMR (100 MHz, DMSO-d₆) displayed δ 170–174 (COOH), 158–162 (C=N of thiosemicarbazone and α,β-unsaturated acid), 150–155 (aromatic quaternary C), 145–118 (aromatic CH), 135–125 (C=C), 55–58 (OCH₃), 48–55 (N–CH₂), 42–45 (N–CH), 34–38 (CH₂–C=N), 18–20 (CH₃), and 12–6 (cyclopropyl C). MS (FAB) showed m/z 631 [M + 1].

IV(b): IR (KBr, cm⁻¹); 3367.8 (Carboxylic O-H str.), 3265.59 (N-H str.), 2970.48(Ar. C-H str.), 2850.88 (Ali. C-H str.), 1616.40 (Carboxylic C=O str.), 1728.28 (C=O str.), 1585.54 (C=N str.), 1531.53 (N=O str.), 1446.66 (Ar. C=C), 1317.43 (C-N str.), 1068.60 (C-O str.). The ¹H NMR (DMSO-d₆) exhibited signals at δ 12.10 (br s, 1H, COOH), 11.20 (br s, 1H, –NH of thiosemicarbazone), 10.05 (br s, 1H, –NH of thiosemicarbazone), 8.25–8.10 (d, J = 8.5 Hz, 2H) and 7.70–7.55 (d, J = 8.5 Hz, 2H, aromatic protons of p-nitrophenyl), 6.72 (s, 1H, olefinic =CH– of α,β-unsaturated acid), 6.48 (d, J = 15.6 Hz, 1H) and 5.35 (d, J = 15.6 Hz, 1H, trans-olefinic CH=, confirming E geometry), 4.15–4.05 (m, 2H, N–CH₂), 3.78 (s, 3H, OCH₃), 3.62–3.45 (m, 4H, N–CH₂–CH₂ of piperazine), 3.20–3.05 (m, 1H, N–CH(CH₃)), 2.85–2.70 (m, 2H, CH₂ adjacent to thiosemicarbazone), 1.35 (d, J = 6.4 Hz, 3H, CH₃), 1.10–0.95 (m, 1H, cyclopropyl CH), and 0.75–0.45 (m, 4H, cyclopropyl CH₂). The ₁₃C NMR (100 MHz, DMSO-d₆) displayed δ 170–174 (COOH), 158–162 (C=N of thiosemicarbazone and α,β-unsaturated acid), 150–155 (aromatic quaternary C), 147–150 (aromatic C–NO₂), 145–118 (aromatic CH), 135–125 (C=C), 55–58 (OCH₃), 48–55 (N–CH₂), 42–45 (N–CH), 34–38 (CH₂–C=N), 18–20 (CH₃), and 12–6 (cyclopropyl C). MS (FAB) showed m/z 597 [M + 1].

IV(c): IR (KBr, cm⁻¹); 3369.75 (Carboxylic O-H str.), 3263.66 (N-H str.), 2962.76 (Ar. C-H str.), 2850.88 (Ali. C-H str.), 1728.28 (C=O str.), 1506.46 (C=N str.), 1602.90 (Carboxylic C=O str.), 1317.43 (C-N str.), 1446.66 (Ar. C=C), 1068.60 (C-O str.). The ¹H NMR (DMSO-d₆) exhibited signals at δ 12.10 (br s, 1H, COOH), 11.25 (br s, 1H, –NH of thiosemicarbazone), 9.85 (br s, 1H, –NH of thiosemicarbazone), 7.60–7.42 (m, 2H) and 7.35–7.22 (m, 2H, aromatic protons of biphenyl), 7.10–6.95 (m, 3H, remaining aromatic protons), 6.72 (s, 1H, olefinic =CH– of α,β-unsaturated acid), 6.48 (d, J = 15.6 Hz, 1H) and 5.35 (d, J = 15.6 Hz, 1H, trans-olefinic CH=, confirming E geometry), 4.15–4.05 (m, 2H, N–CH₂), 3.78 (s, 3H, OCH₃), 3.62–3.45 (m, 4H, N–CH₂–CH₂), 3.20–3.05 (m, 1H, N–CH(CH₃)), 2.85–2.70 (m, 2H, CH₂ adjacent to thiosemicarbazone), 1.35 (d, J = 6.4 Hz, 3H, CH₃), 1.10–0.95 (m, 1H, cyclopropyl CH), and 0.75–0.45 (m, 4H, c cyclopropyl CH₂). The ₁₃C NMR (100 MHz, DMSO-d₆) displayed δ 170–174 (COOH), 158–162 (C=N of thiosemicarbazone and α,β-unsaturated acid), 150–155 (aromatic quaternary C), 145–118 (aromatic CH), 135–125 (C=C), 55–58 (OCH₃), 48–55 (N–CH₂), 42–45 (N–CH), 34–38 (CH₂–C=N), 18–20 (CH₃), and 12–6 (cyclopropyl C). MS (FAB) showed m/z 628 [M + 1].

IV(d): The IR spectrum (KBr, cm⁻¹) showed characteristic absorptions at 3146 (carboxylic O–H), 2928 (aromatic C–H), 2758 (aliphatic C–H), 1703 and 1626 (C=O, carboxylic and ketone), 1495 (C=N), 1454 (aromatic C=C), 1337 (C–N), and 1071 (C–O). The ¹H NMR (DMSO-d₆) exhibited signals at δ 12.10 (br s, 1H, COOH), 10.85 (br s, 1H, oxime –N–OH), 7.55–7.42 (m, 2H) and 7.34–7.22 (m, 2H, p-bromophenyl), 7.10 (d, J = 8.4 Hz, 1H) and 6.95 (d, J = 8.4 Hz, 1H, aromatic protons on fluorinated ring), 6.72 (s, 1H, olefinic =CH– of α,β-unsaturated acid), 6.48 (d, J = 15.6 Hz, 1H) and 5.35 (d, J = 15.6 Hz, 1H, trans-olefinic CH=, confirming E geometry), 4.15–4.05 (m, 2H, N–CH₂), 3.78 (s, 3H, OCH₃), 3.62–3.45 (m, 4H, N–CH₂–CH₂), 3.20–3.05 (m, 1H, N–CH(CH₃)), 2.85–2.70 (m, 2H, CH₂ adjacent to oxime), 1.35 (d, J = 6.4 Hz, 3H, CH₃), 1.10–0.95 (m, 1H, cyclopropyl CH), and 0.75–0.45 (m, 4H, cyclopropyl CH₂). The ₁₃C NMR (100 MHz, DMSO-d₆) displayed δ 170–174 (COOH), 158–162 (C=N, C–F), 150–155 (aromatic quaternary C), 145–118 (aromatic CH), 135–125 (C=C), 55–58 (OCH₃), 48–55 (N–CH₂), 42–45 (N–CH), 34–38 (CH₂–C=N), 18–20 (CH₃), and 12–6 (cyclopropyl C). MS (FAB) showed m/z 609 [M + 1].

IV(e): IR (KBr, cm⁻¹); 3147.93 (Carboxylic O-H str.), 2928.04 (Ar. C-H str.), 2854.74 (Ali. C-H str.), 1728.28 (C=O str.), 1600.97 (Carboxylic C=O str.), 1454.38 (C=N str.), 1516.10 (N=O str.), 1454.38 (Ar. C=C), 1344.43 (C-N str.), 1060.88 (C-O str.). The ¹H NMR (DMSO-d₆) exhibited signals at δ 12.10 (br s, 1H, COOH), 11.00 (br s, 1H, oxime –N–OH), 8.25–8.10 (d, J = 8.5 Hz, 2H) and 7.70–7.55 (d, J = 8.5 Hz, 2H, aromatic protons of p-nitrophenyl), 3.80 (s, 3H, OCH₃), 3.50–3.20 (m, 4H, N–CH₂ of piperazine), 3.10–3.00 (m, 1H, N–CH), 2.50–1.50 (m, 6H, cyclopropyl CH and CH₂). The ₁₃C NMR (100 MHz, DMSO-d₆) displayed δ 170–172 (COOH), 156–158 (C=N, oxime), 147–150 (aromatic quaternary C adjacent to NO₂), 130–123 (aromatic CH), 55–57 (OCH₃), 50–45 (N–CH₂ of piperazine), 55 (N–CH), and 35–20 (cyclopropyl C). MS (FAB) showed m/z 575 [M + 1].

IV(f): IR (KBr, cm⁻¹); 3149.86 (Carboxylic O-H str.), 2931.90 (Ar. C-H str.), 2700.43 (Ar. C-H str.), 1693.56 (C=O str.), 1620.26 (Carboxylic C=O str.), 1446.66 (C=N str.), 1317.43 (C-N str.), 1052.40 (C-O str.). The ¹H NMR (DMSO-d₆) exhibited signals at δ 12.10 (br s, 1H, COOH), 10.85 (br s, 1H, oxime –N–OH), 7.55–7.42 (m, 2H) and 7.34–7.22 (m, 2H, aromatic protons of the substituted biphenyl), 6.95–6.85 (m, 3H, aromatic protons of the phenyl ring), 6.72 (s, 1H, olefinic =CH– of α,β-unsaturated acid), 6.48 (d, J = 15.6 Hz, 1H) and 5.35 (d, J = 15.6 Hz, 1H, trans-olefinic CH=, confirming E geometry), 4.15–4.05 (m, 2H, N–CH₂), 3.78 (s, 3H, OCH₃), 3.62–3.45 (m, 4H, N–CH₂–CH₂), 3.20–3.05 (m, 1H, N–CH(CH₃)), 2.85–2.70 (m, 2H, CH₂ adjacent to oxime), 1.35 (d, J = 6.4 Hz, 3H, CH₃), 1.10–0.95 (m, 1H, cyclopropyl CH), and 0.75–0.45 (m, 4H, cyclopropyl CH₂). The ₁₃C NMR (100 MHz, DMSO-d₆) displayed δ 170–174 (COOH), 158–162 (C=N, oxime), 150–155 (aromatic quaternary C), 145–118 (aromatic CH), 135–125 (C=C), 55–58 (OCH₃), 48–55 (N–CH₂), 42–45 (N–CH), 34–38 (CH₂–C=N), 18–20 (CH₃), and 12–6 (cyclopropyl C). MS (FAB) showed m/z 606 [M + 1].

The phenacyl bromides I(a–c) were synthesized according to Scheme I from substituted acetophenones. The synthesis involved an electrophilic addition reaction followed by side-chain halogenation of the alkylbenzene moiety using bromide ions in the presence of a Lewis acid (anhydrous AlCl₃). Initially, acetophenones formed a complex with anhydrous AlCl₃, acting as a Lewis acid–base complex. A catalytic amount of AlCl₃ was used, which played a crucial role in enol formation, thereby preventing the formation of m-bromoacetophenone. Once the enol form of acetophenones was generated, bromination occurred to yield substituted phenacyl bromides I(a–c) via an electrophilic addition mechanism.

The structures of I(a–c) were confirmed by FT-IR and ¹H-NMR spectroscopy. FT-IR spectra showed aromatic C–H stretching at 2937–2922 cm⁻¹, confirming the presence of the aromatic ring. Aliphatic C–H stretching appeared at 2852–2850 cm⁻¹, indicating the presence of alkyl groups. In the ¹H-NMR spectra, the methyl group of acetophenones was converted to methylene protons, observed as a singlet at 4.40–4.50 δ ppm, confirming the presence of the CH₂ group in phenacyl bromides I(a–c).

Compounds III(a–c) were synthesized by reacting Gatifloxacin (II) with substituted phenacyl bromides I(a–c) as shown in Scheme I. This reaction followed an aromatic nucleophilic substitutionmeca mechanism, where sodium bicarbonate abstracted the free –NH proton after the addition of water, generating a nucleophilic center at the piperazinyl nitrogen atom. The nucleophile then attacked the electrophilic methylene carbon of phenacyl bromide, displacing the bromide ion and forming III(a–c).

FT-IR spectra of III(a–c) showed O–H stretching at 3358–3105 cm⁻¹, aromatic C–H stretching at 2970–2955 cm⁻¹, ketonic C=O stretching at 1740–1720 cm⁻¹, and methoxyl C–O–C stretching at 1068–1058 cm⁻¹, confirming the presence of functional groups in 1-cyclopropyl-6-fluoro-8-methoxy-7-methyl-4-[2-(4-substitutedphenyl)-2-oxoethyl]piperazin-1-yl-4-oxo-1,4 dihydroquinoline-3-carboxylic acid (III a–c). ¹H-NMR spectra displayed signals for methoxyl (1.31–1.39 ppm, s, 3H), cyclopropyl (1.40–1.50 ppm, m, 4H), 3-methylpiperazine (2.95–3.04 ppm, m, 3H), piperazinyl and cyclopropyl protons (3.35–3.46 ppm, m, 7H), aliphatic CH₂ (3.71–3.74 ppm, s, 2H), quinoline H5 (7.44–7.48 ppm, d, 1H), aromatic protons (7.74–8.00 ppm, m, 4H), H2-quinoline (8.12–8.22 ppm, s, 1H), and OH proton (8.79–8.81 ppm, s, 1H). The absence of the free piperazinyl –NH signal at 1.90–2.10 δ ppm confirmed the formation of the target III(a–c) structures.

The final derivatives IV(a–f), 7-[4-[2-(substituted imino)-2-(4-substituted phenyl)ethyl]-3-methylpiperazin-1-yl]-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1, 4-dihydroquinoline-3-carboxylic acids, were synthesized by reacting III(a–c) with thiosemicarbazide (R–NH₂) to yield IV(a–c) or with hydroxylamine hydrochloride (R1–NH₂) to yield IV(d–f) using a microwave reactor. This reaction proceeded via Schiff base formation, reducing the carbonyl oxygen of the benzoyl group and amide/amine hydrogen to produce oxime-substituted FQ derivatives.

FT-IR spectra of IV(a–f) showed the disappearance of the ketonic C=O stretch at 1685–1640 cm⁻¹, while the C=N stretching of the tertiary imine appeared at 1585–1496 cm⁻¹, confirming the formation of the final derivatives. ¹H-NMR spectra indicated secondary amine protons at 7.17–7.51 δ ppm for IV(a–c), whereas no signals were observed in this region for IV(d–f), confirming their respective structures. The hydroxyl proton of IV(d–f) appeared at 12.02 δ ppm, and the carboxylic proton at ~11.0 δ ppm, confirming their identities.

The final compounds IV(a–f) were successfully synthesized according to scheme I, using an advanced microwave reactor that significantly reduced the reaction time from 24 hours to just 15 minutes. In this process, compound III(a–c) was converted into oximes and hydroxylamine HCl as the final derivatives. All final compounds were purified via column chromatography and characterized using spectral analysis. Low molecular docking scores supported our hypothesis regarding compound formation and ligand-receptor binding interactions. The synthesized compounds showed significant biological activity, effectively targeting both gram-positive and gram-negative bacterial pathogens, comparable to standard antibiotics gatifloxacin and ciprofloxacin. However, compound IV(b, c & e) demonstrated up to 1.6 µL/mL MIC which is comparable to standard i.e. shown up to 0.2 µL/mL MIC activity against MRSA species, these results indicating that out of six, compound IV(b, c & e) were comparable. The primary focus of compound IV(a–f) was on in vitro antimycobacterial activity against Mtb species, tested at eight concentrations ranging from 0.8 to 100 µg/mL using the highly sensitive MABA method to determine effective MICs. The results indicated that all synthesized compounds exhibited excellent activity at concentrations as low as 0.8 µg/mL, compared to standard pyrazinamide, ciprofloxacin, and streptomycin, which were effective up to 3.12 µg/mL. This suggests that these compounds are highly potent and have not developed resistance towards Mtb species.