Synthesis and In-Vitro Pharmacological Assessment of Fluoroquinolone Derivatives as Effective Antibacterial and Anti-TB Agents: An Experimental Study

Sourabh D. Jain¹, Kapil M. Agrawal², Sampat Singh Tanwar³ and Seema Sharma³
1. Department of Pharmaceutical Chemistry, Chameli Devi Institute of Pharmacy, Indore, India
2. Department of Pharmaceutical Chemistry, R. C. Patel Institute of Pharmacy, Shirpur, India
3. Department of Pharmacy, Shri Vaishnav Vidhyapeeth Vishwavidhalya, Indore, India
Correspondence to: Sampat Singh Tanwar, sampattanwar1999@gmail.com

DOI: https://doi.org/10.70389/PJS.100266

Additional information

• Ethical approval: Not applicable, as no animals or human subjects were used in this study.
• Consent: N/a
• Funding: No industry funding
• Conflicts of interest: The authors declare that there is no conflict of interest.
• Author contribution: Sourabh D. Jain — Formal analysis, Validation, Visualization, Writing – review & editing, Supervision. Kapil M. Agrawal — Resources, Supervision, Writing – review & editing, Correspondence. Sampat Singh Tanwar — Conceptualization, Project administration, Resources, Supervision, Writing – review & editing, Correspondence. Seema Sharma — Conceptualization, Methodology, Investigation, Data curation, Writing – original draft preparation.
• Guarantor: Sampat Singh Tanwar
• Provenance and peer-review: Unsolicited and externally peer-reviewed
• Data availability statement: N/a

Keywords: C-7 piperazine derivatization, MDR, Phenacyl bromide coupling strategy, piperazinyl-fluoroquinolone oxime hybrids, Thiosemicarbazone quinolone conjugates, XDR-tuberculosis DNA gyrase inhibition.

Peer Review
Received: 30 December 2025
Last revised: 11 February 2026
Accepted: 17 February 2026
Version accepted: 5
Published: 28 February 2026

Plain Language Summary Infographic

Introduction

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.

Material and Method

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).

Table 1: Structural variations in R and R1 groups of target compounds.
Compound	R	R1
IV(a)	–Br	NHC(=S)NH2
IV(b)	–NO2	NHC(=S)NH2
IV(c)	–C6H5	NHC(=S)NH2
IV(d)	–Br	OH.HCl
IV(e)	–NO2	OH.HCl
IV(f)	–C6H5	OH.HCl

Experimental

General Procedure of Phenacyl Bromide I(a–c)

Substituted acetophenones (0.1 mol) were placed in a two-neck round-bottom flask containing an appropriate anhydrous solvent such as ether, chloroform, acetone, or carbon tetrachloride. The reaction was carried out either at room temperature or under cooling conditions (5–10 °C). A catalytic quantity of anhydrous aluminum chloride was added, and the mixture was stirred for 1–4 hours. After this initial activation, bromine (0.1 mol) was added dropwise over approximately 1.5 hours with continuous stirring. During bromination, the reaction temperature typically increased to around 20 °C. Upon completion, the reaction mixture was poured onto crushed ice. The resulting solid was isolated by evaporating the solvent under reduced pressure. The crude phenacyl bromides I(a–c) were purified via recrystallization using rectified spirit, yielding brownish-yellow to nearly colorless crystalline products.^20,21

Melting points (°C): I(a) 109–111 °C, I(b) 92–95 °C, I(c) 123–125 °C.

Yields (%): I(a) 62%, I(b) 69%, I(c) 63%.

General Procedure for the Preparation of III(a–c)

Gatifloxacin (1.00 mmol), sodium bicarbonate (1.28 mmol), and the corresponding substituted phenacyl bromide (1.28 mmol) were combined in a round-bottom flask containing 10 mL of N,N-dimethylformamide. The reaction mixture was stirred at room temperature for approximately 20 hours. After completion, the mixture was poured into ice-cold water and extracted with dichloromethane (DCM). The organic layer was separated, washed thoroughly with water, and dried overnight over anhydrous magnesium sulfate. Removal of the solvent under reduced pressure yielded crude products III(a–c) as brownish-yellow solids. Purification was carried out by recrystallization using a mixed solvent system of ethanol and DCM, affording the pure amorphous derivatives III(a–c).

Melting points (°C): III(a): 145–147 °C, III(b): 110–112 °C, III(c): 140–142 °C.

Yields (%): III(a): 53%, III(b): 61%, III(c): 57%.

General Procedure for Preparation of IV(a–f)

Compound III(a–c) (1 mmol) was reacted with either thiosemicarbazide (2 mmol) or hydroxylamine hydrochloride (2 mmol) in the presence of sodium bicarbonate (2 mmol). The reagents were placed in a sealed reaction vial along with a 1:1 mixture of absolute methanol and DCM (total volume 10 mL). The sealed vial was subjected to microwave-assisted irradiation at 90 W for 15 minutes. After completion, the reaction mixture was concentrated under reduced pressure to obtain the crude products IV(a–f). Purification was performed by recrystallization using a mixed solvent system of ethanol and DCM, furnishing the pure brownish amorphous derivatives IV(a–f).

Melting points (°C): IV(a): 159–161 °C, IV(b): 170–172 °C, IV(c): 140–144 °C, IV(d): 168–170 °C, IV(e): 164–166 °C, IV(f): 181–182 °C

Yields (%): IV(a): 65%, IV(b): 61%, IV(c): 69%, IV(d): 55%, IV(e): 63%, IV(f): 68% (Figure 2; Tables 2 and 3).

Table 2: Physical data of synthesized FQ derivatives.
Compound	R Substituent	R1 Group	Molecular Weight (g/mol)	Molecular Formula	Rf Value
IV(a)	–Br	–NHC(=S)NH2	631	C29H33BrFN5O3S	0.37
IV(b)	–NO2	–NHC(=S)NH2	597	C29H33FN6O5S	0.39
IV(c)	–C6H5	–NHC(=S)NH2	628	C35H38FN5O3S	0.35
IV(d)	–Br	–OH·HCl	609	C28H32BrClFN3O4	0.33
IV(e)	–NO2	–OH·HCl	575	C28H32ClFN4O6	0.41
IV(f)	–C6H5	–OH·HCl	606	C34H37ClFN3O4	0.38

Table 3: Structure table of final compounds IV(a–f).
Compound	Core Scaffold (Constant for All)	R Position (Aromatic Ring of Phenacyl Moiety)	R1 Substitution (Oxime/Thiosemicarbazone Group)	Structural Class
IV(a)	FQ–phenacyl hybrid	–Br (para-bromo phenacyl)	–NHC(=S)NH2 (thiosemicarbazone)	Bromo–thiosemicarbazone hybrid
IV(b)	FQ–phenacyl hybrid	–NO2 (para-nitro phenacyl)	–NHC(=S)NH2	Nitro–thiosemicarbazone hybrid
IV(c)	FQ–phenacyl hybrid	–C6H5 (phenyl phenacyl)	–NHC(=S)NH2	Diphenyl–thiosemicarbazone hybrid
IV(d)	FQ–phenacyl hybrid	–Br	=NOH·HCl (oxime hydrochloride)	Bromo–oxime hybrid
IV(e)	FQ–phenacyl hybrid	–NO2	=NOH·HCl	Nitro–oxime hybrid
IV(f)	FQ–phenacyl hybrid	–C6H5	=NOH·HCl	Diphenyl–oxime hybrid

Pharmacological Screening

In-Vitro Antibacterial Activity of Title Compound IV(a–f)

The synthesized derivatives IV(a–f) were evaluated for in-vitro antibacterial activity against clinically relevant Gram-positive bacteria, Staphylococcus aureus (NCIM 2079) and Bacillus subtilis (NCIM 2250), and Gram-negative bacteria, Escherichia coli (NCIM 2109) and Pseudomonas aeruginosa (NCIM 2036). Ciprofloxacin, gatifloxacin, and streptomycin served as reference standards. All assays were conducted at the Microbiology Department, R. C. Patel Arts and Science College, Shirpur, Maharashtra, India, using nutrient broth for inoculum preparation and nutrient agar (Hi-Media) as the basal medium. Antibacterial activity was assessed via the agar well diffusion method. Test solutions were added to wells on agar plates seeded with standardized bacterial suspensions. After incubation, zones of inhibition were measured in millimeters and compared to those of the standard drugs. This approach provided a qualitative and semi-quantitative assessment of the compounds’ growth-inhibitory potential, effectively differentiating their activity against both Gram-positive and Gram-negative pathogens (Figure 3). The results are presented in Table 4.

Table 4: Zone of inhibition.
Zone of inhibition (mm)
Sr. No.	Compound	B. subtilis	S.aureus	E. coli	P. aeroginosa
1	IV(a)	25.80	20.38	27.56	17.52
2	IV(b)	21.68	19.65	24.97	13.66
3	IV(c)	24.06	19.56	27.19	14.40
4	IV(d)	23.42	20.21	29.16	19.19
5	IV(e)	26.88	18.65	26.77	16.35
6	IV(f)	25.97	23.87	26.34	14.76
7	Ciprofloxacin	25.46	28.17	32.56	26.63
8	Gatifloxacin	34.72	27.22	35.12	30.64
9	Streptomycin	19.17	18.63	18.51	18.45

Antibacterial Result

The antibacterial activity of the synthesized fluoroquinolone derivatives IV(a–f) was evaluated against two Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus) and two Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) using the zone of inhibition method. The results are summarized in Table 4. Among the tested compounds, IV(e) exhibited the highest activity against B. subtilis (26.88 mm), followed closely by IV(f) (25.97 mm) and IV(a) (25.80 mm), showing comparable activity to Ciprofloxacin (25.46 mm), though lower than Gatifloxacin (34.72 mm). Against S. aureus, compound IV(f) demonstrated the strongest inhibition (23.87 mm) among the synthesized derivatives.

However, the standard drugs showed comparatively higher activity, particularly Ciprofloxacin (28.17 mm) and Gatifloxacin (27.22 mm). In the case of E. coli, compound IV(d) exhibited the highest antibacterial effect (29.16 mm), surpassing Streptomycin (18.51 mm) and approaching the activity of Ciprofloxacin (32.56 mm). Compounds IV(a), IV(c), IV(e), and IV(f) also showed notable inhibition (>26 mm), indicating strong activity against Gram-negative bacteria. For P. aeruginosa, compound IV(d) again demonstrated the best activity among the synthesized compounds (19.19 mm), though the inhibition zones were lower compared to Ciprofloxacin (26.63 mm) and Gatifloxacin (30.64 mm). Overall, the synthesized derivatives displayed significant antibacterial activity against both Gram-positive and Gram-negative strains, with compounds IV(d), IV(e), and IV(f) emerging as the most promising candidates.

In-Vitro Antitubercular Activity of Title Compound

The anti-mycobacterial activity of the synthesized derivatives was evaluated against Mtb H37Rv using the broth dilution assay. Minimum inhibitory concentrations (MICs) were determined in Middlebrook 7H9 broth supplemented with 10% ADC (albumin-dextrose-catalase) and 0.2% glycerol. Frozen cultures were used to prepare inoculate at 2 × 10⁵ CFU/mL. Serial dilutions of the test compounds (0.8–100 µg/mL) were prepared in U-tubes to assess growth inhibition (Figures 4 and 5). The results are presented in Table 5.

Table 5: In-vitro anti-TB activity result.
Sr. No.	Compound	100 µg/mL	50 µg/mL	25 µg/mL	12.5 µg/mL	6.25 µg/mL	3.12 µg/mL	1.6 µg/mL	0.8 µg/mL
1	IV(a)	S	S	S	S	S	S	S	S
2	IV(b)	S	S	S	S	S	S	S	R
3	IV(c)	S	S	S	S	S	S	S	S
4	IV(f)	S	S	S	S	S	S	S	S
5	Gatifloxacin	S	S	S	S	S	S	S	S
6	Pyrazinamide	S	S	S	S	S	S	R	R
7	Ciprofloxacin	S	S	S	S	S	S	R	R
8	Streptomycin	S	S	S	S	S	R	R	R

Spectral Data

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

Table 6: In-vitro anti-TB activity result (quantitative MIC).
Sr. No.	Compound	MIC (µg/mL)
1	IV(a)	≤0.8
2	IV(b)	1.6
3	IV(c)	≤0.8
4	IV(f)	≤0.8
5	Gatifloxacin	≤0.8
6	Pyrazinamide	3.12
7	Ciprofloxacin	3.12
8	Streptomycin	6.25

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-Nstr.), 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].

Result and Discussion

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.

Biological Activity

In-Vitro Antibacterial Screening

The antibacterial activity of IV(a–f) was evaluated using the disc diffusion method against Gram-positive bacteria (B. subtilis and S. aureus) and Gram-negative bacteria (E. coli and P. aeruginosa) at 100 µg/mL. Zones of inhibition were measured in millimeters, and MICs were determined for MRSA. IV(a–f) showed moderate activity against Gram-positive pathogens (21.68–26.88 mm for B. subtilis and 18.65–23.87 mm for S. aureus), comparable to ciprofloxacin (25.46 & 28.17 mm) and gatifloxacin (34.72 & 27.22 mm). The activity was influenced by electron-withdrawing substituents (bromo, nitro, phenyl). Against Gram-negative bacteria, IV(a–f) exhibited lower activity due to the double-layered cell wall, requiring higher concentrations to penetrate. Only IV(d), an oxime-containing bromo-substituted analogue, showed comparable activity against E. coli (29.16 mm). MRSA activity showed IV(a–f) were effective up to 3.12 µg/mL, with IV(b, c, e) showing inhibition at 1.6 µg/mL, indicating sensitivity towards Gram-positive MRSA strains.

In-vitro Antimycobacterial Screening

The screening results indicate that most of the synthesized compounds demonstrated consistent sensitivity (S) across the tested concentration range from 100 to 0.8 µg/mL. Compounds IV(a), IV(c), and IV(f) showed activity at all concentrations, comparable to the reference drug gatifloxacin within the tested limits, suggesting strong inhibitory potential up to the lowest evaluated dose (≤0.8 µg/mL). Compound IV(b) remained active down to 1.6 µg/mL but showed resistance (R) at 0.8 µg/mL, indicating slightly lower potency. Among standard drugs, pyrazinamide and ciprofloxacin showed sensitivity up to 3.12 µg/mL but resistance at lower concentrations (1.6 and 0.8 µg/mL), while streptomycin exhibited sensitivity only up to 6.25 µg/mL, becoming resistant at further dilutions. Overall, the data suggest that selected test compounds maintain inhibitory activity comparable to standard drugs within the evaluated concentration range, with effective action observed at ≤0.8–1.6 µg/mL for the most active derivatives.

Structure-Activity Relationship (SAR) Analysis

The antibacterial and antimycobacterial activities of compounds IV(a–f) reveal a consistent dependence on both the electronic nature of the aromatic substituent (R) on the phenacyl moiety and the terminal linker functionality (oxime or thiosemicarbazone). Substituents with strong electron-withdrawing character, such as bromo and nitro groups, generally enhanced antibacterial potency relative to unsubstituted phenyl analogues, particularly against Gram-negative strains (E. coli and P. aeruginosa). This trend can be attributed to increased polarization of the phenacyl–piperazinyl linkage, which may strengthen electrostatic and hydrogen-bonding interactions within the DNA gyrase–quinolone cleavage complex. In contrast, the phenyl-substituted derivatives exhibited balanced activity across Gram-positive and Gram-negative organisms, likely due to increased lipophilicity facilitating membrane penetration, though without the strong electronic activation provided by halogen or nitro groups.

Comparison of the two linker types further highlights the importance of terminal functionality. Thiosemicarbazone-linked derivatives (IVa–c) consistently displayed superior or comparable antibacterial activity relative to their oxime counterparts, particularly against B. subtilis and E. coli. The presence of the thiocarbonyl (C=S) group and additional hydrogen-bond donor sites may enhance binding affinity through metal chelation or interaction with key residues in DNA gyrase, a mechanism previously associated with thiosemicarbazone pharmacophores. Conversely, oxime derivatives (IV d–f), while slightly less potent in some bacterial assays, demonstrated excellent antimycobacterial activity, showing complete inhibition of Mtb H37Rv down to the lowest tested concentration. This suggests that the oxime linker confers an optimal balance of polarity and lipophilicity, favoring penetration of the lipid-rich mycobacterial cell wall and stable target engagement. Overall, the SAR indicates that electron-withdrawing aromatic substituents combined with a chelating thiosemicarbazone linker favor broad-spectrum antibacterial potency, whereas oxime-linked analogues maintain strong anti-TB activity, underscoring the complementary roles of electronic modulation and linker design in optimizing FQ hybrids.

Conclusion

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.

References

1. Fan YL, Wu JB, Ke X, Huang ZP. Design, synthesis and evaluation of oxime-functionalized nitrofuranylamides as novel antitubercular agents. Bioorg Med Chem Lett. 2018;28(18):3064–6. https://doi.org/10.1016/j.bmcl.2018.07.046
2. Mohammed AAM, Suaifan GARY, Shehadeh MB, Okechukwu PN. Design, synthesis and antimicrobial evaluation of novel glycosylated-fluoroquinolone derivatives. Eur J Med Chem. 2020;202:112513. https://doi.org/10.1016/j.ejmech.2020.112513
3. Chen PT, Lin WP, Lee AR, Hu MK. New 7-[4-(4-(un)substituted)piperazine-1-carbonyl]-piperazin-1-yl] derivatives of fluoroquinolone: synthesis and antimicrobial evaluation. Molecules. 2013;18(7):7557–69. https://doi.org/10.3390/molecules18077557
4. Lalavani NH, Gandhi HR, Bhensdadia KA, Patel RK, Baluja SH. Synthesis, pharmacokinetic and molecular docking studies of new benzohydrazide derivatives possessing anti-tubercular activity against Mycobacterium tuberculosis H37Rv. J Mol Struct. 2021;131884.https://doi.org/10.1016/j.molstruc.2021.131884
5. Bhavani GV, Kondapuram SK, Shamsudeen AF, Coumar MS, Selvin J, Kannan T. Synthesis, antitubercular evaluation, and molecular docking studies of hybrid pyridinium salts derived from isoniazid. Drug Dev Res. 2023;84(3):470–83. https://doi.org/10.1002/ddr.22039
6. Swedan HK, Kassab AE, Gedawy EM, Elmeligie SE. Design, synthesis, and biological evaluation of novel ciprofloxacin derivatives as potential anticancer agents targeting topoisomerase II enzyme. J Enzyme Inhib Med Chem. 2023;38(1):118–37. https://doi.org/10.1080/14756366.2022.2136172
7. Jain SD, Jain K, Khan A, Prachand S, Gupta AK. A review on the role of moxifloxacin in the treatment and management of respiratory tract infections. J Pharma Insights Res. 2025;3(4):290–300. https://doi.org/10.69613/ayysyk71
8. Zhang Y, Ma H, Wang H, Xia Q, Wu S, Meng J, et al. Forecasting the trend of tuberculosis incidence in Anhui Province based on machine learning optimization algorithm, 2013–2023. BMC Pulm Med. 2024;24(1):536. https://doi.org/10.1186/s12890-024-03296-z
9. Naidoo K, Perumal R, Cox H, Mathema B, Loveday M, Ismail N, et al. The epidemiology, transmission, diagnosis, and management of drug-resistant tuberculosis—lessons from the South African experience. Lancet Infect Dis. 2024;24(9):e559–75. https://doi.org/10.1016/S1473-3099(24)00144-0
10. Wei S, He C, Xie X, Zhang A, Tang S, Li S, et al. Which fluoroquinolone is safer when combined with bedaquiline for tuberculosis treatment: evidence from FDA Adverse Event Reporting System database from 2013 to 2024. Front Pharmacol. 2024;15:1491921. https://doi.org/10.3389/fphar.2024.1491921
11. Kanchrana M, Gamidi RK, Kumari J, Sriram D, Basavoju S. Design, synthesis, anti-mycobacterial activity, molecular docking and ADME analysis of spiroquinoxaline-1,2,4-oxadiazoles via [3 + 2] cycloaddition reaction under ultrasound irradiation. Mol Divers. 2024;28(6):3979–91. https://doi.org/10.1007/s11030-023-10790-9
12. Jain SD, Prachand S, Gupta AK, Jain S. Fluoroquinolones for the treatment of tuberculosis: an overview. Asian J Res Pharm Sci. 2023;13(4):333–7. https://doi.org/10.52711/2231-5659.2023.00057
13. Chen Z, Wang T, Du J, Sun L, Wang G, Ni R. Decoding the WHO global tuberculosis report 2024: a critical analysis of global and Chinese key data. Zoonoses. 2025;5(1):999.
14. Gutiérrez-Mauricio AM, Trujillo-Paez JV, Trejo-Martinez LA, Rivas-Santiago B, Pérez-García P, Noriega S, et al. Tuberculosis drug development: fluoroquinolone structural tailoring. J Antibiot (Tokyo). 2025;78(9):517–34. https://doi.org/10.1038/s41429-025-00839-2
15. Sharma V, Saini M, Das R, Chauhan S, Sharma D, Mujwar S, et al. Recent updates on antibacterial quinolones: green synthesis, mode of interaction and structure-activity relationship. Chem Biodivers. 2025;22(5):e202401936. https://doi.org/10.1002/cbdv.202401936
16. Poyraz Yılmaz P, Kulabaş N, Bozdeveci A, Vagolu SK, Imran M, Tatar E, et al. Synthesis of novel thiazole/thiadiazole conjugates of fluoroquinolones as potent antibacterial and antimycobacterial agents. Chem Biol Drug Des. 2025;105(5):e70126. https://doi.org/10.1111/cbdd.70126
17. Gudapati VS, Basireddy SR, Gudapati DV, Afzal M, Ayub A, Kolli SK. New 4-thiazolidinone-bearing fluoroquinolone derivatives: synthesis, antimicrobial effectiveness, structural characterization, and in silico docking analysis. Russ J Gen Chem. 2025;95(3):630–643. https://doi.org/10.1134/S1070363224612754
18. Mamolo MG, Carosati E, Pasin D, De Logu A, Cabiddu G, Jukič M, et al. Synthesis, antimycobacterial activity, and computational insight of novel 1,4-benzoxazin-2-one derivatives as promising candidates against multidrug-resistant Mycobacterium tuberculosis. ChemMedChem. 2025;20(14):e202500073. https://doi.org/10.1002/cmdc.202500073
19. Patel UK, Alka, Tiwari P, Tilak R, Joshi G, Kumar R, et al. 1,2,3-Triazole-tethered fluoroquinolone analogues with antibacterial potential: synthesis and in vitro cytotoxicity investigations. RSC Adv. 2025;15(3):1896–914. https://doi.org/10.1039/d4ra08643k
20. Agrawal KM, Talele GS. Synthesis and antibacterial, antimycobacterial activity of 7-[4-{5-(2-oxo-2-p-substituted-phenylethylthio)-1,3,4-thiadiazol-2-yl}-3′-methylpiperazinyl] quinolone derivatives. J Chem. 2013;2013:147565. https://doi.org/10.1155/2013/147565
21. Chen YL, Fang KC, Sheu JY, Hsu SL, Tzeng CC. Synthesis and antibacterial evaluation of certain quinolone derivatives. Journal of Medicinal Chemistry. 2001;44(14):2374–2377. https://doi.org/10.1021/jm0100335

Cite this article as:
Jain SD, Agrawal KM, Tanwar SS and Sharma S. Synthesis and In-vitro Pharmacological Assessment of Fluoroquinolone Derivatives as Effective Antibacterial and Anti-TB Agents: An Experimental Study. Premier Journal of Science 2026;18:100266

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