Design, Synthesis and Pharmacological Evaluation of Some C₃ Heterocyclic-Substituted Ciprofloxacin Derivatives as Chimeric Antitubercular Agents

Abstract

A series of new C₃ heterocyclic-substituted ciprofloxacin derivatives were prepared from ciprofloxacin acid hydrazide as possible chimeric molecules. They were evaluated for their possible in vitro antibacterial (agar cup/bore diffusion method) and antitubercular (Lowenstein–Jensen (LJ) slant method) activities. The results indicated that all the test compounds are highly effective against all the bacterial strains and have shown excellent anti-tubercular activity against normal, multidrug resistant and extensively drug resistant strains of Mycobacterium tuberculosis. They were found to be more potent antibacterial and antitubercular agents than the standard, ciprofloxacin. The minimum inhibitory concentration (MIC)’s of all the compounds against M. tuberculosis were found to be 0.0625 µg/mL as compared to ciprofloxacin (MIC = 2 to > 8 µg/mL). Molecular docking studies were performed by using AUTODOCK 4.2 on the new ciprofloxacin derivatives at the active site of crystal structure of fluoroquinolones target enzyme Mtb DNA gyrase GyrA N-terminal domain (PDB ID: 3ILW) and also on the active site of crystal structure of chosen heterocyclics target enzyme enoyl-acyl carrier protein (ACP) reductase enzyme (PDB ID: 4TZK). Interestingly, almost all the compounds have shown relatively greater binding affinity at both the active sites than ciprofloxacin. Compound 6 exhibited the highest affinity for 3ILW and 4TZK.

Introduction

Bacteria represent an outsized domain or kingdom of prokaryotic microorganisms. Pathogenic bacteria cause severe infectious diseases, widely prevalent throughout the world. One of the bacterial diseases with highest disease burden is tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (Mtb), which kills about 2 million people a year. TB is a chronic infection and its condition is worsened by the existence of multidrug resistant tuberculosis (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB) strains. In view of such a devastating nature of the disease, WHO had declared Tuberculosis (TB) as a “Global Health Emergency.” This particular disease is also known to be one of the most severe health problems as it causes not only ‘morbidity’ leading to loss of human work hours which is detrimental to National Economy, but also culminates in ‘mortality.’²⁾

Fluoroquinolones are the major class of antibiotics useful for the treatment of tuberculosis. They act mainly by DNA gyrase and topoisomerase IV inhibition.³⁾ Isatin is an endogenous indole found in mammalian brain, peripheral tissues, and body fluids. Heterocyclic moieties like isatin, phthalimide and 1,3,4-oxadiazole are also reported to possess antibacterial and antitubercular activities.^4–6) They act by inhibiting the enzyme enoyl-ACP reductase.^7–9)

Ciprofloxacin is one of the widely used fluoroquinolones that exhibits potent in vitro and in vivo antimycobacterial activity. Fluoroquinolones are also found to be active against diverse types of bacteria, including Staphylococcus (S.) aureus, S. epidermis, Bacillus (B.) subtilis, Escherichia (E.) coli and Mtb, at concentrations less than 1 µg/mL. Fluoroquinolones are therapeutically advantageous because of their extended antimicrobial activity, lack of plasmid-mediated resistance, large volume of distribution (or greater amount of tissue distribution) and minimal adverse effects.¹⁰⁾

In view of this, the area of fluoroquinolones has experienced an exponential growth over the last few decades and is still being pursued with more vigor to make available better drugs having multifunctional action.¹¹⁾ Chimeric drugs, a broad class of ‘Multi-functional compounds’ are the single entity molecules that constitute two or more pharmacophoric groups representing different mechanisms of action. They possess advantages such as reduced molecularity, improved pharmacokinetics and pharmacodynamics, devoid of drug–drug interactions etc.^12–14) They are known to produce response by interacting with respective receptors of constituent pharmacophores, thus restoring the efficacy of individual drugs they represent. In this context, chemotherapy is the prime area of attention, hence the emergence of chimeric antibiotics to provide most effective multimechanistic, multimodal, multipotential molecules to treat more effectively the diseases like tuberculosis. Till date there are not many reports on chimeric fluoroquinolones.^15,16) Hence in continuation of our works on developing anti-tuberculosis agents,^17–19) now it is felt worthwhile to make an attempt to bring some potential pharmacophoric moieties of anti-tubercular molecules (viz., fluoroquinolones and isatins/phthalimides/oxadiazoles) acting by different mechanisms, into a single molecular framework (Fig. 1) to produce a new fluoroquinolone-based chimeric compounds, hopefully with improved spectrum, selectivity and efficacy.

Fig. 1. Designing of New Chimeric Ciprofloxacin Antibiotics Containing Two Pharmacophores

Results and Discussion

Molecular Property Prediction

Molecular descriptors like Log P (partition coefficient), Log S (solubility), molecular weight, number of hydrogen bond acceptors and donors in a molecule are used to access the molecular properties like membrane permeability, hydrophobicity and bioavailability. The drug-likeness of the designed molecules has been evaluated using Lipinski’s rule of five.²⁰⁾

Table 1 shows the molecular properties of ciprofloxacin (1), its acid hydrazide (3) and the newly designed compounds (4–8) using Molsoft (version: V.3.7-2), Osiris property explorer (version: 2) and Molinspiration (version: 2018.03) softwares. Except for a few minor acceptable violations,²¹⁾ majority of the compounds obey the Lipinski rule of five. The enzyme inhibitor activity of the test compounds were within the range of a majority of drug-like molecules (−0.5 to +0.5).

Table 1. Molecular Property Prediction of Ciprofloxacin and Its Derivatives Using MOLSOFT (Version: V.3.7-2), OSIRIS Property Explorer and MOLINSPIRATION Softwares

Compd.	Toxicity	M.Wt	C Log P	Solubility	TPSA	HBA	HBD	%ABS	Drug likeness	Enzyme inhibitor score
1	No	331.35	−1.5	—	72.8	4	2	88.72	0.93	0.28
3	Tumorigenic	345.30	1.22	−0.62	90.7	4	4	81.64	1.11	0.12
4	No	564.62	3.41	−6.42	97.3	5	2	81.50	1.12	−0.30
5	No	582.61	3.51	−6.73	97.3	5	2	81.50	1.33	−0.37
6	No	609.62	3.21	−7.42	143.1	7	2	69.98	0.58	−0.50
7	No	475.48	0.46	−4.86	102	5	2	79.30	0.59	0.09
8	No	387.40	0.88	−4.42	113.3	6	2	88.30	0.53	0.03

(M.Wt: molecular weight, C Log P: Calculated Log P, TPSA: Topological polar surface area, HBA: Hydrogen bond acceptors, HBD: Hydrogen bond donors)

Prediction of Activity Spectra for Substances (PASS)

Activity of the new molecules is predicted by PASS software by comparing its structure with that of well-known biologically active substrates existing in the database.²²⁾ All the test compounds have shown good Pa (Probability to be active) values for antimycobacterial, antitubercular and antibacterial activities (Table 2). Amongst them, compound 6 displayed highest Pa values for antimycobacterial and antitubercular activities.

Table 2. PASS Prediction Values of Ciprofloxacin and Its Derivatives

Compound	Antimycobacterial		Antitubercular		Antibacterial
	Pa	Pi	Pa	Pi	Pa	Pi
1	0.639	0.008	0.786	0.004	0.588	0.009
3	0.801	0.004	0.664	0.005	0.576	0.010
4	0.727	0.005	0.587	0.006	0.423	0.025
5	0.700	0.005	0.547	0.008	0.400	0.030
6	0.808	0.004	0.717	0.004	0.444	0.022
7	0.733	0.005	0.592	0.006	0.495	0.017
8	0.385	0.030	0.339	0.008	0.340	0.046

Pa = Probability to be active; Pi = Probability to be inactive.

Molecular Docking

Molecular docking is the most extensively used method for the calculation of protein–ligand interactions. It is commonly performed for predicting the binding modes of ligands to proteins and their binding energies.²³⁾ AUTODOCK is an open-source software for drug discovery, molecular docking. Docking of the new ciprofloxacin derivatives (4–8) into the binding site of Mtb DNA gyrase GyrA N-terminal domain (PDB ID: 3ILW) and Enoyl-ACP reductase enzyme (PDB ID: 4TZK) and estimating the binding affinity of the complexes is a significant part of the structure based drug design process. The structural interactions between 3ILW and 4TZK with 5 inhibitors (4–8) were carried out separately by docking studies and the results were compared with the parent inhibitor, ciprofloxacin (Fig. 2).

Fig. 2. Docking Interactions of Ciprofloxacin, Compounds 5 and 6 with 3ILW and 4TZK

A,B,C-ciprofloxacin, compounds 5 and 6 with 3ILW respectively; D,E,F-ciprofloxacin, compounds 5 and 6 with 4TZK respectively; G,H,I-ciprofloxacin, compounds 5 and 6 with the active site of 3ILW respectively; J,K,L-ciprofloxacin, compounds 5 and 6 with the active site of 4TZK, respectively.

In the present study, compounds 4–8 have been identified as potent antibacterial agents. Experimental activities and predicted values by Lamarckian Genetic Algorithm dockings of the five compounds are summarized in Tables 3, 4. All the derivatives selected for molecular docking have some collective structural features. All the compounds have shown relatively greater binding affinity at the active site of 3ILW than ciprofloxacin. Compounds 4–6 have also exhibited superior affinity at the active site of 4TZK. Compound 6 exhibited hydrogen bond with Leucine137 and has shown highest affinity towards 3ILW with a binding energy of −8.64 kcal/mol and dissociation constant (K_i) of 466.8 nM. This compound interacted with Pro151 of 4TZK by hydrogen bond and also exhibited high affinity with a binding energy of −9.35 kcal/mol and dissociation constant (K_i) of 140.14 nM. Compound 5 also exhibited good binding affinity with 4TZK, having binding energy of −9.46 kcal/mol and dissociation constant (K_i) of 115.72 µM. It also displayed good interaction with 3ILW. Thus the title compounds are predicted to inhibit both the enzymes and can serve as chimeric antibiotics to overcome drug resistance.

Table 3. Molecular Docking Interactions and Their Binding Energy of Compounds 4–8 with 3ILW

Ligand	R	R1	Interacting amino acids	Grid X-Y-Z coordinates	Binding energy ΔG (kcal/mol)	Dissociation constant (Ki)
4	–H	–H	—	9.892, −14.723, 81.543	—	1.99 µM
5	5-F	–H	Gly179	9.892, −14.723, 81.543	−8.01	1.36 µM
6	5-NO2	–H	Leu137	9.892, −14.723, 81.543	−8.64	466.8 nM
7	—	—	Phe116	9.892, −14.723, 81.543	−6.56	15.42 µM
8	—	—	Pro108	9.892, −14.723, 81.543	−6.29	24.03 µM
Ciprofloxacin	—	—	Arg495	9.892, −14.723, 81.543	−5.60	67.29 µM

Table 4. Molecular Docking Interactions and Their Binding Energy of Compounds 4–8 with 4TZK

Ligand	R	R1	Interacting amino acids	Grid X-Y-Z coordinates	Binding energy ΔG (kcal/mol)	Dissociation constant (Ki)
4	–H	–H	—	15.642, 28.949, 59.629	−9.43	122.47 nM
5	5-F	–H	Pro151	15.642, 28.949, 59.629	−9.46	115.72 µM
6	5-NO2	–H	Pro151	15.642, 28.949, 59.629	−9.35	140.14 nM
7	—	—	Lys118	15.642, 28.949, 59.629	−5.22	148.59 µM
8	—	—	Glu62	15.642, 28.949, 59.629	−5.02	210.32 µM
Ciprofloxacin	—	—	Ser176	15.642, 28.949, 59.629	−5.65	71.74 µM

Chemistry

Reaction of ciprofloxacin (1) with thionyl chloride and ethanol afforded ciprofloxacin ethyl ester (2).²⁴⁾ Hydrazinolysis of (2) resulted in ciprofloxacin acid hydrazide (3).²⁵⁾ Condensation of acid hydrazide (3) with N-benzylisatins in presence of ethanol and acetic acid yielded the isatin substituted ciprofloxacin derivatives (4–6).²⁶⁾ In another two different experimental setups, reaction of ciprofloxacin acid hydrazide (3) with phthalic anhydride,²⁷⁾ potassium hydroxide and carbon disulfide²⁸⁾ produced compounds (7) and (8), respectively (Chart 1).

Chart 1. Synthesis of C₃ Heterocyclic-Substituted Ciprofloxacin Derivatives 4–8

Pharmacological Evaluation

The test compounds (4–8) were screened for their possible antibacterial and antitubercular activities against different Gram-positive, Gram-negative, sensitive and resistant strains of mycobacterium.

Antibacterial Activity

In vitro antibacterial activity was done by using cup plate method,²⁹⁾ in triplicate. Compounds 4–6 were tested against E. coli, Klebsiella pneumonia (Gram −ve), B. mirabilus and S. aureus (Gram +ve) at the concentrations of 75 and 100 µg/mL. Compounds 7 and 8 were tested against E. coli, Proteus vulgaris (Gram −ve), S. aureus and Micrococcus luteus (Gram +ve) at the concentrations of 25, 50, 100 µg/mL. Ciprofloxacin was used as reference drug for comparison and dimethyl sulfoxide (DMSO) as control. The results obtained indicate that the ciprofloxacin derivatives (4–6) exhibited potent activity against all the bacterial strains at the concentrations of 75 and 100 µg/mL, and all of them were found to be relatively more potent than the standard ciprofloxacin. Compound 5 with fluoro substitution on isatin moiety showed to be superior in antibacterial activity, comparatively. Compounds 7 and 8 also exhibited significant antibacterial activity against the chosen strains, comparable to the standard. At higher concentrations, they were found to be equipotent to ciprofloxacin against S. aureus and more potent against E. coli and M. luteus. The results are discussed in Tables 5, 6 and represented in Fig. 3 and Fig. 4 (Graphpad Prism version 5.0).

Table 5. In Vitro Antibacterial Activity of Compounds 4–6

Compounds	Concentrations (µg/mL)	Diameter of zone of inhibition (mm)
		Gram-negative		Gram-positive
		E. colia)	K. pneumonia	B. mirabilus	S. aureusa)
4	75	10.5 ± 0.5*	10.8 ± 0.3*	9.5 ± 0.4*	9.8 ± 0.5*
	100	11.1 ± 0.2*	10.6 ± 0.5*	10 ± 0.4*	9.5 ± 0.2*
5	75	12 ± 0.4***	11 ± 0.6***	10 ± 0.3***	11 ± 0.3***
	100	14 ± 0.7****	12 ± 0.4****	11 ± 0.5****	12 ± 0.2****
6	75	9 ± 0.3	10 ± 0.4	9 ± 0.6	8 ± 0.2
	100	11.5 ± 0.3**	11 ± 0.3**	10 ± 0.6**	10 ± 0.4**
Ciprofloxacin	75	8 ± 0.6	9 ± 0.4	8 ± 0.3	8 ± 0.4
	100	10 ± 0.4a)	8 ± 0.3	9 ± 0.4	8 ± 0.4a)

a) Evaluation results mentioned in Table 6 for ciprofloxacin were carried out independently at different times when compared to those of Table 5.

Table 6. In Vitro Antibacterial Activity of Compounds 7 and 8

Compounds	Conc. (µg/mL)	Diameter of zone of inhibition (mm)
		Gram-negative		Gram-positive
		E. colia)	P. vulgaris	S. aureusa)	M. luteus
7	25	8 ± 0.5****	8 ± 0.7****	9 ± 0.6****	8 ± 0.4****
	50	14 ± 0.4*	11 ± 0.6*	13 ± 0.7*	10 ± 0.6*
	100	16 ± 0.6	13 ± 0.8	16 ± 0.5	15 ± 0.2
8	25	6 ± 0.4****	6 ± 0.5****	6 ± 0.6****	6 ± 0.6****
	50	12 ± 0.2*	10 ± 0.6*	13 ± 0.3*	12 ± 0.4*
	100	15 ± 0.3	14 ± 0.2	16 ± 0.3	15 ± 0.4
Ciprofloxacin	25	10 ± 0.2	8 ± 0.4	6 ± 0.5	6 ± 0.4
	50	13 ± 0.9	12 ± 0.7	11 ± 0.4	14 ± 0.3
	100	14 ± 0.2a)	15 ± 0.5	16 ± 0.5a)	14 ± 0.7

Significance values *(p < 0.1), **(p < 0.01), ***(p < 0.001), ****(p < 0.0001). All the values are expressed in mean ± standard deviation. Comparisons are done by oneway ANOVA followed by Dunnet’s test for multiple comparisons.

Fig. 3. Antibacterial Activity of Compounds (4–6)

Fig. 4. Antibacterial Activity of Compounds 7 and 8

Antitubercular Activity

Compounds 4–8 which exhibited significant antibacterial activity and which showed good C log P values were tested in vitro against Mtb H37Rv strain (ATC C 27294 susceptible to rifampicin, pyrazinamide and isoniazid) and against clinical isolates of multidrug resistant strain of Mtb (MDR-TB, 104/14) (resistant to isoniazid and rifampicin), extensively drug resistant strain of Mtb (XDR-TB, 220/19) (resistant to isoniazid, rifampicin and fluoroquinolones) by Lowenstein–Jensen (LJ) slant method.¹⁸⁾ The activity is expressed as minimum inhibitory concentration (MIC), i.e., the lowest concentration of compound that completely inhibited the growth on the culture. The activity for the above said compounds was performed at graded concentrations: 0.0625, 0.125, 0.25, 0.5, 0.75, 1 µg/mL, keeping in view the MIC of ciprofloxacin (0.5–2 µg/mL) against Mtb H37Rv control strain.^30,31) The results indicated that all the compounds (4–8) were promising candidates with uniquely good activity against both sensitive and resistant strains of M. tuberculosis (H37Rv, 104/14 & 220/19) (Table 7). The MIC of all the compounds was found to be 0.0625 µg/mL against H37Rv, MDR-TB (104/14) and XDR-TB (220/19) and they were found to be more potent than the standard, ciprofloxacin.

Table 7. Antitubercular Activity of Compounds 4–8 against Normal and Resistant Strains of Mycobacterium tuberculosis

Compounds	M. tuberculosis (H37Rv)	MDR-TB, 104/14	XDR-TB, 220/19
	MIC (µg mL−1)	MIC (µg mL−1)	MIC (µg mL−1)
4	0.0625	0.0625	0.0625
5	0.0625	0.0625	0.0625
6	0.0625	0.0625	0.0625
7	0.0625	0.0625	0.0625
8	0.0625	0.0625	0.0625
Ciprofloxacin	2	> 8	> 8

Conclusion

The novel fluoroquinolone derivatives were prepared in moderate to high yields and with good purity. These new derivatives were found to be more potent than the parent ciprofloxacin moiety itself against different Gram +ve, Gram −ve bacteria and against normal and multidrug resistant strains of Mtb. Compound 6 exhibited greater binding affinity than the standard ciprofloxacin against both the target enzymes (Mtb DNA gyrase and Enoyl ACP-reductase) in in silico indicating its ability to act by dual mechanism. The modification of carboxylic group at 3 position with isatin substituted derivatives increased the antimicrobial potency of ciprofloxacin. The results of this study revealed the potential of isatin/phthalimide/oxadiazole substituents as promising new antibacterial and antitubercular agents.

Experimental

Molecular Property Prediction

Molsoft, Molinspiration, Osiris property explorer software programmes were used to calculate the molecular properties for ciprofloxacin and its derivatives. The toxicity risks like mutagenicity, tumorigenicity, irritating effects and reproductive effects were calculated by Osiris.

PASS Software

PASS software was used to predict the activity spectra of ciprofloxacin and its derivatives. It predicts the biological potential of organic drug-like molecules, prior to their chemical synthesis. PASS estimates Pa (probability “to be active”) and Pi (probability “to be inactive”) ratio at prediction threshold of Pa > 30%, Pa > 50% and Pa > 70%. Pa of 0.3–0.7 was selected.

Molecular Docking Studies

The binding modes of ciprofloxacin and its derivatives were investigated using Auto Dock software (version 4.2). The protein sequences and crystal structures of Mycobacterial DNA gyrase GyrA N-terminal domain and Enoyl ACP reductase were downloaded from Protein Data Bank (PDB IDs: 3ILW and 4TZK). X,Y,Z coordinates of the proteins 3ILW and 4TZK were selected by using SPDBV. The three dimensional structures of compounds were constructed and optimized by ChemDraw Ultra 12.0 software.

Chemistry

Commercial reagents and solvents were purchased from Sigma-Aldrich chemicals Pvt Ltd. (India). The reactions were monitored by TLC on pre-coated silica gel plates (MERCK). JASCO UV chamber was used for detection of spots in TLC. Melting points were determined in open capillaries using Toshniwal electrical melting point apparatus and are uncorrected. IR spectra were recorded on Bruker FTIR spectrometer. The ¹H-NMR spectra were recorded on a Bruker Avance-300 MHz spectrometer using DMSO as solvent. The chemical shift values relative to tetramethylsilane (TMS) are reported as δ (ppm). Shimadzu LCMS-2010 spectrophotometer was used to obtain the mass spectra. PerkinElmer, Inc. 240B analyzer was used to analyze the elements (C, H, N) of the compounds and the observed values were within ±0.4% of theoretical values.

Compounds 2, 3 and N-benzylisatins were prepared by reported^24–26,32) procedures and their physical and spectral data are in accordance with the earlier reports.^{24–26,32–34)}

General Method for Synthesis of Isatin Substituted Ciprofloxacin Derivatives (4–6)

To a mixture of appropriate N-benzylisatin (1 mmol) and ciprofloxacin acid hydrazide (3,1 mmol, 0.345 g) in ethanol (25 mL), a few drops of glacial acetic acid was added. The reaction mixture was heated under reflux for 4–6 h, and cooled to room temperature. The precipitate was filtered, dried and recrystallized from ethanol or dimethyl formamide.

(Z)-N′-(1-Benzyl-2-oxoindolin-3-ylidine)-1-cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carbohydrazide (4)

Yield: 82%; mp: 118–120 °C; IR (KBr) γ (cm⁻¹): 3105 (N–H), 2853 (C–H), 1720 (C=O), 1688 (C=O amide), 1639 (C=N), 1440 (C=C), 1343 (C–F). ¹H NMR (DMSO, ppm) δ: 1.12–1.32 (m, 5H, cyclopropyl + piperazinyl), 2.05 (t, 4H, H₃, H₅, piperazinyl), 3.65 (s, 2H, –CH₂–C₆H₅), 4.87–4.93 (m, 5H, H₂, H₆ piperazinyl + –N–CH cyclopropyl), 6.46–7.92 (m, 12H, Ar-H), 8.7 (s, 1H, –CONH). MS: m/z: 564.23 (M⁺). Anal. Calcd (%) for C₃₂H₂₉FN₆O₃: C, 68.07; H, 5.17; N, 14.88. Found: C, 68.04; H, 5.14; N, 14.85.

(Z)-N′-(1-Benzyl-5-fluoro-2-oxoindolin-3-ylidine)-1-cyclopropyl-6-fluoro-4-oxo-7-(piperzin-1-yl)-1,4-dihydroquinoline-3-carbohydrazide (5)

Yield: 68%; mp: 216–218 °C; IR (KBr) γ (cm⁻¹): 3406 (N–H), 2920 (C–H), 1761 (C=O), 1666 (C=O amide), 1605 (C=N), 1331 (C–N), 1117 (C–O), 1022 (C–F). ¹H NMR (DMSO, ppm) δ: 0.40–1.89 (m, 4H, –CH₂, –cyclopropyl), 2.80–3.9 (m, 9H, –CH₂piperazinyl + –N–CH cyclopropyl), 4.99 (s, 3H, -NH-piperazinyl + N–CH₂–C₆H₅), 6.83–7.91 (m, 11H, Ar-H), 8.69 (s, 1H, –CONH). MS: m/z: 582.22 (M⁺). Anal. Calcd (%) for C₃₂H₂₈F₂N₆O₃: C, 65.97; H, 4.83; N, 14.42. Found: C, 65.94; H, 4.85; N, 14.40.

(Z)-N′-(1-Benzyl-5-nitro-2-oxoindolin-3-ylidine)-1-cyclopropyl-6-fluoro-4-oxo-7-(piperzin-1-yl)-1,4-dihydroquinoline-3-carbohydrazide (6)

Yield: 85%; mp: 245–248 °C; IR (KBr) γ (cm⁻¹): 3592 (N–H), 2918 (C–H), 1705 (C=O), 1619 (C=O amide), 1513 (C=N), 1386 (N–O), 1327 (C–N), 1146 (C–O). ¹H NMR (DMSO, ppm): δ 0.6–1.5 (m, 4H, –CH₂, cyclopropyl), 2.8–4.0 (m, 11H, –CH₂ piperazinyl + –N–CH cyclopropyl + –CH₂–C₆H₅), 6.5 (s, 1H, –NH–piperazinyl), 6.7–7.7 (m, 5H, Ar-H), 7.87–8.9 (m, 6H, Ar-H), 9.45 (s, 1H, CONH). MS: m/z: 609.21 (M⁺). Anal. Calcd (%) for C₃₂H₂₈FN₇O₅: C, 63.05; H, 4.62; N, 16.08. Found: C, 63.02; H, 4.60; N, 16.10.

Synthesis of 1-Cyclopropyl-N-(1,3-dioxoisoindolin-2-yl)-6-fluoro-4-oxo-7-(piperaziny-1-yl)-1,4-dihydroquinoline-3-carboxamide (7)

Phthalic anhydride (1 g, 7 mmol) was dissolved in glacial acetic acid (8 mL) by heating. It was then cooled to the laboratory temperature and a hot solution of ciprofloxacin acid hydrazide (3) (0.2 gm, 0.6 mmol) in glacial acetic acid (16 mL) was added in small amounts with constant stirring. The crystals which separated immediately were filtered, washed with diethyl ether and recrystallized from acetic acid. Yield: 94%; mp: 158–161 °C; IR (KBr) γ (cm⁻¹): 3428 (N–H), 2827 (C–H), 2366 (C=C), 1576 (C=O), 1290 (C–N), 741 (C–H). ¹H-NMR: 1.2 (s, 1H, –NH piperazinyl), 1.92–1.99 (m, 4H, cyclopropyl), 2.3–3.1 (m, 8H, piperazinyl), 4.03–4.04 (m, 1H, cyclopropyl), 7.50–7.69 (s, 7H, Ar-H), 13.2 (s, 1H, –CONH). MS: m/z: 475.17 (M⁺). Anal. Calcd.(%) for C₂₅H₂₂FN₅O₄: C, 63.13; H, 4.62; N, 14.73. Found: C, 63.10; H, 4.64; N, 14.75.

Synthesis of 1-Cyclopropyl-6-fluoro-3-(5-mercapto-1,3,4-oxadiazol-2-yl)-7-(piperazin-1-yl) Quinoline-4(1H)-one (8)

In a two necked RB flask (250 mL), take ciprofloxacin acid hydrazide (3) (3.45 g, 1 mmol), add 2–3 mL of carbon disulfide while stirring. After the evolution of gases subsided, potassium hydroxide (0.84 g, 1.5 mmol) in absolute ethanol (25 mL) were added and the reaction mixture was heated under reflux with stirring for about 21 h until a clear solution appeared. The product separated on addition of diethyl ether was filtered, dried and recrystallized from ethanol. Yield: 85%; mp: 119–121 °C; IR (KBr) γ (cm⁻¹): 3648 (N–H), 3247 (C–H), 2370 (S–H), 1650 (C=O), 945 (C=C), 784 (C–H). ¹H-NMR: 1.17–1.25 (m, 3H, –CH₂ cyclopropyl + –NH piperazinyl), 2.04 (m, 2H, –CH₂ cyclopropyl), 2.83–3.18 (m, 8H, piperazinyl), 4.03–4.05 (m, 1H, cyclopropyl), 5.75 (s, 2H, Ar-H + –SH), 7.37 (s, 1H, Ar-H), 8.35 (s, 1H, Ar-H). MS: m/z: 387.12 (M⁺). Anal. Calcd (%) for C₁₈H₁₈FN₅O₂S: C, 55.79; H, 4.64; N, 18.08. Found: C, 55.81; H, 4.62; N, 18.10.

Pharmacological Evaluation

Antibacterial Activity in Vitro

The antibacterial activity of the test compounds was evaluated systematically against different strains of bacteria, by cup plate method as per the standard procedure mentioned in Indian Pharmacopoeia.²⁹⁾

Antitubercular Activity in Vitro

The antitubercular activity of the compounds 4–8 was assayed using the conventional LJ slant method, adopting the previously reported procedure.^18,31) The stock solutions (1 mg/mL) of the test compounds were prepared in DMSO. From the above stock, various dilutions were made with DMSO to get the final drug concentrations of 0.0625, 0.125, 0.25, 0.5, 0.75 and 1 µg/mL. Ciprofloxacin was used as reference drug, for comparison.

Acknowledgments

One of the authors (KS) is highly thankful to All India Council for Technical Education (A.I.C.T.E.), New Delhi, India for awarding the CAYT project which enabled her to carry out this work. The authors (MB, DP, NN, KS) thank the Management and Principal of Vaagdevi College of Pharmacy for providing necessary facilities.

Coflict of Interest

The authors declare no conflict of interest.

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