Design, synthesis, and biological evaluation of pyrazole–ciprofloxacin hybrids as antibacterial and antibiofilm agents against Staphylococcus aureus

Abstract

In our continued efforts to tackle antibiotic resistance, a new series of pyrazole–ciprofloxacin hybrids were designed, synthesized, and evaluated for their antibacterial activity against Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa), and Mycobacterium tuberculosis (Mtb). Most of the compounds exhibited good to excellent activities against S. aureus, and six compounds (7a, 7b, 7d, 7g, 7k, and 7p) exhibited higher or comparable activity (MIC = 0.125–0.5 μg mL⁻¹) to ciprofloxacin (0.125 μg mL⁻¹). Further, these selected compounds were non-toxic (CC₅₀ ≥ 1000 μg mL⁻¹) when evaluated for cell viability test against the Hep-G2 cell line. Three compounds (7a, 7d, and 7g) demonstrated excellent activity against ciprofloxacin-resistant S. aureus with MIC values ranging from 0.125–0.5 μg mL⁻¹ and good antibiofilm activity. Among them, 7g displayed remarkable antibiofilm activity with an MBIC₅₀ value of 0.02 μg mL⁻¹, which is 50 times lower than ciprofloxacin (MBIC₅₀ = 1.06 μg mL⁻¹). A time-kill kinetics study indicated that 7g showed both concentration and time-dependent bactericidal properties. In addition, 7g effectively inhibited DNA-gyrase supercoiling activity at 1 μg mL⁻¹ (8× MIC). Two compounds 7b and 7d exhibited the highest activity against Mtb with a MIC of 0.5 μg mL⁻¹, while 7c showed the highest activity against P. aeruginosa with a MIC value of 2 μg mL⁻¹. Molecular docking studies revealed that 7g formed stable interactions at the DNA active site.

A series of pyrazole–ciprofloxacin hybrids were designed, synthesized, and tested for antibacterial activity against Staphylococcus aureus, Pseudomonas aeruginosa, and Mycobacterium tuberculosis, aiming to combat antibiotic resistance.

1. Introduction

Bacterial infections remain a significant public health threat due to the emergence of multiple drug-resistant bacterial strains, and diseases caused by these resistant strains led to 1.27 million deaths in 2019 and are estimated to increase to 10 million by 2050.¹ This rising threat from drug-resistant strains highlights the need to develop novel antibacterial agents with enhanced safety and efficacy.

For over three decades, the fluoroquinolone antibiotic ciprofloxacin has been used to treat a variety of bacterial infections, such as upper and lower respiratory tract infections, skin infections, bone and soft tissue infections,² urinary tract infections,³ and community-acquired pneumonia.⁴ This broad-spectrum antibiotic inhibits bacterial DNA-gyrase and topoisomerase-IV enzymes.⁵ Excess or widespread use of ciprofloxacin led to the emergence of resistant bacterial strains.⁶ Modifications on the ciprofloxacin skeleton have become a promising strategy for developing new and safe antibacterial agents that combat these drug-resistant strains more effectively.⁷ The most common and rational derivatization of ciprofloxacin involves substitution at the N₄-atom of the C-7 piperazinyl ring, which has led to molecules with improved potency, safety and spectrum of activity.⁸ Some of the representatives (A and B) are depicted in Fig. 1, such as ciprofloxacin–uracil conjugate A that displayed potent activity against both methicillin-sensitive and resistant strains of S. aureus⁹ and 3-hydroxy-pyridin-4(1H)-one-ciprofloxacin B, a potent inhibitor of P. aeruginosa 27853 and PAO1 strains.¹⁰ In addition to this, hybridisation of ciprofloxacin with other active pharmacophores via a twin drug/hybridisation approach led to compounds that act on multiple targets and with diverse pharmacological activities, which were also reported in the literature.^8j,11

Fig. 1. Chemical structures of reported ciprofloxacin derivatives (A and B), 1,3-diaryl pyrazole derivatives (C–G) and N-acetyl ciprofloxacin derivative (H) possessing promising antibacterial activity; designed compounds, 7a–q; MIC, minimum inhibitory concentration.

On the other hand, numerous reports have highlighted the importance of 1,3-diaryl pyrazole as a promising scaffold in different antimicrobial agents.¹² In particular, 1,3-diaryl pyrazole derivatives have received considerable attention in the literature as potent anti-MRSA¹³ and antitubercular agents.¹⁴ Recently, Saleh et al. identified the anti-MRSA activity exhibited by N-(trifluoromethyl)phenyl-substituted pyrazole derivatives, and the best compound, C, showed a MIC value of <1 μg mL⁻¹. Mansour and co-workers reported the pyrazole clubbed pyrimidine derivative D with potent anti-MRSA activity (MIC = 521 μM). Additionally, Khonde and co-workers reported the antitubercular activity of 1,3-diaryl pyrazolyl-acyl sulfonamides and the best activity is exhibited by compound E with a MIC value of 1.2 μM.¹⁵ In pursuit of potent antimicrobial agents, recently, we have described the identification of 1,3-diaryl pyrazole based derivatives as potent antimicrobial agents where compound F showed potent inhibition against MRSA¹⁶ and G against Mtb¹⁷ (Fig. 1).

Carboxamide has been used as a linker in developing new pharmacological conjugates owing to its hydrogen bonding capacity, stability and ease of synthesis.¹⁸ Specifically, molecules with acetamide linkers are proven to display a wide range of biological activities.¹⁹ Ciprofloxacin hybrids containing an acetamide linker were proven to exhibit a better pharmacological profile.^8h,9,20 Recently, Struga et al. reported the antibacterial potential of N-acylated ciprofloxacin derivatives. Among them, compound H with a chloroacetyl group proved to exhibit potent antibacterial activity, with MIC values in the range of 0.1–0.2 μg mL⁻¹ against different strains of S. aureus and E. coli.^8l

1.1. Design and SAR strategy

Based on the above considerations and in our continuous attempts to search for effective antibacterial agents, herein we report the design and synthesis of ciprofloxacin–pyrazole hybrids 7a–q by fusing ciprofloxacin and 1,3-diaryl pyrazole moiety through an acetamide linker based on a biology-oriented drug synthesis (BIODS) approach and further evaluation of their antibacterial activities. BIODS refers to the design and synthesis of a new library of compounds via structural modifications to the skeleton of existing marketed drugs and to further explore their biological potential.²¹ Moreover, the rationale was also conceptualised based on a twin drug/hybridisation approach, which infers the combination of two active pharmacophores into a single molecule with the potential to modulate multiple targets and synergistic action.^{8a,l,11a,b,22} In order to achieve compounds with optimal activity, we introduced aryl rings substituted with both electron donating and withdrawing groups and heteroaryl rings at the C-3 position of pyrazole.

2. Results and discussion

2.1. Chemistry

The synthesis of acetamide-linked 1,3-diaryl pyrazole and ciprofloxacin hybrids 7a–q is depicted in Scheme 1. 4-(3-Aryl-1H-pyrazol-1-yl)anilines 5a–q were synthesized according to earlier reported methods.^16,17 Briefly, condensation of commercially available substituted acetophenones 1a–q with N,N-dimethylformamide dimethyl acetal led to intermediate enaminones 2a–q which were subjected to cyclisation with hydrazine hydrate. C-3 aryl substituted pyrazole intermediates 3a–q thus obtained were treated with 1-fluoro-4-nitrobenzene using potassium carbonate as a base to give nitroaryl pyrazole intermediates 4a–q. Nitro intermediates 4a–q were reduced in the next step using iron and a catalytic amount of acetic acid to obtain the pyrazole anilines 5a–q. These anilines were then acylated with chloroacetyl chloride using triethylamine as a base in DCM to afford 6a–q. Finally, the target compounds 7a–q were obtained by heating the acetamide with ciprofloxacin using triethylamine as a base in acetonitrile, and the yields were in the range of 70–90%.

Scheme 1. Synthetic route for the pyrazole–ciprofloxacin hybrids, 7a–q.

2.2. Antibacterial activity

The antibacterial activity of the synthesized compounds was evaluated against two biofilm producing bacterial pathogens, Gram-positive S. aureus ATCC 29213 and Gram-negative P. aeruginosa PAO1 ATCC 15692, in comparison with ciprofloxacin as a reference drug. Moreover, these compounds were also screened for their antimycobacterial activity against Mtb H37Ra ATCC 25177 using isoniazid as a reference drug. Compounds were tested in the concentration range from 128–0.06 μg mL⁻¹. The minimum inhibitory concentration was determined and the results are tabulated in Table 1.

Table 1. MIC values of pyrazole–ciprofloxacin hybrids 7a–q against S. aureus ATCC 29213, P. aeruginosa ATCC 15692, and Mtb H37Ra ATCC 25177.

Compd. code	MIC (μg mL−L)
	S. aureus ATCC 29213	P. aeruginosa ATCC 15692	Mtb H37Ra ATCC 25177
7a	0.125	128	16
7b	0.125	32	0.5
7c	1	2	1
7d	0.125	8	0.5
7e	4	32	8
7f	1	32	4
7g	0.125	64	4
7h	1	128	64
7i	8	64	8
7j	1	16	4
7k	0.125	32	1
7l	32	128	16
7m	8	128	32
7n	16	>128	64
7o	16	128	4
7p	0.5	64	32
7q	1	32	2
Ciprofloxacin	0.25	0.06	NT
Isoniazid	NT	NT	0.034

Detailed analysis of the results revealed that most of the synthesized compounds exhibited good to excellent activities against the tested bacterial strains. Five compounds 7a (R = 3,4-dimethoxyphenyl), 7b (R = 3,4,5-trimethoxyphenyl), 7d (R = phenyl), 7g (R = 2-methoxyphenyl), and 7k (R = 4-fluorophenyl) exhibited the highest potency against S. aureus with a MIC value of 0.125 μg mL⁻¹, a 2-fold greater activity than the standard drug, ciprofloxacin (0.25 μg mL⁻¹). Besides, compound 7p (R = 3,4-methylenedioxyphenyl) showed comparable activity (MIC 0.5 μg mL⁻¹) to ciprofloxacin against S. aureus. Introduction of a bulkier naphthyl ring (7c) and heteroaryl ring like benzofuran (7q) on R resulted in compounds with good activity (MIC = 1 μg mL⁻¹ against S. aureus). SAR study indicated that di- or tri-methoxy substituted phenyl derivatives as R (7a, 7b, and 7p) exhibited better activity against S. aureus compared to mono-methoxy phenyl derivatives except compound 7g (R = 2-methoxyphenyl), whereas mono-halophenyl derivatives exhibited greater activity compared to di-halophenyl derivatives. Among the halogens, the fluorophenyl derivative is more active against S. aureus than chlorophenyl derivatives followed by bromophenyl derivatives (7k > 7h > 7l). Further, almost all the tested derivatives displayed moderate to weak activity against P. aeruginosa with MIC values in the range of 16–128 μg mL⁻¹, except compounds 7c (R = naphthyl) and 7d (R = phenyl) which showed good activities with MICs of 2 and 8 μg mL⁻¹, respectively.

The antimycobacterial results showed that all the hybrids exhibited considerable activity against Mtb H37Ra with MIC values in the range of 0.5–64 μg mL⁻¹. Two compounds 7b (R = 3,4,5-trimethoxyphenyl) and 7d (R = phenyl) exhibited the highest activity with a MIC of 0.5 μg mL⁻¹. Heteroaromatics (7q) and bulkier groups (7c) were tolerated (MIC 1 μg mL⁻¹). Mono-substituted phenyl derivatives (7e, 7f, 7g, 7i, 7j and 7k) exhibited moderate anti-TB activity (MIC = 1–8 μg mL⁻¹). The other derivatives showed only weak anti-TB potency.

2.3. Determination of activity against ciprofloxacin-resistant S. aureus

Potent compounds (MIC ≤ 0.5 μg mL⁻¹) were further studied for their activity against lab-generated one step resistant mutant of S. aureus against ciprofloxacin (Table 2). Compounds 7a, 7d, and 7g demonstrated potent inhibition against ciprofloxacin-resistant S. aureus (MIC = 0.125–0.5 μg mL⁻¹), which is comparable to that against drug-susceptible S. aureus (MIC = 0.125 μg mL⁻¹). Amongst them, 7g was the best, followed by 7a and 7d. Compounds 7b (MIC = 1 μg mL⁻¹) and 7k (MIC = 8 μg mL⁻¹) were found to inhibit the drug-resistant (DR) strain relatively at higher concentrations compared to drug-susceptible S. aureus. 7p showed weak potency (MIC = 32 μg mL⁻¹) on ciprofloxacin-resistant S. aureus.

Table 2. MIC (μg mL⁻¹) of potent compounds (7a, 7b, 7d, 7g, 7k, and 7p) against ciprofloxacin-resistant S. aureus.

Compd. code	S. aureus 29213	S. aureus Ciproa
	MIC (μg mL−L)	MIC (μg mL−L)
7a	0.125	0.25
7b	0.125	1
7d	0.125	0.5
7g	0.125	0.125
7k	0.125	8
7p	0.5	32
Ciprofloxacin	0.25	2.0
Tamoxifen	n.d.	n.d.

^aMutant strain of S. aureus.

2.4. Antibiofilm activity against S. aureus

Numerous studies have shown that bacteria adopt the biofilm mode of growth to survive harsh conditions such as host immune responses and antibiotic treatment which often leads to drug-resistance and prolonged duration of treatment.²³ The effect of compounds 7a, 7b, 7d, 7g, 7k, and 7p on in vitro biofilm formation was tested in the concentration range between 128 and 0.06 μg ml⁻¹ and MBIC₅₀ values were determined. The minimum biofilm inhibition concentration (MBIC₅₀) was defined as the lowest compound concentration that showed 50% or more inhibition on the growth/formation of biofilms. All the tested compounds except 7k & 7p showed MBIC₅₀ values close to their MIC values (Fig. 2; Table 3) and were more potent in reducing biofilm formation compared to standard ciprofloxacin (MBIC₅₀ = 1.06 μg mL⁻¹). Compound 7g demonstrated potent anti-biofilm activity with an MBIC₅₀ value of 0.02 μg mL⁻¹, i.e., six times lower than MIC (0.125 μg mL⁻¹).

Fig. 2. Anti-biofilm activity of compounds 7a, 7b, 7d, 7g, 7k, and 7p against the S. aureus 29213 strain.

Table 3. Minimum biofilm inhibitory concentration (MBIC₅₀) values against S. aureus 29213.

Compd. code	S. aureus 29213	S. aureus 29213
	MIC (μg mL−L)	MBIC50 (μg ml−l)
7a	0.125	0.30
7b	0.125	0.52
7d	0.125	0.27
7g	0.125	0.02
7k	0.125	2.7
7p	0.5	1.84
Ciprofloxacin	0.25	1.06

2.5. Time-kill kinetic study

The three potent non-toxic compounds that were active on the ciprofloxacin-mutant of S. aureus were subjected to a time-kill kinetic assay to determine the rate at which these reduce the bacterial load at three different concentrations (1×, 4×, and 8× MIC). Time-kill kinetics of 7a, 7d, and 7g along with ciprofloxacin, as depicted in Fig. 3, showed ≥3 log₁₀ CFU mL⁻¹ reduction in bacterial load within 9 h at 4× and 8× MIC, which is comparable to ciprofloxacin (1× MIC). In contrast to 7a, compound 7d kept the bacterial load in check within 6 h at 8× MIC. Compound 7g exhibited a higher killing rate reducing the bacterial load ≥3 log units within 9 h at 1× MIC, and within 3 h at 4× and 8× MIC which was superior to ciprofloxacin. Taken together, all the test compounds displayed bactericidal activities in a concentration and time-dependent manner; however, 7g exhibited better time-kill kinetics superior to ciprofloxacin.

Fig. 3. Time-kill kinetics analysis: structures of compounds subjected for analysis (a); time-kill plots of compounds 7a (b), 7d (c), and 7g (d).

2.6. Cytotoxicity assay

The cytotoxicity of the most potent conjugates having MIC values ≤0.5 μg mL⁻¹ on the tested bacteria was evaluated on the Hep-G2 cell line, a human liver cancer cell line, using MTT assay. Tamoxifen was used as a reference standard. CC₅₀ (the lowest compound concentration that causes the death of 50% of viable cells) was determined. The results indicated that all the tested hybrids were non-toxic and safe, with impressive CC₅₀ values exceeding 1000 μg mL⁻¹ (Table 4).

Table 4. Cytotoxicity of compounds 7a, 7b, 7d, 7g, 7k, and 7p against the Hep-G2 cell line (human liver cancer cell line).

Compd. code	S. aureus 29213	CC50a
	MIC (μg mL−L)	(μg mL−L)
7a	0.125	>1000
7b	0.125	>1000
7d	0.125	>1000
7g	0.125	>1000
7k	0.125	>1000
7p	0.5	>1000
Ciprofloxacin	0.25	n.d.
Tamoxifen	n.d.	3.696

^aCC₅₀ = lowest concentration of a compound that causes the death of 50% of viable cells.

2.7. Inhibition of DNA gyrase supercoiling activity

Fluoroquinolones are known to exhibit antimicrobial activity by inhibiting DNA gyrase. Thus, the new ciprofloxacin–pyrazole hybrids are reckoned to exert their action through a similar mechanism. The potent compounds 7a, 7d, and 7g were next assessed for their ability to inhibit DNA gyrase using a DNA supercoiling activity assay. E. coli DH5α pUC19 was treated with 2× (0.25 μg mL⁻¹) and 8× (1 μg mL⁻¹) MIC concentrations of the compounds. The plasmid treated with ciprofloxacin (0.25 μg mL⁻¹) was used as a positive control, and the native plasmid (drug-free DMSO control) served as a negative control. 7g showed a significant reduction in the supercoiled state of the plasmid at 8× MIC, i.e., 1 μg mL⁻¹ compared to native plasmid and other tested compounds. Similar results were obtained in a ciprofloxacin-treated plasmid (Fig. 4).

Fig. 4. DNA supercoiling assay: effect of 7a, 7d, and 7g on E. coli DNA gyrase using the pUC19 plasmid as a substrate shown by gel electrophoresis.

2.8. Molecular docking

Molecular modelling studies were carried out for the representative compounds 7a, 7d, and 7g to explore the binding mode of these compounds within the active site of DNA gyrase (PDB: 2XCT) and to predict the type of interactions between the compounds and enzyme using Schrödinger Release, 2021.^8k,24 The results revealed that the compounds showed a good docking score in the range of −8.591 to −11.881 with the co-crystal (ciprofloxacin) showing a docking score of −11.997. All the three compounds established key interactions with residues in the active pocket such as metal ion coordination between carboxyl and ketone groups of the quinolone ring and Mn²⁺. Further, the planar quinolone ring in all three compounds showed π–π stacking interactions with nucleotides DG-9 and DA-13. In addition, the phenyl ring attached to the nitrogen atom of pyrazole established π–cation interactions with active site residue ARG-458. The carbonyl oxygen of the acetyl linker forms a hydrogen-bonding interaction with the ASN 476 residue. Apart from this, piperazine nitrogen of 7g formed a salt bridge interaction with the GLU-477 residue. Furthermore, several hydrophobic interactions were noticed (Fig. 5). Overall, docking results revealed that the compounds have shown binding modes similar to that of the co-crystal, ciprofloxacin, supporting the results obtained from in vitro enzyme inhibition assay.

Fig. 5. Binding poses of compounds a) 7a, b) 7d, and c) 7g (magenta, ball & stick) in the DNA gyrase active site (PDB ID: 2XCT); DNA gyrase and DNA are represented as red ribbons while Mn²⁺ is shown as a purple sphere; π–π stacking, π–cation and hydrogen bonding.

3. Conclusion

In conclusion, a new series of pyrazole–ciprofloxacin conjugates were synthesized by grafting the C-7 piperazinyl moiety in the antibiotic with 1,3-diaryl pyrazole via an acetamide linker and screened for their antimicrobial potential against S. aureus ATCC 29213, P. aeruginosa PAO1 ATCC 15692 and Mtb H37Ra ATCC 25177. A total of seventeen compounds were tested, out of which five compounds (7a, 7b, 7d, 7g, and 7k) showed a MIC of 0.125 μg mL⁻¹, which was comparable to the MIC value of ciprofloxacin (MIC = 0.25 μg mL⁻¹) against S. aureus. Though compounds were also tested for their activity against Gram-negative P. aeruginosa, all the tested compounds displayed a weak activity with a MIC range of 2–64 μg mL⁻¹. However, when tested against Mtb, two compounds, 7b and 7d, exhibited the highest activity with a MIC of 0.5 μg mL⁻¹. Safety evaluation for these compounds displayed a good selectivity in cytotoxicity study using Hep G2 cell lines (CC₅₀ > 1000). Compound 7g exhibited the same potency against the ciprofloxacin-resistant strain of S. aureus (MIC = 0.125 μg mL⁻¹) and inhibited biofilm formation with a MBIC₅₀ value equal to 0.02 μg mL⁻¹. Further, the time-kill kinetics study revealed that 7g displayed bactericidal properties superior to the standard ciprofloxacin and inhibited DNA gyrase supercoiling activity. The results obtained from the gyrase supercoiling assay were in alignment with our docking studies and confirmed that 7g formed stable complexes at the active site of DNA gyrase as that of co-crystal ciprofloxacin. In this context, 7g exhibits a promising antimicrobial activity even against the lab-generated ciprofloxacin mutant of S. aureus and could be considered for further development.

Data availability

The data supporting this article have been included as part of the ESI.†

Author contributions

Ojaswitha Ommi: conceptualization, design, optimization, synthesis, data analysis & interpretation and writing – original draft. Priyanka Sudhir Dhopatb: synthesis. Shashikanta Sahu: biological studies. Madhu Rekha Estharla: biological studies. Nitin Pal Kalia: supervision and writing – review & editing. Venkata Madhavi Yaddanapudi: project administrator, supervision, and writing – review & editing.

Conflicts of interest

There are no conflicts to declare.