Design, synthesis, and biological investigation of quinoline/ciprofloxacin hybrids as antimicrobial and anti-proliferative agents

Ciprofloxacin-Piperazine C-7 linked quinoline derivatives 6a–c and 8a–c were synthesized and investigated for their antibacterial, antifungal, and anti-proliferative activities. Ciprofloxacin-quinoline-4-yl-1,3,4 oxadiazoles 6a and 6b showed promising anticancer activity against SR- leukemia and UO-31 renal cancer cell lines. The hybrids 8a–c and compound 6b exhibited noticeable antifungal activities against C.Albicans; 8a experienced the most potent antifungal activity compared to Itraconazole with MICs of 21.88 µg/mL and 11.22 µg/mL; respectively. Most of derivatives displayed better antibacterial activity than the parent ciprofloxacin against all the tested strains. Compound 6b was the most potent against the highly resistant Gram-negative K.pneumoniae with MIC 16.96 of µg/mL relative to the parent ciprofloxacin (MIC = 29.51 µg/mL). Docking studies of the tested hydrides in the active site of Topo IV enzyme of K.pneumoniae (5EIX) and S.aureus gyrase (2XCT) indicate that they had stronger binding affinity in both enzymes than ciprofloxacin but have different binding interactions. The hybrid 6b could be considered a promising lead compound for finding new dual antibacterial/anticancer agents. Moreover, Compound 8a could be a lead for discovering new dual antibacterial/antifungal agents.


Introduction
Fluoroquinolones represent the backbone stone among the highly potent antimicrobial agents many decades ago [1]. Fluoroquinolones can eventually result in bacterial cell death via the circumvention of DNA replication and transcription processes in bacterial cell lines. They hamper the topoisomerase II activity (DNA gyrase) or/and prevent the detachment of topoisomerase II from DNA [2][3][4]. Bacterial topoisomerase II is highly different than mammalian topoisomerase; therefore, fluoroquinolones show about 1000fold specific selectivity against bacterial topoisomerase II rather than the same enzyme in human beings [5]. Fluoroquinolones have also been found to abolish the in vitro topoisomerase IV ligase activities that have a higher similarity in structure to the topoisomerase II. This enzyme possesses a critical function represented in partitioning the DNAchromosomes in bacterial strains through cell division. It probably can stand among the fundamental targets for affording the fluoroquinolones' antibacterial activity in specifically Gram-positive bacteria [6]. This technique can be considered related to apoptosis rather than necrosis [6]. Nowadays, fluoroquinolones have enormous applications' clinically, and a vast domain regarding their antimicrobial activity comprises Gram-negative, Gram-positive, aerobic and anaerobic pathogens [7,8]. In relation, fluoroquinolones exhibited highly potent anti-tuberculosis activity with a promising ability to lessen the treatment duration and to decrease the availability of bacterial resistance's existence [9,10]. Moreover, fluoroquinolones are depicted by having a remarkable phagocytic cells' entrance; and a higher comprehensive safety margin [11]. Aside from fluoroquinolones' antibacterial activities [12][13][14], they exert other versatile non-classical biophysiological activities as instances; antifungal [15,16], antitumor [17,18], anti-HCV-NS3 helicase, HIV-1 integrase [19], anti-Alzheimer [20], and antimalarial activities [21]. As a result of the rapid emergence of highly resistant bacterial strains, it becomes a great necessity to ameliorate and develop new fluoroquinolones-based scaffolds that can withstand these virulent pathogens [22]. Brightly, a combination of one of the two well-known approved quinolones, trovafloxacin and ciprofloxacin with antifungal drugs such as fluconazole I or amphotericin B II afforded valuable antifungal activity against Candida albicans than using only quinolones themselves [23]. Currently approved antifungal agents are classified regarding their molecular targets. The main biochemical targets for antifungal drugs are enzymes and another additional bimolecular substances encompassed in biosynthesis of both cell wall (sorbitol), and plasma membrane (ergosterol); in addition to fungal DNA synthesis (metal chelation with DNA), and mitosis (microtubules) ( Fig. 1) [24][25][26]. In relation, Metals play a fundamental role in various biological and infection processes in fungal pathogens as they act as adjuvants in a populace of enzymes-including plurality of direct and indirect roles in redox, catalytic, and malevolence, such as metaldependent superoxide dismutases (SODs), and metalloproteases or melanin-producing laccases. Therefore, metals can affect the organizational, engulfing, and toxin removal systems in fungal strains [27]. As an example, 5-chloro-8hydroxyquinoline and 5-nitro-8-hydroxyquinoline can serve as ligands forming complexes with metals (Cu, Zn, Al, and Ru) for potentiation of their antifungal activity via interfering cell membrane and DNA synthesis [28]. In addition, compounds III and IV (Fig. 2), thiol substituted quinolinium derivatives at 3-and 4-positions were highly potent antifungal agents with 1 μg/mL MIC against C. albicans ATCC 60193 [29].
On the other hand, quinoline derivatives stand among versatile heterocycles that can be considered as an efficient category for subsequent promising antifungal [30], antibacterial [31,32], antimalarial [33,34], anti-tuberculosis [35], anticancer agents [36][37][38]. Molecular hybridization represents a new promising trend utilizing two or more biologically active compounds to afford new scaffolds targeting multiple targets or specific drug targets [39,40]. In literature data, it is reported that quinoline-1,3,4-oxadiazole hybrid V (Fig. 2) showed higher antibacterial activity against Staphylococcus aureus as a Gram-positive strain tested with a MIC value equal to 10 ng/mL, which is threefold lower than that of ciprofloxacin (30 ng/mL), and equal to 25 ng/mL against Escherichia coli Gram-negative bacteria in emulation to ciprofloxacin with MIC value equal to 60 ng/mL [41]. Upon depending on the hybridization strategy in design of this research work, the aim is to synthesize a new ciprofloxacin-linked quinoline hybrids via a quinoline-4-carbonyl linker (8a-c) or quinolin-4-yl-1,3,4oxadiazole linker (6a-c); to study the impact of binding these groups to the C-7-piperazine moiety on antimicrobial and anticancer activities (Fig. 2).

Chemistry
The starting appropriate acids 1a-d and the intermediates esters 2a-c and hydrazides 3a-c were elaborated in the same manner with reported literature procedures [42]. Hydrazides 3a-c converted to 1,3,4-oxadiazole-2-thione derivatives 4a-c were prepared as reported [43] by refluxing with CS 2 in ethanolic solution aside with KOH as a strong base. The structures of oxadiazoles 4b and 4c were elucidated based on their 1 H NMR, 13 C NMR, and DEPT 13 C NMR spectroscopy. The acylated ciprofloxacin 5 was prepared following the reported procedure via the treatment of ciprofloxacin with bromoacetyl bromide in an ice bath along with potassium carbonate as a base [44]. Alkylation of oxadiazole derivatives 4a-c with acylated ciprofloxacin 5 was accomplished in ACN with TEA to give the new hybrids 6a-c in an acceptable output [44] (Scheme 1).
The chemical identity of compounds 6a-c was confirmed upon relying on their 1 H NMRspectroscopy, mass spectrophotometry, and elemental analysis. 1 H NMR spectra revealed the existence the protons of the parent ciprofloxacin scaffold in their expected chemical shifts. Additionally, the singlet signal of the linker CH 2 appeared at δ 4.27-4.78 ppm. Trials to obtain reasonable 13 C NMRspectrums of compounds 6a-c were failed due to the difficulty of solubility of these compounds even in DMSO-d 6 . On the other side, reaction of the  appropriate 2-substituted quinoline-4-carboxylic acid derivatives 1a, 1b, or 1d with excess SOCl 2 affording the corresponding acid chlorides 7a-c that were used in the following step without any purification. The reaction of acid chlorides 7a-c with ciprofloxacin in ACN using TEA afforded the targeted acylated ciprofloxacin derivatives 8a-c (Scheme 2). The structural identity of hybrids 8a-c was affirmed on the basis of their 1 H NMRspectroscopy and elemental analysis. Trials to obtain reasonable 13 C NMRspectrums of compounds 8a and 8b were failed due to the difficulty of solubility of these compounds except compound 8c that showed a good 13

Screening of the antimicrobial activity
The standard agar cup diffusion protocol [45] were used to determine the in vitro antibacterial activity against E. coli (ATCC 8739), Klebsiella pneumoniae (ATCC 10031), Pseudomonas aeruginosa (ATCC 10145), and S. aureus (ATCC 6538) of compounds, 6a-c and 8a-c compared with the reference ciprofloxacin as a positive control and DMSO as a negative control. Also, the antifungal activity of 6a-c and 8a-c was examined against C. albicans by standard agar cup diffusion protocol and using itraconazole as a positive antifungal reference and DMSO as a negative reference [46]. Upon relying on the antibacterial and antifungal in vitro results as shown in Table 1, it is clear that the ciprofloxacinquinoline-4-yl-1,3,4-oxadiazole hybrid 6a and ciprofloxacin-quinoline-4-carbonyl hybrid 8b displayed remarkable On the basis of our investigated outcomes, it becomes clear that most of the tested ciprofloxacin-quinoline compounds showed more robust and broad-magnitude bactericidal activity than ciprofloxacin reference versus most of the tested Gram-positive and Gram-negative strains. It becomes clear that the existence of substitution on N4 of piperazinyl moiety enhances the bactericidal activity through improving their lipophilicity (expressed as logP values calculated via Swiss ADME Prediction web site; mentioned in Table 1) as increasing of logP values due to decrease zwitterion formation which led to increase penetration to bacterial cell [46]. In contrast to ciprofloxacin which exhibited negligible fungicidal activity against C. albicans with MIC equals 169.82 µg/mL, compounds 6b and 8a-8c showed good antifungal activity with MIC values equal to 28.87, 21.88, 32.43, and 27.94 µg/mL, respectively, but less than itraconazole. These promising antifungal activities of compounds 6b and 8a-8c may be due to enhancement of lipophilicity than the parent ciprofloxacin and subsequent increasing the penetration to fungal cell and/or synergistic antifungal activity as a result of gathering ciprofloxacin and quinoline moiety in one compact unit.

Screening of the anticancer activity
The NCI results of new heterocyclic compounds; 6a-c and 8a-c (Shown in Table 2) indicated that the hybrids 6a-c had better anti-proliferative activities than hybrids 8a-c.
For example, compounds 6a and 6b showed promising anti-proliferative activity against SR-leukemia cell lines with growth inhibition percentages; 33.25 and 52.62%, respectively. These results illustrated that electron-donating substitution such as p-methyl group on phenyl ring as in compound 6b was more favorable than being unsubstituted phenyl ring on this position like in compound 6a. On the other hand, replacing p-methyl group with p-methoxy group led to a lack of anti-proliferative activity as in compound 6c. Therefore, this results in a good impact on the anticancer activity of these compounds. Concerning CAKI-1 Renal Cancer, compound 6b had a positive effect on the anticancer activity compared to hybrids 6a and 6c with growth inhibition percentages; 39.81, 26.92 27.31%, respectively. Moreover, the hybrids 6a-c exhibited remarkable anti-proliferative activities against UO-31 renal cancer with growth inhibition percentages; 64.19, 55.49, 40.15%, respectively.
Furthermore, in relation to LOX IMVI Melanoma cancer cells, compounds 6a and 6b showed good anticancer activity with a percentage of growth inhibition of 39.14 and 36.64%, respectively. In contrast, compound 6c showed insignificant anticancer activity with a growth inhibition percentage, 8.95%. Therefore, by addition of a stronger electron-donating group such as methoxy group on phenyl ring in position 2 of quinoline ring in compound 6c had a negative effect on the anticancer activity of this compound compared to compounds 6a and 6b; so being unsubstituted phenyl ring on this position like in compound 6a was better for higher anti-proliferative against LOX IMVI Melanoma, and the same reason reflected on the growth inhibition percentages of compounds 6a-c on A498 renal cancer cell lines.
Notably, compounds 8a-c showed very week anti-proliferative activity versus most of the used cancer cell lines. On balance, the presence of 1,3,4 oxadiazole ring linkage in hybrids 6a-c may be had a striking positive effect on the anticancer activity of these hybrids rather than carbonyl linkage in hybrids 8a-c; this can be accredited to the wellknown intrinsic anticancer activity of the 1,3,4 oxadiazole itself with multiple mechanisms published before in many literatures research works [47][48][49].

Docking studies of antibacterial activity of hybrids 6a-c and 8a-c
Firstly, we investigate both the mode of binding, and binding energies of the hybrids 6a-c, 8a-c, and the parent ciprofloxacin versus the active binding site of K.pneumoniae Topo IV enzyme (PDB: 5EIX) to stand on their efficient binding modes and evaluate their similarity to the standard ligand binding modes [50]. The deprotonated forms of hybrids 6a-c & 8a-c and the zwitterionic forms of ciprofloxacin & levofloxacin were used in this molecular modeling study using Molecular docking software (MOE® version 2014.01). The K. pneumoniae Topo IV X-ray crystallographic structure (PDB ID: 5EIX) was taken from the protein data bank [51]. The binding free energy (ΔG) values of all the new compounds range from − 8.234 to − 7.17 kcal/mol, comparable to the parent reference drug ciprofloxacin (ΔG = − 6.05 kcal/ mol) and the reference levofloxacin downloaded as ligand with enzyme (ΔG = − 5.55 kcal/mol) which means powerful binding, as outlined in Table 3.
From the docking results in K. pneumoniae Topo IV active site as shown in Figs. 3, 4, 5, 6, 7 and 8 and Table 3, we can claim that all the new tested compounds make different interactions to some extent to that of ciprofloxacin or levofloxacin except compound 8c which forms one metal chelation with magnesium ion Mg 4:1501 through the carboxylic group at C-3 and one pi-H bond with His A1077 residue as ciprofloxacin does. Moreover, levofloxacin which is the native ligand with the enzyme, it forms one metal chelation of the carboxylic group at C-3 with magnesium ion Mg 4:1501 as ciprofloxacin does and two hydrogen bonds with DA B5 and Gly A420 residues.
Regarding compound 6b, which had higher antibacterial activity against K. pneumonia, it interacts with 5EIX binding site in different binding mode with various amino acid residues than that of ciprofloxacin Hybrid 6b constructs one pi-H bond with amino acid residue His A1077, and three hydrogen bonds with DT B7, DA B7, and Arg A1029 amino acid residues. Also, the unsubstituted quinoline hybrid 6a forms two pi-cationic interactions with His A1077, and Pro A1076 amino acid residues and one hydrogen bond with amino acid residue DA B7. On the other hand, Compounds Depending on the data obtained from this docking study hybrids 6a-c, and 8a-c in comparison with reference ciprofloxacin (Table 2 supporting information), it becomes clear that formation of metal chelation with Mg 4:1501 as in compound 8c or formation of at least one hydrogen bond with one of the following amino acids; DT B7, DA B7, and Arg A1029 amino acid residues is essential for those hybrids to show good antibacterial activity against K.pneumoniae as in 6a, and 6b.
Secondly, the tested compounds 6a, 6b, 8b, and 8c were docked into the binding pocket of with the active binding site of S. aureus gyrase enzyme (PDB ID: 2XCT) to highlight on their effective binding styles and evaluate their resemblance to the positive control ciprofloxacin;so it is enabled the investigation of both binding style and energies of the final hybrids 6a, and 8b that afford higher bactericidal effects versus S. Aureus comparing with ciprofloxacin with MIC values 19.49, 18.24, and 109.6 µg/mL, respectively. The S. aureus gyrase enzyme X-ray crystallographic structure (PDB ID: 2XCT) was obtained from the protein data bank [51].
Docking outcomes showed robust binding affinity to the S. aureus gyrase which appears on the values of their binding free energy (ΔG); extent from − 7.83 to − 6.88 kcal/ mol, collated to the ciprofloxacin (ΔG = − 6.03 kcal/mol), as shown in Table 4.
On the basis of docking outcomes mentioned in Table 4, Figs. 9, 10, 11 and 12, it can be noted that the new tested compounds afford different binding interactions' styles than that of ciprofloxacin which forms two metal chelation bonds with Mn atom, a hydrogen bond with Ser A1084, two pi-pi  interactions with DG9 residue of DNA nucleotides, and two H-pi bonds with DA H13 and DC H12. Concerning hybrids 6a and 8b, which afford MIC equal to 19.49 μg/ mL and 18.24 μg/mL; respectively, they form interactions with the 2XCT binding site in a different mode with various amino acid residues than that of ciprofloxacin. Docking studies are in a good endorsment with the obtained in vitro antibacterial results. In relation, Compound 6a forms five hydrogen bonds with Lys A1043, Arg A1092, His A1046, DG B9, and DC C14; in addition to one pi-H bond with DNA nucleotides residues DA C13 (Fig. 11). Besides, compound 8b has an ionic bond with Arg A1092, one H-pi bond with DNA nucleotides residues DG B9, and two hydrogen bonds with Lys A1043, and Ser A1085 amino acid residues (Fig. 12). On contrast, the least active compounds 6c and 8a exert weaker interactions with the 2XCT binding site than the parent, and these interactions do not comprise any ionic bonds. Therefore, the more efficient anti-bactericidal effect of the hybrids 6a, and 8b may be due to improvement of the lipophilicity, and subsequently increasing the passage into microbial cells and respectable fitting binding modes on the predictable target site. Additionally, compounds 6a and 8b may had another antibacterial technique in addition to their activity against DNA gyrase of gram positive S. aureus bacteria; that is reflected in more stronger antibacterial activity of these compounds comparing with parent positive control ciprofloxacin.

Conclusion
Ciprofloxacin-quinoline hybrids 6a-c and 8a-c were chemically elaborated, characterized and investigated for their antimicrobial and anticancer activities. Ciprofloxacin-linked quinoline-4-yl-1,3,4 oxadiazoles 6a and 6b showed promising anticancer activity against SR-leukemia and UO-31 renal cancer cell lines and greater bactericidal effect versus S. aureus, highly resistant K. pneumoniae and Ps. aeruginosa than the reference ciprofloxacin. On the other side, the gathering of ciprofloxacin with a quinolone-4-yl nucleus in one compact unit through carbonyl group led to synergistic antifungal activities as shown by compounds 8a-c against C. Albicans. Docking studies on the S. aureus gyrase and K. pneumonia binding sites indicated that the tested compounds interacted in a different manner than that of ciprofloxacin. Therefore, the more efficient antibacterial activity of the tested compounds may be because of improvement of the lipophilicity, and subsequently increasing the passage into microbial cells and respectable fitting binding modes on the predictable target site. Additionally, these compounds may have another antibacterial mechanization in addition to their activity against DNA gyrase or Topo IV enzymes; that is reflected in more efficient antibacterial activity of these compounds comparing with parent positive control ciprofloxacin. Finally, Ciprofloxacin-quinoline-4-yl-1.3.4 oxadiazole hybrids 6a and 6b may be considered promising new dual antibacterial/anticancer lead compound that require further modification and investigation for their mechanism of action. Additionally, compound 8a could be a suitable lead compound for innovating new dual antibacterial/antifungal drugs.

Materials and reagents
• The chemicals used in this study were purchased from Sigma Aldrich. • The reaction progress was monitored using TLC (Kieselgel 60 F 254 pre-coated plates, E. Merck, Darmstadt, Germany) and the spots were visualized by UV lamp at λ 254 and 365 nm. • Melting points detection of the new compounds was done using Stuart electro-thermal melting point apparatus and they were uncorrected.
• Bruker Advance III 400 MHz records 1 H NMR spectra of tested compounds. Chemical shift (δ) in ppm relative to TMS (δ = 0 ppm) as internal standard using DMSOd 6 as solvent, and 13