Synthesis and In Vitro Antimycobacterial and Antibacterial Activity of 8-OMe Ciprofloxacin-Hydrozone/Azole Hybrids

A series of novel 8-OMe ciprofloxacin (CPFX)-hydrazone/azole hybrids were designed, synthesized, and evaluated for their in vitro biological activities. Our results reveal that all of the hydrozone-containing hybrids (except for 7) show potency against Mycobacterium tuberculosis (MTB) H37Rv (minimum inhibitory concentration (MIC): <0.5 μM), which is better than the parent drug CPFX, and comparable to moxifloxacin and isoniazid, some of the tested Gram-positive strains (MIC: 0.06–4 μg/mL), and most Gram-negative strains (MIC: ≤0.03–4 μg/mL).


Introduction
Fluoroquinolones (FQs) have emerged as a family of synthetic broad spectrum antimicrobial drugs, and the development of FQs was initiated in 1962 with the discovery of nalidixic acid. To date, several generations of FQs have been developed based on their antibacterial spectrum, which is getting significantly broader with each new generation, but there is no standard employed to determine which drug belongs to which generation [1]. The second and the third generations of FQs predominately act on Gram-negative bacteria, some Gram-positive bacteria, and intracellular microbes, while the latest fourth generation FQs are highly active against many species of Gram-positive pathogens, and anaerobic bacteria combined with the above mentioned microbes [2]. Currently, FQs are the second most widely used antimicrobial drugs, with extensive indications for infections including upper and lower respiratory infections, gastrointestinal infections, gynecologic infections, sexually transmitted diseases, prostatitis, and some skin, bone, and soft tissue infections, and their value and role in the treatment of bacterial infections continues to expand [1,3,4].
These antimicrobial drugs act by binding two type II bacterial topoisomerase enzymes, DNA gyrase and topoisomerase IV, thereby inhibiting DNA replication and transcription: for most Gram-negative bacteria, DNA gyrase is the target, whereas topoisomerase IV is the target for many Gram-positive bacteria [5]. It is believed that eukaryotic cells do not contain DNA gyrase or topoisomerase IV, while recent evidence has shown eukaryotic topoisomerase II is also a target for a variety of quinolone-based drugs [6]. The fourth generation FQs act at both DNA gyrase and topoisomerase IV, and this dual action slows the development of resistance [7]. Although they share Based on the above research results, and as a part of an ongoing program to optimize 8-OMe FQ derivatives as anti-bacterial/anti-TB agents [24][25][26][27][28], a series of 8-OMe CPFX-hydrozone/azole hybrids were designed, synthesized, and evaluated for their biological activity in this study. Our primary objective was to optimize the potency of these compounds against clinically important pathogens and MTB. A preliminary SAR study is also explored to facilitate the further development of FQs. Azoles, especially imidazole and triazole, are very useful pharmacophores due to their various biological activities, and some of them are currently used in the clinic for the treatment of common diseases [16,17]. It is notable that CPFX-azole hybrids exhibit excellent activity against both drug-susceptible and drug-resistant bacteria, and some of them are far more potent than the parent CPFX [18][19][20][21][22][23]. Moreover, our previous study demonstrated that an incorporation of hydrozone motif into GTFX could lead to an improvement of the activity against both Gram-positive and Gram-negative bacteria, and conjugate 1 (Figure 1), as the most emblematic example, has a broad antimicrobial spectrum with a minimum inhibitory concentration (MIC) in a range of 0.06-1 µg/mL [24].
Based on the above research results, and as a part of an ongoing program to optimize 8-OMe FQ derivatives as anti-bacterial/anti-TB agents [24][25][26][27][28], a series of 8-OMe CPFX-hydrozone/azole hybrids were designed, synthesized, and evaluated for their biological activity in this study. Our primary objective was to optimize the potency of these compounds against clinically important pathogens and MTB. A preliminary SAR study is also explored to facilitate the further development of FQs.

Anti-MTB Activity
The synthesized hybrids were preliminarily screened for in vitro activity against the MTB H37Rv ATCC27294 strain, using the Microplate Alamar Blue Assay (MABA) [29]. The minimum inhibitory concentration is defined as the lowest concentration effecting a reduction in fluorescence of >90% relative to the mean of replicate bacterium-only controls. The MIC values of the compounds along with CPFX, MXFX, and isoniazid (INH) for comparison are presented in μM in Table 1.

Anti-MTB Activity
The synthesized hybrids were preliminarily screened for in vitro activity against the MTB H 37 Rv ATCC27294 strain, using the Microplate Alamar Blue Assay (MABA) [29]. The minimum inhibitory concentration is defined as the lowest concentration effecting a reduction in fluorescence of >90% relative to the mean of replicate bacterium-only controls. The MIC values of the compounds along with CPFX, MXFX, and isoniazid (INH) for comparison are presented in µM in Table 1.

Antibacterial Activity
The target hybrids 1-21 were evaluated for their in vitro antibacterial activity against representative strains using standard techniques [24]. The minimum inhibitory concentration (MIC) is obtained from three independent experiments, defined as the concentration of the compound required to give complete inhibition of bacterial growth, and the MIC values of 1-21 against Gram-positive and Gram-negative strains, along with those of CPFX and levofloxacin (LVFX) for comparison, are listed in Tables 2 and 3, respectively. These data indicate that all of the target hybrids except for 7 and 19-21 have a similar antibacterial spectrum to CPFX and LVFX. These hybrids exhibit considerable potency in inhibiting the growth of some tested Gram-positive strains, such as the methicillin-sensitive Staphylococcus epidermidis (MSSE), the methicillin-sensitive S. aureus (MSSA), MRSA, and Enterococcus faecalis (two strains) (MIC: 0.06-4 μg/mL), as well as most of the tested Gram-negative strains (MIC: ≤0.03-4 μg/mL). It is worth noting that compound 16 shows useful activity (MIC: 0.5 μg/mL) against the CPFX-resistant Stenotrophomonas maltophilia, a common clinical pathogen.
Generally, hybrids 1-21 share a similar antibacterial trend with that of anti-MTB, i.e., the activity order of the (hetero)aromatic rings against both Gram-positive and -negative strains was in the order: acylhydrazones ≥ hydrazones >> azoles. In addition, ethylene imidazole hybrid 18 is much more potent than the corresponding propylidyne imidazole analog 19 and the ethylene triazole hybrids 20 and 21.

Antibacterial Activity
The target hybrids 1-21 were evaluated for their in vitro antibacterial activity against representative strains using standard techniques [24]. The minimum inhibitory concentration (MIC) is obtained from three independent experiments, defined as the concentration of the compound required to give complete inhibition of bacterial growth, and the MIC values of 1-21 against Gram-positive and Gram-negative strains, along with those of CPFX and levofloxacin (LVFX) for comparison, are listed in Tables 2 and 3 Generally, hybrids 1-21 share a similar antibacterial trend with that of anti-MTB, i.e., the activity order of the (hetero)aromatic rings against both Gram-positive and -negative strains was in the order: acylhydrazones ≥ hydrazones >> azoles. In addition, ethylene imidazole hybrid 18 is much more potent than the corresponding propylidyne imidazole analog 19 and the ethylene triazole hybrids 20 and 21.

Antibacterial Activity
The target hybrids 1-21 were evaluated for their in vitro antibacterial activity against representative strains using standard techniques [24]. The minimum inhibitory concentration (MIC) is obtained from three independent experiments, defined as the concentration of the compound required to give complete inhibition of bacterial growth, and the MIC values of 1-21 against Gram-positive and Gram-negative strains, along with those of CPFX and levofloxacin (LVFX) for comparison, are listed in Tables 2 and 3, respectively. These data indicate that all of the target hybrids except for 7 and 19-21 have a similar antibacterial spectrum to CPFX and LVFX. These hybrids exhibit considerable potency in inhibiting the growth of some tested Gram-positive strains, such as the methicillin-sensitive Staphylococcus epidermidis (MSSE), the methicillin-sensitive S. aureus (MSSA), MRSA, and Enterococcus faecalis (two strains) (MIC: 0.06-4 µg/mL), as well as most of the tested Gram-negative strains (MIC: ≤0.03-4 µg/mL). It is worth noting that compound 16 shows useful activity (MIC: 0.5 µg/mL) against the CPFX-resistant Stenotrophomonas maltophilia, a common clinical pathogen.
Generally, hybrids 1-21 share a similar antibacterial trend with that of anti-MTB, i.e., the activity order of the (hetero)aromatic rings against both Gram-positive and -negative strains was in the order: acylhydrazones ≥ hydrazones >> azoles. In addition, ethylene imidazole hybrid 18 is much more potent than the corresponding propylidyne imidazole analog 19 and the ethylene triazole hybrids 20 and 21.

General
Melting points were determined in open capillaries and uncorrected. Clog P was calculated by CLOGP module in sybyl 7.3 software. 1 H-NMR spectra were determined on a Varian Mercury-400 spectrometer (Varian Medical Systems Inc., Palo Alto, CA, USA)in DMSO-d 6 , CD 3 OD, or CDCl 3 using tetra-methylsilane (TMS) as an internal standard. Electro spray ionization (ESI) mass spectra were obtained on a MDSSCIEXQ-Tap mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). Unless otherwise noted, the reagents were obtained from a commercial supplier and were used without further purification. To a solution of I (0.11 mmol) in methanol (2 mL) was added a mixture of hydrazine hydrochloride (0.11 mmol) and NaHCO 3 (0.12 mmol) in H 2 O (1 mL) at room temperature. The reaction mixture was heated to 60 • C and stirred for 5-6 h and concentrated under reduced pressure. The precipitate was filtered and recrystallized from methanol (1 mL) to give targets 1-6 (yield: 45-61%) as off-white solids.

Method 3
A mixture of azole II (1 mmol), 1,2-dibromoethane (5 mmol) or 1,3-dibromopropane (5 mmol), and K 2 CO 3 (10 mmol) in DMF (20 mL) was stirred at room temperature overnight. After filtration, the mixture was diluted with dichloromethane (DCM, 100 mL) and washed with H 2 O (100 mL × 3). After the removal of the solvent, crude N-(2-bromoethyl/3-bromopropyl)azole III (yield: 42-65%) was obtained as a colorless oil, which was used directly in the next step. A mixture of III (0.2 mmol), 8-OMe CPFX (0.2 mmol), and K 2 CO 3 (1 mmol) in DMF (5 mL) was stirred at room temperature overnight. After filtration, the mixture was concentrated under reduced pressure. The residue was purified by silica gel chromatography eluted with DCM to DCM:MeOH = 10:1 to give targets 18-21 (yield: 44-51%) as off-white solids. 20% Tween 80 and 20 µL of Alamar blue were added to all of the wells. After incubation at 37 • C for 16 to 24 h, the fluorescence was read at an excitation of 530 nm and an emission of 590 nm. The MIC was defined as the lowest concentration effecting a reduction in fluorescence of ≥90% relative to the mean of replicate bacterium-only controls. MICs against nonreplicating M. tuberculosis were determined using a low-oxygen-recovery assay (LORA).

Conclusions
In summary, a series of novel 8-OMe CPFX-containing hybrids were designed, synthesized, and evaluated for their in vitro antimycobacterial and antibacterial activity. The results show that all of the 8-OMe CPFX-hydrozone hybrids (except for 7) have potent activity against MTB H 37 Rv (MIC: <0.5 µM) which is better than the parent drug CPFX (MIC: 1.30 µM), some of the tested Gram-positive strains (MIC: 0.06-4 µg/mL), and most of the Gram-negative strains (MIC: ≤0.03-4 µg/mL). However, our results suggest that lipophilicity seems not to be an important parameter affecting both the anti-MTB and antibacterial activity.