Bioactive fluorenes. Part III: 2,7-dichloro-9H-fluorene-based thiazolidinone and azetidinone analogues as anticancer and antimicrobial against multidrug resistant strains agents

Background Thiazoles, thiazolidinones and azetidinones are highly ranked amongst natural and synthetic heterocyclic derivatives due to their great pharmaceutical potential. Results New thiazolidinone and azetidinone class of bioactive agents based on 4-(2,7-dichloro-9H-fluoren-4-yl)thiazole moiety have been successfully synthesized. 4-(2,7-dichloro-9H-fluoren-4-yl)thiazol-2-amine was synthesized and allowed to react with various aryl/heteroaryl aldehydes to afford the corresponding Schiff base intermediates. The target thiazolidinone and azetidinone analogues have derived from Schiff bases by their reactions with thioglycolic acid and chloroacetyl chloride, respectively. The newly synthesized compounds were then evaluated for their antimicrobial activity against some multidrug resistant strains and examined for cytotoxic activity against normal lung fibroblast (WI-38), human lung carcinoma (A549), and human breast carcinoma (MDA-MB-231) cell lines to develop a novel class of fluorene-based bioactive agents. The mode of action and the binding interaction of the synthesized compound with the active sites of dihydrofolate reductase enzyme were well identified by fluorescence-activated cell sorting (FACS) analysis and molecular docking study. Conclusion Some of the synthesized compounds showed remarkable activity against A-549 and MDA-MB-231 when compared to Taxol, which was used as a reference drug. 2,7-dichloro-9H-fluorene-based azetidinones are more efficient as antimicrobial and anticancer agents compared to dichloro-9H-fluorene-based thiazolidinones derivatives.


Introduction
In the last few years, fluorene derivatives exposed effective uses as precursors in broad ranging of synthetic and medical applications [1]. As example, 2,7-dichloro-7H-fluorene considered as a backbone moiety for the synthesis of a well-known antimalarial drug which known as Lumefantrine [2] (Fig. 1). On the other hand, heterocyclic compounds are highly ranked amongst natural and synthetic pharmaceutically significant agents. The fabulous ability of heterocyclic moiety to serve as both biomimetic and active pharmacophores has mainly contributed to their distinctive value as traditional key elements of various drugs. Due to their broad pharmacological profile, the nitrogen and sulfur-containing heterocycles demonstrate an imperative class in the biological research and drug industry areas [3][4][5][6][7][8]. Amongst them, the thiazole ring is a core structural moiety found in a wide range of biologically and medicinally active molecules. The thiazole derivatives are useful for treatment of several diseases such as allergies [9], hypertension [10], microbial [11], human immunodeficiency virus (HIV) infections [12], inflammation [13], and schizophrenia [14]. Moreover, 4-thiazolidinone and its derivatives have considerable attention for the last decades due to their pharmacological potential. These derivatives are known to acquire several promising chemotherapeutical activities such as antihistaminic [15], anti-inflammatory [16], hypolipidaemic [17], antimicrobial [18], anticonvulsant and antipsychotic [19], antimalarial [20], and anti-cancer [21] activities. Numerous drugs containing thiazole or 4-thiazolidinone moieties in their structure used in broad range in the pharmaceutical market such as Niridazole, Abafungin, Fanetinole, Ralitoline and Etozoline (Fig. 1). The traditional synthesis of 4-thiazolidinone derivatives involves cycloaddition of Schiff base with thioglycolic acid [22]. Additionally, the 2-azetidinone moiety is commonly show wide range of biological activities and exist in several β-lactam antibiotics such as penicillins, carbapenems and cephalosporins ( Fig. 1) which are used as broad spectrum antibacterial agents. A large number of 3-chloro monocyclic β-lactam exhibits powerful antimicrobial, anticonvulsant, anti-inflammatory and antitubercular activities [23][24][25]. Conventional synthesis of 3-chloro-2-azetidinones involves [2 + 2] Staudinger's ketene-imine cycloaddition reaction between chloroacetyl chloride and Schiff bases [26].
On the other hand, dihydrofolate reductase (DHFR) is an indispensable enzyme that catalyzes the NADPHdependent reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF), which is the precursor of the cofactors compulsory for the biosynthesis of thymidine and purine nucleotides [27]. Accordingly, inhibition of dihydrofolate reductase lead to the disturbance of deoxyribonucleic acid (DNA) synthesis and the death of the proliferating cells [27,28]. Furthermore, bacteria need DHFR to grow and multiply and consequently inhibitors discerning for bacterial in contradiction of host DHFR have found usage as antibacterial agents [29]. These two remarkable features render DHFR enzyme as one of the main targets for both antimicrobial and anticancer drug design [30,31].

Chemistry
As the inhibition of DHFR is commonly considered as one of the most prominent mechanism in elucidating antimicrobial and anticancer activities [35,36], the compounds synthetic approaches were designed in order to achieve: (i) possess hydrophilic and hydrophobic parts that can interact with the hydrophilic and hydrophobic regions of the DHFR active site, respectively; (ii) comply with the pharmacophores that may interest as DHFR inhibitors, as presented in Fig. 2.
The chemical structures of all synthesized compounds 5a-n and 6a-n were well-confirmed based on spectroscopic data such as Fourier transform infrared (FT-IR), proton nuclear magnetic resonance ( 1 H-NMR), carbon-13 nuclear magnetic resonance ( 13 C-NMR) and The distortionless enhancement by polarization transfer (DEPT-135) data (c.f. "Experimental" section and Additional file 1). The FT-IR spectra of compounds 5a-n revealed the presence of characteristic absorption bands at 1780-1680 cm −1 for (C=O) group, 1636-1600 cm −1 for (C=N) group. Furthermore, to fully establish the chemical structures of the products, intensive 1D ( 1 H, 13 C, and DEPT-135) NMR spectroscopic analysis were recorded. For example, analysis of the 13 C and 13 C-DEPT-135 NMR spectra of 5a indicated the presence of 23 signals representing the 23 of nonequivalent carbons (10 aromatic quaternary carbons, 9 aromatic CH's, 2 methylene carbons, one methine carbon and one carbonyl carbon). Its 1 H-NMR spectrum showed three singlet signals at 7.66, 7.60, 7.28 ppm and two doublets at 7.48 and 6.99 ppm (J = 8.0 Hz) for five protons of the fluorene moiety. A multiplet at 7.20, 6.94 ppm and doublet signals at 7.38, 6.27 ppm (J = 8.0 Hz) appeared for the protons of phenyl moiety. In addition to this, a singlet signal at 6.76 ppm for thiazole moiety. Three singlet signals at 3.98, 3.60 and 3.51 ppm corresponded to two methylene and one methine protons.
On the other hand, the FT-IR spectra of compounds 6a-n showed the presence of characteristic absorption bands at 1792-1697 cm −1 for (C=O) group, 1698-1598 cm −1 for (C=N) group. Indeed, the 13 C and 13 C-DEPT-135 NMR spectra of 6b indicated the presence of 24 signals representing the 24 of nonequivalent carbons (11 aromatic quaternary carbons, 8 aromatic CH's, 2 methine carbons, one methylene carbon, one methyl carbon and one carbonyl carbon). Its 1 H-NMR spectrum showed two doublets at 7.87 and 7.27 ppm (J = 8.0, 8.0 Hz), three singlet signals at 7.70, 7.50 and 7.21 ppm for five protons of the fluorene moiety. Two multiplets at 7.64 and 7.44 ppm appeared for the protons of 4-methoxyphenyl moiety. In addition, a singlet signal at 7.12 ppm for thiazole moiety. Four singlet signals at 4.45, 4.28, 4.20, and 4.01 ppm corresponded to the one methyl, two methine and one methylene protons.

Biological activity Antimicrobial activity
Nowadays, the microbial resistance to currently found antibiotics is considered a precarious problem. Therefore, performing some more trials and efforts to identify novel targets for discovering new antibiotics is supposed to be a strong challenge [37]. The multidrug resistant bacteria have been reported with a diversity of nosocomial and community acquired infections as pneumonia, surgical site infections and urinary tract infections [38].
In the current study, the synthesized fluorene derivatives 5a-n and 6a-n were evaluated for their antimicrobial activity against multidrug resistant strains of Gram-positive bacteria such as staphylococcus aureus (S. aureus), methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pneumoniae (S. pneumoniae) and Gram-negative bacteria such as Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), Pseudomonas aeruginosa (P. aeruginosa) and Acinetobacter baumannii (A. baumannii) as well as three fungal strains such as Aspergillus flavus (A. flavus), Aspergillus niger (A. niger) and Candida albicans (C. albicans). Screening the antimicrobial activity was done by agar well diffusion assay [39] using a concentration of 500 µg/mL of the tested fluorene compounds, the results of the antimicrobial assay are given in Tables 1, 2, 3. It is clearly observed that some of the newly synthesized fluorene derivatives exhibited comparatively high antimicrobial activity when compared to the positive reference drugs; vancomycin for Gram-positive bacteria, gentamicin for Gram-negative bacteria and fluconazole for fungi. It's worthy to mention that, the thiazolidinone derivatives 5g, 5h, 5i and 5l produced relative high activity against S. aureus with a zone of inhibition (ZOI) value 10 mm, 11 mm, 10 mm, and 9 mm, respectively. While the compound 5j showed higher activity against E. coli and P. aeruginosa with a zone of inhibition (ZOI) value 10 mm and 8 mm, respectively (Table 1).
Furthermore, azetidinone derivatives 6a-n achieved relatively high antimicrobial activity against both Gram positive and Gram-negative bacteria, particularly 6h against S. aureus, MRSA, E. coli and P. aeruginosa with a ZOI value 12 mm, 15 mm, 22 mm, and 8 mm, respectively. However, a higher activity was shown against E. coli with ZOI value 27 mm for compound 6m. On the other hand, 6d showed moderate activity against Gramnegative bacteria E. coli, K. pneumoniae and P. aeruginosa with a ZOI value 15 mm, 6 mm, 10 mm, and 8 mm, respectively. However, low activity was shown against S. aureus and no activity was shown against both MRSA and S. pneumoniae. Moreover, the compound 6n showed moderate antimicrobial activity against E. coli and P. aeruginosa with a ZOI value 19 mm and 13 mm, respectively. The rest of the newly synthesized fluorene derivatives display low antimicrobial activity therefore these derivatives have potential for further comprehensive studies ( Table 2).
The minimum inhibitory concentration (MIC) of the most active newly synthesized fluorene derivatives was determined and reported in Table 3. The MIC varied within the range (500 µg/mL-7.8 µg/mL). Compounds 5h and 6e were potent against Gram positive bacteria particularly S. aureus with an MIC value 62.5 µg/ mL. Also, 6h was potent but against both S. aureus and MRSA with an MIC (62.5-31.25 µg/mL, respectively). Furthermore, a lower MIC was observed by the compound 6l against both S. aureus and MRSA as the MIC value was (31.25 µg/mL). On the other hand, the newly synthesized fluorene derivatives showed higher activity against Gram negative bacteria which is clearly achieved by the compound 6j, 6k, 6l and 6 m with MIC ranged from (31.25-15.6 µg/mL) specially against E. coli. All results were compared to vancomycin and Gentamicin as antibacterial reference drug (Table 3).
It's worth to report that, the obtained biological activities make the newly synthesized novel fluorene derivatives 5a-n and 6a-n, interesting molecules for the synthesis of new antibiotics either alone or in combination with other compounds, and subsequently help in fighting the multidrug resistant superbugs.
Fluorescence-activated cell sorting (FACS) analysis for annexin V and PI staining to follow the mechanisms of Table 1 Antimicrobial activity of the newly synthesized thiazolidinone derivatives 5a-n against the multidrug resistant tested microbial strains   Fig. 4.

S. aureus MRSA S. pneumoniae E. coli K. pneumoniae P. aeruginosa A. baumannii A. flavus A. niger C. albicans
Moreover, in vitro anti-proliferative effect of azetidinone derivatives 6a-n on normal lung cells, lung and breast cancer cells and uncover the mechanisms of cell death in selected drugs which show anti-cancer activities.

Docking and molecular modeling study
Molecular Docking study of 28 new synthesized compounds 5a-n and 6a-n has been performed. The main idea was to build molecules that have the ability to intercalate between the DNA base pairs while in the same time be able to stabilize their intercalating complex through formation of different bonding with topoisomerase I amino acids. Molecular Docking study was done in order to comprehend the mechanism of interaction of the synthesized compounds with DNA topoisomerase I and to verify the difference in activity as antibacterial and anticancer between different synthesized analogues. The crystal structure of DNA topoisomerase I was obtained from Protein Data Bank [https ://www.rcsb. org] at 3.0°A resolution (PDB code: 1T8I). It consists of 592 amino acid residues in one chain. After preparation of the enzyme, molecular docking of the cocrystallised Camptothecin ligand was done ( Fig. 9) with different placement protocol in order to choose the best methodology for docking. The Triangle matcher placement method showed RMSD value of less than 2 (1.3581) which indicates the confidence in the produced docking results. As can be seen from the 2D and 3D interaction between Camptothecin and DNA topoisomerase I enzyme, Camptothecin acts mainly through intercalation between DNA base pairs which halts the ability division of DNA double strand. Molecular docking of the conformation database of the 42 synthesized compounds into the active site of DNA topoisomerase I was carried out using the mentioned protocol with the results refinement using force-field based scoring function GBVI/WSA dG which estimates the free energy of binding of the ligand from a given pose. The functional form is a sum of terms: C is represents the average gain/loss of rotational and translational entropy. α, β is constants which were determined during training (along with c) and are forcefielddependent. Ecoul is the columbic electrostatic term,  which is calculated using currently loaded charges, using a constant dielectric of 1. Esol is the solvation electrostatic term which is calculated using the GB/VI solvation model. Evdw is the Van der Waals contribution to binding. SAweighted is the surface area weighted by exposure. The output docking results were arranged according to scoring function and explored using the browser function embedded in MOE software. Representation of 2D and 3D of the ligand interaction between all the synthesized compounds and DNA topoisomerase I enzymes is shown in Fig. 10. The synthesized compounds can be sorted into two different groups 5 and 6 according to the attachment to the (9H-fluoren-4-yl)thiazole; first the attachment is through thiazolino-4-one moiety and second the attachment is through β-lactam ring. Upon examining the scoring results, most of the highest active compounds showed better energy scores. So, compounds 5e, 5h, 5l, 6e and 6h showed high scores in comparison with other analogues. The scores were in the range of − 9.0685 to − 8.4903 kcal/mole.
The details of the interactions are as the following: most of the compounds were able to intercalate between

Conclusion
In this study, various 2,7-dichloro-9H-fluorene-based thiazolidinone and azetidinone derivatives were designed, synthesized, fully characterized and screened in vitro against various multidrug resistant microorganisms as well as against human lung carcinoma (A-549) and human breast carcinoma (MCF-7) cell lines. The results indicated that 2,7-dichloro-9H-fluorene-based azetidinones are more efficacious antimicrobial and anticancer agents compared to dichloro-9H-fluorene-based thiazolidinones analogues. Hence, there is adequate scope for further study in developing such compounds as a good lead activity.

General methods
All Chemicals and solvents used purchased from Sigma-Aldrich are spectroscopic grade and used without further purifications. Melting points were determined on a Stuart SMP3 melting point apparatus and are uncorrected. FT-IR spectra were recorded on a Shimadzu IR-3600 FT-IR spectrometer in KBr pellets. NMR spectra were acquired on a Bruker Avance 400 instrument (400 MHz for 1 H, 100 MHz for 13 C) in DMSO-d 6 solutions, using residual solvent signals as internal standards. Starting materials 2,7-dichloro-9H-fluorene (2) and 2-chloro-1-(2,7-dichloro-9H-fluoren-4-yl)ethanone (3) were prepared according to our previously reported method [31].

Antimicrobial screening Used microorganisms
All microbial strains were kindly provided from the department of Medical Microbiology and Immunology faculty of Medicine Assiut University, these clinical isolates were obtained from clinical cases of infections admitted to Assiut University hospital as urinary tract infections, corneal ulcers, bacterial and fungal pneumonia, otomycosis, oral thrush and wound infections. The clinical isolates were proved by using the VITEK 2 automated microbiology system (BioMérieux).
The clinical isolates used were multidrug resistant strains, they were resistant to β lactam (penicillin, amoxacillin, oxacillin), cephalosporins (cefazolin, cefaclor and cefepime) and macrolides (erythromycin and clarithromycin), they included Gram positive bacteria as

Initial evaluation of the fluorene derivatives antibacterial and antifungal activity
The antimicrobial activity of the fluorene derivatives was initially evaluated by agar well diffusion assay [40]. Mueller-Hinton agar (CM0337) was poured into Petri dishes at 50-60 °C and left to solidify for 15 min. Subsequently, overnight microbial suspensions of tested strains was adjusted to turbidity of 0.5 McFarland Standard, which equals to 1-2 × 10 8 CFU/mL for bacteria and 1-5 × 10 6 for fungi. The microbial inoculums were then diluted in 1:100 ratio in case of bacteria and 1:10 ratio in case of fungi in order to get 1-5 × 10 5 CFU/mL. a sterile cotton swab was dipped into the adjusted microbial suspension and the Mueller-Hinton agar plates were inoculated by evenly streaking cotton swab over the agar medium. Then wells with a diameter of 0.5 cm were cut in the medium with a sterile cork borer. Stock solutions of the flourene derivatives were diluted in DMSO 1% to get 500 μg/ mL concentrations. The tested flourene derivatives and controls (50 μL) were dispensed into the wells. The plates were incubated for 24 h at 37 °C for bacteria and C. albicans while at 25 °C for A. falvus and A. niger. The diameters of zones of inhibition (ZOI) around the wells were measured in mm. Following control agents were used: positive control agents-vancomycin (50 μg/mL) for Gram positive bacteria, gentamicin (10 μg/mL) (for Gram negative bacteria) and fluconazole 25 μg/mL for fungi and negative control agent is 1% DMSO.

Determination of MIC values for the most active fluorene derivatives
Determination of Minimum inhibitory concentrations (MIC) of flourene derivatives was done using broth microdilution method [41]. The procedure involved preparation of twofold dilutions of the fluorene derivatives ranging from (500-7.8 μg/mL) in sterile Mueller-Hinton broth inside the wells of 96-well microplate (Sarstedt, Germany). The inoculums of test strains prepared from fresh overnight cultures were adjusted to 0.5 McFarland standards, which equals to 1-2 × 10 8 CFU/ mL for bacteria, the procedure was done according to CLSI 2012 [42]. The highest dilution of samples (flourene derivatives) without visible growth after 24 h incubation at 37 °C was considered as MIC. For this assay the positive control agents were vancomycin (range: 0.7-50 μg/ mL), gentamicin (range: 0.15-10 μg/mL) and the negative control was 1% DMSO.
For proper determination of the MIC end point resazurin dye has been used. A stock solution of resazurin sodium salt powder (Titan Biotech) was prepared at 0.02% (wt/vol) in distilled water, sterilized by filtration through a 0-2 µm filter into a sterile light protected container then stored protected from light at 4 °C for up to 1 week, or at − 20% for long term use, then 10-15% resazurin solution of the total volume in wells was added to each well and incubation for 1-4 h at 37 °C was done. A change in color from blue to pink indicates the growth of bacteria, and MIC was defined as the lowest concentration of the drug that prevented this change in color.
Data processing All experiments were independently repeated three times. Obtained data were processed; standard deviations were calculated using GraphPad Prism 5.03 (GraphPad Software, Inc.; USA) software.

Cell viability assay
WI-38, A549, and MDA-MB-231 cells were seeded into 96-well plates (at a density of 5000 cells/well). On the following day, cells were treated with different concentrations (0, 1, 10, 31.25, 62.5, 125, 250, 500 µg/mL) of 16 fluorene derivatives in fresh medium and incubated for another 24 h. Cell viability was then assessed using the MTT assay (Sigma Aldrich, St. Louis, MO, USA), and the absorbance was read at 570 nM using an ELISA microplate reader (Molecular Devices, Downingtown, PA, USA).