Design, Synthesis and In Vitro Characterization of Novel Antimicrobial Agents Based on 6-Chloro-9H-carbazol Derivatives and 1,3,4-Oxadiazole Scaffolds

In this paper, we aimed to exploit and combine in the same molecule the carbazole and the 1,3,4-oxadiazole pharmacophores, to obtain novel carprofen derivatives, by using two synthesis pathways. For the first route, the following steps have been followed: (i) (RS)-2-(6-chloro-9H-carbazol-2-yl)propanonic acid (carprofen) treatment with methanol, yielding methyl (RS)-2-(6-chloro-9H-carbazol-2-yl)propanoate; (ii) the resulted methylic ester was converted to (RS)-2-(6-chloro-9H-carbazol-2-yl)propane hydrazide (carprofen hydrazide) by treatment with hydrazine hydrate; (iii) reaction of the hydrazide derivative with acyl chlorides led to N-[(2RS)-2-(6-chloro-9H-carbazol-2-yl)propanoil]-N′-R-substituted-benzoylhydrazine formation, which; (iv) in reaction with phosphorus oxychloride gave the (RS)-1-(6-chloro-9H-carbazol-2-yl)-1-(1,3,4-oxadiazol-2-yl)ethane derivatives. In the second synthesis pathway, new 1,3,4-oxadiazole ring compounds were obtained starting from carprofen which was reacted with isoniazid, in the presence of phosphorus oxychloride to form (RS)-1-(6-chloro-9H-carbazol-2-yl)-1-[5-(4-pyridyl)-1,3,4-oxadiazol-2-yl]ethane. The synthesized compounds were characterized by IR, 1H-NMR and 13C-NMR, screened for their drug-like properties and evaluated for in vitro cytotoxicity and antimicrobial activity. The obtained compounds exhibited a good antimicrobial activity, some of the compounds being particularly active on E. coli, while others on C. albicans. The most significant result is represented by their exceptional anti-biofilm activity, particularly against the P. aeruginosa biofilm. The cytotoxicity assay revealed that at concentrations lower than 100 μg/mL, the tested compounds do not induce cytotoxicity and do not alter the mammalian cell cycle. The new synthesized compounds show good drug-like properties. The ADME-Tox profiles indicate a good oral absorption and average permeability through the blood brain barrier. However, further research is needed to reduce the predicted mutagenic potential and the hepatotoxicity.


Introduction
Carbazole and its derivatives represent an important group of aromatic heterocyclic compounds containing a nitrogen atom. Compounds containing this scaffold have important electronic and charge-transport properties, as well as a conjugated pi-electron system, which facilitate the easy introduction of various functional groups into the structurally rigid carbazole ring [1].
These properties result in the extensive applications of carbazole derivatives in the medical field, such as antitumor, antimicrobial, antihistaminic, antioxidant, anti-inflammatory, and psychotropic agents [2].
The carbazole nucleus can be naturally found in the composition of some alkaloids as ellipticine [3], with antimalarial properties and koenidine, with potential antidiabetic effect [4], or staurosporine, with antibacterial, antifungal, and antitumoral activity [5], among them carbazomycins represent an unprecedented class of antibiotics with a carbazole heterocycle [6]. Carbazomycins A and B have proven to have antibacterial and antifungal activity, inhibiting the growth of phytopathogenic fungi, while Murrayafolin A, a compound isolated from Murraya euchrestifolia Hayata has shown a strong fungicidal effect on Cladosporium cucumerinum [7]. A new carbazole-derived alkaloid (1,8-dimethoxy-3-formylcarbazole) was isolated from the leaves of Clausena heptaphylla, being active against Gram-positive and Gram-negative bacteria, as well as fungi [8]. The carbazole acid ring is also present in many drugs, such as olivacine (antimalarial), rimcazole (antipsychotic and anticonvulsant), carvedilol (antihypertensive) [1,2] (Figure 1). Some members of carbazole series have been shown to exhibit a broad spectrum of antifungal activity, acting as potent fungal plasma membrane proton adenosine triphosphatase inhibitors [9].
Carprofen (2-(6-chloro-9H-carbazol-2-yl)propanoic acid) is a non-steroidal anti-inflammatory drug (NSAID), included in the class of propionic acid derivatives, that inhibits both COX-1 and COX-2 and is used commonly to combat inflammation and pain in animals with osteoarthritis or other inflammatory diseases. Carprofen was previously used in human medicine for over 10 years (1985-1995). It was generally well tolerated, with only mild side effects, such as gastro-intestinal pain and nausea. It is no longer marketed for human usage, after being withdrawn on commercial grounds, but is used by veterinarians as a supportive treatment for the relief of arthritic symptoms in geriatric dogs.
In the present study, we aimed to combine in the same molecular framework the carbazole and the 1,3,4-oxadiazole pharmacophores, to obtain novel carprofen derivatives. The new synthesized compounds were characterized by IR, 1 H-NMR and 13 C-NMR, screened for their drug-like properties and evaluated for in vitro cytotoxicity and antimicrobial activity.

Chemistry
The synthesis routes approached to obtain the target compounds are outlined in Scheme 1. The most commonly used synthetic route for the synthesis of 1,3,4-oxadiazoles includes the reactions of acid hydrazides with acid chlorides or carboxylic acids and cyclization of the formed acylhydrazines using a dehydrating agent, such as phosphorous pentaoxide, thionyl chloride, or phosphorous oxychloride [22].
In the present study, we aimed to combine in the same molecular framework the carbazole and the 1,3,4-oxadiazole pharmacophores, to obtain novel carprofen derivatives. The new synthesized compounds were characterized by IR, 1 H-NMR and 13 C-NMR, screened for their drug-like properties and evaluated for in vitro cytotoxicity and antimicrobial activity.

Chemistry
The synthesis routes approached to obtain the target compounds are outlined in Scheme 1.
The IR spectrum of compounds 4a-c showed characteristic absorption band at 3201-3215 cm −1 due to N-H group and also to carbonyl absorption bands at 1706-1702 and 1649-1651 cm −1 . Its 1 H-NMR spectrum revealed singlets at δ 10.15-10.19 and 10.34-10.66 ppm due to two NH protons of the hydrazide moiety, in addition to singlet at δ 11.38-11.41 ppm assignable for -NH-proton of the carbazole core. The 13 C-NMR spectral data for compounds 4a-c revealed signals in the aromatic region and additional two singlets around δ 172.74-172.90 and in the region 164-165.93 ppm for the two carbonyl groups, which are in agreement with the assigned structures.
The IR spectrum of compounds 5a-d showed absorption bands in the region 3238-3268 cm −1 due to NH group. Its 1 H-NMR spectrum displayed a doublet band at δ 1.80-1.90 ppm and quartet at 4.58-4.80 ppm corresponding to -CH 3 and -CH-groups, respectively. The presence of the ethanyl group was also confirmed by signals around δ 19.50-20.04 and 36.98-38.01 ppm in the 13 C-NMR spectrum of these compounds, which correspond to -CH 3 and -CH-groups, respectively.

Biological Assays
The tested compounds presented MIC values ranging from 0.625 to 10 mg/mL ( Table 1). The most susceptible microbial strains were the Gram-negative bacteria, followed by the fungal C. albicans strain. The best antibacterial effect was recorded for 4a against E. coli, with MIC of 1.25 mg/mL and for 5c against C. albicans, with MIC of 0.625 mg/mL. The minimal bactericidal concentration (MBC) values ranged in the same interval as the MIC ones, being equal or one time higher than that corresponding MIC values, suggesting a microbicidal mechanism of action ( Table 2). All tested compounds exhibited excellent anti-biofilm properties against the bacterial and fungal strains, with Minimal Biofilm Eradication Concentration (MBEC) values generally ranging from 0.009 to 2.5 mg/mL ( Table 3). The MBEC values were in some cases up to 256 times lower than MIC or MBC ( Table 3). The highest susceptibility to all tested compounds was recorded for the P. aeruginosa biofilm. For the in vitro cytotoxicity assay, the results show that the tested compounds become toxic at concentrations higher than 100 µg/mL, with the HeLa cells and HaCaT cells viability gradually decreasing after 48 h of incubation, in a dose-dependent manner ( Figure 3). All tested compounds are highly toxic at 1 mg/mL, the cellular viability decreasing dramatically after 24 h of incubation ( Figure 4). For the in vitro cytotoxicity assay, the results show that the tested compounds become toxic at concentrations higher than 100 μg/mL, with the HeLa cells and HaCaT cells viability gradually decreasing after 48 h of incubation, in a dose-dependent manner ( Figure 3). All tested compounds are highly toxic at 1 mg/mL, the cellular viability decreasing dramatically after 24 h of incubation ( Figure 4).   After 48 h, at 100 μg/mL, the compounds do not alter cell cycle phases of HeLa cells, but a fluctuation of the synthesis (S) phase is observed for HaCaT cells ( Figure 5).

In Silico Analyses
The tested compounds comply with Lipinski rule and are predicted to have a good bioavailability according to the Veber rule (Table 4).  Considering that the structural similarities may induce similarities in biological activities, we identified the possible structural similarities of the analyzed compounds with other ligands (Table  5). Interesting results regarding structural similarity were observed for compounds 5a-c. While compounds 5a and 5b are similar with nociceptive receptor ligand 2- For compound 5d we founded a poor similarity with other know compounds (Table 5).

In Silico Analyses
The tested compounds comply with Lipinski rule and are predicted to have a good bioavailability according to the Veber rule (Table 4). Regarding the predicted structural similarity of compounds 4a-c and 5a-d with other already known chemicals compounds, we noticed that compounds 1a, c recorded a higher similarity with compound 2-(6-chloro-9H-carbazol-2-yl)-N-phenylpropanamide, with biological activity, i.e., inhibition of Faah-fatty acid amide hydrolase active site (Table 5).
* Ligand with higher percent of similarity with analyzed compound.
Considering that the structural similarities may induce similarities in biological activities, we identified the possible structural similarities of the analyzed compounds with other ligands (Table 5). Interesting results regarding structural similarity were observed for compounds 5a-c.
While compounds 5a and 5b are similar with nociceptive receptor ligand 2- For compound 5d we founded a poor similarity with other know compounds (Table 5). Next, ADMET predictions suggest that all molecules have good intestinal absorption, with a value of more than 30% [23] (Table 6). The Caco2 cell line permeability suggesting if a compound is absorbed after oral administration revealed a value greater than 0.9 for all of the tested molecules [23]. The 5a, 5b, and 5c compounds have also shown an average blood brain barrier permeability. No drug-drug interactions affecting the renal clearance have been recorded for the tested compounds ( Table 6).
The AMES tests that predicts the mutagenic potential [23] proved to be positive for all tested compounds (Table 7). The maximum tolerated predicted dose for humans is considered to be low for all the tested molecules, as shown in Table 7. The inhibition of potassium channels encoded by the hERG gene is the main cause of the development of QT interval prolongation, on the electrocardiogram. Predictions in the pkCSM database suggest that all molecules inhibit hERG II, and those in the admetSAR database suggest that all molecules have poor inhibitory activity on hERG I (Table 7). In terms of hepatotoxicity, predictions suggest that the studied molecules exhibit hepatotoxicity.
The biological activity data have shown that the synthesized compounds have a good antimicrobial activity, especially on Gram-negative strains, 4a showing the best activity against E. coli. In contrast, 5c is a potentially antifungal agent. It is to be highlighted that the obtained compounds acted preferentially on biofilm embedded cells, as revealed by the very low MBEC values obtained for some compound-microbial strain pairs, in comparison with the MIC and MBC values. Also, it is to be noticed the very good anti-biofilm activity of all tested compounds against the P. aeruginosa biofilm, well known for its high resistance to all currently available antimicrobial agents. The in vitro cytotoxicity of the tested compounds performed on two cellular lines (i.e., HeLa and HaCaT cells) reveal a dose-and time-related effect, the cellular viability decreasing with the increase of concentration (>100 µg/mL) and incubation time (48 h). However, at concentrations lower than 100 µg/mL, the tested compounds do not induce cytotoxicity and do not alter the mammalian cell cycle.
The new synthesized compounds show good drug-like properties. All studied compounds comply with Lipinski rule and also, have a good predicted bioavailability according to the Veber rule.
To predict a possible biological activity of compounds 4a-c and 5a-d, we evaluated by bioinformatics tools the molecular similarity of our compounds and other compounds, already included into most popular databases. Our results have shown that compounds 4a-c exhibit a molecular similarity with Faah inhibitors, while the compounds 5a-c, appeared to have nociceptive and QPCT-glutaminyl-peptide cyclotransferase inhibitory activities.
The organic cation transporter OCT2 is involved in the renal excretion of cationic drugs and raises the possibility of drug-drug interactions between an inhibitor and a substrate drug, which will have a decreased renal clearance [24].
The ADMET predictions show that the compounds may have good oral absorption. Plus, 5a, 5b, and 5c show an average permeability through the blood brain barrier. Unfortunately, the compounds may also have mutagenic potential and increased hepatotoxicity.
Research may continue in this direction, by synthesizing similar compounds with lower cytotoxic, mutagenic and hepatotoxic potential.

Measurements
Melting points were determined in open glass capillary tube on an Electrothermal 9100 (Bibby Scientific Ltd., Stone, UK) capillary melting point apparatus and are uncorrected.
The FT-IR ATR (attenuated total reflection Fourier transform infrared) spectra were taken in solid state on a Bruker Vertex 70 spectrophotometer (Bruker Corporation, Billerica, MA, USA).
The 1 H-NMR and 13 C-NMR spectra were recorded in dimethylsulfoxide-d6 (DMSO-d 6 ) or deuterated chloroform on a Bruker Fourier 300 MHz or Bruker AvanceIII 500 MHz instrument (Bruker Corporation, Billerica, MA, USA), using tetramethylsilane as the internal standard, the chemical shifts are expressed in δ ppm.
The 1 H-NMR data are reported in the following order: chemical shifts, multiplicity, the coupling constants, number of protons and signal/atom attribution. The apparent resonance multiplicity is described as s (singlet), d (doublet), q (quartet), m (multiplet), dd (double doublet), and br (broad) signal.
For the 13 C-NMR data the order is the following: chemical shifts and signal/atom attribution. The mass spectrum of compound 5d was recorded on a Maxis Bruker 4G spectrometer (Bruker Corporation, Billerica, MA, USA) with an electrospray ionization source (ESI). The samples were dissolved in DMSO 1 mg/1 mL and then the solution was diluted with methanol to a final concentration of 1 µg/1 mL. The scanning range of molecular ions (m/z) was 50-1250.

Chemical Synthesis and Spectral Characterization
Methyl (2RS)-2-(6-chloro-9H-carbazol-2-yl)propanoat (carprofen methyl ester) (2) (RS)-2-(6-Chloro-9H-carbazol-2-yl)propanoic acid (carprofen) (10 g, 0.037 mol) was dissolved with stirring in 400 mL methanol absolute. Then 1.2 mL concentrated sulfuric acid (98%) was dropwise added as a catalyst and stirring was continued at room temperature for 8 h. The reaction mixture was left overnight at room temperature, and then the methanol was evaporated at low pressure until a precipitate appeared. Over the reaction mixture, 200 mL of water was added and the precipitate was filtered off at low pressure and washed well with water on the filter to remove the acid traces. The white or slightly yellow precipitate was air dried for 24 h.
After drying, 1.02 g of N,N'-disubstituted hydrazine were obtained, with a melting point (m.p.) of 262-263 • C at a yield of 87% toward hydrazide. The compound is soluble at room temperature in pyridine, dimethylsulfoxide, dimethylformamide, after heating in methanol, ethanol, isobutanol, and hardly soluble after heating in isopropanol, xylene, insoluble in ethyl acetate, hexane and water.
There were obtained 1.04 g of compound (81% yield toward hydrazide), with m.p. 268-270 • C. The compound is soluble at room temperature in pyridine, dimethylsulfoxide, dimethylformamide, after heating in isobutanol, hardly soluble after heating in methanol, ethanol, isopropanol, ethyl acetate, and insoluble in xylene, hexane and water.
The compound is soluble at room temperature in pyridine, dimethylsulfoxide, dimethylformamide, ethyl acetate, methanol, ethanol, after heating in isopropanol, isobutanol, xylene and insoluble in hexane and water.
An amount of 0.48 g of the compound is obtained, yield 64% toward acyl hydrazide with m.p. 185-186.5 • C, soluble at room temperature in pyridine, ethyl acetate, dimethylsulfoxide, dimethylformamide, after heating in methanol, ethanol, isopropanol, isobutanol, xylene and insoluble in hexane and water.
An amount of 0.56 g of the compound was obtained, yield 69% toward acyl hydrazide with m.p. 200-204 • C, soluble at room temperature in pyridine, ethyl acetate, dimethylsulfoxide, dimethylformamide, after heating in ethanol, isopropanol, isobutanol, xylene, hardly soluble after heating in methanol and insoluble in hexane and water.
An amount of 0.34 g compound (C 21   In order to confirm the structure, the compound 5d was also analyzed by mass spectroscopy, revealing the appearance of a peak corresponding to the molecular ion 375.1 [M + H], which confirms the molecular weight of this compound.

Antimicrobial Activity Assay
The antimicrobial activity of the synthesized compounds was determined using three methods, against a panel of Gram-negative (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853) and Gram-positive (Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212) bacteria, as well as the fungal strain Candida albicans ATCC 90029.
Minimal inhibitory concentration (MIC) assay: The minimum amount of the tested compounds that inhibited the microbial growth was evaluated using the microdilution method in liquid Mueller Hinton medium. The tested binary concentrations in the range of 5-0.009 mg/mL were achieved starting from a stock solution prepared in DMSO of 10 mg/mL, in a 100 µL culture medium final volume, seeded with a 20 µL microbial suspension of 0.5 MacFarland density. In each test, a microbial culture positive control and a sterile medium negative control were performed. The plates were incubated for 24 h at 37 • C. The results are shown in Table 1 [25][26][27].
Minimal bactericidal concentration (MBC) assay: after reading the MIC value, a volume of 10 µL from the well corresponding to the MIC value and from all the previous wells was seeded on solid PCA (plate count agar) medium to determine the MBC value, corresponding to the concentration at which the total inhibition of microbial growth on the solid medium was obtained ( Table 2).
Anti-biofilm activity assay: the plates used to determine the MIC and MBC were washed two to three times with sterile saline to remove free and poorly adherent cells, after which the biofilms formed on the walls of the wells were fixed for 5 min with 100 µL of cold methanol, stained with 1% alkaline crystal violet solution for 15 min and then resuspended in acetic acid solution 33%. After homogenizing the colored suspension, its optical density was measured at 490 nm. The minimal biofilm eradication concentration (CMEB) was determined to be the lowest concentration of the tested compounds at which the decrease in absorbance value, measured at 490 nm, was observed in comparison to the positive control (Table 3) [28].

In Vitro Cytotoxicity Assay
The cell cultures used in this study were HeLa (ATCC ® CCL-2™) and the HaCaT immortalized keratinocyte line. They were maintained in DMEM: F12 medium supplemented with 10% fetal bovine serum.
The compounds 4a-c, 5a-d were diluted in DMSO at a concentration of 20 mg/mL and brought to a concentration of 2 mg/mL in DMEM: F12. The solutions were sterilized by using a 0.22 µm filter.

Toxicity Assessment Using Fluorescein Diacetate Staining (FDA)-Propidium Iodide (PI)
The cells were seeded at a density of 7.5 × 10 5 /well in 24-well plates. The medium contained 1 mg/mL compound. After 24 h, cell viability was assessed by staining with FDA (10 µg/mL) and PI (20 µg/mL). Cells were observed on the Zeiss fluorescence microscope (Gottingen, Germany).

Evaluation of the Cellular Cycle
The cells were seeded at a density of 7.5 × 10 5 /well in 24-well plates and maintained in culture medium containing the tested compounds, at a concentration of 100 µg/mL, for 48 h. After incubation, the cells were trypsinized, washed in phosphate buffer saline, fixed in 70% cold ethanol and stained structures, adding specific substituents and completing valences with hydrogen atoms and passing these structures into three-dimensional space, in order to obtain 3D spatial structures.

Energy Minimization
Minimization of these molecules was done using the Hamiltonian method: Forcefield MMFF94x at a gradient of 0.05. After calculating the minimum energy, the Gasteiger partial loads (PEOE) were applied.

Determination of Drug-Like Character and Bioavailability
For establishing the pharmacological character of the newly synthesized compounds, the compliance with at least two Lipinski rules (drug-like nature) and with the Veber rule (bioavailability) was checked [29,30].

Structural Similarities Using Chemical Compounds Database
By accessing the Expasy/medicinal chemistry database we identified the possible structural similarities of the analyzed compounds with other chemical compounds.