Design, Synthesis and Characterization of Novel Isoxazole Tagged Indole Hybrid Compounds

Abstract Sixteen new isoxazole tagged indole compounds have been synthesized via copper (I) catalyzed click chemistry of the aryl hydroxamoyl chloride and an indole containing alkyne moiety. The chemical structure of the synthesized compounds has been established using various physicochemical techniques. X-ray single crystal analysis of Ethyl 1-((3-phenylisoxazol-5-yl) methyl)-1H-indole-2-carboxylate (8a) has been analyzed. All compounds were tested for their antibacterial and anticancer activities. The activities for the new compounds were weak against both bacterial strains and the cancer cell lines.


Introduction
Indole derivatives, either synthetic or natural, are considered as privileged scaffolds in medicinal chemistry [1][2][3][4]. Many of them are the soul of an important class of therapeutic agents, including anticancer [5], antioxidant [6], antirheumatoidal [7], and anti-HIV [8][9][10] agents. They can also play a vital role in drugs targeting the immune system [11] and as tubulin polymerization inhibitors [12]. In view of the importance of the indole containing compounds, there is an ever-increasing demand to introduce new compounds based on indole moiety.
Isoxazole and their derivatives, on the other hand, have received comparable attention to the indole containing moieties. This is due to their diverse biological activities, which include, but are not limited to, their role as antiplatelet agents [13], antiviral and anti-HIV [14,15], anti-diabetic [16] anti-Alzheimer, anti-cancer and antiinflammatory agents [17,18].
The nature and the fast pace of chemical research forced scientists to cope by creating reaction conditions that subjugate to its nature. Hence, the relatively new "click chemistry" reactions have been originated by Sharpless and coworkers in 2001 [19,20]. Many reactions were found to satisfy the "click" criteria, the most notable of which are the cycloaddition reactions [21]. The superiority of the click methodology over the traditional Huisgen thermal process is the introduction of metal catalysts such as Cu(I), with the possibility of using other metal catalysts (Ru, Ni, Pt, Pd), within the reaction system at room temperature, rather than resorting to elevated temperatures. This introduction led to an enhancement of the regioselectivity of the product with respect to the Huisgen thermal process [22]. Inspired by the above facts, and in pursuit of our research group theme, which targets research in the realms of drug design and discovery, we report herein, the synthesis of new hybrid scaffolds combining indole and substituted isoxazole based on the well-established "click chemistry" methodology. We are hereby successfully reporting potent antimicrobial agents based on the indole moiety hybridization with other heterocyclic systems, such as triazine [23] and imidazole [24]. The latter-mentioned scaffolds have been reported to be active against various bacterial strains as well as cancer cell lines. (Figure 1)

Results, Experimental Procedure, and Discussion
The newly-designed compounds were synthesized from the commercially available 1-H-indole-2-carboxylic acid (1). In detail, we utilized both the carboxylic acid and the NH of the indole moiety to introduce our click precursors (2) and (3), as shown in Scheme 1. The precursor (2) was synthesized in a quantitative yield through the reaction with propargyl bromide in the presence of potassium carbonate in dry DMF. For precursor (3) the carboxylic acid group was protected with ethyl ester before reacting it with sodium hydride in dry DMF, and thereafter with propargyl bromide. Both 1 H and 13 C-NMR analyses for (2) and (3) clearly depict the characteristic features of the propargyl moiety along with the indole part.
Having the first click precursor in hand, the second precursor was synthesized via a two-step process. Initially, a selected set of aldehydes (4a-h) were condensed with hydroxyl amine [25]. This was followed by a reaction with N-chlorosuccinimide in dry DMF to give the pure hydroxyimoyl chloride (6a-h), as shown in Scheme 2.
1,3-dipolar cycloaddition was then realized with the dipolarophile (2) and (3) with the nitrile oxide generated in situ from (6a-h) upon the action of base directly before the reaction. This will serve to prevent dimerization to furoxans. Clicking both precursors shown in Scheme 1 produced the desired new compounds (7a-h) and (8a-h) (Scheme 3). All the new compounds were fully characterized using 1 H, 13 C and 2D-NMR techniques along with HRMS (ESI). In series (7) the 1 H-NMR shows a clear singlet for 2H resonating around δ = 6.0 ppm assigned for the methylene CH 2 protons, where a 0.5-0.7 down field shift were observed for all the synthesized compounds from the propargylated starting compound (2). Additionally, the new singlet signal assigned for CH in the isoxazole appeared around 6.9-7.5 ppm which was correlated with the signal for the carbon at 100-112 ppm in the 13   spectrum. In the other series, the compounds show two CH 2 signals, the first signal belongs to the methylene protons of the ester (OCH 2 CH 3 ) at about 4.3 ppm, while the second one belongs to the methylene protons of the carbon attached to indole nitrogen (NCH 2 -isoxazole) at about 6.0 ppm. It was also observed that the CH of the isoxazole resonated in the same region as in the first series for both proton and carbon.
HRMS-ESI analysis gave exact molecular ions for all the compounds. The structures of the new derivatives were determined from their corresponding 1 H and 13 C-NMR spectra. The formation of the isoxazole moiety was characterized via the methine proton signal, that resonates as a singlet at δ = 7.07-7.43 ppm. The singlet at δ = 7.39 ppm was assigned to H-3 of the indole moiety. All compounds show the features of a para-substituted aromatic system along with the aromatic signals of the indole. 13 C-NMR, on the other hand, clearly indicates all the features of the synthesized compounds. HMQC, HMBC and DEPT experiments allow us to fully assign all protons and carbons for the new compounds. The NH protons in compounds (3) and (7a-h) of the indole moiety appeared at 11.90-12.00 ppm for all the synthesized compounds.
X-ray structure determination was performed to further confirm the indole-hybrid system, with compound (8a) selected as a representative example of this new class of hybrid system ( Figure 2). The molecule crystallizes in a monoclinic chiral space group P2 1 . A summary of data collection and refinement parameters are given in Table  1. Figure 2 of compound (8a) shows that the indole along with the carboxylate moieties is in one plane with the CH2-isoxazole plane. Additionally, the isoxazole ring is connected to the indole via the CH 2 moiety with angle N1C12C13 of 110.7(3)°. An insight into intermolecular interactions can be obtained by electrostatic potential map and two-dimensional fingerprint plots mapped in the Hirschfeld surface, analyzed by CrystalExplorer 17.5 [26][27][28]. The electrostatic potential of compound (8a) (Figure 3a) shows that the isoxazole plane is almost negative towards the oxygen atoms while the indole plane is positive. This makes the molecule neutral with non-favorable intermolecular interactions. The two dimensional fingerprint plots and the contributions of individual interatomic contacts toward the overall crystal packing are shown in Figure 3b. Several directional contacts can be observed, such as C-C (1.8%), C-H (16.4%), N-H (4.2%), O-H (7.8%) along with H-H (66.7%). These intermolecular atom-atom contacts contribute to the stability of the crystal packing of compound (8a).
Ethical approval: The conducted research is not related to either human or animal use.

Biological Activities
Indole derivatives have shown various antibacterial and anticancer activities. In regards to the antimicrobial activity, this study reported weak in vitro antibacterial activity against both Gram positive and Gram negative bacteria. In addition, no significant activity was reported against pathogenic yeast and fungi. By the initial screening against selected pathogenic bacteria including Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa, the minimal inhibitory concentrations of the tested compounds were higher than 1 mg / ml. For the anticancer activity testing of the synthetic compounds, the inhibitory concentrations and percentage of killing of the tested compounds were measured using three human cancer cell lines: Leukemic cells (Hela), breast cancer cells (MCF-7), and epiglottis cancer cells (Hep-2) in addition to a non-human Chinese hamster ovary (CHO) cell lines. A concentration range of 100-500 ug/mL was used for all cells, and cell viability assay MTT was performed. A weak anticancer activity was observed for the tested compounds. Furthermore, Hela cell lines showed the highest sensitivity to all chemicals (% of killing equals to 20-34), whereas Hep-2 and MCF-7 cells were the least sensitive (% of killing equals to 5-11).

Experimental
1 H-NMR spectra were recorded in hexadeuterodimethyl sulfoxide (DMSO-d 6 ) and deuterated chloroform (CDCl 3 ) on a Bruker Avance III-300 MHz and III-500 MHz Spectrometer with TMS as an internal standard. 13 C-NMR spectra were obtained at 75 MHz and 125 MHz on the same instruments, respectively. Chemical shifts were reported as δ-values in ppm. High resolution Mass spectra (HRMS) were recorded in positive ion mode by Electro Spray Ionization (ESI) using a Bruker Daltonics Apex IV, 7.0 T Ultra Shield Plus. The samples were dissolved in chloroform, diluted in spray solution (methanol / water 1:1 v/v + 0.1% formic acid) and infused using a syringe pump with a flow rate of 2 µL / min. External calibration was conducted using arginine cluster in a mass range m/z 175-871. For all HRMS data, mass error falls in the range of 0.00-0.50 ppm. Melting points (m.p.) were determined on an Electrothermal Melting Point Apparatus and were uncorrected in °C. Solvents used in this study were obtained from Scharlau, Fluka, Acros, and Aldrich. All reactions were monitored by thin layer chromatography (TLC) using Merck aluminum plates pre-coated with silica gel PF254; 20 × 20 × 0.25 mm, and detected by visualization of the plate under UV lamp (λ = 254 or 365 nm ). Spots were also detected by spraying with anisaldehyde-sulphuric acid in ethanol, followed by heating to 140°C [35].

X-Ray Crystallography
Compound (8a) was recrystallized from hot DMF/benzene after cooling to room tempetaure. Small amount of ice was added to produce a fine colorless block crystalline solid. The compound crystallized in monoclinic chiral space group P2 1 . A summary of the crystallographic data and structure refinement parameters is given in Table 1.
Single-crystal X-ray diffraction data were collected using an Oxford Diffraction XCalibur, equipped with (Mo) X-ray Source (λ = 0.71073 Å) at room temperature (293(2) K). Data collection, reduction, and cell refinement were performed using the software package CrysAlisPro [29]. Analytical absorption corrections were applied using spherical harmonics implemented in SCALE3 (ABSPACK) scaling algorithm. Crystal structure was solved by direct methods, using the program OLEX2, followed by Fourier synthesis, and refined on F2 with SHELXL implemented in OLEX2 [30,31]. Anisotropic least-squares refinement of non-H atoms was applied. All crystallographic plots were obtained using the CrystalMaker program [32].

Prop-2-yn-1-yl 1H-indole-2-carboxylate (2)
1H-indole-2-carboxylic acid (1) 1.0 g (6.20 mmol) was dissolved in 10 ml DMF, and 5 equivalent of K 2 CO 3 , 4.28 g (31 mmol), were added at 0˚C. After 30 minutes, propargyl bromide (1.5 ml, 19 mmol) was added to the reaction mixture. After two hours, TLC confirmed the completion of the reaction; which was poured over crushed ice. The white precipitate that formed was collected and recrystallized from hot ethyl acetate. The product was produced with a high yield of 90.0%, and a melting point range of 136-138˚C.

General Procedure for the Synthesis of Aryl Oximes (5a-h)
The designed oximes were prepared according to the general procedure described herein. Hydroxylamine hydrochloride (0.55 mol) was added to a mixture of aryl aldehyde (5a-h) (0.25 mol) dissolved in 30 ml H 2 O/EtOH (1:1) and 30 ml of ice. Afterwards, 0.63 mol of 50.0% NaOH was added dropwise and the reaction was stirred at room temperature for approximately one hour, and monitored by TLC in chloroform (CHCl 3 ). After completion of the reaction, neutralization was accomplished using a concentrated HCl solution. Solid oximes were collected by suction filtration while liquid oximes were extracted with CHCl 3 (20mL×2), dried over MgSO 4 . The solvent was evaporator; the aryl oximes were produced with high yields (74 -93%) [27].

General Procedure for the Synthesis of Hydroxamoyl Chloride (6a-h)
To a stirred solution of 1.0 g of oxime (6a-h) in 15 ml DMF at room temperature, 1.3 mol equivalent of NCS was added. The initial addition (about 1/10 of the mass) results in a slight increase of temperature. In case that does not happen, then the solution is heated to 45°C to initiate the reaction. Once the reaction is initiated, the rest of NCS was added portion wise and the temperature was kept under 35°C. The mixture was stirred for 3-6 hours, and monitored by TLC (in CHCl 3 ). After completion of the reaction, the mixture was poured over crushed ice and the resultant product was filtrated as a solid. In the case of liquid products, they were extracted using CHCl 3 (20 mL×2) dried over MgSO 4 , and the solvent was removed using rotary evaporator. All hydroxamoyl chloride compounds (6a-h) were used in situ directly without further purification.

General Procedure for the Synthesis of Series (7) and (8)
A mixture of compound 3 (1.0 mmol) with 0.019 g copper (I) Iodide, CuI, (10 mmol) in the presence of Et 3 N (4.07 mmol) was stirred for 30 minutes. After that, 5-7 ml of toluene was added and the solution was stirred for an additional 30 minutes. To this solution, 1.3 mol equivalent of the corresponding hydroxamoyl chloride (7a-h) was added. The reaction mixture was stirred for 17-24 h. and monitored by TLC. After completion, 25 ml of EtOAc was added with stirring and the copper salt was removed by filtration. The filtrate was extracted and the combined organic layer was dried over Na 2 SO 4 . The solvent was evaporated and the product was purified using normal thin layer chromatography with Hexane/ethyl acetate as mobile phase (the ratio depended on the product polarity). Figure  4 shows the numbering system adopted in this study with compounds 7a and 8a used as representatives for this order.

Grwoth and Maintainance of Microorganisms and Cancer Cell Lines
The synthesized compounds were tested against Grampositive strains namely; Methicillin-sensitive Staphylococcus aureus NCTC 10788 (MSSA), methicillin-resistant Staphylococcus aureus ATCC 33591 (MRSA), two standard Gram-negative bacterial strains namely; Escherichia coli NCTC 12923, Pseudomonas aeruginosa NCTC 12924 along with the two fungi Candida albicans (NCPF 3179) and Aspergillus brasiliences (NCPF 2275). The microorganisms were inoculated into Trypticase soy broth and Sabaroud dextrose broth for bacteria and fungi respectively and the pH of the medium was adjusted to 7.3 with sterile phosphate buffered saline. The cultures were subsequently incubated at 37°C for 24-48 hours except for Aspergillus which was incubated at 25°C for 5-7 days. The optical density (OD) of the bacteria from mid-log phase of growth was measured at 600 nm and to obtain an optical density of 0.4 (corresponding to 1.5×10 8 colony forming units/mL). MCF-7 (human breast adenocarcinoma), Hep2 (human epiglottis cancer), and Hela (cervical cancer cells) cell cultures were kindly provided by Jordan Company for Antibody Production (MONOJO, Amman, Jordan). The cell lines were cultured in growth medium (RPMI-1640medium, pH7.4), supplemented with 15% fetal bovine serum (FBS) and antibiotics [(penicillin (100 units/mL) and streptomycin (100 ug/mL)]. The cell lines were maintained at 37°C in a 5% CO 2 atmosphere with 95% humidity [33].

Antimicrobial Susceptibility Testing
The stock suspensions of microorganisms were prepared to the standard McFarlands (0.5) and subsequently uniformly spread on a solid growth medium in a Petri dish. Sterile paper disks (6 mm in diameter; Becton, Dickinson & Co.) were placed on the agar plates and impregnated with 30 uL solution of each compound. Plates were incubated for the recommended time periods  h and up to 7 days for Aspergillus) under appropriate cultivation conditions. Antimicrobial activity was determined if the tested compound produced an inhibition zone around the impregnated disk. Disks impregnated with sterile DMSO served as negative controls and disks with standard antibiotics (vancomycin, ciprofloxacin, and clarithromycin (Oxoid, UK)) served as positive controls. Measurements at each concentration were performed in triplicates [36]. For MIC determination, 200 µL of diluted microbial suspension was added, (0.2-500 µg/50 µL) of the synthesized compounds and standard antibiotics (Cephtriaxone, Gentamycin and Levofloxacin) in 20% H 2 O/DMSO were added to wells of ELISA plates, and subsequently incubated at conditions described above. The effect of the tested compounds on the growth of microorganisms was monitored by measuring the optical density at 600 nm using an ELISA reader. The MIC was defined as the lowest concentration of the tested compounds allowing no visible growth. All measurements of MIC values were repeated in triplicate.
Antimicrobial interactions between the tested compounds conventional antimicrobial agents like cefzolidin and amoxicillin against MRSA were evaluated by the standard checkerboard titration method [34]. The bacterial suspensions, growth media, and culture conditions were the same as those described for the MIC determination mentioned above. Experiments were performed in triplicate. The fractional inhibitory concentrations (FICs) were calculated as follows:

Cell Cytotoxicity for Anticancer Activity Testing
Microculture tetrazolium (MTT) assay was used to evaluate cell vitality. Briefly, monolayers of each cell line were trypsinized and the cells were seeded in 96-well plates at the density of 5 x10 5 cell/well (100 µL/well) in a culture medium and incubated for 24 h at 37 o C, with 5% CO 2 in a humidified atmosphere. The medium was subsequently removed and fresh growth medium containing different concentrations of tested compounds (concentration range of 5-500 µg/mL) were separately added. Control cells were supplemented only with a medium and colchicine was used as a positive control. 20 µL of 5 mg / mL MTT (pH 7.4) in 180 µL media was added per well and incubated for another 4 h. The plates were then centrifuged and supernatants were removed to all the addition of 100 µL of 1:1 ethanol: DMSO. The absorbance was then measured by an ELISA reader Bio-Rad, USA) at a wavelength of 570 nm. The values are presented as means of duplicate analyses. The activity of the tested compounds on the proliferation of cancer cells was expressed as the % of cytotoxicity [36].

Conclusion
In summary, sixteen new hybrid compounds were synthesized featuring the indole and the isoxazole moieties. All new compounds were identified and tested against three bacterial strains and three cancer cell lines. It was found through this research work that these newlysynthesized compounds did not show any promising activities. Single X-ray crystal structure was determined for a representative compound of these new hybrid systems.