Click synthesis of new 7-chloroquinoline derivatives by using ultrasound irradiation and evaluation of their biological activity

ABSTRACT This study describes the click synthesis of new 7-chloroquinoline derivatives by using ultrasound irradiation and evaluation of their activity as antimicrobial, antimalarial and the anticancer. All the compounds show moderate antimalarial activity with IC50 < 100 μM, six of them showed high antimalarial activity (2, 3, 4, 6, 8 and 9) with IC50 < 50 μM. The most active 7-chloroquinoline derivative is a compound (9). Also, the newly synthesized compounds were screened for their antitumor activity towards three lines of cancer cells, MCF-7 (human breast cancer), HCT-116 (colon carcinoma) and Hela (Cervical carcinoma) cell lines. Compounds (3) and (9) exerted the highest activity on all cell lines and showed special selectivity toward MCF-7 cells and the antibacterial screening data showed moderate to good inhibition zone (12.5 ± 0.63–23.8 ± 1.5) towards all the tested compounds. Elucidation of the structures of these new pure compounds was based on, IR, 1H NMR, 13C NMR, MS and their elemental analysis. GRAPHICAL ABSTRACT


Introduction
Quinolines and their derivatives are present in numerous natural products and have highly antimalaria, antiasthmatic, antiinflammatory, antibacterial and antihypersensitive activities (1). Few methods have been reported for the preparation of quinolines derivatives such as the Skraup, Doebner von Miller and Combes procedures (2,3). Malaria is a contagious disease, caused by protozoa parasites from the genus Plasmodium that is transmitted by mosquitoes of the genus Anopheles. Plasmodium falciparum is responsible for the most lethal form of malaria (4). Chloroquine was the most effective antimalarial clinically used drug but parasite resistance led to its substitution by artemisinin and its semi-synthetic derivatives (artemether, artesunate) (5,6). So, new drugs to treat malaria are critically required. Synthesis of molecular hybrids containing different moieties which are representatives of known or putative antimalarial compounds is presently being extensively explored. Recently, the synthesis of 1,2,3-triazoles by a process known as Cumediated click chemistry (7) has been explored to combine different molecules affording new analogs of chloroquine (8), chalcones (9), naphthoquinones (10) several other hybrid antimalarial molecules have been synthesized (11)(12)(13).
In this sense, ultrasonic irradiation has picked up ubiquity in the previous decades as a useful tool in most of the industrial and academic applications. The utilization of sanction as the energy source of organic synthesis (sonochemistry) is very much recorded (14). This energy source has been proven to be able to accelerate reactions and selectivities through the development of new receptive intermediates and compounds not usually observed under ordinary thermal conditions (14). Furthermore, ultrasound irradiation can be considered as earth amiable procedures, being less vitality concentrated and creating decreased amounts of side items (15).
In this study, the sonochemical technique was used to synthesize new derivatives of 7-chloroquinoline and evaluation as antimicrobial, antimalarial and the anticancer activity of the new compounds.
The first step of the proposed strategy was the synthesis of the key derivatives (2)-(4) which carried out via nucleophilic substitution on the suitably position of 4-7-dihloroquinoline. It should be noted that the chloroquinoline ring can be considered as the prototype electron poor aromatic system. The electronegative nitrogen substituted on the ring has it's aromaticity disrupted through a strong permanent dipole by the inductive polarization that causes fractional positive charge on the C2 and C4 atoms of the chloroquinoline ring. Therefore, 4-7-dihloroquinoline ring can be pictured as experiencing mesomeric electron withdrawal by the nitrogen atom, and more importantly, experiences additional inductive withdrawal from the chlorine atom at the C4 (18)(19)(20)(21). This means that the chloroquinoline preferably undergoes nucleophilic aromatic substitution via an addition of a nucleophile at C4, followed by the elimination of chlorine atom (SNAE). The present research, in combination with prior studies clearly demonstrates that regioselectivity is often achievable, this regioselectivity is also in agreement with that predicted using the 1 H NMR guidelines reported earlier by this group (22). Thus, the chemical shift values for the 7 and 4 positions of quinoline are 7.26 and 8.00, respectively, thereby indicating that C4 should be the more reactive site for SNAE reactions. It is also in agreement with the regioselectivity predicted by the bond dissociation energy calculations reported by Houk and co-workers for the corresponding chloroquinolines (96.2 kcal/Mol for C4 and 97.5 kcal/Mol for C7) (23-25).

Antimalarial activity
The eight 7-chloroquinoline derivatives were assessed for their in vitro antimalarial action against P. falciparum. Table 1 shows the values of IC 50 for the vitro antimalarial assay. All newly synthesized compounds showed moderate antimalarial with IC 50 < 100 μM, six of them showed high antimalarial activity with IC 50 < 50 μM in the range of 11.92-79.71 μM. The compounds (2), (3), (4), (6), (8)  and (9) showed high activities due to the amination of 4,7-dicloroquinoline with o-phenylendiamine, thiosemicarbazide and 3-Amino-1,2,4-triazole via SNAE, then treated with different carbonyl compounds which increase their activities through the substituents on the amino group. The most active 7-chloroquinoline derivative was the compound (9) with an IC 50 of 11.92 μM that is because this compound contains thioxopyrimidinone ring, suggesting that its presence could contribute to the activity (26). Interestingly, the other active compound is the compound (2) with an IC 50 of 35.29 μM, compound (3) with an IC 50 of 25.37 μM, compound (4) with an IC 50 of 42.61 μM, compound (6) with an IC 50 of 49.68 μM and compound (8) with an IC 50 of 38.71 μM, The significant activity was observed for compounds (6) and (8) which having two -OCH 3 group on quinoline moiety, the presence of this group could elevate the activity (27,28) where this functional group can do an electron transfer to the protein of the plasmodium parasite that led to the destruction and death of the parasite (28). However, compounds (5) and (7) showed less antimalarial activity with an IC 50 > 50 μM, the absence of active substituent on the amino group might resulted in less biological activities.

Antitumor activity
Compounds (2)-(9) were screened for their antitumor activity on three tumor cell lines, MCF-7 (human breast cancer) cell lines, HCT-116 (colon carcinoma) cell lines, Hela (Cervical carcinoma) cell lines and as a control cell, the human normal liver cell lines HL-7702 were employed to discern an unspecific of new synthesized compounds. All compounds have been made visible antitumor activity. In general, there is less difference between the antitumor activities of all compounds. Thus, compounds (3), (7) and (9) exerted the highest activities on all cell lines and showed special selectivity toward MCF-7 cells with IC 50 14.68, 14.53 and 7.54 µM, respectively (cf. Table 2 and Figures 1-3). The presence of sulfur and nitrogen atoms in the heterocyclic rings in the compounds (3) and (9) prompts altogether greater selectivity in the direction of MCF-7 cell lines.   The elucidation for this could be either in the ability of these atoms to communicate (bind) with cellular macromolecules (proteins and/or DNA) and to stimulate a similar reaction in tumor cells (e.g. DNA-damage response), or the most mindful usefulness for this activity is the thiosemicarbazide moiety with the quinoline ring. 7-Chloroquinoline derivatives (3), (6) and (9) (3), (8) and (9)

Antibacterial activity
The study examined the antibacterial information of all newly synthesized compounds (2)-(9) which showed a very good to moderately good activities towards different strains of microorganisms (12.5 ± 0.63-23.8 ± 1.5) ( Table 3). The compounds (3), (4), (7) and (9) showed relatively perfect activity with all the bacterial strains. The best activity is attributed to the presence of pharmacologically active groups at position 4 of quinoline, -NHand triazole ring attached to quinoline ring. Introduction of thiosemicarbazide group such as NH-NH -CS-NH 2 to the ring quinoline at position 4 promoted activity while the introduction of o-phenylene diamine derivatives group at quinoline caused a reduction in activity versus most of the bacterial strains. Compounds (7) and (9) demonstrated greater activity against the gram-negative bacterial species Staphylococcus aureus, however, compounds (4) and (8) exhibit a good action versus the gram-negative bacterial species Bacillus subtilis. Fulfillment of gram-negative antibacterial, compounds (4) and (9) demonstrated amazing activity against Salmonella typhimurium, while compounds (7) and (8) showed very good activities against Escherichia coli. DMSO was used as a control solvent and Ampicillin and Ciprofloxacin, as reference drugs.
The compounds showed relatively very good activities against all the fungal strains. These compounds include biologically active groups, -NHand triazole ring attached to quinoline ring. The compounds (3), (4), (7) and (9) exhibited higher activity against all fungal strains. DMSO was used as a control solvent and Amphotericin B, as a reference drug. It is worth noting that the presence of hetero rings at the fourth position of the quinoline system significantly increases the biological effect against fungal strains.

Chemical syntheses-general
Melting points were measured with a Gallen Kamp melting point apparatus. Silica-gel-coated aluminum plates used to test the purity of the compounds. Infrared spectra (λ-cm −1 ) were recorded on Bruker Vector (Germany) and on Mattson FT-IR 1000 (Taibah University, Saudi Arabia), using KBr disks. 1 H NMR spectra were recorded on Gemini 300 MHz, C 13 NMR spectrometer, in DMSO-d 6 using dimethyl sulfoxide as a solvent and tetramethylsilane (TMS) as an internal standard (Chemical shift in δ, ppm); 13 C NMR spectra were recorded on Gemini 50 MHz NMR spectrometer. Mass spectra were measured on GCQ Finnigan MAT and Elemental analyses were performed at the microanalytical Center, Cairo University, Giza, Egypt. Biological activity was determined in a laboratory by the Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Cairo, Egypt. All the chemicals were purchased from Sigma-Aldrich.
3.1.1. General procedure for synthesis 1-(7chloroquinolin-4-yl) derivatives (2)-(4) (0.01 Mol) 4,7-dichloroquinoline (1) was mixed with (0.01 Mol) of the appropriate amine, o-phenylenediamine, thiosemicabazide and 3-amino-1,2,4-triazole in ethanol (15 ml) and refluxed in an ultrasonic bath for 30 min at ambient temperature. TLC monitoring was used to ensure the completion of the reaction. Then we added CH 3 Cl (150 ml) as an organic solvent, then washed the mixture with NaOH (1N, 150 ml) to give an organic layer and aqueous layer. The resulting crude poured into ice water to produce compounds (2)-(4) and the solid formed was collected by filtration and dried.
3.1.3. General procedure for synthesis 3-(7chloroquinolin-4-ylamino)-tetrahydro-6-methyl-2thioxopyrimidin-4(1H)-one (9) (0.01 Mol) of a compound (3) and (0.01 Mol) of ethyl acetoacetate was refluxed in an ultrasonic bath, for 40 min at 90°C, to give brown crystals which recrystallized from ethyl alcohol to give the product (9).  (30). The percentage of Inhibition was studied the 50% inhibitory concentrations (IC 50 ) was measured and the data got from the inhibitor-dependent concentration growth curves were registered into plots with nonlinear retreating analysis from y-axis (inhibition %) to x-axis (inhibitor concentration) (31) (cf. Table 1, Figure 4). P. falciparum isolate NF54 was maintained in continuous culture with gentamicin (40 µg/mL) in Petri dishes (5 cm in diameter) with a gaseous phase of 90% N 2 , 5% O 2 and 5% CO 2 , according to a protocol from Moloney et al. (29) and Trager and Williams (30). P. falciparum parasites were cultured in human erythrocytes (blood group A + at 10% (v/v) hematocrit) in RPMI 1640 medium (Sigma) supplemented with 25 mM HEPES, 20 mM sodium bicarbonate, and 10% heat-inactivated human A + plasma. The culture was routinely monitored through Geimsa staining of the thin blood smears. The parasitemia of the infected erythrocytes was determined in Giemsa-stained smears by light microscopy. Parasitemias and morphological changes detected in the cultures were scored visually with a 100-fold oil-immersion objective, counting at least 1000 erythrocytes to determine the percentage of the infected erythrocytes (32).
Antimalarial activity assay: The experiments were performed in 96-well culture plates (Nunc); compounds were tested at two-fold dilutions in a dose-titration range of 500 μM to 2 μM. One hundred microliters of infected human red blood cell suspension (1% parasitemia, 4% hematocrit), with more than 90% of ring forms, were added to each well containing 100 ml of extracts pre-diluted in RPMI-1640. Test plates were incubated for 48 h. Parasite multiplication was determined microscopically after Giemsa staining and expressed as a percentage of the controls without test compounds. A drug-free control (methanol/water 50:50% v,v) was used in all experiments and CQ (0.01 μM) was used as the standard reference drug. Parasitemia and stage distribution were estimated as triplicates daily from Giemsa-stained smears by counting 1000 erythrocytes (33).

Determination of antitumor activity against MCF-7, HCT-116 and Hela cell lines
The antitumor activities of all new synthesized 7-chloroquinoline derivatives were tested for three cancer cell lines MCF-7 cells (human breast cancer), HCT-116 (colon carcinoma) and Hela (Cervical carcinoma). As control cell, the human normal liver cell line HL-7702 was employed to discern an unspecific cytotoxicity of newly synthesized 7chloroquinoline derivatives. All cell lines were gotten from the VACSERA Tissue Culture Unit. The effect of new compounds on the cell was calculated and the microplate reader (SunRise, TECAN, Inc, U.S.A.) was utilized to gauge the optical density and to determine the number of viable cells and the percentage of viability which calculated as [1-(ODt/ODc)] × 100% where ODt is the mean Optical Density of wells dealed with the tested sample and ODc is the mean Optical Density of untreated Cells (34, 35).

Determination of antimicrobial activity
The antimicrobial activity was measured for the newly synthesized compounds (2)- (9). Each microbial strain of culture collection was given by the RCMB, Al-Azhar University, Cairo, Egypt. The antimicrobial profile was tested against two Gram-positive bacteria species (B. subtilis, S. aureus), two Gram-negative bacterial species (E. coli, S. typhimurium), two fungi (Aspergillus fumigatus and Candida albicans) utilizing an adjusted well dissemination strategy (36)(37)(38) .

Conclusions
The present study describes the synthesis and antimalarials, antimicrobial, and the anticancer activity of a novel series of 7-chloroquinoline derivatives via click chemistry by using ultrasound irradiation energy source. All the compounds exhibit potent antimalarial activity in vitro. Further, the synthesized compounds (2)-(9) were screened for antibacterial activity against two Gram-positive bacteria species (B. subtilis, S. aureus), two Gramnegative bacterial species (E. coli, S. typhimurium), two fungi (A. fumigatus and C. albicans) two pathogenic strains (E. coli and B. subtilis). The results appear clearly that the most of the new compounds have shown very good biological activities.

Disclosure statement
No potential conflict of interest was reported by the authors.

Notes on contributors
Asmaa Aboelnaga was born in Cairo, Egypt. She received a Ph.D. degree from the Ain Shams University, Egypt; she Table 3. Mean zone of inhibition in mm ± standard deviation beyond well diameter (6 mm) produced on a range of environmental and clinical pathogenic microorganisms using (5 mg/ml) concentration of samples. St. worked as a faculty member in chemistry department in Ain shams University, Egypt and Taibah University, KSA. Her research group has been working for the last 10 years and continues to work on various aspects on cycloaddition reaction with the main objective of developing this reaction into a useful and powerful synthetic tool in organic chemistry. Green chemistry is another important area of her research interest.

Tested microorganisms FUNGI
Taghreed H. EL-Sayed graduated from Ain-Shams University in Egypt and received a PhD in 2008. Her research interests cover the field of Green Chemistry and Organometallics. She teaches in Ain-Shams University in Egypt and Taibah University in Saudi Arabia.