Synthesis of the Polyhydroquinoline Derivative 4-(2-Chloro-Phenyl)-2,7,7-Trimethyl5-oxo-1,4,5,6,7,8- Hexahydroquinoline-3-Carboxylic Acid Ethyl Ester: Antimicrobial and Enzyme Modulator

Multicomponent reactions are extremely relevant in green chemistry. They offer better conditions than traditional synthesis and are, therefore, used for many organic modifications. Recently, the synthesis of polyhydroquinolines has received much attention for its high pharmacological potential. a polyhydroquinoline derivative was synthesized without the use of catalysts or solvents. The results of nuclear magnetic resonance and infrared spectroscopy demonstrated that the molecule was successfully synthesized. The molecule presents significant results of antimicrobial activity for the bacteria tested in the serial dilution method. It also increased the clotting time by 25.66 seconds for the highest dose and 12.66 seconds for the other doses tested. Prior incubation with the dose of 125 mg reduced the thrombolytic activity to 73%. The 125, 100, and 50 mg doses previously incubated with Bothrops moojeni venom inhibited approximately 30% of the phospholipase activity. The molecule was also able to reduce the cytotoxicity induced by proteases significantly. In conclusion, the molecule presents several biological properties, which highlights its pharmaceutical potential.


INTRODUCTION
Green chemistry is based on environmental factors and arose from a natural need to adapt previously performed techniques. In order to develop environmentally-friendly strategies that minimize waste production and avoid the use of hazardous chemicals, green chemistry has been used in the pharmaceutical, food, and chemical industries (1,2).
Multicomponent reaction (MCR) corresponds to convergent reactions in which three or more starting materials react in a single step to form a product. In discovering and developing new drugs, MCRs offer several advantages over traditional synthesis, such as shorter experimentation time, fewer laboratory techniques and solvents used, and the generation of smaller amounts of waste (3). Thus, the multicomponent reaction method is in accordance with the principles of green chemistry.
Numerous methods have been reported for the synthesis of polyhydroquinoline derivatives (PHQs). The classical method involves coupling three components under reflux: aldehyde, ethyl acetoacetate, and ammonium acetate in acetic acid or alcohol.
However, new techniques to obtain these derivatives have been developed and studied.
Said methods seek greater efficiency and yield, using reagents that can be recycled and are less toxic (4,5).
PHQs has emerged as one of the most important compounds for the treatment of cardiovascular diseases (e.g., hypertension). These compounds have different medicinal functions, including neuroprotection, platelet aggregation inhibitor, antimicrobial, antiischemic agent, and potentiators in chemotherapy (5,6). Therefore, the objective in the present work was to carry out the multicomponent reaction of a PHQ derivative and perform its structural characterization using the Fourier transform infrared spectroscopy (FTIR) and 1 H-and 13 C-nuclear magnetic resonance (NMR). In addition, the biological activities exerted by the synthesized compound were also evaluated (antimicrobial activity and modulatory potential on enzymes that act in hemostatic and inflammatory processes). experiment. The reactivation of the strains was performed under aerobic conditions by inoculating 100 μL of the culture into tubes containing 10 mL of Brain Heart Infusion (BHI) broth for 24h at 37°C. The inoculum was standardized (McFarland standards) to a cell density corresponding to 10 8 CFU/mL.
A solution containing 500 mg of the compound diluted in 1 mL DMSO was used for broth microdilution. 500 μL of this solution were incubated with 500 μL of BHI broth for 48 hours (stock solution), obtaining a final dose of 250 mg. The cell count was performed three times: 0 hours (preparation of the incubation), 24 hours, and 48 hours.
Dilutions (900 μL of distilled water for each 100 μL of the stock solution) were performed in Eppendorf tubes to count the viable cells. Petri dishes containing TSA were incubated at 37°C for 24h with S. aureus and 37°C for 48h with P. aeruginosa. The spread plate technique was performed at 0, 24, and 48 hours, with serial dilutions to obtain an adequate number of colonies (7). S. aureus or P. aeruginosa in 1000 μL of BHI were used as positive controls, which were performed at 0, 24, and 48 hours.
Both tests and all concentrations were evaluated in triplicate.

Obtaining human biological material
Experimental protocols (coagulation, cytotoxicity on erythrocytes, and thrombolytic) that require the collection and use of human blood were previously approved by the Committee for Ethics in Research on Human Beings (COEP) of the The volunteers (between 18 and 35 years old) were non-smokers and declared that they did not use prescription medication, nor did they work under chemical or biological risk conditions.

Effects on the coagulation of citrated human plasma
The coagulation activity was evaluated following the methodology described by MOURAO et al. (9). Human plasma was collected on a citrate tube (200 µL) and stabilized at 37°C. Treatments were added to the tube, and the time until the formation of a rigid clot was measured. The potential to inhibit coagulation was evaluated by adding different doses of the compound to the citrated plasma and incubating them for 10 minutes. After that, 40µg of Bothrops moojeni venom was added to the tube, and the clotting time was measured. This experiment allows the observation of possible interactions between the compound and proteins of the coagulation cascade.
In addition, the assay was also performed by incubating the compound at different doses with the venom and only then adding citrated plasma. The results obtained allow the observation of possible interactions between the compound and serine proteases present in the venom.
All treatments were evaluated in triplicates.

Effects on human blood thrombi and venom-induced thrombolytic activity
The thrombolytic activity was evaluated on blood clots formed in vitro, according to the methodology described by CINTRA et al. (10). Thrombi were obtained by applying 100 µL of freshly collected blood (without anticoagulant) into the wells of a 96-well microplate. The treatments were added to the thrombi in triplicates, and then the microplates remained in a cell culture chamber for 24 hours at 36°C. The results were evaluated by measuring the volume of liquids released by the thrombi. The compound was evaluated at different doses. The same doses were also previously incubated with Bothrops moojeni (20µg) venom for 30 minutes at 37°C to assess their activity on thrombolytic proteases. 30 µL of phosphate-buffered saline (PBS) was used as the negative control, which corresponds to the same volume as the treatments. Bothrops moojeni (20µg) was used as the positive control. The results were converted into percentages, and the positive control was considered as 100% of activity.

Effects on the activity of phospholipases A2
The phospholipase activity was evaluated according to the methodology described by GUTIÉRREZ et al. (11), with few adaptions. A medium was formed with 0.01 mol L -microorganisms in the medium), and 1% bacteriological agar dissolved in PBS (pH 7.2).
The medium was poured into Petri dishes and, after solidification, the samples were applied to holes made in the gel (~ 0.5 cm diameter). The dishes were kept in a cell culture chamber at 36°C for 18 hours, and the activity was evaluated by measuring the translucent halo formed around the hole (in millimeters -mm). The anti-inflammatory potential of the compound was evaluated by observing its effects on the activity of phospholipases A2 present in Bothrops moojeni venom. Thus, the compound was previously incubated in different doses with the venom (20µg), for 30 minutes at 37°C, with subsequent evaluation of the phospholipase activity. Positive controls were performed using only the venom. All treatments were evaluated in triplicates.

Cytotoxic activity on human erythrocytes
This activity was evaluated using a solid medium, as previously described by Gutiérrez et al. (11). However, the phospholipids were replaced by human erythrocytes.
The blood collected in tubes containing heparin was immediately centrifuged, and the supernatant discarded. 10 mL of PBS was added to the erythrocytes, which were washed twice more in PBS with centrifugations at 1200 g for 10 minutes. The obtained erythrocytes concentrate (1:3 v v -1 in PBS) was used to compose the gel, which also included 0.01 mol L -1 CaCl 2 , 0.005% sodium azide, and 1% bacteriological agar dissolved in PBS (pH 7.2). After solidification, the treatments were applied to ~0.5 cm diameter holes made in the gel. The Petri dishes were kept in a cell culture chamber at 36°C for 18 hours. The formation of a translucent halo around the hole characterizes hemolysis, which was measured (millimeters) with a caliper. The compound was evaluated in different doses to observe if it induced erythrocyte lysis. Then, to verify its anti-cytotoxic potential, the compound was evaluated after pre-incubation with B. moojeni venom (20 µg) for 30 minutes at 36ºC. B. moojeni venom was used as a positive control. All treatments were evaluated in triplicates.

Statistical Analysis
The data obtained were submitted to the Shapiro-Wilk test at a 5% significance level to verify their normality. Dunnett's test was performed for the data that had a normal distribution. A normalization attempt was made using the RANK procedure (PROC RANK) on the data that did not show a normal distribution. Non-normalized data were compared by the Kruskal-Wallis test at a 5% significance level. The statistical software used was SAS (version 9.0).

Synthesis and Structural Characterization
The hexahydroquinoline derivative was synthesized according to the methodology described by Kumar et al. (12), which did not use catalysts and solvents.
The mechanism of the general reaction for obtaining PHQs derivatives (13) begins with the removal of an α-hydrogen from dimedone by a base, forming an enolate ion that has an area of a high concentration negative charge on the α-carbon. This compound is stabilized by the resonance between the electron pairs and the carbonyl groups bonded to the carbon. These electrons on the α-carbon are the ones that attack the carbon of the carbonyl group of benzaldehyde, which has a positive charge density because of the inductive effect caused by the oxygen. The oxygen on the carbonyl group acquires a negative charge after the nucleophilic attack since it breaks one of its bonds with the carbon that was attacked. The carbon captures a hydrogen from the reaction components and forms a secondary alcohol to stabilize this excess negative charge.
The compound still has another α-hydrogen that can be removed by the base present in the reaction. The electron pair that formed the C-C bond now forms a C=C double bond, eliminating the hydroxyl group. Although the -OH group is an unfavorable leaving group, its elimination in this synthesis step is justified by the high stability of the product formed -an α,β-unsaturated carbonyl compound. The electrons of the new double bond can perform resonance with both the carbonyl groups and the aromatic ring (14,15) observed in Figure 1. Since the electrons of the double bond can perform resonance with the carbonyl groups, the β-carbon gets a positive charge density and becomes an electrophilic center.
Another enolate ion, formed from ethyl acetoacetate, reacts with the compound previously formed by a Michael addition. After the attack, the oxygen in the dimedone acquires a negative charge that is stabilized by capturing hydrogen and forming a -OH.
However, the C=O double bond is repaired by tautomerism, and the negative charge passes to the neighboring carbon, which is stabilized by a hydrogen bond. This compound then reacts with ammonia to form an imine.
The mechanism begins with the protonation of the carbonyl group. Consequently,

Antimicrobial activity
No halos were formed in the antibiogram (agar diffusion test), which may be a result of the compound's inability to diffuse into the solid culture medium. However, when evaluated by the pour-plate technique with serial dilution (broth microdilution) in a liquid medium, the compound showed significant antimicrobial activity for both S. aureus and P. aeruginosa, as seen in Tables 1 and 2.   The synthesized molecule proves to be more efficient against Gram-negative bacteria, which are more challenging to treat (16). Thus, it is essential to study the class of polyhydroquinolines and their various biological activities.
Jamale et al. (17) showed that their polyhydroquinoline derivatives had significant potential as an antimicrobial. Some of the compounds were effective against Mycobacterium tuberculosis, which is an extremely difficult microorganism to eradicate. The derivatives were tested at 0.8, 1.6, 3.

Effects on the coagulation of citrated human plasma
The synthesized compound did not induce plasma coagulation in the tested doses (data not shown). When incubated with Bothrops moojeni venom (40µg), the compound prolonged the clotting time. However, when compared to the positive control, a statistically significant difference was observed only for the higher dose -125 mg ( Figure   5A). In contrast, when the venom is added later to plasma previously incubated with the compound, a protective effect was observed at 125, 100, and 50 mg (p<0.05). In this case, there was a significant increase in the clotting time (25.66 seconds for the 125 mg dose and 12.66 seconds for the 100 and 50 mg doses) ( Figure 5B). Thrombi are formed by abnormal control of coagulation factors and clot lysis.
Blood coagulation occurs by the activation of intrinsic and extrinsic pathways (19). It is important to note that the compound did not present coagulant activity but rather has anticoagulant activity.
When previously incubated with the venom, the compound exerted partial

Effects on human blood thrombi and venom-induced thrombolytic activity
The synthesized molecule induced lysis of blood thrombi at all doses tested, which were statistically similar to the positive control (Bothrops moojeni -20µg) ( Figure 6A).
While this result is consistent with the activity observed in the previous experiment, it should be noted that the activities shown here were biologically lower than the control (approximately 20% difference between C+ and treatments).
When incubating the synthesized compound with B. moojeni venom, a significant reduction (p<0.05) in the venom-induced thrombolytic activity was observed for the 125 and 100 mg doses (73% and 79%, respectively). The other doses were not statistically different from the positive control ( Figure 6B). Thus, it is possible to observe that the compound has a thrombolytic activity, which corroborates with the data obtained in the coagulation test using citrated human plasma.
Cardiovascular diseases are caused by disorders of the heart and blood vessels.
Nowadays, endogenous thrombi formation in these vessels is one of the leading causes of death. Therefore, many types of research have been performed in the field of prevention and treatment for these diseases (24). The antithrombotic effects of chlorogenic acid were evaluated using the blood clot assay, which is similar to the test performed in the present work. The acid showed a high level of clot degradation compared to the control (19).
It can be inferred from the results obtained in the present work that the synthesized compound has promising thrombolytic characteristics and a protective effect on human plasma components. However, further testing is needed since there are no complementary data in the literature for polyhydroquinoline derivatives.

Effects on the activity of phospholipases A2
The  Diosmin is a flavone glycoside that is naturally found in many citrus fruits. This substance is known to act on the circulatory system's veins, improving blood circulation and venous tonus. In microcirculation, diosmin normalizes permeability and reinforces the resistance of the capillary vessels, which reduces edema. However, despite the broad therapeutic potential of this substance, it is characterized by low solubility in water and most organic solvents. Since diosmin has low bioavailability, high dosages (500 mg) are required for the oral regimen (33).
Therefore, finding new molecules with protective and anti-inflammatory activities is extremely necessary. As can be seen, the molecule 4-(2-chloro-phenyl)-2,7,7trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylic acid ethyl ester has effects that deserve attention and expanded studies since it is pharmacologically active at a dosage that is considered low when compared to marketed drugs.

CONCLUSION
In the present work, it was possible to synthesize the polyhydroquinoline

Declaration of Competing Interest
The autors declare that they have no Known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.