Synthesis, DFT Studies, and Biological Applications of Some Novel Compounds Containing Lawsone by Using Halo-Reagents

Abstract A novel binary compounds as 3-(1,3-diphenyl-1H-pyrazol-4-yl)-2-phenyl-2H-benzo[f]indazole-4,9-dione (10) and fused compounds as 2-hydroxy-3-(4-hydroxy-7-methoxy-2-oxo-1,2-dihydroquinolin-3-yl)naphthalene-1,4-dione (7) and naphtho[2,3-b]furan-3,4,9(2H)-trione (6) based on lawsone ‘that plays as important precursor’ was synthesized by reaction with 1,3-diphenyl-1H-pyrazole-4-carbaldehyde and halo-compounds, such as 2-chloroacetyl chloride or 3-bromo-4-hydroxy-7-methoxyquinolin-2(1H)-one. Spectra and elemental analysis of newly synthesized compounds were investigated. The target molecules were showed easy access to antioxidant (calorimetrical measurement) and antitumor (Ehrlich ascites carcinoma [EAC] cells) activities. Geometrical isomers (enol, Keto conformers, and syn, anti-conformers) were achieved by density functional theory (DFT) that conformed to the spectral analysis of the investigated compounds. Supplemental data for this article is available online at here.


Introduction
Lawsone is described as a medicinal plant. 1 Therefore, it was considered a common precursor for many synthetic compounds with a number of valuable biological applications like Juglone, Menadione (Vit. K3), Arizonin C1, Alkanin; 2 and its mannich bases ( Figure 1). 3 Lawsone has pharmaceutical activities as anticancer, 4,5 anti-inflammatory, 6 Leishmanicidal activity, 7 antifungal, antiparasitic, 8 antimalarial, 9 and antimicrobial. 10,11 In addition, a wide variety of applications of it, such as anticorrosion 12 and improving the solar cells. 13,14 Halo-reagents characterize an important class of alkylating and/or acylating reagents due to their widespread use as synthetic intermediate. Chloroacetyle chloride and chloroacetic acid are playing important roles in the synthesis of pharmacological compounds like Lidocaine, 15 25hydroxy protopanaxadiol (25-OH-PPD, AD-2) 16 , and its esters. 17 In addition to using these agents in synthesis herbicides as N-alkoxy methyl-N-2,6-diethyl phenyl analogs 18 and indicators in biologically active carbon (BAC) filter backwashing. 19 Based on the aforementioned biological activity and in continuation of the research on literature, 20,21 we described herein to synthesis annulated naphtha[b]furantrione compounds containing lawsone moiety by using chloro and aldehyde reagents, respectively, and investigate their antioxidant and antitumor activities.
Moreover, compound 7 was chemically treatment with hydrazine hydrate to produce compound 8 after aromatization by some drops of hydrogen peroxide (H 2 O 2 ) (Scheme 1). The 1 H NMR spectrum of 8 appeared singlet signals for OCH 3 and NH at 3.85 and 5.55, respectively.

Geometry studies
The geometry optimization of the molecular structure 24 for 3-7, 9, and 11 was performed by the density functional theory (DFT) method. Table 1 is obtained the main quantum data for some characters. Compounds 3, 4, and 6 have two isomers enol and keto conformers (Figures 3-5). From the calculations, the binding energy for compounds 3, 4, and 6 was showed the keto isomers have a more negative value of the binding energy with 0.328, 0.561, and 0.114 ev than the enol one, respectively. Due to the absence of OH peak in IR spectrum analysis for compounds 3, Scheme 2. Synthesis of some binary compounds 9-11. Compound 5 existed in six conformers 5.1-5.6 ( Figure 6). Some conformers showed keto-enol forms like 5.1 and 5.2 among the structure ring and 5.3 and 5.5 in the branched-chain. Also, syn-anti conformers are displayed in 5.3 with 5.4 and 5.5 with 5.6. DFT calculations and spectral data achieved the same conformer 5.1. The DFT calculations showed it has the highest negative binding energy. IR spectrum appeared peaks at 1740 and 3443 for (CO-C 1' ) and (HO-C 2 ), respectively. Therefore, compound 5 exist in conformer 5.1.  Compound 7 existed in twelve conformers 7.1-7.12, shown in Figure 7. According to DFT calculations, conformer 7.1 is more stable than other conformers due to its highest negative value of binding energy. On the other hand, conformer 7.8 has the lowest binding energy, so its stability is the lowest. Spectral data of the conformer 7.1 are confirmed the DFT calculations. IR spectrum of compound 7 showed peaks for OH and NH groups at 2423 and 3100, respectively, and its 1 H NMR also confirmed the suggested structure by existing singlet signals at 12.6, 12, and 11.3 ppm (see the experimental part) approves its existence in conformer 7.1. Figures 8 and 9 show the anti-and syn-conformers for compounds 9 and 11. The binding indicated the syn-isomer for compound 9 has a higher negative value than the anti-one by 1.078 ev. In the same way, the anti-isomer has a higher negative value than syn-one by 0.581 ev for compound 11. Therefore, the stability conforms for compound 9 is syn-conform, while compound 11 is anti-one (Table 1).

Antioxidant activity for ABTS
Different bioassay methods used for utilization the antioxidant activity like calculating free radicals (X') that disbursed as represented in the following reactions: 25 XH þ Y' X' þ YH Calorimetric measurement exhibited a decrease in the intensity of the color of the free radical solution. The addition of compounds led to trapping free radical and hence X 0 concentration was decreased and color changed. The antioxidant behavior for the tested compounds depended on the stable radical cation from '2,2 0 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)' (ABTS).The standard antioxidant sample 'positive control' was ascorbic acid while the solvent without ABTS acted as a blank sample. The antioxidant data is represented in Table 2. The range between 85 and 83% for compounds 1 and 9 showed good antioxidant activity. However, the moderate activity played in the range between 55 and 45%, such as compounds 2, 3, 6, and 10. If we compared the tested compounds with ascorbic acid, compounds 4, 5, 7, 8, and 11 exhibited weak antioxidant activity (Figure 10). The structure-activity relationships (SARs) for tested compounds exhibited the variability of antioxidant activity depending on the existence of naphthoquinone and  pyrazole structure that increases the antioxidant activity. While the incorporation of chloride group into naphthoquinone ring construction decreases the antioxidant activity.

Effect of drugs on the viability of Ehrlich ascites carcinoma (EAC) cells in vitro
Synthesized molecules 1-11 were tested for cytotoxicity against proven model EAC 26 in vitro. Table 3 lists the results of values 100, 50, 25, and IC 50 of the tested compounds. According to the results, it is found that there is a positive relationship between the concentration of compounds and the percentage of dead cells. The most effective one that leads to the death of the cells is 100 mM. The study shows that compounds 1, 2, 3, and 6 are more potent than the other compounds. Otherwise, compounds 4, 5, and 11 are moderate toxic compounds (Figures 11 and 12). SARs postulated that naphthoquinone and linear furanone-naphthoquinone enhance the antitumor activity of the novel compounds. In contrast, introducing the fused pyrazole to the systems decrease their antitumor activity.

Conclusion
The synthetic compounds 2-11 are achieved by a smart, simple synthetic route, and their structures are confirmed by different spectral data analyses. Moreover, all possible isomers for  2-11 were studied by DFT, which confirmed our spectral data analyses. The bioactive synthesized compounds showed a good activity compared with the standard samples.

Chemistry
All melting points were uncorrected that obtained from the capillary melting point apparatus. Infrared (IR) spectra t (cm À1 ) were accomplished on Nicolet NEXUS 470 FTIR spectrophotometer in potassium bromide (KBr). Varian XL-300 spectrometer (300 MHz) was used to obtain 1 H NMR and 13 C NMR spectra. Internal standard tetramethylsilane used as a reference point for chemical shifts was recorded in ppm by using DMSO-d 6 as solvent.  Microanalyses were performed on a Hosli elemental analyzer and the data agreed with the calculated values. Mass spectra (MS) were measured on (Kratos, 70 eV) MS equipment and/or a Varian MAT 311 spectrometer. The aforementioned spectral measurements and elemental microanalysis were carried out at the micro-analytical center, faculty of Science, Cairo University, and Mansoura University, Egypt.
Brown powder; yield (0. Antioxidant screening assay (ABTS method) Reagents. All chemicals used were of the highest quality available. While ABTS was bought from Wak and vitamin C was ordered from Sigma. However, the tested compounds; the ABTS solution (2 mL, 60 mM) and the 3 M MnO 2 solution (25 mg/mL) were prepared in phosphate buffer (pH 7, 0.1 M). The absorbance (A control) of the resulting green-blue solution (ABTS radical solution) is at ca. 0.5 at k 734 mm. Then, the solution of the test compound in spectroscopic grade methanol/ phosphate buffer (1:1) was added. The Inhibition percentage for each compound was calculated from Vitamin C is used as a positive control, while methanol and phosphate (1:1) is a negative one. Also, methanol/phosphate (1:1) without ABTS acts as a blank sample.
Antitumor activity. The viability and percentage of viable cells were calculated by the trypan blue exclusion to know the cytotoxic effect on EAC for the tested compounds in concentration 1-25 mg/mL DMSO. The required dilution that is suitable for the tumor cells was performed by saline solution (0.9%). The cells were discarded if the viability of the tumor cells was found less than 90%. This whole process was done three times.

Disclosure statement
No potential conflict of interest was reported by the author(s).