DFT Study and Antiparasitic Activity of Some Azo Dyes Containing Uracil

In this study, for the first time, molecular modeling and antiparasitic activity studies were carried out on some azo dyes containing uracil, 6-amino-5-[(4-nitrophenyl) diazenyl] pyrimidine-2,4 (1H, 3H)-dione (dye I) and 6-amino-5-[(4-bromophenyl) diazenyl] pyrimidin-2,4 (1H, 3H)-dione (dye II), which were resynthesized using the same method in the literature and whose molecular structures were confirmed using FTIR and 1H-NMR methods. In molecular modeling study, all calculations were performed using DFT/B3LYP/6-311++G(d,p) method. The molecular structures of the possible tautomeric forms of dyes I and II were optimized, and their molecular total energies were calculated in the gas phase and DMSO solvent. IR and 1H-NMR spectral data of the possible tautomeric forms of dyes were obtained, and theoretical spectral data were compared with experimental ones. The evaluations show that, for both dyes, the spectral data of the imine-diketo-hydrazone form, which has the lowest energy and is hence determined to be the most stable form, are in agreement with the experimental ones. In antiparasitic activity study, dyes I and II were tested for the first time against parasites Leishmania infantum, Leishmania major, Leishmania tropica promastigotes, and Trichomonas vaginalis trophozoites. In vitro antileishmanial activities against Leishmania promastigotes were tested by microdilution broth assay with Alamar Blue in RPMI 1640 medium, and in vitro trichomonacidal activities against Trichomonas vaginalis parasite were tested using TYM medium. In tests, antileishmanial and trichomonacidal effects were determined by comparing the obtained minimum inhibitory concentration (MIC) and minimum lethal concentration (MLC) values with those obtained for standard drugs (amphotericin B and metronidazole, respectively).


Introduction
Azo dyes are important colorants with chromosome azo group (-N�N-). Due to their special chemical and biological properties, they have potential applications in medicine and pharmacology, such as antibacterial, antifungal, antitumor, and antioxidant activities [1][2][3]. In addition, they are easy to use and offer strong colors in textile applications as well as being relatively inexpensive. With these aspects, azo dyes attracted considerable attention in scientific studies [4,5]. Uracil derivatives belonging to the pyrimidines group, which have an important role in the binding of ribosomes and phosphate in the synthesis of enzymes required in basic biological processes, show important pharmacological activities [6,7]. Studies on synthetic pyrimidines reveal the presence of remarkable biological and pharmacological properties [8]. erefore, uracil derivatives are used in drugs with antitumor, antibacterial, and antiviral properties [7].
Leishmaniasis is a disease caused by Leishmania parasites and transmitted by the bites of some sandflies, which differs clinically and epidemiologically, and is an important health problem worldwide [9,10]. Visceral leishmaniasis (VL, Kala-Azar), cutaneous leishmaniasis (CL, Oriental Boil), and mucocutaneous leishmaniasis (MCL) are three different clinical forms of leishmaniasis [11]. In the Mediterranean basin, types of Leishmania causing leishmaniasis are as follows: generally Leishmania infantum (L. infantum) for VL and Leishmania major (L. major) and Leishmania tropica (L. tropica) for CL [12]. About 1 billion people are at risk in five continents, and it is estimated that the number of new patients every year is 30 000 for VL and 1 million for CL [13]. e treatment alternatives of the disease are limited, and many of the drugs used are expensive and have side effects that cause significant health problems. erefore, in recent years, many studies have been carried out to develop a drug that is cheap and safe and has a very low side effect, and antileishmanial tests have been applied on a variety of natural and synthetic compounds [14][15][16][17].
Trichomonas vaginalis (T. vaginalis) is a protozoan parasite with flagellates and only human host, found in trophozoite form [18]. It causes infection in the urogenital system of both women and men. Trichomoniasis causes vaginitis, cervicitis, and urethritis, especially in women [19]. 5-Nitroimidazole derivatives (metronidazole, ornidazole, and tinidazole) are currently used in the treatment of trichomoniasis, and these drugs cause DNA damage and cause cell death of the parasite. Although metronidazole is the first choice drug, it has mutagenic and carcinogenic effects.
erefore, alternative treatment methods in the treatment of trichomoniasis are being investigated [20].
In this study, in order to contribute to pharmacological studies, it was aimed to investigate azo dyes containing uracil, which have remarkable pharmacological properties but have not been previously tested for antiparasitic activity. For this reason, some azo dyes given in literature were resynthesized by the same method, and their structures were confirmed by FTIR and 1 H-NMR methods. For the first time, theoretical study and antiparasitic activity study were carried out on the azo dyes containing uracil called as 6-amino-5-[(4-nitrophenyl) diazenyl] pyrimidine-2,4 (1H, 3H)-dione (dye I) and 6-amino-5-[(4-bromophenyl) diazenyl] pyrimidin-2,4 (1H, 3H)-dione (dye II). In the theoretical study, the molecular structures of the possible tautomeric forms of dyes I and II were optimized, and the structural and some spectral parameters of dyes were determined by density functional theory (DFT) method with 6-311++G(d,p) basis set. eoretical IR and 1 H-NMR data of the possible tautomeric forms of dyes I and II were compared with experimental ones.
us, molecular structures of dyes I and II were firstly investigated by DFT methods. In vitro antileishmanial and trichomonacidal activities of these dyes were tested for the first time against parasites L. infantum, L. major, L. tropica, and T. vaginalis, respectively. In vitro antileishmanial activities were tested by microdilution broth assay with Alamar Blue in Roswell Park Memorial Institute (RPMI) 1640 medium, and in vitro trichomonacidal activity was tested using Trypticase Yeast extract Maltose (TYM) medium. As standard control drugs, amphotericin B for Leishmania and metronidazole for Trichomonas were used. Investigation of in vitro antileishmanial and trichomonacidal activities of azo dyes containing uracil, dyes I and II, can make an important contribution to drug design and development studies and can guide the determination of chemicals to be considered in these studies in terms of increasing the variety of compounds that can be used in pharmacological research.

Experimental and Computational Method
2.1. Instrumentation. 1 H-NMR spectra of dyes I and II were recorded on a Varian-Mercury 400 MHz spectrometer. ese measurements used TMS as an internal standard and DMSO-d 6 as solvent. e FTIR spectra of dyes I and II were recorded on a Perkin-Elmer Spectrum One Fourier-transform IR spectrometer (resolution 4) in KBr pellets.

Synthesis.
In this study, azo dyes containing uracil given in the literature, dye I and dye II, were resynthesized by the same method [21,22]. e synthesis reaction of azo dyes containing uracil is shown in Scheme 1. N,N-Dimethylaniline, KOH, 4-bromine, concentrated HCl acid, NaNO 2 , and 6-amino uracil used for the synthesis of dyes I and II were purchased from MERCK and used without further purification. Pure water was obtained in the laboratory.

In Vitro Antiparasitic Activity.
e antiparasitic properties of dyes I and II were determined by testing their antileishmanial and trichomonacidal activities against L. infantum, L. tropica, L. major promastigotes, and T. vaginalis trophozoites, respectively. e study was carried out in vitro using microdilution broth assay. e results of antileishmanial activity against Leishmania promastigotes and trichomonacidal activity against T. vaginalis were evaluated by taking into account minimum inhibitory concentration (MIC) and minimum lethal concentration (MLC) values, respectively.

Antileishmanial Activity.
e axenic standard L. infantum, L. tropica, and L. major promastigotes were used in the study. e promastigotes were propagated on RPMI-1640 medium and maintained by passaging [33,34]. Promastigotes were washed with phosphate buffered saline (PBS). Each of azo dyes containing uracil was dissolved in DMSO/H 2 O (10%) and sterilized by membrane filter with a diameter of 0.45 μm. Final concentrations were prepared by adding RPMI-1640 medium. e stock solution was diluted in 96-well microplates, and 2.5 × 10 7 cells/mL Leishmania promastigotes were added onto the wells. After incubating the microplates at 27°C for 20 hours in the incubator, Alamar Blue was added to the wells and incubated again at 27°C for 4 hours. Microplates were evaluated at 24, 48, and 72 hours, and the results were recorded. In the study, amphotericin B was used as a control drug, and all tests were repeated twice. As expected, the color will change from blue to pink in the positive control well and no color change was observed in the negative control well [35,36].

Trichomonacidal
Activity. T. vaginalis was produced in TYM medium. 1 mL inactivated human serum was added onto the medium under sterile conditions. Penicillin, streptomycin, and triflux were added under sterile conditions and then incubated in a 37°C incubator. Two days later, whether there was reproduction was observed. To test the viability of the trophozoites, 0.4% trypan blue was used and counted in the hemocytometer slide [37].
Azo dyes containing uracil, dyes I and II, were prepared in concentrations of 32, 16, 8, 4, 2, and 1 mg/mL in saline and distributed into sterile Eppendorf tubes. T. vaginalis were incubated at room temperature with 5 × 10 6 trophozoites/ mL. e viability of the parasites was checked at specific times and noted. Recontrol seedlings were performed from the tubes in which the live cells were not detected, and the reproduction was evaluated. Parasites not added to dyes I and II were kept in the same environment as control [38].

Optimized Geometries.
In theoretical study of azo dyes containing uracil called 6-amino-5-[(4-nitrophenyl) diazenyl] pyrimidine-2,4 (1H, 3H)-dione (dye I) and 6-amino-5-[(4-bromophenyl) diazenyl] pyrimidin-2,4 (1H, 3H)dione (dye II), firstly, the molecular structures of dyes I and II in possible tautomeric forms given in Scheme 2 were optimized by DFT/B3LYP/6-311++G(d,p) method in the gas phase and in DMSO solvent, and their molecular total energies, in kcal/mol, were calculated. e most stable forms for dyes I and II in gas phase and in DMSO solvent are the imine-diketo hydrazone forms with the lowest energy. As in the gas phase/in DMSO solvent, molecular total energies of the imine-diketo hydrazone forms are obtained as −637226.06/−637240.19 kcal/mol for dye I and as −2123760.46/−2123771.10 kcal/mol for dye II, respectively. e optimized molecular structures of dyes I and II in iminediketo-hydrazone forms are shown in Figure 1, and their structural parameters are presented in Table 1. e structural parameters obtained by geometry optimization were compared with those obtained from X-ray method for similar single-crystalline compounds given in the literature; thus, the suitability of the method used and that of the selected basis set were discussed.
Since dyes I and II have values compatible with the structural parameters of similar single crystalline compounds in the literature, it can be concluded that the appropriate method and basis set were chosen. is is also an indication that spectral parameters can also be determined correctly.

Vibrational Spectra and 1 H-NMR Spectra.
e IR and 1 H-NMR calculations of dyes I and II were carried out at DFT/B3LYP/6-311++G(d,p) level by considering the possible tautomeric forms to T-VIII from T-I (Scheme 2). eoretical IR and 1 H-NMR data were compared with experimental data, and the compatibility of theoretically obtained values for dyes I and II with experimental data was discussed.
Considering Accordingly, since the NH 2 and CO-H bands given above are not observed in the FTIR spectra of dyes I and II given in Figure 2, the possible tautomeric forms to T-IV from T-I can be eliminated.
Similarly, the bands calculated as dye I/dye II are as follows: for T-VI, C4O-H bands at 3569/3571 cm −1 , respectively; for T-VII, C1O-H band at 3614/3615 cm −1 , respectively; for T-VIII, C1O-H and C4O-H bands at 3612/ 3615 cm −1 and 3568/3570 cm −1 which are absent in FTIR spectra of dyes I and II (Figure 2), and thus possible tautomeric forms to T-VIII from T-VI can be eliminated.
As a result, when the FTIR spectra of dyes I and II are compared with the theoretical ones, it can be said that, in the solid state, dyes I and II are in the imine-diketo-hydrazone form (T-V in Scheme 2). Some vibration bands are considered to confirm the molecular structures of dyes I and II, and theoretical ones obtained in the gas state for iminediketo-hydrazone forms are given in Table 2.
As evaluated in Table 2, N17-H/N18-H stretching bands in the FTIR spectra of dyes I and II (Figures 2(a) and 2(b)) were observed at 3116/3116 cm −1 and 3144/3144 cm −1 , respectively, and they were calculated as 3452/3444 cm −1 for dye I and 3455/3448 cm −1 for dye II. e N21-H imine band was observed as 3116/3099 cm −1 for dyes I/II and was calculated as 3369/3369 cm −1 , respectively. N15-H hydrazone band calculated as 3198/3195 cm −1 for dyes I/II was observed at 3168/3216 cm −1 . e experimental and calculated values of other stretching bands given in Table 2 are quite compatible.
As can be seen from Figure 1, the optimized molecular structures of dyes I and II were obtained in the most stable structure revealing the presence of N15-H· · ·O19 type intramolecular hydrogen bonds. erefore, deviations between experimental and calculated ones are small, and values obtained as 30/21 cm −1 for dyes I/II, respectively, can be considered compatible. As observed for N17-H/N18-N17 stretching bands and N21-H imine bands, the considerable deviations calculated as 336/328 cm −1 and 253 cm −1 for dye I, 311/304 cm −1 and 270 cm −1 for dye II, respectively, can be explained by the presence of N-H· · ·O type strong intermolecular hydrogen bonds. 1 H-NMR spectra of dyes I and II were recorded in DMSO-d 6 (Figures 3(a) and 3(b)). For this reason, primarily the molecular structures of possible tautomeric forms of the dyes were optimized in DMSO solvent by DFT/B3LYP/6-311++G(d,p) method and, for all, 1 H-NMR calculations in DMSO solvent were performed using DFT/B3LYP/6-311++G(d,p) method.
Considering Scheme 2, to T-IV from T-I and as dye I/dye II, NH 2 proton signals are calculated as 5.03, 5.20, 5.32, 5.33 and 7.05, 6.79, 7.07, 6.73 ppm/4.89, 5.04, 5.18, 5.19 and 6.94, 6.64, 6.95, 6.59 ppm, respectively. ese signals are not observed in 1 H-NMR spectra of dyes I and II given in from T-I can be eliminated. Similarly, CO-H proton signals, as dye I/dye II, are obtained at 7.05/6.88 ppm for T-VI, at 6.00/5.91 ppm for T-VII, and 6.08 and 7.18/5.97 and 7.02 ppm for T-VIII which are absent in 1 H-NMR spectra of dyes I and II. erefore, T-VI, T-VII, and T-VIII are eliminated.
us, the 1 H-NMR spectra of dyes I and II are consistent with the theoretical data of dyes I and II in the imine-diketohydrazone form (T-V). eoretical 1 H-NMR spectral data obtained for the imine-diketo-hydrazone form are given in Table 3 together with the experimental values.
As seen in Table 3 As evaluated in the FTIR and 1 H-NMR experimental data (Figures 2 and 3) supporting each other of dyes I and II together with the theoretical data in Tables 2 and 3, the presence of strong intramolecular and intermolecular hydrogen bonds suggested for both dyes is consistent with the literature [22,43].

3.3.1.
e Results of Antileishmanial Activity Test. Antileishmanial activities of dyes I and II against L. infantum, L. major, and L. tropica promastigotes were determined in vitro by microdilution broth assay with Alamar Blue, and MIC values were obtained. In the evaluation of antileishmanial activity, it is expected that Alamar Blue does not change color in the negative control. In addition, in promastigotes added as positive control, the blue color should turn pink, since inhibitory dye is added, and it is expected to continue vitality. In the case of negative and positive controls working, results can be evaluated. In other words, if the blue color does not change in the wells, the absence of vitality and turning pink determine that the vitality continues. e results are shown in Figures 4-6. MIC values obtained in the tests are given in Table 4. According to the data obtained, it was determined that dye I was ineffective against all Leishmania species at the concentrations studied. Dye II was observed to be effective only against L. major promastigotes (MIC: 2500 μg/mL), while it was found that it did not have antileishmanial activity against other Leishmania species. It was determined that amphotericin B, which is used as a control drug, is effective against all Leishmania species even the lowest concentration studied (MIC: 19 μg/mL).

Trichomonacidal Activity Results.
For trichomonacidal activities of dyes I and II against T. vaginalis trophozoites, MLC values were evaluated as in vitro with trypan blue of 0.5% at 16, 24, and 48 hours under invert microscope. For the lethal percent, formula 100 − (number of living cells/ total number of cells × 100) was used.
e MLC values obtained at the 16th, 24th, and 48th hours in the tests are given in Table 5. e microscopic images of the results of dyes I and II obtained against T. vaginalis trophozoites at the end of 48 hours are shown in Figures 7 and 8, respectively.
It was determined that the concentration value of dye I, which has 100% lethal effect against T. vaginalis trophozoites in vitro, is 10,000 μg/mL. At the end of test, the parasites in the well were taken into TYM medium again to determine whether there was growth at this concentration or not. Growth of the parasite was not detected in the medium, and 100% lethal effect was confirmed. In addition, it was determined that dye I was more effective than dye II and     Figure 3: 1 H-NMR spectra of (a) dye I and (b) dye II.

Conclusion
In this study, for the first time, molecular modeling and antiparasitic activity studies were carried out on 6-amino-5-[(4-nitrophenyl) diazenyl] pyrimidine-2,4 (1H, 3H)-dione (dye I) and 6-amino-5-[(4-bromophenyl) diazenyl] pyrimidin-2,4 (1H, 3H)-dione (dye II) given in the literature, which were resynthesized by the same method and whose molecular structures were confirmed using FTIR, 1 H-NMR spectroscopic methods. In molecular modeling study, the molecular structures of the possible tautomeric forms of dyes I and II in the gas state and in DMSO solvent were optimized using the DFT/B3LYP/6-311++G(d,p) method, and their molecular total energies, optimized structures, IR, and 1 H-NMR spectral data were obtained. IR vibration frequencies were calculated in gas state, and 1 H-NMR chemical shift values were calculated in DMSO solvent. eoretical spectral data of the possible tautomeric forms of dyes were compared with experimental data obtained from FTIR and 1 H-NMR spectra of dyes. e results show that, for both dyes, the spectral data of the imine-diketo-hydrazone form, which has the lowest energy and hence is determined as the most stable tautomeric form, are consistent with the experimental ones. e optimized structures of dyes in imine-diketo-hydrazone form were obtained in the most stable form that reveals N-H· · ·O type intramolecular bonds. erefore, the relevant IR and 1 H-NMR spectral data are compatible with the experimental ones. However, significant deviations from experimental values observed in IR and 1 H-NMR spectral data of dyes I and II in imine-diketo-hydrazone forms indicate the presence of N-H· · ·O type strong intermolecular hydrogen bonds in the molecular structure of the dyes.
Antiparasitic activities of the dyes were tested as in vitro against L. infantum, L. major, L. tropica promastigotes, and T. vaginalis trophozoites by microdilution broth assay. Antileishmanial and trichomonacidal activities were evaluated considering MIC and MLC values, respectively. Antileishmanial activities of dyes were evaluated by comparing the MIC values obtained in the tests with those obtained for the standard control drug amphotericin B. Results show that dye I has no antileishmanial activity at the concentrations studied against Leishmania species tested, and dye II is effective against L. major promastigotes, but not against other Leishmania species studied. If higher concentrations are studied, the dyes may be found to be effective as antiparasitic.
In the trichomonacidal activity study, as a standard control drug, metronidazole was used. According to the data obtained, dyes I and II have trichomonacidal activity at some concentrations studied. In tests, it is determined that azo dyes containing uracil show a lethal effect of 33% to 100% and also that dyes tested against T. vaginalis trophozoites show a dosedependent effect in vitro.
As a result, the antiparasitic activity results obtained in the study are promising, and due to in vitro antileishmanial and trichomonacidal activities, azo dyes containing uracil may be considered in pharmacological research, and thus the diversity of compounds used may increase. In order for dyes I and II to be good drug candidates, in vivo control studies and toxicity tests should be performed in experimental animal models.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request. Disclosure e numerical calculations reported in this paper were fully performed at TUBITAK ULAKBIM, High Performance and Grid Computing Center (TRUBA resources). An oral presentation of a part of this article is available (Turkish Physical Society 35th International Physics Congress, Bodrum Municipality-Heredot Culture Center, Konacık-Bodrum/Turkey, September 4-8, 2019).
is article is prepared from Pınar Kubaşık's master's thesis (Graduate School of Natural and Applied Sciences, Advanced Technologies, Gazi University, Turkey).

Conflicts of Interest
e authors declare no conflicts of interest.