Antioxidant Activity of Telmisartan–Cu(II) Nanoparticles Connected 2-Pyrimidinamine and Their Evaluation of Cytotoxicity Activities

Copper nanoparticles (Cu-Nps) are one of the promising materials for the advancement of nanoscience and technology. In this work, we synthesized telmisartan copper nanoparticles and 2-pyrimidinamines via Biginelli reaction using telmisartan copper nanoparticles (Cu-Nps) as a reusable catalyst. The synthesis of 2-pyrimidinamine derivatives (1a-c) was achieved in water and under solvent-free condition (Green chemistry approach). Synthesis of 2-pyrimidinamine with telmisartan copper nanoparticle (Cu-Nps–Pyr) unexpected product was also isolated from synthesis of 2-pyrimidinamine preparation. Antioxidant and cytotoxic activities were carried out both in 2-pyrimidinamine (1a-1c) and 2-pyrimidinamine with telmisartan copper nanoparticles (Cu-Nps–Pyr). The synthesized 2-pyrimidinamine derivatives (1a-c) were characterized from FT-IR, 1H and 13C NMR spectroscopy, mass and elemental analyses. The synthesized telmisartan copper nanoparticles (Cu-Nps) were characterized from UV spectroscopy, XRD, SEM, EDX, AFM (atomic force microscopy), profile, waviness, and roughness analyses. Antioxidant activity was screened based on ABTS·+ radical scavenging and linoleic acid peroxidation performance. Cu-Nps–Pyr-1b showed substantial antioxidant (97.2%) activity against ABTS·+ assay and 91.2% activity against AAPH assays compared with Trolox. Cytotoxicity was evaluated using HepG2, HeLa, and MCF-7 cell lines, the Cu-Nps–Pyr-1a is high in toxicities (GI50 = 0.01 μm) against the HeLa cancel cell line compared with doxorubicin. The developed copper NPs with 2-pyrimidinamine (Cu-Nps–Pyr) could provide promising advances as antioxidant activities; this nanocomposition could be considered an anticancer treatment in future investigations.

Telmisartan is used in the management of hypertension, which acts as an angiotensin II receptor blocker (ARB). The structure of telmisartan is shown in Figure 2 and their orally active direct-acting AII, the AT1receptor antagonist potential of pharmacotherapy of hypertension [12].
Scavenging ROS antioxidants of acetyl cysteine additionally L-carnitine are observed to cure the inflammatory diseases [13,14]. However, it does not performed well against ROS-related diseases due to poor bioavailability and various limitations of clinical screening [15,16]. Recently, studies reported that copper-based nanoparticles improved the ROS-related diseases and higher scavenging ability of this treatment [17]. Copper (Cu) is one of the best elements in recommendation for human usage, which play an important role in tyrosinase and Cu-ZnSOD enzyme [18,19]. Cu enzyme activity is basically a response to many bioactivities such as connective tissue biosynthesis, cellular respiration, neurotransmitter production, iron homeostasis, peptide biogenesis, pigment formation, and antioxidant defense [20,21]. Particularly, Cu-NPs has outstanding catalytic activity to scavenge H 2 O 2 and O 2 [22] but cannot eliminate OH· simultaneously [23] and also can promote electron transfer reactions to inactivate H 2 O 2 or OH· [24]. The catalytic behavior of Cu compounds in organic reaction motivated us to focus our attention on the application of our research. As a result, we assume that the capacity of Cu-Nps acts as catalytic as well as antioxidant activities simultaneously. In addition, the stability of Cu-NPs can be largely enhanced, which is also developing the overall ROS scavenging ability [25,26]. Based on the above observation, we designed to synthesize, high active 2-pyrimidinamine connected with Cu nanoparticles and evaluated the antioxidant of ABTS ⋅+ , linoleic acid peroxidation, and cytotoxic activities.

General Materials and Characterization Techniques
2.1.1. Chemistry. Telmisartan-mediated copper nanoparticle was analyzed from UV, FT-IR, SEM, X-ray with EDX, and AFM methods. FT-IR is recorded via Shimadzu 8201pc (4000-400 cm -1 ). The 1 H and 13 C NMR were recorded via JEOL-300 MHz. An elemental analyzer model (Varian EL III) was used for analysis of elemental presences. Thin layer chromatography was used for purity checking.

Synthesis of Telmisartan-Mediated Copper Nanoparticles.
The mixture of CuCl 2 ·2H 2 O (0.5 mmol, 5 mL) and telmisartan (1 mmol, 10 mL) was added in ethanol; then, the reaction mixture was allowed 1 M NaOH with stirring and maintained pH = 7. The reaction mixture converted blue precipitate followed by adding of water (15 mL). The precipitate was obtained, then filtered all precipitate and purified with suitable techniques.

General Method for Preparation of 2-Pyrimidinamine
Derivatives (1a-1c) via Biginelli Reaction. The compound urea (0.01 mol, 0.6 g), Cu catalyst (0.01 mol, 1.7 g), ethyl acetoacetate (0.01 mol, 1.3 mL), benzaldehyde (0.01 mol, 1 mL), and substituted amine (0.05 mol, 1.6 mL) are mixed well together in a mortar and two drops of the con. HCl were added. The precipitate was dissolved in ethanol. The undissolved catalyst was filtered and dried in vacuum and used for the next cycle. The final product was obtained from the evaporation of ethanol from the filtrate and unexpected product also obtained. The final compound was confirmed by TLC. The pure product was obtained from the using suitable recrystallized method. The above procedure was followed for synthesis of the remaining compounds 1b and 1c.    2.5. Antioxidant-(ABTS ⋅+ ) Scavenging Activity. ABTS + (2,2′ -azino-bis(3-ethylbenzothiazoline-6-sulfonicacid)), the antioxidant radical scavenging activity, was checked with all compounds via spectrophotometric analysis according to the method descripted in Surendra Kumar et al. [27]. The ABTS ⋅+ radical scavenging is established and calculated the ability of antioxidants via characteristic absorption at 734 nm for long-lived ABTS radical cation, which indicated that blue/green chromophore. The results are shown in Table 1. The tested reaction mixture was prepared by 7 mM aqueous ABTS with 2.45 mM potassium per sulfate at 12 h maintained in the dark room with formal temperature. Prior to the experiment, a range of absorbance 0:700 ± 0:025 at 734 nm was obtained by the ABTS ⋅+ solution was diluted with phosphate buffer (0.1 M, pH 7.4) and added with 1 mL of the diluted 1.5 mL ethanolic solutions of 1a-c, Cu-Nps, and Cu-Nps-Pyr (1a-1c) with different concentrations (100 μg/mL). After 30 min, the percentage of inhibition was calculated at 734 nm. Ethanol is used as a blank sample, following the formula used for analysis of the percentage of inhibition.

Inhibition of AAPH Assay Free Radical
Analysis. This linoleic acid peroxidation assay was achieved and followed the methods of Surendra Kumar et al. [27]. All values were carried out in triplicate, with the consistent data, provided in Table 1. The oxidation of linoleic acid production was analyzed by conjugated diene hydroperoxide in an aqueous distribution was observed at 234 nm, [28] with AAPH (2,2 ′ -azobis(2-amidinopropane) dihydrochloride) used as a free radical initiator. For UV cuvette solution preparation, 1 mL aliquot of a 16 mM linoleic acid was added to 1 mL of 0.05 M phosphate buffer(pH 7.4), thermostatic at 37°C and addition of 1 mL of the 40 mM AAPH solution. Oxidation reaction was monitored in occurrence of 1 mL of the tested compounds 1a-c, Cu-Nps, and Cu-Nps-Pyr (1a-1c). In the absence of antioxidants, the lipid oxidation was monitored in DMSO mixed compounds. The rate of oxidation was monitored at 37°C and absorption recorded at 234 nm due to the formation of a conjugated diene hydroperoxide.

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The percentage of linoleic acid oxidation inhibition was calculated as follows: 2.7. Cytotoxic Activity. The synthesized compounds 1a-c, Cu-Nps, and Cu-Nps -Pyr (1a-1c) were screened for cytotoxic activity, and we followed the methods of Surendra Kumar et al. [27,29]. The 100 μM (MTT anticancer assay) concentration treated with test compounds for 48 h was used by three cell lines (HepG2, HeLa, and MCF-7), and standard of doxorubicin was selected in this screening.
The results were analyzed between the growth percentage of treated cells and untreated control cells. Sample preparation, 0.1 mL of the test solution (6.25-100 μg in 1% DMSO), and 0.1 mL aliquot of the cell suspension (5 × 10 6 cells/100 μL) were added and kept in an incubator (5% CO 2 ) at 37°C for 72 h. The blank sample checked only the cell suspension, and the control wells contained 1% DMSO and the cell suspension. After 72 h, 20 μL of MTT was added, and the plates were kept in the CO 2 incubator for 2 h, followed by the addition of propanol (100 μL). The absorption data (562 nm) were obtained by 27-well plates on an ELISA reader; the test sample was prepared by aluminum foil covered with subsequently agitated in a rotary shaker for 10-20 min.

Statistical Analysis. Antioxidant activities results were calculated through 3 independent evaluations, and Microsoft
Excel was used to analysis the standard deviations (SD) of each compound.

Result and Discussion
The one-pot multicomponent 2-pyrimidinamine was synthesized with in high yields. The most important factor was only excellent yield, which was a performance of suitable catalysis with aromatic aldehyde performance including hydroxyl groups reacted efficiently.
A series of compounds were prepared via optimizing the reaction conditions, for instance of reaction involved among benzaldehyde, ethyl acetoacetate, substituted amine, and urea using Cu-Nps as a catalyst in a suitable solvent (Scheme 1). The amount of Cu-Nps was changed subsequently from 0 mol% to 5 mol% under solvent-free conditions, as a result of the optimized mole percentage and the 88% yield performance obtained at 1 mol% concentration. Therefore, the results directed that 1 mol% of Cu-Nps is enough for the synthesis of the 2-pyrimidinamine compound. The synthesized Cu-Nps were characterized by UV, FT-IR spectroscopy, SEM, XRD, EDX, AFM (atomic force microscopy), profile, waviness, and roughness analyses.    Biphenyl group was found in the IR range between 1600 and 1400 cm −1 , respectively. The COOH bends and C-N (Bz) stretching modes was observed at 1235 cm −1, respectively.

UV-Visible Studies.
Spectrophotometric analysis was carried out for the Cu-Np catalyst. 6.25 mM of telmisartan was added with different copper concentrations with the addition of 1 M NaOH (pH 7) for every tested compound. The UVvisible spectra are shown in Figure 4. The Cu-Nps showed absorption bands located at 530 nm (sh). Figure 5 illustrates SEM images of Cu-Nps obtained in water (a) and in formamide mixture (b). The telmisartan-mediated Cu-Nps in the formamide mixture shows cubic in nature with size in the range of 1 μm (c). Figure 6 illustrates the EDX of Cu-Nps that exhibited the elements (Cu, C, and O) in the nanoparticle, thereby confirming the formation of the nanoparticle. Table 2 shows the EDX analysis of percentage of elements present in the Cu-Np catalyst.

Powder X-Ray Diffraction
Studies. The structural analysis and phase crystallinity of the synthesized Cu-Nps were examined via the powder X-ray diffraction method. There are no impurity peaks observed. The strong peaks indicate the highly crystalline nature of the formed nanoparticles. From the observed main diffracted peak, the average crystalline size can be calculated using the Scherer equation.
where D ðhklÞ is the average crystalline size, k is shape constant (0.89), λ is the incident X-ray (CuKα source, λ = 0:15405 nm), β is the full-width half-maximum, and θ is the incident     Figure 8 shows AFM images of Cu-Nsp. AFM image indicates the filtering procedure at cut-off frequency values at 4 μm and Cu-Nsp profile particle size; performance is presented in Figure 8 Figure 10 shows different intensity vs. diameter of the first peak of Cu-Nps.

Atomic Force Microscope Studies.
3.8. Catalyst Recovery Studies. Figure 11 indicated the recovery of the catalyst with salvaged for at least 10 run times and slight loss in catalytic activity. The lessening of activity could be detected with the regenerated catalyst on salvaging due to surface area of the catalyst during the reaction or partial loss of basic sites/regeneration. The values are shown in Table 3. The application of the catalyst was inspected via optimizing the reaction conditions. A number of aromatic aldehydes were selected to go through the condensation reaction with Cu-Nps (1 mol %) catalytic at room temperature in solvent-free settings, the yield reported in Table 3. The Cu-catalyzed performance and reaction mechanism are shown in Scheme 2.
The intermediate of acyl imine formed between urea reacts with primary amine; for the next step, acyl imine reacted with aldehyde to give the guanidine core, which is actuated by copper catalysis.
Step 3 was addition reaction between β-dicarbonyl with 1-benzylidene-2-R-guanidine, then cyclized to afford the 2-aminopyrimidine derivatives. In conclusion, the simple and efficient synthesis of 2-aminopyrimidine uses Cu-Nps under solvent-free conditions. We developed the methodology and environmentally safe synthesis of the 2-aminopyrimidine derivative with good yields.    Compounds 1a-1c were synthesized by condensation method. The compounds 1a-1c physicochemical data are represented in Table 4 and its catalyst performance is shown in Table 5. The compounds were confirmed via FT-IR, 1 H and 3 C NMR spectra, and elemental analyses. The IR absorption bands of compound 1a show the frequency range between 2584 and 2596 and 3239 and 3246 cm -1 corresponding to the C=N and NH groups, respectively.
For compound 1a, the 1 H NMR spectral value obtained a sharp singlet at δ 9.20 for NH proton and a singlet at δ 4.16 for -CH-proton, 13 C NMR spectral values at δ 167.2 and δ 154.0 ppm corresponding to C=O and C=N carbons, respectively. The IR absorption bands show that compound 1b frequency ranges between 3239 and 3246 and 2584 and 2596 cm -1 corresponding to the NH and C=N groups, respectively. For compound 1b, 1 H NMR spectral value obtained a sharp singlet at δ 9.34 for NH proton and a singlet at δ 4.12 for -CH-proton, 13  Scheme 2: Catalysis performance with mechanism of compound preparation (1a-1c).  Cu-Nps 1 88 12 8 BioMed Research International spectral value obtained a sharp singlet at δ 9.26 for NH proton and a singlet at δ 4.10 for -CH-proton; 13 C NMR spectral value obtained the peaks at δ 163.0, 167.2, and 190.0 ppm corresponding to C=N, C=O, and C=S carbons, respectively. Scheme 3 indicates that the mechanism of expected product and unexpected copper nanoparticles connected 2pyrimidine compounds; this mechanism clearly indicated the necessity for evaluation of the biological activity in 1ac, Cu-Nps, and unexpected copper nanoparticles connected 2-pyrimidine Cu-Nps-Pyr (1a-1c) compounds.
3.9. ABTS ⋅+ Radical Scavenging Activity. The antioxidant activity was carried out based on a decolorization technique using the ABTS radical assay, with the generation of the stable blue/green ABTS ⋅+ radical formation. Among the synthesized compounds, Cu-Nps-Pyr-1b showed capable ABTS ⋅+ scavenging activities of 97.2%, compared with Trolox standard (Table 1).
This study, containing both 2-pyrimidinamine and Cu-Nps, shows the capability to scavenge ABTS ⋅+ assays compared with standard; the mechanism is represented in Scheme 4.

Cu-Nps-Pyr-1c-Linoleic Acid Peroxidation-Inhibition.
The Cu-Nsp with 2-pyrimidinamine combination was highly responsive to oxidative changes and inhibition in linoleic acid peroxidation screening. The hydrophilic AAPH initiator was used to measure the conjugated diene hydroperoxide formation at 234 nm by spectrophotometric analysis. Indeed, AAPH produced the free radicals at 37°C, which was an act of spontaneous decomposition. Here, R-N=N-R indicates the radical initiator, and LOO ⋅ denotes a linoleic peroxy radical, and LH stands for linoleic acid. The radicals R ⋅ was produced via immediately reacting with oxygen and causing the oxidation of lipids. The mechanism of reaction is represented in Scheme 5.
The Cu-Nsp with 2-pyrimidinamine derivatives was performed by hydrogen donor antioxidants (AOH), steadying and postponing the free radicals of oxidation lipids. In in vitro studies of free radical fabrication, an azo compound was causing free radicals by impulsive thermal decomposition. Alkylperoxy free radicals were produced via the watersoluble AAPH used as a hygienic and governable source thermally. AAPH assay was achieved to enhance the antioxidant activity of Cu-Nsp with 2-pyrimidinamine; the compound 1c 3.11. Cytotoxic Activity. The results of each compound were reported in Table 6. The results show the contacts of LC 50 , TGI, and GI 50 values. The compound 1a (GI 50 = 0:69 μM) was high, while Cu-Nps-Pyr-1a (GI 50 = 0:01 μm) showed a significant activity against the HeLa cancer cell line. The compound 1b (GI 50 = 02:1 μM) and Cu-Nps-Pyr-1b (GI 50 = 0:01 μM) showed rightful activity against the HepG2 cell line compared with standard doxorubicin. All compounds displayed a moderate activity against all the cell line, whereas when compound with Cu nanoparticles, it is becoming highly responsive in cytotoxic scanning.

Conclusion
In conclusion, for copper nanoparticles mediated by the telmisartan drug, the Cu-Nsp was characterized by UV and FT-IR spectroscopy, scanning electron microscopy, X-Ray, EDX, AFM (atomic force microscopy), profile, waviness, roughness analysis, TGA studies and particle size analyzer. Synthesized Cu-Nps were used as catalysis for preparation of pyrimidinamine via the Biginelli multicomponent cyclocondensation reaction. The Cu-Nsp was a significantly active catalyst and gave excellent yields, and also recycling of the catalyst is very easy in this process. Bioactivity of 2-pyrimidinamine was a significant activity against performance of ABTS ⋅+ and linoleic acid peroxidation, while Cu-Nsp was moderately active in antioxidant screening; therefore, designed 2-pyrimidinamine with Cu-Nsp gave a noble result when compared with commercial drugs. Particularly, Cu-Nps-Pyr-1b was a potential activity (>90%) against ABTS ⋅+ and AAPH assays, as compared with the Trolox standard. In cytotoxicity activity, the compound Cu-Nps-Pyr-1a was perceived for (GI 50 = 0:01 μm) against the HeLa cell line compared with doxorubicin. It is noteworthy that Cu-Nps with 2-pyrimidinamine and their derivatives are medicinally important as anticancer treatment, and future investigation of in vivo screening and the mechanism of action is currently undergoing study.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare that they have no conflict of interest.