Eco-friendly synthesis, characterization, and drug-likeness properties of new uracils and their biological evaluation

2.1 Instruments

The Gallenkamp apparatus was used to record all melting points, and the IR-spectra were obtained using Perkin Elmer Infrared Spectrophotometer Model 157, Grating (KBr; ν/cm⁻¹). The 1H-NMR result were obtained using the Varian (300; 75 MHz) spectrophotometer (TMS; δ/ppm) as an internal reference and the DMSO-d₆ as a solvent. The MS-EI were recorded on 70 eV with the Varian MAT 311Kratos. The elemental analyses were carried out at the micro-analytical center of Cairo University, Giza, Egypt. Results were in good agreement (±0.3%) with the calculated values. Irradiation was performed in a domestic MW oven (E. M-230 M; 800 W). All spectral data were illustrated in Table 1. All chemicals are from Sigma-Aldrich; Molinspiration.com (http://molinspiration.com) is a chemical informatics software vendor based in Slovakia.

2.2 Chemistry

General method for the microwave-assisted synthesis (Method B): The reactants used in method A (conventional method) were placed in a closed Teflon vessel, and were irradiated in microwave at 800 W (see reaction times, Table 2). The reaction mixture was treated in the same way as the conventional method (Method A), thereby obtaining the desired compound.

2.3 Antioxidant activity

3.1 Chemistry

The 11 pyrido[2,3-d]pyrimidine derivatives existing in this work were synthesized through two synthetic routes (conventional and microwave-assisted methods), as illustrated in Schemes 1 and 2, respectively. The starting compound 1 was synthesized by the multicomponent reaction between 6-aminothiouracil, cyclopentanone, and p-anisaldehyde. The IR spectrum of 1 exhibited good absorbance rates (λ=3321, 3096 cm⁻¹) due to both NH groups being in compound 1. On the one hand, the ¹H-NMR spectrum of compound 1 displayed four singlet signals (δ=3.83, 4.0, 8.13, 12.33 ppm), which can be attributed to the OCH₃, N₃-H, CH olefinic, and N₁-H protons, respectively. Furthermore, it showed multiplet signals (δ=6.82–7.81 ppm) due to the aromatic protons. The 2-alkyl thio derivatives 2a–c were prepared through the reactions of compound 1 with different alkyl halides (Scheme 1). All structures 2a – were proven by both elemental and spectral data. The ¹H-NMR spectrum exposed a singlet signal (δ=2.50 ppm) equivalent to the SCH₃ protons.

Scheme 1:

Reaction of pyridopyrimidine derivative 1 with different reagents under microwave irradiation.

Scheme 2:

The reaction of thiomethyl derivative 2a with different amines.

On the other hand, the ¹H-NMR spectrum of compound 2b indicated triplet and quartet signals (δ=1.08, 3.41 ppm) owing to the CH₃ and SCH₂ protons of SC₂H₅ protons, respectively. Additionaly, compound 2c showed triplet signals (δ=0.88 ppm) in its ¹H-NMR spectrum corresponding to the terminal CH₃ of S-C₂H₅ and another one (δ=3.24 ppm) due to SCH₂. Furthermore, the multiple signals (δ=1.25–1.90 ppm) observed were attributable to the alkyl chain (-C₁₄H₂₈) protons. The reaction of 1 with α-haloketone, namely, chloro-acetone or phenacyl-bromide, gave compounds 2d and e. The ¹H-NMR spectrum of 2d displayed two singlet signals (δ=2.28, 4.03 ppm) equivalent to CH₃ and CH₂ of chloroacetonyl moiety, respectively. Compound 2e displayed a singlet signal (δ=4.84 ppm) due to the SCH₂ protons. Compound 4 was obtained through the reaction of compounds 2a and b with hydrazine-hydrate. Compound 4 revealed singlet signals (δ=5.18 ppm) in its ¹H-NMR spectrum owing to the NH₂ protons and δ=6.83 ppm corresponding to the NH proton. Meanwhile, further alkylation at the N-3 of compound 2a when it was treated with methyl-iodide afforded compound 3. Compound 3 exhibited two singlet signals (δ=2.59, 3.45 ppm) attributable to the N-CH₃ and S-CH₃ protons in its ¹H-NMR spectrum. The oxidation of compound 2a with hydrogen peroxide in glacial acetic acid afforded 2-methylsulfone derivative 5. The ¹H-NMR spectrum exposed a singlet signal (δ=3.3 ppm) owing to the 2-methyl sulphone protons.

Additionally, compound 6 was synthesized from the reaction of 2a in dry dioxane with phosphorus oxy-chloride. The ¹H-NMR spectrum of compound 6 displayed a singlet signal (δ=2.50 ppm) due to the S-CH₃ protons. Furthermore, the nucleophilic reactions of compound 6 towards different amines, namely, p-anisidine, piperazine, morphiline, and hydrazine-hydrate were investigated to produce compounds 7, 8a, 8b, 9 and 11, respectively. Two singlet signals (δ=2.44, 8.13 ppm) appeared in the ¹H-NMR spectrum of compound 7 owing to the SCH₃ and NH protons, respectively. The mass spectra of compounds 8a and 8b exhibited the ion peaks (m/z=525 M⁺, 526 M⁺) conforming to the molecular formula of both. Meanwhile, compound 6 reacted with anthranilic acid producing compound 9. The IR spectrum of compound 9 revealed absorption bands at (λ=3510, 3368, 1716 cm⁻¹) owing to NH, OH, and CO, respectively. The ¹H-NMR spectrum exhibited five singlet signals (δ=2.44, 3.81, 7.71, 8.13, and 13.11 ppm) attributable to the SCH₃, two OCH₃, NH, CH= olefinic and OH protons, respectively. Additionaly, compound 9 underwent cyclization in the presence of AcOH/ H₂SO₄ to give the quinazolinone derivative 10. The IR spectrum of compound 10 revealed the absorption band (λ =1708 cm⁻¹) due to the CO function. The ¹H-NMR spectrum also displayed a singlet signal (δ=2.59 ppm) attributable to the SCH₃ protons. The absence of the carboxylic proton signal (δ=13.11 ppm) in compound 9 confirmed the cyclization reaction. The treatment of compound 6 with hydrazine hydrate in dry dioxane gave the dihydazinyl derivative 11. The four singlet signals that appeared in the ¹H-NMR spectrum of compound 11 (δ=4.50, 4.77, 8.15, 10.12 ppm) were due to two NH₂ and two NH protons, respectively. Furthermore, the mass spectrum revealed the molecular ion peak (m/z=455 M⁺) attributable to the formula C₂₅H₂₅N₇O₂ (Scheme 2).

3.2 Biological properties

The products were also tested for compliance to the Lipinski rule of five, and were summarized in Table 3. A molecule is likely to be established as an orally active drug candidate when it obeys the following rules: (a) the hydrogen bond donor’s number “HBD” should be ≤5, (b) the hydrogen bond acceptor’s number “HBA” should be ≤10, (c) its molecular weight should be ≤500 Da, and (d) it should not have an octanol-water partition coefficient (≤5) [21]. The absorption degree is stated by the (%) of absorption, which is computed from the following equation: ABS %=109−(0.345 TPSA) [22], [23]. The scores of all compounds were calculated using the online software [24], [25]. The hydrogen bond donor and acceptor in the synthesized uracils obey the Lipinski rule of five, and thus some of the synthesized compounds have good permeability and absorption properties through the biological membrane. Dissolution is dependent on the influences of aqueous solubility, lipo-philicity (log P), and ignitability (p Ka). The log p values of compounds 1 and 2a–e (Scheme 1) were between 4.53 and 9.99.

Additionally, the molecular weight played an important role in the drug action: if it is higher than the accepted limit, the bulkiness also increases, subsequently affecting the drug action (the interaction between the drug receptor and DNA). The molecular weights of compounds 1 and 2a–e between 443.50 and 667.96 Da showed that compounds 1 and 2a follow Lipinski’s rule of five. Consequently, further reactions have been processed for compound 2a to produce compounds 3, 4 and 5 (Scheme 1). Therefore, the results revealed that compounds 4 and 5 follow Lipinski’s rule of five.

Similarly, compound 1 reacted with POCl₃ to give compound 6 with one violation. Consequently, compound 6 was conducted for further substitution reactions to produce compounds 7–11 (Scheme 2). The log p values for these compounds were between 5.83 and 8.63. Their molecular weights were between 525.68 and 576.68 with more than one violation. PSA is the sum of all surfaces of polar atoms presented in a molecule. The prediction of drug transport property depends mainly on the PSA, which is inversely proportional to the absorption %. Compounds like 3 and 6 have the least polar surface areas among the series so they have the maximum absorption. Meanwhile, compounds 1, 2a, 4 and 5 exhibited violations of the discussed criteria (Table 3). Hence, these compounds have a good potential for eventual development as oral agents and can be potentially active drug candidates. Although the other compounds showed a narrow therapeutic margin, there is chemical space to design and develop more selective and potent compounds.

3.3 Biological activity

3.3.1 Antioxidant activity for erythrocytes hemolysis and ABTS

Most of the synthesized pyridopyrimidine derivatives were examined for their antioxidant activity using ABTS assay. The results showed their ability to inhibit lipid per-oxidation in rat kidney and brain homogenates along with the rate of erythrocyte hemolysis. Compound 1 proved to show an effective antioxidative activity. Additionaly, compounds 4, 5 and 8a showed good activities as well. Meanwhile, compounds 2a, 2b, 2d, 2e, 3 and 6 showed a moderate activity toward the ABTS method. Compounds 2b, 4 and 5 exhibited potent activities toward erythrocyte hemolysis with ascorbic acid (Figures 1 and 2).

Figure 1:

Erythrocyte hemolysis (%).

Figure 2:

ABTS (inhibition %).

3.3.2 Bleomycin-dependent DNA damage

Bleomycins are routinely used as antitumor agents because they are a family of antitumors. Some of the synthesized compounds have been examined. The results indicated that compounds 1 and 2a showed potent protective activity to DNA. Among the tested compounds, compound 5 showed good protection activity against DNA damage (Figure 3).

Figure 3:

Bleomycin-dependent DNA damage.

3.3.3 Anti-inflammatory evaluation

Carrageenan-induced paw edema test in rats was used to evaluate the tested compounds’ activities. The results suggested that all the test compounds protected rats from carrageenan-induced inflammation. On the one hand, compounds 2a and b showed higher activities than diclofenac sodium. On the other hand, compounds 1 and 3 were equipotent to diclofenac sodium (Figure 4).

Figure 4:

Anti-inflammatory activity.