2..1 Chemistry
One-Pot Synthesis of Uracil derivatives (UD-1 to UD-5)
Initially, charged 6-chlorouracil (7.75 g, 0.05 M) and potassium carbonate (9.14 g, 0.06 M) were mixed in 50 mL DMF in 250 mL RBF, followed by halogenated of 1-(bromomethyl) benzene (10 g, 0.05 M), the reaction mixture was stirred for 8 h at RT. Then, with further addition of potassium carbonate (7.31 g, 0.0529 M) and methyl iodide (15.02 g, 0.10 M) the reaction mixture was further stirred overnight at room temperature. After that, the charged piperazine (4.52 g, 0.05 M) or piperidine or 3-aminopiperidine (9.10 g, 0.05 M) was added to the reaction mixture and heated at 80 ˚C for 8 h to get compounds 3, UD-4, and 5 respectively. Finally, the reaction mixture of compound 3 was separately treated with acetone, water, and potassium carbonate with different chloroformates to give UD-1, UD-2, and UD-3. In another reaction, the Moc-L-Valine (9.22g, 0.05 M) was added to the reaction mixture (5) in presence of coupling reagent HATU (40.23 g, 0.10 M) and triethylamine (10.71 g, 0.10M) and stirred the mixture at RT for 25 min. After the completion of the reaction, water was added and the product was extracted in ethyl acetate, the organic layer was dried over sodium sulfate, and the solvent was removed under reduced pressure. The desired product was purified by column chromatography to get the title compound (UD-5). The steps for the synthesis of the uracil derivatives are displayed in Fig. 1 and the chemical structures of the final products are given in Fig. 2.
Heptyl-4-(3-(4-fluorobenzyl)-1-methyl-2,6-dioxohexahydropyrimidin-4-yl)piperazine-1-carboxylate (UD-1)
Yield: 55%, mp; 194–195˚C; White solid powder;1H-NMR: δ = 0.85–0.88 ( t, 3 H, -CH3), 1.27–1.31 (broad, 8 H, -CH2), 1.55–1.63 (m, 2 H, -CH2), 2.80 (broad, 4H, -CH2), 3.32 (s, 3 H, -N-CH3), 3.49–3.63 (broad, 3 H, -CH2), 4.05–4.08 (t, 2 H, -O-CH2), 5.16 ( s, 2 H, Ar-CH2-), 5.33 (s, 1 H, -CH), 7.02–7.07 (t, 1 H, ArH) 7.09–7.13(m, 2 H, ArH), 7.23–7.28 (m, 1 H, ArH) ppm. Mass: (C24H33FN4O4) m/z = 460.25, M + H = 461.1.
Octyl-4-(3-(4-fluorobenzyl)-1-methyl-2,6-dioxohexahydropyrimidin-4-yl)piperazine-1-carboxylate (UD2)
Yield: 57%, mp; 180–191˚C; White solid powder; 1H-NMR: δ = 0.85–0.88 ( t, 3 H, -CH3), 1.02–1.40 (m, 12 H, -CH2), 1.59–1.80 (t, 3 H, -CH2), 2.82 (broad, 4H, -CH2), 3.31 (s, 3 H, -N-CH3), 3.42–3.52(broad, 4 H, -CH2), 4.05–4.09 (t, 2 H, -O-CH2), 5.08 ( s, 2 H, Ar-CH2-), 5.34 (s, 1 H, -CH), 6.89–6.94 (q, 1 H, ArH) 6.96–6.98 (d, 2 H, ArH), 7.26–7.31(q, 1 H, ArH) ppm. Mass: (C25H35FN4O4) m/z = 474.26, M + H = 475.1.
Heptyl-4-(1-methyl-3-(4-nitrobenzyl)-2,6-dioxohexahydropyrimidin-4-yl)piperazine-1 carboxylate (UD-3)
Yield: 59%, mp; 197–198˚C; White solid powder; 1H-NMR: δ = 0.86–0.88 ( t, 3 H, -CH3), 1.21–1.24 (broad, 10 H, -CH2), 1.45–1.57 (m, 3 H, -CH2), 2.80 (broad, 4H, -CH2), 3.23 (s, 3 H, -N-CH3), 3.47–3.56 (broad, 4 H, -CH2), 3.99–4.03 (t, 2 H, -O-CH2), 5.13 ( s, 2 H, Ar-CH2), 5.33 (s, 1 H, -CH), 7.25–7.34 (d, 2 H, ArH) 8.11–8.13(d, 2 H, ArH) ppm. Mass: (C24H33N5O6) m/z = 487.24, M + H = 488.
1-(4-Fluorobenzyl)-3-methyl-6-(piperidin-1-yl)dihydropyrimidine-2,4(1H,3H)-dione (UD-4)
Yield: 57%, mp; 175–176˚C; White solid powder;1H-NMR: δ = 1.63 (m, 6 H,-CH2), 2.86 (s; 4 H, -CH2), 5.02 (s, 2 H, -CH2), 5.32 (s, 1 H, -CH), 6.98 (t, 2 H, Ar-H), 7.22 (m, 3 H, Ar-H) ppm. 13C NMR δ = 23.77, (-CH2), 25.33 (-CH2), 27.84 (-CH3), 47.22 (-CH2), 52.44 (-CH2), 89.78 (-CH), 115 (Ar-C), 128.95 (Ar-C), 132.17 (Ar-C), 152 (-N-CO-N-), 160.37 (-C-), 163.36 (-N-CO-C-) ppm. Mass (C17H22FN3O2) m/z = 319.17
Methyl-1-1-(3-(2-cyanobenzyl)-1-methyl-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)piperidin-3-ylamino)-3-methyl-1-oxobutan-2-ylcarbamate (UD-5)
Yield: 57%, mp; 184–185˚C; White solid powder;1H-NMR: δ = 0.71 (dd, 6 H,-CH3), 1.29 (m; 2 H, -CH2), 1.74–1.79 (m, 3 H, -CH2), 2.3 (m, 1 H, -CH2), 2.46 (s, 3 H, CH2), 2.59 (m, 1 H, -CH2), 2.8 (d, 1 H, -CH2), 3.08 (s, 3 H, -N-CH3), 3.48 (s, 3 H, -O-CH3), 3.71 (t, 2 H, -CH2), 4.92 (q, 2 H, -CH2), 5.25 ( s, 1 H, -CH), 6.97 (m, 3 H, Ar-H), 7.09 (d, 1 H, Ar-H), 7.29 (d, 1 H, Ar-H), 7.95 (d, 1 H, -NH). Mass (C25H32N6O5) m/z = 496.56, M + H = 497.2481.
It is highly noted that in the available literature, generally in benzylation and methylation reactions base like sodium hydride in dimethylformamide have been used which is highly dangerous in the context of safety, environment pollutant and it also results in poor yield, impure compounds. In our simple protocol, we used low-cost, ecofriendly, and safe inorganic base potassium carbonate in dimethylformamide solvent in one pot during four steps which is one of the major advantages of our protocol. In addition, we got higher percentage yields of products. Initially, we have synthesized N-substituted uracil analogs in high yields by simple N-benzylation of 6-chlorouracil with different halogenated benzyl halides and N-methylation by methyl iodide respectively, under catalyst-free conditions in DMF solvent. Then the chlorine atoms of N-substituted uracil derivatives were substituted with piperazine, piperidine, and 3-aminopiperidine, respectively. In the final step, the N-substitution of the piperazine ring was carried out by treating it with different alkyl containing chloroformates in the same pot under similar conditions for the formation of compounds UD-1-UD-4. Simultaneously, under similar conditions in the same solvent amino acid coupling reaction was done in presence of coupling reagent HATU (1-bis(dimethylamino)methylene-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxid hexafluorophosphate) for the formation of target compounds UD-5. The whole synthesis has been done in one solvent in one pot that results in the reduction of industrial waste, affluent generation, and overall good yields (55–59%) of final products.
A reasonable mechanism for the formation of uracil derivatives is shown in Fig. 3. The reaction conditions were optimized using various solvents, bases. The yields ranged from 55 to 59%. A perusal of these indicates that the DMF and inorganic base was the best one; resulting in higher yield and best conversion. During the optimization of organic and inorganic bases were used. It was observed that during organic bases, the reaction was completed at a faster rate, but less conversion was observed. The percentage of yield was increased while using inorganic bases under DMF solvent. Hence, throughout the reaction, potassium carbonate as base and DMF as solvent were used. The proposed structures of uracil derivatives (UD-1 to UD-5) were well supported by analytical and spectroscopic data. All the derivatives were found as solids, stable to air, had good yields, and quite soluble in ethyl acetate, methanol, ethanol, chloroform, DMSO, DMF. The characteristic signal from the CH3N- group in the 1H NMR spectrum was observed as a singlet at ~ 2.90–3.10 ppm, methine proton of uracil ring CH- was observed at 5.00 -5.35 ppm, benzylic proton -CH at 4.86–5.37 ppm, aromatic protons Ar-H appeared in the range of 7.00–8.10 ppm, piperazine protons resonates at 1.50–3.80 ppm, methyl protons appeared in the range of 2.90–3.40 ppm, and methoxy protons -OCH3 appeared in the range of 3.40–3.75 ppm. The 1H NMR spectra of uracil derivatives (UD-1 to UD-5) are shown in Figure S2-S6 (Supplementary Information). Elemental analyses and ESI-MS spectra illustrated the compositions of synthesized compounds. The mass spectra of UD-1 to UD-5 showed the peaks at m/z values of 460.25, 474.26, 487.24, 319.17 and 496.56 corresponding to UD-1, UD-2, UD-3, UD-4 and UD-5.
Drug-likeness
Oral bioavailability (a measure of drug-likeness) is one of the major parameters for developing bioactive molecules as effective pharmaceutical agents. Parameters like good intestinal absorption, reduced molecular flexibility, low polar surface area, or total hydrogen bond count, are essential factors for the prediction of good oral bioavailability (41). Drug properties like membrane permeability and bioavailability are associated with some basic molecular descriptors: molecular mass, hydrogen bond acceptor and donor count, and log P (partition coefficient) in a molecule (12). These properties were originally used by Lipinski (12) to formulate his well-known “Rule of five”. The rule stands, most molecules with good membrane permeability have molecular mass < 500 Da, number of hydrogen bond acceptors < 10, number of hydrogen bond donors < 5, and log P < 5. This rule is commonly used to evaluate the suitability of a drug for oral administration. This rule is widely used as a filter for the screening of molecules with drug-like properties. Generally, a compound that fulfills at least three points out of five is said to follow Lipinski’s rule. A poor permeation or absorption is more likely to occur when there are more than 5 H-bond donors and 10 H-bond acceptors. Therefore, all these molecular properties of the mentioned uracil derivatives were calculated using MarvinSketch and are displayed in Table 1, showing all uracil derivatives have molecular masses lower than 500, contain hydrogen bond acceptors < 10 and number of hydrogen bond donors < 5. Moreover, the uracil derivatives have lower log P, molar refractivity, and polar surface areas than the values recommended by Lipinski’s rule. Thus, the synthesized uracil derivatives fully meet all the five criteria of Lipinski’s rule, hence having good oral bioavailability.
Table 1
Molecular properties of uracil derivatives show their oral bioavailability and drug-likeness.
Derivative | Molecular mass/u | Log P | Hydrogen bond acceptors | Hydrogen bond donors | Molar refractivity/ (m3 M− 1) | Polar surface area/˚A2 |
UD1 | 462.56 | -3.80 | 4 | 0 | 122.75 | 73.40 |
UD2 | 476.5 | -4.24 | 4 | 0 | 127.35 | 73.40 |
UD3 | 487.4 | -4.04 | 6 | 0 | 133.45 | 116.54 |
UD4 | 319.38 | -2.30 | 3 | 0 | 85.27 | 43.86 |
UD5 | 498.5 | -1.62 | 6 | 2 | 131.15 | 135.08 |
2.2 Molecular Docking Studies
Uracil derivatives show a high docking affinity for thymidylate synthase
CB-Dock by default predicts five binding cavities and based on experimental information of the binding site of the co-crystallized substrate of thymidylate synthase, we proved that the correct binding site is among the predicted cavities (Fig. 4A). Pyrimidine analog namely 5-Fluorouracil (5-FU) has been approved by FDA for a variety of cancers including gastric adenocarcinoma and breast cancer (13). Metabolite of 5-FU by name fluorodeoxyuridine monophosphate (FdUMP) competes with normal substrate dUMP and thus prevents its binding with thymidylate synthase (13). As it is clear that FdUMP but not 5-FU binds with thymidylate synthase thus we used the former rather than the latter as a positive control in the docking study and compared the binding affinity of six novel molecules with FdUMP. All the newly tested molecules showed higher binding affinity than FdUMP (− 6.7 kcal/mol). However, two molecules namely UD4 and UD5 exhibited the highest inclination towards the predefined site of thymidylate synthase. While UD- showed a binding affinity value of − 8.3 kcal/mol, UD4 showed a binding affinity of − 8.0 kcal/mol (Fig. 4B). The ligand-receptor interaction is more favorable when the binding affinity values are more negative. Thus, the binding affinity value is more negative for UD5 and UD4 suggests its more inclination towards thymidylate synthase compared to FdUMP.
The interaction profile of the five UDs in the docked state with thymidylate synthase was generated. Expectedly, the two most potent molecules showed relatively more interactions than the third strong binder against thymidylate synthase. UD4 formed four hydrophobic interactions (ILE 108, TRP 109, LEU 192, PHE 225) and two hydrogen bonds with amino acid residues GLN 214 and ASP 218 of binding groove. UD5 which was predicted by flexible docking as the most powerful molecule against the binding groove of thymidylate synthase also showed an extensive interaction profile. It displayed three hydrophobic interactions, two with LEU 192 and one with ASP 218. Furthermore, three hydrogen bonds were seen between ligand 5 and three residues (ARG 50, ASP 218, and TYR 258) of thymidylate synthase (Fig. 4A). Our results align well with the study where enhanced ligand efficiency has been attributed to hydrophobic interactions (8). All the novel molecules and the known binder FdUMP bind at the experimentally certified binding site of dUMP, the substrate of thymidylate synthase, which further authenticates the reliability of our findings Figure S7 (Supplementary Information).
2.3. Expression pattern & prognostic significance of TYMS in BC patients
TYMS is upregulated in BC Patients.
Using the TCGA BrCa dataset of the Gepia2 online portal, the expression of TYMS was analyzed, and it was revealed that TYMS is highly overexpressed in breast cancer patients in contrast to normal women. TYMS was found to be upregulated with a log2 FC of 2.223, (p-value of 1.58e-179) Fig. 5A. Also, we analyzed the TYMS expression within BC subclasses and women with different ethnicities using the UALCAN portal and it was found that among different subclasses of BC, TYMS was highly upregulated in TNBC and women with African-American ethnicity Fig. 5B, C, and D.
High expression of TYMS affects the prognosis of BC patients.
The prognostic significance of the TYMS expression was analyzed using the online open database, the Kaplan-Meier Plotter. The KM plots demonstrated that BC patients with high expression of TYMS had worse relapse-free survival and overall survival (p < 0.05) Fig. 5E, F. Also, BC patients with reduced levels of TYMS had better relapse-free survival and overall survival. These results indicate that targeting TYMS may prolong the survival of breast cancer patients, in particular the TNBC patients who solely rely on chemotherapeutic regimens.
2.4. In vitro anti-cancer activity
Uracil derivatives possess potent anti-proliferative activity
To examine whether UDs, possessed anti-cancer activity, we performed a preliminary assay of cell viability on MDA-MB-231 cells. UD-1, UD-2, UD-3, UD-4 and UD-5 were given at 50 µM conc. and cells were incubated with drugs for 72 h. MTT assay analysis revealed that UD-1, UD-2, and UD-4 possessed high anti-cancer activity compared to UD-3, and UD-5 Fig. 6A. The three derivatives UD-1, UD-2, and UD-4 were selected for further analysis in a panel of breast cancer cell lines.
To investigate the effect of UD-1, UD-2, and UD-4, BC cell lines were treated with different concentrations of the selected UDs. The effects of UDs on the survival of BC cells were assessed by MTT assay. As shown in Fig. 6B, C, D & Fig. 7, UDs inhibited cell proliferation in a time- and concentration-dependent manner. UD1 exhibited a log IC50 value of 1.86 µM, 1.89 µM, 2.0 µM, and 1.89 µM in MDA-MB-231, MDA-MB-468, MCF-7, and 4T1 cells respectively. UD2 exhibited a log IC50 value of 1.51 µM, 1.85 µM, 1.86 µM, and 1.83 µM in MDA-MB-231, MDA-MB-468, MCF-7, and 4T1 cells respectively. UD4 exhibited a log IC50 value of 1.3 µM, 1.7 µM, 1.8 µM, and 1.6 µM in MDA-MB-231, MDA-MB-468, MCF-7, and 4T1 cells respectively Table S8 (Supplementary Information).
Among the three potent UDs, UD-4 demonstrated high anti-proliferative activity in vitro, with the lowest IC50 compared to UD-1 & UD-2. Among different BC cell lines, the TNBC cell lines showed more sensitivity towards UDs and had lower IC50 compared to ER + cell line MCF-7.
Effect of Uracil derivatives on colony formation potential of MDA-MB-231 cells
To further validate the anti-cancer activity of UDs, a colony formation assay was performed in BC cell line MDA-MB-231 Fig. 8. Treatment with UDs showed dose-dependent inhibition of colony formation potential of breast tumor cells. Among the UDs, UD-4 showed the highest reduction in colony formation potential of tumor cells. UD-1 and UD-2 had less effect on colony formation in MDA-MB-231 cells, however, the small size of colonies in the treated wells of UD1 and UD2 demonstrates that UD1 and UD2 restrict the growth of tumor cells. These results demonstrate that UDs can limit the growth of breast tumor metastases and may be a promising approach in reducing the colonization of tumor cells at metastatic niches.