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DOI: 10.1055/a-2456-9620
Some Thiazolopyrimidine Derivatives: Synthesis, DFT, Cytotoxicity, and Pharmacokinetics Modeling Study
The authors received no funding for this work.
Abstract
A pyrimidinethione candidate carrying pyrazole and thiophene scaffolds was produced by a Biginelli cyclocondensation reaction of a pyrazolecarbaldehyde with pentan-2,4-dione and thiourea. To create some heteroannulated thiazolopyrimidines, the pyrimidinethione was subjected to cyclocondensation reactions with ethyl chloroacetate, 1,2-dibromoethane, chloroacetonitrile, and oxalyl chloride. A DFT simulation was performed for a frontier-orbital analysis to determine the molecular geometry. Among the products, 6-acetyl-7-methyl-5-[1-phenyl-3-(2-thienyl)-1H-pyrazol-4-yl]-5H-[1,3]thiazolo[3,2-a]pyrimidine-2,3-dione displayed the highest softness and the lowest energy gap in the DFT calculations. Moreover, it had the highest electrophilicity index, suggesting possible biological impacts. The compounds obtained were evaluated against cell lines of breast adenocarcinoma (MCF7) and hepatocellular carcinoma (HepG2) as antiproliferative agents. A simulation of the molecular docking of our compounds with the epidermal growth factor receptor demonstrated the rationality of our design and identified the binding mode. A model pharmacokinetics analysis showed that the products have the expected and desirable drug-like and bioavailability properties.
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Key words
cytotoxicity - density functional theory - pharmacokinetic modelling - pyrimidinethiones - thiazolopyrimidinesPyrimidines (components of nucleic acids) constitute an influential class of natural and nonnatural products due to their pharmacological properties, such as their anticancer,[1] [2] [3] [4] [5] antiviral,[6,7] antimicrobial,[8,9] and insecticidal activities.[10–12] Pyrimidine and its fused derivatives are promising frameworks in drug development and anticancer efficacy.[13,14] Many pyrimidines and pyrazoles have been shown to inhibit cell proliferation and migration.[15] Several US Food and Drug Administration-approved anticancer drugs that contain a pyrimidine core act as tyrosine kinase inhibitors (TKIs): examples of these include lapatinib, erlotinib, gefitinib, and imatinib.[16]
Derivatives of pyrazole and thiophene exhibit several biological effects, including anticancer, antimicrobial, antiviral, and insecticidal properties.[17] [18] [19] [20] [21] [22] [23] In continuation of our strategy,[24–28] we have prepared several thiazolopyrimidine compounds with pyrazole and thiophene cores with the aim of enhancing their anticancer effects, and we report density functional theory (DFT) computations, in silico molecular-docking studies, and modeling of their pharmacokinetic parameters.
Several pyrimidine compounds have been found to be particular inhibitors of epidermal growth factor receptor (EGFR). It has been shown that the 4,6-dianilinopyrimidine derivative I (Figure [1]) is a selective and powerful inhibitor of EGFR kinase activity in both cells and enzymes.[29] Compound II significantly reduced the levels of phosphorylated EGFR and inhibited the development of HCC827 cells, an EGFR TKI-sensitive NSCLC cell line.[30] Compound III additionally exhibited a substantial antiproliferative activity against HepG-2 and an influential dose-related thymidylate synthase inhibition, comparable to that of 5-fluorouracil.[31] Also, a pyrazole core can simulate the ATP binding site of EGFR. Moreover, the pyrazole, thiophene, and phenyl moieties were expected to create hydrophobic interactions with hydrophobic region II of EGFR active sites.
Based on these premises, the intended pyrimidinethione lead compound 4 was formed by hybridizing pyrazole, thiophene, and pyrimidine moieties in one skeleton (Figure [1]). An ability of the N, S, and C=O groups to form hydrogen bonds with the hinge region was hypothesized on the basis of the mechanisms of binding of EGFR inhibitors to those active pockets. However, we assumed that N-1 and/or N-3 atom of the pyrimidine core might form hydrogen bonds with the EGFR back site through covalent or water-mediated means. To overcome any toxic side effects and resistance to the action of fluoropyrimidines, we replaced the C5-fluoro group with an isosteric acetyl group.[32]
To understand the structure–activity relationship (SAR) associated with EGFR kinase inhibition, three distinct chemical modifications were introduced into the pyrimidine ring. Initially, a thiophenyl-pyrazole group was introduced at position C4 to interact with the hydrophobic areas of the ATP pocket. The second changes involved the insertion of acetyl and methyl groups at positions C5 and C6, respectively. Thirdly, both the N-3 and C2 sites were used to create heteroannulated rings that resembled thiazolopyrimidines. Numerous chemical alterations have been made to these new compounds, which could help us explore their surface chemisorption potential as potential anticancer drugs and multitarget enzyme inhibitors.
The pyrimidinethione candidate 4 containing a thiophenylpyrazole moiety was prepared by a Biginelli cyclocondensation reaction of the pyrazolecarbaldehyde 1 [33] with pentan-2,4-dione (2) and thiourea (3) in N,N-dimethylformamide (DMF) containing concentrated sulfuric acid (Scheme [1]).[34] The IR spectrum of pyrimidinethione 4 showed both NH and C=O absorption bands and suggested the existence of the compound as a mixture of thione and thiol tautomers by exhibiting an absorption band for a thione group and another weak band for a thiol moiety. Moreover, its 1H NMR spectrum showed a singlet signal for the C4-H proton of the pyrimidine moiety, together with two broad singlet signals for the NHCSNH protons and two singlet signals for two methyl protons.
Pyrimidinethione 4 was subjected to cyclocondensation reactions with ethyl chloroacetate, 1,2-dibromoethane, chloroacetonitrile, and oxalyl chloride in efforts to prepare heteroannulated thiazolopyrimidines (Scheme [2]). Thiazolopyrimidine 5 was obtained by treating 4 with ethyl chloroacetate in ethyl alcohol and fused sodium acetate. A carbonyl absorption band was present in the IR spectrum of 5, whereas the NH absorption band was absent. Additionally, the 1H NMR spectrum showed a singlet signal for methylene protons, but no NH singlet signals.
Thiazolopyrimidine 6 was produced by the reaction of pyrimidinethione 4 with 1,2-dibromoethane. It showed no NH or C=S absorption bands in its infrared spectra. The upfield area of the 1H NMR spectrum contained two triplet signals corresponding to the CH2–CH2 group, and was free of NH signals. The formation of compound 6 is thought to have occurred through the removal of two HBr molecules, leading to annulation of the thiazole ring.
Aminothiazolopyrimidine 7 was then prepared by refluxing pyrimidinethione 4 with chloroacetonitrile in DMF (Scheme [2]). An absorption band for an NH group was visible in its IR spectrum. Singlet signals for the protons of the NH2 and methine group were visible in the 1H NMR spectrum. Compound 7 might have been formed by the SN 2 mechanism, with elimination of a molecule of HCl and subsequent cyclization.
Pyrimidinethione 4 was finally transformed into the dioxothiazolopyrimidine 8 by basic treatment with oxalyl chloride. The three carbonyl groups were represented by three singlet peaks in the IR spectrum. The suggested structure was supported by the absence of NH absorption bands in its IR spectrum and of NH singlets in its 1H NMR spectrum. Its mass spectrum showed a molecular-ion peak at m/z = 448.47 (10.70%), as well as other fragment peaks. Additional proof for the designated structures of 4–8 was provided by their 13C NMR spectra.
A DFT simulation was then performed to identify the electrophilic and nucleophilic sites and to demonstrate the courses of reactions. The calculated parameters involved the frontier-molecular orbitals, dipole moment (m), ionization potential (IP), chemical potential (μo), electron affinity (EA), electronegativity (x), global hardness (η), global softness (σ), global electrophilicity index (ω), and nucleophilicity index (n) [see the Supporting Information (SI), Table S1]. ChemBio3D Ultra 14.0 was used to design the frontier molecular orbitals, which included the lowest unoccupied molecular orbitals (LUMOs) and the highest occupied molecular orbitals (HOMOs) (SI, Figure S1).[35] [36]
The sites for electrophiles attack are indicated by the areas of greatest electron density (HOMO), whereas those for nucleophiles attack are shown by LUMOs. The calculated functionalities and electron densities of compounds 4, 6, 7, and 8 were confirmed to be connected to their activities. As shown in Figure [2], compound 8 has the lowest energy gap, the highest softness, and the lowest hardness. Because removing an electron from the HOMO requires less energy and allows the electron to more readily transfer to the hole surface, compounds with lower energy gaps may show strong inhibition efficiencies. The dipole–dipole interactions of the most potent compounds bearing hydrophobic groups correlated closely with their oxidation inhibition efficiencies. The softness values follow the order 8, 5, 4, 6, 7. The highest electrophilicity index (ω) was displayed by compound 8 (134.07 eV), which provides evidence for possible biological effects. Furthermore, 8 displayed the highest EA, suggesting possible enzyme binding. Additionally, compounds with higher binding energies exhibit higher anticancer potency due to their effective interactions with the active sites of the receptor, as discussed below in the molecular-docking simulation.
By using an MTT assay [37] with doxorubicin (a standard anticancer medication) as a positive control, the pyrimidinethione 4 and thiazolopyrimidines 5–8 were tested for their in vitro antiproliferative efficacy against hepatocellular carcinoma (HepG2) and breast adenocarcinoma (MCF7) cell lines.[38] The half maximal inhibitory concentration (IC50) was used to express the anticancer activity. The compounds presented variable degrees of inhibitory properties, from extremely strong to moderate, against the two cell lines (Table [1]). The greatest efficacy against the two cell lines in comparison to doxorubicin was displayed by compounds 8 and 5. Pyrimidinethione 4 showed strong actions against the two cell lines. Conversely, compounds 6 and 7 showed moderate activities.
aIC50 (µM) = 1–10 (very strong); 11–20 (strong); 21–50 (moderate); 51–100 (weak); >100 (noncytotoxic).
Two aspects can demonstrate a potential cytotoxic activity of compounds toward cancer cell lines: (i) the establishment of intermolecular hydrogen bonds with DNA bases and (ii) attraction between a positive charge on the compound and a negative charge on the cell wall. The following SAR was inferred by comparing the experimental cytotoxic activity of the compounds to their structures (Figure [3]). The neighboring location of carbonyl groups in compound 8 greatly increases its anticancer effect, suggesting this is an appropriate ligand for additional hydrogen bonding with enzyme active pockets. Tautomerism (compounds 4 and 5) has a significant role in anticancer reagents by increasing their flexibility (softness) and functionality.[39] The NH2 functionality in compound 7 initiateshydrogen bonding with DNA bases. The absence of a tautomeric structure in compound 6 might decrease the flexibility of its anticancer effect.
An in silico molecular-docking simulation was performed to demonstrate the rationality of our design. All the synthesized compounds 4–8 were investigated in the docking study to determine their mode of binding to EGFR enzyme. The identification of molecular targets implicated in proliferation and cell death is appropriate for the rational design of new antitumor agents. EGFR-TK is a prototypical EGFR group that performs a crucial role in the signal-transduction pathways needed for cell-growth regulation, differentiation, and survival.[40] To establish the pattern by which the inspected substances bind to pocket sites, candidates 4–8 were subjected to a docking analysis with the EGFR enzyme, data for which were obtained from the Protein Data Bank (PDB ID: 1JU6).[41] To develop antitumor agents, we performed structure-guided analyses of the corresponding selective inhibitors. Initially, water molecules were removed from the downloaded complex. The structures of the compounds and reference ligand were drawn using ChemBio3D Ultra 14.0.
The binding affinities of the synthesized ligands 4–8 toward the target protein EGFR were expressed as docking scores S (kcal/mol) (see Tables 2 and 3). A co-crystallized ligand (LYA) displayed an S score of –8.8278 kcal/mol, as a result of the presence of eleven hydrogen bonds and six hydrophobic interactions with diverse amino acids of the EGFR enzyme. The reference ligand, doxorubicin, exhibited an S score of –7.5898 kcal/mol through hydrogen bonding with ILE 108 and MET 311, in addition to a hydrophobic interaction with LEU 221. Notably, compound 8 exhibited the best docking score of –7.0387 kcal/mol through hydrogen bonding with TRP 109 and hydrophobic interactions with TRP 109 and LEU 221. Additionally, compound 5 demonstrated an S score of –6.8892 kcal/mol as a result of hydrophobic interactions with PHE 225, ILE 108, LEU 221, and MET 311, and hydrogen bonding with LYS 77. Compound 4, on the other hand, showed an S score of –6.8051 kcal/mol as a result of hydrophobic contacts with PHE 225 and LEU 221 and the presence of two hydrogen bonds with LYS 77 and 107. The most active substrates 8 and 5, through contacts with LYS 77, TRP 109, LEU 221, and PHE 225 receptors, for example, showed the greatest interactions with the EGFR pockets when the co-crystallized ligand (LYA) was present.
a The amino acids that commonly bind to LYA and compounds 4–8 are highlighted in gray.
b Root mean square deviation.
ADME profiles, involving the physicochemical, lipophilicity, drug-likeness, and pharmacokinetics properties of substrates are projected with the aim of directing the choice of compounds from a vast collection of prepared compounds in the primary stages of drug detection, identification of biological effects, and the development of active drugs.[42] [43] In terms of drug-likeness, compliance with Lipinski’s Rule of Five and with the Ghose, Veber, Egan, and Muegge rules was assessed. All our prepared compounds were completely included in the pink area (SI; Figure 2–6), following Lipinski’s Rule of Five (with no violations), and they exhibited good lipophilicity, expressed by the consensus log P o/w. (Table [4]). This Rule of Five delineates the relationship between parameters of pharmacokinetics and physicochemical properties. Compounds 4–6 showed appropriate physicochemical properties in terms of six parameters: size, lipophilicity, polarity, unsaturation, insolubility, and flexibility, suggesting they are favorable as oral drug candidates. Bioavailability of the compounds 4–6 is also expected, based on their presence in the pink area of the radar chart.
The compounds show a good bioavailability score (0.55) (Table [4] and SI, Figures S2–S6). The topological polar surface area (TPSA) is a key property linked to drug bioavailability. Thus, passively absorbed molecules with a TPSA > 140 Å2 are thought to have a low bioavailability. All the synthesized compounds have TPSA values < 140 Å2 and are therefore predicted to present good passive oral absorption.[17] [44] Compounds 4–6 exhibited a high GI absorption, whereas compounds 7 and 8 showed low absorption. Compound 7 showed the highest skin permeation (log K P) parameter at –6.91 cm/s, thereby permitting accessibility of of the bioactive molecules through the skin. The cytochrome P450 isoenzymes CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4 play major roles in the biotransformation of drugs through O-type oxidation reactions. All the compounds were predicted to be noninhibitors of CYP2D6 and, therefore, side effects (e.g., liver dysfunction) are not expected upon administration of these compounds. The plasma protein-binding model predicts whether a compound is likely to be highly bound to carrier proteins in the blood. There is a high prospect that the synthesized compounds will bind to plasma proteins.
The BOILED-Egg model provides a graphical representation of a compound’s gastrointestinal (GIT) absorption and brain penetration. Compounds 4–6 demonstrated GIT absorption, as evidenced by their presence in the white area of the BOILED-Egg chart (SI, Figure S7). Furthermore, they should be able to cross the blood–brain barrier, as they appear within the yellow area of the chart. The blue color indicates that compounds 4 and 6 are possible candidates for the permeability glycoprotein (PGP). Compound 5, indicated in red, is expected to be excluded from the central nervous system by the PGP. These compounds revealed a surprisingly high degree of lead-likeness. This analysis provides crucial information for prioritizing compounds and for guiding future optimization efforts in the drug-discovery process.
In summary, a pyrimidinethione with a thiophenylpyrazole core was prepared and used in the design and synthesis of several heteroannulated thiazolopyrimidines through its reactions with ethyl chloroacetate, 1,2-dibromoethane, chloroacetonitrile, and oxalyl chloride. An evaluation of the anticancer activity of these compounds against hepatocellular carcinoma (HepG2) and breast adenocarcinoma (MCF7) cell lines revealed that some of the compounds are interesting antitumor agents. The highest potency compared with doxorubicin was displayed by the dioxothiazolopyrimidine 8 and the oxothiazolopyrimidine 5, whereas pyrimidinethione 4 showed strong effects against the two cell lines. DFT calculations showed that compound 8 has the lowest energy gap and the highest softness. Moreover, it shows the highest electrophilicity index, providing evidence for biological effects. Modeling and pharmacokinetics analysis of these compounds confirmed their desirable drug-likeness and bioavailability properties.
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Conflict of Interest
The authors declare no conflict of interest.
Supporting Information
- Supporting information for this article is available online at https://doi-org.accesdistant.sorbonne-universite.fr/10.1055/a-2456-9620.
- Supporting Information
- CSV File
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References and Notes
- 1 Kaur R, Kaur P, Sharma S, Singh G, Mehndiratta S, Bedi PM. S, Nepali K. Recent Pat. Anti-Cancer Drug Discovery 2015; 10: 23
- 2 El-Zahar MI, Abd El-Karim SS, Haiba ME, Khedr MA. Acta Pol. Pharm. 2011; 68: 357
- 3 Ramadan SK, Abd-Rabboh HS. M, Hafez AA. A, Abou-Elmagd WS. I. RSC Adv. 2024; 14: 16584
- 4 Amina LH. T, Shawera TZ, El-Naggar AM, El-Sehrawi HM. A. Bioorg. Chem. 2019; 91: 103159
- 5 Ramadan SK, El-Helw EA. E, Sallam HA. Heterocycl. Commun. 2019; 25: 107
- 6 Parikh KS, Vyas SP. Pharm. Lett. 2012; 4: 638
- 7 Nasr MN, Gineinah M. Archiv der Pharmazie (Weinheim, Germany) 2002; 335: 195
- 8 Parikh KS, Vyas SP. Int. J. Pharm. Sci. Res. 2012; 3: 3425
- 9 Singh NP, Srivastava AN. Asian J. Chem. 2013; 25: 53
- 10 Halim KN. M, Ramadan SK, Rizk SA, El-Hashash MA. Synth. Commun. 2020; 50: 1159
- 11 El-Helw EA. E, Hosni EM, Kamal M, Hashem AI, Ramadan SK. Bioorg. Chem. 2024; 150: 107591
- 12 Ramadan SK, Abd-Rabboh HS, Gad NM, Abou-Elmagd WS. I, Haneen DS. Polycyclic Aromat. Compd. 2023; 43: 7013
- 13 He H.-Y, Zhao J.-N, Jia R, Zhao Y.-L, Yang S.-Y, Yu L.-T, Yang L. Molecules 2011; 16: 10685
- 14 Rostom SA, Ashour HM. A, Abd El Razik HA. Arch. Pharm. 2009; 342: 299
- 15 Wei L, Malhotra SV. MedChemComm 2012; 3: 1250
- 16 Smyth LA, Collins I. J. Chem. Biol. 2009; 2: 131
- 17 Elgubbi AS, El-Helw EA. E, Alzahrani AY, Ramadan SK. RSC Adv. 2024; 14: 5926
- 18 Kaddah MM, Morsy AR. I, Fahmi AA, Kamel MM, Elsafty MM, Rizk SA, Ramadan SK. Synth. Commun. 2021; 51: 3366
- 19 Morsy AR. I, Ramadan SK, Elsafty MM. Med. Chem. Res. 2020; 29: 979
- 20 Ramadan SK, Abou-Elmagd WS. I. Synth. Commun. 2018; 48: 2409
- 21 Ramadan SK, Ibrahim NA, El-Kaed SA, El-Helw EA. E. J. Sulfur Chem. 2021; 42: 529
- 22 El-Helw EA. E, Alzahrani AY, Ramadan SK. Future Med. Chem. 2024; 16: 439
- 23 Elgubbi AS, El-Helw EA. E, Abousiksaka MS, Alzahrani AY. A, Ramadan SK. RSC Adv. 2024; 14: 18417
- 24 Hassaballah AI, Ramadan SK, Rizk SA, El-Helw EA. E, Abdelwahab SS. Polycyclic Aromat. Compd. 2023; 43: 2973
- 25 Hashem AI, Abou-Elmagd WS. I, El-Ziaty AK, Ramadan SK. J. Heterocycl. Chem. 2017; 54: 3711
- 26 Khatab HA, Hammad SF, El-Fakharany EM, Hashem AI, El-Helw EA. E. Sci. Rep. 2023; 13: 15093
- 27 Abdelrahman AM, Fahmi AA, Rizk SA, El-Helw EA. E. Polycyclic Aromat. Compd. 2023; 43: 721
- 28 El-Helw EA. E, Asran M, Azab ME, Helal MH, Alzahrani AY. A, Ramadan SK. Sci. Rep. 2024; 14: 15522
- 29 Zhang Q, Liu Y, Gao F, Ding Q, Cho C, Hur W, Jin Y, Uno T, Joazeiro CA, Gray N. J. Am. Chem. Soc. 2006; 2182
- 30 Song J, Yoo J, Kwon A, Kim D, Nguyen HK, Lee B.-Y, Suh W, Min KH. PLoS One 2015; 10: e0138823
- 31 El-Naggar AM, Abou-El-Regal MM, El-Metwally SA, Sherbiny FF, Eissa IH. Mol. Diversity 2017; 21: 967
- 32 Kitchens ME, Forsthoefel AM, Barbour KW, Spencer HT, Berger FG. Mol. Pharmacol. 1999; 56: 1063
- 33 Kiyani H, Albooyeh F, Fallahnezhad S. J. Mol. Struct. 2015; 1091: 163
- 34 1-{6-Methyl-4-[1-phenyl-3-(2-thienyl)-1H-pyrazol-4-yl]-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl}ethanone (4) A solution of the pyrazolecarbaldehyde 1 (2 mmol), pentan-2,4-dione (2; 2 mmol), and thiourea (3; 2 mmol) in DMF (10 mL) containing 98% H2SO4 (0.1 mL) was heated in an oil bath at 170–180 °C for 5 h, then allowed to cool to r.t. The mixture was then poured onto ice-cold H2O with stirring, and the resulting solid was collected by filtration, dried, and crystallized from EtOH to afford brown crystals; yield: 71%; mp 215–217 °C. FTIR (ν, cm-1): 3285, 3190 (NH), 1706 (C=O), 1612 (C=N), 1180 (C=S) cm–1. 1H NMR (400 MHz, DMSO-d 6): δ = 2.02 (s, 3 H, CH3CO), 2.28 (s, 3 H, CH3), 5.48 (s, 1 H, C4-H pyrimidine), 7.31–7.53 (m, 5 H, Ar-H), 7.81–7.88 (m, 3 H, Ar-H), 8.32 (s, 1 H, C5-H pyrazole), 9.72 (br s, 1 H, NH, exchangeable), 10.23 (br s, 1 H, NH, exchangeable). 13C NMR (100 MHz, DMSO-d 6): δ = 8.82, 30.66, 46.12, 111.71, 118.91, 124.93, 126.95, 128.46, 128.55, 128.88 (2 C), 128.96, 129.97 (2 C), 133.23, 139.71, 144.41, 150.78, 174.26 (C=O), 195.01 (C=S). MS (EI, 70 eV): m/z (%) = 394.43 (11.50) [M+]. Anal. calcd for C20H18N4OS2 (394.51): C, 60.89; H, 4.60; N, 14.20. Found: C, 60.78; H, 4.52; N, 14.19. 6-Acetyl-7-methyl-5-[1-phenyl-3-(2-thienyl)-1H-pyrazol-4-yl]-5H-[1,3]thiazolo[3,2-a]pyrimidin-3(2H)-one (5) A mixture of pyrimidinethione 4 (2 mmol), ethyl chloroacetate (2 mmol), and fused NaOAc (2 mmol) in absolute EtOH (20 mL) was refluxed for 5 h. The solid that precipitated after cooling was collected and crystallized from EtOH to give brown crystals; yield: 74%; mp 243–245 °C. FTIR (ν, cm-1): 1705, 1690 (C=O) cm–1.1H NMR (400 MHz, DMSO-d 6): δ = 2.15 (s, 3 H, CH3CO), 2.21 (s, 3 H, CH3), 3.70 (s, 2 H, CH2), 6.36 (s, 1 H, C4-H pyrimidine), 7.29–7.61 (m, 5 H, Ar-H), 7.78–8.04 (m, 3 H, Ar-H), 8.77 (s, 1 H, C5-H pyrazole). 13C NMR (100 MHz, DMSO-d 6): δ = 23.76, 31.10, 39.38, 48.15, 118.82, 119.90, 122.51, 123.42, 125.10, 126.34, 128.68 (2), 129.01, 129.17 (2), 129.94, 130.09, 134.20, 138.42, 150.31, 165.50, 176.61. Anal. calcd for C22H18N4O2S2 (434.53): C, 60.81; H, 4.18; N, 12.89. Found: C, 60.72; H, 4.13; N, 12.88. 1-{7-Methyl-5-[1-phenyl-3-(2-thienyl)-1H-pyrazol-4-yl]-2,3-dihydro-5H-[1,3]thiazolo[3,2-a]pyrimidin-6-yl}ethanone (6) A mixture of pyrimidinethione 4 (2 mmol), 1,2-dibromoethane (2 mmol), and anhyd NaOAc (2 mmol) in absolute EtOH (20 mL) was refluxed for 6 h. The solid that precipitated after cooling was collected and crystallized from EtOH to give brown crystals; yield: 68%; mp 224–226 °C. FTIR (KBr): 1682 (C=O), 1625 (C=N) cm–1. 1H NMR (400 MHz, DMSO-d 6): δ = 2.04 (s, 3 H, CH3CO), 2.26 (s, 3 H, CH3), 2.91–3.12 (t, J = 7.1 Hz, 2 H, S-CH2), 3.71–3.92 (t, J = 7.1 Hz, 2 H, N–CH2), 5.37 (s, 1 H, C5-H thiazolopyrimidine), 7.20–8.02 (m, 8 H, Ar-H), 8.41 (s, 1 H, C5-H pyrazole). 13C NMR (100 MHz, DMSO-d 6): δ = 21.2, 24.3, 27.3, 52.3, 65.5, 117.1, 118.8, 122.8, 124.2, 126.3, 127.4 (2), 128.6, 129.2 (2), 129.3, 131.2, 132.9, 139.5, 148.9, 150.7, 171.3. Anal. calcd for C22H20N4OS2 (420.55): C, 62.83; H, 4.79; N, 13.32. Found: C, 62.74; H, 4.73; N, 13.31. 1-{3-Amino-7-methyl-5-[1-phenyl-3-(2-thienyl)-1H-pyrazol-4-yl]-5H-[1,3]thiazolo[3,2-a]pyrimidin-6-yl}ethanone (7) A solution of pyrimidinethione 4 (2 mmol) and ClCH2CN (2 mmol) in DMF (10 mL) was refluxed for 4 h. The mixture was allowed to cool to r.t. and then poured onto ice-cold H2O. The precipitated solid was collected and crystallized from EtOH to afford brown crystals; yield: 62%; mp 245–247 °C. IR (ν, cm-1): 3340, 3300 (NH2), 1665 (C=O), 1628 (C=N) cm–1. 1H NMR (400 MHz, DMSO-d 6): δ = 2.74 (s, 3 H, CH3CO), 2.89 (s, 3 H, CH3), 4.94 (s, 1 H, C4-H pyrimidine), 5.02 (s, 1 H, C5–H thiazolo), 7.13 (br s, 2 H, NH2, exchangeable), 7.16–7.55 (m, 5 H, Ar-H), 7.75–8.74 (m, 3 H, Ar-H), 9.23 (s, 1 H, C5-H pyrazole). 13C NMR (100 MHz, DMSO-d 6): δ = 31.2, 36.2, 40.6, 119.1, 124.3, 126.4, 127.4 (2), 128.4, 128.8, 128.9 (2), 129.3, 129.9, 130.1, 131.3, 133.2, 135.4, 149.3, 150.5, 162.7. Anal. calcd for C22H19N5OS2 (433.55): C, 60.95; H, 4.42; N, 16.15. Found: C, 60.81; H, 4.35; N, 16.12. 6-Acetyl-7-methyl-5-[1-phenyl-3-(2-thienyl)-1H-pyrazol-4-yl]-5H-[1,3]thiazolo[3,2-a]pyrimidine-2,3-dione (8) A solution of pyrimidinethione 4 (2 mmol) and oxalyl chloride (2 mmol) in anhyd benzene (20 mL) was refluxed for 6 h in the presence of EtN3 (0.5 mL). The solid that formed on cooling was collected and crystallized from benzene to give pale-brown crystals; yield: 69%; mp 254–256 °C. FTIR (ν, cm-1): 1703, 1673 (C=O), 1655 (C=N) cm–1. 1H NMR (400 MHz, DMSO-d 6): δ = 2.02 (s, 3 H, CH3CO), 2.37 (s, 3 H, CH3), 5.47 (s, 1 H, C5-H thiazolopyrimidine), 7.29–7.58 (m, 5 H, Ar-H), 7.72–8.02 (m, 3 H, Ar-H), 8.31 (s, 1 H, C5-H pyrazole). 13C NMR (100 MHz, DMSO): δ = 1.5, 27.1, 66.8, 117.4, 119.7, 123.1, 126.1, 127.3 (2), 128.8, 129.1, 129.2 (2), 131.8, 133.1, 139.4, 149.7, 151.8, 153.1, 164.2, 171.1, 175.3. MS (EI, 70 eV): m/z (%) = 448.15 (36.70) [M+]. Anal. calcd for C22H16N4O3S2 (448.52): C, 58.91; H, 3.60; N, 12.49. Found: C, 58.82; H, 3.55; N, 12.51.
- 35 Elkholy AE, Rizk SA, Rashad AM. J. Mol. Struct. 2019; 1175: 788
- 36 El-Sewedy A, El-Bordany EA, Mahmoud NF. H, Ali KA, Ramadan SK. Sci. Rep. 2023; 13: 17869
- 37 Mosmann T. J. Immunol Methods 1983; 65: 55
- 38
Cytotoxicity Assay
Cell lines: Hepatocellular carcinoma (HepG2) and mammary gland breast cancer (MCF7) cell lines were obtained from ATCC through a holding company for biological products and vaccines (VACSERA, Cairo, Egypt). Chemical reagents: The reagents used were RPMI-1640 medium, MTT, DMSO, doxorubicin (Sigma Co., St. Louis, USA), and fetal bovine serum (FBS; GIBCO, Paisley, UK). Doxorubicin was used as a standard anticancer drug for comparison. MTT assay: The inhibitory effects of the prepared compounds on the growth of the various cell lines were measured by an MTT assay. Cell lines were cultured in RPMI-1640 medium with 10% FBS containing 100 units/mL penicillin and 100 μg/mL streptomycin at 37 °C in a 5% CO2 incubator. The cell lines were seeded into a 96-well plate at a density of 1.0 × 104 cells/well at 37 °C for 48 h under 5% CO2. After incubation, the cells were treated with various concentrations of the compounds and incubated for 24 h. After 24 h of drug treatment, MTT solution (20 μL) at 5 mg/mL was added and the mixture was incubated for a further 4 h. DMSO (100 μL) was added to each well to dissolve the purple formazan that formed. A colorimetric assay at λ = 570 nm was performed by using a plate reader (EXL 800, Bio-Tech, Winoosky, VT, USA). The relative cell viability as a percentage was calculated by using the expression (A570 treated sample/A570 untreated sample) × 100. - 39 Ramadan SK, Rizk SA. J. Iran. Chem. Soc. 2022; 19: 187
- 40 Kumar M, Nagpal R, Hemalatha R, Verma V, Kumar A, Singh S, Marotta F, Jain S, Yadav H. Acta Bio Med. Atenei Parmensis 2012; 83: 220
- 41 https://doi-org.accesdistant.sorbonne-universite.fr/10.2210/pdb1JU6/pdbb (accessed Nov 12, 2024).
- 42 Daina A, Michielin O, Zoete V. Sci. Rep. 2017; 7: 42717
- 43 Lipinski CA. Drug Discovery Today: Technol. 2004; 1: 337
- 44 Morsy AR. I, Mahmoud SH, Abou Sharma NM, Arafa W, Yousef GA, Khalil AA, Ramadan SK. RSC Adv. 2024; 14: 27935
Corresponding Author
Publication History
Received: 07 August 2024
Accepted after revision: 29 October 2024
Accepted Manuscript online:
29 October 2024
Article published online:
04 December 2024
© 2024. Thieme. All rights reserved
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References and Notes
- 1 Kaur R, Kaur P, Sharma S, Singh G, Mehndiratta S, Bedi PM. S, Nepali K. Recent Pat. Anti-Cancer Drug Discovery 2015; 10: 23
- 2 El-Zahar MI, Abd El-Karim SS, Haiba ME, Khedr MA. Acta Pol. Pharm. 2011; 68: 357
- 3 Ramadan SK, Abd-Rabboh HS. M, Hafez AA. A, Abou-Elmagd WS. I. RSC Adv. 2024; 14: 16584
- 4 Amina LH. T, Shawera TZ, El-Naggar AM, El-Sehrawi HM. A. Bioorg. Chem. 2019; 91: 103159
- 5 Ramadan SK, El-Helw EA. E, Sallam HA. Heterocycl. Commun. 2019; 25: 107
- 6 Parikh KS, Vyas SP. Pharm. Lett. 2012; 4: 638
- 7 Nasr MN, Gineinah M. Archiv der Pharmazie (Weinheim, Germany) 2002; 335: 195
- 8 Parikh KS, Vyas SP. Int. J. Pharm. Sci. Res. 2012; 3: 3425
- 9 Singh NP, Srivastava AN. Asian J. Chem. 2013; 25: 53
- 10 Halim KN. M, Ramadan SK, Rizk SA, El-Hashash MA. Synth. Commun. 2020; 50: 1159
- 11 El-Helw EA. E, Hosni EM, Kamal M, Hashem AI, Ramadan SK. Bioorg. Chem. 2024; 150: 107591
- 12 Ramadan SK, Abd-Rabboh HS, Gad NM, Abou-Elmagd WS. I, Haneen DS. Polycyclic Aromat. Compd. 2023; 43: 7013
- 13 He H.-Y, Zhao J.-N, Jia R, Zhao Y.-L, Yang S.-Y, Yu L.-T, Yang L. Molecules 2011; 16: 10685
- 14 Rostom SA, Ashour HM. A, Abd El Razik HA. Arch. Pharm. 2009; 342: 299
- 15 Wei L, Malhotra SV. MedChemComm 2012; 3: 1250
- 16 Smyth LA, Collins I. J. Chem. Biol. 2009; 2: 131
- 17 Elgubbi AS, El-Helw EA. E, Alzahrani AY, Ramadan SK. RSC Adv. 2024; 14: 5926
- 18 Kaddah MM, Morsy AR. I, Fahmi AA, Kamel MM, Elsafty MM, Rizk SA, Ramadan SK. Synth. Commun. 2021; 51: 3366
- 19 Morsy AR. I, Ramadan SK, Elsafty MM. Med. Chem. Res. 2020; 29: 979
- 20 Ramadan SK, Abou-Elmagd WS. I. Synth. Commun. 2018; 48: 2409
- 21 Ramadan SK, Ibrahim NA, El-Kaed SA, El-Helw EA. E. J. Sulfur Chem. 2021; 42: 529
- 22 El-Helw EA. E, Alzahrani AY, Ramadan SK. Future Med. Chem. 2024; 16: 439
- 23 Elgubbi AS, El-Helw EA. E, Abousiksaka MS, Alzahrani AY. A, Ramadan SK. RSC Adv. 2024; 14: 18417
- 24 Hassaballah AI, Ramadan SK, Rizk SA, El-Helw EA. E, Abdelwahab SS. Polycyclic Aromat. Compd. 2023; 43: 2973
- 25 Hashem AI, Abou-Elmagd WS. I, El-Ziaty AK, Ramadan SK. J. Heterocycl. Chem. 2017; 54: 3711
- 26 Khatab HA, Hammad SF, El-Fakharany EM, Hashem AI, El-Helw EA. E. Sci. Rep. 2023; 13: 15093
- 27 Abdelrahman AM, Fahmi AA, Rizk SA, El-Helw EA. E. Polycyclic Aromat. Compd. 2023; 43: 721
- 28 El-Helw EA. E, Asran M, Azab ME, Helal MH, Alzahrani AY. A, Ramadan SK. Sci. Rep. 2024; 14: 15522
- 29 Zhang Q, Liu Y, Gao F, Ding Q, Cho C, Hur W, Jin Y, Uno T, Joazeiro CA, Gray N. J. Am. Chem. Soc. 2006; 2182
- 30 Song J, Yoo J, Kwon A, Kim D, Nguyen HK, Lee B.-Y, Suh W, Min KH. PLoS One 2015; 10: e0138823
- 31 El-Naggar AM, Abou-El-Regal MM, El-Metwally SA, Sherbiny FF, Eissa IH. Mol. Diversity 2017; 21: 967
- 32 Kitchens ME, Forsthoefel AM, Barbour KW, Spencer HT, Berger FG. Mol. Pharmacol. 1999; 56: 1063
- 33 Kiyani H, Albooyeh F, Fallahnezhad S. J. Mol. Struct. 2015; 1091: 163
- 34 1-{6-Methyl-4-[1-phenyl-3-(2-thienyl)-1H-pyrazol-4-yl]-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl}ethanone (4) A solution of the pyrazolecarbaldehyde 1 (2 mmol), pentan-2,4-dione (2; 2 mmol), and thiourea (3; 2 mmol) in DMF (10 mL) containing 98% H2SO4 (0.1 mL) was heated in an oil bath at 170–180 °C for 5 h, then allowed to cool to r.t. The mixture was then poured onto ice-cold H2O with stirring, and the resulting solid was collected by filtration, dried, and crystallized from EtOH to afford brown crystals; yield: 71%; mp 215–217 °C. FTIR (ν, cm-1): 3285, 3190 (NH), 1706 (C=O), 1612 (C=N), 1180 (C=S) cm–1. 1H NMR (400 MHz, DMSO-d 6): δ = 2.02 (s, 3 H, CH3CO), 2.28 (s, 3 H, CH3), 5.48 (s, 1 H, C4-H pyrimidine), 7.31–7.53 (m, 5 H, Ar-H), 7.81–7.88 (m, 3 H, Ar-H), 8.32 (s, 1 H, C5-H pyrazole), 9.72 (br s, 1 H, NH, exchangeable), 10.23 (br s, 1 H, NH, exchangeable). 13C NMR (100 MHz, DMSO-d 6): δ = 8.82, 30.66, 46.12, 111.71, 118.91, 124.93, 126.95, 128.46, 128.55, 128.88 (2 C), 128.96, 129.97 (2 C), 133.23, 139.71, 144.41, 150.78, 174.26 (C=O), 195.01 (C=S). MS (EI, 70 eV): m/z (%) = 394.43 (11.50) [M+]. Anal. calcd for C20H18N4OS2 (394.51): C, 60.89; H, 4.60; N, 14.20. Found: C, 60.78; H, 4.52; N, 14.19. 6-Acetyl-7-methyl-5-[1-phenyl-3-(2-thienyl)-1H-pyrazol-4-yl]-5H-[1,3]thiazolo[3,2-a]pyrimidin-3(2H)-one (5) A mixture of pyrimidinethione 4 (2 mmol), ethyl chloroacetate (2 mmol), and fused NaOAc (2 mmol) in absolute EtOH (20 mL) was refluxed for 5 h. The solid that precipitated after cooling was collected and crystallized from EtOH to give brown crystals; yield: 74%; mp 243–245 °C. FTIR (ν, cm-1): 1705, 1690 (C=O) cm–1.1H NMR (400 MHz, DMSO-d 6): δ = 2.15 (s, 3 H, CH3CO), 2.21 (s, 3 H, CH3), 3.70 (s, 2 H, CH2), 6.36 (s, 1 H, C4-H pyrimidine), 7.29–7.61 (m, 5 H, Ar-H), 7.78–8.04 (m, 3 H, Ar-H), 8.77 (s, 1 H, C5-H pyrazole). 13C NMR (100 MHz, DMSO-d 6): δ = 23.76, 31.10, 39.38, 48.15, 118.82, 119.90, 122.51, 123.42, 125.10, 126.34, 128.68 (2), 129.01, 129.17 (2), 129.94, 130.09, 134.20, 138.42, 150.31, 165.50, 176.61. Anal. calcd for C22H18N4O2S2 (434.53): C, 60.81; H, 4.18; N, 12.89. Found: C, 60.72; H, 4.13; N, 12.88. 1-{7-Methyl-5-[1-phenyl-3-(2-thienyl)-1H-pyrazol-4-yl]-2,3-dihydro-5H-[1,3]thiazolo[3,2-a]pyrimidin-6-yl}ethanone (6) A mixture of pyrimidinethione 4 (2 mmol), 1,2-dibromoethane (2 mmol), and anhyd NaOAc (2 mmol) in absolute EtOH (20 mL) was refluxed for 6 h. The solid that precipitated after cooling was collected and crystallized from EtOH to give brown crystals; yield: 68%; mp 224–226 °C. FTIR (KBr): 1682 (C=O), 1625 (C=N) cm–1. 1H NMR (400 MHz, DMSO-d 6): δ = 2.04 (s, 3 H, CH3CO), 2.26 (s, 3 H, CH3), 2.91–3.12 (t, J = 7.1 Hz, 2 H, S-CH2), 3.71–3.92 (t, J = 7.1 Hz, 2 H, N–CH2), 5.37 (s, 1 H, C5-H thiazolopyrimidine), 7.20–8.02 (m, 8 H, Ar-H), 8.41 (s, 1 H, C5-H pyrazole). 13C NMR (100 MHz, DMSO-d 6): δ = 21.2, 24.3, 27.3, 52.3, 65.5, 117.1, 118.8, 122.8, 124.2, 126.3, 127.4 (2), 128.6, 129.2 (2), 129.3, 131.2, 132.9, 139.5, 148.9, 150.7, 171.3. Anal. calcd for C22H20N4OS2 (420.55): C, 62.83; H, 4.79; N, 13.32. Found: C, 62.74; H, 4.73; N, 13.31. 1-{3-Amino-7-methyl-5-[1-phenyl-3-(2-thienyl)-1H-pyrazol-4-yl]-5H-[1,3]thiazolo[3,2-a]pyrimidin-6-yl}ethanone (7) A solution of pyrimidinethione 4 (2 mmol) and ClCH2CN (2 mmol) in DMF (10 mL) was refluxed for 4 h. The mixture was allowed to cool to r.t. and then poured onto ice-cold H2O. The precipitated solid was collected and crystallized from EtOH to afford brown crystals; yield: 62%; mp 245–247 °C. IR (ν, cm-1): 3340, 3300 (NH2), 1665 (C=O), 1628 (C=N) cm–1. 1H NMR (400 MHz, DMSO-d 6): δ = 2.74 (s, 3 H, CH3CO), 2.89 (s, 3 H, CH3), 4.94 (s, 1 H, C4-H pyrimidine), 5.02 (s, 1 H, C5–H thiazolo), 7.13 (br s, 2 H, NH2, exchangeable), 7.16–7.55 (m, 5 H, Ar-H), 7.75–8.74 (m, 3 H, Ar-H), 9.23 (s, 1 H, C5-H pyrazole). 13C NMR (100 MHz, DMSO-d 6): δ = 31.2, 36.2, 40.6, 119.1, 124.3, 126.4, 127.4 (2), 128.4, 128.8, 128.9 (2), 129.3, 129.9, 130.1, 131.3, 133.2, 135.4, 149.3, 150.5, 162.7. Anal. calcd for C22H19N5OS2 (433.55): C, 60.95; H, 4.42; N, 16.15. Found: C, 60.81; H, 4.35; N, 16.12. 6-Acetyl-7-methyl-5-[1-phenyl-3-(2-thienyl)-1H-pyrazol-4-yl]-5H-[1,3]thiazolo[3,2-a]pyrimidine-2,3-dione (8) A solution of pyrimidinethione 4 (2 mmol) and oxalyl chloride (2 mmol) in anhyd benzene (20 mL) was refluxed for 6 h in the presence of EtN3 (0.5 mL). The solid that formed on cooling was collected and crystallized from benzene to give pale-brown crystals; yield: 69%; mp 254–256 °C. FTIR (ν, cm-1): 1703, 1673 (C=O), 1655 (C=N) cm–1. 1H NMR (400 MHz, DMSO-d 6): δ = 2.02 (s, 3 H, CH3CO), 2.37 (s, 3 H, CH3), 5.47 (s, 1 H, C5-H thiazolopyrimidine), 7.29–7.58 (m, 5 H, Ar-H), 7.72–8.02 (m, 3 H, Ar-H), 8.31 (s, 1 H, C5-H pyrazole). 13C NMR (100 MHz, DMSO): δ = 1.5, 27.1, 66.8, 117.4, 119.7, 123.1, 126.1, 127.3 (2), 128.8, 129.1, 129.2 (2), 131.8, 133.1, 139.4, 149.7, 151.8, 153.1, 164.2, 171.1, 175.3. MS (EI, 70 eV): m/z (%) = 448.15 (36.70) [M+]. Anal. calcd for C22H16N4O3S2 (448.52): C, 58.91; H, 3.60; N, 12.49. Found: C, 58.82; H, 3.55; N, 12.51.
- 35 Elkholy AE, Rizk SA, Rashad AM. J. Mol. Struct. 2019; 1175: 788
- 36 El-Sewedy A, El-Bordany EA, Mahmoud NF. H, Ali KA, Ramadan SK. Sci. Rep. 2023; 13: 17869
- 37 Mosmann T. J. Immunol Methods 1983; 65: 55
- 38
Cytotoxicity Assay
Cell lines: Hepatocellular carcinoma (HepG2) and mammary gland breast cancer (MCF7) cell lines were obtained from ATCC through a holding company for biological products and vaccines (VACSERA, Cairo, Egypt). Chemical reagents: The reagents used were RPMI-1640 medium, MTT, DMSO, doxorubicin (Sigma Co., St. Louis, USA), and fetal bovine serum (FBS; GIBCO, Paisley, UK). Doxorubicin was used as a standard anticancer drug for comparison. MTT assay: The inhibitory effects of the prepared compounds on the growth of the various cell lines were measured by an MTT assay. Cell lines were cultured in RPMI-1640 medium with 10% FBS containing 100 units/mL penicillin and 100 μg/mL streptomycin at 37 °C in a 5% CO2 incubator. The cell lines were seeded into a 96-well plate at a density of 1.0 × 104 cells/well at 37 °C for 48 h under 5% CO2. After incubation, the cells were treated with various concentrations of the compounds and incubated for 24 h. After 24 h of drug treatment, MTT solution (20 μL) at 5 mg/mL was added and the mixture was incubated for a further 4 h. DMSO (100 μL) was added to each well to dissolve the purple formazan that formed. A colorimetric assay at λ = 570 nm was performed by using a plate reader (EXL 800, Bio-Tech, Winoosky, VT, USA). The relative cell viability as a percentage was calculated by using the expression (A570 treated sample/A570 untreated sample) × 100. - 39 Ramadan SK, Rizk SA. J. Iran. Chem. Soc. 2022; 19: 187
- 40 Kumar M, Nagpal R, Hemalatha R, Verma V, Kumar A, Singh S, Marotta F, Jain S, Yadav H. Acta Bio Med. Atenei Parmensis 2012; 83: 220
- 41 https://doi-org.accesdistant.sorbonne-universite.fr/10.2210/pdb1JU6/pdbb (accessed Nov 12, 2024).
- 42 Daina A, Michielin O, Zoete V. Sci. Rep. 2017; 7: 42717
- 43 Lipinski CA. Drug Discovery Today: Technol. 2004; 1: 337
- 44 Morsy AR. I, Mahmoud SH, Abou Sharma NM, Arafa W, Yousef GA, Khalil AA, Ramadan SK. RSC Adv. 2024; 14: 27935
