Abstract
This study describes the design and synthesis of a novel series of triazole-aryloxyacetyl hydrazide hybrids with furo[2,3-d]pyrimidine scaffold. The structure elucidations were performed using 1 H NMR, 13 C NMR and high-resolution mass spectrometry (HR-MS). The in vitro antitumor activities evaluation indicated that several derivatives exhibited considerable inhibiting effect against HepG2 cell lines, with IC50 values ranging from 4.82 to 69.83 µmol/L for compounds 5a–5l, 18.37 to > 100 µmol/L for compounds 6a–6j, 11.21 and 6.07 µmol/L for compounds 7a and 7b, respectively. Among them, compounds 5d and 5e appeared the most potent activity, along with notably lower cytotoxicity against the normal human liver cell line and the selectivity indices (SIs) were calculated to be 3.36 and 3.02, respectively, underscoring their antitumor potential. Furthermore, compound 5d not only induced concentration-dependent apoptosis but also effectively suppressed HepG2 cell migration. Following molecular docking studies demonstrated that compound 5d interacts with EGFR in a manner similar to gefitinib, suggesting its potential as an EGFR inhibitor. Preliminary comparative structure-activity relationship revealed that the bicyclic furo[2,3-d]pyrimidine core bearing a C-4 aryloxyacetyl hydrazide moiety was crucial for high activity, outperforming the tricyclic triazole-containing analogs (6a–6j). In addition, the introduction of halogen substituents (F, Cl) at the R1 position enhanced potency, as evidenced by compounds 5d, 5e, and 7b. Conversely, the tricyclic series (6a–6j) showed diminished activity. A comparison with the potent compounds 7a and 7b suggests that this may be due to unfavorable steric effects. This insight provides a direction for subsequent molecular optimization efforts.
Supplementary Information
The online version contains supplementary material available at 10.1186/s13065-026-01719-y.
Keywords: Furo[2,3-d]pyrimidine; Aza-wittig reaction; Aryloxyacetyl hydrazide; Triazole; Antitumor
Introduction
Despite considerable progress in cancer therapy, the development of novel antitumor agent remains imperative to combat multidrug resistance, reduce off-target toxicity, and improve outcomes for patients with advanced or treatment-resistant cancers. The triazole scaffold represents a privileged structure component in medicinal chemistry, owing to its favorable pharmacokinetic properties, which include metabolic stability, capacity for hydrogen bonding, and a strong dipole moment [1, 2], with diverse biological activities, including antimalarial [3], anticancer [4, 5], acetylcholinesterase (AChE) inhibitory [6], and antimicrobial effects [7]. Several studies demonstrated that the integration of triazole motifs into hybrid molecular architectures often results in enhanced pharmacological profiles through synergistic mechanisms [8, 9]. Recent studies have further highlighted 1,2,4-triazole derivatives (Fig. 1) as selective inhibitors targeting multiple kinases, including WRN, tubulin polymerization, monocarboxylate transporter 4, VEGFR-2, TB-InhA, ASK1, and PDK1, underscoring their considerable potential as antitumor agents [10–13].
Fig. 1.
Chemical structures of some representative of kinase inhibitors containing 1,2,4-triazole motifs
Over the past decade, our research efforts have been dedicated to the application of the aza-Wittig reaction, focusing on developing efficient synthetic routes for novel fused pyrimidine heterocyclic compounds, aiming to discover innovative antitumor agents [14, 15]. While conventional strategies for modifying the fused pyrimidine core involve substitution at either the C-2 or C-4 position [16–18], and previously reported 2,4-diamino furo[2,3-d]pyrimidine derivatives as lead compounds with promising antitumor activity (IC50 = 5.5 µmol/L) [19]. Furthermore, the molecular hybridization of two or more pharmacologically active substructures represents a powerful strategy in medicinal chemistry for drug discovery. The hydrazide functional group is recognized as a privileged pharmacophore, whose amphipathic properties facilitate cell membrane penetration, contributing to its versatility and remarkable biological activities. Subsequently, the splicing principle was applied to introduce ibuprofen into the furo[2,3-d]pyrimidine skeleton, which achieved the synthesis of furo[2,3-d]pyrimidine-based ibuprofen hydrazide as a potent antitumor agent (IC50 = 1.23 µmol/L) [20]. Aryloxyacetyl hydrazide and ibuprofen hydrazide, as isosteres, exhibit similar electronic properties to serve as hydrogen bond donors/acceptors. In addition, aryloxyacetic group and their derivatives often possess many important chemical properties and biological activities [21, 22]. Herein, we further report an efficient protocol for the preparation of novel triazole-aryloxyacetyl hydrazide hybrids with furo[2,3-d]pyrimidine scaffold as potential anti-tumor agents (Fig. 2).
Fig. 2.
Designing strategy for triazole-aryloxyacetyl hydrazide hybrids with furo[2,3-d]pyrimidine scaffold
Results and discussion
Chemistry
Building upon previous work, we optimized the synthesis of key intermediate 3. Specifically, using CNCH2CO2C2H5 and CH3COCHClCO2C2H5 as starting materials, compound 1 was prepared under the catalysis of NEt3 at room temperature, achieving satisfactory yields. The aza-Wittig reaction is a primary method for synthesizing target compounds, which involve reaction of the products of compound 1 with PPh3 and C2Cl6, and then treating with aromatic isocynates and ammonium hydroxide, affording compound 2. Following, phosphorus oxychloride as a chlorination reagent, 2 converted the functionalized key intermediate 3. Various aryloxyaceatyl hydrazides 4 were efficiently synthesis in a two-step procedure, namely, diverse phenols were alkylated with ethyl bromoacetate under alkaline conditions, and then, the resulting esters were reacted with hydrazine hydrate. At 75–80 °C, treatment of compound 3 with 4 provided compounds 5a–5l in acetonitrile. Subsequently, compounds 5a–5l underwent cyclization in the presence of phosphorus oxychloride, yielding the target compounds 6a–6l (Table 1). To investigate the steric effects of aryloxy methyl groups on the fused tricyclic ring, compounds 7a and 7b were synthesized using the same methodology. The overall synthesis route is outlined in Scheme 1.
Table 1.
Preparation of the compounds 5a–5, 6a–6l, 7a and 7b
| ||||||
|---|---|---|---|---|---|---|
| Enter | Compd. | R 1 | R 2 | IC50 (µmol/L, HepG2) | IC50 (µmol/L, THLE-2) | HepG2, SIa |
| 1 | 5a | m-Methyl | p-Methyl | 54.28 | – | |
| 2 | 5b | p-Methyl | p-Methyl | 28.57 | – | |
| 3 | 5c | p-Methyl | p-Methoxy | 65.16 | – | |
| 4 | 5d | p-Chloro | o-Methyl | 4.82 | 16.20 | 3.36 |
| 5 | 5e | p-Fluoro | p-Methyl | 5.26 | 15.89 | 3.02 |
| 6 | 5f | p-Methyl | p-Methoxy | 64.97 | – | |
| 7 | 5g | 3,5-Dimethyl | p-Methyl | 69.63 | – | |
| 8 | 5h | p-Chloro | p-Methyl | 20.22 | – | |
| 9 | 5i | m-Methyl | m-Methyl | 31.72 | – | |
| 10 | 5j | p-Fluoro | m-Methyl | 20.85 | – | |
| 11 | 5k | 3,5-Dimethyl | o-Chloro | 36.74 | – | |
| 12 | 5l | p-Chloro | p-Methoxy | 11.64 | – | |
| 13 | 6a | p-Methyl | p-Methoxy | 38.91 | – | |
| 14 | 6b | p-Methyl | m-Methyl | 50.78 | – | |
| 15 | 6c | p-Methyl | o-Methyl | 46.15 | – | |
| 16 | 6d | p-Methyl | p-Methyl | 56.28 | – | |
| 17 | 6e | m-Methyl | m-Methyl | 18.37 | – | |
| 18 | 6f | p-Chloro | p-Methyl | 25.52 | – | |
| 19 | 6g | m-Methyl | p-Methoxy | 23.66 | – | |
| 20 | 6h | p-Fluoro | o-Methyl | > 100 | – | |
| 21 | 6i | p-Fluoro | p-Methoxy | > 100 | – | |
| 22 | 6j | m-Methyl | p-Methyl | 44.90 | – | |
| 23 | 7a | p-Methoxy | 11.21 | – | ||
| 24 | 7b | p-Fluoro | 6.07 | – | ||
| Control | Gefitinib | 11.26 | – | |||
“_” indicate no test
aThe in vitro HepG2 selectivity index (THLE-2/HepG2)
Scheme 1.
The synthetic route of target compounds 5a–5l, 6a–6l and 7. Reagents and reaction condition: a Triethylamine, C2H5OH, at room temperature; b) (1) PPh3, C2Cl6 and triethylamine, acetonitrile; (2) aromatic isocyanates, CH2Cl2; (3) ammonium hydroxide, C2H5OH; c) POCl3, 90 °C. d) BrCH2CO2C2H5, K2CO3, C2H5OH, 60–65 °C. e) Hydrazine ydrate, C2H5OH, 60–65 °C. f K2CO3, acetonitrile, 75–80 °C. g POCl3, 80 °C
The structure of 5a–5l, 6a–6l and 7a, 7b were elucidated by 1 H NMR, 13 C NMR and HRMS spectrum data. The ¹H NMR spectrum showed distinct signals corresponding to the NH protons and after the addition of a few drops of D2O and shaking, the spectrum was re-collected, revealing that the intensities of these signals were significantly reduced or had vanished entirely (e.g. 5c, Fig. 3), consistent with the behavior of exchangeable protons. The chemical shifts for all other protons and 13C NMR spectra of 5a–5l, 6a–6l and 7a, 7b are in full agreement with the expected molecular structure. The calculated M+H (Na) ion peak matched with that of the observed M+H (Na) ion peak, which confirmed the structure.
Fig. 3.
1 H NMR spectra of compound 5c before and after D2O exchange in DMSO-d6
In vitro antitumor activities and structure activity relationship
Using gefitinib as a positive control, the antitumor activities of the synthesized compounds 5a–5l, 6a–6j and 7a–7b against HepG2 cancer cell lines were evaluated via the CCK8 assay. As shown in Table 1, these compounds demonstrated potential antitumor activity. Among them, compounds 5d and 5e exhibited superior inhibitory activities (IC50 = 4.82 and 5.26 µmol/L, respectively) compared to other tested compounds and the reference drug gefitinib. Notably, compounds 5d and 5e demonstrated minimal toxicity toward normal human liver cells (THLE-2), with IC50 values of 16.20 µmol/L and 15.89 µmol/L, respectively. The selectivity indices (SIs), as the ratio of the IC50 against the normal cell line to the IC50 against the cancer cell line, for compounds 5d and 5e were calculated to be 3.36 and 3.02, respectively, underscoring their antitumor potential.
Preliminary comparative analysis revealed that bicyclic compounds 5a–5l (IC50 = 4.82–69.63 µmol/L) exhibited better antitumor activity in comparison with tricyclic compounds 6a–6j (IC50 = 18.37 to > 100 µmol/L), and for compound 5a–5l, monosubstitution on the benzene ring at 2-position of the pyrimidine ring is more beneficial to activity than a double substitution (e.g.: 5g and 5k), for 6a–6j was not significantly different, and comparing 6a–6j (IC50 = 18.37 to > 100 µmol/L) and 7a, 7b (IC50 = 11.21 µmol/L and 6.07µmol/L, respectively) shows that steric interactions on the fused triazole ring plays a significant role. Furthermore, further comparison showed that introducing halogen atoms (F, Cl) at the R1 position for bicyclic derivatives 5a–5l, as in compounds 5d, 5e, 5h, 5j, 5l and 7b, positively influenced activity, indicating a promising avenue for drug design modification aimed at developing more potent antitumor agents.
Cell apoptosis induced and cell scratch assay
Given the potent anti-proliferative activity of compound 5d against HepG2 cells, we sought to determine whether its mechanism involved the induction of apoptosis. Therefore, the pro-apoptotic effect of 5d was evaluated in HepG2 cells using a concentration gradient. As quantified in Figs. 4A and 5d treatment induced a marked and dose-dependent increase in apoptotic HepG2 cells. The total apoptotic population in the control group was only 4.02%. In contrast, treatment with 5 µmol/L, 10µmol/L, and 15µmol/L 5d for 72 h resulted in apoptosis rates of 13.79%, 42.11% and 69.23%, respectively.
Fig. 4.
A Effect of compound 5d on apoptosis in HepG2 cells and the representative images of HepG2 cells treated with compound 5d at concentrations of 0, 5, 10, 15µmol/L for 72 h, stained by Annexin V-FITC/PI, and analyzed using flow cytometry. B Effect of compound 5d on HepG2 cells scratch assay. Representative images of HepG2 cells were treated with compound 5d at concentrations of 0, 5, 10, 15µmol/L for 24 and 48 h, respectively, and then photographed by microscopic observation
Fig. 5.
According to prior reports, docking results of 5d (orange) and gefitinib (orange) with EGFR. A, B and C, D are the 3D and 2D docking pose of 5d and gefitinib, respectively, showing similar interaction of some active 5d and gefitinib with EGFR (PDB: 1 M17), respectively
To further confirm the antitumor activity of the compound 5d, cell scratch assay was used to detect the inhibitory effect of compound 5d against HepG2 migration. HepG2 cells were treated with a vehicle control (DMSO), 5 µmol/L, 10µmol/L, and 15µmol/L of 5d immediately after scratching for 24 and 48 h. As shown in Fig. 4B, analysis of the relative wound width revealed that after 24 h, the control group had closed approximately 53.55% of the initial wound. In contrast, the groups treated with 5, 10, and 15µmol/L of 5d achieved only 47.13%, 43.17%, and 36.28% closure, respectively. For 48 h, the control group had closed approximately 79.20% of the initial wound. In contrast, the groups treated with 5, 10, and 15µmol/L of 5d achieved only 52.15%, 50.19%, and 43.87% closure, respectively. These results clearly indicate that 5d effectively inhibits the migration of HepG2 cells in vitro.
Molecular docking
Recently, molecular docking methods have been successfully used to illuminate the mode of action of small molecule inhibitors and their high selectivity between protein kinases. To rationalize the exceptional effectiveness of compound 5d against HepG2 cells, molecular docking comparative studies were completed for 5d and reference drugs gefitinib. According to prior reports [18], compound 5d and reference drugs gefitinib exhibits a similar binding mode with EGFR, occupying the active cavity and forming hydrogen bonds with EGFR hinge residues. As shown in Fig. 5, the results display that the fused furo[2,3-d]pyrimidine moiety of 5d inserts into the ATP-binding pocket of EGFR and forms a hydrogen bond interaction with the hinge region amino acid M769. Meanwhile, the p-Cl-phenyl ring establishes hydrophobic interactions with the side chain of L694, while the o-methyl-phenyl ring extends out of the cavity and forms hydrophobic interactions with the side chain benzene ring of F699 to elucidate its potential mechanism of antitumor action.
Conclusions
In this work, 24 novel triazole and aryloxyacetyl hydrazide derivatives with furo[2,3-d]pyrimidine moiety were synthesized in high yield via a convenient method starting from easily accessible materials. The all synthesized compounds were evaluated for in vitro antitumor activity against HepG2 cells. Among them, 5d and 5e demonstrated the most potent antitumor activity and minimal toxicity toward normal human liver cells (THLE-2), with the selectivity indices (SIs) of 3.36 and 3.02, respectively, underscoring their antitumor potential. Furthermore, concentration-dependent induction of apoptosis and suppression of cell migration were observed in response to compound 5d. Molecular docking results further theoretically demonstrate that compounds 5d have potential inhibitory effects on EGFR. Preliminary structure-activity relationship analysis revealed that bicyclic compounds 5a–5l exhibited better activity in comparison with tricyclic compounds 6a–6j. Meanwhile, for the bicyclic series 5a–5l, monosubstitution at the 2-position of the benzene ring on the pyrimidine moiety appears to be beneficial for activity, as exemplified by compounds 5g and 5k. In contrast, variations within the tricyclic series 6a–6j did not lead to significant differences in potency. However, a comparison between compounds 6a–6j and 7a/7b reveals that steric interactions around the fused triazole ring play a significant role in modulating activity. In addition, the analysis also showed that introducing halogen atoms (F, Cl) at the R1 position for bicyclic derivatives 5a–5l, as in compounds 5d, 5e, 5h, 5j, 5l and 7b, positively influenced activity, indicating a promising avenue for drug design modification aimed at developing more potent antitumor agents.
General procedures of target compounds
Preparation of compounds 5a–5l
Intermediates, 2-arylamino-4-chloro-6-methyl-furo[2,3-d]pyrimidines 3 and aryloxyacetylhydrazides 4, were synthesized following a reported procedure. Subsequently, a mixture of 3 (5 mmol) and 4 (6 mmol) in acetonitrile (30 mL) was refluxed for 6–10 h with a catalytic amount of anhydrous K2CO3. After completion of the reaction (monitored by TLC), the mixed system were cooled to room temperature and poured onto crushed ice and the white precipitate was filtered off, washed with water and absolute ethanol, then recrystallized from absolute ethanol and N,N-dimethyl formamide (v/v = 2:1) to obtain the target compound 5a–5l.
6-methyl-2-(m-tolylamino)-4-(2-(2-(p-tolyloxy)acetyl)hydrazinyl)furo[2,3-d]pyrimidine-5-carboxylate (5a)
White solid, yield: 78%, m.p.: 199–201 °C; Anal. HPLC tR= 14.884, purity 99.07%. 1H NMR (400 MHz, DMSO-d6), δ (ppm): 1.33 (t, J = 8.0 Hz, 3H, CH3), 2.23 (s, 3H, CH3), 2.24 (s, 3H, CH3), 2.64 (s, 3H, CH3), 4.34 (q, J = 8.0 Hz, 2H, OCH2), 4.68 (s, 2H, CH2), 6.71–7.63 (m, 8 H, ArH), 9.55 (bs, 2H, NH), 10.57 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6), δ (ppm): 167.71 (C=O), 166.90 (NC=N), 165.26 (NC=C), 157.82 (C=O), 157.11 (Furan-C), 157.07 (OAr-C), 156.03 (Furan-C), 140.72 (NAr-C), 137.99, 130.37, 130.27 (Ar-C), 128.67, 122.63, 119.77, 116.62, 114.93 (Ar-C), 108.33 (Furan-C), 91.25 (Furan-C), 66.52 (CH2), 62.19 (CH2), 21.76 (CH3), 20.56 (CH3), 14.84 (CH3), 14.41 (CH3). IR (KBr, cm−1): 3507, 3181, 1629, 1509, 1233, 1092 and 779. HRMS for C26H27N5O5 [M+H]+, calcd: 490.2090, found: 490.2095 (100). Elemental analysis % calcd. (found): C, 63.79 (63.71); H, 5.56 (5.59); N, 14.31 (14.28).
Ethyl 6-methyl-2-(p-tolylamino)-4-(2-(2-(p-tolyloxy)acetyl)hydrazinyl)furo[2,3-d]pyrimidine-5-carboxylate (5b)
White solid, yield: 86%, m.p.: 212–213 °C; Anal. HPLC tR= 7.992, purity 99.60%.1H NMR (400 MHz, CDCl3), δ (ppm): 1.41 (t, J = 8.0 Hz, 3H, CH3), 2.23 (s, 3H, CH3), 2.32 (s, 3H, CH3), 2.64 (s, 3H, CH3), 4.41 (q, J = 8.0 Hz, 2H, OCH2), 4.66 (s, 2H, CH2), 6.86–7.44 (m, 9 H, ArH and NH), 8.99 (d, J = 4.0 Hz, 1H, NH), 10.13 (d, J = 4.0 Hz, 1H, NH). 13C NMR (100 MHz, CDCl3), δ (ppm):167.05 (C=O), 165.71 (NC=N), 165.17 (NC=N), 157.34 (C=O), 157.14 (Furan-C), 155.22 (OAr-C), 155.21 (Furan-C), 136.84 (NAr-C), 131.93, 131.70, 130.21, 129.34, 119.62, 114.75 (Ar-C), 108.62 (Furan-C), 92.00 (Furan-C), 67.42 (CH2), 61.74 (CH2), 20.71 (CH3), 20.55 (CH3), 14.62 (CH3), 14.29 (CH3). HRMS for C26H27N5O5 [M+H]+, calcd: 490.2090, found: 490.2093 (100). Elemental analysis % calcd. (found): C, 63.79 (63.74); H, 5.56 (5.51); N, 14.31(14.33).
Ethyl 4-(2-(2-(4-methoxyphenoxy)acetyl)hydrazinyl)-6-methyl-2-(m-tolylamino)furo[2,3-d]pyrimidine-5-carboxylate (5c)
White solid, yield: 79%, m.p.: 215–216 °C; Anal. HPLC tR = 14.014, purity 98.91%. 1H NMR (400 MHz, DMSO-d6), δ (ppm): 1.35 (t, J = 8.0 Hz, 3H, CH3), 2.24 (s, 3H, CH3), 2.67 (s, 3H, CH3), 3.69 (s, 3H, CH3), 4.36 (q, J = 8.0 Hz, 2H, OCH2), 4.65 (s, 2H, CH2), 6.70–7.63 (m, 8 H, ArH), 10.45 (s, 1H, NH), 9.50 (bs, 2H, NH), 10.52 (s, 1H, NH). 13C NMR (100 MHz, CDCl3), δ (ppm): 169.11 (C=O), 165.20 (NC=N), 164.19 (NC=N), 159.19 (C=O), 154.31 (Furan-C), 151.80 (Furan-C), 138.76 (2, OC=C), 136.77 (NAr-C), 128.71 (Ar-C), 124.79, 119.89, 116.46, 116.23, 114.41(2) (Ar-C), 109.16 (Furan-C), 91.09 (Furan-C), 68.05 (CH2), 62.58 (CH2), 55.47 (CH3), 21.40 (CH3), 14.36 (CH3), 14.10 (CH3). IR (KBr, cm−1): 3301, 1630, 1508, 1234, 1093 and 757. HRMS for C26H27N5O6 [M+H]+, calcd: 506.2040, found: 506.2043 (100). Elemental analysis % calcd. (found): C, 61.77 (61.69); H, 5.38 (5.42); N, 13.85 (13.80).
Ethyl 2-((4-chlorophenyl)amino)-6-methyl-4-(2-(2-(o-tolyloxy)acetyl)hydrazinyl)-furo[2,3-d]-pyrimidine-5-carboxylate (5d)
White solid, yield: 85%, m.p.: 203–204 °C; Anal. HPLC tR= 5.695, purity 97.13%. 1H NMR (400 MHz, CDCl3), δ (ppm): 1.41 (t, J = 8.0 Hz, 3H, CH3), 2.34 (s, 3H, CH3), 2.65 (s, 3H, CH3), 4.42 (q, J = 8.0 Hz, 2H, OCH2), 4.73 (s, 2H, CH2), 6.89–7.53 (m, 9 H, ArH and NH), 8.85 (d, J = 4.0 Hz, 1H, NH), 10.15 (d, J = 4.0 Hz, 1H, NH). 13C NMR (100 MHz, CDCl3), δ (ppm): 166.80 (C=O), 166.02 (NC=N), 165.12 (NC=N), 157.49 (C=O), 156.85 (Furan-C), 155.49 (Furan-C), 155.39 (OAr-C), 138.13 (NAr-C), 131.29 (Ar-C), 128.67, 127.28, 127.05, 126.71, 122.27, 120.25, 111.85 (Ar-C), 108.62 (Furan-C), 92.58 (Furan-C), 67.54 (CH2), 61.80 (CH2), 16.39 (CH3), 14.60 (CH3), 14.26 (CH3). IR (KBr, cm−1): 3260, 1695, 1622, 1529, 1423, 1228, 1087 and 757. HRMS for C25H24ClN5O5 [M+H]+, calcd: 510.1544, found: 510.1547 (100). Elemental analysis % calcd. (found): C, 58.88 (58.82); H, 4.74(4.70); 13.73 (13.75).
Ethyl 2-((4-fluorophenyl)amino)-6-methyl-4-(2-(2-(p-tolyloxy)acetyl)hydrazinyl)furo[2,3-d]pyrimid-ine-5-carboxylate (5e)
White solid, yield: 73%, m.p.: 211–212 °C; Anal. HPLC tR = 13.866, purity 97.88%. 1H NMR (400 MHz, DMSO-d6), δ (ppm): 1.36 (t, J = 8.0 Hz, 3H, CH3), 2.24 (s, 3H, CH3), 2.66 (s, 3H, CH3), 4.37 (q, J = 8.0 Hz, 2H, OCH2), 4.67 (s, 2H, CH2), 6.92–7.80 (m, 8 H, ArH), 9.48 (s, 1H, NH), 9.53 (s, 1H, NH), 10.48 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6), δ (ppm): 167.71 (C=O), 166.99 (NC=N), 165.27 (NC=N), 157.74(C=O), 157.37 (Ar-C-F), 156.27 (Furan-C), 156.11 (Furan-C), 137.39 (N-Ar-C), 130.49, 130.29, 120.92, 120.84, 115.32, 115.10, 115.03 (Ar-C), 108.30 (Furan-C), 91.36 (Furan-C), 66.74 (CH2), 62.16 (CH2), 20.56 (CH3), 14.83 (CH3), 14.43 (CH3). IR (KBr, cm−1): 3176, 1690, 1627, 1508, 1217, 1093 and 829. HRMS for C25H24FN5O5 [M+H]+, calcd: 494.1840, found: 494.1838 (100). Elemental analysis % calcd. (found): C, 60.85 (60.78); H, 4.90 (4.97); N, 14.19 (14.16).
Ethyl 4-(2-(2-(4-methoxyphenoxy)acetyl)hydrazinyl)-6-methyl-2-(p-tolylamino)furo[2,3-d]pyrimidine-5-carboxylate (5f)
White solid, yield: 85%, m.p.: 209–210 °C; Anal. HPLC tR = 14.864, purity 99.00%. 1H NMR (400 MHz, DMSO-d6), δ (ppm): 1.35 (t, J = 8.0 Hz, 3H, CH3), 2.16 (s, 3H, CH3), 2.66 (s, 3H, CH3), 3.70 (s, 3H, CH3), 4.36 (q, J = 8.0 Hz, 2H, OCH2), 4.66 (s, 2H, CH2), 6.85–7.65 (m, 8 H, ArH), 9.49 (bs, 2H, NH), 10.48 (s, 1H, NH). 13C NMR (100 MHz, CDCl3), δ (ppm): 166.08 (C=O), 164.63 (NC=N), 158.09 (NC=N), 155.63 (C=O), 153.48 (Furan-C), 138.37 (Furan-C), 130.90, 126.95, 126.91, 124.83, 121.65, 116.96, 111.79, 108.72 (Ar-C), 104.68 (Furan-C), 91.50 (Furan-C), 67.21(CH2), 62.09 (CH2), 21.35 (CH3), 16.38 (CH3), 14.38 (CH3), 14.12 (CH3). IR (KBr, cm−1): 3210, 1702, 1629, 1511, 1232, 1093 and 825. HRMS for C26H27N5O6 [M+H]+, calcd: 506.2040, found: 506.2087 (100). Elemental analysis % calcd. (found): C, 61.77 (61.72); H, 5.38 (5.41); N, 13.85 (13.81).
Ethyl 2-((3,5-dimethylphenyl)amino)-6-methyl-4-(2-(2-(p-tolyloxy)acetyl)hydrazinyl)furo[2,3-d]pyri-midine-5-carboxylate (5g)
White solid, yield: 68%, m.p.: 179–180 °C; Anal. HPLC tR = 6.662, purity 99.18%. 1H NMR (400 MHz, CDCl3), δ (ppm): 1.40 (t, J = 8.0 Hz, 3H, CH3), 2.21 (s, 3H, CH3), 2.27 (s, 6 H, CH3), 2.54 (s, 3H, CH3), 4.33 (q, J = 8.0 Hz, 2H, OCH2), 4.89 (s, 2H, CH2), 6.68 (s, 1H, ArH), 6.91–6.98 (m, 4 H, ArH), 7.21 (s, 2H, ArH), 10.74–10.79 (bd, 3H, NH). 13C NMR (100 MHz, CDCl3), δ (ppm): 169.52 (C=O), 169.00 (NC=N), 165.43 (NC=N), 164.28 (C=O), 159.05 (Furan-C), 155.59 (Furan-C), 152.00 (Ar-C), 138.56, 136.80, 130.82, 129.86, 125.74, 117.20, 114.94 (Ar-C), 109.15 (Furan-C), 91.07 (Furan-C), 67.34 (CH2), 62.59 (CH2), 21.41 (CH3), 20.47 (CH3), 14.36 (CH3), 14.12 (CH3). IR (KBr, cm−1): 3513, 3190, 1691, 1630, 1511, 1235, 1096 and 757. HRMS for C27H29N5O5 [M+H]+, calcd: 504.2247, found: 504.2252(100). Elemental analysis % calcd. (found): C, 64.40 (64.35); 5.81 (5.91); N, 13.91 (13.90).
Ethyl 2-((4-chlorophenyl)amino)-6-methyl-4-(2-(2-(p-tolyloxy)acetyl)hydrazinyl)furo[2,3-d]pyrimid-ine-5-carboxylate (5h)
White solid, yield: 84%, m.p.: 203–204 °C; Anal. HPLC tR= 13.411, purity 99.88%. 1H NMR (400 MHz, DMSO-d6), δ (ppm): 1.35 (t, J = 8.0 Hz, 3H, CH3), 2.24 (s, 3H, CH3), 2.66 (s, 3H, CH3), 4.34 (q, J = 8.0 Hz, 2H, OCH2), 4.66 (s, 2H, CH2), 6.92–7.81 (m, 8 H, ArH), 9.47 (s, 1H, NH), 9.51 (s, 1H, NH), 10.45 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6), δ (ppm): 167.71 (C=O), 167.01 (NC=N), 165.3 (NC=N), 157.72 (C=O), 157.47 (Furan-C), 156.22 (OAr-C), 156.08 (Furan-C), 137.45, 130.51, 130.31, 120.85, 120.78, 115.32, 115.02 (Ar-C), 108.27 (Furan-C), 91.35 (Furan-C), 66.74 (CH2), 62.16 (CH2), 20.56 (CH3), 14.84 (CH3), 14.42 (CH3). HRMS for C25H24ClN5O5 [M+H]+, calcd: 510.1544, found: 510.1546(100). Elemental analysis % calcd. (found): C, 58.88 (58.76); H, 4.74 (4.76); N, 13.73 (13.69).
Ethyl 6-methyl-2-(m-tolylamino)-4-(2-(2-(m-tolyloxy)acetyl)hydrazinyl)furo[2,3-d]pyrimidine-5-carboxylate (5i)
White solid, yield: 59%, m.p.: 172–173 °C; Anal. HPLC tR = 5.533, purity 99.86%. 1H NMR (400 MHz, CDCl3), δ (ppm): 1.41 (t, J = 8.0 Hz, 3H, CH3), 2.30 (s, 3H, CH3), 2.34 (s, 3H, CH3), 2.64 (s, 3H, CH3), 4.39 (q, J = 8.0 Hz, 2H, OCH2), 4.67 (s, 2H, CH2), 6.74–7.50 (m, 9 H, ArH and NH), 9.01 (d, J = 4.0 Hz, 1H, NH), 10.14 (d, J = 4.0 Hz, 1H, NH). 13C NMR (100 MHz, CDCl3), δ (ppm): 166.89 (C=O), 165.62 (NC=N), 165.11 (NC=N), 157.20 (C=O), 157.15 (Furan-C), 155.24 (Furan-C), 139.93, 139.35, 138.52, 129.44 (2), 128.66, 123.20, 123.07, 119.88, 116.46, 115.66, 111.60 (Ar-C), 108.56 (Furan-C), 92.10 (Furan-C), 67.08 (CH2), 61.70 (CH2), 21.50 (CH3), 21.46 (CH3), 14.56 (CH3), 14.22 (CH3). HRMS for C26H27N5O5 [M+H]+, calcd: 490.2090, found: 490.2095 (100). Elemental analysis % calcd. (found): C, 63.79 (63.71); H, 5.56 (5.49); N, 14.31 (14.28).
Ethyl 2-((4-fluorophenyl)amino)-6-methyl-4-(2-(2-(m-tolyloxy)acetyl)hydrazinyl)furo[2,3-d]pyri-midine-5-carboxylate (5j)
White solid, yield: 73%, m.p.: 195–197 °C; Anal. HPLC tR= 5.796, purity 97.42%.1H NMR (400 MHz, DMSO-d6), δ (ppm): 1.34 (t, J = 7.2 Hz, 3H, CH3), 2.27 (s, 3H, CH3), 2.65 (s, 3H, CH3), 4.35 (q, J = 7.2 Hz, 2H, OCH2), 4.70 (s, 2H, CH2), 6.81–7.82 (m, 8 H, ArH), 9.50 (d, J = 4.0, 2H, NH), 10.45 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6), δ (ppm): 167.64 (C=O), 166.99 (NC=N), 165.27 (NC=N), 158.58 (C=O), 158.18 (OAr-C), 157.69 (FAr-C), 157.47 (Furan-OC), 156.22 (Furan-OC), 139.52, 137.48, 129.71, 122.54, 120.82, 115.88, 115.3, 112.13 (Ar-C), 108.25 (Furan-C), 91.37 (Furan-C), 66.56 (CH2), 62.14 (CH2), 21.51 (CH3), 14.80 (CH3), 14.38 (CH3). IR (KBr, cm−1): 3395, 3273, 1698, 1533, 1294, 1086, 830 and 767. HRMS for C25H24FN5O5 [M+H]+, calcd: 494.1840, found: 494.1844 (100). Elemental analysis % calcd. (found): C, 60.85 (60.80); H, 4.90 (4.97); N, 14.19 (14.12).
Ethyl 4-(2-(2-(2-chlorophenoxy)acetyl)hydrazinyl)-2-((3,5-dimethylphenyl)amino)-6-methylfuro[2,3-d] -pyrimidine-5-carboxylate (5k)
White solid, yield: 75%, m.p.: 185–187 °C; Anal. HPLC tR= 6.392, purity 98.58%. 1H NMR (400 MHz, DMSO-d6), δ (ppm): 1.32 (t, J = 8.0 Hz, 3H, CH3), 2.18 (s, 6 H, CH3), 2.43 (s, 3H, CH3), 4.27 (q, J = 8.0 Hz, 2H, OCH2), 4.93 (s, 2H, CH2), 6.57 (s, 1H, ArH), 6.79–7.19 (m, 6 H, ArH), 10.58 (s, 1H, NH), 10.67 (s, 2H, NH). 13C NMR (100 MHz, DMSO-d6), δ (ppm): 168.14 (C=O), 165.53 (NC=N), 164.37 (NC=N), 159.21 (C=O), 153.28 (Furan-C), 152.07 (2, Furan-C and OAr-C), 138.55, 136.70, 130.26, 127.84, 125.69, 123.10, 122.54, 117.13, 114.81 (Ar-C), 109.09 (Furan-C), 91.08 (Furan-C), 67.79 (CH2), 62.67 (CH2), 21.36 (CH3), 14.33 (CH3), 14.14 (CH3). IR (KBr, cm−1): 3303, 3024, 1740, 1633, 1420, 1242, 1099 and 758. HRMS for C26H26ClN5O5 [M+H]+, calcd: 524.1701, found: 524.1767 (100). Elemental analysis % calcd. (found): C, 59.60 (59.56); H, 5.00 (5.04); N, 13.37 (13.31).
Ethyl 2-((4-chlorophenyl)amino)-4-(2-(2-(4-methoxyphenoxy)acetyl)hydrazinyl)-6-methylfuro[2,3-d]-pyrimidine-5-carboxylate (5l)
White solid, yield: 72%, m.p.: 196–197 °C; Anal. HPLC tR = 6.136, purity 98.24%. 1H NMR (400 MHz, DMSO-d6), δ (ppm): 1.34 (t, J = 8.0 Hz, 3H, CH3), 2.66 (s, 3H, CH3), 3.70 (s, 3H, CH3), 4.36 (q, J = 8.0 Hz, 2H, OCH2), 4.65 (s, 2H, CH2), 6.85–7.84 (m, 8 H, ArH), 9.50 (s, 1H, NH), 9.61 (s, 1H, NH), 10.45 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6), δ (ppm): 167.84 (C=O), 166.89 (NC=N), 165.26 (NC=N), 157.91 (C=O), 157.43 (d, C), 156.35 (Furan-C), 140.08, 131.08, 128.59, 127.35, 126.63, 125.10, 121.52, 120.65, 112.07 (Ar-C), 108.25 (Furan-C), 91.64 (Furan-C), 66.70 (CH2), 62.19 (CH2), 16.60 (CH3), 14.84 (CH3), 14.40 (CH3). IR (KBr, cm−1): 3409, 3342, 1688, 1622, 1548, 1426, 1226, 1096 and 827. HRMS for C25H24ClN5O6 [M+H]+, calcd: 526.1493, found: 526.1497(100). Elemental analysis % calcd. (found): C, 57.09 (57.00); H, 4.60 (4.65); N, 13.32 (13.28).
Preparation of compounds 6a–6j
A mixture of compound 5 (5 mmol) and phosphorus oxychloride (2 mL) was refluxed for 6–8 h (monitored by TLC), then the reaction mixture was cooled, and then poured into crushed ice to precipitate the product, the crude product was washed with sodium bicarbonate aqueous solution, filtered and recrystallized from CH2Cl2/EtOH to provide compounds 6a–6j.
Ethyl 3-((4-methoxyphenoxy)methyl)-8-methyl-5-(p-tolylamino)-furo[3,2-e][1,2,4]triazolo-[4,3-c]-pyrimidine-9-carboxylate (6a)
Yellow solid, yield: 83%, m.p.: 179–180 °C; Anal. HPLC tR = 7.073, purity 97.96%. 1H NMR (400 MHz, CDCl3), δ (ppm): 1.43 (t, J = 8.0 Hz, 3H, CH3), 2.33 (s, 3H, CH3), 2.69 (s, 3H, CH3), 3.75 (s, 3H, CH3), 4.43 (s, 2H, CH2), 5.05 (s, 2H, CH2), 6.66 (d, J = 8.0 Hz, 2H, ArH), 7.06 (d, J = 8.0 Hz, 2H, ArH), 7.15 (d, J = 8.0 Hz, 2H, ArH), 7.60 (d, J = 8.0 Hz, 2H, ArH), 12.12 (s, 1H, NH). 13C NMR (100 MHz, CDCl3), δ (ppm): 167.43 (C=O), 165.18 (NC=N), 158.8 (Furan-C), 157.30 (Furan-C), 154.65 (OAr-C), 151.64 (OAr-C), 138.34 (NC=N), 135.68 (NC=N), 129.47 (NAr-C), 127.96 (Ar-C), 120.16, 116.57, 114.76 (Ar-C), 108.84 (Furan-C), 100 (Furan-C), 71.00 (CH2), 62.35 (CH2), 55.68 (OCH3), 20.8 5(CH3), 14.76 (CH3), 14.22 (CH3). IR (KBr, cm−1): 3355, 1684, 1625, 1506, 1206, 1107 and 825. HRMS for C26H25N5O5 [M+H]+, calcd: 488.1934, found: 488.2033(100). Elemental analysis % calcd. (found): C, 64.06 (64.01); H, 5.17 (5.19); N, 14.37 (13.33).
Ethyl 8-methyl-5-(p-tolylamino)-3-((m-tolyloxy)methyl)furo[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine-9-carboxylate (6b)
Yellow solid, yield: 84%, m.p.: 173–175 °C; Anal. HPLC tR = 5.320, purity 97.78%. 1H NMR (400 MHz, CDCl3), δ (ppm): 1.41 (t, J = 8.0 Hz, 3H, CH3), 2.31 (s, 3H, CH3), 2.32 (s, 3H, CH3), 2.66 (s, 3H, CH3), 4.41 (q, J = 8.0 Hz, 2H, CH2), 4.94 (s, 2H, CH2), 6.80–7.57 (m, 8 H, ArH), 11.83 (s, 1H, NH). 13C NMR (100 MHz, CDCl3), δ (ppm): 167.58 (C=O), 165.42 (NC=N), 157.92 (NC=N), 157.70 (C=O), 157.69 (Furan-C), 153.46 (Furan-C), 139.71, 136.96, 131.88, 130.34, 129.42, 129.32, 122.61, 119.28, 116.17, 111.80 (Ar-C), 108.54 (Furan-C), 92.72 (Furan-C), 70.69 (CH2), 61.87 (CH2), 21.53 (CH3), 20.80 (CH3), 14.86 (CH3), 14.28 (CH3). IR (KBr, cm−1): 3332, 1694, 1602, 1295, 1234, 1115, 1020 and 768. HRMS for C26H25N5O4 [M+H] +, calcd: 472.1985, found: 472.2077(100). Elemental analysis % calcd. (found): C, 66.23 (66.15); H, 5.34 (5.36); N, 14.85 (14.80).
Ethyl 8-methyl-5-(p-tolylamino)-3-((o-tolyloxy)methyl)furo[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine-9-carboxylate (6c)
Yellow solid, yield: 78%, m.p.: 184–185 °C; Anal. HPLC tR = 5.690, purity 98.74%. 1H NMR (400 MHz, CDCl3), δ (ppm): 1.42 (t, J = 8.0 Hz, 3H, CH3), 2.29 (s, 3H, CH3), 2.31 (s, 3H, CH3), 2.68 (s, 3H, CH3), 4.42 (q, J = 8.0 Hz, 2H, CH2), 4.97 (s, 2H, CH2), 6.93–6.89 (m, 2H, ArH), 7.17–7.11 (m, 4 H, ArH), 7.57 (d, J = 8.0 Hz, 2H, ArH), 11.86 (s, 1H, NH). 13C NMR (100 MHz, CDCl3), δ (ppm): 167.60 (C=O), 165.47 (NC=N), 157.72 (OAr-C), 156.08 (Furan-C), 153.52 (Furan-C), 136.92 (NC=N), 131.94 (NC=N), 130.93, 130.67, 129.43, 127.45, 126.87, 121.44, 119.18, 119.29, 111.75 (Ar-C), 108.57 (Furan-C), 92.75 (Furan-C), 70.83 (CH2), 61.87 (CH2), 20.80 (CH3), 16.27 (CH3), 14.87 (CH3), 14.29 (CH3). IR (KBr, cm−1): 3332, 1686, 1529, 1235, 1121, 1020 and 770. HRMS for C26H25N5O4 [M + Na] +, calcd: 494.1804, found: 494.1805(100). Elemental analysis % calcd. (found): C, 64.06 (64.00); H, 5.17 (5.19); N, 14.37 (14.31).
Ethyl 8-methyl-5-(p-tolylamino)-3-((p-tolyloxy)methyl)furo[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine-9-carboxylate (6d)
Yellow solid, yield: 82%, m.p.: 182–183 °C; Anal. HPLC tR = 11.024, purity 99.74%. 1H NMR (400 MHz, CDCl3), δ (ppm): 1.42 (t, J = 8.0 Hz, 3H, CH3), 2.28 (s, 3H, CH3), 2.32 (s, 3H, CH3), 2.69 (s, 3H, CH3), 4.43 (q, J = 8.0 Hz, 2H, CH2), 5.04 (s, 2H, CH2), 6.99 (d, J = 8.0 Hz, 2H, ArH), 7.17–7.09 (m, 4 H, ArH), 7.59 (d, J = 8.0 Hz, 2H, ArH), 12.07 (s, 1H, NH). 13C NMR (100 MHz, CDCl3), δ(ppm): 167.45 (C=O), 165.23 (NC=N), 157.14 (OAr-C), 155.54 (OC=C), 148.81 (OC=N), 144.56 (NC=N), 136.61 (NC=N), 131.15, 130.07, 129.47, 119.96, 116.48, 115.18, 108.77 (Ar-C), 106.62 (Furan-C), 95.12 (Furan-C), 70.48 (CH2), 62.26 (CH2), 20.86 (CH3), 20.52 (CH3), 14.81 (CH3), 14.25 (CH3). IR (KBr, cm−1): 3353, 1685, 1625, 1509, 1423, 1207, 1109 and 814. HRMS for C26H25N5O4 [M+H] +,calcd: 472.1985,found: 472.1989(100). Elemental analysis % calcd. (found): C, 64.06 (64.02); H, 5.17 (5.16); N, 14.37 (14.35).
Ethyl 8-methyl-5-(m-tolylamino)-3-((m-tolyloxy)methyl)furo[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine-9-carboxylate (6e)
Yellow solid, yield: 81%, m.p.: 172–173 °C; Anal. HPLC tR = 10.034, purity 99.82%. 1H NMR (400 MHz, CDCl3), δ (ppm): 1.42 (t, J = 8.0 Hz, 3H, CH3), 2.32 (s, 3H, CH3), 2.36 (s, 3H, CH3), 2.69 (s, 3H, CH3), 4.42 (q, J = 8.0 Hz, 2H, CH2), 4.96 (s, 2H, CH2), 6.85–6.80 (m, 3H, ArH), 7.28–7.16 (m, 3H, ArH), 7.52 (d, J = 8.0 Hz, 2H, ArH), 11.85 (s, 1H, NH). 13C NMR (100 MHz, CDCl3), δ (ppm): 167.55 (C=O), 165.43 (NC=N), 157.92 (OAr-C), 157.81 (Furan-C), 157.55 (Furan-C), 153.50 (NC=N), 139.72 (NC=N), 139.48, 138.69, 130.45, 129.32, 128.78, 123.22, 122.63, 119.62, 116.20, 116.17, 111.83 (Ar-C), 108.57 (Furan-C), 92.88 (Furan-C), 70.75 (CH2), 61.89 (CH2), 21.73 (CH3), 21.52 (CH3), 14.87 (CH3), 14.28 (CH3). IR (KBr, cm−1): 3365, 1695, 1611, 1537, 1221, 1114, 1050 and 770. HRMS for C26H25N5O4 [M+H] +, calcd: 472.1985, found: 472.1988(100). Elemental analysis % calcd. (found): C, 64.06 (64.01), H, 5.17 (5.18); N, 14.37 (14.36).
Ethyl 5-((4-chlorophenyl)amino)-8-methyl-3-((p-tolyloxy)methyl)furo[3,2-e][1,2,4]triazolo[4,3-c]-pyrimidine-9-carboxylate (6f)
Yellow solid, yield: 85%, m.p.: 191–192 °C; Anal. HPLC tR = 4.937, purity 99.46%. 1H NMR (400 MHz, CDCl3), δ (ppm): 1.47 (t, J = 8.0 Hz, 3H, CH3), 2.29 (s, 3H, CH3), 2.76 (s, 3H, CH3), 4.47 (q, J = 8.0 Hz, 2H, CH2), 5.40 (s, 2H, CH2), 6.98 (d, J = 8.0 Hz, 2H, ArH), 7.11 (d, J = 8.0 Hz, 2H, ArH), 7.38 (d, J = 8.0 Hz, 2H, ArH), 7.75 (d, J = 8.0 Hz, 2H, ArH), 8.40 (s, 1H, NH). 13C NMR (100 MHz, CDCl3), δ (ppm): 163.74 (C=O), 162.70 (NC=N), 158.10 (NC=N), 157.17 (C=O), 156.18 (Furan-C), 149.57 (Furan-C), 140.39 (Ar-C), 135.34, 130.90, 130.02, 129.64, 129.30, 121.30, 114.70 (Ar-C), 109.81 (Furan-C), 97.28 (Furan-C), 64.36 (CH2), 61.22 (CH2), 20.51 (CH3), 14.35 (CH3), 14.27 (CH3). IR (KBr, cm−1): 3606, 3297, 1706, 1573, 1516, 1406, 1290, 1098 and 826. HRMS for C25H22ClN5O4 [M + Na] +, calcd: 514.1258, found: 514.1262(100). Elemental analysis % calcd. (found): C, 61.04 (60.98); 4.51 (4.54); N, 14.24 (14.19).
Ethyl 3-((4-methoxyphenoxy)methyl)-8-methyl-5-(m-tolylamino)furo[3,2-e][1,2,4]triazolo[4,3-c]-pyrimidine-9-carboxylate (6g)
Yellow solid, yield: 80%, m.p.: 184–185 °C; Anal. HPLC tR = 5.300, purity 95.89%. 1H NMR (400 MHz, CDCl3), δ (ppm): 1.46 (t, J = 8.0 Hz, 3H, CH3), 2.40 (s, 3H, CH3), 2.72 (s, 3H, CH3), 3.79 (s, 3H, CH3), 4.46 (q, J = 8.0 Hz, 2H, CH2), 4.98 (s, 2H, CH2), 7.54–6.85 (m, 8 H, ArH), 11.92 (s, 1H, NH). 13C NMR (100 MHz, CDCl3), δ (ppm): 167.52 (C=O), 165.41 (NC=N), 157.98 (NC=N), 154.61 (C=O), 151.93 (Furan-C), 139.31 (Furan-C), 138.74, 128.80 (2), 123.40, 119.75, 116.48, 116.32, 114.72 (2) (Ar-C), 108.62 (Furan-C), 92.77 (Furan-C), 71.59 (CH2), 61.99 (CH2), 55.70 (CH3), 21.74 (CH3), 14.90 (CH3), 14.30 (CH3). IR (KBr, cm−1): 3367, 1690, 1595, 1280, 1122 and 769. HRMS for C26H25N5O5 [M+H] +, calcd: 488.1934, found: 488.1999(100). Elemental analysis % calcd. (found): C, 64.06 (63.98); 5.17 (5.20); N, 14.37 (14.32).
Ethyl 5-((4-fluorophenyl)amino)-8-methyl-3-((o-tolyloxy)methyl)furo[3,2-e][1,2,4]triazolo[4,3-c]-pyrimidine-9-carboxylate (6h)
Yellow solid, yield: 79%, m.p.: 187–188 °C; Anal. HPLC tR = 7.752, purity 96.64%. 1H NMR (400 MHz, CDCl3), δ (ppm): 1.46 (t, J = 8.0 Hz, 3H, CH3), 2.32 (s, 3H, CH3), 2.72 (s, 3H, CH3), 4.46 (q, J = 8.0 Hz, 2H, OCH2), 5.01 (s, 2H, CH2), 6.92–7.67 (m, 8 H, ArH), 11.94 (s, 1H, NH). 13C NMR (100 MHz, CDCl3), δ (ppm): 167.47 (C=O), 165.43 (NC=N), 159.71 (OAr-C), 157.98 (FAr-C), 156.03 (Furan-C), 153.34 (Furan-C), 135.43 (NC=N), 131.28 (NC=N), 130.97 (NAr-C), 127.46, 126.9, 121.51, 120.94, 120.86, 115.60, 111.78 (Ar-C), 108.64 (Furan-C), 92.94 (Furan-C), 70.78 (CH2), 62.01 (CH2), 16.28 (CH3), 14.89 (CH3), 14.30 (CH3). IR (KBr, cm−1): 3350, 1692, 1622, 1538, 1217, 1116, 1050 and 770. HRMS for C25H22FN5O4 [M+H] +, calcd: 476.1734, found: 476.1815(100). Elemental analysis % calcd. (found): C, 63.15 (63.09); H, 4.66 (4.71); N, 14.73 (14.68).
Ethyl 5-((4-fluorophenyl)amino)-3-((4-methoxyphenoxy)methyl)-8-methylfuro[3,2-e][1,2,4]triazolo-[4,3-c]pyrimidine-9-carboxylate (6i)
Yellow solid, yield: 81%, m.p.: 188–189 °C; Anal. HPLC tR = 9.658, purity 96.69%. 1H NMR (400 MHz, CDCl3), δ (ppm): 1.50 (t, J = 8.0 Hz, 3H, CH3), 2.79 (s, 3H, CH3), 3.80 (s, 3H, CH3), 4.50 (q, J = 8.0 Hz, 2H, OCH2), 5.42 (s, 2H, CH2), 6.86–7.77 (m, 8 H, ArH), 8.33 (s, 1H, NH). 13C NMR (100 MHz, CDCl3), δ (ppm): 163.77 (C=O), 162.74 (NC=N), 160.91 (FAr-C), 158.48 (NC=N), 157.95 (Furan-C), 154.42 (Furan-C), 152.49 (OAr-C), 149.63 (NC=N), 140.85 (NC=N), 132.64, 122.23, 115.96 (2), 114.68 (Ar-C), 109.81 (Furan-C), 97.08 (Furan-C), 65.05 (CH2), 61.22 (CH2), 55.71 (OCH3), 14.38 (CH3), 14.29 (CH3). IR (KBr, cm−1): 3375, 1702, 1615, 1581, 1507, 1227, 1068 and 823. HRMS for C25H22FN5O4 [M+H] +, calcd: 492.1683, found: 492.1766 (100). Elemental analysis % calcd. (found): C, 61.10 (61.05); H, 4.51 (4.56); N, 14.25 (14.21).
Ethyl 8-methyl-5-(m-tolylamino)-3-((p-tolyloxy)methyl)furo[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine-9-carboxylate (6j)
Yellow solid, yield: 83%, m.p.: 176–178 °C; Anal. HPLC tR = 14.034, purity 99.25%. 1H NMR (400 MHz, CDCl3), δ (ppm): 1.42 (t, J = 7.2 Hz, 3H, CH3), 2.28 (s, 3H, CH3), 2.36 (s, 3H, CH3), 2.68 (s, 3H, CH3), 4.43 (q, J = 7.2 Hz, 2H, OCH2), 4.95 (s, 2H, CH2), 6.84 (d, J = 7.6 Hz, 1H, ArH), 6.92 (d, J = 8.8 Hz, 2H, ArH), 7.09 (d, J = 8.4 Hz, 2H, ArH), 7.21 (t, J = 8.0 Hz, 1H, ArH), 7.51–7.52 (m, 2H, ArH), 11.84 (s, 1H, NH). 13C NMR (100 MHz, CDCl3), δ (ppm): 167.54 (C=O), 165.42 (NC=N), 157.8 (Furan-C), 157.54 (OAr-C), 155.78 (Furan-C), 153.48 (NC=N), 139.49 (NC=N), 138.69 (NAr-C), 131.10 (CC=C), 130.58 (CC=C), 130.04 (Ar-C), 128.78, 123.21, 119.62, 116.20, 115.08 (Ar-C), 108.56 (Furan-C), 92.87 (Furan-C), 70.96 (CH2), 61.89 (CH2), 21.73 (CH3), 20.53 (CH3), 14.87 (CH3), 14.28 (CH3). IR (KBr, cm−1): 3356, 1695, 1613, 1537, 1209, 1112, 1024 and 769. HRMS for C26H25N5O4 [M+H] +, calcd: 472.1985, found: 472.1989 (100). Elemental analysis % calcd. (found): C, 64.06 (64.01); H, 5.17 (5.24); N, 14.37 (14.32).
Preparation of compounds 7a, 7b
A mixture of ethyl 4-chloro-2-((4-methoxyphenyl)amino)-6-methylfuro[2,3-d]pyrimidine-5-carboxylate or ethyl 4-chloro-2-((4-fluorophenyl)amino)-6-methylfuro[2,3-d]pyrimidine-5-carboxylate (5 mmol), respectively, and butyral hydrazine (6 mmol) was refluxed in acetonitrile (25 mL) for 6 h in the presence of a catalytic amount of anhydrous potassium carbonate, and then the reaction system was poured into crushed ice and the white precipitate was filtered off, washed with water and absolute ethanol. The dried solid and phosphorus oxychloride (2 mL) was refluxed for 6 h, then similar treatment procedure as 6a–6j to provide compounds 7a, 7b.
Ethyl 5-((4-methoxyphenyl)amino)-8-methyl-3-propylfuro[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine-9-carboxylate (7a)
Yellow solid, yield: 67%, m.p.: 173–174 °C; Anal. HPLC tR = 15.732, purity 98.85%. 1H NMR (400 MHz, CDCl3), δ (ppm): 1.06 (t, J = 8.0 Hz, 3H, CH3), 1.47 (t, J = 8.0 Hz, 3H, CH3), 1.95–2.00 (m, 2H, CH2), 2.76 (s, 3H, CH3), 2.98 (t, J = 8.0 Hz, 2H, CH2), 3.85 (s, 3H, CH3), 4.50 (q, J = 8.0 Hz, 2H, CH2), 6.97 (d, J = 4.0 Hz, 2H, ArH), 7.69 (d, J = 4.0 Hz, 2H, ArH), 8.13 (s, 1H, NH). 13C NMR (100 MHz, CDCl3), δ (ppm): 168.20 (C=O), 162.97 (NC=N), 157.42 (Furan-C), 156.76 (Furan-C), 149.30 (OAr-C), 141.06 (NC=N), 129.78 (NC=N), 122.33 (Ar-C), 114.48 (Ar-C), 109.70 (Furan-C), 96.28 (Furan-C), 61.07 (CH2), 55.56 (OCH3), 31.11 (CH2), 21.42 (CH2), 14.34 (CH3), 14.19 (CH3), 14.11 (CH3). IR (KBr, cm−1): 3269, 1711, 1576, 1247, 1124, 807 and 759. HRMS for C21H23N5O4 [M+H] +, calcd: 410.1828, found: 410.1877 (100). Elemental analysis % calcd. (found): C, 61.60 (61.54); H, 5.66 (5.72); N, 17.10 (17.02).
Ethyl 5-((4-fluorophenyl)amino)-8-methyl-3-propylfuro[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine-9-carboxylate (7b)
Yellow solid, yield: 87%, m.p.: 187–188 °C; HPLC tR = 14.343, purity 99.88%. 1H NMR (400 MHz, CDCl3), δ (ppm): 1.10 (t, J = 8.0 Hz, 3H, CH3), 1.51 (t, J = 8.0 Hz, 3H, CH3), 1.95–2.00 (m, 2H, CH2), 2.79 (s, 3H, CH3), 2.97 (t, J = 8.0 Hz, 2H, CH2), 4.50 (q, J = 8.0 Hz, 2H, CH2), 7.13–7.18 (m, 2H, ArH), 7.77–7.80 (m, 2H, ArH), 8.23 (s, 1H, NH). 13C NMR (100 MHz, CDCl3), δ (ppm): 168.36 (C=O), 162.89 (NC=N), 160.78 (FAr-C), 158.36 (Furan-C), 157.71 (Furan-C), 149.29 (NC=N), 140.60 (NC=N), 132.90 (NAr-C), 121.99 (2, Ar-C), 116.15 (2, Ar-C), 109.77 (Furan-C), 96.79 (Furan-C), 61.12 (CH2), 31.09 (CH2), 21.40 (CH2), 14.34 (CH3), 14.23 (CH3), 14.10 (CH3). IR (KBr, cm−1): 3290, 1714, 1580, 1210, 1020, 832 and 757. HRMS for C20H20FN5O3 [M+H]+, calcd: 198.1628, found: 398.1671 (100). Elemental analysis % calcd. (found): C, 60.45 (60.48); H, 5.07 (5.14); N, 17.62 (17.57).
Associated content
Theoretical calculations and biological evaluation methods in this study are available in supporting information.
Supplementary Information
Below is the link to the electronic supplementary material.
Acknowledgements
Thanks to Dr. Fengxu Wu for Fig. 5.
Author contributions
W.Z. and B.Z conducted molecular synthesis and bioactivity assay. H.G. and C.F. conducted data analysis, results discussion and wrote the main manuscript text. J.M. and Y.H. administration of research group, designed the experiments, carried out the results discussion and writing-review and editing.
Funding
The central government guides local scientific and technological development special fund project of Hubei province (2022BGE260) and the advantages discipline group (Biology and Medicine) project in higher education of Hubei Province (2021–2025, No. 2025BMXKQY7).
Data availability
The data presented in this study are available in this article and the supplementary Information.
Declarations
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Ethical approval and consent to participate
Not applicable.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Junkai Ma, Wenyu Zhao, Haitao Gao and Bingqian Zhang contributed equally to this work.
Contributor Information
Chun Feng, Email: fengchun@hbmu.edu.cn.
Yanggen Hu, Email: huyg@hbmu.edu.cn.
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Data Availability Statement
The data presented in this study are available in this article and the supplementary Information.