GREEN SYNTHESIS OF NOVEL THIENOPYRIMIDINE DERIVATIVES

ABSTRACT

Thieno[2,3-d]pyrimidin-4-one (TP) derivatives constitute a significant class of heterocyclic compounds with diverse biological activities, particularly anticancer properties. In this work, environmentally friendly synthetic approaches were applied to obtain novel derivatives under mild conditions consistent with the principles of green chemistry. Initially, 2-aminothiophene ester was synthesized via an improved Gewald multicomponent reaction and subsequently cyclized with formamide to yield 5,6-disubstituted TP. Further modification at the 6-position afforded a carbohydrazide intermediate, which underwent condensation with 4-methoxybenzaldehyde to give compound 5. Structural elucidation was performed using ¹H and ¹³C NMR spectroscopy together with TLC-MS analysis, confirming the successful formation of the target compound. The developed methodology demonstrates the efficiency of green synthetic strategies, providing access to new thienopyrimidine derivatives with promising potential as bioactive molecules.

АННОТАЦИЯ

Производные тиено[2,3-d]пиримидин-4-она (TП) представляют собой важный класс гетероциклических соединений с широким спектром биологической активности, особенно противораковой. В данной работе применены экологически чистые методы синтеза для получения новых производных в мягких условиях, соответствующих принципам «зелёной химии». На первом этапе был синтезирован сложный эфир 2-аминтиофена с использованием усовершенствованной много-компонентной реакции Гевальда, который затем был циклизован с формиамидом с образованием 5,6-дизамещённого TП. Дальнейшая модификация в положении 6 привела к образованию промежуточного карбогидразида, который подвергли конденсации с 4-метоксибензальдегидом с получением соединения 5. Структурная идентификация была проведена с использованием спектроскопии ¹H и ¹³C ЯМР в сочетании с TLC-MS анализом, что подтвердило успешное образование целевого соединения. Разработанная методология демонстрирует эффективность зелёных синтетических стратегий, обеспечивая доступ к новым производным тиенопиримидина с перспективным потенциалом в качестве биологически активных молекул.

Keywords: thieno[2,3-d]pyrimidin-4-one; green chemistry; Gewald reaction; MW irradiation; carbohydrazide; NMR spectroscopy; TLC MS; anticancer agents.

Ключевые слова: тиено[2,3-d]пиримидин-4-он; зелёная химия; реакция Гевальда; СВЧ-облучение; карбогидразид; ЯМР-спектроскопия; TLC-MS; противораковые агенты.

Introduction

TPs are an important class of heterocyclic compounds with broad pharmacological significance [1-5]. Their biological properties strongly depend on substitution patterns, and many derivatives show remarkable anticancer activity [6-13]. Examples include the drugs relugolix (Orgovyx) and sufugolix (Antigonodatropin). The development of novel thienopyrimidine derivatives using mild, efficient, and environmentally friendly synthetic approaches is therefore of great importance. In this study, new thienopyrimidine derivatives were synthesized and structurally characterized in accordance with green chemistry principles (one-pot reactions, multicomponent reactions, microwave-assisted synthesis, room-temperature conditions, safe and volatile catalysts, solvent-free or non-toxic solvent systems), highlighting their potential as bioactive scaffolds (Fig. 1).

Figure 1. Effective preparations containing TP core

Materials and methods

Solvents. Hexane, petroleum ether (40–70 °C), chloroform, methanol, ethanol, dimethylformamide (DMF), and ethyl acetate were used without further purification. IR spectra were recorded on a Perkin-Elmer FTIR 2000 spectrometer using KBr tablets. ¹H and ¹³C NMR spectra were obtained on JNM-ECZ 400 and JNM-ECZ 600 spectrometers (JEOL, Japan) operating at 400 and 600 MHz. TMS was used as an internal standard (δ scale), and spectra were recorded in deuterated solvents: CDCl₃, C₅D₅N, CD₃OD, and DMSO-d₆. Mass spectra were recorded on a CAMAG TLC-MS system (Germany) equipped with an ACQUITY QDa detector. TLC analyses were performed on silica gel 60 F254 L/W 20 × 20 cm plates (Merck, Germany). Visualization was carried out under UV light at 254 and 366 nm using a CAMAG UV Lamp 4 (Germany). Melting points were measured on a MEL-TEMP electrothermal apparatus (USA).

5-Amino-3-methylthiophene-2,4-diethyldicarboxylate (1).

Acetoacetic ethyl ester (6.32 mL, 6.50 g, ρ=1.029 g/ml, 0.05 mol), cyanoacetic ethyl ester (5.32 mL, 5.65 g, ρ=1.063 g/ml, 0.05 mol), S₈ (1.76 g, 0.055 mol) and absolute ethanol (15 mL) were placed in a 100 mL double-necked round-bottom flask. Morpholine (4.80 mL, 4.78 g, ρ=0.996 g/ml, 0.055 mol) was added dropwise, and the mixture was irradiated in a microwave reactor at 50 °C for 3 min. The resulting solution was kept overnight in a refrigerator, diluted with distilled water (100 mL), and stirred for 3 h. The precipitate was filtered, washed with water, and dried to give compound 1 (10.28 g, 80%). M.p. 104–105 °C (petroleum ether, 40–70 °C). Rf = 0.29 (n-hexane/ethyl acetate, 5:1). IR spectrum (KBr, ν, cm^–1): 3410, 3310 (NH₂), 2998, 2928 (CH₃), 1692, 1667 (O-C=O), 1591, 1542 (C=O), 1225 (C-N), 784 (C-S-C). ¹Н NMR (400 MHz, СDCl₃, δ, ppm, J/Hz): 1.33 (3H, t, J=7.1, 3-СООСН₂CH₃), 1.38 (3H, t, J=7.1, 5-СООСН₂CH₃), 2.70 (3H, s, 4-CH₃), 4.26 (2H, q, J=7.1, 3-СООСН₂CH₃), 4.31 (2H, q, J=7.1, 5-СООСН₂CH₃), 6.52 (2H, s, NH₂). ¹³С NMR (100 MHz, СDCl₃, δ, ppm): 14.48, 14.54, 16.26, 60.23, 60.55, 108.62, 108.71, 148.18, 163.01, 166.20, 166.28.

5-Methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-6-ethylcarboxylate (2).

Compound 1 (7.71 g, 0.03 mol) and formamide (12 mL, 13.56 g, ρ=1.13 g/ml, 0.30 mol) were heated at 150 °C for 4 h in a 100 mL round-bottom flask equipped with a reflux condenser. After cooling, the precipitate was filtered, washed with water, and dried. Recrystallization from ethanol gave compound 2 (6.28 g, 88%). M.p. 243-244 °C (ethanol), Rf=0.35 (benzene/methanol, 5:1). IR spectrum (KBr, ν, cm^–1): 2878, 1704, 1684, 1581, 1462, 1294, 1258, 1168, 1089, 1009, 905, 761, 567. ¹Н NMR (400 MHz, DMSO-d₆+CCl₄, δ, ppm, J/Hz): 1.39 (3H, t, J=7.1, 6-COOCH₂CH₃), 2.84 (3H, s, 5-CH₃), 4.31 (2H, q, 6-COOCH₂CH₃), 7.99 (1H, s, 2-CH), 12.49 (1H, wide s, NH). ¹³С NMR (100 MHz, DMSO-d₆+CCl₄, δ, ppm): 13.99, 14.62, 60.29, 121.56, 123.84, 143.30, 147.03, 158.07, 161.33, 165.69.

3-Butyl-5-methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-6-ethylcarboxylate (3).

Compound 2 (4.76 g, 0.02 mol), n-butyl bromide (2.58 mL, 3.29 g, ρ=1.276 g/ml, 0.024 mol) and K₂CO₃ (3.31 g, 0.024 mol) in DMF (30 mL) were heated at 70 °C for 6 h under reflux. After cooling, the precipitate was filtered, washed with water, and dried. Recrystallization from ethanol afforded compound 3 (3.88 g, 66%). M.p. 143–144 °C (ethanol). Rf = 0.72 (chloroform/methanol, 10:1). ¹Н NMR (600 MHz, СDCl₃+CCl₄, δ, ppm, J/Hz): 1.00 (3H, t, J=7.4, NCH₂(CH₂)₂CH₃), 1.40 (3H, t, J=7.1, COOCH₂CH₃), 1.44 (2H, sext, J=7.5, N(CH₂)₂CH₂CH₃), 1.77 (2H, quin, J=7.6, NCH₂CH₂CH₂CH₃), 2.92 (3H, s, 5-CH₃), 3.96 (2H, t, J=7.4, NCH₂(CH₂)₂CH₃), 4.36 (2H, q, J=7.1, COOCH₂CH₃), 7.97 (1H, s, 2-CH). ¹³С NMR (150 MHz, СDCl₃+CCl₄, δ, ppm): 13.93, 14.65, 15.38, 20.16, 31.82, 46.77, 61.22, 123.95, 123.97, 144.43, 148.37, 158.11, 162.29, 165.38. Mass spectrum m/z 317.4035 [M+Na]⁺ (calcd. for C₁₄H₁₈N₂O₃SNa, 317.3575).

3-Butyl-5-methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-6-carbohydrazide (4).

Compound 3 (2.94 g, 0.01 mol), hydrazine hydrate (10.0 mL, 0.06 mol) and ethanol (15 mL) were refluxed for 4 h, then left overnight. The precipitate was filtered, washed with ethanol (3 × 10 mL) and water, and dried to yield compound 4 (1.56 g, 53%). M.p. 156–158 °C (ethanol). Rf = 0.35 (benzene/methanol, 5:1). ¹Н NMR (600 MHz, СDCl₃+CD₃OD, δ, ppm, J/Hz): 0.82 (3H, t, J=7.4, NCH₂(CH₂)₂CH₃), 1.25 (2H, sext, J=7.4, N(CH₂)₂CH₂CH₃), 1.60 (2H, quin, J=8.1, NCH₂CH₂CH₂CH₃), 2.65 (3H, s, 5-CH₃), 3.84 (2H, t, J=7.4, NCH₂(CH₂)₂CH₃), 3.94 (2H, wide s, NH₂), 7.94 (1H, s, 2-CH). ¹³С NMR (150 MHz, СDCl₃+CD₃OD, δ, ppm): 13.32, 14.92, 19.68, 31.30, 46.67, 123.47, 126.27, 138.16, 148.27, 158.28, 163.63, 163.67. Mass spectrum m/z 223.2639 [M–C₄H₉]⁺ (calcd. for C₈H₇N₄O₂S, 223.2289).

(E)-3-butyl-N'-(4-methoxybenzylidene)-5-methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-6-carbohydrazide (5).

Compound 4 (0.56 g, 0.002 mol), 4-methoxybenzaldehyde (0.30 mL, 0.326 g, ρ=1.12 g/mL, 0.0024 mol), concentrated HCl (0.20 mL, 32%), and distilled water (10 mL) were stirred for 4 h at room temperature. The product was neutralized with saturated NaHCO₃ solution, filtered, washed with ethanol and water, and dried. Recrystallization from ethanol gave compound 5 (0.56 g, 70%). M.p. 187–189 °C (ethanol). Rf = 0.60 (benzene/methanol, 5:1). ¹Н NMR (600 MHz, СDCl₃+CCl₄, δ, ppm, J/Hz): 1.00 (3H, t, J=7.4, NCH₂(CH₂)₂CH₃), 1.43 (2H, sext, J=7.4, N(CH₂)₂CH₂CH₃), 1.79 (2H, quin, J=7.6, NCH₂CH₂CH₂CH₃), 3.06 (3H, s, 5-CH₃), 3.85 (3H, s, OCH₃), 3.99 (2H, t, J=7.5, NCH₂(CH₂)₂CH₃), 6.93 (2H, d, J=8.4, 2ʹ and 6ʹ), 7.68 (2H, d, J=8.2, 3ʹ and 5ʹ), 7.87 (1H, s, HN–N=CH), 8.03 (1H, s, 2-CH), 10.16 (1H, wide s, HN–N=CH). Mass spectrum m/z 421.4538 [M+Na]⁺ (calcd. for C₂₀H₂₂N₄O₃SNa, 421.4685).

Results and discussions

An improved one-pot multicomponent Gewald reaction was applied to obtain 2-aminothiophene ester (1) from ethyl acetoacetate, ethyl cyanoacetate, and morpholine under microwave irradiation at 50 °C for 3 min. The product was subsequently cyclized with excess formamide at 150 °C for 4 h, yielding 5,6-disubstituted TP (2). Compound 2 was alkylated with n-butyl bromide and K₂CO₃ in DMF (respectively in a 1:1.2:1.2 equiv.) at 70 °C for 6 h, giving the corresponding derivative. Further modification at the 6-position was achieved via nucleophilic substitution of the ethyl ester (3) with excess hydrazine hydrate, leading to the formation of carbohydrazide derivative (4). Condensation of compound 4 with 4-methoxybenzaldehyde (1.2 equiv.) was carried out in aqueous medium with concentrated HCl (32%) as catalyst at room temperature for 4 h. This reaction produced (E)-3-butyl-N′-(4-methoxybenzylidene)-5-methyl-4-oxo-3,4-dihydrothieno[2,3-d] pyrimidine-6-carbohydrazide (5) in good yield (Fig. 2).

Figure 2. General scheme of chemical reactions

The ¹H NMR spectrum of compound 5 displayed characteristic signals: a broad singlet at 10.16 ppm (NH of hydrazine fragment), a singlet at 8.03 ppm (aromatic proton of TP core), and a singlet at 7.87 ppm (hydrazomethine proton, –HN–N=CH–). Aromatic protons of the benzene ring were observed as two doublets at 6.93 (2H, d, J=8.4) and 7.68 (2H, d, J=8.2) ppm, consistent with the “roofing effect”. Signals at 3.06 ppm and 3.85 ppm corresponded to methyl (TP core) and methoxy groups, while n-butyl protons appeared a triplet at 3.99 (2H, t, J=7.5; CH₂(CH₂)₂CH₃), a quintet at 1.79 (2H, quin, J=7.6; CH₂CH₂CH₂CH₃), 1.43 a sextet at (2H, sext, J=7.4; (CH₂)₂CH₂CH₃), and a triplet at 1.00 (3H, t, J=7.4; CH₂(CH₂)₂CH₃) ppm (Fig. 3, 4).

Figure 3. ¹H NMR spectrum of compound 5

Figure 4. Multiplicity of protons of compound 5 in ¹H NMR spectra

TLC-MS analysis revealed a molecular ion peak at m/z 421.4538 [M+Na]⁺, in excellent agreement with the calculated value (421.4685), confirming the structure of compound 5.

The experimental results demonstrate the successful application of environmentally friendly synthetic methods for producing novel thienopyrimidine derivatives. Microwave irradiation and multicomponent reactions enabled efficient generation of 2-aminothiophene intermediates under mild conditions. Subsequent cyclization, alkylation, and hydrazinolysis yielded versatile carbohydrazide scaffolds. The condensation of carbohydrazide (4) with 4-methoxybenzaldehyde afforded a novel E-isomeric derivative 5. Its structure was validated by spectroscopic and mass spectrometric data. These findings confirm the utility of green chemistry approaches for synthesizing structurally diverse thienopyrimidines with high potential for biological applications, especially in anticancer research.

Conclusion

A new E-isomeric thienopyrimidine carbohydrazide derivative was synthesized and structurally confirmed. The study highlights the efficiency of green synthetic strategies, offering access to bioactive heterocyclic frameworks with potential pharmaceutical and agrochemical applications.

Acknowledgments

The authors are grateful to the Institute of the Chemistry of Plant Substances of Academy Sciences of Uzbekistan for providing laboratory facilities for spectroscopy and diffraction experiment.

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